Towards Novel Glycoside Hydrolases

当今工业（白色）生物技术的主要挑战之一是以最终必须与化石燃料竞争的成本从生物质生产燃料-但作为提供能源安全的可再生碳中性能源，从长远来看可能更具可持续性。“绿色”（即环境友好型）工业生产线--其特点是减少能源消耗、废物和二氧化碳排放--显然具有吸引力，但关键依赖于发现、改进和适应强大而有效的生物催化剂。这意味着必须开发能够识别合适催化剂的方法和策略。虽然酶用于生物催化的效用是清楚的，但通过酶工程（现在可以说更经常基于达尔文的定向进化循环，而不是设计-尽管这两种方法不是排他性的）来发现或制造用于广泛目的的这种有效的、有用的催化剂仍然不是微不足道的。我们在这个提案中解决了“生物能源管道”中的每一步（从生物质的生长到生物燃料的发酵）都可能成为速率或成本限制的挑战，并且显然需要用于这些目的的酶。植物细胞壁对微生物和酶解的天然抗性是木质纤维素生物质转化的高成本的主要原因。到目前为止，只有一小部分（约40%）的能量含量可从木质纤维素原料（植物材料的农业，工业，家庭和森林残留物形式的不可用部分）可转化为乙醇。我们在多个方面解决了这个问题：使用超高通量筛选系统，我们鉴定了新的蛋白质催化剂（来自宏基因组文库），并通过多轮定向进化改进了这些和已经表征的催化剂。我们的定向进化是通过获得一种新型的文库（模仿自然机制-包括插入和缺失）和通过超高通量筛选非常大的文库（> 10 e7成员）的可用性来推动的。我们希望所选的新型酶的分析将解开纤维素酶/半纤维素酶和底物识别的结构基础之间的进化关系，作为基础，以改善新的有用的生物催化剂的工程，使有效的植物细胞壁水解。我们的最终目标是使强大的和多特异性的生物催化剂，应该是有用的，以及一般规则和实验方法，使这个过程更可控，使其能够被广泛使用的柠檬酸木质纤维素组分的水解。

One of the main challenges of industrial (white) biotechnology today is the production of fuel from biomass at a cost that ultimately must be competitive with fossil fuels - but potentially more sustainable in the long term as a renewable, carbon-neutral energy source providing energy security. "Green" (i.e. environmentally-friendly) industrial production lines - characterised by reduced energy consumption, waste and CO2-emissions - are clearly attractive, but are crucially reliant on the discovery, improvement and adaptation of robust and efficient biocatalysts. This means that methods and strategies have to be developed that allow identification of suitable catalysts. While the utility of enzymes for biocatalysis is clear, it is still not trivial to find or make such efficient, useful catalysts for a wide range of purposes by enzyme engineering (now arguably based more often on Darwinian cylces of 'directed evolution, rather than design - although the two approaches are not exclusive). We tackle in this proposal the challenge that each step in the 'bioenergy pipeline' (from growing biomass to fermentation for biofuels) can potentially become rate- or cost-limiting and that enzymes for those purposes are clearly needed. The natural resistance of plant cell walls to microbial and enzymatic deconstruction is largely responsible for the high cost of lignocellulosic biomass conversion. To date, only a small proportion (approximately 40%) of the energy content available from lignocellulose feedstocks (unusable portions of plant materials in the form of agricultural, industrial, domestic, and forest residues) is convertible to ethanol. We address this problem at a number of fronts: using a ultra-high throughput screening system we identify new protein catalysts (from metagenomic libraries) and we improve these and already characterised catalysts by multiple rounds of directed evolution. Our directed evolution is propelled by access to a new type of libraries (mimicking natural mechanisms - involving insertion and deletions) and by the availability of ultrahigh-throughput screens of very large libraries (>10e7 members). We hope that the analysis of the selected novel enzymes will unravel evolutionary relationships between cellulases/hemicellulases and the structural basis for substrate recognition as a basis to improve the engineering of new useful biocatalysts enabling efficient plant cell wall hydrolysis. Our final goal is to to make robust and multispecific biocatalysts that should be useful for the hydrolysis of recalcitrant lignocellulosic components available as well as general rules and experimental approaches to make this process more controllable and enable it to be widely used.