Endocytosis Is Crucial for Cell Polarity and Apical Membrane Recycling in the Filamentous Fungus Aspergillus oryzae

Establishing the occurrence of endocytosis in filamentous fungi was elusive in the past mainly due to the lack of reliable indicators of endocytosis. Recently, however, it was shown that the fluorescent dye N-(3-triethylammoniumpropyl)-4-(p-diethyl-aminophenyl-hexatrienyl)pyridinium dibromide (FM4-64) and the plasma membrane protein AoUapC (Aspergillus oryzae UapC) fused to enhanced green fluorescent protein (EGFP) were internalized from the plasma membrane by endocytosis. Although the occurrence of endocytosis was clearly demonstrated, its physiological importance in filamentous fungi still remains largely unaddressed. We generated a strain in which A. oryzae end4 (Aoend4), the A. oryzae homolog of Saccharomyces cerevisiae END4/SLA2, was expressed from the Aoend4 locus under the control of a regulatable thiA promoter. The growth of this strain was severely impaired, and its hyphal morphology was altered in the Aoend4-repressed condition. Moreover, in the Aoend4-repressed condition, neither FM4-64 nor AoUapC-EGFP was internalized, indicating defective endocytosis. Furthermore, the localization of a secretory soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) was abnormal in the Aoend4-repressed condition. Aberrant accumulation of cell wall components was also observed by calcofluor white staining and transmission electron microscopy analysis, and several genes that encode cell wall-building enzymes were upregulated, indicating that the regulation of cell wall synthesis is abnormal in the Aoend4-repressed condition, whereas Aopil1 disruptants do not display the phenotype exhibited in the Aoend4-repressed condition. Our results strongly suggest that endocytosis is crucial for the hyphal tip growth in filamentous fungi.The filamentous fungus Aspergillus oryzae has been used in industrial fermentation processes and is regarded to be safe for humans. A. oryzae can secrete several proteins, such as alpha-amylase, into the medium. Thus, A. oryzae is a potential host for heterologous protein production. Since the completion of A. oryzae genome sequencing (18) in recent years, many applied and basic studies have been conducted on A. oryzae using its genome sequencing data. In particular, studies on vesicular trafficking, including the secretory pathway, are of increasing importance because they are closely related to protein production. For example, endoplasmic reticulum and vacuole dynamics and systematic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein analyses have been performed in A. oryzae (16, 19, 23, 30, 31, 32). However, endocytosis, an intracellular trafficking pathway, has not been studied as well in A. oryzae as in other filamentous fungi.Endocytosis is an important cellular process that occurs, for example, in signal transduction and reconstruction of cell polarity and is conserved in eukaryotic cells. The detailed mechanism of endocytosis has been well studied in model organisms such as yeasts. Many proteins are involved in the endocytic process, which is regulated spatiotemporally (12). Saccharomyces cerevisiae END4/SLA2 (synthetic lethal with ABP1) is an endocytosis-associated gene that has been studied in detail (3, 6, 22, 27, 35, 43, 44). End4p/Sla2p is essential for fluid-phase and receptor-mediated endocytosis and actin assembly and polarization (27). The protein has the epsin N-terminal homology (ENTH) and the AP180 N-terminal homology (ANTH) domains, which bind to phosphatidylinositol-4,5-bisphosphate in the plasma membrane in the N-terminal region, and the I/LWEQ domain, which is proposed to be the actin-binding domain in the C-terminal region; it also functions as an adaptor that connects the invaginated plasma membrane and actin cytoskeleton, which plays an important role in endocytosis, to generate force for invaginating the plasma membrane into the intracellular space and forming endocytic pits (13, 33). Abp1p (actin-binding protein) forms actin patches by polymerization of the actin cytoskeleton. It is suggested that endocytosis occurs at the sites in which Abp1p localizes, i.e., cortical actin patches (21, 22). Hence, Abp1p has been used as a tool to investigate the subcellular space in which endocytosis occurs (21).Establishing the existence of endocytosis in filamentous fungi was elusive in the past mainly due to the lack of reliable indicators of endocytosis (28). However, it has been confirmed that the fluorescent dye N-(3-triethylammoniumpropyl)-4-(p-diethyl-aminophenyl-hexatrienyl)pyridinium dibromide (FM4-64) and the plasma membrane protein AoUapC (Aspergillus oryzae UapC [uric acid-xanthine permease]) fused to enhanced green fluorescent protein (EGFP) were internalized from the plasma membrane by endocytosis (8, 25). Moreover, recently, in Aspergillus nidulans, the localization of components required for endocytosis has been analyzed in living hyphae (1, 37, 41). ActA and FimA, which are actin and fimbrin, respectively, are mostly localized in the hyphal tip region (41). Furthermore, AbpA, an actin-binding protein, is primarily localized in the apical region and is used as an endocytic site marker. AmpA, the amphiphysin homolog in A. nidulans, and SlaB, the End4p/Sla2p homolog, are also localized in sites in which AbpA is localized (1). These endocytic components are localized near the hyphal tip regions but slightly away from the apex where exocytosis preferentially occurs (37). Although the occurrence of endocytosis was clearly demonstrated and the localization of endocytic components was analyzed, the physiological importance of endocytosis in filamentous fungi still remains largely unaddressed.In this report, we analyzed the physiological significance of endocytosis by generating strains that conditionally express A. oryzae end4 (Aoend4), the A. oryzae homolog of S. cerevisiae END4/SLA2. Hyphae grown in the Aoend4-repressed condition displayed aberrant morphology; endocytic defects in AoUapC-EGFP and FM4-64; abnormal apical recycling of EGFP-fused AoSnc1, which is a vesicle SNARE required for secretion; and abnormal cell wall synthesis. These results suggest that endocytosis plays crucial roles in the physiology of hyphal growth.     

丝状真菌高效表达异源蛋白研究进展

丝状真菌是具有高效分泌蛋白质潜力的真核表达系统, 能对蛋白质进行翻译后修饰, 如蛋白质糖基化等; 并且比植物、昆虫和哺乳动物细胞具有更快的生长速率。近年来, 随着真菌分子遗传技术和菌种改良策略的进步, 尤其是真菌基因组学的发展, 利用丝状真菌生产异源蛋白越来越受到关注。综述了丝状真菌作为细胞工厂生产异源蛋白的最新探索与进展, 其中包括功能基因组学在蛋白表达与分泌研究中的应用, 同时探讨了异源蛋白表达和生产的改进策略。     

丝状真菌高效表达异源蛋白研究进展

丝状真菌是具有高效分泌蛋白质潜力的真核表达系统, 能对蛋白质进行翻译后修饰, 如蛋白质糖基化等; 并且比植物、昆虫和哺乳动物细胞具有更快的生长速率。近年来, 随着真菌分子遗传技术和菌种改良策略的进步, 尤其是真菌基因组学的发展, 利用丝状真菌生产异源蛋白越来越受到关注。综述了丝状真菌作为细胞工厂生产异源蛋白的最新探索与进展, 其中包括功能基因组学在蛋白表达与分泌研究中的应用, 同时探讨了异源蛋白表达和生产的改进策略。     

A Sandwiched-Culture Technique for Evaluation of Heterologous Protein Production in a Filamentous Fungus

Aspergillus niger is known for its efficient excretion machinery. However, problems have often arisen in obtaining high amounts of heterologous proteins in the culture medium. Here we present a quick method using sandwiched colonies to evaluate transgenic strains for secretion of heterologous proteins. Expressing the ABH1 hydrophobin of Agaricus bisporus in A. niger, we showed that low production levels of the heterologous protein are probably due to extracellular proteolytic degradation of the protein.     

提高蛋白质在米曲霉中表达量的策略

米曲霉是一种重要的微生物,在食品、酿造、商业酶和医用蛋白的生产中具有广泛的应用,该菌被美国食品与药品管理局(FDA)认定为GRAS(generally regarded as safe)级。讨论了提高同源和异源蛋白在米曲霉中表达量的几种策略,包括使用强启动子、多拷贝编码基因、优化培养基和超表达血红素结构域(HBD)等。异源蛋白容易被米曲霉蛋白酶降解,表达量往往较低,因此使用蛋白酶缺陷型宿主菌是非常必要的。另外将外源蛋白与米曲霉高分泌蛋白融合表达也是提高异源蛋白产量的有效途径。     

Truncation Releases Olfactory Receptors from the Endoplasmic Reticulum of Heterologous Cells

Olfactory receptors are difficult to express functionally in heterologous cells. We found that olfactory receptors traffic poorly to the plasma membrane even in cells with neuronal phenotypes, including cell lines derived from the olfactory epithelium. Other than mature olfactory receptor neurons, few cells appear able to traffic olfactory receptors to the plasma membrane. In human embryonic kidney 293 cells and Xenopus fibroblasts, olfactory receptor immunoreactivity overlapped with a marker for the endoplasmic reticulum (ER) but not with markers for the Golgi apparatus or endosomes. Except for the ER, olfactory receptors were therefore absent from organelles normally involved in the plasma membrane trafficking of receptors. Olfactory receptors truncated prior to transmembrane domain VI were expressed in the plasma membrane, however. Co-expression of the missing C-terminal fragment with these truncated receptors prevented their expression in the plasma membrane. Intramolecular interactions between N- and C-terminal domains joined by the third cytoplasmic loop appear to be responsible for retention of olfactory receptors in the ER of heterologous cells. Our results are consistent with misfolding of the receptors but could also be explained by altered trafficking of the receptors.     

Functional Analysis of Sterol Transporter Orthologues in the Filamentous Fungus Aspergillus nidulans

Polarized growth in filamentous fungi needs a continuous supply of proteins and lipids to the growing hyphal tip. One of the important membrane compounds in fungi is ergosterol. At the apical plasma membrane ergosterol accumulations, which are called sterol-rich plasma membrane domains (SRDs). The exact roles and formation mechanism of the SRDs remained unclear, although the importance has been recognized for hyphal growth. Transport of ergosterol to hyphal tips is thought to be important for the organization of the SRDs. Oxysterol binding proteins, which are conserved from yeast to human, are involved in nonvesicular sterol transport. In Saccharomyces cerevisiae seven oxysterol-binding protein homologues (OSH1 to -7) play a role in ergosterol distribution between closely located membranes independent of vesicle transport. We found five homologous genes (oshA to oshE) in the filamentous fungi Aspergillus nidulans. The functions of OshA-E were characterized by gene deletion and subcellular localization. Each gene-deletion strain showed characteristic phenotypes and different sensitivities to ergosterol-associated drugs. Green fluorescent protein-tagged Osh proteins showed specific localization in the late Golgi compartments, puncta associated with the endoplasmic reticulum, or diffusely in the cytoplasm. The genes expression and regulation were investigated in a medically important species Aspergillus fumigatus, as well as A. nidulans. Our results suggest that each Osh protein plays a role in ergosterol distribution at distinct sites and contributes to proper fungal growth.     

Identification of the Augmin Complex in the Filamentous Fungus Aspergillus nidulans

Augmin is a protein complex that binds to spindle microtubules (MTs), recruits the potent MT nucleator, γ-tubulin, and thereby promotes the centrosome-independent MT generation within mitotic and meiotic spindles. Augmin is essential for acentrosomal spindle assembly, which is commonly observed during mitosis in plants and meiosis in female animals. In many animal somatic cells that possess centrosomes, the centrosome- and augmin-dependent mechanisms work cooperatively for efficient spindle assembly and cytokinesis. Yeasts have lost the augmin genes during evolution. It is hypothesized that their robust MT nucleation from the spindle pole body (SPB), the centrosome-equivalent structure in fungi, compensates for the lack of augmin. Intriguingly, however, a gene homologous to an augmin subunit (Aug6/AUGF) has been found in the genome of filamentous fungi, which has the SPB as a robust MT nucleation centre. Here, we aimed to clarify if the augmin complex is present in filamentous fungi and to identify its role in mitosis. By analysing the Aug6-like gene in the filamentous fungus Aspergillus nidulans, we found that it forms a large complex with several other proteins that share weak but significant homology to known augmin subunits. In A. nidulans, augmin was enriched at the SPB and also associated with spindle MTs during mitosis. However, the augmin gene disruptants did not exhibit growth defects under normal, checkpoint-deficient, or MT-destabilised conditions. Moreover, we obtained no evidence that A. nidulans augmin plays a role in γ-tubulin recruitment or in mitotic cell division. Our study uncovered the conservation of the augmin complex in the fungal species, and further suggests that augmin has several functions, besides mitotic spindle MT nucleation, that are yet to be identified.     

利用黑曲霉高效表达外源蛋白策略

黑曲霉Aspergillus niger作为一种广泛存在于自然界的丝状真菌,是极为重要的工业微生物,不仅是重要的柠檬酸生产菌,同时还在表达多种蛋白和生产次级代谢产物等方面应用广泛。虽然黑曲霉用于商业化生产外源蛋白已经有很长的历史,但大多数外源蛋白的表达水平不高,亟需研究高效表达外源蛋白的策略。文中概述了通过选用强启动子、增加基因拷贝数、使用蛋白酶缺陷菌株、采用基因融合表达、增加糖基化修饰等策略来增强黑曲霉表达外源蛋白的进展,旨在为深入开展该领域的研究工作提供参考。     

米曲霉酸性蛋白酶基因在毕赤酵母中的异源表达及酶学性质

酸性蛋白酶作为一类重要的天冬氨酸蛋白酶,被广泛应用于食品、医药和皮革等领域。为推动酸性蛋白酶的研究及应用,通过对发酵豆制品样品进行宏基因组测序,从中获得米曲霉酸性蛋白酶基因pepA,在毕赤酵母GS115中进行异源表达,并对重组酶PepA进行酶学性质分析。结果显示毕赤酵母发酵上清液中酸性蛋白酶的活性为50.62 U/mL。SDS-PAGE验证PepA的分子量约为50 kDa,且发酵上清液几乎无杂蛋白。PepA的最适pH值为4.5,最适温度为50℃,Mn~(2+)和Cu~(2+)对其具有激活作用,而Fe~(3+)、Fe~(2+)与Ca~(2+)则具有抑制作用。上述研究结果可为米曲霉酸性蛋白酶的异源表达及其相关工业应用提供指导。     

Regulation of the acuF Gene, Encoding Phosphoenolpyruvate Carboxykinase in the Filamentous Fungus Aspergillus nidulans

Phosphoenolpyruvate carboxykinase (PEPCK) is a key enzyme required for gluconeogenesis when microorganisms grow on carbon sources metabolized via the tricarboxylic acid (TCA) cycle. Aspergillus nidulans acuF mutants isolated by their inability to use acetate as a carbon source specifically lack PEPCK. The acuF gene has been cloned and shown to encode a protein with high similarity to PEPCK from bacteria, plants, and fungi. The regulation of acuF expression has been studied by Northern blotting and by the construction of lacZ fusion reporters. Induction by acetate is abolished in mutants unable to metabolize acetate via the TCA cycle, and induction by amino acids metabolized via 2-oxoglutarate is lost in mutants unable to form 2-oxoglutarate. Induction by acetate and proline is not additive, consistent with a single mechanism of induction. Malate and succinate result in induction, and it is proposed that PEPCK is controlled by a novel mechanism of induction by a TCA cycle intermediate or derivative, thereby allowing gluconeogenesis to occur during growth on any carbon source metabolized via the TCA cycle. It has been shown that the facB gene, which mediates acetate induction of enzymes specifically required for acetate utilization, is not directly involved in PEPCK induction. This is in contrast to Saccharomyces cerevisiae, where Cat8p and Sip4p, homologs of FacB, regulate PEPCK as well as the expression of other genes necessary for growth on nonfermentable carbon sources in response to the carbon source present. This difference in the control of gluconeogenesis reflects the ability of A. nidulans and other filamentous fungi to use a wide variety of carbon sources in comparison with S. cerevisiae. The acuF gene was also found to be subject to activation by the CCAAT binding protein AnCF, a protein homologous to the S. cerevisiae Hap complex and the mammalian NFY complex.     

Introduction of an N-Glycosylation Site Increases Secretion of Heterologous Proteins in Yeasts

Saccharomyces cerevisiae is often used to produce heterologous proteins that are preferentially secreted to increase economic feasibility. We used N-glycosylation as a tool to enhance protein secretion. Secretion of cutinase, a lipase, and llama V_HH antibody fragments by S. cerevisiae or Pichia pastoris improved following the introduction of an N-glycosylation site. When we introduced an N-glycosylation consensus sequence in the N-terminal region of a hydrophobic cutinase, secretion increased fivefold. If an N-glycosylation site was introduced in the C-terminal region, however, secretion increased only 1.8-fold. These results indicate that the use of N glycosylation can significantly enhance heterologous protein secretion.     

米曲霉外源表达系统研究进展

丝状真菌米曲霉是发酵工业的重要菌种,具有强大的蛋白分泌能力和较高的食品安全性,可作为表达外源蛋白的细胞工厂。近年来,米曲霉全基因组序列的测序完成和基于表达序列标签的基因组学研究,为深入研究米曲霉外源表达系统提供了条件。从基因组学进展、遗传转化体系等方面综述了米曲霉作为外源蛋白表达宿主的研究进展。针对米曲霉在外源蛋白表达中存在的瓶颈,提出构建蛋白酶缺陷株、使用强启动子、融合表达等策略,以提高外源蛋白的表达和产量。最后介绍了米曲霉表达系统的应用,利用米曲霉代谢工程菌生产工业用酶和次级代谢产品具有良好的前景。     

丝状真菌胞外蛋白高效分泌机制的研究进展

丝状真菌由于其胞外蛋白分泌的高效性,成为生产酶制剂的高效细胞工厂.近年来针对真核生物胞外蛋白分泌途径的研究发现,丝状真菌蛋白的分泌途径相比其他真核生物具有高效分泌的特性.为了研究丝状真菌高效分泌的机制,本文总结了近年来丝状真菌分泌途径的最新研究进展,并且选取了分泌途径中关键环节的数种蛋白进行分析,通过与其他真核生物相关蛋白进行结构与序列比对,推测了丝状真菌胞外蛋白高效分泌的可能机制.     

丝状真菌瑞氏木霉生产重组蛋白的分子生物学研究进展

瑞氏木霉是自然界中普遍存在并有重要经济意义的一种丝状真菌,作为工业生产菌株生产多种水解酶类已有多年历史。本文报道了用基因工程手段对瑞氏木霉进行遗传改造,构造具新性状的重组菌株,用以过量产生同源和异源蛋白类物质的分子生物学研究进展。包括利用ＣＢＨＩ基因的强启动子在瑞氏木霉中过量表达瑞氏木霉内切葡聚糖酶、小牛凝乳蛋白酶、人抗体片段、哈茨木霉几丁质酶、Ｈｏｒｍｏｃｏｎｉｓｒｅｓｉｎａｅ葡萄糖淀粉酶等同源和异源蛋白以及利用在葡萄糖上强表达的启动子生产纤维素酶等遗传工程进展情况。     

米曲霉(Aspergillus oryzae)RIB40中烯酮/烯酯还原酶的异源表达及性质分析

以米曲霉(Aspergillus oryzae)RIB40基因组DNA为模版,通过PCR扩增其烯酮烯酯还原酶(AspER)基因(asper)后连接到表达载体pET32a(+)上,在大肠杆菌BL21(DE3)中以可溶形式表达。通过Ni-NTA亲和色谱层析纯化后,蛋白纯度提高1.9倍,回收率为60.62%。根据分子筛凝胶层析结果推算,AspER以二聚体形式存在。性质分析表明此酶为依赖于NADPH的氧化还原酶,最适pH为7.0～8.0,最适温度为40℃。对2-环己烯酮Km和kcat值分别为(2.45±0.36)mmol/L和(4.4±0.4)×103s-1。底物谱分析发现AspER对马来酰亚胺及其衍生物有较高的活性,其中对2-甲基马来酰亚胺的转化率和e.e.值均高于99%。     

Structural Features of Sugars That Trigger or Support Conidial Germination in the Filamentous Fungus Aspergillus niger

The asexual spores (conidia) of Aspergillus niger germinate to produce hyphae under appropriate conditions. Germination is initiated by conidial swelling and mobilization of internal carbon and energy stores, followed by polarization and emergence of a hyphal germ tube. The effects of different pyranose sugars, all analogues of d-glucose, on the germination of A. niger conidia were explored, and we define germination as the transition from a dormant conidium into a germling. Within germination, we distinguish two distinct stages, the initial swelling of the conidium and subsequent polarized growth. The stage of conidial swelling requires a germination trigger, which we define as a compound that is sensed by the conidium and which leads to catabolism of d-trehalose and isotropic growth. Sugars that triggered germination and outgrowth included d-glucose, d-mannose, and d-xylose. Sugars that triggered germination but did not support subsequent outgrowth included d-tagatose, d-lyxose, and 2-deoxy-d-glucose. Nontriggering sugars included d-galactose, l-glucose, and d-arabinose. Certain nontriggering sugars, including d-galactose, supported outgrowth if added in the presence of a complementary triggering sugar. This division of functions indicates that sugars are involved in two separate events in germination, triggering and subsequent outgrowth, and the structural features of sugars that support each, both, or none of these events are discussed. We also present data on the uptake of sugars during the germination process and discuss possible mechanisms of triggering in the absence of apparent sugar uptake during the initial swelling of conidia.