Optimization of docosahexaenoic acid production by heterologous expression in Escherichia coli of pfa gene cluster from Moritella marina

Abstract

Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid, whose usage has risen in the last few years due to its benefits on human health. The main goal of this project is the sustainable production of DHA with a better quality and cheaper production in microorganisms. This molecule is found naturally in many marine bacteria because it is needed to increase the fluidity of cell membrane as a cold-resistance mechanism adaptation. A fatty acid synthase coded by a 25-Kbp PKS-like gene cluster (pfaABCD) converts malonyl-CoA into DHA. Heterologous expression of Moritella marina pfaABCD gene cluster in Escherichia coli produced a 5% DHA of total fatty acids at 15ºC, but no DHA was found in cells grown at 25ºC. Gene expression analysis by RT-qPCR showed a decrease in pfaA, pfaC and pfaD when cultured at higher temperatures, which could partially indicate a cold-mediated gene expression regulation. pfaE increased expression by T7 promoter induction with IPTG does not increase the DHA production, meaning PPTase activity is not a limiting factor in the process. In addition, a diacylglycerol acyl transferase from Thermomonospora curvata (tDGAT) was co-expressed with pfa gene cluster in order to divert DHA from the membrane to triacylglycerols (TAGs) and avoid an increase of membrane fluidity at high temperatures. Results showed a correct formation of TAGs but no DHA was detected at 25ºC. Further studies are needed to determine DGAT ability to incorporate DHA into TAG.