Dataset Information

Characterization of type-2 diacylglycerol acyltransferases in Haematococcus lacustris reveals their functions and engineering potential in triacylglycerol biosynthesis.

ABSTRACT:

Background

Haematococcus lacustris is an ideal source of astaxanthin (AST), which is stored in oil bodies containing esterified AST (EAST) and triacylglycerol (TAG). Diacylglycerol acyltransferases (DGATs) catalyze the last step of acyl-CoA-dependent TAG biosynthesis and are also considered as crucial enzymes involved in EAST biosynthesis in H. lacustris. Previous studies have identified four putative DGAT2-encoding genes in H. lacustris, and only HpDGAT2D allowed the recovery of TAG biosynthesis, but the engineering potential of HpDGAT2s in TAG biosynthesis remains ambiguous.

Results

Five putative DGAT2 genes (HpDGAT2A, HpDGAT2B, HpDGAT2C, HpDGAT2D, and HpDGAT2E) were identified in H. lacustris. Transcription analysis showed that the expression levels of the HpDGAT2A, HpDGAT2D, and HpDGAT2E genes markedly increased under high light and nitrogen deficient conditions with distinct patterns, which led to significant TAG and EAST accumulation. Functional complementation demonstrated that HpDGAT2A, HpDGAT2B, HpDGAT2D, and HpDGAT2E had the capacity to restore TAG synthesis in a TAG-deficient yeast strain (H1246) showing a large difference in enzymatic activity. Fatty acid (FA) profile assays revealed that HpDGAT2A, HpDGAT2D, and HpDGAT2E, but not HpDGAT2B, preferred monounsaturated fatty acyl-CoAs (MUFAs) for TAG synthesis in yeast cells, and showed a preference for polyunsaturated fatty acyl-CoAs (PUFAs) based on their feeding strategy. The heterologous expression of HpDGAT2D in Arabidopsis thaliana and Chlamydomonas reinhardtii significantly increased the TAG content and obviously promoted the MUFAs and PUFAs contents.

Conclusions

Our study represents systematic work on the characterization of HpDGAT2s by integrating expression patterns, AST/TAG accumulation, functional complementation, and heterologous expression in yeast, plants, and algae. These results (1) update the gene models of HpDGAT2s, (2) prove the TAG biosynthesis capacity of HpDGAT2s, (3) show the strong preference for MUFAs and PUFAs, and (4) offer target genes to modulate TAG biosynthesis by using genetic engineering methods.

SUBMITTER: Cui H

PROVIDER: S-EPMC7788937 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Publications

Characterization of type-2 diacylglycerol acyltransferases in Haematococcus lacustris reveals their functions and engineering potential in triacylglycerol biosynthesis.

Cui Hongli H Zhao Chunchao C Xu Wenxin W Zhang Hongjiang H Hang Wei W Zhu Xiaoli X Ji Chunli C Xue Jinai J Zhang Chunhui C Li Runzhi R

BMC plant biology 20210106 1

<h4>Background</h4>Haematococcus lacustris is an ideal source of astaxanthin (AST), which is stored in oil bodies containing esterified AST (EAST) and triacylglycerol (TAG). Diacylglycerol acyltransferases (DGATs) catalyze the last step of acyl-CoA-dependent TAG biosynthesis and are also considered as crucial enzymes involved in EAST biosynthesis in H. lacustris. Previous studies have identified four putative DGAT2-encoding genes in H. lacustris, and only HpDGAT2D allowed the recovery of TAG bio ...[more]

PMID: 33407140

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Project description:BackgroundThe green alga Chlorella zofingiensis has been recognized as an industrially relevant strain because of its robust growth under multiple trophic conditions and the potential for simultaneous production of triacylglycerol (TAG) and the high-value keto-carotenoid astaxanthin. Nevertheless, the mechanism of TAG synthesis remains poorly understood in C. zofingiensis. Diacylglycerol acyltransferase (DGAT) is thought to catalyze the committed step of TAG assembly in the Kennedy pathway. C. zofingiensis genome is predicted to possess eleven putative DGAT-encoding genes, the greatest number ever found in green algae, pointing to the complexity of TAG assembly in the alga.ResultsThe transcription start site of C. zofingiensis DGATs was determined by 5'-rapid amplification of cDNA ends (RACE), and their coding sequences were cloned and verified by sequencing, which identified ten DGAT genes (two type I DGATs designated as CzDGAT1A and CzDGAT1B, and eight type II DGATs designated as CzDGTT1 through CzDGTT8) and revealed that the previous gene models of seven DGATs were incorrect. Function complementation in the TAG-deficient yeast strain confirmed the functionality of most DGATs, with CzDGAT1A and CzDGTT5 having the highest activity. In vitro DGAT assay revealed that CzDGAT1A and CzDGTT5 preferred eukaryotic and prokaryotic diacylglycerols (DAGs), respectively, and had overlapping yet distinctive substrate specificity for acyl-CoAs. Subcellular co-localization experiment in tobacco leaves indicated that both CzDGAT1A and CzDGTT5 were localized at endoplasmic reticulum (ER). Upon nitrogen deprivation, TAG was drastically induced in C. zofingiensis, accompanied by a considerable up-regulation of CzDGAT1A and CzDGTT5. These two genes were probably regulated by the transcription factors (TFs) bZIP3 and MYB1, as suggested by the yeast one-hybrid assay and expression correlation. Moreover, heterologous expression of CzDGAT1A and CzDGTT5 promoted TAG accumulation and TAG yield in different hosts including yeast and oleaginous alga.ConclusionsOur study represents a pioneering work on the characterization of both type I and type II C. zofingiensis DGATs by systematically integrating functional complementation, in vitro enzymatic assay, subcellular localization, yeast one-hybrid assay and overexpression in yeast and oleaginous alga. These results (1) update the gene models of C. zofingiensis DGATs, (2) shed light on the mechanism of oleaginousness in which CzDGAT1A and CzDGTT5, have functional complementarity and probably work in collaboration at ER contributing to the abundance and complexity of TAG, and (3) provide engineering targets for future trait improvement via rational manipulation of this alga as well as other industrially relevant ones.

Dataset Information

Characterization of type-2 diacylglycerol acyltransferases in Haematococcus lacustris reveals their functions and engineering potential in triacylglycerol biosynthesis.

Background

Results

Conclusions

Publications

Characterization of type-2 diacylglycerol acyltransferases in Haematococcus lacustris reveals their functions and engineering potential in triacylglycerol biosynthesis.

Similar Datasets

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