微生物协同降解纤维素的协同因子的鉴定及协同机制研究

Cellulose is the most abundant and renewable biomass resource on Earth. Cellulose can be degraded into glucose by the synergistic action of the three classes of cellulases including endo-1,4-β-glucanase, exo-1,4-β-glucanase and β-glucosidase. These enzymes act synergistically because endoglucanases generate new chain ends for exoglucanses, which release cellobiose that is converted to glucose by β-glucosidase. Glucose can be used to ferment to produce biofuels and other useful products, which is an alternative way to deal with the fossil energy crisis. At present, using recalcitrant lignocellulose as raw material to produce fuel ethanol has not really realize commercialization due to high cost of cellulases and their low efficiencies. It is very necessary to engineer super efficient cellulase-producing strains to circumvent this bottleneck problem. Recent studies revealed that non-hydrolytic cellulose active proteins (NHCAPs) act synergistically with cellulases in lignocellulose hydrolysis. Those NHCAPs contribute to cellulases access and hydrolyze the recalcitrant cellulose. This project plans (1) to use the seven high cellulase-production strain as initiation strain to hydrolyze the alkali-pretreated sugarcane bagasse combination with other isolated microbes (including bacteria and fungi) and obtain the significantly synergistic action strains, (2) to isolate, purify and identify the synergistic factors from the synergistic strains, (3) to clone and express the synergistic factor genes and characterize intensively the biochemical properties of novel synergistic factors, (4) to investigate synergistic mechanisms between cellulases and the novel synergistic factors,(5) to transfer synergistic factors with the high synergy degree into initiation strains to construct higher effeciency cellulase-producing genetic modified strains. The anticipated results of this project will propose novel models of cellulose degradation. From an applied point of view those expected novel synergistic action factors are of interest because they may speed up enzymatic conversion of biomass, thus reducing enzyme loads and processing times.

纤维素是地球上最丰富的可再生的生物质资源，它由纤维素酶协同作用最终水解成葡萄糖，葡萄糖可以发酵用来生产生物燃料或其它有用的产品。但目前由于纤维素酶的成本高使以木质纤维素为原料生产燃料乙醇还没有实现商业化，研发超高效率的产纤维素酶菌株是解决该瓶颈问题的策略之一。近来发现非水解活性蛋白在纤维素降解中与纤维素酶具协同效应，它们有利于纤维素酶接近和水解纤维素。本项目以高产纤维素酶的7株真菌菌株作为出发菌株，将其与其它微生物分别进行酶系的复配，获得对碱预处理甘蔗渣有显著协同降解作用的菌株;分离、纯化和鉴定协同菌株中的协同因子;克隆及异源表达新的或具高协同度的协同因子编码基因，并对它们进行详细的生化鉴定，结合它们对纤维素作用的方式，研究它们的协同机制;将具高协同度的协同因子基因转入出发菌株获得更高效纤维素降解基因工程菌。该项目将为纤维素酶解领域提供新理论，对于降低纤维素酶的成本方面具应用的潜力。

天然纤维素形成了很难被纤维素酶降解的结晶结构，导致以木质纤维素作为原料生成燃料酒精的纤维素酶使用成本很高，解决该问题的一个潜在方法就是如何使难降解的纤维素转变成容易被纤维素酶水解的底物。扩展蛋白、膨胀因子和裂解性多糖单加氧酶等协同因子可以破坏纤维素的结晶结构，进而提高纤维素酶水解纤维素的效率。.本项目主要研究内容有：① 通过对1300余株细菌菌株的筛选，初步获得5株与纤维素酶有协同作用的菌株，测定了2株细菌的基因组并鉴定了该2株细菌中的4个纤维素酶基因；② 对草酸青霉中的7个可能协同因子基因（3个膨胀因子和4个裂解性多糖单加氧酶）进行了遗传学研究，发现裂解性多糖单加氧酶POX09256在草酸青霉降解结晶纤维素中起重要作用，但将POX09256和膨胀因子POX1524异源表达后，并没有发现它们与纤维素酶对滤纸的降解具有协同作用；③ 在纤维素降解细菌哈氏噬纤维菌中鉴定了一个新的碳水化合物结合组件（Carbohydrate binding module, CBM） （CHU_1279）和一个独特的属于糖基水解酶家族9（Glycoside hydrolase family 9, GH9）的1,4-β-D葡聚糖葡萄糖水解酶（CHU_0961）。CHU_1279由2个同源结构域串联形成，分别为CHU_1279-1和CHU_1279-2。全长CHU_1279能结合结晶纤维素，而 CHU_1279-1没有结合多糖的能力， CHU_1279-2与全长一样能结合可溶多糖和单链纤维素，但不能结合结晶纤维素，这说明它属于B型CBM。单体是B型CBM（CHU_1279-2）和另一个无结合能力的同源单体(CHU_1279-1)串联后，串联体（CHU_1279）转变成能结合结晶纤维素的A型CBM，这是在已知的CBM家族没有的新特性。这是在哈氏噬纤维菌中发现的第一个能结合结晶纤维素的CBM，而且也是第一次发现用串联方式获得结合结晶纤维素能力的CBM。CHU_0961虽然属于GH9家族的酶，但是它的催化结构域的氨基酸序列与典型的GH9的酶差异很大，被划分为该家族的一个新群（Group），它的酶学特性也与经典的GH9家族的酶不一样，它是以非连续的方式从糖的非还原端切下葡萄糖，证明它是一个1,4-β-D葡聚糖葡萄糖水解酶。这些独特的CBM和纤维素酶为解释哈氏噬纤维菌未知及独特的纤维素降解机制提供了依据。