三株里氏木霉突变株高产β-葡萄糖苷酶的分子机制研究

Trichoderma reesei is a preferred organism within cellulase industry. It's a widely-used heterologous expression system as well, because of its strong secretary capability. It is concerned how to optimize its expression system and capability of cellualse synthesis and secretary in recent years. Due to the insufficient β-glucosidase produced by T. reesei, additional β-glucosidase is supplemented in its commercial cellulase preparation. In our previous study, we introduced a heterologous β-glucosidase gene in T. reesei by Agrobacterium- mediated T-DNA insertional expression system and obtained mutants A1 and D3. And a mutant U10 with a higher β-glucosidase activity was obtained from D3 after further acclimatization. The β-glucosidase activities were enhanced with different extent in the 3 mutants, but the molecular mechanism behind is unclear yet. In this study, we are planning to verify the insertion site of heterologous gene by Genome Walking in A1 and D3, and unveil the gene mutation in the mutant U10 by comparative genomics analysis.In order to investigate the molecular mechanism of the enhanced production of β-glucosidase, we will delete or overexpress these mutant genes in the parent strain. Following an aggregate analysis with expression profilings, secretome and enzyme activity, the molecular mechanism of different β-glucosidase activities of 3 strains will be elucidated. The revelation of the novel mechanisms will provide clues and foundation for further improvement of the T.reesei expression system and development of new industially applicable strains.

里氏木霉是目前最主要的纤维素酶工业生产菌种，因其强大的蛋白分泌能力常用作异源表达宿主。如何通过遗传改造进一步提高其纤维素酶合成与分泌能力以及优化其表达系统，近来受到普遍关注。我们的前期研究通过T-DNA随机插入于里氏木霉中过表达外源β-葡萄糖苷酶，获得β-葡萄糖苷酶产量提高幅度不同的2株单拷贝插入突变株（A1和D3），进一步通过对D3进行实验室驯化获得了产量最高的U10，但引起它们产量各异的分子机制尚不清楚。在本研究中,首先将利用基因组步移法确认外源β-葡萄糖苷酶于突变株基因组中的插入位点；再通过比较基因组学分析U10基因组的突变位点，并在出发株中敲除和过表达T-DNA随机插入位点及驯化引起的关键突变基因，结合胞外分泌蛋白、酶活、产酶相关基因的表达差异等数据分析确认其功能，阐明这些关键基因的突变引起β-葡萄糖苷酶高产的分子机制。这些研究结果将为里氏木霉的菌种改良或表达系统优化提供研究靶标。

里氏木霉是目前最为主要的纤维素酶工业生产菌种，因其强大的蛋白分泌能力常用作异源表达宿主。本研究通过三株高产β-葡萄糖苷酶的里氏木霉菌株为研究对象，对其高产机制进行解析。首先，通过DNA Walking技术对单拷贝插入突变株D3的异源基因的插入位点，之后通过比较基因组对D3基础上的实验室驯化菌株U10的突变位点进行研究。研究发现D3插入在cbh1的位点，同时还影响了周围序列的缺失；而U10在此基础上还引起了诸多的突变。为了快速的鉴定这些突变基因的功能和产酶相关的机制，我们对里氏木霉CRISPR/Cas9基因组编辑系统进行开发，通过密码子优化和体外表达gRNA相结合的方法，使单敲基因的效率提高到了80%，也成功实现了多基因的敲除的。最终发现两个关键的突变和里氏木霉的高产有关，其中一个关键突变可能和纤维素酶负调控因子ACE1的互作有关。我们利用这些研究还成功应用到异源表达其他基因中，相比随机插入表达或整合到cbh1位点的表达方式，将异源表达框替换到cbh1及其周围毗邻序列能够获得更高的产量。