Homoethanol production from cellobionate and glycerol using recombinant Klebsiella oxytoca strains

Cellobionic acid, an oxidized cellulose degradation product, can be produced from cellulosic biomass. Engineered strains of Klebsiella oxytoca BW21 and WT26 can effectively use cellobionate for ethanol production. However, because cellobionate is a more oxidized substrate than cellobiose, oxidized products such as acetate must be produced as by-products to maintain the overall redox balance, and homoethanol production is not possible. One of the strategies to avoid the redox imbalance problem and achieve homoethanol production is co-fermentation of cellobionate with a more reduced substrate glycerol. When cellobionate and glycerol were used as the carbon source by K. oxytoca BW 21, ethanol yield was about 62%–67%. Co-utilization of cellobionate and glycerol in batch fermentation increased the yield of ethanol to about 77% and substantially decreased the accumulation of by-products (such as acetate and 1,3-propanediol). However, glycerol imposes carbon catabolite repression on cellobionate utilization. The utilization rate mismatch between these two substrates limits the benefits of redox recycling between the two substrates’ utilization pathways. When recombinant strain WT26 (which is disabled in glycerol utilization when fermented alone) was used to co-ferment cellobionate and glycerol, the two substrates’ utilization rates exactly matched, which led to an ethanol yield improvement to 96%.