Involvement of the adaptor protein 3 complex in lignocellulase secretion in Neurospora crassa revealed by comparative genomic screening.
Involvement of the adaptor protein 3 complex in lignocellulase secretion in Neurospora crassa revealed by comparative genomic screening.
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DOI：
10.1186/s13068-015-0302-3
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发表时间：
2015
影响因子：
6.3
通讯作者：
Tian C
中科院分区：
文献类型：
--
作者：
Pei X;Fan F;Lin L;Chen Y;Sun W;Zhang S;Tian C
木质纤维素酶的过度分泌已经在里氏木霉等工业真菌中实现，但与这一过程相关的潜在机制尚不清楚。尽管之前的比较基因组研究表明，与亲本菌株 QM6a 相比，诱变里氏木霉菌株 RUT-C30 存在数百个突变，但这些突变实际上如何导致分泌过多表型仍有待阐明。在这项研究中，我们系统地筛选了纤维素分解真菌粗糙脉孢菌中的基因敲除（KO）突变体，其中含有潜在缺陷的里氏木霉RUT-C30突变基因的直系同源物。在粗糙链孢霉中筛选的 86 个缺失突变体中，12 个的木质纤维素酶产量比野生型 (WT) 菌株高出 25% 以上，4 个则显示出分泌量降低近 25%。我们观察到，Ncap3m (NCU03998)（编码粗糙脉孢菌中接头蛋白 3 (AP-3) 复合物的 μ 亚基）的缺失，导致在 Avicel 和木聚糖培养条件下木质纤维素酶分泌最显着增加。此外，缺乏 Ncap3b (NCU06569) 编码的 AP-3 复合物 β 亚基的菌株具有与 ΔNcap3m 相似的表型，表明 AP-3 复合物参与粗糙脉孢菌中的木质纤维素酶分泌。我们还发现，与WT相比，ΔNcap3m中主要木质纤维素酶基因的转录丰度在发酵后期保持在相对较高的水平，这可能会增加分泌过多的表型。最后，我们发现将里氏木霉 ap3m 直系同源物 Trap3m 导入 ΔNcap3m 可以在遗传上将木质纤维素酶的分泌恢复到正常水平，这表明 AP-3 复合物对木质纤维素酶分泌的影响在纤维素分解子囊菌中是保守的。使用模型纤维素分解真菌 N. crassa，我们探索了里氏木霉菌株 RUT-C30 中潜在的与分泌过多相关的突变。通过对粗糙脉孢菌中 86 个相应的直系同源 KO 突变体进行系统遗传筛选，我们鉴定了几个基因，特别是那些编码有助于木质纤维素酶分泌的 AP-3 复合物的基因。这些发现将有助于未来木质纤维素酶和生物质化学品生产中的菌株改良。本文的在线版本 (doi:10.1186/s13068-015-0302-3) 包含补充材料，可供授权用户使用。
Lignocellulase hypersecretion has been achieved in industrial fungal workhorses such as Trichoderma reesei, but the underlying mechanism associated with this process is not well understood. Although previous comparative genomic studies have revealed that the mutagenic T. reesei strain RUT-C30 harbors hundreds of mutations compared with its parental strain QM6a, how these mutations actually contribute to the hypersecretion phenotype remains to be elucidated. In this study, we systematically screened gene knockout (KO) mutants in the cellulolytic fungus Neurospora crassa, which contains orthologs of potentially defective T. reesei RUT-C30 mutated genes. Of the 86 deletion mutants screened in N. crassa, 12 exhibited lignocellulase production more than 25% higher than in the wild-type (WT) strain and 4 showed nearly 25% lower secretion. We observed that the deletion of Ncap3m (NCU03998), which encodes the μ subunit of the adaptor protein 3 (AP-3) complex in N. crassa, led to the most significant increase in lignocellulase secretion under both Avicel and xylan culture conditions. Moreover, strains lacking the β subunit of the AP-3 complex, encoded by Ncap3b (NCU06569), had a similar phenotype to ΔNcap3m, suggesting that the AP-3 complex is involved in lignocellulase secretion in N. crassa. We also found that the transcriptional abundance of major lignocellulase genes in ΔNcap3m was maintained at a relatively higher level during the late stage of fermentation compared with the WT, which might add to the hypersecretion phenotype. Finally, we found that importation of the T. reesei ap3m ortholog Trap3m into ΔNcap3m can genetically restore secretion of lignocellulases to normal levels, which suggests that the effect of the AP-3 complex on lignocellulase secretion is conserved in cellulolytic ascomycetes. Using the model cellulolytic fungus N. crassa, we explored potential hypersecretion-related mutations in T. reesei strain RUT-C30. Through systematic genetic screening of 86 corresponding orthologous KO mutants in N. crassa, we identified several genes, particularly those encoding the AP-3 complex that contribute to lignocellulase secretion. These findings will be useful for strain improvement in future lignocellulase and biomass-based chemical production. The online version of this article (doi:10.1186/s13068-015-0302-3) contains supplementary material, which is available to authorized users.