Cofactor engineering

In addition, cofactor engineering has been used to deliberately modify the intracellular NADH/NAD+ ratio that plays a predominant role in controlling the Lactococcus lactis fermentation pattern. The introduction of the nox gene, which codes for a NADH oxidase (NOX) that converts molecular oxygen to water at the expense of NADH, to a strain with an inactivated copy of the aldB gene for a-acetolactate decarboxylase led to the efficient metabolism of the na-... 

Wang, Y, San, K.Y., and Bennett, G.N. (2013) Cofactor engineering for advancing chemical biotechnology. Curr. Opin. Biotechnol, 24, 994-999. 

Ji XJ, Xia ZF, Fu NH, Nie ZK, Shen MQ, Tian QQ, Huang H. (2013). Cofactor engineering through heterologous expression of an NADH oxidase and its impact on metabolic flux redistribution in Klebsiella pneumoniae. Biotechnol Biofuels, 6, 7. 

Cofactor Engineering for Xylitol Prodnction in Recombinant Sacchammyces cerevisiae... 

Kragl, 1J., Kruse, W., Hummel, W. and Wandrey, C. (1996) Enzyme engineering aspects of biocatalysis cofactor regeneration as example. Biotechnology and Bioengineering, 52, 309-319. 

Despite the higher selectivity of enzymatic methyl transfer over chemical methylation, where toxic or hazardous reagents are often employed, such as methyl sulfonate and diazomethane, the synthetic applications of these enzymes have been largely ignored primarily as a result of high costs associated with the cofactor SAM. Recent efforts have been directed to in vivo methylation, where SAM may be regenerated inside cells. For example, methyl benzoate production was engineered in recombinant Saccharomyces cerevisiae and in vivo... 

Wichmann, R. and Vasic-Racki, D. (2005) Cofactor regeneration at the lab scale. Advances in Biochemical Engineering/Biotechnology, 92, 225-260. 

To circumvent the cofactor regeneration problem, redox biotransformations are also carried out in whole cells - for example, baker s yeast [28, 29] or engineered Escherichia coli cells [30] - using the intracellular cofactor pool and inherent or recombinant regeneration systems. 

ENGINEERING METAL INSERTION INTO ORGANIC COFACTORS... 

Engineering Metal Insertion into Organic Cofactors.30... 

Biosynthetic production of thymidine is overall a complex process combining the controlled introduction of a novel biotransformation step into a biological system with selective enhancement or knock-out of a series of existing metabolic steps. Metabolic engineering to enhance cofactor recycling at both ribonucleotide reduction and dUMP methylation steps has important parallels in other systems, as whole-cell biotransformations are frequently employed as a means to supply, in situ, high-cost and usually labile cofactors. 

The use of water-miscible organic solvent-water mixtures is a particularly attractive method for use with cofactor-dependent enzymes due to its simphcity. The high water content can allow dissolution of both enzyme and cofactor, whilst the water-miscible solvent can provide a dual role in both substrate dissolution and as a cosubstrate for cofactor recycling (substrate-coupled cofactor recycling).The asymmetric reduction of a ketone intermediate of montelukast using an engineered ADH in the presence of 50 % v/v isopropanol offers a powerful demonstration of this methodology (Scheme 1.55). 

MW 27,500) with no cofactors or metal ions reqnirement for its function, it displays Michaelis-Menten kinetics and it is secreted in large amounts by a wide variety of Bacillus species. Subtilisin is also among the most important industrial enzymes due to its use in laundry detergents. Protein engineering strategies for subtilisin have focused on a number of aspects, namely catalysis, substrate specificity, thermal and oxidative stability and pH profile. We will describe briefly each of these aspects. 

---