Modulation of endogenous pathways enhances bioethanol yield and productivity in Escherichia coli

E. coli is a robust host for various genetic manipulations and has been used commonly for bioconversion of hexose and pentose sugars into valuable products. One of the products that E. coli make under fermentative condition is ethanol. However, availability of limited reducing equivalence and generation of competing co-products undermine ethanol yield and productivity. Here, we have constructed an E. coli strain to produce high yield of ethanol from hexose and pentose sugars by modulating the expression of pyruvate dehydrogenase and acetate kinase and by deleting pathways for competing co-products.

The availability of reducing equivalence in E. coli was increased by inducing the expression of the pyruvate dehydrogenase (PDH) operon under anaerobic condition after replacement of its promoter with the promoters of ldhA, frdA, pflB, adhE and gapA. The SSY05 strain, where PDH operon was expressed under gapA promoter, demonstrated highest PDH activity and maximum improvement in ethanol yield. Deletion of genes responsible for competing products, such as lactate (ldhA), succinate (frdA), acetate (ack) and formate (pflB), led to significant reduction in growth rate under anaerobic condition. Modulation of acetate kinase expression in SSY09 strain regained cell growth rate and ethanol was produced at the maximum rate of 12 mmol/l/h from glucose. The resultant SSY09(pZSack) strain efficiently fermented xylose under microaerobic condition and produced 25 g/l ethanol at the maximum rate of 6.84 mmol/l/h with 97% of the theoretical yield. More importantly, fermentation of mixture of glucose and xylose was achieved by SSY09(pZSack) strain under microaerobic condition and ethanol was produced at the maximum rate of 0.7 g/l/h (15 mmol/l/h), respectively, with greater than 85% of theoretical yield.

The E. coli strain SSY09(pZSack) constructed via endogenous pathway engineering fermented glucose and xylose to ethanol with high yield and productivity. This strain lacking any foreign gene for ethanol fermentation is likely to be genetically more stable and therefore should be tested further for the fermentation of lignocellulosic hydrolysate at higher scale.