Cell-cell interaction in a synthetic co-culture: PHA production from sunlight and CO2 co-cultivating Synechococcus elongatus and Pseudomonas putida

设计合理的合成微生物联合体具有巨大的生物技术应用潜力，因为分工使它们能够完成原本不可能完成的任务。然而，这种联合体在生物过程中的应用需要对所涉及的微生物进行严格的和个人的控制。全面控制的先决条件是彻底了解每个菌株中发生的个别事件及其相互作用。这个项目中正在研究的人工合成联合体是为异养菌恶臭假单胞菌提供原料的蓝藻的联合。将蓝藻碳水化合物作为原料与恶臭假单胞菌的生物合成潜力相结合，为合成生物学开辟了广泛的应用前景。更详细地说，由长聚球藻PCC7942 CSCB和恶臭假单胞菌组成的合成联合体从光和二氧化碳中产生聚羟基烷酸酯(PHA)。在这种共培养中使用的长聚球藻CSCB被改造成在暴露于高盐浓度时分泌蔗糖。然后，这些糖作为恶臭假单胞菌的碳源，被转化为PHA。上一个资助期的起点是这种一锅法，在这个过程中，长链球菌CSCB分泌足够的蔗糖来支持恶臭假单胞菌的生长和PHA的积累，最大效价为~150mgPHA L-1。在第一个资助期，通过使用恶臭假单胞菌cscRABY来改进这一过程，该菌株对蔗糖具有更高的代谢活性。接下来，通过提供不同的氮源来解决共培养伙伴的个体可控性问题，每个氮源只适用于一种菌株。通过这一点，可以单独调节共培养伙伴的生长速度，并可以设定明确的条件。总体而言，流程的简化使PHA的最大滴度提高到393mgL-1，PHA的产率提高到42.1mgL-1day-1。在这项更新项目的建议中，我们致力于从不同层面探索人工合成联合体的潜力。在工艺水平上，我们计划采用高细胞密度培养和更适合的长链球菌UTEX2973来提高PHA产量。在细胞水平上，我们将分析组学技术确定的调控事件，研究细菌表面结构对细胞-细胞相互作用的影响，评估靶向污染对共培养的稳定性，并建立种群模型。在与Kohlheyer小组的合作下，我们将在单细胞水平上跟踪共培养。我们的目标是开发和分析这两个种群的化学计量模型，从而产生一个物种间核心模型，给出代谢通量的第一幅图，并为进一步的工艺改进提供基础。

Rationally designed synthetic microbial consortia carry a vast potential for biotechnological applications, as the division of labor allows them to perform tasks otherwise not possible. However, the application of such a consortium in a bioprocess requires tight and individual controllability of the involved microbes. A prerequisite towards the full control is the thorough understanding of the individual events taking place in each strain and their interaction. The artificial synthetic consortium under examination in this project is the association of a cyanobacterium providing the feedstock for the heterotrophic bacterium, Pseudomonas putida. The combination of the cyanobacterial carbohydrates as feedstock together with the biosynthetic potential of P. putida, an emerging workhorse in Synthetic Biology, opens a wide range of applications. In more detail, the synthetic consortium consisting of Synechococcus elongatus PCC7942 cscB and P. putida produces polyhydroxyalkanoates (PHA) from light and CO2.The cyanobacterium S. elongatus cscB employed in this co-cultivation was engineered to excrete sucrose, when exposed to elevated salt concentrations. This sugar then in turn serves as carbon source for P. putida and is converted to PHA. The starting point of the last funding period was this one-pot process, where S. elongatus cscB secreted sufficient sucrose to support the growth and PHA accumulation of P. putida with a maximal titer of ~150 mg PHA L-1. In the first funding period, this process was improved by employing P. putida cscRABY, a strain with a higher metabolic activity towards sucrose. Next, the individual controllability of the co-culture partners was addressed by providing different nitrogen sources, each exclusively available for one strain. By this, the growth rate of the co-culture partners could be regulated individually and defined conditions could be set. In total, the streamlining of the process resulted in an increased maximal PHA titer of 393 mg L-1 and a PHA production rate of 42.1 mg L -1 day -1.In this proposal for the renewal of the project, we pursue to explore the potential of the artificial synthetic consortium on different levels. On the process level, we plan to employ high cell density cultivations and the better suited S. elongatus UTEX2973 to improve PHA yield. On the cellular level, we will analyse the regulatory events identified by omics technologies, investigate the influence of the bacterial surface structures on the cell-cell interaction, assess the stability of the co-culture by targeted contaminations, and set up a population model. In a collaboration with the Kohlheyer group, we will follow the co-cultivation on single cell level. We aim to to develop and analyze stoichiometric models for both populations that will result in a Inter species core model, giving a first picture of the metabolic fluxes and providing a basis for further process improvements.