Functional analysis of a malonyl-CoA:ACP transacylase gene from Phaeodactylum tricornutum

Abstract:

[Background] Malonyl-CoA:ACP transacylase (MCAT), an important subunit of type II fatty acid synthase (FASII), is associated with fatty acid synthesis. However, little information is available on microalgal MCAT. [Objective] To validate the function of the MCAT gene from Phaeodactylum tricornutum. [Methods] We found a possible mcat gene in the whole genome sequence of the model diatom P. tricornutum and cloned this gene. We performed bioinformatic analysis of this gene and introduced it into the MCAT-deficient E. coli L48 strain. Gas chromatography-mass spectrometry (GC-MS) was employed to analyze the composition and content of fatty acids in the mutant strains. [Results] The MCAT in P. tricornutum had the secondary structure mainly composed of α-helix and random coil and the closest genetic relationship with that in Fragilaria cylindrus, being a protits-type MCAT. To verify the function of the gene, we introduced this gene into the MCAT-deficient E. coli L48 strain and found that the strain recovered the function of fatty acid synthesis. Further, we analyzed the fatty acid composition of the reverting and found that the enzyme had a substrate preference to C14:0. The MCAT promoted the synthesis of medium- and long-chain fatty acids such as C16:0 and C17:1. This feature is largely consistent with the characteristics of protits-type MCATs. [Conclusion] There is a protits-type MCAT in P. tricornutum. This study provides new clues for the study of fatty acid synthesis and metabolism in microalgae, which is beneficial to the research and application of microalgal lipids.

Key words:Phaeodactylum tricornutum;malonyl-CoA:ACP transacylase;fatty acid synthesis;functional verification