Ethanol production from wood hydrolysate using genetically engineered Zymomonas mobilis

Abstract

An ethanologenic microorganism capable of fermenting all of the sugars released from lignocellulosic biomass through a saccharification process is essential for secondary bioethanol production. We therefore genetically engineered the ethanologenic bacterium Zymomonas mobilis such that it efficiently produced bioethanol from the hydrolysate of wood biomass containing glucose, mannose, and xylose as major sugar components. This was accomplished by introducing genes encoding mannose and xylose catabolic enzymes from Escherichia coli. Integration of E. coli manA into Z. mobilis chromosomal DNA conferred the ability to co-ferment mannose and glucose, producing 91 % of the theoretical yield of ethanol within 36 h. Then, by introducing a recombinant plasmid harboring the genes encoding E. coli xylA, xylB, tal, and tktA, we broadened the range of fermentable sugar substrates for Z. mobilis to include mannose and xylose as well as glucose. The resultant strain was able to ferment a mixture of 20 g/l glucose, 20 g/l mannose, and 20 g/l xylose as major sugar components of wood hydrolysate within 72 h, producing 89.8 % of the theoretical yield. The recombinant Z. mobilis also efficiently fermented actual acid hydrolysate prepared from cellulosic feedstock containing glucose, mannose, and xylose. Moreover, a reactor packed with the strain continuously produced ethanol from acid hydrolysate of wood biomass from coniferous trees for 10 days without accumulation of residual sugars. Ethanol productivity was at 10.27 g/l h at a dilution rate of 0.25 h(-1).

Progress and perspective on lignocellulosic hydrolysate inhibitor tolerance improvement in Zymomonas mobilis

“…Compared with the results with pure sugars, ethanol productions in hydrolysates were impeded significantly (Bothast et al 1999;Feng et al 2012;Jennings and Schell 2011;Jeon et al 2010;Joachimsthal et al 1999;Krishnan et al 2000;Mohagheghi et al 2002Mohagheghi et al , 2004Schell et al 2016;Serate et al 2015;Teixeira et al 2000;Yanase et al 2012;Zhao et al 2014). For example, the ethanol yield was much lower in the hydrolysate (15 g/L) than in pure glucose fermentation (44.9 g/L) (Dong et al 2013;Zhao et al 2014).…”

“…Besides the lack of complete information on inhibitory compounds in the hydrolysates due to the detection limitation of current techniques, little is known on potential additional/synergetic effects of these inhibitory compounds on cellular metabolism. In addition, although recombinant strains capable of co-fermentation of pentose and hexose sugars have been achieved (Deanda et al 1996;Dunn and Rao 2014;Yanase et al 2012;Zhang and Eddy 1995), all these strains were sensitive to inhibitor stress, especially for the xylose utilization (Kim et al 2000;Yang et al 2014a). Pentose sugar xylose actually affects cellular metabolism more significantly than hydrolysate inhibitors for Z. mobilis, and the co-fermentation of xylose and glucose in the presence of inhibitor remains the key barrier for economic lignocellulosic biofuel production.…”

Next generation biofuel engineering in prokaryotes

“…It does not produce butanol and is limited in the sugars it can consume, but progress has been made in engineering strains to consume pentose sugars [64,65] and express cellulases [66]. Corynebacterium glutamicum is an amino acid producer, widely used in industry [67].…”

Engineered microbial consortium for the efficient conversion of biomass to biofuels: A preliminary study

“…Other reported efforts include the works of Deanda et al (1996), Dunn and Rao (2014), Yanase et al (2005) and Yanase et al (2012). To the best of the author's knowledge, there has not been any reported microbial consortium involving Z. mobilis and C. cellulolyticum whether as wild types or engineered clones for the production of bioethanol.…”