Recombinant strain, construction method and application thereof, and method for producing total fatty acid

Abstract

The invention relates to the technical field of gene engineering, and discloses a recombinant strain, a construction method and application thereof, and a method for producing total fatty acid. The recombinant strain is obtained by genetic modification of an original strain, and compared with the original strain, the recombinant strain overexpresses citric acid transporter Yhm2p, cis-aconitase E.HA and isocitrate dehydrogenase IDP located in cytoplasm at the same time, and does not express isocitrate dehydrogenase IDH1 and isocitrate dehydrogenase IDH2 located in mitochondria. The recombinant strain provided by the invention forms a high-efficiency pathway of citric acid retention-cytoplasm transport-directional production of NADPH, so that the core bottleneck of insufficient supply of cytoplasm NADPH in fatty acid synthesis is solved, the total fatty acid yield is remarkably increased, the transformation logic is accurate, and a stable and reliable source is provided for industrial high-efficiency production of fatty acid.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a recombinant bacterial strain, its construction method and application, and a method for producing total fatty acids. Background Technology

[0002] Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is an important cofactor and energy source for microbial cells. It directly participates in 1058 biochemical reactions catalyzed by 586 intracellular enzymes as a hydrogen and electron donor / carrier (Updated at Dec 16, 2021; www.kegg.com). These reactions influence microbial metabolism, signal transduction, and substrate transport through NADPH regeneration and competitive utilization. NADPH supply is considered a key factor affecting the synthesis of reducing substances (such as fatty acids and sterols) in microorganisms. However, NADPH is expensive and unstable, making exogenous NADPH addition for efficient synthesis of target products impractical. Therefore, developing a *Yarrowia lipolytica* strain capable of efficiently supplying cytoplasmic NADPH to overcome the limitation of insufficient NADPH supply and achieve efficient synthesis of target products is of great significance.

[0003] Yarrowia lipolytica ( Y. lipolytica Yeast (Yersinia lipolytica) is a widely used yeast in industrial biomanufacturing, possessing highly efficient metabolic capabilities and a good genetic manipulation platform, making it a common host for fatty acid biosynthesis. However, fatty acid production using Yersinia lipolytica as a host is often limited by insufficient NADPH supply, restricting the level of fatty acid synthesis. Therefore, developing a metabolically engineered Yersinia lipolytica strain that can efficiently provide NADPH, thereby promoting efficient fatty acid synthesis, has significant industrial application value. Summary of the Invention

[0004] The purpose of this invention is to overcome the problem in existing technologies where fatty acid production using *Yarrowia lipolyticis* as a host is often limited by insufficient NADPH supply, thus restricting fatty acid synthesis. This invention provides a recombinant strain, its construction method, and its application, as well as a method for producing total fatty acids. This recombinant strain simultaneously overexpresses citrate transporter Yhm2p, cis-aconitase E.HA, and cytoplasmic isocitrate dehydrogenase IDP, while not expressing mitochondrial isocitrate dehydrogenases IDH1 and IDH2. This results in a high cytoplasmic NADPH supply level, and the NADPH generated in the cytoplasm can be used for fatty acid synthesis, thereby increasing the level of fatty acid synthesis.

[0005] To achieve the above objectives, the present invention provides a recombinant strain obtained by genetic modification of a starting strain. Compared with the starting strain, the recombinant strain simultaneously overexpresses citrate transporter Yhm2p, cis-aconitase E.HA, and isocitrate dehydrogenase IDP located in the cytoplasm, and does not express isocitrate dehydrogenase IDH1 and isocitrate dehydrogenase IDH2 located in the mitochondria.

[0006] A second aspect of the present invention provides a method for constructing a recombinant bacterial strain, the method comprising: knocking out the coding genes for mitochondrial isocitrate dehydrogenase IDH1 and isocitrate dehydrogenase IDH2 in a starting strain, and overexpressing the coding genes for citrate transporter Yhm2p, cis-aconitase E.HA and cytoplasmic isocitrate dehydrogenase IDP.

[0007] A third aspect of the present invention provides the application of the recombinant strains as described above and / or the recombinant strains constructed by the methods described above in increasing total fatty acid production.

[0008] A fourth aspect of the present invention provides a method for producing total fatty acids, the method comprising: fermenting and culturing the recombinant strain as described above and / or the recombinant strain constructed by the method described above.

[0009] Through the above technical solutions, the recombinant strain provided by this invention does not express IDH1 and IDH2 located in mitochondria, thus avoiding the consumption of citrate in mitochondria, allowing it to accumulate efficiently and serve as a precursor; the overexpressed Yhm2p can efficiently transport the citrate accumulated in mitochondria to the cytoplasm, and then convert it into isocitrate through E.HA catalysis. Finally, the cytoplasmic IDP enzyme directionally generates NADPH required for fatty acid synthesis, forming an efficient pathway of "citrate retention-cytoplasmic transport-directed NADPH production". This not only solves the core bottleneck of fatty acid synthesis, which is insufficient cytoplasmic NADPH supply, but also significantly increases the total fatty acid yield. Moreover, the modification logic is precise, providing a stable and reliable source for the efficient industrial production of fatty acids. Attached Figure Description

[0010] Figure 1 This is a map of plasmid pUrloxp from Example 1; Figure 2 This is a map of plasmid pUrloxp-IDH1 from Example 1; Figure 3 This is a spectrum of plasmid pUrloxp-IDH2 from Example 1; Figure 4 This is a spectrum of plasmid pYLXP'-Yhm2p-E.HA-IDP from Example 2; Figure 5 This is a schematic diagram of the metabolic pathway in Example 3; Figure 6 This is a comparison of fatty acid production at 120 h between the integrated recombinant strain A and the starting strain in Example 4. Detailed Implementation

[0011] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0012] The first aspect of the present invention provides a recombinant strain obtained by genetic modification of a starting strain. Compared with the starting strain, the recombinant strain simultaneously overexpresses citrate transporter Yhm2p, cis-aconitase E.HA and isocitrate dehydrogenase IDP located in the cytoplasm, and does not express isocitrate dehydrogenase IDH1 and isocitrate dehydrogenase IDH2 located in the mitochondria.

[0013] The recombinant strain provided by this invention does not express IDH1 and IDH2 located in mitochondria, thus avoiding the consumption of citrate in mitochondria and allowing it to accumulate efficiently as a precursor. The overexpressed Yhm2p can efficiently transport the citrate accumulated in mitochondria to the cytoplasm, where it is then converted into isocitrate by E.HA catalysis. Finally, the cytoplasmic IDP enzyme directionally generates NADPH required for fatty acid synthesis, forming an efficient pathway of "citrate retention - cytoplasmic transport - directional NADPH production". This not only solves the core bottleneck of fatty acid synthesis, which is insufficient cytoplasmic NADPH supply, but also significantly increases the total fatty acid yield. Moreover, the modification logic is precise, providing a stable and reliable source for the efficient industrial production of fatty acids.

[0014] According to the present invention, preferably, the recombinant strain originates from *Yersinia lipolytica*. The inventors discovered in their research that using *Yersinia lipolytica* as the originating strain for gene modification better adapts to the remodeling requirements of "IDH1 / IDH2 knockout + Yhm2p / E.HA / IDP overexpression," reducing heterologous host compatibility issues and further ensuring the stability of efficient cytoplasmic NADPH supply and increased total fatty acid production.

[0015] According to the present invention, preferably, the recombinant strain does not express the ku70 gene. The inventors discovered in their research that preferably preventing the recombinant strain from expressing the ku70 gene (this can be achieved by the originating strain itself not expressing the gene or by knocking out the ku70 gene in the originating strain) can inhibit the non-homologous end joining repair pathway, improve the accuracy and stability of IDH1 / IDH2 gene knockout and the integrated expression of Yhm2p, E.HA, and IDP genes, ensure the efficient construction of the cytoplasmic NADPH supply pathway, and thus stably increase total fatty acid production.

[0016] According to the present invention, preferably, the nucleotide sequence of the ku70 gene is as shown in SEQ ID NO. 1. The inventors have found that the preferred nucleotide sequence of the ku70 gene, as shown in SEQ ID NO. 1, avoids the problem of incomplete knockout due to sequence differences, ensuring that the modification effect of the recombinant strain not expressing the ku70 gene is precise and controllable, and providing stable support for efficient supply of cytoplasmic NADPH and high production of total fatty acids.

[0017] According to the present invention, preferably, the nucleotide sequence of the gene encoding the citrate transporter Yhm2p is shown in SEQ ID NO. 2. The inventors have found that the preferred gene sequence encoding the citrate transporter Yhm2p, as shown in SEQ ID NO. 2, ensures that the expressed Yhm2p protein has the function of efficiently transporting mitochondrial citrate to the cytoplasm, thus guaranteeing the stability of high total fatty acid production.

[0018] According to the present invention, preferably, the nucleotide sequence of the gene encoding the aconitase E.HA is shown in SEQ ID NO. 3. The inventors have found that the preferred gene sequence encoding the aconitase E.HA, as shown in SEQ ID NO. 3, ensures that the expressed E.HA enzyme has highly efficient catalytic activity in converting cytoplasmic citrate to isocitrate, providing sufficient substrate for the subsequent targeted NADPH generation by IDP enzymes, and further stabilizing and improving the total fatty acid synthesis efficiency.

[0019] According to the present invention, preferably, the nucleotide sequence of the gene encoding the cytoplasmic isocitrate dehydrogenase IDP is as shown in SEQ ID NO. 4. The inventors have found that the preferred cytoplasmic encoding gene sequence of isocitrate dehydrogenase IDP, as shown in SEQ ID NO. 4, ensures that the expressed IDP enzyme has highly efficient catalytic activity in generating cytoplasmic NADPH from isocitrate, further guaranteeing a high efficiency increase in total fatty acid production.

[0020] According to the present invention, preferably, the encoding genes for the overexpressed citrate transporter Yhm2p, cis-aconitase E.HA, and cytoplasmic isocitrate dehydrogenase IDP are provided in the form of a recombinant vector. The inventors have found that providing the encoding genes for Yhm2p, E.HA, and IDP in the form of a recombinant vector allows for the simultaneous introduction and synergistic expression of the three genes, avoiding the problem of uneven expression levels caused by individual introduction, ensuring efficient synergistic operation, and further improving the stability of total fatty acid production.

[0021] According to the present invention, preferably, the recombinant vector contains the encoding genes for citrate transporter Yhm2p, cis-aconitase E.HA, and isocitrate dehydrogenase IDP located in the cytoplasm, as well as a promoter for overexpression of the encoding genes for citrate transporter Yhm2p, cis-aconitase E.HA, and isocitrate dehydrogenase IDP located in the cytoplasm, and an expression cascade (an expression cascade refers to a complete, independently functional DNA fragment containing the coding sequence of the target gene and the regulatory elements required for its efficient transcription and translation in the host cell). The inventors have found that the preferred recombinant vector simultaneously contains three target genes, a strong promoter, and an adapted vector backbone. This allows for efficient overexpression of the target genes via the promoter and stable gene introduction and inheritance via the vector backbone, ensuring synergistic function at each stage, guaranteeing a continuous and sufficient supply of cytoplasmic NADPH, and further achieving a stable and efficient increase in total fatty acid production.

[0022] According to the present invention, preferably, the promoter is a TEF promoter. The inventors have discovered that the preferred TEF promoter can continuously and strongly drive the overexpression of Yhm2p, E.HA, and IDP genes, ensuring sufficient synthesis of these three key proteins, ensuring the efficient operation of the cytoplasmic NADPH synthesis pathway, and providing stable driving force for high total fatty acid production.

[0023] According to the present invention, preferably, the recombinant vector is pYLXP-Yhm2p-E.HA-IDP. The inventors discovered in their research that the preferred recombinant vector integrates a suitable pYLXP' backbone, a highly efficient TEF promoter, and three target genes, enabling synchronous gene introduction, stable integration, and efficient synergistic expression, thus providing further assurance for high total fatty acid production.

[0024] A second aspect of this invention provides a method for constructing a recombinant bacterial strain. The method includes: knocking out the encoding genes for mitochondrial isocitrate dehydrogenase IDH1 and isocitrate dehydrogenase IDH2 in a starting strain, and overexpressing the encoding genes for citrate transporter Yhm2p, cis-aconitase E.HA, and cytoplasmic isocitrate dehydrogenase IDP. This construction method, through the synergistic operation of precisely knocking out the encoding genes for IDH1 and IDH2 and directionally overexpressing the encoding genes for Yhm2p, E.HA, and IDP, efficiently reconstructs the pathway of "citrate retention-cytoplasmic transport-directed NADPH production," demonstrating strong targeting and clear logic. It can stably obtain recombinant strains with sufficient cytoplasmic NADPH supply, and the operation is highly reproducible, providing a reliable guarantee for the large-scale and efficient production of total fatty acids.

[0025] The preferred technical features of the method provided in the second aspect of the present invention are referenced in the first aspect and will not be repeated here.

[0026] A third aspect of the present invention provides the application of the recombinant strains as described above and / or the recombinant strains constructed by the methods described above in increasing total fatty acid production.

[0027] This application can fully unleash the core characteristic of efficient NADPH supply in the cytoplasm of recombinant strains, precisely overcome the key bottleneck of insufficient reducing power in fatty acid synthesis, and achieve a significant increase in total fatty acid yield.

[0028] A fourth aspect of the present invention provides a method for producing total fatty acids, the method comprising: fermenting and culturing the recombinant strain as described above and / or the recombinant strain constructed by the method described above.

[0029] The method for producing total fatty acids provided by this invention utilizes a recombinant strain with high cytoplasmic NADPH for fermentation. In a culture medium containing 8-12 g / L yeast extract, 16-24 g / L peptone, and 75-85 g / L glucose, fermentation is carried out at 28-35°C and 220-280 rpm for 80-140 h. This method can efficiently overcome the limitations of insufficient reducing power in traditional fermentation, achieving efficient synthesis and stable increase in total fatty acid yield. Furthermore, the method is simple to operate and suitable for industrial production needs.

[0030] The present invention will be described in detail below through examples. In the following examples, Yersinia lipolytica po1f was purchased from Yeastern Biotech Co., Ltd. (Taiwan, China); pUrloxp plasmid and purloxp-ku70 plasmid were preserved in the laboratory of Professor Huang He of Nanjing Normal University; and other reagents and raw materials were all commercially available products.

[0031] Seed culture medium CSM: glucose 20 g / L, YNB 1.7 g / L, (NH4)2SO4 5 g / L, CSM-leu 0.74 g / L.

[0032] Fermentation medium CSM: glucose 40 g / L, YNB 1.7 g / L, (NH4)2SO4 5 g / L, CSM-leu 0.74 g / L.

[0033] Seed culture medium YPD: 10 g / L yeast extract, 20 g / L peptone, 20 g / L anhydrous glucose.

[0034] Fermentation medium YPD: 10 g / L yeast extract, 20 g / L peptone, 80 g / L anhydrous glucose.

[0035] OD 600 Detection: Absorbance value measured by ultraviolet spectrophotometer at 600 nm is expressed as (OD). 600 Biomass.

[0036] Total fatty acid detection: Samples required pretreatment before analysis. First, 1 mL of fermentation broth was centrifuged at 12000 rpm for 10 min, the supernatant was discarded, and the precipitate was freeze-dried for 48 h. 1 mL of 1M potassium hydroxide-methanol solution was added to the freeze-dried precipitate, and the mixture was shaken at 20℃ and 1000 rpm for 6 h. 50 μL of concentrated sulfuric acid was added to terminate the reaction, and 1 mL of n-hexane was added, followed by shaking at 20℃ and 1000 rpm for 0.5 h to extract lipids. The mixture was centrifuged at 12000 rpm for 1 min, and the upper n-hexane phase was filtered through a microporous membrane to remove impurities. The extracted phase was then placed in a gas chromatography vial for gas chromatography analysis. A GC-2010 (Shimadzu, Japan) gas chromatography system was used, equipped with a DB-23 capillary column (60 m * 0.22 mm) and a flame ionization detector (FID). Nitrogen was used as the carrier gas. The injection volume was 1 μL, and the injection temperature was 250℃. The column temperature was increased from 100℃ to 200℃ at a rate of 25℃ / min, then increased to 230℃ at a rate of 4℃ / min and held for 9 min. The FID detector temperature was 280℃. Different fatty acid compositions were identified by comparison with relevant external standards (Sigma, USA). The total fatty acid content was calculated from the peak area on the chromatogram using non-endogenous fatty acids (C17:0) as an internal standard.

[0037] Example 1: Construction of recombinant strain Po1fΔku70-ΔIDH1-ΔIDH2 Construction of knockout plasmids pUrloxp-IDH1 and pUrloxp-IDH2: Using the *Yarrowia lipolyticis* genome as a template, the target gene IDH1-Dw fragment with homologous arms was amplified using primers IDH1-Dw-F / IDH1-Dw-R (nucleotide sequences shown in SEQ ID NO. 5 and SEQ ID NO. 6). The plasmid pUrloxp (plasmid map shown in...) was then deactivated using SalI. Figure 1 The DNA fragment was linearized by restriction enzyme digestion, and the large DNA fragment was recovered by agarose gel electrophoresis. Then, the target gene fragment was inserted into the linearized vector fragment obtained by ClonExpress® rapid cloning technology to obtain the recombinant plasmid pUrloxp-IDH1-Dw. After correct sequencing by a sequencing company, the plasmid pUrloxp-IDH1-Dw was linearized by restriction enzyme digestion using AvrII, and the large DNA fragment was recovered by agarose gel electrophoresis. Simultaneously, using the Yersinia lipolyticis genome as a template, the target gene IDH1-up fragment with homologous arms was amplified using primers IDH1-up-F / IDH1-up-R (nucleotide sequences shown in SEQ ID NO. 7 and SEQ ID NO. 8). Then, the target gene fragment was inserted into the linearized vector fragment obtained by ClonExpress® rapid cloning technology to obtain the recombinant plasmid pUrloxp-IDH1 (plasmid map shown in [link to plasmid map]). Figure 2 ).

[0038] Using the *Yarrowia lipolyticis* genome as a template, the target gene IDH2-Dw fragment with homologous arms was amplified using primers IDH2-Dw-F / IDH2-Dw-R (nucleotide sequences shown in SEQ ID NO. 9 and SEQ ID NO. 10). The plasmid pUrloxp (plasmid map shown in...) was then amplified using SalI. Figure 1 The DNA fragment was linearized by restriction enzyme digestion, and the large DNA fragment was recovered by agarose gel electrophoresis. Then, the target gene fragment was inserted into the linearized vector fragment obtained by ClonExpress® rapid cloning technology to obtain the recombinant plasmid pUrloxp-IDH2-Dw. After correct sequencing by a sequencing company, the plasmid pUrloxp-IDH2-Dw was linearized by restriction enzyme digestion using AvrII, and the large DNA fragment was recovered by agarose gel electrophoresis. Simultaneously, using the Yersinia lipolyticis genome as a template, the target gene IDH2-up fragment with homologous arms was amplified using primers IDH2-up-F / IDH2-up-R (nucleotide sequences shown in SEQ ID NO. 11 and SEQ ID NO. 12). Then, the target gene fragment was inserted into the linearized vector fragment obtained by ClonExpress® rapid cloning technology to obtain the recombinant plasmid pUrloxp-IDH2 (plasmid map shown in [link to plasmid map]). Figure 3 ).

[0039] by Yarrowia lipolytica Po1fΔku70 was used as the starting strain. The strain was cultured in 2 mL of YPD medium to the exponential growth phase (16-24 h). 1 mL of po1fk cells were collected from the fermentation broth, centrifuged, and the supernatant was discarded. Then, 90 μL of 50% PEG4000 solution, 5 μL of lithium acetate (25 M), 5 μL of single-stranded DNA (salmon sperm), and 5 μL of the key target gene knockout frame (obtained by digesting the above recombinant plasmid Purloxp-IDH1 with AvrII) were added. After mixing, the mixture was incubated at 37 °C for 1 h and then plated on a uracil-deficient plate (CSM-URA). The recombinant engineered strain Po1fΔku70-ΔIDH1 with IDH1 knockout was obtained by screening.

[0040] Using Po1fΔku70-△IDH1 as the starting strain, the strain was cultured in 2 mL of YPD medium to the exponential growth phase (16-24 h). The cells in 1 mL of fermentation broth were collected, centrifuged, and the supernatant was discarded. Then, 90 μL of 50% PEG4000 solution, 5 μL of lithium acetate (25 M), 5 μL of single-stranded DNA (salmon sperm), and 5 μL of key target gene knockout frame (obtained by AvrII digestion of the above recombinant plasmid pUrloxp-IDH2) were added. After mixing, the mixture was incubated at 37 °C for 1 h and then plated on uracil-deficient plates (CSM-URA). The recombinant strain Po1fΔku70-△IDH1-△IDH2 with double knockout of IDH1 and IDH2 was obtained by screening.

[0041] Table 1

[0042] Example 2 Construction of recombinant vector pYLXP-Yhm2p-E.HA-IDP The construction process of single-gene recombinant plasmids is illustrated by taking plasmid pYLXP-Yhm2p as an example: plasmid pYLXP' is linearized by enzyme digestion using SnaBI and KpnI. After agarose gel electrophoresis, the large DNA fragment is recovered. Simultaneously, using the Yersinia lipolyticis genome as a template, the target gene Yhm2p fragment with homologous arms is amplified using primers Yhm2p-F / Yhm2p-R (nucleotide sequences shown in SEQ ID NO. 13 and SEQ ID NO. 14). Subsequently, the target gene fragment is inserted into the linearized vector fragment obtained by enzyme digestion using ClonExpress® rapid cloning technology to obtain the recombinant plasmid pYLXP-Yhm2p. Using the same method described above, the target gene E.HA fragment with homologous arms was amplified using primers E.HA-F / E.HA-R (nucleotide sequences shown in SEQ ID NO. 15 and SEQ ID NO. 16, template being the E. coli genome), and inserted into the linearized vector obtained by enzyme digestion to obtain the recombinant plasmid pYLXP-E.HA; the target gene IDP fragment with homologous arms was amplified using primers IDP-F / IDP-R (nucleotide sequences shown in SEQ ID NO. 17 and SEQ ID NO. 18), and inserted into the linearized vector obtained by enzyme digestion to obtain the recombinant plasmid pYLXP-IDP.

[0043] Construction of multi-gene tandem recombinant plasmids: Plasmid pYLXP-Yhm2p was linearized using Nhe I restriction endonuclease. The fragment was recovered after agarose gel electrophoresis. Using plasmid pYLXP-E.HA as a template, the E.HA expression cassette containing homologous arms was amplified using primers E.HA expression cassette-F / E.HA expression cassette-R (nucleotide sequences shown in SEQ ID NO. 19 and SEQ ID NO. 20). Subsequently, the target gene fragment was inserted into the linearized vector fragment using ClonExpress® rapid cloning technology to obtain the recombinant plasmid pYLXP-Yhm2p-E.HA. Plasmid pYLXP-Yhm2p-E.HA was linearized using Not I and Nhe I restriction endonucleases. The large DNA fragment was recovered after agarose gel electrophoresis. Plasmid pYLXP-IDP was linearized using Not I and Avr... Linearization with restriction endonucleases was performed, and small DNA fragments were recovered by agarose gel electrophoresis. The larger of the two recovered DNA fragments was used as a vector and ligated using T4 DNA ligase to obtain the recombinant vector pYLXP-Yhm2p-E.HA-IDP (structure shown in [link to structure]). Figure 4 ).

[0044] Table 2

[0045] Example 3 Construction of recombinant strain A (1) The recombinant vector pYLXP-Yhm2p-E.HA-IDP obtained in Example 2 was linearized using Not I and AvrII restriction endonucleases. The large DNA fragment was recovered after agarose gel electrophoresis. The plasmid pUrloxp-ku70 was linearized using Not I and Nhe I restriction endonucleases. The large DNA fragment was recovered after agarose gel electrophoresis. The larger fragment of the two recovered DNA fragments was used as a vector and ligated using T4 DNA ligase to obtain the recombinant integrated plasmid pUrloxp-ku70-Yhm2p-E.HA-IDP.

[0046] (2) Using the recombinant strain po1FK-△IDH1-△IDH2 obtained in Example 1 as the starting strain, the strain was cultured in 2 mL of YPD medium to the exponential growth phase (16-24 h). The cells in 1 mL of fermentation broth were collected, centrifuged and the supernatant was discarded. Then, 90 μL of 50% volume PEG4000 solution, 5 μL of lithium acetate (25 M), 5 μL of single-stranded DNA (salmon sperm) and 5 μL of key target gene expression cassette (obtained by digesting the above recombinant plasmid pUrloxp-ku70-Yhm2p-E.HA-IDP with AvrII were added. After mixing, the mixture was incubated at 37 °C for 1 h and then plated on a uracil-deficient plate (CSM-URA) to integrate into the ku70 site of Yersinia lipolyticis. Recombinant strain A with integrated cytoplasmic NADPH synthesis pathway genes was obtained by screening. The metabolic pathway diagram is shown in the figure. Figure 5 .

[0047] Example 4: Fermentation production of total fatty acids from recombinant strains (1) Pick the recombinant strain A obtained in Example 3, inoculate it into seed culture medium, and culture it for 24 h at a temperature of 30℃ and a rotation speed of 220 rpm to obtain the seed liquid of recombinant strain A; (2) Transfer 0.5 mL of seed culture from a 250 mL shake flask to 30 mL of YPD fermentation medium. Maintain the temperature at 30 °C throughout the fermentation process and incubate for 120 h.

[0048] (3) The fermentation broth was obtained according to the above method. After fermentation, the total fatty acid content in the fermentation broth of the integrated recombinant strain was detected by the total fatty acid determination method. Figure 6 As shown, the total fatty acid yield of the optimized strain increased by 53.6% compared to the unoptimized strain.

[0049] Comparative Example 1 The total fatty acid production was carried out by fermentation according to the method of Example 4, except that the starting strain po1FK was used as a negative control. The total fatty acid yield is shown in Table 3.

[0050] Comparative Example 2 The fermentation production of total fatty acids was carried out according to the method in Example 4, except that the starting strain was overexpressed with the endogenous glucose-6-phosphate dehydrogenase encoding gene ylzwf1 (NCBI ID: YALI1_E26811g) and the 6-phosphate gluconate dehydrogenase encoding gene ylgnd1 (NCBI ID: YALI1_B20462g). The former can catalyze the production of 6-phosphate gluconate lactone and produce cytoplasmic NADPH, which is the main natural source of yeast cytoplasmic NADPH. The latter catalyzes the dehydrogenation of 6-phosphate gluconate to ribulose-5-phosphate, further enhancing the NADPH production efficiency of the pentose phosphate pathway. The total fatty acid yields are shown in Table 3.

[0051] Comparative Example 3 The total fatty acid production was carried out by fermentation according to the method of Example 4, except that the isocitrate dehydrogenases IDH1 and IDH2 located in the mitochondria were knocked out based on the starting strain po1FK. The total fatty acid yield is shown in Table 3.

[0052] Comparative Example 4 The total fatty acid production was carried out by fermentation according to the method of Example 4, except that only the citrate transporter Yhm2p was overexpressed on the basis of po1FK-△IDH2-△IDH2. The total fatty acid yield is shown in Table 3.

[0053] Comparative Example 5 The total fatty acid production was carried out by fermentation according to the method of Example 4, except that the citrate transporter Yhm2p and cis-aconitase E.HA were overexpressed on the basis of po1FK-△IDH2-△IDH2. The total fatty acid yield is shown in Table 3.

[0054] Table 3

[0055] As can be seen from the results in Table 1, the recombinant strain prepared by the method provided in this invention has a significantly better effect on the production of total fatty acids.

[0056] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A recombinant bacterial strain, characterized in that, The recombinant strain was obtained by genetic modification of the starting strain. Compared with the starting strain, the recombinant strain simultaneously overexpresses citrate transporter Yhm2p, cis-aconitase E.HA and isocitrate dehydrogenase IDP located in the cytoplasm, and does not express isocitrate dehydrogenase IDH1 and isocitrate dehydrogenase IDH2 located in the mitochondria.

2. The recombinant strain according to claim 1, characterized in that, The recombinant strain originated from Yersinia lipophila. And / or, the recombinant strain does not express the ku70 gene; Preferably, the nucleotide sequence of the ku70 gene is shown in SEQ ID NO.

1.

3. The recombinant strain according to claim 2, characterized in that, The nucleotide sequence of the gene encoding the citrate transporter Yhm2p is shown in SEQ ID NO. 2; And / or, the nucleotide sequence of the gene encoding the aconitase E.HA is shown in SEQ ID NO. 3; And / or, the nucleotide sequence of the gene encoding the isocitrate dehydrogenase IDP located in the cytoplasm is shown in SEQ ID NO.

4.

4. The recombinant strain according to claim 3, characterized in that, The genes encoding the overexpressed citrate transporter Yhm2p, cis-aconitase E.HA, and cytoplasmic isocitrate dehydrogenase IDP are provided in the form of a recombinant vector.

5. The recombinant strain according to claim 4, characterized in that, The recombinant vector contains the encoding genes for citrate transporter Yhm2p, cis-aconitase E.HA, and cytoplasmic isocitrate dehydrogenase IDP, as well as a promoter and expression cassette for overexpressing the encoding genes for citrate transporter Yhm2p, cis-aconitase E.HA, and cytoplasmic isocitrate dehydrogenase IDP. Preferably, the promoter is a TEF promoter; Preferably, the carrier skeleton is pYLXP'; More preferably, the recombinant vector is pYLXP-Yhm2p-E.HA-IDP.

6. A method for constructing a recombinant bacterial strain, characterized in that, The method includes: knocking out the coding genes for mitochondrial isocitrate dehydrogenase IDH1 and isocitrate dehydrogenase IDH2 in the starting strain, and overexpressing the coding genes for citrate transporter Yhm2p, cis-aconitase E.HA, and cytoplasmic isocitrate dehydrogenase IDP.

7. The method according to claim 6, characterized in that, The recombinant strain originated from Yersinia lipophila. And / or, the nucleotide sequence of the gene encoding the citrate transporter Yhm2p is shown in SEQ ID NO. 2; And / or, the nucleotide sequence of the gene encoding the aconitase E.HA is shown in SEQ ID NO. 3; And / or, the nucleotide sequence of the gene encoding the cytoplasmic isocitrate dehydrogenase IDP is shown in SEQ ID NO. 4; And / or, the genes encoding the overexpressed citrate transporter Yhm2p, cis-aconitase E.HA, and cytoplasmic isocitrate dehydrogenase IDP are provided in the form of a recombinant vector; Preferably, the recombinant vector contains the encoding genes for overexpressed citrate transporter Yhm2p, cis-aconitase E.HA, and cytoplasmic isocitrate dehydrogenase IDP, as well as a promoter and expression cassette for overexpressing the encoding genes for citrate transporter Yhm2p, cis-aconitase E.HA, and cytoplasmic isocitrate dehydrogenase IDP.

8. The method according to claim 7, characterized in that, The recombinant strain does not express the ku70 gene; And / or, the promoter is a TEF promoter; And / or, the carrier skeleton is pYLXP'; Preferably, the recombinant vector is pYLXP-Yhm2p-E.HA-IDP. Preferably, the nucleotide sequence of the ku70 gene is shown in SEQ ID NO.

1.

9. The use of the recombinant strain according to any one of claims 1-5 and / or the recombinant strain constructed by the method according to any one of claims 6 to 8 in increasing total fatty acid production.

10. A method for producing total fatty acids, characterized in that, The method includes: fermenting and culturing the recombinant strain according to any one of claims 1-6 and / or the recombinant strain constructed by the method according to claim 7 or 8.

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