Zymomonas mobilis genes for pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) were integrated into the Escherichia coli chromosome within or near the pyruvate formate-lyase gene (pif). Integration improved the stability of the Z. mobilis genes in E. coli, but further selection was required to increase expression. Spontaneous mutants were selected for resistance to high levels of chloramphenicol that also expressed high levels of the Z. mobilis genes. Analogous mutants were selected for increased expression of alcohol dehydrogenase on aldehyde indicator plates. These mutants were functionally equivalent to the previous plasmid-based strains for the fermentation of xylose and glucose to ethanol. Ethanol concentrations of 54.4 and 41.6 g/liter were obtained from 10% glucose and 8% xylose, respectively. The efficiency of conversion exceeded theoretical limits (0.51 g of ethanol/g of sugar) on the basis of added sugars because of the additional production of ethanol from the catabolism of complex nutrients. Further mutations were introduced to inactivate succinate production (frd) and to block homologous recombination (recA).

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The conversion of xylose to ethanol by recombinant Escherichia coli has been investigated in pH-controlled batch fermentations. Chemical and environmental parameters were varied to determine tolerance and to define optimal conditions. Relatively high concentrations of ethanol (56 g/L) were produced from xylose with excellent efficiencies. Volumetric productivities of up to 1.4 g ethanol/L h were obtained. Productivities, yields, and final ethanol concentrations achieved from xylose with recombinant E. coli exceeded the reported values with other organisms. In addition to xylose, all other sugar constituents of biomass (glucose, mannose, arabinose, and galactose) were efficiently converted to ethanol by recombinant E. coli. Unusually low inocula equivalent to 0.033 mg of dry cell weight/L were adequate for batch fermentations. The addition of small amounts of calcium, magnesium, and ferrous ions stimulated fermentation. The inhibitory effects of toxic compounds (salts, furfural, and acetate) which are present in hemicellulose hydrolysates were also examined.

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Previously, we reported that GfsA is a novel galactofuranosyltransferase involved in the biosynthesis of O-glycan, the proper maintenance of fungal morphology, the formation of conidia and anti-fungal resistance in Aspergillus nidulans and A. fumigatus (Komachi Y et al., 2013. GfsA encodes a novel galactofuranosyltransferase involved in biosynthesis of galactofuranose antigen of O-glycan in Aspergillus nidulans and Aspergillus fumigatus. Mol. Microbiol. 90:1054-1073). In the present paper, to gain an in depth-understanding of the enzymatic functions of GfsA in A. fumigatus (AfGfsA), we established an in vitro assay to measure galactofuranosyltransferase activity using purified AfGfsA, UDP-α-d-galactofuranose as a sugar donor, and p-nitrophenyl-β-d-galactofuranoside as an acceptor substrate. LC/MS, 1H-NMR and methylation analyses of the enzymatic products of AfGfsA revealed that this protein has the ability to transfer galactofuranose to the C-5 position of the β-galactofuranose residue via a β-linkage. AfGfsA requires a divalent cation of manganese for maximal activity and consumes UDP-α-d-galactofuranose as a sugar donor. Its optimal pH range is 6.5-7.5 and its optimal temperature range is 20-30°C. 1H-NMR, 13C-NMR and methylation analyses of fungal-type galactomannan extracted from the ∆AfgfsA strain revealed that AfGfsA is responsible for the biosynthesis of β1,5-galactofuranose in the galactofuran side chain of fungal-type galactomannan. Based on these results, we conclude that AfGfsA acts as a UDP-α-d-galactofuranose: β-d-galactofuranoside β1,5-galactofuranosyltransferase in the biosynthetic pathway of galactomannans.