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Aurantiochytrium is an important docosahexaenoic acid (DHA) producer containing two kinds of fatty acid synthesis pathways, that is, the fatty acid synthase pathway (FAS) for saturated fatty acid synthesis and the polyketide synthase pathway (PKS) for polyunsaturated fatty acid synthesis. To understand the regulation mechanism between the two pathways, the impacts of six short-chain fatty acids on the fatty acid synthesis of Aurantiochytrium sp. SD116 were studied. All short-chain fatty acids showed little effect on the cell growth, but some of them significantly affected lipid accumulation and fatty acid composition. Pentanoic acid and isovaleric acid greatly inhibited the synthesis of saturated fatty acids, whereas the polyunsaturated fatty acid synthesis was not affected. Analysis of malic enzyme activity, which supplied NADPH for saturated fatty acids biosynthesis, indicated that the two fatty acid synthesis pathways can utilize different substrates and possess independent sources of NADPH.

Background: Triacylglycerol (TAG) is an important storage lipid in organisms, depending on the degree of unsaturation of fatty acid molecules attached to glycerol; it is usually used as the feedstock for nutrition or biodiesel. However, the mechanism of assembly of saturated fatty acids (SFAs) or polyunsaturated fatty acids (PUFAs) into TAGs remains unclear for industrial oleaginous microorganism. Results: Diacylglycerol acyltransferase (DGAT) is a key enzyme for TAG synthesis. Hence, ex vivo (in yeast), and in vivo functions of four DGAT2s (DGAT2A, DGAT2B, DGAT2C, and DGAT2D) in industrial oleaginous thraustochytrid Aurantiochytrium sp. SD116 were analyzed. Results revealed that DGAT2C was mainly responsible for connecting PUFA to the sn-3 position of TAG molecules. However, DGAT2A and DGAT2D target SFA and/or MUFA. Conclusions: There are two specific TAG assembly routes in Aurantiochytrium. The "saturated fatty acid (SFA) TAG lane" primarily produces SFA-TAGs mainly mediated by DGAT2D whose function is complemented by DGAT2A. And, the "polyunsaturated fatty acid (PUFA) TAG lane" primarily produces PUFA-TAGs via DGAT2C. In this study, we demonstrated the functional distribution pattern of four DGAT2s in oleaginous thraustochytrid Aurantiochytrium, and provided a promising target to rationally design TAG molecular with the desired characteristics. [ABSTRACT FROM AUTHOR]

• Metabolomic analysis was performed to explore metabolic changes of microalgaes. • Twenty-two differential metabolites were screened as relevant to the discrepancy. • The potential mechanisms for the optimization of DHA production were explored. • Cell metabolomics could provide valuable insights into the genetic modification. Along with the daily growth of the market requirements for docosahexaenoic acid (DHA) algae oil, a large DHA ingredients are needed to ensure worldwide supply. Undoubtedly a high-productive strain would be the prerequisite for high quality and yield. A comprehensive understanding of the processes of DHA synthesis from glycolysis to the lipid accumulation would be benefit to achieve the final optimization of DHA production. In this study, we comprehensively characterized the metabolic profiles of a Schizochytrium sp. strain, which has higher DHA content and different biomass amino acid composition compared with the wild type to explore the affected pathways and underlying mechanism. Combined with the multivariate statistical analysis, twenty-two differential metabolites were screened as relevant to the discrepancy between two strains. The results showed relatively downregulated glycolysis and saturated fatty acids (SFA) synthesis, and upregulated TCA cycle, amino acids and polyunsaturated fatty acids (PUFA) synthesis in DHA high yield strain. The current study provide a terminal picture of gene regulation from downstream metabolism and demonstrate the advantage of metabolomics in characterizing metabolic status which in turn could provide effective information for the metabolic engineering. [ABSTRACT FROM AUTHOR]

Background: Phosphopantetheinyl transferase (PPTase) can change the acyl-carrier protein (ACP) from an inactive apo-ACP to an active holo-ACP that plays a key role in fatty acids biosynthesis. Currently, the PPTase has been proved to be involved in the biosynthesis of polyunsaturated fatty acids (PUFAs) via a polyketide synthase (PKS) pathway in Thraustochytrids, while its characteristics are not clarified. Results: Here, the heterologous PPTase gene (pfaE) from bacteria was first co-expressed with the PKS system (orfA–orfC) from Thraustochytrid Aurantiochytrium. Then, a new endogenous PPTase (ppt_a) in Aurantiochytrium was identified by homologous alignment and its function was verified in E. coli. Moreover, the endogenous ppt_a was then overexpressed in Aurantiochytrium, and results showed that the production and proportion of PUFAs, especially docosahexaenoic acid (DHA), in the transformant SD116::PPT_A were increased by 35.5% and 17.6%, respectively. Finally, higher DHA and PUFA proportion (53.9% and 64.5% of TFA, respectively) were obtained in SD116::PPT_A using a cerulenin feeding strategy. Conclusions: This study has illustrated a PUFAs-synthase-specific PPTase in PKS system and provided a new strategy to improve the PUFA production in Thraustochytrids. [ABSTRACT FROM AUTHOR]

Microbial fatty acid-producing strains are commonly engineered to improve their performance for industrial applications. However, it is challenging to efficiently and rapidly screen target strains for engineering. This study reported an in situ analytical platform using laser ablation electrospray ionization mass spectrometry (LAESI-MS) for fast profiling of triacylglycerols in cellular lipid droplets of Aurantiochytrium sp. colonies cultured on agar plates. LAESI-MS approach allowed for the direct acquisition of a colony cell's characteristic fingerprint mass spectrum and MS/MS facilitated the identification of triacylglycerol species containing three fatty acyl groups. The fatty acid contents of colony cells were calculated based on the intensities of triacylglycerols from their characteristic fingerprint mass spectrum. A Python package called TAFA-LEMS (Triacylglycerol to Fatty Acid by LAESI-MS) was also developed to process the high-throughput MS data and extract fatty acid contents in colony cells. The results demonstrated that the LAESI-MS platform is fast, stable, and reproducible, with a data acquisition rate of ≤2 s per sampling point and ≤13.69% RSDs of the relative contents of fatty acids. In addition, LAESI-MS was successfully performed on the analysis of P. tricornutum and Y lipolytica strains. This in situ MS platform has the potential to become a common biotechnology platform for microbial strain engineering. [Display omitted] • A high-throughput screening platform for fatty acid-producing strains. • A Python package for the rapid processing of MS data. • This platform exhibited high stability, speed, and reproducibility. • It can serve as the common platform for breeding engineering strains. [ABSTRACT FROM AUTHOR]

Abstract: The growth, lipid content and fatty acid composition of Schizochytrium limacinum OUC88 at different temperatures (16, 23, 30 and 37°C) and salinities (0, 0.9, 1.8, 2.7 and 3.6%, w/v) were analyzed. The strain grew better and lipid contents were higher at 16–30°C and salinity at 0.9–3.6% (w/v). The adaptive responses of this microbe to temperature and salinity were mainly to regulate the degree of fatty acid unstauration to maintain the normal membrane lipid physical state. However, at 37°C and 0 salinity, the growth of the strain was inhibited obviously and the lipid content reduced significantly and, some important changes occurred in fatty acid composition, especially the odd-numbered fatty acids 15:0 and 17:0 which amounts increased greatly. In addition, the ratio of DHA to DPA changed at different temperatures and salinities. [Copyright &y& Elsevier]