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  • 1
    Electronic Resource
    Electronic Resource
    A novel strategy for the isolation of defined pyrG mutants and the development of a site-specific integration system for Aspergillus awamori (1995)
    Springer
    Current genetics 27 (1995), S. 536-540 
    Details
    ISSN: 1432-0983
    Keywords: Recombinant DNA ; Filamentous fungi ; 5-fluoro-orotic acid ; Homologous transformation system
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A homologous gene transfer system for Aspergillus awamori for site-specific integration is described, based on two components. First, a defined A. awamori pyrG mutant strain constructed by a selection strategy for gene-replacement in fungi. Second, a vector with a homologous pyrG selection marker containing a defined mutation at a site different from that of the mutations in the pyrG gene of the defined mutant strain. Defined mutation in the A. awamori pyrG gene, isolated from a genomic library by heterologous hybridisation with the A. niger pyrG gene as a probe, were introduced by specifically altering sequences at restriction sites in the coding region of the gene. After transformation of the A. awamori wild-type strain with vectors containing these mutated pyrG genes, and selection for 5-fluoro-orotic acid resistance (5-FOAR), on the average 60% of the 5-FOAR colonies originated from replacement of the wild-type pyrG gene by the mutated pyrG allele. After transformation of a mutant strain, carrying a mutation near the 5′ end of the pyrG gene with vectors containing a mutation near the 3′ end of the pyrG gene, 35% of the resulting transformants contained one copy of the vector at the pyrG locus.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00314444
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    Paper (German National Licenses)
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  • 2
    Electronic Resource
    Electronic Resource
    The effect of pre- and pro-sequences and multicopy integration on heterologous expression of the Fusarium solani pisi cutinase gene in Aspergillus awamori (1996)
    Springer
    Applied microbiology and biotechnology 45 (1996), S. 755-763 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract  A synthetic derivative of the cutinase cDNA of Fusarium solani pisi was expressed in Aspergillus awamori using the A. awamori endoxylanase II (exlA) promoter and terminator. The influence of the origin of the pre-sequence and the presence of a pro-sequence on the efficiency of extracellular cutinase production was analysed in single-copy transformants containing an expression cassette integrated at the pyrG locus. Transformants containing a construct encoding a direct, in-frame fusion of the xylanase pre-peptide to the mature cutinase showed a 2-fold higher cutinase production level compared to strains containing constructs with an additional cutinase pro-peptide. The effect of multicopy integration of the expression cassette on cutinase production was analysed in strains with different numbers of a cutinase construct containing its own pre-prosequence. The multicopy strains showed a 6- to 12-fold increased production of extracellular cutinase relative to the single-copy strains. No linear dose response relation to the number of expression cassettes present in the strains was observed. The amount of active enzyme produced by the strains correlated with the amount of cutinase-specific mRNA, suggesting that cutinase overproduction is not limited at the level of translation or secretion.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002530050759
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    Paper (German National Licenses)
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  • 3
    Electronic Resource
    Electronic Resource
    An expression system based on the promoter region of the Aspergillus awamori 1,4-β-endoxylanase A gene (1996)
    Springer
    Applied microbiology and biotechnology 46 (1996), S. 28-35 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract  A new, highly inducible fungal promoter derived from the Aspergillus awamori 1,4-β-endoxylanase A (exlA) gene is described. Induction analysis, carried out with the wild-type strain in shake flasks, showed that exlA expression is regulated at the transcriptional level. Using a β-glucuronidase (uidA) reporter strategy, D-xylose was shown to be an efficient inducer of the exlA promoter, whereas sucrose or maltodextrin were not. Upon D-xylose induction, the exlA promoter was threefold more efficient than the frequently used A. niger glucoamylase (glaA) promoter under maltodextrin induction. Detailed induction analyses demonstrated that induction was dependent on the presence of D-xylose in the medium. Carbon-source-limited chemostat cultures with the uidA reporter strain showed that D-xylose was also a very good inducer in a fermenter, even in the presence of sucrose.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002530050779
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    Paper (German National Licenses)
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  • 4
    Electronic Resource
    Electronic Resource
    Efficient production of secreted proteins by Aspergillus : progress, limitations and prospects (1997)
    Springer
    Applied microbiology and biotechnology 47 (1997), S. 1-11 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002530050880
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    Paper (German National Licenses)
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