Host cell and expression engineering for development of an E. coli ketoreductase catalyst: enhancement of formate dehydrogenase activity for regeneration of NADH.

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  • Author(s): Mädje K;Mädje K; Schmölzer K; Nidetzky B; Kratzer R
  • Source:
    Microbial cell factories [Microb Cell Fact] 2012 Jan 11; Vol. 11, pp. 7. Date of Electronic Publication: 2012 Jan 11.
  • Publication Type:
    Journal Article; Research Support, Non-U.S. Gov't
  • Language:
    English
  • Additional Information
    • Source:
      Publisher: BioMed Central Country of Publication: England NLM ID: 101139812 Publication Model: Electronic Cited Medium: Internet ISSN: 1475-2859 (Electronic) Linking ISSN: 14752859 NLM ISO Abbreviation: Microb Cell Fact Subsets: MEDLINE
    • Publication Information:
      Original Publication: London : BioMed Central, [2002-
    • Subject Terms:
      Aldehyde Reductase/*biosynthesis ; Escherichia coli/*enzymology ; Formate Dehydrogenases/*biosynthesis ; NAD/*metabolism; Aldehyde Reductase/genetics ; Biocatalysis ; Candida/enzymology ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Formate Dehydrogenases/genetics ; Protein Engineering ; Recombinant Proteins/biosynthesis ; Recombinant Proteins/genetics ; omega-Chloroacetophenone/metabolism
    • Abstract:
      Background: Enzymatic NADH or NADPH-dependent reduction is a widely applied approach for the synthesis of optically active organic compounds. The overall biocatalytic conversion usually involves in situ regeneration of the expensive NAD(P)H. Oxidation of formate to carbon dioxide, catalyzed by formate dehydrogenase (EC 1.2.1.2; FDH), presents an almost ideal process solution for coenzyme regeneration that has been well established for NADH. Because isolated FDH is relatively unstable under a range of process conditions, whole cells often constitute the preferred form of the biocatalyst, combining the advantage of enzyme protection in the cellular environment with ease of enzyme production. However, the most prominent FDH used in biotransformations, the enzyme from the yeast Candida boidinii, is usually expressed in limiting amounts of activity in the prime host for whole cell biocatalysis, Escherichia coli. We therefore performed expression engineering with the aim of enhancing FDH activity in an E. coli ketoreductase catalyst. The benefit resulting from improved NADH regeneration capacity is demonstrated in two transformations of technological relevance: xylose conversion into xylitol, and synthesis of (S)-1-(2-chlorophenyl)ethanol from o-chloroacetophenone.
      Results: As compared to individual expression of C. boidinii FDH in E. coli BL21 (DE3) that gave an intracellular enzyme activity of 400 units/g(CDW), co-expression of the FDH with the ketoreductase (Candida tenuis xylose reductase; XR) resulted in a substantial decline in FDH activity. The remaining FDH activity of only 85 U/g(CDW) was strongly limiting the overall catalytic activity of the whole cell system. Combined effects from increase in FDH gene copy number, supply of rare tRNAs in a Rosetta strain of E. coli, dampened expression of the ketoreductase, and induction at low temperature (18°C) brought up the FDH activity threefold to a level of 250 U/g(CDW) while reducing the XR activity by just 19% (1140 U/g(CDW)). The E. coli whole-cell catalyst optimized for intracellular FDH activity showed improved performance in the synthesis of (S)-1-(2-chlorophenyl)ethanol, reflected in a substantial, up to 5-fold enhancement of productivity (0.37 g/g(CDW)) and yield (95% based on 100 mM ketone used) as compared to the reference catalyst. For xylitol production, the benefit of enhanced FDH expression was observed on productivity only after elimination of the mass transfer resistance caused by the cell membrane.
      Conclusions: Expression engineering of C. boidinii FDH is an important strategy to optimize E. coli whole-cell reductase catalysts that employ intracellular formate oxidation for regeneration of NADH. Increased FDH-activity was reflected by higher reduction yields of D-xylose and o-chloroacetophenone conversions provided that mass transfer limitations were overcome.
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    • Grant Information:
      V 191 Austria FWF_ Austrian Science Fund FWF
    • Accession Number:
      0 (Recombinant Proteins)
      0U46U6E8UK (NAD)
      88B5039IQG (omega-Chloroacetophenone)
      EC 1.1.1.21 (Aldehyde Reductase)
      EC 1.17.1.9 (Formate Dehydrogenases)
    • Publication Date:
      Date Created: 20120113 Date Completed: 20120531 Latest Revision: 20211021
    • Publication Date:
      20221213
    • Accession Number:
      PMC3278346
    • Accession Number:
      10.1186/1475-2859-11-7
    • Accession Number:
      22236335