Research Of The Redox Balance For Isobutanol Production In Escherichia Coli

As environmental pollution and the contradiction between the energy supply and demand has been increasing,looking for bioenergy that could replace the fossil fuel have been attracting more and more people's attention.Although ethanol has been the most widely used biofuel,this compound has several severe problems as an alternative to gasoline,such as low energy density and high hygroscopicity leading to storage and transportation problems.Isobutanol is better than ethanol because it have higher energy densities similar to gasoline,can be transported using current petroleum pipelines and have a low hygroscopicity and vapor pressure so that they are compatible with existing engines and can be mixed with gasoline.The biosynthetic process of isobutanol requires a reducing equivalent of NADPH,but under anaerobic conditions,glycolysis mainly produce NADH,so the redox imbalance has been regarded as the key limitation for anaerobic isobutanol production in metabolically engineered Escherichia coli strains.In this work,two different strategies were used to optimize the balance between demand and supply of the reducing equivalent.The first strategy is to recruite the ethanol synthetic pathway to solve the NADH redundant problem while the pentose phosphate pathway was activated to solve the NADPH deficient problem for anaerobic isobutanol production.Recruiting the ethanol synthetic pathway in strain AS 108 decreased isobutanol yield from 0.66 to 0.29 mol/mol glucose.It was found that there was a negative correlation between aldehyde/alcohol dehydrogenase(AdhE)activity and isobutanol production,and decreasing AdhE activity increased isobutanol yield from 0.29 to 0.6 mol/mol.On the other hand,activation of the glucose 6-phosphate dehydrogenase gene of the pentose phosphate pathway increased isobutanol yield from 0.29 to 0.41 mol/mol.Combination of these two strategies had a synergistic effect on improving isobutanol production.Isobutanol titer and yield of the best strain ZL021 was 53 mM and 0.74 mol/mol,which was 51%and 12%higher than the starting strain AS 108,respectively.The total alcohol yield of strain ZL021 was 0.81 mol/mol,which was 23%higher than strain AS 108.The second strategy is to use NADH type of ketol-acid reductoisomerase(KARI)instead of the original NADPH type IlvC.After replacing the KARI enzyme in AS 108,the genes of isobutanol biosynthetic pathway was modulated by RBS library one by one.Isobutanol titer and yield of strain ZL114 was 83mM and 0.85 mol/mol,which was 137%and 29%higher than the starting strain AS 108,respectively.By knocking out different types of alcohol dehydrogenase,the production of isobutanol was reduced 40%,show the NADPH type of alcohol dehydrogenase play a more improtant role in the production of isobutanol.Then we modulated the transhydrogenase gene pntAB by RBS library,in order to increase the supply of NADPH,and sobutanol titer and yield of the best strain ZL116 was 137 mM and 0.88 mol/mol,which was 290%and 33%higher than the starting strain AS 108,respectively.Through these two strategy,we relieve the problem of redox imbalance for anaerobic isobutanol production in metabolically engineered Escherichia coli strains,and obtained a strain that have a high titer and yield of isobutanol.