No abstract

Metabolically active resting (i.e., nongrowing) bacterial cells have a high potential in cofactor-dependent redox biotransformations. Where growing cells require carbon and energy for biomass production, resting cells can potentially exploit their metabolism more efficiently for redox biocatalysis allowing higher specific activities and product yields on energy source. Here, the potential of resting recombinant E. coli containing the styrene monooxygenase StyAB was investigated for enantioselective styrene epoxidation in a two-liquid phase setup. Resting cells indeed showed twofold higher specific activities as compared to growing cells in a similar setup. However, product formation rates decreased steadily resulting in lower final product concentrations. The low intrinsic stability of the reductase component StyB was found to limit overall biocatalyst stability. Such limitation by enzyme stability was overcome by increasing intracellular StyB levels. Beyond that, product inhibition was identified as a limiting factor, whereas complete toxification of the bacterial cells, as it was observed with growing cells, and deactivation of the multicomponent enzyme system did not occur. The resting cell setup allowed high product yields on glucose of more than 5 mol mol(glucose)(-1), which makes the use of resting cells a promising approach for ecologically as well as economically sustainable oxygenase-based whole-cell biocatalysis.

Selection of the ideal microbe is crucial for whole-cell biotransformations, especially if the target reaction intensively interacts with host cell functions. Asymmetric styrene epoxidation is an example of a reaction which is strongly dependent on the host cell owing to its requirement for efficient cofactor regeneration and stable expression of the styrene monooxygenase genes styAB. On the other hand, styrene epoxidation affects the whole-cell biocatalyst, because it involves toxic substrate and products besides the burden of additional (recombinant) enzyme synthesis. With the aim to compare two fundamentally different strain engineering strategies, asymmetric styrene epoxidation by StyAB was investigated using the engineered wild-type strain Pseudomonas sp. strain VLB120ΔC, a styrene oxide isomerase (StyC) knockout strain able to accumulate (S)-styrene oxide, and recombinant E. coli JM101 carrying styAB on the plasmid pSPZ10. Their performance was analyzed during fed-batch cultivation in two-liquid phase biotransformations with respect to specific activity, volumetric productivity, product titer, tolerance of toxic substrate and products, by-product formation, and product yield on glucose. Thereby, Pseudomonas sp. strain VLB120ΔC proved its great potential by tolerating high styrene oxide concentrations and by the absence of by-product formation. The E. coli-based catalyst, however, showed higher specific activities and better yields on glucose. The results not only show the importance but also the complexity of host cell selection and engineering. Finding the optimal strain engineering strategy requires profound understanding of bioprocess and biocatalyst operation. In this respect, a possible negative influence of solvent tolerance on yield and activity is discussed.

Microbial biocatalysis has emerged to a standard technology in the food, feed, pharmaceutical, and fine chemical industries. Since microorganisms are optimized by nature to maximize survival and typically not for the high‐level accumulation of any sort of product, effective engineering strategies are required to satisfy the growing demand of new, economically competitive, and environmentally friendly products and processes. Random mutagenesis and subsequent selection used to be successful strateges, despite the fact that genetic changes were often not clear and include potentially unwanted detrimental alterations. The recent advances in systems biology research allow a genome‐scale characterization of the complex composition and function of cells. We consider it important to recognize that the comprehensive understanding of a bioprocess is not restricted to the quantitative description of cellular functions but, in addition, includes analyses and evaluations on the reaction and process level. Using today's unprecedented toolbox, new whole‐cell catalyst design targets can be systematically identified and the properties of microbial hosts can be specifically fine‐tuned according to the requirements of a given bioprocess. This review proposes an integrated strategy as a holistic knowledge‐driven approach for systems biotechnology and summarizes recent advances on the basis of selected examples.