Maximizing Photosynthesis-Driven Baeyer–Villiger Oxidation Efficiency in Recombinant Synechocystis sp. PCC6803

Adrian Tüllinghoff,Jörg Toepel,Magdalena B Uhl,Friederike E H Nintzel,Andreas Schmid,Bruno Bühler

doi:10.3389/fctls.2021.780474

Abstract

Photosynthesis-driven whole-cell biocatalysis has great potential to contribute to a sustainable bio-economy since phototrophic cells use light as the only energy source. It has yet to be shown that phototrophic microorganisms, such as cyanobacteria, can combine the supply of high heterologous enzyme levels with allocation of sufficient reduction equivalents to enable efficient light-driven redox biocatalysis. Here, we demonstrated that the heterologous expression of an NADPH-dependent Baeyer–Villiger monooxygenase (BVMO) gene from Acidovorax sp. CHX100 turns Synechocystis sp. PCC6803 into an efficient oxyfunctionalization biocatalyst, deriving electrons and O2 from photosynthetic water oxidation. Several expression systems were systematically tested, and a PnrsB-(Ni2+)–controlled expression based on a replicative plasmid yielded the highest intracellular enzyme concentration and activities of up to 60.9 ± 1.0 U gCDW−1. Detailed analysis of reaction parameters, side reactions, and biocatalyst durability revealed—on the one hand—a high in vivo BVMO activity in the range of 6 ± 2 U mgBVMO−1 and—on the other hand—an impairment of biocatalyst performance by product toxicity and by-product inhibition. Scale-up of the reaction to 2-L fed-batch photo-bioreactors resulted in the stabilization of the bioconversion over several hours with a maximal specific activity of 30.0 ± 0.3 U g CDW−1, a maximal volumetric productivity of 0.21 ± 0.1 gL−1 h−1, and the formation of 1.3 ± 0.1 gL−1 of ε-caprolactone. Process simulations based on determined kinetic data revealed that photosynthesis-driven cyclohexanone oxidation on a 2-L scale under high-light conditions was kinetically controlled and not subject to a limitation by photosynthesis.

Highlights

Biotechnological processes have been developed in the past decades to produce materials, chemicals, and pharmaceuticals (Schmid et al, 2001)
This was further confirmed by light intensity variation (250 μmolphotons m−2 s−1 vs. 700 μ μmolphotons m−2 s−1) under otherwise identical conditions as in the HLHC experiment (Figure 4C). This low-light high-carbon (LLHC) experiment resulted in a similar initial volumetric productivity as in the HLHC experiment corroborating that light did not limit the biocatalytic activity (Supplementary Table S2). These results indicate that neither inhibition by C-one or C-ol nor photosynthesis limited the biocatalytic activity
Photosynthesis is the central biochemical transformation process converting light energy into chemical energy and a highly attractive module for environmentally friendly industrial processes. It can be exploited for biotechnology by making use of photosynthetically active microorganisms such as cyanobacteria (Barber, 2009); (Angermayr et al, 2009)

Summary

Introduction

Biotechnological processes have been developed in the past decades to produce materials, chemicals, and pharmaceuticals (Schmid et al, 2001). While there are numerous feasible processes using enzymes as Photosynthesis-Driven BVMO Catalysis catalysts or microbes as whole-cell factories, redox biocatalysis remains challenging (Schrewe et al, 2013). This is due to the dependence of redox enzymes on co-substrates such as electron carriers and O2 (Law et al, 2006). When using isolated enzymes as catalysts, process efficiencies suffer high enzyme and cofactor regeneration costs (Kadisch et al, 2017). Respective costs and high O2 demands still often hinder efficient, stable, and cheap redox biocatalysis in heterotrophic cell factories (Baldwin and Woodley, 2006; Kadisch et al, 2017)

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