Unravelling molecular mechanisms of fungal hypersecretion

本建议的目的是阐明真菌高分泌蛋白质的分子机制。丝状真菌由于其健壮性和分泌蛋白质的能力而被用于酶的商业生产。工业酶市场规模达数十亿美元。遗传和基因组分析显示，丝状真菌拥有酵母中发现的所有分泌机制的关键成分。然而，赋予丝状真菌比酵母更倾向于分泌蛋白质的机制在很大程度上仍然未知。我建议使用正向和反向遗传学结合全基因组测序来确定黑曲霉高分泌的分子基础。除了是生产胞外酶和有机酸最常用的细胞工厂外，黑曲霉是第一个也是唯一一个由人类管理员完全管理和注释的真菌基因组。下面将阐述导致分泌过多的机制。1)产生和识别高分泌突变。随机诱变仍然是菌株改良的有力工具，因为它可以很容易地引起功能丧失和功能获得突变。为了确定哪些突变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。具体来说，我们将随机诱变黑孢杆菌并筛选总蛋白分泌过多。突变将通过基因组测序来确定。虽然随机突变可以影响代谢的任何部分，但我们将重点关注预测涉及菌丝形态，分泌途径和调节功能的突变。2)确定先前确定的突变在高分泌中的作用。先前对丝状真菌的高分泌突变菌株的表征已经发现了许多突变。预测参与调控功能、菌丝形态和分泌机制的基因是高分泌表型的候选者。然而，这些突变中很少有实验证实与高分泌有关。使用基因组编辑方法CRISPR/Cas9，将黑曲霉中这些基因的同源物删除（功能丧失）或替换（功能获得），每个突变体将进行表型测试。3)高分泌突变组合效应的表征及突变的验证。高分泌表型预计是多种突变共同作用的结果。从方法1和方法2中鉴定出的突变将被汇总并转化为黑曲霉，从而形成一个突变基因组文库，这将有助于鉴定突变的组合效应。了解丝状真菌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.