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

诸如菊粉和levans之类的果聚糖是基于果糖的低聚物和从蔗糖中提取的多糖，已在各种工业，农业和医疗保健应用中得到应用，最明显的是作为益生元膳食补充剂。以前，人们认为果糖的生物合成仅限于细菌和植物。然而，最近的研究结果首次证明了古细菌生物合成果糖，特别是嗜盐古细菌（haloarchaea）。该博士项目将重点研究盐古菌合成果聚糖的酶和机制，以期建立可用于工业生物技术的果聚糖生物合成新模式。盐古菌是生物技术应用的一个特别有吸引力的平台。它们是无害的，遗传上易于处理（允许容易的异源基因表达），在中温高盐培养基中直接生长，并产生在高盐中具有活性且在高温下通常稳定的酶。该项目最初将侧重于卤古菌GH68家族果糖基转移酶（FT）酶，主要目标有四个：[a]设计进化多样性的盐古菌GH68酶在具有良好特征的遗传易感（但非构造性）的火山盐古菌中的异源表达，[b]开发分子遗传学工具，允许盐古菌GH68酶在其天然宿主生物中受调控的高水平表达，[c]在体外纯化和表征选定的进化多样性的盐古菌GH68酶的活性。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学工具和方案通过反向遗传方法分析GH68酶的细胞功能。该项目将麦克尼尔实验室在培养和基因操纵盐古菌方面的专业知识与Gloster实验室在酶学和碳水化合物加工酶结构生物学方面的专业知识结合在一起。

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.