【作者】 刘恩凯；

【导师】 马平生；

【作者基本信息】 天津大学 ， 生物化工， 2010， 博士

【摘要】 D-木糖是自然界中含量仅次于葡萄糖的碳水化合物,在全球化石能源日渐枯竭,环境污染日益严重形势下,将木糖高效转化为乙醇将对全球经济和环境产生巨大的影响。Saccharomyces cerevisiae在己糖发酵上具有天然的优势,但是S. serevisiae不具备代谢木糖能力。本研究采用重组DNA技术,化学诱变和适应性进化筛选结合起来构建工业木糖发酵菌株。首先通过重组DNA技术将P. stipitis的木糖还原酶基因XYL1和木糖醇脱氢酶基因XYL2以及酵母内源的木酮糖激酶基因XKS1,整合到经修饰的单倍体酵母工业菌株KAM-2中,所得重组菌株LEK122具备基本的代谢木糖能力,再通过化学诱变(EMS)和适应性进化筛选方法结合作用于基因工程改造菌株,获得在木糖上的生长能力,木糖代谢能力大幅提高的优势菌株LEK513。在好氧条件下木糖作为单一碳源时,LEK513最大比生长速率是0.225h-1,而LEK122是0.055h-1。当在微好氧条件下,LEK513最大比生长速率是0.205h-1,与好氧条件下相比无显著差距。在100小时分批发酵中,LEK513的最终OD可以到达60,而LEK122仅为7.5。在相同时间内,LEK513消耗掉培养基中95%的木糖,而LEK122仅消耗20%。同时,LEK513在微好氧条件下比在好氧条件下可以多生产11%的乙醇。这表明基于理性设计的基因工程改造与随机筛选方法相结合可以更有效的构建符合工业要求的木糖发酵生产乙醇的S. cerevisiae菌株。S. serevisiae中的非氧化磷酸戊糖途径(PPP途径)的代谢通量较低,以PGK1启动子过表达PPP全部四个基因(TAL1, TKL1, RKI1, RPE1),同时缺失促进木糖醇产生的转醛酶基因GRE3。以此作为出发菌株(LEK625),通过Western blot实验,获得在木糖为单一碳源时,调控表达强度较高的启动子作为引入木糖代谢通路的首选,将调控XYL1的启动子更换为TPI1启动子,将调控XYL2和内源XKS1的启动子更换为HXT7 truncated启动子,更换了启动子的表达系统整合到出发菌株染色体获得菌株LEK631,对比先前使用的启动子系统的菌株LEK630,结果如下:两株菌均以OD600=0.6接入YPX(50g/L)液体培养基中进行分批发酵培养,在216h发酵过程中,测得LEK630 OD600最高为7.0,而LEK631是31.2;发酵过程消耗总木糖比例LEK630为11.9%,而LEK631是96.8%;最高乙醇浓度LEK630为1.052g/L,而LEK631是7.575g/L。发酵结果显示,更换了木糖还原酶、木糖醇脱氢酶以及木酮糖激酶的启动子后,重组菌株的生长能力、木糖消耗能力和乙醇产量都大幅提高。使用融合PCR技术合成Piromyces的木糖异构酶基因xylA,将其与木酮糖激酶基因XKS1在HXT7 truncated启动子调控下整合到LEK625染色体上实现过量表达,所得菌株具备木糖代谢、生长能力,但是在发酵液中没有检测到乙醇的产生。更多还原

【Abstract】 D-Xylose is the second most abundant carbohydrate in nature, which is considered to be of great economic and environmental significance for future biofuels. While Saccharomyces cerevisiae has natural advantage in ethanolic fermentation of hexose, it is incapable of xylose utilization.In the first part of the work, we describe the construction of a S. cerevisiae strain via combined approaches of recombinant DNA technology, chemical mutagenesis and evolutionary adaptation for an efficient xylose utilization and ethanol fermentation. A haploid derivative of an industrial ethanol fermenting strain KAM-2 was first engineered to functionally express the XYL1 and XYL2 genes from Pichia stipitis, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, and the endogenous XKS1 gene, encoding xylulokinase (XK). The resulting recombinant strain LEK122, which has acquired basic xylose-utilizing ability, was then subjected to EMS mutagenesis followed by adaptive evolution, resulting a single isolate, LEK513, that displayed significantly improved xylose-utilizing property. The specific growth rate of the LEK513 strain was 0.225h-1 under aerobic condition with xylose as the sole carbon source, while that of the LEK122 was 0.055h-1. When cultured under oxygen-limited condition, the specific growth rate of LEK513 was 0.205h-1 comparable to that under aerobic condition. During 100 hour batch cultivation, the optical density of LEK513 reached 60, while LEK122 only grew to 7.5. In the same time period, LEK513 consumed 95% of the xylose in the medium, while LEK122 only consumed 20% of the xylose in the medium. The LEK513 strain produced 11% more ethanol in oxygen-limited fermentation than it did in aerobic fermentation. The significantly improved xylose-utilizing property demonstrated the feasibility of the combination of rational and random approaches in construction of efficient xylose-utilizing, ethanol fermenting S. cerevisiae strains for industrial application.The flux through the nonoxidative PPP in S. cerevisiae is insufficient, so the four genes involved in nonoxidative PPP (TAL1, TKL1, RKI1, RPE1) were overexpressed by the PGK1 promoter and the GRE3 gene encoding endogenous xylose (aldose) reductase which mediates unwanted production of xylitol was deleted to construct a original strain. In consideration of Western blot results when xylose as sole carbon source, the relatively strong promoter TPI1 promoter and HXT7 truncated promoter were chosed to introduce the xylose assimilation pathway into the yeast. The new xylose assimilating cassette with ADH1 promoter replaced by TPI1 promoter and PGK1 promoter replaced by HXT7 truncated promoter was inserted into genome of original strain to generate the strain LEK631, compared with strain LEK630 in which using the ADH1 & PGK1 promoter to express xylose assimilating pathway, the fermentation results as follows: both of them were taken to inoculate YPX(50g/L) with an initial cell concentration of OD600=0.6, in 216h batch cultivation, the final OD600 of LEK630 was 7.0, whereas LEK631 was 31.2; LEK630 consumed 11.9% of total xylose, whereas LEK631 consumed 96.8%; and the highest ethanol concentration during fermentation period in LEK630 was 1.052g/L, whereas in LEK631 that was 7.575g/L. From the results, the changing of the promoters significantly improved growth ability, xylose-utilizing and ethanol-yielding property. The Piromyces xylose isomerase gene xylA that synthesized by fusion PCR and endogenous XKS1 gene were placed under the control of HXT7 truncated promoter. Both of them were inserted into original strain genome, the resulting strain could grow on the medium that xylose as sole carbon source, but no ethanol was detected in fermentation broth.更多还原