Kinetic improvement of an algal diacylglycerol acyltransferase 1 via fusion with an acyl‐CoA binding protein

Abstract

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 structureÀfunction relationship, as well as the improvement of DGAT1 performance via protein engineering, remain largely untapped. Here, we explored the structureÀfunction 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.

The oleaginous astaxanthin-producing alga Chromochloris zofingiensis: potential from production to an emerging model for studying lipid metabolism and carotenogenesis

“…In general, microalgae harbor a much larger number of DGAT isoforms than vascular plants (e.g., one versus eleven for the type II), pointing to more complex regulations of microalgal TAG synthesis. Although why microalgae need such high dose of DGATs remains less understood, functional characterization of DGATs from multiple aspects has been conducted for many species including C. reinhardtii [ 183 , 187 , 188 ], C. zofingiensis [ 189 – 191 ], H. pluvialis [ 192 , 193 ], N. oceanica [ 194 – 196 ], and P. tricornutum [ 197 – 199 ]. C. zofingiensis harbors ten putative DGAT isoforms, two type I (DGAT1A and DGAT1B) and eight type II (DGTT1 through DGTT8); all are predicted to be extrachloroplast-targeted [ 189 ].…”

Characterization of a Haematococcus pluvialis Diacylglycerol Acyltransferase 1 and Its Potential in Unsaturated Fatty Acid-Rich Triacylglycerol Production

“…The AST-binding sites were also predicted in HpDGAT1 protein sequences ( Figure 5 ). Moreover, structure–function analysis of the hydrophilic N-terminal domain of HpDGAT1 indicated that the N-terminal domain of HpDGAT1 may play important roles in maintaining enzyme performance, like the case of CzDGAT1 in C. zofingiensis ( Xu et al, 2020 ). All these results provide a new clue for functional role of HpDGAT1 in AST esterification despite further biological experiments, such as the expression of HpDGAT1 in AST-producing yeast, algal, and bacteria strains, are necessary to support this hypothesis.…”

Biochemistry and Biotechnology of Lipid Accumulation in the Microalga Nannochloropsis oceanica

“…For another example, protein fusion also represents a promising strategy to improve lipid enzyme performance and lipid production. With the fusion of DGAT to its substrate carrier protein (acyl-CoA-binding protein), improved enzyme activity and kinetics and increased lipid yield were achieved . Because lipid enzymes might form local metabolomes to facilitate lipid channeling, , bringing lipid players catalyzing consecutive reactions together by protein fusion may enable efficient substrate channeling and lead to enhanced lipid production.…”

“…With the fusion of DGAT to its substrate carrier protein (acyl-CoAbinding protein), improved enzyme activity and kinetics and increased lipid yield were achieved. 71 Because lipid enzymes might form local metabolomes to facilitate lipid channeling, 72,73 bringing lipid players catalyzing consecutive reactions together by protein fusion may enable efficient substrate channeling and lead to enhanced lipid production. In addition to lipid enzymes, master regulators of lipid metabolism might be more effective targets.…”