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.
Historically, heterologous protein production has been challenging using the hyper-cellulolytic fungus, Trichoderma reesei. T. reesei is known for poor transformation efficiency, low homologous recombination frequency, and marginal screening systems for identification of successful transformants. In this work, we have applied the 2A-peptide multi-gene expression system to co-express four enzymes, which include three cellulases, a cellobiohydrolase (CBH1), an endoglucanase (EG1), and a β-D-glucosidase (BGL1); as well as the enhanced Green Fluorescent Protein, (eGFP), used here as a marker for monitoring expression levels. We designed a new chassis vector, pTrEno-4X-2A for this work. Expression of these cellulase enzymes was confirmed by real-time quantitative reverse transcription PCR and immunoblot analysis. The activity of each of the cellulases was assessed using pNP glycosides and the chromogenic substrate, AZCL-HE-cellulose, which confirmed the functionality of the enzymes. Expression and activity of these enzymes was proportional to the level of eGFP fluorescence, thereby validating the reliability of this screening technique. Although all three cellulase proteins were successfully expressed, their expression levels varied significantly. Specifically, up to an 18-fold difference was observed between the first and the third gene within the 2A-peptide construct, based on protein quantitation. The availability of this new screening tool is expected to greatly impact multi-enzyme applications, such as production of complex commercial enzyme formulations and the study of metabolic pathway enzymes, especially those destined for cell free applications.
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