Abstract
Genetic manipulation was undertaken in order to understand the mechanism involved in the heterologous synthesis of lycopene in Escherichia coli. Knockout of the central carbon metabolic gene zwf (glucose-6-phosphate dehydrogenase) resulted in the enhancement of lycopene production (above 130 % relative to control). The amplification and overexpression of rate-limiting steps encoded by idi (isopentenyl diphosphate isomerase), dxs (1-deoxyxylulose-5-phosphate synthase) and ispDF (4-diphosphocytidyl-2C-methyl-d-erythritol synthase and 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase) genes improved lycopene synthesis from 0.89 to 5.39 mg g−1 DCW. The combination of central metabolic genes knockout with the amplification of MEP pathway genes yielded best amounts of lycopene (6.85–7.55 mg g−1 DCW). Transcript profiling revealed that idi and dxs were up-regulated in the zwf knock-out strain, providing a plausible explanation for the increase in lycopene yield observed in this strain. An increase in precursor availability might also have contributed to the improved lycopene production.
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Ajikumar PK, Xiao WH, Tyo KE, Wang Y, Simeon F, Leonard E, Mucha O, Phon TH, Pfeifer B, Stephanopoulos G (2010) Isoprenoid pathway optimization for taxol precursor overproduction in Escherichia coli. Science 330:70–74
Albermann C (2011) High versus low level expression of the lycopene biosynthesis genes from Pantoea ananatis in Escherichia coli. Biotechnol Lett 33:313–319
Albrecht M, Misawa N, Sandmann G (1999) Metabolic engineering of the terpenoid biosynthetic pathway of Escherichia coli for production of the carotenoids b-carotene and zeaxanthin. Biotechnol Lett 21:791–795
Alper H, Jin YS, Moxley JF, Stephanopoulos G (2005a) Identifying gene targets for the metabolic engineering of lycopene biosynthesis in Escherichia coli. Metab Eng 7:155–164
Alper H, Miyaoku K, Stephanopoulos G (2005b) Construction of lycopene-overproducing E. coli strains by combining systematic and combinatorial gene knockout targets. Nat Biotechnol 23:612–616
Bignotto L, Rocha J, Sepodes B et al (2009) Anti-inflammatory effect of lycopene on carrageenan-induced paw oedema and hepatic ischaemia-reperfusion in the rat. Br J Nutr 102:126–133
Choi HS, Lee SY, Kim TY, Woo HM (2010) In silico identification of gene amplification targets for improvement of lycopene production. Appl Environ Microbiol 76:3097–3105
Cunningham FX Jr, Sun Z, Chamovitz D, Hirschberg J, Gantt E (1994) Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942. Plant Cell 6:1107–1121
Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97:6640–6645
Erdman JW, Ford NA, Lindshield BL (2009) Are the health attributes of lycopene related to its antioxidant function? Arch Biochem Biophys 483:229–235
Farmer WR, Liao JC (2001) Precursor balancing for metabolic engineering of lycopene production in Escherichia coli. Biotechnol Prog 17:57–61
Hua Q, Yang C, Baba T, Mori H, Shimizu K (2003) Responses of the central metabolism in Escherichia coli to phosphoglucose isomerase and glucose-6-phosphate dehydrogenase knockouts. J Bacteriol 185:7053–7067
Jin YS, Stephanopoulos G (2007) Multi-dimensional gene target search for improving lycopene biosynthesis in Escherichia coli. Metab Eng 9:337–347
Kajiwara S, Fraser PD, Kondo K, Misawa N (1997) Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid biosynthesis in Escherichia coli. Biochem J 324:421–426
Kim SW, Keasling JD (2001) Metabolic engineering of the nonmevalonate isopentenyl diphosphate synthesis pathway in Escherichia coli enhances lycopene production. Biotechnol Bioeng 72:408–415
Kim SW, Kim JB, Ryu JM, Jung JK, Kim JH (2009) High-level production of lycopene in metabolically engineered E. coli. Process Biochem 44:899–905
Leonard E, Ajikumar PK, Thayer K, Xiao WH, Mo JD, Tidor B, Stephanopoulos G, Prather KLJ (2010) Combining metabolic and protein engineering of a terpenoid biosynthetic pathway for overproduction and selectivity control. Proc Natl Acad Sci USA 107:13654–13659
Lim SJ, Jung YM, Shin HD, Lee YH (2002) Amplification of the NADPH-related genes zwf and gnd for the oddball biosynthesis of PHB in an E. coli transformant harboring a cloned phbCAB operon. J Biosci Bioeng 93:543–549
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408
Martin VJJ, Pitera DJ, Withers ST, Newman JD, Keasling JD (2003) Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat Biotechnol 21:796–802
Martinez I, Zhu J, Lin H, Bennett GN, San KY (2008) Replacing Escherichia coli NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridium acetobutylicum facilitates NADPH dependent pathways. Metab Eng 10:352–359
Matthews PD, Wurtzel ET (2000) Metabolic engineering of carotenoid accumulation in Escherichia coli by modulation of the isoprenoid precursor pool with expression of deoxyxylulose phosphate synthase. Appl Microbiol Biotechnol 53:396–400
Mein JR, Lian FZ, Wang XD (2008) Biological activity of lycopene metabolites: implications for cancer prevention. Nutr Rev 66:667–683
Mickus BE (2009) Transcriptomic and proteomic analysis of lycopene-overproducing Escherichia coli strains. US, Massachusetts Institute of Technology
Misawa N, Nakagawa M, Kobayashi K, Yamano S, Izawa Y, Nakamura K, Harashima K (1990) Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli. J Bacteriol 172:6704–6712
Reiling KK, Yoshikuni Y, Martin VJJ, Newman J, Bohlmann J, Keasling JD (2004) Mono and diterpene production in Escherichia coli. Biotechnol Bioeng 87:200–212
Rodríguez-Villalón A, Perez-Gil J, Rodríguez-Concepción M (2008) Carotenoid accumulation in bacteria with enhanced supply of isoprenoid precursors by upregulation of exogenous or endogenous pathways. J Biotechnol 135:78–84
Rude MA, Schirmer A (2009) New microbial fuels: a biotech perspective. Curr Opin Microbiol 12:274–281
Sauer U, Lasko DR, Fiaux J, Hochuli M, Glaser R, Szyperski T, Wüthrich K, Bailey JE (1999) Metabolic flux ratio analysis of genetic and environmental modulations of Escherichia coli central carbon metabolism. J Bact 181:6679–6688
Sitthithaworn W, Wungsintaweekul J, Sirisuntipong T, Charoonratana T, Ebizuka Y, De Eknamkul W (2010) Cloning and expression of 1-deoxy-d-xylulose 5-phosphate synthase cDNA from Croton stellatopilosus and expression of 2C-methyl-d-erythritol 4-phosphate synthase and geranylgeranyl diphosphate synthase, key enzymes of plaunotol biosynthesis. J Plant Physiol 167:292–300
Vadali RV, Fu YC, Bennett GN, San KY (2005) Enhanced lycopene productivity by manipulation of carbon flow to isopentenyl diphosphate in Escherichia coli. Biotechnol Prog 21:1558–1561
Wang CW, Oh MK, Liao JC (1999) Engineered isoprenoid pathway enhances astaxanthin production in Escherichia coli. Biotechnol Bioeng 62:235–241
Wang CW, Oh MK, Liao JC (2000) Directed evolution of metabolically engineered Escherichia coli for carotenoid production. Biotechnol Prog 16:922–926
Ye VM, Bhatia SK (2012) Pathway engineering strategies for production of beneficial carotenoids in microbial hosts. Biotechnol Lett 34:1405–1414
Yuan LZ, Rouvière PE, LaRossa RA, Suh W (2006) Chromosomal promoter replacement of the isoprenoid pathway for enhancing carotenoid production in E. coli. Metab Eng 8:79–90
Zhao J, Baba T, Mori H, Shimizu K (2004a) Effect of zwf gene knockout on the metabolism of Escherichia coli grown on glucose or acetate. Metab Eng 6:164–174
Zhao J, Baba T, Mori H, Shimizu K (2004b) Global metabolic response of Escherichia coli to gnd or zwf gene-knockout, based on 13C-labeling experiments and the measurement of enzyme activities. Appl Microbiol Biotechnol 64:91–98
Acknowledgments
This work was financially supported by National Basic Research Program of China (973 Program) (2012CB721101), National Special Fund for State Key Laboratory of Bioreactor Engineering (2060204), and partially supported by Shanghai Leading Academic Discipline Project (B505). We thank one of the reviewers for his/her valuable comments.
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Zhou, Y., Nambou, K., Wei, L. et al. Lycopene production in recombinant strains of Escherichia coli is improved by knockout of the central carbon metabolism gene coding for glucose-6-phosphate dehydrogenase. Biotechnol Lett 35, 2137–2145 (2013). https://doi.org/10.1007/s10529-013-1317-0
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DOI: https://doi.org/10.1007/s10529-013-1317-0