Kinetic improvement of an algal diacylglycerol acyltransferase 1 via fusion with an acyl-CoA binding protein
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Xu, Y., Caldo, K. M. P., Falarz, L., Jayawardhane, K., & Chen, G. (2020). Kinetic improvement of an algal diacylglycerol acyltransferase 1 via fusion with an acyl‐CoA binding protein. The Plant Journal, 102(4), 856–871. Portico. https://doi.org/10.1111/tpj.14708
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Microalgal oils in the form of triacylglycerols (TAGs) are broadly used as nutritional supplements and biofuels. Diacylglycerol acyltransferase (DGAT) catalyzes the final step of acyl-CoA-dependent biosynthesis of TAG and is considered a key target for manipulating oil production. Although a growing number of DGAT1s have been identified and over-expressed in some algal species, the detailed structure-function relationship, as well as the improvement of DGAT1 performance via protein engineering, remain largely untapped. Here, we explored the structure-function features of the hydrophilic N-terminal domain of DGAT1 from the green microalga Chromochloris zofingiensis (CzDGAT1). The results indicated that the N-terminal domain of CzDGAT1 was less disordered than those of the higher eukaryotic enzymes and its partial truncation or complete removal could substantially decrease enzyme activity, suggesting its possible role in maintaining enzyme performance. Although the N-terminal domains of animal and plant DGAT1s were previously found to bind acyl-CoAs, replacement of CzDGAT1 N-terminus by an acyl-CoA binding protein (ACBP) could not restore enzyme activity. Interestingly, the fusion of ACBP to the N-terminus of the full-length CzDGAT1 could enhance the enzyme affinity for acyl-CoAs and augment protein accumulation levels, which ultimately drove oil accumulation in yeast cells and tobacco leaves to higher levels than the full-length CzDGAT1. Overall, our findings unravel the distinct features of the N-terminus of algal DGAT1 and provide a strategy to engineer enhanced performance in DGAT1 via protein fusion, which may open a vista in generating improved membrane-bound acyl-CoA-dependent enzymes and boosting oil biosynthesis in plants and oleaginous microorganisms.
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http://purl.org/coar/resource_type/c_6501 http://purl.org/coar/version/c_970fb48d4fbd8a85
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en