Biorefining Research Institute and Department of Biology, Lakehead University, Thunder Bay, Ontario, Canada, P7E 5E1.
Microb Cell Fact. 2012 May 20;11:63. doi: 10.1186/1475-2859-11-63.
Trichoderma reesei is a widely used industrial strain for cellulase production, but its low yield of β-glucosidase has prevented its industrial value. In the hydrolysis process of cellulolytic residues by T. reesei, a disaccharide known as cellobiose is produced and accumulates, which inhibits further cellulases production. This problem can be solved by adding β-glucosidase, which hydrolyzes cellobiose to glucose for fermentation. It is, therefore, of high vvalue to construct T. reesei strains which can produce sufficient β-glucosidase and other hydrolytic enzymes, especially when those enzymes are capable of tolerating extreme conditions such as high temperature and acidic or alkali pH.
We successfully engineered a thermostable β-glucosidase gene from the fungus Periconia sp. into the genome of T. reesei QM9414 strain. The engineered T. reesei strain showed about 10.5-fold (23.9 IU/mg) higher β-glucosidase activity compared to the parent strain (2.2 IU/mg) after 24 h of incubation. The transformants also showed very high total cellulase activity (about 39.0 FPU/mg) at 24 h of incubation whereas the parent strain almost did not show any total cellulase activity at 24 h of incubation. The recombinant β-glucosidase showed to be thermotolerant and remains fully active after two-hour incubation at temperatures as high as 60°C. Additionally, it showed to be active at a wide pH range and maintains about 88% of its maximal activity after four-hour incubation at 25°C in a pH range from 3.0 to 9.0. Enzymatic hydrolysis assay using untreated, NaOH, or Organosolv pretreated barley straw as well as microcrystalline cellulose showed that the transformed T. reesei strains released more reducing sugars compared to the parental strain.
The recombinant T. reesei overexpressing Periconia sp. β-glucosidase in this study showed higher β-glucosidase and total cellulase activities within a shorter incubation time (24 h) as well as higher hydrolysis activity using biomass residues. These features suggest that the transformants can be used for β-glucosidase production as well as improving the biomass conversion using cellulases.
里氏木霉是一种广泛用于纤维素酶生产的工业菌株，但它β-葡萄糖苷酶产量低，限制了其工业价值。在里氏木霉水解纤维素残留物的过程中，会产生一种二糖，即纤维二糖，并积累，从而抑制进一步的纤维素酶的生产。这个问题可以通过添加β-葡萄糖苷酶来解决，β-葡萄糖苷酶可以将纤维二糖水解成葡萄糖用于发酵。因此，构建能够产生足够的β-葡萄糖苷酶和其他水解酶的里氏木霉菌株具有很高的价值，特别是当这些酶能够耐受高温、酸性或碱性 pH 等极端条件时。
我们成功地从真菌 Periconia sp. 中构建了一个耐热β-葡萄糖苷酶基因，并将其导入里氏木霉 QM9414 菌株的基因组中。与亲本菌株（2.2 IU/mg）相比，经过 24 小时的培养，工程化的里氏木霉菌株的β-葡萄糖苷酶活性提高了约 10.5 倍（23.9 IU/mg）。转化子在 24 小时的培养中也表现出非常高的总纤维素酶活性（约 39.0 FPU/mg），而亲本菌株在 24 小时的培养中几乎没有表现出任何总纤维素酶活性。重组β-葡萄糖苷酶表现出耐热性，在高达 60°C 的温度下孵育两小时后仍保持完全活性。此外，它在很宽的 pH 范围内都具有活性，并在 25°C 下 pH 范围为 3.0 到 9.0 时孵育四小时后保持约 88%的最大活性。使用未经处理、NaOH 或 Organosolv 预处理的大麦秸秆以及微晶纤维素进行的酶水解测定表明，与亲本菌株相比，转化的里氏木霉菌株释放出更多的还原糖。
本研究中过表达 Periconia sp. β-葡萄糖苷酶的重组里氏木霉在更短的培养时间（24 小时）内表现出更高的β-葡萄糖苷酶和总纤维素酶活性，以及使用生物质残余物更高的水解活性。这些特性表明，转化子可用于β-葡萄糖苷酶的生产以及提高纤维素酶对生物质的转化效率。
