PDF(Pubmed)
Abstract：
BACKGROUND: Members of the fungal kingdom are heterotrophic eukaryotes encased in a chitin containing cell wall. This polymer is vital for cell wall stiffness and, ultimately, cell shape. Most fungal genomes contain numerous putative chitin synthase encoding genes. However, systematic functional analysis of the full chitin synthase catalogue in a given species is rare. This greatly limits fundamental understanding and potential applications of manipulating chitin synthesis across the fungal kingdom.
RESULTS: In this study, we conducted in silico profiling and subsequently deleted all predicted chitin synthase encoding genes in the multipurpose cell factory Aspergillus niger. Phylogenetic analysis suggested nine chitin synthases evolved as three distinct groups. Transcript profiling and co-expression network construction revealed remarkably independent expression, strongly supporting specific role(s) for the respective chitin synthases. Deletion mutants confirmed all genes were dispensable for germination, yet impacted colony spore titres, chitin content at hyphal septa, and internal architecture of submerged fungal pellets. We were also able to assign specific roles to individual chitin synthases, including those impacting colony radial growth rates (ChsE, ChsF), lateral cell wall chitin content (CsmA), chemical genetic interactions with a secreted antifungal protein (CsmA, CsmB, ChsE, ChsF), resistance to therapeutics (ChsE), and those that modulated pellet diameter in liquid culture (ChsA, ChsB). From an applied perspective, we show chsF deletion increases total protein in culture supernatant over threefold compared to the control strain, indicating engineering filamentous fungal chitin content is a high priority yet underexplored strategy for strain optimization.
CONCLUSIONS: This study has conducted extensive analysis for the full chitin synthase encoding gene repertoire of A. niger. For the first time we reveal both redundant and non-redundant functional roles of chitin synthases in this fungus. Our data shed light on the complex, multifaceted, and dynamic role of chitin in fungal growth, morphology, survival, and secretion, thus improving fundamental understanding and opening new avenues for biotechnological applications in fungi.
RESULTS: In this study, we conducted in silico profiling and subsequently deleted all predicted chitin synthase encoding genes in the multipurpose cell factory Aspergillus niger. Phylogenetic analysis suggested nine chitin synthases evolved as three distinct groups. Transcript profiling and co-expression network construction revealed remarkably independent expression, strongly supporting specific role(s) for the respective chitin synthases. Deletion mutants confirmed all genes were dispensable for germination, yet impacted colony spore titres, chitin content at hyphal septa, and internal architecture of submerged fungal pellets. We were also able to assign specific roles to individual chitin synthases, including those impacting colony radial growth rates (ChsE, ChsF), lateral cell wall chitin content (CsmA), chemical genetic interactions with a secreted antifungal protein (CsmA, CsmB, ChsE, ChsF), resistance to therapeutics (ChsE), and those that modulated pellet diameter in liquid culture (ChsA, ChsB). From an applied perspective, we show chsF deletion increases total protein in culture supernatant over threefold compared to the control strain, indicating engineering filamentous fungal chitin content is a high priority yet underexplored strategy for strain optimization.
CONCLUSIONS: This study has conducted extensive analysis for the full chitin synthase encoding gene repertoire of A. niger. For the first time we reveal both redundant and non-redundant functional roles of chitin synthases in this fungus. Our data shed light on the complex, multifaceted, and dynamic role of chitin in fungal growth, morphology, survival, and secretion, thus improving fundamental understanding and opening new avenues for biotechnological applications in fungi.
摘要：
背景：真菌界的成员是包裹在含几丁质的细胞壁中的异养真核生物。这种聚合物对细胞壁的刚度至关重要，最终,细胞形状。大多数真菌基因组包含许多推定的几丁质合酶编码基因。然而,对给定物种中的完整几丁质合酶目录进行系统功能分析是罕见的。这极大地限制了操纵整个真菌界的几丁质合成的基本理解和潜在应用。
结果：在这项研究中，我们进行了硅谱分析，随后删除了多功能细胞工厂黑曲霉中所有预测的几丁质合酶编码基因。系统发育分析表明，九种几丁质合酶进化为三个不同的群体。转录谱和共表达网络构建显示出明显的独立表达，强烈支持各自的几丁质合酶的特定作用。缺失突变体证实所有基因对发芽都是可有可无的，但影响了菌落孢子滴度，菌丝间隔的几丁质含量，和浸没的真菌颗粒的内部结构。我们还能够为单个几丁质合酶分配特定的角色，包括影响菌落径向生长速率的那些(ChsE，ChsF),侧细胞壁几丁质含量（CsMA），与分泌的抗真菌蛋白(CsMA，CsmB,ChsE,ChsF),对疗法的抗性（ChsE），以及那些在液体培养中调节颗粒直径的物质(ChsA，ChsB).从应用的角度来看，我们显示，与对照菌株相比，chsF缺失使培养上清液中的总蛋白增加了三倍以上，表明工程丝状真菌几丁质含量是菌株优化的高度优先但未充分开发的策略。
结论：这项研究对黑曲霉的全部几丁质合酶编码基因库进行了广泛的分析。我们首次揭示了几丁质合酶在这种真菌中的冗余和非冗余功能作用。我们的数据揭示了这个建筑群，多方面,以及几丁质在真菌生长中的动态作用，形态学,生存,和分泌，从而提高对真菌生物技术应用的基本认识并开辟新的途径。
结果：在这项研究中，我们进行了硅谱分析，随后删除了多功能细胞工厂黑曲霉中所有预测的几丁质合酶编码基因。系统发育分析表明，九种几丁质合酶进化为三个不同的群体。转录谱和共表达网络构建显示出明显的独立表达，强烈支持各自的几丁质合酶的特定作用。缺失突变体证实所有基因对发芽都是可有可无的，但影响了菌落孢子滴度，菌丝间隔的几丁质含量，和浸没的真菌颗粒的内部结构。我们还能够为单个几丁质合酶分配特定的角色，包括影响菌落径向生长速率的那些(ChsE，ChsF),侧细胞壁几丁质含量（CsMA），与分泌的抗真菌蛋白(CsMA，CsmB,ChsE,ChsF),对疗法的抗性（ChsE），以及那些在液体培养中调节颗粒直径的物质(ChsA，ChsB).从应用的角度来看，我们显示，与对照菌株相比，chsF缺失使培养上清液中的总蛋白增加了三倍以上，表明工程丝状真菌几丁质含量是菌株优化的高度优先但未充分开发的策略。
结论：这项研究对黑曲霉的全部几丁质合酶编码基因库进行了广泛的分析。我们首次揭示了几丁质合酶在这种真菌中的冗余和非冗余功能作用。我们的数据揭示了这个建筑群，多方面,以及几丁质在真菌生长中的动态作用，形态学,生存,和分泌，从而提高对真菌生物技术应用的基本认识并开辟新的途径。
