Development of a cellulolytic Saccharomyces cerevisiae strain with enhanced cellobiohydrolase activity
Development of a cellulolytic Saccharomyces cerevisiae strain with enhanced cellobiohydrolase activity
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DOI：
10.1007/s11274-014-1726-9
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发表时间：
2014-11-01
影响因子：
4.1
通讯作者：
Zhang, Minhua
中科院分区：
文献类型：
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作者：
Hong, Jiefang;Yang, Huajun;Zhang, Minhua
联合生物处理技术是一种很有前途的木质纤维素乙醇生产技术，其关键是高效利用纤维素的微生物工程。开发用于CBP的酿酒酵母需要高水平表达纤维素酶，特别是纤维二糖水解酶（CBH）。在这项研究中，为了构建具有增强的CBH活性的CBP使能酵母，构建了三个包含组成型表达的CBH编码基因（来自棘孢曲霉的cbh 1，来自里氏木霉的cbh 1和cbh 2）的盒。T. reesei eg 2、白纹伊蚊A. aculeatus bgl 1，然后将这三个编码CBH的基因依次整合到S.酿酒酵母W303-1A染色体通过δ序列介导的整合。所得菌株W1、W2和W3分别表达单、双和三功能纤维素酶，表现出相应的纤维素酶活性。此外，活性和葡萄糖产生活性均上升。在含纤维素平板上的生长试验表明，CBH是成功利用结晶纤维素的必要组分。在酸和碱预处理的含有玉米芯的培养基中，以5 FPU外源纤维素酶/g生物量加载来评估三种重组菌株和对照菌株W303-1A和AADY。7天内的最高乙醇滴度（g/l）分别为5.92 +/- A 0.51、18.60 +/- A 0.81、28.20 +/- A 0.84、1.40 +/- A 0.12和2.12 +/- A 0.35。与对照菌株相比，W3菌株能高效发酵预处理后的玉米芯生产乙醇。据我们所知，这是第一个旨在通过将三种类型的CBH编码基因与强组成型启动子Ptpi整合来创建具有增强CBH活性的纤维素分解酵母的研究。
Consolidated bioprocessing (CBP) is a promising technology for lignocellulosic ethanol production, and the key is the engineering of a microorganism that can efficiently utilize cellulose. Development of Saccharomyces cerevisiae for CBP requires high level expression of cellulases, particularly cellobiohydrolases (CBH). In this study, to construct a CBP-enabling yeast with enhanced CBH activity, three cassettes containing constitutively expressed CBH-encoding genes (cbh1 from Aspergillus aculeatus, cbh1 and cbh2 from Trichoderma reesei) were constructed. T. reesei eg2, A. aculeatus bgl1, and the three CBH-encoding genes were then sequentially integrated into the S. cerevisiae W303-1A chromosome via delta-sequence-mediated integration. The resultant strains W1, W2, and W3, expressing uni-, bi-, and trifunctional cellulases, respectively, exhibited corresponding cellulase activities. Furthermore, both the activities and glucose producing activity ascended. The growth test on cellulose containing plates indicated that CBH was a necessary component for successful utilization of crystalline cellulose. The three recombinant strains and the control strains W303-1A and AADY were evaluated in acid- and alkali-pretreated corncob containing media with 5 FPU exogenous cellulase/g biomass loading. The highest ethanol titer (g/l) within 7 days was 5.92 +/- A 0.51, 18.60 +/- A 0.81, 28.20 +/- A 0.84, 1.40 +/- A 0.12, and 2.12 +/- A 0.35, respectively. Compared with the control strains, W3 efficiently fermented pretreated corncob to ethanol. To our knowledge, this is the first study aimed at creating cellulolytic yeast with enhanced CBH activity by integrating three types of CBH-encoding gene with a strong constitutive promoter Ptpi.