Metabolic Engineering of Carotenoid Biosynthesis in Escherichia coli by Ordered Gene Assembly in Bacillus subtilis

We attempted to optimize the production of zeaxanthin inEscherichia coli by reordering five biosynthetic genes in the natural carotenoid cluster ofPantoea ananatis . Newly designed operons for zeaxanthin production were constructed by the ordered gene assembly inBacillus subtilis (OGAB) method, which can assemble multiple genes in one step using an intrinsicB. subtilis plasmid transformation system. The highest level of production of zeaxanthin inE. coli (820 μg/g [dry weight]) was observed in the transformant with a plasmid in which the gene order corresponds to the order of the zeaxanthin metabolic pathway (crtE-crtB-crtI-crtY-crtZ ), among a series of plasmids with circularly permuted gene orders. Although two of five operons using intrinsic zeaxanthin promoters failed to assemble inB. subtilis , the full set of operons was obtained by repressing operon expression during OGAB assembly with ap R promoter-c I repressor system. This result suggests that repressing the expression of foreign genes inB. subtilis is important for their assembly by the OGAB method. For all tested operons, the abundance of mRNA decreased monotonically with the increasing distance of the gene from the promoter inE. coli , and this may influence the yield of zeaxanthin. Our results suggest that rearrangement of biosynthetic genes in the order of the metabolic pathway by the OGAB method could be a useful approach for metabolic engineering.