产甘油假丝酵母甘油分解途径关键基因及其应用研究

发布时间：2018-02-26 03:11

本文关键词： 产甘油假丝酵母甘油分解代谢甘油脱氢酶二羟丙酮激酶木糖醇　出处：《江南大学》2017年硕士论文　论文类型：学位论文

【摘要】：甘油是生物柴油生产过程中产生的主要副产物,是一种发酵行业可用的碳源原料。与酿酒酵母(Saccharomyces cerevisiae)等不能很好地利用甘油的工业微生物相比,产甘油假丝酵母(Candida glycerinogenes)能够利用甘油大量生长菌体,而有机酸、醇等其它代谢产物积累很少,是潜在的优良宿主细胞。为研究C.glycerinogenes甘油分解代谢,分别以甘油和葡萄糖为唯一碳源进行发酵性能比较,结果表明:C.glycerinogenes以甘油为碳源时大量生长菌体而有机酸、醇等其它代谢产物积累很少。以甘油为共底物进行木糖发酵,木糖醇产量和转化率达到39.4g·L~(-1)和88%,与以葡萄糖为共底物相比,分别提高了79%和32%,表明甘油作为共底物可有效提高木糖醇的合成和转化率。对甘油分解代谢预测相关甘油脱氢酶基因(CgGCY1、CgGCY2)和二羟基丙酮激酶基因(CgDAK)进行克隆,并在S.cerevisiae中表达进行功能验证。在此基础上,通过qRT-PCR及酶活测定考察甘油的分解代谢途径,结果显示与葡萄糖相比,甘油培养下细胞通过强化糖异生、HMP途径积累生物量,下调EMP途径和副产物合成关键酶表达以弱化有机酸、醇的合成,同时上调TCA循环以补偿EMP途径下调带来的能量和还原力不足,使得生物量提高25%而很少积累有机酸、醇等代谢物。为进一步在分子水平研究C.glycerinogenes甘油分解代谢途径,利用同源重组“Ura-Blaster”敲除盒分别构建了缺失突变菌C.g gcy1?、C.g gcy2?、C.g gcy1?/gcy2?和C.g dak?。结果显示编码甘油脱氢酶的基因CgGCY1、CgGCY2单独缺失后,缺失菌C.g gcy1?和C.g gcy2?利用甘油的能力会有不同程度的降低,但并未完全丧失分解代谢甘油的能力;而双基因缺失菌C.g gcy1?/gcy2?和缺失菌C.g dak?均不能在甘油培养基中生长。结果表明C.glycerinogenes甘油分解代谢仅依赖于二羟基丙酮(DHA)途径,并且在甘油分解过程中基因CgGCY2扮演着比CgGCY1更加重要的角色。为探索C.glycerinogenes甘油分解代谢途径在工业菌株改造的应用,研究以木糖醇为目标产物考察强化甘油分解代谢途径对目标产物合成的影响。过表达甘油分解途径关键基因CgGCY1、CgGCY2和CgDAK后,重组菌C.g-pUR-CgGCY1-CgDAK和C.g-pUR-CgGCY2-CgDAK甘油和木糖消耗加快,发酵结束时木糖醇产量分别达到44.0g·L~(-1)和45.0 g·L~(-1),与野生菌相比,提高了12%和13%。敲除木糖还原酶基因CgXYL2后,阻断了木糖醇的下游代谢。重组菌C.g xyl2?-p UR-CgGCY1-CgDAK和C.g xyl2?-pUR-CgGCY2-CgDAK的木糖醇产量分别为49.2 g·L~(-1)和49.5 g·L~(-1),相比出发菌提高了23%和24%;木糖醇转化率分别达到了97%和98%,提高了9%和10%,单位菌体产量分别为2.89 g·g~(-1)和2.91 g·g~(-1),提高了34%和35%。这为进一步利用甘油作为共底物代谢改造该菌株合成其它高附加值产品奠定了基础。
[Abstract]:Glycerol is the main by-product produced in the production of biodiesel and is a kind of carbon source used in fermentation industry. Compared with the industrial microorganisms such as Saccharomyces cerevisiae, which can not make good use of glycerin, Candida glycerinogenes-producing Candida glycerinogenes can make use of glycerin to grow bacteria, but other metabolites such as organic acids, alcohols and other metabolites accumulate little, so it is a potential good host cell to study the catabolism of C. glycerinogenes glycerinogenes. When glycerol was used as carbon source, glycerinogenes was used as carbon source to grow a large number of bacteria, but other metabolites such as organic acids and alcohols accumulated little, while glycerin was used as co-substrate for xylose fermentation. The yield and conversion rate of xylitol were 39.4 g 路L ~ (-1) and 88g 路L ~ (-1), compared with glucose. The results showed that glycerol as a co-substrate could effectively improve the synthesis and conversion of xylitol. The gene of glycerol catabolism related to glycerol catabolism prediction gene (CgGCY1) and dihydroxyacetone kinase gene (CgDAK) were cloned. On the basis of the functional verification in S. cerevisiae, the catabolism pathway of glycerol was investigated by qRT-PCR and enzyme activity. The results showed that compared with glucose, cells cultured with glycerol accumulated biomass through enhanced glycosylated homogenate pathway. The expression of key enzymes in EMP pathway and by-product synthesis was down-regulated to weaken the synthesis of organic acids and alcohols, and the TCA cycle was up-regulated to compensate for the lack of energy and reductive power caused by the down-regulation of the EMP pathway, which resulted in a biomass increase of 25% and little accumulation of organic acids. In order to further study the catabolism pathway of C.glycerinogenes glycerinogenes at molecular level, the deletion mutant C.gcy1 was constructed using homologous recombinant "Ura-Blaster" knockout box. ,C.g gcy2? ,C.g gcy1? /gcy2? And C.g dak? The results showed that the gene encoding glycerol dehydrogenase, CgGCY1, CgGCY2, was deleted alone, and the deletion strain C.ggcy1? And C.g Gcy2? The ability to use glycerol decreased in varying degrees, but it did not completely lose the ability to catabolize glycerol. /gcy2? And C. The results showed that the catabolism of glycerinogenes was only dependent on the dihydroxyacetone (DHA) pathway. In order to explore the application of glycerinogenes glycerinogenes in the transformation of industrial strains, the gene CgGCY2 plays a more important role than CgGCY1 in the process of glycerol decomposition. The effects of enhanced glycerol catabolism pathway on the synthesis of target products were studied by using xylitol as the target product. After overexpressing the key genes of glycerol decomposition pathway, CgGCY1, CgGCY2 and CgDAK, the consumption of glycerol and xylose was accelerated by the recombinant bacteria C.g-pUR-CgCY1-CgDAK and C.g-pUR-CgGCY2-CgDAK. The xylitol production reached 44.0 g 路L ~ (-1) and 45.0 g 路L ~ (-1) respectively at the end of fermentation, which was 12% and 13 ~ (th) higher than that of wild bacteria. The downstream metabolism of xylitol was blocked by knockout of xylose reductase gene CgXYL2. -p UR-CgGCY1-CgDAK and C.g xyl2? The xylitol yields of -pUR-CgGCY2-CgDAK were 49.2 g 路L ~ (-1) and 49.5 g 路L ~ (-1) respectively, which increased by 23% and 24g / L, respectively, and the conversion rates of xylitol reached 97% and 98 respectively, increased by 9% and 10 respectively, and the yield per cell was 2.89 g 路g 路g ~ (-1)) and 2.91 g 路L ~ (-1), 34% and 35.1, respectively. Glycerol was used as a common substrate to transform the strain into other high added value products.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ926;TQ645.5

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