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  

Effect of differential expression of transaldolase gene on xylose fermentation and acetic acid tolerance of Saccharomyces cerevisiae strain

作　　者：肖琴[1] 曾维怡 汤岳琴[1] 木田建次 

机构地区：[1]四川大学建筑与环境学院,四川成都610065

出　　处：《微生物学通报》2014年第6期1094-1108,共15页Microbiology China

基　　金：国家自然科学基金项目(No.31170093)

摘　　要：【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 （TALl）, one key gene in oxidative pentose phosphate pathway （PPP）, on xylose utilization as well as acetic acid tolerance ofgenetically engineered xylose-fermenting strain NAPX37 was studied. [Methods] The promoter of TALl gene （PTALl） 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 PTALl were replaced with PTDH3, PAHP1 or PUBI4 were constructed. The fermentation capacity and acetic acid tolerance of the three homozygotes and theoriginal strain NAPX37 were compared through batch fermentation using xylose or the mixture of glucose and xylose as carbon source. [Results] Three promoters, PTDrt3, PAHP1 and PUBI4, increased the transcription level of TALl gene differentially, which not only improved xylose consumption rate and acetic acid tolerance significantly, but also imnroved glucoseconsumpiton 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 PAHPl-controlled TALl gene showed better fermentation results than strains with PTDH3- or PuBx4-controlled TALl gene, indicating the expression level of PAHpl-controlled TALl gene was most aDorooriate. When xvlose was used assole carbon source under the condition of 30 mmol/L of acetic acid, the strain with PuBI4-controlled TALl gene showed best fermentation results among all strains, indicating the most suitable expression level of PUBI4-controlled TALl gene. [Conclusion] Three promoters, PTDH3, PAHP1 and PUBI4, overexpressed TALl gene, which improved xylose fermentation rate and acetic acid tolerance of strain NAPX37 differentially. However, the fer

关 键 词：纤维素燃料乙醇 TAL1基因 木糖发酵 乙酸耐受性 

分 类 号：TQ223.122[化学工程—有机化工] TQ920.6[轻工技术与工程—发酵工程]