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Abstract：
UNASSIGNED: Fungal 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.
UNASSIGNED: In 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 factory Aspergillus niger based on CRISPR-Cas technology. First, we interrogated a previously published co-expression networks for A. niger to 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 conditional aplD expression mutant under control of the titratable Tet-on system. Reduced aplD expression 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.
UNASSIGNED: The 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 gene aplD plays a crucial role in A. niger filamentous growth and pellet formation during submerged culture. This suggests that endocytic components are underexplored targets for engineering fungal cell factories.
UNASSIGNED: In 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 factory Aspergillus niger based on CRISPR-Cas technology. First, we interrogated a previously published co-expression networks for A. niger to 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 conditional aplD expression mutant under control of the titratable Tet-on system. Reduced aplD expression 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.
UNASSIGNED: The 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 gene aplD plays a crucial role in A. niger filamentous growth and pellet formation during submerged culture. This suggests that endocytic components are underexplored targets for engineering fungal cell factories.
摘要：
■真菌发酵用于产生多种酶，化学品,和各种行业的药物。在水下种植期间，丝状真菌形成一系列宏观形态，包括分散的菌丝体，结块的骨料，或颗粒，这对发酵过程中的流变方面有重要影响，气体/营养转移，and,因此，产品滴度。菌株工程努力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带来新的线索。
■在这项研究中，我们开发了一个自动图像分析管道，以量化颗粒状和分散生长（MPD）的形态，快速，可重复地测量任何浸没真菌培养物的分散和颗粒状宏观形态。它（i）可以捕获和分析每个用户/天的数百个图像，（ii）旨在定量评估由分散和颗粒状形式组成的异质培养物，(iii)给出了文化异质性的定量测量，（iv）自动生成单个真菌结构的关键欧几里得参数，包括颗粒直径，纵横比,area,和坚固，它们也被组装成先前描述的无量纲形态数MN，（v）具有内置的质量控制检查，使最终用户能够轻松确认自动呼叫的准确性，和(vi)容易适应用户指定的放大倍数和宏观形态定义。同时为该图像分析管道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技术在黑曲霉细胞工厂中产生了形态突变体。首先,我们询问了先前发表的A.niger共表达网络,以鉴定一种推定的γ-适应素编码基因(aplD),该基因被预测在内体货物运输中起作用.基因编辑用于在可滴定的Tet-on系统的控制下产生条件aplD表达突变体。apro表达降低导致超支化生长表型和颗粒形成的多种缺陷，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有关，颗粒直径和MN均减小。
■MPD图像分析管道是一个简单的，快速,和灵活的方法来量化不同的真菌形态。作为一个例子，我们已经证明，推定的内体转运基因aplD在水下培养过程中在黑曲霉丝状生长和颗粒形成中起着至关重要的作用。这表明胞吞成分是工程化真菌细胞工厂的未充分开发的靶标。
■在这项研究中，我们开发了一个自动图像分析管道，以量化颗粒状和分散生长（MPD）的形态，快速，可重复地测量任何浸没真菌培养物的分散和颗粒状宏观形态。它（i）可以捕获和分析每个用户/天的数百个图像，（ii）旨在定量评估由分散和颗粒状形式组成的异质培养物，(iii)给出了文化异质性的定量测量，（iv）自动生成单个真菌结构的关键欧几里得参数，包括颗粒直径，纵横比,area,和坚固，它们也被组装成先前描述的无量纲形态数MN，（v）具有内置的质量控制检查，使最终用户能够轻松确认自动呼叫的准确性，和(vi)容易适应用户指定的放大倍数和宏观形态定义。同时为该图像分析管道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技术在黑曲霉细胞工厂中产生了形态突变体。首先,我们询问了先前发表的A.niger共表达网络,以鉴定一种推定的γ-适应素编码基因(aplD),该基因被预测在内体货物运输中起作用.基因编辑用于在可滴定的Tet-on系统的控制下产生条件aplD表达突变体。apro表达降低导致超支化生长表型和颗粒形成的多种缺陷，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有关，颗粒直径和MN均减小。
■MPD图像分析管道是一个简单的，快速,和灵活的方法来量化不同的真菌形态。作为一个例子，我们已经证明，推定的内体转运基因aplD在水下培养过程中在黑曲霉丝状生长和颗粒形成中起着至关重要的作用。这表明胞吞成分是工程化真菌细胞工厂的未充分开发的靶标。
