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

果聚糖如菊糖和果聚糖是衍生自蔗糖的基于果糖的寡糖和多糖，其已被发现用于各种工业、农业和医疗保健应用，最值得注意的是作为益生元膳食补充剂。以前，人们认为果聚糖的生物合成仅限于细菌和植物。然而，最近的研究结果首次证明果聚糖生物合成的古菌，特别是嗜盐古菌（haloarchaea）。这个博士项目将专注于描述盐古菌果聚糖生物合成的酶和机制，以期建立可由工业生物技术部署的果聚糖生物合成的新模式。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，遗传上易处理的（允许容易的异源基因表达），直接在中温温度下在高盐培养基中生长，并产生在高盐中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），主要目标有四个：[a]在充分表征的遗传上易处理的盐古菌中工程化进化上多样的盐古菌GH68酶的异源表达，（但不产果）盐古菌Haloferax volcano ii，[B]开发允许盐古菌GH 68酶在其天然宿主生物体中调节高水平表达的分子遗传工具，[c]在体外纯化和鉴定所选进化上多样化的盐古菌GH 68酶的活性，直至并包括通过X射线晶体学确定蛋白质结构，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GH 68酶的细胞功能。该项目将MacNeill实验室在培养和遗传操纵盐古菌方面的专业知识与光泽者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.