Functional Characterization of Long-Chain Acyl-CoA Synthetase Gene Family from the Oleaginous Alga Chromochloris zofingiensis

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.