Communities of niche-optimized strains (CoNoS) – a novel concept for improving biotechnological production of small molecules

大多数小分子的生物技术生产过程是基于单一的、工程生产菌株的纯培养。这些菌株通常具有中心代谢蛋白的过剩能力，这是应对不断变化的环境条件的自然机制。在生物反应器的相对明确的环境中，这些未使用的过剩产能代表生产所需产品所损失的碳和能量。为了使这些过剩能力用于小分子的生产，我们计划设计生态位优化菌株（CoNoS）的合成群落。一个CoNoS至少由同一物种的两个菌株组成，每个菌株对一种或多种氨基酸缺乏营养。这些菌株应该相互交叉喂养，从而通过更有效地利用自然可用的生物合成能力来节省碳和能源。相似的群落中，营养不良基因组减少的细菌相互交叉喂养，在自然界中经常进化，表明与技术上使用的原生营养纯培养物相比，一定有显著的适应性优势。我们项目的总体目标是产生比目前最佳生产菌株的纯培养更有效地产生选定氨基酸的cono。为此，我们将从最近构建的基因组减少的谷氨酸棒状杆菌基底菌株C1*开始，产生在选定的氨基酸生物合成途径中携带广泛基因缺失的谷氨酸棒状杆菌新菌株。这些菌株的性能将在纯培养物和相互交叉饲养群落中进行定量分析。为了优化群落的交叉饲养和生长性能，将使用适应性实验室进化（ALE），然后对这些实验中富集的突变进行详细的分子分析。因此，我们将更好地了解菌株在这种环境下如何相互作用。此外，关于合理工程进一步目标的现有信息将用于构建第二代CoNoS菌株，然后进入进料批工艺开发。菌株将用特定的基因组标记进行标记，这将允许在整个生产过程开发中对合成混合培养物进行定量表征。后者还将通过应用最先进的13C/15N代谢通量分析，对生产条件下的CoNoS动力学进行基于模型的分析。我们的项目需要分子生物学家和生物过程工程师的紧密结合，这是Interzell项目的理想代表。

The majority of biotechnological production processes for small molecules is based on pure cultures of single, engineered production strains. These strains often possess overcapacities of central, metabolic proteins, which is a natural mechanism to cope with changing environmental conditions. In the comparably well-defined environment of a bioreactor, these unused overcapacities represent lost carbon and energy for production of the desired product. To make these overcapacities available for the production of small molecules, we plan to design synthetic communities of niche-optimized strains (CoNoS). A CoNoS consists of at least two strains of the same species, each auxotrophic for one or more amino acid. The strains are supposed to cross-feed each other and thereby saving carbon and energy by using the naturally available biosynthesis capacity for one amino acid more efficiently. Similar communities, where auxotrophic genome reduced bacteria cross-feed each other, have frequently evolved in nature, demonstrating that there must be a significant fitness advantage compared to the technically used prototrophic pure cultures. The overall aim of our project is to generate CoNoS that produce selected amino acids more efficiently than pure cultures of current best producer strains. For this purpose, we will generate novel Corynebacterium glutamicum strains carrying broad gene deletions in the selected amino acid biosynthesis pathways, starting from the recently constructed genome-reduced C. glutamicum chassis strain C1*. The performance of these strains will be quantitatively analyzed both in pure cultures with external supplementation of the respective amino acid and in reciprocal cross-feeding communities. To optimize cross feeding and growth performance of the communities, adaptive laboratory evolution (ALE) will be used, followed by a detailed molecular analysis of the mutations enriched during these experiments. Thereby we will gain a better understanding how the strains interact in such a setting. Additionally, existing information on further targets for rational engineering will be utilized to construct 2nd generation CoNoS strains, which will then enter fed-batch process development. Strains will be labelled with specific genomic markers that will allow a quantitative characterization of the synthetic mixed cultures throughout the whole production process development. The latter will also be supported by model-based analyses of CoNoS dynamics under production conditions by applying state of the art 13C/15N metabolic flux analysis. Our project requires a tight integration of molecular biologists and bioprocess engineers, an approach which is ideally represented by the Interzell program.