Background
As a potential source of polyunsaturated fatty acids (PUFA), Schizochytrium sp. has been widely used in industry for PUFA production. Polyketide synthase (PKS) cluster is supposed to be the primary way of PUFA synthesis in Schizochytrium sp. As one of three open reading frames (ORF) in the PKS cluster, ORFC plays an essential role in fatty acid biosynthesis. However, the function of domains in ORFC in the fatty acid synthesis of Schizochytrium sp. remained unclear.
Results
In this study, heterologous expression and overexpression were carried out to study the role of ORFC and its domains in fatty acid accumulation. Firstly, ORFC was heterologously expressed in yeast which increased the PUFA content significantly. Then, the dehydratase (DH) and enoyl reductase (ER) domains located on ORFC were overexpressed in Schizochytrium limacinum SR21, respectively. Fatty acids profile analysis showed that the contents of PUFA and saturated fatty acid were increased in the DH and ER overexpression strains, respectively. This indicated that the DH and ER domains played distinct roles in lipid accumulation. Metabolic and transcriptomic analysis revealed that the pentose phosphate pathway and triacylglycerol biosynthesis were enhanced, while the tricarboxylic acid cycle and fatty acids oxidation were weakened in DH-overexpression strain. However, the opposite effect was found in the ER-overexpression strain.
Conclusion
Therefore, ORFC was required for the biosynthesis of fatty acid. The DH domain played a crucial role in PUFA synthesis, whereas the ER domain might be related to saturated fatty acids (SFA) synthesis in Schizochytrium limacinum SR21. This research explored the role of ORFC in the PKS gene cluster in Schizochytrium limacinum and provided potential genetic modification strategies for improving lipid production and regulating PUFA and SFA content.
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.
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