Functions of Enyolreductase (ER) Domains of PKS Cluster in Lipid Synthesis and Enhancement of PUFAs Accumulation in Schizochytrium limacinum SR21 Using Triclosan as a Regulator of ER.

Xueping Ling,Shengyang Yu,Qinghua Yang,Zhipeng Li,Cuixue Chen,Yinghua Lu,Ning He,Jun Li,Hao Zhou

doi:10.3390/microorganisms8020300

Abstract

The polyketide synthase (PKS) cluster genes are supposed to synthesize polyunsaturated fatty acids (PUFAs) in S. limacinum. In this study, two enyolreductase (ER) genes located on PKS cluster were knocked out through homologous recombination to explore their functions. The knock-out of OrfB-ER (located on OrfB subunit) decreased lipid content and had obvious decrease on PUFAs content, indicating OrfB-ER domain played a vital role on PUFAs synthesis; the knock-out of OrfC-ER (located on OrfC subunit) decreased SFAs content and increased total lipid content, indicating OrfC-ER domain was likely to be related with SFAs synthesis, and lipid production could be improved by down-regulating OrfC-ER domain expression. Therefore, the addition of triclosan as a reported regulator of ER domain induced the increase of PUFAs production by 51.74% and lipids yield by 47.63%. Metabolic analysis indicated triclosan played its role through inhibiting the expression of OrfC-ER to reduce the feedback inhibition of SFAs and further to enhance NADPH synthesis for lipid production, and by weakening mevalonate pathway and tricarboxylic acid (TCA) cycle to shift precursors for lipid and PUFAs synthesis. This research illuminates functions of two ER domains in S. limacinum and provides a potential targets for improving lipid production.

Highlights

The polyunsaturated fatty acids (PUFAs), mainly including docosahexaenoic acid (DHA; C22:6) and eicosapentaenoic acid (EPA; C20:5), are beneficial to human health
The zeocin gene was used to serve as a screening marker, and S. limacinum were cultured in seed medium containing 50 μg/mL zeocin
The addition of triclosan caused the decrease of citrate, succinate, and oxaloacetate, which were some intermediate metabolites of tricarboxylic acid (TCA) cycle. Some amino acids, such as oxoproline and arginine, were reduced, which were synthesized by further catalysis of α-ketoglutarate from TCA cycle. These results suggested that triclosan treatment promoted the metabolic flux from TCA cycle to the fatty acids biosynthesis

Summary

Introduction

The polyunsaturated fatty acids (PUFAs), mainly including docosahexaenoic acid (DHA; C22:6) and eicosapentaenoic acid (EPA; C20:5), are beneficial to human health. PUFAs may be beneficial in prevention of cardiovascular disease and treatment of mild Alzheimer’s disease [1,2]. S. limacinum has been widely studied due to its ability to produce significant amounts of total lipids rich in PUFAs. It was reported that S. limacinum was able to achieve 40%~70% of the lipid content and 20%~50% of the DHA production [4,5]. The heterotrophic microalga S. limacinum have been approved to commercially produce DHA mainly used for pharmaceutical products and infant formula, which represent an industrially important source of PUFAs [11]

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Results

Conclusion