Xylitol Or Ethanol Production At High Temperature By Engineered Kluyveromyces Marxianus

Xylitol and ethanol are two important chemicals. Xylitol has important application value in medicine and food. However, the industrial xylitol production is mainly through chemical conversion method which brings food security and environmental pollution problems. Therefore, using microorganisms to produce xylitol has attracted more and more attention. On the other hand, liquid fuel is mainly dependent on oil, but the limited fossil fuel and global environmental problems focus people’s attention on the sustainable development. The production of transportation fuels such as bioethanol by microorganisms not only meet the demand of transportation fuel on the current and future, but also a way out to deal with the serious environmental problems. Therefore, it becomes an urgent demand for bioethanol production by microorganism.In the aspect of xylitol production from xylose, first of all, we constructed three engineered strains which were disrupted the xylose reductase gene (Xyll) or the xylitol dehydrogenase gene (Xyl2) individually or simultaneously. Then the Neurospora crassa xylose reductase gene (NcXyll) or Pichia pastoris xylose reductase gene (PsXyll) were overexpressed in the three engineered strains. YZJ015, which maintained the original xylitol dehydrogenase gene, produced xylitol with the highest productivity (1.49g/L/h) from100g/L xylose at42℃. Even at45℃, YZJ015was still able to produce60.03g/L xylitol from100g/L xylose with a productivity of1.25g/L/h. In addition, for20rounds of cell recycling at42℃, YZJ015produced71.35g/L xylitol from100g/L xylose with a productivity of4.43g/L/h per cycle. YZJ017, in which the xylitol dehydrogenase gene was disrupted, produced100.02g/L xylitol at a yield of1.01g/g from100g/L xylose with40g/L glycerol as co-substrate at42℃. The xylitol production of YZJ074which harbored CiGXFl on the basis of YZJ017was improved to147.62g/L in Erlenmeyer flask at42℃. In fermenter,99.29and149.60g/L xylitol were produced from99.55and151.91g/L xylose with productivity of4.14and3.40g/L/h respectively at42℃. Even at45℃, YZJ074could produce101.30g/L xylitol from101.41g/L xylose with productivity of2.81g/L/h. Using fed-batch fermentation through repeatedly adding non-sterilized substrate directly, YZJ074could produce312.05g/L xylitol which is the highest yield reported to date. These engineered strains provided an excellent foundation for xylitol production at elevated temperatures.In the aspect of ethanol production from xylose, a combination of NADPH-preferring xylose reductase (XR) from Neurospora crassa and NADP+-preferring xylitol dehydrogenase (XDH) mutant from Scheffersomyces stipitis was constructed. The xylose fermentation ability and redox balance of the recombinant strains was improved significantly further by over-expressing several metabolic downstream genes. The intracellular concentrations of coenzymes and the ratio of reduced coenzyme/oxidized coenzyme were increased significantly in metabolic vigorous strains. The byproduct such as glycerol and acetic acid were significantly reduced by the disruption of the glycerol-3-phosphate dehydrogenase (GPD1) gene. The obtained engineered strain K. marxianus YZJ088produced44.95g/L ethanol from118.39g/L xylose with the productivity of2.49g/L/h at42℃. Also YZJ088obtained the simultaneous glucose and xylose co-fermentation ability and produced51.43g/L ethanol from the mixture of103.97g/L xylose and40.96g/L glucose with a productivity of2.14g/L/h. These promising results validate the strain YZJ088as an excellent producer of ethanol from xylose by the synthetical xylose assimilation pathway.In summary, we finally obtained engineered strains YZJ015, YZJ074or YZJ088which have the ability of using xylose to produce xylitol or ethanol at higher temperature (>42℃). Compared with those previously reported yeasts which produce xylitol by xylose, YZJ015and YZJ074had faster productivity rate, higher yield and higher production. Compared with those previously reported thermotolerant yeasts which produce ethanol by xylose, YZJ088has faster rate, higher yield and higher production. These strains provided a very good metabolic route modification idea and actual production platform for using xylose to produce xylitol or ethanol in industry.