Long-chain acyl-coenzyme A (CoA) synthetase (LACS) catalyzes the formation of acyl-CoAs from free fatty acids, which is pivotal for lipid metabolism. Here, we confirmed the presence of six CzLACS genes in Chromochloris zofingiensis. Functional complementation and in vitro enzymatic assay indicated that CzLACS2 through CzLACS5 rather than CzLACS1 or CzLACS6 are bona fide LACS enzymes and they have overlapping yet distinct substrate preference. The results of the subcellular colocalization experiment and different expression patterns under three triacylglycerol (TAG)-inducing conditions showed that CzLACS2 through CzLACS4 reside at endoplasmic reticulum (ER) and are involved in TAG biosynthesis, while CzLACS5 resides in peroxisome and participates in fatty acid β-oxidation. The yeast one-hybrid assay using a library of 50 transcription factors (TFs) constructed in our study identified 12 TFs potentially involved in regulating the expression of CzLACSs. Moreover, heterologous expression of CzLACSs demonstrated their engineering potential for modulating TAG synthesis in yeast and algal cells.

中文翻译：

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产油藻藻类中的长链酰基辅酶A合成酶基因家族的功能表征

长链酰基辅酶A（CoA）合成酶（LACS）催化游离脂肪酸形成酰基辅酶A，这对于脂质代谢至关重要。在这里，我们确定的六个存在CzLACS基因Chromochloris zofingiensis。功能互补和体外酶促测定表明，CzLACS2至CzLACS5而非CzLACS1或CzLACS6是真正的LACS酶，它们具有重叠但明显的底物偏好。亚细胞共定位实验的结果和在三种诱导三酰甘油（TAG）的条件下不同表达模式的结果表明CzLACS2至CzLACS4驻留在内质网（ER）并参与TAG生物合成，而CzLACS5驻留在过氧化物酶体中并参与脂肪酸β -氧化。使用我们研究中构建的50个转录因子（TFs）库进行的酵母单杂交试验鉴定出了12个可能参与调控CzLACS表达的TF。此外，CzLACS的异源表达证实了它们在调节酵母和藻类细胞中TAG合成方面的工程潜力。