Biocatalytic Redox Cascades Using Baeyer-Villiger Monooxygenases

This cooperative research project aims at the development of artifical metabolic pathways. First, simple model systems will be used to determine properties of enzyme cascades, which could not develop in an evolutionary context, but will be de novo created. In order to study as many parameters as possible in the model systems, cofactor-dependent redox biotransformations have been explicitly chosen to combine them in the host organism (E. coli). In a first 3-enzyme system (reductase, oxygenase, cofactor recycling) the basic relationship between the energy demand of the host will be investigated. Based on the obtained data and the performed optimizations, the complexity of the system will be enhanced to evaluate the applicability of this research concept with respect to the production of chemical target compounds.

Cell metabolism consists of an efficient system of enzyme cascades enabling life, per se. Within this network substrates are converted to products via various intermediates in a highly efficient fashion without intermediate isolation. Decreasing reaction time, costs and waste products by avoiding intermediate compound purification as well as the prospect to directly convert toxic and/or unstable intermediates towards products represent particular benefits of cascade reactions, which puts them in focus of current research and development in biotechnology. Within a cooperative effort by Greifswald University and TU Wien a proof-of-concept enzyme cascade could be established in vivo for the first time consisting of oxidoreductases for the preparation of optically active lactones via desymmetrization or enantioconvergent reactions. A number of suitable enzymes was identified and functionally expressed in E. coli to compose the particular in vivo cascade type containing reductases and oxygenases. Two dehydrogenases of complementary selectivity enabled oxidation of chiral cyclohexenol derivaitves and carveols to the corresponding prochiral ketones. Three novel enoate reductases were also identified and biochemically characterized and employed in the conversion of aliphatic as well as cyclic ketones and aldehydes in excellent stereoselectivity. Elaborate studies in molecular biology enabled expression of all enzyme combinations in E. coli whole-cells in soluble and active form. By combining biocatalysts of unrelated metabolic origin the conversion of cyclohexenol and mono-methyl-substituted cyclohexenol derivatives was achieved to produce chiral lactones. The modular design principle of the cascade allowed for high flexibility for the conversion of various substrates in diverse stereopreferences. Conversion rates were significantly improved by designing fusion proteins. Conversion of limonene to dihydrocarvo-lactones was achieved by application of a mixed-culture approach using R. equi and E. coli expressing the artificial synthetic cascade. Subsequent studies on the combination of an in-situ extraction process of limonene from orange peel utilizing ionic liquids with the above mixed-culture system succeeded in the production of the target lactones; this represent the first coupling of an artificial enzyme cascade for the sustainable utilization of renewable resources in the formation of future platform chemicals. Consequently, all milestones of the research program were achieved and the design of modular in vivo enzyme cascades for the synthesis of chiral lactones was successfully exemplified.