【作者】 査健；

【导师】 元英进；

【作者基本信息】 天津大学 ， 制药工程， 2013， 博士

【摘要】 木糖是木质纤维素水解液中含量仅次于葡萄糖的单糖，高效利用木糖是纤维素乙醇进行产业化生产的必要条件。前人在构建高效代谢木糖的酿酒酵母菌株的研究中已经取得了一些成果，但是菌株的木糖利用效率还不能达到工业生产的要求，需要进一步的改善和提高。在本研究中设计、构建了由木糖还原酶基因XYL1、木糖醇脱氢酶突变体基因mXYL2和木酮糖激酶基因XKS1构成的木糖代谢模块，并通过优化XYL1和mXYL2的表达、强化非氧化磷酸戊糖途径功能，优化木糖代谢模块和酵母底盘细胞间的适配性，获得了木糖代谢速率为0.260g/L/h、乙醇得率为0.20g/g的酵母菌株SyBE004。通过启动子工程组合优化设计和构建由木糖异构酶基因XylA和XKS1构成的木糖代谢模块，获得了木糖代谢速率为0.081g/L/h的菌株SyBE002。基于传代限氧培养的进化工程改造，在SyBE002和SyBE004的基础上，分别获得了进化菌株SyBE003和SyBE005。与SyBE004相比，SyBE005的木糖代谢速率和乙醇产率提高2.20倍、2.67倍，乙醇得率提高到0.33g/g；SyBE003的木糖比消耗速率比SyBE002提高了4倍。以SyBE003和SyBE005为出发菌株，通过恒浊进化获得了SyBE006和SyBE007。SyBE007的木糖比消耗速率提高了34%，达到0.98g/g DW/h；SyBE006的木糖比消耗速率提高了54%，达到1.27g/gDW/h。为了解析进化过程中木糖代谢模块发生的重构变化，运用实时定量RT-PCR检测了SyBE002和SyBE003中相关基因表达差异。结果表明：XylA的表达显著降低，但是酶的活性保持不变；XKS1的表达增加了3倍；三羧酸循环中的基因表达显著降低，可能与增强的乙醇代谢功能有关。对菌株SyBE004、SyBE005和SyBE007的分析表明：初始木糖代谢途径基因XYL1、XYL2、XKS1和基因ZWF1的表达量显著增加，揭示强化的初始木糖代谢途径和NADPH供给可以加强木糖代谢。另外，在酵母中构建纤维二糖（葡萄糖二聚体）代谢途径，从而消除乙醇发酵过程中葡萄糖抑制效应的影响；并通过组合优化纤维二糖转运酶CDT-1和XYL1的表达，实现同时高效共利用纤维二糖和木糖。更多还原

【Abstract】 Xylose is the second most abundant monosaccharide after glucose in lignocellulosichydrolysates. Efficient utilization of xylose is required for economic lignocellulosicethanol production. Some achievements in construction of xylose-fermentingSacchromyces cerevisiae have been obtained. However, the xylose fermentationcapacities of these strains cannot satisfy the industrial applications and need furtherimprovement.We successfully designed and expressed a xylose-utilizing module in S.cerevisiae consisting of xylose reductase gene XYL1, mutated xylitol dehydrogenasegene mXYL2and xylulose kinase gene XKS1. To optimize the compatibility of thexylose-utilizing module and host chassiss, the expression of XYL1and mXYL2wasoptimized, coupling with strengthening the function of the non-oxidative pentosephosphate pathway, enabling the construction of strain SyBE004with a xyloseconsumption rate of0.260g/L/h and a ethanol yield of0.20g/g. Moreover, axylose-utilizing strain SyBE002with a xylose consumption rate of0.081g/L/h wasconstructed by combinatorial optimization of the expression of xylose isomerase geneXylA and XKS1through promoter engineering. Strains SyBE002and SyBE004werethen subjected to repetive adaptation under oxygen-limited conditions, enabling theisolation of improved strains SyBE003and SyBE005, respectively. The xylose uptakerate and ethanol production rate of SyBE005increased by2.20-and2.67-foldcompared with strain SyBE004. Accordingly, the ethanol yield increased to0.30g/g.As for SyBE003, it showed a4-fold higher specific xylose consumption rate thanSyBE002. On the basis of strains SyBE003and SyBE005, evolved isolates ofSyBE006and SyBE007were further obtained throught continuous chemostatevolution. The specific xylose uptake rate of SyBE007increased by37%comparedwith SyBE005, reaching0.98g/gDW/h. Similarly, the specific xylose uptake rate ofSyBE006(1.27g/gDW/h) was54%higher than that of SyBE003.In order to understand the reconfiguration in xylose metabolic pathway, thequantitative RT-PCR was applied to investigate the transcriptional changes inSyBE003compared to SyBE002. The expression of XylA in SyBE003wassignificantly down-regulated but the exzymatic activity kept consistent. The geneXKS1showed a3-fold higher expression level in SyBE005. Meanwhile, thetranscriptional levels of genes in the TCA cycle decreased significantly, which might contribute to enhanced ethanol production. The transcriptional analysis of genes inSyBE004, SyBE005and SyBE007demonstrated that the expression of XYL1, XYL2,XSK1and ZWF1was significantly enhanced, suggesting that increasing theexpression of XYL1, XYL2, XKS1and enhancing NADPH supply were promisingstrategies to improve xylose fermentation in recombinant S. cerevisiae.In addition, a cellobiose metabolic pathway was introduced into S. cerevisiae toeliminate the inhibitory effect of glucose repression during ethanol fermentation fromlignocellulosic hydrolysates. Through combinatorial optimization of cellobiosetransporter gene CDT-1and XYL1, efficient and simultaneous co-metabolism ofcellobiose and xylose was implemented.更多还原