Photosynthetic Reduction of Xylose to Xylitol Using Cyanobacteria

Photosynthetic generation of reducing power makes cyanobacteria an attractive host for biochemical reduction compared to cell‐free and heterotrophic systems, which require burning of additional resources for the supply of reducing equivalent. Here, using xylitol synthesis as an example, efficient uptake and reduction of xylose photoautotrophically in Synechococcus elongatus PCC7942 are demonstrated upon introduction of an effective xylose transporter from Escherichia coli (Ec‐XylE) and the NADPH‐dependent xylose reductase from Candida boidinii (Cb‐XR). Simultaneous activation of xylose uptake and matching of cofactor specificity enabled an average xylitol yield of 0.9 g g^?1 xylose and a maximum productivity of about 0.15 g L^?1 day^?1 OD^?1 with increased level of xylose supply. While long‐term cellular maintenance still appears challenging, high‐density conversion of xylose to xylitol using concentrated resting cell further pushes the titer of xylitol formation to 33 g L^?1 in six days with 85% of maximum theoretical yield. While the results show that the unknown dissipation of xylose can be minimized when coupled to a strong reaction outlet, it remains to be the major hurdle hampering the yield despite the reported inability of cyanobacteria to metabolize xylose.     

Polylysine Produced by Streptomyces

The xylitol dehydrogenase gene (xdh) of Bacillus pallidus was cloned and overexpressed in Escherichia coli using pQE60 vector, for the first time. The open reading frame of 759 bp encoded a 253 amino acid protein with a calculated molecular mass of 27,333 Da. The recombinant xylitol dehydrogenase (XDH) was purified to homogeneity by three-step column chromatography, producing a single SDS–PAGE band of 28 kDa apparent molecular mass. The enzyme exhibited maximal activity at 55 °C in glycine-NaOH buffer pH 11.0, with 66% of initial enzyme activity retained after incubation at 40 °C for 1 h. In further application of the recombinant bacterium to L-xylulose production from xylitol (initial concentration 5%) using a resting cell reaction, 35% L-xylulose was produced within 24 h. This result indicates that this recombinant XDH is applicable in the large-scale production of L-xylulose.     

Increased xylitol production rate during long-term cell recycle fermentation of Candida tropicalis

Long-term cell recycle fermentations of Candida tropicalis were performed over 14 rounds of fermentation. The average xylitol concentrations, fermentation times, volumetric productivities and product yields for 14 rounds were 105 g l^–1, 333 h, 4.4 g l^–1 h^–1 and 78%, respectively, in complex medium; and 110 g l^–1, 284 h, 5.4 g l^–1 h^–1 and 81%, respectively, in a chemically defined medium. These productivities were 1.7 and 2.4 times those with batch fermentation in the complex and chemically defined media, respectively. The xylitol yield from xylose with cell recycle fermentation using the chemically defined medium was 81% (w/w), which was 7% greater than the xylitol yield with batch fermentation (74%); both modes of fermentation gave the same yield using the complex medium. These results suggest that the chemically defined medium is more suitable for production of xylitol than complex medium.     

Metabolic behavior of immobilized Candida guilliermondii cells during batch xylitol production from sugarcane bagasse acid hydrolyzate

Candida guilliermondii cells, immobilized in Ca-alginate beads, were used for batch xylitol production from concentrated sugarcane bagasse hydrolyzate. Maximum xylitol concentration (20.6 g/L), volumetric productivity (0.43 g/L. h), and yield (0.47 g/g) obtained after 48 h of fermentation were higher than similar immobilized-cell systems but lower than free-cell cultivation systems. Substrates, products, and biomass concentrations were used in material balances to study the ways in which the different carbon sources were utilized by the yeast cells under microaerobic conditions. The fraction of xylose consumed to produce xylitol reached a maximum value (0.70) after glucose and oxygen depletion while alternative metabolic routes were favored by sub-optimal conditions.     

Xylitol production from high xylose concentration: evaluation of the fermentation in bioreactor under different stirring rates

AIMS: To investigate the production of xylitol by the yeast Candida guilliermondii FTI 20037, in a bioreactor, from rice straw hemicellulosic hydrolysate with a high xylose concentration. METHODS AND RESULTS: Batch fermentation was carried out with rice straw hemicellulosic hydrolysate containing about 85 g xylose l(-1), in a stirred-tank bioreactor at 30 degrees C, under aeration of 1.3 vvm (volume of air per volume of medium per min) and different stirring rates (200, 300 and 500 rev min(-1)). The bioconversion of xylose into xylitol by the yeast depended on the stirring rate, the maximum xylitol yield (YP/S = 0.84 g g(-1)) being achieved at 300 rev min-1, with no need to pretreat the hydrolysate for purification. CONCLUSIONS: To determine the most adequate oxygen transfer rate is fundamental to improving the xylose-to-xylitol bioconversion by C. guilliermondii. SIGNIFICANCE AND IMPACT OF THE STUDY: For the microbial production of xylitol to be economically viable, the initial concentration of xylose in the lignocellulosic hydrolysate should be as high as possible, as with high substrate concentrations it is possible to increase the final product concentration. Nevertheless, there are few reports on the use of high xylose concentrations. Considering a process in bioreactor, from rice straw hemicellulosic hydrolysate, this is an innovator work.     

一种基因工程改造的NADH高亲和力木糖还原酶突变基因可促进酿酒酵母发酵木糖生成乙醇

在导入表达毕赤酵母(Pichia stipitis)木糖还原酶(xylose reductase,XR)和木糖醇脱氢酶(xylitol dehydrogenase,XDH)基因的重组酿酒酵母中,木糖还原酶活性主要依赖辅酶NADPH,木糖醇脱氢酶活性依赖辅酶 NAD+,两者的辅助因子不同导致细胞内电子氧化还原的不平衡,是造成木糖醇积累,影响木糖代谢和乙醇产量的主要原因之一.将经过基因工程改造获得的NADH高亲和力的木糖还原酶突变基因m1,与毕赤酵母木糖醇脱氢酶(PsXDH)基因xyl2共转染酿酒酵母AH109,以转染毕赤酵母木糖还原酶(PsXR)基因xyl1和xyl2重组质粒的酵母细胞为对照菌株,在SC/-Leu/-Trp营养缺陷型培养基中进行筛选,获得的阳性转化子分别命名为AH-M-XDH和AH-XR-XDH.重组酵母在限制氧通气条件下对木糖和葡萄糖进行共发酵摇瓶培养,HPLC检测发酵底物的消耗和代谢产物的产出情况.结果显示,与对照菌株AH-XR-XDH相比,AH-M-XDH的木糖利用率明显提高,乙醇得率增加了16%,木糖醇产生下降了41.4%.结果证实,通过基因工程改造的木糖代谢关键酶,可用于酿酒酵母发酵木糖生产乙醇,其能通过改善酿酒酵母细胞内氧化还原失衡的问题,提高木糖利用率和乙醇产率.     

Genetic analysis of D-xylose metabolism by endophytic yeast strains of Rhodotorula graminis and Rhodotorula mucilaginosa

Two novel endophytic yeast strains, WP1 and PTD3, isolated from within the stems of poplar (Populus) trees, were genetically characterized with respect to their xylose metabolism genes. These two strains, belonging to the species Rhodotorula graminis and R. mucilaginosa, respectively, utilize both hexose and pentose sugars, including the common plant pentose sugar, D-xylose. The xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) genes were cloned and characterized. The derived amino acid sequences of xylose reductase (XR) and xylose dehydrogenase (XDH) were 32%～41% homologous to those of Pichia stipitis and Candida. spp., two species known to utilize xylose. The derived XR and XDH sequences of WP1 and PTD3 had higher homology (73% and 69% identity) with each other. WP1 and PTD3 were grown in single sugar and mixed sugar media to analyze the XYL1 and XYL2 gene regulation mechanisms. Our results revealed that for both strains, the gene expression is induced by D-xylose, and that in PTD3 the expression was not repressed by glucose in the presence of xylose.     

Purification of Xylitol from Fermented Hemicellulosic Hydrolyzate Using Liquid–Liquid Extraction and Precipitation Techniques

Xylitol was produced by Candida guilliermondii by fermentation of sugarcane bagasse hemicellulosic hydrolysate. Undesirable impurities were extracted from the broth using either ethyl acetate, chloroform or dichloromethane. The best results on clarification of the broth without xylitol loss were obtained with ethyl acetate. When ethanol, acetone or tetrahydrofuran were used for precipitation of impurities, only tetrahydrofuran clarified the fermented broth, but a high xylitol loss (~30%) was observed.     

Immobilized cells cultivated in semi-continuous mode in a fluidized bed reactor for xylitol production from sugarcane bagasse

Summary Xylitol production from sugarcane bagasse hemicellulosic hydrolyzate was evaluated in a fluidized bed reactor operated in semi-continuous mode, using cells immobilized on porous glass. The fermentative process was performed during five successive cycles of 72 h each one. The lowest xylitol production occurred in the first cycle, where a high cell concentration (12 g l⁻¹) was observed. In the subsequent cycles the xylitol concentration was ever increasing due to the cells adaptation to the medium. In the last one, 18 g xylitol l⁻¹ was obtained with a yield factor of 0.44 g g⁻¹ and volumetric productivity of 0.32 g l⁻¹ h⁻¹.