Genetic Engineering of Zymobacter palmae for Production of Ethanol from Xylose

Abstract

Its metabolic characteristics suggest that Zymobacter palmae gen. nov., sp. nov. could serve as a useful new ethanol-fermenting bacterium, but its biotechnological exploitation will require certain genetic modifications. We therefore engineered Z. palmae so as to broaden the range of its fermentable sugar substrates to include the pentose sugar xylose. The Escherichia coli genes encoding the xylose catabolic enzymes xylose isomerase, xylulokinase, transaldolase, and transketolase were introduced into Z. palmae, where their expression was driven by the Zymomonas mobilis glyceraldehyde-3-phosphate dehydrogenase promoter. When cultured with 40 g/liter xylose, the recombinant Z. palmae strain was able to ferment 16.4 g/liter xylose within 5 days, producing 91% of the theoretical yield of ethanol with no accumulation of organic acids as metabolic by-products. Notably, xylose acclimation enhanced both the expression of xylose catabolic enzymes and the rate of xylose uptake into recombinant Z. palmae, which enabled the acclimated organism to completely and simultaneously ferment a mixture of 40 g/liter glucose and 40 g/liter xylose within 8 h, producing 95% of the theoretical yield of ethanol. Thus, efficient fermentation of a mixture of glucose and xylose to ethanol can be accomplished by using Z. palmae expressing E. coli xylose catabolic enzymes.

METABOLIC ADAPTATION STRATEGY USING Zymomonas mobilis CP4 CELLS FOR THE PRODUCTION OF SECOND GENERATION ETHANOL

“…After metabolic adaptation in xylose-containing medium, the microorganism reached 95% efficiency from the fermentation of glucose and xylose [12], higher than the efficiency reported in the study by Mohagheghi et al [13], who achieved 76% efficiency with the use of 75% (v/v) of the pentose-derived hemicellulosic hydrolysate by Z. mobilis. Thus, Zhang et al [14] demonstrated that the co-expression of XI, XK, TAL and TKT, derived from Escherichia coli, allowed Z. mobiles to coferment xylose and glucose, reaching 11 g/L ethanol and resulting in a yield of 0.44 g ethanol/g xylose consumed.…”

“…Therefore, although improvements regarding fermentation with this genetically modified Z. mobilis are still needed, the fermentation time and the ability to ferment xylose were both improved. Figure 3 summarizes the metabolic adaptation process insynthetic medium, suggesting that the recombinant Z. mobilis colonies increased ethanol production in the earlier cycles (1-30)but maintained such levels in subsequent cycles (31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50). This strategy allowed the recombinant to produce ethanol from xylose more efficiently becausehigher participation in ethanol production is derived from this pentose in the latter cycles.…”

“…Thus, some authors, e.g., Zhang et al [33] and Viitanem et al [34], have successfully developed the methodology of metabolic adaptation to circumvent this problem. Yanase et al [35] reported the need for successive adaptation in recombinant species of Zymobacter palmae to increase ethanol yields.…”

A review of pentose utilizing bacteria from bagasse hemicellulose

“…Therefore, efforts have been made to obtain recombinant strains of bacteria and yeast able to meet the requirements of industrial lignocellulose fermentation. Escherichia coli, Klebsiella oxytoca and Zymomonus mobilis have all been genetically engineered to produce ethanol and biofuel efficiently from all hexose and pentose sugars present in the polymers of hemicellulose (Attfield and Philip, 2006;Hahn-Hagerdal et al, 2007;Kim et al, 2007;Yanase et al, 2007). Others mentioned in the literature included Klebsiella species, Pichia stipitis, Bacillus species and Kluveromyces species.…”

Versatile microbial surface-display for environmental remediation and biofuels production

“…A strain of Zymobacter palmae was engineered to express xylose catabolic enzymes intracellularly, and produce approximately 45 g/l of ethanol from pure xylose [45]. The ability of the strain to utilize corn starch or more complex carbohydrates as a carbon source was not explored.…”