Phylogenetics-based identification and characterization of a superior 2,3-butanediol dehydrogenase for Zymomonas mobilis expression

Abstract

Background Zymomonas mobilis has recently been shown to be capable of producing the valuable platform biochemical, 2,3-butanediol (2,3-BDO). Despite this capability, the production of high titers of 2,3-BDO is restricted by several physiological parameters. One such bottleneck involves the conversion of acetoin to 2,3-BDO, a step catalyzed by 2,3-butanediol dehydrogenase (Bdh). Several Bdh enzymes have been successfully expressed in Z. mobilis, although a highly active enzyme is yet to be identified for expression in this host. Here, we report the application of a phylogenetic approach to identify and characterize a superior Bdh, followed by validation of its structural attributes using a mutagenesis approach. Results Of the 11 distinct bdh genes that were expressed in Z. mobilis, crude extracts expressing Serratia marcescens Bdh (SmBdh) were found to have the highest activity (8.89 µmol/min/mg), when compared to other Bdh enzymes (0.34–2.87 µmol/min/mg). The SmBdh crystal structure was determined through crystallization with cofactor (NAD+) and substrate (acetoin) molecules bound in the active site. Active SmBdh was shown to be a tetramer with the active site populated by a Gln247 residue contributed by the diagonally opposite subunit. SmBdh showed a more extensive supporting hydrogen-bond network in comparison to the other well-studied Bdh enzymes, which enables improved substrate positioning and substrate specificity. This protein also contains a short α6 helix, which provides more efficient entry and exit of molecules from the active site, thereby contributing to enhanced substrate turnover. Extending the α6 helix to mimic the lower activity Enterobacter cloacae (EcBdh) enzyme resulted in reduction of SmBdh function to nearly 3% of the total activity. In great contrast, reduction of the corresponding α6 helix of the EcBdh to mimic the SmBdh structure resulted in ~ 70% increase in its activity. Conclusions This study has demonstrated that SmBdh is superior to other Bdhs for expression in Z. mobilis for 2,3-BDO production. SmBdh possesses unique structural features that confer biochemical advantage to this protein. While coordinated active site formation is a unique structural characteristic of this tetrameric complex, the smaller α6 helix and extended hydrogen network contribute towards improved activity and substrate promiscuity of the enzyme.

Efficient 1-Hydroxy-2-Butanone Production from 1,2-Butanediol by Whole Cells of Engineered E. coli

“…The effects of different temperature and pH value on (2R, 3R)-BDH and (2S, 3S)-BDH activity were investigated by using 50 mM 1,2-BD mixture and 0.3 mM NAD + as substrate and coenzyme, respectively. The influence of the buffer pH (6-12) were investigated at room temperature using 50 mM potassium phosphate buffers (pH 6-8), glycine-NaOH buffers (pH 9-10), and Na2HPO4-NaOH buffers (pH [11][12]. The results indicated that the activities of two enzymes were obviously increased with the increase of pH values (Figure 1AB).…”

“…Previous studies showed that diol dehydrogenases such as 2,3-butanediol dehydrogenase (BDH) could efficiently catalyze the interconversion of 2,3-BD and AC [8][9][10][11][12]. Substrate specificity assays indicated that the BDH enzyme could oxidate 1,2-pentadiol and 1,2-propanediol into the corresponding secondary ketone in the presence of NAD + , suggesting the BDH enzyme might have potential for the conversion of 1,2-BD to HB [13,14].…”

“…Fortunately, various diol dehydrogenases have shown the abilities in the oxidation of diol to the corresponding ketone alcohol [6,7]. Further, 2,3-Butanediol dehydrogenase (BDH), one of diol dehydrogenases, can catalyze the dehydrogenation of 2,3-butanediol (2,3-BD) into acetoin (AC, a stereoisomer of HB) [8][9][10][11][12]. Furthermore, the BDH enzymes from Serratia marcescens H30 and Paenibacillus polymyxa ATCC 12321 also showed the activities for 1,2-pentadiol and 1,2-propanediol as substrates [13,14].…”

Recent advances in metabolic engineering of microorganisms for advancing lignocellulose-derived biofuels

“…Other than ethanol, Z. mobilis possesses endogenous abilities to produce other fuel alcohols, such as isobutanol and 2, 3-butanediol. For the 2, 3-butanediol production, recombinant strains were successfully developed by expression of acetolactate synthase, acetolactate decarboxylase, and butanediol dehydrogenase encoding genes [ 96 , 97 ]. The best-enhanced strain was able to produce more than 10 g/l of 2, 3-butanediol from glucose and xylose as well as sugar streams derived from deacetylation and mechanical refining [ 96 ].…”

Zymo-Parts: A Golden Gate Modular Cloning Toolbox for Heterologous Gene Expression in Zymomonas mobilis

“… 3 Second, it has only a low tolerance toward inhibitory substances, arising, for example, from treatment of lignocellulosic biomass. 4 Lastly, despite some recent developments to expand the extraordinary capacity for ethanol synthesis toward other products such as lactate, 5 isobutanol, 5 , 6 poly-3-hydroxybutyrate, 7 and 2,3-butanediol, 8 there is an increasing need for efficient genetic tools for metabolic engineering.…”