Modulating redox metabolism to improve isobutanol production in Shimwellia blattae

Abstract

Background Isobutanol is a candidate to replace gasoline from fossil resources. This higher alcohol can be produced from sugars using genetically modified microorganisms. Shimwellia blattae (p424IbPSO) is a robust strain resistant to high concentration of isobutanol that can achieve a high production rate of this alcohol. Nevertheless, this strain, like most strains developed for isobutanol production, has some limitations in its metabolic pathway. Isobutanol production under anaerobic conditions leads to a depletion of NADPH, which is necessary for two enzymes in the metabolic pathway. In this work, two independent approaches have been studied to mitigate the co-substrates imbalance: (i) using a NADH-dependent alcohol dehydrogenase to reduce the NADPH dependence of the pathway and (ii) using a transhydrogenase to increase NADPH level. Results The addition of the NADH-dependent alcohol dehydrogenase from Lactococcus lactis (AdhA) to S. blattae (p424IbPSO) resulted in a 19.3% higher isobutanol production. The recombinant strain S. blattae (p424IbPSO, pIZpntAB) harboring the PntAB transhydrogenase produced 39.0% more isobutanol than the original strain, reaching 5.98 g L−1 of isobutanol. In both strains, we observed a significant decrease in the yields of by-products such as lactic acid or ethanol. Conclusions The isobutanol biosynthesis pathway in S. blattae (p424IbPSO) uses the endogenous NADPH-dependent alcohol dehydrogenase YqhD to complete the pathway. The addition of NADH-dependent AdhA leads to a reduction in the consumption of NADPH that is a bottleneck of the pathway. The higher consumption of NADH by AdhA reduces the availability of NADH required for the transformation of pyruvate into lactic acid and ethanol. On the other hand, the expression of PntAB from E. coli increases the availability of NADPH for IlvC and YqhD and at the same time reduces the availability of NADH and thus, the production of lactic acid and ethanol. In this work it is shown how the expression of AdhA and PntAB enzymes in Shimwellia blattae increases yield from 11.9% to 14.4% and 16.4%, respectively.

Biosynthesis of 1,3-Propanediol via a New Pathway from Glucose in Escherichia coli

“…In addition, the pentose phosphate pathway was upregulated according to Figure E, which likely occurred because 3-hydroxy acid dehydrogenase and alcohol dehydrogenase in the pathway are both NADPH-dependent. Therefore, various NADPH systems were introduced to supply NADPH including the Entner–Doudoroff (ED) pathway and pntAB , , but this did not improve production as expected (Supporting Figure 5).…”

Isobutanol Production by Autotrophic Acetogenic Bacteria

“…The change of coenzyme specificity from NADPH to NADH has already been achieved for IlvC from C. ljungdahlii and can thus easily be combined with the aforementioned alterations. The benefit of a NADH-depending alcohol dehydrogenase replacement in the natural, decarboxylase-based isobutanol formation pathway of Shimwellia blattae has recently been shown by Acedos et al (2021). Finally, growing the strains in gassed and stirred bioreactors will certainly improve the fermentation outcome.…”

Bioisobutanol as a Promising Feedstock for Production of “Green” Hydrocarbons and Petrochemicals (A Review)

“…The addition of NADH-dependent alcohol dehydrogenase isolated from Lactococcus lactis (AdhA) to Shimwellia blattae (p424IbPSO) led to a 19.3% increase in the isobutanol yield. The recombinant Shimwellia blattae strain (p424IbPSO, PIZPN TAB) containing transhydrogenase PntAB allowed production of 39.0% larger isobutanol amount compared to the initial strain; the productive capacity of 5.98 g L -1 was reached [30]. Both strains showed a considerably decreased yield of byproducts, lactic acid and ethanol.…”