Directing enzyme devolution for biosynthesis of alkanols and 1,n-alkanediols from natural polyhydroxy compounds

- The promiscuity of GDHt and DDHt for catalyzing long-chain polyols was systematically characterized.
- Novel pathways for 1,4-BD production from erythritol and 1-pentanol production from 1,2-PD were established.
- The mutants with enhanced promiscuity may serve as a platform for the production of various alcohols and acids.
- Exploiting enzyme promiscuity is proposed as a promising strategy that enriches the toolbox for biotechnologists.

Primordial enzymes are proposed to possess broad specificities. Through divergence and evolution, enzymes have been refined to exhibit specificity towards one reaction or substrate, and are thus commonly assumed as “specialists”. However, some enzymes are “generalists” that catalyze a range of substrates and reactions. This property has been defined as enzyme promiscuity and is of great importance for the evolution of new functions. The promiscuities of two enzymes, namely glycerol dehydratase and diol dehydratase, were herein exploited for catalyzing long-chain polyols, including 1,2-butanediol, 1,2,4-butanetriol, erythritol, 1,2-pentanediol, 1,2,5-pentanetriol, and 1,2,6-hexanetriol. The specific activities required for catalyzing these six long-chain polyols were studied via in vitro enzyme assays, and the catalytic efficiencies were increased through protein engineering. The promiscuous functions were subsequently applied in vivo to establish 1,4-butanediol pathways from lignocellulose derived compounds, including xylose and erythritol. In addition, a pathway for 1-pentanol production from 1,2-pentanediol was also constructed. The results suggest that exploiting enzyme promiscuity is promising for exploring new catalysts, which would expand the repertoire of genetic elements available to synthetic biology and may provide a starting point for designing and engineering novel pathways for valuable chemicals.