固态发酵系统中微生物扩张蛋白参与木质纤维素降解的基因行为及蛋白功能研究

The structure of natural lignocellulose, such as agricultural and forestry waste, is tight and extremely difficult to degrade. As reported, expansins can loosen the structure of natural lignocellulose and synergize cellulase to degrade substrates. However, most related studies deviated from microbial self-metabolism, which lead to many limitations in the results and conclusions. As previously found in our research, Trichoderma asperellum T-1 could degrade the untreated waste straw and secrete swollenin-like protein. This project aims to clarify how swollenins in T. asperellum T-1participate in lignocellulose degradation through the following studies: 1) The first section aims to clarify the characteristics of cellulase production and its influencing factors through solid-state fermentation; 2) Based on cellulase production metabolism of T. asperellum T-1, transcriptome analysis will be carried out to identify key swollenins involved in lignocellulose degradation, and to analyze the dynamic expression of their coding genes, as well as the co-expression with cellulase genes and regulatory factor genes; 3) Elucidate the function of key swollenin coding genes, especially their correlations with lignocellulose degradability of T. asperellum T-1; 4) Explore the substrate binding ability, structure loosening ability and hydrolysis synergizing ability of key swollenins by in vitro experiments. Based on the above results, this project will systematically elucidate the mechanism of T. asperellum T-1 swollenins participating in lignocellulose degradation from two aspects: gene behaviors at transcriptional level and protein functions at expression level. Research results of this project will enrich the microbial mechanism of lignocellulose degradation, and provide theoretical guidance for the application of expansin-like proteins.

农林废弃木质纤维素结构紧密、极难降解。已知扩张蛋白可疏松木质纤维素结构，协同纤维素酶降解底物，但相关研究多脱离微生物自身代谢，存在诸多局限。申请人前期发现棘孢木霉T-1可降解未经预处理的废弃秸秆，且可分泌扩张蛋白类大分子。基于此，项目拟从降解代谢及其动态演变角度入手，针对棘孢木霉T-1扩张蛋白展开如下研究：1）结合固态发酵，明确菌株产酶特征及其影响因素；2）以产酶代谢为基础进行转录组分析，发掘参与降解代谢的关键扩张蛋白，解析其编码基因动态表达规律，及其与纤维素酶基因、调控因子基因的共表达规律；3）解析关键扩张蛋白基因功能及其与菌体木质纤维素降解能力的相关性；4）体外探究关键扩张蛋白结合、疏松底物及协同酶解的作用方式。项目旨在从转录水平的基因行为和表达水平的蛋白功能两方面，系统阐释扩张蛋白参与木质纤维素降解的作用机制。研究结果可丰富木质纤维素降解的微生物机制，为扩张蛋白实际应用提供理论指导。

废弃秸秆的资源化利用是农业农村可持续发展和实现“双碳”目标的重要组成部分。具有木质纤维素降解能力的微生物是秸秆生物质转化的关键因素。针对天然秸秆内部结构紧密，难以被打破，从而阻碍其有效转化的问题，本项目以优势木质纤维素降解菌的产酶代谢表型为基础，从动态演变的视角出发，系统探究丝状真菌扩张蛋白参与木质纤维素降解的微生物机制。主要内容及结论如下：.（1）通过小麦秸秆固态和液态发酵明确了棘孢木霉T-1和里氏木霉QM6a的产酶动力学特征，确定了转录组测序取样点；相较于里氏木霉QM6a，棘孢木霉T-1发酵小麦秸秆时可迅速出现代谢响应，且具备有效降解天然小麦秸秆的微生物活性。.（2）转录组学研究分析了碳源不同、发酵方式不同及发酵阶段不同时，丝状真菌的基因表达差异，并进行了差异显著性分析和关键基因的生物学功能富集分析；揭示了棘孢木霉T-1水解酶基因和扩张蛋白类基因的共同响应是其有效降解未经预处理的天然小麦秸秆的关键；被明显富集的扩张蛋白类大分子包括多糖结合蛋白、碳水化合物结合蛋白、纤维素结合蛋白和碳水化合物衍生物结合蛋白等。.（3）棘孢木霉T-1中被显著富集的蛋白功能涉及碳水化合物代谢、糖基化合物水解代谢和淀粉和蔗糖代谢等，其均与木质纤维素降解相关，且其编码基因呈现共表达（上调）规律；菌株T-1扩张蛋白编码基因的差异表达和富集强度远高于里氏木霉QM6a，且其与多种水解酶、有机物代谢关键酶均存在明显的蛋白质互作关系。.（4）棘孢木霉T-1对小麦秸秆的预发酵有效提高炭化产物的污染物吸附性能；其对小麦秸秆的解构效应导致炭化产物含氧官能团的进一步暴露和比表面积的增加，进而提高秸秆炭吸附性能；该结果得益于菌株T-1纤维素酶解基因和扩张蛋白基因的共表达。.本项目研究成果丰富了木质纤维素降解的微生物机制，同时为开发基于“扩张蛋白-纤维素酶/纤维素降解菌”协同降解转化废弃木质纤维素的新模式提供了理论指导。.