Engineering of Glycolytic Pathway for Production of Isobutanol  in Cofactor-Balanced Manner

Isobutanol is considered as one of the promising next-generation biofuels to replace petroleum based conventional fuels. We developed an isobutanol producing Enterobacter aerogenes through L-valine biosynthesis pathway. Naturally, Enterobacter aerogenes is well known as 2,3-butanediol producer using glucose as a carbons source. Therefore large amount of metabolic flux can be driven from glucose to pyruvate and alpha-acetolactate which are precursors of isobutanol. Firstly, we tried to increase intracellular pyruvate pool and reduce major by-products such as 2,3-butanediol and lactate by deleting ldhA, budA and pflB sequentially. Additionally, transformation of mutant strain was conducted to overexpress isobutanol synthesis pathway. The plasmids harboring isobutanol pathway genes, acetolactate synthase (budB),  keto-acid reductoisomerase (ilvC), dihydroxyacid dehydratase (ilvD) of K. pneumoniae, alpha-ketoisovalerate decarboxylase (kivD), alcohol dehydrogenase (adhA) of L. lactis and alcohol dehydrogenase (yqhD) of Escherichia coli were constructed and introduced to mutant strains. We constructed engineered strain producing 12.5g/L of isobutanol (0.25g/g glucose) by metabolic pathway engineering.

When it comes to reducing equivalents generated in native glycolytic pathway, two molecules of NADH are regenerated by glyceraldehyde 3-phosphate dehydrogenase (GAPDH, GapA). However wild type keto-acid reductoisomerase (KARI, IlvC) oxidizes cofactor NADPH. In the aspects of enzymatic cofactor balance, additional NADPH should be supplied to approach theoretical maximum yield. We tried to increase intracellular NADPH pools by substituting NAD(H)-dependent GAPDH, GapA, to NADP(H)-dependent GAPDH, GapC, from Clostridium acetobutylicum together with deleting phosphofructokinase (PfkA) for further increasing NADPH generation through pentose phosphate pathway. By reference to the research implemented in S. Bastian et al., NAD(H)-dependent KARI was also constructed. Then four types of isobutanol production pathway were constructed such as 2 NADH dependent pathway (ilvC^WT, adhA), 1 NADH + 1 NADPH dependent pathway (ilvC^WT yqhD, ilvC^Mut adhA) and 2 NADPH dependent pathway (ilvC^WT yqhD). These four pathways were introduced in the host microorganisms that have NADPH generating glycolytic pathway. The production yield increased about 32% in NADPH dependent pathway as the NADPH generating glycolytic pathway applied. This study suggests that engineering glycolytic pathway related to the generation of reducing equivalent can satisfy the cofactor requirement in isobutanol production pathway. Furthermore, 2,3-butanediol-producing Enterobacteriaceae have the potentiality as production host of isobutanol in the bio-fuel industry.