Exploring the biotechnological potential of GH68 family fructosyltransferases from evolutionary diverse haloarchaea

菊粉和果聚糖等果聚糖是源自蔗糖的果糖寡糖和多糖，已在各种工业、农业和医疗保健应用中得到应用，最引人注目的是作为益生元膳食补充剂。此前，人们认为果聚糖生物合成仅限于细菌和植物。然而，最近的结果首次证明了古菌生物，特别是嗜盐古菌（haloarchaea）的果聚糖生物合成。该博士项目将重点研究盐古菌果聚糖生物合成的酶和机制，以期建立可通过工业生物技术部署的果聚糖生物合成新模式。对于生物技术应用来说，盐古菌是一个特别有吸引力的平台。它们无害，遗传上易于处理（允许轻松的异源基因表达），可以直接在中温高盐培养基中生长，并产生在高盐中具有活性且在高温下通常稳定的酶。该项目最初将重点关注盐古菌 GH68 家族果糖基转移酶 (FT)，有四个主要目标：[a] 在已充分表征的遗传易处理（但非果糖）盐古菌 Haloferax volcanii 中工程化进化上多样化的盐古菌 GH68 酶的异源表达，[b] 开发允许盐古菌高水平表达的分子遗传工具[c] 在体外纯化和表征选定的进化多样化盐古菌 GH68 酶的活性，直至并包括通过 X 射线晶体学测定蛋白质结构，以及 [d] 利用新开发的分子遗传工具和方案通过反向遗传方法分析 GH68 酶的细胞功能。该项目汇集了麦克尼尔实验室在盐古菌培养和基因操作方面的专业知识与格洛斯特实验室在碳水化合物加工酶的酶学和结构生物学方面的专业知识。

Fructans such as inulins and levans are fructose-based oligo- and polysaccharides derived from sucrose that have found use in a variety of industrial, agricultural and healthcare applications, most notably as prebiotic dietary supplements. Previously, it was thought that fructan biosynthesis was limited to bacteria and plants. However, recent results have for the first time demonstrated fructan biosynthesis by archaeal organisms, specifically halophilic archaea (haloarchaea). This PhD project will focus on characterising the enzymes and mechanisms of fructan biosynthesis by haloarchaea with a view to establishing new modalities for fructan biosynthesis that can be deployed by industrial biotech. The haloarchaea are a particularly attractive platform for biotech applications. They are non-hazardous, genetically tractable (allowing facile heterologous gene expression), straightforward to grow in high salt medium at mesophilic temperatures and produce enzymes that are active in high salt and generally stable at elevated temperatures. The project will focus initially on haloarchaeal GH68 family fructosyltransferase (FT) enzymes, with four primary objectives: [a] to engineer heterologous expression of evolutionarily diverse haloarchaeal GH68 enzymes in the well-characterised genetically tractable (but non-fructogenic) haloarchaeon Haloferax volcanii, [b] to develop molecular genetic tools allowing regulated high level expression of haloarchaeal GH68 enzymes in their natural host organisms, [c] to purify and characterise in vitro the activities of selected evolutionarily diverse haloarchaeal GH68 enzymes, up to and including protein structure determination by X-ray crystallography, and [d] to utilise the newly developed molecular genetic tools and protocols to analyse the cellular function of GH68 enzymes via reverse genetic approaches. The project brings together the expertise of the MacNeill lab in culturing and genetically manipulating haloarchaea with the expertise of the Gloster lab in the enzymology and structural biology of carbohydrate processing enzymes.