Unravelling molecular mechanisms of fungal hypersecretion

这项提议的目的是阐明真菌高分泌蛋白质的分子机制。丝状真菌因其健壮和分泌蛋白质的能力而被用于商业生产酶。工业酵素市场规模达数十亿美元。遗传和基因组分析表明，丝状真菌拥有酵母中发现的分泌机制的所有关键组件。然而，丝状真菌比酵母菌更倾向于分泌蛋白质的机制在很大程度上仍不清楚。我建议使用正向和反向遗传学与全基因组测序相结合的方法来确定黑曲霉高分泌的分子基础。除了是最常用的生产胞外酶和有机酸的细胞工厂外，黑曲霉还是第一个也是唯一一个由人类策展人全面管理和注释的真菌基因组。解释高分泌的机制如下所示。1)产生和鉴定高分泌突变。随机突变仍然是菌株改良的有力工具，因为它很容易引起功能丧失和功能获得突变。为了确定哪些突变更有利于蛋白质高分泌表型，我们将使用自上而下和自下而上的方法。具体地说，我们将随机诱变黑曲霉，并筛选总蛋白高分泌。突变将通过基因组测序来识别。虽然随机突变可以影响新陈代谢的任何部分，但我们将重点关注与菌丝形态、分泌途径和调节功能有关的突变。2)确定先前发现的突变在高分泌中的作用。以前对丝状真菌高分泌突变菌株的表征已经发现了许多突变。被预测参与调节功能、菌丝形态和分泌机制的基因是高分泌表型的候选基因。然而，这些突变中很少有实验证实与高分泌有关。这些基因在黑曲霉中的同源基因将被删除(功能丧失)或替换(功能获得)，使用基因组编辑方法CRISPR/Cas9，将对每个突变体进行表型测试。3)高分泌突变组合效应的表征和突变的验证。高分泌表型被认为是多个突变共同作用的结果。从方法1和方法2中确定的突变将被汇集并转化到黑曲霉中，从而产生一个突变体基因组文库，这将有助于识别突变的组合效应。了解丝状真菌中的分泌机制对于设计真菌宿主菌株以降低工业和环境应用中的酶生产成本至关重要。

The goal of this proposal is to elucidate the molecular mechanism that underlie fungal hypersecretion of proteins. Filamentous fungi are used for commercial production of enzymes due to their robustness and capacity to secrete proteins. The industrial enzymes market ranges in billions USD. Genetic and genomic analysis revealed that filamentous fungi possess all the key components of the secretion machinery found in yeast. However, the mechanisms that confer filamentous fungi with a higher propensity to secrete proteins than yeast remain largely unknown. I propose to use forward and reverse genetics coupled with whole-genome sequencing to identify the molecular underpinnings of hypersecretion in Aspergillus niger. In addition to being the most commonly used cell factory for the production of extracellular enzymes and organic acids, A. niger is the first and only fungal genome, fully curated and annotated by human curators. Elucidation of the mechanisms responsible for hypersecretion will be done as following. 1) Generating and identifying hypersecretion mutations. Random mutagenesis remains a powerful tool for strain improvement as it can readily elicit both loss-of-function and gain-of-function mutations. To identity which mutations favor a protein hypersecretion phenotype we will use a top-down and a bottom-up approach. Specifically, we will randomly mutagenize A. niger and screen for total protein hypersecretion. Mutations will be identified by genome sequencing. Although random mutagenesis can affect any part of the metabolism, we will focus on mutations predicted to be involved in hyphal morphology, the secretory pathway and regulatory functions. 2) Establishing the role of previously identified mutations in hypersecretion. Previous characterization of hypersecretion mutant strains of filamentous fungi has uncovered numerous mutations. Genes predicted to be involved in regulatory functions, hyphal morphology and secretion machinery are candidates for a hypersecretion phenotype. However, very few of these mutations have experimental confirmation of being involved in hypersecretion. Orthologues of these genes in A. niger will be deleted (loss of function) or replaced (gain of function) using the genome editing method CRISPR/Cas9, each mutant will be tested phenotypically. 3) Characterization of the combinatorial effect of hypersecretion mutations and validation of the mutations. The hypersecretion phenotype is expected to be the result of a combined action of multiple mutations. The mutations identified from approach 1 and 2 will be pooled and transformed into A. niger resulting in a genomic library of mutants that will help identifying the combinatorial effect of the mutations. Understanding the secretion machinery in filamentous fungi is crucial to engineer fungal host strains to reduce enzyme production cost for industrial and environmental applications.