17-ERACoBioTech Thermophilic bacteria and archaeal chassis for extremolyte production -HotSolute

嗜热生物由细菌和古细菌组成。从这些物种和其他极端栖息地分离的酶对高温、有机溶剂和其他蛋白水解酶的分解更稳健。它们通常具有独特的底物特异性，并来源于新的代谢途径。嗜热菌及其稳定酶该项目将建立嗜热的体外酶级联以及两个新的底盘，即嗜热细菌嗜热栖热菌（Tth）和嗜热嗜酸古菌酸热硫化叶菌（Saci），细菌和古细菌平台，用于生产新的高附加值-极端分子是在嗜热物种的细胞中天然发现的小分子相容性溶质，其在细胞中积累以响应多个环境压力和帮助稳定细胞成分（包括蛋白质和膜）。极端分子为工业应用提供了迄今为止尚未开发的惊人潜力，包括食品，健康，消费者护理和化妆品。然而，它们在常见的嗜温生物如真菌和大肠杆菌中的生产目前受到各自代谢途径的超嗜热起源的阻碍，这些代谢途径需要嗜热细胞工厂。新设计的“细胞工厂”的开发将用于生产三种极端微生物，即环状2，3二磷酸甘油酸酯（cDPG），二-myo-1，1 '-肌醇-磷酸（DIP）和甘露糖基甘油酸（MG）。这些极端微生物（除了少数例外的MG）只在超嗜热菌中发现，迄今为止还没有在嗜温宿主中产生。极端微生物的生物合成途径已被确定，许多涉及的酶已被鉴定。在该项目中，除了这些成熟的酶外，还将通过在从（超）嗜热栖息地新分离的宏基因组菌株中寻找新的合成酶来提供新的候选酶。所有这三个extrexites来自中央糖酵解中间体的几个步骤，并在Saci中不存在，只有MG已在Tth中报道。体外嗜热酶级联和体内酶平台的建立将用于极端微生物的生产。Saci和Tth这两种微生物都易于生长（最小或复杂培养基，需氧生长）。许多其他嗜热生物需要厌氧或专门的条件才能在实验室或工业环境中成功生长。已经为Tth和Saci建立了重要的先进遗传工具，这将允许使用合成生物学方法将新模块插入细胞中。对于酶级联和菌株设计、构建、优化和产物回收，将采用基于模型的系统生物学和合成生物学方法，包括最先进的遗传学、生物化学、转录组学、蛋白质组学、建模、数据管理和生命周期评估。

Thermophilic organisms are composed of both bacterial and archaeal species. The enzymes isolated from these species and from other extreme habitats are more robust to high temperature, organic solvents and to breakdown from other proteolytic enzymes. They often have unique substrate specificities and originate from novel metabolic pathways. Thermophiles as well as their stable enzymes ('thermozymes') are receiving increased attention for biotechnological applications.The proposed project will establish thermophilic in vitro enzyme cascades as well as two new chassis, the thermophilic bacterium Thermus thermophilus (Tth) and the thermoacidophilic archaeon Sulfolobus acidocaldarius (Saci) as new thermophilic, bacterial and archaeal platforms for the production of novel high added-value products called 'extremolytes'.Extremolytes are small molecule compatible solutes found naturally in the cells of thermophilic species that accumulate in the cell in response to multiple environmental stresses and help to stabilize cellular components (including proteins and membranes). Extremolytes offer an amazing so far unexploited potential for industrial applications including food, health, consumer care and cosmetics. However, their production in common mesophilic organisms such as fungi and Escherichia coli is currently hampered by the hyperthermophilic origin of the respective metabolic pathways which require a thermophilic cell factory.The development of the newly designed 'cell factories' will be used for the production of three extremolytes, cyclic 2,3 di-phosphoglycerate (cDPG), di-myo-1,1'-inositol-phosphate (DIP) and mannosylglycerate (MG). These extremolytes (with few exceptions for MG) are exclusively found in hyperthermophiles, and have not been produced in a mesophilic host to date. The extremolyte biosynthetic pathways have been identified and many of the enzymes involved have been characterized. Within the project in addition to these well established enzymes, new candidates will be provided by searching for new synthetic enzymes in metagenome newly isolated strains from (hyper)thermophilic habitats. All three extremolytes are derived in a few steps from central glycolytic intermediates and are absent in Saci and only MG has been reported in Tth. The establishment of thermophilic in vitro enzyme cascades as well as in vivo enzyme platforms will be used for extremolyte production. Both organisms, Saci and Tth are easy to grow (minimal or complex media, aerobic growth). Many other thermophilic organisms require anaerobic or specialised conditions to achieve successful growth in the laboratory or in an industrial setting. Importantly advanced genetic tools have been established for both Tth and Saci that will allow the insertion of new modules into the cells using a synthetic biology approach. For enzyme cascade and strain design, construction, optimization and product recovery a model-based systems biology and synthetic biology approach will be employed including state of the art genetics, biochemistry, transcriptomics, proteomics, modelling, data management and life cycle assessment.