【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 crucial in lignocellulosic ethanol production. Acetic acid generated during pretreatment process seriously inhibits xylose fermentation of yeast strain. The effect of differential expression of transaldolase gene (TAL1), one key gene in oxidative pentose phosphate pathway (PPP), on xylose utilization as well as acetic acid tolerance of genetically engineered xylose-fermenting strain NAPX37 was studied. [Methods] The promoter of TAL1 gene (PTAL1) of the strain NAPX37 was separately replaced with three promoters, PTDH3, PAHP1 and PUBI4, through homologous recombination. By subsequent sporulation, spore segregation and mating, three homozygotes in which PTAL1 were replaced with PTDH3, PAHP1 or PUBI4 were constructed. The fermentation capacity and acetic acid tolerance of the three homozygotes and the original strain NAPX37 were compared through batch fermentation using xylose or the mixture of glucose and xylose as carbon source. [Results] Three promoters, PTDH3, PAHP1 and PUBI4, increased the transcription level of TAL1 gene differentially, which not only improved xylose consumption rate and acetic acid tolerance significantly, but also improved glucose consumption rate under the condition of 60 mmol/L of acetic acid. When xylose was used as sole carbon source without acetic acid or when mixed sugar was used, the strain with PAHP1-controlled TAL1 gene showed better fermentation results than strains with PTDH3- or PUBI4-controlled TAL1 gene, indicating the expression level of PAHP1-controlled TAL1 gene was most appropriate. When xylose was used as sole carbon source under the condition of 30 mmol/L of acetic acid, the strain with PUBI4-controlled TAL1 gene showed best fermentation results among all strains, indicating the most suitable expression level of PUBI4-controlled TAL1 gene. [Conclusion] Three promoters, PTDH3, PAHP1 and PUBI4, overexpressed TAL1 gene, which improved xylose fermentation rate and acetic acid tolerance of strain NAPX37 differentially. However, the fermentation condition affected the level of improvement.
肖琴,曾维怡,汤岳琴,木田建次. 转醛醇酶基因差异表达影响酵母发酵木糖及耐受乙酸性能[J]. 微生物学通报, 2014, 41(6): 1094-1108复制