Acedos Miguel G, de la Torre Isabel, Santos Victoria E, García-Ochoa Félix, García José L, Galán Beatriz
Chemical and Materials Engineering Department, Chemical Sciences School, Universidad Complutense de Madrid, 28040, Madrid, Spain.
Department of Microbial and Plant Biotechnology, Centro de Investigaciones Biológicas, CSIC, 28040, Madrid, Spain.
Biotechnol Biofuels. 2021 Jan 6;14(1):8. doi: 10.1186/s13068-020-01862-1.
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.
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 of isobutanol. In both strains, we observed a significant decrease in the yields of by-products such as lactic acid or ethanol.
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.
异丁醇是一种有望替代化石资源汽油的物质。这种高级醇可以利用基因改造的微生物从糖类生产得到。嗜蜚希姆氏菌（p424IbPSO）是一种对高浓度异丁醇具有抗性的健壮菌株，能够实现这种醇的高产率。然而，与大多数为生产异丁醇而开发的菌株一样，该菌株在其代谢途径上存在一些局限性。厌氧条件下异丁醇的生产会导致NADPH的消耗，而NADPH是代谢途径中两种酶所必需的。在这项工作中，研究了两种独立的方法来缓解共底物失衡：（i）使用依赖NADH的醇脱氢酶来降低途径对NADPH的依赖性，以及（ii）使用转氢酶来提高NADPH水平。
向嗜蜚希姆氏菌（p424IbPSO）中添加来自乳酸乳球菌的依赖NADH的醇脱氢酶（AdhA），使得异丁醇产量提高了19.3%。携带PntAB转氢酶的重组菌株嗜蜚希姆氏菌（p424IbPSO，pIZpntAB）比原始菌株多产生39.0%的异丁醇，达到5.98 g/L的异丁醇产量。在这两种菌株中，我们都观察到副产物如乳酸或乙醇的产量显著下降。
嗜蜚希姆氏菌（p424IbPSO）中的异丁醇生物合成途径利用内源性依赖NADPH的醇脱氢酶YqhD来完成该途径。添加依赖NADH的AdhA导致作为该途径瓶颈的NADPH消耗减少。AdhA对NADH的更高消耗降低了丙酮酸转化为乳酸和乙醇所需的NADH的可用性。另一方面，大肠杆菌PntAB的表达增加了IlvC和YqhD可利用的NADPH，同时降低了NADH的可用性，从而减少了乳酸和乙醇的产生。在这项工作中表明，嗜蜚希姆氏菌中AdhA和PntAB酶的表达分别将产量从11.9%提高到14.4%和16.4%。
