Impact of host metabolism and oxygen levels on heterologous pathway engineering for anaerobic 2,3-BDO production in Zymomonas mobilis

To develop pathways for advanced biofuel production, and to understand the impact of host metabolism and environmental conditions on heterologous pathway engineering for economic advanced biofuels production from biomass, we seek to redirect the carbon flow of the model ethanologen Zymomonas mobilis to produce desirable hydrocarbon intermediate 2,3 butanediol (2,3-BDO). 2,3-BDO is a bulk chemical building block, and can be upgraded in high yields to gasoline, diesel, and jet fuel. 2,3-BDO biosynthesis pathways from various bacterial species were examined, which includes three genes encoding acetolactate synthase (Als), acetolactate decarboxylase (AldC), and butanediol dehydrogenase (Bdh). Bioinformatics analysis was carried out to pinpoint potential bottlenecks for high 2,3-BDO production. Different combinations of 2,3-BDO biosynthesis metabolic pathways using genes from different bacterial species have been constructed. Our results demonstrated that carbon flux can be deviated from ethanol production into 2,3-BDO biosynthesis, and all three heterologous genes are essential to efficiently redirect pyruvate from ethanol production for high 2,3-BDO production in Z. mobilis. The down-selection of best gene combinations up to now enabled Z. mobilis to reach the 2,3-BDO production of more than 10 g/L from glucose and xylose; as well as mixed C6/C5 sugar streams derived from the Deacetylation and Mechanic Refining (DMR) process. This study confirms the value of integrating bioinformatics analysis and systems biology data during metabolic engineering endeavors, and reveals the interactions among host metabolism, oxygen levels, and heterologous 2,3-BDO biosynthesis pathway, which will provide guidance for future metabolic engineering efforts to continue to boost 2,3-BDO titer anaerobically.