Revisiting overexpression of a heterologous β-glucosidase in Trichoderma reesei: fusion expression of the Neosartorya fischeri Bgl3A to cbh1 enhances the overall as well as individual cellulase activities.
Revisiting overexpression of a heterologous β-glucosidase in Trichoderma reesei: fusion expression of the Neosartorya fischeri Bgl3A to cbh1 enhances the overall as well as individual cellulase activities.
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DOI：
10.1186/s12934-016-0520-9
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发表时间：
2016-07-11
影响因子：
6.4
通讯作者：
Yao B
中科院分区：
文献类型：
--
作者：
Xue X;Wu Y;Qin X;Ma R;Luo H;Su X;Yao B
丝状真菌里氏木霉具有分泌大量纤维素酶的能力，广泛应用于各种行业。然而，里氏木霉纤维素酶的β-葡萄糖苷酶活性较弱，导致纤维二糖的积累抑制了内外纤维素酶的活性。通过表达外源β-葡萄糖苷酶基因，重组里氏木霉纤维素酶有望更有效地将纤维素降解为葡萄糖。β-葡萄糖苷酶NfBgl3A具有较强的活性，因此被选为里氏新丝核菌中高效表达的酶。在体外，毕赤酵母表达的NfBgl3A有助于里氏曲霉纤维素酶从结晶纤维素中释放更多的葡萄糖和显著减少的纤维二糖。因此，将NfBgl3A基因与cbh1结构基因融合，并将其组装在cbh1强启动子和cbh1终止子之间，通过DNA组装的方法获得了pRS-NfBgl3A。将pRS-NfBgl3A转化里氏木霉尿苷营养缺陷菌TU-6。6个阳性转化子的β-葡萄糖苷酶活性为2.3~6 9.7U/m L(比野生型高出175倍)。转化子中β-葡萄糖苷酶活性的差异可能与NfBgl3A或其整合位点的基因拷贝数有关。里氏锥虫表达的NfBgl3A与巴斯德毕赤酵母表达的NfBgl3A具有高度相似的生化性质。不出所料，NfBgl3A的过表达提高了里氏木霉的纤维素酶活性。所有转化子的CBHI活性都提高了，这可能是由于cbh1基因的额外拷贝，而三个转化子的内切葡聚糖酶活性也大大增加，这在任何其他过表达β-葡萄糖苷酶的研究中都没有观察到。NfBgl3A具有显著的转糖基化活性，能从葡萄糖和纤维二糖中产生有效的纤维素酶诱导剂苦参糖和其他寡糖。在此，我们成功地在里氏锥虫中过表达了一种嗜热裂殖酵母β-葡萄糖苷酶。与此同时，NfBgl3A与cbh1基因的融合引入了纤维二糖水解酶1基因的额外拷贝。结果，我们观察到β-葡萄糖苷酶和纤维二糖水解酶的活性以及纤维素酶的总体活性都得到了提高。此外，部分转化子的内切葡聚糖酶活性也有所提高。我们的结果可能会为设计更健壮的里氏木霉纤维素酶提供参考。
The filamentous fungus Trichoderma reesei has the capacity to secret large amounts of cellulase and is widely used in a variety of industries. However, the T. reesei cellulase is weak in β-glucosidase activity, which results in accumulation of cellobiose inhibiting the endo- and exo-cellulases. By expressing an exogenous β-glucosidase gene, the recombinant T. reesei cellulase is expected to degrade cellulose into glucose more efficiently. The thermophilic β-glucosidase NfBgl3A from Neosartorya fischeri is chosen for overexpression in T. reesei due to its robust activity. In vitro, the Pichia pastoris-expressed NfBgl3A aided the T. reesei cellulase in releasing much more glucose with significantly lower amounts of cellobiose from crystalline cellulose. The NfBgl3A gene was hence fused to the cbh1 structural gene and assembled between the strong cbh1 promoter and cbh1 terminator to obtain pRS-NfBgl3A by using the DNA assembler method. pRS-NfBgl3A was transformed into the T. reesei uridine auxotroph strain TU-6. Six positive transformants showed β-glucosidase activities of 2.3–69.7 U/mL (up to 175-fold higher than that of wild-type). The largely different β-glucosidase activities in the transformants may be ascribed to the gene copy numbers of NfBgl3A or its integration loci. The T. reesei-expressed NfBgl3A showed highly similar biochemical properties to that expressed in P. pastoris. As expected, overexpression of NfBgl3A enhanced the overall cellulase activity of T. reesei. The CBHI activity in all transformants increased, possibly due to the extra copies of cbh1 gene introduced, while the endoglucanase activity in three transformants also largely increased, which was not observed in any other studies overexpressing a β-glucosidase. NfBgl3A had significant transglycosylation activity, generating sophorose, a potent cellulase inducer, and other oligosaccharides from glucose and cellobiose. We report herein the successful overexpression of a thermophilic N. fischeri β-glucosidase in T. reesei. In the same time, the fusion of NfBgl3A to the cbh1 gene introduced extra copies of the cellobiohydrolase 1 gene. As a result, we observed improved β-glucosidase and cellobiohydrolase activity as well as the overall cellulase activity. In addition, the endoglucanase activity also increased in some of the transformants. Our results may shed light on design of more robust T. reesei cellulases.