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对化学废物流的环境影响的担忧增加，工业生物技术专家正在探索在“生物精炼厂”中使用植物原料来生产生物燃料和制造聚合物，药品和商品化学品的方法。生物精炼的长期成功取决于开发用于加工植物生物质的经济方法，以利用纤维素中富含能量的多糖进行发酵。存在于木质素中的复杂酚类聚合物在植物细胞壁的解构中产生主要瓶颈，因为它们不容易降解。目前的生物精炼需要使用酸和蒸汽爆破来处理木质纤维素，这是低效的并且依赖于能量，然后用纤维素酶的混合物消化释放的纤维素。将生物质转化为可发酵糖所涉及的成本目前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，但它在自然环境中通过微生物群落有效地进行。这种群落存在于堆肥系统和土壤中。确定木质纤维素降解过程中微生物群落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，绝大多数微生物生物多样性仍然未被表征，因为在大多数环境中，只有不到1%的微生物适合纯培养，因此，迄今为止，木质纤维素降解主要是在少数表征良好且可培养的微生物中研究的。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，并采用综合蛋白质组学和元转录组学方法对其进行鉴定。我们蛋白质组学方法的创新之处在于，微生物细胞不能摄取未消化的木质纤维素，而必须首先将这种物质转化为单糖，然后再将其输入细胞，这就需要它们分泌适当的消化酶。因此，参与木质纤维素动员的大多数酶和辅助蛋白质与参与管家活动的酶和辅助蛋白质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素紧密结合，并且需要严格的提取条件来释放它们。类似地，参与木质纤维素消化的许多微生物也结合到底物上并且不能被洗掉。其结果是，如果使用足够严格的提取来使蛋白质离开底物，这不可避免地导致细胞裂解和提取物被细胞酶污染;而较温和的提取仅释放小比例的靶蛋白。为了克服这个问题，我们将使用非侵入性的细胞外蛋白质标记来识别微生物群落产生的分泌酶。其关键是使用蛋白质亲和标签，该标签不能穿过生物膜，因此只能访问和标记细胞外蛋白。一旦标记反应被淬灭，木质纤维素和微生物培养物可以在严格条件下提取，在蛋白质组学分析之前通过亲和纯化简单地分离标记的细胞外蛋白。结合细胞外蛋白质组学和元转录组学的力量将使我们能够专注于从微生物群落中解构木质纤维素的关键蛋白质。这将大大提高我们识别全新类型的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，又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.