SiMBal 2.0: Quantifying co-culture performance as a function of condition-dependent inter-cell mass exchange

合成微生物共培养物执行复杂的催化任务，并受益于合作的相互作用。合理设计促进生物技术生产的有益相互作用的共培养物可以将催化模块化带入生物过程，并具有比纯培养物更有效地催化级联反应的潜力。然而，合理的代谢和过程工程对他们的有效的生物技术应用，需要定量和时空分辨的数据微生物性能。相关数据通常包括特定生长、摄取和生产速率、产率系数和作为共培养相互作用动力学的函数的化学计量。这种知识不能从平均读数中产生，基于摇瓶或生物反应器中的群体实验。SiMBal 2.0将分析具有单细胞分辨率的共培养物中的性能决定动力学和化学计量指标，以应用基本的生物化学工程原理，例如材料平衡。由于相互作用动力学和共培养化学计量学强烈受细胞外条件的影响，我们将扩展已建立的材料平衡框架，以进行条件依赖性共培养生理学分析。为此，我们将利用灌注培养，微流体批次在室和液滴，结合延时显微镜，质谱成像和质谱底物和产品分析。这些数据将用于物料平衡，并为分离的动态共培养模型提供饲料，以揭示调整机会。基于单细胞数据的动态建模将用于预测生长、生产率和物种比方面的共培养性能，并考虑表型异质性。然后，我们将通过基于种群的共培养实验来验证所获得的模型的有效性。我们不仅将推进我们的技术概念，而且还将朝着真共生共培养的分析方向发展。作为合成共培养物的模型，我们将研究谷氨酸棒杆菌菌株之间的细胞-细胞相互作用，每种菌株对一种必需氨基酸具有营养缺陷型，但能够相互补充这些营养缺陷型。我们将专注于相关参数，如文化的pH值，底物的可用性，氨基酸，空间文化的安排，和最初的播种比例作为共培养生理的主要决定因素。我们将推进对共培养相互作用的理解，以实现合理的菌株和工艺开发。我们的跨学科项目涵盖了生物化学工程，微流体单细胞分析和培养建模等学科。SiMBal 2.0的首要目标概括为合成微生物共培养物的细胞间相互作用的量化。所获得的这些知识对于未来基于共培养的生物技术是非常宝贵的。

Synthetic microbial co-cultures perform complex catalytic tasks and benefit from cooperative interactions. The rational design of co-cultures facilitating beneficial interactions for biotechnological production could bring catalytic modularity into bioprocesses and hold the potential to catalyse a cascade of reactions more efficiently than axenic cultures. Rational metabolic and process engineering towards their efficient biotechnological application, however, requires quantitative and spatiotemporally resolved data on microbial performance. Relevant data typically includes specific growth, uptake and production rates, yield coefficients, and stoichiometry as a function of the co-culture interaction kinetics. Such knowledge cannot be generated from averaged readouts, based on population experiments in shake flasks or bioreactors. SiMBal 2.0 will analyse the performance-determining kinetic and stoichiometric indicators in co-cultures with single-cell resolution to apply fundamental biochemical engineering principles, such as material balancing. As interactions kinetics and co-culture stoichiometry are strongly governed by extracellular conditions, we will extend the established material-balancing framework towards condition-dependent co-culture physiology analyses. To this end, we will exploit perfusion cultivations, microfluidic batches in chambers and droplets, combined with time-lapse microscopy, mass imaging, and mass spectrometry for substrate and product analysis. The data will be used for material balancing and feed a segregated dynamic co-culture model for unravelling tuning opportunities. Dynamic modelling based on single-cell data will be used to predict the co-culture performance in terms of growth, productivity, and species ratios and also take phenotypic heterogeneity into account. The obtained model will then be tested for validity with population-based co-culture experiments.We will not only advance our technological concepts but also progress towards the analyses of eusymbiotic co-cultures. As a model for synthetic co-cultures, we will study cell-cell interactions between Corynebacterium glutamicum strains each auxotrophic for one essential amino acid, but capable of mutually complementing these auxotrophies. We will focus on relevant parameters such culture pH, substrate availability, amino acids, spatial culture arrangement, and the initial seeding ratios as the main determinants for co-culture physiology. We will push forward the understanding of co-culture interactions for rational strain and process development. Our interdisciplinary project spans the disciplines of biochemical engineering, microfluidic single-cell analysis, and culture modelling. The overarching goal of SiMBal 2.0 summarizes as the quantification of intercellular interactions of synthetic microbial co-cultures. This knowledge obtained is invaluable for future co-culture-based biotechnology.