里氏木霉中调控Caldicellulosiruptor bescii双功能木聚糖酶/纤维素酶CbXyn10C高效表达的关键分子的筛选与鉴定

Cellulose and xylan both as the main non-starch polysaccharides are deconstructed by xylanase and cellulase which are added in feed raw materials resulting in improving the utilization rate of raw materials. When industrializing the difunctional enzyme with ability to hydrolysis cellulose and xylan, the cost of feed enzymes can be reduced. Accordingly, to express bifunctional xylanase/cellulase CbXyn10C from C. bescii has been tried in Trichoderma reesei, but there are some key molecules blocking the efficient expression of CbXyn10C. In this study, two stains with different expression levels of CbXyn10C and two fermentation time points of efficient expression strain are performed to analysis their transcriptome and proteome aiming to screen distinct molecules. Then the key molecules which may inhibit efficient expression of CbXyn10C will be identified by CRISPR/Cas9 or RNAi in the high expression strain. Futher more, the groups of the key molecules with synergistic effects will be identified by CRISPR/Cas9 and the more higher expression strain of CbXyn10C can be obtained as well. Lastly, the mutant strains are about to be constructed by knocking out/down two or more key molecule genes in TU-6. Other multimodular and bifunctional cellulases/xylanases from C. bescii are prepared to express in the mutant strains, respectively. The groups of the key molecules with synergistic effects can be further confirmed, and more higher expression strains of multimodular and bifunctional cellulases/xylanases are obtained simultaneously.

木聚糖和纤维素是主要的非淀粉多糖，在饲料原料中添加木聚糖酶和纤维素酶可提高原料利用率。实现具有木聚糖和纤维素降解能力的双功能酶的工业化生产，可降低饲料用酶的成本。据此将C. bescii双功能木聚糖酶/纤维素酶CbXyn10C在里氏木霉中表达，但其中存在关键分子阻碍CbXyn10C的高效表达。选取CbXyn10C表达量差异的两株菌株和高表达量菌株的2个发酵时间点进行转录组和蛋白质组分析，筛选阻碍CbXyn10C高效表达的分子；以高表达量菌株为研究对象，利用CRISPR/Cas9或RNAi技术鉴定关键分子以及具有协同作用的关键分子组合，并获得高效表达CbXyn10C的菌株。以TU-6为研究对象，构建具有协同作用关键分子组合缺失的菌株，用于表达C. bescii的其它多结构域双功能纤维素酶/木聚糖酶，再次鉴定具有协同作用的关键分子组合以及获得高效表达多结构域双功能纤维素酶/木聚糖酶的菌株。

木聚糖和纤维素是主要的非淀粉多糖，在饲料原料中添加木聚糖酶和纤维素酶可提高原料利用率。实现具有木聚糖和纤维素降解能力的双功能酶的工业化生产，可降低饲料用酶的成本。据此本项目以成功表达 CbXyn10C 的菌株作为核心材料，分别从CbXyn10C表达差异的菌株和CbXyn10C高表达量菌株的不同发酵时间点的比较两方面来展开研究，发现影响里氏木霉高效表达CbXyn10C的关键分子，其中包括21个蛋白降解相关的蛋白酶基因，42个调控转录因子基因，转运及分泌相关蛋白基因67个基因；其次，利用CRISPR/Cas9或RNAi技术成功对Peptidase_A4(TRIREDRAFT_69555)、Peptidase_A4(TRIREDRAFT_106661)和 Peptidase M50 family proteion (TRIREDRAFT_107913)及3 个天冬氨酸蛋白酶 Aspartyl proteases (TRIREDRAFT_108686/TRIREDRAFT_74156/TRIREDRAF T_68662) 进行了敲除或RNA干扰，过表达全局转录调控因子Spt7(TRIREDRAFT_3737)及蛋白分泌相关基因SNARE associated Golgi protein(TRIREDRAFT_46613)、Snc1(TRIREDRAFT_53601)、Rav1p_C (TRIRED RAFT_67281)、 Vps8 (TRIREDRAFT_64654) 、 v-SNARE protein(TRIREDRAFT _104490)。并构建8种蛋白酶敲除的里氏木霉菌株，转录因子Spt7敲除及过表达菌株，12种蛋白转运及分泌相关蛋白基因的菌株，以及蛋白酶组合基因敲除或过表达菌株，最终获得的Tr-89菌株较原始菌株发酵液CbXyn10C酶活力提高8.5倍；以里氏木霉TU-6为出发菌株构建蛋白酶基因组合敲除和蛋白分泌相关基因的组合过表达菌株，并成功表达C. besseii来源的甘露聚糖酶、Neosartorya fischeri P1来源的β-葡萄糖苷酶 NfBgl3A等糖苷水解酶。本项目的实施，有助于加深影响里氏木霉高效表达（细菌）异源基因的关键分子机制的认知，为基因工程改良工业菌株里氏木霉提供更深入的科学依据，并寄予实现新酶工业化生产更大的希望。