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

Publication Type

Journal contribution (peer reviewed)

Authors

Ghanegaonkar, S.; Conrad, J.; Beifuss, U.; Sprenger, G. A.; Albermann, C.

Year of publication

2012

Published in

J. Biotechnol.

Band/Volume

164/

DOI

10.1016/j.jbiotec.2012.08.010

Page (from - to)

238-247

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 E. coli strains for an efficient synthesis of 2-methyl-6-geranylgeranyl-benzoquinone (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 pyrophosphate 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 ¹H- and ¹³C-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.