Identifying, characterising and engineering fungal plant cell wall degrading enzymes for enhanced biocatalysts in biofuel production

真菌和卵菌基因组是能够降解植物细胞壁的碳水化合物加工酶的丰富来源。这些酶的开发是有吸引力的，因为它们具有将植物的废木质纤维素生物质转化为可用于生物燃料生产的简单糖的能力。我们将重点关注真菌/卵菌物种，包括Rhynchosporium spp., Magnaporthe spp.和Phytophthora spp.这些物种具有一系列碳水化合物加工特异性，在生物催化方面具有潜力。该项目的第一部分将涉及对一系列真菌/卵菌物种的基因组进行生物合成，以确定碳水化合物降解酶。重点将放在具有天然和工程底物多样性潜力的酶家族上，这些酶家族为进一步的工程设计提供了有希望的基础。在此之后，编码这些酶的基因将被克隆并在大肠杆菌中过表达，并研究它们对一系列碳水化合物底物的活性和特异性。此外，酶的结构将用x射线晶体学测定。这些结构和功能的见解将用于确定候选的工程，我们将使用合理的方法来设计突变体，包括那些相关酶的自然序列多样性可以被纳入的突变体，以探索增强活性和/或更广泛的底物特异性的潜力。工程可能需要突变体和迭代循环的组合，以获得可用于生物催化应用的酶。

BBSRC Theme: Industrial Biotechnology and BioenergyFungal and oomycete genomes are a rich source of carbohydrate processing enzymes that are capable of plant cell wall degradation. Exploitation of these enzymes is attractive given they have the capability of converting waste lignocellulosic biomass from plants into simpler sugars, which can be utilised in biofuel production.We will focus on fungal/oomycete species including Rhynchosporium spp., Magnaporthe spp., and Phytophthora spp. These species possess a range of carbohydrate processing specificities with potential in biocatalysis. The first part of the project will involve biomining the genomes of a range of fungal/oomycete species to identify carbohydrate degrading enzymes. The focus will be on enzyme families that have potential for natural and engineered substrate diversity, and which present a promising basis for further engineering.Following this, the genes encoding a number of these enzymes will be cloned and over-expressed in Escherichia coli, and their activity and specificity against a range of carbohydrate substrates investigated. In addition, the structures of the enzymes will be determined using X-ray crystallography. These structural and functional insights will be used to identify candidates to take forward for engineering, where we will use a rational approach to design mutants, including those where the natural sequence diversity of related enzymes can be incorporated, to explore the potential for enhanced activity and/or a wider substrate specificity. Engineering may require combinations of mutants and iterative cycles in order to obtain enzymes that can be applied in biocatalytic applications.