Towards the in vivo production of tocotrienol compounds: Engineering of a plasmid-free Escherichia coli strain for the heterologous synthesis of 2-methyl-6-geranylgeranyl-benzoquinol
The vitamin E family consists of 4 tocopherol and 4 tocotrienol compounds. During recent years, tocotrienols have gained increased interest due to their biological activities that are beyond the vitamin E activity. Here we report the engineering of plasmid-free Escherichia coli strains for an efficient synthesis of 2-methyl-6-geranylgeranyl-benzoquinol (MGGBQ), the central precursor for all four natural tocotrienol compounds.Heterologous genes needed for the in vivo synthesis of MGGBQ in E. coli (crtE, hpd, and hpt) were individually integrated into the chromosome of E. coli. The yield of MGGBQ after cultivation of the plasmid-free recombinant E. coli strain was significantly higher (604 μg/g cdw) compared to an E. coli strain that carries these biosynthesis genes on a multi-copy expression plasmid (325 μg/g cdw). Further chromosomal integration of an additional copy of the isopentenyl-diphosphate isomerase gene (idi) and a subsequent increase in expression level of the deoxy-xylulose synthase gene (dxs) increased the MGGBQ yield by 80% (1110 μg/g cdw) and 135% (1425 μg/g cdw), respectively. MGGBQ which accumulated in the membrane fraction of the recombinant E. coli cells was isolated and its structure was completely elucidated by 1D and 2D NMR and MS measurements. The engineered, plasmid-free E. coli strain is a promising host for the heterologous in vivo production of tocotrienol and its derivatives.
► Construction of a plasmid-free E. coli strain for the synthesis of MGGBQ. ► Chromosomal integration led to higher production of MGGBQ than multi-copy plasmid. ► Formation of MGGBQ increased by enhanced expression of isoprenoid synthesis genes. ► Isolation and structural elucidation of the heterologously produced MGGBQ. ► The system has potential applications for the efficient formation of tocotrienols.
Journal: Journal of Biotechnology - Volume 164, Issue 2, March 2013, Pages 238–247