Here we report a new robust nicotinamide dinucleotide phosphate cofactor analog (carba‐NADP+) and its acceptance by many enzymes in the class of oxidoreductases. Replacing one ribose oxygen with a methylene group of the natural NADP+ was found to enhance stability dramatically. Decomposition experiments at moderate and high temperatures with the cofactors showed a drastic increase in half‐life time at elevated temperatures since it significantly disfavors hydrolysis of the pyridinium‐N−glycoside bond. Overall, more than 27 different oxidoreductases were successfully tested, and a thorough analytical characterization and comparison is given. The cofactor carba‐NADP+ opens up the field of redox‐biocatalysis under harsh conditions.
Successful directed evolution examples span a broad range of improved enzyme properties. Nevertheless, the most challenging step for each single directed evolution approach is an efficient identification of improved variants from a large genetic library. Thus, the development and choice of a proper high-throughput screening is a central key for the optimization of enzymes. The detection of low enzymatic activities is especially complicated when they lead to products that are present in the metabolism of the utilized genetic host. Coupled enzymatic assays based on colorimetric products have enabled the optimization of many of such enzymes, but are susceptible to problems when applied on cell extract samples. The purpose of this study was the development of a high-throughput screening for D-glycerate dehydratase activity in cell lysates. With the aid of an automated liquid handling system, we developed a high-throughput assay that relied on a pre-treatment step of cell extract prior to performing the enzymatic and assay reactions. We could successfully apply our method, which should also be transferable to other cell extract-based peroxidase assays, to identify an improved enzyme for the dehydration of D-glycerate.
Vibrio natriegens is an emerging host for biotechnology due to its high growth and substrate consumption rates. In industrial processes typically fed‐batch processes are applied to obtain high space‐time yields. In this study, we established an aerobic glucose‐limited fed‐batch fermentation with the wild type (wt) of V. natriegens which yielded biomass concentrations of up to 28.4 gX L−1. However, we observed that the viscosity of the culture broth increased by a factor of 800 at the end of the cultivation due to the formation of 157 ± 20 mg exopolysaccharides (EPS) L−1. Analysis of the genomic repertoire revealed several genes and gene clusters associated with EPS formation. Deletion of the transcriptional regulator cpsR in V. natriegens wt did not reduce EPS formation, however, it resulted in a constantly low viscosity of the culture broth and altered the carbohydrate content of the EPS. A mutant lacking the cps cluster secreted two‐fold less EPS compared to the wt accompanied by an overall low viscosity and a changed EPS composition. When we cultivated the succinate producer V. natriegens Δlldh Δdldh Δpfl Δald Δdns::pycCg (Succ1) under anaerobic conditions on glucose, we also observed an increased viscosity at the end of the cultivation. Deletion of cpsR and the cps cluster in V. natriegens Succ1 reduced the viscosity five‐ to six‐fold which remained at the same level observed at the start of the cultivation. V. natriegens Succ1 ΔcpsR and V. natriegens Succ1 Δcps achieved final succinate concentrations of 51 and 46 g L−1 with a volumetric productivity of 8.5 and 7.7 gSuc L−1 h−1, respectively. Both strains showed a product yield of about 1.4 molSuc molGlc−1, which is 27% higher compared with that of V. natriegens Succ1 and corresponds to 81% of the theoretical maximum.
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