Enhanced heterologous production of eicosapentaenoic acid in Escherichia coli cells that co-express eicosapentaenoic acid biosynthesis pfa genes and foreign DNA fragments including a high-performance catalase gene, vktA

Abstract

Cellular eicosapentaenoic acid (EPA) makes up approximately 3% of total fatty acids in Escherichia coli DH5α, a strain that carries EPA biosynthesis genes (pEPAΔ1). EPA was increased to 12% of total fatty acids when the host cell coexpressed the vector pGBM3::sa1(vktA), which carried the high-performance catalase gene, vktA. Where this vector was coexpressed, the transformant accumulated a large amount of VktA protein. However, the EPA production of cells carrying the vector that included the insert lacking almost the entire vktA gene was approximately 6%. This suggests that the retention of a large DNA insert in the vector and the accumulation of the resulting protein, but not the catalytic activity of VktA catalase, would potentially be able to increase the content of EPA.

Enhanced production of polyunsaturated fatty acids by enzyme engineering of tandem acyl carrier proteins

“…In the case of the aerobic desaturase/elongase pathway, improvement of reducing equivalent (NADPH) flux, utilization of inhibiters of the acyl-exchange reaction between phosphatidylcholine and acyl-CoA substrates, and prevention of PUFA degradation by β-oxidation were shown to be effective for titre improvement 6 23 . For anaerobic PUFA synthase, co-expression of catalase 24 , addition of cerulenin 25 26 , an inhibiter of de novo fatty acid synthesis, and metabolic engineering to increase the substrate supply 27 have been employed. However, all these attempts have focussed only on the metabolic flow and no examples of activation of enzyme activity have been reported.…”

Exogenous Catalase Gene Expression as a Tool for Enhancing Metabolic Activity and Production of Biomaterials in Host Microorganisms

“…Catalase activity was increased to 535 U/mg protein for DH5α(pEPAΔ1)(pGBM3::vktA). However, there was no enhancement of catalase activity in DH5α(pEPAΔ1)(pGBM3::ΔvktA) Orikasa et al (2007).…”

pfaBproducts determine the molecular species produced in bacterial polyunsaturated fatty acid biosynthesis

“…Fatty acids were converted to methyl esters and then analyzed as described (Orikasa et al , 2006a). DHA and EPA were identified by comparing their retention time on gas–liquid chromatography with standards and by GC‐MS as described previously (Orikasa et al , 2006a, 2007).…”

“…Various constructs are available that contain the pfa genes of S. pneumatophori SCRC‐2738. In addition to cosmid and plasmid vectors that carry all five pfa genes, deletion constructs lacking one of the five pfa genes and clones containing individual pfa genes have been prepared (Yazawa et al , 1998; Orikasa et al , 2004, 2007). These clones have been used to increase the heterologous production of EPA (Orikasa et al , 2004, 2007) and to determine the physiological functions of EPA in E. coli recombinant systems (Nishida et al , 2006a, b; Okuyama et al , 2008).…”