Bilateral BBSRC-FAPESP / Targeted analysis of microbial lignocellulolytic secretomes - a new approach to enzyme discovery.

随着化石燃料供应的减少和人们对化学废物对环境影响的担忧加剧，工业生物技术专家正在探索如何利用生物炼油厂的植物原料来生产生物燃料，并制造聚合物、药品和日用化学品。生物精炼的长期成功依赖于开发经济的植物生物质加工方法，以开发纤维素中富含能量的多糖进行发酵。木质素中存在的复杂的酚类聚合物是植物细胞壁解构的主要瓶颈，因为它们不能降解。目前的生物精炼需要使用酸和蒸汽爆破法来处理木质纤维素，效率低且依赖能源，然后用纤维素酶的鸡尾酒消化释放的纤维素。目前，将生物质转化为可发酵糖的成本太高，使得纤维素发酵过于昂贵。虽然木质纤维素的糖化仍然是工业上的一个问题，但在自然环境中，微生物群落可以有效地进行糖化。这样的群落在堆肥系统和土壤中都能找到。确定微生物群落在木质纤维素降解过程中使用的酶和其他蛋白质的范围的主要挑战在于过程本身的复杂性。目前，绝大多数微生物的生物多样性仍然不具特征，因为在大多数环境中，只有不到1%的微生物适合无菌培养，因此，到目前为止，木质纤维素的降解主要是在少数特征良好和可培养的微生物中进行的。本研究旨在从腐烂的谷物秸秆和甘蔗渣中发现新的木质纤维素酶和相关蛋白质，并采用蛋白质组学和转录后基因组学相结合的方法进行鉴定。我们蛋白质组方法的创新方面源于这样一个事实，即微生物细胞不能摄取未消化的木质纤维素片段，而必须首先将这种物质转化为单糖，然后再输入细胞，这要求它们分泌适当的消化酶。因此，大多数参与木质纤维素动员的酶和辅助蛋白与那些参与家务活动的酶和辅助蛋白的区别在于它们是分泌的。主要的挑战是木质纤维素活性蛋白与木质纤维紧密结合，需要严格的提取条件才能释放它们。同样，许多参与木质纤维素消化的微生物也会结合到底物上，不能被洗掉。其结果是，如果使用足够严格的提取方法将蛋白质从底物上去除，这将不可避免地导致细胞裂解和提取液受到细胞酶的污染；而较温和的提取只释放一小部分目标蛋白。为了克服这个问题，我们将使用非侵入性的胞外蛋白标记来识别微生物群落产生的分泌酶。关键是使用一种蛋白质亲和标签，这种标签不能跨越生物膜，因此只能接触和标记细胞外蛋白质。一旦标记反应结束，木质纤维素和微生物培养物可以在严格的条件下提取，标记的胞外蛋白在蛋白质组分析之前通过亲和纯化简单地分离出来。结合细胞外蛋白质组学和转录组学的力量，将使我们能够专注于从微生物群落中分解木质纤维素的关键蛋白质。这将极大地提高我们识别全新类型木质纤维素活性蛋白的能力，既拓宽了我们对这一过程的基本理解，也为研究和工业应用提供了新的活动。

As fossil fuel supplies dwindle and concerns increase about the environmental impact of chemical waste streams, industrial biotechnologists are exploring ways to use plant based feedstocks in 'biorefineries' to generate biofuels and manufacture polymers, pharmaceuticals and commodity chemicals. The long-term success of biorefining is dependent on the development of economical methods for processing plant biomass to exploit the energy rich polysaccharides in cellulose for fermentation. The complex phenolic polymers present in lignin create a major bottleneck in the deconstruction of plant cell walls, as they are recalcitrant to degradation. Currently biorefineries require the use of acid and steam explosion to treat lignocellulose, which is inefficient and energy dependent, the released cellulose is then digested with a cocktail of cellulases. The costs involved in converting biomass into fermentable sugars currently make cellulosic fermentation too expensive. While the saccharification of lignocellulose remains a problem for industry, it is carried out effectively in the natural environment by microbial communities. Such communities are found in composting systems and soils. The major challenge in identifying the range of enzymes and other proteins used by communities of microorganisms during lignocellulose degradation lies in the complexity of the process itself. At present the vast majority of microbial biodiversity remains uncharacterised, because less than 1% of microorganisms in most environments are amenable to axenic cultivation, therefore, to date lignocellulose degradation has largely been studied in a few well characterised and culturable microorganisms. The research proposed here is concerned with discovering new enzymes and associated proteins for lignocellulose digestion from rotting cereal straw and sugar cane bagasse, and takes an integrated proteomics and metatranscriptomic approach for their identification. The innovative aspect to our proteomic approach arises from the fact that microbial cells cannot ingest pieces of undigested lignocellulose, but must first convert this material to simple sugars that can then be imported in to the cell, and this requires that they secrete the appropriate digestive enzymes. Thus, the majority of enzymes and accessory proteins involved in lignocellulose mobilisation are distinguished from those involved in housekeeping activities by the fact that they are secreted. The major challenge is that lignocellulose active proteins bind tightly to lignocellulose and require stringent extraction conditions to release them. Similarly, many of the microbes involved in lignocellulose digestion also bind to the substrate and cannot be washed out. The result of this is that if sufficiently stringent extraction is used to get the proteins off the substrate this inevitably leads to cell lysis and contamination of the extract with cellular enzymes; whilst milder extractions only release a small proportion of the target proteins. To overcome this problem we will use non-invasive extracellular protein tagging to identify secreted enzymes produced by microbial communities. The key to this is the use of a protein affinity tag that cannot cross biological membranes and can therefore only access and tag extracellular proteins. Once the tagging reaction has been quenched, the lignocellulose and microbial culture can be extracted under stringent conditions, with the tagged extracellular proteins simply separated by affinity purification prior to proteomic analysis. Combining the power of extracellular proteomics and metatranscriptomics will allow us to focus in on the proteins critical for lignocellulose deconstruction from microbial communities. This will greatly enhance our ability to identify completely new types of lignocellulose active proteins, both broadening our fundamental understanding of this process, as well as providing novel activities for research and industrial applications.