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

嗜热生物由细菌和古生菌组成。从这些物种和其他极端栖息地分离出来的酶对高温、有机溶剂和从其他蛋白水解酶中分解的能力更强。它们通常具有独特的底物特异性，并起源于新的代谢途径。嗜热菌及其稳定的酶(热酶)在生物技术应用中正受到越来越多的关注。拟议的项目将建立体外嗜热酶级联，以及两个新的底盘，嗜热细菌Thermus thermophilus(TTH)和嗜热嗜酸考古菌Sulfolobus acidocaldarius(SACI)作为新的嗜热、细菌和古生物平台，用于生产称为极端分子的新型高附加值产品。极端分子是在嗜热物种的细胞中自然积累的小分子兼容溶质，可响应多种环境压力而积累在细胞中，有助于稳定细胞成分(包括蛋白质和膜)。在食品、保健、消费者护理和化妆品等工业应用方面，超钼矿提供了令人惊叹的迄今尚未开发的潜力。然而，在真菌和大肠杆菌等常见的中温生物中，它们的生产目前受到各自代谢途径的高温起源的阻碍，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分子，环状2，3-二磷酸甘油酸酯(CDPG)、二肌醇-1，1‘-磷酸肌醇(DIP)和甘露糖甘油酸酯(MG)。这些极端分子(MG除外)只在超嗜热菌中发现，到目前为止还没有在中温宿主中产生。极端分子的生物合成途径已经被确定，涉及的许多酶已经被表征。在该项目中，除了这些公认的酶之外，还将通过在(超)嗜热生境中新分离的超基因组菌株中寻找新的合成酶来提供新的候选酶。所有这三个极端分子都是从中心糖酵解中间体几个步骤中衍生出来的，在SACI中不存在，在TTH中只有MG被报道。高温体外酶的建立以及体内酶平台的建立将被用于极端分子的生产。这两种生物，SACI和TTH都很容易生长(最少或复杂的培养基，好氧生长)。许多其他嗜热生物需要厌氧或特殊条件才能在实验室或工业环境中成功生长。重要的是，已经为TTH和SACI建立了先进的遗传工具，将允许使用合成生物学方法将新模块插入细胞中。对于酶的级联和菌株的设计、构建、优化和产品回收，将采用基于模型的系统生物学和合成生物学方法，包括最新的遗传学、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.