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Overexpression of peanut diacylglycerol acyltransferase 2 in Escherichia coli.

ABSTRACT: Diacylglycerol acyltransferase (DGAT) is the rate-limiting enzyme in triacylglycerol biosynthesis in eukaryotic organisms. Triacylglycerols are important energy-storage oils in plants such as peanuts, soybeans and rape. In this study, Arachis hypogaea type 2 DGAT (AhDGAT2) genes were cloned from the peanut cultivar 'Luhua 14' using a homologous gene sequence method and rapid amplification of cDNA ends. To understand the role of AhDGAT2 in triacylglycerol biosynthesis, two AhDGAT2 nucleotide sequences that differed by three amino acids were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli Rosetta (DE3). Following IPTG induction, the isozymes (AhDGAT2a and AhDGAT2b) were expressed as 64.5 kDa GST fusion proteins. Both AhDGAT2a and AhDGAT2b occurred in the host cell cytoplasm and inclusion bodies, with larger amounts in the inclusion bodies. Overexpression of AhDGATs depressed the host cell growth rates relative to non-transformed cells, but cells harboring empty-vector, AhDGAT2a-GST, or AhDGAT2b-GST exhibited no obvious growth rate differences. Interestingly, induction of AhDGAT2a-GST and AhDGAT2b-GST proteins increased the sizes of the host cells by 2.4-2.5 times that of the controls (post-IPTG induction). The total fatty acid (FA) levels of the AhDGAT2a-GST and AhDGAT2a-GST transformants, as well as levels of C12:0, C14:0, C16:0, C16:1, C18:1n9c and C18:3n3 FAs, increased markedly, whereas C15:0 and C21:0 levels were lower than in non-transformed cells or those containing empty-vectors. In addition, the levels of some FAs differed between the two transformant strains, indicating that the two isozymes might have different functions in peanuts. This is the first time that a full-length recombinant peanut DGAT2 has been produced in a bacterial expression system and the first analysis of its effects on the content and composition of fatty acids in E. coli. Our results indicate that AhDGAT2 is a strong candidate gene for efficient FA production in E. coli.

SUBMITTER: Peng Z

PROVIDER: S-EPMC3623910 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Researchers have long endeavored to accumulate triacylglycerols (TAGs) or their derivatives in easily managed microbes. The attempted production of TAGs in Escherichia coli has revealed barriers to the broad applications of this technology, including low TAG productivity and slow cell growth. We have demonstrated that an acyl-CoA-independent pathway can divert phospholipid flux into TAG formation in E. coli mediated by Chlamydomonas reinhardtii phospholipid:diacylglycerol acyltransferase (CrPDAT) without interfering with membrane functions. We then showed the synergistic effect on TAG accumulation via the acyl-CoA-independent pathway mediated by PDAT and the acyl-CoA-dependent pathway mediated by wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT). Furthermore, CrPDAT led to synchronous TAG accumulation during cell growth, and this could be enhanced by supplementation of arbutin. We also showed that rationally mutated CrPDAT was capable of decreasing TAG lipase activity without impairing PDAT activity. Finally, ScPDAT from Saccharomyces cerevisiae exhibited similar activities as CrPDAT in E. coli Our results suggest that the improvement in accumulation of TAGs and their derivatives can be achieved by fine-tuning of phospholipid metabolism in E. coli Understanding the roles of PDAT in the conversion of phospholipids into TAGs during the logarithmic growth phase may enable a novel strategy for the production of microbial oils.IMPORTANCE Although phospholipid:diacylglycerol acyltransferase (PDAT) activity is presumed to exist in prokaryotic oleaginous bacteria, the corresponding gene has not been identified yet. In this article, we have demonstrated that an acyl-CoA-independent pathway can divert phospholipid flux into TAG formation in Escherichia coli mediated by exogenous CrPDAT from Chlamydomonas reinhardtii without interfering with membrane functions. In addition, the acyl-CoA-independent pathway and the acyl-CoA-dependent pathway had the synergistic effect on TAG accumulation. Overexpression of CrPDAT led to synchronous TAG accumulation during cell growth. In particular, CrPDAT possessed multiple catalytic activities, and the rational mutation of CrPDAT led to the decrease of TAG lipase activity without impairing acyltransferase activity. The present findings suggested that applying PDAT in E. coli or other prokaryotic microbes may be a promising strategy for accumulation of TAGs and their derivatives.

Project description:Microalgae are promising sources of lipid triacylglycerol (TAG) for biodiesel production. However, to date, microalgal biodiesel is technically feasible, but not yet economically viable. Increasing TAG content and productivity are important to achieve economic viability of microalgal biodiesel. To increase TAG content, oleaginous microalga Neochloris oleoabundans was genetically engineered with an endogenous key enzyme diacylglycerol acyltransferase 2 (NeoDGAT2) responsible for TAG biosynthesis.The integration of NeoDGAT2 expression cassettes in N. oleoabundans transformant was confirmed by PCR. The neutral lipid accumulation in the transformant detected by Nile red staining was accelerated and 1.9-fold higher than in wild type; the lipid bodies in the transformant visualized under fluorescence microscope were also larger. The NeoDGAT2 transcript was two-fold higher in the transformant than wild type. Remarkably higher lipid accumulation was found in the transformant than wild type: total lipid content increased 1.6-to 2.3-fold up to 74.5 ± 4.0% dry cell weight (DCW) and total lipid productivity increased 1.6- to 3.2-fold up to 14.6 ± 2.0 mg/L/day; while TAG content increased 1.8- to 3.2-fold up to 46.1 ± 1.6% DCW and TAG productivity increased 1.6- to 4.3-fold up to 8.9 ± 1.3 mg/L/day. A significantly altered fatty acid composition was detected in the transformant compared to wild type; the levels of saturated fatty acid C16:0 increased double to 49%, whereas C18:0 was reduced triple to 6%. Long-term stability was observed in the transformant continuously maintained in solid medium over 100 generations in a period of about 4 years.Our results demonstrate the increased TAG content and productivity in N. oleoabundans by NeoDGAT2 overexpression that may offer the first step towards making microalgae an economically feasible source for biodiesel production. The strategy for genetically improved microalga presented in this study can be applied to other microalgal species possessing desired characteristics for industrial biofuel production.

Project description:BackgroundFatty acid (FA) composition is the most important parameter affecting the flavor and nutritional value of the meat. The final and the only committed step in the biosynthesis of triglycerides is catalyzed by diacylglycerol acyltransferase 2 (DGAT2). The role of DGAT2 in lipid accumulation has been demonstrated in adipocytes, However, little is known about the effect of DGAT2 on the FA composition of these cells.MethodsTo investigate the role of DGAT2 in regulating lipid accumulation, FA composition and the expression of adipogenic genes, we cloned the open reading frame of the porcine DGAT2 gene and established 3T3-L1 cells that overexpressed DGAT2. Cells were then cultured in differentiation medium (DM) without FA, with a mixture of FAs (FA-DM), or containing a (13)C stable isotope-labeled FA mixture (IFA-DM). The FA composition of adipocytes was analyzed by gas chromatography-mass spectrometry and gas chromatography-isotope ratio mass spectrometry. Quantitative PCR and western blotting were employed to detect expression of adipogenic genes in 3T3-L1 adipocytes cultured with FA-DM for 12 d.ResultsThe triacylglyceride (TAG) content was significantly higher in 3T3-L1 adipocytes overexpressing DGAT2 than in control cells. When cultured in DM or FA-DM for 12 d, cells overexpressing DGAT2 showed a higher proportion of unsaturated FAs (C16:1 and C18:1). However, when cells overexpressing DGAT2 were cultured with FA-DM for 30 min, the FA composition was almost identical to that of controls. Further, the proportion of stable isotope-labeled FAs were similar in 3T3-L1 adipocytes overexpressing DGAT2 and control cells cultured in IFA-DM for 12 d. These results collectively indicate that the higher proportion of mono-unsaturated FAs, C16:1 and C18:1, may originate from de novo FA synthesis but not from the uptake of specific FAs from the medium. This hypothesis is further supported by evidence that both mRNA and protein expression of genes involved in FA synthesis (ACACA, FASN, SCD1, and A-FABP) were significantly higher in cells overexpressing DGAT2 than in control cells.ConclusionsIn conclusion, our study revealed that TAG accumulation, the proportion of MUFAs, and the expression of adipogenic genes were higher in 3T3-L1 cells overexpressing DGAT2 than in control cells.

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Overexpression of peanut diacylglycerol acyltransferase 2 in Escherichia coli.

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