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工艺是基于单一工程生产菌株的纯培养物。这些菌株通常具有过量的中心代谢蛋白，这是科普不断变化的环境条件的自然机制。在生物反应器的严格限定的环境中，这些未使用的过剩容量代表了用于生产所需产物的碳和能量的损失。为了使这些过剩的产能可用于生产小分子，我们计划设计生态位优化菌株（CoNoS）的合成社区。CoNoS由相同物种的至少两个菌株组成，每个菌株对一个或多个氨基酸具有营养缺陷型。这些菌株应该相互交叉喂养，从而通过更有效地利用一种氨基酸的天然生物合成能力来节省碳和能量。营养缺陷型基因组减少的细菌相互交叉喂养的类似群落在自然界中经常进化，这表明与技术上使用的原养型纯培养物相比，必须有显着的适应性优势。我们项目的总体目标是产生比目前最好的生产菌株的纯培养物更有效地生产选定氨基酸的CoNoS。为此，我们将从最近构建的基因组减少的谷氨酸棒杆菌开始，产生在所选氨基酸生物合成途径中携带广泛基因缺失的新型谷氨酸棒杆菌菌株。谷氨酸底盘菌株C1*。这些菌株的性能将在外部补充相应氨基酸的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），然后对这些实验期间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如何在这种环境中相互作用。此外，关于合理工程的进一步目标的现有信息将用于构建第二代CoNoS菌株，然后将进入补料分批工艺开发。将使用特定基因组标记物标记菌株，以便在整个生产工艺开发过程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13 C/15 N代谢通量分析，在生产条件下对CoNoS动力学进行基于模型的分析来支持。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，这种方法理想地由Interzell程序代表。

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.