isopropyl-thiogalactoside and isopentenyl-pyrophosphate

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 elucidated. 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 by dxs. The second reaction in the pathway is the reduction of DXP to 2-C-methyl-D-erythritol-4-phos- phate, catalyzed by DXP reductoisomerase and encoded 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 expression vectors under the control of three different promoters and transformed into three E. coli strains (DH5alpha, XL1-Blue, and JM101) that had been engineered to produce lycopene. Lycopene production was improved significantly in strains transformed with the dxs expression vectors. When the dxs gene was expressed from the arabinose-inducible araBAD promoter (P(BAD)) on a medium-copy plasmid, lycopene production was twofold higher than when dxs was 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 densities of cells expressing dxs from IPTG-inducible promoters, the low lycopene production was probably due to the metabolic burden of plasmid maintenance and an excessive drain of central metabolic intermediates. At arabinose 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 than cells expressing dxs only. However, at higher arabinose concentrations lycopene production in cells expressing both dxs and dxr was lower than in cells expressing dxs only. A comparison of the three E. coli strains transformed with the arabinose-inducible dxs on a medium-copy plasmid revealed that lycopene production was highest in XL1-Blue.

Topics: Aldose-Ketose Isomerases; Arabinose; Carotenoids; Escherichia coli; Gene Expression Regulation, Bacterial; Genetic Vectors; Hemiterpenes; Isopropyl Thiogalactoside; Lycopene; Multienzyme Complexes; Organophosphorus Compounds; Oxidoreductases; Pentosephosphates; Promoter Regions, Genetic; Recombinant Fusion Proteins; Transferases; Transformation, Bacterial

A biosynthetic 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1. 1.1.34), the rate-limiting enzyme of the mevalonate pathway for isopentenyl diphosphate biosynthesis, had previously been purified from Streptomyces sp. strain CL190 and its corresponding gene (hmgr) had been cloned (S. Takahashi, T. Kuzuyama, and H. Seto, J. Bacteriol. 181:1256-1263, 1999). Sequence analysis of the flanking regions of the hmgr gene revealed five new open reading frames, orfA to -E, which showed similarity to those encoding eucaryotic and archaebacterial enzymes for the mevalonate pathway. Feeding experiments with [1-(13)C]acetate demonstrated that Escherichia coli JM109 harboring the hmgr gene and these open reading frames used the mevalonate pathway under induction with isopropyl beta-D-thiogalactopyranoside. This transformant could grow in the presence of fosmidomycin, a potent and specific inhibitor of the nonmevalonate pathway, indicating that the mevalonate pathway, intrinsically absent in E. coli, is operating in the E. coli transformant. The hmgr gene and orfABCDE are thus unambiguously shown to be responsible for the mevalonate pathway and to form a gene cluster in the genome of Streptomyces sp. strain CL190.

Topics: Antigens, Bacterial; Escherichia coli; Fosfomycin; Hemiterpenes; Hydroxymethylglutaryl CoA Reductases; Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent; Isopropyl Thiogalactoside; Magnetic Resonance Spectroscopy; Mevalonic Acid; Models, Chemical; Open Reading Frames; Organophosphorus Compounds; Sequence Analysis, DNA; Streptomyces

Multicopy plasmids are often chosen for the expression of recombinant genes in Escherichia coli. The high copy number is generally desired for maximum gene expression; however, the metabolic burden effects that usually result from multiple plasmid copies could prove to be detrimental for maximum productivity in certain metabolic engineering applications. In this study, low-copy mini-F plasmids were compared to high-copy pMB1-based plasmids for production of two metabolites in E. coli: polyphosphate (polyP) and lycopene derived from isopentenyl diphosphate (IPP). The stationary-phase accumulation of polyP on a per cell basis was enhanced approximately 80% when either high- or low-copy plasmids were used, from 120 micromol/g DCW without augmented polyP kinase (PPK) activity to approximately 220 micromol/g DCW. The cell density of the high-copy plasmid-containing culture at stationary phase was approximately 24% lower than the low-copy culture and 30% lower than the control culture. This difference in cell density is likely a metabolic burden effect and resulted in a lower overall product concentration for the high-copy culture (approximately 130 micromol/L culture) relative to the low-copy culture (approximately 160 micromol/L culture). When the gene for DXP (1-deoxy-D-xylulose 5-phosphate) synthase, the first enzyme in the IPP mevalonate-independent biosynthetic pathway, was expressed from the tac promoter on multicopy and low-copy plasmids, lycopene production was enhanced two- to threefold over that found in cells expressing the chromosomal copy only. Cell growth and lycopene production decreased substantially when isopropyl beta-D-thiogalactosidase (IPTG) was added to the high-copy plasmid-containing culture, suggesting that overexpression of DXP synthase was a significant metabolic burden. In the low-copy plasmid-containing culture, no differences in cell growth or lycopene production were observed with any IPTG concentrations. When dxs was placed under the control of the arabinose-inducible promoter (P(BAD)) on the low-copy plasmid, the amount of lycopene produced was proportional to the arabinose concentration and no significant changes in cell growth resulted. These results suggest that low-copy plasmids may be useful in metabolic engineering applications, particularly when one or more of the substrates used in the recombinant pathway are required for normal cellular metabolism.

Topics: Acid Anhydride Hydrolases; Arabinose; Carotenoids; Cell Division; Escherichia coli; Gene Dosage; Gene Expression Regulation, Bacterial; Genetic Engineering; Hemiterpenes; Isopropyl Thiogalactoside; Lycopene; Mutation; Organophosphorus Compounds; Phosphotransferases (Phosphate Group Acceptor); Plasmids; Polyphosphates; Transferases