Activating transhydrogenase and NAD kinase in combination for improving isobutanol production.

Isobutanol is an excellent alternative biofuel. Fermentative production of isobutanol had been realized in several microorganisms by combining branched-chain amino acids synthetic pathway and Ehrlich pathway. In contrast to using plasmid overexpression and inducible promoters, genetically stable Escherichia coli strains for isobutanol production were constructed in this work by integrating essential genes into chromosome. A chromosome-based markerless gene modulation method was then developed for fine-tuning gene expression with multiple regulatory parts to improve isobutanol production. There was also a cofactor imbalance problem for anaerobic isobutanol synthesis. NADPH is the reducing equivalent required for isobutanol production, while the common reducing equivalent under anaerobic condition is NADH. Two strategies were used to modulate expression of transhydrogenase (pntAB) and NAD kinase (yfjB) genes to increase NADPH supply for improving isobutanol production. Plasmid overexpression of pntAB and yfjB genes either individually or in combination had little effect on isobutanol production. In contrast, modulating pntAB and yfjB gene expression in chromosome with multiple regulatory parts identified optimal modulators under aerobic and anaerobic conditions, respectively, and improved isobutanol production. Modulating pntAB gene alone led to 20% and 8% increase of anaerobic isobutanol titer and yield. Although modulating yfjB gene alone had nearly no effect, modulating pntAB and yfjB genes in combination led to 50% and 30% increase of isobutanol titer and yield in comparison with modulating pntAB gene alone. It was also found that increasing pntAB gene expression alone had a threshold for improving anaerobic isobutanol production, while activating NAD kinase could break through this threshold, leading to a yield of 0.92mol/mol. Our results suggested that transhydrogenase and NAD kinase had a synergistic effect on increasing NADPH supply and improving anaerobic isobutanol production. This strategy will be useful for improving production of target compounds using NADPH as reducing equivalent within their synthetic pathways. In addition, combined activation of PntAB and YfjB led to 28% and 22% increase of aerobic isobutanol titer and yield, resulting in production of 10.8g/L isobutanol in 24h with a yield of 0.62mol/mol.