Abstract
BackgroundAs 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.ResultsIn 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.ConclusionTherefore, 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.
Highlights
As a potential source of polyunsaturated fatty acids (PUFA), Schizochytrium sp. has been widely used in industry for PUFA production
Gas chromatography/mass spectrometry (GC–MS) based metabolomic analysis and an RNA-seq analysis were conducted to explain the Effects of ORFC heterologous expression on fatty acid analysis in S. cerevisiae To study the effects of ORFC on fatty acid accumulation, heterologous expression of ORFC was conducted in S. cerevisiae YSG50
As shown in Additional file 1: Fig. S2, single clones grew in the Ura-depletion plate, and ORFC gene was detected in the engineered strain while not in the wild-type strain by genomic polymerase chain reaction (PCR) analysis, revealed that ORFC was successfully heterologously expressed in S. cerevisiae YSG50
Summary
As a potential source of polyunsaturated fatty acids (PUFA), Schizochytrium sp. has been widely used in industry for PUFA production. As one of three open reading frames (ORF) in the PKS cluster, ORFC plays an essential role in fatty acid biosynthesis. The function of domains in ORFC in the fatty acid synthesis of Schizochytrium sp. Hayashi et al investigated the function of ACPs and found that the number of activated ACPs increased the productivity of PUFA [13]. They investigated the role of the KS domains in PUFA synthases by in vivo and in vitro analysis, and found that that two KS domains in subunit A and subunit C (KSA and KSC) were verified to catalyze the condensation from C18-C20 and C20-C22, respectively [14]
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