Skip to main content
SpringerLink
Log in

Improvement of NADPH bioavailability in Escherichia coli through the use of phosphofructokinase deficient strains

  • Applied genetics and molecular biotechnology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

NADPH-dependent reactions play important roles in production of industrially valuable compounds. In this study, we used phosphofructokinase (PFK)-deficient strains to direct fructose-6-phosphate to be oxidized through the pentose phosphate pathway (PPP) to increase NADPH generation. pfkA or pfkB single deletion and double-deletion strains were tested for their ability to produce lycopene. Since lycopene biosynthesis requires many NADPH, levels of lycopene were compared in a set of isogenic strains, with the pfkA single deletion strain showing the highest lycopene yield. Using another NADPH-requiring process, a one-step reduction reaction of 2-chloroacrylate to 2-chloropropionic acid by 2-haloacrylate reductase, the pfkA pfkB double-deletion strain showed the highest yield of 2-chloropropionic acid product. The combined effect of glucose-6-phosphate dehydrogenase overexpression or lactate dehydrogenase deletion with PFK deficiency on NADPH bioavailability was also studied. The results indicated that the flux distribution of fructose-6-phosphate between glycolysis and the pentose phosphate pathway determines the amount of NAPDH available for reductive biosynthesis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • 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(3):155–164. doi:10.1016/j.ymben.2004.12.003

    Article  PubMed  CAS  Google Scholar 

  • 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(5):612–616. doi:10.1038/nbt1083

    Google Scholar 

  • Bartek T, Blombach B, Zonnchen E, Makus P, Lang S, Eikmanns BJ, Oldiges M (2010) Importance of NADPH supply for improved L-valine formation in Corynebacterium glutamicum. Biotechnol Prog 26(2):361–371. doi:10.1002/btpr.345

    Google Scholar 

  • Chemler JA, Fowler ZL, McHugh KP, Koffas MA (2010) Improving NADPH availability for natural product biosynthesis in Escherichia coli by metabolic engineering. Metab Eng 12(2):96–104. doi:10.1016/j.ymben.2009.07.003

    Article  PubMed  CAS  Google Scholar 

  • Chin JW, Cirino PC (2011) Improved NADPH supply for xylitol production by engineered Escherichia coli with glycolytic mutations. Biotechnol Prog 27(2):333–341. doi:10.1002/btpr.559

    Article  PubMed  CAS  Google Scholar 

  • Chin JW, Khankal R, Monroe CA, Maranas CD, Cirino PC (2009) Analysis of NADPH supply during xylitol production by engineered Escherichia coli. Biotechnol Bioeng 102(1):209–220. doi:10.1002/bit.22060

    Article  PubMed  CAS  Google Scholar 

  • 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(8):1107–1121. doi:10.1105/tpc.6.8.1107

    Google Scholar 

  • Farmer WR, Liao JC (2001) Precursor balancing for metabolic engineering of lycopene production in Escherichia coli. Biotechnol Prog 17(1):57–61. doi:10.1021/bp000137t

    Article  PubMed  CAS  Google Scholar 

  • Kim S, Lee CH, Nam SW, Kim P (2011a) Alteration of reducing powers in an isogenic phosphoglucose isomerase (pgi)-disrupted Escherichia coli expressing NAD(P)-dependent malic enzymes and NADP-dependent glyceraldehyde 3-phosphate dehydrogenase. Lett Appl Microbiol 52(5):433–440. doi:10.1111/j.1472-765X.2011.03013.x

    Article  PubMed  CAS  Google Scholar 

  • Kim YM, Cho HS, Jung GY, Park JM (2011b) Engineering the pentose phosphate pathway to improve hydrogen yield in recombinant Escherichia coli. Biotechnol Bioeng 108(12):2941–2946. doi:10.1002/bit.23259

    Article  PubMed  CAS  Google Scholar 

  • Kurata A, Kurihara T, Kamachi H, Esaki N (2005) 2-Haloacrylate reductase, a novel enzyme of the medium chain dehydrogenase/reductase superfamily that catalyzes the reduction of a carbon-carbon double bond of unsaturated organohalogen compounds. J Biol Chem 280(21):20286–20291. doi:10.1074/jbc.M414605200

    Article  PubMed  CAS  Google Scholar 

  • Kurata A, Fujita M, Mowafy AM, Kamachi H, Kurihara T, Esaki N (2008) Production of (S)-2-chloropropionate by asymmetric reduction of 2-chloroacrylate with 2-haloacrylate reductase coupled with glucose dehydrogenase. J Biosci Bioeng 105(4):429–431. doi:10.1263/jbb.105.429

    Google Scholar 

  • Lee WH, Park JB, Park K, Kim MD, Seo JH (2007) Enhanced production of epsilon-caprolactone by overexpression of NADPH-regenerating glucose 6-phosphate dehydrogenase in recombinant Escherichia coli harboring cyclohexanone monooxygenase gene. Appl Microbiol Biotechnol 76(2):329–338. doi:10.1007/s00253-007-1016-7

    Article  PubMed  CAS  Google Scholar 

  • Lovingshimer MR, Siegele D, Reinhart GD (2006) Construction of an inducible, pfkA and pfkB deficient strain of Escherichia coli for the expression and purification of phosphofructokinase from bacterial sources. Protein Expr Purif 46(2):475–482. doi:10.1016/j.pep.2005.09.015

    Article  PubMed  CAS  Google Scholar 

  • 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(6):352–359. doi:10.1016/j.ymben.2008.09.001

    Article  PubMed  CAS  Google Scholar 

  • Phillips GJ, Park SK, Huber D (2000) High copy number plasmids compatible with commonly used cloning vectors. Biotechniques 28(3):400–402, 404, 406 passim

    Google Scholar 

  • Sanchez AM, Andrews J, Hussein I, Bennett GN, San KY (2006) Effect of overexpression of a soluble pyridine nucleotide transhydrogenase (UdhA) on the production of poly(3-hydroxybutyrate) in Escherichia coli. Biotechnol Prog 22(2):420–425. doi:10.1021/bp050375u

    Article  PubMed  CAS  Google Scholar 

  • Siedler S, Bringer S, Bott M (2011) Increased NADPH availability in Escherichia coli: improvement of the product per glucose ratio in reductive whole-cell biotransformation. Appl Microbiol Biotechnol 92(5):929–937. doi:10.1007/s00253-011-3374-4

    Article  PubMed  CAS  Google Scholar 

  • Siedler S, Bringer S, Blank LM, Bott M (2012) Engineering yield and rate of reductive biotransformation in Escherichia coli by partial cyclization of the pentose phosphate pathway and PTS-independent glucose transport. Appl Microbiol Biotechnol 93(4):1459–1467. doi:10.1007/s00253-011-3626-3

    Article  PubMed  CAS  Google Scholar 

  • Vinopal RT, Clifton D, Fraenkel DG (1975) PfkA locus of Escherichia coli. J Bacteriol 122(3):1162–1171

    PubMed  CAS  Google Scholar 

  • Wakao S, Benning C (2005) Genome-wide analysis of glucose-6-phosphate dehydrogenases in Arabidopsis. Plant J 41(2):243–256. doi:10.1111/j.1365-313X.2004.02293.x

    Article  PubMed  CAS  Google Scholar 

  • Walton AZ, Stewart JD (2004) Understanding and improving NADPH-dependent reactions by nongrowing Escherichia coli cells. Biotechnol Prog 20(2):403–411. doi:10.1021/bp030044m

    Article  PubMed  CAS  Google Scholar 

  • Yoon KW, Doo EH, Kim SW, Park JB (2008) In situ recovery of lycopene during biosynthesis with recombinant Escherichia coli. J Biotechnol 135(3):291–294. doi:10.1016/j.jbiotec.2008.04.001

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgement

This work was supported in part by National Science Foundation CBET0828516. Y. Wang was partially supported by a postdoctoral fellowship from the HHMI Beyond Traditional Borders program and by a John S. Dunn Foundation Collaborative Research Award. The authors want to thank Prof. Kurata for providing plasmid pET101-D-topo-CAA43 and Prof. Benning for the gift of pASK-IBA3-G6PD1.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Cell Biology, MS-140, Rice University, 6100 Main Street, Houston, TX, 77005-1892, USA

    Yipeng Wang & George N. Bennett

  2. Department of Bioengineering, Rice University, Houston, TX, 77005, USA

    Ka-Yiu San

  3. Department of Chemical and Biomolecular Engineering, Rice University, Houston, TX, 77251, USA

    Ka-Yiu San

Authors
  1. Yipeng Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ka-Yiu San
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. George N. Bennett
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to George N. Bennett.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, Y., San, KY. & Bennett, G.N. Improvement of NADPH bioavailability in Escherichia coli through the use of phosphofructokinase deficient strains. Appl Microbiol Biotechnol 97, 6883–6893 (2013). https://doi.org/10.1007/s00253-013-4859-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-013-4859-0

Keywords

Search

Navigation