13C Metabolic Flux Analysis of Enhanced Lipid Accumulation Modulated by Ethanolamine in Crypthecodinium cohnii.

Jinyu Cui,Fangzhong Wang,Mengliang Shi,Lei Chen,Jinjin Diao,Tao Sun,Weiwen Zhang,Liangsen Liu

doi:10.3389/fmicb.2018.00956

Abstract

The heterotrophic microalga Crypthecodinium cohnii has attracted considerable attention due to its capability of accumulating lipids with a high fraction of docosahexaenoic acid (DHA). In our previous study, ethanolamine (ETA) was identified as an effective chemical modulator for lipid accumulation in C. cohnii. In this study, to gain a better understanding of the lipid metabolism and mechanism for the positive effects of modulator ETA, metabolic flux analysis was performed using 13C-labeled glucose with and without 1 mM ETA modulator. The analysis of flux distribution showed that with the addition of ETA, flux in glycolysis pathway and citrate pyruvate cycle was strengthened while flux in pentose phosphate pathway was decreased. In addition, flux in TCA cycle was slightly decreased compared with the control without ETA. The enzyme activity of malic enzyme (ME) was significantly increased, suggesting that NADP+-dependent ME might be the major source of NADPH for lipid accumulation. The flux information obtained by this study could be valuable for the further efforts in improving lipid accumulation and DHA production in C. cohnii.

Highlights

Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid (PUFA) belonging to the ω-3 group
We first developed a synthetic medium suitable for 13C metabolic flux analysis based on the American Type Culture Collection (ATCC) 460 A2E6 medium (Pleissner and Eriksen, 2012)
The results showed that growth rates of C. cohnii were 0.051 and 0.054 h−1 in the medium supplied with 2 g/L glutamate and yeast extract, respectively, suggesting that C. cohnii was able to grow normally in the medium containing glutamate as nitrogen source

Summary

INTRODUCTION

Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid (PUFA) belonging to the ω-3 group. In order to systematically understand the mechanisms underlying lipid accumulation as well as quantitative metabolic information in C. cohnii, in this study, we utilized gas chromatographymass spectrometry to analyze the 13C labeling patterns of the amino acids in biomass hydrolysates of C. cohnii grown in an optimized chemically defined medium with and without 1 mM ETA addition. By integrating these labeling measurement data with metabolite balancing, the intracellular flux distributions in C. cohnii were further quantitated. The study provided valuable information to promote the future modification on lipid accumulation and DHA production in C. cohnii

MATERIALS AND METHODS

RESULTS AND DISCUSSION

CONCLUSION