Modulating redox metabolism to improve isobutanol production in Shimwellia blattae

Miguel G Acedos,Beatriz Galán,Victoria E Santos,Félix García-Ochoa,Isabel De La Torre,José L García

doi:10.1186/s13068-020-01862-1

Abstract

BackgroundIsobutanol 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.ResultsThe 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.ConclusionsThe 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.

Highlights

Isobutanol is a candidate to replace gasoline from fossil resources
Testing the effect of alcohol dehydrogenase from Lactococcus lactis (AdhA) from L. lactis in isobutanol production To determine the effect of the AdhA aldehyde reductase from L. lactis in the production of isobutanol by S. blattae (p424IbPSO), we have cloned the adhA gene in the wide host range compatible expression vector pIZ2 to construct pIZadhA and transformed S. blattae (p424IbPSO) to render the new recombinant strain S. blattae (p424IbPSO, pIZadhA) (Table 2)
The new recombinant strain produced 18.6% and 48.3% less lactate and ethanol, respectively, than the strain harboring the empty pIZ2 plasmid (Fig. 5). This result suggests that the NADHdependent AdhA of L. lactis is competing for the NADH pool to transform the overproduced isobutyraldehyde into isobutanol with the NADH-dependent LdhA and AdhE enzymes of S. blattae which transform pyruvate and acetyl-CoA into lactate and ethanol, respectively

Summary

Introduction

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. This strain, like most strains developed for isobutanol production, has some limitations in its metabolic pathway. To achieve isobutanol production from glucose the most common strategy has been the derivation of intermediates from amino acid biosynthesis pathways to alcohol production. Most of the modifications carried out are based on the last two steps in the Ehrlich pathway for 2-keto acid degradation and the valine biosynthesis pathway. 2-Keto acids are converted to aldehydes by heterologous broad-substrate-range

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