A quantitative image analysis pipeline for the characterization of filamentous fungal morphologies as a tool to uncover targets for morphology engineering: a case study using aplD in Aspergillus niger
A quantitative image analysis pipeline for the characterization of filamentous fungal morphologies as a tool to uncover targets for morphology engineering: a case study using aplD in Aspergillus niger
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用于表征丝状真菌形态的定量图像分析管道，作为发现形态工程目标的工具：在黑曲霉中使用 aplD 的案例研究
DOI：
10.1186/s13068-019-1473-0
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发表时间：
2019-06-15
影响因子：
6.3
通讯作者：
Meyer, Vera
中科院分区：
文献类型：
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作者：
Cairns, Timothy C.;Feurstein, Claudia;Meyer, Vera
背景真菌发酵用于为各个行业生产各种酶、化学品和药物。在深层培养过程中，丝状真菌形成一系列宏观形态，包括分散的菌丝体、成团的聚集体或颗粒，这对发酵过程中的流变学、气体/营养物转移以及产品滴度具有重要影响。菌株工程工作的一个重要组成部分是定量评估真菌生长表型的能力，这将为形态优化的生产菌株提供新的线索。结果在这项研究中，我们开发了一种自动图像分析管道来量化颗粒和分散生长（MPD）的形态，它可以快速、可重复地测量任何水下真菌培养物的分散和颗粒宏观形态。它 (i) 能够捕获和分析每个用户/天的数百张图像，(ii) 旨在定量评估由分散和颗粒形式组成的异质培养物，(iii) 定量测量培养物异质性，(iv) 自动生成单个真菌结构的关键欧几里德参数，包括粒径、长宽比、面积和坚固性，这些参数也组装成先前描述的无量纲形态数 MN，(v) 具有内置质量控制检查功能，使最终用户能够轻松确认自动化分析的准确性调用，并且（vi）很容易适应用户指定的放大倍数和宏观形态定义。为了同时为该图像分析流程的实用性提供原理证明，并为形态优化的真菌菌株提供新的线索，我们基于 CRISPR-Cas 技术在细胞工厂黑曲霉中生成了形态突变体。首先，我们询问了之前发布的 A 共表达网络。 nigerto 发现了一个假定的 γ-适应素编码基因 (aplD)，预计该基因在内体货物运输中发挥作用。使用基因编辑在可滴定Tet-on系统的控制下生成条件alaplD表达突变体。 daplD表达减少导致超支化生长表型和沉淀形成中的多种缺陷，并推定蛋白质分泌增加。这种可能的蛋白质分泌过多表型可能与分散菌丝体的增加以及颗粒直径和 MN 的减小有关。结论 MPD 图像分析流程是一种简单、快速且灵活的方法，可用于量化不同的真菌形态。作为一个例子，我们已经证明假定的内体转运基因aplD在A中发挥着至关重要的作用。深层培养期间黑丝的生长和沉淀的形成。这表明内吞成分是真菌细胞工厂工程中尚未开发的目标。
BackgroundFungal fermentation is used to produce a diverse repertoire of enzymes, chemicals, and drugs for various industries. During submerged cultivation, filamentous fungi form a range of macromorphologies, including dispersed mycelia, clumped aggregates, or pellets, which have critical implications for rheological aspects during fermentation, gas/nutrient transfer, and, thus, product titres. An important component of strain engineering efforts is the ability to quantitatively assess fungal growth phenotypes, which will drive novel leads for morphologically optimized production strains.ResultsIn this study, we developed an automated image analysis pipeline to quantify the morphology of pelleted and dispersed growth (MPD) which rapidly and reproducibly measures dispersed and pelleted macromorphologies from any submerged fungal culture. It (i) enables capture and analysis of several hundred images per user/day, (ii) is designed to quantitatively assess heterogeneous cultures consisting of dispersed and pelleted forms, (iii) gives a quantitative measurement of culture heterogeneity, (iv) automatically generates key Euclidian parameters for individual fungal structures including particle diameter, aspect ratio, area, and solidity, which are also assembled into a previously described dimensionless morphology number MN, (v) has an in-built quality control check which enables end-users to easily confirm the accuracy of the automated calls, and (vi) is easily adaptable to user-specified magnifications and macromorphological definitions. To concomitantly provide proof of principle for the utility of this image analysis pipeline, and provide new leads for morphologically optimized fungal strains, we generated a morphological mutant in the cell factoryAspergillus nigerbased on CRISPR-Cas technology. First, we interrogated a previously published co-expression networks forA. nigerto identify a putative gamma-adaptin encoding gene (aplD) that was predicted to play a role in endosome cargo trafficking. Gene editing was used to generate a conditionalaplDexpression mutant under control of the titratable Tet-on system. ReducedaplDexpression caused a hyperbranched growth phenotype and diverse defects in pellet formation with a putative increase in protein secretion. This possible protein hypersecretion phenotype could be correlated with increased dispersed mycelia, and both decreased pellet diameter and MN.ConclusionThe MPD image analysis pipeline is a simple, rapid, and flexible approach to quantify diverse fungal morphologies. As an exemplar, we have demonstrated that the putative endosomal transport geneaplDplays a crucial role inA. nigerfilamentous growth and pellet formation during submerged culture. This suggests that endocytic components are underexplored targets for engineering fungal cell factories.