CN113025634A

CN113025634A - Gene sequence containing sugar alcohol and ATP binding domain and expression and function verification method of gene sequence in yarrowia lipolytica

Info

Publication number: CN113025634A
Application number: CN202110247016.2A
Authority: CN
Inventors: 裴疆森; 李红娇; 周芸琪; 金玮鋆; 董建辉
Original assignee: China National Research Institute of Food and Fermentation Industries
Current assignee: China National Research Institute of Food and Fermentation Industries
Priority date: 2021-03-05
Filing date: 2021-03-05
Publication date: 2021-06-25
Anticipated expiration: 2041-03-05
Also published as: CN113025634B

Abstract

The invention discloses a gene sequence containing a sugar alcohol and ATP binding domain and a construction method of a yarrowia lipolytica recombinant strain containing the sequence, belonging to the field of microbial genetic engineering. The nucleotide sequence of the gene is shown as SEQ ID NO.1, and after the gene is expressed in yarrowia lipolytica, the assimilation performance difference of the recombinant strain to carbohydrate such as erythritol, ribose and the like of a yeast strain is obvious, so that the gene is presumed to be a new gene with erythritol synthesis regulation and control capacity. The recombined yarrowia lipolytica strain also shows the assimilation capability of various sugar alcohols and some non-fermentable disaccharides in the mother liquor extracted from the erythritol fermentation liquor of the normal yarrowia lipolytica, and has practical application value for improving the substrate utilization capability of the yarrowia lipolytica and comprehensively utilizing waste liquor in the erythritol production process.

Description

Gene sequence containing sugar alcohol and ATP binding domain and expression and function verification method of gene sequence in yarrowia lipolytica

Technical Field

The invention belongs to the technical field of yeast genetic engineering and microorganisms, and particularly relates to a gene sequence containing a sugar alcohol and ATP binding domain, a recombinant strain construction method containing the sequence and a recombinant strain function verification method.

Background

Erythritol widely exists in microorganisms such as yeast, algae, mushrooms and the like in nature, belongs to a sugar alcohol natural sweetener, has extremely low heat, is difficult to absorb moisture, is stable at high temperature and has certain antioxidant activity, does not cause insulin level change in human metabolism, and does not cause great fluctuation of blood sugar after being eaten. Bacteria in the oral cavity generally do not utilize such sweeteners and do not cause dental caries on long-term consumption. Therefore, the erythritol can be used as a functional sugar substitute to be added into non-calorie or low-calorie foods, and is more suitable for patients with obesity and diabetes when being applied to medical products. The common microbial fermentation method for producing erythritol mainly uses starchy raw materials of wheat, corn and the like to be degraded into glucose by enzyme, and then uses hypertonic resistant yeast such as Pichia (Pichia) Yarrowia genus (Yarrowia) Candida genus (Candida) Torulopsis genus (A)Torulopsis) Trigonopsis genus (A)Trigonopsis) The erythritol is obtained by fermentation with glucose as a substrate and subsequent steps of filtering, separating and purifying and the like as a production strain, the average yield is about 50%, and industrial mass production is performed due to high production efficiency, mild reaction and low cost.

The yarrowia lipolytica mainly takes glucose and glycerol as fermentation substrates to synthesize erythritol. In an anabolic pathway taking glucose as a carbon source, through glycolysis, hexokinase catalyzes glucose to generate glucose-6-phosphate, glucose-6-phosphate dehydrogenase catalyzes glucose-6-phosphate to enter a pentose phosphate pathway to generate erythrose-4-phosphate, and erythritol is generated under the catalysis of erythrose-4-phosphate kinase and erythrose reductase. Host bacterium yarrowia lipolytica (Yarrowia lipolytica) Is a safe non-pathogenic strain, hasComplete the sequencing of the whole genome and mitochondria, complete gene operation means, can utilize cheap substrates and various carbon sources to secrete metabolites, can produce compounds with certain industrial value through a directional shunt metabolic pathway, gradually becomes a yeast expression system and is widely applied to food and medicine production.

Disclosure of Invention

The invention discloses a gene sequence containing a sugar alcohol and an ATP binding domain, which comprises the following steps:

the nucleotide sequence of the gene is shown in SEQ ID NO.1 and consists of 1831 nucleotides.

② the nucleotide sequence shown by SEQ ID NO.1 is substituted, added and deleted with one or more nucleotides to obtain the sequence of coding the same functional gene.

And the sequence of the coding same functional gene with homology of more than 75 percent with the sequence shown in SEQ ID NO. 1. The gene sequence of the present invention can be mutated by a known method such as point mutation by a person skilled in the art, and a nucleotide sequence having 75% or more identity to the nucleotide sequence provided by the present invention which has been artificially modified is derived from the gene sequence of the present invention and is equivalent to the sequence of the present invention as long as it has the same function as the protein encoded by the gene sequence.

"identity" refers to similarity to a nucleotide sequence of the present invention. "identity" includes nucleotide sequences having 75% or more homology with the sequence shown in SEQ ID No.1 of the present invention. Identity can be assessed by computer sequence alignment software or by the BLAST algorithm for sequence homology, and identity between two or more sequences is expressed as a percentage.

The amino acid sequence of the gene sequence expression protein is shown as SEQ ID NO.2 and consists of 603 amino acids. The protein with the same function obtained by substituting, deleting and adding no more than 10 amino acid residues to the amino acid sequence shown in SEQ ID No.2, or the protein with 75 percent or higher homology and the same function with the amino acid sequence shown in SEQ ID No.2 is derived from the amino acid sequence of the invention and is identical with the sequence of the invention.

The invention provides biological materials containing the gene sequence, including recombinant vectors and recombinant strains. In the present invention, the cloning Vector is a pMD18-T Vector-spx recombinant cloning Vector (FIG. 1) obtained by inserting a cloned target gene into a pMD18-T cloning Vector; the recombinant expression vector is pINA1269-spx recombinant expression vector obtained by inserting target gene spx into pINA1269 plasmid, specifically is a recombinant expression vector obtained by inserting target fragment shown by 1-1831 bit nucleotide from 5' end of SEQ ID No.1 into polyclonal enzyme cutting site (between Pml I and Kpn I enzyme cutting recognition site) of pINA1269 vector, and the promoter of the target gene in the expression vector is hp4d promoter carried on pINA1269 plasmid (figure 2); the recombinant strain is obtained by transforming a recombinant expression vector pINA1269-spx into a host yarrowia lipolytica yeast and screening and identifying.

The gene sequences of the invention are derived fromZygoascus hellenicusAn ORF sequence with sugar phosphorylation function obtained by yeast in whole genome sequencing analysis, named spx, is predicted by nucleic acid conserved domain alignment and structural domain (FIG. 3), and contains a sugar binding domain and an ATP binding domain, and is related to pentose or hexokinase gene families. The functional gene sequence of the invention has the effects that: compared with an unmodified parent strain, the difference of the recombinant strain for over-expressing the gene in the assimilation performance of erythritol, ribose and other carbohydrates is obvious, which shows that the gene is related to erythritol synthesis and pentose metabolic pathway, the gene is a new gene capable of regulating erythritol synthesis, and a new direction is provided for further modifying a sugar alcohol metabolic pathway, expanding a fermentation available carbon source and improving erythritol yield; in addition, the erythritol crystallization mother liquor contains unused glucose, uncrystallized erythritol, residual mannitol, ribitol, glycerol and other complex components, the viscosity of the mother liquor is reduced after the recombinant strain is fermented, the components of the mother liquor are changed, and a new scheme is provided for the treatment and comprehensive utilization of fermentation waste liquor.

Drawings

FIG. 1 is a schematic representation of the recombinant cloning Vector pMD18-T Vector-spx;

FIG. 2 is a schematic representation of the recombinant expression vector pINA 1269-spx;

FIG. 3 is a screenshot of an alignment of the conserved domain of the spx sequence;

FIG. 4 is a comparison graph of high performance liquid chromatography for reusing erythritol crystallization mother liquor by a recombinant yarrowia lipolytica strain overexpressing spx gene and a parent strain.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Materials, reagents and the like used in examples are commercially available unless otherwise specified. In the examples, the symbol "%" is used in percent by mass unless otherwise specified.

Example 1: cloning of the Gene of interest

Extracting original strain by using Tiangen yeast genome extraction kitZygoascus hellenicusThe target fragment was amplified from the genomic DNA of yeast using the template and Primer Premier 5 designed specific primers. Wherein the forward primer for amplifying the target fragment is spx-F: 5' -AGCACGTGATGTCAGATTCAAACGGCCCTCTTTAT-3' carrying a Pml I cleavage site. The reverse primer is spx-R: 5' -CGGGTACCTTATACAAGCCGAGACTCCATCTG-3', carrying a Kpn I cleavage site. The PCR reaction system is 0.5 mu L TaKaRaTaqPolymerase (5U/. mu.L), 5.0. mu.L 10 XPCR Buffer (Mg)²⁺Plus), 4.0. mu.L dNTP mix (2.5mM each), 1.0. mu.L Forward Primer (20. mu.M), 1.0. mu.L Reverse Primer (20. mu.M), 2.5ng template DNA, up to 50. mu.L sterile distilled water. The PCR reaction program is: 3min at 94 ℃; 94 ℃ 30sec, 56 ℃ 30sec, 72 ℃ 1min45s, 30 cycles; 7min at 72 ℃; forever at 4 ℃.

Example 2: construction of pMD18-T Vector-spx recombinant cloning Vector

Detecting PCR product by agarose gel electrophoresis, and recovering target fragment by OMEGA gel recovery kit. The recovered product was ligated into pMD18-T Vector Cloning Kit (TaKaRa), reaction system: mu.L of pMD18-T Vector, 4. mu.L of recovered product, 5. mu.L of Solution I, up to 10. mu.L of ddH₂O, mix well and attach overnight at 16 ℃. The ligation mixture was transformed into DH 5. alpha. competent cells and the ligation mixture was added to 150. mu.L of DH 5. alpha. competent EP tube in ice bath for 30min, at 42 ℃ for 90s and in ice bath for 5 min. Adding intomu.L of LB liquid medium (total volume 1 mL), resuscitated at 37 ℃ and 100r/min for 1 h. Centrifuging the resuscitating bacterial liquid for 2min at 5000r/min, discarding the supernatant, leaving 100 mu L of suspended bacteria, coating the resuscitating bacterial liquid on an LB (lysogeny broth) plate containing Amp resistance, carrying out inversion culture at 37 ℃ overnight, selecting a single colony on a transformation plate, extracting plasmid DNA (deoxyribonucleic acid) after the LB liquid culture, carrying out double enzyme digestion identification, verifying the size of an enzyme digestion fragment by agarose gel electrophoresis, and sending the recombinant clone T vector successfully constructed by the verification to Shanghai biological engineering company Limited for sequencing.

Example 3: construction of pINA1269-spx recombinant expression vector

Extracting pINA1269 plasmid DNA by using an OMEGA plasmid miniprep kit, carrying out double digestion by Pml I and Kpn I, carrying out agarose gel electrophoresis, and then recovering. Similarly, the target fragment is obtained and recovered by Pml I and Kpn I double enzyme digestion construction success recombination clone T carrier, the purified carrier and the target fragment are connected by TaKaRa T4 DNA ligase, the connection system is: mu. L T4 ligase, 2. mu.L 10 XBuffer, 10. mu.L recovered spx target fragment, 4. mu.L recovered pINA1269 vector fragment (molar ratio of target fragment to vector fragment 7: 1), up to 20. mu.L ddH₂And O, transforming to DH5 alpha competence, coating on an LB plate containing Amp resistance, culturing overnight at 37 ℃, selecting a transformant, carrying out colony PCR verification by using primers spx-F and spx-R used in amplification of an integration target fragment, transferring the verified recombinant into an LB liquid culture medium, extracting plasmid DNA after culture, carrying out double digestion identification, and sending to Shanghai biological engineering corporation Limited for sequencing after double verification to obtain a successfully constructed pINA1269-spx expression vector.

Example 4: recombinant expression vector transformation of yarrowia lipolytica

Preparing host strain yeast competence, streaking yarrowia lipolytica strain on YPD solid culture medium, culturing at 30 deg.C for 2 days, selecting single colony, shake culturing at 30 deg.C and 220r/min in YPD liquid culture medium for 18h, and inoculating to 50mLYPD culture medium (initial OD)₆₀₀0.1-0.15), shaking and culturing at 30 deg.C for 4 hr (to OD)₆₀₀0.4-0.6), subpackaging the bacterial liquid at 25 ℃, centrifuging at 4000r/min for 5min, discarding the supernatant, collecting the thalli, and resuspending the thalli by using a 100mM LiAc solution to obtain the yeast competent cells.

The recombinant expression vector pINA1269-spx is treated by a restriction enzyme Not1 in a linear mode by adopting a lithium acetate chemical transformation method and is introduced into host bacteria yarrowia lipolytica competent cells, and the transformation system is as follows: 100ng of enzyme digestion purification vector, 600 mu L of 50% PEG4000, 10 mu L of inactivated salmon sperm DNA, 100 mu L of yeast competed, incubation is carried out for 30min at 30 ℃ after gentle mixing, heat shock is carried out for 15min at 42 ℃ after gentle inversion and uniform mixing is carried out every 5-10min, the mixed liquor of the processed yeast transformation system is centrifuged, supernatant is discarded, 100 mu L of culture medium is used for suspension coating on YNB screening culture medium (2% glucose, 0.67% non-amino yeast nitrogen source, 2% agar, pH5.5), inverted culture is carried out for 3-5 days at 28 ℃, yeast transformants growing on the screening culture medium are picked and inoculated on liquid culture medium, bacterial liquid is collected to extract yeast genome, primers spx-F and spx-R during amplification of integration target fragments are used as templates for PCR verification, and the transformants which are verified to be positive are the successful lipolysis yarrowia yeast recombinant strain.

Example 5: experiment for utilizing and transforming different carbon sources by recombinant strain

The host strain and the recombinant strain are respectively inoculated into 2.5mL of liquid culture medium (0.3% yeast extract powder, 0.5% peptone and 5% glucose), and shake-cultured at 28 ℃ for 24h to obtain seed liquid. Then inoculating the seed liquid into 8 fermentation culture media with different carbon sources, namely 1% xylitol, 1% sorbitol, 1% glycerol, 1% erythritol, 1% xylose, 1% ribose, 1% arabinose and 1% mannitol in an inoculation amount of 10%, wherein the other components of the fermentation culture media are the same as: 10 percent of glucose, 0.5 percent of yeast extract powder, 0.5 percent of triammonium citrate, 0.025 percent of magnesium sulfate, 0.025 percent of monopotassium phosphate, and shaking culture at 28 ℃ and 280 r/min for 72 hours to obtain fermentation liquor.

The fermentation broth is diluted and filtered, and the sugar and sugar alcohol contents are detected by an HPLC method. HPLC chromatographic conditions, chromatograph: agilent 1260; a detector: RID-G1362A shows a difference detector; a chromatographic column: carbomix Ca-Np, sepax technologies, Inc.; detecting the temperature of the cell: room temperature; separating column temperature: 80 ℃; mobile phase: ultrapure water; flow rate: 0.7 mL/min; sample injector: G1329A/B autosampler; sample introduction volume: 10 μ L. Observing the growth characteristics of the recombinant engineering strains, and detecting and analyzing fermentation substrates and products by using high performance liquid chromatography.

Example 6: recycling of erythritol crystallization mother liquor by recombinant strain

The host strain and the recombinant strain are respectively inoculated into 2.5mL of liquid culture medium (0.3 percent of yeast extract powder, 0.5 percent of peptone and 5 percent of glucose), and shake-cultured for 24 hours at 28 ℃ to obtain seed liquid. Transferring the seed solution into an erythritol mother solution fermentation medium (20% erythritol crystallization mother solution, 0.5% yeast extract powder and 0.5% triammonium citrate) with the inoculation amount of 10% at 28 ℃, shaking and culturing at 280 r/min for 72 h to obtain a fermentation solution, diluting and filtering, and detecting the contents of sugar and sugar alcohol in the metabolite by an HPLC method (figure 4).

Sequence listing

<110> research institute of food fermentation in China, Ltd

<120> a gene sequence comprising a sugar alcohol and an ATP binding domain and a method for expression and functional verification thereof in yarrowia lipolytica

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<213> Zygoascus hellenicus

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atgtcagatt caaacggccc tctttatctt gtatgtgttt gcgggactca cttaaggggc 60

ccttttggtg ttcttttttt tttttttagc taagaaaacg attttacatt ctactatagg 120

ctcaattact ataaggcttg tttcagcaac taacataatc agggactgga tttgtctacg 180

cagcagctca aaggtgtcgt tgtcgatgcc aatttgatga ctgttagcag ctccaaagtt 240

gagtttgatg tggatattcc ttcgtacaag accatcaaag gagtctatgc aaaagggaaa 300

gaaatcgtgg ctccagtagc catgtggcta gaagctctgg atctactcat gcaacgatta 360

tctgagactc ctgggatatt agagagactg gcagcagtat ctggagcttg ccagcaacat 420

gggagtgtgt ttttcaactc caatgctgag caagcgttga agaacatggg acacgcggag 480

agcttgcgtt cggctttgga agattctttt gcatgggaaa tgtcacccaa ttggcaggat 540

cattcgactg gtatggagtg ctccaagttt gaagatgcgg ttggtggtaa gcagaagctt 600

gctgatatca caggatcacg agctcattat cggttcacgg gaccgcagat tctcaagctc 660

aagactcaac agcccgaaaa atacgcacag acgaaaagga tatcactggt gagcagtttt 720

ctgacatctg ttctagccgg gaaaatcagc gatattgaaa tatcagatgc atgtggcatg 780

aatctgtgga atgttgaggg tcgacactgg gaaccgaggc tgatccaagt gattggtgca 840

gaagttgaaa acaaactcgg ctcagtcgaa accaaaggag gagagtttgc gggactgatc 900

agcagctatt tcaccaaaaa gtatggcgtt tctcatgagt gccaagtcaa tttctgtact 960

ggtgataatc ctgggactat tttggcttta ccgttgcaga tgaatgatgt gatggttagc 1020

cttggaacca gtaccactgc actgatcgta accgagaact atcggcccag tcctttgtac 1080

cacttattca gccacccaac tggaagggga tatatgggaa tgctttgtta ttgtaatgga 1140

gctctcgcca gagagaaggt tcgggatagc ttgctaacca taggcagcag tagtgtctca 1200

ggagattcgg cttgggatga ttttaatgag attgcgattt cgggggctcc tctaggagcc 1260

gatgacaaag tcaaagctcg cctaggtgta tacttcccct tgggagaaat tatccccgat 1320

gtggggagcc aaacccgacg gttcgtggta tggcctgatg attccctcga agaagtgaaa 1380

tctggtgatg atgtaggcga tcccgatagt agcaatttgt ggggaaaaga gcaggatgtg 1440

gtgtgtattt tggaaagtca ggctctgagt atccgagaga gacttgagcc aatgttaaca 1500

acggccgaca aatgcccatc aaggatattt ttcgtcggcg gagggtccaa gaaccgtgca 1560

atttgtcaag taatgtccag ggtgctttca ccagccaacg gtgcttatcg gctcgagtta 1620

agcgatgcct gtgccatggg agctgctcac aaggctgcat gggccgatgt agctgatacc 1680

atcagctgga gctgatacca tcagctggga agcatttatt gatagcaagt ggaatatggc 1740

aaatgttcag gcggtgccgc agctagccgc agcttgtgac tatggagaaa ttgtgccgat 1800

atttgagcag atggagtctc ggcttgtata a 1831

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Met Ser Ala Ser Ala Gly Pro Leu Thr Leu Val Cys Val Cys Gly Thr

1 5 10 15

His Leu Ala Gly Pro Pro Gly Val Leu Pro Pro Pro Pro Ser Gly Ala

20 25 30

Ala Pro Thr Pro Thr Thr Ala Leu Ala Thr Thr Leu Ala Cys Pro Ser

35 40 45

Ala His Ala Gly Gly Leu Ala Leu Ser Thr Gly Gly Leu Leu Gly Val

50 55 60

Val Val Ala Ala Ala Leu Met Thr Val Ser Ser Ser Leu Val Gly Pro

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

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Gly Leu Gly Ile Val Ala Pro Val Ala Met Thr Leu Gly Ala Leu Ala

100 105 110

Leu Leu Met Gly Ala Leu Ser Gly Thr Pro Gly Ile Leu Gly Ala Leu

115 120 125

Ala Ala Val Ser Gly Ala Cys Gly Gly His Gly Ser Val Pro Pro Ala

130 135 140

Ser Ala Ala Gly Gly Ala Leu Leu Ala Met Gly His Ala Gly Ser Leu

145 150 155 160

Ala Ser Ala Leu Gly Ala Ser Pro Ala Thr Gly Met Ser Pro Ala Thr

165 170 175

Gly Ala His Ser Thr Gly Met Gly Cys Ser Leu Pro Gly Ala Ala Val

180 185 190

Gly Gly Leu Gly Leu Leu Ala Ala Ile Thr Gly Ser Ala Ala His Thr

195 200 205

Ala Pro Thr Gly Pro Gly Ile Leu Leu Leu Leu Thr Gly Gly Pro Gly

210 215 220

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

225 230 235 240

Ser Val Leu Ala Gly Leu Ile Ser Ala Ile Gly Ile Ser Ala Ala Cys

245 250 255

Gly Met Ala Leu Thr Ala Val Gly Gly Ala His Thr Gly Pro Ala Leu

260 265 270

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

275 280 285

Thr Leu Gly Gly Gly Pro Ala Gly Leu Ile Ser Ser Thr Pro Thr Leu

290 295 300

Leu Thr Gly Val Ser His Gly Cys Gly Val Ala Pro Cys Thr Gly Ala

305 310 315 320

Ala Pro Gly Thr Ile Leu Ala Leu Pro Leu Gly Met Ala Ala Val Met

325 330 335

Val Ser Leu Gly Thr Ser Thr Thr Ala Leu Ile Val Thr Gly Ala Thr

340 345 350

Ala Pro Ser Pro Leu Thr His Leu Pro Ser His Pro Thr Gly Ala Gly

355 360 365

Thr Met Gly Met Leu Cys Thr Cys Ala Gly Ala Leu Ala Ala Gly Leu

370 375 380

Val Ala Ala Ser Leu Leu Thr Ile Gly Ser Ser Ser Val Ser Gly Ala

385 390 395 400

Ser Ala Thr Ala Ala Pro Ala Gly Ile Ala Ile Ser Gly Ala Pro Leu

405 410 415

Gly Ala Ala Ala Leu Val Leu Ala Ala Leu Gly Val Thr Pro Pro Leu

420 425 430

Gly Gly Ile Ile Pro Ala Val Gly Ser Gly Thr Ala Ala Pro Val Val

435 440 445

Thr Pro Ala Ala Ser Leu Gly Gly Val Leu Ser Gly Ala Ala Val Gly

450 455 460

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

465 470 475 480

Ile Leu Gly Ser Gly Ala Leu Ser Ile Ala Gly Ala Leu Gly Pro Met

485 490 495

Leu Thr Thr Ala Ala Leu Cys Pro Ser Ala Ile Pro Pro Val Gly Gly

500 505 510

Gly Ser Leu Ala Ala Ala Ile Cys Gly Val Met Ser Ala Val Leu Ser

515 520 525

Pro Ala Ala Gly Ala Thr Ala Leu Gly Leu Ser Ala Ala Cys Ala Met

530 535 540

Gly Ala Ala His Leu Ala Ala Thr Ala Ala Val Ala Ala Thr Ile Ser

545 550 555 560

Thr Ser Thr His Gly Leu Gly Ser Ile Thr Gly Val Gly Thr Gly Leu

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Cys Ser Gly Gly Ala Ala Ala Ser Ala Ser Leu Leu Thr Ala Ala Cys

580 585 590

Ala Ala Ile Ala Ala Gly Val Ser Ala Cys Ile

595 600

Claims

1. A gene sequence comprising a sugar alcohol and an ATP-binding domain, the sequence having:

[0001] (1) a nucleotide sequence shown as SEQ ID NO. 1; or

(2) The nucleotide sequence shown in SEQ ID NO.1 is a sequence which is obtained by replacing, adding or deleting one or more nucleotides and encoding the same functional gene;

(3) a sequence which has homology of more than 75 percent with the sequence shown in SEQ ID NO.1 and encodes the same functional gene.

2. The gene sequence comprising a sugar alcohol and an ATP-binding domain according to claim 1, wherein the amino acid sequence of the expressed protein is as shown in SEQ ID No. 2.

3. Biological material comprising the gene sequence of claim 1, including recombinant vectors and recombinant strains;

in the recombinant vector, the vector can be a cloning vector and an expression vector;

in the recombinant strain, the strain can be bacteria and saccharomycetes.

4. The biomaterial according to claim 3, wherein the recombinant vector and the recombinant strain are constructed by a method comprising the steps of:

(1) constructing a recombinant cloning Vector pMD18-T Vector-spx containing the gene sequence;

(2) constructing a recombinant expression vector pINA1269-spx containing the gene sequence;

(3) transforming the recombinant expression vector into host yarrowia lipolytica;

(4) culturing, screening and identifying to obtain the recombinant strain.

5. The use of the nucleotide sequence according to claim 1 or the amino acid sequence according to claim 2 in erythritol fermentation production and erythritol waste liquor treatment processes.