高山被孢霉二酰基甘油酰基转移酶的功能和底物选择性。
Functions and substrate selectivity of diacylglycerol acyltransferases from Mortierella alpina.
机构信息
State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, Jiangsu, 214122, People's Republic of China.
School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, People's Republic of China.
出版信息
Appl Microbiol Biotechnol. 2023 Sep;107(18):5761-5774. doi: 10.1007/s00253-023-12694-y. Epub 2023 Jul 27.
Mortierella alpina produces various polyunsaturated fatty acids in the form of triacylglycerols (TAG). Diacylglycerol acyltransferase (DGAT) catalyzes the binding of acyl-CoA to diacylglycerol to form TAG and is the key enzyme involved in TAG synthesis. A variety of DGATs are present in M. alpina; however, comparative analysis of the functional properties and substrate selectivity of these DGATs is insufficient. In this study, DGAT1 (MaDGAT1A/1B/1C) and DGAT2 (MaDGAT2A/2B) isoforms from M. alpina were analyzed and heterologously expressed in S. cerevisiae H1246. The results showed that MaDGAT1A/1B/2A/2B were able to restore TAG synthesis, and the corresponding TAG content in recombinant yeasts was 2.92 ± 0.42%, 3.62 ± 0.22%, 0.86 ± 0.34%, and 0.18 ± 0.09%, respectively. In S. cerevisiae H1246, MaDGAT1A preferred C16:1 among monounsaturated fatty acids, MaDGAT1B preferred C16:0 among saturated fatty acids (SFAs), and MaDGAT2A/2B preferred C18:0 among SFAs. Under exogenous addition of polyunsaturated fatty acids (PUFAs), MaDGAT1A and 2A preferentially assembled linoleic acid into TAG, and MaDGAT2B had substrate selectivity for eicosapentaenoic and linoleic acids in ω-6 PUFAs. In vitro, MaDGAT1A showed no obvious acyl-CoA selectivity and MaDGAT1B preferred C20:5-CoA. MaDGAT1A/1B preferred C18:1/C18:1-DAG compared with C20:4/C20:4-DAG. This study indicates that MaDGATs have the potential to be used in the production of LA/EPA-rich TAG and provide a reference for improving the production of TAGs in oleaginous fungi. KEY POINTS: • MaDGAT1A preferred C16:1 among MUFAs, MaDGAT1B and MaDGAT2A/2B preferred C16:0 and C18:0 among SFAs, respectively • MaDGAT1A/2A preferentially assembled linoleic acid into TAG, and MaDGAT2B has substrate selectivity for eicosapentaenoic acid and linoleic acid in ω-6 PUFAs • MaDGAT1A showed no obvious acyl-CoA selectivity, and MaDGAT1B preferred C20:5-CoA. MaDGAT1A/1B preferred to select C18:1/C18:1-DAG compared with C20:4/C20:4-DAG.
被膜霉菌能以三酰基甘油（TAG）的形式合成各种多不饱和脂肪酸。二酰基甘油酰基转移酶（DGAT）催化酰基辅酶 A 与二酰基甘油结合形成 TAG，是 TAG 合成中涉及的关键酶。被膜霉菌中存在多种 DGAT；然而，对这些 DGAT 的功能特性和底物选择性的比较分析还不够充分。在本研究中，对被膜霉菌中的 DGAT1（MaDGAT1A/1B/1C）和 DGAT2（MaDGAT2A/2B）同工型进行了分析，并在酿酒酵母 H1246 中进行了异源表达。结果表明，MaDGAT1A/1B/2A/2B 能够恢复 TAG 合成，重组酵母中的相应 TAG 含量分别为 2.92±0.42%、3.62±0.22%、0.86±0.34%和 0.18±0.09%。在酿酒酵母 H1246 中，MaDGAT1A 优先选择单不饱和脂肪酸中的 C16:1，MaDGAT1B 优先选择饱和脂肪酸（SFAs）中的 C16:0，MaDGAT2A/2B 优先选择 SFAs 中的 C18:0。在外源添加多不饱和脂肪酸（PUFAs）的情况下，MaDGAT1A 和 2A 优先将亚油酸组装成 TAG，MaDGAT2B 对 ω-6 PUFAs 中的二十碳五烯酸和亚油酸具有底物选择性。在体外，MaDGAT1A 对酰基辅酶 A 没有明显的选择性，而 MaDGAT1B 则优先选择 C20:5-CoA。MaDGAT1A/1B 优先选择 C18:1/C18:1-DAG 而不是 C20:4/C20:4-DAG。本研究表明，MaDGAT 具有用于生产富含亚油酸/二十碳五烯酸的 TAG 的潜力，并为提高油脂真菌中 TAG 的生产提供了参考。
关键点
MaDGAT1A 在 MUFA 中优先选择 C16:1，MaDGAT1B 和 MaDGAT2A/2B 分别在 SFA 中优先选择 C16:0 和 C18:0；
MaDGAT1A/2A 优先将亚油酸组装成 TAG，MaDGAT2B 对 ω-6 PUFAs 中的二十碳五烯酸和亚油酸具有底物选择性；
MaDGAT1A 对酰基辅酶 A 没有明显的选择性，MaDGAT1B 优先选择 C20:5-CoA。MaDGAT1A/1B 优先选择 C18:1/C18:1-DAG 而不是 C20:4/C20:4-DAG。
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