Engineered Saccharomyces cerevisiae for producing 10-hydroxystearic acid, its construction method and application

Abstract

The present invention provides an engineered Saccharomyces cerevisiae strain for producing 10-hydroxystearic acid, thereby increasing the expression of the FAD cofactor in the Saccharomyces cerevisiae strain and / or expressing a FAD-dependent hydratase in the Saccharomyces cerevisiae strain. The present invention also provides methods for constructing and using the engineered Saccharomyces cerevisiae strain. The engineered Saccharomyces cerevisiae strain can be used for fermentation to produce 10-hydroxystearic acid, with a maximum yield of 41.93 mg / L.

Description

Technical Field

[0001] This invention belongs to the fields of metabolic engineering, genetic engineering and fermentation engineering technology. Specifically, it relates to the engineered strain of Saccharomyces cerevisiae for producing 10-hydroxystearic acid, its construction method and application. Background Art

[0002] The increased demand for fuels and chemicals has led to higher oilseed crop yields and raised concerns about the sustainability and environmental impact of oilseed production, which has damaged biodiversity and caused other ecological problems. With the continuous improvement of metabolic engineering and synthetic biology technologies, microbial cell factories, as an environmentally friendly process for producing renewable bio-based products, are receiving increasing attention. This production method not only effectively avoids the harsh conditions such as high temperature and high pressure required in chemical production processes, but also solves the problem of uncontrollable stereoenantiomerism in product production.

[0003] As a safe model eukaryote, Saccharomyces cerevisiae not only has advantages such as short growth cycle, easy cultivation, and high tolerance to harsh industrial reaction conditions, but its production is not limited by season or raw material supply. It has played a huge role in the synthesis of a variety of natural products, including fatty acid derivatives, isoprene-like compounds, alkaloids, and polyketides, and has been widely used in food, medicine, and industrial products.

[0004] Hydroxy fatty acids are important fatty acid derivatives, widely used in surfactants, lubricants, cosmetics, antibacterial agents, and pharmaceutical intermediates. Although the properties of a large number of hydroxy fatty acids have been identified, only a small number are currently used in industrial production. 10-hydroxystearic acid (10-HSA), as an industrially applicable chemical, can be used not only as a lubricant component but also as a precursor in the production of γ-dodecanolide, a flavoring agent with a buttery taste. However, the temperature fluctuations during chemical synthesis can lead to a mixture of isomers, significantly reducing the purity of 10-HSA in the product and limiting its applications. Current reports on microbial production of 10-HSA mainly utilize prokaryotes such as *Escherichia coli*, *Stenotrophomonas maltophilia*, and *Corynebacterium glutamicum* to produce 10-HSA through the conversion of oleic acid under the action of hydratase. However, high concentrations of oleic acid can inhibit bacterial growth, reducing production efficiency. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides an engineered Saccharomyces cerevisiae strain for producing 10-hydroxystearic acid (10-HSA), its construction method, and its applications. This invention uses glucose as a substrate and utilizes Saccharomyces cerevisiae to produce 10-HSA, providing a green, sustainable, and inexpensive method for producing 10-HSA.

[0006] The first objective of this invention is to provide an engineered strain of *Saccharomyces cerevisiae* that produces 10-hydroxystearic acid, thereby increasing the expression level of the FAD cofactor in *Saccharomyces cerevisiae*; and / or, to express FAD-dependent hydratases in *Saccharomyces cerevisiae*.

[0007] Preferably, the increase in the expression level of FAD cofactor in Saccharomyces cerevisiae is achieved by targeting the FAD1 gene in Saccharomyces cerevisiae.

[0008] The expression of FAD-dependent hydratase in Saccharomyces cerevisiae involves expressing the EmFAH10 protein in Saccharomyces cerevisiae.

[0009] The nucleotide sequence of the FAD1 gene is SEQ ID No. 8 in the sequence listing;

[0010] The nucleotide sequence of the EmFAH10 protein is SEQ ID No. 3 in the sequence listing.

[0011] Preferably, the Saccharomyces cerevisiae is an integrated strain of Saccharomyces cerevisiae; the integrated strain of Saccharomyces cerevisiae is obtained by integrating a plasmid containing a gene encoding a transporter protein into the XI2 site of chromosome GL1 of Saccharomyces cerevisiae; the transporter protein is PkFAT or PcFAT; preferably, the transporter protein is PkFAT.

[0012] The second objective of this invention is to provide a method for constructing an engineered strain of Saccharomyces cerevisiae that produces 10-hydroxystearic acid, by constructing a gene encoding a fatty acid hydratase onto a plasmid, and then introducing the recombinant plasmid into Saccharomyces cerevisiae.

[0013] The fatty acid hydratase is SmFAH10, EmFAH10 or PaFAH10, preferably EmFAH10.

[0014] The nucleotide sequence of the SmFAH10 protein is SEQ ID No. 2 in the sequence listing;

[0015] The nucleotide sequence of the EmFAH10 protein is SEQ ID No. 3 in the sequence listing;

[0016] The nucleotide sequence of the PaFAH10 protein is SEQ ID No. 5 in the sequence listing.

[0017] Preferably, the plasmid is pOH, and the pOH is constructed as follows: using plasmid PRS426 as a vector, the vector fragment is obtained by double digestion with KpnI and HindIII and gel recovery; using plasmid Y33-PGKCYC as a template, PCR amplification is performed using 426-PGK-F and 426-CYC-R as primers, and the gene fragment is obtained after gel recovery; finally, the gene fragment and the vector fragment are homologously ligated to obtain plasmid pOH.

[0018] The 426-PGK-F is: aggaattcgatatcaagcttTATTTTAGATTCCTGACTTCAACTCAAG

[0019] The 426-CYC-R is: tatagggcgaattgggtaccGCAAATTAAAGCCTTCGAGCG.

[0020] Preferably, the fatty acid hydratase gene and the FAD1 gene are constructed onto the same plasmid, and then the recombinant plasmid is introduced into Saccharomyces cerevisiae GL1.

[0021] Preferably, the plasmid is pOH or PUGG1; more preferably, the plasmid is PUGG1.

[0022] The plasmid is pOH, and the construction method of pOH is as follows: using plasmid PRS426 as a vector, the vector fragment is obtained by double digestion with KpnI and HindIII and gel recovery; using plasmid Y33-PGKCYC as a template, PCR amplification is performed using 426-PGK-F and 426-CYC-R as primers, and the gene fragment is obtained after gel recovery; finally, the gene fragment and the vector fragment are homologously ligated to obtain plasmid pOH.

[0023] The 426-PGK-F is: aggaattcgatatcaagcttTATTTTAGATTCCTGACTTCAACTCAAG

[0024] The 426-CYC-R is: tatagggcgaattgggtaccGCAAATTAAAGCCTTCGAGCG.

[0025] Preferably, the fatty acid hydratase gene and the FAD1 gene are constructed onto the same plasmid, and then the recombinant plasmid is introduced into the Saccharomyces cerevisiae integrative strain.

[0026] The integrated Saccharomyces cerevisiae strain is obtained by integrating a plasmid containing a gene encoding a transporter protein into the XI2 site of the Saccharomyces cerevisiae GL1 chromosome; the transporter protein is PkFAT or PcFAT; preferably, the transporter protein is PkFAT.

[0027] The nucleotide sequence of the transporter protein PkFAT is SEQ ID No. 12 in the sequence listing;

[0028] The nucleotide sequence of the transporter protein PcFAT is SEQ ID No. 13 in the sequence listing.

[0029] A third objective of this invention is to provide the above-described engineered Saccharomyces cerevisiae, or the engineered Saccharomyces cerevisiae prepared by the above-described method, for the fermentation production of 10-hydroxystearic acid.

[0030] The fourth objective of this invention is to provide a method for fermenting and producing 10-hydroxystearic acid, wherein the engineered Saccharomyces cerevisiae of claims 1-3, or the engineered Saccharomyces cerevisiae prepared by the method described in claims 4-8, is inoculated into SC-URA auxotrophic liquid culture medium and fermented at 26-30°C and 180-250 rpm.

[0031] The engineered Saccharomyces cerevisiae of the present invention can be used to produce 10-hydroxystearic acid, with a maximum yield of 41.93 mg / L. Attached Figure Description

[0032] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0033] Figure 1 This is a diagram of the metabolic pathway for the synthesis of 10-HSA in Saccharomyces cerevisiae.

[0034] Figures 2-12 The diagrams shown are, in order, schematic representations of the construction of plasmids pOH and pOH1-pOH10.

[0035] Figure 13 In the figure, A: hydratase conversion of oleic acid; B: 10-HSA production in GL7, GL8 and GL8+ oleic acid; C: free fatty acid content in CEN.PK 113-11C and GL1 bacteria; D: 10-HSA production in GL1 when expressing hydratases from 6 different sources.

[0036] Figure 14 To increase the content of FAD cofactor and the copy number of plasmid in Saccharomyces cerevisiae GL1, the yield of 10-HSA was improved.

[0037] Figure 15 The yield of 10-HSA in yeast after expressing different transport proteins. Detailed Implementation

[0038] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent companies. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0039] Overview of the technical solution of this invention:

[0040] (1) A method for synthesizing 10-HSA using glucose as a substrate and Saccharomyces cerevisiae was established for the first time. The synthetic pathway is as follows: Figure 1 As shown.

[0041] Figure 1 This is a diagram of the metabolic pathway for the synthesis of 10-HSA in Saccharomyces cerevisiae.

[0042] (2) Fatty acid hydrases from different sources were screened in Saccharomyces cerevisiae strain GL1 (fatty acid hydrase information is shown in Table 1). Then, Beijing Qingke Biotechnology Co., Ltd. performed codon optimization to optimize the exogenous nucleotide sequence into a nucleotide sequence preferred by Saccharomyces cerevisiae. The optimized nucleotide sequences are listed in SEQ ID No. 1-6 in the sequence listing. EmFAH10 hydrase, which can be expressed in yeast and has relatively high activity, was obtained.

[0043] Table 1 Information on fatty acid hydratases from different sources

[0044]

[0045]

[0046] (3) Since EmFAH10 hydratase is a FAD-dependent enzyme, we increased the supply of FAD cofactor in yeast by overexpressing the FAD1 gene. The yield of 10-HSA increased from 5.98 mg / L to 9.04 mg / L. After expression in a multi-copy vector, the yield reached 29.05 mg / L.

[0047] (4) Hydroxy fatty acids are toxic to cells, thus affecting cell growth. Therefore, by expressing a transporter protein that is specific to hydroxy fatty acids (the transporter protein information is shown in Table 2, and then Beijing Qingke Biotechnology Co., Ltd. performed codon optimization to optimize the exogenous nucleotide sequence into a nucleotide sequence preferred by Saccharomyces cerevisiae, and the optimized nucleotide sequences are listed in the sequence listing as SEQ ID No. 9-13), the product was transported to the extracellular space, reducing the influence of intracellular 10-HSA. The final yield of 10-HSA reached 41.93 mg / L, which is about 85 times higher than that of wild-type yeast (0.49 mg / L), and is also the highest yield reported in yeast to date.

[0048] Table 2 Information on transport proteins from different sources

[0049]

[0050] The following is the specific experimental procedure of this invention:

[0051] Example 1

[0052] 1. Experimental Materials

[0053] 1.1 Strains, plasmids, and primers

[0054] The plasmids used in this experiment are shown in Table 3.

[0055] Table 3. Plasmids used in the experiment

[0056]

[0057]

[0058] The strains used in this experiment are shown in Table 4.

[0059] Table 4. Strains used in the experiment

[0060]

[0061]

[0062] The primers used to construct the plasmid are shown in Table 5.

[0063] Table 5 Primers used for plasmid construction.

[0064]

[0065] The bolded and underlined parts in Table 5 are homologous arms.

[0066] 1.2 Culture medium and culture conditions

[0067] YPD medium (1% yeast extract, 2% peptone, 2% anhydrous glucose) was used for activation and culture of Saccharomyces cerevisiae strains, while LBA medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride, ampicillin 100ug / mL) was used for culture of Escherichia coli. All cultures were autoclaved at 121℃ for 20 min and stored at room temperature.

[0068] Yeast transformation, strain screening, streak plating, and fermentation experiments were conducted using SC-URA auxotrophic medium with glucose as the carbon source. The culture temperature for Saccharomyces cerevisiae was 30℃, and the culture temperature for Escherichia coli was 37℃.

[0069] 2. Plasmid and bacterial strain construction methods and detection methods used in the experiment

[0070] 2.1 Plasmid Construction

[0071] Gene fragments were amplified using Primer STAR high-fidelity DNA polymerase (Takara). The amplification system and procedure are shown in Tables 6 and 7, and the vector digestion system is shown in Table 8.

[0072] Table 6 Primer STAR Ultra-Fidelity DNA Polymerase Reaction System

[0073]

[0074] Table 7 Primer STAR Ultra-Fidelity DNA Polymerase Reaction Procedure

[0075]

[0076] Table 8. Vector Enzyme Digestion System

[0077]

[0078] (1) Construction of plasmid pOH: Using plasmid PRS426 as a vector, the fragment was obtained by double digestion with KpnI and HindIII and gel extraction. Using plasmid Y33-PGKCYC as a template, PCR amplification was performed using primers 426-PGK-F and 426-CYC-R, and the gene fragment was obtained after gel extraction. Finally, the gene fragment and the vector fragment were homologously ligated to obtain plasmid pOH, as shown in the schematic diagram of the plasmid map. Figure 2 As shown.

[0079] (2) Construction of plasmids pOH1, pOH2, pOH3, pOH4, pOH5, and pOH6: Using the codon-optimized synthetic genes LfFAH10, SmFAH10, EmFAH10, SnFAH10, PaFAH10, and BbFAH10 as templates (the corresponding gene sequences are listed in SEQ ID No. 1-6 in the sequence listing), PCR amplification was performed using primers 426-Lf-F and 426-Lf-R, 426-Sm-F and 426-Sm-R, 426-Em-F and 426-Em-R, 426-Sn-F and 426-Sn-R, 426-Pa-F and 426-Pa-R, and 426-Bb-F and 426-Bb-R, respectively. The six gene fragments were obtained after gel recovery. The pOH plasmid was used as a vector, digested with SaII, and the fragments were obtained after gel recovery. Finally, the gene fragment and the vector fragment were homologously ligated to obtain plasmids pOH1, pOH2, pOH3, pOH4, pOH5, and pOH6. A schematic diagram of the plasmid maps is shown below. Figure 3-8 As shown.

[0080] (3) Construction of plasmids pOH7 and pOH8: Using the synthetic genes FMN1 and FAD1 as templates (the corresponding gene sequences are shown in SEQ ID No. 7 and 8 in the sequence listing, respectively), PCR amplification was performed using primers 426-TEF1-F and 426-ADH1-R, and the two gene fragments were obtained after gel recovery. Using plasmid pOH3 as a vector, the fragments were digested with BamHI and EcoRI, and the fragments were recovered from the gel to obtain the vector fragment. Finally, the gene fragments and the vector fragment were homologously ligated to obtain plasmids pOH7 and pOH8, as shown in the schematic diagram of the plasmid map. Figure 9 and 10 As shown.

[0081] (4) Construction of plasmids pOH9 and pOH10: Using plasmids pOH3 and pOH8 as templates, PCR amplification was performed using PUGG-Em-F and PUGG-Em-R, and PUGG-EF-F and PUGG-EF-R as primers. The two gene fragments were obtained after gel extraction. Using plasmid PUGG1 as a vector, the fragments were digested with BamHI and EcoRI, and then extracted using a gel to obtain the vector fragments. Finally, the gene fragments and vector fragments were homologously ligated to obtain plasmids pOH9 and pOH10. A schematic diagram of the plasmid map is shown below. Figure 11 and 12 As shown.

[0082] Figures 2-12 The diagrams shown are, in order, schematic representations of the construction of plasmids pOH and pOH1-pOH10.

[0083] 2.2 Construction of integrated strains

[0084] Construction of five strains GL2-GL6: Plasmids containing the transporter genes PaFAT, GbFAT, SdFAT, PkFAT, and PcFAT (corresponding gene sequences are shown in SEQ ID Nos. 9-13 in the sequence listing) were used as templates for PCR amplification using XI2-F and XI2-R primers. The five gene fragments were obtained after gel recovery and used as DNA donors. Then, the plasmid pCas9_XI2 (containing gRNA to guide Cas9 protein cleavage) containing the Cas9 protein gene and the DNA donors were added to the system shown in Table 9. The mixture was heat-shocked at 42℃ for 30 min, followed by centrifugation at 9000 rpm for 30 s. The supernatant was discarded, and the bacterial cells were resuspended and plated on SC-URA plates. After 3 days of growth, PCR verification was performed. Verified single clones were transferred to YPD plates and shaken for 48 h before discarding the plasmids. Finally, the cells were streaked onto YPD plates, single clones were picked, shaken, and stored at -80℃.

[0085] 2.2.1 Chemical transformation of Saccharomyces cerevisiae

[0086] The plasmid was transformed into *Saccharomyces cerevisiae* using a lithium acetate / polyethylene glycol (LiOAc / PEG) chemical transformation method. The yeast chemical transformation reagents and system are shown in Table 9.

[0087] Table 9. Transformation system of yeast chemical transformation method

[0088]

[0089] 2.2.2 Fermentation culture of brewer's yeast

[0090] After streaking enrichment culture of genotype-verified single colonies from the plates, they were inoculated into 4 mL of SC-URA auxotrophic liquid medium and incubated overnight at 30°C and 220 rpm in a shaker. The next day, the culture was carried out according to the initial OD... 600 The culture medium was transferred to a 100 mL shake flask at a concentration of 0.2 μL and fermented for 72 h. Three replicates were set up for each sample. After 72 h, the cells were collected to determine the fatty acid content and cell dry weight.

[0091] 2.3 Fatty acid sample pretreatment

[0092] To detect the content of hydroxy fatty acids in the fermentation broth, the fatty acids first need to be silanized. The specific steps for fatty acid silanization are as follows:

[0093] (1) Take 200 μL of fermentation broth, add glass beads and shake to break it up for 30 min;

[0094] (2) Add 10 μL of tetradecenoic acid (1 mg / mL) as an internal standard, and simultaneously add 400 μL of ethyl acetate. Shake, extract, and centrifuge at 14000 rpm for 5 min. Then transfer the upper organic phase to a new EP tube. Add another 400 μL of ethyl acetate and repeat the above operation. Concentrate and evaporate the obtained upper organic phase to dryness for further processing.

[0095] (3) Add 40 μL of n-hexane, 40 μL of pyridine and 20 μL of silanizing reagent to the EP tube that has been evaporated to dryness in (2), mix well and place in an oven at 60 °C for 1 h. Then add 100 μL of n-hexane, mix well and take an appropriate amount into a liquid chromatography vial for GC-MS detection.

[0096] 2.4 GC-MS Detection Conditions

[0097] Samples were analyzed using gas chromatography-mass spectrometry (GC-MS) (QP2020, Shimadzu, Japan). A DB-5MS column (30m × 0.25mm × 0.25um, Agilent) was used, with a carrier gas flow rate of 3.0 mL / min, an injection volume of 1 μL, and a split ratio of 10:1. Data were acquired in full detection mode (50-650 m / z), analyzed using GCMS 4.4 software, and quantified using a standard curve. The temperature program is shown in Table 10.

[0098] Table 10 GC-MS Temperature Program

[0099]

[0100] 2.5 Plasmids pOH and pOH1-pOH10 were constructed and introduced into the Saccharomyces cerevisiae strain.

[0101] Construction of GL7 and GL8 strains: Plasmids pOH and pOH1 were added to CEN.PK113-11C competent cells according to the system shown in Table 9. The cells were heat-shocked in a water bath at 42℃ for 30 min, then centrifuged at 9000 r / min for 30 s. The supernatant was discarded, the cells were resuspended and plated on SC-URA plates. After 3 days of growth, single clones were selected for fermentation.

[0102] Construction of GL9-GL19 strains: Plasmids pOH and pOH1-pOH10 were added to GL1 competent cells according to the system shown in Table 9. The cells were heat-shocked in a water bath at 42℃ for 30 min, then centrifuged at 9000 r / min for 30 s. The supernatant was discarded, the cells were resuspended and plated on SC-URA plates. After 3 days of growth, single clones were selected for fermentation.

[0103] Construction of GL20-GL24 strains: Plasmid pOH10 was added to GL2-GL6 competent cells according to the system shown in Table 9. The cells were heat-shocked in a water bath at 42℃ for 30 min, then centrifuged at 9000 r / min for 30 s. The supernatant was discarded, the cells were resuspended and plated on SC-URA plates. After 3 days of growth, single clones were selected for fermentation.

[0104] 3. Experimental Procedure and Results

[0105] Unless otherwise specified, all fermentation conditions below are as follows: the seed culture is prepared according to the initial OD... 600 The sample was transferred to a 100 mL shake flask containing SC-ura medium at 0.2°C and incubated at 220 rpm for 72 h at 30°C. Three replicates were set up for each sample.

[0106] 3.1 Screening of hydratases

[0107] According to research reports, the applicant has screened out six hydration enzymes with good specificity and conversion efficiency, as shown in Table 1, for oleic acid hydration reaction. Figure 13 As shown in Figure A.

[0108] First, to verify whether the screened hydratase could be expressed in yeast, we expressed the pOH and pOH1 plasmids in CEN.PK 113-11C and performed shake-flask fermentations. Since the free fatty acid content in wild-type yeast is very low, and the 10-HSA yield in GL8 was very low, 2 mM oleic acid was added to the GL8 culture medium as a substrate for fermentation. The results are as follows... Figure 13 As shown in B, the yield of 10-HSA in the bacteria with added oleic acid and expressing the pOH1 plasmid was 4.8 mg / L, indicating that LfFAH10 can be expressed in yeast, but the conversion rate of oleic acid is not high, possibly due to the low utilization rate of exogenously added oleic acid.

[0109] Then, the applicant selected GL1 bacteria, which are capable of producing a high content of free fatty acids, the free fatty acid content of which is as follows: Figure 13 As shown in C (Note: GL1 is a publicly reported bacterium, and subsequent experiments were conducted based on this bacterium). The results of fermenting the six hydratases screened in Table 1 in GL1 are shown below. Figure 13 As shown in Figure D, the results indicate that 10-HSA was detected in four bacteria: GL10, GL11, GL12, and GL14. GL12 (whose hydratase is EmFAH10) exhibited the highest yield, approximately 6 mg / L. Compared to the oleic acid content in GL1, the current conversion rate remains relatively low. Therefore, to further increase 10-HSA, the applicant is attempting to increase the content of the FAD cofactor required in the hydratase reaction, thereby improving the oleic acid conversion rate.

[0110] Figure 13 Figure A: The reaction of hydratase to oleic acid; Figure B: 10-HSA yield in GL7, GL8 and GL8+ oleic acid; Figure C: Free fatty acid content in CEN.PK 113-11C and GL1 bacteria, where C10:0 is decanoic acid, C12:0 is dodecanoic acid, C14:0 is tetradecanoic acid, C16:1 is palmitoleic acid, C16:0 is palmitic acid, C18:1 is oleic acid and C18:0 is stearic acid; Figure D: 10-HSA yield in GL1 when expressing hydratases from 6 different sources.

[0111] 3.2 Increase the supply of FAD cofactors

[0112] Since EmFAH10 is a FAD-dependent hydratase, the presence of FAD cofactors can improve protein stability and catalytic activity. Therefore, the applicant further increased the content of FAD cofactors in yeast by expressing two genes involved in FAD synthesis, FMN1 and FAD1, and investigated the effect of FAD cofactors on the hydration reaction (generating GL16 and GL17 strains as shown in Table 4). Then, the applicant increased the copy number of the plasmid to further increase the yield of 10-HSA (generating GL18 and GL19 strains as shown in Table 4), and the experimental results are as follows. Figure 14 As shown.

[0113] Figure 14 To increase the content of FAD cofactor and the copy number of plasmid in Saccharomyces cerevisiae GL1, the yield of 10-HSA was improved.

[0114] The results showed that, compared with GL12, expression of the FMN1 and FAD1 genes increased the production of 10-HSA in both GL16 and GL17 bacteria. However, FMN1 expression had a smaller effect on 10-HSA, while FAD1 expression resulted in a 10-HSA production of 9.04 mg / L. Furthermore, using a high-copy vector significantly increased the 10-HSA production in both GL18 and GL19 bacteria, with GL19 showing a production of 29.05 mg / L, 5.67 times the original level. These results indicate that FAD1 is the main gene affecting FAD cofactor production, and increasing the FAD cofactor content in yeast can enhance the activity of EmFAH10 hydratase and increase 10-HSA production.

[0115] 3.3 Expression of hydroxy fatty acid transport proteins reduces cytotoxicity.

[0116] Fatty acids produced in yeast can be transported extracellularly via transport proteins near the cell membrane. For example, proteins such as Tpo1, Fat1, Faa1, and Faa4 are all involved in fatty acid uptake and secretion. Since most hydrophobic matrices are toxic to cells, the accumulation of 10-HSA inside the cell negatively impacts cell growth and transformation, which is a major reason for the low yield of this substance during biotransformation.

[0117] Currently, there are few reports on proteins capable of transporting hydroxy fatty acids. To reduce the cytotoxicity of 10-HSA, we screened five transport proteins that might secrete 10-HSA extracellularly (as shown in Table 2) and integrated these five genes into the XI2 locus of chromosome 2, resulting in five bacterial strains, GL2-GL6. Then, plasmid pOH10 was expressed in each of the five strains. GC-MS analysis of the fermented samples showed the following results: Figure 15 As shown.

[0118] Figure 15 The yield of 10-HSA was calculated by expressing different transport proteins in different yeasts.

[0119] from Figure 15 It can be seen that, compared with GL19, the expression of transport proteins in GL20, GL21, and GL22 bacteria did not increase the yield of the target product, especially in GL21 bacteria where the decrease was more significant. This may be because transport proteins are bidirectional, and the three transport proteins PaFAT, GbFAT, and SdFAT have poor effects on the secretion of hydroxy fatty acids. The yields of 10-HSA in GL23 and GL24 bacteria were 41.93 mg / L and 39.11 mg / L, respectively, showing a significant increase. Compared with the 0.49 mg / L yield of 10-HSA in wild-type bacteria, the yield of 10-HSA in GL23 increased by approximately 85 times, which is also the highest yield reported in yeast to date.

[0120] The sources of plasmids and strains mentioned in the article:

[0121] [1] Plasmid PRS426: SHI S, SI T, LIU Z, et al. Metabolic engineering of asynergistic pathway for n-butanol production in Saccharomyces cerevisiae[J]. Sci Rep, 2016, 6: 25675.

[0122] [2] Plasmid Y33-PGKCYC: DING W, MENG Q, DONG G, et al. Metabolic engineering ofthreonine catabolism enables Saccharomyces cerevisiae to produce propionateunder aerobic conditions[J]. Biotechnol J, 2022, 17(3):e2100579.

[0123] [3] Plasmid PUGG1 and plasmid pCas9_XI2: ZHANG Y, SU M, QIN N, et al. Expressing acytosolic pyruvate dehydrogenase complex to increase free fatty acid production in Saccharomyces cerevisiae[J]. Microb Cell Fact, 2020, 19(1):226.

[0124] [4] Saccharomyces cerevisiae CEN.PK 113-11C and GL1: ZHOU YJ, BUIJS NA, ZHU Z, et al. Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast cell factories[J]. Nat Commun, 2016, 7:11709. In this article, Saccharomyces cerevisiae GL1 is named Saccharomyces cerevisiae YJZ45.

[0125] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A brewer's yeast strain for producing 10-hydroxystearic acid, characterized in that: To increase the expression level of FAD cofactor in Saccharomyces cerevisiae; and to express FAD-dependent hydratase in Saccharomyces cerevisiae; The method of increasing the expression level of FAD cofactor in Saccharomyces cerevisiae involves expressing the FAD1 gene in Saccharomyces cerevisiae. The expression of FAD-dependent hydratase in Saccharomyces cerevisiae involves expressing the EmFAH10 protein in Saccharomyces cerevisiae. The nucleotide sequence of the FAD1 gene is SEQ ID No. 8 in the sequence listing; The nucleotide sequence of the EmFAH10 protein is SEQ ID No. 3 in the sequence listing; The *Saccharomyces cerevisiae* is an integrated strain of *Saccharomyces cerevisiae*; the integrated strain of *Saccharomyces cerevisiae* is obtained by integrating a plasmid containing a gene encoding a transport protein into the XI2 site of chromosome GL1 of *Saccharomyces cerevisiae*; the transport protein is PkFAT or PcFAT. The nucleotide sequence of the transporter protein PkFAT is SEQ ID No. 12 in the sequence listing; The nucleotide sequence of the transporter protein PcFAT is SEQ ID No. 13 in the sequence listing.

2. The engineered Saccharomyces cerevisiae strain for producing 10-hydroxystearic acid according to claim 1, characterized in that: The transporter protein is PkFAT.

3. A method for constructing engineered Saccharomyces cerevisiae strains for producing 10-hydroxystearic acid, characterized in that: The fatty acid hydratase gene and the FAD1 gene were constructed onto the same plasmid, and then the recombinant plasmid was introduced into the Saccharomyces cerevisiae integrative strain. The fatty acid hydratase is EmFAH10; the nucleotide sequence of the EmFAH10 protein is SEQ ID No. 3 in the sequence listing; The integrated strain of *Saccharomyces cerevisiae* is obtained by integrating a plasmid containing a gene encoding a transporter protein into the XI2 site of chromosome GL1 of *Saccharomyces cerevisiae*; the transporter protein is PkFAT or PcFAT. The nucleotide sequence of the transporter protein PkFAT is SEQ ID No. 12 in the sequence listing; The nucleotide sequence of the transporter protein PcFAT is SEQ ID No. 13 in the sequence listing.

4. The construction method according to claim 3, characterized in that: The plasmid is pOH or PUGG1; The pOH was constructed as follows: using plasmid PRS426 as a vector, the vector fragment was obtained by double digestion with KpnI and HindIII and gel recovery; using plasmid Y33-PGKCYC as a template, PCR amplification was performed using 426-PGK-F and 426-CYC-R as primers, and the gene fragment was obtained after gel recovery; finally, the gene fragment and the vector fragment were homologously ligated to obtain plasmid pOH. The 426-PGK-F is: aggaattcgatatcaagcttTATTTTAGATTCCTGACTTCAACTCAAG The 426-CYC-R is: tatagggcgaattgggtaccGCAAATTAAAGCCTTCGAGCG.

5. The construction method according to claim 4, characterized in that: The plasmid is PUGG1.

6. The construction method according to claim 3, characterized in that: The transporter protein is PkFAT.

7. The use of the engineered Saccharomyces cerevisiae according to claim 1 or 2, or the engineered Saccharomyces cerevisiae prepared by the method according to any one of claims 3-6, in the fermentation production of 10-hydroxystearic acid.

8. A method for producing 10-hydroxystearic acid by fermentation, characterized in that: The engineered Saccharomyces cerevisiae according to any one of claims 1-2, or the engineered Saccharomyces cerevisiae prepared by the method according to any one of claims 3-6, is inoculated into SC-URA auxotrophic liquid medium and fermented at 26-30 °C and 180-250 rpm.

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