Integrated gene engineering synergistically improved substrate-product transport, cofactor generation and gene translation for cadaverine biosynthesis in E. coli

Several approaches for efficient production of cadaverine, a bio-based diaminewith broad industrial applications have been explored. Here, Serratia marcescens lysine decarboxylase (SmcadA) was expressed in E. coli; mild surfactants added in biotransformation reactions; the E. coli native lysine/cadaverine antiporter cadB, E. coli pyridoxal kinases pdxK and pdxY overexpressed and synthetic RBS libraries screened. Addition of mild surfactants and overexpression of antiporter cadB increased cadaverine biosynthesis of SmcadA. Moreover, expression of pdxY gene yielded 19.82 g/L in a reaction mixture containing added cofactor precursor pyridoxal (PL), without adding exogenous PLP. The screened synthetic RBS1, applied to fully exploit pdxY gene expression, ultimately resulted in PLP self-sufficiency, producing 27.02 g/L cadaverine using strain T7R1_PL. To boost SmcadA catalytic activity, the designed mutants Arg595Lys and Ser512Ala had significantly improved cumulative cadaverine production of 219.54 and 201.79 g/L respectively compared to the wild-typeWT(181.62 g/L), after 20 h reaction. Finally, molecular dynamics simulations for WT and variants indicated that increased flexibility at the binding sites of the protein enhanced residue-ligand interactions, contributing to high cadaverine synthesis. This work demonstrates potential of harnessing different pull factors through integrated gene engineering of efficient biocatalysts and gaining insight into the mechanisms involved through MD simulations. (C) 2020 Published by Elsevier B.V.

Automated summary

Various approaches have been explored to efficiently produce cadaverine, a bio-based diaminewith broad industrial applications. By expressing SmcadA in E. coli, addition of mild surfactants, overexpressing antiporter cadB, pyridoxal kinases pdxK and pdxY, and screening synthetic RBS libraries, the biosynthesis of cadaverine was enhanced. Mutants design and molecular dynamics simulations helped understand the mechanisms involved in high cadaverine synthesis.