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

如菊粉和Levans等果聚糖是以果糖为基础的寡糖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发现了用途，最著名的是作为益生菌膳食补充剂。此前，人们认为果聚糖的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生菌生物，特别是嗜盐古生菌(盐生古生菌)合成果聚糖。这一博士项目将重点研究嗜盐古生菌生物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的新模式。对于生物技术应用来说，海洋生物是一个特别有吸引力的平台。它们是无害的，遗传上容易处理(允许轻松的异源基因表达)，在中温下直接在高盐介质中生长，并产生在高盐中活性和在高温下通常稳定的酶。该项目最初将专注于嗜盐古菌GH68家族果糖基转移酶(FT)，有四个主要目标：[a]设计进化上多样化的嗜盐古菌GH68酶在具有良好特征的遗传易处理(但非果源)的嗜盐古生菌火山II中的异源表达，[b]开发允许在其自然宿主生物体中高水平表达嗜盐古菌GH68酶的分子遗传工具，[c]在体外纯化和表征选定的进化多样化的古嗜盐菌GH68酶的活性，包括通过X射线结晶学确定蛋白质结构，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遗传操作盐生细菌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.