Acid-resistant pichia kudriavzevii and application thereof

Abstract

The invention discloses acid-resistant pichia kudriavzevii and an application of the acid-resistant pichia kudriavzevii. The acid-resistant pichia kudriavzevii is Pichia kudriavzevii S9 and is preserved in the Guangdong Microbial Culture Collection Center on September 20, 2024, and the preservation number of the acid-resistant pichia kudriavzevii S9 is GDMCC.No: 65148. The acid-resistant pichia kudriavzevii S9 has the advantages that the acid-resistant pichia kudriavzevii S9 is used for According to the invention, a low-pH-resistant pichia kudriavzevii strain is screened from surface microorganisms of grapes, and the pichia kudriavzevii strain can tolerate an environment with pH of 1.8 and ferment organic acid under the condition of not adding a neutralizing agent; besides, genetic modification is carried out based on pichia kudriavzevii, including knocking out succinic acid degradation related genes, knocking out ethanol and glycerol production pathways and enhancing succinic acid transporter expression, so that engineering bacteria for high-yield succinic acid are obtained, and the fermentation yield of succinic acid reaches 31.74 g / L under the condition that a neutralizer is not added.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to acid-resistant Pichia kudriavzi and application thereof. Background Art

[0002] Succinic acid, also known as succinic acid, is an important C4 platform compound. As the starting material for the synthesis of general chemicals, succinic acid is widely used in food, chemistry, medicine and other fields.

[0003] At present, the production of succinic acid mainly includes chemical synthesis and microbial fermentation. The chemical synthesis method includes paraffin oxidation, methyl chloroacetate cyanide hydrolysis and vanadium pentoxide catalytic hydrogenation. However, due to the reduction of petroleum resources and the increasingly serious environmental pollution, the disadvantages of the chemical synthesis method are becoming increasingly apparent. The production of succinic acid by fermentation can utilize renewable resources, has little environmental pollution, and shows a good development prospect. The succinic acid-producing bacteria that have been studied more in the microbial fermentation method include succinic acid-producing actinomycetes, succinic acid-producing anaerobic spirillium, Escherichia coli and glutamicum. However, most bacterial strains need a pH environment close to neutral to obtain the best fermentation performance. A large amount of organic acid will be produced during succinic acid fermentation, and the accumulation of organic acid will cause certain toxicity to the strain, thereby inhibiting the growth and production of the strain. A common method is to add a neutralizing additive during fermentation to keep the fermentation environment neutral. At present, solid magnesium carbonate is considered to be the most favorable neutralizing additive for succinic acid fermentation. It will react with the fermentation product to form succinate, and the subsequent complicated and cumbersome separation and purification process of succinic acid basic salt has always been a problem that hinders its large-scale production. Therefore, how to achieve fermentation with little or no addition of neutralizer is of great significance to the production of succinic acid.

[0004] Currently, the main strategy for achieving fermentation production of succinic acid with little or no addition of neutralizer is to develop strains capable of fermenting and producing succinic acid at low pH. For example, CN116656514A discloses an acid-resistant yeast and its application, which can ferment and produce succinic acid at a relatively low pH.

[0005] In summary, developing new strains capable of producing succinic acid by fermentation at low pH and expanding the tools and methods for succinic acid production are of great significance for the production and application of succinic acid. Summary of the invention

[0006] In view of the deficiencies in the prior art and actual needs, the present invention provides an acid-resistant Pichia kudriavzi and applications thereof, screens and obtains low pH-resistant Pichia kudriavzi, and further develops engineered bacteria, in order to achieve efficient production of organic acids such as succinic acid.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides an acid-resistant Pichia kudriavzevii, wherein the acid-resistant Pichia kudriavzevii is Pichia kudriavzevii S9, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 20, 2024, with a deposit number of GDMCC.No: 65148.

[0009] In the present invention, a new Pichia kudriavzevii (Pichiakudriavzevii) is screened from grape epidermal microorganisms and named as Pichia kudriavzevii S9. The new Pichia kudriavzevii S9 can tolerate a low pH environment (pH 1.8). During the fermentation process, it is not necessary to add a pH neutralizer. The new Pichia kudriavzevii S9 can grow and efficiently produce acidic substances under acidic conditions, which is conducive to achieving high-concentration accumulation of products without affecting production bacteria, and provides a new strain for the production of organic acids (such as succinic acid).

[0010] In a second aspect, the present invention provides use of the acid-resistant Pichia kudriavzii described in the first aspect in producing acidic substances.

[0011] In a third aspect, the present invention provides a method for producing an acidic substance by fermentation, the method comprising:

[0012] The acid-resistant Pichia kudriavzii described in the first aspect is used for fermentation production.

[0013] It is to be understood that the fermentation methods of acid-producing strains and product purification methods commonly used in the art are applicable to the present invention.

[0014] Preferably, the acidic substance comprises an organic acid.

[0015] Preferably, the organic acid comprises at least one of succinic acid, lactic acid or malic acid.

[0016] In a fourth aspect, the present invention provides an engineered bacterium, wherein the starting strain of the engineered bacterium is the acid-resistant Pichia kudriavzii described in the first aspect, and the engineered bacterium undergoes any of the following genetic modifications:

[0017] (1) Loss of glycerol metabolism genes;

[0018] (2) lack of glycerol metabolism genes and ethanol metabolism genes;

[0019] (3) lack of glycerol metabolism genes, ethanol metabolism genes, and succinate degradation genes;

[0020] (4) Deficiency of glycerol metabolism genes, ethanol metabolism genes, and succinate degradation genes, and overexpression of succinate transport genes.

[0021] In the present invention, genetic modification is performed based on the screened acid-resistant Pichia kudriavzi, including knocking out genes related to succinate degradation, knocking out ethanol and glycerol production pathways, and enhancing the expression of succinate transporters to obtain an engineered bacterium with high succinate production.

[0022] Preferably, the succinate degradation gene includes at least one of the SDH5 gene, the SDH2 gene, the SDH3 gene or the SDH4 gene.

[0023] Preferably, the glycerol metabolism gene includes the GPD gene.

[0024] Preferably, the ethanol metabolism gene includes the PDC gene and / or the ADH1 gene.

[0025] Preferably, the succinate transporter gene comprises the SpMAE1 gene.

[0026] The present invention designs a genetic modification strategy for increasing the yield of succinic acid. It is understandable that the strategies commonly used in the art for knocking out genes or reducing gene expression can be applied to the present invention to modify the strain.

[0027] In a fifth aspect, the present invention provides a method for constructing the engineering bacteria according to the fourth aspect, the method for constructing the engineering bacteria comprising:

[0028] The acid-resistant Pichia kudriavzii described in the first aspect is used as a starting strain, the succinate degradation gene, the glycerol metabolism gene and the ethanol metabolism gene therein are knocked out, and the succinate transport gene is overexpressed to obtain the engineered bacteria.

[0029] In a sixth aspect, the present invention provides the use of the engineered bacteria described in the fourth aspect in producing acidic substances.

[0030] In a seventh aspect, the present invention provides a method for producing an acidic substance by fermentation, the method comprising:

[0031] The engineered bacteria described in the fourth aspect are used for fermentation production.

[0032] Preferably, the acidic substance comprises an organic acid.

[0033] Preferably, the organic acid comprises at least one of succinic acid, lactic acid or malic acid.

[0034] The invention carries out genetic modification based on the screened acid-resistant Pichia kudriavzia to obtain an engineered bacterium with high succinic acid production, and the succinic acid fermentation yield reaches 31.74 g / L without adding a neutralizing agent.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention screens out a low-pH-resistant Pichia kudriavzii from grape surface microorganisms. The Pichia kudriavzii can tolerate an environment of pH 1.8 and ferment organic acids without adding a neutralizer. In addition, genetic modification is performed based on the Pichia kudriavzii, including knocking out genes related to succinic acid degradation, knocking out ethanol and glycerol production pathways, and enhancing the expression of succinic acid transporters, to obtain an engineered bacterium with high succinic acid production, and the succinic acid fermentation yield reaches 31.74 g / L without adding a neutralizer. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The graph shows the results of succinic acid production in different strains. DETAILED DESCRIPTION

[0038] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0039] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0040] In a specific embodiment of the present invention, Pichia kudriavzevii S9 was deposited in Guangdong Microbiological Culture Collection Center (GDMCC) on September 20, 2024, with a deposit number of GDMCC.No: 65148, and the address is 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou; strains and plasmids: Escherichia coli TOP10 competent cells were purchased from Qingke Bio, plasmid pYSE2 was purchased from Biowind Company, and plasmid pV1081 was purchased from Miaoling Bio; LB medium: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L; YPD medium: peptone 20g / L, yeast powder 10g / L, glucose 20g / L (concentration in fermentation medium is 100g / L); Martin Bengal red medium: potassium dihydrogen phosphate 1g / L, magnesium sulfate heptahydrate 0.5g / L, peptone 5g / L, glucose 10g / L, 1% Bengal red aqueous solution 3.3mL / L, add 0.3mL of 1% streptomycin solution to every 100mL of culture medium before use.

[0041] The reaction system used in the specific embodiment is as follows:

[0042] PCR reaction system:

[0043] Reagents Volume (μL) KOD FX Neo 1 2×KOD buffer 25 2mM dNTPs 10 Upstream primer F 1.5 Downstream primer R 1.5 template X <![CDATA[ddH2O]]> 11-X Total volume 50

[0044] When the template is plasmid: the amount added is 50ng; when the template is genome: the amount added is 10-200ng, and when the template is cDNA: 200ng. Overlap PCR reaction system:

[0045] Reagents Volume (μL) KOD FX Neo 1 2×KOD buffer 25 2mM dNTPs 10 Upstream primer F 1.5 Downstream primer R 1.5 Template (left arm) X Template (right arm) X <![CDATA[ddH2O]]> 11-2X Total volume 50

[0046] PCR reaction program settings:

[0047]

[0048] PCR recovery product digestion system:

[0049] 10× digestion buffer (TAKARA) 5 μL, Dpn I 1 μL, purified DNA product 20 μL, distilled water 24 μL, total volume 50 μL, react at 37°C for 30 min.

[0050] Example 1

[0051] This example is used to isolate and identify strains.

[0052] The sampling vineyard is located in the Yinying Grape Plantation in Nansha District, Guangzhou. The plate dilution method is used to isolate microorganisms. The harvested grapes are soaked in sterile water for 2 hours, and the soaked sterile water is diluted in different gradients. Take 0.2mL of each dilution of different gradients and spread it on different isolation medium plates, and culture at 30℃ for 72 hours; pick the yeast colonies with dominant numbers and certain differences in morphology on the plate, and after streaking separation and purification, number the strains and freeze them for storage. The isolation medium is yeast isolation medium: Martin Bengal red medium.

[0053] The 13 screened strains were numbered and named S1-S13, and their acid resistance was tested. Among them, S9 had good growth under pH 1.8 conditions. Sequencing analysis showed that it was Pichia kudriavzevii, named Pichia kudriavzevii S9, and preserved.

[0054] The growth conditions of strains S1-S13 in pH 2 to pH 6 are shown in Table 1, where + represents that the strain can grow, and - represents that the strain cannot grow.

[0055] Table 1

[0056] serial number S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 pH 6.0 + + + + + + + + + + + + + pH5.0 + + + + + + + + + + + + + pH4.0 + + + + + + + + + + + + + pH 3.0 + - - - - + - + + - + - + pH 2.0 - - - - - - - - + - - - - pH 1.9 - - - - - - - - + - - - - pH 1.8 - - - - - - - - + - - - - pH 1.7 - - - - - - - - - - - - -

[0057] Example 2

[0058] This example constructs an engineering bacterium based on the screened Pichia kudriavzi S9.

[0059] The transformation strategy includes knocking out the succinate degradation-related gene SDH5 in Pichia kudriavzii S9, knocking out the ethanol and glycerol production pathways, and enhancing the expression of succinate transporter to construct the engineered bacterium Pichia kudriavzii S9-105.

[0060] (1) The Pichia kudriavzii S9 strain produced high ethanol in YPD fermentation. First, the glycerol metabolism-related gene GPD was knocked out in the S9 strain.

[0061] The upstream fragment P1 and downstream fragment T1 of the GPD gene were knocked out by constructing the upstream and downstream fragments of the GPD gene:

[0062] P1-S: 5'-acacgtcatcattcatttctgtgtcaagagagg-3';

[0063] P1-A: 5'-ggttataataaacgatatcaaaattcaaagggttcaaagtgggaattcc-3';

[0064] T1-S: 5'-gaattttgatatcgtttattataacctcaaggagaactttggcattgtactctcc-3';

[0065] T1-A: 5'-taatgttgatgctggcagtgacagtagtgc-3';

[0066] The genome of Pichia kudriavzi S9 was used as a template for PCR (polymerase chain reaction) in vitro amplification, and the upstream fragment P1 of the GPD gene was obtained using primer pair P1-S and P1-A, and the downstream fragment of the GPD gene was obtained using primer pair T1-S and T1-A. After digestion with DpnI endonuclease, the homologous recombinant fragments were recovered, purified and concentrated.

[0067] Using P1 and T1 fragments as templates, overlapping extension PCR was performed using primers P1-S and T1-A to obtain the GPD gene knockout repair template GPD-H1. After digestion with DpnI endonuclease, the homologous recombinant fragments were recovered, purified and concentrated.

[0068] Using pV1081 as template, primers GPD-sg-S and GPD-sg-A were used for circular amplification to construct the MDH2 knockout plasmid p-dGPD;

[0069] After amplification, the pV1081 template was degraded with DpnI, and the product was recovered and transformed into the competent E. coli TOP10. After positive transformants were selected with ampicillin, the knockout plasmid p-dGPD was obtained after extraction and sequencing.

[0070] GPD-sg-S:5'-ttaatttggatccgcggttgttggttccggtaacgttttagagctagaaatagcaagtt-3'.

[0071] GPD-sg-A:5'-gttaccggaaccaacaaccgcggatccaaattaaaaatagtttacgcaag-3'.

[0072] The GPD knockout plasmid p-dGPD and the repair template GPD-H1 were co-transformed into S9 competent cells and screened with nourseothricin resistance to obtain a GPD knockout strain named S9-101.

[0073] (2) Knockout the ethanol metabolism-related gene PDC.

[0074] The upstream and downstream fragments of the PDC gene were constructed to knock out the upstream fragment P2 and the downstream fragment T2 of the PDC gene:

[0075] P2-S: 5'-atgggatactcttgacgtggaaaattc-3';

[0076] P2-A: 5'-caatttttgtgttttgctgtgttttgttttattttgttttattg-3';

[0077] T2-S: 5'-acacagcaaaacacaaaaattgacatctgaatgtaaaatgaac-3';

[0078] T2-A: 5'-taatgttgatgctggcagtgacagtagtgc-3';

[0079] The genome of Pichia kudriavzi S9 was used as a template for PCR (polymerase chain reaction) in vitro amplification, and the upstream fragment P2 of the PDC gene was obtained using primer pair P2-S and P2-A, and the downstream fragment T2 of the PDC gene was obtained using primer pair T2-S and T2-A. After digestion with DpnI endonuclease, the homologous recombinant fragments were recovered, purified and concentrated.

[0080] Using P2 and T2 fragments as templates, overlapping extension PCR was performed using primers P2-S and T2-A to obtain the PDC gene knockout repair template PDC-H1. After digestion with DpnI endonuclease, the homologous recombinant fragments were recovered, purified and concentrated.

[0081] Using pV1081 as template, primers PDC-sg-S and PDC-sg-A were used for circular amplification to construct the PDC knockout plasmid p-dPDC;

[0082] After amplification, the pV1081 template was degraded with DpnI, and the product was recovered and transformed into the competent E. coli TOP10. After positive transformants were selected with ampicillin, the knockout plasmid p-dPDC was obtained after extraction and sequencing.

[0083] PDC-sg-S:5'-ttaatttggatccgcttgttgttacaccatacctgttttagagctagaaatagcaagtt-3'.

[0084] PDC-sg-A:5'-aggtatggtgtaacaacaagcggatccaaattaaaaatagtttacgcaag-3'.

[0085] The PDC knockout plasmid p-dPDC and the repair template PDC-H1 were co-transformed into S9-101 competent cells, and the cells were screened for resistance to nourseothricin to obtain a PDC knockout strain named S9-103.

[0086] (3) Knockout genes related to succinate degradation.

[0087] The upstream fragment P3 and downstream fragment T3 of the SDH5 gene were knocked out by constructing the upstream and downstream fragments of the SDH5 gene:

[0088] P3-S: 5'-tatggatacaaatggtttgggatttggc-3';

[0089] P3-A: 5'-gcaaacacctaagttttgtcgaacttcaactacggaa-3';

[0090] T3-S: 5'-gttcgacaaaacttaggtgtttgcattttatctctcaaggcctt-3';

[0091] T3-A: 5'-tgaacaagagggtggagagaattccg-3';

[0092] The genome of Pichia kudriavzi S9 was used as a template for PCR (polymerase chain reaction) in vitro amplification, and the upstream fragment P3 of the SDH5 gene was obtained using primer pair P3-S and P3-A, and the downstream fragment T3 of the SDH5 gene was obtained using primer pair T3-S and T3-A. After digestion with DpnI endonuclease, the homologous recombinant fragments were recovered, purified and concentrated.

[0093] Using P3 and T3 fragments as templates, overlapping extension PCR was performed with primers P3-S and T3-A to obtain the SDH5 gene knockout repair template SDH5-H1. After digestion with DpnI endonuclease, the homologous recombinant fragments were recovered, purified and concentrated.

[0094] Using pV1081 as template, primers SDH5-sg-S and SDH5-sg-A were used for circular amplification to construct the SDH5 knockout plasmid p-dSDH5;

[0095] After amplification, the pV1081 template was degraded with DpnI, and the product was recovered and transformed into the competent E. coli TOP10. After positive transformants were selected with ampicillin, the knockout plasmid p-dSDH5 was obtained after extraction and sequencing.

[0096] SDH5-sg-S:5'-ttaatttggatccggacaagacagcaactccacagttttagagctagaaatagcaagtt-3'.

[0097] SDH5-sg-A:5'-tgtggagttgctgtcttgtccggatccaaattaaaaatagtttacgcaag-3'.

[0098] The SDH5 knockout plasmid p-dSDH5 and the repair template SDH5-H1 were co-transformed into S9-103 competent cells, and the cells were screened with nourseothricin as resistance to obtain the SDH5 knockout strain, which was named S9-105.

[0099] (4) Screening of clones of the NAT marker fragment.

[0100] The pV1081 vector was used as a template for PCR amplification in vitro. The expression cassette fragment of the Knowles resistance gene NAT was obtained using the primer pair primer P4-S and primer P4-A. The linearized fragment was recovered, purified and concentrated.

[0101] P4-S: 5'-cgagtgtttaaggataatgataactgaa-3';

[0102] P4-A: 5'-aacaaaagctgaggcctgcatgcgagctc-3'.

[0103] Cloning of plasmid vector-derived fragments

[0104] The pYSE2 plasmid was used as a template for PCR amplification in vitro, and the vector fragment of pYSE2 was obtained using the primer pair primer P5-S and primer P5-A. The linearized vector fragment was recovered, purified and concentrated.

[0105] pYSE2-S:5'-atttaaacagatttaaatgattcttacacaaagatttgatacatg-3';

[0106] pYSE2-A:5'-ctttctttgtatgacgccatccttgacaaacaaactactttattaaagc-3'.

[0107] (5) Plasmid assembly.

[0108] Take 6 μL of linearized pYSE2 fragment, 4 μL of NAT cloned fragment and 10 μL of assembly reaction system (Gibson assembly cloning kit; Gibson After mixing with 50 DEG C of 1% dapoxetine monophosphate (DDP) Cloning Kit, the mixture was reacted at 50 DEG C for 30 min, and the competent cells of Escherichia coli TOP10 were transformed and spread on ampicillin resistance plates. The newly constructed vector was pYSE2-NAT.

[0109] (6) Construction of SpMAE1 overexpression plasmid vector.

[0110] The overexpression plasmid was constructed by connecting the restriction fragment of the above vector and the PCR product of the target gene using the Gibson assembly cloning kit.

[0111] 1) Obtaining the SpMAE1 expression cassette

[0112] The SpMAE1 gene expression cassette was synthesized by Nanjing GenScript, in which the promoter of SpMAE1 was TEF1, the terminator was TEF1, and the SpMAE1 gene sequence was optimized by Nanjing GenScript.

[0113] 2) Cloning of SpMAE1 gene expression cassette

[0114] The SpMAE1 expression cassette synthesized by Nanjing GenScript was used as a template for PCR in vitro amplification, and the SpMAE1 gene expression cassette fragment was obtained using the primer pair primer P6-S and primer P6-A. The linearized fragment was recovered, purified, and concentrated.

[0115] P6-S:5'-gacggaggttacccggcctggatatac-3';

[0116] P6-A:5'-gagatcaatgagttgcagctaaatgtccatt-3';

[0117] 3) Cloning of SpMAE1 gene expression vector

[0118] The overexpression vector plasmid pYSE2-NAT was used as a template for PCR amplification in vitro, and the SpMAE1 gene expression cassette fragment was obtained using the primer pair primer P7-S and primer P7-A. The linearized vector fragment was recovered, purified and concentrated.

[0119] P7-S:5'-caggccgggtaacctccgtcGGCCCTGCATTAATGAATCGGCCAACGC-3';

[0120] P7-A:5'-gctgcaactcattgatctcGCAAATTAAAGCCTTCGAGCGTCC-3'.

[0121] Take 6 μL of linearized pYSE2-NAT fragment, 4 μL of SpMAE1 cloned fragment and mix with 10 μL assembly reaction system, react at 50°C for 30 min, transform Escherichia coli TOP10 competent cells, spread on ampicillin resistance plates, and the constructed new vector is pYSE2-NAT-SpMAE1.

[0122] The constructed pYSE2-URA3-SpMAE1 was transformed into the competent cell of S9-105 strain, and the SpMAE1 overexpressing strain was obtained by screening with nourseoin resistance plates and named S9-106.

[0123] (7) Preparation of competent cells and chemical transformation of Pichia kudriavzia.

[0124] Competent cell preparation:

[0125] 1) Use a sterilized toothpick to streak the Pichia kudriavzii strain on a YPD solid plate to activate the strain, and place the streaked plate in a 30°C incubator for 2-3 days to grow a single colony;

[0126] 2) Select a single yeast colony with good growth and inoculate it into 10 mL of YPD yeast liquid medium, and culture it overnight at 30°C and 200 rpm;

[0127] 3) Transfer the overnight cultured seed solution to 100 mL of YPD yeast liquid medium at an initial OD600≈0.3, and culture it at 30°C and 200 rpm for about 4 h until the bacterial OD reaches about 1.0;

[0128] 4) The cultured bacterial solution was dispensed into sterilized 50 mL centrifuge tubes, centrifuged at 6000 rpm for 3 min at room temperature, and the supernatant was discarded and the bacterial cells were collected;

[0129] 5) Wash once with 5 mL of sterile water;

[0130] 6) Resuspend the cells with 25 mL of LT solution and incubate at 30 °C, 100 rpm for 1 h;

[0131] 7) Collect the cells by centrifugation, resuspend them in 1 mL of 100 mM LiAc and aliquot them into 175-200 μL tubes;

[0132] Conversion:

[0133] 1) Centrifuge the competent cells and remove the supernatant. Boil 2 mg / mL salmon sperm DNA for 5 min and immediately place on ice.

[0134] 2) Gently resuspend the cells with 240 μL 50% PEG3350, and then add 36 μL 1M LiAc, 3) 50 μL salmon sperm DNA, 34 μL plasmid (pYSE2-URA3-SpMAE1), DNA and ddH2O;

[0135] 4) Vortex and shake for 1 min to mix, and let stand at 30°C for 30 min;

[0136] 5) Heat shock at 42°C for 30 min;

[0137] 6) Centrifuge to remove the supernatant, add 1 mL of YPD medium and incubate for 2 h (30°C, 250 rpm), and finally coat.

[0138] TE buffer: TE (Tris-EDTA buffer) is Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 8.0).

[0139] LT solution: TE buffer + 100 mM lithium acetate (66), pH = 7.5, filter sterilized.

[0140] SpMAE1 protein sequence (SEQ ID NO.1):

[0141] MGELKEILKQRYHELLDWNVKAPHVPLSQRLKHFTWSWFACTMATGGVGLIIGSFPFRFYGLNTIGKIVYILQIFLFSLFGSCMLFRFIKYPSTIKDSWNHHLEKLFIATCLLSISTFIDMLAIYAYPDTGEWMVWVIRILYYIYVAVSFIYCVMAFFTIFNNHVYTIETASPAWILPIFPPMICGVIAGAVNSTQPAHQLKNMVIFGILFQGLGFWVYLLLFAVNVLRFFTVGLAKPQDRPGMFMFVGPPAFSGLALINIARGAMGSRPYIFVGANSSEYLGFVSTFMAIFIWGLAAWCYCLAMVSFLAGFFTRAPLKFACGWFAFIFPNVGFVNCTIEIGKMIDSKAFQMFGHIIGVILCIQWILLMYLMVRAFLVNDLCYPGKDEDAHPPPKPNTGVLNPTFPPEKAPASLEKVDTHVTSTGGESDP PSSEHESV。

[0142] SpMAE1 gene sequence (SEQ ID NO.2):

[0143]

[0144] TEF1 promoter sequence (SEQ ID NO.3):

[0145] gacggaggttacccggcctggatatacggtctttttcctaaaagagaaacggaaaatgtttttttgtttttttgttgtttttttgttgtttttccgtgttttctgaaaatttgaaaaataaaaaaataaaataaaataaaataaaaaataaaataaaataaaaaataaaataaaataaaataaaaaatagagtaaaaaataaaataaaataaaaaaatttttcaaaaaaaaaaaacaacaataaaatcaaaaaatttcaaagaaaaaaaaagaaaaaaaaattttttctttcaaaagaattttcccccccttttttttcgccactcaaatttttttcttggaaattgtatttctacaataagggctggagttggtgctctattctatttaatctgtttgtatttcccttccacttgagaaattctttctttgcttgtgtttaatgaaatttttgtttctcttcttttctccttttcttttctcttcttttcttttctcttctttcccttattatcaactttatatagtactgcaaa.

[0146] TEF1 terminator sequence (SEQ ID NO.4):

[0147] acatgttctattttaggctgtgtttaataaactttgtatttccatctctttgattgttacagcatgtctgtaatctcacaagaagcaaaaacaacaacaaaaaacccacttttcatttattttatttgtaaaaagcactttttttcttctttttctttttttttggctggtgaaaccaattgtatattttttttttattgtcagggcacacctgcaattcatcactttaatatagaacatttcatttgtcaaaagacgtaaaggagtgtggattcgaatagtttttttttttcctttaaagagaaaaaaaatgacgtttcttaaaagatataaattgatatatacgagaccggctaaggatcactaaccattacatcgaccaaatcatccaatggtccatagtctttccgctgtaacaattggaactcgttggcaaacaggacaaaatgttttagacttgtgttcaaatggacatttagctgcaactcattgatctc。

[0148] NAT gene sequence (SEQ ID NO.5)

[0149]

[0150] NAT expression cassette sequence (SEQ ID NO.6)

[0151]

[0152] Example 3

[0153] This example uses the engineered bacteria constructed in Example 2 for fermentation.

[0154] Seed solution: The strain stored in a frozen glycerol tube was streaked onto a YPD plate and cultured at 30°C for 30 h.

[0155] Fermentation: The single colony grown on the above plate was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of YPD liquid culture medium and cultured for activation at 30°C and 220 rpm with shaking.

[0156] The culture solution was transferred to a 250 mL Erlenmeyer flask containing 50 mL of fermentation medium (2% peptone, 1% yeast powder, 10% glucose) with an inoculation amount of 2% and cultured at 30° C. and 200 rpm for 60 h.

[0157] Succinic acid detection: After the fermentation broth was centrifuged to remove the bacteria, it was filtered using a 0.45 μm aqueous filter membrane. The filtrate was used to detect the succinic acid concentration using a Waters 2695 high performance liquid chromatograph (HPLC). The mobile phase was 2.5 mM sulfuric acid, the detector was a Milford RI-2414 differential refractometer, the chromatographic column was Bio-Rad's Aminex HPX-87H (300×7.8 mm), the injection volume was 10 μL, the mobile phase flow rate was maintained at 0.6 mL / min, the column oven temperature was maintained at 60°C, and the analysis time was 25 min; the detector operating temperature was 40°C, and the sensitivity was 32.

[0158] The succinic acid production of the wild-type strain of Pichia kudriavzi S9 is 0.32 g / L, and the succinic acid production of the modified engineered strain Pichia kudriavzi S9-106 reaches 31.74 g / L.

[0159] In summary, the present invention screened out a low-pH-resistant Pichia kudriavzii S9 from grape surface microorganisms, which can tolerate an environment of pH 1.8 and ferment organic acids without adding a neutralizer; in addition, genetic modification was performed based on Pichia kudriavzii S9, including knocking out succinic acid degradation-related genes, knocking out ethanol and glycerol production pathways, and enhancing the expression of succinic acid transporters, to obtain an engineered bacterium S9-106 with high succinic acid production, and the succinic acid fermentation yield reached 31.74 g / L without adding a neutralizer.

[0160] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An acid-tolerant Pichia kudriavzi yeast, characterized in that The acid-resistant Pichia kudriavzevii is Pichia kudriavzevii S9, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 20, 2024, with a deposit number of GDMCC.No: 65148.

2. Use of the acid-resistant Pichia kudriavzii described in claim 1 in producing acidic substances.

3. A method for producing acidic substances by fermentation, characterized in that: The method comprises: The acid-resistant Pichia kudriavzii described in claim 1 is used for fermentation production.

4. The method for producing acidic substances by fermentation according to claim 3, characterized in that: The acidic substance includes an organic acid; Preferably, the organic acid comprises at least one of succinic acid, lactic acid or malic acid.

5. An engineered bacterium, characterized in that: The starting strain of the engineered bacteria is the acid-resistant Pichia kudriavzii described in claim 1, and the engineered bacteria has undergone any of the following genetic modifications: (1) Loss of glycerol metabolism genes; (2) lack of glycerol metabolism genes and ethanol metabolism genes; (3) lack of glycerol metabolism genes, ethanol metabolism genes, and succinate degradation genes; (4) Deficiency of glycerol metabolism genes, ethanol metabolism genes, and succinate degradation genes, and overexpression of succinate transport genes.

6. The engineered bacteria according to claim 5, characterized in that The succinate degradation gene includes at least one of the SDH5 gene, the SDH2 gene, the SDH3 gene or the SDH4 gene; Preferably, the glycerol metabolism gene includes the GPD gene; Preferably, the ethanol metabolism gene includes PDC gene and / or ADH1 gene; Preferably, the succinate transporter gene comprises the SpMAE1 gene.

7. The method for constructing an engineered bacterium according to claim 5 or 6, characterized in that: The construction method of the engineering bacteria comprises: The acid-resistant Pichia kudriavzii described in claim 1 is used as a starting strain, the succinate degradation gene, the glycerol metabolism gene and the ethanol metabolism gene therein are knocked out, and the succinate transport gene is overexpressed to obtain the engineered bacteria.

8. Use of the engineered bacteria according to claim 5 or 6 in producing acidic substances.

9. A method for producing acidic substances by fermentation, characterized in that: The method comprises: The fermentation production is carried out using the engineered bacteria described in claim 5 or 6.

10. The method for producing acidic substances by fermentation according to claim 9, characterized in that: The acidic substance includes an organic acid; Preferably, the organic acid comprises at least one of succinic acid, lactic acid or malic acid.

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