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（白人）生物技术的主要挑战之一是生物量的燃料生产，最终必须与化石燃料竞争，但从长远来看，作为一种可再生的，可再生的碳独立能源提供了能源安全的可能性更高。 “绿色”（即环保的）工业生产线（以减少的能源消耗，废物和CO2排放为特征）显然很有吸引力，但至关重要的是依赖于发现，改进和适应可靠，有效的生物催化剂。这意味着必须制定允许识别合适催化剂的方法和策略。尽管酶对生物催化的实用性很明显，但通过酶工程来找到或制造出如此高效，有用的催化剂并不是微不足道的（现在可以说，现在可以说更频繁地基于达尔文式的“定向进化”，而不是设计 - 尽管这两种方法不是排他性的）。我们在这项提案中应对“生物能管道”（从生物量到生物燃料的发酵）中的每个步骤都可能成为速率或成本限制，并且显然需要出于这些目的的酶。植物细胞壁对微生物和酶促解构的自然耐药性在很大程度上是造成木质纤维素生物量转化率的高成本。迄今为止，只有一小部分（约40％）可从木质纤维素原料（以农业，工业，家庭和森林残留物的形式使用的植物材料的无法使用的植物材料）提供的能源含量可转换为乙醇。我们在多个方面解决了这个问题：使用超高的吞吐量筛选系统，我们识别出新的蛋白质催化剂（来自元基因组库），我们通过多个定向进化来改善这些蛋白质催化剂（来自元基因组库）。通过访问新型的库（模仿自然机制 - 涉及插入和删除）以及可用的非常大的库（> 10E7成员）的超高通量屏幕（> 10E7成员），我们的定向演变是通过访问新类型的库（模仿自然机制）来推动的。我们希望对所选新型酶的分析能够揭示纤维素酶/半纤维素酶之间的进化关系，以及底物识别的结构基础，作为改善新有用的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可靠的固定和多特异性生物催化剂，这些生物催化剂对于可用的顽固​​木质纤维素组件的水解以及一般规则和实验方法，以使该过程更具可控性并启用其可广泛使用。

项目成果

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

• DOI: 10.1038/ncomms10008
• 发表时间: 2015-12-07
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Colin PY;Kintses B;Gielen F;Miton CM;Fischer G;Mohamed MF;Hyvönen M;Morgavi DP;Janssen DB;Hollfelder F
• 通讯作者: Hollfelder F

In vitro Evolution of Antibody Affinity via Insertional Scanning Mutagenesis of an Entire Antibody Variable Region

通过整个抗体可变区的插入扫描诱变进行抗体亲和力的体外进化

• DOI: 10.17863/cam.57648
• 发表时间: 2020
• 作者: Hollfelder F

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: 10.17863/cam.6859
• 发表时间: 2016
• 作者: Gielen F

Functional Trade-Offs in Promiscuous Enzymes Cannot Be Explained by Intrinsic Mutational Robustness of the Native Activity.

• DOI: 10.1371/journal.pgen.1006305
• 发表时间: 2016-10
• 期刊: PLoS genetics
• 影响因子: 4.5
• 作者: Kaltenbach M;Emond S;Hollfelder F;Tokuriki N
• 通讯作者: Tokuriki N

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: 10.17863/cam.72969
• 发表时间: 2020
• 作者: Emond S