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【目的】木糖发酵是纤维素燃料乙醇生产的一个关键瓶颈,同时木质纤维素水解液中的乙酸严重抑制酿酒酵母的木糖发酵过程,因此通过基因工程手段提高菌株对木糖的利用以及对乙酸的耐受性具有重要意义。本研究以非氧化磷酸戊糖途径(PPP途径)中关键基因转醛醇酶基因(TAL1)为研究对象,探讨了3种不同启动子PTDH3、PAHP1和PUBI4,控制其表达对菌株利用木糖和耐受乙酸的影响。【方法】通过同源重组用3种启动子替换酿酒酵母基因工程菌NAPX37的TAL1基因的启动子PTAL1,再通过孢子分离和单倍体交配构建了纯合子,利用批次发酵比较了在以木糖为唯一碳源和混合糖(葡萄糖和木糖)为碳源条件下,3种启动子控制TAL1基因表达导致的发酵和乙酸耐受能力的差异。【结果】启动子PTDH3、PAHP1和PUBI4在不同程度上提高了TAL1基因的转录水平,提高了菌株对木糖的利用速率及乙酸耐受能力,提高了菌株在60 mmol/L乙酸条件下的葡萄糖利用速率。在以木糖为唯一碳源且无乙酸存在、以及混合糖为碳源的条件下,PAHP1启动子控制TAL1表达菌株的发酵结果优于PTDH3和PUBI4启动子的菌株,PAHP1启动子控制的TAL1基因的转录水平比较合适。在木糖为唯一碳源且乙酸为30 mmol/L时,PUBI4启动子控制TAL1基因表达的菌株发酵结果则优于PAHP1和PTDH3启动子菌株,此时PUBI4启动子控制的TAL1的转录水平比较合适。【结论】启动子PTDH3、PAHP1和PUBI4不同程度地提高TAL1基因的表达,在不同程度上改善了酵母菌株的木糖发酵速率和耐受乙酸性能,改善程度受发酵条件的影响。
【Objective】 Xylose fermentation is a key bottleneck in the production of cellulosic fuel ethanol. At the same time, acetic acid in lignocellulosic hydrolyzate severely inhibits the xylose fermentation process of Saccharomyces cerevisiae, therefore, the utilization of xylose by the strain is improved by genetic engineering Acetic acid tolerance is of great importance. In this study, the key gene transaldolase gene (TAL1) in non-oxidative pentose phosphate pentose pathway (PPP pathway) was studied. Three different promoters, PTDH3, PAHP1 and PUBI4, were explored to control the expression of xylose Resistant to acetic acid. 【Method】 The promoter of TAL1 gene of Saccharomyces cerevisiae genetic engineering strain NAPX37 was replaced by three kinds of promoters through homologous recombination. Then the homozygote was constructed by spore separation and haploid mating. By using batch fermentation, Three promoters controlled the expression of TAL1 gene and resulted in the differences in the ability of fermentation and acetic acid tolerance when sugar was the sole carbon source and mixed sugar (glucose and xylose) as carbon source. 【Result】 The promoter PTDH3, PAHP1 and PUBI4 increased the transcript level of TAL1 gene to some extent, increased the utilization rate of xylose and the tolerance ability of acetic acid to the strain, increased the glucose content of the strain under the condition of 60 mmol / L acetic acid Utilization rate. Under the conditions of xylose as sole carbon source and no acetic acid, and mixed sugar as carbon source, the fermentation result of PAHP1 promoter controlling TAL1 expression strain was superior to that of PTDH3 and PUBI4 promoters. The PAHP1 promoter-controlled TAL1 gene The transcription level is more appropriate. The results showed that the PUBI4 promoter controlled the TAL1 gene expression when the xylose was the sole carbon source and the acetic acid was 30 mmol / L, but the transcriptional level of TAL1 under the control of PUBI4 promoter was better than that of the PAHP1 and PTDH3 promoters . 【Conclusion】 The promoters PTDH3, PAHP1 and PUBI4 can enhance the expression of TAL1 gene to different extents, and improve the xylose fermentation rate and acetic acid tolerance of yeast strains in varying degrees, and the improvement degree is affected by the fermentation conditions.