Multiple mechanisms contribute to increased neutral lipid accumulation in yeast producing recombinant variants of plant diacylglycerol acyltransferase 1

Abstract

The apparent bottleneck in the accumulation of oil during seed development in some oleaginous plant species is the formation of triacylglycerol (TAG) by the acyl-CoA-dependent acylation of -1,2-diacylglycerol catalyzed by diacylglycerol acyltransferase (DGAT, EC 2.3.1.20). Improving DGAT activity using protein engineering could lead to improvements in seed oil yield ( in canola-type ). Directed evolution of DGAT1 (BnaDGAT1) previously revealed that one of the regions where amino acid residue substitutions lead to higher performance in BnaDGAT1 is in the ninth predicted transmembrane domain (PTMD9). In this study, several BnaDGAT1 variants with amino acid residue substitutions in PTMD9 were characterized. Among these enzyme variants, the extent of yeast TAG production was affected by different mechanisms, including increased enzyme activity, increased polypeptide accumulation, and possibly reduced substrate inhibition. The kinetic properties of the BnaDGAT1 variants were affected by the amino acid residue substitutions, and a new kinetic model based on substrate inhibition and sigmoidicity was generated. Based on sequence alignment and further biochemical analysis, the amino acid residue substitutions that conferred increased TAG accumulation were shown to be present in the DGAT1-PTMD9 region of other higher plant species. When amino acid residue substitutions that increased BnaDGAT1 enzyme activity were introduced into recombinant DGAT1, they also improved enzyme performance. Thus, the knowledge generated from directed evolution of DGAT1 in one plant species can be transferred to other plant species and has potentially broad applications in genetic engineering of oleaginous crops and microorganisms.

Properties and Biotechnological Applications of Acyl‐CoA:diacylglycerol Acyltransferase and Phospholipid:diacylglycerol Acyltransferase from Terrestrial Plants and Microalgae

“…DGAT1 has been shown to be modulated by its substrate, acyl‐CoA, which binds to an allosteric site at the N‐terminus (Caldo et al, ; Weselake et al, ). The presence of this allosteric site for acyl‐CoA agrees with kinetic studies of microsomal and purified plant DGAT1 showing that DGAT1 exhibits positive cooperativity with acyl‐CoA (Caldo et al, ; Roesler et al, ; Xu et al, ). Interestingly, CoA was identified as a feedback inhibitor of BnaDGAT1 and was shown to bind to the same allosteric site for acyl‐CoA.…”

“…Many improved BnaDGAT1 variants were generated using the aforementioned method and the two most promising ones were used to increase the oil content of tobacco leaves (Chen et al, ). Kinetic analysis indicated that one of the BnaDGAT1 variants exhibited apparent decreased substrate inhibition at concentrations of acyl‐CoA beyond 5 μM (Xu et al, ). The possible role of the ninth and tenth predicted TMD in enzyme regulation was also identified as a considerable number of beneficial mutations were localized near and within this region (Chen et al, ).…”

“…Recent advances in genome‐editing techniques, such as, CRISPR (Belhaj et al, ) and targeting‐induced local lesions in genomes (TILLING; Till et al, ), open up new perspectives on improving the enzyme action in planta . For instance, the “super DGAT” variants with single amino‐acid residue substitutions generated in B. napus DGAT1 provide valuable candidates for genome editing of DGAT1 in different species using CRISPR and TILLING (Chen et al, ; Xu et al, ). The knowledge obtained from directed evolution, in turn, can provide novel and valuable insights into structure–function relationships of DGAT1 (Chen et al, ), especially since no detailed three‐dimensional structure is available for the entire DGAT1 enzyme.…”

Diacylglycerol acyltransferase 1 is activated by phosphatidate and inhibited by SnRK1‐catalyzed phosphorylation

“…The plots of reaction velocity as a function of increasing oleoyl‐CoA concentration were fitted with the Michaelis–Menten equation or the Hill model. Based on R 2 values, the Hill equation was the preferred model, suggesting that BnaDGAT1 exhibits cooperative substrate binding behavior with oleoyl‐CoA (Table ), as recently described for recombinant BnaDGAT1 in yeast microsomes (Caldo et al ., ; Xu et al ., ). The microsomal preparation, however, is subject to possible interference with other proteins that interact with acyl‐CoA, but the current results obtained with purified protein are similar to that observed using microsomes containing recombinant enzyme.…”

“…This result suggests that BnaDGAT1 undergoes substrate inhibition. Once oleoyl‐CoA concentrations greater than 5 μM are reached, the plot prefers a combined model for the Hill equation and substrate inhibition (Table S1), which was recently used to account for the effects of increasing oleoyl‐CoA concentration on the activity of recombinant BnaDGAT1 in yeast microsomes (Xu et al ., ). The combined model was previously proposed for another acyltransferase that exhibited the same kinetic behavior (Dovala et al ., ).…”