A yeast engineering bacterium for fermentative production of triacetin and application thereof

Abstract

The application discloses a kind of yeast engineering bacteria for fermenting production triacetin and application thereof.The engineering bacteria uses Pichia pastoris GS115 as starting strain, imports 2-pyrone synthetase coding gene Gh2PS, simultaneously overexpresses acetyl-coenzyme A carboxylase gene ScACC1, further improves Gh2PS copy number, and obtains the Pichia pastoris engineering bacteria.The Pichia pastoris engineering bacteria developed in the application can efficiently produce triacetin, and the yield of shake flask fermentation reaches 2.72g / L, and the highest yield of 1L fermenter fermentation reaches 9.72g / L.The application constructs a kind of Pichia pastoris engineering bacteria for fermenting production triacetin, and lays a foundation for subsequent development of Pichia pastoris chassis strain for efficiently synthesizing high-value platform compound triacetin.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, specifically to an engineered yeast strain for fermenting triacetin and its applications. Background Technology

[0002] Triacetic acid lactone (TAL), chemically known as 4-hydroxy-6-methyl-2-pyranone, with the molecular formula C6H6O3, is a polyketide compound found in African daisies. Gerbera hybrida Within plant hosts. Triacetin, as a potential renewable platform compound, can generate more high-value-added compounds through a series of reactions, such as patchouli and sorbic acid. It is a direct precursor to many food additives, antibiotics, and fuel additives, as well as an intermediate for some bifunctional compounds. It is mainly used in chemical, materials, food, and pharmaceutical fields.

[0003] Currently, the synthesis of triacetin mainly involves plant extraction, chemical synthesis, and biosynthesis. Plant extraction yields triacetin directly from African daisies, but this method requires large amounts of raw materials, occupies a large area, and has low extraction yield and small production volume. Chemical synthesis mainly involves the pyrolysis of acetic acid; however, the harmful catalysts used and the toxic byproducts produced hinder its industrial application. Biosynthesis mainly involves microbial fermentation, offering advantages such as environmental friendliness, fewer byproducts, and lower production costs. Therefore, developing suitable engineered host strains for the biosynthesis of triacetin is of great significance in meeting current sustainable development requirements. The biosynthesis of triacetin is catalyzed by a type III polyketide synthase (PKS)-2-pyranone synthase (2-PS) derived from African daisies. Gh2PS The gene encodes a catalyst that catalyzes the condensation of two molecules of malonyl-CoA and one molecule of acetyl-CoA to produce a one-molecule triacetin lactone. Numerous studies have reported the introduction of this catalyst into different hosts. Gh2PS Recombinant genetically engineered strains are constructed for the biosynthesis of triacetin, including but not limited to *Escherichia coli*, *Saccharomyces cerevisiae*, *Yersinia lipolytica*, and *Rhodotorula glutinis*. For example, patent application CN113403213A discloses a recombinant *Yersinia lipolytica* strain that produces triacetin from xylose and its application. The engineered strain uses *Yersinia lipolytica* as the starting strain and incorporates a 2-pyranone synthase encoding gene. gh2psMeanwhile, the xylose reductase-encoding gene, the xylitol dehydrogenase-encoding gene and the xylulokinase-encoding gene are overexpressed to obtain the recombinant Yarrowia lipolytica engineering strain. The fermentation process is further optimized to develop the optimal production process of the triacetin produced by the Yarrowia lipolytica using xylose, and the yield reaches the highest level reported in the flask, which is between 3 g / L and 6 g / L. Up to now, the highest yield of triacetin is produced by the fermentation of the Yarrowia lipolytica, and the highest yield reaches 35.9 g / L, which is as high as 43% of the theoretical yield of the glucose fermentation.

[0004] Pichia pastoris is a kind of unconventional methylotrophic yeast, and has the characteristics of high expression of excreted proteins and high-density fermentation. In recent years, with the characterization of many constitutive promoters of Pichia pastoris and the development of CRISPR / Cas9 gene editing technology, more and more attention has been paid to the construction of Pichia pastoris engineering strains for the synthesis of high-value biotechnological products. However, the introduction of Gh2PS and the acetyl-CoA carboxylase-encoding gene ScACC1 There is no report on the construction of yeast engineering strains for the biosynthesis of triacetin. SUMMARY

[0005] To solve the above technical problems, the present application develops a yeast engineering strain for the fermentation production of triacetin, and provides a method for producing triacetin by the yeast engineering strain using glucose as a carbon source.

[0006] One of the technical solutions provided by the present application is to provide a yeast engineering strain for the fermentation production of triacetin, which is obtained by introducing genes into a yeast as a starting strain. The introduced genes include any one of the following groups: (1) the coding gene of 2-pyrone synthetase Gh2PS; (2) the coding gene of 2-pyrone synthetase Gh2PS and the coding gene of acetyl-CoA carboxylase ScACC1.

[0007] The coding gene of 2-pyrone synthetase Gh2PS is introduced by using gene editing technology, and 2-pyrone synthetase is expressed to realize the intracellular synthesis of triacetin in Pichia pastoris. Then, the coding gene of acetyl-CoA carboxylase ScACC1 is introduced by using the same method, and acetyl-CoA carboxylase is overexpressed to improve the synthesis of the precursor malonyl-CoA. Finally, the copy number of the gene is increased Gh2PS to remove the rate-limiting step in the triacetin synthesis pathway, and a recombinant yeast engineering strain is constructed. Preferably, the coding gene of 2-pyrone synthetase Gh2PS in the introduced gene is 1 or more copies. More preferably, the coding gene of 2-pyrone synthetase Gh2PS in the introduced gene is 1 to 6 copies.

[0008] Preferably, the starting strain is Pichia pastoris, Saccharomyces cerevisiae, or Yersinia lipolytica. More preferably, the starting strain is Pichia pastoris, such as Pichia pastoris strain GS115. The gene encoding the 2-pyranone synthase is a plant-derived gene, including but not limited to those derived from African daisies (…). Gerbera hybrida The gene encoding 2-pyranone synthase Gh2PS, as shown in SEQ ID NO.1, is a yeast heterologous gene, including but not limited to those derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae The gene encoding acetyl-CoA carboxylase ScACC1 is shown in SEQ ID NO.2.

[0009] This invention also provides a method for constructing the yeast engineered strain for fermenting triacetin production, wherein the introduced genes include the coding gene for 2-pyranone synthase Gh2PS and the coding gene for acetyl-CoA carboxylase ScACC1. The construction method includes the following steps: (1) integrating the coding gene sequence for 2-pyranone synthase Gh2PS into the genome of Pichia pastoris GS115-Cas9 to obtain strain GTAL-1; (2) integrating the coding gene sequence for acetyl-CoA carboxylase ScACC1 into strain GTAL-1 to obtain strain GTAL-2; (3) integrating a single copy of the coding gene sequence for 2-pyranone synthase Gh2PS into strain GTAL-2 to obtain... (4) Integrate a single copy of the coding gene sequence of 2-pyranone synthase Gh2PS into strain GTAL-3 to obtain strain GTAL-4; then integrate a single copy of the coding gene sequence of 2-pyranone synthase Gh2PS into strain GTAL-4 to obtain strain GTAL-5; then integrate a single copy of the coding gene sequence of 2-pyranone synthase Gh2PS into strain GTAL-5 to obtain strain GTAL-6; (5) Integrate a single copy of the coding gene sequence of 2-pyranone synthase Gh2PS into strain GTAL-6 to obtain strain GTAL-7, which is the yeast engineered strain for fermenting triacetin. The promoter used in the construction process is pTEF1 The nucleotide sequence is shown in SEQ ID NO.3; the terminator is... t0547 or tAOX1 The terminator t0547 The nucleotide sequence is shown in SEQ ID NO.4; the terminator tAOX1 The nucleotide sequence is shown in SEQ ID NO.5.

[0010] The application further provides application of the yeast engineering bacteria for fermentatively producing triacetin in preparation of triacetin.

[0011] The application has the following advantages based on the prior art:

[0012] (1) The application develops Pichia pastoris genetic engineering bacteria capable of efficiently producing triacetin, and the shake flask fermentation yield of the bacteria is nearly 2.72 g / L, and the fermentation yield of the bacteria in a 1 L fermenter is up to 9.72 g / L.

[0013] (2) The application improves the metabolic flux of triacetin biosynthesis and synthesis pathway by overexpressing rate-limiting enzymes of the synthesis pathway, improving precursor supply and increasing the gene copy number of the rate-limiting enzymes.

[0014] (3) The application utilizes Pichia pastoris to efficiently produce triacetin, and has the advantages of short cycle, environmental friendliness and low production cost. The application lays a foundation for subsequent development of Pichia pastoris chassis strains for efficiently synthesizing high-value platform compound triacetin. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 It is a schematic diagram of triacetin biosynthesis pathway in Pichia pastoris.

[0016] Figure 2 It is a schematic diagram of an expression cassette of a key enzyme introduced from an exogenous source.

[0017] Figure 3 It is a map of plasmid pZ-panARS- hcas9 -sgRNA (ADE2).

[0018] Figure 4 It is a map of plasmid pGAP-HsCas9-SV40 .

[0019] Figure 5 It is a map of plasmid Int- pTEF1 - tAOX1 .

[0020] Figure 6 It is a map of plasmid Int- pTEF1 - t0547 .

[0021] Figure 7 It is a map of plasmid HZP-sgRNA.

[0022] Figure 8 This is the HPLC absorption peak diagram of triacetin.

[0023] Figure 9 This is a graph showing the yield of Pichia pastoris engineered strains fermented in shake flasks.

[0024] Figure 10 The figure shows the fermentation yield of the engineered Pichia pastoris strain GTAL-7 in a 1 L fermenter. Detailed Implementation

[0025] The biosynthetic pathway of triacetin is as follows: Figure 1 As shown, the exogenous key enzyme expression cassette is as follows: Figure 2 As shown, the detailed synthesis process is detailed in the embodiments.

[0026] Table 1 Primer sequences involved in this invention

[0027]

[0028] Example 1: Construction of Pichia pastoris recombinant strain GS115-Cas9

[0029] Build Cas9 Recombinant vectors for gene expression pGAP-Cas9 The strain was then transferred into Pichia pastoris GS115 to obtain the recombinant Pichia pastoris strain GS115-Cas9. The specific implementation method is as follows:

[0030] (1) Recombinant vector pGAP-Cas9 Construction

[0031] Human codon optimized from Streptococcus pyogenes Cas9 The gene nucleotide sequence is shown in SEQ ID NO.6, using laboratory-preserved pZ-panARS-hcas9-sgRNA (ADE2) (laboratory-preserved, plasmid map see [link]). Figure 3 Using plasmids as templates, primers were designed. pGAP-Cas9-F / pGAP-Cas9-R Amplification Cas9 Gene expression cassette pGAP-hCas9-tDAS1 The GibsonAssembly method was used to combine it with a universal backbone (ampicillin resistance gene expression cassette plus the replication origin site ori of a prokaryotic gene plasmid). His4 Genes were assembled, transformed into E. coli DH5α strain, and constructed to obtain Cas9 Gene expression recombinant vector, named pGAP-HsCas9-SV40 (See plasmid map) Figure 4 ).

[0032] (2) Construction of Pichia pastoris recombinant strain GS115-Cas9

[0033] Recombinant vector pGAP-Cas9 Using restriction endonucleases Sal I. The vector was digested into a linearized fragment, which was then transferred into laboratory-preserved Pichia pastoris GS115 competent cells using the Lin-Cereghino electroporation method to construct Pichia pastoris GS115-Cas9.

[0034] Example 2: Construction of Pichia pastoris engineered strain GTAL-1 with triacetin

[0035] The Int values ​​refer to the Pichia pastoris GS115 genome insertion site, and the helper plasmids Int used are also mentioned. - pTEF1-tAOX1 Int -pTEF1-t0547 Composed of a universal backbone (ampicillin resistance gene expression cassette and prokaryotic gene replication origin site) ori ), upstream and downstream 500 bp homologous arms of the site and pTEF1-BamH Ⅰ -tAOX1 , pTEF1-Hind III - Nde Ⅰ- t0547 Composition (see plasmid map) Figure 5 , Figure 6 The helper plasmid HZP-sgRNA consists of a universal backbone, an antibiotic resistance gene expression cassette, the sgRNA sequence corresponding to the site, and two... Bsa I. Enzyme restriction site composition, Z represents the bleomycin resistance gene (see plasmid map). Figure 7 ); P stands for plasmid.

[0036] Constructing a gene encoding 2-pyranone synthase with high expression Gh2PS The expression cassette and corresponding guide plasmid were transformed into Pichia pastoris GS115-Cas9 to construct the Pichia pastoris triacetin engineered strain GTAL-1. The specific implementation is as follows:

[0037] (1) Int1- pTEF1-Gh2PS-t0547 Construction

[0038] The gene encoding 2-pyranone synthase Gh2PS Synthesized by Genscript Biotech Co., Ltd. Gh2PS Design primers using templates. Gh2PS -F / Gh2PS -R, amplification obtained Gh2PS (The sequence is shown in SEQ ID NO.1), using the GibsonAssembly method to... Gh2PS Fragments and Hind III , Nde I. Helper plasmid Int1 after double enzyme digestion-pTEF1-t0547 (Laboratory-preserved genes PAS_FragB_0066 on the left and PAS_FragB_0067 on the right of Int1) were ligated to construct the gene encoding 2-pyranone synthase. Gh2PS The auxiliary plasmid Int1- used for integration pTEF1-Gh2PS-t0547 .

[0039] (2) Construction of HZP-sgRNA-Int1

[0040] Using plasmid HZP-sgRNA as a template, primers sgRNA-Int1-F / sgRNA-Int1-R were designed. The primers annealed to form a doublet DNA fragment sgRNA-Int1 (sgRNA sequence 5'-tatctgaagtatttactggg-3'). sgRNA-Int1 was then ligated with... Bsa I. The digested vector was ligated to construct HZP-sgRNA-Int1.

[0041] (3) Construction of Pichia pastoris engineered strain GTAL-1 with triacetin

[0042] With Int1- pTEF1-Gh2PS-t0547 Design primers Int1- for the template Gh2PS -F and Int1- Gh2PS -R, PCR amplification of the target gene expression cassette used for homologous recombination, named Int1 -Gh2PS-donor . Int1 -Gh2PS-donor The triacetin-lactone Pichia pastoris strain GTAL-1 was constructed by co-transferring it with HZP-sgRNA-Int1 into Pichia pastoris GS115-Cas9.

[0043] Example 3: Construction of Pichia pastoris engineered strain GTAL-2 with triacetin

[0044] Constructing a gene encoding acetyl-CoA carboxylase with high expression ScACC1 The expression cassette and corresponding guide plasmid were transformed into the above-mentioned Pichia pastoris engineered strain GTAL-1, ultimately obtaining the Pichia pastoris engineered strain GTAL-2. The specific implementation is as follows:

[0045] (1) Int39- pTEF1-ScACC1-tAOX1 Construction

[0046] Primers were designed using the Saccharomyces cerevisiae genome as a template. ScACC1 -F / ScACC1 -R, amplification obtained ScACC1(The sequence is shown in SEQ ID NO.2), using the Gibson Assembly method to... ScACC1 Fragments and BamH I. Helper plasmid Int39 after enzyme digestion -pTEF1-tAOX1 (The genes PAS_chr1-4_0294 on the left and PAS_chr1-4_0295 on the right of Int39, preserved in the laboratory, were ligated to construct the gene encoding acetyl-CoA carboxylase.) ScACC1 The auxiliary plasmid Int39- used for integration pTEF1-ScACC1-tAOX1 .

[0047] (2) Construction of HZP-sgRNA-Int39

[0048] Using plasmid HZP-sgRNA as a template, primers sgRNA-Int39-F / sgRNA-Int39-R were designed. The primers annealed to form a doublet DNA fragment sgRNA-Int39 (sgRNA sequence 5'-gattcagtagagtcctattg-3'). sgRNA-Int39 was then ligated with... Bsa I. The digested vector was ligated to construct HZP-sgRNA-Int39.

[0049] (3) Construction of Pichia pastoris engineered strain GTAL-2 with triacetin

[0050] With Int39- pTEF1-ScACC1-tAOX1 Design primers Int39- for the template ScACC1 -F / Int39- ScACC1 -R, PCR amplification of the target gene expression cassette used for homologous recombination, named Int39 -ScACC1-donor Int39 - ScACC1-donor The triacetin-lactone Pichia pastoris engineered strain GTAL-1 was co-transformed with HZP-sgRNA-Int39 to construct the triacetin-lactone Pichia pastoris engineered strain GTAL-2.

[0051] Example 4: Construction of Pichia pastoris engineered strain GTAL-3 with triacetin

[0052] Add another copy Gh2PS The gene encoding 2-pyranone synthase was obtained with high expression. Gh2PS The expression cassette and corresponding guide plasmid were constructed and transformed into Pichia pastoris GTAL-2 to construct the triacetin-lactone Pichia pastoris engineered strain GTAL-3. The specific implementation is as follows:

[0053] (1) Construction of HZP-sgRNA-Int11

[0054] Using plasmid HZP-sgRNA as a template, primers sgRNA-Int11-F / sgRNA-Int11-R were designed. The primers annealed to form a doublet DNA fragment sgRNA-Int11 (sgRNA sequence 5'-tattaaaaaagacgatcccg-3', with PAS_chr3_0154 on the left and PAS_chr3_1156 on the right). The sgRNA-Int11 was then ligated with... Bsa I. The digested vector was ligated to construct HZP-sgRNA-Int11.

[0055] (2) Construction of Pichia pastoris engineered strain GTAL-3 with triacetin

[0056] The gene encoding 2-pyranone synthase constructed using the method described in Example 1 Gh2PS The auxiliary plasmid Int1- used for integration pTEF1-Gh2PS-t0547 Design primers Int11- for template Gh2PS -F / Int11- Gh2PS -R, PCR amplification of the target gene expression cassette used for homologous recombination, named Int11 -Gh2PS-donor Int11 -Gh2PS-donor The triacetin-lactone Pichia pastoris strain GTAL-3 was constructed by co-transferring it with HZP-sgRNA-Int11 into Pichia pastoris GTAL-2.

[0057] Example 5: Construction of Pichia pastoris engineered strain GTAL-4 with triacetin lactone

[0058] Add another copy Gh2PS The gene encoding 2-pyranone synthase was obtained with high expression. Gh2PS The expression cassette and corresponding guide plasmid were constructed and transformed into Pichia pastoris GTAL-3 to construct the triacetin-lactone Pichia pastoris engineered strain GTAL-4. The specific implementation is as follows:

[0059] (1) Construction of HZP-sgRNA-Int20

[0060] Using plasmid HZP-sgRNA as a template, primers sgRNA-Int20-F / sgRNA-Int20-R were designed. The primers annealed to form a doublet DNA fragment sgRNA-Int20 (sgRNA sequence 5'-agaagaaaatgcgaaacagg-3', with PAS_chr4_0467 on the left and PAS_chr4_0465 on the right). The sgRNA-Int20 was then ligated with... Bsa I. The digested vector was ligated to construct HZP-sgRNA-Int20.

[0061] (2) Construction of Pichia pastoris engineered strain GTAL-4 with triacetin

[0062] The gene encoding 2-pyranone synthase constructed using the method described in Example 1 Gh2PS The auxiliary plasmid Int1- used for integration pTEF1-Gh2PS-t0547 Design primers Int20- for the template Gh2PS -F / Int20- Gh2PS -R, PCR amplification of the target gene expression cassette used for homologous recombination, named Int20. -Gh2PS-donor Int20 -Gh2PS-donor The triacetin-lactone Pichia pastoris strain GTAL-4 was constructed by co-transferring it with HZP-sgRNA-Int20 into Pichia pastoris GTAL-3.

[0063] Example 6: Construction of Pichia pastoris engineered strain GTAL-5 with triacetin lactone

[0064] Add another copy Gh2PS The gene encoding 2-pyranone synthase was obtained with high expression. Gh2PS The expression cassette and corresponding guide plasmid were constructed and transformed into Pichia pastoris GTAL-4 to construct the triacetin-lactone Pichia pastoris engineered strain GTAL-5. The specific implementation is as follows:

[0065] (1) Construction of HZP-sgRNA-Int32

[0066] Using plasmid HZP-sgRNA as a template, primers sgRNA-Int32-F / sgRNA-Int32-R were designed. The primers annealed to form a doublet DNA fragment sgRNA-Int32 (sgRNA sequence 5'-gtgacgaaagagatgaggtg-3', with PAS_chr2-2_0142 on the left and PAS_chr2-2_0143 on the right). The sgRNA-Int32 was then ligated to [a specific DNA fragment] using the T4-PNK enzyme ligation method. BsaI. The digested vector was ligated to construct HZP-sgRNA-Int32.

[0067] (2) Construction of Pichia pastoris engineered strain GTAL-5 with triacetin

[0068] The gene encoding 2-pyranone synthase constructed using the method described in Example 1 Gh2PS The auxiliary plasmid Int1- used for integration pTEF1-Gh2PS-t0547 Design primers Int32- for the template Gh2PS -F / Int32- Gh2PS -R, PCR amplification of the target gene expression cassette used for homologous recombination, named Int32 -Gh2PS-donor Int32 -Gh2PS-donor The triacetin-lactone Pichia pastoris strain GTAL-5 was constructed by co-transferring it with HZP-sgRNA-Int32 into Pichia pastoris GTAL-4.

[0069] Example 7: Construction of Pichia pastoris engineered strain GTAL-6 with triacetin lactone

[0070] Add another copy Gh2PS The gene encoding 2-pyranone synthase was obtained with high expression. Gh2PS The expression cassette and corresponding guide plasmid were constructed and transformed into Pichia pastoris GTAL-5 to construct the triacetin-lactone Pichia pastoris engineered strain GTAL-6. The specific implementation is as follows:

[0071] (1) Construction of HZP-sgRNA-Int33

[0072] Using plasmid HZP-sgRNA as a template, primers sgRNA-Int33-F / sgRNA-Int33-R were designed. The primers annealed to form a doublet DNA fragment sgRNA-Int33 (sgRNA sequence 5'-ccgtcactatgaggacaaag-3', with PAS_chr1-1_0054 on the left and PAS_chr1-1_0053 on the right). The sgRNA-Int33 was then ligated with... Bsa I. The digested vector was ligated to construct HZP-sgRNA-Int33.

[0073] (2) Construction of Pichia pastoris engineered strain GTAL-6 with triacetin

[0074] The gene encoding 2-pyranone synthase constructed using the method described in Example 1 Gh2PS The auxiliary plasmid Int1- used for integration pTEF1-Gh2PS-t0547 Design primers Int33- for the templateGh2PS -F / Int33- Gh2PS -R, PCR amplification of the target gene expression cassette used for homologous recombination, named Int33 -Gh2PS-donor Int33 -Gh2PS-donor The triacetin-lactone Pichia pastoris strain GTAL-6 was constructed by co-transferring it with HZP-sgRNA-Int33 into Pichia pastoris GTAL-5.

[0075] Example 8: Construction of Pichia pastoris engineered strain GTAL-7 with triacetin lactone

[0076] Add another copy Gh2PS The gene encoding 2-pyranone synthase was obtained with high expression. Gh2PS The expression cassette and corresponding guide plasmid were constructed and transformed into Pichia pastoris GTAL-6 to construct the triacetin-lactone Pichia pastoris engineered strain GTAL-7. The specific implementation is as follows:

[0077] (1) Construction of HZP-sgRNA-Int34

[0078] Using plasmid HZP-sgRNA as a template, primers sgRNA-Int34-F / sgRNA-Int34-R were designed. The primers annealed to form a doublet DNA fragment sgRNA-Int34 (sgRNA sequence 5'-gatcagttcattgatagaca-3', with PAS_chr3_0054 on the left and PAS_chr3_0053 on the right). The sgRNA-Int34 was then ligated with... Bsa I. The digested vector was ligated to construct HZP-sgRNA-Int34.

[0079] (2) Construction of Pichia pastoris engineered strain GTAL-7 with triacetin

[0080] The gene encoding 2-pyranone synthase constructed using the method described in Example 1 Gh2PS The auxiliary plasmid Int1- used for integration pTEF1-Gh2PS-t0547 Design primers Int34- for the template Gh2PS -F / Int34- Gh2PS -R, PCR amplification of the target gene expression cassette used for homologous recombination, named Int34 -Gh2PS-donor Int34 -Gh2PS-donor The Pichia pastoris strain GTAL-7 was constructed by co-transferring it with HZP-sgRNA-Int34 into Pichia pastoris GTAL-6.

[0081] Example 9: High-efficiency production of triacetin by engineered Pichia pastoris strain

[0082] The Pichia pastoris engineered strains obtained in Examples 1-7 were activated by streaking on YPD plates. Single colonies were picked and inoculated into 5 mL YPD liquid test tubes and fermented at 30°C and 250 rpm for 12 h. The engineered strain seed culture was then inoculated into 50 mL YPD shake flasks at an inoculation rate of 1-5% and fermented at 30°C and 250 rpm for 72 h. The processed samples were analyzed by HPLC. The Pichia pastoris engineered strain GTAL-7 was fed-batch fermented in a 1 L fermenter to obtain high-concentration products.

[0083] The specific implementation is as follows:

[0084] (1) Shake-flask fermentation of engineered Pichia pastoris strains

[0085] Single colonies of activated GS115-Cas9, GTAL-1, GTAL-2, GTAL-3, GTAL-4, GTAL-5, GTAL-6, and GTAL-7 were picked from YPD plates and inoculated into 5 mL YPD tubes. The colonies were incubated at 30°C and 250 rpm for 12 h. Seed culture was then inoculated at a 1% inoculum into 50 mL YPD shake flasks, with three replicates for each colony. The cultures were incubated at 30°C and 250 rpm for 72 h. After fermentation, the fermentation broth was collected for sample processing and subsequent HPLC product analysis.

[0086] (2) Feed-in batch fermentation of Pichia pastoris engineered strain GTAL-7 in a 1 L fermenter

[0087] Activated GTAL-7 single colonies were picked from YPD plates and inoculated into 5 mL YPD tubes. The colonies were incubated at 30°C and 250 rpm for 12 h on a shaker. The seed culture was then inoculated at a 1% inoculum into 50 mL YPD shake flasks, with three replicates per colony. The inoculum was incubated at 30°C and 250 rpm for 12 h to obtain the seed culture. This seed culture was then pumped into a 1 L fermenter (600 mL capacity). After inoculation, the dissolved oxygen level was calibrated to 100%. The feed bottle (YPD), acid bottle (acetic acid), and alkali bottle (ammonia) were then connected. During fermentation, the culture temperature was maintained at 30°C, the fermenter pressure at 0.8 atm, and the aeration rate at 3 vvm. The pH was maintained between 5.5 and 6.0 by adding 25% ammonia or 18% acetic acid. The dissolved oxygen (DO) was maintained at 10% to 20% by controlling the stirring speed at 200–800 rpm. Dissolved oxygen and pH were monitored using DO and pH electrodes, respectively. Fermentation broth samples were taken every 4 hours to determine sugar content and OD. 600The value is used to determine the feed flow rate. When the glucose mass fraction in the culture medium drops to 1%, a glucose solution with a mass fraction of 40% (v / v) is added. The above conditions are controlled until the fermentation is completed.

[0088] (3) HPLC detection of triacetin

[0089] Sample preparation: Transfer 1 mL of fermentation broth from the shake flask to a 1.5 mL EP tube, centrifuge at 12000 rpm for 1 min, take 10 μL of the fermentation broth supernatant and dilute 100 times with 990 μL ddH2O, then vortex to mix. Use a syringe to draw up the sample dilution, filter impurities through a 0.22 μm aqueous filter membrane, and transfer to a liquid chromatography vial for subsequent HPLC analysis.

[0090] HPLC detection conditions: PDA detector set to 280 nm wavelength, column type C18, column temperature 35℃, flow rate 0.6 mL / min, mobile phase A was 0.1% acetic acid solution, and mobile phase B was pure methanol.

[0091] HPLC detection procedure: Initial mobile phase A is 95% and mobile phase B is 5%; 0.0~6.8 min, mobile phase A decreases from 95% to 75% and mobile phase B increases from 5% to 25%; 6.8~7.8 min, mobile phase A decreases from 75% to 5% and mobile phase B increases from 25% to 95%; 7.8~16.0 min, mobile phase A increases from 5% to 95% and mobile phase B decreases from 95% to 5%.

[0092] (4) Yield of Pichia pastoris engineered strain in shake flask fermentation

[0093] Table 2. Yields of triacetin produced by fermentation of various strains

[0094]

[0095] Pichia pastoris engineered strains were fermented according to the methods described in (1) and (2), and the products were detected by HPLC using the detection method in (3). The absorption peak diagram for triacetin detection is shown below. Figure 8 As shown, the yield of Pichia pastoris engineered strains during shake-flask fermentation is as follows: Figure 9 As shown in Table 2, the fermentation yield and OD curve of the engineered Pichia pastoris strain GTAL-7 in a 1 L fermenter are as follows: Figure 10 As shown.

[0096] Fermentation culture was carried out on strains GS115-Cas9, GTAL-1, GTAL-2, GTAL-3, GTAL-4, GTAL-5, GTAL-6, and GTAL-7 according to the culture method described in (1). The results showed that, except for strain GS115-Cas9 which did not ferment to produce triacetin, all other strains fermented to produce triacetin. In particular, strain GTAL-7 produced the highest yield of triacetin, reaching 2.72 g / L.

[0097] The engineered Pichia pastoris strain GTAL-7 was subjected to fed-batch fermentation in a 1 L fermenter according to the method described in (2), and the results are as follows. Figure 10 The results showed that the final OD measured after 88 hours of fermentation... 600 The OD value reached 65, and the highest yield of triacetin reached 9.72 g / L. Compared with the shake-flask fermentation results of this strain, the OD value of the fermenter culture was significantly higher. 600 The yield of triacetin increased by 2 times, and the highest yield of triacetin increased by 2.33 times. This invention constructs a Pichia pastoris engineered strain for fermentation production of triacetin, laying the foundation for subsequent development of Pichia pastoris chassis strains for efficient synthesis of the high-value platform compound triacetin.

Claims

1. A yeast strain for fermenting and producing triacetin, characterized in that, Using Pichia pastoris as the starting strain, the engineered yeast strain was obtained by introducing genes. The introduced genes were the encoding genes for 2-pyranone synthase Gh2PS and acetyl-CoA carboxylase ScACC1. During construction, the introduced genes were integrated into the strain genome using CRISPR / Cas9 technology. The method for constructing engineered yeast strains for fermentation production of triacetin includes the following steps: (1) The gene sequence encoding 2-pyranone synthase Gh2PS was integrated into the genome of Pichia pastoris GS115-Cas9 to obtain strain GTAL-1. The sgRNA sequence used in this step was 5'-tatctgaagtatttactggg-3'. (2) The coding gene sequence of acetyl-CoA carboxylase ScACC1 was integrated into strain GTAL-1 to obtain strain GTAL-2. The sgRNA sequence used in this step was 5'-gattcagtagagtcctattg-3'. (3) Integrate the coding gene sequence of a single copy of 2-pyranone synthase Gh2PS into strain GTAL-2 to obtain strain GTAL-3. The sgRNA sequence used in this step is 5'-tattaaaaaagacgatcccg-3'. (4) Integrate the coding gene sequence of a single copy of 2-pyranone synthase Gh2PS into strain GTAL-3 to obtain strain GTAL-4. The sgRNA sequence used in this step is 5'-agaagaaaatgcgaaacagg-3'. Then, integrate the coding gene sequence of a single copy of 2-pyranone synthase Gh2PS into strain GTAL-4 to obtain strain GTAL-5. The sgRNA sequence used in this step is 5'-gtgacgaaagagatgaggtg-3'. Then, integrate the coding gene sequence of a single copy of 2-pyranone synthase Gh2PS into strain GTAL-5 to obtain strain GTAL-6. The sgRNA sequence used in this step is 5'-ccgtcactatgaggacaaag-3'. (5) Integrate the coding gene sequence of a single copy of 2-pyranone synthase Gh2PS into strain GTAL-6 to obtain strain GTAL-7, namely the yeast engineered strain for fermentation to produce triacetin. The sgRNA sequence used in this step is 5'-gatcagttcattgatagaca-3'. The gene sequence encoding the 2-pyranone synthase Gh2PS is shown in SEQ ID NO.1, and the gene sequence encoding the acetyl-CoA carboxylase ScACC1 is shown in SEQ ID NO.

2. The promoter used during the build process is pTEF1 ;Terminator is t0547 or tAOX1 .

2. The application of the engineered yeast strain for fermenting triacetin as described in claim 1 in the preparation of triacetin.

3. A method for preparing triacetin, characterized in that, The yeast engineered strain for fermentation production of triacetin according to claim 1 is fermented and cultured, and triacetin is extracted and obtained.

4. The method for preparing triacetin according to claim 3, characterized in that, The fermentation culture method is as follows: the engineered yeast is inoculated into a 50 mL YPD shake flask at a volume percentage of 1% to 5% and cultured at 30℃ and 250 rpm for 72 h.

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