Towards Novel Glycoside Hydrolases

迈向新型糖苷水解酶

• 批准号: BB/L002469/1
• 负责人: Florian Hollfelder
• 金额: $ 46.05万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FL002469%2F1
• 关键词: Towards; Novel; Glycoside; Hydrolases

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.

当今工业（白色）生物技术的主要挑战之一是用生物质生产燃料，其成本最终必须与化石燃料相竞争，但从长远来看，作为提供能源的可再生、碳中性能源，可能更具可持续性安全。 “绿色”（即环保）工业生产线——其特点是减少能源消耗、废物和二氧化碳排放——显然很有吸引力，但关键依赖于强大而高效的生物催化剂的发现、改进和适应。这意味着必须开发能够识别合适催化剂的方法和策略。虽然酶在生物催化中的效用是显而易见的，但通过酶工程（现在可以说更多地基于达尔文循环的“定向进化，而不是设计”）来寻找或制造用于多种目的的高效、有用的催化剂仍然不是一件容易的事。 - 尽管这两种方法并不排斥）。我们在该提案中解决了“生物能源管道”中的每一步（从生物质生长到生物燃料发酵）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构的天然抵抗力是木质纤维素生物质转化成本高昂的主要原因。迄今为止，木质纤维素原料（以农业、工业、家庭和森林残留物形式存在的植物材料中无法使用的部分）提供的能量中只有一小部分（约 40%）可转化为乙醇。我们从多个方面解决这个问题：使用超高通量筛选系统，我们识别新的蛋白质催化剂（来自宏基因组库），并通过多轮定向进化改进这些催化剂和已经表征的催化剂。我们的定向进化是通过访问新型文库（模仿自然机制 - 涉及插入和删除）以及非常大的文库（> 10e7 成员）的超高通量筛选的可用性来推动的。我们希望对所选新型酶的分析将揭示纤维素酶/半纤维素酶和底物识别的结构基础之间的进化关系，作为改进新型有用生物催化剂工程的基础，从而实现有效的植物细胞壁水解。我们的最终目标是制造坚固且多特异性的生物催化剂，用于水解顽固的木质纤维素成分，并制定一般规则和实验方法，使该过程更加可控并使其得到广泛应用。

项目成果

Accessing unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis

通过插入/删除诱变访问定向酶进化中序列空间中未探索的区域

• DOI: http://dx.10.17863/cam.51305
• 发表时间: 2020
• 作者: Emond S

Accessing unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis.

通过插入/删除诱变访问定向酶进化中序列空间中未探索的区域。

• DOI: http://dx.10.1038/s41467-020-17061-3
• 发表时间: 2020
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Emond S

Ultrahigh-throughput-directed enzyme evolution by absorbance-activated droplet sorting (AADS).

通过吸光度激活液滴分选 (AADS) 进行超高通量定向酶进化。

• DOI: http://dx.10.1073/pnas.1606927113
• 发表时间: 2016
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Gielen F

Accessing unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis

通过插入/删除诱变访问定向酶进化中序列空间中未探索的区域

• DOI: 10.1038/s41467-020-17061-3
• 发表时间: 2020-07-10
• 期刊: Nature Communications
• 影响因子: 16.6
• 作者: S. Emond;Maya Petek;Emily Kay;Brennen Heames;S. Devenish;N. Tokuriki;F. Hollfelder
• 通讯作者: F. Hollfelder

Ultrahigh-throughput discovery of promiscuous enzymes by picodroplet functional metagenomics.

通过皮液滴功能宏基因组学超高通量发现混杂酶。

• DOI: http://dx.10.1038/ncomms10008
• 发表时间: 2015
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Colin PY