CN112680367B - Aspergillus niger strain for producing transglucosidase - Google Patents

Abstract

The invention discloses an Aspergillus niger strain for producing transglucosidase, which comprises a saccharifying enzyme coding gene and an alpha-glucosidase coding gene with higher hydrolytic activity in the Aspergillus niger high transglucosidase activity transglucosidase coding gene to replace the saccharifying enzyme coding gene and the alpha-glucosidase coding gene with higher hydrolytic activity in the Aspergillus niger strain, so as to obtain a new transglucosidase high-yield strain. The strain can synthesize and secrete high-level transglucosidase, the glucose group hydrolysis activity in the produced enzyme preparation is low, the produced enzyme preparation can be used for efficient and low-cost preparation of the transglucosidase, and the prepared enzyme preparation can be used for production of isomaltooligosaccharide and remarkably improves the quality of the isomaltooligosaccharide.

Description

Aspergillus niger strain for producing transglucosidase

Technical Field

The invention belongs to the field of genetic engineering and enzyme engineering, and particularly relates to an aspergillus niger strain capable of highly producing transglucosidase and application thereof.

Background

Isomaltooligosaccharides (IMO) are functional oligosaccharides with the largest production scale and wide industrial application range in functional oligosaccharides, mainly comprise isomaltobiose, isomaltotriose and panose (shortly: ditripan), and are prepared by using starch as a raw material and performing enzymatic conversion. The general production process is as follows: the starch is converted into maltodextrin under the action of starch liquefying enzyme, and then into isomaltose hypgather under the action of transglucosidase. The key enzyme preparation prepared by the isomaltooligosaccharide enzyme method is transglucosidase, and the transglucosidase is used for catalyzing and converting starch hydrolysates such as maltose, malto-oligosaccharide and the like to form the isomaltooligosaccharide. The quality and the enzyme activity purity of the transglucosidase preparation directly determine the final ratio and the preparation efficiency of main components of isomaltobiose, isomaltotriose and panose in the preparation process of the maltooligosaccharide.

Aspergillus niger is the main enzyme source strain of transglucosidase, but Aspergillus niger also synthesizes and secretes higher levels of saccharifying enzymes, amylases, etc. simultaneously. Due to the starch hydrolase synthesized by Aspergillus niger, especially saccharifying enzyme and a plurality of alpha-glucosidase, maltodextrin after starch liquefaction can be directly hydrolyzed into glucose. Therefore, if the transglucosidase preparation prepared by aspergillus niger contains glucoamylase and alpha-glucosidase enzyme activity, the conversion efficiency of isomaltooligosaccharide in the production process is reduced, the product quality does not reach the standard, and the product quality can reach the standard only by subsequent steps of separation and purification and the like, so that the conversion rate from a substrate to a product is reduced, and the production cost is also obviously increased.

According to the invention, firstly, through molecular cloning and expression, the hydrolytic activity and the transglycosylation activity of a plurality of alpha-glucosidase in Aspergillus niger are determined, the synthesis level of the transglucosidase in Aspergillus niger is further enhanced through a gene recombination technology, simultaneously, the glucoamylase and the alpha-glucosidase only having the hydrolytic activity are deleted, the Aspergillus niger production strain with high yield of the transglucosidase is obtained, the prepared enzyme preparation has high transglucosidase activity, the effect of being applied to preparing the oligomeric maltose from starch is obvious, and the utilization rate of raw materials and the quality of products are obviously improved.

Disclosure of Invention

The invention aims to provide an Aspergillus niger high-yield strain with high transglucosidase yield and application thereof, and the Aspergillus niger high-yield strain with remarkably reduced hydrolytic activity and remarkably improved transglucosidase synthesis capacity is obtained and is used for efficiently preparing high-quality transglucosidase and applied to the enzymatic preparation of isomaltooligosaccharide.

In order to achieve the aim, the invention adopts the technical scheme that,

by molecular cloning and expression, against Aspergillus nigerAspergillus niger) Performing functional identification on alpha-glucosidase in An-F308 to obtain transglucosidase with the best transglycosidation activity and a coding gene thereof; the glucoamylase encoding gene and the alpha-glucosidase encoding gene with hydrolytic activity in the Aspergillus niger genome are replaced by the transglucosidase encoding gene with the best transglycosidase activity through a gene replacement method, so that the Aspergillus niger strain with high transglucosidase yield is obtained.

Said Aspergillus nigerAspergillus niger) An-F308, obtained by natural isolation culture and identification, has the capability of synthesizing transglucosidase, and is preserved in China general microbiological culture Collection center (CGMCC) at 11/26/2020 with the address: no.3 of the Xilu No.1 Beijing, Chaoyang, the preservation number is CGMCC NO. 21028.

The transglucosidase and the coding gene thereof are from Aspergillus niger (A.niger)Aspergillus niger) An-F308 is obtained by gene cloning, expression, function confirmation and nucleotide sequence determination, and has the characteristics of a nucleotide sequence SEQ ID NO.1, and An amino acid sequence of the An-F308 has the characteristics of a sequence SEQ ID NO. 2.

The aspergillus niger strain for high yield of transglucosidase is prepared by a traceless self-gene replacement method by means of a saccharifying enzyme gene in An a-F308 genome of aspergillus nigerglaA) And alpha-glucosidase-encoding gene (a)agF) The upstream and downstream sequences (nucleotide sequence SEQ ID number 3 and nucleotide sequence SEQ ID number 4) of the gene of Aspergillus niger transglucosidase (nucleotide sequence SEQ ID number 1) are integrated and replaced in the genome of Aspergillus niger An-F308glaAAndagFthe obtained aspergillus niger strain with high yield of transglucosidase is An-agDIII; on the level of shake flask fermentation, the highest transglucosidase fermentation enzyme activity of An-agDIII strain can reach 4882U/mL, only trace glycosylation enzyme activity (115U/mL) is produced, and the transglycosidation activity is more than 97.6%;

the fermentation production process of the transglucosidase comprises the following steps of: 3-20% of starch dextrin, 1-8% of bean cake powder, 0-5% of corn steep liquor, 0-3% of ammonium sulfate and 0.01-2% of calcium chloride; the fermentation temperature is 25-36 ℃, the dissolved oxygen is maintained to be 5-60% in the fermentation process, the pH is controlled to be 4-6 by using sulfuric acid or ammonia water in the fermentation process, and the fermentation time is 60-150 hours. Under the condition, the transglucosidase-producing enzyme activity of the An-agDIII strain reaches 49986-50820U/mL in a 30L fermentation system.

The isomaltose hypgather prepared by the transglucosidase obtained by the method contains 40-49% of isomaltose (IG 2), panose (P) and isomaltotriose (IG 3) in dry matter.

The invention has the beneficial effects that:

1. the invention is prepared byagDGene integration expression in Aspergillus nigerglaAAndagFgene locus and synchronous deletionglaAAndagFthe gene successfully constructs a transglucosidase high-yield strain, and the transglucosidase activity ratio is improved to 97.6 percent under the condition of shaking the bottle;

2. the highest enzyme production level of the transglucosidase high-yield strain obtained by the invention reaches 50820U/mL, and only contains lower starch hydrolase activity (-246U/mL);

3. the prepared transglucosidase is used for carrying out industrial production of isomaltooligosaccharide, and the content of IG2+ P + IG3 in the prepared isomaltooligosaccharide syrup is up to 49% of dry matter.

Drawings

FIG. 1 recombinant plasmid pZA: cagD.

FIG. 2 shows the recombinant plasmid pZF: cagD.

FIG. 3 HPLC chromatogram of the resulting isomaltooligosaccharide syrup, (DP 1 glucose, DP2 maltose, IG₂Isomaltose, DP3 maltotriose, P panose, IG₃Isomaltotriose, IG₄Isomaltotetraose).

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail with reference to specific embodiments below. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not intended to limit the present invention.

The present invention is further illustrated by the following examples.

(1) Separation and purification of Aspergillus niger total RNA

Aspergillus niger An-F308 is inoculated in YPD medium (2% yeast extract, 2% glucose, 2% peptone), is cultivated for 16 h at 32 ℃, collects the thalli and freezes the back at-70 ℃, grinds in dry ice, adopts the TRIZol Plus RNA purification kit of Thermofisiher company to retrieve total RNA again.

(2) Gene cloning and functional identification of transglucosidase and related enzymes

Synthesizing cDNA from the purified Aspergillus niger total RNA by using a Reverse Transcriptase Kit EnzChek Reverse Transcriptase Assay Kit, using the cDNA as a template and using primer pairs AGA-1/AGA-2, AGB-1/AGB-2, AGC-1/AGC-2, AGD-1/AGD-2, AGE-1/AGE-2 and AGF-1/AGF-2 (table 1) respectively, and amplifying to obtain related coding genesagA，agB，agC，agD，agEAndagF。

TABLE 1 nucleic acid sequence List of primers used in the present invention

The obtained encoding gene of alpha-glucosidaseagA，agB，agC，agD，agEAndagFcloned into the pPIC9K (Invitrogen) plasmidSnaBI/AvrII site obtaining recombinant plasmid pPIC-agA，pPIC-agB，pPIC-agC，pPIC-agD，pPIC-agEAnd pPIC-agF(ii) a Warp beamSacI linearization the manner of shock transformation was integrated in pichia pastoris GS115 (Invitrogen). And positive transformants are obtained by histidine auxotrophy screening and G418 resistance screening; the cells were inoculated into 50 mL of BMGY medium (1% yeast powder, 2% peptone, 100 mM potassium phosphate buffer, 1.34% YNB, 4X 10 cells) at 1% inoculum size^-5% biotin, 1% glycerol; pH =6), 30 ℃, 180 r/min to OD₆₀₀Centrifuge (ca. 16-18 h), using 1/5-1/10 volumes of BMMY (1% yeast powder, 2% peptone, 100 mM potassium phosphate buffer, 1.34% YNB, 4X 10) in original medium^-5% biotin, 0.5% methanol; pH =6) suspension of the cells to OD₆₀₀=1, culturing, 0.5% methanol induction, fermenting for 144 h; after the fermentation is finishedCentrifuging and taking the supernatant to obtain recombinant enzyme liquid for subsequent enzyme activity determination and analysis.

(3) Enzyme activity assay

Transglucosidase enzyme activity was measured according to GB 1886.174-2016. Alpha-methyl glucose is used as a substrate. The enzymatic method is defined as: under the conditions of 40 ℃ and pH 5.0, 1 mL of enzyme sample reacts with a substrate alpha-methyl glucose, and 1 mu g of glucose is generated in 60 min, namely 1 enzyme activity unit expressed by U/mL or U/g.

And (3) measuring the enzyme activity of the saccharifying enzyme and the alpha-glucosidase according to the national standard GB 1886.174-2016. Soluble starch is used as a substrate. The enzyme activity is defined as: 1 mL of enzyme solution or 1 g of enzyme powder hydrolyzes soluble starch for 1 hour under the conditions of 40 ℃ and pH 4.6 to generate 1 mg of glucose, namely an enzyme activity unit expressed by U/mL (or U/g).

(4) Construction of high-yield transglucosidase Aspergillus niger

Molecular cloning, DNA ligation, transformation, nucleotide sequence determination, restriction mapping, preparation of Aspergillus niger genome, PCR gene amplification, etc. were performed according to the conventional Laboratory methods (Zhu Jian, King Zheng Xiang. Industrial microbial Experimental technical Manual [ M ]. Beijing: China light industry Press, 1994: 413-450.; Sambrook J, Russell D W. Molecular cloning: a Laboratory Manual, 2001, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY.). Genetic transformation of Aspergillus niger was carried out by the electrotransformation method according to [ Pinus salicinae, king, J.Microbiol., 2010, 01: 11-15 ] study of electrotransformation conditions of Aspergillus niger.

The construction of the recombinant plasmid pAUR-kan was carried out by the following procedure. By usingEcoRI andPstplasmid pRS305K (Taxis C, Knop M. System of centromeric, epidemic, and integral vector based on drug resistance markers for)Saccharomyces cerevisiaeBiotechniques, 2006: 40, 73-78) and usePfuDNA polymerase filling-up, gel recovering 1.25 kb G418 resistance coding gene sequence fragment, and mixing with the obtained extractPstI digestion to remove the resistance coding gene of aureobasidin (Aba) and filling in plasmid pAUR135 (TaKaRa company, Japan) for connection, thus obtaining recombinant plasmid pAUR-kan.

The construction of the gene-traceless replacement recombinant plasmid was carried out as follows. Aspergillus niger An-F308 genome DNA is taken as a template, primer GlaA-1p/GlaA-2p is adopted, PCR amplification is carried outglaA(Yaotong et al. Microbiol. Productions, 2005) promoter region (PglaA) and PCR amplification Using primers GlaA-1t/GlaA-2tglaATerminator and its downstream sequence (TglaA), then through PCR amplification technology, adopting primer GlaA-1p/GlaA-2t to make the obtained PglaA and TglaA fragments andagDfusion was performed to obtain PglaA-cagD-TglaA (nucleotide sequence SEQ ID NO: 3), and this fragment was cloned into pAUR-kanSmaI site, obtaining recombinant plasmid pZA as shown in the specification, cagD. Similarly, the Aspergillus niger genome is amplified by PCR technologyagFThe promoter (primer is AgF-1p/AgF-2 p) and the terminator sequence (primer is AgF-1t/AgF-2 t), and PCR technology and the primers AgF-1p/AgF-2t andagDcarrying out fusion to obtain PagF-cagD-TagF (nucleotide sequence SEQ ID NO: 4), and carrying out gene recombination to obtain a recombinant plasmid pZF:: cagD.

Recombinant expression plasmids transformation of Aspergillus niger and selection of positive transformants were performed according to the instructions of pAUR135, TaKaRa. The constructed recombinant plasmid pZA is transformed into An Aspergillus niger An-F308 strain and screened, the screening is firstly carried out on YPD plates (1 percent of yeast extract, 2 percent of peptone and 2 percent of glucose) containing 0.5 g/L G418, and then is carried out on YPG plates (1 percent of yeast extract, 2 percent of peptone and 1 percent of galactose) containing 1wt.% of galactose to obtain the recombinant plasmid pZAagDReplacement in the A.niger genomeglaARecombining strains; then, the constructed recombinant plasmid pZF is transformed into cagD and screened, and screening is firstly carried out on YPD plates (1% of yeast extract, 2% of peptone and 2% of glucose) containing 0.5 g/L G418, and then is carried out on YPG plates (1% of yeast extract, 2% of peptone and 1% of galactose) containing 1wt.% of galactose, so that agD recombinant strains replacing agF in an Aspergillus niger genome are obtained. The enzyme production level of the new strain was evaluated by fermentation in a shake flask and in a 30L fermenter.

(5) Fermentation test

The shake flask fermentation is carried out in a 250 mL triangular flask, the liquid loading amount is 25-50 mL, the culture medium is PDA (2% glucose, 2% peptone and 1% yeast extract), the initial pH value is 4.5-5.5, the fermentation temperature is 28-32 ℃, and the rotation speed is 180-250 r/min.

The fermentation production on the fermentation tank is carried out on a 30L full-automatic fermentation tank, and the fermentation process comprises the following steps: the fermentation medium comprises the following components: 3-20% of starch dextrin, 1-8% of bean cake powder, 0-5% of corn steep liquor, 0-3% of ammonium sulfate, 0.01-2% of calcium chloride, the fermentation temperature is 25-36 ℃, the ventilation is 0.1-1.2 vvm, the dissolved oxygen is maintained at 5-60% in the fermentation process, the pH is controlled to be 4-6 by sulfuric acid or ammonia water in the fermentation process, and the fermentation time is 60-150 hours. Sampling at regular time for fermentation, detecting and analyzing enzyme activity level, and centrifuging/filtering to remove thallus after fermentation is finished to obtain transglucosidase enzyme liquid.

(6) Preparation and analysis of isomaltooligosaccharide

The reaction was carried out in a 30L sugar production system. The preparation process flow of the isomaltooligosaccharide comprises the following steps: liquefying by using high-temperature alpha-amylase, wherein the DE value of liquefied starch reaches 10-30, adding beta-amylase, pullulanase, medium-temperature alpha-amylase and transglucosidase, wherein the addition amounts are 0-1500U/g, 0-10U/g, 0-30U/g and 100-5000U/g respectively, and keeping the temperature at 50-70 ℃ for 5-80 h.

(15) Sugar profile analysis

The total dry matter (solids) was determined as 6.2 in GB/T20885-2007. And reading the refractive index of the sample to be measured by using a handheld refractometer, and comparing with a syrup brix and relative density relation table at 20 ℃ to obtain the dry matter content. The DE value of starch liquefaction is determined according to GB/T22428.1-2008 by direct titration (calculated as glucose).

The content and the composition of the isomaltose hypgather are analyzed and determined according to GB/T20881-2017 and by adopting a high performance liquid chromatography. The chromatographic column is a Prevail @ Carbohydrate ES 5u sugar column, the mobile phase is 65% acetonitrile, the flow rate is 1 mL/min, the detection temperature of the chromatographic column is 30 ℃, the detector uses an ELSD 2000ES evaporation light detector, the gas flow rate of the detector is 2.2L/min, and the temperature of the detector is 90 ℃. According to the requirements of national standard GB/T20881-2007, the contents of isomaltose (IG 2), isomaltotriose (IG 3) and panose (P) are calculated.

Example 1: transglycosidation Activity of An-F308 alpha-glucosidase from Aspergillus niger

Adopting PCR amplification technology, cloning and expressing 6 genes related to transglucoside activity in Aspergillus niger An-F308 one by cDNA cloning method. The related gene is amplified to obtain the gene with the size of 1815 bpagA2892 bp ofagB2229 bp ofagC2958 bp ofagD1764 bp ofagEAnd 1968 bpagFA gene fragment. Integrating them in Pichia yeast under the mediation of pPIC9k plasmid to respectively obtain recombinant strain GS115-agA，GS115-agB，GS115-agC，GS115-agD，GS115-agEAnd GS115-agF. Further carrying out shake flask fermentation on the strains to prepare enzyme liquid, and further analyzing the hydrolysis activity and the transglycosylation activity of the prepared enzyme liquid by taking maltose as a substrate. The results show that all 6 aspergillus niger glycosidases tested had varying degrees of transglycosidic activity, with AgD having the highest glucosyl transfer activity and AgF having a relatively high hydrolytic activity (table 2).

TABLE 2 hydrolysis and transglycosidation characteristics of Aspergillus niger transglucosidase

Example 2: construction of high-yield transglucosidase Aspergillus niger strains

By usingEcoRI andPsti digestion of plasmid pRS305K and usePfuDNA polymerase filling-up, gel recovering 1.25 kb G418 resistance coding gene sequence fragment, and mixing with the obtained extractPstI, digesting and removing a resistance coding gene of aureobasidin (Aba) by enzyme and connecting the filled-in plasmid pAUR135 to obtain a recombinant plasmid pAUR-kan.

Aspergillus niger An-F308 genome DNA is taken as a template, and PCR technology is adopted to amplifyglaAThe promoter region (PglaA) and the terminator and the downstream sequence (TglaA) of the promoter region (PglaA), and then the PglaA and TglaA fragments obtained are mixed with the PCR amplification technologyagDFusion was performed to obtain PglaA-cagD-TglaA (nucleotide sequence SEQ ID NO: 3), and this fragment was cloned into pAUR-kanSmaI site, obtaining recombinant plasmid pZA (shown in figure 1). Similarly, the PCR technology is used for amplifyingAspergillus niger genomeagFThe obtained PagF and TagF fragments are then combined with the promoter and terminator sequences of (1) by PCR amplification technologyagDThe fusion was performed to obtain PagF-cagD-TagF (nucleotide sequence SEQ ID NO: 4), and this fragment was cloned into pAUR-kanSmaI site, obtaining a recombinant plasmid pZF (shown in figure 2).

The plasmid pZA:: cagD was transformed into Aspergillus niger An-F308, transformants were selected on YPD plates (yeast extract 1%, peptone 2%, glucose 2%) containing 0.5 g/L G418, single colonies were picked, and then on YPG plates (yeast extract 1%, peptone 2%, galactose 1%) containing 1wt.% galactose, chromosomal DNA was further extracted from the grown colonies, and it was confirmed by PCR techniqueagDCorrect replacementglaAThe transformant of (1) having the genotype ofagD, glaA::agD，This strain was named An-agDII. Further, plasmid pZF agD was transformed into Aspergillus niger An-agDII, which was first performed on YPD plates (yeast extract 1%, peptone 2%, glucose 2%) containing 0.5 g/L G418, and then on YPG plates (yeast extract 1%, peptone 2%, galactose 1%) containing 1wt.% galactose to obtain plasmid pZFagDReplacement ofagFThe new strain of (1) genotype isagD, glaA::agD, agF::agDThis strain was named An-agDIII. The new strain An-agDIII is fermented for 5 days under the condition of shake flask fermentation, the enzyme activity of the fermentation liquor from the transglucosidase reaches 4882U/mL, and the activity level of the starch hydrolase is greatly reduced (Table 3).

TABLE 3 Aspergillus niger An-agDIII transglucosidase Shake flask enzyme Activity

Example 3: transglucosidase producing aspergillus niger An-agDIII fermentation production of transglucosidase

Fermentation preparation is carried out in a 30L full-automatic fermentation tank, the inoculation amount is 2%, and the fermentation medium comprises the following components: 1% of corn steep liquor, 10% of starch dextrin, 2.5% of bean cake powder, 1% of ammonium sulfate, 0.5% of calcium chloride, the initial pH value of 5.5, the fermentation temperature of 30 ℃, ventilation of 1 vvm, and centrifugation/filtration to remove thalli after the fermentation is finished, namely the transglucosidase enzyme solution. The Aspergillus niger An-agDIII reaches the highest enzyme production level after being fermented for 120 h, the enzyme activity of the transglucosidase reaches 50820U/mL, and only the enzyme activity of the amylolytic enzyme is low (-246U/mL).

Example 4: transglucosidase for producing isomaltooligosaccharide

The transglucosidase prepared in example 3 was used, and the reaction was carried out in a 30L sugar production system. Taking starch liquefied liquid with DE value of 20 as a raw material, adding 500U/g transglucosidase, 1U/g pullulanase, 20U/g moderate temperature alpha-amylase and 500U/g beta-amylase, and reacting for 13 h at 60 ℃. After the enzymatic catalysis is finished, the composition of the oligoisomaltose in the product is quantitatively analyzed by HPLC, the characteristic sugar spectrum composition is shown in FIG. 3, and the functional sugar composition is summarized in Table 4. The content of the dimipropan and the total IMO content in the prepared isomaltooligosaccharide respectively reach 49.1 percent and 59.5 percent.

TABLE 4 comparison of IMOs compositions

SEQUENCE LISTING

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gcggcgtcag cttcggcggg agcttccagt caggctgcag caaccgcgac caccacgtcg 1680

acttcggtat catatctgcg gacaacgccc acgcctggtg tccgcaatgt tgagcaccca 1740

ccctatgtga tcaaccatga ccaagaaggc catgatctca gtgtccatgc ggtgtcgccg 1800

aatgcaacgc atgttgatgg tgttgaggag tatgatgtgc acggtctcta cggacatcaa 1860

ggattgaacg ctacctacca aggtctgctt gaggtctggt ctcataagcg gcggccattt 1920

attattggcc gctcaacctt cgctggctct ggcaaatggg caggccactg gggcggcgac 1980

aactattcca aatggtggtc catgtactac tccatctcgc aagccctctc cttctcactt 2040

ttcggcattc cgatgtttgg tgcggacacc tgtgggttta acggaaactc cgatgaggag 2100

ctctgcaacc gatggatgca actgtccgca ttcttcccat tctaccgaaa ccacaatgag 2160

ctctccacaa tcccacagga gccttatcgg tgggcttctg ttattgaagc aaccaagtcc 2220

gccatgagaa ttcggtacgc catcctacct tacttttata cgttgtttga cctggcccac 2280

accacgggct ccactgtaat gcgcgcactt tcctgggaat tccctaatga cccaacattg 2340

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gattggtaca cccaggctgc agttgatgcg aagcccgggg tcaacacgac catttcggca 2520

ccattgggcc acatcccagt ttatgtacga ggtggaaaca tcttgccgat gcaagagccg 2580

gcattgacca ctcgtgaagc ccggcaaacc ccgtgggctt tgctagctgc actaggaagc 2640

aatggaaccg cgtcggggca gctctatctc gatgatggag agagcatcta ccccaatgcc 2700

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Ala Tyr Gly Ala Ser Gln Ser Leu Leu Ser Thr Thr Ala Pro Ser Gln

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Pro Gln Phe Thr Ile Pro Ala Ser Ala Asp Val Gly Ala Gln Leu Ile

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Ala Asn Ile Asp Asp Pro Gln Ala Ala Asp Ala Gln Ser Val Cys Pro

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Gly Tyr Lys Ala Ser Lys Val Gln His Asn Ser Arg Gly Phe Thr Ala

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Ser Leu Gln Leu Ala Gly Arg Pro Cys Asn Val Tyr Gly Thr Asp Val

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Glu Ser Leu Thr Leu Ser Val Glu Tyr Gln Asp Ser Asp Arg Leu Asn

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Ile Gln Ile Leu Pro Thr His Val Asp Ser Thr Asn Ala Ser Trp Tyr

115 120 125

Phe Leu Ser Glu Asn Leu Val Pro Arg Pro Lys Ala Ser Leu Asn Ala

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Phe Asn Phe Lys Val Ile Arg Lys Ala Thr Gly Asp Ala Leu Phe Ser

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Thr Ala Leu Pro Glu Glu Tyr Asn Leu Tyr Gly Leu Gly Glu His Ile

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Thr Gln Phe Arg Leu Gln Arg Asn Ala Asn Leu Thr Ile Tyr Pro Ser

210 215 220

Asp Asp Gly Thr Pro Ile Asp Gln Asn Leu Tyr Gly Gln His Pro Phe

225 230 235 240

Tyr Leu Asp Thr Arg Tyr Tyr Lys Gly Asp Arg Gln Asn Gly Ser Tyr

245 250 255

Ile Pro Val Lys Ser Ser Glu Ala Asp Ala Ser Gln Asp Tyr Ile Ser

260 265 270

Leu Ser His Gly Val Phe Leu Arg Asn Ser His Gly Leu Glu Ile Leu

275 280 285

Leu Arg Ser Gln Lys Leu Ile Trp Arg Thr Leu Gly Gly Gly Ile Asp

290 295 300

Leu Thr Phe Tyr Ser Gly Pro Ala Pro Ala Asp Val Thr Arg Gln Tyr

305 310 315 320

Leu Thr Ser Thr Val Gly Leu Pro Ala Met Gln Gln Tyr Asn Thr Leu

325 330 335

Gly Phe His Gln Cys Arg Trp Gly Tyr Asn Asn Trp Ser Asp Leu Ala

340 345 350

Asp Val Val Ala Asn Phe Glu Lys Phe Glu Ile Pro Leu Glu Tyr Ile

355 360 365

Trp Thr Asp Ile Asp Tyr Met His Gly Tyr Arg Asn Phe Asp Asn Asp

370 375 380

Gln His Arg Phe Ser Tyr Ser Glu Gly Asp Glu Phe Leu Ser Lys Leu

385 390 395 400

His Glu Ser Gly Arg Tyr Tyr Val Pro Ile Val Asp Ala Ala Leu Tyr

405 410 415

Ile Pro Asn Pro Glu Asn Ala Ser Asp Ala Tyr Ala Thr Tyr Asp Arg

420 425 430

Gly Ala Ala Asp Asp Val Phe Leu Lys Asn Pro Asp Gly Ser Leu Tyr

435 440 445

Ile Gly Ala Val Trp Pro Gly Tyr Thr Val Phe Pro Asp Trp His His

450 455 460

Pro Lys Ala Val Asp Phe Trp Ala Asn Glu Leu Val Ile Trp Ser Lys

465 470 475 480

Lys Val Ala Phe Asp Gly Val Trp Tyr Asp Met Ser Glu Val Ser Ser

485 490 495

Phe Cys Val Gly Ser Cys Gly Thr Gly Asn Leu Thr Leu Asn Pro Ala

500 505 510

His Pro Ser Phe Leu Leu Pro Gly Glu Pro Gly Asp Ile Ile Tyr Asp

515 520 525

Tyr Pro Glu Ala Phe Asn Ile Thr Asn Ala Thr Glu Ala Ala Ser Ala

530 535 540

Ser Ala Gly Ala Ser Ser Gln Ala Ala Ala Thr Ala Thr Thr Thr Ser

545 550 555 560

Thr Ser Val Ser Tyr Leu Arg Thr Thr Pro Thr Pro Gly Val Arg Asn

565 570 575

Val Glu His Pro Pro Tyr Val Ile Asn His Asp Gln Glu Gly His Asp

580 585 590

Leu Ser Val His Ala Val Ser Pro Asn Ala Thr His Val Asp Gly Val

595 600 605

Glu Glu Tyr Asp Val His Gly Leu Tyr Gly His Gln Gly Leu Asn Ala

610 615 620

Thr Tyr Gln Gly Leu Leu Glu Val Trp Ser His Lys Arg Arg Pro Phe

625 630 635 640

Ile Ile Gly Arg Ser Thr Phe Ala Gly Ser Gly Lys Trp Ala Gly His

645 650 655

Trp Gly Gly Asp Asn Tyr Ser Lys Trp Trp Ser Met Tyr Tyr Ser Ile

660 665 670

Ser Gln Ala Leu Ser Phe Ser Leu Phe Gly Ile Pro Met Phe Gly Ala

675 680 685

Asp Thr Cys Gly Phe Asn Gly Asn Ser Asp Glu Glu Leu Cys Asn Arg

690 695 700

Trp Met Gln Leu Ser Ala Phe Phe Pro Phe Tyr Arg Asn His Asn Glu

705 710 715 720

Leu Ser Thr Ile Pro Gln Glu Pro Tyr Arg Trp Ala Ser Val Ile Glu

725 730 735

Ala Thr Lys Ser Ala Met Arg Ile Arg Tyr Ala Ile Leu Pro Tyr Phe

740 745 750

Tyr Thr Leu Phe Asp Leu Ala His Thr Thr Gly Ser Thr Val Met Arg

755 760 765

Ala Leu Ser Trp Glu Phe Pro Asn Asp Pro Thr Leu Ala Ala Val Glu

770 775 780

Thr Gln Phe Met Val Gly Pro Ala Ile Met Val Val Pro Val Leu Glu

785 790 795 800

Pro Leu Val Asn Thr Val Lys Gly Val Phe Pro Gly Val Gly His Gly

805 810 815

Glu Val Trp Tyr Asp Trp Tyr Thr Gln Ala Ala Val Asp Ala Lys Pro

820 825 830

Gly Val Asn Thr Thr Ile Ser Ala Pro Leu Gly His Ile Pro Val Tyr

835 840 845

Val Arg Gly Gly Asn Ile Leu Pro Met Gln Glu Pro Ala Leu Thr Thr

850 855 860

Arg Glu Ala Arg Gln Thr Pro Trp Ala Leu Leu Ala Ala Leu Gly Ser

865 870 875 880

Asn Gly Thr Ala Ser Gly Gln Leu Tyr Leu Asp Asp Gly Glu Ser Ile

885 890 895

Tyr Pro Asn Ala Thr Leu His Val Asp Phe Thr Ala Ser Arg Ser Ser

900 905 910

Leu Arg Ser Ser Ala Gln Gly Arg Trp Lys Glu Arg Asn Pro Leu Ala

915 920 925

Asn Val Thr Val Leu Gly Val Asn Lys Glu Pro Ser Ala Val Thr Leu

930 935 940

Asn Gly Gln Ala Val Phe Pro Gly Ser Val Thr Tyr Asn Ser Thr Ser

945 950 955 960

Gln Val Leu Phe Val Gly Gly Leu Gln Asn Leu Thr Lys Gly Gly Ala

965 970 975

Trp Ala Glu Asn Trp Val Leu Glu Trp

980 985

<210> 3

<211> 4758

<212> DNA

<213> PglaA-cagD-TglaA

<400> 3

gacctgctgc aggtgttcta tgggatcaag ccaaactatg cagctagttc tagccacacg 60

tactatctga gctttgtgta tacgctggat ccgaactcca accgggggga gtacattgag 120

tggccgcagt ggaaggaatc gcggcagttg atgaatttcg gagcgaacga cgccagtctc 180

cttacggatg atttccgcaa cgggacatat gagttcatcc tgcagaatac cgcggcgttc 240

cacatctgat gccattggcg gaggggtccg gacggtcagg aacttagcct tatgagatga 300

atgatggacg tgtctggcct cggaaaagga tatatgggga tcatgatagt actagccata 360

ttaatgaagg gcatatacca cgcgttggac ctgcgttata gcttcccgtt agttatagta 420

ccatcgttat accagccaat caagtcacca cgcacgaccg gggacggcga atccccggga 480

attgaaagaa attgcatccc aggccagtga ggccagcgat tggccacctc tccaaggcac 540

agggccattc tgcagcgctg gtggattcat cgcaatttcc cccggcccgg cccgacaccg 600

ctataggctg gttctcccac accatcggag attcgtcgcc taatgtctcg tccgttcaca 660

agctgaagag cttgaagtgg cgagatgtct ctgcaggaat tcaagctaga tgctaagcga 720

tattgcatgg caatatgtgt tgatgcatgt gcttcttcct tcagcttccc ctcgtgcaga 780

tgaggtttgg ctataaattg aagtggttgg tcggggttcc gtgaggggct gaagtgcttc 840

ctccctttta gacgcaactg agagcctgag cttcatcccc agcatcatta cacctcagca 900

atggtgaagt tgacgcatct ccttgccaga gcatggcttg tccctctggc ttatggagcg 960

agccagtcac tcttatccac cactgcccct tcgcagccgc agtttaccat tcctgcttcc 1020

gcagatgtcg gtgcgcagct gattgccaac atcgatgatc ctcaggctgc cgacgcgcag 1080

tcggtttgtc cgggctacaa ggcttcaaaa gtgcagcaca attcacgtgg attcactgcc 1140

agtcttcagc tcgcgggcag gccatgtaac gtatacggca cagatgttga gtccttgaca 1200

ctgtctgtgg agtaccagga ttcggatcga ctgaatattc agattctccc cactcatgtt 1260

gactccacaa acgcttcttg gtactttctt tcggaaaacc tggtccccag acccaaggct 1320

tccctcaatg catctgtatc ccagagcgac ctttttgtgt catggtcaaa tgagccgtcg 1380

ttcaatttca aggtgatccg aaaggctaca ggcgacgcgc ttttcagtac agaaggcact 1440

gtgctcgtat atgagaatca gttcatcgaa tttgtgaccg cgctccctga agaatataac 1500

ttgtatggcc ttggggagca tatcacgcaa ttccgcctcc agagaaatgc taatctgacc 1560

atatatcctt cggatgatgg aacacctatt gaccaaaacc tctacggcca acatcccttc 1620

tatctggata caagatatta caaaggagat aggcagaatg ggtcttatat tcccgtcaaa 1680

agcagcgagg ctgatgcctc gcaagattat atctccctct ctcatggcgt gtttctgagg 1740

aactctcatg gacttgagat actcctccgg tctcaaaaat tgatctggcg gaccctaggt 1800

ggaggaatcg atctcacctt ctactcaggt cccgccccgg ccgatgttac caggcaatat 1860

cttaccagca ctgtgggatt accggccatg cagcaataca acactcttgg attccaccaa 1920

tgtcgttggg gctacaacaa ctggtcggat ctggcggacg ttgttgcgaa ctttgagaag 1980

tttgagatcc cgttggaata tatctggacc gatattgact acatgcacgg atatcgcaac 2040

tttgacaacg atcaacatcg cttttcctac agtgagggcg atgaatttct cagcaagcta 2100

catgagagtg gacgctacta tgtacccatt gttgatgcgg cgctctacat tcctaatccc 2160

gaaaatgcct ctgatgcata cgctacgtat gacagaggag ctgcggacga cgtcttcctc 2220

aagaatcccg atggtagcct ctatattgga gccgtttggc caggatatac agtcttcccc 2280

gattggcatc atcccaaggc agttgacttc tgggctaacg agcttgttat ctggtcgaag 2340

aaagtggcgt tcgatggtgt gtggtacgac atgtctgaag tttcatcctt ctgtgtcggg 2400

agctgtggca caggtaacct gactttgaac ccggcacacc catcgtttct tctccccggt 2460

gagcctggtg atatcatata tgattaccca gaggctttca atatcaccaa cgctacagag 2520

gcggcgtcag cttcggcggg agcttccagt caggctgcag caaccgcgac caccacgtcg 2580

acttcggtat catatctgcg gacaacgccc acgcctggtg tccgcaatgt tgagcaccca 2640

ccctatgtga tcaaccatga ccaagaaggc catgatctca gtgtccatgc ggtgtcgccg 2700

aatgcaacgc atgttgatgg tgttgaggag tatgatgtgc acggtctcta cggacatcaa 2760

ggattgaacg ctacctacca aggtctgctt gaggtctggt ctcataagcg gcggccattt 2820

attattggcc gctcaacctt cgctggctct ggcaaatggg caggccactg gggcggcgac 2880

aactattcca aatggtggtc catgtactac tccatctcgc aagccctctc cttctcactt 2940

ttcggcattc cgatgtttgg tgcggacacc tgtgggttta acggaaactc cgatgaggag 3000

ctctgcaacc gatggatgca actgtccgca ttcttcccat tctaccgaaa ccacaatgag 3060

ctctccacaa tcccacagga gccttatcgg tgggcttctg ttattgaagc aaccaagtcc 3120

gccatgagaa ttcggtacgc catcctacct tacttttata cgttgtttga cctggcccac 3180

accacgggct ccactgtaat gcgcgcactt tcctgggaat tccctaatga cccaacattg 3240

gctgcggttg agactcaatt catggttggg ccggccatca tggtggtccc ggtattggag 3300

cctctggtca atacggtcaa gggcgtattc ccaggagttg gacatggcga agtgtggtac 3360

gattggtaca cccaggctgc agttgatgcg aagcccgggg tcaacacgac catttcggca 3420

ccattgggcc acatcccagt ttatgtacga ggtggaaaca tcttgccgat gcaagagccg 3480

gcattgacca ctcgtgaagc ccggcaaacc ccgtgggctt tgctagctgc actaggaagc 3540

aatggaaccg cgtcggggca gctctatctc gatgatggag agagcatcta ccccaatgcc 3600

accctccatg tggacttcac ggcatcgcgg tcaagcctgc gctcgtcggc tcaaggaaga 3660

tggaaagaga ggaacccgct tgctaatgtg acggtgctcg gagtgaacaa ggagccctct 3720

gcggtgaccc tgaatggaca ggccgtattt cccgggtctg tcacgtacaa ttctacgtcc 3780

caggttctct ttgttggggg gctgcaaaac ttgacgaagg gcggcgcatg ggcggaaaac 3840

tgggtattgg aatggtagac aatcaatcca tttcgctata gttaaaggat ggggatgagg 3900

gcaattggtt atatgatcat gtatgtagtg ggtgtgcata atagtagtga aatggaagcc 3960

aagtcatgtg attgtaatcg accgacggaa ttgaggatat ccggaaatac agacaccgtg 4020

aaagccatgg tctttccttc gtgtagaaga ccagacagac agtccctgat ttacccttgc 4080

acaaagcact agaaaattag cattccatcc ttctctgctt gctctgctga tatcactgtc 4140

attcaatgca tagccatgag ctcatcttag atccaagcac gtaattccat agccgaggtc 4200

cacagtggag cagcaacatt ccccatcatt gctttcccca ggggcctccc aacgactaaa 4260

tcaagagtat atctctaccg tccaatagat cgtcttcgct tcaaaatctt tgacaattcc 4320

aagagggtcc ccatccatca aacccagttc aataatagcc gagatgcatg gtggagtcaa 4380

ttaggcagta ttgctggaat gtcggggcca gttggcccgg tggtcattgg ccgcctgtga 4440

tgccatctgc cactaaatcc gatcattgat ccaccgccca cgaggcgcgt ctttgctttt 4500

tgcgcggcgt ccaggttcaa ctctctctgc agctccagtc caacgctgac tgactagttt 4560

acctactggt ctgatcggct ccatcagagc tatggcgtta tcccgtgccg ttgctgcgca 4620

atcgctatct tgatcgcaac cttgaactca ctcttgtttt aatagtgatc ttggtgacgg 4680

agtgtcggtg agtgacaacc aacatcgtgc aagggagatt gatacggaat tgtcgctccc 4740

atcatgatgt tcttgccg 4758

<210> 4

<211> 4878

<212> DNA

<213> PagF-cagD-TagF

<400> 4

cggagctccg ggagatgcaa tctttgatca tatgagagcc attgcttcta gttttgttgt 60

cactcccgcc attgccgttg tcgaatgacg agtctgtttt acagactctg atggggttca 120

tacacctgat cagcgtagcg cgccgtcgct tcgcagtatg atgggtaacc ttaccgggtt 180

gtcgccccga cgttcctgct gggcttaaac cactgcgata ttgaatacca ctcgtagata 240

tcgttgtagc gtaaaagtgt aagtgatccg aaatttagag cgaacatcat ggtagattag 300

tctacattga cattgaaggc cgtctacagc aacaacggct accgccggct tcttttcaag 360

acagaaccgt ggatccatcc acctaggata atatgatata gatcattcta ataatctagc 420

aaagaagcga tgatgggtcg actcaaggta gagtcaagat agcattaatg caatcttgac 480

cagatgcagg cctgtgttcc cgccttcccc gcttcttgtc atctcgacgc ttccccggaa 540

cttcacctcc tcattcaagg aatgtggcgg ttaagattag gctatattgc catattccag 600

tccgatgaat gctattcgtg gaccaaaacg acttccgatc tcttggccgg aacaagtcac 660

tcgctttaga gcagtcgttt ctctacgctg ccttttttac tggacgaaga tgaagctggt 720

cagcagcatg cccgtcgtgg cgttggtaac accgtgacgc aggcagcgat gcggccatag 780

cagaatactt tgtgtccttc tcctaattgg actgcatctg ttcaactgtt agtgtcaatg 840

ctcttgcctt gggttagacc ggtcatcgtg aggggctgca attgcacagc atgttcaaac 900

acccctctca gtctcatttc tagactgaca gatttaaatc aattagaacc cttcccatac 960

tcctgtgacc ttcagagcct acattcttcg cacacccacc cagacaatca catcaacaca 1020

atggtgaagt tgacgcatct ccttgccaga gcatggcttg tccctctggc ttatggagcg 1080

agccagtcac tcttatccac cactgcccct tcgcagccgc agtttaccat tcctgcttcc 1140

gcagatgtcg gtgcgcagct gattgccaac atcgatgatc ctcaggctgc cgacgcgcag 1200

tcggtttgtc cgggctacaa ggcttcaaaa gtgcagcaca attcacgtgg attcactgcc 1260

agtcttcagc tcgcgggcag gccatgtaac gtatacggca cagatgttga gtccttgaca 1320

ctgtctgtgg agtaccagga ttcggatcga ctgaatattc agattctccc cactcatgtt 1380

gactccacaa acgcttcttg gtactttctt tcggaaaacc tggtccccag acccaaggct 1440

tccctcaatg catctgtatc ccagagcgac ctttttgtgt catggtcaaa tgagccgtcg 1500

ttcaatttca aggtgatccg aaaggctaca ggcgacgcgc ttttcagtac agaaggcact 1560

gtgctcgtat atgagaatca gttcatcgaa tttgtgaccg cgctccctga agaatataac 1620

ttgtatggcc ttggggagca tatcacgcaa ttccgcctcc agagaaatgc taatctgacc 1680

atatatcctt cggatgatgg aacacctatt gaccaaaacc tctacggcca acatcccttc 1740

tatctggata caagatatta caaaggagat aggcagaatg ggtcttatat tcccgtcaaa 1800

agcagcgagg ctgatgcctc gcaagattat atctccctct ctcatggcgt gtttctgagg 1860

aactctcatg gacttgagat actcctccgg tctcaaaaat tgatctggcg gaccctaggt 1920

ggaggaatcg atctcacctt ctactcaggt cccgccccgg ccgatgttac caggcaatat 1980

cttaccagca ctgtgggatt accggccatg cagcaataca acactcttgg attccaccaa 2040

tgtcgttggg gctacaacaa ctggtcggat ctggcggacg ttgttgcgaa ctttgagaag 2100

tttgagatcc cgttggaata tatctggacc gatattgact acatgcacgg atatcgcaac 2160

tttgacaacg atcaacatcg cttttcctac agtgagggcg atgaatttct cagcaagcta 2220

catgagagtg gacgctacta tgtacccatt gttgatgcgg cgctctacat tcctaatccc 2280

gaaaatgcct ctgatgcata cgctacgtat gacagaggag ctgcggacga cgtcttcctc 2340

aagaatcccg atggtagcct ctatattgga gccgtttggc caggatatac agtcttcccc 2400

gattggcatc atcccaaggc agttgacttc tgggctaacg agcttgttat ctggtcgaag 2460

aaagtggcgt tcgatggtgt gtggtacgac atgtctgaag tttcatcctt ctgtgtcggg 2520

agctgtggca caggtaacct gactttgaac ccggcacacc catcgtttct tctccccggt 2580

gagcctggtg atatcatata tgattaccca gaggctttca atatcaccaa cgctacagag 2640

gcggcgtcag cttcggcggg agcttccagt caggctgcag caaccgcgac caccacgtcg 2700

acttcggtat catatctgcg gacaacgccc acgcctggtg tccgcaatgt tgagcaccca 2760

ccctatgtga tcaaccatga ccaagaaggc catgatctca gtgtccatgc ggtgtcgccg 2820

aatgcaacgc atgttgatgg tgttgaggag tatgatgtgc acggtctcta cggacatcaa 2880

ggattgaacg ctacctacca aggtctgctt gaggtctggt ctcataagcg gcggccattt 2940

attattggcc gctcaacctt cgctggctct ggcaaatggg caggccactg gggcggcgac 3000

aactattcca aatggtggtc catgtactac tccatctcgc aagccctctc cttctcactt 3060

ttcggcattc cgatgtttgg tgcggacacc tgtgggttta acggaaactc cgatgaggag 3120

ctctgcaacc gatggatgca actgtccgca ttcttcccat tctaccgaaa ccacaatgag 3180

ctctccacaa tcccacagga gccttatcgg tgggcttctg ttattgaagc aaccaagtcc 3240

gccatgagaa ttcggtacgc catcctacct tacttttata cgttgtttga cctggcccac 3300

accacgggct ccactgtaat gcgcgcactt tcctgggaat tccctaatga cccaacattg 3360

gctgcggttg agactcaatt catggttggg ccggccatca tggtggtccc ggtattggag 3420

cctctggtca atacggtcaa gggcgtattc ccaggagttg gacatggcga agtgtggtac 3480

gattggtaca cccaggctgc agttgatgcg aagcccgggg tcaacacgac catttcggca 3540

ccattgggcc acatcccagt ttatgtacga ggtggaaaca tcttgccgat gcaagagccg 3600

gcattgacca ctcgtgaagc ccggcaaacc ccgtgggctt tgctagctgc actaggaagc 3660

aatggaaccg cgtcggggca gctctatctc gatgatggag agagcatcta ccccaatgcc 3720

accctccatg tggacttcac ggcatcgcgg tcaagcctgc gctcgtcggc tcaaggaaga 3780

tggaaagaga ggaacccgct tgctaatgtg acggtgctcg gagtgaacaa ggagccctct 3840

gcggtgaccc tgaatggaca ggccgtattt cccgggtctg tcacgtacaa ttctacgtcc 3900

caggttctct ttgttggggg gctgcaaaac ttgacgaagg gcggcgcatg ggcggaaaac 3960

tgggtattgg aatggtaggt ctcgtgaata atttaggttg cctgaaggtg atggctggat 4020

gtatcgacgg ggaggtatag ccggagcagg gttggatgta tcgatttgca aattgtcaga 4080

gaagggagaa attagttcag ccaacattca ctaagtaatc acaaatgcaa tctgtcagca 4140

tgtgtatgtg atggactcta caccttaggt aacttacaat aagtcgctgg gatatcgtag 4200

ccaaagtgca tcgatccatc aggtagcaag aaacagtaac tagggagggg caaatgacaa 4260

attcaaaggc gacttagatg accctttttt ttcaatactt gggtcaatgg tttatttcgt 4320

ttcttttttt ctgcttcatt ttcagcctcg ccaaggaaga aaagcaagaa agtcgcatgg 4380

atggatgctt cgacgactga atctcatggc aaaagcgtgg ccactatcga agactagaca 4440

agtagcgaac attctacaag atgagtctgg aagctctctt agatttcttc attttcctct 4500

tcgatcgatc tagtggcgac cagggtctcc atgaagatgt cgccagtagc cccaagcgta 4560

gccacaaaat tgagtccagc ttagccacat ggcccaaggt ttgggttctg gcaggaaaaa 4620

gaaaagggta tgtatggctc agcccagcca cagacgtatg gatttatcaa taggaggaat 4680

agtttctagt ctgtaatagg cggaaacagg gaagacgcag gaatttgtcc agactgaaca 4740

gagtttgtgt gttctcccta ttcgcttcct tcttcctttc cagccaagaa gacagttggc 4800

cgtctcaccg ttggatatac gaacgggatc gattggcccc cagaagaagt accaatgtca 4860

caagtggaag tctgtgca 4878

<210> 5

<211> 25

<212> DNA

<213> Artificial sequence

<400> 5

atgtcaacca tgtgtaacaa gtcca 25

<210> 6

<211> 25

<212> DNA

<213> Artificial sequence

<400> 6

ctaactacaa cacccaacga cacct 25

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence

<400> 7

atggccggaa ctcggccaat g 21

<210> 8

<211> 28

<212> DNA

<213> Artificial sequence

<400> 8

ctaaaactca atccgccatg tctttcca 28

<210> 9

<211> 23

<212> DNA

<213> Artificial sequence

<400> 9

atgccgtcaa cttatttggg agc 23

<210> 10

<211> 21

<212> DNA

<213> Artificial sequence

<400> 10

tcacgcatac agcaccgttc c 21

<210> 11

<211> 23

<212> DNA

<213> Artificial sequence

<400> 11

atggtgaagt tgacgcatct cct 23

<210> 12

<211> 25

<212> DNA

<213> Artificial sequence

<400> 12

ctaccattcc aatacccagt tttcc 25

<210> 13

<211> 18

<212> DNA

<213> Artificial sequence

<400> 13

atggccaaat ccgcctcc 18

<210> 14

<211> 22

<212> DNA

<213> Artificial sequence

<400> 14

tcaagcctcc accaaaagaa ca 22

<210> 15

<211> 22

<212> DNA

<213> Artificial sequence

<400> 15

atgctgtacg ccgaagacaa ca 22

<210> 16

<211> 22

<212> DNA

<213> Artificial sequence

<400> 16

tcagaccctg acaaataccg gc 22

<210> 17

<211> 22

<212> DNA

<213> Artificial sequence

<400> 17

gacctgctgc aggtgttcta tg 22

<210> 18

<211> 44

<212> DNA

<213> Artificial sequence

<400> 18

ggagatgcgt caacttcacc attgctgagg tgtaatgatg ctgg 44

<210> 19

<211> 52

<212> DNA

<213> Artificial sequence

<400> 19

ggaaaactgg gtattggaat ggtagacaat caatccattt cgctatagtt aa 52

<210> 20

<211> 20

<212> DNA

<213> Artificial sequence

<400> 20

cggcaagaac atcatgatgg 20

<210> 21

<211> 18

<212> DNA

<213> Artificial sequence

<400> 21

cggagctccg ggagatgc 18

<210> 22

<211> 45

<212> DNA

<213> Artificial sequence

<400> 22

ggagatgcgt caacttcacc attgtgttga tgtgattgtc tgggt 45

<210> 23

<211> 49

<212> DNA

<213> Artificial sequence

<400> 23

ggaaaactgg gtattggaat ggtaggtctc gtgaataatt taggttgcc 49

<210> 24

<211> 22

<212> DNA

<213> Artificial sequence

<400> 24

tgcacagact tccacttgtg ac 22

Claims (2)

1. A transglucosidase producing Aspergillus niger strain, characterized in that: the strain is prepared by using Aspergillus niger (Aspergillus niger) by using gene cloning and gene replacement technologyAspergillus niger) An-F308 transglucosidase coding gene replaces the glucoamylase coding gene and the alpha-glucosidase coding gene therein to obtain; the transglucosidase coding gene isagDThe gene has a sequence shown in SEQ ID NO. 1; the saccharifying enzyme coding gene isglaAThe gene has a sequence shown in SEQ ID NO. 3; the alpha-glucosidase coding gene isagFThe gene has a sequence shown in SEQ ID NO. 4; said Aspergillus nigerAspergillus niger) An-F308, which has been preserved in China general microbiological culture Collection center (CGMCC) at 26.11.2020, with the preservation number of CGMCC NO. 21028.

2. The transglucosidase producing aspergillus niger strain of claim 1, wherein: the genotype of the Aspergillus niger strain is characterized in that:agD，glaA：：agD，agF：：agD。

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