Accelerated triacylglycerol production without growth inhibition by overexpression of a glycerol-3-phosphate acyltransferase in the unicellular red alga Cyanidioschyzon merolae

Satoshi Fukuda,Tokiaki Takemura,Imran Pancha,Kaumeel Chokshi,Eri Hirasawa,Kan Tanaka,Sota Takahashi,Sousuke Imamura

doi:10.1038/s41598-018-30809-8

Abstract

Microalgae accumulate triacylglycerols (TAGs), a promising feedstock for biodiesel production, under unfavorable environmental or stress conditions for their growth. Our previous analyses revealed that only transcripts of CmGPAT1 and CmGPAT2, both encoding glycerol-3-phosphate acyltransferase, were increased among fatty acid and TAG synthesis genes under TAG accumulation conditions in the red alga Cyanidioschyzon merolae. In this study, to investigate the role of these proteins in TAG accumulation in C. merolae, we constructed FLAG-fused CmGPAT1 and CmGPAT2 overexpression strains. We found that CmGPAT1 overexpression resulted in marked accumulation of TAG even under normal growth conditions, with the maximum TAG productivity increased 56.1-fold compared with the control strain, without a negative impact on algal growth. The relative fatty acid composition of 18:2 in the TAGs and the sn−1/sn−3 positions were significantly increased compared with the control strain, suggesting that CmGPAT1 had a substrate preference for 18:2. Immunoblot analysis after cell fractionation and immunostaining analysis demonstrated that CmGPAT1 localizes in the endoplasmic reticulum (ER). These results indicate that the reaction catalyzed by the ER-localized CmGPAT1 is a rate-limiting step for TAG synthesis in C. merolae, and would be a potential target for improvement of TAG productivity in microalgae.

Highlights

Microalgae produce biomass using sunlight, water and carbon dioxide by the process of photosynthesis
We investigated the role of the endoplasmic reticulum (ER)-localized glycerol-3-phosphate acyltransferases (GPATs), CmGPAT1 and CmGPAT2, in TAG accumulation in C. merolae, and found that the reaction catalyzed by the ER-localized CmGPAT1 is a rate-limiting step for TAG synthesis in this alga
CmGPAT1 and CmGPAT2 were categorized into group IV, which contains Chlamydomonas reinhardtii GPAT (CrGPAT) and Arabidopsis thaliana GPAT9 (AtGPAT9)

Summary

Introduction

Microalgae produce biomass using sunlight, water and carbon dioxide by the process of photosynthesis. With respect to improving TAG production, it was reported that TAG production was increased by overexpression of CrDGTT4, encoding a type-2 diacylglycerol acyltransferase (DGAT), under phosphorus-depletion conditions in C. reinhardtii[8]. The complete genome sequences of these three organelles were determined[11], and it was revealed that they have simple, minimally redundant gene contents Because these biological characteristics have been elucidated and various tools have been established for analysis of C. merolae[12,13,14], this alga is considered a good model organism to understand various molecular functions of photosynthetic eukaryotes, including the fundamental regulation of TAG production. We investigated the role of the endoplasmic reticulum (ER)-localized glycerol-3-phosphate acyltransferases (GPATs), CmGPAT1 and CmGPAT2, in TAG accumulation in C. merolae, and found that the reaction catalyzed by the ER-localized CmGPAT1 is a rate-limiting step for TAG synthesis in this alga. We suggest that ER-localized GPATs are potential targets for increasing the TAG productivity in microalgae

Methods

Results

Conclusion