Citrate As Cost-Efficient NADPH Regenerating Agent

Overview

Journal Front Bioeng Biotechnol

Date 2019 Jan 12

PMID 30631764

Citations 4

Authors

Reinhard Oeggl

Timo Neumann

Jochem Gatgens

Diego Romano

Stephan Noack

Dorte Rother

Affiliations

Soon will be listed here.

Abstract

The economically efficient utilization of NAD(P)H-dependent enzymes requires the regeneration of consumed reduction equivalents. Classically, this is done by substrate supplementation, and if necessary by addition of one or more enzymes. The simplest method thereof is whole cell NADPH regeneration. In this context we now present an easy-to-apply whole cell cofactor regeneration approach, which can especially be used in screening applications. Simply by applying citrate to a buffer or directly using citrate/-phosphate buffer NADPH can be regenerated by native enzymes of the TCA cycle, practically present in all aerobic living organisms. Apart from viable-culturable cells, this regeneration approach can also be applied with lyophilized cells and even crude cell extracts. This is exemplarily shown for the synthesis of 1-phenylethanol from acetophenone with several oxidoreductases. The mechanism of NADPH regeneration by TCA cycle enzymes was further investigated by a transient isotopic labeling experiment feeding [1,5-C]citrate. This revealed that the regeneration mechanism can further be optimized by genetic modification of two competing internal citrate metabolism pathways, the glyoxylate shunt, and the glutamate dehydrogenase.

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References

Liese A, Filho M . Production of fine chemicals using biocatalysis. Curr Opin Biotechnol. 1999; 10(6):595-603. DOI: 10.1016/s0958-1669(99)00040-3. View

Noronha S, Yeh H, Spande T, Shiloach J . Investigation of the TCA cycle and the glyoxylate shunt in Escherichia coli BL21 and JM109 using (13)C-NMR/MS. Biotechnol Bioeng. 2000; 68(3):316-27. View

Schwaneberg U, Otey C, Cirino P, Farinas E, Arnold F . Cost-effective whole-cell assay for laboratory evolution of hydroxylases in Escherichia coli. J Biomol Screen. 2001; 6(2):111-7. DOI: 10.1177/108705710100600207. View

Straathof A, Panke S, Schmid A . The production of fine chemicals by biotransformations. Curr Opin Biotechnol. 2002; 13(6):548-56. DOI: 10.1016/s0958-1669(02)00360-9. View

van der Donk W, Zhao H . Recent developments in pyridine nucleotide regeneration. Curr Opin Biotechnol. 2003; 14(4):421-6. DOI: 10.1016/s0958-1669(03)00094-6. View

Burton S . Oxidizing enzymes as biocatalysts. Trends Biotechnol. 2003; 21(12):543-9. DOI: 10.1016/j.tibtech.2003.10.006. View

Phue J, Shiloach J . Transcription levels of key metabolic genes are the cause for different glucose utilization pathways in E. coli B (BL21) and E. coli K (JM109). J Biotechnol. 2004; 109(1-2):21-30. DOI: 10.1016/j.jbiotec.2003.10.038. View

Wichmann R, Vasic-Racki D . Cofactor regeneration at the lab scale. Adv Biochem Eng Biotechnol. 2005; 92:225-60. DOI: 10.1007/b98911. View

Phue J, Noronha S, Hattacharyya R, Wolfe A, Shiloach J . Glucose metabolism at high density growth of E. coli B and E. coli K: differences in metabolic pathways are responsible for efficient glucose utilization in E. coli B as determined by microarrays and Northern blot analyses. Biotechnol Bioeng. 2005; 90(7):805-20. DOI: 10.1002/bit.20478. View

10.

Studier F . Protein production by auto-induction in high density shaking cultures. Protein Expr Purif. 2005; 41(1):207-34. DOI: 10.1016/j.pep.2005.01.016. View

11.

Csonka L, FRAENKEL D . Pathways of NADPH formation in Escherichia coli. J Biol Chem. 1977; 252(10):3382-91. View

12.

Goldberg K, Schroer K, Lutz S, Liese A . Biocatalytic ketone reduction--a powerful tool for the production of chiral alcohols-part II: whole-cell reductions. Appl Microbiol Biotechnol. 2007; 76(2):249-55. DOI: 10.1007/s00253-007-1005-x. View

13.

Bennett B, Kimball E, Gao M, Osterhout R, Van Dien S, Rabinowitz J . Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli. Nat Chem Biol. 2009; 5(8):593-9. PMC: 2754216. DOI: 10.1038/nchembio.186. View

14.

Chemler J, Fowler Z, McHugh K, Koffas M . Improving NADPH availability for natural product biosynthesis in Escherichia coli by metabolic engineering. Metab Eng. 2009; 12(2):96-104. DOI: 10.1016/j.ymben.2009.07.003. View

15.

Blank L, Ebert B, Buehler K, Buhler B . Redox biocatalysis and metabolism: molecular mechanisms and metabolic network analysis. Antioxid Redox Signal. 2010; 13(3):349-94. DOI: 10.1089/ars.2009.2931. View

16.

Hall M, Bommarius A . Enantioenriched compounds via enzyme-catalyzed redox reactions. Chem Rev. 2011; 111(7):4088-110. DOI: 10.1021/cr200013n. View

17.

Paczia N, Nilgen A, Lehmann T, Gatgens J, Wiechert W, Noack S . Extensive exometabolome analysis reveals extended overflow metabolism in various microorganisms. Microb Cell Fact. 2012; 11:122. PMC: 3526501. DOI: 10.1186/1475-2859-11-122. View

18.

Gerhards T, Mackfeld U, Bocola M, von Lieres E, Wiechert W, Pohl M . Influence of Organic Solvents on Enzymatic Asymmetric Carboligations. Adv Synth Catal. 2013; 354(14-15):2805-2820. PMC: 3549479. DOI: 10.1002/adsc.201200284. View

19.

Kulig J, Frese A, Kroutil W, Pohl M, Rother D . Biochemical characterization of an alcohol dehydrogenase from Ralstonia sp. Biotechnol Bioeng. 2013; 110(7):1838-48. DOI: 10.1002/bit.24857. View

20.

Kara S, Schrittwieser J, Hollmann F, Ansorge-Schumacher M . Recent trends and novel concepts in cofactor-dependent biotransformations. Appl Microbiol Biotechnol. 2013; 98(4):1517-29. DOI: 10.1007/s00253-013-5441-5. View