Mutant of delta 6-fatty acid desaturase Ma delta 6D and application thereof

Abstract

The invention relates to the technical field of biology, and provides a mutant of delta 6 fatty acid desaturase Ma delta 6D and application of the mutant. Compared with a wild type amino acid sequence, at least one of the 34th site, the 277th site and the 418th site of the mutant is mutated. The fatty acid desaturase mutant provided by the invention improves the catalytic performance of the existing delta 6 fatty acid desaturase Ma delta 6D. The invention also provides a method for preparing gamma-linolenic acid in oleaginous yeast by using the Ma delta 6D mutant. The dominant mutant provided by the invention can be used for constructing engineering bacteria for high-yield production of gamma-linolenic acid and downstream fatty acids and fatty acid derivatives thereof, and is applied to the fields of medicines, foods, health care, feeds, cosmetics and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of mutants of the Δ6-fatty acid desaturase MaΔ6D in improving the ability of oil-producing yeasts to synthesize γ-linolenic acid. Background Technology

[0002] The information disclosed in this background section is intended to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] γ-linolenic acid (GLA) is a cis-6,9,12-octadecanoic acid with the molecular formula C1. 18 H 30 O2 is an ω-6 series long-chain polyunsaturated essential fatty acid. GLA is a structural component of the biological membranes of various tissues in the human body, and is also a precursor for the synthesis of bioactive components such as prostaglandins, leukotrienes, and thromboxanes. It has multiple functions such as vasodilation, effective prevention of thrombotic cardiovascular and cerebrovascular diseases, immune regulation, and anti-inflammation (Savini M, et al. Nat Cell Biol, 2022,24(6):906-16; Dierge E, et al. Cell Metab, 2021, 33(8):1701-15.e1705; Ramsden CE, et al. BMJ, 2021, 374:n1448). In clinical practice, evening primrose oil capsules with GLA as the main component have significant effects on indications such as atherosclerosis and hyperlipidemia. In addition, GLA can also be used as a treatment for conditions such as allergic eczema, rheumatoid arthritis, diabetic complications, menstrual breast pain and menopausal syndrome (Bamford JT, et al. Cochrane Database Syst Rev, 2013, 2013(4):CD004416; Macfarlane GJ, et al. Rheumatology (Oxford), 2011, 50(9):1672-83).

[0004] In nature, evening primrose seeds have the highest content of GLA, but the extraction of GLA from plants is limited by many factors such as arable land and season, and cannot meet market demand. With the development of metabolic engineering and synthetic biology, the advantages of microbial fermentation have become increasingly significant. In 1948, Bernhard and Albercht first extracted GLA from *Gastrodia elata*. Phycomyces blakesleeanusThe identification of GLA in bacterial cells (Bernhard K, et al. Helv Chim Acta, 1948, 31(4):977-88) opened the prelude to the production of GLA by microbial fermentation, and a large number of studies followed. Currently, the microorganisms found to accumulate GLA are mainly lower fungi of the Zygomycetes phylum, such as strains of *Morchella* and *Fazili* (Fazili ABA, et al. Microb Cell Fact, 2022, 21(1):29). Genetic manipulation of molds is difficult, the probability of positive mutations in mutagenesis breeding is usually low, and the fermentation process is greatly affected by mycelial morphology, which greatly limits the production efficiency of the target product. *Yarrowia lipolytica* Yarrowia lipolytica It is an important oil-producing microorganism with advantages such as high oil content, fast growth, easy genetic modification and high safety, and is a highly promising GLA synthesis chassis strain.

[0005] Δ6-fatty acid desaturase is the primary rate-limiting step in the synthesis of ω-6 / ω-3 polyunsaturated fatty acids, mainly due to its substrate nonspecificity; both linoleic acid (LA) and α-linolenic acid (ALA) can serve as its catalytic substrates (Cui J, et al. Appl Microbiol Biotechnol, 2020, 104(23):9947-63; Gao L, et al. Plant Cell Physiol, 2020, 61(7):1335-47). Known natural fatty acid desaturase systems tend to catalyze the synthesis of ALA. The scarcity of high-content biological resources for GLA and Δ6-fatty acid desaturase resources suggests that the activity of natural Δ6-fatty acid desaturases is not high. Therefore, exploring more Δ6-fatty acid desaturase resources or improving the catalytic efficiency of enzymes for the substrate LA is an important aspect of high-yield GLA research. In 2015, Shi et al. discovered that *Morchella alpina* ( Mortierella alpina ) and microcystis ( Micromonas pusillaThe Δ-6 fatty acid desaturase (Δ6D) of Saccharomyces cerevisiae exhibits significant substrate preference in Saccharomyces cerevisiae. MaΔ6D from Morphyra alpineum prefers LA, while Δ6D from Microcystis aeruginosa prefers ALA (Shi H, et al. J Lipid Res, 2015, 56(12):2309-21). Sun et al. constructed a GLA synthesis pathway by expressing MaΔ6D in the *Yersinia lipolytica* strain Po1f, and applied a temperature-changing culture strategy to increase the GLA content of the strain to 6.1% of the total oil (total oil yield 1.16 g / L, GLA titer 71.6 mg / L) after 7 days of shake-flask fermentation (Sun M, et al. Biochem Eng J, 2017, 117:172-80); Chuang et al. obtained a strain with GLA accounting for 20% of the total oil by co-expressing MaΔ12D (Δ12-fatty acid desaturase from *Morchella alpina*) and MaΔ6D in the *Yersinia lipolytica* strain Po1g, with the titer unknown (Chuang L, et al. N Biotechnol, 2010, 27(4):277-82); Wang et al. introduced the Δ6 pathway of *Morchella alpina* and *Isochrysis galbana* into the chassis of *Yersinia lipolytica* Po1f (… Isochrysis galbana The Δ8 pathway synthesizes ω-6 polyunsaturated fatty acids, and the content of GLA in the recombinant strain can reach 22.58% of the total oil, but the titer is only 386.59 mg / L (Wang J, et al. BioresourTechnol, 2023, 383:129231).

[0006] In summary, current research demonstrates the potential of MaΔ6D in GLA synthesis; however, the catalytic performance of this enzyme in high-yielding oil-producing bacteria remains unclear, and current GLA yields are still below industrialization requirements. Utilizing AI-assisted modern enzyme engineering techniques to modify and enhance the catalytic performance of MaΔ6D and develop high-yielding strains of GLA and its downstream derivatives holds significant market potential. Summary of the Invention

[0007] To further increase the yield of GLA in oil-producing yeast, the purpose of this invention is to provide the application of seven MaΔ6D mutant genes in yeast GLA synthesis.

[0008] To achieve the above objectives, the present invention adopts the following technical solution.

[0009] A mutant of the Δ6-fatty acid desaturase MaΔ6D, the amino acid sequence of which is formed by at least one of the following mutations in the wild-type amino acid sequence shown in SEQ ID NO:1: (1) The 34th position is mutated from methionine to valine; (2) The 277th position is changed from cysteine ​​to tyrosine; (3) The 418th position is mutated from threonine to glutamic acid.

[0010] The present invention also provides the nucleotide sequence of the above-mentioned mutant, which can be codon optimized according to different expression strains. Preferably, the nucleotide sequence of the above-mentioned mutant is formed by at least one of the following mutations in the wild-type nucleotide sequence shown in SEQ ID NO:2: (1) The mutation at positions 100-102 is GTG; (2) The mutation at positions 829-831 is TAG; (3) The mutation at positions 1252-1254 is GAG.

[0011] The present invention also provides an expression cassette, recombinant vector, and engineered bacteria capable of expressing the above-mentioned mutants.

[0012] The preferred scaffold carrier for the recombinant vector is pUC57.

[0013] The engineered bacteria are selected from Escherichia coli (Escherichia coli) Escherichia coli ) or yeast.

[0014] The above mutant can enhance the activity of the Δ6-fatty acid desaturase that catalyzes the synthesis of γ-linolenic acid (GLA) from linoleic acid, thereby increasing the GLA content. Linoleic acid can be derived from endogenous or exogenous sources. Therefore, it can be used for the fermentation production of GLA. The above mutant can also be used for the fermentation production of highly active Δ6-fatty acid desaturase. The fermenting strain is preferably yeast, especially oil-producing yeast, including but not limited to *Yersinia* genus (…). Yarrowia ) yeast, Rhodotorula buergerianum ( Rhodosporidium toruloides ), Stirryl oil yeast ( Lipomyces starkeyi ), Oil-producing mycorrhizal yeast ( Cutaneotrichosporon oleaginosus ), dermal filamentous yeast ( Trichosporon cutaneum ), Red Hair Fertilizer ( Phaffia rhodozyma More preferably, it is Yersinia genus ( Yarrowia Yeast, such as Yeastia lipolytica (Yeastia lipolytica) Yarrowia lipolytica The strains used in the above fermentation can also be Escherichia coli.

[0015] To enhance GLA production, the strain can overexpress Δ12-fatty acid desaturase, Δ6-fatty acid desaturase, and acetyl-CoA carboxylase ( ). ACC1 ), diacylglycerol acyltransferase ( DGA1 ) and stearoyl-CoA desaturase ( SCD One or more of them.

[0016] The present invention also provides a method for producing GLA-rich oils, comprising the following steps: (1) Constructing engineered yeast expressing Δ-12 fatty acid desaturase and the above-mentioned Δ6-fatty acid desaturase mutant; (2) The engineered yeast was fermented and cultured to obtain cell cells; (3) GLA-rich oil was extracted and isolated from the bacterial cells.

[0017] Preferably, the yeast is an oil-producing yeast, including but not limited to Yersinia lipolytica (Yersinia lipophila). Yarrowia lipolytica ), Rhodotorula buergerianum ( Rhodosporidium toruloides ), Stirryl oil yeast ( Lipomyces starkeyi ), Oil-producing mycorrhizal yeast ( Cutaneotrichosporon oleaginosus ), dermal filamentous yeast ( Trichosporon cutaneum ), Red Hair Fertilizer ( Phaffia rhodozyma ).

[0018] In step (1), the construction of engineered yeast includes the following steps: a) Construct homologous recombinant plasmids expressing Δ-12 fatty acid desaturase and the above-mentioned Δ6-fatty acid desaturase mutant; b) Linearize the homologous recombinant plasmid and transform it into oil-producing yeast, then screen to obtain positive transformants, i.e., engineered yeast.

[0019] In step b, the transformation of the lipophilic yeast employs a site-directed integration method. In some embodiments, the lipophilic yeast is *Yarrowia lipophila*, and the site-directed integration site includes, but is not limited to, D17.

[0020] The engineered Yersinia lipophila obtained by the above method has a GLA content of more than 20% in total lipids compared with strains expressing MaΔ6D wild type; preferably more than 30%; and more preferably more than 40%.

[0021] The present invention also provides a GLA-rich oil prepared by the above method, wherein the GLA accounts for at least 7% of the total oil content; preferably, at least 8%.

[0022] The present invention has the following advantages: The application of the mutant Δ6-fatty acid desaturase MaΔ6D provided by this invention in enhancing yeast GLA synthesis: The expression of the MaΔ6D mutant in high-oil-producing Yeast Yeast can improve the conversion efficiency of LA to GLA, effectively enhancing the yeast's ability to synthesize GLA. Under unoptimized shake-flask fermentation conditions, the GLA content in recombinant strains YLGLA2-MaΔ6D / M34V, YLGLA2-MaΔ6D / C277Y, YLGLA2-MaΔ6D / T418E, YLGLA2-MaΔ6D / M34V / C277Y, YLGLA2-MaΔ6D / M34V / T418E, YLGLA2-MaΔ6D / C277Y / T418E, and YLGLA2-MaΔ6D / M34V / C277Y / T418E was 8.3%, 7.9%, 8.0%, 7.3%, 7.4%, 7.5%, and 6.9% of the total lipids, respectively, representing increases of 40.7%, 33.9%, 35.6%, 23.7%, 25.4%, 27.1%, and 16.9% compared to strains expressing MaΔ6D. The seven mutants of the Δ6-fatty acid desaturase MaΔ6D provided by this invention can be used to construct engineered bacteria that produce high levels of GLA and its downstream fatty acids and fatty acid derivatives, and have broad application prospects in the fields of medicine, food, health care, feed, and cosmetics. Attached Figure Description

[0023] Figure 1 This is the design pattern of the recombinant plasmid pYL-D17-MaΔ12D-MaΔ6D / M34V; Figure 2 These are the diagnostic PCR results of recombinant strains with single-point mutants of MaΔ6D; where A: design of homologous recombination target sequences and PCR primers; B: diagnostic PCR electrophoresis images of different transformants of recombinant strains YLGLA2-MaΔ6D / WT, YLGLA2-MaΔ6D / M34V, YLGLA2-MaΔ6D / V104Y, YLGLA2-MaΔ6D / L110E, YLGLA2-MaΔ6D / E221H, YLGLA2-MaΔ6D / C277Y, YLGLA2-MaΔ6D / N342F, YLGLA2-MaΔ6D / G376T, YLGLA2-MaΔ6D / G376S and YLGLA2-MaΔ6D / T418E; CK is the control strain YLGLA1-ΔKu70-ΔURA; Figure 3 These are the results of the fermentation endpoint biomass and oil yield of the MaΔ6D single-point mutant recombinant strain; Figure 4 These are the results of fatty acid gas chromatography analysis of the MaΔ6D single-point mutant recombinant strain after 168 h of shake-flask fermentation. Figure 5 These are the results of the analysis of the content of each fatty acid component in the MaΔ6D single-point mutant recombinant strain; Figure 6 These are the diagnostic PCR results of recombinant strains with MaΔ6D multipoint mutants; the images show the diagnostic PCR electrophoresis diagrams of different transformants of recombinant strains YLGLA2-MaΔ6D / M34V / C277Y, YLGLA2-MaΔ6D / M34V / T418E, YLGLA2-MaΔ6D / C277Y / T418E, and YLGLA2-MaΔ6D / M34V / C277Y / T418E; CK is the control strain YLGLA1-ΔKu70-ΔURA. Figure 7 The results are the fermentation endpoint biomass and oil yield of recombinant strains of MaΔ6D single-point dominant mutant and multi-point combined mutant; Figure 8 These are the results of fatty acid gas chromatography analysis of recombinant strains of MaΔ6D single-point dominant mutant and multi-point combined mutant after shake-flask fermentation for 168 h. Figure 9 These are the results of the analysis of the content of each fatty acid component in the MaΔ6D single-point dominant mutant and multi-point combined mutant recombinant strains. Detailed Implementation

[0024] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0025] Example 1: Construction of a recombinant yeast strain expressing Δ6-fatty acid desaturase MaΔ6D and a single-point mutant The culture medium used in the examples can be obtained commercially or through formulations and methods known in the art.

[0026] 1. Design of MaΔ6D mutation sites The Δ6-fatty acid desaturase MaΔ6D (GenBank: AF110510.1) from *Morchella alpineensis* has been shown to catalyze the conversion of linoleic acid to GLA in *Yarrowia lipolytica* (Chuang L, et al. N Biotechnol, 2010, 27(4):277-82.). Using this as a control, and further using a protein AI large language model to predict favorable mutants, the performance of MaΔ6D was modified. Nine mutation sites were selected, corresponding to mutants M34V, V104Y, L110E, E221H, C277Y, N342F, G376T / S, and T418E. The coding gene of MaΔ6D was codon optimized and mutants were designed according to the preferences of *Yarrowia lipolytica*, as shown in Table 1.

[0027] Table 1. MaΔ6D and its mutation sites 2. Preparation of recombinant yeast strains expressing the MaΔ6D single-point mutant (1) Preparation of YLGLA1-ΔKu70-ΔURA lipophilic yeast The Yersinia lipophila strain used in the examples was Y. lipolytica Po1g (MATa, leu2-270, ura3-302::URA3, xpr2-3) was purchased from Yeastern Biotech Co., Ltd. This was achieved by overexpressing endogenous genes from Yeastern lipase. ACC1 , DGA1 and SCD The strain Po1g-G3 (Qiao K, et al. Metab Eng, 2015, 29:56-65) was obtained. A uracil auxotrophic strain, Po1g-G3-ΔURA, was obtained through reverse selection using the 5-FOA method. Subsequently, a MaΔ12D and a MaΔ6D expression cassette were integrated into the XPR2 (YALI1_F39494g) site in its genome, yielding a first-generation strain expressing GLA, named YLGLA1. The sequences of the homologous arms required for XPR2 site integration are shown in SEQ ID NO:3-4. The strain was then knocked out using the CRISPR / Cas9 system. Ku70 Genes were extracted to obtain strain YLGLA1-ΔKu70, and then the uracil auxotrophic strain YLGLA1-ΔKu70-ΔURA was obtained through reverse screening using the 5-FOA method.

[0028] (2) Preparation of mutant recombinant vector and transformation of Yersinia lipophila Will MaΔ12D and MaΔ6D The gene was codon-optimized for Yersinia lipophila, and the sequences are shown in SEQ ID NO:5 and SEQ ID NO:2, respectively; the hp4d promoter sequence, VMA2t terminator sequence, GPD promoter sequence, and XPR2 terminator sequence are shown in SEQ ID NO:6-9, respectively; it was commercially synthesized and inserted into the EcoRV site of pUC57 in the order pGPD-MaΔ12D-tXPR2-php4d-MaΔ6D-tVMA2t to construct the shuttle plasmid pUC57-MaΔ12D-MaΔ6D-AMP.

[0029] Using the shuttle plasmid pUC57-MaΔ12D-MaΔ6D-AMP as a template, the vector backbone and sequences containing the Amp expression cassette and the *E. coli* origin of replication were amplified using primer pairs pYL-F / pYL-R and Amp-PF / Amp-PR, respectively. Using the genome of *Yarrowia lipolytica* Po1g-G3 as a template, the URA3 expression cassette sequence, downstream homologous sequence of D17, and upstream homologous arm sequence of D17 were amplified using primer pairs URA3-F / URA3-R, D17DdF / D17DdR, and D17UdF / D17UdR, respectively. Finally, the five fragments containing homologous sequences were assembled using the Gibson assembly method, and the shuttle plasmid pYL-D17-MaΔ12D-MaΔ6D / WT was obtained after sequencing verification.

[0030] Using shuttle plasmid pYL-D17-MaΔ12D-MaΔ6D / WT as a template, the backbone DNA fragment was amplified using primer pair M12-6-PF / M12-6-PR, and the mutation site Met was amplified using primer pairs D17U-M12-F / M6-M34V-R and M6-M34V-F / D17D-M6-R. 34 The upstream and downstream target fragments. The three fragments with homologous sequences were assembled using the Gibson assembly method. The assembly product was transformed into competent *E. coli* DH5α cells and plated on LB agar plates containing 100 μg / mL ampicillin. The obtained single clones were identified by PCR using primers M12-F-cx / URA3-R-jj, and finally, positive recombinant expression plasmids were obtained through sequencing verification. The recombinant plasmid pYL-D17-MaΔ12D-MaΔ6D / M34V expressing the MaΔ6D mutant M34V is shown in the image. Figure 1 As shown.

[0031] Table 2 Primers used for plasmid construction and PCR diagnosis of strains The same method was used to obtain the backbone DNA fragments, and then the fragments were analyzed using primer pairs D17U-M12-F / M6-V104Y-R and M6-V104Y-F / D17D-M6-R, D17U-M12-F / M6-L110E-R and M6-L110E-F / D17D-M6-R, D17U-M12-F / M6-E221H-R and M6-E221H-F / D17D-M6-R, and D17U-M12-F / M6-C277Y-R and M6-C277Y-F / D17D-M6-R. D-M6-R, D17U-M12-F / M6-N342F-R and M6-N342F-F / D17D-M6-R, D17U-M12-F / M6-G376T-R and M6-G376T-F / D17D-M6-R, D17U-M12-F / M6-G376S-R and M6-G376S-F / D17D-M6-R, D17U-M12-F / M6-T418E-R and M6-T418E-F / D17D-M6-R amplify the mutation site Val, respectively. 104 Leu 110 Glu 221 Cys 277 Asn 342 Gly 376 and Thr 418 The target fragments upstream and downstream of the mutation site were assembled using the Gibson assembly method, and positive recombinant expression plasmids were obtained and screened. They were named pYL-D17-MaΔ12D-MaΔ6D / V104Y, pYL-D17-MaΔ12D-MaΔ6D / L110E, pYL-D17-MaΔ12D-MaΔ6D / E221H, pYL-D17-MaΔ12D-MaΔ6D / C277Y, pYL-D17-MaΔ12D-MaΔ6D / N342F, pYL-D17-MaΔ12D-MaΔ6D / G376T, pYL-D17-MaΔ12D-MaΔ6D / G376S, and pYL-D17-MaΔ12D-MaΔ6D / T418E, respectively.

[0032] All PCR amplifications were performed using high-fidelity Phanta Max Super-Fidelity DNA polymerase. The amplification volume was 50 µL (2 × Phanta Max Master Mix 25 µL, 10 µM primers 2 µL each, template 1 ng / 1 µL, water added to 50 µL). The amplification conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60-72℃ annealing for 15 s, 72℃ extension (30-60 s / kb), 30 cycles; 72℃ extension for 5 min.

[0033] The recombinant expression plasmids pYL-D17-MaΔ12D-MaΔ6D / WT, pYL-D17-MaΔ12D-MaΔ6D / M34V, D17-MaΔ12D-MaΔ6D / V104Y, D17-MaΔ12D-MaΔ6D / L110E, D17-MaΔ12D-MaΔ6D / E221H, D17-MaΔ12D-MaΔ6D / C277Y, and D17-MaΔ12D-MaΔ6D / C277Y were created using NotI restriction endonuclease to express MaΔ6D and its single-point mutations. -MaΔ12D-MaΔ6D / N342F, D17-MaΔ12D-MaΔ6D / G376T, D17-MaΔ12D-MaΔ6D / G376S, and D17-MaΔ12D-MaΔ6D / T418E were linearized by enzyme digestion and confirmed by agarose gel electrophoresis. After gel recovery, the linear template was obtained and then transformed into Yeast Rice YLGLA1-ΔKu70-ΔURA using the LiAc transformation method. The transformation system is shown in Table 3.

[0034] Table 3. Transformation system of Yersinia lipophila The transformation system was vortexed after being bathed in a 30℃ water bath for 1 h, and then heat-shocked at 39℃ for 10 min. The resulting mixture was then plated onto YNB-Δura selection plates and cultured at 28℃ for approximately 2 days to obtain single colonies. The YNB-Δura medium formulation consisted of: 0.17% YNB (amino-free yeast nitrogen source), 0.5% (NH4)2SO4, 2% glucose, 0.067% CSM-URA, 2.5% agar powder, and the remainder being water.

[0035] Diagnostic PCR verification was performed on the different transformants of the recombinant strains obtained above: single colonies of the recombinant strains were picked from the screening plates and cultured on YNB-Δura plates for about 24 h. After that, bacterial cells were picked, and genomic DNA was extracted using a commercial fungal genome rapid extraction kit. Using this DNA as a template, identification PCR was performed using primer pairs D17-UF / D17-DR (Table 2), with YLGLA1-ΔKu70-ΔURA as a control. Identification PCR was performed using Phanta Flash Super-Fidelity DNA polymerase. The amplification volume was 20 µL (10 µL of 2×Phanta Flash Master Mix, 1 µL each of 10 µM primers, 1 ng / µL template, and water to a final volume of 20 µL). Amplified fragments were identified by agarose gel electrophoresis. Amplification conditions were: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, primer melting temperature + 5℃ annealing for 5 s, 72℃ extension (5-10 s / kb), cycle number 35; 72℃ extension for 1 min.

[0036] Agarose gel electrophoresis of PCR products as follows Figure 2 As shown: The size of the bacterial PCR product of the control group YLGLA1-ΔKu70-ΔURA was consistent with the theoretical value for a negative result, at 2591 bp. Figure 2 The positive PCR product (from lane B's CK lane) was 9184 bp in size. Sequencing of the positive PCR product confirmed the positive recombinant strains, which were named YLGLA2-MaΔ6D / WT, YLGLA2-MaΔ6D / M34V, YLGLA2-MaΔ6D / V104Y, YLGLA2-MaΔ6D / L110E, YLGLA2-MaΔ6D / E221H, YLGLA2-MaΔ6D / C277Y, YLGLA2-MaΔ6D / N342F, YLGLA2-MaΔ6D / G376T, YLGLA2-MaΔ6D / G376S, and YLGLA2-MaΔ6D / T418.

[0037] Table 4. Recombinant strains and their transformants (underlined transformants correspond to PCR positive bands) Three transformants from each strain were selected (underlined in Table 4) for subsequent experiments.

[0038] Example 2: Fermentation of MaΔ6D and single-point mutant recombinant Yersinia lipolytica to produce γ-linolenic acid The recombinant strain screened in Example 1 was inoculated into 5 mL of YNB-Δura liquid medium and cultured at 28°C and 220 rpm for approximately 24 h to obtain the fermentation seed culture. The seed culture was inoculated into 30 mL of fermentation medium (15% glucose, 0.6% yeast extract, 1.2% (NH4)2SO4, and the remainder being water) and cultured at 28°C and 220 rpm for 168 h; with strain YLGLA1 as the control, three replicates were set up for each group.

[0039] After fermentation, total cell lipids were extracted using an acid-heat method: 1 mL of fermentation broth was placed in a glass centrifuge tube, centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the cells were collected; 3 mL of 4 M HCl was added, the mixture was shaken and mixed, and the mixture was shaken on a shaker at 28℃ for 1.5-2 h; the mixture was then boiled in a water bath for 10 min, and cooled at -20℃ for 30 min; 6 mL of a 1:1 chloroform:methanol solution was added, the mixture was shaken and mixed, and the mixture was centrifuged at 4000 rpm for 10 min; the lower chloroform layer was transferred to a new centrifuge tube, 3 mL of 0.15% NaCl was added, the mixture was mixed, and the mixture was centrifuged at 4000 rpm for 10 min; the lower chloroform layer was transferred to a new, pre-dried and weighed glass centrifuge tube, and the chloroform was dried under nitrogen in a fume hood to obtain the lipids. The total lipid yield was calculated by weighing. The results are as follows: Figure 3 As shown, compared with the control strain YLGLA1, the biomass of the nine MaΔ6D single-point mutant strains was not significantly reduced, and the OD... 600 The values ​​ranged from 79 to 101; except for YLGLA2-MaΔ6D / V104Y, whose oil production was significantly reduced, the oil production of the other strains was relatively similar, concentrated in the range of 12-16 g / L.

[0040] The extracted oil was methylated: 3.9 mL of methanol:sulfuric acid solution (98:2, volume ratio) was added to a glass tube, mixed well, and reacted at 85 °C for 3 h, shaking every 30 min during the reaction until the oil droplets disappeared; 1.5 mL of saturated NaCl solution and 1.5 mL of n-hexane were added, shaken well, and centrifuged at 4000 rpm for 5 min; 800 µL of the supernatant was transferred to a clean 1.5 mL EP tube, centrifuged at 12000 rpm for 5 min, and 200 µL of the supernatant was gently aspirated, sealed, and stored for further gas chromatography analysis.

[0041] The methylated fatty acids were analyzed by gas chromatography (Agilent 7890B-GC). Chromatographic conditions: HP-INOWAX column (30 m × 0.32 mm × 0.5 μm); injection temperature: 250℃; detector temperature: 250℃; injection volume: 1 μL; initial column temperature: 140℃, held for 1 min, increased to 180℃ at 10℃ / min, held for 2 min, increased to 210℃ at 5℃ / min, held for 4 min, then increased to 250℃ at 5℃ / min, held for 4 min; the relative content of each fatty acid component was obtained by area normalization.

[0042] The results of gas chromatography analysis of fatty acids from single-point mutant recombinant strains fermented in shake flasks for 168 h are as follows: Figure 4 As shown, the content of each fatty acid component is as follows: Figure 5As shown, compared with the starting strain YLGLA1, the GLA2 series strains with increased expression of MaΔ12D and MaΔ6D showed significantly increased GLA synthesis. Furthermore, compared with the control strain YLGLA2-MaΔ6D / WT, the GLA content of strains YLGLA2-MaΔ6D / M34V, YLGLA2-MaΔ6D / C277Y, and YLGLA2-MaΔ6D / T418E was significantly increased, while the GLA content of strains YLGLA2-MaΔ6D / V104Y, YLGLA2-MaΔ6D / L110E, YLGLA2-MaΔ6D / E221H, YLGLA2-MaΔ6D / N342F, YLGLA2-MaΔ6D / G376T, and YLGLA2-MaΔ6D / G376S showed no significant difference. This indicates that the mutants M34V, C277Y, and T418V significantly improved the catalytic performance of the Δ6-fatty acid desaturase MaΔ6D.

[0043] Example 3 Construction of a recombinant yeast strain expressing the MaΔ6D multipoint mutation Based on the lipid component analysis results of the single-point mutants, the designed combined mutants are shown in Table 5. Referring to the method in Example 1, pYL-D17-MaΔ12D-MaΔ6D / M34V obtained in Example 1 was used as a template to prepare pYL-D17-MaΔ12D-MaΔ6D / M34V / C277Y and pYL-D17-MaΔ12D-MaΔ6D / M34V / T418E. The pYL-D17-MaΔ12D-MaΔ6D / T418E obtained in Example 1 was also used as a template. Using / C277Y as a template, pYL-D17-MaΔ12D-MaΔ6D / C277Y / T418E was prepared, and using pYL-D17-MaΔ12D-MaΔ6D / M34V / C277Y as a template, pYL-D17-MaΔ12D-MaΔ6D / M34V / C277Y / T418E was prepared, resulting in a total of 4 shuttle plasmids carrying MaΔ6D multi-site combination mutants.

[0044] Table 5 Mutation sites of MaΔ6D multi-site combination mutants Table 6. Recombinant strains and their transformants (underlined transformants correspond to PCR-positive bands) The recombinant strain was then prepared and verified according to the method in Example 1. Figure 6 Finally, three transformants from each strain were selected (underlined in Table 6) for subsequent experiments.

[0045] Example 4: Fermentation of γ-linolenic acid by MaΔ6D multi-point mutant recombinant Yersinia lipolytica. The recombinant strains screened in Example 3 and the dominant strain with the MaΔ6D single point mutation were tested in the same batch for fermentation. Each strain was inoculated into 5 mL of YNB-Δura liquid medium and cultured at 28 °C and 220 rpm for approximately 24 h to obtain the fermentation seed culture. The seed culture was inoculated into 30 mL of fermentation medium (15% glucose, 0.6% yeast extract, 1.2% (NH4)2SO4, and the remainder water) and cultured at 28 °C and 220 rpm for 168 h; with the YLGLA1 strain as a control, three replicates were set up for each group. After fermentation, the total lipids were extracted from the cells using the acid-heat method as described in Example 2, and the total lipid yield was calculated by weighing. The extracted lipids were methylated, and the relative content of each fatty acid component was determined by gas chromatography.

[0046] The results of the fermentation endpoint strain biomass and oil yield determination are as follows: Figure 7 As shown, compared with the control strain YLGLA1 and the MaΔ6D single-point mutant strain, the biomass and oil production of the MaΔ6D combined mutant strain were similar, and the OD... 600 The values ​​ranged from 89 to 94, and the oil yield ranged from 15 to 16 g / L. The gas chromatographic analysis results of fatty acids from the dominant MaΔ6D single-point mutant strain and the combined mutant strain after 168 h of fermentation are as follows: Figure 8 As shown, the content of each fatty acid component is as follows: Figure 9 As shown in the figure, compared with the control strain YLGLA2-MaΔ6D / WT, the GLA content of both the MaΔ6D single-point mutant and the combined mutant strains was significantly increased. The GLA contents of the MaΔ6D single mutant strains YLGLA2-MaΔ6D / M34V, YLGLA2-MaΔ6D / C277Y, and YLGLA2-MaΔ6D / T418E were 8.3%, 7.9%, and 8.0% of total fatty acids, respectively, representing increases of 40.7%, 33.9%, and 35.6% compared to the control strain. The GLA contents of the MaΔ6D combined mutant strains YLGLA2-MaΔ6D / M34V / C277Y, YLGLA2-MaΔ6D / M34V / T418E, YLGLA2-MaΔ6D / C277Y / T418E, and YLGLA2-MaΔ6D / M34V / C277Y / T418E were 7.3%, 7.4%, 7.5%, and 6.9% of total fatty acids, respectively, representing increases of 23.7%, 25.4%, 27.1%, and 16.9% compared to the control strain. There was no significant difference in GLA content between single-point mutant and combined mutant strains of MaΔ6D. This indicates that mutants M34V, C277Y, and T418V significantly improved the catalytic performance of the Δ6-fatty acid desaturase MaΔ6D, but combined mutations did not produce an additive effect.

[0047] The mutants involved in this invention were obtained through protein AI large language model prediction, which, compared with rational / semi-rational design methods based on protein structure, rapidly achieves full sequence space optimization. This invention enriches the resources of Δ6-fatty acid desaturases, providing enzyme elements for better utilization and modification of the Yersinia lipolytica chassis. The seven mutants of the Δ6-fatty acid desaturase MaΔ6D obtained effectively enhance the ability of high-oil-producing Yersinia lipolytica to convert linoleic acid, and are expected to improve the ability of yeast fermentation to produce GLA and its downstream derivatives, thus contributing to the industrial application of GLA and its downstream derivatives.

Claims

1. A mutant of the Δ6-fatty acid desaturase MaΔ6D, characterized in that, Its amino acid sequence is formed by at least one of the following mutations in the wild-type amino acid sequence shown in SEQ ID NO:1: (1) The 34th position is mutated from methionine to valine; (2) The 277th position is changed from cysteine ​​to tyrosine; (3) The 418th position is mutated from threonine to glutamic acid.

2. A coding nucleotide sequence of the mutant as described in claim 1.

3. The encoding nucleotide sequence according to claim 2, characterized in that, Its nucleotide sequence is formed by at least one of the following mutations in the wild-type nucleotide sequence shown in SEQ ID NO:2: (1) The mutation at positions 100-102 is GTG; (2) The mutation at positions 829-831 is TAG; (3) The mutation at positions 1252-1254 is GAG.

4. An expression cassette comprising the encoding nucleotide sequence as described in claim 1, a recombinant vector, and an engineered bacterium.

5. The expression cassette, recombinant vector, and engineered bacteria according to claim 4, characterized in that, The recombinant vector's backbone vector is pUC57; the engineered bacteria are selected from *Escherichia coli* (…). Escherichia coli or yeast; Preferably, the engineered strain is Yersinia lipophila, and compared with the strain expressing the wild type MaΔ6D, the content of GLA in the total oil is increased by more than 20%; or more than 30%; or more than 40%.

6. The use of a mutant as described in claim 1, the encoding nucleotide sequence as described in claim 2 or 3, the expression cassette, recombinant vector, and engineered bacteria as described in any one of claims 4-6 in the synthesis of GLA and the fermentation production of Δ6-fatty acid desaturase.

7. The application according to claim 6, characterized in that, The strains used in the fermentation method were selected from Escherichia coli (E. coli) Escherichia coli ) or yeast; the yeast is selected from the genus Yersinia ( Yarrowia ) yeast, Rhodotorula buergerianum ( Rhodosporidium toruloides ), Stirryl oil yeast ( Lipomyces starkeyi ), Oil-producing mycorrhizal yeast ( Cutaneotrichosporon oleaginosus ), dermal filamentous yeast ( Trichosporon cutaneum ) or red yeast ( Phaffia rhodozyma ); preferably, Yersinia lipophila ( Yarrowia lipolytica ); The fermentation strains overexpress one or more of the following: Δ12-fatty acid desaturase, Δ6-fatty acid desaturase, acetyl-CoA carboxylase, diacylglycerol acyltransferase, and stearoyl-CoA desaturase.

8. A method for producing GLA-rich oils, characterized in that, Includes the following steps: (1) Constructing engineered yeast expressing Δ-12 fatty acid desaturase and the above-mentioned Δ6-fatty acid desaturase mutant; (2) The engineered yeast was fermented and cultured to obtain cell cells; (3) GLA-rich oil was extracted and isolated from the bacterial cells.

9. The method according to claim 8, characterized in that the yeast is an oil-producing yeast, selected from *Yarrowia lipophila* (…). Yarrowia lipolytica ), Rhodotorula buergerianum ( Rhodosporidium toruloides ), Stirryl oil yeast ( Lipomyces starkeyi ), Oil-producing mycorrhizal yeast ( Cutaneotrichosporon oleaginosus ), dermal filamentous yeast ( Trichosporon cutaneum ) or red yeast ( Phaffia rhodozyma ); In step (1), the construction of engineered yeast includes the following steps: a) Construct homologous recombinant plasmids expressing Δ-12 fatty acid desaturase and the above-mentioned Δ6-fatty acid desaturase mutant; b) Linearize the homologous recombinant plasmid and transform it into oil-producing yeast, then screen to obtain positive transformants, i.e., engineered yeast.

10. A GLA-rich oil prepared by the method of claim 8 or 9, characterized in that, The GLA content is at least 7% or at least 8% of the total oil content.

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