Classifications

• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/50—Improvements relating to the production of bulk chemicals
  • Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts

Definitions

• the invention relates to the fields of molecular biology, bacteriology and industrial fermentation. More specifically, the invention relates to the identification and isolation of nucleic acid sequences and proteins for subunits of a novel, membrane bound sorbitol dehydrogenase in Gluconobacter suboxydans. The invention further relates to the fermentative production of L-sorbose from D- sorbitol and the subsequent production of 2-keto-L-gulonic acid.
• Sorbitol dehydrogenase is an enzyme responsible for the efficient conversion of D-sorbitol into L-sorbose during sorbose fermentation in the process of the manufacturing of 2-keto-L-gulonic acid (2-KLG), an important precursor for vitamin C synthesis.
• Gluconobacter possesses several SDHs, which may be categorized according to their cofactor requirement: ( 1 ) NAD-dependent, (2) NADP-dependent and (3) NAD(P)-independent types. Among them, NAD(P)-independent enzyme is believed to be directly involved in efficient production of sorbose during industrial sorbose fermentation (Cummins, J. T. et al., J. Biol. Chem., 224, 323; 226, 3 01 (1957)).
• L-sorbose from D-sorbitol is typically performed by fermentation with an acetic acid bacterium such as Gluconobacter suboxydans and Acetobacter xylinum. At room temperature, 96-99% of conversion is made in less than 24 hours (Liebster, J. et al., Chem. List., 50:395 (1956)).
• L-sorbose produced by the action of SDH is a substrate in the production of 2-keto-L-gulonic acid (2-KLG).
• 2KLG 2-keto-L-gulonic acid
• a variety of processes for the production of 2KLG are known.
• the fermentative production of 2-KLG via oxidation of L-sorbose to 2-KLG via a sorbosone intermediate is described for processes utilizing a wide range of bacteria: Gluconobacter oxydans (U.S. Pat.
• U.S. Patent Nos. 5,88,786; 5,861,292; 5,834,263 and 5,753,481 disclose nucleic acid molecules encoding and/or isolated proteins for L-sorbose dehydrogenase and L-sorbosone dehydrogenase; and U.S. Patent No. 5,747,301 discloses an enzyme with specificity for sorbitol dehydrogenase.
• the sorbitol dehydrogenase identified in U.S. Patent No.5,747,301 is distinguished on the basis of subcellular location (membrane-bound) and a haloenzyme molecular weight of 800 ⁇ 50 kDa (10 homologous subunits of 79 ⁇ 5 kDa).
• the membrane-bound D-sorbitol dehydrogenase isolated by Shinagawa et al.(E. Shinagawa, K. Matsushita, O. Adachi and M. Ameyama (Agric. Biol. Chem., 46, 135-141, 1982)) consisted of three kinds of subunits with molecular weights of 63,000, 51,000 and 17,000.
• This invention pertains to a novel, membrane-bound sorbitol dehydrogenase of Gluconobacter suboxydans.
• the isolated sorbitol dehydrogenase enzyme comprises three subunits: a first subunit of 75 kDa containing pyrroloquinoline quinone (PQQ) as cofactor; a second subunit of 50 kDa being a cytochrome c; and a third subunit of 29 kDa playing a very important role in the stability and the catalytic activity of the enzyme.
• PQQ pyrroloquinoline quinone
• the present invention provides nucleic acid molecules for the 3 protein subunits of the Gluconobacter sorbitol dehydrogenase described herein.
• the invention provides an isolated nucleic acid molecule drawn to the first SDH subunit (75 kDA) identified by SEQ ID NO: 1.
• the invention provides an isolated nucleic acid molecule drawn to the second SDH subunit (50 kDA) identified by SEQ ID NO:2.
• the invention provides an isolated nucleic acid molecule drawn to the third SDH subunit (29 kDA) identified by SEQ ID NO:3.
• Other related embodiments are drawn to vectors, processes for producing the same and host cells carrying said vectors.
• the invention also provides isolated nucleic acid molecules encoding the three subunits of the SDH of the invention.
• the invention provides a cloned nucleic acid molecule encoding the 75 kDa and 50 kDa subunits.
• the structural genes for the first and the second subunit of sorbitol dehydrogenase are 2,265 bp and 1,437 bp, respectively, in size and are clustered in the cloned nucleic acid molecule which is a 5.7 kb Pstl DNA fragment that defines the operon.
• the invention provides a cloned nucleic acid molecule encoding the third, 29 kDa, SDH subunit protein.
• the structural gene coding for the third subunit is 921 bp in size and found in a 4.5 kb Clal DNA fragment.
• Other related embodiments are drawn to vectors, processes to make the same and host cells containing said vectors.
• the invention is also drawn to isolated polypeptides for the three subunits of the SDH described herein.
• the invention also provides a method for the production of D-sorbose comprising: (a) transforming a host cell with at least one isolated nucleotide sequence selected from the group consisting of a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: l ; a polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2; and a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 3; and (b) selecting and propagating said transformed host cell.
• Another aspect of the invention is drawn to a method for production of 2- KLG comprising: (a) transforming a host cell with at least one isolated nucleotide sequence selected from the group consisting of a polynucleotide comprising the polynucleotide sequence of SEQ ID NO:l ; a polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2; and a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 3 ; and (b) selecting and propagating said transformed host cell.
• Figure 1 presents DEAE-TSK column chromatography using a sodium acetate buffer of pH 5.0 (A) and pH 6.0 (B).
• Figure 2 presents SDS-PAGE analysis of peak I (A) and peak II fractions (B) separated by DEAE-TSK column chromatography.
• Figure 3 presents DEAE-TSK column chromatography using a sodium phosphate buffer of pH 6.5.
• Figure 4 presents SDS-PAGE analysis of column fractions of peak I (lane 1), peak II (lane 3) and peak III (lane 2) separated by DEAE-TSK column chromatography at pH 6.5.
• Lane M denotes molecular weight standard markers.
• Figure 5 presents an HPLC chromatogram of a tryptic digest of peak II protein from DEAE-TSK column chromatography at pH 6.5. The dotted line indicates the concentration gradient of acetonitrile in the mobile phase.
• Figure 6 presents a restriction enzyme map of the Lambda GEM 5- 1.
• the 1.53 kb DNA fragment (#SDH 2- 1 ) used as probe is shown as solid bar.
• Figure 7 presents the locations of SI, S2 and S3 DNA fragments generated with different sets of restriction enzymes from 5.7 kb Pstl fragment of Lambda GEM 5-1.
• Figure 8 presents the nucleotide sequence of 4,830 bases (SEQ ID NO:7) of the 5.7 kb Pstl fragment.
• the deduced amino acid sequence for the first and the second subunit is shown below the nucleotide sequence.
• the N-terminal amino acid sequence obtained by the N-terminal amino acid sequencing of the purified sorbitol dehydrogenase is underlined.
• Signal sequence cleavage site is marked as a triangle.
• the heme-binding sequences are underlined with dotted lines.
• Potential ribosome-binding sequences are enclosed in boxes.
• the transcription termination stem-and-loop structure is indicated by arrows.
• the complete coding sequence for the first subunit gene is located at position 665- 2,929 (SEQ ID NO: 1), with the signal sequence located at position 665-766, and the coding sequence for the mature protein of the SDH first subunit located at position 767-2,929 (SEQ ID NO:22).
• the complete coding sequence for the second subunit gene is located at position 2,964-4,400 (SEQ ID NO:2), with the signal sequence located at position 2,964-3,071 , and the coding sequence for the mature protein of the SDH second subunit located at position 3,072-4,400 (SEQ ID NO:23).
• Figure 9 presents a restriction enzyme map of ClaI-#69.
• the closed box represents the coding region of the third subunit gene of sorbitol dehydrogenase.
• Figure 10 presents the nucleotide sequence (SEQ ID NO:8) of DNA fragment containing the third subunit gene of sorbitol dehydrogenase.
• the deduced amino acid sequence is shown below the nucleotide sequence.
• Signal sequence cleavage site is marked as a triangle.
• Potential ribosome-binding sequence (SD) is enclosed in box.
• the complete coding sequence for the third subunit gene is located at position 1,384-2.304 (SEQ ID NO:3), with the signal sequence located at position 1 ,384- 1 ,461. and the coding sequence for the mature protein of the SDH third subunit located at position 1,462-2,304 (SEQ ID NO:24).
• Cloning Vector A plasmid or phage DNA or other DNA sequence which is able to replicate autonomously in a host cell, and which is characterized by one or a small number of restriction endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without loss of an essential biological function of the vector, and into which a DNA fragment may be spliced in order to bring about its replication and cloning.
• the cloning vector may further contain a marker suitable for use in the identification of cells transformed with the cloning vector. Markers, for example, provide tetracycline resistance or ampicillin resistance.
• Expression is the process by which a polypeptide is produced from a structural gene. The process involves transcription of the gene into mRNA and the translation of such mRNA into polypeptide(s).
• Expression Vector A vector similar to a cloning vector but which is capable of enhancing the expression of a gene which has been cloned into it, after transformation into a host. The cloned gene is usually placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences. Promoter sequences may be either constitutive or inducible.
• Gene A DNA sequence that contains information needed for expressing a polypeptide or protein.
• Host Any prokaryotic or eukaryotic cell that is the recipient of a replicable expression vector or cloning vector.
• a "host,” as the term is used herein, also includes prokaryotic or eukaryotic cells that can be genetically engineered by well known techniques to contain desired gene(s) on its chromosome or genome. For examples of such hosts, see Sambrook et al,
• Homologous/Nonhomologous Two nucleic acid molecules are considered to be "homologous” if their nucleotide sequences share a similarity of greater than 50%, as determined by HASH-coding algorithms (Wilber, W.J. and
• nucleic acid molecules are considered to be "nonhomologous" if their nucleotide sequences share a similarity of less than 50%.
• Mutation refers to a single base pair change, insertion or deletion in the nucleotide sequence of interest.
• Mutagenesis refers to a process whereby a mutation is generated in DNA. With “random" mutatgenesis, the exact site of mutation is not predictable, occurring anywhere in the chromosome of the microorganism, and the mutation is brought about as a result of physical damage caused by agents such as radiation or chemical treatment.
• Operon As used herein, the term refers to a unit of bacterial gene expression and regulation, including the structural genes and regulatory elements in DNA.
• Parental Strain As used herein, the term refers to a strain of microorganism subjected to some form of mutagenesis to yield the microorganism of the invention.
• Phenotype As used herein, the term refers to observable physical characteristics dependent upon the genetic constitution of a microorganism.
• Promoter A DNA sequence generally described as the 5 ' region of a gene, located proximal to the start codon. The transcription of an adjacent gene(s) is initiated at the promoter region. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter.
• Recombinant Host According to the invention, a recombinant host may be any prokaryotic or eukaryotic cell which contains the desired cloned genes on an expression vector or cloning vector.
• Recombinant Vector Any cloning vector or expression vector which contains the desired cloned gene(s).
• the present invention isolates and purifies SDH from the cytoplasmic membrane of G. suboxydans KCTC (Korea Culture Type Collection) 2111 (equivalent to ATCC 621) using a series of column chromatographic steps.
• Biochemical properties of the purified enzyme are provided, as well as the isolation of each subunit and a determination of the N-terminal amino acid sequence of each subunit using an amino acid sequence analyzer (Applied Biosystems, 411 A).
• the newly characterized enzyme is different from the reported FAD- dependent SDH from G. suboxydans IFO 3254 strain (Shinagawa, E. etal, Agric. Biol. Chem., 46: 135 (1982)), containing pyrroloquinoline quinone (PQQ) as a cofactor and comprising three subunits (Choi, E. S. et al. , FEMS Microbiol Lett., 125:45 (1995)).
• the SDH of the invention may be isolated using standard protein techniques. Briefly, G.
• suboxydans KCTC 2111 is cultured in SYP medium (5% D-sorbitol, 1% Bacto-Peptone, 0.5% yeast extract) and the cells are lysed in a 10 mM sodium acetate buffer solution (pH 5.0). After centrifuged at 12,000 g to remove cell debris, the supernatant is centrifuged in an ultracentrifuge to recover cytoplasmic membrane fraction.
• SYP medium 5% D-sorbitol, 1% Bacto-Peptone, 0.5% yeast extract
• a 10 mM sodium acetate buffer solution pH 5.0
• the purified enzyme is active towards polyols such as D-sorbitol (100%), D-mannitol (68%) and D-ribitol (70%). Activity of the enzyme increases up to nine fold when pyrroloquinoline quinone (PQQ) is added, suggesting that PQQ is a cofactor for the enzyme; fluorescence spectrum analysis confirmed that the purified enzyme contains pyrroloquinoline quinone (PQQ). The absorption spectrum analysis of the purified enzyme demonstrates that this enzyme contains cytochrome c.
• PAGE poly aery lamide gel electrophoresis
• the initial SDS-PAGE analysis of the purified enzyme showed that the enzyme comprised three subunits of 75 kDa, 50 kDa and 14 kDa which were named the first subunit, the second subunit, and the third subunit, respectively (Choi, E. S. et al, FEMSMicrobiol Lett., 125:45 (1995)).
• the enzyme comprisesd three subunits of 75 kDa, 50 kDa and 14 kDa which were named the first subunit, the second subunit, and the third subunit, respectively (Choi, E. S. et al, FEMSMicrobiol Lett., 125:45 (1995)).
• another subunit of 29 kDa played a very important role in the stability and the catalytic activity of SDH.
• This additional subunit of 29 kDa was renamed the third subunit: it is uncertain whether the 14 kDa subunit previously assigned the third subunit is a true subunit of the enzyme when comparing the relative amount with other subunits on the acrylamide gel. It was also found that a further increase of the pH of the elution buffer to pH 6.5 resulted in a complete separation of three subunits into individual subunits.
• DCIP Dichlorophenol indophenol
• PMS phenazine methosulfate
• Example 1 Further details of the purification and characterization of the SDH enzyme of the invention are provided in Example 1.
• the invention provides isolated nucleic acid molecules encoding one or more of the three subunits of the SDH enzyme described herein.
• Methods and techniques designed for the manipulation of isolated nucleic acid molecules are well known in the art. For example, methods for the isolation, purification and cloning of nucleic acid molecules, as well as methods and techniques describing the use of eukaryotic and prokaryotic host cells and nucleic acid and protein expression therein, are described by Sambrook, et al, Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor, N.Y., 1989, and Current Protocols in Molecular Biology, Frederick M. Ausubel et al. Eds., John Wiley & Sons, Inc., 1987, the disclosure of which is hereby incorporated by reference.
• the invention provides several isolated nucleic acid molecules encoding the individual 75 kDa, 50 kDa, and 29 kDa subunit proteins of SDH enzyme of the invention. Additionally, the invention provides several isolated nucleic acid molecules encoding one or more of subunit proteins of the SDH enzyme of the invention. For the purposes of clarity, the particular isolated nucleic molecules of the invention are described. Thereafter, specific properties and characteristics of these isolated nucleic acid molecules are described in more detail.
• nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373A from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
• a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
• the invention provides an isolated nucleic acid molecule for the first (75 kDa) subunit of the SDH enzyme of the invention comprising a polynucleotide sequence selected from the group consisting of (a) the polynucleotide of SEQ ID NO: 1 : (b) a polynucleotide fragment at least about 20 nucleotides in length of the polynucleotide of SEQ ID NOT ; (c) a polynucleotide encoding the amino acid sequence of SEQ ID NO:4; and (d) a polynucleotide encoding a fragment at least about 10 amino acids in length of the amino acid sequence of SEQ ID NO:4.
• the invention provides an isolated nucleic acid molecule for the first (75 kDa) subunit of the SDH enzyme of the invention comprising a polynucleotide at least about 95% identical to the isolated nucleic acid sequence for the first (75 kDa) subunit of the SDH enzyme of the invention described above.
• Another embodiment of the invention provides an isolated nucleic acid molecule for the second (50 kDa) subunit of the SDH enzyme of the invention comprising a polynucleotide sequence selected from the group consisting of: (a) the polynucleotide, or fragment thereof, of SEQ ID NO:2; (b) a polynucleotide fragment at least about 20 nucleotides in length of the polynucleotide of SEQ ID NO:2; (c) a polynucleotide encoding the amino acid sequence of SEQ ID NO:5; and (d) a polynucleotide encoding a fragment at least about 10 amino acids in length of the amino acid sequence of SEQ ID NO:5.
• the invention provides an isolated nucleic acid molecule for the second (50 kDa) subunit of the SDH enzyme of the invention comprising a polynucleotide at least about 95% identical to the isolated nucleic acid sequence for the second (50 kDa) subunit of the SDH enzyme of the invention described above.
• Another embodiment of the invention provides an isolated nucleic acid molecule for the third (29 kDa) subunit of the SDH enzyme of the invention comprising a polynucleotide sequence selected from the group consisting of: (a) the polynucleotide of SEQ ID NO: 3 ; (b)a polynucleotide fragment at least about 20 nucleotides in length of the polynucleotide of SEQ ID NO:3; (c) a polynucleotide encoding the amino acid sequence of SEQ ID NO:6; and (d) a polynucleotide encoding a fragment at least about 10 amino acids in length of the amino acid sequence of SEQ ID NO:6.
• the invention provides an isolated nucleic acid molecule for the third (29 kDa) subunit of the SDH enzyme of the invention comprising a polynucleotide at least about 95% identical to the isolated nucleic acid sequence for the third (29 kDa) subunit of the SDH enzyme of the invention described above.
• the invention provides an isolated nucleic acid molecule encoding both the first (75 kDa) and second (50 kDa) subunit proteins of the SDH enzyme of the invention comprising a polynucleotide sequence selected from the group consisting of: (a) the polynucleotide of SEQ ID NO:7; and (b) a polynucleotide fragment at least about 20 nucleotides in length of the polynucleotide of SEQ ID NO:7.
• the invention provides an isolated nucleic acid molecule for the first (75 kDa) and second (50 kDa) subunit proteins of the SDH enzyme of the invention comprising a polynucleotide at least about 95% identical to the isolated nucleic acid sequence for the first (75 kDa) and second (50 kDa) subunit proteins of the SDH enzyme of the invention.
• Another embodiment of the invention provides an isolated nucleic acid molecule for the third (29 kDa) subunit of the SDH enzyme of the invention comprising a polynucleotide sequence selected from the group consisting of: (a) the polynucleotide of SEQ ID NO:8; and (b) a polynucleotide fragment at least about 20 nucleotides in length of the polynucleotide of SEQ ID NO: 8.
• the invention provides an isolated nucleic acid molecule for the third (29 kDa) subunit of the SDH enzyme of the invention comprising an isolated nucleic acid molecule at least about 95% identical to the isolated nucleic acid molecule for the third (29 kDa) subunit of the SDH enzyme of the invention described above.
• isolated nucleic acid molecule is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment.
• recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention.
• Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
• Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
• RNA vectors may also be utilized with the SDH nucleic acid molecules disclosed in the invention. These vectors are based on positive or negative strand RNA viruses that naturally replicate in a wide variety of eukaryotic cells (Bredenbeek, P.J. and Rice, CM., Virology 3:297-310 (1992)). Unlike retroviruses, these viruses lack an intermediate DNA life-cycle phase, existing entirely in RNA form.
• alpha viruses are used as expression vectors for foreign proteins because they can be utilized in a broad range of host cells and provide a high level of expression; examples of viruses of this type include the Sindbis virus and Semliki Forest virus (Schlesinger, S., TIBTECH 77:18-22 (1993); Frolov, I., et al, Proc. Natl Acad. Sci. (USA) 93:11371-11377 (1996)).
• Sindbis virus and Semliki Forest virus Schond, S., TIBTECH 77:18-22 (1993); Frolov, I., et al, Proc. Natl Acad. Sci. (USA) 93:11371-11377 (1996).
• the investigator may conveniently maintain the recombinant molecule in DNA form (pSinrep5 plasmid) in the laboratory, but propagation in RNA form is feasible as well.
• the vector containing the gene of interest exists completely in RNA form and may be continuously propagated in that state if desired.
• the invention further provides variant nucleic acid molecules that encode portions, analogs or derivatives of the isolated nucleic acid molecules described herein.
• Variants include those produced by nucleotide substitutions, deletions or additions, which may involve one or more nucleotides.
• the variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions.
• Variants of the isolated nucleic acid molecules of the invention may occur naturally, such as a natural allelic variant.
• an allelic variant is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed.. John Wiley & Sons, New York (1985). Non-naturally occurring variants may be produced using art-known mutagenesis techniques.
• Isolated nucleic acid molecules of the invention also include polynucleotide sequences that are 95%. 96%, 97%, 98% and 99% identical to the isolated nucleic acid molecules described herein.
• Computer programs such as the BestFit program (Wisconsin Sequence Analysis Package, Nersion 10 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 5371 1) may be used to determine whether any particular nucleic acid molecule is at least 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequences disclosed herein or the the nucleotides sequences of the deposited clones described herein.
• BestFit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981 ), to find the best segment of homology between two sequences.
• a computer alignment program such as BestFit is utilized to determine 95% identity to a reference nucleotide sequence
• the percentage of identity is calculated over the full length of the reference nucleotide sequence and gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
• 95% identity indicates that as many as 5 of 100 nucleotides in the subject sequence may vary from the reference nucleotide sequence.
• the invention also encompasses fragments of the nucleotide sequences and isolated nucleic acid molecules described herein.
• the invention provides for fragments that are at least 20 bases in length.
• the length of such fragments may be easily defined algebraically.
• SEQ ID NO:l provides for an isolated nucleotide molecule that is 2, 265 bases in length.
• the isolated nucleic acid sequence fragments of the invention may single stranded or double stranded molecules.
• the invention discloses isolated nucleic acid sequences encoding the three subunit proteins of the SDH enzyme of the invention.
• Computer analysis provides information regarding the open reading frames, putative signal sequence and mature protein forms of each subunit.
• Genes encoding the first (75 kDa) and second (50 kDa) subunits are completely contained in a 5.7 kb Pst I fragment of the lambda GEM 5-1 clone, which is deposited in bacteria under the accession number KCTC 0593BP and as DNA under accession number KCTC 0597BP with the Korean Collection for Type Cultures (KCTC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), 52, Oun-Dong, Yusong-Ku, Taejon 305-33, Republic of Korea.
• the third (29 kDa) subunit gene is contained in a sequence 4.5 kb in length, referred to as Cla I-#69, which is deposited in bacteria under the accession number KCTC 0594BP and as DNA under accession number KCTC 0598BP with the Korean Collection for Type Cultures (KCTC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), 52, Oun-Dong, Yusong-Ku, Taejon 305-33, Republic of Korea.
• the invention provides an isolated nucleic acid molecule (KCTC 0597BP) carried in the novel strain KCTC 0593BP, and the invention also provides an isolated nucleic acid molecule (KCTC 0598BP) carried in the novel strain KCTC 0594BP.
• Sequence obtained from the lambda GEM 5-1, 5.7 kb Pst I fragment is presented in Figure 8 and assigned SEQ ID NO:7.
• the complete coding sequence for the first subunit gene is located at position 665-2,929 (SEQ ID NOT), with the signal sequence located at position 665-766, and the coding sequence for the mature protein of the SDH first subunit located at position 767-2,929 (SEQ ID NO:22).
• the complete coding sequence for the second subunit gene is located at position 2,964-4,400 (SEQ ID NO:2), with the signal sequence located at position 2,964-3,071, and the coding sequence for the mature protein of the SDH second subunit located at position 3,072-4,400 (SEQ ID NO:23).
• nucleic acid molecules derived from sequence obtained from the lambda GEM 5-1 , 5.7 kb Pst I fragment that is presented in Figure 8 and identified as SEQ ID NO:7.
• isolated nucleic acid molecules include the following: (1) nucleotides 1-664 of
• the sequence obtained from the Cla I-#69 clone is presented in Figure 10 and assigned SEQ ID NO: 8.
• the complete coding sequence for the third subunit gene is located at position 1 ,384-2,304 (SEQ ID NO:3), with the signal sequence located at position 1 ,384- 1 ,461 , and the coding sequence for the mature protein of the SDH third subunit located at position 1,462-2,304 (SEQ ID NO:24).
• nucleic acid molecules derived from sequence obtained from the Cla I-#69 clone that is presented in Figure 10 and assigned SEQ ID NO:8.
• isolated nucleic acid molecules include the following: (1) nucleotides 1-1,383 of SEQ ID NO:8 identified as SEQ ID NO:51 ; (2) nucleotides 50-1 ,383 of SEQ ID NO:8 identified as SEQ ID NO:52; (3) nucleotides 100-1.383 of SEQ ID NO:8 identified as SEQ ID NO:53; (4) nucleotides 150-1,383 of SEQ ID NO:8 identified as SEQ ID NO:54; (5) nucleotides 200-1,383 of SEQ ID NO:8 identified as SEQ ID NO:55; (6) nucleotides 250-1,383 of SEQ ID NO:8 identified as SEQ ID NO:56; (7) nucleotides 300-1,383 of SEQ ID NO:8 identified as SEQ ID NO:57; (8) nucleo
• the invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
• the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
• the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
• a promoter operably linked to the sequence.
• the following vectors are provided by way of example: Bacterial- pET (Novagen), pQE70, pQE60, pQE-9 (Qiagen), pBs, phagescript, psiX174, pBlueScript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); and Eukaryotic- pWLneo, pSV2cat, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).
• these and any other plasmids or vectors may be used as long as they are replicable and viable in the host.
• Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
• Two appropriate vectors are pKK232-8 and pCM7.
• Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda P R , P L and trp.
• Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
• the invention provides processes for producing the vectors described herein which comprises: (a) inserting the isolated nucleic acid molecule of the invention into a vector; and (b) selecting and propagating said vector in a host cell.
• Suitable hosts include, but are not limited to, bacterial cells, such as Gluconobacter. Brevibacterium, Corynebacterim, E. coli, Streptomyces, Salmonella typhimurium, Acetobacter, Pseudomonas,
• Pseudogluconobacter Bacillus and Agrobacterium cells
• fungal and yeast organisms including Saccharomyces, Kluyveromyces, Aspergillus and Rhizopus
• insect cells such as Drosophila S2 and Spodoptera Sf9 cells
• animal cells such as
• CHO, COS and Bowes melanoma cells CHO, COS and Bowes melanoma cells; and plant cells.
• Appropriate culture mediums and conditions for the above-described host cells are known in the art.
• the invention provides isolated polypeptide molecules for the SDH enzyme of the invention. Methods and techniques designed for the manipulation of isolated polypeptide molecules are well known in the art. For example, methods for the isolation and purification of polypeptide molecules are described
• the invention provides several isolated polypeptide molecules encoding the individual 75 kDa, 50 kDa, and 29 kDa subunit proteins of SDH enzyme of the invention.
• the particular isolated polypeptide molecules of the invention are described. Thereafter, specific properties and characteristics of these isolated polypeptide molecules are described in more detail.
• the invention provides an isolated polypeptide comprising a polypeptide sequence selected from the group consisting of: (a) the polypeptide sequence encoded in the polynucleotide sequence of SEQ ID NO: 1 ;
• polypeptide sequence of SEQ ID NO:4 (b) the polypeptide sequence of SEQ ID NO:4; and (c) a polypeptide at least about 10 amino acids long from the polypeptide sequence of SEQ ID NO:4.
• the invention provides an isolated polypeptide comprising a polypeptide sequence selected from the group consisting of: (a) the polypeptide sequence encoded in the polynucleotide sequence of SEQ ID NO:2;
• polypeptide sequence of SEQ ID NO:5 (b) the polypeptide sequence of SEQ ID NO:5; and (c) a polypeptide at least about 10 amino acids long from the polypeptide sequence of SEQ ID NO:5.
• the invention provides an isolated polypeptide comprising a polypeptide sequence selected from the group consisting of: (a) the polypeptide sequence encoded in the polynucleotide sequence of SEQ ID NO:3;
• polypeptide sequence of SEQ ID NO:6 (b) the polypeptide sequence of SEQ ID NO:6; and (c) a polypeptide at least about 10 amino acids long from the polypeptide sequence of SEQ ID NO:6.
• inventions include an isolated polypeptide sequence comprising the polypeptide encoded by the isolated nucleic acid sequence SEQ ID NO:7; an isolated polypeptide sequence comprising the polypeptide encoded by the isolated nucleic acid sequence SEQ ID NO:8; an isolated polypeptide sequence comprising the polypeptide encoded by the DNA clone contained in KCTC Deposit No. 0593BP; and an isolated polypeptide sequence comprising the polypeptide encoded by the DNA clone contained in KCTC Deposit No. 0594BP.
• isolated polypeptide is used herein to mean a polypeptide removed from its native environment. Thus, a polypeptide produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention. Also intended as an “isolated polypeptide” are polypeptides that have been purified, partially or substantially, from a recombinant host cell.
• Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
• the isolated polypeptides of the invention also include variants of those polypeptides described above.
• variants is meant to include natural allelic variant polypeptide sequences possessing conservative or nonconservative amino acid substitutions, deletions or insertions.
• variants is also meant to include those isolated polypeptide sequences produced by the hand of man, through known mutagenesis techniques or through chemical synthesis methodology. Such man-made variants may include polypeptide sequences possessing conservative or non-conservative amino acid substitutions, deletions or insertions.
• Amino acids in the protein of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081- 085 ( 989)).
• Isolated polypeptide molecules of the invention also include polypeptide sequences that are 95%, 96%, 97%. 98% and 99% identical to the isolated polypeptide molecules described herein.
• Computer programs such as the BestFit program (Wisconsin Sequence Analysis Package, Version 10 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711) may be used to determine whether any particular polypeptide molecule is at least 95%, 96%, 97%, 98% or 99% identical to the polypeptide sequences disclosed herein or the the polypeptide sequences encoded by the isolated DNA molecule of the deposited clones described herein.
• BestFit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 ( 1981 ), to find the best segment of homology between two sequences.
• a computer alignment program such as BestFit is utilized to determine 95% identity to a reference polypeptide sequence
• the percentage of identity is calculated over the full length of the reference polypeptide sequence and gaps in homology of up to 5% of the total number of amino acids in the reference sequence are allowed.
• 95% identity indicates that as many as 5 of 100 amino acids in the subject sequence may vary from the reference polypeptide sequence.
• the invention also encompasses fragments of the polypeptide sequences and isolated polypeptide molecules described herein.
• the invention provides for fragments that are at least 10 amino acids in length.
• the length of such fragments may be easily defined algebraically.
• SEQ ID NO:4 provides for an isolated polypeptide molecule that is 754 amino acids in length.
• Particularly preferred embodiments of the invention provide isolated polypeptides such as the following: (1) the full length polypeptide the SDH subunit 1 of the invention, encoded by the isolated nucleic acid molecule of SEQ ID NO: 1 and identified by SEQ ID NO:4; (2) the full length polypeptide the SDH subunit 2 of the invention, encoded by the isolated nucleic acid molecule of SEQ ID NO:2 and identified by SEQ ID NO:5; (3) the full length polypeptide the SDH subunit 3 of the invention, encoded by the isolated nucleic acid molecule of SEQ ID NO:3 and identified by SEQ ID NO:6; (4) the mature form of the SDH subunit 1 polypeptide of the invention, encoded by the isolated nucleic acid molecule of SEQ ID NO:22 and identified by SEQ ID NO:25; (5) the mature form of the SDH subunit 2 polypeptide of the invention, encoded by the isolated nucleic acid molecule of SEQ ID NO:24 and identified as SEQ ID NO:26; and the mature form of the
• the invention also provides a process for producing a polypeptide comprising: (a) growing the host cell containing the isolated nucleic acid molecule of SEQ ID NOT or variants thereof; (b) expressing the polypeptide encoded by said isolated nucleic acid molecule; and (c) isolating said polypeptide.
• the invention provides a process for producing a polypeptide comprising: (a) growing the host cell containing the isolated nucleic acid molecule of SEQ ID NO:2 or variants thereof; (b) expressing the polypeptide encoded by said isolated nucleic acid molecule; and (c) isolating said polypeptide.
• Another process provided by the invention is for the production of a polypeptide which comprises: (a) growing the host cell containing the isolated nucleic acid molecule of SEQ ID NO: 3 or variants thereof; (b) expressing the polypeptide encoded by said isolated nucleic acid molecule; and (c) isolating said polypeptide.
• bacterial cells such as Gluconobacter, Brevibacterium, Corynebacterim, E. coli, Streptomyces, Salmonella typhimurium, Acetobacter, Pseudomonas, Pseudogluconobacter, Bacillus and Agrobacterium cells; fungal and yeast organisms including Saccharomyces, Kluyveromyces, Asperg ⁇ llus and Rhizopus; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells.
• bacterial cells such as Gluconobacter, Brevibacterium, Corynebacterim, E. coli, Streptomyces, Salmonella typhimurium, Acetobacter, Pseudomonas, Pseudogluconobacter, Bacillus and Agrobacterium cells
• fungal and yeast organisms including Saccharomyces, Kluyveromyces, Asperg ⁇ llus
• the polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
• Polypeptides of the invention may be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography. hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
• HPLC high performance liquid chromatography
• the invention provides processes for the production of L-sorbose and
• the invention provides a process for the production of L-sorbose from D-sorbitol comprising: (a) transforming a host cell with at least one isolated nucleotide sequence selected from the group consisting of: (i) a polynucleotide comprising the polynucleotide sequence of SEQ ID NOT ; (ii) a polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2; and (iii) a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 3; and (b) selecting and propagating said transformed host cell.
• the invention provides a process for the production of 2-keto-L-gulonic acid comprising: (a) transforming a host cell with at least one isolated nucleotide sequence selected from the group consisting of: (i) a polynucleotide comprising the polynucleotide sequence of SEQ ID NO : 1 ; (ii) a polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2; and
• Suitable bacteria for use as host cells in the processes provided herein for the production of L-sorbose and 2-keto-L-gulonic acid are known to those skilled in the art.
• Such bacteria include, but are not limited to, Escherichia coli,
• Other host cells for expression of the SDH enzyme of the invention include: strains identified in U.S. Patent NO. 5,834,231 ; Gluconobacter oxydans DSM 4025 (U.S. Patent No. 4,960,695); Gluconobactor oxydans TIOO (Appl.
• L-sorbose and 2-keto-L-gulonic acid may be produced by fermentation processes such as the batch type or of the fed-batch type.
• batch type fermentations all nutrients are added at the beginning of the fermentation.
• fed-batch or extended fed-batch type fermentations one or a number of nutrients are continuously supplied to the culture, right from the beginning of the fermentation or after the culture has reached a certain age, or when the nutrient(s) which are fed were exhausted from the culture fluid.
• a variant of the extended batch of fed-batch type fermentation is the repeated fed-batch or fill-and-draw fermentation, where part of the contents of the fermenter is removed at some time, for instance when the fermenter is full, while feeding of a nutrient is continued.
• the continuous fermentation or chemostat culture uses continuous feeding of a complete medium, while culture fluid is continuously or semi-continuously withdrawn in such a way that the volume of the broth in the fermenter remains approximately constant.
• a continuous fermentation can in principle be maintained for an infinite time.
• an organism grows until one of the essential nutrients in the medium becomes exhausted, or until fermentation conditions become unfavorable (e.g. the pH decreases to a value inhibitory for microbial growth).
• measures are normally taken to maintain favorable growth conditions, e.g. by using pH control, and exhaustion of one or more essential nutrients is prevented by feeding these nutrient(s) to the culture.
• the microorganism will continue to grow, at a growth rate dictated by the rate of nutrient feed. Generally a single nutrient, very often the carbon source, will become limiting for growth. The same principle applies for a continuous fermentation, usually one nutrient in the medium feed is limiting, all other nutrients are in excess. The limiting nutrient will be present in the culture fluid at a very low concentration, often unmeasurably low. Different types of nutrient limitation can be employed. Carbon source limitation is most often used. Other examples are limitation by the nitrogen source, limitation by oxygen, limitation by a specific nutrient such as a vitamin or an amino acid (in case the microorganism is auxotrophic for such a compound), limitation by sulphur and limitation by phosphorous.
• Suitable supplemental carbon sources include, but are not limited to: other carbohydrates, such as glucose, fructose, mannitol, starch or starch hydrolysate, cellulose hydrolysate and molasses; organic acids, such as acetic acid, propionic acid, lactic acid, formic acid, malic acid, citric acid, and fumaric acid; and alcohols, such as glycerol.
• other carbohydrates such as glucose, fructose, mannitol, starch or starch hydrolysate, cellulose hydrolysate and molasses
• organic acids such as acetic acid, propionic acid, lactic acid, formic acid, malic acid, citric acid, and fumaric acid
• alcohols such as glycerol.
• suitable nitrogen sources include, but are not limited to: ammonia, including ammonia gas and aqueous ammonia; ammonium salts of inorganic or organic acids, such as ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium sulfate and ammonium acetate; urea; nitrate or nitrite salts, and other nitrogen-containing materials, including amino acids as either pure or crude preparations, meat extract, peptone, fish meal, fish hydrolysate, corn steep liquor, casein hydrolysate, soybean cake hydrolysate, yeast extract, dried yeast, ethanol-yeast distillate, soybean flour, cottonseed meal, and the like.
• ammonia including ammonia gas and aqueous ammonia
• ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium sulfate and ammonium acetate
• urea nitrate
• suitable inorganic salts include, but are not limited to: salts of potassium, calcium, sodium, magnesium, manganese, iron, cobalt, zinc, copper and other trace elements, and phosphoric acid.
• suitable trace nutrients, growth factors, and the like include, but are not limited to: coenzyme A, pantothenic acid, biotin, thiamine, riboflavin, flavine mononucleotide, flavine adenine dinucleotide, other vitamins, amino acids such as cysteine, sodium thiosulfate, p-aminobenzoic acid, niacinamide, and the like, either as pure or partially purified chemical compounds or as present in natural materials. Cultivation of the inventive microorganism strain may be accomplished using any of the submerged fermentation techniques known to those skilled in the art, such as airlift, traditional sparged-agitated designs, or in shaking culture.
• the culture conditions employed may be determined empirically by one of skill in the art to maximize L-sorbose and 2-keto-L-gulonic acid production.
• the selection of specific culture conditions depends upon factors such as the particular inventive microorganism strain employed, medium composition and type, culture technique, and similar considerations.
• Illustrative examples of suitable methods for recovering 2-KLG are described in U.S. Pat. Nos. 5,474,924; 5.312,741 ; 4,960,695; 4,935,359; 4,877,735; 4,933,289; 4,892,823; 3,043,749; 3,912,592; 3,907,639and3,234,105.
• the microorganisms are first removed from the culture broth by known methods, such as centrifugation or filtration, and the resulting solution concentrated in vacuo.
• Crystalline 2-KGL is then recovered by filtration and, if desired, purified by recrystallization.
• 2-KGL can be recovered using such known methods as the use of ion-exchange resins, solvent extraction, precipitation, salting out and the like.
• 2-KGL is recovered as a free acid, it can be converted to a salt, as desired, with sodium, potassium, calcium, ammonium or similar cations using conventional methods.
• 2-KGL when 2-KGL is recovered as a salt, it can be converted to its free form or to a different salt using conventional methods.
• G suboxydans KCTC 21 1 1 was inoculated into 5 ml of SYP medium (5% D-sorbitol, 1% BactoPeptone and 0.5% yeast extract) and incubated at 30°C for 20 hours. One milliliter (ml) of this culture were transferred to 50 ml of the same medium in a 500 ml flask and cultivated at 30°C for 20 hours on a rotary shaker
• the culture thus prepared was used as an inoculum for a 5 L jar fermentor containing 3 L of the same medium, and the 3 L culture was grown to early stationary phase.
• Cells were harvested by centrifugation at 12,000 g for 10 min, washed once with 10 mM sodium acetate buffer (pH 5.0) and disrupted with glass beads (0.1 mm in diameter) in a bead beater (Edmund Buhler, Vi 4) for 90 sec at 4°C. The homogenate thus prepared was centrifuged at 12,000 g for 5 min to remove cell debris and glass beads. The resulting supernatant was centrifuged at 100,000 g for 60 min, and a crude membrane fraction was obtained as a precipitate.
• Enzyme activity was assayed spectrophotometrically using 2,6- dichlorophenol indophenol (DCIP) as an artificial electron acceptor and phenazine methosulphate (PMS) as an electron mediator.
• the reaction mixture contained 50 mM sodium acetate buffer (pH 5.0), 10 mM MgCl 2 , 5 mM CaCl 2 ,
• Enzyme activity was determined also by the Ferric-Dupanol method (Wood, W.A. etal, Meth. Enzymol. 5:287 (1962)) for the reconstituted subunits described in step 6 of Example 1. Subunit proteins were preincubated either singly or in different combinations for 5 min at 25°C. The reaction was started by the addition of 10 mM (final concentration) of potassium ferricyanide and 250 mM (final concentration) D-sorbitol.
• the reaction was stopped by adding the ferric sulfate-Dupanol solution (Fe 2 (SO 4 ) 3 nH 2 O 5g/L, Kunol (sodium lauryl sulfate) 3g/L and 85% phosphoric acid 95ml/L), and the absorbance of the Prussian color was determined at 660 nm in a spectrophotometer.
• CM-TSK column was eluted with a linear gradient (from 10 mM to 500 mM) of sodium acetate. Active fractions were pooled and concentrated by ultrafiltration using a membrane filter (Amicon, YM 10) and loaded onto a DEAE-TSK 650 (S) (Merck) column (2.5 x 20 cm). The DEAE-TSK column was eluted isocratically with a 10 mM sodium acetate buffer of either pH 5.0 or pH 6.0.
• Figure 2-A shows fractions of peak I.
• Column M shows standard molecular weight markers. As shown in columns 1 through 4, the 75 kDa first subunit band and the 50 kDa second subunit band could be observed but the 29 kDa third subunit band was not observed. In this case, one hour after elution, the enzyme activity was reduced by more than ten fold.
• Figure 2-B shows the fractions of peak II. Column M shows standard molecular weight markers.
• fractions of peak II contained the 29 kDa third subunit band in addition to the 75 kDa and 50 kDa subunits. This observation indicated that the 29 kDa third subunit might play an important role in the stability of SDH.
• the purified SDH prepared in Example 1 was subjected to SDS-PAGE (12.5% gel), and the separated proteins were electroblotted onto a polyvinylidene difluoride (PNDF) membrane (Bollag, D. M. and Edelstein, S. J., Protein Methods, Wiley-Liss, Inc., 8 (1991)). After visualization with ponceau S stain, the section of membrane containing each SDH subunit was cut into pieces, and the membrane pieces for each subunit were applied directly to an amino acid sequence analyzer (Applied Biosystems, Model 477 A) for ⁇ -terminal amino acid sequence analysis.
• PNDF polyvinylidene difluoride
• the resultant data for the first and the third subunits are shown in Table 3 (SEQ ID ⁇ os:9 and 11).
• the N-terminal amino acid sequence of the second subunit could not be obtained, most likely because of a blocked N-terminus.
• a similar finding of a blocked N-terrninus was reported for the cytochrome c subunit of the alcohol dehydrogenase complex of another acetic acid bacterium, Acetobacter pasteurianus (Takemura, H. et al., J.
• the isolated SDH protein was first treated with pyroglutamate aminopeptidase to release the potentially blocked N-terminus. Briefly, about 50 pg of purified SDH from Example 1 was dissolved in a digestion buffer ( 100 mM sodium phosphate buffer, 10 mM EDTA.
• the third subunit internal amino acid sequences were also determined in addition to the N-terminal amino acid sequence for the facilitated cloning of the corresponding gene.
• the tryptic digest was separated by HPLC using a Brownlee SPHERI-5 58-
• primer 1 5' - CCGGAATTC GAA(G) GAT(C) ACI GGI ACI GC-3') (SEQ ID NO: 15) and primer 2 (5'-ATT(C,A) ACI AAT(C) GCI GAT(C) CAAG) CAT(C) CC-3')(SEQ ID NO: 16), were synthesized.
• the genomic DNA isolated from G. suboxydans KCTC 21 11 using the method of Takeda and Shimizu was partially digested with BamHI.
• the plasmid pBluescript SK (Stratagene) was restricted withNael and BamHI and the BamHI partial digest of genomic DNA was ligated to the BamHI site of the plasmid.
• PCR polymerase chain reaction
• the ligation reaction mixture prepared above was amplified with a gene specific primer, the primer 1 , and the T7 primer of the vector.
• the generic T7 primer anneals to the ends of all ligated fragments, the resulting products increase only linearly.
• simultaneous annealing of the gene specific primer 1 and T7 primer to the specific product results in exponential amplification of the specific primary product.
• Secondary PCR carried out with a nested primer, the gene specific primer 2, and the T7 primer generated a specific secondary product, which confirmed the specificity of the primary PCR.
• the PCR product was ligated to plasmid pT7 Blue (Novagen) and transformed into Escherichia coli DH5a by SEM protocol (Inoue, H. et al., Gene, 96:23 (1990)). Transformants were cultivated in an LB medium (1% Bacto- Tryptone, 0.5% yeast extract and 1% NaCl) supplemented with 100 ⁇ g/ml of ampicillin. Subsequently, the plasmid was extracted by alkaline lysis method
• #SDH2-1 contained only a part of the first subunit gene.
• the #SDH2-1 isolated in Example 4 was labeled with DIG Labeling and Detection Kit (The DIG System User's Guide for Filter Hybridization, p. 6-9, Boehringer Mannheim (1993)).
• DIG Labeling and Detection Kit The DIG System User's Guide for Filter Hybridization, p. 6-9, Boehringer Mannheim (1993)
• To construct a genomic DNA library of G suboxydans KCTC 2111 the genomic DNA isolated from G suboxydans KCTC 21 1 1 using the method described in Example 4 was partially digested with Sau3AI. Partially digested DNA was electrophoresed in a 0.8% agarose gel and the DNA of 15 to 23 kb in size was eluted using QLAEX II Gel Extraction Kit (QIAGEN) .
• the eluted DNA was then ligated into the BamHI site of Lambda GEM- 1 1 vector (Promega).
• the ligation mixture was packaged into phage lambda particles using the Packagene In Vitro Packaging System (Promega) according to the instruction manual.
• E. coli LE392 cells were grown in TB medium ( 1 % Bacto-Tryptone and 0.5% NaCl) supplemented with 0.2% maltose and 10 mM MgSO 4 , at 30°C and stored at 4°C when the OD 600 had reached 0.6.
• the packaging mixture was added to the cell suspension, and the mixture was incubated for 30 min at 37°C to allow infection.
• the lambda phage plaques were immobilized on nylon membranes
• the lambda DNAs were isolated from positive lambda clones and purified with Lambda DNA Purification Kit (Stratagene).
• the isolated lambda DNAs were digested with BamHI and subjected to a 0.7% agarose gel electrophoresis.
• the DNA fragments separated on the gel were transferred onto a nylon membrane and analyzed again by Southern hybridization with #SDH2- 1 as probe under the same condition as described above.
• a clone which gave a positive signal was selected, and the insert DNA of 15 kb was excised with Xhol from this clone and subsequently cloned into the Xhol site of pBluescript SK give Lambda GEM 5-1.
• the Lambda GEM 5-1 clone was mapped by digestion with several different restriction enzymes.
• Figure 6 presents the restriction enzyme map of Lambda GEM 5-1.
• the positive clone, Lambda GEM 5-1 , obtained in Example 5 was mapped with several different restriction enzymes and analyzed by Southern hybridization as described in Example 5.
• a 5.7 kb Pstl fragment hybridizing with #SDH2-1 was subcloned, and the nucleotide sequence was determined.
• three overlapping subclones, SI, S2 and S3, were constructed using restriction enzyme sets, Kpnl-Rs l, Notl-Sacll and Rstl-Sacl, respectively, as shown in Figure 7.
• a set of deletion clones was prepared for each subclone using Exo III-Mungbean Deletion Kit (Stratagene).
• the nucleotide sequencing reaction was done by Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems), and the sequence was determined in an automatic DNA sequencer (Applied Biosystems, Model 373 A).
• Figure 8 shows the nucleotide sequence of 4,830 bp in the 5.7 kb Pstl fragment (SEQ ID NO:7).
• the sequenced DNA contains two open reading frames (ORFs) of 2,265 and 1,437 nucleotides.
• the first ORF encodes the first subunit.
• the first subunit gene is preceded by a Shine-Dalgarno sequence, "AGGA" positioned at 651-654 bp.
• the 34 amino acid signal sequence of the first subunit is positioned at 665-766 bp of SEQ ID NO:7.
• the coding sequence of the mature part of the first subunit protein is positioned at 767-2,929 bp of SEQ ID NO:7, which encodes a 720 amino acid polypeptide whose derived N- terminal amino acid sequence is in perfect agreement with 15 amino acid residues obtained by N-terminal amino sequence analysis.
• the first ORF was followed by the second ORF, the two ORF's being interrupted by a short intergenic region.
• the second ORF encodes the second subunit.
• the Shine-Dalgarno sequence (AGGA) of the second subunit gene is found at 2,950-2,953 bp SEQ ID NO:8. and the structural gene is positioned at 2,964-4,400 bp of SEQ ID NO:8.
• the second subunit gene encodes a 478 amino acid polypeptide, including a signal sequence of 36 amino acids.
• the derived amino acid sequence of the mature polypeptide showed a perfect match with the experimentally obtained sequence for the sample treated with pyroglutamate aminopeptidase. Downstream of the stop codon of the second subunit gene, inverted repeat sequences are found.
• the calculated molecular weights of the mature proteins of the first and the second subunit are 79 kDa and 48 kDa, respectively, which are in good agreement with the experimental values of 75 kDa and 50 kDa, respectively, as determined by SDS-PAGE.
• signature PQQ-binding sequences consensus sequences appearing characteristically in the amino- and carboxy- terminals of PQQ-dependent dehydrogenases, are present in the first subunit gene.
• the signature PQQ-binding sequence in amino-terminal part is shown as Sequence ID NO: 17, and the signature sequence existing in the carboxy- terminal part is shown as Sequence ID No: 18 (Here, X represents an arbitrary amino acid.).
• the amino-terminal signature sequence occurs at position 812-898 bp, and the carboxy-terminal signature sequence occurs at position 1,490-1,555 bp.
• X A represents an arbitrary amino acid.
• X B is an arbitrary amino acid different from X A ).
• a database search for homologous sequences determined that the DNA sequence of the first subunit gene showed a great degree of similarity to many dehydrogenases containing pyrroloquinoline quinone (PQQ) as cofactor:
• PQQ pyrroloquinoline quinone
• the alcohol dehydrogenases of Acetobacter polyoxogenes Tamaki, T. et al, Biochim. Biophys. Ada, 1088, 292 (1991)
• Acetobacter aceti Inoue, T. et al, J. Bacteriol , 171 , 3115 ( 1989)
• a search of the database with the second subunit gene provided the greatest degree of similarity, matching the cytochrome c of G. suboxydansIFO 12528 (Takeda and Shimizu, J Eerw. Bioeng, 72: ⁇ (1991)) with a nucleotide sequence identity of 83% and an amino acid sequence identity of 88%.
• a PCR reaction was done with primers 3 and 4 using genomic DNA of G suboxydans KCTC 2111 prepared by the method of Takeda and Shimizu (Takeda and Shimizu, J. Ferm. Bioeng.. 72 1 (1991)) as template.
• the reaction generated a 320 bp DNA fragement.
• This 320 bp PCR product was ligated to pBluescript SK (Stratagene) and transformed into E. coli DH5ar by the SEM protocol (Inoue, H. et al, Gene, 96, 23 (1990)). Transformants were cultivated in an LB medium supplemented with 100 ⁇ g/ml of ampicillin. Subsequently, the plasmid was extracted by the alkaline lysis method (Sambrook, J. et al,
• the membrane was prehybridized in a hybridization oven (Hybaid) using a prehybridization solution (5X SSC, 1% (w/v) blocking reagent, 0.1% N-lauroylsarcosine, 0.2% SDS and 50% (v/v) formamide) at 42 °C for 2 hours. Then the membrane was hybridized using a hybridization solution (DIG-labeled probe diluted in the prehybridization solution) at 42 °C for 12 hours. Southern hybridization gave a strong discrete signal for each enzyme used. DNA corresponding to the positive signal at 4.5 kb Clal was eluted and cloned into pBluescript SK to construct a mini-library. The mini-library was screened for the positive clone by repeating the Southern hybridization as described above. A clone which gave a positive signal was selected and designated Clal-#69.
• Figure 9 presents the restriction enzyme map of CM-#69.
• the nucleotide sequence of the third subunit gene in the 4.5 kb Clal-#69 clone was determined and analyzed. To facilitate the DNA sequencing, several overlapping restriction fragments were subcloned, and the nucleotide sequence of each clone determined. The nucleotide sequencing reaction was done by Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems), and the sequence was determined in an automatic DNA sequencer (Applied Biosystems, Model 373A).
• Figure 10 shows the nucleotide sequence of 2700 bp of the 4.5 kb Clal fragment (SEQ ID NO:8).
• the sequenced DNA contains an open reading frame (ORF) of 921 nucleotides, which encodes the third subunit polypeptide.
• the third subunit gene is preceded by a potential Shine-Dalgarno sequence (AGG) positionedat l,375-l,377 bp.
• AAG potential Shine-Dalgarno sequence
• the amino acid signal sequence of the third subunit polypeptide is positioned at 1,384-1,461 bp.
• the coding sequence of the mature part of the third subunit protein is positioned at 1,462-2,304 bp, encoding a 280 amino acid polypeptide whose derived N-terminal amino acid sequence was in perfect agreement with the 25 amino acid residues obtained by N-terminal amino sequence analysis.
• the calculated molecular weight of the mature protein of the third subunit is 29,552 Da, which was in good agreement with the experimental value of 29 kDa determined by SDS-PAGE.

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