牦牛瘤胃真菌木聚糖酶和乙酰酯酶协同降解机制及功效研究

Due to the special ecological environment and long-term natural selection on Qinghai-Tibet Plateau, yaks have evolved some special physiological adaptations to adapt to the harsh environment with severe cold, less oxygen, strong ultra-violet radiation, and poor forage quality. Our previous studies revealed that yaks fed with roughages can produce high level of cell wall degrading enzymes, and we also found that there exist strong positive synergistic effects among these enzymes. The objectives of this project are to research the diversity of anaerobic fungi from the rumen fluids of yaks, to explore the synergetic degradation mechanism and efficacy of xylanase and acetyl esterase co-expression in Pichia pastoris on the crosslinking network structure of cell walls. With the assistance of Yak Comprehensive Experiment Station in Qinghai and based on our previous studies, anaerobic fungi metagenomic DNA from the rumen fluids of yaks will be prepared to construct the Fosmid library by using gradient centrifugation and to study the diversity of these fungi by using high-throughput sequencing and bioinformatics. Xylanase and acetyl esterase will be amplified and co-expressed into Pichia pastoris by specific primers. The synergetic degradation mechanism and efficacy of high enzymes yield on crop stalks will be evaluated after optimizing the fermentation conditions. This proposed project may promote the study and utilization of microbial genetic resources and fungi enzyme resources to improve the efficiency in degrading/decomposing agricultural by-products.

青藏高原特殊的生态环境和长期的自然选择，使我国牦牛形成了耐寒、耐低氧、耐强紫外线和耐粗饲等生态生理特性。申请者前期研究结果表明，长期以粗饲料为生的牦牛瘤胃真菌含有更高的细胞壁降解酶活性，且降解酶间存在协同效应。为探究牦牛瘤胃真菌多样性、真菌源木聚糖酶和乙酰酯酶协同降解机制及双酶共表达毕赤酵母工程菌对细胞壁致密网状交联结构的疏解功效，本项目拟在青海大通牦牛综合试验站的协助下，在申请者前期研究基础上，采集放牧饲养条件下青海牦牛瘤胃食糜样品，梯度离心后提取真菌元基因组DNA，高通量测序技术结合生物信息学方法研究牦牛真菌多样性，构建Fosmid克隆文库，设计特异性引物获得木聚糖酶和乙酰酯酶基因克隆，将其整合至毕赤酵母中高效共表达，筛选并优化工程菌发酵条件，研究双酶协同降解机制，评价其对农作物秸秆的降解功效，为今后有效开发牦牛瘤胃微生物遗传种质资源和提高粗饲料在反刍家畜的利用提供基础依据。

青藏高原特殊的生态环境和长期的自然选择，使我国牦牛形成了耐寒、耐低氧、耐强紫外线和耐粗饲等生态生理特性。本项目团队研究表明，（1）相同放牧条件下，牦牛瘤胃液细胞壁降解酶活性显著高于犏牛和黄牛（P<0.05）。（2）采用亨盖特厌氧滚管技术从瘤胃中分离获得12株具有细胞壁降解酶活性的厌氧真菌，12株瘤胃厌氧真菌均为Piromyces属，分别命名为Piromyces sp. CN1- CN12（Genbank 登录号为KY368100~KY368111）。其中，Piromyces sp. CN6的木聚糖酶、羧甲基纤维素酶和乙酰酯酶活性显著高于其他菌株（P<0.05）。木聚糖酶与羧甲基纤维素酶、乙酰酯酶存在极显著正相关（P<0.01），与微晶纤维素酶呈显著正相关（P<0.05）。本研究从瘤胃内容物筛选获得的Piromyces sp. CN6 具有较高的植物细胞壁降解酶活性，通过酶活测定和酶学特性分析，丰富和完善瘤胃真菌的酶系信息，为深入应用提供依据。（3）瘤胃真菌Piromyces sp. CN6作为青贮添加剂能够可显著降低发酵30 d青贮饲料pH、乙酸含量和NH3-N/TN（P<0.05），显著降低发酵10 d、30 d、60 d青贮饲料NDF和ADF含量（P<0.05），显著提高发酵30 d青贮饲料可溶性碳水化合物含量、粗蛋白含量、乳酸含量和乳酸/乙酸比值（P<0.05），显著提高发酵30 d青贮饲料DM、NDF和ADF体外降解率（P<0.05）。瘤胃真菌Piromyces sp. CN6能够改善青贮饲料的发酵品质和营养成分，提高粗纤维的降解率。（4）体内和体外试验均表明，复合酶制剂能够提高反刍动物饲料消化率和乳品质，维持动物健康和生产性能。