Templates for rapid directed evolution of biocatalysts

酶生物催化剂现在涉及许多工业过程，并且在药物合成和将生物质转化为可再生燃料中特别有用。这是因为天然存在的酶可以是高度活性和非常选择性的（对映体选择性、区域选择性和化学选择性）。生物催化剂的潜力已经变得越来越容易获得，这主要是由于定向进化和产生优质突变体文库的进展，即有效地探索序列空间，以及功能变体的高通量筛选（HTP）。通常定向进化已被用于改善低酶活性，增加热稳定性或对工业条件的耐受性，以及扩大对酶具有低活性的底物的活性。也有很好的例子，定向进化用于开发新的酶功能。然而，用于没有生物背景的过程的生物催化剂仍然是蛋白质工程的挑战。从头设计已被用于试图弥合这一差距，但以这种方式获得新的生物催化剂所花费的时间是不理想的。祖先蛋白质的基于系统发育的创建表明，这些蛋白质比它们当前的对应物更混杂，并且具有更广泛的底物特异性。考虑到这一点，任何朝向真正新型生物催化剂的定向进化的合理起点应该是对几种底物具有低活性的酶。蛋白质的折叠单位可以追溯到共同的祖先。常见的蛋白质折叠包括但不限于：Rossman折叠、α/β折叠、TIM桶和免疫球蛋白折叠。这个项目的目的是探索一个去进化的酶作为一个模板，向新的功能进化的想法。其重点将是产生具有最小功能装饰的蛋白质支架，祖先序列重建和利用中性漂移结合HTP筛选方法。

Enzyme biocatalysts are now involved in lots of industrial processes and are particularly useful in drug synthesis and conversion of biomass to renewable fuels This is because naturally occurring enzymes can be highly active and very selective (enantio-, regio- and chemoselectivity). The potential of biocatalysts has become increasingly accessible largely due to directed evolution and advances in generating libraries of quality mutants i.e effectively exploring sequence space, as well as high throughput screening (HTP) for functional variants. Often directed evolution has been employed to improve low enzyme activity, increase thermostability or tolerance to industrial conditions, as well as expanding activity towards substrates which an enzyme has low activity. There are also good examples of directed evolution used to develop novel enzyme function. However, biocatalysts for processes that have no biological context still remains a challenge for protein engineering. De novo design has been used in attempt to bridge this gap but the time taken to achieve novel biocatalysts in this way is less than ideal. Phylogeny based creation of ancestral proteins suggests that these were more promiscuous and had broader substrate specifity than their current day counterparts. With that in mind a rational place to start any directed evolution towards a truly novel biocatalyst ought to be an enzyme with low activity towards several substrates. Folding units of proteins can be traced to common ancestors. Commonly seen protein folds include but are not limited to; the Rossman fold, alpha/beta plait, TIM barrels and the immunoglobulin fold. This project aims to explore the idea of a de-evolved enzyme as a template for evolvability towards new functions. The focus of this will be on generating a protein scaffold with minimal functional decoration, ancestral sequence reconstruction and capitalising on neutral drift combined with HTP screening methods.