CN113388537A - Application of GSH synthesis and circulation related protein and recombinant saccharomyces cerevisiae strain - Google Patents

Abstract

The invention relates to the technical field of microorganisms, and discloses application of GSH (glutathione) synthesis and circulation related genes and recombinant saccharomyces cerevisiaeAnd (4) strain. The invention selects GSH synthesis and circulation related genes from Kluyveromyces marxianus, acid-fast marine bacterium Wallemia mellicol and extreme thermophilic bacterium Thermus thermophilus HB27 to construct a recombinant saccharomyces cerevisiae strain with excellent stress resistance, compared with wild saccharomyces cerevisiae, the recombinant saccharomyces cerevisiae has H resistance according to different exogenous genes₂O₂The yeast strain has different effects on heat resistance, high temperature resistance, acid resistance and salt resistance, provides an excellent yeast strain for industrial fermentation production, and provides a cell model for researching the generation or protection of ROS in eukaryotes.

Description

Application of GSH synthesis and circulation related protein and recombinant saccharomyces cerevisiae strain

Technical Field

The invention relates to the technical field of microorganisms, in particular to application of GSH synthesis and circulation related protein and a recombinant saccharomyces cerevisiae strain.

Background

Under unfavorable survival conditions of high temperature, high salt, acid and alkali and the like, the saccharomyces cerevisiae can generate excessive Reactive Oxygen Species (ROS), so that the saccharomyces cerevisiae suffers from oxidative damage and even dies. Saccharomyces cerevisiae, when exposed to ROS, initiates an intracellular oxidative stress response, and thus its stress resistance is closely related to oxidative stress. The major site of ROS production is the mitochondria, the most oxidatively damaged organelles, and thus protection of mitochondria from oxidative damage is the focus of antioxidant defense. The anti-oxidation defense system of the saccharomyces cerevisiae consists of an enzymatic defense system and a non-enzymatic defense system. Antioxidant protective enzymes are important mechanisms for dealing with oxidative stress, including superoxide dismutase (SOD), Catalase (CAT), and peroxidase (Prx). The non-enzymatic defense system consists of small molecules capable of scavenging free radicals, mainly consisting of thioredoxin (Trx), ascorbic acid, Glutathione (GSH) and other reducing agents.

SOD is the most common and important antioxidant that will catalyze superoxide anion (O)²-) is reduced to (H₂O₂) Then CAT or Prx catalyzes H₂O₂Then reduced to water (H)₂O). The thioredoxin system includes thioredoxin reductase (TRR1) and two thioredoxins (TRX1 and TRX2), and in yeast cells, TRX has antioxidant activity and plays a key role in the protection against oxidative damage by the thioredoxin system. The alkylene glycol group in ascorbic acid molecule is easily oxidized, and can bind and scavenge oxygen, thus having antioxidant effect. However, Saccharomyces cerevisiae does not produce L-AA, and it can synthesize D-erythro ascorbic acid (D-EAA), a functional analogue thereof. Research has shown that D-EAA plays a certain role in resisting stress, and yeast strains of gene ALO1 for catalyzing last step enzyme of D-EAA synthesis are deleted, and the strain can be used for H₂O₂And O²-sensitive. Monteiro et alIt was suggested that ascorbic acid may be involved in the reduction of 1-Cys Prx, revealing additional antioxidant capacity of ascorbic acid.

The cysteine residue of GSH has redox activity, and can be used for scavenging free radicals and helping cells resist oxidative damage in various life activities of organisms. In addition, GSH also plays a key role in the response of saccharomyces cerevisiae to NaCl stress. ROS-mediated oxidative damage, one of the earliest responding antioxidant stresses, is the oxidation of sulfhydryls, suggesting that GSH plays a very important role in antioxidant as a cofactor for stress defense enzymes. Glutathione peroxidase (GPx) catalyzes the oxidation of GSH to its disulfide bond form (GSSG) with concomitant H-stimulation due to oxidative stress caused by conditions such as high temperature₂O₂And reduction of lipid peroxides. Conversely, under the action of GSSG reductase (Glr1), GSSG can also be reduced to GSH with concomitant consumption of NADPH, thus forming a GSH redox cycle. CAT can also reduce H₂O₂But only in the peroxisome. Mitochondria are the most oxidatively damaged organelles, and thus the GSH system plays a key role in protecting mitochondria from oxidative damage. Qiu et al increase the copy number of GCSGS of GSH synthase gene by Cre-LoxP system, integrate it into Saccharomyces cerevisiae ribosomal DNA, increase the accumulation of GSH by three times, thus make the yeast able to bear 40 degrees of high temperature. This indicates that stress resistance can be improved by increasing GSH content in saccharomyces cerevisiae.

Disclosure of Invention

In view of the above, the present invention aims to provide a recombinant saccharomyces cerevisiae strain, which starts with optimization of GSH synthesis and recycling, so that the constructed recombinant saccharomyces cerevisiae strain can show better H-tolerance₂O₂One or more of heat resistance, salt resistance, acid resistance, high temperature resistance and the like;

another object of the present invention is to provide the application of GSH synthesis and cycle related protein or its coding gene in constructing stress-resistant recombinant Saccharomyces cerevisiae.

In order to achieve the above purpose, the invention provides the following technical scheme:

a recombinant saccharomyces cerevisiae strain with improved stress resistance capable of expressing any one or more of exogenous GSH synthesis and circulation-related proteins (achieved by transforming a vector expressing exogenous GSH synthesis and circulation-related protein genes, or chromosomally integrating exogenous GSH synthesis and circulation-related protein genes):

(1) superoxide dismutase derived from Kluyveromyces marxianus, wherein the coding gene of the superoxide dismutase is sod 1;

(2) superoxide dismutase derived from Thermus thermophilus HB27, wherein the coding gene is ttc 0189;

(3) gamma-glutamylcysteine synthetase from Kluyveromyces marxianus, the coding gene of which is gsh 1;

(4) glutathione synthetase derived from Kluyveromyces marxianus, and encoding gene of the glutathione synthetase is gsh 2;

(5) glutathione synthetase derived from Wallemia mellicol, and a coding gene of the glutathione synthetase is gs 61512;

(6) glutathione peroxidase derived from Kluyveromyces marxianus, wherein the coding gene of the glutathione peroxidase is gpx.km;

(7) glutathione peroxidase derived from Wallemia mellicol, and a coding gene of the glutathione peroxidase is gpx.wm;

(8) cytoplasmic glutaredoxin derived from Kluyveromyces marxianus, encoding a gene grx 2;

(9) cytoplasmic glutaredoxin derived from Wallemia mellicol, encoding a gene grx 35095;

(10) glutathione oxidoreductase originated from Kluyveromyces marxianus in cytoplasm and mitochondria, and the encoding gene is glr 1;

(11) NADH kinase derived from Kluyveromyces marxianus, and encoding gene thereof is pos 5;

(12) NADH kinase derived from Wallemia mellicol, and the coding gene thereof is nk 60672;

the stress resistance comprises H resistance₂O₂Heat resistance, high temperature resistance, salt resistance and acid resistance.

Twelve genes related to GSH synthesis and antioxidation from Kluyveromyces marxianus, acid-fast marine bacteria Wallemia mellicol and extreme thermophilic bacteria Thermus thermophilus HB27 are selected, are cloned on a carrier after being optimized by a saccharomyces cerevisiae codon, and are transformed into a chassis cell to obtain a recombinant saccharomyces cerevisiae strain for optimizing GSH synthesis and circulation; in a specific embodiment of the invention, the amino acid sequences of the exogenous GSH synthesis and circulation related proteins in the items (1) to (12) are sequentially shown as SEQ ID NO.1-12, and the corresponding coding gene sequences are sequentially shown as SEQ ID NO. 13-24; the vector is preferably pRS series plasmid (such as pRS 416);

preferably, the starting Saccharomyces cerevisiae used for recombination may be selected from the BY series Saccharomyces cerevisiae (e.g., BY4741) and the CEN. PK series Saccharomyces cerevisiae.

Preferably, the recombinant saccharomyces cerevisiae is H-resistant₂O₂A recombinant Saccharomyces cerevisiae expressing one or more of the exogenous GSH synthesis and circulation associated proteins of (1) and (4) to (12) above. More preferably, the H resistance₂O₂A recombinant Saccharomyces cerevisiae expressing one or more of the exogenous GSH synthesis and circulation associated proteins of (4) and (8) to (9) above.

Preferably, the recombinant saccharomyces cerevisiae is heat shock resistant recombinant saccharomyces cerevisiae, and expresses one or more exogenous GSH synthesis and circulation related proteins in the aforementioned items (1) - (8) and (10) - (12). More preferably, one or more of the exogenous GSH synthesis and circulation-related proteins of (1), (4) - (5) above are expressed.

Preferably, the recombinant saccharomyces cerevisiae is a high-temperature resistant recombinant saccharomyces cerevisiae and expresses one or more exogenous GSH synthesis and circulation related proteins in the (10) th and (11) th steps.

Preferably, the recombinant saccharomyces cerevisiae is salt-tolerant recombinant saccharomyces cerevisiae and expresses one or more exogenous GSH synthesis and circulation-related proteins in the steps (1) to (12). More preferably, one or more of the exogenous GSH synthesis and circulation-related proteins of (4), (6) - (10), and (12) above are expressed.

Preferably, the recombinant saccharomyces cerevisiae is acid-tolerant recombinant saccharomyces cerevisiae and expresses one or more exogenous GSH synthesis and circulation-related proteins in the aforementioned items (1) - (2), (4), (6) - (10) and (12). More preferably, one or more of the exogenous GSH synthesis and circulation-related proteins of (6), (8), and (10) above are expressed.

Meanwhile, the invention also provides the recombinant saccharomyces cerevisiae with various excellent stress resistance, which is H-resistant₂O₂And two or more of heat-resistant, salt-resistant and acid-resistant recombinant saccharomyces cerevisiae, and expresses the exogenous GSH synthesis and circulation-related protein of the item (4).

Or it has H resistance₂O₂And (3) the recombinant saccharomyces cerevisiae with two or more of heat resistance and salt resistance expresses the exogenous GSH synthesis and circulation related protein of the item (5).

Or it has H resistance₂O₂And two or more of heat-resistant, salt-resistant, high-temperature-resistant and acid-resistant recombinant saccharomyces cerevisiae, and expresses the exogenous GSH synthesis and circulation related protein of the item (10).

Or it has H resistance₂O₂And two or more of heat-resistant, salt-resistant and high-temperature-resistant recombinant saccharomyces cerevisiae, and expresses the exogenous GSH synthesis and circulation related protein of the item (11).

The invention also provides a construction method of the recombinant saccharomyces cerevisiae, fragment enzyme cutting sites 1-promoter-enzyme cutting sites 2-enzyme cutting sites 3-terminator-enzyme cutting sites 4 are obtained through an overlap extension PCR technology, enzyme cutting is carried out on a vector by utilizing the enzyme cutting sites 1 and the enzyme cutting sites 4, and the fragments are accessed to the vector to obtain a vector box;

and then, assembling twelve genes of the above-mentioned foreign GSH synthesis and circulation related proteins by Gibson, respectively cloning on a vector box which is linearized after enzyme digestion by an enzyme digestion site 2 and an enzyme digestion site 3, verifying a transformed host bacterium to obtain a positive clone, extracting a plasmid vector, and selecting the plasmid vector containing the foreign genes to transform saccharomyces cerevisiae according to requirements to obtain the recombinant saccharomyces cerevisiae.

In a specific embodiment of the present invention, the construction method is:

firstly, a fragment XhoI-TDH3p-HindIII-BamHI-GPM1T-SpeI is obtained by an overlap extension PCR technology, then the fragment is cut by restriction enzymes XhoI and SpeI, and the fragment is cloned on a vector pRS416 linearized by the same endonuclease through T4 connection, so that a plasmid box EGSH-HE is obtained. The twelve obtained exogenous GSH synthesis and circulation related protein genes were then assembled by Gibson, cloned into HindIII and BamHI linearized vector EGSH-HE, and transformed into E.coli competent Trans-T1. Positive clones are obtained through PCR verification of escherichia coli colonies, and escherichia coli strains EGSH-1-12 are obtained. After extracting the large intestine plasmids 416-sod1, 416-ttc0189, 416-gsh1, 416-gsh2, 416-gs61512, 416-gpx.km, 416-gpx.wm, 416-grx2, 416-grx35095, 416-glr1, 416-pos5 and 416-nk60672, respectively, Saccharomyces cerevisiae BY4741 is transformed. And obtaining optimized GSH synthesis and circulating yeast strains YGSH-1-12 through saccharomyces cerevisiae colony PCR verification.

In addition, the invention also provides the application of one or more of the exogenous GSH synthesis and circulation related proteins or the coding genes thereof in the construction of stress-resistant recombinant saccharomyces cerevisiae in the items (1) to (12); the stress resistance comprises H resistance₂O₂Heat resistance, high temperature resistance, salt resistance and acid resistance.

According to the technical scheme, GSH synthesis and circulation related genes from Kluyveromyces marxianus, acid-fast marine bacterium Wallemia mellicol and extreme thermophilic bacterium Thermus thermophilus HB27 are selected to construct the recombinant saccharomyces cerevisiae strain with excellent stress resistance, and compared with wild saccharomyces cerevisiae, the recombinant saccharomyces cerevisiae is resistant to H according to different exogenous genes₂O₂The yeast strain has different effects on heat resistance, high temperature resistance, acid resistance and salt resistance, provides an excellent yeast strain for industrial fermentation production, and provides a cell model for researching the generation or protection of ROS in eukaryotes.

Drawings

FIG. 1 is a schematic diagram showing the construction of recombinant Saccharomyces cerevisiae;

FIG. 2 shows that the strains are resistant to H₂O₂The characterization result of (1);

FIG. 3 shows the results of cell survival of heat shock injury of strains;

FIG. 4 shows the characterization results of the strains resistant to high temperature;

FIG. 5 shows the results of characterization of the strains for salt tolerance;

FIG. 6 shows the results of the characterization of the strains with respect to their resistance to mineral acids.

Detailed Description

The embodiment of the invention discloses application of GSH synthesis and circulation related protein and a recombinant saccharomyces cerevisiae strain, and a person skilled in the art can realize the synthesis and circulation related protein by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to those skilled in the art are deemed to be included within the invention. While the use of the invention and recombinant strains of Saccharomyces cerevisiae have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the techniques of the invention can be practiced and applied by modifying or appropriately modifying and combining the use and recombinant strains of Saccharomyces cerevisiae described herein without departing from the spirit, scope and spirit of the invention.

The use of the GSH synthesis and cycle-related proteins provided by the invention and recombinant Saccharomyces cerevisiae strains are further described below.

Example 1: obtaining of foreign Gene

The present invention provides 12 genes encoding GSH synthesis and circulation-related enzymes, each of which is: superoxide dismutase (SOD) encoding gene SOD1, gamma-glutamylcysteine synthetase (GSH1) encoding gene GSH1, glutathione synthetase (GSH2) encoding gene GSH2, Glutathione Peroxidase (GPX) encoding gene gpx.km, Glutaredoxin (GRX) encoding gene GRX2 in cytoplasm, NADH kinase (NADHK) encoding gene pos5, glutathione oxidoreductase (GLR) encoding gene GLR1 in cytoplasm and mitochondria, derived from Kluyveromyces marxianus; from Wallemia mellicol a gene GPX. wm encoding GPX, a gene GRX35095 encoding GRX, a gene NK60672 encoding NADHK, a gene gs61512 encoding glutathione synthetase; gene ttc0189 encoding SOD derived from Thermus thermophilus HB 27. Twelve exogenous genes related to GSH synthesis and circulation are obtained by PCR after codon optimization.

Example 2: construction of recombinant Saccharomyces cerevisiae

Firstly, a fragment XhoI-TDH3p-HindIII-BamHI-GPM1T-SpeI is obtained by an overlap extension PCR technology, then the fragment is cut by restriction enzymes XhoI and SpeI, and the fragment is cloned on a vector pRS416 linearized by the same endonuclease through T4 connection, so that a plasmid box EGSH-HE is obtained. The twelve foreign genes obtained were then assembled by Gibson, cloned into the HindIII and BamHI linearized vector EGSH-HE, and transformed into E.coli competent Trans-T1. Positive clones are obtained through PCR verification of escherichia coli colonies, and escherichia coli strains EGSH-1-12 are obtained. After extracting the large intestine plasmids 416-sod1, 416-ttc0189, 416-gsh1, 416-gsh2, 416-gs61512, 416-gpx.km, 416-gpx.wm, 416-grx2, 416-grx35095, 416-glr1, 416-pos5 and 416-nk60672, respectively, Saccharomyces cerevisiae BY4741 is transformed. And obtaining optimized GSH synthesis and circulating yeast strains YGSH-1-12 through saccharomyces cerevisiae colony PCR verification.

Example 3: strain tolerance to H₂O₂Is characterized by

Experimental materials:

YGSH-1-12 strains and BY 4741;

the experimental method comprises the following steps:

SC medium: 6.7g/L of synthetic yeast nitrogen source YNB, 20g/L of glucose, 2g/L of mixed amino acid powder of uracil, histidine, leucine and tryptophan, 0.1g/L of histidine, 0.1g/L of leucine and 0.02g/L of tryptophan, and pH is adjusted to 6.0 by 1M NaOH. (or adjusting the pH to 3.0 with HCL);

YPD medium: 20g/L of glucose, 20g/L of peptone and 10g/L of yeast powder.

YGSH-1 to YGSH-12 and BY4741 single colonies were picked from the solid streaking plate and inoculated into 3ml of the corresponding SC auxotrophic liquid medium, and cultured overnight at 220rpm at 30 ℃. Then at the initial OD₆₀₀Inoculating 0.2 to 5ml of SC liquid culture medium, culturing at 30 deg.C and 220rpm for 10-12 hr until thallus grows to middle logarithmic phase to obtain secondary seed, and culturing at initial OD₆₀₀0.2 inoculation in a seed containing 40mMH₂O₂The YPD medium of (5) was cultured for 48 hours, and the influence of the transformed foreign gene on the acid resistance of the strain was analyzed.

The experimental results are as follows:

strain H₂O₂The tolerance of the gene is characterized in FIG. 2, the corresponding data are shown in Table 1, and it is evident that YGSH-4 derived from K.marxianus gene GSH2 is expressed in twelve yeast strains from H₂O₂The oxidative damage of the cells has the fastest recovery speed and the highest cell concentration, and presents an obvious secondary growth phenomenon. anti-H₂O₂The best of the capacity were the strains YGSH-1, YGSH-9 and YGSH-8, the worst being the strain YGSH-3, the remaining strains were compared with BY4741 against H₂O₂The sensitivity difference was not significant.

TABLE 1

Example 4: survival of Heat-shock-injured cells of Strain

Experimental materials:

YGSH-1-12 strains and BY 4741;

the experimental method comprises the following steps:

after obtaining secondary seeds as described in example 3, the seeds were diluted to OD with sterile water₆₀₀The test group is thermally shocked in a metal bath at 55 ℃ for 15min, BY4741 is not subjected to any treatment, three biological parallels are made for each group, and then the yeast cell number under two conditions is obtained BY a gradient dilution plating method, and the cell lethality caused BY the thermal shock at 55 ℃ is calculated.

The experimental results are as follows:

the results of cell lethality caused BY heat shock of the strain at 55 ℃ for 15min are shown in FIG. 3, and the remaining eleven strains except the strain YGSH-9 have lower cell lethality than that of the wild-type yeast BY4741 under the heat shock injury at 55 ℃ and have higher cell survival number. Among the eleven strains, the strain with the highest heat shock tolerance at 55 ℃ is YGSH-5, then YGSH-4 and YGSH-1, and the lethality of the remaining strains is about 50%.

Example 5: characterization of strains resistant to high temperatures

Experimental materials:

YGSH-1-12 strains and BY 4741;

the experimental method comprises the following steps:

after secondary seeds were obtained as described in example 3, the final OD was used₆₀₀The culture was inoculated in YPD liquid medium at 0.2 and cultured at 40 ℃ and 220rpm for 48 hours.

The experimental results are as follows:

the results of the high temperature tolerance of the strain are shown in fig. 4, the corresponding data are shown in table 2, the heat resistance of the strain YGSH-10 is obviously better than that of other strains, and the specific manifestations are short lag phase, long log phase and high cell concentration, which proves that the recovery speed from high temperature injury is fast, and the strain can keep longer higher cell activity at the high temperature of 40 ℃. Although the cell recovery activity of the strain YGSH-11 from high temperature was slow, its log phase was shorter than that of YGSH-10, demonstrating that it maintained the cell activity higher than that of YGSH-10 at high temperature and its final OD₆₀₀Second only to YGSH-10. In addition, the strains YGSH-12, YGSH-9, YGSH-1, YGSH-6 and YGSH-4 (in order from fast to slow) were able to recover the cell viability at a faster rate under high temperature and pressure and to reach the cell concentration comparable to the level of wild-type yeast. While the remaining strains YGSH-3, YGSH-7, YGSH-8, YGSH-2 and YGSH-5 failed to restore cell viability at high temperature, which was significantly more sensitive to high temperature than BY 4741.

TABLE 2

Example 6: characterization of the osmotic tolerance (salt tolerance) of the strains

Experimental materials:

YGSH-1-12 strains and BY 4741;

the experimental method comprises the following steps:

after secondary seeds were obtained as described in example 3, the final OD was used₆₀₀When the culture medium is 0.2, the medium is inoculated into YPD liquid medium with NaCl content of 5% for 48 h.

The experimental results are as follows:

the results are shown in the figure5 and corresponding data Table 3, twelve yeast strains can be divided into two categories by their salt tolerance: YGSH-12, YGSH-8, YGSH-9, YGSH-6, YGSH-10, YGSH-7 and YGSH-4 (in the order from strong to weak) have improved salt tolerance compared with wild type strains; the salt tolerance of YGSH-2, YGSH-11, YGSH-3, YGSH-5 and YGSH-1 is slightly better than or equal to BY 4741. In addition, 5% NaCl brought about the stress on the yeast compared to H₂O₂Lesser and thermal stress, terminal OD₆₀₀Both of these pressures were higher, but it is clear that 5% NaCl had a greater effect on the yeast in the log phase, with a significantly slower growth rate. In addition, twelve strains all showed a tendency to secondary growth during the stationary phase.

TABLE 3

Example 7: characterization of inorganic acid tolerance of strains

Experimental materials:

YGSH-1-12 strains and BY 4741;

the experimental method comprises the following steps:

after secondary seeds were obtained as described in example 3, the final OD was used₆₀₀0.2 was inoculated into SC-Ura broth adjusted to pH 3.0 with HCl for 48 h.

The experimental results are as follows:

the results are shown in FIG. 6 and the corresponding data Table 4, where it is clear that YGSH-10 is able to recover cell viability faster from inorganic acid stress and is the only secondary growth tendency among the twelve strains. Next, the strains YGSH-6, YGSH-8, YGSH-12, YGSH-4 and YGSH-7, which had a final OD600 of about 4.0 at pH 3.0, were used. And the sensitivity of the rest strains to inorganic acid is basically the same as BY 4741.

TABLE 4

The foregoing is only for the purpose of understanding the method of the present invention and the core concept thereof, and it will be understood by those skilled in the art that various changes and modifications may be made without departing from the principle of the invention, and the invention also falls within the scope of the appended claims.

Sequence listing

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<120> application of GSH synthesis and cycle-related protein and recombinant saccharomyces cerevisiae strain

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Gly Gln Leu His Gln Tyr Leu Asn Asp Ser Gly Arg Tyr Ser Ala Asp

165 170 175

Gly Glu Ser Phe Phe Asn Glu Glu Ile Pro Val Ser Pro Ser Ala Glu

180 185 190

Leu Leu Ala Glu Gly Leu Thr Lys Ala Ile Gln His Phe Lys Pro Ala

195 200 205

Ala Ser Asp Lys Lys Pro Ile Val Ala Phe Ile Val Gln Glu Gly Glu

210 215 220

Arg Asn Val Phe Asp Gln Ser Val Leu Ala Phe Asn Leu Leu Ser Lys

225 230 235 240

His Gly Ile Gln Ser Cys Arg Leu Thr Met Gln Glu Ile His Leu Lys

245 250 255

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

260 265 270

Lys Glu Ile Gly Leu Val Tyr Phe Arg Ser Gly Tyr Ala Pro Asn Asp

275 280 285

Phe Val Thr Gln Gln Asp Trp Glu Asn Arg Phe Thr Leu Glu Ile Ser

290 295 300

Tyr Ala Ile Lys Ala Pro Asn Leu Leu Thr Gln Leu Ser Gly Thr Lys

305 310 315 320

Lys Ile Gln Gln Leu Leu Thr Asn Lys Asn Ile Leu Thr Lys Phe Leu

325 330 335

Pro Asp Ala Asn Ser Ala Asp Ala Leu Leu Gln Thr Phe Val Arg Ile

340 345 350

Tyr Pro Leu Asp Ser Ser Glu Leu Gly Glu Met Gly Lys Lys Leu Ala

355 360 365

Phe Thr Asn Pro Glu Asn Phe Val Leu Lys Pro Gln Arg Glu Gly Gly

370 375 380

Gly Asn Asn Ile Tyr Lys Glu Asp Ile Pro Thr Phe Leu Lys Ser Ile

385 390 395 400

Asp Glu Lys Asp Trp Pro Ala Tyr Ile Leu Met Glu Leu Ile Asn Pro

405 410 415

Arg Pro Thr Glu Glu Asn Ile Val Ile Arg Gly Glu Gln Thr Phe Gln

420 425 430

Val Pro Ile Leu Ser Glu Leu Gly Val Phe Gly Thr Ile Leu Phe Asp

435 440 445

Asn Lys Thr Val His Ser Asn Asp Tyr Ala Gly Trp Leu Leu Arg Ser

450 455 460

Lys Phe Ser Ala Ser Asn Glu Gly Gly Val Ala Ala Gly Phe Gly Cys

465 470 475 480

Val Asp Ser Val Val Leu Tyr

485

<210> 5

<211> 480

<212> PRT

<213> Wallemia mellicola

<400> 5

Met Glu Phe Pro Pro Lys Ile Glu Glu Ser Phe Glu Lys Glu Leu Ile

1 5 10 15

Glu Asn Leu Arg Ala Phe Cys Leu Gly Asn Gly Leu Val Leu Leu Pro

20 25 30

Pro Ile Lys Ala Gly Glu Thr Thr Val Ser Ser Ser Glu Gly Val Gln

35 40 45

Ala Pro Val Ser Leu Phe Pro Thr Pro Phe Pro Arg Lys Leu Tyr Asn

50 55 60

Gln Ala Leu Ser Ile Gln Pro Val Phe Asn Glu Leu Tyr Ala Asn Val

65 70 75 80

Ala Arg Asp Val Glu Phe Leu Asp Lys Val Met Ser Asp Val Ser Arg

85 90 95

Phe Asp Thr Phe Gln Ala Ser Leu Trp Asp Gln Trp Lys Ser Ile Arg

100 105 110

Asp Ser Ile Val Gln Pro Asn Gln Leu Leu Val Ala Arg Ser Asp Tyr

115 120 125

Leu Cys Asn Thr Ser Val Ser Ser Lys Gly Asp Val Lys Gly Leu Ser

130 135 140

Gln Val Glu Phe Asn Thr Ile Ala Ala Ser Phe Gly Ala Leu Ser Asn

145 150 155 160

Gln Val His Gln Leu His Asp Tyr Leu Ser Lys Asp Val Arg Tyr Ser

165 170 175

Gly Leu His Pro Leu Leu Lys Asn Asp Ile Ala Glu Asn Asn Thr Leu

180 185 190

Asn Gly Leu Ile Asp Gly Phe Glu Ala Ala His Lys Ile Tyr Lys Ser

195 200 205

Gly Tyr Leu Arg Ser Lys His Ser Pro Tyr Ile Leu Phe Val Val Gln

210 215 220

Asp Asn Glu Arg Asn Val Phe Asp Gln Arg Leu Leu Glu Phe Glu Leu

225 230 235 240

Ser Arg Arg Gly Ile Arg Thr Ile Arg Arg Thr Leu Thr Gln Leu Thr

245 250 255

Gln Gln Ala Ser Leu Ser Asp Glu Asn Ala Leu Lys Val Asn Gly Ile

260 265 270

Glu Ile Ser Val Val Tyr Tyr Arg Ser Gly Tyr Ser Pro Asp Asp Tyr

275 280 285

Thr Ser Lys Lys Glu Trp Asp Val Arg Lys Leu Ile Glu Leu Ser Leu

290 295 300

Ala Ile Lys Cys Pro Thr Leu Ala Leu Gln Leu Ala Gly Cys Lys Lys

305 310 315 320

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

325 330 335

Pro Ser Gln Val Arg Lys Ile Lys Thr Tyr Phe Val Glu Ile Leu Pro

340 345 350

Phe Asp Glu Ser Asp Arg Gly Lys Leu Ala His Ser Ile Val Asn Asn

355 360 365

Val Glu Lys Cys Arg Asp Phe Val Leu Lys Pro Gln Arg Glu Gly Gly

370 375 380

Gly Asn Asn Ile Tyr Lys Glu Gln Ile Lys Asp Phe Val Lys Gly Val

385 390 395 400

Lys Lys Glu Glu Leu Ser Gln Tyr Ile Leu Met Glu Leu Ile Lys Pro

405 410 415

Pro Ala Asn Leu Asn Asn Tyr Leu Val Arg Tyr Thr Asp Asp Arg Val

420 425 430

Ile Pro Ser Asp Val Val Ser Glu Leu Gly Ile Tyr Gly Thr Val Ile

435 440 445

Phe Asn Asn His Thr Ile Thr Arg Asn Glu Thr Ala Gly His Leu Leu

450 455 460

Arg Thr Lys Ser Lys Glu Ser Asp Glu Gly Gly Val Ala Val Gly Ile

465 470 475 480

<210> 6

<211> 165

<212> PRT

<213> Kluyveromyces marxianus

<400> 6

Met Ser Gly Asp Phe Tyr Ser Tyr Thr Cys Glu Glu Arg Asp Gly Ser

1 5 10 15

Glu Phe Pro Met Ser Lys Leu Arg Gly Lys Val Val Leu Ile Val Asn

20 25 30

Val Ala Ser Lys Cys Gly Phe Arg Met Gln Phe Gly Glu Leu Gln Asp

35 40 45

Leu Tyr Arg Glu Phe Gln Asn Gln Gly Leu Glu Ile Leu Ala Phe Pro

50 55 60

Cys Asn Gln Phe Ile Asn Gln Glu Pro Gly Thr Asp Glu Glu Ile Gly

65 70 75 80

Lys Phe Cys Gln Glu Tyr Tyr Gly Val Thr Tyr Lys Val Met Lys Lys

85 90 95

Cys Lys Val Asn Gly Gly Asp Ala Ile Pro Leu Tyr Asn Tyr Leu Lys

100 105 110

Asp Gln Lys Pro Gly Thr Leu Ser Phe Lys Phe Ile Arg Trp Asn Phe

115 120 125

Glu Lys Phe Leu Val Asp Arg Ser Gly Lys Val Ile Lys Arg Tyr Ser

130 135 140

Thr Leu Val Lys Pro Lys Asp Met Arg Asp Asp Ile Ile Lys Leu Leu

145 150 155 160

Asn Gly Glu Glu Val

165

<210> 7

<211> 160

<212> PRT

<213> Wallemia mellicola

<400> 7

Met Ser Ala Ser Glu Phe Tyr Asn Leu Lys Ala Glu Lys Pro Asn Gly

1 5 10 15

Gln Glu Tyr Ser Phe Asp Thr Leu Lys Asp Lys Val Val Leu Val Val

20 25 30

Asn Thr Ala Ser Lys Cys Gly Phe Thr Pro Gln Tyr Thr Gly Leu Glu

35 40 45

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

50 55 60

Pro Cys Asn Gln Phe Gly Gly Gln Glu Pro Gly Thr Asp Asp Asp Ile

65 70 75 80

Asp Asn Phe Cys Lys Ile Asn His Gly Val Ser Phe Gln Leu Phe Lys

85 90 95

Lys Ser Asp Val Asn Gly Asp Lys Thr Asn Glu Val Phe Lys Tyr Leu

100 105 110

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

115 120 125

Glu Lys Phe Leu Val Asp Lys Gln Gly Asn Val Val Asn Arg Tyr Ser

130 135 140

Ser Met Ala Lys Pro Ser Asp Ile Gly Asn Asp Val Glu Lys Leu Leu

145 150 155 160

<210> 8

<211> 107

<212> PRT

<213> Kluyveromyces marxianus

<400> 8

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

1 5 10 15

Ser Lys Ile Phe Val Ala Ala Lys Thr Tyr Cys Pro Tyr Cys Gln Ala

20 25 30

Thr Leu Lys Thr Leu Phe Asp Asp Lys Lys Val Asn Lys Asp Ile Ala

35 40 45

Thr Val Leu Gln Leu Asn Glu Leu Asp Asp Gly Ala Glu Ile Gln Asp

50 55 60

Ala Leu Leu Glu Ile Ser Gly Gln Arg Thr Val Pro Asn Ile Tyr Ile

65 70 75 80

Asn Gly Lys His Ile Gly Gly Asn Ser Asp Leu Gln Glu Leu Asn Ser

85 90 95

Ser Gly Glu Leu Asp Lys Leu Leu Ala Ala Leu

100 105

<210> 9

<211> 151

<212> PRT

<213> Wallemia mellicola

<400> 9

Met Met Phe Asn Leu Leu Arg Asn Ser Ala Ser Thr Arg Ala Ala Lys

1 5 10 15

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

20 25 30

Phe Arg Gln Leu Ser Thr Glu Ser Arg Lys Leu Ile Asp Gln Ala Val

35 40 45

Asn Ala His Pro Ile Val Leu Phe Met Lys Gly Lys Pro Ser Ala Pro

50 55 60

Leu Cys Gly Phe Ser Arg Ala Val Val Gln Ile Leu Asp Val Gln Gly

65 70 75 80

Ala Asp Pro Glu Lys Ile Arg Ala Tyr Asp Cys Leu Glu Asp Asp Glu

85 90 95

Leu Arg Asn Gly Ile Lys Glu Tyr Ser Asp Trp Pro Thr Ile Pro Gln

100 105 110

Val Tyr Val Asn Gly Glu Phe Val Gly Gly Cys Asp Ile Leu Leu Ser

115 120 125

Met His Gln Ser Gly Glu Leu Ser Gln Leu Leu Lys Asp Lys Asn Leu

130 135 140

Leu Leu Glu Phe Ala Asn Glu

145 150

<210> 10

<211> 469

<212> PRT

<213> Kluyveromyces marxianus

<400> 10

Met Ser Ala Ala Thr Asn His Tyr Asp Tyr Leu Val Ile Gly Gly Gly

1 5 10 15

Ser Gly Gly Val Ala Ser Ser Arg Arg Ala Ala Lys Tyr Gly Ala Lys

20 25 30

Thr Leu Leu Ile Glu Gly Lys Ala Met Gly Gly Thr Cys Val Asn Lys

35 40 45

Gly Cys Val Pro Lys Lys Val Met Trp Tyr Ala Ser Asp Leu Ala Thr

50 55 60

Arg Ile Thr His Ala His Ser Tyr Asn Leu Phe Glu Asp Leu Pro Leu

65 70 75 80

Thr Lys Asp Asn Leu Thr Phe Asn Trp Pro Glu Phe Lys Lys Lys Arg

85 90 95

Asp Ala Tyr Ile His Arg Leu Asn Gly Ile Tyr Glu Arg Asn Leu Thr

100 105 110

Lys Glu Gly Val Asp Phe Ile Tyr Gly Trp Ala Ser Phe Thr Glu Asp

115 120 125

Gly Lys Val Gln Val Arg Lys Ala Asp Asn Ser Val Glu Thr Tyr Thr

130 135 140

Ala Glu His Ile Leu Ile Ala Thr Gly Gly Lys Pro Val Phe Pro Ser

145 150 155 160

Lys Val Pro Gly Tyr Glu Tyr Gly Ile Ser Ser Asp Gly Phe Phe Glu

165 170 175

Leu Glu Lys Gln Pro Glu Lys Val Val Val Val Gly Ala Gly Tyr Ile

180 185 190

Gly Val Glu Leu Ala Gly Val Phe Asn Gly Leu Gly Ser Asp Ser His

195 200 205

Leu Val Ile Arg Gly Glu Thr Val Leu Arg Lys Phe Asp Gln Cys Ile

210 215 220

Gln Asp Thr Val Thr Asp Thr Tyr Ile Lys Glu Gly Val Asn Ile His

225 230 235 240

Lys Ser Ser Asn Val Thr Lys Val Glu Lys Asp Glu Lys Thr Gly Lys

245 250 255

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

260 265 270

Leu Ile Trp Thr Ile Gly Arg Arg Ser Met Leu Gly Leu Gly Leu Glu

275 280 285

Asn Ile Gly Val Lys Leu Asn Glu Arg Glu Gln Ile Ile Val Asp Glu

290 295 300

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

305 310 315 320

Gly Lys Val Glu Leu Thr Pro Val Ala Ile Ala Ala Gly Arg Lys Leu

325 330 335

Ala Asn Arg Leu Phe Gly Pro Asp Gln Phe Lys Asn Gln Lys Gln Asp

340 345 350

Tyr Glu Asn Val Pro Ser Val Ile Phe Ser His Pro Glu Ala Gly Ser

355 360 365

Ile Gly Leu Ser Glu Lys Glu Ala Ile Glu Lys Phe Gly Lys Glu Asn

370 375 380

Val Lys Val Tyr Asn Ser Lys Phe Asn Ala Met Tyr Tyr Ala Met Met

385 390 395 400

Asn Glu Glu Asn Lys Thr Pro Thr Arg Tyr Lys Leu Val Cys Ala Gly

405 410 415

Glu Glu Glu Lys Val Val Gly Leu His Ile Val Gly Asp Ser Ser Ala

420 425 430

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

435 440 445

Ala Asp Phe Asp Ser Cys Val Ala Ile His Pro Thr Ser Ala Glu Glu

450 455 460

Ile Val Thr Leu Thr

465

<210> 11

<211> 411

<212> PRT

<213> Kluyveromyces marxianus

<400> 11

Met Ile His Phe Trp Arg Lys Glu Cys Asn Ala Phe Ser Gln His Ile

1 5 10 15

Gly Leu Leu Gln Arg Arg Phe Tyr Thr Glu Val Lys Leu Arg Asp Ser

20 25 30

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

35 40 45

Leu Pro Asp Phe Ile Ser Ser Pro Ser Ser Lys Leu Gln Ser Leu Ile

50 55 60

Trp His Arg Pro Met Gln Asn Val Phe Leu Met Lys Lys Pro Trp Thr

65 70 75 80

Asn Thr Thr Arg Lys Ala Met Val Glu Phe Ile Thr His Leu His Asp

85 90 95

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

100 105 110

Ile Ser Gln Asp Phe Arg Ser Met Pro Lys Ser Asn Pro Asn Gln Pro

115 120 125

His Val Leu Tyr Thr Gly Pro Asn Ser Glu Ile Ile Lys Lys Thr Asp

130 135 140

Leu Leu Val Thr Leu Gly Gly Asp Gly Thr Ile Leu Arg Ser Val Ser

145 150 155 160

Leu Phe Ser His Thr Gln Val Pro Pro Val Leu Ala Phe Ser Leu Gly

165 170 175

Thr Leu Gly Phe Leu Leu Pro Phe Asp Phe Lys Asp His Lys Glu Val

180 185 190

Phe Glu Arg Val Leu Thr Ser Arg Ala Lys Cys Leu His Arg Thr Arg

195 200 205

Leu Glu Cys His Leu Val Arg Asn Gly Gln Thr Lys Gln Thr Lys Ser

210 215 220

Leu His Ala Met Asn Asp Ile Phe Leu His Arg Gly Asp Ser Ser His

225 230 235 240

Leu Thr Asn Leu Asp Ile Tyr Ile Asp Gly Glu Phe Leu Thr Arg Thr

245 250 255

Thr Ala Asp Gly Val Thr Leu Ala Thr Pro Thr Gly Ser Thr Ala Tyr

260 265 270

Ser Leu Ser Ala Gly Gly Ser Ile Val Ser Pro Leu Val Pro Ser Ile

275 280 285

Leu Leu Thr Pro Ile Cys Pro Arg Ser Leu Ser Phe Arg Pro Leu Ile

290 295 300

Leu Pro Glu Thr Ser His Ile Lys Ile Lys Ile Gly Ser Lys Asn Asn

305 310 315 320

Gly Gly Pro Asp Ser Ser Val Val Lys Leu Ser Ile Asp Gly Ile Pro

325 330 335

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

340 345 350

Gly Thr Ile Tyr Val Asn Gly Thr Lys Leu Pro Leu Pro Gln Glu Ala

355 360 365

Leu Lys Ser Ala Lys Pro Ser Val Lys Asn Ala Gly Ile Tyr Cys Val

370 375 380

Ala Lys Thr Glu Asn Asp Trp Thr Lys Gly Ile Asn Glu Leu Leu Gly

385 390 395 400

Phe Asn Ser Ser Phe Arg Phe Ala Ser Lys Pro

405 410

<210> 12

<211> 405

<212> PRT

<213> Wallemia mellicola

<400> 12

Met Leu Arg Ser Leu Val Arg Tyr Thr Lys Ser Ala Leu Asp Lys Leu

1 5 10 15

Pro Asn Lys Val Asp Phe Glu Leu Lys Pro Leu Gly Arg Leu Asn Lys

20 25 30

Gln Val Thr Ser Lys Arg Val Pro His Val Thr Gln Ser Val Gly Ser

35 40 45

Met Phe Gly Gly Asn His Ser Leu Lys Trp Thr Ser Gln Pro Arg Asn

50 55 60

Val Leu Ile Val Lys Lys Ser Asp Ser Glu Cys Ser Thr Arg Ala Met

65 70 75 80

Glu Glu Val Ile Ala His Leu Arg Thr Asn Tyr Cys Asp Thr Asn Ile

85 90 95

Ile Val Glu Asn Gly Val Lys Glu Glu Leu Lys Ala Ser Arg Glu Leu

100 105 110

Tyr Thr Thr Thr Glu Thr Asp Glu Phe Thr Leu Ser Ser Lys Val Asp

115 120 125

Phe Ala Ile Thr Leu Gly Gly Asp Gly Thr Ala Leu His Thr Ala Ser

130 135 140

Leu Phe Pro Thr Gly Pro Val Pro Pro Val Leu Ser Phe Ser Thr Gly

145 150 155 160

Thr Leu Gly Phe Leu Leu Pro Phe His Ile Asn Ser Tyr Lys Ser Ala

165 170 175

Ile Asp Asp Val Leu Asn Ser Asn Val Ser Val Ile Lys Arg Met Arg

180 185 190

Leu Met Cys Thr Leu His Asp Ala Ser Gly Gly Leu Ile Asp Asp Leu

195 200 205

Asp Val Thr His Val Leu Asn Glu Val Ala Leu His Arg Gly Arg Tyr

210 215 220

Pro His Leu Val Gln Ile Glu Ile Tyr Val Asp Gly Met Pro Leu Thr

225 230 235 240

Glu Thr Val Ala Asp Gly Leu Ile Val Ser Thr Pro Thr Gly Ser Ser

245 250 255

Ala Tyr Ser Leu Ser Ala Gly Gly Pro Leu Val His Pro Cys Val Gln

260 265 270

Ser Ile Val Leu Thr Pro Ile Cys Pro Arg Ser Leu Ser Phe Arg Pro

275 280 285

Val Ile Leu Pro Ser Asp Ser Thr Val Gln Leu Arg Met Ser Thr Lys

290 295 300

Ala Arg Ser Lys Pro Asp Val Ser Leu Asp Gly Arg Glu Val Met Gln

305 310 315 320

Leu Asp Ser Asp Asn Tyr Ile Gln Ile Ser Met Ser Pro Phe Pro Leu

325 330 335

Pro Ser Ile Asn Arg Ala Ala Ile Tyr Asp Pro Glu Ser Arg Gln Gly

340 345 350

Thr Pro Arg Leu Pro Ser Glu Lys Gln Leu Ala Gln Ser Ala Leu Asp

355 360 365

Arg Leu Gly Arg Ala Gln Asp Asp Trp Val Arg Asp Ile Asn Asp Leu

370 375 380

Leu Asn Phe Asn Ser Arg Phe Glu Ser Lys Gly Gln Glu Ile Tyr His

385 390 395 400

Gly Gly Gln Asp Asp

405

<210> 13

<211> 465

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atggttaaag ctattgcagt tttgaagggt gactctaatg tttcaggtat cgttagattc 60

gaacaagaat ctgaagatca atcaactaaa atttcttggg aaatcactgg taacgatgct 120

aacgcattga gaggtttcca tatccatgaa tttggtgaca actcaaatgg ttgtacttct 180

gctggtccac atttcaaccc atacaagaaa actcatggtg ctccatcaga tgaaacaaga 240

catgttggtg acttgggtaa tatttcaact gatgctcaag gtgttgcaaa gggttctgtt 300

acagataagc atgttaaatt gttgggtcca ttgtctgtta ttggtagaac tgttgttgtt 360

catggtggtc aagatgattt gggtaaaggt ggtaacgaag aatcattaaa gactggtaat 420

gctggtggta gagttgcatg tggtgttatt ggtatttcta attaa 465

<210> 14

<211> 615

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

atgccatatc cttttaaatt gccagatttg ggttatccat acgaagcatt ggaaccacat 60

atcgatgcaa agactatgga aatccatcat caaaagcatc atggtgcata cgttacaaat 120

ttgaacgctg cattggaaaa gtacccatac ttacatggtg ttgaagttga agttttgttg 180

agacatttgg ctgcattacc acaagatatt caaactgctg ttagaaacaa tggtggtggt 240

catttgaacc attctttatt ttggagattg ttaacaccag gtggtgctaa agaaccagtt 300

ggtgaattga agaaagcaat cgatgaacaa tttggtggtt tccaagcatt gaaggaaaaa 360

ttgactcaag ctgcaatggg tagatttggt tctggttggg catggttggt taaagatcca 420

ttcggtaaat tgcatgtttt atcaactcca aaccaagata acccagttat ggaaggtttt 480

acaccaattg ttggtattga tgtttgggaa catgcttact acttgaagta ccaaaacaga 540

agagctgatt acttgcaagc aatctggaac gttttgaact gggatgttgc tgaagaattt 600

ttcaagaaag cataa 615

<210> 15

<211> 2007

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atgggtttgt tatcattggg tactccattg ccatggttgg aatctagaca atacaacgaa 60

catgttagag ataacggtat cgaacaattg atccaatctt ttagaaaagc tggtggtaga 120

gataatgatg aattatactg gggtgacgaa gttgaataca tgatctgtga atttgatgaa 180

gcaggtgaaa atgttgtttt gtctgttgaa catgatgaaa ttttggaaca attgaacaga 240

gaataccatg aagaatgtga aagattaaac gttcatttcc atccagaata tggtagattc 300

atgttggaag ctacaccagc aagaccatac catttgtatg aaggtgttga agttgaaaag 360

aatatgagaa ctagaagagt tgttgctgaa gaaaaattgc aaaaattgaa cagaaagtct 420

ggtaatgaag ttgttccatt gtctttaaca gtttttccaa gaatgggtag agatggtttt 480

actaatttga aagatccatg ggatcataaa aattctgctt ctagatcatt gtttttgcca 540

gatgaagtta ttaatagaca tgctagattc ccaactttga ctgcaaacat cagaacaaga 600

agaggtgaaa aggtttgtat caacgttcca atgtaccaag attcaagaac tgctccaaga 660

gatgaatcta tctatgaaag agattggttt gttccagaag atttggaatc agctaaagca 720

tctaaaccag gtcatatcta tatggattca atgggttttg gtatgggttg ttcttgtttg 780

caattgacat tccaagctcc aaacatcgat aaggcaagat acttgtacga tacattggct 840

aactttgcac cagttttctt ggctgcaact gctgcatctc cagtttttaa aggtttcttg 900

gctgatcaag atgttagatg gaatgttatt tcaggtgcag ttgatgatag aactccatac 960

gaaagatcag aagaaccatt gttgccaaag tacaacaacg gttacggttc aattgctcca 1020

gaagaagttc catctgttag aagaattaat aagtcaagat attctgttgt tgatttgttt 1080

ttaggtggta acggtttctt tgatgcaaag tttaatgata cagaagttcc aattaatgaa 1140

aaggttttta aaagattgac tacaaacgat attcatccaa tggataaaga tttggctaga 1200

catttcgcac atttgtttat tagagatcca ttagttattt ttgaagaaag aatcgaacaa 1260

gataacatga ctgaaacaga tcatttcgaa aacatccaat caacaaactg gcaatctttg 1320

agattcaaag ttccagctca agatgcaact ccaaataata agaaagctcc aggttggaga 1380

gttgagttta gaccaatgga agttcaattg acagatttcg aaaacgctgc atactcaaac 1440

ttcatatatt tggttattga atcaatttta actttttctg ataagattaa tgcatacatg 1500

tacatgtctg aaatttggga aaacatggaa acagctcata gaagagatgc aactttgaag 1560

gaaaagttct actggaagtc agatttcgct gatactcaag gtaaaacaga attgttgact 1620

attaatgaaa tttttcataa tgcacataat ggtattttct ctgtttttat taacccaatc 1680

ttggttcata agggtttagt ttcaaagtct tggactgaat tgttgcataa ttcaggtgac 1740

aatgctgatt tgatcagatt atactactat ttgaaattaa tttcagatag agcttctggt 1800

gttttgccat cagaagcatc ttttattaga tcatacgttt tgaaccatcc agaatacgat 1860

catacatcta acgttactaa gaaaattaat tacgatttgt tacatttgtc atacagaatt 1920

tctcattacg ataactcaaa gggtgaatta acagctttgt tgggtcaaga aatcgcaact 1980

tatttgatta ataattctat tttataa 2007

<210> 16

<211> 1464

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

atgtcttcat tgaagggtta tccaaagttc ccagatgtta ctaaggaaaa attggaaaga 60

gaattgttgc cagaagtttt ccaatgggct atttctaatg gtttgacaat ctatccacca 120

gatttcaaaa tttcagaagc tcaagttgca ccaattactt tgtttccaac accattacca 180

agaaaagcat ttgaagatgc aatcgatgtt caaaaatctt ttaattcatt gtatgctaaa 240

atttctcaaa accataacgg ttggttggaa gaagaatctg aaaaattggc aaaatcagat 300

ccagagttta ctggtaaatt atgggaaata tataagagag ctaaggaaca tggtatttct 360

caagatttgg cattaggtgt ttttagatca gattacttga ttaatgataa cgataaccaa 420

attaaacaag ttgagtttaa tactgtttct gtttcatttg ctggtttatc tacaaaggtt 480

ggtcaattgc atcaatattt gaacgattct ggtagatact cagcagatgg tgaatctttc 540

tttaacgaag aaatcccagt ttctccatca gctgaattgt tagcagaagg tttgactaag 600

gctatccaac atttcaaacc agctgcatct gataagaaac caatcgttgc ttttattgtt 660

caagaaggtg aaagaaacgt tttcgatcaa tcagttttgg cttttaattt gttgtctaag 720

catggtattc aatcatgtag attaacaatg caagaaatcc atttgaagac tacattggat 780

aaatctacta aaagattgta cttaaattct actggtaaag aaatcggttt agtttacttc 840

agatcaggtt acgctccaaa tgatttcgtt actcaacaag attgggaaaa cagattcact 900

ttggaaattt cttacgctat taaagcacca aatttgttga ctcaattgtc aggtactaag 960

aaaattcaac aattgttgac taataagaac atcttgacaa agttcttacc agatgctaat 1020

tctgctgatg cattgttaca aactttcgtt agaatctatc cattggattc ttcagaatta 1080

ggtgaaatgg gtaaaaagtt ggcttttact aacccagaaa acttcgtttt gaaaccacaa 1140

agagaaggtg gtggtaataa tatctataag gaagatattc caacattttt aaagtctatt 1200

gatgaaaaag attggccagc ttacatcttg atggaattaa ttaatccaag accaactgaa 1260

gaaaacatcg ttatcagagg tgaacaaaca ttccaagttc caatcttgtc agaattaggt 1320

gttttcggta ctatcttgtt cgataataag acagttcatt ctaatgatta tgctggttgg 1380

ttgttgagat caaaattttc tgcatcaaac gaaggtggtg ttgctgctgg tttcggttgt 1440

gttgattctg ttgttttgta ctaa 1464

<210> 17

<211> 1443

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

atggaatttc caccaaaaat tgaagaatct tttgaaaagg aattaatcga aaatttgaga 60

gctttctgtt tgggtaacgg tttggttttg ttaccaccaa ttaaagctgg tgaaactaca 120

gtttcttcat ctgaaggtgt tcaagcacca gtttcattat tcccaactcc attcccaaga 180

aaattgtaca accaagcatt gtctatccaa ccagttttta atgaattata cgctaatgtt 240

gcaagagatg ttgaattttt ggataaagtt atgtcagatg tttctagatt cgatacattc 300

caagcatctt tgtgggatca atggaagtca atcagagatt ctatcgttca accaaaccaa 360

ttgttggttg caagatcaga ttatttgtgt aacacttctg tttcatctaa aggtgacgtt 420

aagggtttgt ctcaagttga gtttaataca attgctgcat catttggtgc tttatctaac 480

caagttcatc aattgcatga ttatttgtca aaagatgtta gatactctgg tttgcatcca 540

ttgttgaaaa atgatattgc agaaaacaac actttgaatg gtttgattga tggtttcgaa 600

gctgcacata aaatctataa gtcaggttac ttaagatcaa agcattctcc atacatcttg 660

ttcgttgttc aagataacga aagaaacgtt ttcgatcaaa gattgttaga atttgaatta 720

tctagaagag gtattagaac tatcagaaga actttgacac aattgacaca acaagcatct 780

ttgtctgatg aaaacgcatt gaaggttaac ggtatcgaaa tttctgttgt ttactacaga 840

tcaggttatt ctccagatga ttacacttct aagaaagaat gggatgttag aaaattgatc 900

gaattgtctt tagctattaa atgtccaaca ttggctttac aattggcagg ttgtaagaaa 960

gttcaacaag ttttggttga tgaaaacact ttgtcaaagt atttgaaccc atctcaagtt 1020

agaaagatta aaacatactt cgttgaaatt ttaccatttg atgaatcaga tcgtggtaaa 1080

ttggctcatt ctatcgttaa caacgttgaa aagtgtagag atttcgtttt gaagccacaa 1140

agagaaggtg gtggtaacaa catctataag gaacaaatta aagattttgt taaaggtgtt 1200

aagaaagaag aattgtctca atacatcttg atggaattga ttaaaccacc agctaatttg 1260

aacaactatt tggttagata cactgatgat agagttattc catcagatgt tgtttctgaa 1320

ttgggtatct atggtacagt tatttttaat aaccatacta tcacaagaaa tgaaactgct 1380

ggtcatttgt tgagaacaaa gtcaaaggaa tctgatgaag gtggtgttgc agttggtatt 1440

taa 1443

<210> 18

<211> 498

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

atgtctggtg acttctactc atacacttgt gaagaaagag atggttctga atttccaatg 60

tcaaaattgc gtggtaaagt tgttttgatc gttaacgttg cttctaagtg tggttttaga 120

atgcaatttg gtgaattgca agatttgtac agagaatttc aaaaccaagg tttggaaatt 180

ttagcattcc catgtaacca attcattaat caagaaccag gtactgatga agaaatcggt 240

aaattctgtc aagaatacta cggtgttaca tacaaagtta tgaagaaatg caaggttaat 300

ggtggtgacg ctatcccatt gtacaactac ttaaaggatc aaaagccagg tactttgtct 360

tttaagttta ttagatggaa cttcgaaaag ttcttggttg atagatcagg taaagttatt 420

aaaagatatt caacattggt taaaccaaaa gatatgagag atgatattat taaattgtta 480

aatggtgaag aagtttaa 498

<210> 19

<211> 483

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

atgtctgctt cagaatttta caatttgaag gcagaaaaac caaacggtca agaatactct 60

ttcgatactt tgaaggataa ggttgtttta gttgttaaca cagcttcaaa gtgtggtttt 120

actccacaat acacaggttt ggaagaatta catcaaaagt acaaggatag aggtttggtt 180

gttttgggtt tcccatgtaa ccaattcggt ggtcaagaac caggtactga tgatgatatt 240

gataacttct gtaagattaa tcatggtgtt tcttttcaat tgtttaagaa atctgatgtt 300

aacggtgaca agactaacga agtttttaaa tatttgaagg ataagaaatc tcaattgggt 360

ttaacaagaa ttaaatggaa cttcgaaaag ttcttagttg ataaacaggg taacgttgtt 420

aacagatact cttcaatggc aaaaccatca gatattggta atgatgttga aaaattgtta 480

taa 483

<210> 20

<211> 324

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

atggctccag tttctactgt ttcaagagtt caaggtttga ttaattcttc aaagattttt 60

gttgctgcaa agacatattg tccatactgt caagctactt tgaagacatt gttcgatgat 120

aagaaagtta ataaggatat tgcaactgtt ttgcaattga acgaattaga tgatggtgct 180

gaaatccaag atgcattgtt ggaaatttct ggtcaaagaa cagttccaaa catctatatc 240

aacggtaaac atattggtgg taattcagat ttgcaagaat taaattcttc tggtgaattg 300

gataaattgt tagctgcatt ataa 324

<210> 21

<211> 456

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

atgatgttca atttgttgag aaattctgct tcaactagag ctgcaaaatt gttacaacca 60

gtttacacac catcaacttt gacaagattg aacaacttca gacaattatc tactgaatca 120

agaaaattga tcgatcaagc tgttaacgca catccaatcg ttttgtttat gaagggtaaa 180

ccatcagctc cattgtgtgg tttttctaga gcagttgttc aaattttaga tgttcaaggt 240

gctgatccag aaaagattag agcatacgat tgtttggaag atgatgaatt gagaaacggt 300

attaaagaat actctgattg gccaacaatt ccacaagttt acgttaatgg tgaatttgtt 360

ggtggttgtg atattttgtt gtctatgcat caatctggtg aattgtctca attgttaaaa 420

gataaaaatt tgttattgga atttgcaaat gaataa 456

<210> 22

<211> 1407

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

atgtctgctg caacaaacca ttacgattac ttagttattg gtggtggttc aggtggtgtt 60

gcttcttcaa gaagagctgc aaagtacggt gctaagacat tgttgatcga gggtaaagca 120

atgggtggta cttgtgttaa taagggttgt gttccaaaga aagttatgtg gtatgcttct 180

gatttggcaa caagaatcac tcatgctcat tcatacaatt tgttcgaaga tttgccattg 240

acaaaggata atttgacttt taattggcca gagtttaaaa agaaaagaga tgcttacatc 300

catagattaa atggtatcta tgaaagaaat ttgacaaagg aaggtgttga tttcatctat 360

ggttgggctt cttttactga agatggtaaa gttcaagtta gaaaggcaga taactcagtt 420

gaaacttaca cagctgaaca tatcttgatt gcaacaggtg gtaaaccagt ttttccatct 480

aaagttccag gttatgaata cggtatttct tcagatggtt tctttgaatt agaaaagcaa 540

ccagaaaaag ttgttgttgt tggtgctggt tatattggtg ttgaattggc aggtgttttt 600

aatggtttag gttctgattc acatttggtt attagaggtg aaactgtttt gagaaagttc 660

gatcaatgta tccaagatac tgttacagat acttacatta aagaaggtgt taacatccat 720

aaatcttcaa acgttacaaa agttgaaaag gatgaaaaga ctggtaaatt aactattttc 780

ttggatacag gtaaaaatat cactgatgtt gattctttga tctggactat cggtagaaga 840

tcaatgttgg gtttaggttt ggaaaatatt ggtgttaaat tgaatgaaag agaacaaatt 900

attgttgatg aataccaaaa cacaaacgtt aaaaatgttt actctttagg tgacgttgtt 960

ggtaaagttg aattgactcc agttgctatt gctgctggta gaaaattggc aaatagattg 1020

ttcggtccag atcaattcaa aaaccaaaag caagattacg aaaatgttcc atctgttatt 1080

ttctctcatc cagaagctgg ttctatcggt ttatcagaaa aggaagcaat tgaaaaattt 1140

ggtaaagaaa acgttaaggt ttacaactct aagtttaatg ctatgtacta cgcaatgatg 1200

aacgaagaaa ataagacacc aactagatac aaattggttt gtgctggtga agaagaaaag 1260

gttgttggtt tacatattgt tggtgactct tcagctgaaa ttttgcaagg ttttggtgtt 1320

gcaattaaaa tgggtgctac aaaagcagat tttgattctt gtgttgctat tcatccaact 1380

tcagcagaag aaattgttac attaact 1407

<210> 23

<211> 1236

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

atgatccatt tctggagaaa ggaatgtaac gctttttctc aacatatcgg tttgttacaa 60

agaagattct acacagaagt taaattgaga gattcttcaa gattcgttac attgaaggat 120

gttggtaatt tgagagcaac ttctttacca gatttcatct cttcaccatc ttcaaaattg 180

caatcattaa tctggcatag accaatgcaa aatgttttct tgatgaagaa accatggaca 240

aatactacaa gaaaggctat ggttgagttt attactcatt tgcatgattc ttacccagaa 300

attaatgtta tcgttcaacc agatgttgca gaagaaattt cacaagattt cagatcaatg 360

ccaaagtcaa acccaaacca accacatgtt ttgtacacag gtccaaattc tgaaattatt 420

aagaaaactg atttgttagt tacattaggt ggtgacggta ctatcttgag atcagtttct 480

ttgttttctc atactcaagt tccaccagtt ttggcttttt cattgggtac tttaggtttc 540

ttgttaccat tcgatttcaa ggatcataag gaagtttttg aaagagtttt gacatctaga 600

gcaaagtgtt tgcatagaac tagattggaa tgtcatttgg ttagaaacgg tcaaactaag 660

caaacaaaat cattgcatgc aatgaacgat attttcttgc atcgtggtga ctcttcacat 720

ttgactaatt tggatatata tattgatggt gaatttttga ctagaactac agctgatggt 780

gttacattag caactccaac aggttctact gcttactctt tgtcagcagg tggttctatt 840

gtttcaccat tagttccatc aatcttgttg acaccaattt gtccaagatc attgtctttt 900

agaccattga tcttaccaga aacttctcat atcaagatta aaatcggttc taaaaataat 960

ggtggtccag attcttcagt tgttaaattg tctatcgatg gtattccaca agatgatgtt 1020

tacgttaacg atgaaatcca tatcgttaat gaaatcggta ctatctatgt taacggtaca 1080

aaattgccat taccacaaga agcattgaag tctgcaaagc catcagttaa aaatgctggt 1140

atctattgtg ttgcaaagac tgaaaacgat tggacaaagg gtattaatga attgttaggt 1200

tttaattctt cttttagatt tgcttctaaa ccataa 1236

<210> 24

<211> 1218

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

atgttgagat cattagttag atacacaaag tctgctttgg ataaattgcc aaataaggtt 60

gatttcgaat tgaagccatt gggtagattg aataagcaag ttacatctaa gagagttcca 120

catgttactc aatctgttgg ttcaatgttc ggtggtaacc attctttgaa atggacatca 180

caaccaagaa acgttttgat cgttaagaaa tctgattcag aatgttcaac tagagctatg 240

gaagaagtta ttgcacattt gagaacaaac tactgtgata ctaacatcat cgttgaaaat 300

ggtgttaagg aagaattaaa agcatctaga gaattgtaca ctacaactga aacagatgag 360

tttactttat cttcaaaggt tgattttgca attacattgg gtggtgacgg tactgcttta 420

catactgcat cattgtttcc aactggtcca gttccaccag ttttatcttt ttcaacaggt 480

actttgggtt tcttgttgcc attccatatc aattcttata aatcagcaat tgatgatgtt 540

ttaaattcta atgtttcagt tattaaaaga atgagattaa tgtgtacatt gcatgatgct 600

tctggtggtt taatcgatga tttggatgtt actcatgttt tgaacgaagt tgcattgcat 660

agaggtagat acccacattt ggttcaaatc gaaatatatg ttgatggtat gccattaaca 720

gaaactgttg ctgatggttt gattgtttca acaccaactg gttcttcagc ttactcttta 780

tcagcaggtg gtccattggt tcatccatgt gttcaatcta tcgttttgac accaatctgt 840

ccaagatcat tgtcttttag accagttatt ttgccatctg attcaacagt tcaattgaga 900

atgtctacta aagcaagatc aaaaccagat gtttctttag atggtagaga agttatgcaa 960

ttggattctg ataactacat ccaaatttct atgtcaccat ttccattgcc atcaattaat 1020

agagctgcaa tctatgatcc agaatctaga caaggtactc caagattacc atcagaaaaa 1080

caattggctc aatctgcatt agatagattg ggtagagcac aagatgattg ggttagagat 1140

attaacgatt tgttgaactt caactctaga tttgaatcaa aaggtcaaga aatatatcat 1200

ggtggtcaag atgattaa 1218

Claims (12)

1. A recombinant strain of saccharomyces cerevisiae with improved stress resistance, capable of expressing any one or more of the following exogenous GSH synthesis and circulation-related proteins:

(1) superoxide dismutase derived from Kluyveromyces marxianus;

(2) superoxide dismutase derived from Thermus thermophilus HB 27;

(3) gamma-glutamylcysteine synthetase derived from Kluyveromyces marxianus;

(4) glutathione synthetase derived from Kluyveromyces marxianus;

(5) glutathione synthetase derived from Wallemia mellicol;

(6) glutathione peroxidase derived from Kluyveromyces marxianus;

(7) glutathione peroxidase from Wallemia mellicol;

(8) cytoplasmic glutaredoxin derived from Kluyveromyces marxianus;

(9) cytoplasmic glutaredoxin derived from Wallemia mellicol

(10) Glutathione oxidoreductase from Kluyveromyces marxianus in cytoplasm and mitochondria;

(11) NADH kinase derived from Kluyveromyces marxianus;

(12) NADH kinase derived from Wallemia mellicol;

the stress resistance comprises H resistance₂O₂Heat resistance, high temperature resistance, salt resistance and acid resistance.

2. The recombinant Saccharomyces cerevisiae according to claim 1, characterized in that it is H-tolerant₂O₂A recombinant Saccharomyces cerevisiae expressing one or more of the exogenous GSH synthesis and circulation associated proteins of (1), (4) - (12) of claim 1.

3. The recombinant Saccharomyces cerevisiae of claim 1, which is a thermotolerant recombinant Saccharomyces cerevisiae expressing one or more of the exogenous GSH synthesis and circulation associated proteins of (1) - (8) and (10) - (12) of claim 1.

4. The recombinant Saccharomyces cerevisiae of claim 1, which is a high temperature resistant recombinant Saccharomyces cerevisiae, expressing one or more exogenous GSH synthesis and cycle associated proteins of claim 1 (10), (11).

5. The recombinant Saccharomyces cerevisiae of claim 1, which is a salt tolerant recombinant Saccharomyces cerevisiae expressing one or more exogenous GSH synthesis and circulation associated proteins of (1) - (12) of claim 1.

6. The recombinant Saccharomyces cerevisiae of claim 1, which is acid-tolerant recombinant Saccharomyces cerevisiae transformed to express one or more of the exogenous GSH synthesis and circulation-associated proteins of claim 1 (1) - (2), (4), (6) - (10), (12).

7. The recombinant Saccharomyces cerevisiae according to claim 1, characterized in that it is H-tolerant₂O₂A recombinant saccharomyces cerevisiae resistant to two or more of heat, salt and acid, expressing the exogenous GSH synthesis and circulation-associated protein of claim 1, item (4).

8. The recombinant Saccharomyces cerevisiae according to claim 1, characterized in that it is H-tolerant₂O₂A recombinant saccharomyces cerevisiae resistant to two or more of heat shock and salt, expressing the exogenous GSH synthesis and cycle-associated protein of claim 1 (5).

9. The recombinant Saccharomyces cerevisiae according to claim 1, characterized in that it is H-tolerant₂O₂Recombinant Saccharomyces cerevisiae resistant to two or more of heat, salt, heat and acid, expressing the exogenous GSH synthesis and circulation associated protein of claim 1, item (10).

10. The recombinant Saccharomyces cerevisiae according to claim 1, characterized in that it is H-tolerant₂O₂A recombinant saccharomyces cerevisiae resistant to two or more of heat shock, salt and high temperature, expressing the exogenous GSH synthesis and circulation related protein of claim 1 (11).

11. The construction method of the recombinant saccharomyces cerevisiae as claimed in claim 1, characterized in that, by the overlap extension PCR technique, fragment restriction sites 1-promoter-restriction sites 2-restriction sites 3-terminator-restriction sites 4 are obtained, the restriction sites 1 and 4 are utilized to perform enzyme cutting on the vector, and the fragment is inserted into the vector to obtain a vector cassette;

and then, the twelve genes of the exogenous GSH synthesis and circulation related proteins recorded in the claim 1 are assembled by Gibson, respectively cloned on a linearized vector box after enzyme digestion by an enzyme digestion site 2 and an enzyme digestion site 3, a positive clone is obtained by verification of a transformed host bacterium, a plasmid vector containing the exogenous genes is selected to transform saccharomyces cerevisiae according to requirements after the plasmid vector is extracted, and the recombinant saccharomyces cerevisiae is obtained.

12. Use of the exogenous GSH synthesis and circulation-associated protein of claim 1 in items (1) to (12) or one or more of its encoding genes in the construction of stress-resistant recombinant saccharomyces cerevisiae; the stress resistance comprises H resistance₂O₂Heat resistance, high temperature resistance, salt resistance and acid resistance.

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