Sunday, July 26, 2009
P119

Genomic Engineering of Escherichia coli for Stable- and High-Level Production of Lycopene

Yun-Peng Chao, Chemical Engineering, Feng Chia University, 100 Wenhwa Road, Taichung, Taiwan and Chung-Jen Chiang, Department of Medical Laboratory Science and Biotechnology, China Medical University, Taiwan.

Lycopene is a potent antioxidant with a high potential for the health-care product. To be a “clean” production process, a replicon-free and markerless method was developed for chromosomal insertion of genes and controlled expression of genomic genes in Escherichia coli. To approach the former, the integration vectors of conditional replication were incorporated with the prophage attachment site and duplicated FRT sites. For the latter, the template vectors were constructed to carry a DNA cassette containing the T7 promoter and a marker flanked with the FRT site. Therefore, with the aid of integration vectors, an artificial operon, consisting of three heterologous genes gps, crtI, and crtB, controlled by the T7 promoter was first inserted into E. coli genome in a site-specific manner. The antibiotic marker and plasmid replicon associated with the inserted DNA were subsequently removed by FLP recombinase. Subsequently, by a similar approach the rate-limiting step in the lycopene-synthesis pathway was enhanced by insertion of an extra copy of the endogenous dxs gene into E. coli genome. Moreover, to redirect the carbon flux to supplement the precursors, the native promoter of chromosomal pckA was replaced by the T7 promoter using the template vector. Similarly, the selective marker in the integrated DNA was later eliminated from integrants with FLP. Consequently, the expression of these five genes was subject to control by one response regulator, T7 RNA polymerase, in a regulon way, leading to a high and stable production of lycopene in the cell.

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