【作者】 沈煜；

【导师】 曲音波； 鲍晓明；

【作者基本信息】 山东大学 ， 微生物学， 2005， 博士

【摘要】 燃料乙醇的生产和应用因其在经济发展和战略安全上的重大意义,越来越受到各国政府的重视。自然界中含有丰富的植物纤维资源,而目前只有3-4%的植物纤维资源被有效地利用。木糖是木质纤维素水解物中含量仅次于葡萄糖的一种单糖,在利用葡萄糖的同时,利用木糖发酵生产乙醇将有助于充分利用木质纤维素资源,降低乙醇生产成本。 酿酒酵母(Saccharomyces cerevisiae)是传统上食品及化工工业的最佳生产菌株,在工业生产中有上百年的酒精发酵应用历史,具有乙醇产量高、抗逆性强等许多优良特性,是酿酒工业生产的首选菌株。酿酒酵母菌缺乏有效转化木糖生成木酮糖的酶而不能利用木糖,但能利用木糖的异构体形式——木酮糖。 自然界中由木糖转化为木酮糖的代谢途径有两条:其一,主要在某些酵母及丝状真菌中,木糖经木糖还原酶(xylose reductase,XR)催化生成木糖醇,然后由木糖醇脱氢酶(xylitol dehydrogenase,XDH)作用生成木酮糖,XR和XDH分别需要NADPH和NAD~+作为辅酶。其二,在某些细菌和低等真菌中,木糖通过木糖异构酶(xylose isomerase,XI)催化,直接转化为木酮糖。木酮糖经过木酮糖激酶(xylulokinase,XK)磷酸化生成5-磷酸木酮糖,从而进入磷酸戊糖途径(pentose phosphate pathway,PPP)。 通过代谢工程(metabolic engineering)手段,包括本试验室在内的多个工作小组已经分别在酿酒酵母中表达Pichia stipitis来源的木糖还原酶基因XYL1和木糖醇脱氢酶基因XYL2,Thermus thermophilus来源的木糖异构酶基因xylA。在酿酒酵母中建立自然界存在的两种木糖代谢途径,即,木糖还原酶—木糖醇脱氢酶(XR-XDH)途径和木糖异构酶(XI)途径。由于具有XR-XDH途径或者XI途径的重组酿酒酵母木糖利用率低且发酵副产物木糖醇产生量较高,因而需要对代谢途径下游进行疏通。同时,由于在自然界中酿酒酵母本身不利用木糖,因此,其运输系统向细胞内运输木糖的能力也不能满足利用木糖发酵生产乙醇的需要。本文的主要研究内容在此基础上开展。 木酮糖激酶(XK)催化木酮糖磷酸化形成5-磷酸木酮糖,是木糖代谢的限速步骤之一,处于木糖代谢物进入磷酸戊糖途径(PPP)的节点位置。超表达酿酒酵母自身的木酮糖激酶基因XKS1可以加速木糖代谢流,提高酿酒酵母的木糖利用率和乙醇得率。更多还原

【Abstract】 The production and application of fuel ethanol is paid much attention to by many governments for the importance of economic development and strategic significance. There is abundance of lignocellulosic materials in the nature, however only 3-4% of lignocellulosic materials are utilized. D-xylose is the most abundant monosaccharide in lignocellulose hydrolysates after glucose. To adequately utilize xylose can make the production of ethanol more economical.Saccharomyces cerevisiae has been traditionally used in producing ethanol, which has acquired qualities such as high ethanol productivity, tolerance to process hardiness, tolerance to fermentation by-products and is, therefore, preferred for ethanol production from crops. Although a few xylose-fermenting yeasts were found, the preferred organism for industrial ethanol production is the yeast S. cerevisiae which can not utilize D-xylose, only its isomer D-xylulose.In the naturally xylose-utilizing yeast, the first two-enzymatic steps in xylose metabolism are catalysed by NADPH-dependent xylose reductase (XR) and NAD+-dependent xylitol dehydrogenase (XDH). First, xylose is reduced by XR to xylitol, then xylitol oxidized by XDH to xylulose. In bacteria, D-xylose isomerase catalyses the reversible isomerization of D-xylose and D-xylulose. XI does not require redox cofactors and cannot generate cofactor imbalance during anaerobic xylose utilization. Xylulose is converted to xylulose 5-phosphate by the xylulokinase, then enter the pentose phosphate pathway (PPP).Metabolic engineering can be used to extend the substrate range for growth and product formation of an organism. Many work groups, including us, established the xylose metabolic pathway in the S. cerevisiae. The recombinant S. cerevisiae which expressing the xylose reductase and xylitol dehydrogenase from Pichia stipitis or xylose isomerase from Thermits thermophilus can ferment xylose to produce ethanol in a correspondingly low-level. The downstream of the pathway is influential. And because the xylose is not the nature carbon source of S. cerevisiae, the transport of it is not easy for the S. cerevisiae.The main objectives of this research are as follows:Xylulokinase converts the five-carbon sugar D-xylulose and ATP to xylulose 5-phosphate and ADP. The expression level of xylulokinase can limit xylose metabolism. Over-expression of the xylulokinase gene XKS1 can increase the D-xylose utilization and ethanol yield of recombinant yeast strain.The xylulokinase gene XKS1 was cloned from S. cerevisiae NAN-27 and ligated into plasmids pMA91 and YEp24, producing pMA-xy203 and YEpP-xy204, respectively. In both plasmids, XKS1 was under the control of PGK promoter, which is strong and constitutive promoter. pMA-xy203 was transferred into the pre-constructed recombinant yeast strain H158-XR-XDH, which contains the XYL1 and XYL2 genes from P. stipitis, encoding xylose reductase and xylitol dehydrogenase respectively, in an episomal plasmid vector. This new recombinant strain was named HSXY-251. YEpP-xy204 was transferred into the pre-constructed recombinant yeast strain H158-XI, which contains the xylA gene from Thermus thermophilus encoding xylose isomerase in an episomal plasmid vector, resulting in recombinant strain HSXY-252. The xylulokinase activities in HSXY-251 and HSXY-252 were 14 and 6.7 times higher than in the parent strain. Glucose and xylose co-fermentation carried out with HSXY-251 under oxygen-limited conditions at 30℃ resulted in 9.4 g/L ethanol concentration with 12.4 g/L xylose consumed. Xylose consumption and ethanol production were 120.9% and 36% higher, respectively, than in the parent strain. Furthermore, the production of xylitol was 0.7 g/L, a decrease of 84.9%.Xylose uptake might be one of limiting step for xylose fermentation by recombinant xylose-utilizing S. cerevisiae cells. The problem of xylose uptake may be slided over by expressing the XI outside the cells. The signal peptide of S. cerevisiae invertase or agglutinin can lead the protein secretion out of the cell. A genetic system has been exploited to immobilize proteins in their active and functional forms on the cell surface of S. cerevisiae. DNAs encoding proteins with a secretion signal peptide were fused with the genes encoding yeast agglutinins, a- and alpher-type proteins involved in mating.First, the xylose isomerase (XI) gene from T. thermophilus was fused with the sequence encoding the C-terminal of Aga2p contained in the yeast a-agglutinin surface display vector pYD1. This system is a commercial one. The fused gene was under the control of GAL1 promoter. The recombinant plasmid was transformed into the yeast stain EBY100 by the lithium acetate method. The strain EBY100/pYD-xylA containing the recombinant plasmid was induced by the galactose, and the result of immunofluorescence microscopy confirmed that the fused protein was displayed on the cell wall. The XI activity detected on cell wall was 1.52 U. The results showed that the XI was displayed on the cell wall actively but the utilizing of xylose was not improved.The XI was fused with the C-terminal of Agap and the signal peptide of S.cerevisiae invertase. The fuse protein was under the control of PGK promoter. The recombinant strain expressing the fuse protein was named H158-SXI. The XI activity is mainly detected on the cell wall of H158-SXI. Glucose and xylose co-fermentation carried out with H158-SXI consumed 2.7 g/L xylose, which is 45.7% higer than parent strain H158 but as the same level as H158-XI. Likewise, the XI was fused with the signal peptide of S. cerevisiae invertase only and under the control of PGK promoter. The recombinant strain expressing the fuse protein was named H158-IXI. Glucose and xylose co-fermentation carried out with H158-IXI consumed 3.2 g/L xylose, which is 69.5% higer than parent strain HI58. All the recombinant strains, H158-XI, H158-SXI and H158-IXI, can grow slowly on the plant that xylose is the only carbon source. All the result above suggested that xylose uptake will not be the limiting step when the XI activity is not strong enough.The xylA gene actively expressed in S. cerevisiae was cloned from the thermophilic bacterium, T. thermophilus. The temperature optimum of this enzyme is around 85 ℃ and the temperature optimum of S. cerevisiae is around 30℃. DNA sequence and amino acid analysis revealed that the 137th amino acid of XI from T. thermophilus is praline (Pro) but the amino acid of XI from other microorganism in the same location is glycin (Gly). The amino acid sequences of the domains in different XI are high similarity with each other and important for the 3D-structure. We designed the PCR primer to do the site-directed mutagenesis to lower the temperature optimum of this enzyme.The gene we store was sequenced and compared with the initial xylA that reported. The result showed that the 372nd amino acid of XI were changed (glutamic to glycin) but the temperature optimum was no different between the XIs. We did the site-directed mutagenesis twice. Two amino acids of XI were changed in mutentl, 137th amino acid (praline to glycin), 141st amino acid (glutamine to arginine) and only one amino acid of XI were changed in mutent2, 137th amino acid (praline to glycin).The XI activity of recombinate strain H158-XI137-1 expressing the XI mutantl is higher than H158-XI at 30℃, but is quite lower than H158-XI at high temperature. The XI temperature optimum of recombinate strain H158-XI137-2 expressing the XI mutant2 is 70℃, but the XI activity of H158-XI137-2 is as lower as H158-XI at 30℃.Recently, the xylA gene encoding the Piromyces sp. E2 xylose isomerase was functionally expressed in S. cerevisiae. That encouraged us to find new XI. Sorangium cellulosum is a kind of advanced procaryotae, and it is closer to the eukaryotes. We designed the PCR primer based on the high similarity amino acid sequence ofdifferent XI to clone a part of DNA sequence from the chromosome of S. cellulosum 157-2. The production of PCR was sequenced and had a high degree of sequence similarity with xylA from Piromyces sp. E2. The the full sequence cloning work of xylA from S. cellulosum are going.更多还原