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1.
米曲霉作为一种重要的工业微生物,在异源蛋白表达方面已有广泛应用,受限于被表达蛋白的修饰及分泌过程,目前实际生产使用的基因供体主要局限于其他真菌,尤其是丝状真菌。当外源基因来源于植物、昆虫和哺乳动物时,米曲霉所生产的异源蛋白产量及生物活性往往不尽如人意。本文综述了米曲霉作为宿主表达异源蛋白的研究进展,包括其现有的遗传操作手段及异源表达方面的应用及探索,重点介绍了应用过程中面临的挑战和解决策略,另外,对米曲霉表达异源蛋白的应用前景及发展方向进行了展望。 相似文献
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Ward OP 《Biotechnology advances》2012,30(5):1119-1139
The initial focus of recombinant protein production by filamentous fungi related to exploiting the extraordinary extracellular enzyme synthesis and secretion machinery of industrial strains, including Aspergillus, Trichoderma, Penicillium and Rhizopus species, was to produce single recombinant protein products. An early recognized disadvantage of filamentous fungi as hosts of recombinant proteins was their common ability to produce homologous proteases which could degrade the heterologous protein product and strategies to prevent proteolysis have met with some limited success. It was also recognized that the protein glycosylation patterns in filamentous fungi and in mammals were quite different, such that filamentous fungi are likely not to be the most suitable microbial hosts for production of recombinant human glycoproteins for therapeutic use. By combining the experience gained from production of single recombinant proteins with new scientific information being generated through genomics and proteomics research, biotechnologists are now poised to extend the biomanufacturing capabilities of recombinant filamentous fungi by enabling them to express genes encoding multiple proteins, including, for example, new biosynthetic pathways for production of new primary or secondary metabolites. It is recognized that filamentous fungi, most species of which have not yet been isolated, represent an enormously diverse source of novel biosynthetic pathways, and that the natural fungal host harboring a valuable biosynthesis pathway may often not be the most suitable organism for biomanufacture purposes. Hence it is expected that substantial effort will be directed to transforming other fungal hosts, non-fungal microbial hosts and indeed non microbial hosts to express some of these novel biosynthetic pathways. But future applications of recombinant expression of proteins will not be confined to biomanufacturing. Opportunities to exploit recombinant technology to unravel the causes of the deleterious impacts of fungi, for example as human, mammalian and plant pathogens, and then to bring forward solutions, is expected to represent a very important future focus of fungal recombinant protein technology. 相似文献
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Efficient production of secreted proteins by Aspergillus : progress, limitations and prospects 总被引:4,自引:0,他引:4
R. J. Gouka P. J. Punt C. A. M. J. J. van den Hondel 《Applied microbiology and biotechnology》1997,47(1):1-11
Filamentous fungi are widely used for the production of homologous and heterologous proteins but, compared to homologous proteins,
the levels of production of heterologous proteins are usually low. During the last 5 years, the levels of production of heterologous
proteins have been drastically improved by fusing the corresponding gene to the 3' end of a homologous gene, encoding a well-secreted
protein such as glucoamylase. Nevertheless, little research has been carried out to determine the limitations that hamper
heterologous protein production. Recently we have carried out a detailed analysis of the levels of production of several proteins
and glucoamylase fusion proteins in defined recombinant Aspergillus awamori strains. In this review we will focus on the use of filamentous fungi for the production of heterologous, especially non-fungal,
proteins. In particular, the effect of gene-fusion strategies will be reviewed. Furthermore, the remaining limitations in
heterologous protein production and suggestions for improvement strategies for overproduction of these protein will be discussed.
Received: 5 July 1996 / Accepted: 6 September 1996 相似文献
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Dissecting cellular components of the secretory pathway in filamentous fungi: insights into their application for protein production 总被引:1,自引:0,他引:1
Studies on protein production using filamentous fungi have mostly focused on improvement of the protein yields by genetic
modifications such as overexpression. Recent genome sequencing in several filamentous fungal species now enables more systematic
approaches based on reverse genetics and molecular biology of the secretion pathway. In this review, we summarize recent molecular-based
advances in our understanding of vesicular trafficking in filamentous fungi, and discuss insights into their high secretion
ability and application for protein production. 相似文献
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Panjideh H Coelho V Dernedde J Fuchs H Keilholz U Thiel E Deckert PM 《Bioprocess and biosystems engineering》2008,31(6):559-568
Recombinant antibody fusion constructs with heterologous functional domains are a promising approach to new therapeutic targeting strategies. However, expression of such constructs is mostly limited to cost and labor-intensive mammalian expression systems. Here we report on the employment of Pichia pastoris for the expression of heterologous antibody fusion constructs with green fluorescent protein, A33scFv::GFP, or with cytosine deaminase, A33scFv::CDy, their production in a biofermenter and a modified purification strategy. Combined, these approaches improved production yields by about thirty times over established standard protocols, with extracellular secretion of the fusion construct reaching 12.0 mg/l. Bifunctional activity of the fusion proteins was demonstrated by flow cytometry and an in-vitro cytotoxicity assay. With equal amounts of purified protein, the modified purification method lead to higher functional results. Our results demonstrate the suitability of methylotrophic Pichia expression systems and laboratory-scale bioreactors for the production of high quantities of bifunctionally active heterologous single-chain fusion proteins. 相似文献
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While Escherichia coli expression systems have been widely utilized for the production of heterologous proteins, these systems have limitations with regard to the production of particular protein products, including poor expression, expression of insoluble proteins into inclusion bodies, and/or expression of a truncated product. Using the surface protein expression (SPEX) system, chromosomally integrated heterologous genes are expressed and secreted into media by the naturally competent gram-positive organism Streptococcus gordonii. After E. coli turned out to be an inappropriate expression system to produce sufficient quantities of intact product, we successfully utilized SPEX to produce the heterologous antigen BH4XCRR that is designed from sequences homologous to the S. pyogenes M-protein C-repeat region. To further enhance production of this product by S. gordonii, we sought to develop a novel system for the production and secretion of heterologous proteins. We observed that under various growth conditions, S. gordonii secreted high levels of a 172 kDa protein, which was identified by N-terminal sequence analysis as the glucosyltransferase GTF. Here we report on the development of a plasmid-based expression system, designated as PLEX, which we used to enhance production of BH4XCRR by S. gordonii. A region from the S. gordonii chromosome that contains the positive regulatory gene rgg, putative gtfG promoter, and gtfG secretion-signal sequence was cloned into the E. coli/Streptococcus shuttle plasmid pVA838. Additionally, the bh4xcrr structural gene was cloned into the same plasmid downstream and in-frame with rgg and gtfG. This plasmid construct was transformed into S. gordonii and BH4XCRR was detected in culture supernatants from transformants at greater concentrations than in supernatants from a SPEX strain expressing the same product. BH4XCRR was easily purified from culture supernatant using a scalable two-step purification process involving hydrophobic-interaction and gel-filtration chromatography. 相似文献
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《Trends in biotechnology》1986,4(11):285-288
Recently, considerable research effort has focused on the molecular genetics of filamentous fungi of industrial importance. Intense research was initiated following reports of transformation systems for the non-commercial filamentous fungi Neurospora crassa and Aspergillus nidulans, and was prompted by two principle considerations: (1) the possibility of exploiting the inherent ability of many filamentous fungi to secrete copious quantities of protein in submerged culture, and (2) the disappointing yields of many heterologous proteins when secreted from prokaryotic and yeast expression systems. 相似文献
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Ruchika Sharma Meenu Katoch P. S. Srivastava G. N. Qazi 《World journal of microbiology & biotechnology》2009,25(12):2083-2094
Fungi combine the advantages of a microbial system such as a simple fermentability with the capability of secreting proteins
that are modified according to a general eukaryotic scheme. Filamentous fungi such as Aspergillus niger efficiently secrete genuine proteins but the secretion of recombinant proteins turned out be a difficult task. Aspergillus niger is an attractive organism because of its high secretion capacity and is frequently used as a model organism. Whereas high
production yields can be obtained when homologous proteins are expressed, much lower amounts are obtained with the production
of heterologous proteins. To fully exploit the potential of filamentous fungi, understanding of the molecular genetics, their
physiology, and the glycosylation metabolism has to be investigated and clarified in more detail. This review summarizes recent
developments in heterologous protein production by filamentous fungi and also generalizes the possibilities of improving the
protein production by various genetic and bioprocessing approaches, thereby easing recognition of filamentous fungi as a relevant
and reliable expression platform. 相似文献
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Although filamentous fungi are used extensively for protein expression, their use for the production of heterologous glycoproteins
is constrained by the types of N-glycan structures produced by filamentous fungi as compared to those naturally found on the
glycoproteins. Attempts are underway to engineer the N-glycan synthetic pathways in filamentous fungi in order to produce
fungal expression strains which can produce heterologous glycoproteins carrying specific N-glycan structures. To fully realize
this goal, a detailed understanding of the genetic components of this pathway in filamentous fungi is required. In this review,
we discuss the characterization of the α-mannosidase gene family in filamentous fungi and its implications for the elucidation
of the N-glycan synthetic pathway. 相似文献
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Filamentous fungi have long been used for the production of metabolites and enzymes. With developments in genetic engineering and molecular biology, filamentous fungi have also achieved increased attention as hosts for recombinant DNA. However, the production levels of non-fungal proteins are usually low. Despite the achievements obtained using molecular tools, the heterologous protein loss caused by extracellular fungal protease degradation persists. This review provides an overview of the potential bioprocessing strategies that can be applied to inhibit protease activity thereby enhancing heterologous protein production. 相似文献
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Kimura S Maruyama J Kikuma T Arioka M Kitamoto K 《Biochemical and biophysical research communications》2011,(3):1197-470
Autophagy is a conserved intracellular degradation process of eukaryotic cells. In filamentous fungi, although autophagy has been reported to have multiple physiological roles, it is not clear whether autophagy is involved in the degradation of misfolded proteins. Here, we investigated the role of autophagy in the degradation of misfolded secretory proteins accumulated in endoplasmic reticulum (ER) in the filamentous fungus Aspergillus oryzae. In late-phase cultures, a disulfide bond-deleted mutant of the secretory protein α-amylase AmyB fused with mDsRed that had accumulated in the ER was subsequently delivered to vacuoles, whereas wild-type AmyB-mDsRed was predominantly located at cell walls and septa. To examine the involvement of autophagy in the delivery of mutant AmyB to vacuoles, mutant AmyB-EGFP was expressed in an A. oryzae autophagy-deficient strain (ΔAoatg8). Microscopic examination revealed that the protein delivery to vacuoles did not occur in the absence of autophagic activity, with mutant AmyB-mDsRed forming large spherical structures surrounded by ER membrane. Hence, we conclude that autophagy is responsible for the delivery of misfolded secretory proteins accumulated in the ER to vacuoles for degradation during late-growth phase in A. oryzae. This is the first study to provide evidence that autophagy plays a role in the degradation of misfolded secretory proteins in filamentous fungi. 相似文献
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丝状真菌,俗称霉菌,在食品工业中被用于生产多种生物酶和有机酸。近年来,人们发现丝状真菌具有分泌量大、表达的蛋白有天然活性等特点,非常适合作为同源和异源重组蛋白的表达宿主,因此被广泛研究和探讨。简要综述了以黑曲霉为代表的几种常被用作蛋白表达宿主的丝状真菌的特点、应用中的主要问题和基本解决方案,以及近年关于丝状真菌表达系统的最佳培养条件和发酵条件的研究进展。 相似文献
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A small protein that fights fungi: AFP as a new promising antifungal agent of biotechnological value
Meyer V 《Applied microbiology and biotechnology》2008,78(1):17-28
As fungal infections are becoming more prevalent in the medical or agricultural fields, novel and more efficient antifungal
agents are badly needed. Within the scope of developing new strategies for the management of fungal infections, antifungal
compounds that target essential fungal cell wall components are highly preferable. Ideally, newly developed antimycotics should
also combine major aspects such as sustainability, high efficacy, limited toxicity and low costs of production. A naturally
derived molecule that possesses all the desired characteristics is the antifungal protein (AFP) secreted by the filamentous
ascomycete Aspergillus giganteus. AFP is a small, basic and cysteine-rich peptide that exerts extremely potent antifungal activity against human- and plant-pathogenic
fungi without affecting the viability of bacteria, yeast, plant and mammalian cells. This review summarises the current knowledge
of the structure, mode of action and expression of AFP, and highlights similarities and differences concerning these issues
between AFP and its related proteins from other Ascomycetes. Furthermore, the potential use of AFP in the combat against fungal
contaminations and infections will be discussed. 相似文献
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Saccharomyces cerevisiae is frequently used in biotechnology, including fermentative processes in food production, heterologous protein production
and high throughput developments for biomedicine. Accurate expression of selected genes is essential for all these areas.
Systems that can be regulated are particularly useful because they allow controlling the timing and levels of gene expression.
We examine here new expression systems that have been described, including improvements of classical ones and new strategies
of artificial gene control that have been applied in functional genomics. 相似文献
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Jin FJ Watanabe T Juvvadi PR Maruyama J Arioka M Kitamoto K 《Applied microbiology and biotechnology》2007,76(5):1059-1068
In this study, we investigated the effects of proteinase gene disruption on heterologous protein production by Aspergillus oryzae. The human lysozyme (HLY) was selected for recombinant production as a model for the heterologous protein. A tandem HLY construct
fused with α-amylase (AmyB) was expressed by A. oryzae in which the Kex2 cleavage site was inserted at the upstream of HLY. HLY was successfully processed from AmyB and produced
in the medium. We performed a systematic disruption analysis of five proteinase genes (pepA, pepE, alpA, tppA, and palB) in the HLY-producing strain with the adeA selectable marker. Comparative analysis indicated that disruption of the tppA gene encoding a tripeptidyl peptidase resulted in the highest increase (36%) in the HLY production. We further deleted the
tppA gene in the pepE or palB disruptant with another selectable marker, argB. Consequently, a double disruption of the tppA and pepE genes led to a 63% increase in the HLY production compared to the control strain. This is the first study to report that
the double disruption of the tppA and pepE genes improved the production level of a heterologous protein by filamentous fungi.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献