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

酶是白色生物技术的核心，它将创造更环保、更高效的过程。将我们的碳源从化石转变为可再生能源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”（定向进化中的人制造或功能宏基因组学中的环境）中寻找新的催化剂是一个数字游戏：成功很少，因此能够筛选大量图书馆成员的技术将更成功。这种组合活动的选择标准也很重要：分析反应与实际应用越相似，筛选活动产生有用酶的可能性就越大。我们已经开发了一个系统，通过使用耦合反应系统，可以在一天内测试超过1000万种变体，以分解天然底物（而不是模型底物），所以我们希望能更快地找到正确的“大海捞针”。在对碳水化合物活性酶的耦合分析的首次应用中，我们将进化用于降解木材主要成分（碳水化合物，主要以纤维素的形式）的酶，并尝试在温泉，南极和一些国内环境（当地堆肥堆，池塘，土壤）中收集的宏基因组样品中发现新的酶。定向进化的结果将使用我们最近开发的测序技术进行解释（“UMIC-Seq”：Nat comm 2020, 11(1), 6023），该技术使我们能够获得每轮进化的100万个序列的全长读数（每个序列的价格低于1便士）。有了这一见解，我们希望更好地理解酶的合作相互作用如何使进化进行（或使其在其他情况下变得困难），基于序列景观的重建，其中高活性区域将被探索。

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.