多基因协同调控里氏木霉高效分泌表达蛋白的研究

The hyperproducing ability of mutated Trichoderma reesei strains are from the actions of multiple genes. However, whether there is synergistic regulation and how it works among multiple genes are not clear. Thus the identification of the combinations of important genes and their expression styles, which can synergistically regulate high efficiency production of endogenous or heterologous proteins, can largely speed up the breeding of T. reesei by genetic engineering. In the hyperproducing strain CL847, the expression styles of four key transcription regulators, that are cre1, ace1, ace2 and xyr1, are changed not by random. Based on this, we choose these four key transcription regulators as a model to explore the synergistic roles among multiple genes in regulating high efficiency endogenous gene expression and secretion. For each of these four genes, either a mutated promoter library (by inserting the gpd box which has a transcription-enhancing effect) or an RNAi plasmid will be constructed, which will allow up- or down-regulation of the expression of these genes at multiple levels. Then different numbers and combinations of the mutated promoter libraries and/or RNAi plasmids will be used to transform T. reesei simultaneously. This will introduce abundant biodiversity regarding the combinations of the expression of the four genes. By screening transformants for largely improved cellulase producing ability, the synergistic effect among special gene combinations and their expression styles will be revealed. In addition, for the heterologous genes, we will choose the Aspergillus niger lipase as a model and three key regulators hac1, ftt1 and ftt2, as the target genes and use the same method as described above.

里氏木霉诱变菌纤维素酶高产性状来自于多个基因的共同作用，但基因间是否存在协同调控及作用机理并不清楚。辨识出能协同调控高效分泌内、外源蛋白的重要基因组合及表达方式可大大加快里氏木霉的基因工程育种。高产菌CL847中四个关键转录因子(cre1、ace1、ace2和xyr1)的表达发生了有规律的变化。据此选定这四个关键因子作为模型对基因间能否协同调控内源纤维素酶的高效分泌表达进行研究。对每个基因，构建突变启动子文库（插入能增强转录的gpd box）或RNAi质粒分别多水平上调或下调基因的表达。将不同数目或组合的突变启动子文库和/或RNAi质粒同时转化木霉，在转化子中引入这四个基因表达水平组合的丰富生物多样性，筛选纤维素酶分泌极大提高的转化子，从而揭示所研究的调控基因间是否存在协同作用及它们的作用方式。对外源基因的表达选定黑曲霉脂肪酶为模型、关键因子hac1、ftt1和ftt2为靶点同法进行研究。

里氏木霉具有强大的蛋白分泌能力，但对其的研究多为单个基因的研究，是否存在协同调控并不为人所知。本研究内容从三个方面对协同调控里氏木霉提高其产酶能力展开研究，分别为1）对多个转录因子同时进行调控（协同调控）提高里氏木霉内源纤维素酶的表达；2）对分泌途径的关键基因进行改造，结合对转录关键因子的改造，协同调控里氏木霉内源纤维素酶的表达；3）选取表达细菌和真菌来源的酶基因作为报告基因，通过改造里氏木霉的转录和分泌，可能能够提高外源基因的表达。在研究中，建立了里氏木霉同时转化多个基因单拷贝定点整合的技术方法；同时操控转录激活和抑制因子Xyr1和Cre1的表达使得转化子的纤维素酶酶活提高了2.5倍。选取的模型外源基因Neosartorya fischeri GH3 beta-glucosidase的过表达提高了里氏木霉的纤维素酶表达，其原因可能与参与槐二糖的形成有关。从转录因子和蛋白折叠、分泌途径中的关键因子互作、协同调控角度，也证明同时操作多因子可以协同调控里氏木霉产酶能力的提升。另外成功表达了黑曲霉葡萄糖氧化酶和甘露聚糖酶、Caldicellulosiruptor bescii来源的第10家族木聚糖酶CbXyn10C均可作为表达的外源基因的模型。