Search

CN-121991905-A - Mutant of delta 6-fatty acid desaturase Madelta 6D and application thereof

CN121991905ACN 121991905 ACN121991905 ACN 121991905ACN-121991905-A

Abstract

The invention relates to the technical field of biology, and provides a mutant of delta 6 fatty acid desaturase Madelta 6D and application thereof. The mutant has a mutation at least one of positions 34, 277 and 418 compared with the wild-type amino acid sequence. The fatty acid desaturase mutant provided by the invention improves the catalytic performance of the existing delta 6 fatty acid desaturase Madelta 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 gamma-linolenic acid and downstream fatty acid and fatty acid derivatives thereof, and is used for the fields of medicine, food, health care, feed, cosmetics and the like.

Inventors

  • WANG SHIAN
  • SU JUANJUAN
  • ZHENG JIAWEN
  • BAI YU
  • MIAO RUNZE
  • WANG YEFEI
  • LI FULI

Assignees

  • 中国科学院青岛生物能源与过程研究所

Dates

Publication Date
20260508
Application Date
20241104

Claims (10)

  1. 1. A mutant of delta 6-fatty acid desaturase Ma delta 6D, characterized in that the amino acid sequence thereof is at least one of the following mutations in the wild-type amino acid sequence as shown in SEQ ID No. 1: (1) Mutation from methionine to valine at position 34; (2) Mutation from cysteine to tyrosine at position 277; (3) Mutation from threonine to glutamic acid at position 418.
  2. 2. A coding nucleotide sequence of the mutant of claim 1.
  3. 3. The coding nucleotide sequence of claim 2, wherein the nucleotide sequence is at least one of the following mutations in the wild-type nucleotide sequence as set forth in SEQ ID No. 2: (1) Mutation at positions 100-102 to GTG; (2) Mutations 829-831 to TAG; (3) Mutation at positions 1252-1254 is GAG.
  4. 4. An expression cassette, a recombinant vector and an engineering bacterium for expressing the mutant according to claim 1, comprising the coding nucleotide sequence according to claim 2 or 3.
  5. 5. The expression cassette, the recombinant vector and the engineering bacterium according to claim 4, wherein the skeleton vector of the recombinant vector is pUC57, and the engineering bacterium is selected from Escherichia coli (ESCHERICHIA COLI) or saccharomycetes; preferably, the engineering bacteria are yarrowia lipolytica, and compared with a strain expressing MaΔ6D wild type, GLA accounts for more than 20%, or more than 30%, or more than 40% of total oil content.
  6. 6. Use of a mutant according to claim 1, a coding nucleotide sequence according to claim 2 or 3, an expression cassette according to any one of claims 4 to 6, a recombinant vector and an engineering bacterium for the synthesis of GLA and the fermentative production of Δ6-fatty acid desaturase.
  7. 7. Use according to claim 6, characterized in that the strain of the fermentation process is selected from the group consisting of E.coli (ESCHERICHIA COLI) or yeasts selected from the group consisting of Yarrowia (Yarrowia) yeast, rhodosporidium toruloides (Rhodosporidium toruloides), oleaginous stonecrop (Lipomyces starkeyi), candida oleaginous (Cutaneotrichosporon oleaginosus), candida pinnata (Trichosporon cutaneum) or rhodotorula rubra (Phaffia rhodozyma), preferably Yarrowia lipolytica (Yarrowia lipolytica); The strain of the fermentation process overexpresses one or more of Δ12-fatty acid desaturase, Δ6-fatty acid desaturase, acetyl-CoA carboxylase, diacylglycerol acyltransferase, and stearoyl-CoA desaturase.
  8. 8. A method of producing GLA-rich grease comprising the steps of: (1) Constructing engineering yeast for expressing delta-12 fatty acid desaturase and delta 6 fatty acid desaturase mutant; (2) Fermenting and culturing engineering yeast to obtain thalli; (3) Extracting and separating the thallus to obtain GLA-rich grease.
  9. 9. The method of claim 8, wherein the yeast is oleaginous yeast selected from yarrowia lipolytica (Yarrowia lipolytica), rhodosporidium toruloides (Rhodosporidium toruloides), oleaginous yeast (Lipomyces starkeyi), oleaginous yeast (Cutaneotrichosporon oleaginosus), candida utilis (Trichosporon cutaneum) or rhodotorula rubra (Phaffia rhodozyma); In the step (1), the construction of the engineering yeast comprises the following steps: a) Constructing homologous recombinant plasmids expressing delta-12 fatty acid desaturase and delta 6 fatty acid desaturase mutants; b) Linearizing the homologous recombinant plasmid, then transforming into oleaginous yeast, and screening to obtain positive transformant, i.e. engineering yeast.
  10. 10. GLA-rich oil and fat produced according to the method of claim 8 or 9, wherein GLA comprises at least 7%, or at least 8% of the total oil and fat content.

Description

Mutant of delta 6-fatty acid desaturase Madelta 6D and application thereof Technical Field The invention belongs to the technical field of biology, and particularly relates to application of a mutant of delta 6-fatty acid desaturase Madelta 6D in improving the capacity of oleaginous yeast for synthesizing gamma-linolenic acid. Background This background information is disclosed to enhance an understanding of the general background of the invention and is not necessarily to be considered an admission or any form of suggestion that this information constitutes prior art already known to a person of ordinary skill in the art. Gamma-linolenic acid (GLA) is all-cis 6,9, 12-octadecatrienoic acid, the molecular formula is C 18H30O2, and omega-6 series long chain polyunsaturated essential fatty acid. GLA is a structural component for forming biological membranes of various tissues of human bodies, is a precursor for synthesizing bioactive components such as prostaglandin, leukotriene, thromboxane and the like, has multiple effects （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）. of dilating blood vessels, effectively preventing thrombotic cardiovascular and cerebrovascular diseases, regulating immunity, resisting inflammation and the like, and has remarkable effects on adaptation diseases such as atherosclerosis, hyperlipidemia and the like in clinic, and the evening primrose capsule taking GLA as a main component. In addition, GLA can be used as therapeutic agent for allergic eczema, rheumarthritis, diabetes complication, menstrual breast pain and climacteric syndrome （Bamford JT, et al. Cochrane Database Syst Rev, 2013, 2013(4):CD004416; Macfarlane GJ, et al. Rheumatology (Oxford), 2011, 50(9):1672-83）. GLA is the highest in seed content of evening primrose in nature, but GLA extraction from plants is limited by a plurality of factors such as cultivated land, seasons and the like, and can not meet market demands. With the development of metabolic engineering and synthetic biology, the advantages of microbial fermentation methods are becoming more pronounced. In 1948 Bernhard and Albercht identified GLA for the first time from the cells of Mortierella beljakovae Phycomyces blakesleeanus (Bernhard K, et al HELV CHIM ACTA, 1948, 31 (4): 977-88), uncovered a preinstalled for GLA production by microbial fermentation, and a number of studies were carried out successively. Microorganisms found to accumulate GLA have been focused mainly on lower fungi of the phylum zygomycota, such as strains of the genus Mortierella, the genus Hantaenia (Fazili ABA, et al Microb Cell Fact, 2022, 21 (1): 29). The mould genetic operation is not easy, the probability of positive mutation generated by mutation breeding is usually small, and the influence of mycelium morphology in the fermentation process is large, so that the production efficiency of a target product is greatly limited. Yarrowia lipolytica Yarrowia lipolytica is an important oleaginous microorganism, has the advantages of high grease content, fast growth, easy genetic transformation, high safety and the like, and is a GLA synthetic chassis strain with great potential. Delta 6-fatty acid desaturase is the primary rate limiting step in omega-6/omega-3 polyunsaturated fatty acid synthesis, mainly because the enzyme has substrate non-specificity, and natural fatty acid desaturases known as alpha-linolic acid (ALA) and alpha-linolenic acid (linoleic acid, LA) can be used as 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）., and tend to catalyze the synthesis of ALA, and GLA has high content biological resources and deficiency of delta 6-fatty acid desaturase resources, which indicates that the activity of the natural delta 6-fatty acid desaturase is not high. Therefore, the excavation of more delta 6-fatty acid desaturase resources or the improvement of the catalytic efficiency of enzymes on substrate LA is an important link in the study of high-yield GLA. In 2015, shi et al found that Mortierella alpina (Mortierella alpina) and Microcystis parvum (Micromonas pusilla) delta-6 fatty acid desaturases (delta 6D) have significant substrate preference in Saccharomyces cerevisiae, mortierella alpina-derived Ma delta 6D prefers LA, and Microcystis parvum-derived delta 6D prefers ALA (Shi H, et al J liquid Res, 2015, 56 (12): 2309-21). Sun et al constructed the GLA synthesis pathway by expressing MaΔ6D in yarrowia lipolytica strain Po1f and applied a temperature change culture strategy to raise the GLA content of the strain to 6.1% of total oil after shaking fermentation 7D (total oil production 1.16 g/L, GLA titer 71.6 mg/L) (Sun M, et al Biochem Eng J, 2017, 117:172-80), chuang et al obtained a strain with GLA content of 20% of total oil by coexpression of MaΔ1D (Mortierella alpina-derived Δ12-