Seventeen species of facultative anaerobic bacteria belonging to three genera (Serratia, Cellulomonas, and Corynebacterium) were screened for the production of xylitol; a sugar alcohol used as a sweetener in the pharmaceutical and food industries. A chromogenic assay of both solid and liquid cultures showed that 10 of the 17 species screened could grow on D-xylose and produce detectable quantities of xylitol during 24-96 h of fermentation. The ten bacterial species were studied for the effect of environmental factors, such as temperature, concentration of D-xylose, and aeration, on xylitol production. Under most conditions, Corynebacterium sp. NRRL B 4247 produced the highest amount of xylitol. The xylitol produced by Corynebacterium sp. NRRL B 4247 was confirmed by mass spectrometry. Corynebacterium sp. NRRL B 4247 was studied for the effect of initial D-xylose concentration, glucose, glyceraldehyde, and gluconate, aeration, and growth medium. Corynebacterium sp. NRRL B 4247 produced xylitol only in the presence of xylose, and did not produce xylitol when gluconate or glucose was the substrate. The highest yield of xylitol produced in 24 h (0.57 g/g xylose) was using an initial D-xylose concentration of 75 g/l. Under aerobic conditions the highest xylitol yield was 0.55 g/g while under anaerobic conditions the highest yield was 0.2 g/g. Glyceraldehyde in concentrations greater than 1 g/l inhibited Corynebacterium sp. B 4247 growth and xylitol production. Corynebacterium sp. NRRL B 4247 culture grown in the presence of potassium gluconate (96 g/l) for 48 h and on addition of D-xylose to the media increased accumulation to 10.1 g/l of xylitol after 150 h. Corynebacterium sp. NRRL B 4247 exhibited both NADH and NADPH-dependent xylose reductase activity in cell-free extracts. The NADPH-dependent activity was substrate dependent. The activity was 2.2-fold higher when DL-glyceraldehyde was used as substrate than with D-xylose. In cell-free extracts the difference in xylose reductase and xylitol dehydrogenase activity was highest at 24 h, whereas for cell cultures that were grown in gluconate and xylose, the difference in the reductase and dehydrogenase activities was highest at 12 h after xylose addition. The NAD+ dependent xylitol dehydrogenase activity was low compared to the cells grown without gluconate. The molecular weight of NADPH-dependent xylose reductase protein obtained by gel filtration chromatography was 58 kDa. Initial purification was performed on a DE-52 anion exchange column. Purification using Red Sepharose affinity column resulted in a 58 kDa protein on the SDS PAGE gel and was further purified on a Mono-Q column. The activity stained band on the native gel yielded 58, 49, 39 and 30 kDa bands on the denaturing gel. The peptides of the 58 kDa protein of Corynebacterium sp. B 4247 sequenced by mass spectrometry, identified with E2 and E3 (Bacillus subtilis) components of multi-enzyme system consisting of pyruvate dehydrogenase complex, 2-oxoglutarate dehydrogenase complex and oxo-acid dehydrogenase complex. A 75% match was shown by the peptide "QMSSLVTR" with E-value of 8e-04 to the Saccharomyces cerevisiae protein that was capable of reducing xylose to xylitol. The peptide "LLNDPQLILMEA" had conserved match "LL + DP" over several aldose reductases. The xylose reductase of the yeast Candida tropicalis ATCC 96745 was also purified. The molecular weight of the yeast NADPH-dependent xylose reductase was about 37 kDa on an SDS PAGE / Ph. D.

Ethanol from lignocellulosic biomass is a promising alternative to rapidly depleting oil reserves. However, natural recalcitrance of lignocelluloses to biological and chemical treatments presents major engineering challenges in designing an ethanol conversion process. Current methods for pretreatment and hydrolysis of lignocelluloses generate a mixture of pentose (C5) and hexose (C6) sugars, and several microbial growth inhibitors such as acetic acid and phenolic compounds. Hence, an efficient ethanol production process requires a fermenting microorganism not only capable of converting mixed sugars to ethanol with high yield and productivity, but also having high tolerance to inhibitors. Although recombinant bacteria and yeast strains have been developed, ethanol yield and productivity from C5 sugars in the presence of inhibitors remain low and need to be further improved for a commercial ethanol production. The overarching objective of this work is to transform Zymomonas mobilis into an efficient whole-cell biocatalyst for ethanol production from lignocelluloses. Z. mobilis, a natural ethanologen, is ideal for this application but xylose (a C5 sugar) is not its 'natural' substrate. Back in 1995, researches at National Renewable Energy Laboratory (NREL) had managed to overcome this obstacle by metabolically engineering Z. mobilis to utilize xylose. However, even after more than a decade of research, xylose fermentation by Z. mobilis is still inefficient compared to that of glucose. For example, volumetric productivity of ethanol from xylose fermentation is 3- to 4- fold lower than that from glucose fermentation. Further reduction or complete inhibition of xylose fermentation occurs under adverse conditions. Also, high concentrations of xylose do not get metabolized completely. Thus, improvement in xylose fermentation by Z. mobilis is required. In this work, xylose fermentation in a metabolically engineered Z. mobilis was markedly improved by applying the technique of adaptive mutation. The adapted strain was able to grow on 10% (w/v) xylose and rapidly ferment xylose to ethanol within 2 days and retained high ethanol yield. Similarly, in mixed glucose-xylose fermentation, the strain produced a total of 9% (w/v) ethanol from two doses of 5% glucose and 5% xylose (or a total of 10% glucose and 10% xylose). Investigation was done to identify the molecular basis for efficient biocatalysis. An altered xylitol metabolism with reduced xylitol formation, increased xylitol tolerance and higher xylose isomerase activity were found to contribute towards improvement in xylose fermentation. Lower xylitol production in adapted strain was due to a single mutation in ZMO0976 gene, which drastically lowered the reductase activity of ZMO0976 protein. ZMO0976 was characterized as a novel aldo-keto reductase capable of reducing xylose, xylulose, benzaldehyde, furfural, 5-hydroxymethyl furfural, and acetaldehyde, but not glucose or fructose. It exhibited nearly 150-times higher affinity with benzaldehyde than xylose. Knockout of ZMO0976 was found to facilitate the establishment of xylose fermentation in Z. mobilis ZM4. Equipped with molecular level understanding of the biocatalytic process and insight into Z. mobilis central carbon metabolism, further genetic engineering of Z. mobilis was undertaken to improve the fermentation of sugars and lignocellulosic hydrolysates. These efforts culminated in construction of a strain capable of fermenting glucose-xylose mixture in presence of high concentration of acetic acid and another strain with a partially operational EMP pathway.