Model-Driven Redox Pathway Manipulation for Improved Isobutanol Production in Bacillus subtilis Complemented with Experimental Validation and Metabolic Profiling Analysis

To rationally guide the improvement of isobutanol production, metabolic network and metabolic profiling analysis were performed to provide global and profound insights into cell metabolism of isobutanol-producing Bacillus subtilis. The metabolic flux distribution of strains with different isobutanol production capacity (BSUL03, BSUL04 and BSUL05) drops a hint of the importance of NADPH on isobutanol biosynthesis. Therefore, the redox pathways were redesigned in this study. To increase NADPH concentration, glucose-6-phosphate isomerase was inactivated (BSUL06) and glucose-6-phosphate dehydrogenase was overexpressed (BSUL07) successively. As expected, NADPH pool size in BSUL07 was 4.4-fold higher than that in parental strain BSUL05. However, cell growth, isobutanol yield and production were decreased by 46%, 22%, and 80%, respectively. Metabolic profiling analysis suggested that the severely imbalanced redox status might be the primary reason. To solve this problem, gene udhA of Escherichia coli encoding transhydrogenase was further overexpressed (BSUL08), which not only well balanced the cellular ratio of NAD(P)H/NAD(P)(+), but also increased NADH and ATP concentration. In addition, a straightforward engineering approach for improving NADPH concentrations was employed in BSUL05 by overexpressing exogenous gene pntAB and obtained BSUL09. The performance for isobutanol production by BSUL09 was poorer than BSUL08 but better than other engineered strains. Furthermore, in fed-batch fermentation the isobutanol production and yield of BSUL08 increased by 11% and 19%, up to the value of 6.12 g/L and 0.37 C-mol isobutanol/C-mol glucose (63% of the theoretical value), respectively, compared with parental strain BSUL05. These results demonstrated that model-driven complemented with metabolic profiling analysis could serve as a useful approach in the strain improvement for higher bio-productivity in further application.