Templates for rapid directed evolution of biocatalysts

酶生物催化剂现在涉及到许多工业过程，在药物合成和生物质转化为可再生燃料方面特别有用，这是因为自然产生的酶具有高度的活性和非常高的选择性(对映体、区域和化学选择性)。生物催化剂的潜力已经变得越来越容易获得，这在很大程度上是由于定向进化和在产生高质量突变体文库方面的进展，即有效地探索序列空间，以及高通量筛选(HTP)功能变体。通常定向进化被用来改善低酶活性，提高热稳定性或对工业条件的耐受性，以及向酶活性较低的底物扩展活性。也有用于开发新酶功能的定向进化的很好的例子。然而，没有生物背景的过程的生物催化剂仍然是蛋白质工程的一个挑战。从头开始的设计已经被用来试图弥合这一差距，但以这种方式获得新型生物催化剂所需的时间并不理想。基于系统发育的祖先蛋白质的产生表明，这些蛋白质比当今的同类更混杂，具有更广泛的底物特异性。考虑到这一点，开始任何定向进化到真正新颖的生物催化剂的合理地点应该是一种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罗斯曼折叠、α/β折叠、TIM桶折叠和免疫球蛋白折叠。该项目旨在探索将去进化的酶作为新功能进化的模板的想法。这项工作的重点将是构建一种功能修饰最少的蛋白质支架，重建祖先序列，并利用中性漂移结合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.