CN1950392A - Lactobacillus acidophilus nucleic acid sequences encoding carbohydrate utilization-related proteins and uses therefor - Google Patents

Abstract

The present invention discloses carbohydrate utilization-related and multidrug transporter nucleic acids and polypeptides, and fragments and variants therof, are disclosed in the current invention. In addition, carbohydrate utilization-related and multidrug transporter fusion proteins, antigenic peptides, and anti-carbohydrate utilization-related and antimultidrug transporter antibodies are encompassed. The invention also provides vectors containing a nucleic acid of the invention and cells into which the vector has been introduced. Methods for producing the polypeptides and methods of use for the polypeptides of the invention are further disclosed.

Description

Encoding carbohydrate utilizes lactobacillus acidophilus nucleic acid sequences of associated protein and uses thereof

The mutual reference of related application

The application requires to submit on March 7th, 2005, autograph for " encoding carbohydrate utilizes lactobacillus acidophilus nucleic acid sequences of associated protein and uses thereof ", listed invention people for Todd R.Klaenhammer, Eric Altermann, Rodolphe Barrangu, W.MichaelRussell and Tri Duong, attorney docket be 5051-693 the U. S. application series number _ _ _ _ right of priority, it has required the U.S. Provisional Application series number 60/551 of submission on March 8th, 2004,121 rights and interests, the content of each piece all is incorporated herein by reference at this.

Invention field

The present invention relates to separate from milk-acid bacteria, promptly the polynucleotide of Lactobacterium acidophilum (lactobacillusacidophilus) by they encoded polypeptides, and use this polypeptide and express the method for their biology.

Background of invention

Lactobacterium acidophilum is Gram-positive, shaft-like, the homofermentation bacterium that do not form gemma, and it is that the normal habit of gi tract and urogenital tract occupies bacterium.Since from infant faeces, being separated to this bacterium by Moro (1900) at first, the people, with breast-fed infant with consume in people's the enteron aisle of high breast, lactose or dextrin diet and found this " having a liking for acid " biology.In history, Lactobacterium acidophilum be often be believed to gi tract germ thing group cause lactobacillus species in the beneficial bacteria of intestinal tract of beneficial effect (Klaenhammer and Russell (2000) " Species ofthe Lactobacillus acidophilus complex; " Encyclopedia of FoodMicrobiology, 2:1151-1157.Robinson et al., eds. (AcademicPress, San Diego, California).Lactobacterium acidophilum can zymohexose, comprises lactose and complex oligosaccharide more, to produce lactic acid and to reduce and cultivate biological environment pH.Acidifying environment (for example food, vagina and gi tract inner compartment) can disturb undesirable bacterium, pathogenic agent and zymic growth.This biology is because its acid resistance, the survival in cultivating milk-product and be known by the viability in stomach and the gi tract process.There are some to be considered to the probiotic bacterium of " helping life " in Bacterium lacticum and other symbiotic bacterium, broad research they to the influence of human health, the especially influence in prevention or treatment intestinal tract infections, diarrhea disease, preventing cancer and stimulating immune system.Also studied the influence of Bacterium lacticum to flavor of dairy products and function and constitutional features.The hereditary feature (for example Lactobacillus johnsonii (L johnsonii) and lactobacillus rhamnosus (L.rhamnosus)) of having described other lactobacillus species is (referring to for example United States Patent (USP) 6,476,209; United States Patent (USP) 6,544,772; U.S. Patent Publication No. 20020159976,2003013882 ﹠amp; 20040009490; PCT publication number WO 2004/031389; PCT publication number 2003/084989; PCT publication number WO 2004/020467).

Bacterial growth needs the specialized transport system to introduce nutrient substance from external environment.Milk-acid bacteria by three systems with molecule transport into and transport out cell: elementary transhipment, secondary transhipment and group translocation.In elementary transhipment, use chemical agent (mainly being ATP), electricity or Driven by Solar Energy transhipment.ATP is an elementary movement system type the most plentiful in the milk-acid bacteria in conjunction with box (ABC) translocator.In this system, film is passed in the transportation of ATP hydrolysis link substrate, introduces sugar and compatible solutes and medicine or the toxin of output product as not conforming to the cell needs, or the cellular constituent that works in the extracellular, as cell wall polysaccharides.Usually, abc transport albumen is relative specificity for their substrate, but some are arranged is polyspecifics, as multidrug transporter.

The secondary movement system uses electrochemical gradient so that the energy of sugar transportation to be provided.They comprise symport albumen (two or more solutes of its co-transport), uniport albumen (it transports a molecule) and antiport albumen (it transports two or more solutes on the contrary).Symport albumen moves upward substrate usually and proton (or ion) moves downward coupling, antiport albumen uses the ion gradient secretory product, and uniport albumen does not use coupling ion (Poolman (2002) Antonie vanLeeuwenhoek 82:147-164).

Group translocation comprises phosphoenolpyruvic acid (PEP) dependency phosphotransferase system (PTS), and it is with the picked-up and its phosphorylation coupling of carbohydrate or sugar alcohol get up (Poolman (2002), above-mentioned).Phosphate derives from the transformation of PEP to pyruvic acid, and phosphorylation subsequently relates to energy coupling albumen, enzyme I and HPr and substrate specificity phosphoryl transfer protein IIA, IIB and IIC.

Multidrug transporter can be divided into two kinds of main types, secondary multidrug transporter and abc transport albumen.The secondary multidrug transporter can be further divided into different families, comprise main promotion superfamily protein (major facilitator superfamily, MFS), little multi-medicine resistance (the small multidrug resistance family of family, SMR), resistance-(the resistance-nodulation-cell division family of brief summary formation-cell fission family, RND) and multiple medicines and toxic chemical discharge family (multidrug and toxic compoundextrusion family, MATE) (Putman et al. (2000) Microbiol.Mol.Biol.Reviews 64:672-693).The secondary multidrug transporter uses as electrochemical gradient described here is discharged cell with medicine.ABC type multidrug transporter uses the energy from the ATP hydrolysis that medicine is pumped cell (Putman et al. (2000), above-mentioned).

Bacterium can be by utilizing translocator and the enzyme with different carbohydrate specifics, and use different regulation mechanisms, as catabolite repression, thus the various carbohydrate of metabolism.These proteic separation and sign make can develop the essential probiotic bacterium product with many application, includes the product that benefits people and/or animal health, and the product that relates to foodstuffs production and safety.This albumen can also be used to develop the transgenic plant of growth or survival ability change.

The invention summary

The composition and the method that change microorganism and plant are provided.Composition of the present invention comprises that the encoding carbohydrate from Lactobacterium acidophilum utilizes the isolating nucleic acid of associated protein, this carbohydrate utilizes associated protein to comprise phosphotransferase system (PTS), the proteic albumen of abc transport and relates to other albumen of transhipment, degraded and/or synthetic of sugar in the Lactobacterium acidophilum.Composition also comprises the isolating nucleic acid from the coding multidrug transporter of Lactobacterium acidophilum.Particularly, the invention provides isolated nucleic acid molecule, wherein comprise the described nucleotide sequence of odd number among the SEQ ID NOS:1-363 individually and/or with any array mode, basically formed by them, and/or form, and provide the isolated nucleic acid molecule of the described aminoacid sequence of even number among the coding SEQ ID NOS:2-364 (individually or with any array mode) by them.The polypeptide that separates and/or recombinate also is provided, wherein comprise by nucleic acid molecule encoding described here and/or as the described aminoacid sequence of even number among the SEQ ID NOS:2-364 (individually and/or with any array mode), it is gone up and is made up of them, and/or is made up of them.Variant nucleic acid and the polypeptide enough same with the described nucleotide sequence of sequence table and aminoacid sequence comprise in the present invention.In addition, the fragment of this nucleotide sequence and aminoacid sequence and enough same fragment have also been contained.Also comprise with nucleic acid array complementation of the present invention or with the nucleotide sequence of nucleotide sequence hybridization of the present invention.

Composition further comprises carrier and protokaryon, eucaryon and the vegetable cell that is used for recombinant expressed nucleic acid described here, and the transgenic microorganism and the plant population that comprise this carrier.The present invention also comprises the method for recombinant production polypeptide of the present invention and their using method.The method and the test kit that further comprise the existence of nucleic acid of the present invention in the test sample and/or peptide sequence, and with polypeptide bonded antibody of the present invention.The biology pure growth that also comprises the bacterium that comprises Nucleotide of the present invention or aminoacid sequence.Comprise the food that contains these cultures, comprise breast, sour milk, curdled milk, cheese, fermented milk, ice-creams, based on the product of fermented cereal, pulvis, infant formala, tablet, liquid bacterial suspension, dry oral enriching substance and liquid oral enriching substance based on breast.

Carbohydrate of the present invention utilizes dependency and multidrug transporter molecule to can be used for selecting and producing recombinant bacteria, the bacterium that especially production fermentation capacity improves.This bacterium includes but not limited to synthesize, transport, gather and/or utilize the bacterium of the ability change of various carbohydrate, the bacterium that local flavor or quality (texture) change, the bacterium of mutagenic carbohydrate, with the bacterium that under stress condition, can better survive, as the bacterium that runs in food-processing and/or the animal gastrointestinal tract.Multidrug transporter molecule of the present invention comprises the molecule that allows bacterium better survival when contact antimicrobial polypeptide such as bacteriocin or other toxin.These carbohydrate utilizations are relevant also to can be used for changing plant variety with the multidrug transporter molecule.The transgenic plant that comprise one or more sequences of the present invention economically may be useful, because they have more resistance to described environmental stress, described environmental stress includes but not limited to phytopathogen, high salt concentration or dehydration.They also may can better stand food-processing and storage condition.

The invention provides the isolating nucleic acid that is selected from down group, described group by comprising following nucleotide sequence, form and/or basically by its nucleic acid of forming by it: SEQ ID NOS:1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175,177,179,181,183,185,187,189,191,193,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,289,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337,339,341,343,345,347,349,351,353,355,357,359,361 and/or 363 (can exist in any combination, comprise a plurality of identical sequences) and/or its complementary strand, comprise the nucleotide sequence that has at least 90% sequence identity with following nucleotide sequence, form and/or basically by its nucleic acid of forming by it: SEQ ID NOS:1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175,177,179,181,183,185,187,189,191,193,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,289,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337,339,341,343,345,347,349,351,353,355,357,359,361 and/or 363 described nucleotide sequences (can the arbitrary combination mode exist, comprise a plurality of identical sequences), and/or its complementary strand, the fragment that comprises following nucleotide sequence, form and/or basically by its nucleic acid of forming by it: SEQ ID NOS:1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175,177,179,181,183,185,187,189,191,193,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,289,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337,339,341,343,345,347,349,351,353,355,357,359,361 and/or 363 (can the arbitrary combination mode, comprise a plurality of identical sequences) and/or its complementary strand, coding comprises the nucleic acid of the polypeptide of following aminoacid sequence: SEQ ID NO:2,4,6,8,10,12, i4,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362 and/or 364 described aminoacid sequences (can the arbitrary combination mode, comprise a plurality of identical sequences) and/or by the aminoacid sequence of nucleic acid molecule encoding described here, comprise the nucleic acid of nucleotide sequence that coding and following aminoacid sequence have the polypeptide of at least 90% amino acid sequence identity: SEQ ID NO:2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362 and/or 364 described aminoacid sequences (can the arbitrary combination mode, comprise a plurality of identical sequences) and/or by the aminoacid sequence of nucleic acid molecule encoding described here, and under stringent condition with the nucleic acid of above-mentioned any nucleic acid hybridization.

Composition further comprises the carrier that comprises nucleic acid described here, further comprise the heterologous polypeptide of encoding nucleic acid carrier and contain the cell of described carrier, comprise bacterium, plant and eukaryotic cell.The present invention also comprises the method for recombinant production polypeptide of the present invention and their using method.The method and the test kit that further comprise the existence of nucleic acid of the present invention in the test sample or peptide sequence, and with polypeptide bonded antibody of the present invention.

The present invention further provides the isolated polypeptide that is selected from down group: a) comprise following aminoacid sequence (, comprising a plurality of identical sequences) in the arbitrary combination mode, form and/or basically by its polypeptide of forming by it: SEQ ID NO:2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362 and/or 364 described aminoacid sequences and/or by the aminoacid sequence of nucleic acid molecule encoding described here; B) comprise following aminoacid sequence (, comprising a plurality of identical sequences) and/or by the fragment of the aminoacid sequence of nucleic acid molecule encoding described herein in the arbitrary combination mode, form and/or basically by its polypeptide of forming by it: SEQ ID NO:2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362 and/or 364 described aminoacid sequences; C) comprise the aminoacid sequence that the aminoacid sequence with following aminoacid sequence (with any combination, comprising a plurality of identical sequences) and/or nucleic acid molecule encoding described herein has at least 90% sequence identity, form and/or basically by its polypeptide of forming by it: SEQ ID NO:2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362 and/or 364 described aminoacid sequences; D) by the nucleotide sequence coded polypeptide that has at least 90% sequence identity with following nucleotide sequence: SEQ ID NOS:1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175,177,179,181,183,185,187,189,191,193,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,289,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337,339,341,343,345,347,349,351,353,355,357,359,361 and/or 363 described nucleotide sequences (with any combination); And e) by the following nucleotide sequence encoded polypeptides: SEQ ID NOS:1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175,177,179,181,183,185,187,189,191,193,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,289,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337,339,341,343,345,347,349,351,353,355,357,359,361 and/or 363 (with any combinations).

Also provide the polypeptide of the present invention that further comprises one or more allogeneic amino acid sequences and with the antibody of polypeptide selective binding described here.

The method for preparing polypeptide is provided in addition, described method is included under the condition expressed of nucleic acid of this polypeptide of coding and cultivates cell of the present invention, described polypeptide is selected from down group: the polypeptide that a) comprises following aminoacid sequence: SEQ ID NO:2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362 and/or 364 described aminoacid sequences are (with any combination, comprise a plurality of identical sequences), and/or by the aminoacid sequence of nucleic acid molecule encoding described here; B) comprise the segmental polypeptide of following aminoacid sequence: SEQ ID NO:2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362 and/or 364 described aminoacid sequences are (with any combination, comprise a plurality of identical sequences), and/or by the aminoacid sequence of nucleic acid molecule encoding described here; C) comprise the polypeptide that has the aminoacid sequence of at least 90% sequence identity with following aminoacid sequence: SEQ ID NO:2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362 and/or 364 described aminoacid sequences are (with any combination, comprise a plurality of identical sequences), and/or by the aminoacid sequence of nucleic acid molecule encoding described here; D) by the nucleotide sequence coded polypeptide that has at least 90% sequence identity with following nucleotide sequence: SEQ IDNOS:1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175,177,179,181,183,185,187,189,191,193,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,289,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337,339,341,343,345,347,349,351,353,355,357,359,361 described nucleotide sequences (with any combination); And e) by the following nucleotide sequences encoded polypeptides: SEQ ID NOS:1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175,177,179,181,183,185,187,189,191,193,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,289,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337,339,341,343,345,347,349,351,353,355,357,359,361 and/or 363 described nucleotide sequences (with any combination).

The method that has situation of polypeptide in the test sample also is provided, and described method comprises the compound that makes this polypeptide of sample contact selective binding and determines whether this compound combines with polypeptide in the sample; Wherein said polypeptide is selected from down group: a) by the following nucleotide sequence encoded polypeptides: SEQ IDNOS:1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175,177,179,181,183,185,187,189,191,193,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,289,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337,339,341,343,345,347,349,351,353,355,357,359,361 and/or 363 described nucleotide sequences (with any combination); B) comprise the segmental polypeptide of the aminoacid sequence of following nucleic acid sequence encoding: SEQ ID NOS:1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175,177,179,181,183,185,187,189,191,193,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,289,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337,339,341,343,345,347,349,351,353,355,357,359,361 and/or 363 described nucleotide sequences (with any combination); C) by the nucleotide sequence coded polypeptide that has at least 90% sequence identity with following Nucleotide: SEQ ID NOS:1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175,177,179,181,183,185,187,189,191,193,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,289,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337,339,341,343,345,347,349,351,353,355,357,359,361 and/or 363 described nucleotide sequences (with any combination); D) comprise the polypeptide that has the aminoacid sequence of at least 90% sequence identity with following aminoacid sequence: SEQ ID NO:2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362 and/or 364 described aminoacid sequences (with any combination); And e) comprises the polypeptide of following aminoacid sequence: SEQ ID NO:2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362 and/or 364 described aminoacid sequences (with any combination).

Provide the method that has situation of polypeptide in the test sample in addition, described method comprises the compound that makes sample contact and polypeptide selective binding and measures this compound whether combine with polypeptide of the present invention in the sample, be antibody with polypeptide bonded compound wherein.Also provide and comprised with in the methods of the invention the compound and the test kit of working instructions.

The present invention also provides nucleic acid molecule of the present invention and/or its segmental method that has situation in the test sample, comprising: the nucleic acid probe or the primer that a) make sample contact and this nucleic acid molecule and/or its fragment selective cross; And b) measure nucleic acid probe or primer whether with sample in making nucleic acid molecular hybridization, nucleic acid molecule of the present invention and/or its segmental situation that exists in the test sample thus.Nucleic acid molecule of the present invention and/or the segmental method that has situation in the test sample also are provided, and wherein sample comprises the mRNA molecule and makes it to contact nucleic acid probe.Provide in addition at this and to have comprised and the compound of nucleic acid selective cross of the present invention and the test kit of working instructions.

Provide 1 in addition) change the ability that organism entered or transported out cell with the carbohydrate transhipment; 2) change the ability that organism gathers carbohydrate; 3) change organism and utilize the ability of carbohydrate as the energy; 4) change the biological ability that produces the carbohydrate of modifying; 5) change is by the flavours in food products of microbial fermentation; 6) change is by the quality of the food of microbial fermentation; 7) change the ability that organism is stood food-processing and storage condition; 8) change the ability of microorganism in the survival of stomach and intestine (GI) road; 9) change the ability that organism enters drug transport or transport out cell; With 10) change the method that organism produces the ability of carbohydrate, comprise carrier introduced described organism and/or microorganism that this carrier comprises at least one nucleotide sequence of the present invention and/or at least one is selected from down the nucleotide sequence of group: a) SEQ ID NOS:1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175,177,179,181,183,185,187,189,191,193,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,289,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337,339,341,343,345,347,349,351,353,355,357,359,361 and/or 363 described nucleotide sequences (with any combination); B) comprise the segmental nucleotide sequence of following nucleotide sequence: SEQ ID NOS:1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175,177,179,181,183,185,187,189,191,193,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,289,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337,339,341,343,345,347,349,351,353,355,357,359,361 and/or 363 described nucleotide sequences (with any combination), the polypeptide of wherein said fragment coding retentive activity; C) nucleotide sequence same: SEQ ID NOS:1 with following sequence at least 90%, 3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175,177,179,181,183,185,187,189,191,193,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,289,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337,339,341,343,345,347,349,351,353,355,357,359,361 and/or 363 described sequences (with any combination), the polypeptide of wherein said nucleotide sequence coded retentive activity; And d) coding comprises the nucleotide sequence of polypeptide that has the aminoacid sequence of at least 90% sequence identity with following aminoacid sequence: SEQ ID NO:2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362 and/or 364 described aminoacid sequences (with any combination), wherein said polypeptide retentive activity; And e) coding comprises the nucleotide sequence of the polypeptide of following aminoacid sequence: SEQ ID NO: 2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362 and/or 364 aminoacid sequences (with any combination).

Further provide 1 at this) compare with the wild-type Lactobacterium acidophilum, the carbohydrate transhipment is entered or transports out the lactobacillus acidophilus strains of the ability change of cell; 2) compare with the wild-type Lactobacterium acidophilum, gather the lactobacillus acidophilus strains of the ability change of carbohydrate; 3) compare with the wild-type Lactobacterium acidophilum, utilize the lactobacillus acidophilus strains of carbohydrate as the ability change of the energy; 4) compare with the wild-type Lactobacterium acidophilum, the lactobacillus acidophilus strains that makes the food of local flavor change owing to fermentation is provided; 5) compare with the wild-type Lactobacterium acidophilum, the lactobacillus acidophilus strains that makes the food of quality change owing to fermentation is provided; 6) compare with the wild-type Lactobacterium acidophilum, produce the lactobacillus acidophilus strains that the carbohydrate ability changes; 7) compare with the wild-type Lactobacterium acidophilum, stand the lactobacillus acidophilus strains of the ability change of food-processing and storage condition; With 8) compare with the wild-type Lactobacterium acidophilum, the lactobacillus acidophilus strains that the ability of surviving in the GI road changes, the ability of wherein said change, local flavor and/or quality are because SEQ ID NO:2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360, described at least one carbohydrate of 362 and/or 364 (with any combinations) utilizes due to the polypeptide expression.

Provide in addition with the wild-type Lactobacterium acidophilum and compared, stand to contact the lactobacillus acidophilus strains that the ability of antimicrobial polypeptide or toxin changes, the ability of wherein said change is because SEQ IDNOs:78-88,92-94,124-126,132,282-288,308 and/or 312-322 in due to the described at least a multiple medicines transhipment of the even number polypeptide expression.

The DNA construct and/or stable its genomic plant, vegetable cell and/or the plant seed of mixing of at least one nucleotide sequence of the present invention that will comprise at least one nucleotide sequence of the present invention also are provided, this sequence is selected from down group: a) among the SEQ ID NOs:1-363 (separately and/or with any combination) any one described nucleotide sequence, or its complementary strand; B) with SEQID NOs:1-363 in (separately and/or with any combination) any one described nucleotide sequence has the nucleotide sequence of at least 90% sequence identity, or its complementary strand; C) the segmental nucleotide sequence that comprises among the SEQ IDNOs:1-363 (separately and/or with any combination) any one described nucleotide sequence, or its complementary strand; D) coding comprises the nucleotide sequence of the polypeptide of arbitrary described aminoacid sequence among the SEQ ID NOs:2-364; E) coding comprise with SEQ IDNOs:2-364 in any one described aminoacid sequence have the nucleotide sequence and the f of polypeptide of the aminoacid sequence of at least 90% sequence identity) under stringent condition with a)-e) nucleotide sequence of any one hybridization.

The accompanying drawing summary

Fig. 1. interested gene locus.The layout that has shown the site of discussing in the text: man: glucose-seminose site; Fru: fructose site; Suc: sucrose site; The fos:FOS site; Raff: raffinose site; Lac: lactose-semi-lactosi site; Tre: trehalose site; CCR: carbon katabolic product site.

Fig. 2. the carbohydrate utilization in the Lactobacterium acidophilum.The figure illustrates as transcribing the carbohydrate translocator and the lytic enzyme of spectrum prediction.The protein name of each element and EC number have been described.The PTS translocator is with black display.The GPH translocator shows with light gray.Abc transport albumen shows with lead.

Detailed Description Of The Invention

The present invention relates to the relevant and multidrug transporter of carbohydrate utilization from lactobacillus acidophilus. Nucleotides and the amino acid sequence of the relevant and multidrug transporter of carbohydrate utilization are provided. This sequence can be used for changing microorganism, cell and plant, makes it have the performance of raising.

" one (kind) " and " being somebody's turn to do " can be plural number or odd number as used herein, as run through in specification and the claim book and use. For example " one (kind) " cell can refer to perhaps a plurality of (kind) cell of single (kind) cell.

Equally as used herein " and/or " refer to and comprise any and all possible combination of one or more listed relevant projects, and do not comprise combination when a mode ("or") is used to select.

" the carbohydrate utilization is relevant " molecule or the gene meaning refer to the new sequence from lactobacillus acidophilus, and its coding relates to the albumen that carbohydrate molecule is utilized, and include but not limited to synthesizing, transport or degraded of carbohydrate. The molecule of " multidrug transporter " molecule meaning reference and antibacterial polypeptide transhipment, described antibacterial polypeptide for example are bacterium element or other medicine or toxin. Concrete carbohydrate of the present invention utilizes correlation multidrug transporter molecule referring to table 1. Full-length gene order is expressed as " carbohydrate utilization be correlated with sequence " or " multidrug transporter sequence " and shows that they utilize phase correlation gene or multidrug transporter gene to have similitude with carbohydrate respectively. The present invention further provides fragment and the variant of the relevant sequence of these carbohydrate utilizations or multidrug transporter sequence, it also can be used for implementing method of the present invention.

" carbohydrate " refers to contain the organic compound of carbon, hydrogen and oxygen, and ratio is 1: 2: 1 usually. Carbohydrate includes but not limited to sugar, starch, cellulose and natural gum. Term " gene " and " restructuring gene " refer to comprise the relevant albumen of nucleic acid, especially encoding carbohydrate utilization of open frame or the nucleic acid of multidrug transporter as used herein. The nucleic acid of separation of the present invention comprises the described nucleotide sequence of odd number and variant and fragment in the nucleotide sequence, SEQ ID NOS:1-363 of the described amino acid sequence of even number in the nucleotide sequence, coding SEQ ID NOS:2-364 of the relevant albumen of encoding carbohydrate utilization or multidrug transporter. The present invention also comprises antisensenucleic acids, and is following described.

In addition, comprise having polypeptide and albumen and variant and the fragment that carbohydrate utilizes the separation of related activity or multidrug transporter activity, and the method that produces these polypeptide. For the purposes of the present invention, term " albumen " and " polypeptide " can Alternates. Polypeptide of the present invention has the relevant protein active of carbohydrate utilization or multidrug transporter activity. The relevant protein active of carbohydrate utilization or multidrug transporter activity refer to according to the code test technology in body or the biology of external test or functional activity. These activity include but not limited to synthetic carbohydrate ability, with carbohydrate transhipment enter or transport out ability, the degraded carbohydrate of cell ability, regulate the ability of carbohydrate concentration in the cell, in conjunction with the ability of carbohydrate and ability that medicine or toxin transhipment were entered or transported out cell.

The structure of various types of bacterium transhipment albumen is well known in the art. The ATP of transhipment albumen forms (Higgins et al. (1986) Nature 323:448-450 in conjunction with box (ABC) superfamily by the albumen with four Core domains; Hyde et al. (1990) Nature 346:362-365; Higgins (2001) Res.Microbiol.152:205-210). Typically, two cross-film domains (PFAM accession number PF00664) are arranged, wherein each domain has six cross-film α spirals, with two ATP binding structural domains, it contains the core amino acid (Higgins (2001) is above-mentioned .) of determining transhipment albumen, and another conservative motif, comprise Walker A and Walker B motif (Walker et al. (1982) EMBO is Ref.No.PDOC00185 J.1:945-951:Prosite).

Abc transport albumen of the present invention comprises SEQ ID NOS:40,42,44,48,52,54,56,58,62,64,66,68,70,72,74,110,112,114,116,122,124,126,128,130,132,134,136,144,146,148,152,154,160,236,262,274,278,280,294, albumen in 296,298,300,302,306,338,340 and 360. SEQ ID NOS:126 and 144 is members (PFAM accession number PF 00664) of abc transport albumen cross-film district family.

TOBE domain (the relevant OB of transhipment) (PFAM accession number PF03459) always exists with dimer, because the terminal chain of the C of each domain is supplied with (Koonin et al. (2000) Adv.Protein Chem.54:245-75) by the partner. It probably relates to the identification of little part such as molybdenum and sulfate. In abc transport albumen, find that it is just after ATP enzymatic structure territory. TOBE domain protein of the present invention comprises the albumen among the SEQ ID NO:110.

Secondary transhipment systematic protein comprises that galactoside-pentose of transport albumen-hexuronic acid acid anhydride (hexuronide) organizes (Poolman et al. (1996) Mol.Microbiol.19:911-922). These albumen form (Poolman et al. (1989) J.Bacteriol. 171:244-253) by the hydrophobic structure territory that comprises the terminal enzyme IIA of 12 cross-film domains and carboxyl domain usually.

Phosphoric acid shifts enzyme system (PTS) catalysis sugar substrate and passes phosphorylation in the cell membrane process in displacement. This mechanism comprises that the enzyme I (EI) (Prosite Ref.No. PDOC00527) by the PTS system is transferred to enzyme II (EII) (Prosite Ref.Nos.PDOC00528 with the phosphorus acyl group from phosphoric acid enol pyruvic acid (PEP); PDOC00795), then transfer them to phosphoric acid carrier albumen (HPr) (Prosite Ref.No.PDOC00318) (PFAM accession number PF00381). HPr albumen contain two conservative phosphorylation sites-be positioned at the amino terminal side, by the histidine residues of enzyme I phosphorylation, one be at this albumen the carboxyl end side, can be by the serine residue of ATP dependence protein tyrosine phosphorylation (de Vos (1996) Antonie vanLeeuwenhoek 70:223-242). SEQ ID NO:178 is the member of PTS HPr component phosphorylation site family (PFAM accession number PF00381).

The penetrating enzyme of sugared specificity (enzyme II) of PTS system is comprised of at least three different domains (IIA, IIB and IIC) of structure, and it can merge with single polypeptide chain form, and perhaps the chain as two or three interactions exists. The IIA domain carries first penetrating enzyme spcificity phosphorylation site, namely by the histidine of phosphoric acid base-HPr phosphorylation. In cysteinyl-or histidine acyl residue place phosphorylation, this depends on penetrating enzyme to second domain (IIB) by phosphoric acid base-IIA. At last, the phosphorus acyl group is transferred to sugared substrate from the IIB domain in by the process of IIC domain catalysis, and this process is transported coupling with the cross-film of sugar. Phosphoric acid enol pyruvic acid dependence sugar phosphotransferase of the present invention system, EIIA1 family (PFAM accession number PF00358) albumen comprises SEQ ID NOS:6, the albumen in 12,14,34,102,104,174,176,268 and 290. Phosphoric acid enol pyruvic acid dependence sugar phosphotransferase of the present invention system, EIIA 2 (PFAM accession number PF00359) albumen comprises the albumen among the SEQ ID NO:36. SEQ ID NO:36 also is the member of PTS system, the inferior unit (PFAM accession number PF02379) of fructose specificity IIB. Phosphoric acid of the present invention shifts the enzyme system, and EIIB family (PFAM accession number PF00367) albumen comprises SEQ ID NOS:12, the albumen in 14,32,34,102,104268 and 290. Phosphoric acid of the present invention shifts the enzyme system, and EIIC family (PFAM accession number PF02378) albumen comprises SEQ ID NOS:12, the albumen in 14,16,28,30,32,34,36,102,104,174,268,270,272 and 290.

Lactose/fiber disaccharides specificity family is one of colony different on four 26S Proteasome Structure and Functions. IIA PTS system enzyme (PFAM accession number PF02255) normally works with the homotype tripolymer of the metal ion stabilisation that is positioned at the center. PTS of the present invention system, the inferior unit of lactose/fiber disaccharides specificity IIA family protein comprises the albumen of SEQ ID NO:4. The inferior family of unit of lactose/fiber disaccharides specificity IIB (PFAM accession number PF02302) is the kytoplasm enzyme. The Folding display of IIB fiber disaccharides goes out the structure similar to mammal tyrosine phosphatase enzyme. PTS of the present invention system, the inferior unit of lactose/fiber disaccharides specificity IIB family protein comprises the albumen of SEQ ID NO:170.

Mannose family is unique aspect several in the middle of the penetrating enzyme family of PTS. It is the PTS family that unique wherein member has IID albumen; It is the PTS family of unique wherein IIB component phosphorylation on the group aminoacyl rather than on the cysteinyl residue; And its penetrating enzyme member demonstrates the wide in range specificity of sugar on a large scale, rather than only to a kind of or some sugaredly have a specificity. SEQ ID NOS:20 and 264 is members of the fructose IIA of PTS system component (PFAM accession number PF03610) family. SEQ ID NO:168 is the member of the PTS system mannose/fructose/IID of sorbose family component (PFAM accession number PF03613). SEQ ID NO:264 is the member of the inferior IIB of the family component of PTS system sorbose (PFAM accession number PF03830). SEQ ID NO:166 is the member of sorbose specificity iic component family of PTS system (PFAM accession number PF03609).

Many enzymes that catalysis phosphorus acyl group shifts by phosphoric acid base * histidine intermediate from phosphoric acid enol pyruvic acid (PEP) structurally relevant (Reizer et al. (1993) Protein Sci.2:506-21) have been proved. All these enzymes are shared identical catalyst mechanism: they are transferred to histidine residues in conjunction with PEP and with the phosphorus acyl group from it. Sequence around that residue is very conservative. Usually find PEP utilize enzyme TIM cylindrical structure territory (PFAM accession number PF02896) and pyruvate phosphate two kinases PEP/ pyruvic acid binding structural domains (INTERPRO：IPR002192) terminal and PEP utilizes enzyme removable frame territory (PFAM accession number PF00391) to combine at its N-. It is " rotation " β/beta/alpha domain that PEP utilizes enzyme removable frame territory, is considered to known and all can moves (Cosenza et al. (2002) J.Mol.Biol. 318:1417-32) in containing its all albumen. Usually find it and pyruvate phosphate two kinases PEP/ pyruvic acid binding structural domains (INTERPRO：IPR002192) combine at its N-end. PEP of the present invention utilizes enzyme TIM cylindrical structure territory albumen to comprise the albumen of SEQ ID NO:180. PEP of the present invention utilizes enzyme removable frame territory albumen to comprise the albumen of SEQ ID NO:180 and 258. PEP of the present invention utilizes the terminal family (PFAM accession number PF05524) of enzyme N-albumen to comprise the albumen of SEQ ID NO:180.

(major facilitatorsuperfamily, member MFS) have 12 or 14 TMDs to the main promotion superfamily protein of multidrug transporter.The member of little multi-medicine resistance family (SMR) of multidrug transporter is considered to form four tightly compacted spiral antiparallel bundles.They give the resistance to various toxic chemicals (by remove them from cell).Resistance brief summary-member of cell fission family (RND) is contained terminal TMD of single N-and the big terminal pericentral siphon structural domain (Putman et al. (2000) Microbiol.Mol.Biol.Reviews 64:672-693) of C-.The multidrug transporter of these types and whole multidrug transporters of MFS, SMR and RND family have been described, and from the conservative motif (having accession number) in each albumen in the specific proteins of various bacteriums (Putman et al. (2000) supra).Multidrug transporter of the present invention comprises SEQ ID NOS:78,80,82,84,86,88,92,94,282, and 284,286,288 and 322 albumen.

Sugar (reaching other) translocator family (PFAM accession number PF00083) is The main albumen that promotes SuperfamilyThe member of family.The MFS translocator is only can reply the chemosmosis ion gradient and single polypeptide two grade carriers of transporting little solute.All MFS permeases of approving at present keep two six TMDs (TMS) unit in single polypeptide chain, although 3 in 17 MFS families have found two other TMSs (Paulson et al. (1996) Microbio1.Rev.60:575-608).In addition, relevant but be not in fact whole proteic features of MFS more than 300 kinds that very conservative motif (Henderson and Maiden (1990) Philos.Trans.R.Soc.Lond.B.Biol.Sci.326:391-410) is proved to be to be identified between the very conservative special motif of MFS and TMS8 and the TMS9 between TMS2 and the TMS3.Sugar of the present invention (reaching other) translocator comprises the albumen of SEQ ID NOS:8O and 282.

The conjugated protein dependency movement system of bacterium is usually by with energy and activation movement system pericentral siphon substrate conjugated protein, one or two mutual homologous integration inner membrane protein (PFAM accession number PF00528) and the conjugated protein multicomponent system formed of one or two periphery film ATP of coupling mutually.This integration inner membrane protein passes film with the substrate displacement.Shown and contained the conserved regions that is positioned at terminal about 80 to 100 residues of their C-by these albumen of great majority (Dassa and Hofnung (1985) EMBO J.4:2287-93; Saurin et al. (1994) Mol.Microbiol.12:993-1004).As if this zone kytoplasm ring (Pearce et al. (1992) Mol.Microbiol.6:47-57) between two membrane spaning domains.These albumen can be divided into seven families, are called as respectively: araH, cysTW, fecCD, hisMQ, livHM, malFG and oppBC.Conjugated protein dependency movement system inner membrance component protein of the present invention comprises SEQ ID NOS:42, the albumen in 44,62,64,112,114 and 294.Branched-chain amino acid movement system/permease component family (PFAM accession number PF 02653) is the extended familys that mainly comprise high affinity branched-chain amino acid translocator.In this family, also found albumen from semi-lactosi movement system permease and ribose movement system.Branched-chain amino acid movement system of the present invention/permease component protein comprises SEQ ID NOS:54, the albumen in 72 and 74.

SEQ ID NO:184 is the member of HPr serine kinase N-terminal family (PFAM accession number PF02603) and the member of HPr serine kinase C-terminal family (PFAM accession number PF07475).N-terminal family represents the N-stub area of Hpr serine-threonine kinase PtsK.C-terminal family represents the C-terminal kinase domain of Hpr serine-threonine kinase PtsK.This kinases is the transmitter (Marquez et al. (2002) Proc.Natl.Acad.Sci.U.S.A.99:3458-63) of the polycomponent phosphorylation system of control carbon catabolite repression in the bacterium.This kinases is unusual, because its discerns the tertiary structure of its target, and is member with the irrelevant new family of any previously described protein phosphorylation enzyme.1.95A resolving power under, show that from the x-ray analysis of the total length crystal enzyme of staphylococcus xylosus this enzyme is made up of two structural domains that obviously separate, they are assemblied in six poly structures of similar three bladed propeller.Respectively each forms blade by two N-end structure territories, closely the terminal kinase domain (Reizer et al. (1998) Mol.Microbiol.27:1157-69) of wheel hub assembling C-.

The periplasmic binding protein and the sugared binding domains of LacI family (PFAM accession number PF00532) comprise periplasmic binding protein and LacI family transcription regulatory protein.Periplasmic binding protein is chemotaxis and many primary receptors of transporting based on the solute of sugar.The albumen of LacI family is made up of the relevant transcription regulatory protein of lac repressor.In this case, the usually sugar binding domains is in conjunction with the sugar of the dna binding activity that changes repressor structural domain (lacI).The periplasmic binding protein of LacI family protein of the present invention and sugared binding domains comprise the albumen in SEQ ID NOS:38 and 98.

Bacterium high affinity movement system relates to the active transport that solute passes cytoplasmic membrane.The protein ingredient of these haulage system comprises one or two transmembrane protein composition, conjugated protein and high affinity pericentral siphon solute-conjugated protein (the PFAM accession number PF01547) of the related ATP of one or two film.Gram positive bacterium is centered on by single film, does not therefore have the pericentral siphon zone, and in this bacterium, equal albumen combines with film by the terminal lipid anchor of N-.These homologous proteins do not play complete effect in transport process itself, but probably serve as acceptor to trigger or to start solute by combining the transhipment of striding film with the proteic outer portion of the conformity membrane of outflow system.In addition, at least some solutes are conjugated protein works in the startup of sensory transduction approach.The outer conjugated protein SEQ ID NOS:40 that comprises of solute of bacterium born of the same parents of the present invention, the albumen in 66,116,262,274 and 296.

HUCEP-8 family (PFAM accession number PF06800) is the family of the long bacterium HUCEP-8 of about 300 residues.The member comprises the membranin of glucose uptake albumen (Fiegler et al. (1999) J.Bacteriol.181:4929-36), ribose translocator and several that infer and imagination that may be relevant with the sugar transport of striding bacterial film.HUCEP-8 of the present invention comprises the albumen among the SEQ ID NO:234.

MIP (major intrinsic protein) family protein (PFAM accession number PF00230) demonstrates distinct basically two class channel performances: (1) is by the special moisture transhipment of aquaporins, (2) little neutral solute is transported, as promoting protein transport glycerine (Froger et al. (1998) Protein Sci.7:1458-68) by glycerine.The MIP family protein is considered to contain 6 TM structural domains.Perhaps, it is to produce (Wisow et al. (1991) TrendsBiochem.Sci.16:170-1) by the original protein that contains 3 TM structural domains by duplicating in series connection, the gene that these albumen are pointed out in sequential analysis.

General translocator of the present invention comprises SEQ ID NOS:76, the albumen in 90,96 and 194.

The nucleic acid that the present invention includes separates with protein ingredient or purifying basically." separation " or " purifying basically " meaning be described nucleic acid or protein molecular or its biological active fragment or variant in fact or abnormal basically that find and nucleic acid or protein bound composition state of nature.This composition comprises other cellular material, from the various chemical reagent that use in the substratum of recombinant products and/or chemosynthesis albumen or the nucleic acid.Preferably, " isolating " of the present invention nucleic acid does not contain the nucleotide sequence (as 5 ' or the 3 ' terminal encoding sequence that exists) of nucleic acid side joint interested in the genomic dna with the organism that obtains this nucleic acid.Yet this molecule may comprise the other base or the part of the essential characteristic that some can the harmful effect component.For example, in various embodiments, isolating nucleic acid contains in normal and the cell that obtains it the genomic dna bonded less than the nucleotide sequence of 5kb, 4kb, 3kb, 2kb, 1kb, 0.5kb or 0.1kb.Similarly, the contaminating protein or the non-carbohydrate that have less than about 30%, 20%, 10%, 5% or 1% (calculating with dry weight) of the albumen of purifying utilizes associated protein basically.When this albumen of recombinant production, preferred culture medium accounts for below 30%, 20%, 10% or 5% of volume of protein formulation, when this albumen of chemical production, have chemical reagent precursor or the relevant chemical reagent of non-carbohydrate utilization in the preferred formulation less than about 30%, 20%, 10% or 5% (calculating) with dry weight.

The compositions and methods of the invention can be used for regulating the function of the relevant or multidrug transporter molecule of the carbohydrate utilization of Lactobacterium acidophilum." adjusting ", " change " or " improvement " meaning refer to bioactive rise of target or downward modulation.Albumen of the present invention can be used for improving the biologic activity of milk-acid bacteria, and improves nutrition or health-promoting feature by the food of this fermentation using bacteria.Nucleic acid molecule of the present invention can be used for regulating milk-acid bacteria and expresses the relevant or multidrug transporter of carbohydrate utilization.Comprise the rise or the downward modulation of expression of nucleic acid of the present invention.Rise can be for example by the polygene copy being provided, regulating and express, promote to transcribe or translating mechanism or alternate manner are finished by modifying regulatory element.Downward modulation can be for example by using known antisense and gene silent technology to finish.

" milk-acid bacteria " is meant from the bacterium that is selected from down dependent of dead military hero: Aerococcus (Aerococcus), meat Bacillaceae (Carnobacterium), enterococcus spp (Enterococcus), lactococcus (Lactococcus), lactobacillus (Lactobacillus), leuconos toc (Leuconostoc), wine Coccus (Oenococcus), Mycosphaerella (Pediococcus), streptococcus (Streptococcus), honeybee Coccus (Melissococcus), difference Coccus (Alloiococcus), deceitful Coccus (Dolosigranulum), lactic acid producing Coccus (Lactosphaera), Tetracoccus (Tetragenococcus), roaming Coccus (Vagococcus) and Wei Si Bordetella (Weissella) (Holzapfel et al. (2001) Am.J Clin.Nutr.73:365S-373S; Bergey ' s Manual of SystematicBacteriology, Vol.2 (Wiliams and Wilkins, Baltimore; (1986)) pp.1075-1079).

Polypeptide of the present invention or express their microorganism can be effectively as accessory substance or additive and as the enriching substance in dairy products and the fermenting process.Nucleotide sequence, coded polypeptide and the microorganism of expressing them are at dairy by product, and be useful in the preparation as cheese, yogourt, fermented milk prod, yogurt milk and buttermilk.The microorganism of expressing polypeptide of the present invention can be a probiotic organism." probiotic bacterium " meaning refers to can survive and the experimenter be had by gi tract the live microorganism of useful influence." experimenter " meaning abutment is expressed the organism of the proteic microorganism of the present invention.The experimenter can refer to people and other animal.

Except that carbohydrate utilization disclosed herein relevant and multidrug transporter nucleotide sequence and fragment and the variant, nucleic acid of the present invention also comprises by with and multidrug transporter nucleotide sequence relevant available from carbohydrate utilization disclosed herein or its variant and segmental all or part of sequence hybridization and from other organism or cell evaluation and isolating homologous nucleotide sequence.

Fragment and variant

The invention provides the isolating nucleic acid of the nucleotide sequence that comprises the relevant and multidrug transporter of encoding carbohydrate utilization, and by the relevant and multidrug transporter of carbohydrate utilization of its coding." carbohydrate utilizes associated protein " is meant the albumen with described aminoacid sequence of even number among the SEQ ID NOS:2-364.These nucleotide sequences and coded proteic fragment and variant also are provided.Nucleotide sequence or proteic " fragment " are meant the part of described Nucleotide or aminoacid sequence.

The fragment of nucleic acid disclosed herein can be utilized the nucleic acid of associated protein or the nucleic acid of coding multidrug transporter with the identification code carbohydrate as hybridization probe, maybe can as the amplification scheme for example [for example polymerase chain reaction (PCR)] or carbohydrate utilization are relevant or the multidrug transporter nucleic acid mutation in primer.Nucleic acid fragment of the present invention can also combine with physical matrix with comprise can regard big or microarray as material (referring to for example United States Patent (USP) 5,837,832; United States Patent (USP) 5,861,242; WO 89/10977; WO 89/11548; WO 93/17126; United States Patent (USP) 6,309,823).This nucleic acid array or " chip " can be used to study the nucleic acid that genetic expression or evaluation and target sequence have enough identity.

Nucleic acid array or chip have been the present invention further provides, be that numerous nucleic acid (for example DNA) are as accurately organizing or the molecular probe of array on solid support, it can be used for the research and/or the gene expression analysis of gene sequencing, wherein contained sudden change, have large vol owing to the small-sized of them and at the analysis quantitative aspects, array like this and chip are interesting at present.

The function of these nucleic acid array/chips is based on molecular probe, mainly is oligonucleotide, and it is attached to has common several square centimeters or according to requiring to be larger sized carrier.In order to analyze, carrier as the carrier in DNA array/chip, is wrapped quilt by dna probe (for example oligonucleotide), and these probes are arranged in place or position predetermined on the carrier.Make the sample that contains target nucleic acid to be analyzed and/or its fragment (for example the DNA of mark or RNA or cDNA) in advance contact this DNA array/chip, cause forming duplex by hybridization.Behind the washing step, the analysis of chip surface makes the signal that can send by labels targets and any hybridization is positioned.Produce the hybridization fingerprint, by Computer Processing, the existence, sequencing and/or the sudden change that can be used for specific fragment in information retrieval such as genetic expression, the sample are identified.

In one embodiment of the invention, target nucleic acid and can be by fluorescence, radioactivity, electric detective technology or the like are determined as known in the art with the hybridization that the probe form is used and placement or original position are synthesized between the nucleic acid of the present invention on DNA chip/array.

In another embodiment, nucleotide sequence of the present invention can use by DNA array/chip form, to carry out the analysis of Lactobacterium acidophilum genetic expression.This analysis is based on DNA array/chip, has the probe of selecting, be used to characterize given gene or nucleotide sequence at its specificity on it.Target sequence to be analyzed is being labeled before the hybridization on the chip.After the washing, the mixture of detection and quantitative mark is hybridized at least in duplicate.Same probe can be used to analyze the differential transcription of the RNA that for example derives from sample at the comparative analysis of the strength of signal of same sample acquisition at different samples and/or different probe.

In another embodiment, the array/chip that contains nucleotide sequence of the present invention can comprise the nucleotide sequence special to other microorganism, and its permission is carried out series of tests and Rapid identification to the situation that exists of microorganism in the sample.

In further embodiment, the principle of DNA array/chip can also be used to prepare protein arrays/chip, and the upholder on it is by polypeptide of the present invention and/or antibody or its array replacement nucleic acid bag quilt.These protein arrays/chips make might by for example surface element resonance (SPR) for example analyze the bag quilt on the proteic carrier affinity of target catch the inductive bio-molecular interaction.Can the specificity combining source can be used in the protein arrays/chip that detects and/or identify albumen in the sample and/or peptide in the polypeptide of the present invention of the antibody of sample to be analyzed or polypeptide or antibody.

Therefore, the invention provides and comprise the various nucleic acid of the present invention microarray or the microchip of (, comprising repetition), and comprise each peptide species of the present invention microarray of (, comprising repetition) with any combination with any combination.The microarray that comprises with the antibody of each peptide species of the present invention (with any combination, comprising repetition) specific reaction also is provided.

" nucleic acid " is meant dna molecular (for example cDNA or genomic dna) and RNA molecule (for example mRNA) and uses the DNA of nucleotide analog deposits yields or the analogue of RNA.Nucleic acid can be strand or two strands, but double-stranded DNA normally.The protein fragments that encoding carbohydrate utilizes the fragment of the nucleic acid of associated protein or multidrug transporter to encode and has biologic activity maybe can be used as hybridization probe described here or PCR primer.The biological active fragment of polypeptide disclosed herein can prepare by the following method: separate the part of one of nucleotide sequence of the present invention, express the activity of coded protein part (for example passing through in-vitro recombination expression) and the coded protein part of assessment.The fragment of the nucleic acid of the relevant or multidrug transporter of encoding carbohydrate utilization comprises at least about 5,10,15,20,25,30,35,40,45,50,75,100,200,250,300,350,400,450,500,550,600,650,700,750,800,850,900,950,1000,1050,1100,1150,1200,1250,1300,1350,1400,1450,1500,1600,1700,1800,1900,2000,2200 or 2500 continuous nucleotides, comprise between 5 and 2500 at this concrete surplus any number of stating, maybe can reach the Nucleotide sum that exists in the relevant or multidrug transporter nucleotide sequence of total length carbohydrate disclosed herein utilization (is 432 for SEQ ID NO:1 for example, for SEQ ID NO:3 is 369, or the like).

The fragment of aminoacid sequence comprises and is suitable as immunogen to produce the polypeptide fragment of the relevant or anti-multidrug transporter antibody of anti-carbohydrate utilization.Fragment comprises that to comprise or multidrug transporter relevant with carbohydrate utilization of the present invention or the proteic aminoacid sequence of partial-length enough identical or derive from this aminoacid sequence and demonstrate at least a activity of the relevant or multidrug transporter of carbohydrate utilization, still comprises than full-length proteins disclosed herein amino acid whose peptide still less.Typically, biologic activity partly comprises and has carbohydrate utilization relevant or multidrug transporter at least a active structures territory or motif.Carbohydrate utilization biologic activity part relevant or multidrug transporter can be following polypeptide, for example length be between 10,25,50,100,150,200,250,300,350,400,450,500,550,6005 continuous acid 10 and 650 in this any numeral of concrete narration not, can be up to the amino acid sum that exists in the full-length proteins of the present invention (being 144 for SEQ ID NO:2 for example, be 123 or the like for SEQ ID NO:4).This biologic activity part can by the recombinant technology preparation and the utilization of evaluation natural carbohydrate be relevant or one or more functionally activies of multidrug transporter.As used herein, fragment comprises at least 5 continuous amino acids of any even number sequence among the SEQ ID NOS:2-364.Yet, the present invention includes other fragment, as proteinic greater than 6,7,8 or 9 amino acid whose any fragments.

The variant of Nucleotide and aminoacid sequence comprises in the present invention." variant " is meant enough identical sequence.Therefore, the present invention includes with the SEQ ID NOS:2-364 that encodes in the carbohydrate of even number utilize the enough identical isolating nucleic acid of nucleotide sequence of associated protein and multidrug transporter, or under stringent condition with SEQ ID NOS:1-363 in the nucleic acid of odd-numbered or the nucleic acid of its complementary strand hybridization.Variant also comprises by the nucleotide sequence coded polypeptide of variation of the present invention.In addition, polypeptide of the present invention have with SEQ ID NOS:2-364 in the enough identical aminoacid sequence of the described aminoacid sequence of even number." enough identical " is meant that first amino acid or nucleotide sequence compare with second amino acid or nucleotide sequence, contains being equal to or the same amino acid residue of enough or minimum, thereby the common structural domain is provided and/or shows the common functionally active.Conservative variant comprises because genetic code degeneracy and those different sequences.

Usually, respectively with SEQ ID NOS:2-364 among any aminoacid sequence of even number or the SEQ ID NOS:1-363 any nucleotide sequence of odd-numbered have at least about 45%, 55% or 65% identity, preferably at least about 70% or 75% identity, more preferably at least about 80%, 85% or 90%, most preferably be listed in this and be defined as enough identical at least about the amino acid of 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98% or 99% sequence identity or nucleotides sequence.The variant protein that comprises among the present invention has biologic activity, that is to say that they keep the expectation biological activity of native protein, and carbohydrate promptly described here utilizes related activity or multidrug transporter activity.The proteic biologic activity variant of the present invention can with described albumen have few to as 1-15 amino-acid residue, few to as 1-10, as 6-10, few to as 5, lack extremely as 4,3,2 or even the difference of 1 amino-acid residue.

Naturally occurring variant may reside in (for example Lactobacterium acidophilum group) in the colony.This variant can use well-known Protocols in Molecular Biology to identify, as polymerase chain reaction (PCR) and hybridization as described below.The synthetic nucleotide sequence that obtains, for example produce by site-directed mutagenesis or PCR mediated mutagenesis, still encoding carbohydrate utilizes the sequence of associated protein or multidrug transporter to be also included within the variant scope.One or more Nucleotide or aminoacid replacement, interpolation or disappearance can be introduced in Nucleotide disclosed herein or the aminoacid sequence, thereby this replacement, interpolation or disappearance are introduced encoded protein.Can be at the N-terminal or the C-terminal of native protein, or add (insertion) or disappearance (brachymemma) in one or more sites of native protein.Similarly, one or more Nucleotide or amino acid whose replacement can be carried out in one or more sites of native protein.

For example, conserved amino acid replaces and can carry out at nonessential amino-acid residue one or more predictions, preferred place." nonessential " amino-acid residue is and not change bioactive residue by proteic wild-type sequence change, and " essential " amino acid is that biological activity is needed." conserved amino acid replacement " is the displaced replacement of amino-acid residue that amino-acid residue is had similar side chain.Amino-acid residue family with similar side chain is known in the art.These families comprise amino acid with basic side chain (Methionin for example, arginine, Histidine), acid side-chain (aspartic acid for example, L-glutamic acid), uncharged polar side chain (glycine for example, l-asparagine, glutamine, Serine, Threonine, tyrosine, halfcystine), non-polar sidechain (L-Ala for example, Xie Ansuan, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met), tryptophane), β-branched building block (Threonine for example, Xie Ansuan, Isoleucine) and aromatic side chain (tyrosine for example, phenylalanine, tryptophane, Histidine).This replacement should not carried out to conservative amino acid residues or to the amino-acid residue that is positioned at conservative motif (this there residue is that protein-active is essential).

Alternatively, sudden change can all or part of length relevant along the carbohydrate utilization randomly or the multidrug transporter encoding sequence be carried out, as passes through saturation mutagenesis.Mutant can be opened and use the relevant or active mutant that screens retains biological activity of multidrug transporter of standard test technology for detection carbohydrate utilization by recombinant expressed.The method that mutagenesis and nucleotide sequence change is known in the art.Referring to for example Kunkel (1985) Proc.Natl.Acad.Sci.USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol.Molecular Biology (MacMillan Publishing Company, New York) and the reference of wherein quoting.Obviously, the sudden change of carrying out in the DNA of coding variant should not destroy the reading frame, preferably can not produce the complementation district that can form secondary mRNA structure.Referring to EP patent application publication number 75,444.The relevant guidance that does not influence the bioactive suitable aminoacid replacement of proteins of interest can be at Dayhoff et al. (1978) Atlas of ProteinSequence and Structure (Natl.Biomed.Res.Found., Washington, D.C.) find in the model, be hereby incorporated by.

Expectation does not produce radical change in disappearance, insertion and the replacement of this protein sequence that comprises to proteic characteristic.Yet, when before doing like this, being difficult to predict definite influence of replacement, disappearance or insertion, it will be understood by those skilled in the art that this influence can be by conventional shaker test assessment.That is to say that this activity can be estimated with the modification sequence specific activity by original series is active.Referring to " using method " of lower section, for example can be used for measuring carbohydrate and utilize related activity or the active test of multidrug transporter.

Variation Nucleotide of the present invention and aminoacid sequence also comprise the sequence that derives from mutagenesis and reorganization program such as DNA reorganization.Use this program, the relevant or multidrug transporter coding region of one or more different carbohydrate utilizations can be used for producing the new carbohydrate with desired characteristic and utilizes associated protein or new multidrug transporter.In such a way, from the correlated series polynucleotide group who comprises sequence area with basic sequence identity produce the recombination of polynucleotide library and can be in external or body homologous recombination.For example, use this method, the sequence motifs in coding structures of interest territory can be in carbohydrate utilization of the present invention relevant or multidrug transporter gene and other carbohydrate utilization is relevant or the multidrug transporter gene between reorganize, to obtain the proteic new gene that the coding properties of interest improves, increase as Km under the situation of enzyme.The strategy of this DNA reorganization is known in the art.Referring to for example Stemmer (1994) Proc.Natl.AGad.Sci.USA 91:10747-10751; Stemmer (1994) Nature370:389-391; Crameri et al. (1997) Nature Biotech.15:436-438; Moore et al. (1997) J Mol.Biol.272:336-347; Zhang et al. (1997) Proc.Natl.Acad.Sci.USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; With United States Patent (USP) 5,605,793 and 5,837,458.

Carbohydrate utilization variant relevant and multidrug transporter can play agonist (simulation) or antagonist.Protein agonist can keep the biologic activity substantially the same with the albumen of natural existence form or its subgroup.Protein antagonist can suppress proteic one or more activity of natural existence form, by for example competitive in conjunction with comprising that the carbohydrate utilization is correlated with or the downstream or the upstream member of the cell signal cascade reaction of multidrug transporter.

The carbohydrate utilization variant relevant or multidrug transporter that plays agonist or antagonist action can be by the mutant of or multidrug transporter relevant at the carbohydrate utilization, and for example the agonist of truncated mutant or antagonistic activity screening combinatorial library identifies.In one embodiment, carbohydrate utilizes the diversified library of related variants by producing at the nucleic acid level combinatorial mutagenesis and being encoded by diversified gene library.Carbohydrate utilization diversified library relevant or the multidrug transporter variant can followingly produce, by for example enzyme process the synthetic oligonucleotide mixture is connected into gene order, make potential carbohydrate utilization degeneracy group relevant or the multidrug transporter sequence can be used as independent expression of polypeptides, perhaps alternatively, one group of bigger fusion rotein (for example for phage display) relevant as the carbohydrate containing one utilization or the multidrug transporter sequence set is expressed.The whole bag of tricks that can be used for producing from degenerate oligonucleotide sequence the library of the relevant or multidrug transporter variant of potential carbohydrate utilization is arranged.The chemosynthesis of degeneracy gene order can be carried out in automatic dna synthesizer, synthetic gene can be connected with suitable expression then.The use of gene degeneracy group allows to provide coding to expect that potential carbohydrate utilization is correlated with or all sequences of multidrug transporter sequence set with mixture.The synthetic method that annexs oligonucleotide be known in the art (referring to for example, Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu.Rev.Biochem.53:323; Itakura et al. (1984) Science 198:1056; Ikeet al. (1983) Nucleic Acids Res.11:477).

In addition, the relevant or segmental library of multidrug transporter encoding sequence of carbohydrate utilization can be used for producing screening and selects segmental diversified group of the relevant or multidrug transporter of carbohydrate utilization carbohydrate utilization relevant or the multidrug transporter variant subsequently.In one embodiment, the segmental library of encoding sequence can be by following generation: only take place at per molecule under the condition of an about otch, handle the double-stranded PCR fragment of the relevant or multidrug transporter encoding sequence of carbohydrate utilization with nuclease, denatured double stranded dna, to the DNA renaturation with form double-stranded DNA (double-stranded DNA can comprise from the different incisions product have justice/antisense to), by handling with the S1 nuclease and removing the strand part and the fragment that is produced is connected into expression vector from the duplex that forms again.Use this method, people can obtain encoding carbohydrate utilization relevant or all size N-terminal of multidrug transporter and the expression library of interior segments.

Screening is known in the art by the combinatorial library gene product of point mutation or brachymemma preparation and several technology in the cDNA library of screening the gene product with selected performance.This technology can be adjusted to the rapid screening of the gene library of the combinatorial mutagenesis generation that is used for or multidrug transporter relevant by the carbohydrate utilization.The most widely used technology that is suitable for high throughput analysis, the big gene library of screening generally includes gene library is cloned into the rf expression vector, transform suitable cell with the vector library that produced, with to expecting that active detection can promote the carrier condition following table of separating the detected gene of coded product to reach combination gene.Whole mutagenesis (the REM)-a kind of technology that increases functional mutants frequency in the library that circulates can be used in combination to identify relevant or multidrug transporter variant (Arkin and Yourvan (1992) the Proc.Natl.Acad.Sci.USA 89:7811-7815 of carbohydrate utilization with shaker test; Delgrave etal. (1993) Protein Engineering 6 (3): 327-331).

Sequence identity

Relevant and the multidrug transporter sequence of carbohydrate utilization is the member of family with molecule of conservative functional character." family " is meant two or more albumen or the nucleic acid with enough Nucleotide or amino acid sequence identity.The family of containing highly different groups can be divided into subfamily.Clan (clan) is the one group of family that is considered to have the common ancestor.The member of clan usually has similar tertiary structure." sequence identity " meaning refers to when at least one is compared two sequences identical Nucleotide or amino-acid residue when obtaining maximum consistence at comparison window.Continuous fragment when " comparison window " is meant two Nucleotide or the comparison of aminoacid sequence the best, wherein second sequence compared with first sequence to contain and added or disappearance (being breach).Usually, for the nucleic acid comparison, at least 20 continuous nucleotides of comparison window length, optional can be 30,40,50,100 or longer.For the aminoacid sequence comparison, at least 6 continuous amino acids of comparison window length, optional can be 10,15,20,30 or longer.It will be appreciated by those skilled in the art that for fear of owing to comprise the high similarity that breach causes, introduce the breach point penalty usually and deduct this point penalty from mating numeral.

The family member can be from identical or different species, and can comprise homologue and different albumen.Usually the member of a family demonstrates the common functional character.Homologue can be relevant with Lactobacterium acidophilum carbohydrate disclosed herein utilization according to them or the identity of multidrug transporter nucleotide sequence, use cDNA or its part as hybridization probe, under following disclosed stringent hybridization condition, separate according to the standard hybridization technique.

For determining the identity per-cent of two amino acid or nucleotide sequence, compare.The identity per-cent of two sequences is functions (being identity per-cent=identical residue number/residue sum * 100) of the identical residue number that two sequences are shared in the comparison window.In one embodiment, sequence length is identical.And mention that below the method similar methods can be used for determining the identity per-cent between two sequences.This method can be used under the situation that allows or do not allow breach.Comparison also can be undertaken by checking manually.

When aminoacid sequence aspect the conservative replacement not simultaneously, identity per-cent can be to adjusted to revise the conservative character that replaces.The method of carrying out this adjustment is known in the art.Usually conservative the replacement as part rather than complete mispairing, mark, and increases sequence identity per-cent thus.

Mathematical algorithm can be used for determining the identity per-cent of two sequences.The limiting examples of mathematical algorithm is the algorithm of Karlin and Altschul (1990) Proc.Natl.Acad.Sci.USA87:2264, revises as Karlin and Altschul (1993) Proc.Natl.Acad.Sci.USA 90:5873-5877; The algorithm of Myers and Miller (1988) CABIOS 4:11-17; The local alignment algorithm of Smith et al. (1981) Adv.Appl.Math.2:482; Comprehensive alignment algorithm of Needleman and Wunsch (1970) J.Mol.Biol.48:443-453; With the local comparison method of the retrieval of Pearson and Lipman (1988) Proc.Natl.Acad.Sci.USA 85:2444-2448.

Various executive programs have been designed, so that can determine sequence identity based on these mathematical algorithms.The blast program of Altschul et al. (1990) J.Mol.Biol.215:403 is based on the algorithm of above-mentioned Karlin and Altschul (1990).Obtain to use BLASTN program, score=100, word length=12 to implement with the retrieval of nucleotide sequence homologous nucleotide sequence of the present invention.For the aminoacid sequence of acquisition, can use the BLASTX program, score=50, word length=3 with the sequence homology of coding albumen of the present invention or polypeptide.By using the Gapped BLAST (BLAST 2.0) that describes among Altschul et al. (1997) the Nucleic acids Res.25:3389 can obtain the breach comparison.For the distance relation between detection molecules, can use PSI-BLAST.Referring to above-mentioned Altschul et al. (1997).For all blast programs, can use the default parameters of each program.Comparison also can be undertaken by checking manually.

Can be used for determining that another program of sequence identity per-cent is ALIGN program (2.0 version), it uses the mathematical algorithm of above-mentioned Myers and Miller (1988).When the comparing amino acid sequence, PAM120 weighting residue table, notch length point penalty be 12 and the breach point penalty be 4 can use with this program.

Except that ALIGN and blast program, BESTFIT, GAP, FASTA and TFASTA program are that GCG Wisconsin genetics software package the 10th version (can be from Accelrys Inc., 9685Scranton Rd., San Diego, California, the USA acquisition) a part, and can be used to carry out sequence alignment.This preferable procedure is a GAP version 10, and it uses the algorithm of above-mentioned Needleman and Wunsch (1970).Except as otherwise noted, be meant by use to have those values that the GAP version 10 of following parameters obtains in this sequence identity value that provides: nucleotide sequence homology % and similarity % use GAP weight be 50 and length weight be 3, and nwsgapdna.cmp gets sub matrix; Amino acid sequence identity % and similarity % use GAP weight be 8 and length weight be 2 and BLOSUM62 get sub matrix; Or its any equivalent procedures." equivalent procedures " meaning refers to in question any two any sequences of sequence, when comparing with the corresponding comparison that GAP version 10 produces, produces the comparison program of the comparison with identical Nucleotide or amino-acid residue coupling and identical sequence identity per-cent.

The comparison of sequence and inquiry sequence is described to " hitting " (hit) usually in the database that BLASTN, FASTA, BLASTP or similar algorithm produce.The one or more database sequences that produce by BLASTN, FASTA, BLASTP or similar algorithm are to being compared and the similar part of identifying sequence by hitting of inquiry sequence.To the sequence length part of hitting ordinary representation and inquiry sequence of database sequence, promptly the part of inquiry sequence or fragment are overlapping.Yet, the overlapping whole length that can represent inquiry sequence.Inquiry sequence that BLASTN, FASTA or BLASTP algorithm produce and the common series arrangement of hitting of database sequence comparison with similarity and sequence overlap length.

Polynucleotide and polypeptide by BLASTN, FASTA or BLASTP algorithm and inquiry sequence comparison hit generation " expection " value.Desired value (E value) expression is when retrieving the database of a certain size, and people's " expection " can see on the continuous sequence of certain number at random hits " number ".Desired value is used as the significance threshold value of determining database such as GenBank or EMBL database that whether shows true similarity of hitting.For example, hit to polynucleotide and to be appointed as 0.1 E value and to be interpreted as in the database of GenBank database size, people can be expected on the sequence alignment part with similar score and see 0.1 coupling at random.By this standard, the comparison of the polynucleotide sequence probability identical with compatible portion is 90%.Have 0.01 or littler E value sequence for comparison and compatible portion, use BLASTN or fasta algorithm find the probability of coupling at random in the GenBank database be 1% or littler.

According to embodiment of the present invention, " variation " of the present invention polynucleotide and polypeptide comprise produce about 0.01 or the sequence of littler E value when comparing with polynucleotide of the present invention or peptide sequence.That is to say, variation polynucleotide or polypeptide are to have at least 99% identical possibility with polynucleotide of the present invention or polypeptide, are set to BLASTN, the FASTA of parameter described here or BLASTP algorithm as use and are measured to and have 0.01 or any sequence of littler E value.In other embodiments, the variation polynucleotide are to have at least 99% identical possibility with polynucleotide of the present invention, and BLASTN that is set at this characterising parameter as use or fasta algorithm are measured to has 0.01 or the polynucleotide sequence of the present invention of littler E value has equal amts or than the sequence of its smaller amounts nucleic acid.Similarly, the variation polypeptide is to have at least 99% identical possibility with polypeptide of the present invention, and the BLASTP algorithm that is set at this characterising parameter as use is measured to has 0.01 or the peptide sequence of the present invention of littler E value has equal amts or than the amino acid whose sequence of its smaller amounts.

As mentioned above, identity per-cent is following definite: use one of BLASTN, the FASTA be set to working parameter described here or BLASTP algorithm aligned sequences, and identify identical nucleic acid or amino acid whose quantity on the comparison part; With identical nucleic acid or amino acid whose quantity nucleic acid or amino acid sum divided by polynucleotide of the present invention or peptide sequence; Multiply by 100 then to determine identity per-cent.For example, use in the comparison that parameter described here carries out at the BLASTN algorithm, the polynucleotide sequence that has 520 nucleic acid in polynucleotide with 220 nucleic acid of the present invention and the GenBank database has one and hits on the fragment of 23 Nucleotide.23 Nucleotide hit and comprise 21 identical Nucleotide, breach and a different Nucleotide.Therefore the identity per-cent that hits in polynucleotide of the present invention and the GenBank library is 21/220 to multiply by 100 again, or 9.5%.Therefore the polynucleotide sequence in the GenBank database is not the variant of polynucleotide of the present invention.

The evaluation of homologous sequence with separate

The carbohydrate that sequence identity relevant with carbohydrate utilization described herein based on them or multidrug transporter nucleotide sequence or its fragment and variant is identified utilizes related nucleotide sequences to comprise in the present invention.Method such as PCR or hybridization can be used for identifying sequence, for example identical with sequence of the present invention basically sequence from cDNA or genomic library.Referring to for example Sambrook et al. (1989) Molecular Cloning.Laboratory Manual (2ded., Cold Spring Harbor Laboratory Press, Plainview, NewYork) and Innis, et al. (1990) PCR Protocols:A Guide to Methodsand Applications (Academic Press, New York).The method that makes up this cDNA and genomic library is normally known in the art, and also open in the above referred-to references.

In hybridization technique, hybridization probe can be genomic DNA fragment, cDNA fragment, RNA fragment or other oligonucleotide, and can be made up of all or part of of known nucleotide sequence disclosed herein.In addition, but can with detection moiety as ³²P or any other detectable label, as other radio isotope, fluorescent chemicals, enzyme or enzyme cofactor come mark they.The probe that is used to hybridize can carry out mark by the synthetic oligonucleotide to or multidrug transporter nucleotide sequence relevant based on known carbohydrate utilization disclosed herein and prepare.Can use the conservative Nucleotide of or multidrug transporter nucleotide sequence or coded aminoacid sequence relevant or the degenerated primer of amino-acid residue design in addition based on known carbohydrate utilization.Hybridization probe typically be included under the stringent condition with nucleotide sequence of the present invention or its fragment or variant at least about 10, preferred about 20, the nucleotides sequence column regions of 50,75,100,125,150,175,200,250,300,350 or 400 continuous nucleotide hybridization more preferably from about.For finishing specific hybrid under various conditions, this probe comprises the relevant or central unique sequence of multidrug transporter sequence of carbohydrate utilization.The preparation of hybridization probe is known in the art, and at Sambrook et al. (1989) Molecular Cloning:A Laboratory Manual (2ded., Cold Spring Harbor Laboratory Press, Plainview, NewYork) open in, be hereby incorporated by.

In one embodiment, encoding carbohydrate utilization complete nucleotide sequence relevant or multidrug transporter is used as and identifies that new carbohydrate utilization is correlated with or the probe of multidrug transporter sequence and messenger RNA(mRNA) s.In another embodiment, this probe is the fragment of nucleotide sequence disclosed herein.In some embodiments, under stringent condition, can be at least about 300,325,350,375,400,425,450,500,550,600,650,700,800,900,1000,1200,1400,1600,1800 or 2000 Nucleotide with the nucleotide sequence length of probe hybridization.

Substantially the same sequence will hybridization each other under stringent condition." stringent condition " be meant probe will with it target sequence than with the condition (for example surpass background at least 2 times) of other sequence with detectable hybridization more.Usually, stringent condition comprises those conditions of hybridization and washing, has with this understanding at least about 60%, 65%, 70%, and the Nucleotide of preferred 75% sequence identity keeps hybridization usually each other.Stringent condition is known in the art and can finds (John Wiley ﹠amp in the Current of Molecular Biology Protocols; Sons, New York (1989)), 6.3.1-6.3.6.Hybridization is carried out in about 24 hours usually, about 4 to about 12 hours usually.

Stringent condition is sequence-dependent, and under different environment with difference.Total length or part nucleotide sequence can be used for the homologue and/or the orthologous gene that obtain the present invention includes." orthologous gene " is meant the gene that is got by the common ancestral gene and find in different plant species because species form.The gene of finding in different plant species is when their nucleotide sequence and/or their coding protein sequence is considered to orthologous gene when being shared in this basic identity that defines Anywhere.The function of orthologous gene is usually very conservative in the middle of each species.

When using probe, hybridization conditions will be that salt concn is less than about 1.5M, common about 0.01 to 1.0M Na ionic concn (or other salt), pH 7.0 to 8.3, and for short probe (for example 10 to 50 Nucleotide) temperature at least about 30 ℃, for long probe (for example greater than 50 Nucleotide) temperature at least about 60 ℃ condition.

Post-hybridization washing helps to control specificity.Two ionic strength and temperature that key factor is final washing soln.For the sequence that the target sequence of detection and total length or approximate total length is hybridized, the temperature under the stringent condition is chosen to hang down about 5 ℃ than this concrete sequence in ionic strength of determining and the melting temperature(Tm) under the pH (Tm).Yet stringent condition will comprise low 1 ℃ of temperature to 20 ℃ of scopes than Tm, and this depends on the strict degree of expectation, or other qualified herein temperature.For DNA-DNA hybridization, Tm can use the equation of Meinkoth and Wahl (1984) Anal.Biochem.138:267-284 to determine: Tm=81.5 ℃+16.6 (logM)+0.41 (%GC)-0.61 (% methane amide)-500/L; Wherein M is that volumetric molar concentration, the %GC of monovalent cation are that per-cent in DNA of guanine and cytidylic acid(CMP), per-cent, the L that the % methane amide is methane amide in the hybridization solution are the crossbred length of representing with base pair.Tm is the temperature (under ionic strength that limits and pH) of the probe hybridization of 50% complementary target sequence and Perfect Matchings.

Detection has in various degree, and the ability of the sequence of homology can obtain by the severity that changes hybridization and/or wash conditions.For the target sequence of 100% identical (homology detection), must acquisition do not allow the stringent condition of mispairing.If allow the nucleotide residue mispairing to take place, then can detect sequence (allos detection) with low similarity.Mispairing for per 1%, Tm reduces about 1 ℃; Therefore, can handle hybridization and/or wash conditions to allow to have the sequence hybridization of target identity per-cent.For example, if preferably have 〉=sequence of 90% sequence identity, then Tm can reduce by 10 ℃.Two nucleotide sequences can be substantially the same, and can not hybridize each other under stringent condition, but their encoded polypeptides are substantially the same.For example, if use the maximum codon degeneracy generation nucleic acid copy of genetic code, this situation can take place.

Exemplary low stringency condition be included in hybridize in 30-35% methane amide, 1M NaCl, 1%SDS (sodium lauryl sulphate), 37 ℃ the damping fluid and 1 * to 2 * SSC (20 * SSC=3.0M NaCl/0.3M Trisodium Citrate), 50 to 55 ℃ of washings.The medium stringent condition of example is included in 40 to 45% methane amides, 1.0M NaCl, 1%SDS, 37 ℃ of hybridization and 0.5 * to 1 * SSC, 55 to 60 ℃ of washings.The high stringent condition of example is included in 50% methane amide, 1MNaCl, 1%SDS, 37 ℃ of hybridization and in 0.1 * SSC, 60 to 65 ℃ of washings.Randomly, lavation buffer solution can comprise about 0.1% to about 1%SDS.Hybridize the time length usually less than about 24 hours, about 4 to about 12 hours usually.The extension instructs of nucleic acid hybridization is at Tijssen (1993) Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, Part I, Chapter2 (Elsevier, New York); With Ausubel et al., eds. (1995) CurrentProtocols in Molecular Biology finds among the Chapter 2 (Greene Publishing andWiley-Interscience, New York).Referring to Sambrook et al. (1989) Molecular Cloning:A Laboratory Manual (2d ed.; Cold SpringHarbor Laboratory Press, Plainview, New York).

In PCR method, can the design oligonucleotides primer, be used in the PCR reaction from the cDNA of interested any biological extraction or the genomic dna corresponding dna sequence dna that increases.Preferred PCR primer length is at least about 10 Nucleotide, most preferably at least about 20 Nucleotide.Design PCR primer and PCR clone's method is that this area is known usually, and at Sambrook etal. (1989) Molecular Cloning:A Laboratory Manual (2d ed.; ColdSpring Harbor Laboratory Press, Plainview, New York) in open.Also referring to Innis et al., eds. (1990) PCR Protocols:A Guide to Methodsand Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); With Innisand Gelfand, and eds. (1999) PCR Methods Manual (Academic Press, NewYork).The currently known methods of PCR includes but not limited to use the method for pairing primer, nested primers, monospecific primer, degenerated primer, gene-specific primer, carrier specificity primer, part mispairing primer or the like.

Test

Disclosed polypeptide described in the test sample and/or expression of nucleic acids and disclosed their active diagnostic test are disclosed.Exist in the test sample or do not exist the disclosed nucleic acid or the proteic exemplary method that comprise disclosed polypeptide to comprise from food/milk-product/feeds product, starting culture (mother liquor, seed, on a large scale/supporting, concentrate, dry, freeze-drying, freezing), food/milk-product/feeds product of cultivating, food supplement, the biological processing fermented product or the experimenter of administration of probiotic material obtain sample, make sample contact can detect the compound or the reagent of disclosed polypeptide or nucleic acid (for example comprising disclosed nucleic acid or its segmental mRNA or genomic dna), make the situation that exists that in sample, detects disclosed sequence.The result who obtains by sample from food, enriching substance, culture, product or experimenter can with by comparing from control cultures, product or the result that tried to obtain.

A kind of reagent that detection comprises the mRNA of disclosed nucleotide sequence or genomic dna be can with the openly labeling nucleic acid probe of nucleotide sequence hybridization of mRNA or genomic dna.Nucleic acid probe can be for example disclosed nucleic acid, as nucleic acid or its part of odd number among the SEQ ID NOS:1-363, as length at least 15,30,50,100,250 or 500 Nucleotide and under stringent condition, be enough to comprise as described in the mRNA of disclosed nucleotide sequence or the nucleic acid of genomic dna specific hybrid.At this other proper probes that is used for diagnostic test of the present invention has been described.

Proteic a kind of reagent that detection comprises open peptide sequence is preferably to have the antibody of detectable label in conjunction with the antibody of the disclosure polypeptide.Antibody can be polyclonal, or more preferably monoclonal.Can use complete antibody or its fragment (for example Fab or F (ab ') 2).Be directed to term " mark " that probe or antibody uses and mean and comprise by with detectable substance and probe or direct label probe of antibody coupling (being physical connection) or antibody, and by indirect labelling probe or antibody with another reagent react of direct mark.The example of indirect labelling comprises use fluorescent mark two anti-detection of primary antibody and with vitamin H end mark dna probe, makes probe to detect with fluorescently-labeled streptavidin.

Term " sample " meaning comprise exist among the experimenter or from its isolating tissue, cell and biological fluid, and from the cell of starting culture or the food that carries this culture or use this culture to get.That is to say that detection method of the present invention can be used for comprising in the test sample in external and the body mRNA, albumen or the genomic dna of open sequence.The ex vivo technique that detection comprises the mRNA of open sequence comprises Northern hybridization and in situ hybridization.The proteic ex vivo technique that detection comprises open polypeptide comprises enzyme linked immunosorbent assay (ELISAs), Western trace, immuno-precipitation and immunofluorescence technique.The ex vivo technique that detection comprises the genomic dna of open nucleotide sequence comprises Southern hybridization.In addition, detect the interior technology of the proteic body that comprises open polypeptide and comprise the traget antibody introducing experimenter that will resist open polypeptide.For example, this antibody can come mark by the radio-labeling that the standard imaging technique detects with its existence and the location in the experimenter.

In one embodiment, this sample contains the protein molecular from the test experimenter who consumes the probiotic bacterium material.Alternatively, this sample can contain mRNA or the genomic dna from starting culture.

The present invention also comprises open nucleic acid or the proteic test kit that has situation that comprises open polypeptide in the test sample.This test kit can be used for measuring the microorganism of expressing the concrete polypeptide of the present invention and whether is present in food or starting culture or consumes among the experimenter of probiotic bacterium material.For example, this test kit can comprise the amount of disclosed polypeptide in the tagged compound of open polypeptide in can test sample or mRNA or reagent and the working sample approach (for example the antibody of the open polypeptide of identification or with the open polypeptide of coding, for example SEQ ID NOS:2-364 in the DNA bonded oligonucleotide probe of even number sequence).Test kit can also comprise the specification sheets of the use that this compound is described in detail in detail.

For the test kit based on antibody, this test kit for example can comprise: (1) and open polypeptide bonded first antibody (for example being attached to solid support); Randomly (2) but with open polypeptide or first antibodies and put together detection reagent second different antibodies.For the test kit based on oligonucleotide, this test kit for example can comprise: the oligonucleotide of (1) and disclosed nucleic acid array hybridizing, for example the oligonucleotide of detectable label or (2) can be used for the increasing primer of disclosed nucleic acid is right.

This test kit can also comprise for example buffer reagent, sanitas or protein stabiliser.But this test kit can also comprise the required composition of detection detection reagent (for example enzyme or substrate).This test kit can also contain control sample or control sample series, can to its test and with contained specimen relatively.Each composition of test kit is sealed in the independent container usually, and all various containers are in working instructions are contained in individual packaging.

In one embodiment, this test kit comprises a plurality of probes of array format, and the probe as describing among for example United States Patent (USP) 5,412,087 and 5,545,531 and the international publication number WO 95/00530 is hereby incorporated by.Supply the probe of the usefulness of array directly to synthesize on array surface, disclosed as international publication number WO 95/00530, or go forward that earlier synthetic (Gait compiles (1984) Oligonucleotide Synthesis aPractical Approach IRL Press being fixed to array surface, Oxford, England).This probe can, use technology well known to those skilled in the art, as United States Patent (USP) 5,412, the technology of describing in 087 is fixing from the teeth outwards.Probe can be nucleic acid or peptide sequence, the form of preferred purifying, or antibody.

Array can be used for screening organism, sample or the product difference aspect their genome, cDNA, polypeptide or anti-body contg, whether comprises concrete sequence or proteic existence, and the concentration of those materials.Detect by the signal that produces from the mark that is attached to the nucleic acid, the polypeptide that comprises disclosed aminoacid sequence or the antibody that comprise described open nucleotide sequence with for example combining of capture probe.This method can comprise makes the molecule contact that comprises open nucleic acid, polypeptide or antibody have first array and second array with different polynary capture probes of polynary capture probe.The result that can compare each hybridization is to analyze expression difference between first and second samples.First polynary capture probe can be from control sample, for example wild-type milk-acid bacteria or contrast experimenter, for example food, food supplement, starting culture sample or biofluid.Second polynary capture probe can be from laboratory sample, for example mutant milk-acid bacteria or consume the experimenter of probiotic bacterium material, for example starting culture sample or biofluid.

These tests may be selected with the quality control procedure that must detect unwanted material particularly useful to microorganism.The genetic composition that the detection of special nucleus nucleotide sequence or polypeptide also can be used for determining food, leavened prod or industrial microorganism or be present in the microorganism of the animal or human's Digestive tract that consumes probiotic bacterium.

The antisense nucleoside enzyme sequence

The present invention also comprises antisense nucleic acid, promptly with proteins encoded phosphorothioate odn complementary molecule arranged, for example with double-stranded cDNA molecule encoding chain complementary or with mRNA sequence complementary molecule.Therefore, antisense nucleic acid can with phosphorothioate odn is arranged with hydrogen bonded.Antisense nucleic acid can or multidrug transporter coding strand complementation relevant with whole carbohydrate utilization, or only complementary with its part, for example all or part of protein-coding region (or open reading frame).Antisense nucleic acid can be relevant with the encoding carbohydrate utilization or the non-coding region antisense of the nucleotide sequence coded chain of multidrug transporter.Non-coding region is to be positioned at the coding region flank and not to be translated into amino acid whose 5 ' and 3 ' sequence.Antisense base sequences is useful in destroying expression of target gene.Can use with corresponding sequence to have 70%, preferred 80%, the more preferably antisense constructs of 85%, 90% or 95% sequence identity.

Under the situation of the coding strand sequence that the relevant or multidrug transporter (for example even number sequence among the SEQ ID NOS:2-364) of coding carbohydrate utilization disclosed herein is arranged, can design antisense nucleic acid of the present invention according to the base pairing rules of Watson and Crick.Antisense nucleic acid can or multidrug transporter mRNA whole coding region complementation relevant with the carbohydrate utilization, but is more preferably the oligonucleotide of only a part of antisense of the coding of or multidrug transporter mRNA relevant with the carbohydrate utilization or non-coding region.For example, antisense oligonucleotide can with the regional complementarity around the relevant or multidrug transporter mRNA translation initiation site of carbohydrate utilization.Antisense oligonucleotide length can be for example about 5,10,15,20,25,30,35,40,45 or 50 Nucleotide, or it can be 100,200 Nucleotide or longer length.Antisense nucleic acid of the present invention can use chemosynthesis known in the art and enzymatic Connection Step to make up.

For example, can use naturally occurring Nucleotide or be designed to increase the biological stability of this molecule or increase antisense and the various modified nucleotides of the double-helical physical stability that forms between the phosphorothioate odn are arranged, include but not limited to that for example phosphorothioate derivative and acridine substituted nucleotide come chemosynthesis antisense nucleic acid (for example antisense oligonucleotide).Alternatively, can use its amplifying nucleic acid to come biological length of schooling to be equipped with antisense nucleic acid with antisense orientation (promptly the RNA from the transcribed nucleic acid that inserted will be an antisense orientation to interested target nucleic acid) subclone expression of nucleic acids carrier.

Antisense nucleic acid of the present invention can be a α-different nucleic acid.α-different nucleic acid and complementary RNA form special double-stranded heterozygote, and be wherein opposite with common β unit, this chain (Gaultier et al. (1987) Nucleic Acids Res.15:6625-6641) parallel to each other.Antisense nucleic acid can also comprise 2 '-O-methyl ribonucleotides (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or chimeric RNA-DNA analogue (Inoueet al. (1987) FEBSLett.215:327-330).

The present invention also comprises ribozyme, and it is to have to cut the catalytic RNA molecule that has the ribonuclease activity of complementary single-chain nucleic acid of distinguishing such as mRNA with it.Ribozyme (for example hammerhead ribozyme (Haselhoff and Gerlach (1988) Nature 334:585-591) in describe) can be used for the catalyze cleavage carbohydrate and utilize associated protein mRNA transcript, suppresses the translation of the relevant or multidrug transporter mRNA of carbohydrate utilization thus.Can have specific ribozyme by nucleotide sequence (for example odd number sequence among the SEQ IDNOS:1-363) design relevant based on carbohydrate utilization disclosed herein or multidrug transporter cDNA nucleic acid relevant to the encoding carbohydrate utilization or multidrug transporter.United States Patent (USP) 4,987,071 referring to for example Cech etc.; United States Patent (USP) 5,116,742 with Cech etc.Alternatively, the relevant or multidrug transporter mRNA of carbohydrate utilization can be used for selecting to have from the RNA library of molecules catalytic RNA of special ribozyme activity.Referring to for example Bartel and Szostak (1993) Science 261:1411-1418.

The present invention also comprises the nucleic acid that forms triple-helix structure.For example, or multidrug transporter regulatory region (for example carbohydrate utilization relevant or multidrug transporter promotor and/or enhanser) the complementary nucleotide sequence relevant by target with the carbohydrate utilization, form the triple-helix structure that the utilization of prevention carbohydrate is relevant or the multiple medicines transporter gene is transcribed in target cell, can suppress the relevant or multidrug transporter genetic expression of carbohydrate utilization.Usually referring to Helene (1991) Anticancer Drug Des.6 (6): 569; Helene (1992) Ann.N.Y.Acad.Sci.660:27; And Maher (1992) Bioassays 14 (12): 807.

In some embodiments, nucleic acid of the present invention can be modified at base portion, sugar moieties or phosphoric acid ester main chain, to improve for example stability, hybridization or the solubleness of this molecule.For example, the deoxyribose phosphate ester main chain of nucleic acid can be modified, to produce peptide nucleic acid(PNA) (referring to Hyrup etal. (1996) Bioorganic ﹠amp; Medicinal Chemistry 4:5).Term " peptide nucleic acid(PNA) " or " PNAs " are meant nucleic acid mimics as used herein, dna analog for example, and wherein deoxyribose phosphate ester main chain is replaced by false peptide main chain, and only keeps four kinds of natural nucleus bases.The neutral main chain that has shown PNAs allows under conditions of low ionic strength and DNA and RNA specific hybrid.The synthetic of PNA oligomer can be used for example Hyrup et al. (1996) supra; Standard solid-phase polypeptide synthetic schemes described in Perry-O ' Keefe et al. (1996) the Proc.Natl.Acad Sci.USA 93:14670 carries out.

PNAs can be used as antisense or anti-gene reagent, stops or suppressing to duplicate the sequence-specific regulation and control that cause genetic expression by for example inducible transcription or translation.PNAs of the present invention can also be used in the analysis of single base-pair mutation in the gene for example, and the PCR clamping plate that instruct by for example PNA carry out; When with other enzyme, for example S1 nuclease (above-mentioned Hyrup (1996)) when being used in combination, can be used as artificial restriction enzyme; Or as the probe or primer (the above-mentioned Hyrup (1996) of dna sequence dna and hybridization; Above-mentioned Perry-O ' Keefe et al. (1996).

In another embodiment, carbohydrate utilization PNAs relevant or the multidrug transporter molecule can be modified, so that, form the PNA-DNA mosaic or pass through use liposome or other medicines delivery technique known in the art by lipotropy or other auxiliary group being connected with PNA, passing through as improving their stability, specificity or cellular uptake.The chimeric formation of PNA-DNA can be as Hyrup (1996) supra; Finn et al. (1996) NucleicAcids Res.24 (17): 3357-63; Mag et al. (1989) Nucleic AcidsRes.17:5973; With described the carrying out of Peterson et al. (1975) Bioorganic Med.Chem.Lett.5:1119.

Fusion rotein

The present invention comprises that also the carbohydrate utilization is relevant or multidrug transporter is chimeric or fusion rotein.Relevant or the multidrug transporter " chimeric protein " of carbohydrate utilization or " fusion rotein " comprise the relevant or multiple medicines of carbohydrate utilization that relevant with the non-carbohydrate utilization or non-multiple medicines transhipment polypeptide can be operatively connected respectively and transport polypeptide." carbohydrate utilizes related polypeptide " or " multiple medicines transhipment polypeptide " is meant to have the polypeptide that utilizes the aminoacid sequence of associated protein or multidrug transporter corresponding to carbohydrate respectively, and " non-carbohydrate utilizes related polypeptide " or " non-multiple medicines transhipment polypeptide " is meant to have respectively corresponding to utilizing the proteic aminoacid sequence that associated protein or multidrug transporter are not substantially the same, stem from identical or different biology with carbohydrate.In the carbohydrate utilization in the relevant or multidrug transporter fusion rotein, relevant or the multiple medicines transhipment polypeptide of carbohydrate utilization can be equivalent to all or part of of the relevant or multidrug transporter of carbohydrate utilization, preferably includes at least a biologic activity part of the relevant or multidrug transporter of carbohydrate utilization.The term relevant with fusion rotein " can be operatively connected " meaning and show that the carbohydrate utilization is relevant or multiple medicines is transported polypeptide and the non-carbohydrate utilization is relevant or multiple medicines transhipment polypeptide merges in in-frame mode each other.The non-carbohydrate utilization is correlated with or multiple medicines transhipment polypeptide can N-terminal or C-terminal relevant with the carbohydrate utilization or multiple medicines transhipment polypeptide merge.

The expression of the encoding sequence that connects produces the allogeneic amino acid sequence of two connections that form fusion rotein.The carrier polypeptide that carrier sequence (the relevant or non-multiple medicines transhipment of non-carbohydrate utilization polypeptide) can be encoded and be strengthened or increase the expression of fusion rotein in host bacterium.The fusion rotein part of carrier sequence encoding, promptly carrier polypeptide can be protein fragments, the partial or complete protein sequence of full functionality.Carrier zone or polypeptide can be designed to purified fusion protein in addition, comprise with antibody or the affinity purification special to carrier polypeptide.Similarly, can utilize the physicals of carrier polypeptide to allow the selectivity purifying of fusion rotein.

Interested special carrier polypeptide comprises superoxide dismutase (SOD), maltose binding protein (MBP), glutathione-S-transferase (GST), terminal Histidine (His) mark of N-or the like.This list is not restrictive, utilizes associated protein or multiple drug resistance albumen all can use in the method for the invention as any carrier polypeptide of expressing fusion protein because strengthen carbohydrate.

In one embodiment, fusion rotein is the relevant fusion rotein of GST-carbohydrate utilization, and wherein carbohydrate utilizes correlated series and the terminal fusion of GST sequence C.In another embodiment, fusion rotein is that carbohydrate utilizes associated protein-domain-immunoglobulin fusion proteins, and wherein carbohydrate utilizes all or part of of associated protein and the sequence that derives from the immunoglobulin (Ig) family member to merge.In other embodiments, fusion rotein comprises multidrug transporter of the present invention.Carbohydrate utilization of the present invention is correlated with or multidrug transporter-domain-immunoglobulin fusion proteins can be used as immunogen, in the experimenter, produce the relevant or anti-multidrug transporter associated antibodies of anti-carbohydrate utilization, relevant or the multidrug transporter associated ligands of purified carbon hydrate utilization, be used in shaker test in to identify that to suppress the carbohydrate utilization relevant or multidrug transporter is relevant with the carbohydrate utilization or the molecule of multidrug transporter ligand interaction.

Those skilled in the art will recognize that, can select the specific support polypeptide according to purification scheme.For example, His mark, GST and maltose binding protein representative have the carrier polypeptide of easy acquisition affinity column, and carrier polypeptide can combine and elute with this post.Therefore, at carrier polypeptide is under the situation of the terminal His mark of N-as six Histidines (His6 mark), can use to comprise metal-resin, for example nickel nitrilotriacetic acid(NTA) (Ni-NTA), nickel iminodiethanoic acid (Ni-IDA) and contain the relevant or multidrug transporter fusion rotein of matrix purified carbon hydrate utilization of cobalt resin (Co-resin).Referring to for example Steinert et al. (1997) QIAGEN News 4:11-15, all be incorporated herein by reference at this.At carrier polypeptide is under the situation of GST, can use the relevant or multidrug transporter fusion rotein of the matrix purified carbon hydrate utilization that comprises gsh-sepharose 4B (Sigma or Pharmacia Biotech); At carrier polypeptide is under the situation of maltose binding protein (MBP), can use the relevant or multidrug transporter fusion rotein of matrix purified carbon hydrate utilization that comprises with amylose starch deutero-agarose resin.

Preferably, produce chimeric or fusion rotein of the present invention by the standard recombinant dna technology.For example, the dna fragmentation of the different peptide sequences of encoding links together with can meeting frame, maybe can synthesize fusion gene, as uses automatic dna synthesizer.Alternatively, can use the anchor primer that between two consecutive gene fragments, causes complementary overhang to carry out the pcr amplification of gene fragment, can make this gene fragment annealing and amplification more subsequently, produce chimeric gene sequence (referring to for example Ausubel et al., eds. (1995) Current Protocols inMolecular Biology (Greene Publishing and Wiley-Interscience, New York).In addition, encoding carbohydrate utilization nucleic acid relevant or multidrug transporter can be cloned in the commercially available expression vector so that it partly meets frame ground ways of connecting with existing fusion.

Fusion protein expression vector is designed to be convenient to remove carrier polypeptide usually, to allow the natural bioactive that the carbohydrate utilization is relevant or the multidrug transporter reservation is relevant with it.The method of cleavage of fusion proteins is known in the art.Referring to for example Ausubel et al., eds. (1998) Current Protocols in Molecular Biology (John Wiley ﹠amp; Sons, Inc.).The chemical chop of fusion rotein can be finished with reagent such as cyanogen bromide, 2-(2-nitrophenyl sulphenyl)-3-methyl-3 '-bromo pseudo-indole, azanol or low pH.Chemical chop is usually finished under the sex change condition, with the insoluble fusion rotein of cutting script.

Expectation with the carbohydrate utilization relevant or multiple medicines transhipment polypeptide separates with carrier polypeptide and these fusion polypeptide between the cleavage site of joint be not under the naturally occurring situation, fusion constructs can be designed to contain the specific protease cleavage site, to promote removing of enzyme cutting and carrier polypeptide.In such a way, comprise enzyme interested the specificity cleavage site peptide-coding sequence joint sequence can carrier polypeptide encoding sequence (for example terminal His label of MBP, GST, SOD or N-) and the carbohydrate utilization is relevant or multiple medicines transhipment polypeptid coding sequence between meet the fusion of frame ground.Have the specific suitable enzymes of cleavage site and include but not limited to factor Xa, zymoplasm, enteropeptidase, remin, collagenase and marmor erodens (tobacco etchvirus, TEV) proteolytic enzyme.The cleavage site of these enzymes is well known in the art.Therefore, for example under the situation that usage factor Xa is relevant with the carbohydrate utilization with carrier polypeptide or the cutting of multiple medicines transhipment polypeptide is come, fusion constructs can be designed to comprise the joint sequence of coding factor Xa susceptibility cleavage site, for example sequence IEGR is (referring to for example Nagai and Thegersen (1984) Nature309:810-812, Nagai and Th φ gersen (1987) Meth.Enzymol.153:461-481 and Pryor and Leiting (1997) ProteinExpr.Purif.10 (3): 309-319 is hereby incorporated by).Using under the situation that zymoplasm is relevant with the carbohydrate utilization with carrier polypeptide or the cutting of multiple medicines transhipment polypeptide is come, fusion constructs can be designed to comprise the joint sequence of coding zymoplasm susceptibility cleavage site, for example sequence LVPRGS or VIAGR (respectively referring to for example Pryor and Leiting (1997) Protein Expr.Purif10 (3): 309-319 and Hong et al. (1997) Chin.Med.Sci.J.12 (3): 143-147 is hereby incorporated by).TEV proteolytic enzyme cutting site is known in the art.Cleavage site referring to describing in the United States Patent (USP) 5,532,142 for example all is incorporated herein by reference at this.Also referring to Ausubel et al., eds. (1998) Current Protocols in Molecular Biology (John Wiley ﹠amp; Sons, Inc.), the discussion among the Chapter16.

Antibody

Isolated polypeptide of the present invention can be used as immunogen, with the antibody that produces the relevant or multidrug transporter of specificity combined carbon hydrate utilization or stimulate in the body and produce antibody.Relevant or the multidrug transporter of total length carbohydrate utilization can be used as immunogen, or replacedly can use carbohydrate utilization described here to be correlated with or the antigenic peptide fragment of multidrug transporter.Carbohydrate utilization antigenic peptide relevant or multidrug transporter comprises among the SEQ ID NOS:1-320 shown in the even number at least 8 of aminoacid sequence, preferred 10,15,20 or 30 amino-acid residues, and comprise that the carbohydrate utilization is correlated with or the epi-position of multidrug transporter, the antibody of feasible anti-this peptide or multidrug transporter formation specific immunity mixture relevant with the carbohydrate utilization.The preferred epi-position that antigenic peptide comprises is that to be positioned at the carbohydrate utilization of protein surface relevant or multidrug transporter is regional, for example hydrophilic area.

Recombinant expression vector and cell

Nucleic acid of the present invention can be included in the carrier, the preferred expression carrier." carrier " is meant the nucleic acid that can transport another nucleic acid that it connects.Expression vector comprises one or more adjusting sequences, and guides the expression of gene that can be operatively connected with them." can be operatively connected " and be meant nucleotide sequence interested and regulate sequence and be connected, make to allow this nucleotide sequence to express (for example in in-vitro transcription/translation system during maybe when this carrier importing host cell, in host cell).Term " adjusting sequence " meaning comprises that promotor that may command transcribes, operon, enhanser, transcription terminator and other express controlling elements as translation control sequence (for example Shine-Dalgarno consensus sequence, initial sum terminator codon).These regulate sequence can be different, and this depends on for example employed cell.

Carrier can be in cell self-replicating (free build carrier), maybe can be incorporated in the cellular genome, duplicate (non-free build Mammals carrier) with host genome.Integrating vector contains at least one and the bacterial chromosome homologous sequence that allows to take place between the homologous dna and bacterial chromosome in the carrier reorganization usually.Integrating vector also can comprise phage or transposon sequence.Free build carrier or plasmid are the circular double-stranded DNA rings that wherein can be connected into extra dna fragmentation.When using recombinant DNA technology, the plasmid that can stablize maintenance in the host is the preferred form of expression vector normally.

Expression construct that the present invention includes or carrier comprise the nucleic acid construct of the present invention of the form that is suitable for express nucleic acid in cell.Expression in prokaryotic cell prokaryocyte and the vegetable cell comprises in the present invention.The design that those skilled in the art will figure out expression vector can be depended on the factor of selection, desirable protein expression level of cell to be transformed for example or the like.Expression vector of the present invention can be introduced into cell, produce the albumen or the peptide of nucleic acid encoding described here thus, comprise fusion rotein or peptide, for example the carbohydrate utilization is relevant or the carbohydrate utilization of multidrug transporter, mutant form is relevant or multidrug transporter, fusion rotein or the like).

Bacterial expression vector

Regulate sequence and comprise the sequence that instructs the nucleotide sequence constitutive expression, and the sequence that only under some envrionment conditions, instructs the nucleotide sequence inducible expression.The bacterium promotor is and to start any dna sequence dna that encoding sequence (for example structure gene) downstream (3 ') is transcribed into mRNA in conjunction with the bacteria RNA polysaccharase.Promotor will have transcripting starting area, and it is in the position near encoding sequence 5 ' end usually.This transcription initiation region generally includes RNA polymerase binding site and transcription initiation site.The bacterium promotor also can have second structural domain that is called operon, and it can be overlapping with the synthetic contiguous RNA polymerase binding site that begins to locate of RNA.This operon allows negative regulate (induction type) to transcribe, because gene inhibition albumen can and suppress transcribing of special gene thus in conjunction with this operon.Constitutive expression can take place under the situation as operon there not being negative regulatory element.In addition, just regulating and can realize by the gene activation protein binding sequence, if this sequence exists, then common (5 ') is near the RNA polymerase binding sequence.

The proteic example of gene activation is meta-bolites activated protein (CAP), and it helps to start the lac operon and transcribe (Raibaud et al. (1984) Annu.Rev.Genet.18:173) in intestinal bacteria.Therefore the expression of being regulated can be forward or negative sense, increases thus or reduces and transcribe.Other example of positive and negative regulatory element is well known in the art.The various promotors that can be included in the protein expression system include but not limited to T7/LacO hybrid promoter, trp promotor, T7 promotor, lac promotor and phage promotor.Can use any suitable promotor to implement the present invention, comprise natural promoter or allogeneic promoter.Allogeneic promoter can be constitutive activity or induction type.United States Patent (USP) 6,242 has provided the limiting examples of allogeneic promoter in 194.

The sequence of encoding metabolic pathway enzyme provides especially effectively promoter sequence.Example comprises the promoter sequence that derives from the carbohydrate metabolism enzyme, as semi-lactosi, lactose (lac) (Chang etal. (1987) Nature 198:1056) and maltose.Other example comprises promoter sequence (Goeddel et al. (1980) the NucleicAcids Res.8:4057 that derives from biosynthetic enzyme such as tryptophane (trp); Yelverton et al. (1981) Nucleic AcidsRes.9:731; United States Patent (USP) 4,738,921; EPO publication number 36,776 and 121,775).β-Nei Xiananmei (bla) promoter systems (Weissmann, (1981) " The Cloning ofInterferon and Other Mistakes, " in Interferon 3 (ed.I.Gresser); Phage PL (Shima take et al. (1981) Nature 292:128); Pectinose induction type araB promotor (United States Patent (USP) 5,028,530) and T5 (United States Patent (USP) 4,689,406) promoter systems also provide effective promoter sequence.Also, coli expression carrier has been discussed wherein referring to Balbas (2001) Mol.Biotech.19:251-267.

In addition, natural non-existent synthetic promoter also can play the bacterium promotor.For example, the transcription-activating sequence of bacterium or phage promoter can combine with the operon sequence of another bacterium or phage promoter, produces synthetic hybrid promoter (United States Patent (USP) 4,551,433).For example, tac (Amann et al. (1983) Gene 25:167; De Boeret al. (1983) Proc.Natl.Acad.Sci.80:21) and trc (Brosius etal. (1985) J Biol.Chem.260:3539-3541) promotor be heterozygosis trp-lac promotor, it is formed by the trp promotor with by the lac operon sequence that the lactose repressor is regulated.The tac promotor has the other feature of regulating sequence as induction type.Therefore, for example, the expression of the encoding sequence that can be operatively connected with the tac promotor can be induced by adding sec.-propyl-1-sulfo--β-D-galactoside (IPTG) in cell cultures.In addition, the bacterium promotor can comprise having in conjunction with the natural promoter of bacteria RNA polysaccharase with the non-bacterial origin that starts the ability of transcribing.The natural promoter of non-bacterial origin can also with compatible RNA polymerase coupling, make some genes in prokaryotic organism, produce high level expression.Phage t7 RNA polymerase/promoter systems is example (Studier et al. (1986) J.Mol.Biol.189:113 of coupling promoter systems; Tabor et al. (1985) Proc.Natl.Acad.Sci.82:1074).In addition, hybrid promoter can also be formed (EPO publication number 267,851) by phage promoter and intestinal bacteria manipulation subarea.

This carrier can contain the gene of coding at the repressor (or inductor) of this promotor in addition.For example, induction type carrier of the present invention can be regulated transcribing from Lac operon (LacO) by the gene of expressing coding Lad repressor.Other example comprises that use lexA generegulation pRecA expresses and use trpO adjusting ptrp.Can use increases the allelotrope that checks degree (for example lacIq) or modify this gene of inducing mode (for example λ CI857 provides λ pL thermal induction type, or λ CI+, and λ pL chemical induction type is provided).

Except that functional promoter sequence, effectively ribosome bind site also can be used for expressing fusion constructs.In prokaryotic organism, ribosome bind site is called as Shine-Dalgarno (SD) sequence and comprises initiator codon (ATG) and be positioned at the long sequence (Shine et al. (1975) Nature 254:34) of 3-9 Nucleotide of a upstream from start codon 3-11 Nucleotide.The SD sequence be considered to by the base pairing between SD sequence and the bacterial 16 S rRNA 3 ' end promote mRNA combine with rrna (Steitz et al. (1979) " Genetic Signals andNucleotide Sequences in Messenger RNA; " in BiologicalRegulation and Development:Gene Expression (ed.R.F.Goldberger, Plenum Press, NY).

Carbohydrate utilizes the associated protein can also be by the chimeric dna molecule that produces proteins encoded from emiocytosis, this albumen comprises provides the carbohydrate utilization the relevant and excretory signal peptide sequence fragment (United States Patent (USP) 4 of multiple medicines transhipment polypeptide in bacterium, 336,336).The signal sequence fragment is encoded usually by the signal peptide that instructs albumen to form from the hydrophobic amino acid of emiocytosis.This protein excretion is to growth medium (gram positive bacterium) or the periplasmic space between cell inner membrance and adventitia (gram negative bacterium).Preferably have the processing site, it can be in vivo or external being cut, and is coded between the relevant or multidrug transporter of signal peptide fragment and carbohydrate utilization.

The DNA of coding appropriate signals sequence can stem from the proteic gene of secreting bacteria, as escherichia coli outer membrane protein gene (ompA) (Masuiet al. (1983) FEBS Lett.151 (1): 159-164; Ghrayeb et al. (1984) EMBO is J.3:2437-2442) and escherichia coli alkaline phosphatase signal sequence (phoA) (Oka et al. (1985) Proc.Natl.Acad.Sci.82:7212).Other protokaryon signal for example comprises signal sequence Ipp or the heat-stable toxin II leader sequence from penicillinase.

Bacterium such as Lactobacterium acidophilum utilize initiator codon ATG usually, and it determines methionine(Met) (it is modified to N-formylmethionine in prokaryotic organism).Bacterium also discerns selectable initiator codon, as codon GTG and TTG, and their encode respectively Xie Ansuan and leucines.Yet when they are used as the initiation codon period of the day from 11 p.m. to 1 a.m, these codons will instruct mixing of methionine(Met), rather than the amino acid of their normal encodings.Lactobacterium acidophilum NCFM discerns these alternative initiation sites, and mixes methionine(Met) as first amino acid.

Typically, the transcription termination sequence of bacterium identification is the regulatory region that is positioned at translation stop codon 3 ' side, therefore with promotor side joint encoding sequence.The mRNA that these are sequence-directed can be translated as by the polypeptide of dna encoding transcribes.Transcription termination sequence usually comprises can form the dna sequence dna (about 50 Nucleotide) that help to stop the stem-ring structure of transcribing.Example comprises and derives from the gene transcription terminator sequence with strong promoter, as the trp gene in the intestinal bacteria and other biosynthesis gene.

Expression vector will have many restriction sites for inserting the relevant or multidrug transporter sequence of carbohydrate utilization, so that be under the transcriptional regulatory of regulatory region.Guarantee that the selected marker that carrier keeps also can be included in the expression vector in cell.Preferred selected marker comprises the mark of giving the resistance of medicine such as penbritin, paraxin, erythromycin, kantlex (Xin Meisu) and tsiklomitsin (Davies et al. (1978) Annu.Rev.Microbiol.32:469).Selected marker can also allow cell at minimum medium, or growth in the presence of toxic metabolite, and can comprise biosynthesis gene, as the gene in Histidine, tryptophane and the leucine biosynthetic pathway.

Regulatory region can be natural (homologous) or can be external (allogenic) for cell and/or nucleotide sequence of the present invention.Regulatory region also can be natural or synthetic.When this zone pair cell is under the situation of " external " or " allogenic ", means in introducing this regional n cell to find this zone.Relevant or multidrug transporter nucleotide sequence is " external " or " allogenic " to carbohydrate utilization of the present invention when this zone, means that this zone is not natural for the carbohydrate utilization of the present invention that can be operatively connected the relevant or multidrug transporter nucleotide sequence or is not natural existence zone.For example, this zone can derive from phage.Although preferably use allos regulatory region expressed sequence, still can use natural zone.Expect that this construct will change the relevant or expression level of multidrug transporter in cell of carbohydrate utilization sometimes.Therefore, the phenotype of cell can be changed.

When this expression cassette of preparation, can operate various dna fragmentations, making provides dna sequence dna with proper orientation, and according to circumstances, they is in the suitable frame.For this reason, can use connexon or joint to connect dna fragmentation, maybe can comprise other operation with the restriction site of providing convenience, remove unnecessary DNA, remove restriction site or the like.For this purpose, can comprise vitro mutagenesis, primer reparation, restriction enzyme digestion, annealing, replace for example conversion and transposition again.

The present invention further provides and comprised the recombinant expression vector that is cloned into the dna molecular of the present invention of expression vector with antisense orientation.That is to say, dna molecular with regulate the mode of sequence and can be operatively connected with the RNA developed by molecule (transcribing) that allows or multidrug transporter mRNA antisense relevant by dna molecular with the carbohydrate utilization.The adjusting sequence that the nucleic acid that can select and clone with antisense orientation can be operatively connected is to instruct the continuous or inducible expression of antisense rna molecule.Antisense expression vector can be recombinant plasmid or phagemid form, and wherein antisense nucleic acid produces under the control of high-level efficiency regulatory region, and its activity can be measured by the cell type of introducing carrier.For the discussion of using the inverted defined gene regulatory gene to express referring to Weintraub et al. (1986) Reviews-Trends in Genetics, Vol.1 (1).

Perhaps, some said components can be placed in the conversion carrier together.As mentioned above, conversion carrier contains selected marker usually, described mark or remain in the replicon or enter in the integrative vector.

Plant expression vector

With regard to the expression in the vegetable cell, expression cassette should comprise the transcription initiation region that is connected with nucleotide sequence operability of the present invention.Can comprise various restricted point of contacts in these expression vectors, so that can insert nucleotide sequence with under the transcriptional control that is in control region.In addition, described expression cassette can comprise the selected marker, comprises that those make its gene with weedicide or antibiotics resistance, such as tetracyclin resistance, hygromycin resistance, amicillin resistance or glyphosate resistance.

Described expression cassette will be included in by 5 ' to 3 ' transcriptional orientation transcribing with translation initiation district, nucleotide sequence of the present invention and transcribing with the translation termination district (being the terminator) of function in the plant.Described terminator can be the natural zone that contains the transcription initiation region of promotor nucleotide sequence, can be the natural zone of nucleotide sequence of the present invention, perhaps can be from other source.Many terminators easily are known in the art, including, but not limited to, from the terminator of the Ti-plasmids of Agrobacterium tumefaciems (A.tumefaciens), such as octopine synthase and nopaline synthase terminator.Also can consult (1991) Mol.Gen.Genet.262:141-144 such as Guerineau; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev.5:141-149; Mogen et al. (1990) Plant Cell2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas etal.1989) Nucleic Acids Res.17:7891-7903; And Joshi et al. (1987) Nucleic Acid Res.15:9627-9639.

The expression cassette that contains nucleotide sequence of the present invention also can comprise at least a other nucleotide sequence, so that gene can be entered in the organism by cotransformation.Perhaps, described other sequence can provide on another expression cassette.

Described expression cassette can comprise 5 ' untranslated leader or 5 ' non-coding sequence in addition.When being used for herein, " 5 ' leader sequence ", " translation leader sequence " or " 5 ' non-coding sequence " refer to the dna sequence dna part between the promotor and encoding sequence in the gene, it is transcribed into RNA and is present in the mRNA upstream that processes fully translation initiation codon upstream (5 ').5 ' untranslated leader is characterized by such mRNA molecular moiety usually, and it is to extend to AUG protein translation initiator codon from 5 ' CAP site the most at large.The translation leader sequence may influence stability or the translation efficiency (Turner etc. (1995) Molecular Biotechnology 3:225) that the primary transcription product is processed into mRNA, mRNA.Therefore, the translation leader sequence plays an important role in gene expression regulation.

The translation leader sequence is known in the art, including, but not limited to: picornavirus leader sequence, for example EMCV leader sequence (encephalomyocarditis 5 ' non-coding region) (Elroy-Stein etc. (1989) Proc.Nat.Acad.Sci.USA 86:6126-6130); Marmor upsilon group leader sequence, for example TEV leader sequence (tobacco Etch virus) (Allison etc. (1986) Virology 154:9-20); MDMV leader sequence (the short and small mosaic virus of corn); Human immunoglobulin heavy chain conjugated protein (BiP) (Macejak etc. (1991) Nature353:90-94); Untranslated leader (AMV RNA 4) (Jobling etc. (1987) Nature 325:622-625) from alfalfa coat protein for mosaic virus mRNA; Tobacco mosaic virus (TMV) leader sequence (TMV) (Gallie etc. (1989) MolecularBiology of RNA, page number 237-256); And corn chlorotic mottle poison leader sequence (MCMV) (Lommel etc. (1991) Virology 81:382-385).

Also can use other method of known enhancing translation and/or mRNA stability, intron for example, such as corn ubiquitin intron (Christesen and Quail (1996) TransgenicRes.5:213-218 and Christensen etc. (1992) Plant MolecularBiology 18:675-689) or corn AdhI intron ((1990) Maydica 35:353-357 such as (1991) Mol.Gen.Genet.228:40-48 such as Kyozuka and Kyozuka), or the like.Demonstrated various intron sequences and can strengthen expression, especially in monocot plant cell.Found that corn Adh1 gene intron significantly strengthens the expression of wild type gene under can the control at its homologous promoter when introducing maize cell.Discover the especially effective and enhancing expression (Callis etc. (1987) Genes Develop.1:1183-1200) of introne 1 in the fusion constructs that contains the paraxin acetyl transferase gene.In same experimental system, has the effect of similar enhancing expression from the intron of corn bronze 1 gene.The expression that the AdhI intron also demonstrates CAT has strengthened 12 times (Mascarenhas etc. (1990) Plant Mol.Biol.6:913-920).The intron sequences routine is impregnated in the plant conversion carrier, usually in untranslated leader.

The expression cassette that contains promoter sequence of the present invention can comprise 3 ' non-coding sequence in addition." 3 ' non-coding sequence " or " 3 ' non-translational region " refers to be positioned at the nucleotide sequence of encoding sequence 3 ' side (downstream), and comprises that poly-adenosine signal sequence and coding can influence other sequence that the polyadenylic acid segment is added into the adjustment signal of mRNA precursor 3 ' end.3 ' non-translational region comprises the zone of mRNA, and this zone originates in translation stop codon usually and extends at least after the poly-adenosine site.Found that the non-translated sequence that is positioned at gene 3 ' end influences the expression of gene level.Ingelbrecht etc. (consulting Plant Cell, 1:671-680,1989) assess the importance of these elements, find to stablize endophytic expression very big difference is arranged along with not coexisting of 3 ' non-translational region source.Utilize the 3 ' non-translational region relevant with following albumen: octopine synthase, from the 2S seed albumen of Arabidopis thaliana, from the rbcS small subunit of Arabidopis thaliana, from the extension segment of Radix Dauci Sativae with from the chalkone synthase of antirrhinum, observing to express between the minimum construct (containing chalkone synthase 3 ' zone) of best construct (containing rbcS 3 ' non-translational region) and expression has 60 times difference.

Also can improve transcriptional level by utilizing the enhanser of uniting with promoter region of the present invention.Enhanser is to be used to increase the nucleotide sequence that promoter region is expressed.Enhanser is known in the art, comprises that SV40 strengthens subarea, 35S enhancer element or the like.

In the preparation of expression cassette, can operate various dna fragmentations and be in the correct reading frame so that dna sequence dna is provided and is convenient in due course with proper orientation.Can use adapter or connexon and connect dna fragmentation, the processing that perhaps can carry out other is to obtain restricted easily point of contact.Can add or remove restriction site, unnecessary DNA can be removed, and perhaps can carry out other modification to sequence of the present invention.For this reason, can carry out vitro mutagenesis, primer repair, restriction enzyme digestion, annealing, replace again, for example change and transversion.

Except the selected marker that microbiotic or Herbicid resistant are provided, as mentioned above, in transgenic event reclaims, come in handy but in end product, may include, but are not limited to GUS (b-glucuronidase by optional other gene; Jefferson (1987) PlantMol.Biol.Rep.5:387), GFP (green fluorescent protein; Chalfie etc. (1994) Science 263:802), the corn gene (Ludwig etc. (1990) Science247:449) of luciferase (Riggs etc. (1987) Nucleic Acids Res.15 (19): 8115 and (1992) MethodsEnzymol.216:397-414 such as Luehrsen) and coding anthocyanin product.

Nucleic acid of the present invention can be used at expressing in the method for nucleotide sequence in plant.By transforming the purpose vegetable cell with the expression cassette that contains the promotor that is connected with the determined nucleotide sequence operability of this paper and, can achieving the above object from the regenerate plant of stable conversion of said vegetable cell.The expression cassette that contains the promoter sequence that is connected with nucleotide sequence operability of the present invention can be used to transform any plant.Can obtain genetic modification in this way, i.e. transgenosis or plant transformed, vegetable cell, plant tissue, seed, root or the like.

Microorganism or bacterial cell

Contain the generation of the bacterium of allos phage resistance gene, described bacterium starting culture preparation and fermentation substrate, especially can carry out according to known technology such as the method for food substrates such as milk.

" introducing " speech means by routine conversion or rotaring dyeing technology or the infection by the phage mediation and imports protokaryon or eukaryotic cell when relevant with nucleic acid.When being used for herein, mean term " conversion ", " transduction ", " puting together " and " protoplastis fusion " and generally acknowledge the technology that is used for exogenous nucleic acid (for example DNA) is introduced cell in multiple this area, comprise transfection, liposome transfection or the electroporation of calcium phosphate or calcium chloride co-precipitation, the mediation of DEAE-dextran.

The suitable conversion or the method for transfectional cell can be consulted (1989) molecular clonings such as books Sambrook: laboratory manual Molecular Cloning:A Laboratory Manual (second edition, press of cold spring harbor laboratory, Plainview, New York) and other laboratory manual.When " introducing " speech is relevant with polypeptide of the present invention or microorganism, means by picked-up, topical application, nasal feeding, suppository, urogenital tract or oral application polypeptide or microorganism are introduced the host.

As (1989) Molecular Cloning such as document Sambrook, ALaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainvew, described usually among the New York, will be used for producing carbohydrate utilization of the present invention bacterial cell relevant or the multidrug transporter polypeptide and be incubated at suitable medium.

The bacterial isolates that the present invention involves comprises the biologically pure bacterial cultures of the bacterium that comprises at least a Nucleotide of the present invention or aminoacid sequence.These bacterial strains comprise: have the lactobacillus acidophilus bacteria strains that carbohydrate advances or go out the ability of cell that transports than the improvement of wild-type Lactobacterium acidophilum, the ability of wherein said improvement causes owing at least a carbohydrate of the present invention utilizes the dependency polypeptide expression; Compare with the wild-type Lactobacterium acidophilum, have the Lactobacterium acidophilum bacterial strain system of the accumulation carbohydrate ability of improvement, wherein said ability improvement is because at least a carbohydrate of the present invention utilizes the dependency polypeptide expression to cause; Compare with the wild-type Lactobacterium acidophilum, the carbohydrate that utilizes with improvement is that wherein said ability improvement is because at least a carbohydrate of the present invention utilizes the dependency polypeptide expression to cause as the Lactobacterium acidophilum bacterial strain of the ability of the energy; Compare with the wild-type Lactobacterium acidophilum, the Lactobacterium acidophilum bacterial strain system of the foodstuff products of smell improvement is provided by fermentation, wherein said smell improvement is because at least a carbohydrate of the present invention utilizes the dependency polypeptide expression to cause; Compare with the wild-type Lactobacterium acidophilum, provide the system of the Lactobacterium acidophilum bacterial strain with the foodstuff products that improves structure by fermentation, wherein said texture improvement causes owing at least a carbohydrate of the present invention utilizes the dependency polypeptide expression; Compare with the wild-type Lactobacterium acidophilum, have the Lactobacterium acidophilum bacterial strain system of the ability of improved generation carbohydrate, wherein said ability causes owing at least a carbohydrate of the present invention utilizes the dependency polypeptide expression; Compare with the wild-type Lactobacterium acidophilum, have the improved Lactobacterium acidophilum bacterial strain system that stands the ability of food-processing and storage requirement, wherein said improvement ability causes owing at least a carbohydrate of the present invention utilizes the dependency polypeptide expression; Compare with the wild-type Lactobacterium acidophilum, have the Lactobacterium acidophilum bacterial strain system of improved ability of surviving in the GI road, wherein said improvement ability causes owing at least a carbohydrate of the present invention utilizes the dependency polypeptide expression; Compare with the wild-type Lactobacterium acidophilum, have the Lactobacterium acidophilum bacterial strain system of the ability of improved tolerance antimicrobial polypeptide or toxin, wherein said improvement ability causes owing at least a carbohydrate of the present invention utilizes the dependency polypeptide expression.

Transgenic plant and vegetable cell

When being used for herein, term " conversion plant " and " transgenic plant " refer to contain at its genome the plant of heterologous polynucleotide.Usually, the heterologous polynucleotide stable integration is gone into transgenosis or is transformed in the genome of plant so that polynucleotide pass in the suceeding generation.

Heterologous polynucleotide can be separately or is integrated in the genome as the part of recombinant expression cassettes.Should understand, when being used for herein, term " transgenosis " comprises because the existence of heterologous nucleic acids and any cell, clone, callus, plant part or plant that its genotype has changed, comprises those initial transgenosis types that change like this and by sexual hybridization or vegetative propagation generation those types from initial transgenosis type.Term " transgenosis " does not comprise when being used for herein and utilizes conventional plant cultivation method or because genome (the chromosomal or the karyomit(e) body) change that causes such as naturally occurring incidents such as hybridization pollination, non-recombinant virus infect at random, non-recombinant bacteria transforms, non-reorganization swivel base or spontaneous mutations.

Transgenosis " incident " is allogeneic dna sequence DNA construct transformed plant cells (described construct comprise contain the genetically modified expression of nucleic acid box of purpose), regenerate and insert the flora that Plant Genome generates and select to be characterized as the special plant that is inserted into specific gene group position because of transgenosis.Described incident shows as genetically modified expression on phenotype.On hereditary level, incident is the part that plant genetic is formed.Term " incident " also refers to transformant and contains the descendant of the sexual outcross generation of another kind of allogeneic dna sequence DNA.

When being used for herein, term " plant " comprises complete plant, plant organ (for example leaf, stem, root etc.), seed, vegetable cell and their offspring.The part of transgenic plant is understood to include in the scope of the invention, for example, derive from and transformed in advance the transgenic plant of dna molecular of the present invention or their descendant's vegetable cell, protoplastis, tissue, callus, embryo and flower, pollen, pollen sac, stem, fruit, ovule, leaf or root, therefore form by transgenic cell to small part.

When being used for herein, term " vegetable cell " includes, but are not limited to seed suspension culture, embryo, meristem zone, callus, leaf, root, bud, gametophyte, sporophyte, pollen and sporule.The kind with the higher plant that can handle with method for transformation is the same extensive in general to can be used for floristics in the inventive method, comprises monocotyledons and dicotyledons.

The present invention can be used for transforming arbitrary plant species, including, but not limited to, unifacial leaf and dicotyledons.The example of purpose plant comprises, but be not limited to, corn (Zeamays), rape kind (Brassica sp.) (B.napus for example, B.rapa, B.juncea), especially those rape kind of plant that can be used as the seed oil source, alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cereale), Chinese sorghum (Sorghumbicolor, Sorghum vulgare), grain (pearl millet (Pennisetum glaucum) for example, broomcorn millet (Panicum miliaceum), millet (Setaria italica), grain (Eleusinecoracana)), Sunflower Receptacle (Helianthus annuus), safflower (Carthamustinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), Semen arachidis hypogaeae (Arachis hypogaea), cotton (Gossypium barbadense, Gossypiumhirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.) coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea tree (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), Fructus Fici (Ficus casica), piscidia (Psidium guajava), mango (Mangiferaindica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamiaintegrifolia), apricot (Prunus amygdalus), beet (Beta vulgaris), sugarcane (Saccharum spp.), oat, barley, vegetables, ornamental plant and softwood tree.

Vegetables comprise tomato (Lycopersicon esculentum), Caulis et Folium Brassicae capitatae (e.g., Lactuca sativa), green French beans (Phaseolus vulgaris), lima bean (Phaseolus limensis), pea (Lathyrus spp.), and Cucumis member, for example cucumber (C.sativus), muskmelon (C.cantalupensis) and muskmelon (C.melo).Ornamental plant comprises that rhododendron (Rhododendron spp.), Seven barks (Macrophylla hydrangea), lotus belong to (Hibiscus rosasanensis), rose (Rosa spp.), turmeric (Tulipa spp.), flower of Chinese Narcissus (Narcissus spp.), petunia (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima) and chrysanthemum.Can be used for carrying out softwood tree of the present invention comprises, for example, pine tree is such as torch pine (Pinus taeda), slash pine (Pinus elliotii), yellow oregon pine (Pinus ponderosa), korean pine (Pinus contorta) and pine (Pinusradiata); Pseudotsuga menziesii (Mirbel) Franco (Pseudotsugamenziesii); California hemlock spruce (Tsugacanadensis); Picea sitchensis (Piceaglauca); Chinese larch (Sequoiasempervirens); Real fir wood is such as silver fir (Abies amabilis) and glue fir (Abiesbalsamea); And such as Pacific red cedar (Thuja plicata) and yellow cedar cdears such as (Chamaecyparis nootkatensis).

Method of the present invention does not depend on certain concrete grammar with the constructs introduced plant, as long as described constructs can enter at least one cell interior of plant.With the method in the constructs introduced plant is known in the art, including, but not limited to, stable conversion method, instantaneous conversion method and virus-mediated method.

" instantaneous conversion " means that the constructs unconformability of introduced plant goes in the genome of plant.The constructs that " stable conversion " means introduced plant is integrated into the genome of plant and can be inherited by its offspring." elementary transformant " and " T0 generation " transgenic plant belong to same genetic algebra (that is, not passing through reduction division and insemination after the conversion) with the tissue that is transformed at first." secondary transformant " and " T1, T2, T3 and follow-up algebraically " refers to from elementary transformant and through one or many reduction division and insemination round-robin transgenic plant.They can being transformed or the hybridization of unconverted plant from body insemination or elementary or secondary transformant and other from elementary or secondary transformant.

The conversion scheme and with nucleotide sequence introduce plant method can according to transform at the plant or the type of vegetable cell and change (being unifacial leaf or dicotyledons).Constructs of the present invention can be by in arbitrary method introduced plant known in the art, including, but not limited to, (for example, United States Patent (USP) 5,889 with virus or viral nucleic acid contact plant, 191,5,889,190,5,866,785,5,589,367 and 5,316,931; Incorporate into own forces as a reference at this), microinjection (Crossway etc. (1986) Biotechniques 4:320-334), electroporation (Riggs etc. (1986) Proc.Natl.Acad.Sci.USA 83:5602-5606), agriculture bacillus mediated conversion (United States Patent (USP) 5,981,840 and 5,563,055), directly transgenosis (Paszkowski etc. (1984) EMBO J.3:2717-2722) and trajectory particle quicken (to consult, for example United States Patent (USP) 4,945,050,5,879,918,5,886,244 and 5,932,782), all these methods are all incorporated into own forces as a reference at this.

Utilize methods known in the art to make by cell transformed and be grown to serve as plant.Consult, for example, McCormick etc. (1986) Plant Cell Reports 5:81-84.Can cultivate these plants then, perhaps with same being transformed strain or, identify the heterozygote that is produced with expection phenotypic characteristic with different strain pollinations.Can cultivate expression stable maintenance and the heredity of two generations or more generations, gather in the crops seed then to guarantee to reach the expression of expection phenotypic characteristic to guarantee the expection phenotypic characteristic.Therefore, when being used for herein, " seed that is transformed " refers to contain the seed that stable integration is gone into the constructs of Plant Genome.

Using method

Provide the method for the carbohydrate utilization that can be used for improving organism relevant or multidrug transporter gene or protein expression at this.In a certain embodiment, the characteristic of employed microorganism is carried out and modifies so that plant can utilize the source of alternative carbohydrate as energy or carbon in the fermentation.These modifications can cause synthesizing, transport, gather or the new ability of degradable carbon hydrate.The ability of still surviving after perhaps, these modifications can cause bacterial strain and antimicrobial polypeptide (comprising microbiotic and toxin) contacts.

These new abilities also make microorganism better to survive in being full of the environment of pressure, such as the environment that runs in processing of digestive tube or food and the storage process etc., they can increase the effectiveness of these microorganisms in the various food of fermentation, and make them that more persistent beneficial liveliness proof can be provided after picked-up.These new abilities also make microorganism by different smell or the qualities (texture) of generation in product of fermenting.In addition, described new ability also makes bacterium can produce carbohydrate, exo polysaccharides or the cell surface polysaccharide of modification.In another embodiment, the characteristic of plant is carried out to modify and makes it have similar ability.These abilities are that Nucleotide and the aminoacid sequence of being announced by the present invention provided.

Generally speaking, described method comprises introducing or crosses and express one or more related in carbohydrate utilization or multi-medicine resistance protein." introducing " means target protein matter and expresses in the cell of modified and do not express in the cell that unmodified is crossed." expression excessively " means the expression amount of target protein matter in the organism of modified to be increased to some extent than its expression in the wild-type organisms of unmodified.Particularly, homo-fermentative lactic-acid-bacterium has simple relatively metabolism, between energy metabolism and biosynthetic metabolism, almost do not have overlapping, this make they become metabolism engineered desirable object (Hugenholz and Kleerebezem (1999) Current Opin.Biotech.10:492-497).The expression of bacterial gene in plant is well-known in the art.Consult, for example, Shewmaker etc. (1994) PlantPhysiol.104:1159-1166; Shen etc. (1997) Plant Physiol.113:1177-1183; Blaszczyk etc. (1999) Plant J.20:237-243.

Express the ability that the relevant or multidrug transporter of one or more carbohydrate utilizations can make organism have the transhipment carbohydrate of improvement or advance or go out cell such as antimicrobial polypeptides such as bacteriocins.Comprise the abc transport albumen system component (for example HisJ and MalE) that contains the substrate conjugated protein with the relevant carbohydrate of transhipment application albumen or multidrug transporter matter, such as film related components such as permease (for example LacF and LacG) and such as cytoplasmic protein matter (for example msmK) such as ATP-conjugated proteins.The carbohydrate that transhipment is relevant utilizes protein or multidrug transporter matter also to comprise secondary delivery system albumen, such as main promotion superfamily protein (MFS) member, and glucoside/pentoside/biose aldehydic acid glycosides family member.In group translocation system albumen is also included within, comprise enzyme I, enzyme II and HPr protein.

Carbohydrate utilizes dependency protein also to comprise desaturase.Acetaldehyde dehydrogenase (EC:1.2.1.3 and EC:1.2.1.5) (the PFAM number of including PF00171) is with the enzyme of NADP as multiple aliphatics of cofactor oxidation and aromatic aldehyde.Having four kinds of multi-form enzymes in Mammals at least is known (Hempel etc. (1989) Biochemistry 28:1160-7): type-1 (or Ald C)-four poly-cytoplasm enzyme, type 2 (or Ald M)-four poly-cyclophorase, type 3 (or Ald D)-dimerization cytoplasm enzyme and type 4-microsomal enzymes.Acetaldehyde dehydrogenase to fungi and bacterial species source checks order in addition.Many enzymes are known evolves relevant with acetaldehyde dehydrogenase.

Relate to L-glutamic acid and cysteine residues in the catalytic activity of Mammals acetaldehyde dehydrogenase.These residues are guarded in all enzymes of this family.Acetaldehyde dehydrogenase protein of the present invention comprises the protein among the SEQ ID NO:228.The 2-hydroxy acid dehydrogenase that the D-isomer is special, catalyst structure domain protein of the present invention (the PFAM number of incorporating into own forces PF00389) sequence is as shown in SEQ ID NO:242.The 2-hydroxy acid dehydrogenase that the D-isomer is special, NAD binding domains of the present invention (the PFAM number of incorporating into own forces PF02826) protein comprises those sequences among the SEQ ID NO:242.Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) changes into 1 by reversibility catalysis D-glyceraldehyde-3-phosphate oxidation and phosphoric acid, 3-diphosphoglyceric acid and play an important role in glycolysis-and gluconeogenesis (Huang etc. (1989) J.Mol.Biol.206:411-24).

Described enzyme exists with the tetramer form of same subunit, and each comprises 2 conservative functional structure territories: the catalyst structure domain of NAD-binding domains and high conservative.SEQ ID NO:248 has Glycerose 3-phosphate dehydrogenase C-end structure territory (the PFAM number of incorporating into own forces PF02800) and Glycerose 3-phosphate dehydrogenase NAD binding domains (the PFAM number of incorporating into own forces PF00044).

The L-serum lactic dehydrogenase is the metabolic enzyme that catalysis L-lactic acid is converted into pyruvic acid, and this is the final step in the anaerobism glycolysis-.The change of malate dehydrogenase enzyme catalysis oxysuccinic acid and oxaloacetic acid.Described enzyme participates in tricarboxylic acid cycle.The proteinic lactic acid of the present invention/malate dehydrogenase (malic acid dehydrogenase) α/β C-end structure territory (the PFAM number of incorporating into own forces PF02866) and lactic acid/malate dehydrogenase (malic acid dehydrogenase) NAD binding domains (the PFAM number of incorporating into own forces PF00056) albumen are as shown in SEQ ID NOS:222 and 246.The method that is used to detect dehydrogenase activity is (consults, for example, Ercolani etc. (1988) J.Biol.Chem.263:15335-41) well-known in the art.

Carbohydrate utilizes related protein also to comprise the O-glycosyl hydrolase.O-glycosyl hydrolase (EC 3.2.1.-) is the widely distributed enzyme of a class, the glycosidic link between glycosidic link between the two or more carbohydrate of their hydrolysis or carbohydrate and the non-carbohydrate part.The test that detects hydrolytic enzyme activities be well-known in the art (consult, for example, Avigad and Bauer (1966) Methods Enzymol.8:621-628; Neumann and Lampen (1967) Biochemistry 6:468-475; Henry and Darbyshire (1980) Phytochemistry 19:1017-1020).

αDian Fenmei catalyst structure domain protein (the PFAM number of incorporating into own forces PF00128) is classified as the 13rd family of glycosyl hydrolase.αDian Fenmei catalyst structure domain protein sequence of the present invention is shown in SEQ IDNOS:108,196,240,292 and 332.

Beta-galactosidase enzymes family (the PFAM number of incorporating into own forces PF02449) belongs to glycosyl hydrolase 42 families.The hydrolysis of the terminal non reducing end beta-D-galactosidase of described enzyme catalysis residue.Beta-galactosidase enzymes protein of the present invention comprises the protein shown in the SEQ ID NO:210.Beta galactosidase enzyme chainlet C-terminal structural domain (the PFAM number of incorporating into own forces PF02930) is found in the C-terminal part that is present in dimer beta-galactosidase enzymes (EC:3.2.1.23) chainlet.Beta galactosidase enzyme chainlet N-terminal structural domain (the PFAM number of incorporating into own forces PF02929) is found in the N-terminal part that is present in dimer beta-galactosidase enzymes (EC:3.2.1.23) chainlet.These structural domains also are found and are present in the strand beta galactosidase enzyme.Beta galactosidase enzyme chainlet C-terminal domain protein white matter of the present invention is as shown in SEQ ID NO:214.Beta galactosidase enzyme chainlet N-terminal domain protein white matter of the present invention comprises sequence as shown in SEQ ID NO:214.

Glycoside hydrolysis enzyme family 1 (the PFAM number of incorporating into own forces PF00232) comprises the enzyme with many known activity: beta-glucosidase enzyme (EC:3.2.1.21); P-tilactase (EC:3.2.1.23); 6-phosphoric acid-beta-galactosidase enzymes (EC:3.2.1.85); 6-phosphoric acid-beta-glucosidase enzyme (EC:3.2.1.86); Sumylact L-Phlorizin hydrolase (EC:3.2.1.62), (EC:3.2.1.108); P-mannosidase (EC:3.2.1.25); Myrosin (EC:3.2.1.147).Glucoside hydrolase family 1 protein sequence of the present invention is as shown in SEQ ID NOS:10 and 220.

Glycoside hydrolysis enzyme family 2 comprises the enzyme with many known activity: beta-galactosidase enzymes (EC:3.2.1.23); Beta-Mannosidase (EC:3.2.1.25); β-glucuronidase (EC:3.2.1.31).These enzymes comprise conservative glutaminic acid residue, and it is acid/alkaline catalysts (Gebler etc. (1992) J.Biol.Chem.267:11126-30) common in the enzyme active sites that this residue has demonstrated in intestinal bacteria lacZ.Immunoglobulin-like β-sandwich structure territory has been described in glycosyl hydrolase family 2 immunoglobulin-like β sandwich structure territories (the PFAM number of incorporating into own forces PF00703).Sugar binding domains (the PFAM number of incorporating into own forces PF02837) has the jellyroll pleated sheet structure.From colibacillary beta-galactosidase enzymes have by four other big beta structure territories around TIM-tubbiness core (the PFAM number of incorporating into own forces PF02836).SEQ ID NO:212 has each in these structural domains.

Glycoside hydrolysis enzyme family 31 (the PFAM number of incorporating into own forces PF01055) comprises the enzyme with several known activity: alpha-glucosidase (EC:3.2.1.20), alpha-galactosidase (EC:3.2.1.22), glucoamylase (EC:3.2.1.3), Sucrase-isomaltase (EC:3.2.1.48) (EC:3.2.1.10), α-xylosidase (EC:3.2.1) and alpha-glucan lyase (EC:4.2.2.13).Glycoside hydrolysis enzyme family 31 is the basis with the sequence similarity to many glycosyl hydrolases (Henrissat (1991) Biochem.J 280:309-16 that carried out dividing into groups; Naim etc. (1991) FEBS Lett.294:109-12).In the catalytic activity of sucrase, isomaltase and lysosmal, all related to aspartic acid (Hermans etc. (1991) J.Biol.Chem.266:13507-12).Glycoside hydrolysis enzyme family 31 protein of the present invention comprise the protein as shown in SEQ ID NO:226.Glycoside hydrolase family 32 (the PFAM number of incorporating into own forces PF00251) comprises the enzyme with several known activity: saccharase (EC:3.2.1.26), inulinase (EC:3.2.1.7), levanase (EC:3.2.1.65), outer inulinase (EC:3.2.1.80), sucrose: Sucrose 1-fructosyltransferase (EC:2.4.1.99) and Polylevulosan: Polylevulosan 1-fructosyl transferase (EC:2.4.1.100).Glycoside hydrolase family 32 protein of the present invention comprises sequence as shown in SEQ ID NOS:46 and 100.

The enzyme that comprises isoamylase N-terminal structural domain (the PFAM number of incorporating into own forces PF02922) belongs to glycosyl hydrolase family 13.Found to have this structural domain in acting on a series of enzymes of branch's substrate, said enzyme is isoamylase, Starch debranching enzyme and q enzyme.In isoamylase hydrolysis glycogen, amylopectin and the dextrin 1,6-α-D-glucoside branch connects; 1,4-alpha-glucan q enzyme works during the connection of 6-glucoside forms at 1 of glycogen; And Starch debranching enzyme is a starch debranching enzyme.Isoamylase N-terminal domain protein white matter of the present invention comprises sequence shown in the SEQ ID NO:240.

Alpha-galactosidase (EC:3.2.1.22) (melibiose) (the PFAM number of incorporating into own forces PF02065) catalysis melibiose hydrolysis becoming semi-lactosi and glucose (Dey and Pridham (1972) Adv.Enzymol.Relat.AreasMol.Biol.36:91-130).Alpha-galactosidase is present in the multiple organism.There is similarity significantly in α tilactase from various eucaryon species on sequence.

Intestinal bacteria α tilactase (gene melA) needs NAD and magnesium as cofactor, and it is structurally irrelevant with the enzyme of eucaryon state; On the contrary, the alpha-galactosidase (gene rafA) of escherichia coli plasmid coding contains about 50 the amino acid whose zones similar to eucaryon α tilactase structural domain (Aslanidis etc. (1989) J.Bacteriol.171:6753-63).Melibiose protein of the present invention comprises sequence as shown in SEQ ID NO:198.

Carbohydrate utilizes dependency protein also including, but not limited to the enzyme of following type: aldose 1-epimerase (EC:5.1.3.3) (mutarotase) (the PFAM number of incorporating into own forces PF01263), this be responsible for D-glucose and other aldose its α-and β-form between the enzyme that exchanges of different head.Detect the active method of aldose 1-epimerase and be (consult, for example, Majumdar etc. (2004) Eur.J Biochem.271:753-9) well-known in the art.Aldose 1-epimerization enzyme sequence of the present invention is as shown in SEQ ID NO:200.

Enolase (2-phospho-D-glycerate lytic enzyme) is the basic carbohydrate-splitting enzyme of the exchange between catalysis 2-phosphoglyceric acid and the phosphoenolpyruvic acid.

The method that detects the phosphoenolpyruvic acid hydrolytic enzyme activities is (consults, for example, Fox etc. (1995) Plant Physio.109:433-43) well-known in the art.SEQ ID NO:254 comprises the terminal TIM barrel-like structure territory (the PFAM number of incorporating into own forces PF00113) of enolase C-and enolase N-end structure territory (the PFAM number of incorporating into own forces PF03952).

Fructose-bis phosphoric acid zymohexase (EC:4.1.2.13) is the aldehyde alcohol cracking or the fructose-1 of catalyzed reversible, the carbohydrate-splitting enzyme of 6-bis phosphoric acid condensation becoming otan phosphoric acid and Glycerose 3-phosphoric acid.There are two classes to have the fructose-bis phosphoric acid zymohexase of different catalyst mechanisms.Class1 1 zymohexase (the PFAM number of incorporating into own forces PF01116) (Marsh and Lebherz (1992) Trends Biochem.Sci.17:110-3) mainly is found in prokaryotic organism and the fungi, they are homodimer enzymes, its activity needs divalent-metal ion, generally is zinc.This family also comprises intestinal bacteria melampyrum operon protein gatY, and its catalysis tagatose 1,6-bis phosphoric acid change into glycerone phosphoric acid and D-Glycerose 3-phosphoric acid, and the identical reaction of intestinal bacteria N-acetylgalactosamine operon albumen agaY catalysis.In preceding half section sequence of these enzymes, there are two histidine residues, demonstrated relevant with the combination of zine ion.The method that detects fructose bis phosphoric acid aldolase activity is (consults, for example, Alefounder etc. (1989) Biochem.J.257:529-534) well-known in the art.Fructose of the present invention-bis phosphoric acid zymohexase Type II protein comprises sequence as shown in SEQ IDNO:260.

Galactose-1-phosphate uridyl transferring enzyme catalysis UTP and α-D-semi-lactosi 1-phosphoric acid is transformed into UDP-glucose and pyrophosphate salt between semi-lactosi metabilic stage.The C-terminal of C-end structure territory (the PFAM number of incorporating into own forces PF02744) the galactose-1-phosphate uridyl transferring enzyme that refers to.N-end structure territory (the PFAM number of incorporating into own forces PF01087) refers to the N-terminal of galactose-1-phosphate uridyl transferring enzyme.SEQID NO:202 has this two structural domains.Detect UTP-sugar-active method of 1-phosphoric acid uridine acyltransferase be (consult, for example, Lobelle-Rich and Reeves (1983) Mol.Biochem.Parasitol.7:173-182) well-known in the art.

Semi-lactosi-(EC:2.7.1.6), homoserine (EC:2.7.1.39), mevalonic acid (EC:2.7.1.36) and phosphoric acid mevalonic acid (EC:2.7.4.2) kinases partly contain conservative rich Gly/Ser district at its N-terminal, and this zone may relate to the combination of ATP.SEQID NO:204 is the conjugated protein family member of ATP (the PFAM number of incorporating into own forces PF00288) that the GHMP kinases is inferred.The method that detects kinase activity is (consults, for example, Tsay and Robinson (1991) Mol.Cell Biol.11:620-31) well-known in the art.

NAD dependency epimerase/dehydratase family (the PFAM number of incorporating into own forces PF01370) protein utilization NAD is as cofactor.The protein utilization Nucleotide of this family-sugared substrate carries out number of chemical reaction (Thoden etc. (1997) Biochemistr 36:6294-304).NAD dependency epimerase of the present invention/dehydratase protein matter comprises sequence described in the SEQ ID NO:216.

The bacteriocin of lantibiotics and non-lantibiotics is that this extension area ripening period is cut off as the precursor peptide synthetic (leading peptide) that contains the N-terminal extension area.The leading peptide that most of non-lantibiotics and some lantibioticss have so-called double-glycine type.The common processing site that these leading peptides are enjoyed consensus sequence and have two conservative glycine residues in position-1 and-2.Double-glycine type leading peptide with instruct protein to have nothing to do by the N-terminal signal sequence that the sec approach passes through cytoplasmic membrane.Their processing site also is different from typical signal peptidase cleavage site, shows wherein to have related to different processive enzymes.Lanthocin is to pass through cytoplasmic membrane output by special ATP in conjunction with box (ABC) translocator.Abc transport albumen is sophisticated proteolytic enzyme, and its proteolysis structural domain is positioned at this proteinic N-terminal (Havarstein etc. (1995) Mol.Microbiol.16:229-40).PEPC C 39 families (the PFAM number of incorporating into own forces PF03412) structural domain is found and is present in a series of abc transport albumen.But, the catalytic halfcystine of supposition and Histidine are not all conservative in all members of this family.PEPC C 39 family proteins of the present invention comprise the protein shown in the SEQ ID NO:144.The activity of peptase can be assessed (consult, for example, Sasaki etc. (1995) J Dairy Res.62:601-610, and Machuga and Ives (1984) Biochim.Biophys.Acta 789:26-36) by detecting its hydrolytic activity.

Carbohydrate kinases family of PfkB family (the PFAM number of incorporating into own forces PF00294) comprises multiple kinds of carbohydrate and pyrimidine kinases.Described family comprises phosphomethylpyrimidine kinase (EC:2.7.4.7), fructokinase (EC:2.7.1.4) and ribokinase (ribokinase) (EC:2.7.1.15) (rbsK gene).This enzyme is the part of diphosphothiamine (TPP) route of synthesis, and TPP is a basic cofactor of many enzymes.The method that detects kinase activity is (consults, for example, Sato etc. (1993) J.Gen.Microbiol.139:921-7) well-known in the art.PfkB of the present invention family carbohydrate kinase protein comprises the protein shown in SEQ ID NOS:60 186,224 and 238.

It is an important reaction many biological procedureses that enzymatic phosphate group shifts from ATP.A kind of enzyme that utilizes this reaction is phosphofructokinase (PFK) (the PFAM number of incorporating into own forces PF00365), and its catalysis fructose-6-phosphate phosphorylation becomes fructose-1, the 6-bis phosphoric acid, and this is a crucial regulation and control step in the glycolytic pathway.PFK length is approximately 300 amino acid, about the structural research of this bacterial enzyme show it contain two similar (a/ (β) leaflet: one relate to ATP in conjunction with and another has substrate binding site and allosteric site (completely different but influence the regulation and control binding site of enzymic activity with avtive spot) concurrently.Same tetramer subunit has been taked two kinds of different conformations: under " closing " state, combined magnesium ion gets up the phosphoryl group bridge joint of enzyme product (ADP and fructose-1,6-diphosphate); And under " opening " state, magnesium ion is only in conjunction with ADP, because these two kinds of products are at this moment by farther separating.These conformations are considered to the successive stage of reaction path, and described approach needs subunit to close to make two molecule close enoughs so that reaction (Shirakihara and Evans (1988) J.Mol.Biol.204:973-94).The method that detects the fructose-1, 6-diphosphate kinase activity is (consults, for example, Wegener and Krause (2002) Biochem.Soc.Trans.30:264-70) well-known in the art.Phosphofructokinase albumen of the present invention comprises protein shown in the SEQ ID NO:256.

Glucose phosphate isomerase (EC:5.3.1.9) (PGI) (the PFAM number of incorporating into own forces PF00342) is the isomerized dimer enzyme of catalysis G-6-P and fructose-6-phosphate reversibility.PGI is relevant with different approach: in most of higher organism bodies, it is relevant with glycolysis-; It is relevant with gluconeogenesis in Mammals; It is relevant with the carbohydrate biosynthesizing in plant; It provides inlet for fructose enters the Entner-Doudouroff approach in some bacterium.Also known multifunctional protein PGI is neuroleukin (neurotrophic factor of mediation neurone differentiation), the autocrine mobility factor (the tumour secrete cytokines of regulating cell mobility), differentiation and ripe medium and sarcostyle bonded serpin, and has different effects inside and outside cell.In tenuigenin, glucolytic second step of its catalysis, and it plays nerve growth factor and effect of cytokines in the extracellular.The method that detects the G-6-P isomerase activity is (consults, for example, Nozue etc. (1996) DNA Seq.6:127-35) well-known in the art.Phosphoglucose isomerase albumen of the present invention comprises albumen shown in the SEQID NO:252.

Phosphoglyceric kinase (EC:2.7.2.3) (PGK) (the PFAM number of incorporating into own forces PF00162) is the enzyme that catalysis ATP forms ADP and inverse process.In second step of glycolysis-subordinate phase, 1, the 3-diphosphoglyceric acid is transformed into 3-phoshoglyceric acid, forms a part ATP.If opposite situation, then will form the ADP of a part.This is reflected in most of cell is necessary, and generation, the anaerobe that is used for ATP in aerobiont is used for fermenting and plant is used for carbon fixation.PGK is found in all organisms that live, and its sequence is very conservative in whole evolutionary process.Described enzyme exists with monomeric form, contains corresponding to the almost equal structural domain of two sizes of this albumen N-and C-end (N-end structure territory is gone in the wraparound of last 15 C-terminal residues).3-phoshoglyceric acid (3-PG) combines with the N-terminal of this enzyme, and Nucleotide substrate MgATP or MgADP are then in conjunction with the C-end structure territory of this enzyme.The geminus domain structure of this extension is relevant with large-scale " hinge bending " conformational change, the structure (Kumar etc. (1999) Cell Biochem.Biophys.31:141-64) that is similar in the hexokinase to be found.In the core of each structural domain by 6 chain parallels of α spiral surrounding.Structural domain 1 is that order is 342156 the parallel beta-pleated sheet of 6 chains, is 321456 the parallel beta-pleated sheet of 6 chains and structural domain 2 is order.The reversibility of yeast phosphoglyceric kinase is separated the folding analysis revealed that carries out, described two leaflets can independently fold, this with folding pathway in the folding intermediate in single structure territory to occur having be consistent (Yon etc. (1990) Biochimie 72:417-29).Detect the active method of phosphoglyceric kinase and be (consult, for example, Pal etc. (2004) Biochim.Biophys.Acta.1699:277-80) well-known in the art.Phosphoglyceric kinase albumen of the present invention comprises albumen shown in the SEQ ID NO:250.

Phosphoglycerate phosphomutase (EC:5.4.2.1) (PGAM) and diphosphoglycerate mutase (EC:5.4.2.4) be involved enzyme on the structure (BPGM), their catalysis relates to the reaction of the transfer of phosphate group between three carbon atoms of phosphorylglyceric acid.Two kinds of enzymes all can be with not three kinds of differential responses of homospecificity catalysis, with 2,3-diphospho glycerate acid isomer ester (2,3-DPG) 2-phosphoglyceric acid (2-PGA) isomery is turned to 3-phoshoglyceric acid (3-PGA) as start material, with 3-PGA as initiator from 1, the 3-DPG Synthetic 2,3-DPG and with 2,3-DPG is degraded into 3-PGA (phosphoesterase EC:3.1.3.13 activity).Many other protein comprise, catalysis fructose-2, bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2 that α-ribazole-5 ' of 6-bisphosphate and bacterium-monophosphate monophosphate esterase synthesizes and degrades, 6-bisphosphatase, it is relevant with the cobalami biosynthesizing, belongs to this family.The method that detects the catalytic activity of these enzymes is (consults, for example, Rigden etc. (2001) ProteinSci.10:1835-46) well-known in the art.Phosphoglycerate phosphomutase family of the present invention (the PFAM number of incorporating into own forces PF00300) albumen comprises the described protein of SEQ ID NO:244.

Pyruvate kinase (EC:2.7.1.40) is the final step in the catalysis glycolysis-(PK), and promptly phosphoenolpyruvic acid is to the transformation of pyruvic acid and be accompanied by the ADP phosphorylation and become ATP.This kind of enzyme that most of bacterium and low etc. eukaryote have a kind of form, and except some bacterium, intestinal bacteria for example, it just has two kinds of isozymes.The tetramer of the same subunit of seemingly about 500 residues of all isozymes.PK helps to control glucolytic speed with phosphofructokinase and hexokinase.Detect the active method of pyruvate kinase and be (consult, for example, Boles etc. (1997) J.Bacteriol.179:2987-93) well-known in the art.Pyruvate kinase α of the present invention/beta structure territory (the PFAM number of incorporating into own forces PF02887) albumen comprises protein shown in the SEQ IDNO:258.Pyruvate kinase barrel-like structure of the present invention territory (the PFAM number of incorporating into own forces PF00224) albumen comprises protein shown in the SEQ ID NO:258.

Many enzyme require diphosphothiamines (TPP) (VITMAIN B1) are as cofactor.Shown that in these enzymes some structurally is (Green (1989) the FEBS Lett.246:1-5) that is correlated with.The Rossman that 2 foldings are contained in diphosphothiamine enzyme central construct territory (the PFAM number of incorporating into own forces PF00205) folds.SEQ ID NO:232 comprises diphosphothiamine enzyme central construct territory and the terminal TPP binding domains (the PFAM number of incorporating into own forces PF02776) of diphosphothiamine enzyme N-.

Enzyme 3beta-Hydroxysteroid dehydrogenase/5-alkene-4-bisabolene isomer enzyme (3 β-HSD) catalysis 5-alkene-3 beta-hydroxy pregnene (hydroxypregnene) and 5-alkene-hydroxyl androstene steroid precursor oxidation and be isomerizated into to all types steroid hormone and form essential corresponding 4-alkene-ketosteroid.3-beta hydroxysteroid dehydrogenase of the present invention/isomerase family (the PFAM number of incorporating into own forces PF01073) albumen comprises protein described in the SEQ ID NO:216.Detect 3-beta-hydroxy-active method of δ 5-steroid dehydrogenase and be (consult, for example, Moisan etc. (1999) J Clin Endocrinol Metab.84:4410-25) well-known in the art.

Under the condition that ATP exists, the glycerone phosphorylation is become glycerone phosphoric acid in (EC:2.7.1.29) catalysis glycerol of dihydroxyacetone kinase (glycerone kinases) application approach.Dak1 structural domain (the PFAM number of incorporating into own forces PF02733) is the kinase domain of dihydroxyacetone kinase family.Dak1 domain protein of the present invention comprises protein shown in the SEQ ID NO:190.DAK2 structural domain (the PFAM number of incorporating into own forces PF02734) is the Phosphoric acid esterase structural domain of dihydroxyacetone kinase family prediction.Dak2 domain protein of the present invention comprises protein shown in the SEQ ID No:192.The method that detects the glycerone kinase activity is (consults, for example, Sellinger and Miller (1957) Fed.Proc.16:245-246) well-known in the art.

The biosynthesizing of disaccharides, oligosaccharides and polysaccharide relates to the effect of hundreds of different glycosyltransferases.These enzymes are that the catalysis glycosyl part is transferred to the enzyme that special acceptor molecule forms glycosidic link from the donor molecule that is activated.Described the glycosyltransferase (EC:2.4.1) that will utilize Nucleotide bisphosphate-sugar, nucleotide monophosphate one sugar and sucrose phosphoric acid and relevant albumen and be divided into family based on unique sequences.Identical three dimensional fold expection is present in each family.Because 3-D texture ratio sequence is more conservative, the several families based on the sequence similarity definition in the described family may have similar 3-D structure, and therefore form " clique ".The member of glycosyltransferase group 1 family (the PFAM number of incorporating into own forces PF00534) shifts the sugar that UDP, ADP, GDP or CMP connect.Bacterial enzyme is relevant with various biosynthetic processes, comprises the biosynthesizing of exo polysaccharides biosynthesizing, the biosynthesizing of lipopolysaccharides core and mucopolysaccharide colanic acid.Glycosyltransferase group 1 albumen of the present invention comprises the protein shown in the SEQ ID NO:328.

Kinase protein of the present invention comprises the albumen shown in the SEQ ID NOS:224,230,190,192,186 and 238.Protein hydrolysate of the present invention comprises the protein shown in the SEQ ID NOS:10,46,50,60,100,108,196,198,200,202,204,210,212,214,216,218,220,222,226,276,292,342,344,346,356,358,362 and 364.

Related protein of the present invention comprises the protein shown in the SEQ ID NOS:60,186,200,202,216,218,228,342,344,346,356 and 358 in the metabolism.Glycolysis-albumen of the present invention comprises the albumen shown in the SEQ ID NOS:242,244,246,248,250,252,254,256,258,260.Glycogen Metabolism albumen of the present invention comprises the albumen shown in the SEQ ID NOS:240,324,326,328,330 and 332.The protein of the present invention that relates in the EPS metabolism comprises the albumen shown in the SEQ ID NOS:348,350,352 and 354.

Clone and to express the method that carbohydrate utilizes the dependency method of protein and assess those proteinic functions be known in the art (consulting in microorganism and plant, for example, de Vos (1996) Antonie vanLeeuwenhoek 70:223-242; Yeo etal. (2000) Mol.Cells 10:263-268; Goddijn etc. (1997) Plant Physio.113:181-190).For example, can pass through the function that assessment primary and secondary delivery system related protein is measured in enzymatic determination, fermentation mensuration and transhipment.For example, can measure the function of assessing group translocation system associated protein by sugared phosphorylation.Consult, for example, Russell etc. (Russell etc. (1992) J Biol.Chem.267:4631-4637) have wherein identified from the gene of elementary delivery system (msm) in the Streptococcus mutans and have expressed in intestinal bacteria; Leong-Morgenthaler etc. (Leong-Morgenthalr et al. (1991) J.Bacteriol.173:1951-1957) have wherein cloned from two kinds of genes of the secondary delivery system of lactobacillus bulgaricus (lactose) and have expressed in intestinal bacteria; Vaughan etc. (Vaughan etc. (1996) Appl.Env.Microbiol.62:1574-1582) wherein clone from secondary delivery system (lacS) gene of leuconostoc lactis (Leuconostoc lactis) and express in intestinal bacteria; De Vos etc. ((1990) J Biol.Chem.265:22554-22560 such as de Vos) wherein identify, clone and expressed two kinds of PTS system genes from Lactococcus lactis in intestinal bacteria and lactobacillus lactis (Lactobacillus lactis); Sato etc. (Sato etc. (1989) J.Bacteriol.171:263-271) wherein will go into also to find to show the sucrose PTS activity in the intestinal bacteria from the scrA gene clone of Streptococcus mutans; Alpert and Chassy (Alpert and Chassy (1990) J.Biol.Chem.265:22561-22568), wherein cloned the coding lactobacterium casei lactose specific enzymes II gene and be expressed in the intestinal bacteria; Boyd etc. (Boyd etc. (1994) Infect.Immun.62:1156-1165) have wherein cloned the gene of the HPr of PTS delivery system of coding Streptococcus mutans and enzyme I and have been expressed in the intestinal bacteria; Garg etc. (Garg etc. (2002) Proc.Natl.Acad.Sci.USA 99:15898-15903), wherein cross expressing of intestinal bacteria trehalose biosynthetic genes otsA and otsB causes transgenic plant that the tolerance of inanimate pressure is improved and strengthened productivity; And Grinius and Goldberg (Grinius and Goldberg (1994) J.Biol.Chem.269:29998-30004), wherein expressed the effect that drug efflux pump has been brought into play in multi-medicine resistance protein and confirmation.

One or more carbohydrate utilize the expression of related protein to make organism have the ability of gathering carbohydrate in cell cytoplasm of improvement.For example, introduce or cross and express enzyme related in the sugar decomposition metabolism and do not express corresponding translocator matter and may cause carbohydrate in tenuigenin, to gather.Perhaps, introducing or cross expression carbohydrate transfer related protein may cause increasing carbohydrate and transport in the outside atmosphere.Introduce carbohydrate dependency gene in the organism or the method for expressing therein is known in the art.

Carbohydrate gathering in cell is appreciable, for example, and by chromatography or enzyme detection method.Consult, for example, Chaillou etc. (1998) J.Bacteriol.180:4011-4014 andGoddijn et al. (1997) together above.

One or more carbohydrate utilize the expression of associated protein to make organism may have the utilization of improvement or produce the ability of carbohydrate as the energy.In organism the clone and express method that carbohydrate utilizes the method for related protein and assess those protein functions be known in the art (consult, for example, de Vos (1996) AntonievanLeeuwenhoek 70:223-242; Hugenholz etc. (2002) AntonievanLeeuwenhoek 82:217-235).For example, the gene of lactose metabolism can be introduced in the bacterium improving the utilization of lactose, and produce the not more acceptable product of crowd of anti-lactose (Hugenholz etc. (2002) see above).In the bacterium of these improvement, can further modify, so that glucose is not degraded, but be released into the substratum, thereby the sweet taste of nature is provided from cell such as the blocking-up glucose metabolism.Consult, for example, (Hugenholz etc. (2002) see above).Perhaps, the gene that can introduce metabolic gene of semi-lactosi and α-Pu Taotang transphosphorylase is to improve the semi-lactosi fermentation capacity of microorganism, consume high-caliber semi-lactosi thereby help prevent, (Hugenholz etc. (2002) see above because the latter may cause health problem; Hirasuka and Li (1992) J.Stud.Alcohol 62:397-402).A gene relevant with the semi-lactosi metabolism is alpha-galactosidase, and its expression may be useful for the carbohydrate of removing the raffinose type from tunning, their (Hugenholz etc. (2002) see above) because monogastric animal can not be degraded.Mannitol-1-phosphate dehydrogenase (mtlD) gene of successful expression bacterium causes the synthetic of N.F,USP MANNITOL and gathers (Tarczynski etc. (1992) Proc.Natl.Acad.Sci.USA 89:2600-2604) in tobacco plant.

The function of various carbohydrate related proteins can be assessed with following method, for example, by microorganism growth measure, transhipment mensuration, enzymatic determination or analyze by chromatography and NMR.Consult, for example, Djordjevic etc. (2001) J.Bacteriol.183:3224-3236; Chaillou etc. (1998) J.Bacteriol.180:4011-4014; See above with (1992) such as Tarczynski.

Generally speaking, permease, membrane-associated enzyme and may need to be expressed in the cell so that best application carbohydrate such as regulators such as transcription repression or anti-terminators.The effect of transcribing anti-terminator can detect (consult, for example, Alpert and Siebers (1997) J.Bacteriol.179:1555-1562) by the activity of measuring anti-terminator in the reporting system.Function such as repressors such as lacR can assess by enzymic activity or growth measurement method (consult, for example, (1993) Protein Eng.6:201-206 such as van Rooijen; VanRooijen and deVos (1990) J Biol.Chem.265:18499-18503).

Bacterium regulation protein of the present invention comprises the sequence shown in the SEQ ID NOS:8,38,98,104,118,150,178,180,182,184,188,266,290,304 and 336.

Bacterium modulin lacI family of the present invention (the PFAM number of incorporating into own forces PF00356) albumen comprises the sequence shown in the SEQ ID NOS:38,98 and 182.

Bacterium modulin gnt R family of the present invention (the PFAM number of incorporating into own forces PF00392) comprises the sequence shown in the SEQID NO:106.Helix-turn-helix protein matter AraC family (the PFAM number of incorporating into own forces the PF00165) albumen of bacterium regulation and control of the present invention comprises the protein shown in the SEQ ID NO:118.Bacterium modulin deoR family of the present invention (the PFAM number of incorporating into own forces PF00455) albumen comprises the protein shown in SEQ ID NO:188 and 336.

PRD structural domain (at the adjusted and controlled territory of PTS) (the PFAM number of incorporating into own forces PF00874) but be that the bacterium that is found in BglG family is transcribed in the anti-terminator and such as the adjusted and controlled territory of the phosphorylation in the activators such as MtlR and LevR.The PRD structural domain on conservative histidine residues by phosphorylation.The protein that contains PRD is relevant with the adjusting of katabolism operon in Gram-positive and the gram negative bacterium, its feature usually be with the short N-terminal effector structure domain of RNA (with regard to anti-terminator (CAT RBD) with regard to is CAT-RBD) or DNA (with regard to activator) bonded and by sugar phosphotransferase system (PTS) in response to Gong the usability of carbon source on the Histidine of guarding the repetition PRD module of phosphorylation.Therefore phosphorylation is considered to change the stability of protein dimer, and changes the RNA-of effector structure domain or DNA-in conjunction with activity.This is a bacterioprotein family relevant with intestinal bacteria bglG albumen.Intestinal bacteria bglG albumen has mediated the just regulation and control (Houman etc. (1990) Cell 62:1153-63) of β-glucoside (bgl) operon by the effect of performance transcription terminator.BglG is that to discern the RNA-of the distinguished sequence that is positioned at two termination site upstreams of operon just conjugated protein.The activity of bglG is checked (Amster-Choder and Wright (1990) Science249:540-2) by the permease bglF (EII-bgl) from β-glucoside PTS system to its phosphorylation.BglG is similar to other protein height, and it also may be as transcribing anti-terminator.The protein of the PRD of containing structural domain of the present invention comprises the sequence of SEQ ID NOS:8 and 266.

AraC-sample ligand binding domains family (the PFAM number of incorporating into own forces PF02311) has represented the structural domain in conjunction with pectinose and dimerization of bacterial gene modulin AraC.Described structural domain is found and combines primitive HTH AraC associating with helix turn helix (HTH) DNA.This structural domain and Cupin structural domain relation are far away.AraC-sample ligand binding domains family protein of the present invention comprises the protein described in the SEQ ID NO:118.

CAT RNA binding domains (the PFAM number of incorporating into own forces PF03123) is found and is present in the N-terminal of transcribing the anti-terminator protein family.This structural domain has been called as CAT (Co-AntiTerminator is anti-terminator altogether) structural domain.This structural domain forms dimer in known structure.BglG/SacY family to transcribe anti-ending at be the modulin of the inducing action of carbohydrate metabolism operon in mediation Gram-positive and the gram negative bacterium.By activation, these protein combine with special target among the newborn mRNA, thereby prevent that RNA polymerase is from the dna profiling failure (Declerck etc. (1999) J.Mol.Biol.294:389-402) of dissociating.CAT RNA binding domains albumen of the present invention comprises the protein described in SEQ ID NOS:8 and 266.

SEQ ID NO:184 is the member of HPr serine kinase N-terminal family (the PFAM number of incorporating into own forces PF02603), and the member of HPr serine kinase C-terminal family (the PFAM number of incorporating into own forces PF07475).N-terminal family has represented the N-terminal zone of Hpr serine/threonine kinase PtsK.C-terminal family has represented the C-terminal kinase domain of Hpr serine/threonine kinase PtsK.This kinases is that the polycomponent phosphoric acid under the regulation and control of carbon katabolism restraining effect transmits the interior inductor block (Marquez etc. (2002) Proc.Natl.Acad.Sci.U.S.A.99:3458-63) of system in the bacterium.The tertiary structure of distinguished its target of identification of this kinases, and be the member of the new family that all has nothing to do with arbitrary aforementioned protein phosphorylation enzyme.Total length crystalloid enzyme from Staphylococcus xylosus is carried out x-ray analysis with the resolving power of 1.95  show that this enzyme is made up of two obvious separated structures territories, is assemblied in the six aggressiveness structures of similar three leaf propeller.Each blade is made up of two N-terminal structural domains, and compact central axle has been gathered C-terminal kinase domain (Reizer etc. (1998) Mol.Microbiol.27:1157-69).

Thereby sequence of the present invention also can change smell or quality that the ability of organism changes food.Modifying glucose metabolism is a kind of method that can cause smell or quality characteristic to change to produce alternative carbohydrate.The destruction of lactate dehydrogenase gene and the expression that is accompanied by from N.F,USP MANNITOL or sorbyl alcohol operon gene cause producing N.F,USP MANNITOL and sorbyl alcohol (Hugenholz etc. (2002) see above).The generation of diacetyl causes plant butter fragrance between yeast phase, this effect can be by serum lactic dehydrogenase destruction or nadh oxidase cross express and in conjunction with the destruction of alpha-acetolactate decarboxylase strengthened (Hugenholz and Kleerebezem, (1999) see above; Hugenholtz etc. (2000) Appl.Environ.Microbiol.66:4112-4114).Perhaps, the excessive production of alpha-acetolactate synthase or the acetohydroxy acid synthase destruction that is accompanied by alpha-acetolactate decarboxylase has caused increase (Swindell etc. (1996) Appl.Environ.Microbiol.62:2641-2643 of diacetyl output; Platteeuw etc. (1995) Appl.Enviro.Microbiol.61:3967-3971).The expression of crossing of alanine dehydrogenase has caused the generation L-Ala but not lactic acid, for leavened food provides odorant and sweetener (Hols etc. (1999) Nat.Biotechno l.17:588-592).

Be used to improve organism produce improvement carbohydrate ability method also within the scope of the present invention, comprise at least a nucleotide sequence of the present invention introduced in the organism.The method that relates to the carbohydrate that produces improvement in addition comprises with polypeptide contact of the present invention carbohydrate to be finished.The method that produces the carbohydrate of improvement is known in the art.Consult, for example, Kim etc. (2001) Biotechnol.Prog.17:208-210.

Sequence of the present invention also can be improved the ability that organism survives in food system or Mammals gi tract, perhaps improve stability and the viability of organism at food processing and lay up period.For example, the increase of trehalose output may make the freshness of leavened prod and taste hold-time prolong (consult, for example, www.nutracells.com).The disease that trehalose also can help to prevent protein aggregation or protein morbid state conformation to be caused is such as Creutzfeld-Jacob disease.In plant, the gathering of trehalose produced antagonism such as environmental stresss such as arid, salt and cold provide protection (consult, for example, Jang etc. (2003) Plant Physiol.131:516-524; Penna (2003) Trends Plant Sci.8:355-357; Garg etc. (2002) Proc.Natl.Acad.Science 99:15898-15903; Yeo etc. (2000) see above).In addition, plant is transformed by the fructose transferase gene, and it makes this plant can gather high-caliber Polylevulosan ((1994) Plant Cell 6:561-570 such as van der Meer).Except effect as the carbohydrate deposit, Polylevulosan also can make the plant tolerance do and cold condition (Pontis and delCampillo (1985) " Fructans " in Biochemistry ofStorage Carbohydrates in Green Plants, Day and Dixon, eds. (London:Academic Press), pp.810-816; Pilon-Smits etc. (1995) Plant Physiol.107:125-130).The bacterial gene mannitol-1-phosphate dehydrogenase also has been expressed in the plant, causes N.F,USP MANNITOL to produce, and this has been considered to give favourable proterties, comprise osmoregulation and in and hydroxide radical (Tarczynski etc. (1992) see above).

Multidrug transporter sequence of the present invention can make organism still can survive behind contact antimicrobial polypeptide or other toxin.This may be because the ability that medicine or toxin are transported out cell increases causes.

In the variant of these nucleotide sequences was also included within, such as those maintenances or change the sequence of carbohydrate or toxin being advanced or go out the transporting power of cell, and the sequence of the ability of carbohydrate was gathered or is utilized in those maintenances or change.

Preparation is well-known in the art with the method that detects carbohydrate variant relevant or multidrug transporter.Consult, for example, Poolman etc. (Poolman etc. (1996) Mol.Microbiol.19:911-911) wherein separate or have made up the secondary delivery system protein variant (mellibiose and lactose) of the substrate specificity with change and test.In these mutant, the transhipment of sugar is and positively charged ion symport link coupled mutually not.Also can consult, for example, Djorovevic etc. (2001) see above, and have wherein made up the mutant HPr protein of the regulation activity with change; And Adams etc. (Adams etc. (1994) J.Biol.Chem.269:5666-5672), wherein produce and identified cold responsive type beta-galactosidase gene variant from Lactobacillusdelbr ü eckiisubsp.Bulgaricus.The gene of these sudden changes is Vma at low temperatures _xReduce, therefore can be used for preventing tunning acidifying during refrigerating (Mainzer etc. (1990) " Pathway engineering of lactobacillus bulgaricus forimproved yoghurt; " in Yoghurt:Nutritional and Health Properties, Chandan, ed., (National Yoghurt Association, Virginia, US), pp.41-55.Also can consult, Bettenbrock etc. (Bettenbrock etc. (1999) J Bacteriol.181:225-230) have wherein separated the mutant of the semi-lactosi specificity PTS gene with improvement.Also can consult, vanRooijen etc. (1990) see above, and have wherein separated the lacR repressor variant that activity is not had influence.Also can consult Kroetz etc., wherein analyzed the polymorphism (Kroetz etc. (2003) Pharmacogenetics 13:48-94) of people MDR1 gene, and Mitomo etc., wherein analyzed the variant (Mitomo etc. (2003) Biochem.J.373:767-74) of abc transport albumin A BCG2.

Any above modification all can combine with other metabotic change of having been transformed in lactic-acid-bacterium or propose.These variations comprise the generation of B-VITAMIN, such as folic acid (Bll), riboflavin (B2) or cobalami (B12); The generation of polyol compound or low calory sugar can replace sucrose, lactose, glucose or fructose as sweeting agent; Tagatose-another kind of sucrose substitute-the generation of the various exo polysaccharides of generation, the blocking-up glucose metabolism reduces the output of semi-lactosi so that natural sweet taste to be provided; The generation of the food that alpha galactosides such as stachyose and raffinose level are lower; And the increase of trehalose output, these change the characteristic all kept food and may be relevant with the prevention of disease (Hugenholz etc. (2002) see above; VanRoojen etc. (1991) J.Biol.Chem.266:7176-7178).

The present invention also provides the method for removing or change undesirable carbohydrate from food or chemical products.Described method comprises with purified polypeptide of the present invention and contacts described product.It is well-known in the art measuring the removal of carbohydrate or the method for change.

Table 1. carbohydrate of the present invention is used albumen

ORF #	SEQ ID NO：	Identity/function	COG
				452	1，2	PTS system seminose specific factor IIAB	3444
877	3，4	Phosphotransferase system (PTS) lichenan specific enzymes IIA component	1447
				609	5，6	The special translocator of β-glucoside	2190
1479	7，8	Transcribe anti-terminator	3711
				1574	9，10	Phosphoric acid β Polyglucosidase	2723
1707	11，12	β glucoside permease IIABC component	1263
				725	13，14	The PTS system, the special IIABC component of β-glucoside	1263
491	15，16	Phosphotransferase system (PTS) albumen, lichenan specific enzymes IIC component	1455

ORF #	SEQ ID NO：	Identity/function	COG
1369	17，18	Phosphotransferase system enzyme II	1455
				1684	19，20	Phosphotransferase system IIA component	2893
146	21，22	PTS system enzyme IIBC component (melampyrum/fructose is special)	1762
				227	23，24	The special component I IC of PTS cellobiose	1455
989	25，26	PTS cellobiose specific enzymes IIC	1455
				884	27，28	The special PTS system of cellobiose IIC component	1455
618	29，30	The PTS system, cellobiose specific enzymes IIC
				606	31，32	Phosphotransferase system (PTS) arbutin sample enzyme IIBC component	1263
1705	33，34	The special PTS system of sucrose IIBC component	1263
				1777	35，36	PTS system albumen	1299
500	37，38	Sucrose operon inhibition	1609
				502	39，40	The abc transport protein substrate is conjugated protein	1653
503	41，42	Abc transport albumen is striden film permease-HUCEP-8	1175
				504	43，44	Abc transport albumen is striden film permease-HUCEP-8	395
505	45，46	Sucrose-6-phosphohydrolase	1621
				506	47，48	Multiple sugared binding transport albumin A TP is conjugated protein	3839
507	49，50	GtfA albumen	366

ORF #	SEQ ID NO：	Identity/function	COG
				1481	51，52	Ribose abc transport albumen (ribose is conjugated protein)	1879
1482	53，54	Ribose abc transport albumen (permease)	1172
				1483	55，56	TP is conjugated protein for ribose abc transport albumin A	1129
1484	57，58	Ribose permease (RbsD)	1869
				1485	59，60	Ribokinase (RbsK)	524
1864	61，62	Maltose abc transport albumen permease albumen	3833
				1865	63，64	Maltose abc transport albumen permease albumen	1175
1866	65，66	Maltose abc transport protein substrate is conjugated protein	2182
				1867	67，68	Multiple sugared binding transport ATP is conjugated protein	3839
1944	69，70	Sugar abc transport albumen	3845
				1945	71，72	Sugar abc transport albumen permease albumen	4603
1946	73，74	Sugar abc transport albumen permease albumen	1079
				45	75，76	HUCEP-8	477
552	77，78	Translocator	477
				566	79，80	Translocator	477
567	81，82	Medicine flows out translocator	477
				753	83，84	Translocator	477
1446	85，86	Medicine output albumen	477
				1471	87，88	Flow out albumen	477

ORF #	SEQ ID NO：	Identity/function	COG
				1616	89，90	Translocator	2244
1621	91，92	The effluent translocator	477
				1853	93，94	Medicine flows out translocator	477
1917	95，96	The polysaccharide translocator	2244
				399	97，98	Sucrose operon modulin	1609
400	99，100	Sucrose-6-phosphohydrolase	1621
				401	101，102	Phosphotransferase system enzyme II	1263
1012	103，104	The special PTS system of β-glucoside IIABC component	1263
				1013	105，106	Trehalose operon transcription repressor	2188
1014	107，108	The dextran glucoside	366
				1439	109，110	Abc transport albumin A TP is conjugated protein-multiple HUCEP-8	3839
1440	111，112	Multiple sugared binding transport system permease albumen	395
				1441	113，114	Abc transport albumen is striden film permease-multiple sugar	1175
1442	115，116	Multiple carbohydrate-binding protein precursor	1653
				1443	117，118	The Raffinose Operon transcriptional regulation protein	2207
73	119，120	Carbohydrate utilizes associated protein
				74	121，122	Abc transport albumen bacteriocin	1132
75	123，124	Abc transport albumen	1132
				1131	125，126	Abc transport albumen	1132
1132	127，128	Abc transport albumen	1132

ORF #	SEQ ID NO：	Identity/function	COG
				1357	129，130	Abc transport albumen	1132
1358	131，132	Abc transport albumen	1132
				1679	133，134	Permease	577
1680	135，136	Translocator	1136
				1681	137，138	Carbohydrate utilizes associated protein
1793	139，140	Carbohydrate utilizes associated protein
				1794	141，142	Carbohydrate is used associated protein mutually
1796	143，144	plnG	2274
				1838	145，146	Abc transport albumen	1136
1839	147，148	Permease	577
				1840	149，150	Regulator	1309
1913	151，152	Abc transport albumen	1136
				1914	153，154	Abc transport albumen	577
1915	155，156	Carbohydrate utilizes associated protein
				1938	157，158	Carbohydrate utilizes associated protein	1434
1939	159，160	Abc transport albumen	1136
				453	161，162	The special phosphotransferase system component of seminose
454	163，164	PTS system seminose specific factor IIAB
				455	165，166	PTS system seminose specific factor IIC	3715
456	167，168	PTS system seminose specific factor IID	3716
				876	169，170	PTS system enzyme II albumen	1440

ORF #	SEQ ID NO：	Identity/function	COG
				879	171，172	The special EII component of phosphotransferase system sugar	1455
1575	173，174	PTS system β glucoside specific enzymes II, the ABC component	1263
				1463	175，176	LacS	2211
639	177，177	ptsH	1925
				640	179，180	ptsI	1080
431	181，182	ccpA	1609
				676	183，184	ptsK	1493
1778	185，186	FruK	1105
				1779	187，188	FruR	1349
1433	189，190	The Protosol kinases	2376
				1434	191，192	The Protosol kinases	2376
1436	193，194	The glycerol picked-up promotes albumen	580
				1437	195，196	gtfAII	366
1438	197，198	melA	3345
				1457	199，200	GalM	2017
1458	201，202	GalT	4468
				1459	203，204	GalK	153
1460	205，206	Surface protein
				1461	207，208	Transcriptional control	1309
1462	209，210	LacZ	1874
				1467	211，212	Beta-galactosidase enzymes	3250
1468	213，214	Beta-galactosidase enzymes	3250

ORF #	SEQ ID NO：	Identity/function	COG
				1469	215，216	GalE	1087
1719	217，218	UDP glucose phosphorylation enzyme	1210
				874	219，220	Beta galactosidase enzyme	2723
910	221，222	L-LDH	39
				1007	223，224	Pyridoxal kinase	2240
1812	225，226	The α Polyglucosidase	1501
				1632	227，228	Acetaldehyde dehydrogenase	1012
1401	229，230	The NADH peroxidase	446
				1974	231，232	Pyruvic oxidase	28
1102	233，234	Amino acid permease	4975
				1783	235，236	Abc transport albumen	4152
1879	237，238	The pyrimidine kinases	351
				680	239，240	glgB	296
55	241，242	D-LDH	1052
				185	243，244	Phosphoglycerate phosphomutase	588
271	245，246	L-LDH	39
				698	247，248	GPDH	57
699	249，250	Phosphoglyceric kinase	126
				752	251，252	Glucose 6-phosphoric acid isomerase	166
889	253，254	2-Phosphoglycerate dehydratase	148
				956	255，256	Phosphofructokinase	205
957	257，258	Pyruvate kinase	469
				1599	259，260	Fructose-bis phosphate aldolase	191

ORF #	SEQ ID NO：	Identity/function	COG
				1641	261，262	Glyceraldehyde-3 phosphate abc transport albumen	1653
452	263，264	Seminose; PTS system seminose specific factor IIAB	3444
				1479	265，266	The β glucoside; Transcribe anti-terminator	3711
725	267，268	The β glucoside; The PTS system, the special IIABC component of β glucoside	1263
				1369	269，270	Cellobiose; Phosphotransferase system enzyme II	1455
227	271，272	Cellobiose; The special component I I of PTS cellobiose	1455
				502	273，274	HUCEP-8; The abc transport protein substrate is conjugated protein	1653
507	275，276	GtfA	366
				1483	277，278	RbsA; TP is conjugated protein for ribose abc transport albumin A	1129
1484	279，280	Ribose permease RbsD	1869
				552	281，282	Multidrug transporter	477
567	283，284	Multidrug transporter	477
				1471	285，286	Multidrug transporter	477
1853	287，288	Multidrug transporter	477
				1012	289，290	TreB; The β glucoside; The special PTS system of β glucoside IIABC component	1263
1014	291，292	treC	366
				1440	293，294	msmG	395
1442	295，296	msmE	1653
				1132	297，298	Abc transport albumen	1132

ORF #	SEQ ID NO：	Identity/function	COG
				1358	299，300	Abc transport albumen	1132
1838	301，302	Abc transport albumen	1136
				1840	303，304	Transcriptional regulatory (TetR/AcrR family)	1309
1913	305，306	Abc transport albumen	1136
				1938	307，308	Carbohydrate utilizes associated protein	1434
164	309，310	Multidrug transporter	3590
				251	311，312	Multidrug transporter	477
252	313，314	Multidrug transporter	477
				253	315，316	Multidrug transporter	477
1062	317，318	Multidrug transporter	477
				597	319，320	The ABC multidrug transporter	1132
1854	321，322	Multidrug transporter	477
				681	323，324	Cori ester adenylyl transferase glgC	448
682	325，326	Cori ester adenylyl transferase glgD	448
				683	327，328	Glycogensynthase	297
685	329，330	Glycogen phosphorylase	58
				686	331，332	Starch debranching enzyme	366
1356	333，334	The arsenic acid reductase enzyme	1393
				1465	335，336	The transcription repressor of tilactase	1609
1643	337，338	Sugar abc	395
				1645	339，340	Multiple sugar is in conjunction with abc transport albumen	3839

ORF #	SEQ ID NO：	Identity/function	COG
				1731	341，342	Glycosyltransferase	1216
1732	343，344	Galactosyltransferase
				1733	345，346	Phosphoglucose based transferase epsE	2148
1734	347，348	epsD	4464
				1735	349，350	epsC	489
1736	351，352	epsB	3944
				1737	353，354	epsA	1316
1738	355，356	Gtp binding protein Hf1X	2262
				1739	357，358
1782	359，360	Abc transport albumen permease albumen	1668
				1869	361，362	β-phosphoglucomutase	637
1870	363，364	Maltose phosphorylase	1554

Following examples are only for illustrating but not limitation of the present invention.

* the breach BlastP result of embodiment 1. aminoacid sequences

Breach BlastP sequence alignment shows the 1-140 amino acids of SEQ ID NO:2 (144 amino acid) and the protein (number of incorporating into own forces the NP469488.1 that also comes from PTS system seminose specific factor IIAB from harmless Listera together; NC003212) have about 61% identity, the 1-140 amino acids with from Listeria monocytogenes and with the protein (number of incorporating into own forces the NP463629.1 that comes from PTS system seminose specific factor IIAB; NC～003210) have about 60% identity, the 1-139 amino acids with from clostridium acetobutylicum and be the protein (number of incorporating into own forces NP～149230.1 of the special phosphotransferase system component of seminose IIAB; NC～001988) have about 63% identity, the 1-139 amino acids with from clostridium perfringens (Clostridium perfringens) and be the proteic protein of the PTS system (number of incorporating into own forces NP561737.1; NC003366) have about 62% identity, the 2-141 amino acids with from streptococcus pyogenes (Streptococcuspyogenes) and be the protein (number of incorporating into own forces the NP269761.1 of the special phosphotransferase system component of seminose IIAB; NC～002737) has about 50% identity.

Lack the BlastP sequence alignment and show SEQ ID NO:4 (123 amino acid) 20-109 amino acids and the protein (number of incorporating into own forces NP～471165.1 that also come from phosphotransferase system (PTS) lichenin (lichenan) specific enzymes IIA component from harmless Listera together; NC～003212) have about 60% identity, the 20-110 amino acids with from harmless Listera and with the protein (number of incorporating into own forces NP～472161.1 that come from cellobiose phosphotransferase IIA component; NC～003212) have about 57% identity, the 1-112 amino acids with from Lactococcus lactis breast subspecies and be the protein (number of incorporating into own forces the NP266570.1 of the special PTS system of cellobiose IIA component (EC 2.7.1.69); NC002662) have about 46% identity, the 9-112 amino acids with from Bacillushalodurans and be the protein (number of incorporating into own forces the NP241776.1 of PTS system cellobiose specific enzymes IIA component; NC_002570) have about 44% identity, and the 16-112 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP607437.1 that comes from PTS enzyme III; NC_003485) has about 51% identity.

Lack the BlastP sequence alignment show SEQ ID NO:6 (161 amino acid) 6-143 amino acids with from faecium and be the protein (number of incorporating into own forces the gb1AAD28228.1 of the protein (BglS) of the special translocator of β glucoside; AF121254) have about 53% identity, the 13-159 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP_345256.1 of PTS system IIABC component; NC 003028) have about 48% identity, the 13-159 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP 358262.1 of PTS glucose specific enzymes IIABC component; NC_003098) have about 48% identity, the 13-159 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP608025.1 that comes from PTS system enzyme IIA component; NC_003485) have about 46% identity, and the 13-159 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP269950.1 that comes from PTS enzyme IIA component; NC_002737) has about 46% identity.

Breach BlastP sequence alignment show SEQ ID NO:8 (291 amino acid) 11-282 amino acids with from subtilis and be that the protein (number of incorporating into own forces sp/P39805/LICT_BACSU) of transcribing anti-terminator (licT) has about 36% identity, the 11-282 amino acids with from subtilis and be the protein (number of incorporating into own forces the NP_391787.1 that transcribes anti-terminator (BglG family); NC_000964) have about 36% identity, the 11-282 amino acids with from intestinal bacteria and relate to the protein (number of incorporating into own forces the NP-418179.1 that the bgl operon is just being regulated and control; NC～000913) has about 37% identity, the 11-282 amino acids with from chrysanthemum Erwinia (Erwinia chrysanthemi) and be β glucoside operon anti-terminator protein (number of incorporating into own forces sp1P26211 1ARBG～ERWCH) has about 33% identity, and the 9-288 amino acids with from clostridium acetobutylicum and be the protein (number of incorporating into own forces the Nu-347062.1 that transcribes anti-terminator (licT); NC～003030) has about 34% identity.

Breach BlastP sequence alignment shows the 8-473 amino acids of SEQ ID NO:10 (480 amino acid) and the protein (number of incorporating into own forces the NP463849.1 that also comes from phosphoric acid β Polyglucosidase from Listeria monocytogenes together; NC～003210) have about 59% identity, the 8-473 amino acids with from harmless Listera and with the protein (number of incorporating into own forces the NP469689.1 that comes from phosphoric acid β Polyglucosidase; NC～003212) have about 58% identity, the 7-473 amino acids with from clostridium acetobutylicum and be protein (NP～347379.1 of 6-phosphoric acid-beta-glucosidase enzyme; NC003030) has about 57% identity, the 8-473 amino acids with from Clostridium longisporum and be 6-phosphoric acid-beta-glucosidase enzyme protein (number of incorporating into own forces sp1Q461301ABGA～CLOLO) has about 57% identity, the 1-473 amino acids with from subtilis and be the protein (number of incorporating into own forces NP～391805.1 of beta-glucosidase enzyme; NC～000964) has about 55% identity.

Breach BlastP sequence alignment show the 1-624 amino acids of SEQ ID NO:12 (625 amino acid) with from streptococcus pyogenes and be the protein (number of incorporating into own forces the NP 268836.1 of β-glucoside permease IIABC component; NC～002737) have about 38% identity, the 1-624 amino acids with from streptococcus pyogenes and be the protein (number of incorporating into own forces the NP606826.1 of β-glucoside permease IIABC component; NC～003485) have about 38% identity, the 1-605 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces NP～358099.1 of the special EII component of phosphotransferase system sugar; NC003098) have about 38% identity, the 1-605 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces NP～345091.1 of the special IIABC component of PTS system β-glucoside; NC～003028) have about 38% identity, the 1-622 amino acids with from Bacillus halodurans and be the protein (number of incorporating into own forces the NP 241162.1 of the special IIABC component of PTS system β-glucoside; NC～002570) has about 38% identity.

Lack the BlastP sequence alignment show the 17-648 amino acids of SEQ ID NO:14 (675 amino acid) with from clostridium acetobutylicum and be the protein (number of incorporating into own forces the NP_348035.1 of the special IIABC component of PTS system β-glucoside; NC_003030) have about 50% identity, the 17-656 amino acids with from Bacillus halodurans and be the protein (number of incorporating into own forces NP～241461.1 of PTS system β-glucoside specific enzymes IIABC; NC～002570) have about 50% identity, the 17-656 amino acids with from Listeria monocytogenes and with the protein (number of incorporating into own forces the NP_463560.1 that comes from PTS system β-glucoside specific enzymes IIABC; NC_003210) have about 50% identity, the 17-654 amino acids with from Clostridium longisporum and be the protein (number of incorporating into own forces the gb/AAC05713.1 of PTS dependent form enzyme II; L49336) about 48% identity is arranged, the 13-654 amino acids with from Streptococcus mutans and be the protein (number of incorporating into own forces the gb/AAF89975.1 of β-glucoside specificity EII permease; AF206272) about 48% identity is arranged.

Breach BlastP sequence alignment show SEQ ID NO:16 (445 amino acid) 10-443 amino acids with from subtilis and be the protein (number of incorporating into own forces the NP_391737.1 of phosphotransferase system (PTS) albumen lichenin specific enzymes IIC component; NC_000964) about 41% identity is arranged, the 14-442 amino acids with from subtilis and with the protein (number of incorporating into own forces sp/P39584/YWBA_BACSU) that comes from PTS system IIBC component (ywbA) component about 42% identity is arranged, the 14-441 amino acids with from bacstearothermophilus and be that the protein (number of incorporating into own forces SP/Q45400/PTCC_BACST) of cellobiose phosphotransferase IIC component has about 41% identity, the 12-441 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP_358015.1 of phosphotransferase system sugar specificity EII component; NC_003098) about 41% identity is arranged, and the 12-441 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP_344993.1 of PTS system cellobiose specificity IIC component; NC_003028) about 40% identity is arranged.

Breach BlastP sequence alignment show SEQ ID NO:18 (422 amino acid) 9-417 amino acids with from subtilis and with the protein (number of incorporating into own forces the NP_391718.1 that comes from phosphotransferase system enzyme II; NC_000964) have about 34% identity, the 17-414 amino acids with from subtilis and be protein (the contraction NP_391737.1 of phosphotransferase system (PTS) lichenin specific enzymes IIC component; NC_000964) has about 33% identity, the 10-417 amino acids with from bacstearothermophilus and be that the protein (number of incorporating into own forces SP/Q45400/PTCC_BACST) of cellobiose phosphotransferase IIC component has about 34% identity, the 9-414 amino acids with from nontoxic Listera and with the protein (number of incorporating into own forces the NP_470241.1 that comes from PTS system cellobiose specificity IIC component; NC_003212) about 33% identity is arranged, and the 11-415 amino acids with from B. burgdorferi and be the protein (number of incorporating into own forces the NP_046990.1 of PTS system cellobiose specificity IIC component (CelB); NC_001903) about 31% identity is arranged.

Breach BlastP sequence alignment show SEQ ID NO:20 (130 amino acid) with from Bacterium melitense and be the protein (number of incorporating into own forces the NP_540949.1 of phosphotransferase system IIA component; NC_003317) about 33% identity is arranged, the 2-102 amino acids with from lactobacillus curvatus and be the protein (number of incorporating into own forces the 96/AAB 04153.1 of EIIA-Mannoproteins; U28163) about 32% identity is arranged, the 3-96 amino acids with from clostridium perfringens and with coming from the proteic protein of the PTS system (number of incorporating into own forces NP_563545.1; NC_003366) about 32% identity is arranged, the 3-123 amino acids with from clostridium perfringens and with coming from the proteic protein of the PTS system (number of incorporating into own forces NP_561737.1; NC_003366) about 25% identity is arranged, and the 3-123 amino acids with from clostridium acetobutylicum and be the protein (number of incorporating into own forces the NP_149230.1 of seminose specificity phosphotransferase system component IIAB; NC_001988) about 25% identity is arranged.

Breach BlastP sequence alignment show SEQ ID NO:22 (162 amino acid) 8-159 amino acids with from clostridium acetobutylicum and be the protein (number of incorporating into own forces the NP_349560.1 of PTS system enzyme IIBC component (melampyrum/fructose specificity); NC_003030) about 38% identity is arranged, the 7-158 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP_358156.1 of phosphotransferase system sugar specificity EII composition; NC_003098) about 36% identity is arranged, the 7-158 amino acids with from streptococcus pneumoniae and with the protein (number of incorporating into own forces the NP_345152.1 that comes from PTS system IIA component; NC_003028) about 36% identity is arranged, the 20-134 amino acids with from streptococcus agalactiae and be the proteic protein of the GatA (number of incorporating into own forces gb/AAG 09977.1; AF248038) about 38% identity is arranged, and the 16-159 amino acids with from Bacillus halodurans and be the protein (number of incorporating into own forces the NP_241058.1 of PTS system melampyrum specific enzymes IIA component; NC_002570) about 33% identity is arranged,

Breach BlastP sequence alignment show SEQ ID NO:24 (466 amino acids) 30-461 amino acids with from clostridium acetobutylicum and be the protein (number of incorporating into own forces NP～347026.1 of PTS cellobiose specificity component IIC; NC～003030) have about 47% identity, the 26-465 amino acids with from Lactococcus lactis breast subspecies and be the protein (number of incorporating into own forces the NP266974.1 of the special PTS system of cellobiose IIC component (EC 2.7.1.69); NC～002662) have about 45% identity, the 82-465 amino acids with from Lactococcus lactis breast subspecies and be the protein (number of incorporating into own forces NP～266572.1 of the special PTS system of cellobiose IIC component (EC 2.7.1.69); NC～002662) have about 46% identity, the 34-466 amino acids with from streptococcus pyogenes and be the protein (number of incorporating into own forces the NP269994.1 of PTS system enzyme IIC component; NC～002737) have about 41% identity, and the 34-466 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces NP～608069.1 that come from PTS system enzyme IIC component; NC～003485) has about 40% identity.

Breach BlastP sequence alignment shows the 25-420 amino acids of SEQ ID NO:26 (428 amino acid) and the protein (number of incorporating into own forces the NP-472233.1 that also comes from PTS cellobiose specific enzymes IIC component from harmless Listera together; NC 003212) have about 28% identity, the 115-415 amino acids with from lactobacterium casei and be the LacE protein (number of incorporating into own forces emblCAB02556.1; Z80834) have about 27% identity, the 137-425 amino acids with from harmless Listera and with the protein (number of incorporating into own forces the NP-472184.1 that comes from PTS system cellobiose specific enzymes IIC; NC 003212) have about 26% identity, the 137-425 amino acids with from Listeria monocytogenes and with the protein (number of incorporating into own forces the NP466230.1 that comes from PTS system cellobiose specific enzymes IIC; NC003210) have about 26% identity, and the 115-415 amino acids with from lactobacterium casei and be that the protein (number of incorporating into own forces pirllB23697) of phosphotransferase system enzyme II (EC 2.7.1.69) has about 26% identity.

Breach BlastP sequence alignment show the 10-471 amino acids of SEQ ID NO:28 (475 amino acid) with from Lactococcus lactis breast subspecies and be the protein (number of incorporating into own forces NP～266974.1 of the special PTS system of cellobiose IIC component (EC 2.7.1.69); NC～002662) have about 57% identity, the 71-475 amino acids with from Lactococcus lactis breast subspecies and be the protein (number of incorporating into own forces the NP266572.1 of the special PTS system of cellobiose IIC component (EC 2.7.1.69); NC～002662) have about 45% identity, the 13-470 amino acids with from the special component I IC protein (number of incorporating into own forces the Nu-347026.1 of the PTS cellobiose of clostridium acetobutylicum; NC003030) have about 42% identity, the 17-468 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP269994.1 that comes from PTS system enzyme IIC component; NC～002737) have about 41% identity, the 17-468 amino acids is with (number of incorporating into own forces NP～608069.11 (NC～003485) has about 41% identity from streptococcus pyogenes and with the protein that comes from PTS system enzyme IIC component.

Breach BlastP sequence alignment shows the 1-428 amino acids of SEQ ID NO:30 (441 amino acid) and the protein (number of incorporating into own forces the Nu-472184.1 that also comes from PTS system cellobiose specific enzymes IIC from harmless Listera together; NC～003212) have about 46% identity, the 1-428 amino acids with from Listeria monocytogenes and with the protein (number of incorporating into own forces the NP466230.1 that comes from PTS system cellobiose specific enzymes IIC; NC～003210) have about 46% identity, the 10-427 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP 607435.1 that comes from PTS system IIC component; NC～003485) has about 39% identity, 1-428 amino acids and the protein (number of incorporating into own forces NP266330.1 from the special PTS system of the cellobiose IIC component (EC 2.7.1.69) of Lactococcus lactis breast subspecies; NC 002662) have about 36% identity, the 1-421 amino acids with from Listeria monocytogenes and with the protein (number of incorporating into own forces the NP466206.1 that comes from cellobiose phosphotransferase IIC component; NC～003210) has about 31% identity.

1-532 amino acids that breach BlastP sequence alignment shows SEQ ID NO:32 (626 amino acid) and the protein (number of incorporating into own forces NP-388701.1 from phosphotransferase system (PTS) the arbutin sample enzyme IIBC component of subtilis; NC000964) has about 54% identity, 2-530 amino acids and the protein (number of incorporating into own forces NP561112.1 from the PTS arbutin sample enzyme IIBC component of clostridium perfringens (Clostridiumperfringens); NC～003366) have about 51% identity, the 1-533 amino acids with from fusobacterium mortiferum and be the protein (number of incorporating into own forces the gb1AAB63014.2 of pts protein EII; U81185) have about 52% identity, the 1-533 amino acids with from clostridium acetobutylicum and be the proteic protein of the MalP (number of incorporating into own forces gb1AAK69555.1; AF290982) have about 51% identity, the 1-533 amino acids with from clostridium acetobutylicum and be the protein (number of incorporating into own forces NP～347171.1 of PTS system arbutin sample IIBC component; NC～003030) has about 51% identity.

Breach BlastP sequence alignment show the 1-456 amino acids of SEQ ID NO:34 (663 amino acid) with from Lactococcus lactis breast subspecies and be the protein (number of incorporating into own forces the NP267287.1 of the special PTS system of sucrose IIBC component (EC2.7.1.69); NC～002662) have about 58% identity, the 5-471 amino acids with from the golden yellow subspecies of golden yellow glucose coccus and with the protein (number of incorporating into own forces Nu～373429.1 that come from sucrose phosphotransferase II; NC002745) have about 54% identity, the 5-472 amino acids with from Bacillushalodurans and be the protein (number of incorporating into own forces NP～244441.1 of PTS system sucrose phosphotransferase IIBC component; NC～002570) have about 46% identity, the 4-468 amino acids with from Salmonella enterica subsp.enteric serovar Typhi and with the protein (number of incorporating into own forces the Nu-457099.1 that comes from PTS system IIBC component; NC003198) have about 39% identity, and the 4-468 amino acids with from Salmonella typhimurium (Salmonellat yphimurium) and with the protein (number of incorporating into own forces the NP461505.1 that comes from phosphotransferase IIB component; NC～003197) has about 39% identity.

Breach BlastP sequence alignment show the 1-661 amino acids of SEQ ID NO:36 (665 amino acid) with from clostridium perfringens and be the protein (number of incorporating into own forces the .NP561500.1 of PTS systematic protein; NC～003366) have about 44% identity, the 1-657 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP269062.1 that comes from the PTS system BC component of fructose specific enzymes II; NC 002737) have about 46% identity, the 1-657 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP607065.1 that comes from fructose specific enzymes II PTS system BC component; NC_003485) have about 46% identity, the 1-657 amino acids with from Lactococcus lactis breast subspecies and be the protein (number of incorporating into own forces the NP267115.1 of the special PTS system enzyme IIBC component (EC 2.7.1.69) of fructose; NC_002662) have about 45% identity, and the 1-660 amino acids with from Bacillus halodurans and be the protein (number of incorporating into own forces the NP241694.1 of PTS system fructose specific enzymes IIB component; NC_002570) has about 43% identity.

Breach BlastP sequence alignment show SEQ ID NO:38 (334 amino acid) 4-334 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP-359213.1 of sucrose operon repressor (Scr operon modulin); NC_003098) have about 48% identity, the 4-334 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP-346232.1 that sugar combines transcriptional regulatory in the LacI family; NC 003028) have about 46% identity, the 13-332 amino acids with from Pediococcus pentosaceus and be that the protein (number of incorporating into own forces sp1P434721SCRR_PEDPE) of sucrose operon repressor (Scr operon modulin) has about 35% identity, the 10-334 amino acids with from Bacillus halodurans and be the protein (number of incorporating into own forces the NP244594.1 of ribose operon transcription repressor; NC_002570) have about 35% identity, and the 10-332 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP-346162.1 of sucrose operon repressor; NC003028) has about 35% identity.

Breach BlastP sequence alignment show SEQ ID NO:40 (415 amino acid) 3-415 amino acids with from streptococcus pneumoniae and the protein-bonded protein of the abc transport protein substrate (number of incorporating into own forces NP～359212.1; NC 003098) have about 50% identity, the 19-389 amino acids with from Agrobacterium tumefaciens and be the protein (number of incorporating into own forces the NP-535638.1 of carbohydrate-binding protein; NC～003306) have about 27% identity, the 11-396 amino acids with belong to PCC 7120 from the beads cyanobacteria and be the protein-bonded protein of the abc transport protein sugar (number of incorporating into own forces NP-488317.1; NC 003272) have about 25% identity, the 76-353 amino acids with from streptomyces coelicolor (Streptomycescoelicolor) and with the protein (number of incorporating into own forces the emblCAB95275.1 that comes from the HUCEP-8 carbohydrate-binding protein; AL359779) have about 26% identity, and the 1-324 amino acids with from harmless Listera and with the protein (number of incorporating into own forces the Nu-470104.1 that comes from sugared abc transport albumen pericentral siphon carbohydrate-binding protein; NC～003212) has about 26% identity.

Breach BlastP sequence alignment show SEQ ID NO:42 (294 amino acid) 10-285 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP 359211.1 that abc transport albumen is striden film permease HUCEP-8; NC～003098) have about 56% identity, the 7-285 amino acids with from Listeria monocytogenes and with coming from the proteic protein of the HUCEP-8 permease (number of incorporating into own forces NP464293.1; NC003210) have about 38% identity, the 7-285 amino acids with from harmless Listera and with coming from the proteic protein of the sugared abc transport albumen permease (number of incorporating into own forces NP-470102.1; NC003212) have about 38% identity, the 12-286 amino acids with belong to PCC6803 from collection born of the same parents cyanobacteria and be the protein (number of incorporating into own forces the NP-440703.1 of lactose movement system permease albumen (LacF); NC_000911) have about 36% identity, and the 11-281 amino acids with from xyllela fastidiosa and be the protein (number of incorporating into own forces the NP299726.1 of abc transport protein sugar permease; NC_002488) has about 36% identity.

Breach BlastP sequence alignment show SEQ ID NO:44 (285 amino acid) 12-285 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP_359210.1 that abc transport albumen is striden film permease HUCEP-8; NC_003098) has about 59% identity, 30-281 amino acids and the protein (number of incorporating into own forces NP 356672.1 from Agrobacterium tumefaciens; NC 003063) have about 32% identity, the 30-281 amino acids with from Agrobacterium tumefaciens and be the protein (number of incorporating into own forces the NP-534455.1 of abc transport albumen transmembrane protein [sugar]; NC_003305) have about 32% identity, the 10-281 amino acids with from Listeria monocytogenes and with coming from the proteic protein of the sugared abc transport albumen permease (number of incorporating into own forces NP463711.1; NC 003210) have about 33% identity, and the 13-281 amino acids with from harmless Listera and with coming from the proteic protein of the sugared abc transport protein sugar permease (number of incorporating into own forces NP469564.1; NC_003212) has about 34% identity.

Breach BlastP sequence alignment show SEQ ID NO:46 (430 amino acid) 2-429 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP-359209.1 of sucrose-6-phosphohydrolase; NC 003098) have about 36% identity, the 2-429 amino acids with from streptococcus pneumoniae and with the protein (number of incorporating into own forces the NP-346228.1 that comes from sucrose-6-phosphohydrolase; NC_003028) have about 36% identity, the 18-373 amino acids with from Thermotoga maritima and be the protein (number of incorporating into own forces the NP229215.1 of β fructosidase; NC_000853) has about 36% identity, the 21-405 amino acids with from zymomonas mobilis and be that the protein (number of incorporating into own forces pir11JU0460) of invertase (EC 3.2.1.26) has about 31% identity, and the 21-362 amino acids with from intestinal bacteria and be the protein (number of incorporating into own forces the NP-311270.1 of sucrose-6-phosphohydrolase; NC_002695) has about 35% identity.

Breach BlastP sequence alignment show SEQ ID NO:48 (368 amino acid) 1-366 amino acids with from Streptococcus mutans and be that the protein (number of incorporating into own forces sp1Q007521MSMK_STRMU) of multiple sugared binding transport ATP conjugated protein (msmK) has about 65% identity, the 1-366 amino acids with from streptococcus pyogenes and be the protein (number of incorporating into own forces the NP269942.1 that multiple sugar combines abc transport system (ATP is conjugated protein); NC_002737) have about 65% identity, the 1-367 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP-359030.1 of the conjugated protein multiple HUCEP-8 of abc transport albumin A TP; NC_003098) have about 66% identity, the 1-366 amino acids with from streptococcus pyogenes and be the protein (number of incorporating into own forces the NP608016.1 that combines the abc transport system (ATP is conjugated protein) of multiple sugar; NC003485) have about 65% identity, and the 1-367 amino acids with from streptococcus pneumoniae and be the protein-bonded protein of the sugared abc transport albumin A TP (number of incorporating into own forces NP-346026.1; NC003028) has about 66% identity.

Breach BlastP sequence alignment show SEQ ID NO:50 (490 amino acid) 11-489 amino acids with from Streptococcus mutans and be that the proteic protein of gtfA (number of incorporating into own forces pirllBWSOGM) has about 63% identity, the 11-490 amino acids with from Streptococcus mutans and be that the protein (number of incorporating into own forces pirllA27626) of sucrose phosphorylase (EC 2.4.1.7) has about 63% identity, the 11-489 amino acids with from Streptococcus mutans and be that the protein (number of incorporating into own forces sp1P102491SUCP_STRMU) of sucrose phosphorylase (sucrose glucosyltransferase) has about 63% identity, the 11-484 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP-359301.1 of Sucrose:glucan alpha1 (dextransucrase); NC 003098) have about 63% identity, and the 11-484 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP-346325.1 of sucrose phosphorylase; NC_003028) has about 63% identity.

Breach BlastP sequence alignment show SEQ ID NO:52 (328 amino acid) 47-316 amino acids with from subtilis and be the protein (number of incorporating into own forces the NP-391477.1 of ribose abc transport albumen (ribose is conjugated protein); NC_000964) have about 55% identity, the 5-323 amino acids with from Lactococcus lactis breast subspecies and be the protein-bonded protein of the ribose abc transport protein substrate (number of incorporating into own forces NP_267791.1; NC_002662) have about 45% identity, the 4-278 amino acids with from having a liking for the salt tetrads and being the protein-bonded protein of the ribose (number of incorporating into own forces dbj BAA31869-1; AB009593) have about 42% identity, the 15-316 amino acids with from Bacillushalodurans and be the protein (number of incorporating into own forces the NP244599.1 of ribose abc transport albumen (ribose is conjugated protein); NC_002570) have about 39% identity, and the 4-315 amino acids with from multocida (Pasteurella multocida) and be the proteic protein of the RbsB (number of incorporating into own forces NP245090.1; NC_002663) has about 42% identity.

Breach BlastP sequence alignment show SEQ ID NO:54 (285 amino acid) 1-277 amino acids with from subtilis and be the protein (number of incorporating into own forces the NP_391476.1 of ribose abc transport albumen (permease); NC_000964) has about 60% identity, the 1-277 amino acids with from subtilis and be that the protein (number of incorporating into own forces sp1P369481RBSC_BACSU) of ribose delivery system permease albumen (rbcS) has about 59% identity, the 4-277 amino acids with from Bacillus halodurans and be the protein (number of incorporating into own forces the NP244598.1 of ribose abc transport albumen (permease); NC002570) have about 57% identity, the 4-277 amino acids with from Lactococcus lactis breast subspecies and be the proteic protein of the ribose abc transport albumen permease (number of incorporating into own forces NP267792.1; NC002662) have about 58% identity, and the 4-278 amino acids with from Haemophilus influenzae and be the protein (number of incorporating into own forces NP～438661.1 of D-ribose abc transport albumen permease albumen (rbsC); NC～000907) has about 54% identity.

Breach BlastP sequence alignment show SEQ ID NO:56 (496 amino acid) 5-496 amino acids with from Lactococcus lactis breast subspecies and be the protein-bonded protein of the ribose abc transport albumin A TP (number of incorporating into own forces NP267793.1; NC～002662) has about 59% identity, the 5-496 amino acids with from subtilis and be that the protein-bonded protein of ATP (number of incorporating into own forces pirllI40465) has about 51% identity, the 5-495 amino acids with from Bacillushalodurans and be the protein (number of incorporating into own forces NP～244597.1 of ribose abc transport albumen (ATP is conjugated protein); NC002570) have about 49% identity, and the 7-494 amino acids with from Agrobacterium tumefaciens and be the protein (number of incorporating into own forces NP～533484.1 of abc transport albumen, bind nucleic acid/ATPase albumen [ribose]; NC003304) has about 45% identity.

Breach BlastP sequence alignment show SEQ ID NO:58 (134 amino acid) 4-134 amino acids with from lactobacillus sake i and be the protein (number of incorporating into own forces the gb1AAD34337.1 of ribose permease (RbsD); AF115391) have about 58% identity, the 4-134 amino acids with from clostridium perfringens (Clostridium perfringens) and with coming from the proteic protein of the ribose abc transport (number of incorporating into own forces Nos.NP562547.1; NC～003366) have about 51% identity, the 4-132 amino acids with from Lactococcus lactis breast subspecies and be the protein (number of incorporating into own forces the NP267794.1 of ribose abc transport albumen permease; NC～002662) have about 50% identity, the 4-134 amino acids with from Bacillus halodurans and be the protein (number of incorporating into own forces the NP244596.1 of ribose abc transport albumen (permease); NC～002570) have about 45% identity, and the 4-134 amino acids with from the golden yellow subspecies of golden yellow glucose coccus and be the protein (number of incorporating into own forces the NP-370793.1 of ribose permease; NC002758) has about 51% identity.

Breach BlastP sequence alignment show SEQ ID NO:60 (308 amino acid) 4-301 amino acids with from lactobacillus sake i and be the protein (number of incorporating into own forces the gb1AAD34338.1 of ribokinase (RbsK); AF115391) have about 51% identity, the 1-303 amino acids with from the golden yellow subspecies of golden yellow glucose coccus and with the protein (number of incorporating into own forces the NP-370792.1 that comes from ribokinase; NC～002758) have about 48% identity, the 3-305 amino acids with from Clostridium perfringens and be the protein (number of incorporating into own forces NP～562548.1 of ribokinase; NC003366) have about 45% identity, the 1-299 amino acids with from Haemophilus influenzae and be the protein (number of incorporating into own forces NP～438663.1 of ribokinase (RbsK); NC～000907) have about 41% identity, and the 2-300 amino acids with from Yersinia pestis and be the protein (number of incorporating into own forces the NP403674.1 of ribokinase; NC～003143) has about 38% identity.

Breach BlastP sequence alignment show SEQ ID NO:62 (285 amino acid) 1-285 amino acids with from Lactococcus lactis breast subspecies and be the proteic protein of the maltose abc transport albumen permease (number of incorporating into own forces NP267841.1; NC002662) have about 63% identity, the 6-284 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP269423.1 that comes from maltose/maltodextrin abc transport system albumen (permease); NC～002737) has about 54% identity, the 12-284 amino acids with have about 38% identity from acid-producing Klebsiella bacterium and with coming from the proteic protein of malG (number of incorporating into own forces pirllS63616), the 9-285 amino acids with from Bacillus halodurans and be the protein (number of incorporating into own forces the NP243790.1 of maltose/maltodextrin movement system (permease); NC～002570) have about 39% identity, and the 7-285 amino acids with from subtilis and with the protein (number of incorporating into own forces NP～391294.1 that come from maltodextrin movement system permease; NC～000964) has about 36% identity.

Breach BlastP sequence alignment show SEQ ID NO:64 (452 amino acid) 1-452 amino acids with from Lactococcus lactis breast subspecies and be the proteic protein of the maltose abc transport albumen permease (number of incorporating into own forces NP267840.1; NC002662) have about 63% identity, the 3-452 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP269422.1 that comes from maltose/maltodextrin abc transport system albumen (permease); NC～002737) have about 52% identity, the 3-452 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP607422.1 that comes from maltose/maltodextrin abc transport system (permease); NC003485) has about 52% identity, the 28-451 amino acids with have about 34% identity from acid-producing Klebsiella bacterium and with coming from the proteic protein of malF (number of incorporating into own forces pirllS63615), and the 23-451 amino acids with from Bacillus halodurans and be the protein (number of incorporating into own forces the NP243791.1 of maltodextrin movement system permease; NC002570) has about 33% identity.

Breach BlastP sequence alignment show SEQ ID NO:66 (408 amino acid) 1-407 amino acids with from Lactococcus lactis breast subspecies and be the protein-bonded protein of the maltose abc transport protein substrate (number of incorporating into own forces NP267839.1; NC 002662) have about 49% identity, the 1-405 amino acids with from streptococcus pyogenes and with coming from the protein-bonded protein of the maltose/maltodextrin (number of incorporating into own forces NP607421.1; NC 003485) have about 37% identity, the 1-405 amino acids with from streptococcus pyogenes and with coming from the protein-bonded protein of the maltose/maltodextrin (number of incorporating into own forces NP_269421.1; NC_002737) have about 36% identity, the 1-393 amino acids with from harmless Listera and with the protein (number of incorporating into own forces the NP_471563.1 that comes from maltose/maltodextrin abc transport albumen (conjugated protein); NC_003212) have about 27% identity, and the 1-403 amino acids with from subtilis and with coming from the protein-bonded protein of the maltose/maltodextrin (number of incorporating into own forces NP_391296.1; NC_000964) has about 26% identity.

Breach BlastP sequence alignment show SEQ ID NO:68 (368 amino acid) 1-366 amino acids with from Streptococcus mutans and be that the protein (number of incorporating into own forces sp1QO07521MSMK_STRMU) of multiple sugared binding transport albumin A TP conjugated protein (msmK) has about 64% identity, the 1-366 amino acids with from streptococcus pyogenes and be that multiple sugar combines the proteic protein (number of incorporating into own forces the NP269942.1 of abc transport system (ATP combination); NC_002737) have about 64% identity, the 1-366 amino acids with from streptococcus pyogenes and be that multiple sugar combines the proteic protein (number of incorporating into own forces the NP608016.1 of abc transport system (ATP in conjunction with); NC_003485) have about 64% identity, the 1-366 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP-359030.1 of the conjugated protein multiple HUCEP-8 of abc transport albumin A TP; NC_003098) have about 64% identity, and the 1-368 amino acids with from Lactococcus lactis breast subspecies and be the protein-bonded protein of the multiple sugared abc transport albumin A TP (number of incorporating into own forces NP266577.1; NC_002662) has about 62% identity.

Breach BlastP sequence alignment show SEQ ID NO:70 (512 amino acid) 1-510 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP269365.1 that comes from sugared abc transport albumen (ATP is conjugated protein); NC002737) have about 60% identity, the 1-510 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP607296.1 that comes from sugared abc transport albumen (ATP is conjugated protein); NC_003485) have about 60% identity, the 5-503 amino acids with from Lactococcus lactis breast subspecies and be the protein-bonded protein of the sugared abc transport albumin A TP (number of incorporating into own forces NP_267484.1; NC002662) have about 59% identity, the 7-503 amino acids with from streptococcus pneumoniae and be the protein-bonded protein of the sugared abc transport albumin A TP (number of incorporating into own forces NP_345337.1; NC003028) have about 61% identity, and the 7-503 amino acids with from streptococcus pneumoniae and be abc transport albumin A TP conjugated protein-protein (number of incorporating into own forces the NP_358342.1 of ribose/semi-lactosi translocator; NC_003098) has about 60% identity.

Breach BlastP sequence alignment show SEQ ID NO:72 (383 amino acid) 7-351 amino acids with from Lactococcus lactis breast subspecies and be the proteic protein of the sugared abc transport albumen permease (number of incorporating into own forces NP267485.1; NC～002662) has about 49% identity, 4-351 amino acids and the protein (number of incorporating into own forces NP～358343.1 of striding film permease (ribose/semi-lactosi translocator) from streptococcus pneumoniae and abc transport albumen; NC003098) have about 47% identity, the 4-351 amino acids with from streptococcus pneumoniae and with coming from the proteic protein of the sugared abc transport albumen permease (number of incorporating into own forces NP-345338.1; NC～003028) have about 47% identity, the 4-342 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP269364.1 that comes from sugared abc transport albumen (permease albumen); NC002737) have about 49% identity, and the 4-342 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP607295.1 that comes from sugared abc transport albumen (permease albumen); NC～003485) has about 49% identity.

Breach BlastP sequence alignment show SEQ ID NO:74 (318 amino acid) 1-318 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP607294.1 that comes from sugared abc transport albumen (permease albumen); NC～003485) have about 67% identity, the 1-318 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP269363.1 that comes from sugared abc transport albumen (permease albumen); NC～002737) have about 66% identity, the 1-318 amino acids with from streptococcus pneumoniae and with coming from the proteic protein of the sugared abc transport albumen permease (number of incorporating into own forces NP-345339.1; NC～003028) have about 65% identity, the 1-318 amino acids with from Lactococcus lactis breast subspecies and be the proteic protein of the sugared abc transport albumen permease (number of incorporating into own forces NP267486.1; NC002662) have about 63% identity, and the 6-318 amino acids with from harmless Listera and be the protein (number of incorporating into own forces the NP-470764.1 of sugared abc transport albumen (permease albumen); NC～003212) has about 61% identity.

Breach BlastP sequence alignment show SEQ ID NO:76 (450 amino acid) 11-448 amino acids with from Neisseria meningitidis (Neisseria meningitidis) and with the protein (number of incorporating into own forces NP～273437.1 that come from HUCEP-8; NC～003112) have about 68% identity, the 11-448 amino acids with from Neisseriameningitidis and with the protein (number of incorporating into own forces the NP284797.1 that comes from inherent protein called membrane transporters; NC～003116) have about 68% identity, the 17-229 amino acids with from crescent handle bacillus (Caulobacter crescentus) and with the protein (number of incorporating into own forces the NP_421086.1 that comes from translocator; NC_002696) about 39% identity is arranged.The 31-450 amino acids with from Lycopersicon esculentum and be the protein (number of incorporating into own forces the gb|AAG09270.1 of sucrose transporter; AF176950) have about 21% identity, and the 31-442 amino acids with from Arabidopis thaliana (Arabidopsis thaliana) and be the protein (number of incorporating into own forces the gblAAG09191.1 of sucrose transporter; AF175321) has about 21% identity.

Breach BlastP sequence alignment show SEQ ID NO:78 (495 amino acid) 8-482 amino acids with from Lactococcus lactis breast subspecies and be the protein (number of incorporating into own forces the NP266394.1 of translocator; NC002662) have about 32% identity, the 8-482 amino acids with from Listeria monocytogenes and with the protein (number of incorporating into own forces the NP464506.1 that comes from the effluent translocator; NC～003210) have about 34% identity, the 8-482 amino acids with from harmless Listera and be the protein (number of incorporating into own forces the NP470317.1 of effluent translocator; NC～003212) have about 34% identity, the 7-422 amino acids with from clostridium acetobutylicum and be the protein (number of incorporating into own forces the NP_149294.1 of the relevant permease of MDR; NC_001988) have about 30% identity, and the 8-425 amino acids with from streptomyces coelicolor and with the protein (number of incorporating into own forces the emb|CAB89031.1 that comes from protein called membrane transporters; AL353870) has about 29% identity.

Breach BlastP sequence alignment show SEQ ID NO:80 (471 amino acid) 1-440 amino acids with from Lactococcus lactis breast subspecies and be the protein (number of incorporating into own forces the NP266394.1 of translocator; NC 002662) have about 32% identity, the 1-464 amino acids with from Listeria monocytogenes and with the protein (number of incorporating into own forces the NP464506.1 that comes from the effluent translocator; NC～003210) have about 34% identity, the 1-464 amino acids with from harmless Listera and with the protein (number of incorporating into own forces the NP-470317.1 that comes from the effluent translocator; NC003212) have about 34% identity, the 1-412 amino acids with from clostridium acetobutylicum and be the protein (number of incorporating into own forces the NP_149294.1 of the relevant permease of MDR; NC001988) have about 29% identity, and the 4-459 amino acids with have about 28% identity from streptomyces coelicolor and with coming from the proteic protein of output (number of incorporating into own forces pirllT36377).

Breach BlastP sequence alignment show SEQ ID NO:82 (412 amino acid) 18-400 amino acids with from harmless Listera and with coming from the protein (number of incorporating into own forces the NP-472212.1 that drug flow goes out translocator; NC003212) have about 49% identity, the 18-400 amino acids with from Listeria monocytogenes and with coming from the protein (number of incorporating into own forces the NP466263.1 that drug flow goes out translocator; NC_003210) have about 49% identity, the 18-397 amino acids with from intestinal bacteria and with the protein (number of incorporating into own forces the NP_415571.1 that comes from translocator; NC_000913) have about 48% identity, the 15-399 amino acids with from Lactococcus lactis breast subspecies and be the protein (number of incorporating into own forces the Nu266282.1 of multi-medicine resistance effluent pump; NC_002662) have about 47% identity, and the 18-399 amino acids with from Salmonella typhimurium (Salmonella typhimurium) and with the protein (number of incorporating into own forces the NP460125.1 that comes from MFS family translocator; NC_003197) has about 48% identity.

Breach BlastP sequence alignment shows SEQ ID NO:84 (462 amino acid) 9-413 amino acids and the ORFC (number of incorporating into own forces emblCAB61253.1 from Oenococcus Oeni; AJ250422) have about 38% identity, the 2-378 amino acids with from Lactococcus lactis breast subspecies and be the protein (number of incorporating into own forces the NP267695.1 of translocator; NC_002662) have about 38% identity, the 6-411 amino acids with from streptococcus pyogenes and be the proteic protein of the drug resistance (number of incorporating into own forces NP606824.1; NC_003485) have about 34% identity, the 6-411 amino acids with from streptococcus pyogenes and be with coming from the proteic protein of the drug resistance (number of incorporating into own forces NP268834.1; NC002737) have about 33% identity, and the 2-454 amino acids with from Lactococcus lactis breast subspecies and be that medicine is exported the proteic protein (number of incorporating into own forces NP267504.1; NC002662) has about 34% identity.

Breach BlastP sequence alignment shows SEQ ID NO:86 (490 amino acid) 3-476 amino acids and exports the proteic protein (number of incorporating into own forces NP466111.1 from Listeria monocytogenes and with coming from medicine; NC_003210) have about 55% identity, the 3-476 amino acids with export the proteic protein (number of incorporating into own forces NP-472062.1 from harmless Listera and with coming from medicine; NC_003212) have about 54% identity, the 6-478 amino acids with from Lactococcus lactis breast subspecies and be the proteic protein of the multi-medicine resistance (number of incorporating into own forces NP267065.1; NC_002662) have about 45% identity, the 8-484 amino acids with from subtilis and be with coming from the proteic protein of the multi-medicine resistance (number of incorporating into own forces NP_388266.1; NC000964) have about 49% identity, and the 18-425 amino acids with from subtilis and with coming from the proteic protein of the multi-medicine resistance (number of incorporating into own forces NP_388782.1; NC_000964) has about 44% identity.

Breach BlastP sequence alignment shows SEQ ID NO:88 (416 amino acid) 17-408 amino acids and the protein (number of incorporating into own forces gb1AAL87781.1 from Desulftobacterium hafniense; AF403184) have about 26% identity, the 26-408 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the Nu-359046.1 that mainly promotes the translocator in the superfamily protein; NC～003098) have about 25% identity, the 61-399 amino acids with from campylobacter jejuni and with coming from the proteic protein of the effluent (number of incorporating into own forces NP282813.1; NC002163) have about 21% identity, the 25-368 amino acids with from Agrobacterium tumefaciens and be with the protein (number of incorporating into own forces the NP-533033.1 that comes from the MFS permease; NC～003304) have about 19% identity, and the 19-205 amino acids with from Bacillus halodurans and be the proteic protein of the multi-medicine resistance (number of incorporating into own forces NP244175.1; NC002570) has about 25% identity.

Breach BlastP sequence alignment show SEQ ID NO:90 (548 amino acid) 17-546 amino acids with from harmless Listera and with the protein (number of incorporating into own forces NP～471001.1 that come from translocator; NC～003212) have about 38% identity, the 17-546 amino acids with from Listeria monocytogenes and with the protein (number of incorporating into own forces the NP465149.1 that comes from translocator; NC～003210) have about 37% identity, the 1-534 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP-358976.1 of polysaccharide translocator; NC～003098) have about 36% identity, the 17-534 amino acids with from streptococcus pneumoniae and be with coming from the proteic protein of the polysaccharide biosynthesizing (number of incorporating into own forces NP-345978.1; NC～003028) have about 36% identity, and 12-546 amino acids and the putative protein matter (number of incorporating into own forces NP267962.1 from Lactococcus lactis breast subspecies; NC002662) has about 35% identity.

Breach BlastP sequence alignment show SEQ ID NO:92 (485 amino acid) 1-484 amino acids with from Listeria monocytogenes and with the protein (number of incorporating into own forces the NP464506.1 that comes from the effluent translocator; NC～003210) have about 44% identity, the 1-484 amino acids with from harmless Listera and with the protein (number of incorporating into own forces the NP-470317.1 that comes from the effluent translocator; NC～003212) have about 44% identity, the 9-420 amino acids with from clostridium acetobutylicum and be the protein (number of incorporating into own forces the NP_149294.1 of the relevant permease of MDR; NC_001988) have about 34% identity, the 12-475 amino acids with from Lactococcus lactis breast subspecies and be the protein (number of incorporating into own forces the NP_266394.1 of translocator; NC_002662) have about 33% identity, and 1-457 amino acids and the putative protein matter (number of incorporating into own forces emblCAB37973.1 from yellow myxococcus; X76640) has about 34% identity.

Breach BlastP sequence alignment shows SEQ ID NO:94 (199 amino acid) 23-173 amino acids and goes out the proteic protein of the carrier (number of incorporating into own forces NP-472212.1 from harmless Listera and with coming from drug flow; NC_003212) have about 46% identity, the 23-173 amino acids with go out the proteic protein of the carrier (number of incorporating into own forces NP466263.1 from Listeria monocytogenes and with coming from drug flow; NC003210) have about 45% identity, the 23-173 amino acids with from Lactococcus lactis breast subspecies and be the protein (number of incorporating into own forces the NP266282.1 of multi-medicine resistance efflux pump; NC002662) have about 49% identity, the 23-173 amino acids with from Salmonella entericasubsp.enterica serovar Typhi and be with the protein (number of incorporating into own forces the NP-454977.1 that comes from the effluent pump; NC_003198) have about 46% identity, and the 23-173 amino acids with from Salmonella typhimurium and with the protein (number of incorporating into own forces the NP_459377.1 that comes from permease; NC003197) has about 46% identity.

Breach BlastP sequence alignment show SEQ ID NO:96 (538 amino acid) 4-525 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP-358976.1 of polysaccharide translocator; NC_003098) have about 32% identity, the 5-525 amino acids with from streptococcus pneumoniae and with coming from the proteic protein of the polysaccharide biosynthesizing (number of incorporating into own forces NP-345978.1; NC_003028) has about 32% identity, 5-526 amino acids and the conservative putative protein matter (number of incorporating into own forces NP-606680.1 from streptococcus pyogenes; NC_003485) has about 33% identity, 5-526 amino acids and the conservative putative protein matter (number of incorporating into own forces NP268708.1 from streptococcus pyogenes; NC002737) have about 33% identity, and 4-526 amino acids and the putative protein matter (number of incorporating into own forces NP267962.1 from Lactococcus lactis breast subspecies; NC_002662) has about 30% identity.

Breach BlastP sequence alignment show SEQ ID NO:98 (328 amino acid) 1-323 amino acids with from Pediococcus pentosaceus and be sucrose operon modulin (scrR) protein (number of incorporating into own forces sp1P434721SCRR～PEDPE) has about 57% identity, the 1-322 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP-346162.1 of sucrose operon repressor; NC003028) has about 51% identity, the 1-326 amino acids with from Streptococcus mutans and be sucrose operon modulin (scrR) protein (number of incorporating into own forces sp1Q544301SCRR～STRMU) has about 49% identity, the 1-322 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP607889.1 that comes from sucrose operon repressor; NC～003485) have about 49% identity, and the 1-322 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP269821.1 that comes from sucrose operon repressor; NC～002737) has about 49% identity.

Breach BlastP sequence alignment show SEQ ID NO:100 (485 amino acid) 1-466 amino acids with from Streptococcus sobrinus (Streptococcus sobrinus) and be that the protein (number of incorporating into own forces pirllS68598) of sucrose-6-phosphohydrolase (ScrB) has about 50% identity, the 1-461 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces NP～359160.1 of sucrose-6-phosphohydrolase; NC003098) have about 49% identity, the 1-461 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces NP～346161.1 of sucrose-6-phosphohydrolase; NC003028) have about 49% identity, the 1-466 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP607888.1 that comes from sucrose-6-phosphohydrolase; NC003485) have about 49% identity, and the 1-466 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP269820.1 that comes from sucrose-6-phosphohydrolase; NC～002737) has about 49% identity.

Breach BlastP sequence alignment show SEQ ID NO:102 (649 amino acid) 1-645 amino acids with from Streptococcus mutans and be that (number of incorporating into own forces sp1P126551PTSA～STRMU) has about 65% identity for the protein of the special IIABC component of sucrose of phosphotransferase system II type enzyme (EC2.7.1.69), the 1-647 amino acids with from Pediococcus pentosaceus and be the sucrose specific enzymes IIABC of phosphotransferase system II type enzyme (EC 2.7.1.69) protein (number of incorporating into own forces sp1P434701PTSA～PEDPE) has about 56% identity, the 1-643 amino acids with from Lactococcus lactis and be the proteic protein of the II type enzyme sucrose (number of incorporating into own forces emblCAB09690.1; Z97015) have about 52% identity, the 114-647 amino acids with from lactobacillus sake and be the protein (number of incorporating into own forces the gb1AAK92528.1 of the sucrose specificity II type enzyme of PTS; AF401046) have about 52% identity, and the 1-621 amino acids with from Corynebacterium glutamicum (Corynebacterium glutamicum) and be the protein (number of incorporating into own forces the NP601842.1 of phosphotransferase system IIB component; NC003450) has about 45% identity.

Breach BlastP sequence alignment show SEQ ID NO:104 (667 amino acid) 192-661 amino acids with from Lactococcus lactis breast subspecies and be the protein (number of incorporating into own forces the NP266583.1 of β-glucoside specificity PTS system IIABC component (EC 2.7.1.69); NC～002662) have about 42% identity, the 191-652 amino acids with from Listeria monocytogenes and with the protein (number of incorporating into own forces the NP464560.1 that comes from phosphotransferase system (PTS) β glucoside specific enzymes IIABC; NC003210) have about 39% identity, the 191-662 amino acids with from Clostridium longisporum and be the protein (number of incorporating into own forces the gb1AAC05713.1 of PTS dependent form enzyme II; L49336) have about 37% identity, the 191-666 amino acids with from Bacillushalodurans and be the protein (number of incorporating into own forces the NP241461.1 of PTS system β glucoside specificity II type enzyme ABC component; NC～002570) have about 36% identity, and the 191-650 amino acids with from harmless Listera and with the protein (number of incorporating into own forces the NP469373.1 that comes from PTS system β glucoside specific enzymes IIABC; NC003212) has about 36% identity.

Breach BlastP sequence alignment show SEQ ID NO:106 (241 amino acid) 1-238 amino acids with from subtilis and be trehalose operon transcription repressor protein (number of incorporating into own forces sp1P397961TRER～BACSU) has about 47% identity, the 4-238 amino acids with from Bacillus halodurans and be the protein (number of incorporating into own forces the NP241739.1 of the transcription repressor of trehalose operon; NC002570) have about 41% identity, the 9-237 amino acids with from harmless Listera and with the protein (number of incorporating into own forces NP～470558.1 that come from the sub-GntR of transcriptional control family; NC003212) have about 44% identity, the 9-237 amino acids with from Listeria monocytogenes and with the protein (number of incorporating into own forces the NP464778.1 that comes from the sub-GntR of transcriptional control family; NC003210) have about 44% identity, and the 5-238 amino acids with from Lactococcus lactis breast subspecies and be the protein (number of incorporating into own forces the NP266581.1 of GntR family transcriptional control; NC002662) has about 41% identity.

Breach BlastP sequence alignment show SEQ ID NO:108 (570 amino acid) 22-566 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP608103.1 that comes from the dextran Polyglucosidase; NC 003485) have about 56% identity, the 23-568 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces NP～359290.1 of dextran Polyglucosidase; NC003098) have about 57% identity, the 22-566 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP270026.1 that comes from the dextran Polyglucosidase; NC～002737) have about 56% identity, the 23-568 amino acids with from streptococcus pneumoniae and with the protein (number of incorporating into own forces the NP-346315.1 that comes from dextran Polyglucosidase DexS; NC003028) have about 57% identity, and the 17-570 amino acids with from clostridium perfringens and be the protein (number of incorporating into own forces the NP561478.1 of α Polyglucosidase; NC003366) has about 54% identity.

Breach BlastP sequence alignment show SEQ ID NO:110 (370 amino acid) 1-368 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP-359030.1 of the conjugated protein multiple HUCEP-8 of abc transport albumin A TP; NC～003098) have about 67% identity, the 1-368 amino acids with from streptococcus pneumoniae and be the protein-bonded protein of the sugared abc transport albumin A TP (number of incorporating into own forces NP-346026.1; NC～003028) has about 67% identity, the 1-368 amino acids with from Streptococcus mutans and be multiple sugared binding transport albumin A TP conjugated protein (msmK) protein (number of incorporating into own forces sp1Q007521MSMK～STRMU) has about 66% identity, the 1-365 amino acids with from harmless Listera and with coming from the protein-bonded protein of the sugared abc transport albumin A TP (number of incorporating into own forces NP469649.1; NC～003212) have about 68% identity, and the 1-365 amino acids with from Listeria monocytogenes and with coming from the protein-bonded protein of the sugared abc transport albumin A TP (number of incorporating into own forces NP463809.1; NC～003210) has about 67% identity.

Breach BlastP sequence alignment show SEQ ID NO:112 (278 amino acid) 2-278 amino acids with from Streptococcus mutans and be that (number of incorporating into own forces sp1Q007511MSMG～STRMU) has about 81% identity for the protein of multiple sugared binding transport system permease albumen (msmG), the 1-278 amino acids with from streptococcus pneumoniae and be the proteic protein of the sugared abc transport albumen permease (number of incorporating into own forces NP～346326.1, NC～003028) have about 73% identity, the 2-278 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP-359302.1 that abc transport albumen is striden the multiple sugar of film permease; NC003098) has about 72% identity, the 72-278 amino acids with have about 85% identity from the putative protein matter fragment (number of incorporating into own forces pirllB27626) of Streptococcus mutans, and the 4-278 amino acids with from clostridium acetobutylicum and be the protein (number of incorporating into own forces NP～350251.1 of sugared permease; NC～003030) has about 44% identity.

Breach BlastP row comparison show SEQ ID NO:114 (291 amino acid) 4-290 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the Nu-359303.1 that abc transport albumen is striden film permease-multiple sugar; NC003098) have about 73% identity, the 4-290 amino acids with from streptococcus pneumoniae and be the proteic protein of the sugared abc transport albumen permease (number of incorporating into own forces NP-346327.1; NC～003028) has about 73% identity, the 1-290 amino acids with from Streptococcus mutans and be multiple sugared binding transport system permease albumen (msmF) protein (number of incorporating into own forces sp1Q007501MSMF～STRMU) has about 73% identity, the 6-291 amino acids with from clostridium acetobutylicum and be the protein (number of incorporating into own forces the NP-350252.1 of ABC type sugar transport system permease component; NC～003030) have about 53% identity, and the 2-291 amino acids with come self-heating to produce the hot anaerobic bacillus(cillus anaerobicus) of sulphur (Thermoanaerobacterium thermosulfurigenes) and be that (number of incorporating into own forces sp1P377301AMYD～THETU) has about 32% identity to the proteic protein of potential starch decomposition products delivery system permease.

Breach BlastP sequence alignment show SEQ ID NO:116 (423 amino acid) 8-421 amino acids with from Streptococcus mutans and be multiple carbohydrate-binding protein precursor protein (number of incorporating into own forces sp1Q007491MSME～STRMU) has about 60% identity, the 9-421 amino acids with from streptococcus pneumoniae and be the protein-bonded protein of the sugared abc transport protein sugar (number of incorporating into own forces NP-346328.1; NC～003028) have about 56% identity, the 9-421 amino acids with from streptococcus pneumoniae and be the abc transport protein substrate conjugated protein-protein (number of incorporating into own forces NP～359304.1 of multiple sugar; NC003098) have about 56% identity, the 9-420 amino acids with from clostridium acetobutylicum and be the protein (number of incorporating into own forces NP～350253.1 that ABC type sugar transport system pericentral siphon sugar combines component; NC～003030) have about 29% identity, and the 6-412 amino acids with from subtilis and with the protein (number of incorporating into own forces the NP-391140.1 that comes from multiple carbohydrate-binding protein; NC～000964) has about 24% identity.

Breach BlastP sequence alignment show SEQ ID NO:118 (279 amino acid) 1-273 amino acids with from Pediococcus pentosaceus and be that (number of incorporating into own forces sp1P434651RAFR～PEDPE) has about 57% identity for the protein of raffinose operon transcriptional regulation protein (rafR), the 5-273 amino acids with have about 35% identity from Streptococcus mutans and with the protein (number of incorporating into own forces pirllA42400) that comes from transcriptional control (msmR), the 5-273 amino acids with from Streptococcus mutans and be msm operon modulin protein (number of incorporating into own forces sp1Q007531MSMR～STRMU) has about 35% identity, the 19-273 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces NP～346330.1 of msm operon modulin; NC003028) have about 36% identity, and the 19-273 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP-359306.1 of msm (multiples ugarmetabolism, multiple carbohydrate metabolism) operon modulin; NC～003098) has about 36% identity.

Breach BlastP sequence alignment show SEQ ID NO:120 (277 amino acid) 37-141 amino acids with from Treponoma palladium (Treponema pallidum) and with the protein (number of incorporating into own forces the NP218549.1 that comes from the rRNA methylase; NC～000919) have about 28% identity, the 74-141 amino acids with from Guillardi theta and be the protein (number of incorporating into own forces NP～113408.1 of GTP syncaryon albumen RAN; NC～002753) has about 32% identity, the 75-141 amino acids with from Dictyosteliumdiscoideum and be GTP syncaryon albumen RAN/TC4 protein (number of incorporating into own forces sp1P335191RAN～DICDI) has about 29% identity, and the 140-190 amino acids with infer the protein (number of incorporating into own forces NP～191798.1 from Arabidopis thaliana (Arabidopsis thaliana); NM～116104) has about 25% identity.

Breach BlastP sequence alignment show SEQ ID NO:122 (530 amino acid) 8-524 amino acids with from the newborn subspecies of Lactococcus lactis and be abc transport albumen and the proteic protein of the permease (number of incorporating into own forces the NP267678.1 that combines ATP; NC002662) have about 26% identity, the 49-518 amino acids with from streptococcus pneumoniae and be the protein-bonded protein of the abc transport albumin A TP (number of incorporating into own forces NP-344680.1; NC～003028) have about 25% identity, the 49-518 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP357731.1 that abc transport albumin A TP combined/strode the film permease; NC003098) have about 25% identity, the 47-511 amino acids with belong to PCC6803 from collection born of the same parents cyanobacteria and be the proteic protein of the abc transport (number of incorporating into own forces Nu-440626.1; NC000911) have about 24% identity, and the 7-511 amino acids with from subtilis and with the protein (number of incorporating into own forces NP～388852.1 that come from abc transport albumen (ATP is conjugated protein); NC000964) has about 24% identity.

Breach BlastP sequence alignment show SEQ ID NO:124 (530 amino acid) 4-524 amino acids with from the newborn subspecies of Lactococcus lactis and be abc transport albumen and the proteic protein of the permease (number of incorporating into own forces the NP267678.1 that combines ATP; NC～002662) have about 24% identity, the 55-508 amino acids with from streptococcus pneumoniae and be the protein-bonded protein of the abc transport albumin A TP (number of incorporating into own forces NP-344680.1; NC～003028) have about 25% identity, the 55-508 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces NP～357731.1 that abc transport albumin A TP combined/strode the film permease; NC003098) have about 25% identity, the 1-511 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP-345800.1 that the medicine that combines ATP flows out ABC type translocator/permease; NC～003028) have about 24% identity, and the 1-511 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces NP～358796.1 that combine abc transport albumen/transmembrane protein of ATP; NC003098) has about 24% identity.

Breach BlastP sequence alignment show SEQ ID NO:126 (527 amino acid) 8-527 amino acids with from the newborn subspecies of Lactococcus lactis and be abc transport albumen and the proteic protein of the permease (number of incorporating into own forces the NP267678.1 that combines ATP; NC～002662) have about 25% identity, the 13-520 amino acids with from streptococcus pneumoniae and be the abc transport albumen that combines ATP/the stride proteic protein of the film permease (number of incorporating into own forces NP～357731.1; NC～003098) have about 24% identity, the 13-520 amino acids with from streptococcus pneumoniae and be the protein-bonded protein of the abc transport albumin A TP (number of incorporating into own forces NP-344680.1; NC～003028) have about 24% identity, the 22-511 amino acids with from streptococcus pneumoniae and be that the medicine that combines ATP flows out the proteic protein of the ABC type translocator/permease (number of incorporating into own forces NP-345800.1; NC～003028) have about 22% identity, and the 22-511 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces NP～358796.1 that combine abc transport albumen/transmembrane protein of ATP; NC003098) has about 22% identity.

Breach BlastP sequence alignment show SEQ ID NO:128 (534 amino acid) 14-512 amino acids with from streptococcus pneumoniae and be that the proteic protein of comA (number of incorporating into own forces pirllA39203) has about 23% identity, the 3-512 amino acids with from Lactococcus lactis and be Lactococcin A translocator ATP conjugated protein (lcnC) protein (number of incorporating into own forces sp1Q005641LCNC～LACLA) has about 26% identity, the 14-512 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP-357637.1 of the translocator (ComA) that combines ATP; NC～003098) have about 23% identity, the 113-509 amino acids with from streptococcus-salivarius (Streptococcus salivarius) and be the protein (number of incorporating into own forces the gb1AAC72026.1 of ABC type translocator; AF043280) have about 25% identity, and the 14-512 amino acids with from streptococcus pneumoniae and be that transhipment ATP combines/protein (number of incorporating into own forces the NP-344591.1 of the proteic competence factor of permease (ComA); NC～003028) has about 22% identity.

Breach BlastP sequence alignment show SEQ ID NO:130 (527 amino acid) 16-524 amino acids with from the newborn subspecies of Lactococcus lactis and be abc transport albumen and the proteic protein of the permease (number of incorporating into own forces the NP267678.1 that combines ATP; NC～002662) has about 23% identity, the 6-520 amino acids with from streptococcus pneumoniae and be the protein-bonded protein of the abc transport albumin A TP (number of incorporating into own forces NP～344680.1, NC～003028) have about 25% identity, the 6-520 amino acids with from streptococcus pneumoniae and be the abc transport albumen that combines ATP/the stride protein (number of incorporating into own forces NP～357731.1 of film permease; NC003098) have about 25% identity, the 105-511 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces NP～358796.1 that combine ABC type translocator/transmembrane protein of ATP; NC003098) have about 24% identity, and the 99-511 amino acids with belong to PCC7120 from the beads cyanobacteria and be the protein-bonded protein of the abc transport albumin A TP (number of incorporating into own forces NP-490403.1; NC～003276) has about 25% identity.

Breach BlastP sequence alignment show SEQ ID NO:132 (529 amino acid) 10-526 amino acids with from the newborn subspecies of Lactococcus lactis and be abc transport albumen and the proteic protein of the permease (number of incorporating into own forces the NP267678.1 that combines ATP; NC～002662) have about 25% identity, the 112-525 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP_357731.1 that abc transport albumin A TP combined/strode the film permease; NC_003098) have about 26% identity, the 112-525 amino acids with from streptococcus pneumoniae and be the proteic protein of the protein-abc transport (number of incorporating into own forces the NP-344680.1 that combines ATP; NC～003028) has about 26% identity, the 107-518 amino acids with have about 24% identity from Brevibacillus brevis and with the protein (number of incorporating into own forces pirllT31077) that comes from abc transport albumen (TycD), and the 83-521 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP-345800.1 that combines medicine outflow ABC type translocator/permease of ATP; NC～003028) has about 24% identity.

Breach BlastP sequence alignment show SEQ ID NO:134 (600 amino acid) 2-600 amino acids with from harmless Listera and with the protein (number of incorporating into own forces the NP-471553.1 that comes from abc transport albumen (permease); NC～003212) have about 23% identity, the 1-598 amino acids with from monocyte hyperplasia Li Site Salmonella and be with the protein (number of incorporating into own forces the NP465271.1 that comes from abc transport albumen (permease); NC～003210) have about 23% identity, the 1-599 amino acids with from clostridium perfringens and with coming from the proteic protein of the abc transport (number of incorporating into own forces NP_561767.1; NC_003366) have about 22% identity, the 1-564 amino acids with from clostridium perfringens and with coming from the proteic protein of the abc transport (number of incorporating into own forces NP_561039.1; NC_003366) have about 22% identity, and the 4-593 amino acids with from clostridium acetobutylicum and with the protein (number of incorporating into own forces the NP_346868.1 that comes from permease; NC_003030) has about 22% identity.

Breach BlastP sequence alignment show SEQ ID NO:136 (249 amino acid) 1-242 amino acids with from clostridium perfringens and with coming from the proteic protein of the abc transport (number of incorporating into own forces NP_561766.1; NC003366) have about 58% identity, the 3-242 amino acids with from clostridium perfringens and with coming from the proteic protein of the abc transport (number of incorporating into own forces NP561038.1; NC_003366) have about 55% identity, the 1-242 amino acids with from Listeria monocytogenes and with the protein (number of incorporating into own forces the NP465638.1 that comes from abc transport albumen (ATP is conjugated protein); NC_003210) have about 51% identity, the 1-242 amino acids with from harmless Listera and with the protein (number of incorporating into own forces the NP-471552.1 that comes from abc transport albumen (ATP is conjugated protein); NC_003212) have about 50% identity, and the 3-242 amino acids with from clostridium acetobutylicum and be the protein-bonded protein of the ABC type translocator-ATP (number of incorporating into own forces NP-346867.1; NC003030) has about 54% identity.

Breach BlastP sequence alignment shows SEQ ID NO:138 (423 amino acid) 2-391 amino acids and the putative protein matter (number of incorporating into own forces NP270004.1 from streptococcus pyogenes; NC002737) has about 21% identity, 2-383 amino acids and the putative protein matter (number of incorporating into own forces NP608080.1 from streptococcus pyogenes; NC_003485) have about 21% identity, the 9-166 amino acids with from subtilis and be the proteic protein of the yvbJ (number of incorporating into own forces NP_391268.1; NC000964) has about 26% identity, the 92-281 amino acids with from caprine arthritis-encephalitis virus and be that the protein (number of incorporating into own forces pirllVCLJC6) of env polyprotein precursor has about 25% identity, and the 92-281 amino acids with from caprine arthritis-encephalitis virus and be the protein (number of incorporating into own forces the gb AAD 14661.1 of envelope glycoprotein; AF105181) has about 24% identity.

Breach BlastP sequence alignment show SEQ ID NO:140 (438 amino acid) 86-216 amino acids with from bird rape rope silk bacterium and be the attached proteic protein (number of incorporating into own forces the gb1AAC95141.1 of transhipment; AF075600) have about 27% identity, the 107-219 amino acids with from streptococcus pneumoniae and be that bacterocin transports the attached proteic protein (number of incorporating into own forces NP-345950.1; NC003028) have about 26% identity, the 107-219 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the gb1AAD56628.1 of Bta; AF165218) has about 26% identity, 88-201 amino acids and the putative protein matter (number of incorporating into own forces NP052783.1 from Bacillus anthracis (Bacillus anthracis); NC_001496) have about 23% identity, and the 144-214 amino acids with from Neisseria meningitidis (Neisseria meningitidis) and be the protein (number of incorporating into own forces the NP274384.1 of Trx; NC_003112) has about 32% identity.

Breach BlastP sequence alignment show SEQ ID NO:142 (196 amino acid) 1-196 amino acids with from the protein of Lactobacillus gasseri (Lactoacillus gasseri) (number of incorporating into own forces dbj! BAA82351.1; AB029612) has about 56% identity, the 10-196 amino acids with have about 49% identity from the putative protein matter (number of incorporating into own forces sp1P294701YLA1_LACAC) of Bacterium lacticum, the 41-196 amino acids with from lactobacterium casei and be the protein (number of incorporating into own forces the NP_542220.1 of the attached factor of abc transport; NC003320) have about 28% identity, the 90-196 amino acids with from plant lactobacillus and be the protein (number of incorporating into own forces the emblCAA64190.1 of the attached factor of abc transport albumen (PlnH); X94434) have about 35% identity, and the 41-196 amino acids with have about 30% identity from lactobacillus sake (Lactobacillus sake) and with the protein (number of incorporating into own forces pirllA56973) that comes from the ABC output attached factor of albumen (SapE).

Breach BlastP sequence alignment show SEQ ID NO:144 (720 amino acid) 9-720 amino acids with from plant lactobacillus and be the protein (number of incorporating into own forces the emblCAA64189.1 of abc transport albumen (PlnG); X94434) has about 62% identity, the 6-720 amino acids with have about 62% identity from lactobacillus sake i and with the protein (number of incorporating into own forces pirllS57913) that comes from ATP dependent form transposition albumen (sppT), the 2-720 amino acids with from lactobacillus sake i and be that the protein (number of incorporating into own forces pirllI56273) of ATP dependent form translocator (SapT) has about 62% identity, the 9-720 amino acids with from lactobacterium casei and be the proteic protein of the abc transport (number of incorporating into own forces NP_542219.1; NC003320) have about 62% identity, and the 25-718 amino acids with from Lactobacterium acidophilum and be the proteic protein of the abc transport (number of incorporating into own forces NP604412.1; NC003458) has about 57% identity.

Breach BlastP sequence alignment show SEQ ID NO:146 (234 amino acid) 13-228 amino acids with from the golden yellow subspecies of golden yellow glucose coccus and with coming from the protein-bonded protein of the abc transport albumin A TP (number of incorporating into own forces NP-370833.1; NC～002758) have about 52% identity, the 11-234 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces NP～606994.1 that come from abc transport albumen (ATP is conjugated protein); NC003485) have about 50% identity, the 11-234 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP268993.1 that comes from abc transport albumen (ATP is conjugated protein); NC～002737) have about 50% identity, the 13-232 amino acids with from Lactococcus lactis breast subspecies and be the protein-bonded protein of the abc transport albumin A TP (number of incorporating into own forces NP266815.1; NC～002662) have about 50% identity, and the 11-233 amino acids with from Lactococcus lactis breast subspecies and be the protein-bonded protein of the abc transport albumin A TP (number of incorporating into own forces NP268413.1; NC002662) has about 53% identity.

Breach BlastP sequence alignment shows SEQ ID NO:148 (353 amino acid) 1-352 amino acids and the putative protein matter (number of incorporating into own forces NP268412.1 from Lactococcus lactis breast subspecies; NC002662) has about 40% identity, 1-352 amino acids and the conservative putative protein matter (number of incorporating into own forces NP-370832.1 from the golden yellow subspecies of golden yellow glucose coccus; NC～002758) has about 38% identity, 1-352 amino acids and the conservative putative protein matter (number of incorporating into own forces NP268992.1 from streptococcus pyogenes; NC～002737) has about 33% identity, 1-352 amino acids and the conservative putative protein matter (number of incorporating into own forces NP606993.1 from streptococcus pyogenes; NC～003485) have about 33% identity, and the 1-352 amino acids with from Lactococcus lactis breast subspecies and be the proteic protein of the abc transport albumen permease (number of incorporating into own forces NP-266816.1; NC～002662) has about 34% identity.

Breach BlastP sequence alignment show SEQ ID NO:150 (188 amino acid) 14-85 amino acids with from Lactococcus lactis breast subspecies and be the protein (number of incorporating into own forces the NP266817.1 of transcriptional regulatory; NC002662) have about 47% identity, the 21-90 amino acids with from Aquifex aeolicus and be the protein (number of incorporating into own forces the NP213195.1 of transcriptional regulatory in the TetR/AcrR family; NC～000918) have about 28% identity, the 14-75 amino acids with from clostridium acetobutylicum and be the protein (number of incorporating into own forces the NP-348163.1 of transcriptional regulatory in the AcrR family; NC～003030) have about 30% identity, the 25-109 amino acids with from streptomyces coelicolor and with the protein (number of incorporating into own forces the emblCAB93030.1 that comes from transcriptional regulatory; AL357432) have about 29% identity, and the 27-88 amino acids with from clostridium acetobutylicum and be the protein (number of incorporating into own forces NP～347457.1 of transcriptional regulatory in the TetR/AcrR family; NC～003030) has about 41% identity.

Breach BlastP sequence alignment show SEQ ID NO:152 (236 amino acid) 3-236 amino acids with from streptococcus pneumoniae and be the protein-bonded protein of the abc transport albumin A TP (number of incorporating into own forces Nu-359090.1; NC～003098) have about 65% identity, the 4-236 amino acids with from streptococcus pneumoniae and be the protein-bonded protein of the abc transport albumin A TP (number of incorporating into own forces NP-346092.1; NC～003028) have about 66% identity, the 4-236 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP607321.1 that comes from abc transport albumen (ATP is conjugated protein); NC003485) have about 65% identity, the 4-236 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP269390.1 that comes from abc transport albumen (ATP is conjugated protein); NC～002737) have about 65% identity, and the 4-236 amino acids with from Listeria monocytogenes and with coming from the protein-bonded protein of the abc transport albumin A TP (number of incorporating into own forces NP464748.1; NC003210) has about 62% identity.

Breach BlastP sequence alignment show the 6-846 amino acids of SEQ ID NO:154 (846 amino acid) with from Lactococcus lactis breast subspecies and be the proteic protein of the abc transport albumen permease (number of incorporating into own forces NP267260.1; NC～002662) has about 41% identity, 2-846 amino acids and the putative protein matter (number of incorporating into own forces NP-359089.1 from streptococcus pneumoniae; NC003098) has about 34% identity, 2-846 amino acids and the putative protein matter (number of incorporating into own forces NP346091.1 from streptococcus pneumoniae; NC～003028) has about 34% identity, 4-846 amino acids and the putative protein matter (number of incorporating into own forces NP269389.1 from streptococcus pyogenes; NC002737) have about 33% identity, and 4-846 amino acids and the putative protein matter (number of incorporating into own forces NP607320.1 from streptococcus pyogenes; NC～003485) has about 33% identity.

Breach BlastP sequence alignment shows SEQ ID NO:156 (78 amino acid) 12-70 amino acids and the protein (number of incorporating into own forces gb1AAF 19707.1 from Arabidopis thaliana; AC008047) have about 30% identity, the 12-70 amino acids with from Arabidopis thaliana and with coming from the white protein (number of incorporating into own forces the NP-176533.1 of ATP dependent form copper transport protein; NM-105023) has about 30% identity, 1-65 amino acids and the putative protein matter (number of incorporating into own forces NP-579673.1 from fierce hot-bulb bacterium; NC～003413) have about 32% identity, and 21-55 amino acids and the protein (number of incorporating into own forces gb1AAK 11712.1 from hepatitis TT virus; AF345529) has about 37% identity.

Breach BlastP (version) sequence alignment shows SEQ ID NO:158 (379 amino acid) 32-368 amino acids and the conservative putative protein matter (number of incorporating into own forces NP-470340.1 from harmless Listera; NC～003212) has about 36% identity, 32-353 amino acids and the conservative putative protein matter (number of incorporating into own forces NP464529.1 from monocyte hyperplasia Li Site bacterium; NC～003210) has about 37% identity, 87-370 amino acids and the protein (number of incorporating into own forces emblCAA68042.1 from Lactococcus lactis; X99710) has about 36% identity, 28-372 amino acids and the putative protein matter (number of incorporating into own forces NP267885.1 from Lactococcus lactis breast subspecies; NC～002662) have about 31% identity, and 32-348 amino acids and the protein (number of incorporating into own forces gb1AAC14002.1 from precious synnema actinomycetes tangerine orange subspecies; U33059) has about 30% identity.

Breach BlastP sequence alignment show SEQ ID NO:160 (779 amino acid) 1-308 amino acids with from Streptococcus mutans and be the protein (number of incorporating into own forces the gb1AAD09218.1 of abc transport albumin A TP binding subunit; U73183) have about 61% identity, the 1-362 amino acids with from Lactococcus lactis breast subspecies and be that abc transport albumin A TP combines and the proteic protein of the permease (number of incorporating into own forces NP266870.1; NC～002662) have about 37% identity, the 1-295 amino acids with from monocyte hyperplasia Li Site bacterium and with coming from the protein-bonded protein of the abc transport albumin A TP (number of incorporating into own forces NP464271.1; NC003210) have about 39% identity, the 1-221 amino acids with from the ancient green-ball bacterium of flicker and be the protein-bonded protein of the abc transport albumin A TP (number of incorporating into own forces NP070298.1; NC000917) have about 47% identity, and the 1-218 amino acids with from the ancient green-ball bacterium of flicker and be the protein-bonded protein of the abc transport albumin A TP (number of incorporating into own forces NP069851.1; NC000917) has about 49% identity.

Breach BlastP sequence alignment show SEQ ID NO:162 (38 amino acid) 1-27 amino acids with from clostridium acetobutylicum and be the protein (number of incorporating into own forces NP～149230.1 of the special phosphotransferase system component of seminose; NC001988) have about 66% identity, the 3-27 amino acids with from Listeria monocytogenes and with the protein (number of incorporating into own forces the NP463629.1 that comes from PTS system seminose specific factor IIAB; NC～003210) have about 72% identity, the 3-27 amino acids with from harmless Listera and with the protein (number of incorporating into own forces the NP469488.1 that comes from PTS system seminose specific factor IIAB; NC～003212) have about 72% identity, the 1-27 amino acids with from clostridium perfringens and be the proteic protein of the PTS system (number of incorporating into own forces NP561737.1; NC003366) have about 66% identity, and the 2-27 amino acids with from streptococcus pyogenes and be the protein (number of incorporating into own forces the NP269761.1 of the special phosphotransferase system component of seminose IIAB; NC002737) has about 65% identity.

Breach BlastP sequence alignment show SEQ ID NO:164 (105 amino acid) 1-103 amino acids with from Listeria monocytogenes and with coming from the protein (number of incorporating into own forces the NP463629.1 of PTS system seminose specific factor IIAB; NC～003210) have about 60% identity, the 1-103 amino acids with from harmless Listera and with the protein (number of incorporating into own forces the NP469488.1 that comes from PTS system seminose specific factor IIAB; NC～003212) have about 59% identity, the 1-104 amino acids with from clostridium perfringens and be the proteic protein of the PTS system (number of incorporating into own forces NP 561737.1; NC003366) have about 57% identity, the 1-104 amino acids with from clostridium acetobutylicum and be the protein (number of incorporating into own forces the NP149230.1 of the special phosphotransferase system component of seminose IIAB; NC001988) have about 53% identity, and the 1-96 amino acids with from streptococcus pyogenes and be the protein (number of incorporating into own forces the NP607831.1 of the special phosphotransferase system component of seminose IIAB; NC003485) has about 54% identity.

Breach BlastP sequence alignment show SEQ ID NO:166 (269 amino acid) 1-269 amino acids with from harmless Listera and with the protein (number of incorporating into own forces the NP469489.1 that comes from PTS system seminose specific factor IIC; NC～003212) have about 69% identity, the 1-269 amino acids with from Listeria monocytogenes and with the protein (number of incorporating into own forces the NP463630.1 that comes from PTS system seminose specific factor IIC; NC003210) have about 69% identity, the 1-269 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces NP～344821.1 of the special IIC component of PTS system seminose; NC～003028) have about 67% identity, the 1-269 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP269762.1 that comes from the special phosphotransferase system component of seminose IIC; NC～002737) have about 65% identity, and the 1-269 amino acids with from clostridium acetobutylicum and be the protein (number of incorporating into own forces NP～149231.1 of the special phosphotransferase system component of seminose/fructose IIC; NC001988) has about 64% identity.

Breach BlastP sequence alignment show SEQ ID NO:168 (307 amino acid) 5-307 amino acids with from harmless Listera and with the protein (number of incorporating into own forces the NP469490.1 that comes from PTS system seminose specific factor IID; NC003212) have about 67% identity, the 5-307 amino acids with from Listeria monocytogenes and with the protein (number of incorporating into own forces the NP463631.1 that comes from PTS system seminose specific factor IID; NC003210) have about 67% identity, the 6-303 amino acids with from clostridium acetobutylicum and be the protein (number of incorporating into own forces NP～149232.1 of the special phosphotransferase system component of seminose IID; NC001988) have about 64% identity, the 4-300 amino acids with from Lactococcus lactis breast subspecies and be the protein (number of incorporating into own forces the NP267864.1 of the special PTS system component of seminose IID (EC 2.7.1.69); NC002662) have about 64% identity, and the 5-307 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP-344820.1 of the special IID component of PTS system seminose; NC～003028) has about 64% identity.

Breach BlastP sequence alignment show SEQ ID NO:170 (111 amino acid) 4-105 amino acids with from streptococcus pyogenes and with coming from the proteic protein of the PTS system enzyme II (number of incorporating into own forces NP269441.1; NC002737) have about 51% identity, the 4-110 amino acids with from Listeria monocytogenes and with the protein (number of incorporating into own forces the NP_466205.1 that comes from cellobiose phosphotransferase IIb component; NC_003210) have about 54% identity, the 4-110 amino acids with from harmless Listera and with the protein (number of incorporating into own forces the NP_472159.1 that comes from cellobiose phosphotransferase IIB component; NC_003212) have about 54% identity, the 4-105 amino acids with from streptococcus pyogenes and with the protein (number of incorporating into own forces the NP_607438.1 that comes from PTS system enzyme II; NC_003485) have about 50% identity, and the 1-109 amino acids with from Lactococcus lactis breast subspecies and be that the protein (EC2.7.1.69) (number of incorporating into own forces NP_266569.1NC_002662) of the special PTS system of cellobiose IIB component has about 50% identity.

Breach BlastP sequence alignment show SEQ ID NO:172 (256 amino acid) 1-250 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP-357876.1 of the special EII component of phosphotransferase system sugar; NC03098) have about 53% identity, the 1-250 amino acids with from streptococcus pneumoniae and be the protein (number of incorporating into own forces the NP-344847.1 of PTS system IIC component; NC003028) have about 53% identity, the 1-255 amino acids with from clostridium acetobutylicum and be the protein (number of incorporating into own forces the NP-347026.1 of the special component I IC of PTS system cellobiose; NC003030) have about 43% identity, the 1-249 amino acids with from Lactococcus lactis breast subspecies and be the protein (number of incorporating into own forces NP～266572.1 of the special PTS system of cellobiose IIC component (EC 2.7.1.69); NC002662) have about 38% identity, and the 1-255 amino acids with from harmless Listera and with the protein (number of incorporating into own forces NP～470241.1 that come from the special IIC component of PTS system cellobiose; NC～003212) has about 37% identity.

Breach BlastP sequence alignment show SEQ ID NO:174 (560 amino acid) 1-551 amino acids with from Bacillus halodurans and be the protein (number of incorporating into own forces the NP241162.1 of PTS system β glucoside specific enzymes IIABC component; NC～002570) have about 39% identity, the 1-551 amino acids with from Listeria monocytogenes and with the protein (number of incorporating into own forces the NP464265.1 that comes from phosphotransferase system (PTS) β glucoside specific enzymes IIABC component; NC003210) have about 39% identity, the 1-554 amino acids with from subtilis and be the protein (number of incorporating into own forces the NP391806.1 of phosphotransferase system (PTS) β glucoside specific enzymes IIABC component; NC～000964) has about 38% identity, the 1-554 amino acids with from subtilis and be the PTS system β special IIABC component of glucoside (EIIABC-BGL) (β glucoside permease IIABC component) protein (number of incorporating into own forces sp1P407391PTBA～BACSU) has about 38% identity, and the 1-554 amino acids with from Bacillushalodurans and be the protein (number of incorporating into own forces the NP241461.1 of PTS system β glucoside specific enzymes IIABC component; NC002570) has about 37% identity.

The optimal sequence comparison result of even number sequence is as shown in table 2 among the relevant SEQ ID NOS:176-364.

The optimal sequence comparison result of table 2.SEQ ID NOS:176-308

SEQ ID NO：	ORF	The identity percentage	The amino acid scope	Organism	Describe	The number of incorporating into own forces
							176	1463	83	3 to 639	Lactobacterium helveticus	The lactose permease	emb|CAD55501. 1
178	639	90	1 to 88	Lactobacillus johnsonii NCC 533	Phosphate carrier albumen HPr	ref|NP_964671.1
							180	640	83	1 to 576	Lactobacillus johnsonii NCC 533	Phosphoenolpyruvic acid-protein phosphotransferase (enzyme 1)	ref|NP_964672.1
182	431	77	1 to 333	Lactobacillus delbruockii subspecies bulgaricus	pepR1	emb|CAB76946. 1
							184	676	71	1 to 314	Lactobacillus johnsonii NCC 533	HPr (Ser) kinases/Phosphoric acid esterase	ref|NP_964704.1
186	1778	79	1 to 303	Lactobacillus johnsonii NCC 533	Fructose-1-phosphokinase	ref|NP_965684.1
							188	1779	54	1 to 251	Lactobacillus johnsonii NCC 533		ref|P_965685.1
190	1433	77	1 to 331	Plant lactobacillus WCFS1	The glycerone kinases	ref|NP_784000.1
							192	1434	64	3 to 194	Plant lactobacillus WCFS1	The Protosol kinases, Phosphoric acid esterase structural domain dak2	ref|NP_784001.1
194	1436	73	1 to 231	Plant lactobacillus WCFS1	The glycerol picked-up promotes albumen	ref|NP_784003.1
							196	1437	100	1 to 480	Lactobacterium acidophilum	Sucrose phosphorylase	gb|AAO21868.1
198	1438	100	1 to 732	Lactobacterium acidophilum	The α tilactase	gb|AAO21867.1
							200	1457	74	1 to 327	Lactobacillus johnsonii NCC 533	Aldose 1-epimerase	ref|NP964716.1
202	1458	84	1 to 486	Lactobacterium helveticus	Semi-lactosi-1-P-uridine transferring enzyme	emb|CAA40526. 1
							204	1459	89	1 to 387	Lactobacterium helveticus	Galactokinase	emb|CAA40525. 1

SEQ ID NO：	ORF	The identity percentage	The amino acid scope	Organism	Describe	The number of incorporating into own forces
							206	1460	31	79 to 305	Plant lactobacillus WCFS1	The cell surface protein precursor	ref|NP_784891.1
208	1461	27	2 to 201	Lactobacillus johnsonii NCC 533		ref|NP_964254.1
							210	1462	74	1 to 665	Lactobacillus johnsonii NCC 533	Beta galactosidase enzyme	ref|NP_964713.1
212	1467	99	1 to 628	Lactobacterium acidophilum	Beta galactosidase enzyme	dbj|BAA20536.1
							214	1468	100	1 to 316	Lactobacterium acidophilum	BGAM LACAC beta galactosidase enzyme small subunit (LACTASE)	sp|O07685
216	1469	95	1 to 330	Lactobacillus helveticus	UDP-semi-lactosi 4-epimerase	emb|CAD55502. 1
							218	1719	80	1 to 294	Lactobacillus johnsonii NCC 533	UTP-1-Cori ester uridine transferring enzyme	ref|NP_965397.1
220	874	87	6 to 481	Lactobacillus gasseri	JE0395 phosphoric acid-beta-galactosidase enzymes I-Lactobacillus gasseri	pir||JE0395
							222	910	66	3 to 308	Lactobacillus gasseri	COG0039: oxysuccinic acid/serum lactic dehydrogenase	ref|ZP_00046547 .1
224	1007	55	13 to 279	Lactobacillus gasseri	COG2240: pyridoxal/pyridoxol/Pyridoxylamine kinases	ref|ZP_00046499 .1
							226	1812	71	3 to 766	Lactobacillus johnsonii NCC 533	The α Polyglucosidase	ref|NP_965686.1
228	1632	69	1 to 457	Lactobacillus johnsonii NCC 533	Succsinic acid-semialdehyde desaturase	ref|NP_965584.1
							230	1401	89	1 to 454	Lactobacillus gasseri	COG0446: not the NAD of Biao Zhenging (FAD)-	ref|ZP_00046159 .1

SEQ ID NO：	ORF	The identity percentage	The amino acid scope	Organism	Describe	The number of incorporating into own forces
												The dependent form desaturase
232	1974	72	1 to 601	Acetolactate synthase, pyruvic oxidase (cytopigment), the oxoethanoic acid carboligase, the phosphopyruvate decarboxylase	COG0028: diphosphothiamine indispensable enzyme	ref|ZP_00047198 .1
							234	1102	56	1 to 269	Lactobacterium helveticus	Transmembrane protein	emb|CAA05490. 1
236	1783	68	1 to 298	Lactobacillus johnsonii NCC 533	Abc transport albumin A TPase component	ref|NP_965688.1
							238	1879	72	9 to 268	Lactobacillus gasseri	COG0351: oxymethylpyrimidine/phosphomethylpyrimidine kinase	ref|ZP_00046866 .1
240	680	56	8 to 633	Streptococcus agalactiae NEM316		ref|NP_735321.1
							242	55	96	8 to 349	Lactobacillus gasseri	COG1052: serum lactic dehydrogenase and relevant desaturase	ref|ZP_00046778 .2
244	185	97	1 to 230	Lactobacillus gasseri	COG0588: phosphopyruvate mutase 1	ref|ZP_00047243 .1
							246	271	91	1 to 323	Lactobacterium helveticus	Serum lactic dehydrogenase	emb|CAB03618. 1
248	698	92	1 to 338	Lactobacillus johnsonii NCC 533	Glycerose 3-phosphate dehydrogenase	ref|NP_964727.1
							250	699	93	1 to 403	Lactobacillus johnsonii NCC 533	Phosphoglyceric kinase	ref|NP_964728.1

SEQ ID NO：	ORF	The identity percentage	The amino acid scope	Organism	Describe	The number of incorporating into own forces
							252	752	83	3 to 445	Lactobacillus gasseri	COG0166: glucose-6-phosphoric acid isomerase	ref|ZP_00046229 .1
254	889	93	1 to 428	Lactobacillus gasseri	C0G0148: enolase	ref|ZP_00046557 .1
							256	956	78	1 to 319	Lactobacillus johnsonii NCC 533	The fructose-1, 6-diphosphate kinases	ref|NP_964935.1
258	957	88	1 to 589	Lactobacillus gasseri	COG0469: pyruvate kinase	ref|ZP_00046514 .1
							260	1599	81	1 to 303	Lactobacillus johnsonii NCC 533	Fructose-bis phosphate aldolase	ref|NP_964539.1
262	1641	71	1 to 433	Lactobacillus gasseri	COG1653:ABC type sugar transport system, the pericentral siphon component	ref|ZP_00046816 .2
							264	452	69	1 to 335	Lactobacillus johnsonii NCC 533	Phosphoenolpyruvic acid dependent form sugar phosphotransferase system	ref|NP_965752.1
266	1479	71	1 to 278	Lactobacillus johnsonii NCC 533		ref|NP_965117.1
							268	725	62	1 to 655	Lactobacillus gasseri	COG 1263: phosphotransferase system IIC component	ref|ZP_00046302 .1
270	1369	81	1 to 411	Lactobacillus johnsonii NCC 533	Phosphoenolpyruvic acid dependent form sugar phosphotransferase system EIIC,	ref|NP_964585.1
							272	227	52	1 to 436	Enterococcus faecalis V583	The PTS system, the IIC component	ref|NP_814084.1
274	502	100	1 to	Lactobacterium acidophilum	Substrate is conjugated protein	gb|AAO21856.1

SEQ ID NO：	ORF	The identity percentage	The amino acid scope	Organism	Describe	The number of incorporating into own forces
										431		MsmE
276	507	100	1 to 480	Lactobacterium acidophilum	Sucrose phosphorylase	gb|AAO21861.1
							278	1483	59	1 to 492	Streptococcus agalactiae NEM316		ref|NP_734585.1
280	1484	75	1 to 131	Lactobacillus johnsonii NCC 533	High-affinity ribose translocator	ref|NP_965069.1
							282	552	76	1 to 487	Lactobacillus johnsonii NCC 533	The main superfamily protein permease that promotes	ref|NP_964553.1
284	567	79	3 to 400	Lactobacillus gasseri	COG0477: the permease that mainly promotes superfamily protein	ref|ZP_0004599 .1
							286	1471	74	79 to 405	Lactobacillus johnsonii NCC 533		ref|NP_965113.1
288	1853	80	4 to 163	Lactobacillus gasseri	COG0477: the permease that mainly promotes superfamily protein	ref|ZP_00046596 .1
							290	1012	77	9 to 643	Lactobacillus johnsonii NCC 533	Phosphoenolpyruvic acid dependent form sugar phosphotransferase system	ref|NP_964612.1
292	1014	77	1 to 552	Lactobacillus gasseri	COG0366: Glycosylase	ref|ZP_00045981 .1
							294	1440	100	1 to 277	Lactobacterium acidophilum	Stride film permease MsmG2	gb|AAO21865.1
296	1442	100	1 to 418	Lactobacterium acidophilum	The conjugated protein MsmE2 of substrate	gb|AAO21863.1
							298	1132	62	1 to 525	Lactobacillus gasseri	COG1132:ABC type multiple medicines transports system, ATPase and	ref|ZP_00045932 .1

SEQ ID NO：	ORF	The identity percentage	The amino acid scope	Organism	Describe	The number of incorporating into own forces
												Permease
300	1358	37	1 to 525	Lactobacillus gasseri	COG1132:ABC type multiple medicines transports system, ATPase and permease	ref|ZP_00045932 .1
							302	1838	71	1 to 224	Lactobacillus johnsonii NCC 533	Abc transport albumin A TPase component	ref|NP_965714.1
304	1840	50	1 to 172	Lactobacillus johnsonii NCC 533		ref|NP_965716.1
							306	1913	72	1 to 233	Lactobacillus gasseri	COG1136:ABC type antimicrobial peptide delivery system, ATPase	ref|ZP_00045892 .1
308	1938	59	19 to 364	Lactobacillus johnsonii NCC 533		ref|NP_965786.1
							310	165	74	7 to 650	Lactobacillus gasseri	COG3590: the Zinc metalloproteinase of prediction	ref|ZP_00046938 .1
312	251	43	9 to 184	Bacillus cereus ATCC 14579	Multi-medicine resistance albumen	ref|NP_832953.1
							314	252	39	1 to 117	Breast galactococcus breast subspecies Il1403	Multi-medicine resistance albumen	ref|NP_267065.1
316	253	35	1 to 57	Staphylococcus epidermidis ATCC12228	Multi-medicine resistance albumen	ref|NP_765487.1
							318	1062	86	1 to 173	Lactobacillus johnsonii NCC 533		ref|NP_965077.1
320	597	73	15 to 585	Lactobacillus johnsonii NCC 533	Abc transport albumin A TPase and permease component	ref|NP_965013.1
							322	1854	67	1 to 211	Lactobacillus gasseri	COG0477: the permease that mainly promotes superfamily protein	ref|ZP_0046596 .1
324	681	66	1 to 380	Breast galactococcus breast subspecies	The Cori ester adenylyl transferase	ref|NP_266853.1
							326	682	40	1 to 377	Streptococcus pneumoniae	The glycogen biosynthesizing is necessary	ref|NP_358625.1

SEQ ID NO：	ORF	The identity percentage	The amino acid scope	Organism	Describe	The number of incorporating into own forces
							328	683	52	1 to 475	Streptococcus pneumoniae	Glycogensynthase	ref|NP_345595.1
330	685	56	1 to 797	Breast galactococcus breast subspecies	Glycogen phosphorylase	ref|NP_266856.1
							332	686	42	5 to 548	Breast galactococcus breast subspecies	Starch debranching enzyme	ref|NP_266857.1
334	1356	71	1 to 118	Lactobacillus johnsonii		ref|NP_965359.1
							336	1465	80	1 to 333	Lactobacillus johnsonii	The lactose operon repressor	ref|NP_964711.1
338	1643	82	2 to 273	Lactobacillus gasseri	ABC type multiple medicines delivery system permease component	ref|ZP_00046332 .1
							340	1645	74	1 to 361	Lactobacillus johnsonii	Abc transport albumin A TPase component	ref|NP_965601.1
342	1731	40	3 to 293	Lactobacillus johnsonii		ref|NP_964886.1
							344	1732	75	1to 257	Lactobacillus johnsonii		ref|NP_964882.1
346	1733	73	4 to 217	Lactobacillus gasseri	C0G2148: related sugared transferring enzyme during lipopolysaccharides is synthetic	ref|ZP_00045843 .2
							348	1734	68	1 to 255	Lactobacillus delbruockii subspecies bulgaricus	EpsD	gb|AAG44708.1
350	1735	66	5 to 227	Lactobacillus johnsonii	Tyrosine protein kinase	ref|NP_964879.1
							352	1736	54	1 to 288	Lactobacillus johnsonii		ref|NP_964878.1
354	1737	52	5 to 340	Lactobacillus delbruockii subspecies bulgaricus	EpsA	gb|AAG44705.1
							356	1738	75	1 to 417	Lactobacillus gasseri	COG2262： GTPases	ref|ZP_00046671 .1
358	1739	42	25 to 331	Lactobacillus johnsonii		ref|MNP_64123.1
							360	1782	55	4 to 405	Lactobacillus johnsonii	Abc transport albumen permease component	ref|NP_965687.1
362	1869	87	1 to 220	Lactobacillus johnsonii	β-glucophosphomutase	ref|NP_964230.1
							364	1870	88	1 to 756	Lactobacillus gasseri	COG1554: trehalose and maltose lytic enzyme (possible Starch phosphorylase)	ref|ZP_00047083 .1

The PFAM result of embodiment 2. relevant aminoacid sequences

Table 3 has shown the best PFAM result of relevant aminoacid sequence of the present invention.

The PFAM result of table 3. aminoacid sequence

SEQ ID NO：	ORF	Structural domain	The amino acid scope is initial, stops	Family	The PFAM number of incorporating into own forces	The E value
							4	877	PTS_IIA	16，111	The PTS system, the special IIA subunit of lactose/cellobiose	PF02255	8.20E-40
6	609	PTS_EIIA_1	30，134	Phosphoenolpyruvic acid dependent form sugar phosphotransferase system, EIIA 1	PF00358	6.00E-55
							8	1479	PRD	76，171； 181，282	The PRD structural domain	PF00874	9.90E-52
8	1479	CAT_RBD	6，67	CAT RNA binding domains	PF03123	1.10E-16
							10	1574	Glyco_hydro_1	4，471	Glycosyl hydrolase family 1	PF00232	2.90E-133
12	1707	PTS_EIIA_1	491，595	Phosphoenolpyruvic acid dependent form sugar phosphotransferase system, EIIA 1	PF00358	6.10E-53
							12	1707	PTS_EIIC	105，387	The phosphotransferase system, EIIC	PF02378	3.10E-33
12	1707	PTS_EIIB	7，41	The phosphotransferase system, EIIB	PF00367	8.50E-19
							14	725	PTS_EIIA_1	528，632	Phosphoenolpyruvic acid dependent form sugar phosphotransferase system, EIIA 1	PF00358	4.10E-60

The PFAM result of table 3. aminoacid sequence

SEQ ID NO：	ORF	Structural domain	The amino acid scope is initial, stops	Family	The PFAM number of incorporating into own forces	The E value
							14	725	PTS_EIIC	122，419	The phosphotransferase system, EIIC	PF02378	3.80E-35
14	725	PTS_EIIB	21，55	The phosphotransferase system, EIIB	PF00367	8.90E-17
							16	491	PTS_EIIC	35，368	The phosphotransferase system, EIIC	PF02378	6.90E-80
20	1684	EIIA-man	1，115	PTS system fructose IIA component	PF03610	1.20E-13
							28	884	PTS_EIIC	34，392	The phosphotransferase system, EIIC	PF02378	7.70E-86
30	618	PTS_EIIC	29，360	The phosphotransferase system, EIIC	PF02378	8.70E-40
							32	606	PTS_EIIC	9，351	The phosphotransferase system, EIIC	PF02378	3.10E-48
32	606	PTS_EIIB	457，491； 551，585	The phosphotransferase system, EIIB	PF00367	1.40E-22
							34	1705	PTS_EIIA_I	531，636	Phosphoenolpyruvic acid dependent form sugar phosphotransferase system, EIIA1	PF00358	3.90E-48
34	1705	PTS_EIIC	131，412	The phosphotransferase system, EIIC	PF02378	2.80E-38
							34	1705	PTS_EIIB	10，44	The phosphotransferase system, EIIB	PF00367	2.00E-13
36	1777	PTS_IIB_fruc	183，285	The PTS system, the special IIB subunit of fructose	PF02379	2.40E-45
							36	1777	PTS_EIIC	313，597	The phosphotransferase system, EIIC	PF02378	5.20E-34
36	1777	PTS_EIIA_2	5，149	Phosphoenolpyruvic acid dependent form sugar phosphotransferase system, EIIA 2	PF00359	2.60E-26

The PFAM result of table 3. aminoacid sequence

SEQ ID NO：	ORF	Structural domain	The amino acid scope is initial, stops	Family	The PFAM number of incorporating into own forces	The E value
							38	500	Peripla_BP_1	68，331	The periplasmic binding protein of LacI family and sugared binding domains	PF00532	2.60E-10
38	500	LacI	11，36	The bacterium modulin, lacI family	PF00356	6.40E-10
							40	502	SBP_bacterial_1	28，403	The outer solute of bacterium born of the same parents is conjugated protein	PF01547	1.50E-51
42	503	BPD_transp_1	66，287	Membrane component in the conjugated protein dependent form delivery system	PF00528	2.30E-19
							44	504	BPD_transp_1	80，279	Membrane component in the conjugated protein dependent form delivery system	PF00528	1.00E-19
46	505	Glyco_hydro_32	24，409	Glycosyl hydrolase family 32	PF00251	5.50E-72
							48	506	ABC_tran	31，212	Abc transport albumen	PF00005	2.70E-58
54	1482	BPD_transp_2	5，274	Branched-chain amino acid delivery system/permease component	PF02653	6.40E-73
							56	1483	ABC_tran	32，219； 280，472	Abc transport albumen	PF00005	8.70E-88
60	1485	PfkB	4，297	PfkB family carbohydrate kinases	PF00294	4.70E-73
							62	1864	BPD_transp_1	70，280	Membrane component in the conjugated protein dependent form delivery system	PF00528	5.805-13

The PFAM result of table 3. aminoacid sequence

SEQ ID NO：	ORF	Structural domain	The amino acid scope is initial, stops	Family	The PFAM number of incorporating into own forces	The E value
							64	1865	BPD_transp_1	213，451	Membrane component in the conjugated protein dependent form delivery system	PF00528	2.90E-13
66	1866	SBP_bac_1	7，322	The outer solute of bacterium born of the same parents is conjugated protein	PF01547	1.40E-22
							68	1867	ABC_tran	31，212	Abc transport albumen	PF00005	8.00E-58
70	1944	ABC_tra	34，220； 287，481	Abc transport albumen	PF00005	8.20E-64
							72	1945	BPD_transp_2	53，338	Branched-chain amino acid delivery system/permease component	PF02653	1.40E-43
74	1946	BPD_transp_2	10，297	Branched-chain amino acid delivery system/permease component	PF02653	9.40E-44
							80	566	Sugar_tr	25，93	Sugar (and other) translocator	PF00083	1.10E-10
90	1616	Polysacc_synt	16，329	Polysaccharide biosynthesizing albumen	PF01943	1.70E-08
							98	399	Peripla_BP_1	60，325	The periplasmic binding protein of LacI family and sugared binding domains	PF00532	1.60E-18
98	399	LacI	3，28	Bacterium 1 modulin, lacI family	PF00356	8.50E-11
							100	400	Glyco_hydro_32	37，449	Glycosyl hydrolase family 32	PF00251	5.40E-158
102	401	PTS_EIIA_1	517，621	Phosphoenolpyruvic acid dependent form sugar phosphoric acid	PF00358	2.00E-70

The PFAM result of table 3. aminoacid sequence

SEQ ID NO：	ORF	Structural domain	The amino acid scope is initial, stops	Family	The PFAM number of incorporating into own forces	The E value
											The transferring enzyme system, EIIA1
102	401	PTS_EIIC	111，403	The phosphotransferase system, EIIC	PF02378	4.60E-68
							102	401	PTS_EIIB	7，40	The phosphotransferase system, EIIB	PF00367	5.50E-14
104	1012	PTS_EIIA_1	49，153	Phosphoenolpyruvic acid dependent form sugar phosphotransferase system, EIIA1	PF00358	4.00E-45
							104	1012	PTS_EIIC	301，587	The phosphotransferase system, EIIC	PF02378	1.20E-43
104	1012	PTS_EIIB	197，231	The phosphotransferase system, EIIB	PF00367	2.40E-16
							106	1013	GntR	5，68	The bacterium modulin, gntR family	PF00392	2.50E-15
108	1014	Alpha-amylase	28，429	αDian Fenmei, catalyst structure domain	PF00128	1.50E-110
							110	1439	ABC_tran	31，212	Abc transport albumen	PF00005	2.20E-58
110	1439	TOBE	301，359	The TOBE structural domain	PF03459	6.80E-09
							112	1440	BPD_transp_1	162，235	Membrane component in the conjugated protein dependent form delivery system	PF00528	2.90E-27
114	1441	BPD_transp_1	66，290	Membrane component in the conjugated protein dependent form delivery system	PF00528	1.60E-29
							116	1442	SBP_bacterial_1	48，411	The outer solute of bacterium born of the same parents is conjugated protein	PF01547	1.20E-61
118	1443	AraC_binding	16，159	AraC sample ligand binding domains	PF02311	6.80E-30

The PFAM result of table 3. aminoacid sequence

SEQ ID NO：	ORF	Structural domain	The amino acid scope is initial, stops	Family	The PFAM number of incorporating into own forces	The E value
							118	1443	HT_ AraC	229，273	Bacterium regulation and control helix-turn-helix protein, AraC family	PF00165	8.40E-20
122	74	ABC_tran	346，527	Abc transport albumen	PF00005	2.80E-36
							124	75	ABC_tran	346，527	Abc transport albumen	PF00005	9.20E-35
126	1131	ABC_tran	346，527	Abc transport albumen	PF00005	4.50E-35
							126	1131	ABC_membrane	14，280	Abc transport albumen is striden the film district	PF00664	1.00E-08
128	1132	ABC_tran	347，528	Abc transport albumen	PF00005	4.80E-36
							130	1357	ABC_tran	346，527	Abc transport albumen	PF00005	3.50E-33
132	1358	ABC_tran	348，529	Abc transport albumen	PF00005	1.30E-35
							134	1679	FtsX	85，182	The permease of prediction	PF02687	1.40E-08
136	1680	ABC_tran	35，221	Abc transport albumen	PF00005	1.90E-60
							144	1796	ABC_membrane	164，440	Abc transport albumen is striden the film district	PF00664	5.10E-68
144	1796	Peptidase_C39	10，145	PEPC C 39 families	PF03412	3.30E-64
							144	1796	ABC_tran	512，696	Abc transport albumen	PF00005	1.60E-46
146	1838	ABC_tran	43，228	Abc transport albumen	PF00005	2.10E-56
							148	1839	FtsX	192，347	The permease of prediction	PF02687	4.80E-16
152	1913	AB_tran	36，217	Abc transport albumen	PF00005	1.50E-57

The PFAM result of table 3. aminoacid sequence

SEQ ID NO：	ORF	Structural domain	The amino acid scope is initial, stops	Family	The PFAM number of incorporating into own forces	The E value
							154	1914	FtsX	246，441； 668，838	The permease of prediction	PF02687	7.60E-48
160	1939	ABC_tran	31，215	Abc transport albumen	PF00005	7.60E-57
							160	1939	FtsX	594，772	The permease of prediction	PF02687	3.00E-35
166	455	EII-Sor	1，238	The special iic component of PTS system sorbose	PF03609	8.00E-124
							168	456	EIID-AGA	7，307	The PTS system seminose/fructose/IID of sorbose family component	PF03613	4.80E-184
170	876	PTS_IIB	5，107	The PTS system, the special IIB subunit of lactose/cellobiose	PF02302	1.40E-31
							174	1575	PTS_EIIA_1	425，529	Phosphoenolpyruvic acid dependent form sugar phosphotransferase system, EIIA 1	PF00358	6.70E-63
174	1575	PTS_EIIC	42，322	The phosphotransferase system, EIIC	PF02378	1.40E-39
							176	1463	PTS_EIIA_1	516，608	Phosphoenolpyruvic acid dependent form sugar phosphotransferase system, EIIA 1	PF00358	2.10E-28
178	639	PTS-HPr	1，84	PTS HPr component phosphorylation site	PF00381	7.10E-52
							180	640	PEP-utilizers_C	252，544	PEP utilizes enzyme, TIM barrel-like structure territory	PF02896	8.30E-182
180	640	PEP-utilizers_N	5，129	PEP utilizes enzyme, and N does not hold	PF05524	3.50E-57

The PFAM result of table 3. aminoacid sequence

SEQ ID NO：	ORF	Structural domain	The amino acid scope is initial, stops	Family	The PFAM number of incorporating into own forces	The E value
							180	640	PEP-utilizers	146，227	PEP utilizes enzyme, the removable frame territory	PF00391	4.60E-37
182	431	LacI	6，31	The bacterium modulin, lacI family	PF00356	1.90E-11
							184	676	Hpr_kinase_C	133，313	HPr serine kinase C-terminal	PF07475	3.50E-86
184	676	Hpr_kinase_N	3，132	HPr serine kinase N-terminal	PF02603	7.90E-26
							186	1778	PfkB	7，292	PfkB family carbohydrate kinases	PF00294	1.50E-37
188	1779	DeoR	6，231	The bacterium modulin, deoR family	PF00455	1.30E-64
							190	1433	Dak1	16，331	The Dak1 structural domain	PF02733	2.30E-104
192	1434	Dak2	32，189	The DAK2 structural domain	PF02734	4.10E-71
							194	1436	MIP	1，231	Main integrated protein	PF00230	2.00E-39
196	1437	Alpha-amylase	10，423	αDian Fenmei, catalyst structure domain	PF00128	3.60E-07
							198	1438	Melibiase	293，690	Melibiose	PF02065	9.00E-252
200	1457	Aldose_epim	18，326	Aldose 1-epimerase	PF01263	7.30E-63
							202	1458	GalP_UDP_tr_C	222，430	Galactose-1-phosphate uridy1 transferase, the C-terminal structural domain	PF02744	9.30E-106
202	1458	GalP_UDP_transf	15，220	Galactose-1-phosphate uridy1 transferase, the N-terminal structural domain	PF01087	2.30E-95
							204	1459	GHMP_kinases	112，351	The ATP that the GHMP kinases is inferred is conjugated protein	PF00288	2.00E-50

The PFAM result of table 3. aminoacid sequence

SEQ ID NO：	ORF	Structural domain	The amino acid scope is initial, stops	Family	The PFAM number of incorporating into own forces	The E value
							210	1462	Glyco_hydro_42	192，605	Beta galactosidase enzyme	PF02449	1.90E-150
212	1467	Glyco_hydro_2_C	333，628	Glycosyl hydrolase family 2, TIM barrel-like structure territory	PF02836	2.60E-146
							212	1467	Glyco_hydro_2_N	39，227	Glycosyl hydrolase family 2, sugared binding domains	PF02837	2.00E-86
212	1467	Glyco_hydro_2	229，331	Glycosyl hydrolase family 2, immunoglobulin-like β sandwich structure territory	PF00703	2.90E-21
							214	1468	Bgal_small_N	4，197	The beta galactosidase enzyme chainlet, the N-terminal structural domain	PF02929	3.30E-94
214	1468	Bgal_small_C	206，315	The beta galactosidase enzyme chainlet, the C-terminal structural domain	PF02930	8.40E-61
							216	1469	Epimerase	3，324	NAD dependent form epimerase/dehydratase family	PF01370	2.00E-142
216	1469	3Beta_HSD	2，324	3-beta hydroxysteroid dehydrogenase/isomerase family	PF01073	1.00E-07
							218	1719	NTP_transferase	5，272	Nucleotidyl transferase	PF00483	8.30E-28
220	874	Glyco_hydro_1	2，479	Glycosyl hydrolase family 1	PF00232	1.20E-136

The PFAM result of table 3. aminoacid sequence

SEQ ID NO：	ORF	Structural domain	The amino acid scope is initial, stops	Family	The PFAM number of incorporating into own forces	The E value
							222	910	Ldh_1_N	3，142	Lactic acid/malate dehydrogenase (malic acid dehydrogenase), the NAD binding domains	PF00056	1.60E-59
222	910	Ldh_1_C	144，308	Lactic acid/malate dehydrogenase (malic acid dehydrogenase), α/β C-terminal binding domains	PF02866	1.90E-32
							224	1007	PfkB	130，187； 217，247	PfkB family carbohydrate kinases	PF00294	3.10E-07
226	1812	Glyco_hydro_31	105，757	Glycosyl hydrolase family 31	PF01055	2.10E-120
							228	1632	Aldedh	3，456	Acetaldehyde dehydrogenase family	PF00171	1.40E-98
230	1401	Pyr_redox	5，294	Pyridine nucleotide-disulphide oxydo-reductase	PF00070	2.10E-65
							232	1974	TPP_enzyme_N	4，174	The diphosphothiamine enzyme, N-terminal TPP binding domains	PF02776	4.00E-34
232	1974	TPP_enzyme_M	193，340	The diphosphothiamine enzyme, the central construct territory	PF00205	1.40E-32
							234	1102	Sugar_transport	16，280	HUCEP-8	PF06800	1.60E-114
236	1783	ABC_tran	27，204	Abc transport albumen	PF00005	2.20E-44
							238	1879	PfkB	129，178；	PfkB family carbohydrate kinases	PF00294	5.50E-07

The PFAM result of table 3. aminoacid sequence

SEQ ID NO：	ORF	Structural domain	The amino acid scope is initial, stops	Family	The PFAM number of incorporating into own forces	The E value
										212，240
240	680	Isoamylase_N	25，102	Isoamylase N-end structure territory	PF02922	8.80E-19
							240	680	Alpha-amylase	146，495	αDian Fenmei, catalyst structure domain	PF00128	3.30E-08
242	55	2-Hacid_dh_C	119，309	The special 2-alcohol acid of D-isomer desaturase, the NAD binding domains	PF02826	1.70E-100
							242	55	2-Hacid_dh	16，113	The special 2-alcohol acid of D-isomer desaturase, catalyst structure domain	PF00389	1.50E-23
244	185	PGAM	2，226	Phosphoglycerate phosphomutase family	PF00300	4.60E-117
							246	271	Ldh_1_N	8，147	Lactic acid/malate dehydrogenase (malic acid dehydrogenase), the NAD binding domains	PF00056	9.40E-76
246	271	Ldh_1_C	149，317	Lactic acid/malate dehydrogenase (malic acid dehydrogenase), α/β C-terminal structural domain	PF02866	2.00E-75
							248	698	Gp_dh_C	157，318	Glycerose 3-phosphate dehydrogenase, the C-terminal structural domain	PF02800	9.70E-88
248	698	Gp_dh_N	3，156	Glycerose 3-phosphate dehydrogenase, the NAD binding domains	PF00044	1.10E-82
							250	699	PGK	1，403	Phosphoglyceric kinase	PF00162	1.20E-218

The PFAM result of table 3. aminoacid sequence

SEQ ID NO：	ORF	Structural domain	The amino acid scope is initial, stops	Family	The PFAM number of incorporating into own forces	The E value
							252	752	PGI	7，442	Phosphoglucose isomerase	PF00342	3.80E-136
254	889	Enolase_C	139，427	Enolase, the terminal TIM barrel-like structure of C-territory	PF00113	2.30E-126
							254	889	Enolase_N	5，135	Enolase, the N-terminal structural domain	PF03952	1.40E-58
256	956	PFK	2，277	Phosphofructokinase	PF00365	1.70E-174
							258	957	PK	1，346	Pyruvate kinase, the barrel-like structure territory	PF00224	4.30E-228
258	957	PK_C	360，475	Pyruvate kinase, α/beta structure territory	PF02887	2.20E-64
							258	957	PEP-utilizers	490，579	PEP-utilizes enzyme, the removable frame territory	PF00391	2.50E-32
260	1599	F_bP_aldolase	4，285	Fructose-bis phosphate aldolase II type	PF01116	7.40E-97
							262	1641	S8P_bac_1	7，343	The outer solute of bacterium born of the same parents is conjugated protein	PF01547	1.70E-27
264	452	PTSIIB_sorb	169，319	PTS system sorbose subfamily IIB component	PF03830	6.20E-76
							264	452	EIIA-man	2，120	PTS system fructose IIA component	PF03610	1.60E-47
266	1479	PRD	76，164； 181，275	The PRD structural domain	PF00874	3.00E-39
							266	1479	CAT_RBD	2，60	CAT RNA binding domains	PF03123	3.10E-16
268	725	PTS_EIIA_1	516，620	Phosphoenolpyruvic acid dependent form sugar phosphotransferase system, EIIA 1	PF00358	9.10E-60

The PFAM result of table 3. aminoacid sequence

SEQ ID NO：	ORF	Structural domain	The amino acid scope is initial, stops	Family	The PFAM number of incorporating into own forces	The E value
							268	725	PTS_EIIC	111，406	The phosphotransferase system, EIIC	PF02378	1.40E-33
268	725	PTS_EIIB	9，43	The phosphotransferase system, EIIB	PF00367	2.00E-16
							270	1369	PTS_EIIC	31，336	The phosphotransferase system, EIIC	PF02378	1.30E-60
272	227	PTS_EIIC	26，364	The phosphotransferase system, EIIC	PF02378	1.40E-82
							274	502	SBP_bac_1	6，344	The outer solute of bacterium born of the same parents is conjugated protein	PF01547	5.30E-35
278	1483	ABC_tran	28，215； 276，468	Abc transport albumen	PF00005	6.40E-88
							280	1484	RbsD_FucU	1，131	RbsD/FucU translocator family	PF05025	5.90E-59
282	552	Sugar_tr	8，111	Sugar (and other) translocator	PF00083	1.80E-09
							290	1012	PTS_EIIA_1	25，129	Phosphoenolpyruvic acid dependent form sugar phosphotransferase system, EIIA 1	PF00358	8.70E-45
290	1012	PTS_EIIC	278，562	The phosphotransferase system, EIIC	PF02378	5.10E-40
							290	1012	PTS_EIIB	173，207	The phosphotransferase system, EIIB	PF00367	5.40E-16
292	1014	Alpha-amylase	11，413	αDian Fenmei, catalyst structure domain	PF00128	1.00E-112
							294	1440	BPD_transp_1	69，273	Membrane component in the conjugated protein dependent form delivery system	PF00528	2.40E-27
296	1442	SBP_bac_1	8，337	The outer solute of bacterium born of the same parents is conjugated protein	PF01547	2.90E-45

The PFAM result of table 3. aminoacid sequence

SEQ ID NO：	ORF	Structural domain	The amino acid scope is initial, stops	Family	The PFAM number of incorporating into own forces	The E value
							298	1132	ABC_tran	342，523	Abc transport albumen	PF00005	5.60E-35
300	1358	ABC_tran	344，525	Abc transport albumen	PF00005	2.60E-35
							302	1838	ABC_tran	33，218	Abc transport albumen	PF00005	2.40E-56
306	1913	ABC_tran	33，214	Abc transport albumen	PF00005	3.10E-57
							308	1938	DUF218	177，331	The DUF218 structural domain	PF02698	1.10E-45
310	165	Peptidase_M13_N	22，401	Peptide enzyme family M13	PF05649	7.00E-142
							310	165	Peptidase_M13	458，647	Peptide enzyme family M13	PF01431	8.80E-75
320	597	ABC_tran	372，557	Abc transport albumen	PF00005	1.10E-58
							320	597	ABC_membrane	36，302	Abc transport albumen is striden the film district	PF00664	2.10E-44
324	681	NTP_transferase	6，261	Nucleotidyl transferase	PF00483	4.60E-85
							326	682	NTP_transferase	19，80	Nucleotidyl transferase	PF00483	7.50E-05
328	683	Glycos_transf_1	280，454	Glycosyltransferase group 1	PF00534	1.20E-08
							332	686	Alpha-amylase	132，505	αDian Fenmei, catalyst structure domain	PF00128	2.40E-61
334	1356	ArsC	5，118	ArsC family	PF03960	1.80E-37
							336	1465	LacI	2，27	The bacterium modulin, lacI family	PF00356	7.50E-07
338	1643	BPD_transp_1	66，269	Membrane component in the conjugated protein dependent form delivery system	PF00528	4.50E-16
							340	1645	ABC_tran	33，216	Abc transport albumen	PF00005	4.80E-66
342	1731	Glycos_transf_2	5，171	Glycosyltransferase	PF00535	1.40E-26
							346	1733	Bac_transf	24，217	Bacterium sugar transferring enzyme	PF02397	2.30E-94
352	1736	Wzz	9，186	Chain length decision albumen	PF02706	3.20E-16

The PFAM result of table 3. aminoacid sequence

SEQ ID NO：	ORF	Structural domain	The amino acid scope is initial, stops	Family	The PFAM number of incorporating into own forces	The E value
							354	1737	LytR_cpsA_psr	105，252	Cell envelope associated retroviral attenuator structural domain	PF03816	2.50E-57
362	1869	Hydrolase	2，192	Hydracid dehalogenation enzyme sample lytic enzyme	PF00702	6.80E-29
							364	1870	Glyco_hydro_65N	11，266	Glycosyl hydrolase family 65, the N-terminal structural domain;	PF03636	1.90E-86
364	1870	Glyco_hydro_65m	320，692	Glycosyl hydrolase family 65 central catalyst structure domains;	PF03632	4.10E-203
							364	1870	Glyco_hydro_65C	696，748	Glycosyl hydrolase family 65, the C-terminal structural domain	PF03633	1.10E-15

Embodiment 3: glucose metabolism genes

API50 sugar-fermenting pattern as it is shown, and Lactobacterium acidophilum has the ability to utilize various carbohydrate, comprises monose, disaccharides and polysaccharide.Especially, can utilize not by the complicated meals carbohydrate of top enteral digestion, such as (Gibson etc. (1995) J.Nutr.125:1401-1412 such as raffinose and fructo-oligosaccharides; Barrangou etc. (2003) Proc.Natl.Acad.Sci.U.S.A 100:8957-8962).The gene that the many kinds of NCFM genome encoding are relevant with the carbohydrate utilization comprises that 20 kinds of phosphoenolpyruvic acids sugar transferring enzyme systems (PTS) of translocator and 5 ATP are in conjunction with box (ABC) family.

Identified the PTS translocator of inferring of following carbohydrate: trehalose (ORF 1012) (SEQ ID NOS:103 and 289), fructose (ORF 1777) (SEQ ID NO:35), sucrose (ORF 401) (SEQ ID NO:101), glucose and seminose (ORF 452 (SEQ IDNOS:1 and 263), ORF 453 (SEQ ID NO:161), ORF 454 (SEQ ID NO:163), ORF 455 (SEQ ID NO:165) and ORF 456 (SEQ ID NO:167)), melibiose (ORF 1705) (SEQ ID NO:33), gentiobiose and cellobiose (ORF 1369) (SEQ ID NOS:17 and 269), saligenin (ORF 876) (SEQ ID NO:169), ORF 877 (SEQ ID NO:3), ORF 879 (SEQ ID NO:171)), arbutin (ORF 884) (SEQ ID NO:27) and N-acetyl-glucosamine (ORF 146) (SEQ IDNO:21).The abc transport albumen of inferring of following carbohydrate: FOS (ORF 502 (SEQID NOS:39 and 273), ORF 504 (SEQ ID NO:43), ORF 506 (SEQ IDNO:47)), raffinose (ORF 1439 (SEQ ID NO:109), ORF 1440 (SEQ IDNOS:111 and 293), ORF 1441 (SEQ ID NO:113), ORF 1442 (SEQ IDNOS:115 and 295) and maltose (ORF 1854-ORF 1857) have been identified.The lactose of inferring-semi-lactosi permease has also been identified (ORF 1463) (SEQ ID NO:175).Most of these translocators and Glycosylase and the total a certain genetic locus of transcriptional control make transcriptional control to localize.

Genomic In silico analyzes and shows the gene that has the whole glycolytic pathway of representative.In addition, comprehensive carbohydrate utilizes the regulated and control network member also to be determined, promptly, HPr (ORF 639 (SEQ ID NO:177), ptsH), EI (ORF 640 (SEQ ID NO:179), ptsI), CcpA (ORF 431 (SEQ ID NO:181), ccpA) and HPrK/P (ORF 676 (SEQ IDNO:183), ptsK), shown that the carbon metabolism that enlivens based on the operability of sugar suppresses network.

Embodiment 4: difference expression gene

Utilize Ward ' s hierarchical clustering method, volcano shape figure and isogram to manifest the comprehensive gene expression pattern (Eisen etc. (1998) Proc.Natl.Acad.Sc i.USA 95:14863-14868) that from 8 kinds of different carbohydrate are cultivated, obtains by the clustering analysis.Substantially, be differential expression (the p value is lower than the Bonferroni correction value) between the treatment condition 23-379 gene being arranged in pairs, represented genomic 1%-20% respectively.Considered all possible processing, and if certain gene at least a processing relatively, demonstrated and induced, then this gene is regarded as being induced and has surpassed a certain special level.For under more than one situation, demonstrating the inductive gene, select the highest level of inducing.Induce level high more than 2 times although there are 342 kinds of genes (genomic 19%) to demonstrate, have only 63 kinds of genes (genomic 3%) to demonstrate and induce level high more than 4 times, show that the gene of relatively small number amount is highly induced.Although no matter grow which kind of used substratum is, the comprehensive representation level of most gene is consistent (genomic 80%), and selected gene cluster demonstrates the differential transcription of gene and operon.But, for every kind of sugar, have only the gene of limited quantity to demonstrate special inducing.

Compare with other monose (fructose, semi-lactosi) and disaccharides (sucrose, lactose, trehalose), under the situation that glucose exists, ORF 1679 (SEQ ID NO:133) and ORF 1680 (SEQ ID NO:135) are highly induced.Compare with other sugar, the level of inducing changes between 3.5 and 6.3 times for ORF 1679 (SEQ ID NO:133), and the level of inducing changes between 3.7 and 4.7 times for ORF 1680 (SEQ ID NO:135).ORF 1679 (SEQ ID NO:133) coding ABC nucleotide binding protein comprises the Nucleotide binding domains primitive that finds usually, i.e. WalkerA, WalkerB, ABC signal sequence and Linton and Higgins primitive.ORF1680 (SEQ ID NO:135) coding ABC permease has the membrane spaning domain of 10 predictions.Near the solute of not encoding them is conjugated protein, shows that its effect may be follower but not input unit.Several genes and operon are arranged by special the checking of glucose, comprise ORFs 680 (SEQ ID NO:239)-ORF 686, they are relevant with Glycogen Metabolism.Because cell carries out Glycogen Metabolism with storing energy, so when existing such as preferred carbon sources such as glucose, energy storage is just dispensable.Other gene that is checked under the situation that glucose exists comprises the enzyme that relates in protein related in other carbohydrate source material picked-up and the described carbohydrate inversion.

At three gene ORF 1777 at the fructose locus place of inferring (SEQ ID NO:35) (FruA, fructose PTS translocator EIIABCP ^Fru), ORF 1778 (SEQ ID NO:185) (FruK, phosphofructokinase EC 2.7.1.56) and ORF 1779 (SEQ ID NO:187) (FruR, transcriptional regulatory) be differential expression.FruA, fruK and fruR induce level respectively up to 3.9,4.3 and 4.6 times.These results show that fructose enters cell by the transhipment of PTS translocator, convert fructose-6-phosphate to, and phosphofructokinase FruK becomes glucolytic intermediate product fructose-1,6-diphosphate with its phosphorylation.

Having under the situation of sucrose, three genes on the sucrose locus are differential expressions, be ORF 399 (SEQ ID NO:97) (ScrR, transcriptional regulatory), ORF 400 (SEQ IDNO:99) (ScrB, sucrose-6-phosphohydrolase EC 3.2.1.26) and ORF 401 (SEQID NO:101) (ScrA, sucrose PTS translocator EIIBCA ^Suc).Compare with the situation that glucose exists, scrR, scrB and scrA induce level respectively up to 3.1,2.8 and 17.2 times.Compare with the situation that disaccharides exists with monose, ORF 401 (SEQ ID NO:101) especially shows height and induces level, between 8.0 to 17.2.These results show that sucrose transports by the PTS translocator and enter cell that be transformed into sucrose-6-phosphoric acid, it is hydrolyzed into G-6-P and fructose by ScrB subsequently.

6 genes of FOS operon are differential expressions, be ORF 502 (SEQ ID NOS:39 and 273), ORF 503 (SEQ ID NO:41), ORF 504 (SEQ ID NO:43), ORF 506 (SEQ ID NO:47) (MsmEFGK abc transport albumen), ORF 505 (SEQID NO:45) (BfrA, beta-fructosidase enzyme EC 3.2.1.26) and ORF 507 (SEQ IDNOS:49 and 275) (GtfA, sucrose phosphorylase EC 2.7.1.4).Compare with the situation that disaccharides exists with monose, their level of inducing changes between 15.1 to 40.6 times, compares with the situation that raffinose exists, and their level of inducing changes between 5.5 to 8.9 times.These results show that FOS transports by abc transport albumen and enter cell and be hydrolyzed into fructose and sucrose by fructosidase subsequently.Sucrose might be hydrolyzed into fructose and glucose-1-P by sucrose phosphorylase subsequently.Except the FOS operon, FOS also induces fructose operon, sucrose PTS translocator, trehalose operon and abc transport albumen (ORF 1679-ORF 1680) (being respectively SEQ ID NOS:133 and 135).

Having under the situation of raffinose, 6 genes of raffinose operon are induced by special.The raffinose locus is by ORF 1442 (SEQ ID NOS:115 and 295), ORF 1441 (SEQ IDNO:113), ORF 1440 (SEQ ID NOS:111 and 293), ORF1439 (SEQ ID NO:109) (MsmEFGK ₂Abc transport albumen), ORF1438 (SEQ ID NO:197) (MelA alpha-galactosidase EC 3.2.1.22) and ORF1437 (SEQ ID NO:195) (GtfA ₂, sucrose phosphorylase EC 2.7.1.4) form.Compare with all other conditions, their level of inducing changes between 15.1 to 45.6 times.In addition, compare with other condition, ORFs 1433 (SEQID NO:189), 1434 (SEQ ID NO:191) (Protosol kinases EC 2.7.1.29) and ORF 1436 (SEQ ID NO:193) (the glycerine picked-up promotes albumen) induce level between 1.9 and 24.7 times.

Under the situation that lactose and semi-lactosi are arranged, be distributed in 10 kinds of gene differential expressions of two locus, be ORF 1463 (SEQ ID NO:175) (the LacS permease of GPH transposon family), ORF 1462 (SEQ ID NO:209) (LacZ, beta-galactosidase enzymes EC 3.2.1.23), ORF 1461 (SEQ ID NO:207), ORF 1460 (SEQ ID NO:205) (surface protein), ORF 1459 (SEQ ID NO:203) (GalK, galactokinase EC 2.7.1.6), ORF 1458 (SEQ ID NO:201) (Ga lT, galactose-1-phosphate uridyl transferring enzyme EC 2.7.7.10), ORF 1457 (SEQ ID NO:199) (GalM, galactose epimerase EC 5.1.3.3), ORFs 1467 (SEQ ID NO:211), 1468 (SEQID NO:213) (La cLM, the big small subunit of beta-galactosidase enzymes EC 3.2.1.23) and 1469 (SEQ ID NO:215) (GalE, UDP-glucose epimerase EC 5.1.3.2).LacS (SEQ ID NO:175) is similar to the previous GPH permease of determining in lactic-acid-bacterium.Although LacS (SEQ ID NO:175) contains EIIA at carboxyl terminal, it is not the PTS translocator.In addition, LacS (SEQ ID NO:175) contains a His at the 553rd, and this may relate to the interaction with HPr, in S.salivarius (Lessard etc. (2003) J.Bacteriol.185:6764-6772).Compare with the situation that other carbohydrate that does not contain semi-lactosi (being glucose, fructose, sucrose, trehalose and FOS) is arranged, under the situation that lactose and semi-lactosi are arranged, galKTM (SEQ ID NOS:199,201 and 203) induces level between 3.7 and 17.6 times; LacSZ (SEQ ID NOS:175 and 209) induces level between 2.8 and 17.6 times; LacL (SEQ ID NO:213) and galE (SEQID NO:215) induce level between 2.7 to 29.5 times.These results show that semi-lactosi is entered cell by the LacS permease of galactoside-pentose and hexose aldehydic acid glucosides transposon family by transhipment.In cell, lactose is hydrolyzed into glucose and semi-lactosi by LacZ.Semi-lactosi is changed into galactose-1-phosphate by GalK phosphoric acid then, is further changed into the UDP-semi-lactosi by GalT.The UDP-semi-lactosi is changed into UDP-glucose by the GalE epimerization subsequently.UDP-glucose may be transformed into glucose-IP by ORF 1719 (SEQ ID NO:217), its encode UDP-glucose phosphorylation enzyme EC 2.7.7.9 of consistent high expression level.At last, phosphoglucomutase EC 5.4.2.2 acts on glucose-1P probably, produces glucose-6P, a kind of glucolytic substrate.

Three genes of the trehalose locus of inferring also are differential expressions.The trehalose locus is by ORF 1012 (SEQ ID NOS:103 and 289) (coding TreB trehalose PTS translocator EIIABC ^TreEC 2.7.1.69), ORF1013 (SEQ ID NO:105) (TreR, trehalose regulator) and ORF1014 (SEQ ID NOS:107 and 291) (TreC, trehalose-6 phosphohydrolase EC 3.2.1.93) form.Compare with the situation that glucose, sucrose, raffinose and semi-lactosi are arranged, induce level between 4.3 to 18.6 times about treB (SEQ ID NOS:103 and 289), relevant treR (SEQ ID NO:105) induces level between 2.3 to 7.3 times, and induces level between 2.7 to 18.5 times about treC (SEQ ID NOS:107 and 291).These results show that trehalose transports by the PTS translocator and enter cell that phosphoric acid changes into trehalose-6-phosphate and is hydrolyzed into glucose and G-6-P by TreC.

In addition, the gene that demonstrates differential expression comprises the gene ORF 874 (SEQ ID NO:219) (beta galactosidase enzyme EC 3.2.1.86) relevant with energy with sugar, ORF 910 (SEQ IDNO:221) (L-LDH EC 1.1.1.27), ORF 1007 (SEQ ID NO:223 (pyridoxal kinase 2.7.1.35), ORF 1812 (SEQ ID NO:225) (α Polyglucosidase EC3.2.1.3), ORF 1632 (SEQ ID NO:227) (acetaldehyde dehydrogenase EC 1.2.1.16), ORF 1401 (SEQ ID NO:229) (NADH peroxidase EC 1.11.1.1), ORF1974 (SEQ ID NO:231) (pyruvic oxidase EC 1.2.3.3); Adherance is because of ORF555, ORF 649, ORF 1019; Aminopeptidase ORF 911, ORF 1086; Amino acid permease, ORF 1102 (SEQ ID NO:233) (membranin), ORF 1783 (SEQ ID NO:235) (abc transport albumen) and ORF 1879 (SEQ ID NO:237) (pyrimidine kinases EC 2.7.4.7).

Embodiment 5. real-time RT-PCRs

For the level of inducing that confirms to detect with microarray, 5 genes that are chosen in differential expression in the microarray experiment carry out the real-time quantitative RT-PCR experiment.According to they wide expression level scope (LSM-1.52 and+3.87 between) and the level (inducing multiple) of inducing between various carbohydrates up to 34 select these genes.All selected genes all demonstrate under at least a situation and are higher than 6 times the level of inducing.In addition, all the utilization with carbohydrate is relevant on function for the note of selected gene.5 kinds of selected genes are beta-fructosidase enzyme (ORF 505) (SEQ ID NO:45), trehalose PTS (ORF 1012) (SEQ ID NOS:103 and 289), glycerine picked-up promotion albumen (ORF 1436) (SEQ ID NO:193), beta galactosidase enzyme (ORF 1467) (SEQ ID NO:211) and abc transport albumen (ORF 1679) (SEQ ID NO:133).

For these 5 kinds of selected genes, compare the level of inducing of 6 kinds of different treatment, cause having 15 kinds to induce level to every kind of gene.In order to confirm the data of microarray, the level of inducing that Q-PCR detects is mapped with the level of inducing of microarray assay.Changed (data of weighing with log2 are between 0.652 and 0.978) for every kind between 0.642 and 0.883 by each R-side's value for the cls gene.When data combination, total R-side's value is 0.78 (data of weighing with log2 is 0.88).(Cary NC) carries out correlation analysis, demonstrates with regard to Spearman, Hoeffding and Kendall test, and the P value of two kinds of methods is less than 0.001, thereby has dependency with SAS.In addition, (Microsoft CA) carries out regression analysis, demonstrates significant high correlation (p＜1.02 * 10 on the statistics between the result of microarray and Q-PCR with excel ^-25).But, the Q-PCR observed value demonstrates the higher level of inducing, and this may be owing to compare with Q-PCR circulation instrument, and the microarray scanner dynamicrange is less to be caused.

Existing report (Wagner etc. (2003) J.Bacteriol.185:2080-2095) in the similar result document formerly.

Embodiment 6: comparative analysis

Total transcripting spectrum of being measured with 8 kinds of carbohydrate cultivations is compared analysis determined carbohydrate transhipment and catabolic basis in the Lactobacterium acidophilum.Clear and definite says, three kinds of dissimilar carbohydrate translocators are by differential expression, be phosphoenolpyruvic acid: sugar phosphotransferase system (PTS), ATP have illustrated the diversity of the carbohydrate translocator that Lactobacterium acidophilum is used in conjunction with box (ABC) and galactoside-pentose and hexose aldehydic acid acid anhydride (GPH) transposon.Transcripting spectrum shows galactoside by the transhipment of GPH transposon, and monose and disaccharides are transported by the PTS family member, and polysaccharide is transported by the ABC family member.

Microarray results shows that fructose, sucrose and trehalose are respectively by PTS translocator EIIABC ^Fru(ORF 1777) (SEQ ID NO:35), EIIBCA ^SuC(ORF 401) (SEQID NO:101) and EIIABC ^Tre(ORF1012) (SEQ ID NOS:103 and 289) transports.Those genes encodings on typical PTS locus (Fig. 1), together with and in other organism by the regulon enzyme of fine evaluation.Different therewith is that FOS and raffinose are respectively by the abc transport albumen ORFs 502 (SEQ ID NOS:39 and 273), 503 (SEQ ID NO:41) of MsmEFGK family, 504 (SEQ ID NO:43) and 505 (SEQ IDNO:45); And ORFs 1437 (SEQ ID NO:195), ORF 1438 (SEQ ID NO:197), 1439 (SEQ ID NO:109), ORF 1440 (SEQ ID NOS:111 and 293), ORF 1441 (SEQ ID NO:113) and ORF 1442 (SEQ ID NO:115 and 295) transhipment.Under the situation of trehalose and FOS, microarray results has good dependency with the functional study that fixed point knocks out the sugar decomposition potential of carbohydrate translocator and lytic enzyme improvement Lactobacterium acidophilum NCFM.

EIIABC ^TreDifferential expression be with the research unanimity of nearest relevant Lactobacterium acidophilum, show that wherein ORF 1012 (SEQ ID NOS:103 and 289) relates to the picked-up of trehalose.Similarly, the differential expression of fos operon is with the work unanimity of previous relevant Lactobacterium acidophilum, what this was previous studies show that those genes are relevant with picked-up and the katabolism of FOS, and when FOS is arranged, induced, and when glucose is arranged, checked (Barrangou etc. (2003) Proc.Natl.Acad.Sci.USA 100:8957-8962).In addition, inducing of raffinose msm locus is that work with previous relevant Streptococcus mutans (Russell etc. (1992) J.Biol.Chem.267:4631-4637) and streptococcus pneumoniae (Rosenow etc. (1999) Genome Res.9:1189-1197) is consistent.

Many lactic-acid-bacteriums are taken in glucose by the PTS translocator.EII ^ManThe PTS translocator has the two ability (Cochu etc., 2003) of input seminose and glucose.The seminose PTS system of Lactobacterium acidophilum is similar to thermophilus streptococcus (Streptococcus thermophilus), and its protein has the identity of 53-65% and the similarity of 72-79%.Clear and definite says, EII ^ManContain three kinds of protein IIAB ^Man, IIC ^ManAnd IID ^Man, respectively by ORF 452 (SEQ IDNOS:1 and 263) (manL), ORF455 (SEQ ID NO:165) (manM) and ORF 456 (SEQID NO:167) (manN) encode (Fig. 1).

Induce the gene that they relate in that most of carbohydrate of this inspection is special in body transporting and hydrolysis, but glucose not like this.The analysis revealed of relevant seminose PTS, regardless of the carbohydrate source, coding EIIABCD ^ManThe all consistent high expression level of gene.Show as previous institute about plant lactobacillus, this expression map shows that glucose is a kind of preferred carbohydrate, and Lactobacterium acidophilum also is designed to effectively utilize different carbohydrate sources (Kleerebezem etc. (2003) Proc.Natl.Acad.Sci.USA 100:1990-1995).

The gene of differential expression comprises the enzymatic system of permease (LacS) and Leloir approach when semi-lactosi and lactose are arranged.In multiple lactic-acid-bacterium, all record LacS subfamily member galactoside-pentose-hexuronic acid acid anhydride (GPH) transposon, comprise breast bright string coccus (Vaughan etc. (1996) Appl.Environ.Microbiol.62:1574-1582), S.thermophilus (2000) J.Bacteriol.182:5982-5989 such as () van den Bogaard, streptococcus-salivarius (Lessard etc. (2003) JBacteriol.185:6764-6772) and lactobacillus delbruckii (Lapierre etc. (2002) J.Bacteriol.184:928-935).Although LacS contains PTS EIIA at C-terminal, it is not the member of PTS family translocator.It is reported that in some selected organism, LacS can import semi-lactosi and lactose (Vaughan etc. (1996) App l.Environ.Microbiol.62:1574-1582; (2000) J.Bacteriol.182:5982-5989 such as van den Bogaard).Although uniting in plant lactobacillus (Kleerebezem etc. 2003) and the bright string coccus of breast (Vaughan etc. (1996) Appl.Environ.Microbiol.62:1574-1582) of LacS lactose permease and two beta galactosidase enzyme subunit LacL and LacM all has description, the relevant report arranged never in Lactobacterium acidophilum.Though previous existing report (Vaughan etc. (1996) Appl.Environ.Microbiol.62:1574-1582) about lacS and lacLM constitutive expression, these results show that related gene is the specificity inductive in semi-lactosi and the two picked-up of lactose and the katabolism.The operon composition of using about galactoside is variable and unsettled (Lapierre etc. (2002) J BacterioL184:928-935 between gram positive bacterium; Vaillancourt etc. (2002) J.Bacteriol.184:785-793; Boucher etc. (2003) Appl.Environ.Microbiol.69:4149-4156; Fortina etc. (2003) Appl.Environ.Microbiol.69:3238-3243; Grossiord etc. (2003) J.Bacteriol.185:870-878).Even closely-related lactobacillus species, i.e. Lactobacillus johnsonii, Lactobacillus gasseri and Lactobacterium acidophilum, (Pridmore etc. (2004) the Proc.Natl.Acad.Sci.USA 101:2512-2517) that lactose-semi-lactosi locus neither be very conservative.

Although before shown phosphoenolpyruvic acid: the phosphotransferase system is that the main sugar of gram positive bacterium transports system (Ajdic etc. (2002) Proc.Natl.Acad.Sci.USA99:14434-14439; Warner and Lolkema (2003) Microbiol.MoL Rev.67:475-490), present microarray data shows that the abc transport system also is very important.And the PTS translocator is relevant with the disaccharides absorption with monose, and those carbohydrate are digested on digestive tube top.On the contrary, oligosaccharides then arrives more following enteron aisle part, thereby symbiotic microorganism might be competed more mixture and insufficient nutrition.May be under such condition abc transport albumen even more important than PTS, comprise that they show tangible effect in the transporting of oligosaccharides such as FOS and raffinose.In this, the nutraceutical ability of utilizing the host not digest is the competitiveness relevant with persistence (Schell etc. (2002) Proc.Natl.Acad.Sci.USA 99:14422-14427) with the useful intestinal microflora of colonic.

The gene transcription collection of illustrative plates of differential expression shows under test condition, and except glucose, all carbohydrate capturing systems and their glycosylhydrolases are separately induced by the special of its substrate all.And, but the gene in those induced gene seats when having glucose to exist, checked, and identify the cre sequence in their promotor-manipulation subarea.Search the difference expression gene of relevant degradation production response element of inferring and the promotor-manipulation subarea of operon according to consensus sequence TGNNWNCGNNWNCA (SEQ ID NO:365) (Miwa etc. (2000) Nucleic AcidsRes.28:1206-1210) with TGWAANCGNTNWCA (SEQ ID NO:366) (Weickert and Chambliss (1990) Proc.Natl.Acad.Sc i.USA 87:6238-6242).Generally speaking, these results have disclosed carbohydrate picked-up and the metabolism regulation and control at transcriptional level, and show the comprehensive regulator control system that may relate to the inhibition of carbon degradation production.The carbon degradation production suppresses (CCR) and is controlling related proteinic transcribing (Miwa etc. (2000) Nucleic Acids Res.28:1206-1210) in carbohydrate transhipment and the metabolism.It is the mechanism that is distributed widely in the gram positive bacterium that degradation production suppresses, and is subjected to degradation production response element cis mediation (Miwa etc. (2000) Nucleic Acids Res.28:1206-1210; Wickert and Chambliss (1990) Proc.Natl.Acad.Sci.USA87:6238-6242), and be subjected to the trans mediation of LacI family repressor, the gene transcription that this family is responsible for the nonessential products of sugar decomposition of coding in the presence of preferred substrate checks (Wickert and Chambliss (1990) Proc.Natl.Hcad.Sci.USA 87:6238-6242; Viana etc. (2000) Mol.Microbiol.36:570-584; Muscariello etc. (2001) Appl.Environ.Microbiol.67:2903-2907; Warner and Lolkema (2003) Microbiol.Mol.Rev.67:475-490).This regulatory mechanism makes cell can coordinate to utilize different carbohydrate, mainly concentrates on the preferred energy derive.CCR is based on several crucial enzymes, be HPr (ORF 639 (SEQ ID NO:177), ptsH), EI (ORF 640 (SEQ ID NO:179), ptsI), CcpA (ORF 431 (SEQ ID NO:181), ccpA) and HPrK/P (ORF 676 (SEQ ID NO:183), ptsK), all these enzymes are all encoded in Lactobacterium acidophilum karyomit(e).

Carbon degradation production restraining effect is existing report (Mahr etc. 2000) in Bacterium lacticum.PTS is characterised in that the phosphoric acid that relates to PEP, EI, HPr, EIIABC shifts cascade reaction, thereby makes phosphate finally be transferred to (Saier, 2000 on the carbohydrate substrate; Warner and Lolkema, 2003).HPr is the important component of CCR, and it is subjected to the regulation and control of the phosphorylation of enzyme I and HPrK/P.When HPr at His15 during by phosphorylation, PTS activates, and the carbohydrate by the PTS transhipment is by the EIIABC phosphorylation.On the contrary, when HPr at the Ser46 place during by phosphorylation, PTS mechanism then do not play a role (Mijakovic etc. (2002) Proc.Natl.Acad.Sci.USA 99:13442-13447).

Although the phosphorylation cascade reaction has shown the regulation and control at protein level, also have several researchs to report the modulation of transcribing of ccpA and ptsHI.In S.thermophilus, it is (the van den Bogaart etc. 2000) that are subjected to glucose induction that CcpA produces.In several bacteriums, the transcriptional level (Luesink etc. 1999) of ptsHI is being regulated in the carbohydrate source.On the contrary, the expression level of ccpA, ptsH, ptsI and ptsK does not change with the existence of different carbohydrate in the Lactobacterium acidophilum.These results are consistent with the regulation and control of phosphorylation on the protein level.About transcribe (Cochu etc. (2003) Appl.Environ.Microbiol.69:5423-5432) of ptsHI in the expression level (Mahr etc. (2000) Appl.Environ.Microbiol.66:277-283) of the interior ccpA of Lactobacillus pentosus (Lactobacillus pentosus) and the S.thermophilus also has similar report.

See that comprehensively microarray results makes the investigator can rebuild carbohydrate transhipment and catabolic pathway (Fig. 2).Although transcribing of carbohydrate translocator and lytic enzyme is to be subjected to their substrate specificity inductive separately, these glycolysis-genes still are consistent high expression level: D type serum lactic dehydrogenase (D-LDH, ORF 55 (SEQ ID NO:241)), phosphoglycerate phosphomutase (PGM, ORF 185 (SEQ ID NO:243)), L type serum lactic dehydrogenase (L-LDH, ORF271 (SEQ ID NO:245)), Glycerose 3-phosphate dehydrogenase (GPDH, ORF 698 (SEQID NO:247)), phosphoglyceric kinase (PGK ORF 699 (SEQ ID NO:249)), G-6-P isomerase (GPI, ORF 752 (SEQ ID NO:251)), 2-Phosphoglycerate dehydratase (PGDH, ORF 889 (SEQ ID NO:253)), phosphofructokinase (PFK, ORF 956 (SEQ ID NO:255)), pyruvate kinase (PK, ORF 957 (SEQID NO:257)) and fructose bis phosphoric acid zymohexase (FBPA, ORF 1599 (SEQ ID NO:259)).Glycerol-3-phosphate abc transport albumen (ORF 1641 (SEQ ID NO:261)) also is one of gene that continues high expression level.Coordinating the carbohydrate picked-up might cancel energy derive and make other bacterium can not be near these resources from intestinal environment.Thereby Lactobacterium acidophilum may good and other symbiotic microorganism competition nutritive ingredient.

In a word, with regard to the expression map when existing, there is the multiple kinds of carbohydrate capturing system to be identified and to identify, comprises PTS, ABC and GHP translocator with regard to different carbohydrate.Picked-up and catabolic mechanism are highly regulated and control at transcriptional level, show that it is flexibly, dynamically and be suitable for effectively utilizing carbohydrate that Lactobacterium acidophilum is transcribed group.Differential gene expression shows that total carbon degradation production of existence suppresses regulated and control network.The consistent high expression level of modulin shows that regulation and control are at protein level, but not transcriptional level.In a word, Lactobacterium acidophilum seems effectively to change its metabolic mechanism with available carbohydrate source in the change small intestine nutrition compound environment.Particularly, in the picked-up of FOS and raffinose the abc transport albumen of related MsmEFG family might Lactobacterium acidophilum and enteron aisle Symbiont compete host people play an important role in the ability of indigested compounding sugar.

At last, this data provides new viewpoint for how indigested compound in the understanding diet influences intestinal microbial balance.This research is the comparison transcription analysis model that is suitable for being exposed to the bacterium in the growth medium of continuous variation.

Embodiment 7: multidrug transporter

Developed the whole bag of tricks that opposing microbiotic and other hazardous compound toxic action such as microorganisms such as Lactobacterium acidophilums.One of these means relate to initiatively effusive translocator of promotion medicine, and drug resistance may be affected for special microorganism thus.

Two kinds of main multidrug transporters are arranged: utilize the membrane electrochemical gradient of striding of proton or sodium ion to order about the secondary multidrug transporter that medicine is extruded from cell; Utilize the free energy of ATP hydrolysis medicine to be pumped the ATP of cell in conjunction with box (ABC) formula multidrug transporter.

Secondary multidrug transporter is subdivided into several distinct translocator family: main promotion superfamily protein (MFS, Pao etc. (1998) Microbiol.Mol.Biol.Rev.62:1-34), small-sized multi-medicine resistance (smallmultidrug resistance, SMR) family (Paulsen etc. (1996) Mol.Microbiol.19:1167-1175), oncocyte division (resistance-nodulation-celldivision is given birth in opposing, RND) family (Saier etc. (1994) Mol.Microbiol.11:841-847) and multiple medicines and toxic chemical are discharged (multidrug and toxic compound extrusion, MATE) family (Brown etc. (1999) Mol.Microbiol.31:394-395).These families are not only relevant with multiple medicines output, but also comprise other proton motive force dependency transport process or the related protein of other function.

The MFS protein called membrane transporters relate to the cotransporting of various substrates, antiport or uniport, comprising microbiotic (Marger and Saier (1993) Trends Biochem.Sci.18:13-20).The medicine that makes organism have resistance is flowed out analysis and the sequence alignment that proteic conservative primitive carries out show that these protein can be separated into two independent monoids, have 12 or 14 and stride diaphragm disconnected (Paulsen and Skurry (1993) Gene 124:1-11).The NCFM genome comprises several genes that coding causes the MFS translocator of multiple medicines thing transhipment.Comprising ORFs 552 (SEQ ID NO:77), 566 (SEQ ID NO:79), 567 (SEQ IDNO:81), 1446 (SEQ ID NO:85), 1471 (SEQ ID NO:87), 1621 (SEQ ID NO:91), 1853 (SEQ ID NO:93), 1854 (SEQ ID NO:321), 164 (SEQ ID NO:309), 251-253 (SEQ ID NO s:311,313,315) and 1062 (SEQ ID NO:317) in the coding translocator.

4 different structural domains of abc transport protein requirement: two hydrophobic membrane structural domains, wherein each is made up of 6 film α spirals of striding of inferring usually, and two hydrophilic Nucleotide binding domainss (NBD), comprise WalkerA and B primitive (Walker etc. (1982) EMBO is J.1:945-951) and ABC signal (Hyde etc. (1990) Nature 346:362-365).Each structural domain can be used as isolating protein expression or can incorporate (Faith and Kolter (1993) Microbiol.Rev.57:995-1017 in the Multidomain polypeptide in many ways; Higgens (1992) Annu.Rev.CellBio.8:67-113; Hyde etc. (1990) Nature 346:362-365).Encode by ORF 597 (SEQ ID NO:320) in the NCFM genome to from the ABC multidrug transporter ImrA of Lactococcus lactis and from the similar multiple medicines thing abc transport albumen of the multidrug transporter horA of short lactobacillus.

The level of the person skilled in the art in the affiliated field of the present invention is all indicated in mentioned all publications, patent and patent application in this specification sheets.All publications, patent and patent application are all incorporated into own forces as a reference at this, its scope and each independent publication or patent application by clear and definite and individually indication incorporate into own forces the same as a reference.

Although aforementioned invention is described on some details so that clearly understanding obviously can be carried out some change and modification in appended claim scope in the mode of diagram and embodiment.

Claims (20)

1. be selected from down the isolating nucleic acid of group:

A) comprise the nucleic acid of following nucleotide sequence: SEQ ID NO:289,1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175,177,179,181,183,185,187,189,191,193,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337,339,341,343,345,347,349,351,353,355,357,359,361 and/or 363, can be any combination, or its complementary strand;

B) comprise the nucleic acid that has the nucleotide sequence of at least 90% sequence identity with following nucleotide sequence: SEQ ID NO:289,1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175,177,179,181,183,185,187,189,191,193,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337,339,341,343,345,347,349,351,353,355,357,359,361 and/or 363, can be any combination, or its complementary strand;

C) comprise the segmental nucleic acid of following nucleotide sequence: SEQ ID NO:289,1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175,177,179,181,183,185,187,189,191,193,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337,339,341,343,345,347,349,351,353,355,357,359,361 and/or 363, can be any combination, or its complementary strand;

D) coding comprises the nucleic acid of the polypeptide of following aminoacid sequence: SEQ ID NO:290,2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362 and/or 364, can be any combination;

E) comprise the nucleic acid of nucleotide sequence that coding and following aminoacid sequence have the polypeptide of at least 90% amino acid sequence identity: SEQ ID NO:290,2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362 and/or 364, can be any combination; With

F) under stringent condition with a-e in the nucleic acid of any nucleic acid hybridization.

2. the carrier that comprises the nucleic acid of claim 1.

3. the carrier of claim 2 further comprises the nucleic acid of the heterologous polypeptide of encoding.

4. the cell that comprises the carrier of claim 2.

5. isolated polypeptide is selected from down group:

A) comprise the polypeptide of following aminoacid sequence: SEQ ID NO:290,2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362 and/or 364, can be any combination;

B) comprise the segmental polypeptide of following aminoacid sequence: SEQ ID NO:290,2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362 and/or 364, can be any combination;

C) comprise the polypeptide that has the aminoacid sequence of at least 90% sequence identity with following aminoacid sequence: SEQ ID NO:290,2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362 and/or 364, can be any combination;

D) by the following nucleotide sequence encoded polypeptides: SEQ ID NO:289,1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175,177,179,181,183,185,187,189,191,193,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337,339,341,343,345,347,349,351,353,355,357,359,361 and/or 363, can be any combination, or its complementary strand; With

E) by the nucleotide sequence coded polypeptide that has at least 90% sequence identity with following nucleotide sequence: SEQ ID NO:289,1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175,177,179,181,183,185,187,189,191,193,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337,339,341,343,345,347,349,351,353,355,357,359,361 and/or 363, can be any combination, or its complementary strand, wherein said polypeptide retentive activity.

6. the polypeptide of claim 5 further comprises the allogeneic amino acid sequence.

7. with the antibody of the polypeptide selective binding of claim 5.

8. method for preparing polypeptide is included in the cell of cultivating claim 4 under the condition that the nucleic acid of this polypeptide of coding expressed, and described polypeptide is selected from down group:

A) comprise the polypeptide of following aminoacid sequence: SEQ ID NO:290,2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,118,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362 and/or 364, can be any combination;

B) comprise the segmental polypeptide of following aminoacid sequence: SEQ ID NO:290,2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362 and/or 364, can be any combination;

C) comprise the polypeptide that has the aminoacid sequence of at least 90% sequence identity with following aminoacid sequence: SEQ ID NO:290,2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362 and/or 364, can be any combination;

D) by the nucleotide sequence coded polypeptide that has at least 90% sequence identity with following nucleotide sequence: SEQ ID NO:289,1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175,177,179,181,183,185,187,189,191,193,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337,339,341,343,345,347,349,351,353,355,357,359,361 and/or 363, can be any combination; With

E) by the following nucleotide sequence encoded polypeptides: SEQ ID NO:289,1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175,177,179,181,183,185,187,189,191,193,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337,339,341,343,345,347,349,351,353,355,357,359,361 and/or 363, can be any combination.

9. one kind changes organism carbohydrate transhipment is entered or transport out the method for the ability of cell, comprises the nucleic acid of claim 1 is introduced described organism.

10. one kind changes the method that organism gathers the ability of carbohydrate, comprises the nucleic acid of claim 1 is introduced described organism.

11. one kind changes organism and utilizes the method for carbohydrate as the ability of the energy, comprises the nucleic acid of claim 1 is introduced described organism.

12. a change is comprised the nucleic acid of claim 1 is introduced described microorganism by the method for the flavours in food products of microbial fermentation.

13. a change is comprised the nucleic acid of claim 1 is introduced described microorganism by the method for the quality of the food of microbial fermentation.

14. one kind changes the method that organism produces the ability of the carbohydrate of modifying, comprises the nucleic acid of claim 1 is introduced described organism.

15. one kind changes the method that organism is stood the ability of food-processing and storage condition, comprises the nucleic acid of claim 1 is introduced described organism.

16. a method that changes organism in the ability of GI road survival comprises the nucleic acid of claim 1 is introduced described microorganism.

17. one kind changes the method that organism produces the ability of carbohydrate, comprises the nucleic acid of claim 1 is introduced described organism.

18. one kind changes the method that organism enters drug transport or transport out the ability of cell, comprises the nucleic acid of claim 1 is introduced described organism.

19. comprise the vegetable cell of nucleic acid construct, this nucleic acid construct comprises the nucleic acid of claim 1.

20. the plant that produces by the vegetable cell of claim 19.

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