Phaeodactylum Tricornutum Chloroplast Expression System For The Overexpression Of Acetyl-CoA Carboxylase

The marine diatom Phaeodactylum tricornutum, a widely used forage species, has astorage lipid content of up to30%dry cell weight. To explore the mechanism behindthe high storage lipid accumulation in this diatom, acetyl-CoA carboxylase (ACCase),which catalyzes the first committed step of the fatty acid biosynthetic pathway, wascharacterized in this study. A homogeneous type of ACCase (PtACC) was identifiedfrom P. tricornutum by homology searches. The first exon of the ACCase genedesignated PtACC-1was cloned to the pre-constructed plasmid pPtc-T, a highefficiency chloroplast transformation vector which contains a rbcL promoter from P.tricornutum chloroplast genome. The resultant chloroplast transformation vectorpPtc-PtACC-1was transferred into the chloroplast of P. tricornutum byelectroporation. Transplastomic P. tricornutum cells were obtained uponchloramphenicol selection, and confirmed by genomic PCR screening of CAT gene.High expression level of PtACC-1was demonstrated by real-time quantitative PCRand Western blot analysis. The neutral lipid content in transformed E. coli and P.tricornutum increased substantially by2.36-fold and1.74-fold, respectively, asdetermined by Nile red fluorescent dye staining and the observation of oil bodiesunder a laser scanning confocal microscope. Concomitantly, enzyme activity ofACCase increased by3.3-fold. The fatty acid composition, analyzed by GC-MS,showed significant changes with a56%decrease in polyunsaturated fatty acids and a25.4%increase in monounsaturated fatty acids. This study represents the firstcharacterization of the key domains of ACCase from a diatom and demonstrates highneutral lipid accumulation in P. tricornutum expressing PtACC-1, providing anadditional genetic resource with potential for biodiesel development.