Filamentous co-cultures: from screening to bioprocess development (Co-Pilot)

纤维素水解丝状真菌和丝状土壤细菌在纤维素基质上的共同培养为发现和生产天然产物提供了一条有前途的途径。由于其复杂性，丝状共培养很少被研究和表征。在拟议的项目中，纤维素降解木霉里氏木霉与各种链霉菌菌株合作，系统地解锁后者属新型天然产物的生产。这项工作是基于我们对T. reesei和色链霉菌模型共培养的经验，众所周知，色链霉菌可以产生色素和抗生素。对于这种共培养，我们建立了一种基于荧光的方法来跟踪种群动态和非侵入性方法来分析代谢和色素形成。为了筛选使用非标记链霉菌菌株的许多不同共培养的种群动态，将建立基于自体荧光分析与荧光标记T. reesei相结合的方法。通过对不同链霉菌菌株单株和共培养的meta分析，可以建立单株性能指标（如生长率）与共培养行为之间的相关性。因此，我们特别期待不同形态的性能差异。为了系统地分析形态的影响，我们将模型细菌S. colelicolor分散生长敲除菌株的共培养与我们建立的S. colelicolor颗粒生长野生菌株的共培养进行比较。因此，将监测种群稳定性、产物形成、形态和流变性，并将其与链霉菌筛选的不同形态表现结果相关联。这将有助于估计链霉菌共培养行为可以推广和预测的程度，这将有助于未来设计丝状共培养物。随后，将对筛选过程中发现的最有希望的共培养进行从微滴板到搅拌发酵罐的系统放大。由于形态的复杂性，共培养的形态和性能被认为对流体力学的变化很敏感。因此，在仔细研究形态和流变学的基础上，通过评估不同的叶轮，根据最大局部和平均体积功率输入来比较放大。所有已经应用于小尺度的测量技术都将用于研究丝状共培养物的生物合成性能如何在不同尺度上发生变化。有了这些信息，我们将能够引导共同培养伙伴之间的互动，并使基于形态的直接规模扩大到中试规模。

The co-cultivation of cellulolytic filamentous fungi and filamentous soilbacteria on cellulosic substrates represents a promising approach forthe discovery and production of natural products. Due to thecomplexity, filamentous co-cultures are poorly studied andcharacterized. In the proposed project, the cellulose degraderTrichoderma reesei is partnered with various Streptomyces strains tosystematically unlock the production of novel natural products in thelatter genus. The work is based on our experience with a model cocultureof T. reesei and Streptomyces coelicolor, which is well knownto produce pigments and antibiotics. For this co-culture, weestablished a fluorescence-based method to follow populationdynamics and non-invasive methods for analysis of metabolism andpigment formation. To enable the screening of population dynamics ofmany different co-cultures using non-tagged Streptomyces strains, amethod based on autofluorescence profiling in combination withfluorescently-tagged T. reesei will be established. By meta-analysis ofaxenic and co-cultures of different Streptomyces strains, correlationsbetween the axenic performance indicators (e.g. growth rate) and coculturebehavior can be established. Thereby, we especially expectgreat performance differences depending on morphology. Tosystematically analyze the influence of morphology, co-cultures of adispersed growing knockout strain of our model bacterium S.coelicolor will be compared to our established co-culture of the pelletgrowing wildtype strain of S. coelicolor. Thereby, population stability,product formation, morphology, and rheology will be monitored andcorrelated to the different morphology:performance results of theStreptomyces screening. This will allow to estimate to what extentStreptomyces co-culture behavior can be generalized and predicted,which will aid in future design of filamentous co-cultures.Subsequently, a systematic up-scaling from microtiter plate to stirredfermenter will be performed for the most promising co-cultivationsdiscovered during the screening. Due to the morphologicalcomplexity, the co-culture morphology and performance is assumed to be sensitive towards changes in fluid dynamics. Therefore, scaleupwill be compared based on maximum local and average volumetricpower input by assessment of different impellers under closeinvestigation of morphology and rheology. All measurementtechniques already applied in small scale will be used to study howthe biosynthetic performance of filamentous co-cultures changeacross scales. With this information, we will be able to steer theinteractions of co-culture partners and enable a straightforwardmorphology-based scale-up to the pilot scale.