Computationally Guided Enzymatic Studies on Schizochytrium-Sourced Malonyl-CoA:ACP Transacylase

Abstract

The malonyl-CoA:ACP transacylase (MAT) domain is responsible for the selection and incorporation of malonyl building blocks in the biosynthesis of polyunsaturated fatty acids (PUFAs) in eukaryotic microalgae (Schizochytrium) and marine bacteria (Moritella marina, Photobacterium profundum, and Shewanella). Elucidation of the structural basis underlying the substrate specificity and catalytic mechanism of the MAT will help to improve the yield and quality of PUFAs. Here, a methodology guided by molecular dynamics simulations was carried out to identify and mutate specificity-conferring residues within the MAT domain of Schizochytrium. Combining mutagenesis, cell-free protein synthesis, and in vitro biochemical assay, we dissected nearby interactions and molecular mechanisms relevant for binding and catalysis and found that the reorientation of the Ser154 Cβ–Oγ bond establishes distinctive proton-transfer chains (His153-Ser154 and Asn235-His153-Ser154) for catalysis. Gln66 can be replaced by tyrosine to shorten the distance between His153 (Nε2) and Ser154 (Oγ), which facilitates a faster proton-transfer rate, allowing better use of acyl substrates than the wild type. Furthermore, we screened a mutant that displayed an 18.4% increase in PUFA accumulation. These findings provide important insights into the study of MAT through protein engineering and will benefit dissecting the molecular mechanisms of other PUFA-related catalytic domains.

Function of the Polyketide Synthase Domains of Schizochytrium sp. on Fatty Acid Synthesis in Yarrowia lipolytica

“…7 In another report, saturation scanning mutagenesis was performed by substituting the Gln66 residue of the MAT domain of Schizochytrium, when placed by tyrosine, the PUFA and DHA production was increased by 18.4 and 16.7%, respectively compared with the wild type. 32 Compared with the CK strain, the C16:0 and C18:0 content were both reduced in the ADH strain while C18:0 content was reduced in CDH2 strain. On the contrary, the C18:1Δ 9 , C18:2Δ 9,12 , and C20:5Δ 5,8,11,14,17 contents were increased in ADH, CDH1, and CDH2 strains (Figure 4b).…”

“…resulted in an increase of 172.5% in EPA production compared to the wild strain . In another report, saturation scanning mutagenesis was performed by substituting the Gln66 residue of the MAT domain of Schizochytrium, when placed by tyrosine, the PUFA and DHA production was increased by 18.4 and 16.7%, respectively compared with the wild type . Compared with the CK strain, the C16:0 and C18:0 content were both reduced in the ADH strain while C18:0 content was reduced in CDH2 strain.…”

Enhancing the biomass and docosahexaenoic acid‐rich lipid accumulation of Schizochytrium sp. in propionate wastewater

“…Schizochytrium sp., a heterotrophic marine organism, has been used industrially to produce docosahexaenoic acid (DHA). [1][2][3] The typical condition of laboratory scale cultivation for Schizochytrium sp. is artificial seawater, which requires a large amount of water resources and costs a lot.…”

“…Schizochytrium sp., a heterotrophic marine organism, has been used industrially to produce docosahexaenoic acid (DHA) 1–3 . The typical condition of laboratory scale cultivation for Schizochytrium sp.…”

Integrated Analysis of lncRNA–mRNA Regulatory Networks Related to Lipid Metabolism in High-Oleic-Acid Rapeseed

“…It begins with the synthesis of fatty acids: Acetyl-CoA carboxylase (ACC) catalyzes the first committed step of de novo fatty acid biosynthesis via the carboxylation of acetyl-CoA to malonyl-CoA [ 11 ]. Next, Mal-CoA:ACP S-malonyltransferase (MAT) converts malonyl-CoA to malonyl-ACP, which is the primary substrate for a subsequent series of condensation reactions [ 12 ]. The fatty acid synthetase system further extends the carbon chain through condensation, reduction, dehydration, and further reduction to obtain various types of fatty acids [ 13 ].…”