CAZyme evolution and discovery: Ultrahigh throughput screening of carbohydrate-active enzymes in modular assays modular based on coupled reactions

CAZyme 的演变和发现：基于耦合反应的模块化测定中碳水化合物活性酶的超高通量筛选

• 批准号: BB/W006391/1
• 负责人: Florian Hollfelder
• 金额: $ 59.11万
• 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: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FW006391%2F1
• 关键词: CAZyme; evolution; discovery; Ultrahigh; throughput

Enzymes are at the core of the white biotechnologies that will create greener, more efficient processes. The environmental challenge of switching our carbon source from fossil to renewable will require many more enzymes than are currently available, so enzyme discovery and evolution are necessary. Finding new catalysts in 'libraries' (person-made in directed evolution or environmental in functional metagenomics) is a numbers game: hits are rare, so technologies that can screen large numbers of library members will be more successful. The selection criterion in such combinatorial campaigns also matters: the more similar the assay reaction is to the actual application, the more likely is the screening campaign to yield useful enzymes. We have developed a system in which more than 10 million variants can be tested in a day for breakdown of natural substrates (and not for model substrates), by using coupled reaction systems, so we hope to find the right "needle in a haystack", more quickly. In a first application of coupled assays to carbohydrate-active enzymes, we will evolve enzymes used for the degradation of the main components of wood (carbohydrates, mainly in the form of cellulose) and also try to discover new enzymes in metagenomic samples collected in hot springs, the Antarctic and several domestic settings (local compost heaps, ponds, soils). The results of directed evolution will be interpreted with a sequencing technology that we have recently developed ('UMIC-Seq': Nat Commun 2020, 11 (1), 6023), that allows us to obtain full-length readouts of > 10,000 sequence per round of evolution (at a price of less than 1 penny per sequence). Equipped with this insight we hope to understand better how cooperative interactions in the enzymes enable evolution to proceed (or make it difficult in other cases), based on the reconstruction of sequence landscapes, in which areas of high activity are to be explored.

酶是白色生物技术的核心，它将创造更绿色、更高效的工艺。将我们的碳源从化石碳源转变为可再生碳源所面临的环境挑战将需要比目前可用的更多的酶，因此酶的发现和进化是必要的。在“图书馆”（定向进化中的人造或功能宏基因组学中的环境）中寻找新的催化剂是一场数字游戏：命中很少，因此能够筛选大量图书馆成员的技术将更加成功。此类组合活动中的选择标准也很重要：测定反应与实际应用越相似，筛选活动就越有可能产生有用的酶。我们开发了一个系统，通过使用耦合反应系统，一天可以测试超过 1000 万个变体，以了解天然底物（而不是模型底物）的分解情况，因此我们希望找到正确的“大海捞针” ，更快。在碳水化合物活性酶的耦合测定的首次应用中，我们将进化用于降解木材主要成分（碳水化合物，主要以纤维素形式）的酶，并尝试在热收集的宏基因组样本中发现新的酶。泉水、南极洲和一些家庭环境（当地堆肥、池塘、土壤）。定向进化的结果将通过我们最近开发的测序技术（“UMIC-Seq”：Nat Commun 2020, 11 (1), 6023）进行解释，该技术使我们能够获得每个序列> 10,000 个序列的全长读数。一轮进化（每个序列的价格不到 1 美分）。有了这种洞察力，我们希望更好地了解酶中的合作相互作用如何使进化得以进行（或在其他情况下使其变得困难），基于序列景观的重建，其中将探索高活性区域。

项目成果

Versatile Product Detection via Coupled Assays for Ultrahigh-Throughput Screening of Carbohydrate-Active Enzymes in Microfluidic Droplets.

通过耦合分析进行多功能产品检测，用于微流体液滴中碳水化合物活性酶的超高通量筛选。

• DOI: http://dx.10.1021/acscatal.3c01609
• 发表时间: 2023
• 期刊: ACS catalysis
• 影响因子: 12.9
• 作者: Ladeveze S

Sub-single-turnover quantification of enzyme catalysis at ultrahigh throughput via a versatile NAD(P)H coupled assay in microdroplets

通过微滴中的多功能 NAD(P)H 偶联测定，以超高通量对酶催化进行亚单周转定量

• DOI: http://dx.10.1101/2023.11.22.568356
• 发表时间: 2023
• 作者: Penner M

Functional metagenomic screening identifies an unexpected ß-glucuronidase.

功能宏基因组筛选发现了一种意想不到的α-葡萄糖醛酸酶。

• DOI: http://dx.10.17863/cam.84202
• 发表时间: 2022
• 作者: Neun S

Functional metagenomic screening identifies an unexpected ß-glucuronidase.

功能宏基因组筛选发现了一种意想不到的α-葡萄糖醛酸酶。

• DOI: http://dx.10.1038/s41589-022-01071-x
• 发表时间: 2022
• 期刊: Nature chemical biology
• 影响因子: 14.8
• 作者: Neun S

Versatile Product Detection via Coupled Assays for Ultra-high-throughput Screening of Carbohydrate-Active-Enzymes in Microfluidic Droplets

通过耦合分析进行多功能产品检测，用于微流体液滴中碳水化合物活性酶的超高通量筛选

• DOI: http://dx.10.1101/2023.03.29.534725
• 发表时间: 2023
• 作者: Ladeveze S