A1 Refereed original research article in a scientific journal
Sucrose as an electron source for cofactor regeneration in recombinant Escherichia coli expressing invertase and a Baeyer Villiger monooxygenase
Authors: Sovic, Lucija; Malihan-Yap, Lenny; Tóth, Gabor Szilveszter; Siitonen, Vilja; Alphand, Veronoque; Allahverdiyeva, Yagut; Kourist, Robert
Publisher: BioMed Central Ltd
Publication year: 2024
Journal: Microbial Cell Factories
Journal name in source: Microbial Cell Factories
Article number: 227
Volume: 23
Issue: 1
ISSN: 1475-2859
eISSN: 1475-2859
DOI: https://doi.org/10.1186/s12934-024-02474-2
Web address : https://doi.org/10.1186/s12934-024-02474-2
Self-archived copy’s web address: https://research.utu.fi/converis/portal/detail/Publication/457657676
Background: The large-scale biocatalytic application of oxidoreductases requires systems for a cost-effective and efficient regeneration of redox cofactors. These represent the major bottleneck for industrial bioproduction and an important cost factor. In this work, co-expression of the genes of invertase and a Baeyer–Villiger monooxygenase from Burkholderia xenovorans to E. coli W ΔcscR and E. coli BL21 (DE3) enabled efficient biotransformation of cyclohexanone to the polymer precursor, ε-caprolactone using sucrose as electron source for regeneration of redox cofactors, at rates comparable to glucose. E. coli W ΔcscR has a native csc regulon enabling sucrose utilization and is deregulated via deletion of the repressor gene (cscR), thus enabling sucrose uptake even at concentrations below 6 mM (2 g L−1). On the other hand, E. coli BL21 (DE3), which is widely used as an expression host does not contain a csc regulon.
Results: Herein, we show a proof of concept where the co-expression of invertase for both E. coli hosts was sufficient for efficient sucrose utilization to sustain cofactor regeneration in the Baeyer–Villiger oxidation of cyclohexanone. Using E. coli W ΔcscR, a specific activity of 37 U gDCW−1 was obtained, demonstrating the suitability of the strain for recombinant gene co-expression and subsequent whole-cell biotransformation. In addition, the same co-expression cassette was transferred and investigated with E. coli BL21 (DE3), which showed a specific activity of 17 U gDCW− 1. Finally, biotransformation using photosynthetically-derived sucrose from Synechocystis S02 with E. coli W ΔcscR expressing BVMO showed complete conversion of cyclohexanone after 3 h, especially with the strain expressing the invertase gene in the periplasm.
Conclusions: Results show that sucrose can be an alternative electron source to drive whole-cell biotransformations in recombinant E. coli strains opening novel strategies for sustainable chemical production.
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Funding information in the publication:
Open access funding provided by Graz University of Technology. This project has received funding from the FET Open grant agreement 899576 (FuturoLEAF) and by the Novo Nordisk Foundation project “PhotoCat” (project no. NNF20OC0064371 to YA). The funding bodies had no role in the design of the study, analysis of the data, or writing of the manuscript.