后基因组时代的丝状真菌基因组学与代谢工程

丝状真菌不仅是传统发酵工业中抗生素、酶制剂和有机酸的主要生产者,而且也是代谢工程育种中异源蛋白表达的重要细胞工厂。丝状真菌的遗传修饰和代谢工程研究是现代工业生物技术领域最具活力的研究方向之一。特别是与细菌和酵母相比,丝状真菌在细胞生长、营养需求、环境适应性、翻译后修饰、蛋白分泌能力和生物安全性等方面具有显著的优势。文章综述了丝状真菌作为异源蛋白表达系统在基因组学技术研究和代谢工程研究方面的最新进展。作者在分析丝状真菌基因组结构、特点的基础上,阐述了比较基因组学、蛋白质组学、转录组学和代谢组学等对丝状真菌的代谢途径重构、新型蛋白挖掘和代谢工程育种中的作用和意义。另一方面,作者分析了丝状真菌在表达外源蛋白时遇到的瓶颈问题,总结了丝状真菌代谢工程育种中的常用策略包括异源基因的融合表达、反义核酸技术、蛋白分泌途径改造、密码子优化和蛋白酶缺陷宿主的选育等技术和手段。最后,对该领域的发展趋势进行了展望。     

The major cellulases CBH‐1 and CBH‐2 of Neurospora crassa rely on distinct ER cargo adaptors for efficient ER‐exit

Filamentous fungi are native secretors of lignocellulolytic enzymes and are used as protein‐producing factories in the industrial biotechnology sector. Despite the importance of these organisms in industry, relatively little is known about the filamentous fungal secretory pathway or how it might be manipulated for improved protein production. Here, we use Neurospora crassa as a model filamentous fungus to interrogate the requirements for trafficking of cellulase enzymes from the endoplasmic reticulum to the Golgi. We characterized the localization and interaction properties of the p24 and ERV‐29 cargo adaptors, as well as their role in cellulase enzyme trafficking. We find that the two most abundantly secreted cellulases, CBH‐1 and CBH‐2, depend on distinct ER cargo adaptors for efficient exit from the ER. CBH‐1 depends on the p24 proteins, whereas CBH‐2 depends on the N. crassa homolog of yeast Erv29p. This study provides a first step in characterizing distinct trafficking pathways of lignocellulolytic enzymes in filamentous fungi.     

Genetic engineering of filamentous fungi--progress, obstacles and future trends

Filamentous fungi are widely used in biotechnology as cell factories for the production of chemicals, pharmaceuticals and enzymes. In order to improve their productivities, genetic engineering strategies can be powerful approaches. Different transformation techniques as well as DNA- and RNA-based methods to rationally design metabolic fluxes have been developed for industrially important filamentous fungi. However, the lack of efficient genetic engineering approaches still forms an obstacle for a multitude of fungi producing new and commercially interesting metabolites. This review summarises the variety of options that have recently become available to introduce and control gene expression in filamentous fungi and discusses their advantages and disadvantages. Furthermore, important considerations that have to be taken into account to design the best engineering strategy will be discussed.     

丝状真菌细胞凋亡研究进展

细胞凋亡是真核生物中保守而重要的细胞死亡机制,与癌症、艾滋病等多种疾病密切相关.与酵母菌这一细胞凋亡模式生物相比,丝状真菌凋亡研究起步较晚但具有其独特的优势.近年来丝状真菌细胞凋亡的内外源诱因、细胞凋亡的特征以及信号传导通路等方面的研究进展迅速.丝状真菌,尤其是构巢曲霉和烟曲霉有望成为细胞凋亡研究新的模式物种.此外,研究丝状真菌细胞凋亡现象在农业和医疗领域也具有重要的应用价值,可为生物防治和人类真菌病的治疗提供新的思路.工业丝状真菌细胞凋亡研究有助于构建性状更加优良的工程菌株.     

Yeast glucoamylases: molecular-genetic and structural characterization

Glucoamylase is an extracellular enzyme produced mainly by microorganisms. It belongs to the commercially frequently exploited biocatalysts. The major application of glucoamylase is in the starch bioprocessing to produce glucose and in alcoholic fermentations of starchy materials. Filamentous fungi have been the source of glucoamylases for industrial purposes as well as an object of numerous research studies. Some yeasts also secrete a large amount of glucoamylase with biochemical characteristics slightly different from those of filamentous fungi. Modern biotechnological applications require glucoamylases of certain properties optimal for a given process. Novel biocatalysts can be prepared from already existing enzymes using techniques of protein engineering or directed evolution. Tailoring of a commercial glucoamylase requires knowledge, on a molecular level, of structure/function relationships of enzymes originating from various sources and having different catalytic properties. Sequences of the cloned genes, their recombinant expression and the tertiary structure determination of glucoamylase are prerequisite to obtain such information. The presented review focuses on molecular-genetic and structural aspects of yeast glucoamylases, supplemented with the basic biochemical characterization of the given enzymes.     

The fungal α‐aminoadipate pathway for lysine biosynthesis requires two enzymes of the aconitase family for the isomerization of homocitrate to homoisocitrate

Fungi produce α‐aminoadipate, a precursor for penicillin and lysine via the α‐aminoadipate pathway. Despite the biotechnological importance of this pathway, the essential isomerization of homocitrate via homoaconitate to homoisocitrate has hardly been studied. Therefore, we analysed the role of homoaconitases and aconitases in this isomerization. Although we confirmed an essential contribution of homoaconitases from Saccharomyces cerevisiae and Aspergillus fumigatus, these enzymes only catalysed the interconversion between homoaconitate and homoisocitrate. In contrast, aconitases from fungi and the thermophilic bacterium Thermus thermophilus converted homocitrate to homoaconitate. Additionally, a single aconitase appears essential for energy metabolism, glutamate and lysine biosynthesis in respirating filamentous fungi, but not in the fermenting yeast S. cerevisiae that possesses two contributing aconitases. While yeast Aco1p is essential for the citric acid cycle and, thus, for glutamate synthesis, Aco2p specifically and exclusively contributes to lysine biosynthesis. In contrast, Aco2p homologues present in filamentous fungi were transcribed, but enzymatically inactive, revealed no altered phenotype when deleted and did not complement yeast aconitase mutants. From these results we conclude that the essential requirement of filamentous fungi for respiration versus the preference of yeasts for fermentation may have directed the evolution of aconitases contributing to energy metabolism and lysine biosynthesis.     

Fungus-Specific Sirtuin HstD Coordinates Secondary Metabolism and Development through Control of LaeA

The sirtuins are members of the NAD⁺-dependent histone deacetylase family that contribute to various cellular functions that affect aging, disease, and cancer development in metazoans. However, the physiological roles of the fungus-specific sirtuin family are still poorly understood. Here, we determined a novel function of the fungus-specific sirtuin HstD/Aspergillus oryzae Hst4 (AoHst4), which is a homolog of Hst4 in A. oryzae yeast. The deletion of all histone deacetylases in A. oryzae demonstrated that the fungus-specific sirtuin HstD/AoHst4 is required for the coordination of fungal development and secondary metabolite production. We also show that the expression of the laeA gene, which is the most studied fungus-specific coordinator for the regulation of secondary metabolism and fungal development, was induced in a ΔhstD strain. Genetic interaction analysis of hstD/Aohst4 and laeA clearly indicated that HstD/AoHst4 works upstream of LaeA to coordinate secondary metabolism and fungal development. The hstD/Aohst4 and laeA genes are fungus specific but conserved in the vast family of filamentous fungi. Thus, we conclude that the fungus-specific sirtuin HstD/AoHst4 coordinates fungal development and secondary metabolism via the regulation of LaeA in filamentous fungi.     

High diversity and complex evolution of fungal cytochrome P450 reductase: cytochrome P450 systems

Cytochrome P450 reductase (CPR) is the redox partner of P450 monooxygenases, involved in primary and secondary metabolism of eukaryotes. Two novel CPR genes, sharing 34% amino acid identity, were found in the filamentous ascomycete Cochliobolus lunatus. Fungal genomes were searched for putative CPR enzymes. Phylogenetic analysis suggests that multiple independent CPR duplication events occurred in fungi, whereas P450-CPR fusion occurred before the diversification of Dikarya and Zygomycota. Additionally, losses of methionine synthase reductase were found in certain fungal taxa; a truncated form of this enzyme was conserved in Pezizomycotina. In fungi, high numbers of cytochrome P450 enzymes, multiple CPRs, and P450-CPR fusion proteins were associated with filamentous growth. Evolution of multiple CPR-like oxidoreductases in filamentous fungi might have been driven by the complexity of biochemical functions necessitated by their growth form, as opposed to yeast.     

High diversity and complex evolution of fungal cytochrome P450 reductase: cytochrome P450 systems.

Cytochrome P450 reductase (CPR) is the redox partner of P450 monooxygenases, involved in primary and secondary metabolism of eukaryotes. Two novel CPR genes, sharing 34% amino acid identity, were found in the filamentous ascomycete Cochliobolus lunatus. Fungal genomes were searched for putative CPR enzymes. Phylogenetic analysis suggests that multiple independent CPR duplication events occurred in fungi, whereas P450-CPR fusion occurred before the diversification of Dikarya and Zygomycota. Additionally, losses of methionine synthase reductase were found in certain fungal taxa; a truncated form of this enzyme was conserved in Pezizomycotina. In fungi, high numbers of cytochrome P450 enzymes, multiple CPRs, and P450-CPR fusion proteins were associated with filamentous growth. Evolution of multiple CPR-like oxidoreductases in filamentous fungi might have been driven by the complexity of biochemical functions necessitated by their growth form, as opposed to yeast.     

The occurrence of fungi in hospital and community potable waters

The prevalence of fungi was investigated in 126 potable water samples (84 hospital and 42 community samples), in parallel with the standard pollution indicator micro-organisms. Filamentous fungi were isolated from 104 of 126 (82.5%) samples and yeasts from 14 (11.1%), whereas their mean counts were 36.6 and 4.4, respectively. Fungi were isolated from 95.2% of community and 76.2% of hospital water samples, with the difference being statistically significant (P < 0.05), while yeasts were isolated from 9.5 and 11.9%, respectively. Prevailing genera were Penicillium spp., isolated from 64, Aspergillus spp., from 53, and Candida, from nine of the examined samples. Colony-forming units of yeasts were significantly correlated with those of total and faecal coliforms, whereas the counts of filamentous fungi were significantly correlated with total heterotrophic bacteria counts. These results suggest that tap water is a potential transmission route for fungi both in hospitals and the community in the examined region and may pose a health hazard mainly for the immunocompromised host.     

Improving extracellular production of food-use enzymes from Aspergillus nidulans

Filamentous fungi, and particularly those of the genus Aspergillus, are major producers of enzymatic activities that have important applications in the food and beverage industries. Prior to the availability of transformation systems improvement of industrial production strains was largely restricted to the strategy of mutagenesis, screening and selection. Aspergillus nidulans is a genetically amenable filamentous fungus the ease of handling and analysis of which has led to its use as a model system for the investigation of eukaryotic gene regulation. Although not used industrially it is able to produce a wide variety of extracellular enzymatic activities. As a consequence of half a century of study a considerable resource of characterised mutants has been generated in conjunction with extensive genetic and molecular information on various gene regulatory systems in this micro-organism. Investigation of xylanase gene regulation in A. nidulans as a model for the production of food-use extracellular enzymes suggests strategies by which production of these enzymes in industrially useful species may be improved.     

The use of secondary metabolite profiling in chemotaxonomy of filamentous fungi

A secondary metabolite is a chemical compound produced by a limited number of fungal species in a genus, an order, or even phylum. A profile of secondary metabolites consists of all the different compounds a fungus can produce on a given substratum and includes toxins, antibiotics and other outward-directed compounds. Chemotaxonomy is traditionally restricted to comprise fatty acids, proteins, carbohydrates, or secondary metabolites, but has sometimes been defined so broadly that it also includes DNA sequences. It is not yet possible to use secondary metabolites in phylogeny, because of the inconsistent distribution throughout the fungal kingdom. However, this is the very quality that makes secondary metabolites so useful in classification and identification. Four groups of organisms are particularly good producers of secondary metabolites: plants, fungi, lichen fungi, and actinomycetes, whereas yeasts, protozoa, and animals are less efficient producers. Therefore, secondary metabolites have mostly been used in plant and fungal taxonomy, whereas chemotaxonomy has been neglected in bacteriology. Lichen chemotaxonomy has been based on few biosynthetic families (chemosyndromes), whereas filamentous fungi have been analysed for a wide array of terpenes, polyketides, non-ribosomal peptides, and combinations of these. Fungal chemotaxonomy based on secondary metabolites has been used successfully in large ascomycete genera such as Alternaria, Aspergillus, Fusarium, Hypoxylon, Penicillium, Stachybotrys, Xylaria and in few basidiomycete genera, but not in Zygomycota and Chytridiomycota.     

Microparticle based morphology engineering of filamentous microorganisms for industrial bio-production

Filamentous microorganisms are important work horses in industrial biotechnology and supply enzymes, antibiotics, pharmaceuticals, bulk and fine chemicals. Here we highlight recent findings on the use of microparticles in the cultivation of filamentous bacteria and fungi, with the aim of enabling a more precise control of their morphology towards better production performance. First examples reveal a broad application range of microparticle based processes, since multiple filamentous organisms are controllable in their growth characteristics and respond by enhanced product formation.     

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。