In vivo multistep biocatalysis

The heterologous expression of metabolically related enzymes represents one way to overcome several of the limitations and drawbacks of the above-described mixed culture strategies. Within a biocatalytic context, such an approach is appeahng only when all enzymes involved display sufHdently broad substrate tolerance in order to ultimately form a system capable of converting various starting materials. 

The group of Bornscheuer [29] engineered a potential metabolic pathway from P. Jluorescens in E. coli. The aim of the work was to prove the metaboUc connection of the used enzymes and to investigate the importance and meaning of the metabolic pathway in which they are involved in vivo. 

The authors exploited this three-step oxidation system to transform pseudocumenes (0.46 mM substrate concentration) into the corresponding carboxylic acids. 

Buhler, Schmid, and coworkers [36] described the development of a recombinant whole-cell biocatalyst for the direct terminal alkylamino-functionalization of fatty acid methyl esters (e.g., dodecanoic acid methyl ester). The model substrate was dodecanoic acid methyl ester, which was oxidized by an alkane monooxygenase (AlkBGT) from Pseudomonas putida GPol to the corresponding 

The prime objective and major aim of this study was to combine the efficiency of biosynthetic redox pathways and the modularity of synthesis by functional group transformations applied to diverse substrates based on the promiscuity of enzymes. Designing and evaluating the feasibility of a multi-enzyme-catalyzed cascade process in Hving microbial cells enabled the creation of an artificial mini -metaboHc pathway connected to the primary metabolism via redox-cofactor 

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