Unmarked insertional inactivation in the gfo gene improves growth and ethanol production by Zymomonas mobilis ZM4 in sucrose without formation of sorbitol as a by-product, but yields opposite effects in high glucose

Abstract

Sorbitol, one of the main by-products of growth on high sucrose concentrations, is catalyzed by glucose-fructose oxidoreductase (GFOR, EC 1.1.99.28) in Zymomonas mobilis, which decreases the ethanol yield. In this study, an unmarked gfo mutant from Z. mobilis ZM4 was constructed using a site-specific FLP recombinase, and growth and ethanol production were evaluated with or without the addition of sorbitol to the media. The inactivation of gfo had contrasting effects in different substrates, especially at high concentrations. The maximum specific growth rate (μm) and theoretical ethanol yield value (Ym) increased from 0.065h−1 and 60.56% to 0.094h−1 and 83.87% in 342g/L sucrose, respectively. Conversely, in 200g/L glucose, gfo inactivation decreased μm and Ym from 0.15h−1 and 89.85% to 0.10h−1 and 67.59%, respectively, and prolonged the lag period from 16h to 40h. The addition of sorbitol slightly accelerated growth and sucrose hydrolysis by the gfo mutant in 342g/L sucrose; however, addition of sorbitol restored the μm and Ym of the gfo mutant in 200g/L glucose to 0.14h−1 and 82.50%, respectively. Inactivation of gfo had a small effect on fructose utilization, and a positive one on mixture of glucose and fructose similar to that on sucrose. These results provide further understanding of the osmoregulation mechanisms in Z. mobilis and may help to exploit the biotechnological applications of this industrially important bacterium.