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.

中文翻译：

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通过与酰基辅酶A结合蛋白融合改善藻类二酰基甘油酰基转移酶 1 的动力学。


三酰甘油（TAG）形式的微藻油被广泛用作营养补充剂和生物燃料。二酰基甘油酰基转移酶 (DGAT) 催化酰基辅酶 A 依赖性 TAG 生物合成的最后一步，被认为是操纵石油生产的关键目标。尽管越来越多的 DGAT1 已在一些藻类物种中被鉴定并过度表达，但详细的结构-功能关系以及通过蛋白质工程改进 DGAT1 的性能在很大程度上仍未得到开发。在这里，我们探索了绿色微藻 Chromocholis zofingiensis (CzDGAT1) 的 DGAT1 亲水性 N 端结构域的结构-功能特征。结果表明，CzDGAT1 的 N 端结构域比高等真核酶的 N 端结构域的无序程度要低，其部分截断或完全去除可显着降低酶活性，表明其可能在维持酶性能方面发挥作用。尽管之前发现动物和植物 DGAT1 的 N 端结构域可以结合酰基辅酶 A，但用酰基辅酶 A 结合蛋白 (ACBP) 替换 CzDGAT1 N 端并不能恢复酶活性。有趣的是，ACBP 与全长 CzDGAT1 N 端的融合可以增强酶对酰基辅酶 A 的亲和力并增加蛋白质积累水平，最终推动酵母细胞和烟草叶中的油脂积累达到比全长 CzDGAT1 更高的水平。长度CzDGAT1。总体而言，我们的研究结果揭示了藻类 DGAT1 N 末端的独特特征，并提供了一种通过蛋白质融合增强 DGAT1 性能的策略，这可能为产生改进的膜结合酰基辅酶 A 依赖性酶和促进油的产生开辟了前景。植物和产油微生物中的生物合成。