Study On The Molecular Mechanisms Of Lipid Accumulation During Batch Culture Of Phaeodactylum Tricornutum

Microalgae have garnered a great deal of interest as renewable and sustainable biofuel feedstock because of their advantageous characteristics such as rapid growth and photosynthesis rate, higher lipid content etc. It is well known that extending batch culture would result in elevated lipid content. It has been established that lipid content of microalgal cell was found to be higher during stationary phase than lag phase and logarithmic phase of their growth period. However the molecular mechanism underlying the lipid elevation during lipid accumulating in batch culture remains unclear. Hence, we attempt to study the gene transcription level of Phaeodactylum tricornutum during batch culture. In this study, we used high-through sequencing technology in order to elucidate the genes and metabolic network related to lipid biosynthesis in P. tricornutum under different growth conditions. We found that the gene expression profile of P. tricornutum during lag phase, logarithmic phase and stationary phase was varied significantly. It was proposed that elevated lipid content of P. tricornutum during stationary phase was not only related to lipid biosynthetic pathway, but also due to the combinatorial impact of various metabolic pathways.Moreover, earlier studies showed that fatty acid biosynthesis have been influenced by multiple genes instead of a single rate-limiting gene. Recent studies have been involved in expression of one key gene to generate engineered strain with superior lipid content and suitable for commercial purpose, whereas the attempt have not fulfill the aim of generating superior lipogenic strain with industrial potential. Hence, in this study we attempted to simultaneously overexpress dual rate limiting genes using linker peptide in order to generate lipid-enriched microalgal strain. For this, we chose diacylglycerol acyltransferase 1(DGAT1) and glycerol-3-phosphate acyltransferase(GPAT) which catalyze the critical reaction in fatty acid biosynthetic pathway in diatom P. tricornutum. The former gene(DGAT1) catalyzes the final and committed step in TAG biosynthetic pathway, whereas the later catalyze the first and rate limiting step of key enzyme in the same TAG biosynthetic pathway. These two genes were fused by a linker protein sequence from foot and mouth disease virus in order to influence the self-lysis of the linker; therefore these two genes fused with a linker peptide were expressed under a same promoter. The results showed that content of neutral lipid; EPA and DHA of engineered algae were increased up to 89%, 29.7% and 76.0%, respectively than that of wild type. Total fatty acid content was also found to be increased in engineered lines. Moreover, engineered lines overexpressing dual genes exhibited similar growth and physiological characteristics as wild type cells.Altogether, this study revealed the impact of various gene expression profiles and elucidated the role of several metabolic pathways in lipogenesis of P. tricornutum during stationary phase. The data demonstrate the potential role of dual rate limiting genes in elevating lipid content and generating microalgal strains with commercial potential.