title: Low-copy plasmids can perform as well as or better than high-copy plasmids for metabolic engineering of bacteria.
abstract: 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.
authors: Department of Chemical Engineering, University of California, Berkeley, California 94720-1462, USA.
authors: K L
authors: KL
authors: Jones
authors: S W
authors: SW
authors: Kim
authors: J D
authors: JD
authors: Keasling
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000235:genetics / Q000378:metabolism; D001089:Arabinose / Q000378:metabolism; D002338:Carotenoids / Q000378:metabolism; D002455:Cell Division; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering; D045782:Hemiterpenes; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D000077276:Lycopene; D009154:Mutation / Q000235:genetics; D009943:Organophosphorus Compounds / Q000378:metabolism; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D011122:Polyphosphates / Q000378:metabolism; D014166:Transferases / Q000235:genetics / Q000378:metabolism
chemical_list: D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D011122:Polyphosphates; C004809:isopentenyl pyrophosphate; D002338:Carotenoids; D007544:Isopropyl Thiogalactoside; D001089:Arabinose; D014166:Transferases; C109461:deoxyxylulose-5-phosphate synthase; D017856:Phosphotransferases (Phosphate Group Acceptor); C022424:polyphosphate kinase; D017766:Acid Anhydride Hydrolases; C022563:endopolyphosphatase; D000077276:Lycopene