Recombinant pichia kudriavzevii strain and application thereof

Abstract

The invention provides a recombinant pichia kudriavzevii strain and application thereof, pichia kudriavzevii is taken as an original strain, and a reduced TCA pathway is introduced and comprises the following steps: overexpressing fumarate reductase FRD in the strain; weakening or inactivating an ethanol pathway, comprising: knocking out any one or a combination of at least two of encoding genes of ethanol dehydrogenase ADH1, ADH3 or ADH6; enhancing the PPP pathway in the pentose phosphate pathway of the strain, including: overexpressing glucose-6-phosphate dehydrogenase ZWF1 and / or 6-phosphogluconate dehydrogenase GND1; the method for reducing the activity of the malate dehydrogenase MDH2 in the TCA circulation pathway comprises the step of knocking out the coding gene of the malate dehydrogenase MDH2 in the TCA pathway. The pichia kudriavzevii is modified by using genetic engineering and metabolic engineering methods, so that the yield and the yield of succinic acid are increased, and the pichia kudriavzevii has important application prospects in industrial production.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a recombinant Pichia kudriavzevii strain and an application thereof, and in particular to a method for producing succinic acid by using the Pichia kudriavzevii strain having a reductive TCA pathway and fermenting the same. Background Art

[0002] There are two pathways in the microbial synthesis of succinic acid: aerobic fermentation and anaerobic fermentation. In the aerobic fermentation process, microorganisms mainly synthesize succinic acid through the oxidative TCA pathway, which involves the release of CO2, and the theoretical succinic acid yield is 0.65g / g glucose. In nature, there is a reductive TCA pathway to synthesize succinic acid, which consists of three enzymes: malate dehydrogenase, fumarase, and fumarate reductase, of which fumarate reductase is the key rate-limiting enzyme. In the reductive TCA pathway, not only is there no release of CO2, but CO2 can also be carboxylated and fixed for succinic acid synthesis, and its theoretical yield is higher than that of the oxidative TCA pathway, so the reductive TCA pathway is more suitable for succinic acid production.

[0003] However, the reductive TCA pathway usually exists in specific strict anaerobes or facultative anaerobes, and it is particularly difficult to construct and express the reductive TCA pathway in yeast strains. The lack of a reductive TCA pathway in Pichia kudriavzi has limited the improvement of its succinic acid production efficiency and its industrial production application prospects, and has become a bottleneck in the research of succinic acid synthesis.

[0004] In the process of Pichia kudriazie producing succinic acid through the reduced TCA pathway, the accumulation of the intermediate metabolite malic acid was found. Malate dehydrogenase is a bidirectional enzyme that can catalyze the mutual conversion of oxaloacetic acid and malic acid. Malic acid has a similar structure to succinic acid, which will affect the separation and purification of succinic acid and increase production costs.

[0005] Therefore, using genetic engineering and metabolic engineering methods to transform Pichia kudriavzii and provide a recombinant Pichia kudriavzii strain with high succinic acid production has important application prospects in the industrial production of succinic acid. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide a recombinant Pichia kudriavzi strain and its application. The present invention transforms Pichia kudriavzi using genetic engineering and metabolic engineering methods, exogenously introduces and enhances the pathway of the reduced TCA pathway, increases the theoretical yield and yield of succinic acid, and has important application prospects in the industrial production of succinic acid.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a recombinant Pichia kudriavzi strain, wherein the strain is based on Pichia kudriavzi as a starting strain and a reduced TCA pathway is introduced, comprising: overexpressing fumarate reductase FRD in the strain;

[0009] Weakening or inactivating the ethanol pathway, including: knocking out any one or a combination of at least two of the genes encoding alcohol dehydrogenase ADH1, ADH3 or ADH6;

[0010] Enhancing the PPP pathway in the pentose phosphate pathway of the strain, including: overexpressing glucose-6-phosphate dehydrogenase ZWF1 and / or 6-phosphogluconate dehydrogenase GND1;

[0011] Reducing the activity of malate dehydrogenase MDH2 in the TCA cycle pathway, including: knocking out the encoding gene of malate dehydrogenase MDH2 in the TCA pathway.

[0012] The present invention utilizes genetic engineering and metabolic engineering methods to transform Pichia kudriavzi, introduces and enhances the pathway of exogenous reduced TCA pathway, and increases the theoretical yield and yield of succinic acid. At the same time, since the reduced TCA pathway is introduced into Pichia kudriavzi, the pathway needs to consume a large amount of reducing power, therefore, the present invention also enhances the PPP pathway of the strain to provide more reducing power; the present invention also knocks out the malate dehydrogenase MDH2 in the TCA pathway to effectively reduce the accumulation of the intermediate metabolite malic acid, and further improves the yield of succinic acid.

[0013] Preferably, the introduction of the reduced TCA pathway further comprises overexpressing any one or a combination of at least two of pyruvate carboxylase PYC, malate dehydrogenase MDH or fumarase FUM in the strain.

[0014] Preferably, the introduction of the reduced TCA pathway is achieved by integrating a homologous recombination donor sequence containing a gene encoding a target enzyme into the genome of the Pichia kudriavzii strain using the CRISPR-Cas9 gene editing system, wherein the integration site is selected from the alcohol dehydrogenase gene ADH1, ADH3 or ADH6.

[0015] Preferably, the PPP pathway in the pentose phosphate pathway of the enhanced strain is achieved by any one of the following (a) and (b):

[0016] (a) increasing the copy number of the gene encoding the target enzyme;

[0017] (b) Replacing the transcriptional or translational regulatory element of the gene encoding the target enzyme with a regulatory element with higher activity.

[0018] Preferably, the increase in the copy number of the gene encoding the target enzyme is achieved by introducing a plasmid carrying the gene encoding the target enzyme and / or integrating the gene encoding the target enzyme into the genome.

[0019] Preferably, the transcription or translation regulatory element is selected from any one or more combinations of promoters, ribosome binding sites or enhancers.

[0020] Preferably, the PPP pathway in the pentose phosphate pathway of the strain is enhanced by replacing the transcription or translation regulatory element of the gene encoding the target enzyme with a regulatory element with higher activity.

[0021] Preferably, the CRISPR-Cas9 gene editing system is used to replace the promoter of the gene encoding glucose-6-phosphate dehydrogenase ZWF1 and / or 6-phosphogluconate dehydrogenase GND1 with a promoter with higher activity.

[0022] Preferably, the method of introducing the reduced TCA pathway includes: using the CRISPR-Cas9 gene editing system to insert the gene encoding fumarate reductase FRD into the position of alcohol dehydrogenase gene ADH1.

[0023] Preferably, the CRISPR-Cas9 gene editing system for editing the ADH1 gene comprises: a promoter-FRD-terminator expression cassette, an ADH1 knockout plasmid, and repair templates P9 and P10, wherein the ADH1 knockout plasmid comprises a Cas9 element and an ADH1-sgRNA element.

[0024] Preferably, the DNA sequence corresponding to the ADH1-sgRNA element includes that shown in SEQ ID NO.41.

[0025] Preferably, the repair template P9 includes that shown in SEQ ID NO.9, and P10 includes that shown in SEQ ID NO.10.

[0026] In the present invention, the promoter-FRD-terminator expression cassette is integrated with the repair template in the strain, and works together with the Cas9 element and the sgRNA element to complete homologous recombination.

[0027] SEQ ID NO. 41: ttagtcaaggttctgtactc.

[0028] P9: 5'-accccccctccccctattagaaaaaggaatagacctttctttaaacaatt-3' (SEQ ID NO. 9).

[0029] P10: 5'-gcggggaggtatacactctctagaccaatgagttgcagctaaatgtccat-3' (SEQ ID NO. 10).

[0030] Preferably, the method of enhancing the PPP pathway in the pentose phosphate pathway of the strain comprises:

[0031] The CRISPR-Cas9 gene editing system is used to insert the gene encoding glucose-6-phosphate dehydrogenase ZWF1 into the ADH3 position of the alcohol dehydrogenase gene; and / or the gene encoding 6-phosphogluconate dehydrogenase GND1 is inserted into the ADH6 position of the alcohol dehydrogenase gene.

[0032] Preferably, the CRISPR-Cas9 gene editing system for editing ADH3 comprises: a promoter-ZWF1-terminator expression cassette, an ADH3 knockout plasmid, and repair templates P15 and P16, wherein the ADH3 knockout plasmid comprises a Cas9 element and an ADH3-sgRNA element.

[0033] Preferably, the DNA sequence corresponding to the ADH3-sgRNA element includes that shown in SEQ ID NO.42.

[0034] Preferably, the repair template P15 includes that shown in SEQ ID NO.15, and P16 includes that shown in SEQ ID NO.16.

[0035] SEQ ID NO. 42: tgtgaacatgccgatttgag.

[0036] P15: 5'-aagtttgctgttttcttcatcttcagataacatgaaaatggggttctggt-3' (SEQ ID NO. 15).

[0037] P16: 5'-cctatatagatacgccttgccaaatacaaattatttacctcatttcccat-3' (SEQ ID NO. 16).

[0038] Preferably, the CRISPR-Cas9 gene editing system for editing ADH6 comprises: a promoter-GND1-terminator expression cassette, an ADH6 knockout plasmid, and repair templates P21 and P22, wherein the ADH6 knockout plasmid comprises a Cas9 element and an ADH6-sgRNA element.

[0039] Preferably, the DNA sequence corresponding to the ADH6-sgRNA element includes that shown in SEQ ID NO.43.

[0040] Preferably, the repair template P21 includes SEQ ID NO.21, and P22 includes SEQ ID NO.22.

[0041] SEQ ID NO. 43: gatatcatgacagctagagg.

[0042] P21: 5'-ctaatcatctaatcattgaattataaattcatgtctatcacggtatccgt-3' (SEQ ID NO. 21).

[0043] P22: 5'-aaaaataagggttggtttagctcatatgaatagtgttatcaatatcgaca-3' (SEQ ID NO. 22).

[0044] Preferably, the CRISPR-Cas9 gene editing system for knocking out the gene encoding malate dehydrogenase MDH2 comprises: an MDH2 knockout plasmid and repair templates P25 and P26, wherein the MDH2 knockout plasmid comprises a Cas9 element and an MDH2-sgRNA element.

[0045] Preferably, the DNA sequence corresponding to the MDH2-sgRNA element includes that shown in SEQ ID NO.44.

[0046] Preferably, the repair template P25 includes that shown in SEQ ID NO.25, and P26 includes that shown in SEQ ID NO.26.

[0047] SEQ ID NO. 44: agaaagcccggcatgactag.

[0048] P25: 5'-tttgttgaaaagtcaagatacaaaccaaccaactcaacgaaaaacaaatt-3' (SEQ ID NO. 25).

[0049] P26: 5'-gatcaaaggaggcaaagaaggccggcaaaatcagtagaaaagattcaacg-3' (SEQ ID NO. 26).

[0050] In a second aspect, the present invention provides a method for constructing the recombinant Pichia kudriavzii strain described in the first aspect, the construction method comprising:

[0051] (1) Using Pichia kudriavzii as the starting strain, fumarate reductase FRD was overexpressed in the strain;

[0052] (2) knocking out any one or a combination of at least two of the genes encoding alcohol dehydrogenase ADH1, ADH3 or ADH6;

[0053] (3) overexpression of glucose-6-phosphate dehydrogenase ZWF1 and / or 6-phosphogluconate dehydrogenase GND1;

[0054] (4) Knock out the gene encoding malate dehydrogenase MDH2 in the TCA pathway.

[0055] In a third aspect, the present invention provides a method for producing succinic acid, the method comprising: fermenting the recombinant Pichia kudriavzi strain described in the first aspect, and separating succinic acid from the fermentation broth.

[0056] Preferably, the fermentation process further comprises adding a neutralizing agent into the culture medium.

[0057] Preferably, the neutralizing agent is selected from any one of sodium hydroxide, calcium carbonate, magnesium carbonate or magnesium hydroxide, or a combination of at least two thereof.

[0058] In a fourth aspect, the present invention provides use of the recombinant Pichia kudriavzii strain described in the first aspect in succinic acid synthesis.

[0059] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0060] Compared with the prior art, the present invention has the following beneficial effects:

[0061] The present invention utilizes genetic engineering and metabolic engineering methods to transform Pichia kudriavzii, introduces and enhances the pathway of exogenous reduced TCA pathway, enhances the PPP pathway of the strain, and knocks out the malate dehydrogenase MDH2 in the TCA pathway. After the above transformation, the theoretical yield and yield of succinic acid are improved. Compared with wild strains, the succinic acid and malic acid yields of the S9-1 strain constructing the reduced TCA pathway are 7.38 g / L and 0.62 g / L, respectively, and the succinic acid and malic acid yields of the S9-3 strain overexpressing the ZWF1 and GND1 genes in the pentose phosphate pathway (PPP pathway) are 10.87 g / L and 0.97 g / L, respectively; after further knocking out MDH2, the succinic acid yield is increased from 10.87 g / L to 11.34 g / L, and the malic acid yield is reduced from 0.97 g / L to 0.28 g / L, and knocking out MDH2 can reduce the yield of malic acid, a byproduct in fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1is the yield of succinic acid and malic acid in different strains. DETAILED DESCRIPTION

[0063] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0064] 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.

[0065] The materials involved in the following examples are as follows:

[0066] Bacterial strains and plasmids: Escherichia coli TOP10 competent cells and plasmid pV1081 were purchased from Miaoling Biotechnology.

[0067] The Pichia kudriavzevii strain S9 is a strain screened by the laboratory. The Pichia kudriavzevii strain is named Pichia kudriavzevii S9 and is preserved in the Guangdong Provincial Microbiological Culture Collection Center, located on the 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The preservation number is GDMCC.No: 65148, and the preservation date is September 20, 2024.

[0068] LB medium: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L.

[0069] YPD medium: peptone 20g / L, yeast powder 10g / L, glucose 20g / L, the glucose concentration in the fermentation medium is 100g / L, if necessary, the concentration of nourseoinzyme is 20μg / L, and the concentration of agar powder is 2%.

[0070] The amplification reaction system used in the following examples is as follows:

[0071] The PCR reaction system is shown in Table 1.

[0072] Table 1

[0073] 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

[0074] When the template is a plasmid: the amount added is 50ng; when it is a genome: the amount added is 10-200ng, and when it is cDNA: 200ng.

[0075] The Overlap PCR reaction system is shown in Table 2.

[0076] Table 2

[0077]

[0078]

[0079] The PCR reaction program settings are shown in Table 3.

[0080] Table 3

[0081]

[0082] PCR recovery product digestion system:

[0083] 5 μL of 10× enzyme digestion buffer (TAKARA), 1 μL of Dpn I, 20 μL of purified DNA product, 24 μL of distilled water, total volume of 50 μL, react at 37°C for 30 min.

[0084] The preparation and transformation methods of Pichia kudriavzi competent cells in the embodiment are as follows:

[0085] Use a sterilized toothpick to streak the S9 strain on a YPD solid plate for strain activation, and place the streaked plate in a 30°C incubator for 2-3 days to grow a single colony.

[0086] A single yeast colony with good growth was selected and inoculated into 10 mL of YPD yeast liquid culture medium and cultured overnight at 30°C and 220 rpm.

[0087] The seed solution cultured overnight was 600 Transfer to 100 mL of YPD yeast liquid medium at 0.1 and culture at 30°C and 220 rpm for about 4-6 hours until the bacterial OD 600 The growth reaches about 1.2.

[0088] The cultured bacterial solution was dispensed into sterilized 50 mL centrifuge tubes, centrifuged at 8000 rpm at room temperature for 5 min, the supernatant was discarded and the bacteria were collected.

[0089] Wash the cells twice with 25 mL of sterile water.

[0090] Resuspend the cells with 25 mL of LT solution and incubate at 30 °C with a shaker at 100 rpm for 1 h.

[0091] The cells were collected by centrifugation, resuspended in 1 mL 100 mM LiAc and aliquoted into 175-200 μL tubes.

[0092] The conversion method is as follows:

[0093] Centrifuge the competent cells to remove the supernatant, boil 2mg / mL salmon sperm DNA for 5 minutes and immediately place on ice. Gently resuspend the cells with 240μL 50% PEG3350, then add 36μL 1M LiAc, 50μL salmon sperm DNA, 34μL plasmid, DNA and ddH2O in sequence. Vortex for 1min to mix, let stand at 30℃ for 30min. Heat shock at 42℃ for 30min. Centrifuge to remove the supernatant, add 1mL YPD medium and incubate for 2h (30℃, 250rpm), and finally apply.

[0094] The buffer solution used in the competent cell preparation process is as follows:

[0095] TE buffer: TE (Tris-EDTA buffer) is Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 8.0).

[0096] LT solution: TE buffer + 100 mM lithium acetate, pH = 7.5, filter sterilized.

[0097] The Pichia kudriavzia shake flask fermentation method in the embodiment is as follows:

[0098] The Pichia kudriavzii S9 strain stored in a frozen glycerol tube was streaked onto a YPD plate and cultured at 30°C for 30 h.

[0099] The transformed single colony was inoculated into a 50 mL Erlenmeyer flask containing 10 mL of YPD liquid culture medium and cultured for activation at 30°C and 220 rpm for 24 h.

[0100] The activated bacterial liquid was inoculated into a 500 mL Erlenmeyer flask containing 50 mL of YPD liquid culture medium at a 2% inoculation rate, and cultured at 30° C. and 220 rpm for 48 h.

[0101] The fermentation broth was centrifuged and the supernatant was filtered, and the production of succinic acid and malic acid in the fermentation broth was detected by liquid phase; the liquid mobile phase was 2.5 mM sulfuric acid.

[0102] Example 1 Insertion of TbFRD gene into Pichia kudriavzi S9

[0103] TbFRD (KT026107.1) from Trypanosoma brucei was codon-optimized and synthesized by Qingke Biotechnology; a TbFRD expression cassette was constructed, whose promoter was PTEF1 and terminator was TTEF1.

[0104] First, primers P1 and P2 were used to amplify the TbFRD gene, primers P3 and P4 were used to amplify the promoter PTEF1, and primers P5 and P6 were used to amplify the terminator TTEF1.

[0105] P1:5'-AATGGTCGATGGTAGATCTTCTGCATCCATG-3'(SEQ ID NO.1)

[0106] P2:5'-ATGTTTAAGAACCGGATGTGTTCAGTTTCGT-3'(SEQ ID NO.2)

[0107] P3:5'-ccccctattagaaaaaggaatagacctttcttt-3'(SEQ ID NO.3)

[0108] P4:5'-GAAGATCTACCATCGACCATtttgcagtactatataaagttgat-3'(SEQ ID NO.4)

[0109] P5:5'-CGAAACTGAACCATCCGGTTCTTAAacatgttctattttaggctgtg-3'(SEQ ID NO.5)

[0110] P6:5'-caatgagttgcagctaaatgtccatttg-3'(SEQ ID NO.6)

[0111] By overlapping extension PCR technology, the PTEF1 and TbFRD fragments were used as templates, and primers P2 and P3 were used to amplify the PTEF1-TbFRD fragment. Then, the PTEF1-TbFRD and TTEF1 fragments were used as templates, and primers P3 and P6 were used to amplify the PTEF1-TbFRD-TTEF1 expression cassette.

[0112] Among them, the gene sequence of the TbFRD fragment is shown in SEQ ID NO.27, and the amino acid sequence is shown in SEQ ID NO.28.

[0113] The PTEF1-TbFRD-TTEF1 expression cassette was inserted into the alcohol dehydrogenase gene ADH1 position, and pV1081 (Miaoling Biotechnology, Catalog No. P49399) was used as a template and primers P7 and P8 were used for circular amplification to construct the ADH1 knockout plasmid p-dADH1.

[0114] Among them, the gene sequence of the alcohol dehydrogenase gene ADH1 is shown in SEQ ID NO.29, and the amino acid sequence is shown in SEQ ID NO.30.

[0115] After amplification, the pV1081 template was degraded with DpnI, and the product was recovered and transformed into Escherichia coli Top10 competent cells. After positive transformants were selected with ampicillin, the knockout plasmid p-dADH1 was obtained after extraction and sequencing.

[0116] P7:5'-Ttaatttggatccgttagtcaaggttctgtactcgttttagagctagaaatagcaagtt-3' (SEQ ID NO.7)

[0117] P8:5'-gagtacagaaccttgactaacggatccaaattaaaaatagtttacgcaag-3'(SEQ IDNO.8)

[0118] The resulting knockout plasmid p-dADH1 contains a Cas9 element and an ADH1-sgRNA element, wherein the ADH1-sgRNA is as shown in SEQ ID NO.41, ttagtcaaggttctgtactc.

[0119] The characteristics and advantages of the gene editing system of the present invention include: the knockout plasmid contains Cas9 elements and sgRNA elements, and only the sgRNA sequence needs to be replaced for different targets; the strain has a high homologous recombination ability, and the repair template only needs to contain a homologous arm of about 50bp to complete homologous recombination in vivo.

[0120] The PTEF1-TbFRD-TTEF1 expression cassette, ADH1 knockout plasmid p-dADH1, and repair templates P9 and P10 were co-transformed into S9 competent cells, and nourseothricin was used for resistance screening to obtain the TbFRD overexpression strain, which was named S9-1.

[0121] P9:5'-accccccctccccctattagaaaaaggaatagacctttctttaaacaatt-3' (SEQ IDNO.9)

[0122] P10:5'-gcggggaggtatacactctctagaccaatgagttgcagctaaatgtccat-3'(SEQ IDNO.10)

[0123] Example 2: Overexpression of the ZWF1 gene in Pichia kudriavzi S9-1

[0124] The ZWF1 expression cassette was amplified using the S9 genome as a template.

[0125] P11:5'-acaaattatttacctcatttcccatatccacg-3'(SEQ ID NO.11)

[0126] P12:5'-gataacatgaaaatggggttctggttcg-3'(SEQ ID NO.12)

[0127] The ZWF1 expression cassette was inserted into the alcohol dehydrogenase gene ADH3 position, and pV1081 was used as a template and primers P13 and P14 were used for circular amplification to construct the ADH3 knockout plasmid p-dADH3.

[0128] The ZWF1 gene sequence is shown in SEQ ID NO.31, and the amino acid sequence is shown in SEQ ID NO.32.

[0129] The sequence of the alcohol dehydrogenase gene ADH3 is shown in SEQ ID NO.33, and the amino acid sequence is shown in SEQ ID NO.34.

[0130] After amplification, the pV1081 template was degraded with DpnI, and the product was recovered and transformed into Escherichia coli Top10 competent cells. After positive transformants were selected with ampicillin, the knockout plasmid p-dADH3 was obtained after extraction and sequencing.

[0131] P13:5'-ttaatttggatccgtgtgaacatgccgatttgaggttttagagctagaaatagcaagtt-3' (SEQ ID NO.13)

[0132] P14:5'-ctcaaatcggcatgttcacacggatccaaattaaaaatagtttacgcaag-3'(SEQ IDNO.14)

[0133] The resulting knockout plasmid p-dADH3 contains a Cas9 element and an ADH3-sgRNA element, wherein the ADH3-sgRNA is as shown in SEQ ID NO.42, tgtgaacatgccgatttgag.

[0134] The ZWF1 expression cassette, ADH3 knockout plasmid p-dADH3, and repair templates P15 and P16 were co-transformed into S9-1 competent cells, and nourseothricin was used for resistance screening to obtain the ZWF1 overexpression strain, which was named S9-2.

[0135] P15:5'-aagtttgctgttttcttcatcttcagataacatgaaaatggggttctggt-3'(SEQ IDNO.15)

[0136] P16:5'-cctatatagatacgccttgccaaatacaaattatttacctcatttcccat-3'(SEQ IDNO.16)

[0137] Example 3: Overexpression of the GND1 gene in Pichia kudriavzi S9-2

[0138] Using the S9 genome as a template, the GND1 expression cassette was amplified;

[0139] P17:5'-aattcatgtctatcacggtatccgtgaatata-3'(SEQ ID NO.17)

[0140] P18:5'-atgaatagtgttatcaatatcgacactctc-3'(SEQ ID NO.18)

[0141] The GND1 expression cassette was inserted into the position of the alcohol dehydrogenase gene ADH6, and the ADH6 knockout plasmid p-dADH6 was constructed by circular amplification using primers P19 and P20 using pV1081 as a template.

[0142] The GND1 gene sequence is shown in SEQ ID NO.35, and the amino acid sequence is shown in SEQ ID NO.36.

[0143] The sequence of the alcohol dehydrogenase gene ADH6 is shown in SEQ ID NO.37, and the amino acid sequence is shown in SEQ ID NO.38.

[0144] After amplification, the pV1081 template was degraded with DpnI, and the product was recovered and transformed into Escherichia coli Top10 competent cells. After positive transformants were selected with ampicillin, the knockout plasmid p-dADH6 was obtained after extraction and sequencing.

[0145] P19:5'-ttaatttggatccggatatcatgacagctagagggttttagagctagaaatagcaagtt-3'(SEQ ID NO.19)

[0146] P20:5'-cctctagctgtcatgatatccggatccaaattaaaaatagtttacgcaag-3'(SEQ IDNO.20)

[0147] The knockout plasmid p-dADH6 comprises a Cas9 element and an ADH6-sgRNA element, wherein the ADH6-sgRNA is as shown in SEQ ID NO.43, gatatcatgacagctagagg.

[0148] The GND1 expression cassette, ADH6 knockout plasmid p-dADH6, and repair templates P21 and P22 were co-transformed into S9-2 competent cells, and screened with nourseothricin as resistance to obtain a GND1 overexpression strain named S9-3.

[0149] P21:5'-ctaatcatctaatcattgaattataaattcatgtctatcacggtatccgt-3'(SEQ IDNO.21)

[0150] P22:5'-aaaaataagggttggtttagctcatatgaatagtgttatcaatatcgaca-3'(SEQ IDNO.22)

[0151] Example 4: Knockout of the MDH2 gene in Pichia kudriavzi S9-3

[0152] Malic acid accumulation was found during the fermentation process of S9-3. The presence of malic acid would affect the purification of succinic acid. Malic dehydrogenase is a bidirectional catalytic enzyme. Knocking out MDH2 in this strain could effectively reduce malic acid accumulation.

[0153] Using pV1081 as a template, primers P23 and P24 were used for circular amplification to construct the MDH2 knockout plasmid p-dMDH2; after amplification, the pV1081 template was degraded with DpnI, the product was recovered and transformed into Escherichia coli Top10 competent cells, and the positive transformants were selected with ampicillin, and then the knockout plasmid p-dMDH2 was obtained after extraction and sequencing.

[0154] P23:5'-ttaatttggatccgagaaagcccggcatgactaggttttagagctagaaatagcaagtt-3' (SEQ ID NO.23)

[0155] P24:5'-ctagtcatgccgggctttctcggatccaaattaaaaatagtttacgcaag-3'(SEQ IDNO.24)

[0156] The MDH2 gene sequence is shown in SEQ ID NO.39, and the amino acid sequence is shown in SEQ ID NO.40.

[0157] The knockout plasmid p-dMDH2 comprises a Cas9 element and an MDH2-sgRNA element, wherein the MDH2-sgRNA is as shown in SEQ ID NO.44, agaaagcccggcatgactag.

[0158] The MDH2 knockout plasmid p-dMDH2 and the repair templates P25 and P26 were co-transformed into S9-3 competent cells, and the cells were screened for resistance to nourseothricin to obtain the MDH2 knockout strain, which was named S9-4.

[0159] P25:5'-tttgttgaaaagtcaagatacaaaccaaccaactcaacgaaaaacaaatt-3'(SEQ IDNO.25)

[0160] P26:5'-gatcaaaggaggcaaagaaggccggcaaaatcagtagaaaagattcaacg-3'(SEQ IDNO.26)

[0161] S9, S9-1, S9-2, S9-3 and S9-4 were fermented in shake flasks to identify the yields of succinic acid and malic acid in the fermentation broth. The results are as follows: Figure 1 As shown, the production of succinate and malate of the wild-type S9 strain was 0.30 g / L and 0.14 g / L, respectively; the production of succinate and malate of the S9-1 strain constructing the reduced TCA pathway was 7.38 g / L and 0.62 g / L, respectively; the production of succinate and malate of the S9-2 strain was 9.59 g / L and 0.78 g / L, respectively; the production of succinate and malate of the S9-3 strain was 10.87 g / L and 0.97 g / L, respectively; the production of succinate and malate of the S9-4 strain was 11.34 g / L and 0.28 g / L, respectively; after knocking out MDH2, the production of succinate increased from 10.87 g / L to 11.34 g / L, and the production of malate decreased from 0.97 g / L to 0.28 g / L. Knocking out MDH2 can reduce the production of malic acid, a byproduct in fermentation.

[0162] In summary, the present invention utilizes genetic engineering and metabolic engineering methods to transform Pichia kudriavzii, introduces and enhances the pathway of exogenous reduced TCA pathway, weakens or inactivates alcohol dehydrogenase ADH1, ADH3 and ADH6 in the ethanol pathway, overexpresses ZWF1 and GND1 genes in the pentose phosphate pathway (PPP pathway), and knocks out the malate dehydrogenase MDH2 gene in the TCA cycle pathway; the yield of succinic acid in the transformed strain is improved, and the yield of malic acid, a byproduct in fermentation, can also be reduced; the obtained recombinant bacteria have important application value in the production of succinic acid.

[0163] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A recombinant Pichia kudriavzia strain, characterized in that: The strain uses Pichia kudriavzia as a starting strain and introduces a reduced TCA pathway, including: overexpressing fumarate reductase FRD in the strain; Weakening or inactivating the ethanol pathway, including: knocking out any one or a combination of at least two of the genes encoding alcohol dehydrogenase ADH1, ADH3 or ADH6; Enhancing the PPP pathway in the pentose phosphate pathway of the strain, including: overexpressing glucose-6-phosphate dehydrogenase ZWF1 and / or 6-phosphogluconate dehydrogenase GND1; Reducing the activity of malate dehydrogenase MDH2 in the TCA cycle pathway, including: knocking out the encoding gene of malate dehydrogenase MDH2 in the TCA pathway.

2. The recombinant Pichia kudriavzii strain according to claim 1, characterized in that The introduction of the reduced TCA pathway also includes overexpressing any one or a combination of at least two of pyruvate carboxylase PYC, malate dehydrogenase MDH or fumarase FUM in the strain.

3. The recombinant Pichia kudriavzii strain according to claim 1 or 2, characterized in that: The introduction of the reduced TCA pathway is achieved by: using the CRISPR-Cas9 gene editing system, integrating a homologous recombination donor sequence containing a gene encoding a target enzyme into the genome of the Pichia kudriavzi strain, wherein the integration site is selected from the alcohol dehydrogenase gene ADH1, ADH3 or ADH6; Preferably, the PPP pathway in the pentose phosphate pathway of the enhanced strain is achieved by any one of the following (a) and (b): (a) increasing the copy number of the gene encoding the target enzyme; (b) Replacing the transcriptional or translational regulatory element of the gene encoding the target enzyme with a regulatory element with higher activity.

4. The recombinant Pichia kudriavzii strain according to claim 3, characterized in that The increase in the copy number of the gene encoding the target enzyme is achieved by introducing a plasmid carrying the gene encoding the target enzyme and / or integrating the gene encoding the target enzyme into the genome; Preferably, the transcription or translation regulatory element is selected from any one or more combinations of promoters, ribosome binding sites or enhancers; Preferably, the PPP pathway in the pentose phosphate pathway of the strain is enhanced by replacing the transcription or translation regulatory element of the gene encoding the target enzyme with a regulatory element with higher activity; Preferably, the CRISPR-Cas9 gene editing system is used to replace the promoter of the gene encoding glucose-6-phosphate dehydrogenase ZWF1 and / or 6-phosphogluconate dehydrogenase GND1 with a promoter with higher activity.

5. The recombinant Pichia kudriavzii strain according to any one of claims 1 to 4, characterized in that The method of introducing the reduced TCA pathway includes: using the CRISPR-Cas9 gene editing system to insert the gene encoding fumarate reductase FRD into the position of the alcohol dehydrogenase gene ADH1; Preferably, the CRISPR-Cas9 gene editing system for editing the ADH1 gene comprises: a promoter-FRD-terminator expression cassette, an ADH1 knockout plasmid, and repair templates P9 and P10, wherein the ADH1 knockout plasmid comprises a Cas9 element and an ADH1-sgRNA element; Preferably, the DNA sequence corresponding to the ADH1-sgRNA element includes that shown in SEQ ID NO.41; Preferably, the repair template P9 includes that shown in SEQ ID NO.9, and P10 includes that shown in SEQ ID NO.

10.

6. The recombinant Pichia kudriavzii strain according to any one of claims 1 to 5, characterized in that The method of enhancing the PPP pathway in the pentose phosphate pathway of the strain comprises: The CRISPR-Cas9 gene editing system is used to insert the gene encoding glucose-6-phosphate dehydrogenase ZWF1 into the position of alcohol dehydrogenase gene ADH3; and / or the gene encoding 6-phosphogluconate dehydrogenase GND1 is inserted into the position of alcohol dehydrogenase gene ADH6; Preferably, the CRISPR-Cas9 gene editing system for editing ADH3 comprises: a promoter-ZWF1-terminator expression cassette, an ADH3 knockout plasmid, and repair templates P15 and P16, wherein the ADH3 knockout plasmid comprises a Cas9 element and an ADH3-sgRNA element; Preferably, the DNA sequence corresponding to the ADH3-sgRNA element includes that shown in SEQ ID NO.42; Preferably, the repair template P15 includes SEQ ID NO.15, and P16 includes SEQ ID NO.16; Preferably, the CRISPR-Cas9 gene editing system for editing ADH6 comprises: a promoter-GND1-terminator expression cassette, an ADH6 knockout plasmid, and repair templates P21 and P22, wherein the ADH6 knockout plasmid comprises a Cas9 element and an ADH6-sgRNA element; Preferably, the DNA sequence corresponding to the ADH6-sgRNA element includes that shown in SEQ ID NO.43; Preferably, the repair template P21 includes SEQ ID NO.21, and P22 includes SEQ ID NO.

22.

7. The recombinant Pichia kudriavzii strain according to any one of claims 1 to 6, characterized in that The CRISPR-Cas9 gene editing system for knocking out the gene encoding malate dehydrogenase MDH2 comprises: an MDH2 knockout plasmid and repair templates P25 and P26, wherein the MDH2 knockout plasmid comprises a Cas9 element and an MDH2-sgRNA element; Preferably, the DNA sequence corresponding to the MDH2-sgRNA element includes that shown in SEQ ID NO.44; Preferably, the repair template P25 includes that shown in SEQ ID NO.25, and P26 includes that shown in SEQ ID NO.

26.

8. The method for constructing a recombinant Pichia kudriavzii strain according to any one of claims 1 to 7, characterized in that: The construction method comprises: (1) Using Pichia kudriavzii as the starting strain, fumarate reductase FRD was overexpressed in the strain; (2) knocking out any one or a combination of at least two of the genes encoding alcohol dehydrogenase ADH1, ADH3 or ADH6; (3) overexpression of glucose-6-phosphate dehydrogenase ZWF1 and / or 6-phosphogluconate dehydrogenase GND1; (4) Knock out the gene encoding malate dehydrogenase MDH2 in the TCA pathway.

9. A method for producing succinic acid, characterized in that: The method comprises: fermenting with the recombinant Pichia kudriavzi strain according to any one of claims 1 to 7, and separating succinic acid from the fermentation liquid.

10. Use of the recombinant Pichia kudriavzii strain according to any one of claims 1 to 7 in the synthesis of succinic acid.

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