Kinetic improvement of an algal diacylglycerol acyltransferase 1 via fusion with an acyl‐CoA binding protein. (23rd February 2020)

• Record Type: Journal Article
• Authors: Xu, Yang; Caldo, Kristian Mark P.; Falarz, Lucas; Jayawardhane, Kethmi; Chen, Guanqun
• Is Part Of: Plant journal. Volume 102:Number 4(2020)
• Page Start: 856
• Page End: 871
• Publication Date: 2020-02-23
• Subjects: triacylglycerol biosynthesis -- diacylglycerol acyltransferase -- acyl‐CoA binding protein -- algal lipid -- enzyme kinetics -- tobacco -- yeast -- Chromochloris zofingiensis; Plant molecular biology -- Periodicals; Plant cells and tissues -- Periodicals; Botany -- Periodicals; 580
• DOI: 10.1111/tpj.14708
• Languages: English
• ISSNs: 0960-7412

Abstract

Summary: 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 DGAT1 s 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. Significance Statement: Here, we explored the N‐terminus of a microalgal DGAT1, a membrane‐bound enzyme determining oil biosynthesis, using in silico analysis, truncation mutagenesis, protein fusion, and in vitro and in vivo characterization, and demonstrated its distinct structure−function features from the higher eukaryotic enzymes. We further engineered enhanced performance in DGAT1 via N‐terminal fusion of ACBP, and obtained a kinetically improved enzyme with augmented protein production levels, which could boost oil accumulation in yeast and plant vegetative tissues.