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This paper describes the development of a biocatalytic process on the multi-dozen gram scale for the synthesis of a precursor to Nylon-9, a specialty polyamide. Such materials are growing in demand, but their corresponding monomers are often difficult to synthesize, giving rise to biocatalytic approaches. Here, we implemented cyclopentadecanone monooxygenase as an Escherichia coli whole-cell biocatalyst in a defined medium, together with a substrate feeding-product removal concept, and an optimized downstream processing (DSP). A previously described hazardous peracid-mediated oxidation was thus replaced with a safe and scalable protocol, using aerial oxygen as oxidant, and water as reaction solvent. The engineered process converted 42 g (0.28 mol) starting material ketone to the corresponding lactone with an isolated yield of 70% (33 g), after highly efficient DSP with 95% recovery of the converted material, translating to a volumetric yield of 8 g pure product per liter. Biotechnol. Bioeng. 2017;114: 1670-1678. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

This paper describes the measurement and analysis of in vivo activity and stability of cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMO), a model Baeyer-Villiger monooxygenase, in the recombinant host Escherichia coli. This enzyme was often described as poorly stable in vitro, and has recently been found to deactivate rapidly in the absence of its essential cofactors and antioxidants. Its stability in vivo was scarcely studied, so far. Under conditions common for the overexpression of CHMO we investigated the ability of the host to support these properties using metabolomics. Our results showed that E. coli failed to provide the intracellular levels of cofactors required to functionally stabilize the enzyme, although the biocatalyst was produced in high concentration, and was invariably detected after protein synthesis had stopped. We thus infer that biotechnological applications of CHMO with this host relied on a residual activity of approximately 5-10%. Other microorganisms might offer a more efficient solution for recombinant production of CHMO and related enzymes. [ABSTRACT FROM AUTHOR]

Synthetic enzyme cascades in living cells often lack efficiency owing to the formation of byproducts by endogenous enzymes or toxicity of the cascade intermediates. Highly reactive aldehyde species can trigger a metabolic stress response, and this leads to undesired side reactions and decreased yields. Owing to the metabolic background of Escherichia coli ( E. coli), aldehydes may be irreversibly oxidized to carboxylic acids or reduced to the corresponding alcohols. Herein, we applied an approach to equilibrate the aldehyde concentration in vivo. We oxidized primary alcohols to the corresponding aldehydes by AlkJ, an alcohol dehydrogenase from Pseudomonas putida. Introduction of a carboxylic acid reductase from Nocardia iowensis allowed the target compound to be retrieved from the carboxylate sink. Further reduction of the aldehydes to alcohols by endogenous E. coli enzymes completed the equilibration between alcohols, aldehydes, and carboxylic acids. Thus, the aldehyde concentrations remained below nonviable concentrations. We demonstrated the concept on several primary alcohols, which reached the redox equilibrium within 6 h and persisted up to 24 h. Subsequent combination with a dihydroxyacetone-dependent aldolase (Fsa1-A129S, E. coli) demonstrated that the reactive aldehyde species were freely available and gave the aldol product, (3 S,4 R)-1,3,4-trihydroxy-5-phenylpentan-2-one, in 70 % yield within short reaction times. [ABSTRACT FROM AUTHOR]

An immense number of chemical reactions are carried out simultaneously in living cells. Nature's optimization approach encompasses the assembly of reactions in cascades and to embed them in finely tuned metabolic networks. With the vast progress in the field of biocatalysis, man-made cascades, especially redox cascades, have reached a degree of complexity that needs tools for improved control and optimization. Combined strategies from biocatalysis, metabolic engineering and synthetic biology lead to the establishment of artificial metabolic pathways with minimized interference with the cellular host environment. This review will focus on genetic and metabolic engineering tools for the assembly and introduction of de novo redox pathways into the host Escherichia coli and will present state of the art redox cascades performed by tailor-made microbial cell factories. [ABSTRACT FROM AUTHOR]