Metabolic engineering of the nonmevalonate isopentenyl diphosphate synthesis pathway in Escherichia coli enhances lycopene production

Isopentenyl diphosphate (IPP) is the common, five-carbon building block in the biosynthesis of all carotenoids. IPP in Escherichia coli is synthesized through the nonmevalonate pathway, which has not been completely elucidate d. The first reaction of IPP biosynthesis in E. coli is the formation of 1- deoxy-D-xylulose-5-phosphate (DXP), catalyzed by DXP synthase and encoded b y dxs. The second reaction in the pathway is the reduction of DXP to 2-C-me thyl-D-erythritol-4-phosphate, catalyzed by DXP reductoisomerase and encode d by dxr. To determine if one or more of the reactions in the nonmevalonate pathway controlled flux to IPP, dxs and dxr were placed on several express ion vectors under the control of three different promoters and transformed into three E. coli strains (DH5 alpha, XL1-Blue, and JM101) that had been e ngineered to produce lycopene. Lycopene production was improved significant ly in strains transformed with the dxs expression vectors. When the dxs gen e was expressed from the arabinose-inducible araBAD promoter (P-BAD) on a m edium-copy plasmid, lycopene production was twofold higher than when dxs wa s expressed from the IPTG-inducible trc and lac promoters (P-trc and P-lac' respectively) on medium-copy and high-copy plasmids. Given the low final d ensities of cells expressing dxs from IPTG-inducible promoters, the low lyc opene production was probably due to the metabolic burden of plasmid mainte nance and an excessive drain of central metabolic intermediates. At arabino se concentrations between 0 and 1.33 mM, cells expressing both dxs and dxr from P-BAD on a medium-copy plasmid produced 1.4-2.0 times more lycopene th an cells expressing dxs only. However, at higher arabinose concentrations l ycopene production in cells expressing both dxs and dxr was lower than in c ells expressing dxs only. A comparison of the three E. coli strains transformed with the arabinose-in ducible dxs on a medium-copy plasmid revealed that lycopene production was highest in XL1-Blue. (C) 2001 John Wiley & Sons, Inc.