Low-copy plasmids can perform as well as or better than high-copy plasmids for metabolic engineering of bacteria.
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.
Jones
K L
KL
Department of Chemical Engineering, University of California, Berkeley, California 94720-1462, USA.
Kim
S W
SW
Keasling
J D
JD
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
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