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

合成共培养中的细胞间相互作用：利用阳光和 CO2 共培养细长聚球藻和恶臭假单胞菌生产 PHA

• 批准号: 427887573
• 负责人: Professor Dr.-Ing. Andreas Kremling
• 金额: --
• 依托单位: Professur für Systembiotechnologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/427887573?language=en
• 关键词: Cell; cell; interaction; synthetic; co

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.

合理设计的合成微生物联合体具有巨大的生物技术应用潜力，因为劳动分工使它们能够执行其他方式无法执行的任务。然而，在生物过程中应用这样的聚生体需要对所涉及的微生物进行严格和单独的控制。全面控制的先决条件是彻底了解每种菌株中发生的单个事件及其相互作用。本项目中研究的人工合成聚生体是为异养细菌恶臭假单胞菌提供原料的蓝藻菌群。作为原料的蓝藻碳水化合物与恶臭假单胞菌（P. putida）的生物合成潜力（合成生物学中的新兴主力）的组合开启了广泛的应用。更详细地说，由细长聚球藻PCC 7942 cscB和恶臭假单胞菌组成的合成聚生体利用光和CO2产生聚羟基链烷酸酯（PHA）。在该共培养中使用的细长体cscB被改造为当暴露于升高的盐浓度时分泌蔗糖。这种糖然后反过来作为恶臭假单胞菌的碳源并转化为PHA。上一个融资期的起点是这种一锅法的过程，S。细长体cscB分泌足够的蔗糖以支持恶臭假单胞菌的生长和PHA积累，最大滴度为~150 mg PHA L-1。在第一个资助期内，通过采用恶臭假单胞菌cscRABY（一种对蔗糖具有较高代谢活性的菌株）改进了该工艺。接下来，通过提供不同的氮源来解决共培养伴侣的个体可控性，每种氮源仅可用于一种菌株。由此，可以单独调节共培养伙伴的生长速率，并且可以设定限定的条件。总的来说，工艺的简化导致PHA的最大滴度增加到393 mg L-1，PHA的生产速率增加到42.1 mg L-1·d-1。在本项目的更新建议中，我们寻求在不同水平上探索人工合成聚生体的潜力。在工艺水平上，我们计划采用高细胞密度培养和更适合的S。elongatus UTEX 2973提高PHA产量。在细胞水平上，我们将分析组学技术鉴定的调控事件，研究细菌表面结构对细胞-细胞相互作用的影响，评估靶向污染物共培养的稳定性，并建立种群模型。在与Kohlheyer小组的合作中，我们将在单细胞水平上进行共培养。我们的目标是开发和分析两个群体的化学计量模型，这将导致物种间的核心模型，给出了第一张代谢通量的图片，并为进一步的工艺改进提供了基础。