里氏木霉VPS13基因缺失对菌丝分支、生孢和纤维素酶产量的影响

Effects of VPS13 deletion on hyphal branch, sporulation and cellulase production in Trichoderma reesei

摘要:

[目的]丝状真菌里氏木霉是纤维素酶生产的主要工业真菌。纤维素酶分泌过程中的蛋白运输途径是控制大量纤维素酶成功输出的重要环节，因此，研究蛋白分泌途径的特定靶标基因功能将有助于鉴定纤维素酶运输分泌过程的关键调控因子。本研究借助基因敲除方法将里氏木霉液泡蛋白分选相关基因VPS13缺失，分析了该基因缺失对菌株生长、生孢尤其是纤维素酶分泌的影响。[方法]利用Double-jointPCR技术和同源重组策略构建里氏木霉VPS13基因缺失突变株，通过菌丝培养、显微观察、生孢检测、蛋白与酶活测定，系统比较VPS13基因敲除前后菌株的生长特征、菌丝形态、孢子形成、蛋白分泌以及纤维素酶活等。[结果]成功获得两株VPS13基因缺失株。与出发菌株相比，该基因突变后菌丝蔓延速率明显减慢，但菌体生物量在对数生长期后显著增多。通过显微观察，发现该基因缺失株菌丝更加密集，分支明显增多。此外，该基因缺失也导致菌株生孢延迟。纤维素底物平板分析发现VPS13基因缺失株菌落周围透明圈更加清晰，且透明圈圈径比是出发菌株的4倍，说明降解纤维素的能力有明显提高。进一步的液体发酵实验结果显示，该基因缺失导致蛋白产量及纤维素酶活力分别提高16.4%和21.9%。[结论]里氏木霉VPS13基因在菌丝生长、生孢、蛋白分泌等不同生物学过程中具有功能多样性，且该基因在菌种改良上可以作为提高纤维素酶产量的重要靶点。

Abstract:

[Objective] Trichoderma reesei is one of the major industrial fungi for cellulase production. A large amount of cellulases are secreted from the fungal cell through the protein secretion pathway. Therefore, knowledge of specific gene functions related to protein secretion would contribute to identification of key factors controlling cellulase production. In this study, the vacuolar protein sorting-associated gene VPS13 was deleted by gene knockout strategy in T. reesei and effects of VPS13 deletion on fungal growth and cellulase production were analyzed.[Methods] Double-joint PCR technique and homologous recombination method were used to delete VPS13 gene in T. reesei QP4 strain. Fungal cultivation, microscopic observation, sporulation detection, protein content and enzyme activity determination were applied to compare the growth characteristics, morphology, spore formation, and cellulase activity between the deletion strains and the parental strain.[Results] Two VPS13 deletion strains, ΔVPS13-1 and ΔVPS13-2, were obtained successfully. Compared with the parental strain, the mutants showed slower hyphae spreading, but their biomass increased significantly after the logarithmic growth phase. In addition, VPS13 deletion led to delay of sporulation. Cellulose plate cultivation revealed that VPS13 mutants produced clearer transparent halos around colonies than the parental strain, indicating that VPS13 deletion increased the ability to degrade cellulose. Furthermore, liquid fermentation showed that VPS13 deletion also improved protein yield and cellulase activity.[Conclusion] T. reesei VPS13 gene can be used as an important target to improve cellulase yield.