本文關(guān)鍵詞： 產(chǎn)甘油假絲酵母 甘油分解代謝 甘油脫氫酶 二羥丙酮激酶 木糖醇　出處：《江南大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：甘油是生物柴油生產(chǎn)過程中產(chǎn)生的主要副產(chǎn)物,是一種發(fā)酵行業(yè)可用的碳源原料。與釀酒酵母(Saccharomyces cerevisiae)等不能很好地利用甘油的工業(yè)微生物相比,產(chǎn)甘油假絲酵母(Candida glycerinogenes)能夠利用甘油大量生長菌體,而有機(jī)酸、醇等其它代謝產(chǎn)物積累很少,是潛在的優(yōu)良宿主細(xì)胞。為研究C.glycerinogenes甘油分解代謝,分別以甘油和葡萄糖為唯一碳源進(jìn)行發(fā)酵性能比較,結(jié)果表明:C.glycerinogenes以甘油為碳源時大量生長菌體而有機(jī)酸、醇等其它代謝產(chǎn)物積累很少。以甘油為共底物進(jìn)行木糖發(fā)酵,木糖醇產(chǎn)量和轉(zhuǎn)化率達(dá)到39.4g·L~(-1)和88%,與以葡萄糖為共底物相比,分別提高了79%和32%,表明甘油作為共底物可有效提高木糖醇的合成和轉(zhuǎn)化率。對甘油分解代謝預(yù)測相關(guān)甘油脫氫酶基因(CgGCY1、CgGCY2)和二羥基丙酮激酶基因(CgDAK)進(jìn)行克隆,并在S.cerevisiae中表達(dá)進(jìn)行功能驗(yàn)證。在此基礎(chǔ)上,通過qRT-PCR及酶活測定考察甘油的分解代謝途徑,結(jié)果顯示與葡萄糖相比,甘油培養(yǎng)下細(xì)胞通過強(qiáng)化糖異生、HMP途徑積累生物量,下調(diào)EMP途徑和副產(chǎn)物合成關(guān)鍵酶表達(dá)以弱化有機(jī)酸、醇的合成,同時上調(diào)TCA循環(huán)以補(bǔ)償EMP途徑下調(diào)帶來的能量和還原力不足,使得生物量提高25%而很少積累有機(jī)酸、醇等代謝物。為進(jìn)一步在分子水平研究C.glycerinogenes甘油分解代謝途徑,利用同源重組“Ura-Blaster”敲除盒分別構(gòu)建了缺失突變菌C.g gcy1?、C.g gcy2?、C.g gcy1?/gcy2?和C.g dak?。結(jié)果顯示編碼甘油脫氫酶的基因CgGCY1、CgGCY2單獨(dú)缺失后,缺失菌C.g gcy1?和C.g gcy2?利用甘油的能力會有不同程度的降低,但并未完全喪失分解代謝甘油的能力;而雙基因缺失菌C.g gcy1?/gcy2?和缺失菌C.g dak?均不能在甘油培養(yǎng)基中生長。結(jié)果表明C.glycerinogenes甘油分解代謝僅依賴于二羥基丙酮(DHA)途徑,并且在甘油分解過程中基因CgGCY2扮演著比CgGCY1更加重要的角色。為探索C.glycerinogenes甘油分解代謝途徑在工業(yè)菌株改造的應(yīng)用,研究以木糖醇為目標(biāo)產(chǎn)物考察強(qiáng)化甘油分解代謝途徑對目標(biāo)產(chǎn)物合成的影響。過表達(dá)甘油分解途徑關(guān)鍵基因CgGCY1、CgGCY2和CgDAK后,重組菌C.g-pUR-CgGCY1-CgDAK和C.g-pUR-CgGCY2-CgDAK甘油和木糖消耗加快,發(fā)酵結(jié)束時木糖醇產(chǎn)量分別達(dá)到44.0g·L~(-1)和45.0 g·L~(-1),與野生菌相比,提高了12%和13%。敲除木糖還原酶基因CgXYL2后,阻斷了木糖醇的下游代謝。重組菌C.g xyl2?-p UR-CgGCY1-CgDAK和C.g xyl2?-pUR-CgGCY2-CgDAK的木糖醇產(chǎn)量分別為49.2 g·L~(-1)和49.5 g·L~(-1),相比出發(fā)菌提高了23%和24%;木糖醇轉(zhuǎn)化率分別達(dá)到了97%和98%,提高了9%和10%,單位菌體產(chǎn)量分別為2.89 g·g~(-1)和2.91 g·g~(-1),提高了34%和35%。這為進(jìn)一步利用甘油作為共底物代謝改造該菌株合成其它高附加值產(chǎn)品奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：TQ926;TQ645.5

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