title: Application of a delta-6 desaturase with α-linolenic acid preference on eicosapentaenoic acid production in Mortierella alpina.
abstract: BACKGROUND
  Delta-6 desaturase (FADS6) is a key bifunctional enzyme desaturating linoleic acid (LA) or α-linolenic acid (ALA) in the biosynthesis of polyunsaturated fatty acids (PUFAs). In previous work, we analyzed the substrate specificity of two FADS6 enzymes from Mortierella alpina ATCC 32222 (MaFADS6) and Micromonas pusilla CCMP1545 (MpFADS6), which showed preference for LA and ALA, respectively. We also clarified the PUFA profiles in M. alpina, where these lipids were synthesized mainly via the ω6 pathway and rarely via the ω3 pathway and as a result contained low ALA and eicosapentaenoic acid (EPA) levels.
  
  
  RESULTS
  To enhance EPA production in M. alpina by favoring the ω3 pathway, a plasmid harboring the MpFADS6 gene was constructed and overexpressed in a uracil-auxotrophic strain of M. alpina using the Agrobacterium tumefaciens-mediated transformation (ATMT) method. Our results revealed that the EPA production reached 80.0 ± 15.0 and 90.4 ± 9.7 mg/L in MpFADS6 transformants grown at 28 and at 12 °C, respectively. To raise the level of ALA, free form fatty acid was used as exogenous substrate, which increased the EPA production up to 114.5 ± 12.4 mg/L. To reduce the cost of EPA production in M. alpina, peony seed oil (PSO) and peony seed meal (PSM) were used as source of ALA, and EPA production was improved to 149.3 ± 7.8 and 515.29 ± 32.66 mg/L by supplementing with 0.1 % PSO and 50 g/L PSM, respectively. The EPA yield was further increased to 588.5 ± 29.6 mg/L in a 5-L bioreactor, which resulted in a 26.2-fold increase compared to EPA production in wild-type M. alpina. In this work, we have significantly enhanced EPA production through overexpression of a FADS6 desaturase with preference for ALA, combined with supplementation of its substrate.
  
  
  CONCLUSIONS
  An ALA-preferring FADS6 from M. pusilla CCMP1545 was applied to enhance EPA production in M. alpina. By exogenous addition of peony seed oil or peony seed meal, EPA production was further increased in flasks and fermenters. This research also highlights the value of peony seed meal which can be converted to a high value-added product containing EPA, and as a way to increase the EPA/AA ratio in M. alpina.
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, People's Republic of China.
authors.forename: Haisu
authors.initials: H
authors.lastname: Shi
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, People's Republic of China. haiqinchen@jiangnan.edu.cn.|Synergistic Innovation Center for Food Safety and Nutrition, Wuxi, 214122, People's Republic of China. haiqinchen@jiangnan.edu.cn.
authors.forename: Haiqin
authors.initials: H
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, People's Republic of China.|Synergistic Innovation Center for Food Safety and Nutrition, Wuxi, 214122, People's Republic of China.
authors.forename: Zhennan
authors.initials: Z
authors.lastname: Gu
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, People's Republic of China.|Synergistic Innovation Center for Food Safety and Nutrition, Wuxi, 214122, People's Republic of China.
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, People's Republic of China.|Synergistic Innovation Center for Food Safety and Nutrition, Wuxi, 214122, People's Republic of China.
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, People's Republic of China. yqchen@jiangnan.edu.cn.|Synergistic Innovation Center for Food Safety and Nutrition, Wuxi, 214122, People's Republic of China. yqchen@jiangnan.edu.cn.|Departments of Cancer Biology and Biochemistry, Wake Forest School of Medicine, Winston-Salem, NC, 27157, USA. yqchen@jiangnan.edu.cn.
authors.forename: Yong Q
authors.initials: YQ
authors.lastname: Chen
mesh_terms: D003470:Culture Media / Q000737:chemistry / Q000378:metabolism; D015118:Eicosapentaenoic Acid / Q000096:biosynthesis*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D050602:Linoleoyl-CoA Desaturase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020094:Mortierella / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D017962:alpha-Linolenic Acid / Q000378:metabolism*
chemical_list: D003470:Culture Media; D005656:Fungal Proteins; D017962:alpha-Linolenic Acid; D015118:Eicosapentaenoic Acid; D050602:Linoleoyl-CoA Desaturase
keywords: Delta 6 desaturase; Eicosapentaenoic acid; Mortierella alpina; Peony seed meal; Peony seed oil