Expression Of Xylose Metabolism Related Genes In Engineering Strains And Optimization Of Ethanol Production By Fermentation Of Glucose / Xylose

With the consumption of fossil energy, clean and renewable energy has been the main trend. Ethanol have been identified as the most promising renewable energy because of its clean, renewable and easy to transport and other advantages. Ethanol production from lignocellulosic biomass source broadens the raw material for fuel ethanol, and solves environmental pollution caused by agricultural and forestry wastes. Lignocellulosic feedstock after hydrolysis to produce a large number of monosaccharides, the highest content of glucose and xylose. To achieve effective conversion of lignocellulose to ethanol, xylose utilization is particularly important. Nature about 100 kinds of microorganisms can utilize xylose, a larger proportion of studies in yeast. Saccharomyces cerevisiae, The ethanol fermentation predominant strains, however, can not effectively utilize xylose. Saccharomyces cerevisiae is often used as the recipient strain, accept other xylose metabolism genes, so as to adapt to the requirements of industrialization.The laboratory had been successfully constructed a series of recombinant engineering strains which can utilize xylose well by protoplast fusion and genetic engineering. These strains had high xylose utilization, ethanol production and ethanol yields. Whether these engineered strains can be used for industrial production or not, which remains to be verified whether the full flow of xylose metabolism.ZLYRHZ7 that obtained by protoplast fusion had the highest ethanol production, up to 21.37g/L. When the shaking speed of 200 r/min, pinhole rubber plug for fermentation, ethanol production reached 25.48±0.23 g/L. It is necessary to test validation of ZLYRHZ7 xylose metabolism genes in DNA, RNA and protein levels. The results show that ZLYRHZ7 could amplifiy xy11(972 bp), xyl2(1113 bp) and xks(1831 bp) by PCR testing. Xylose reductase gene (xyll) whose maximun transcription level was 266.37, and whose enzyme activity was 7.49±0.03 U/mg. Highest transcriotion level of xylitol dehydrogenase gene(xyl2) was 418.61, whose enzyme activity was 18,62±0.25 U/mg. Xylulose kinase gene transcription level was 317.38, and the enzyme activity was 136.96±0.35 U/mg. This suggests that ZLYRHZ7 does exist xylose metaolism-related gene, and these gene does work.For verification the xylose flux of ZLYRHZ7, different intermediate metabolites were used as sole carbon source. The results of ethanol production and ethanol yield from xylose (10 g/L) 2.419±0.027 g/L and 0.420 ±0.003 g/g. The production and yield from xylitol (10 g/L) were1.672±0.0027 g/L and 0.287±0.003 g/g. When the carbon source was xylulose(10 g/L),the production and yield were 0.444±0.003 g/L and 0.149 ±0.005 g/g.This research provided the basis for the to build efficient use of xylose recombinant strains. It provided a reference for lignocellulosic biomass sources as raw material for the production of bio-ethanol.