<br/>The filamentous fungus <em>Aspergillus niger</em> is capable of producing and secreting large amounts of homologous proteins (2040 g/l; Montenecourt and Eveleigh, 1985; Nevalainen et al., 1991). Because of this very efficient secretion large effort is being made to use <em>Aspergilli</em> as hosts for expression of many heterologous products. Especially <em>A. niger</em> is an interesting option because of the very high secretion capacity and because it has GRAS (Generally Regarded As Safe) status, which enables food-grade applications. Unfortunately, in spite of the very high expression and secretion capacity of <em>A. niger</em> , it has been shown both in homologous and heterologous expression studies that overexpression of gene products is often hampered by homologous proteases. The <em>Aspergillus</em> expression strains produce several proteases which cause partial or complete degradation of target products. In order to study these protease-related problems and to construct <em>Aspergillus</em> strains in which proteolytic degradation is decreased or absent, the protease project was started. The results of these elaborate studies to improve protein production in relation to proteolytic degradation are presented in this thesis.<p><em>Aspergillus</em> strains express many different proteolytic activities as discussed in the introduction (chapter I). Therefore, the protease spectra of <em>A.</em><em>nidulans and A. niger</em> have been characterized (chapter III). The very different protease spectra, the differences in acidification of the growth medium and glycosylation of the proteins produced, caused large differences in expression yields, stability and activities of the expressed products. This clearly proved the importance of the choice of expression strain for heterologous expression. Many different proteolytic activities, comprising acid, semi-alkaline and alkaline proteases and serine carboxpeptidases, have been isolated from <em>A. niger</em> . Some of these activities were cloned ( <em>pep</em> A; Berka et al., 1990; <em>pep</em> B, Inoue et al., 1991 and <em>pep</em> C, Frederick et al., 1993) at the beginning of the protease project in which five additional <em>A. niger</em> protease genes were cloned, <em>pep</em> D, <em>pep</em> E, <em>pep</em> F, <em>pep</em> H and <em>pal</em> B (van den Hombergh et al., unpublished results). In chapter IV the cloning of a pepstatin-repressible intracellular acid protease, <em>pep</em> E, is described. The PEPE protein is probably located in the vacuole and involved in the cascade activation of two additional vacuolar proteolytic enzymes (PEPC and CPY, carboxypeptidase Y). After cloning and characterization of <em>pep</em> F an extracellular serine carboxypeptidase it was shown that this extracellular exo-protease was regulated in a very complex manner (chapter V). Both carbon catabolite repression and nitrogen metabolite repression are involved in the regulation of <em>pep</em> F at the level of mRNA amounts. Furthermore, pathway specific induction by protein and regulation by external pH have been described. Additionally, an extracellular metallo protease, <em>pep</em> H, was cloned from <em>A.</em><em>niger.</em> This endo-protease was cloned after the observation that in the residual activities of some isolated protease ( <em>prt</em> ) mutants, metallo protease activities were still present (see also chapter VIII). Cloning of the responsible gene(s) and subsequent disruption enables specific strain improvement of these protease mutants. Gene disruption of the three cloned acid proteases (two extracellular ( <em>pep</em> A and <em>pep</em> B) and one intracellular ( <em>pep</em> E), resulted in a significant reduction of degradation of 'a-specific' proteins (chapter VII). Also, for specific tester-proteins such as PELB a similar reduced <em>in vitro</em> degradation was observed. Furthermore, it was shown that disruption of <em>pep</em> E inactivated the cascade activation of Pro-PEPC and Pro-CPY, resulting in a strongly reduced vacuolar proteolytic activity. Besides generating specific gene disruptions, a large number of protease ( <em>prt</em> ) deficient mutants were isolated (chapter VIII). Classical genetic analyses of the <em>prt</em> mutants identified at least seven complementation groups ( <em>prt</em> A-G). Residual activities in some of these <em>prt</em> mutants varied from 2-80 % compared to wild type and <em>in</em> vitro degradation of tester proteins was reduced 500-1000 fold in some of these mutants. Construction of multiple <em>prt</em> deficient mutants, either by recombination of characterized mutations or by additional mutagenesis in existing <em>prt</em> mutants, resulted in further reduction of degradation. For six <em>prt</em> complementation groups transformable strains were constructed and subsequently expression of a <em>pkipel</em> B expression-secretion <em></em> cassette was studied (chapter IX). These experiments proved that <em>in vivo</em> degradation of PELB was also significantly reduced and overall expression yields were improved. The highest expression was observed in the <em>prt</em> F28 mutant and the overall expression was improved by a factor of 13 compared to expression in wild type. The regulation of several proteases in <em>A. niger</em> is very complex, as already mentioned in chapters I, IV, V and VI. The observed pH regulation of several extracellular proteases combined with the fact that <em>A. niger</em> acidifies its medium very rapidly, initiated analysis of pH regulation which finally resulted in the isolation of the wide domain pH regulatory gene, <em>pac</em> C, in <em>A.</em><em>niger</em> (chapter X). The <em>pac</em> C gene encodes a zinc-finger regulator protein which binds to specific sequences (5'-GCCA/GG-3') in the promoters of target genes. A disruption construct, designed in a way that upon integration at the homologous <em>pac</em> C locus a C-terminally truncated PacC protein is produced, resulted in constitutive expression of <em>pac</em> C (chapter XI). From expression studies with this <em>pac</em> 2C <sup><font size="-2">c</font></SUP>disruptant it was concluded that the <em>pac</em> C gene is auto-regulated and that the C-terminal part of PacC (deleted in the <em>pac</em> 2C <sup><font size="-2">c</font></SUP>strain) has a negative regulatory effect. Expression of several phosphatases identified at least three acid phosphatases (PI, PII and PIII) which are regulated both by phosphate and by pH. The observed pH regulatory effects were shown to be <em>pac</em> C mediated, as expression of PI, PII and PIII decreased severely in the <em>pac</em> 2C <sup><font size="-2">c</font></SUP>disruptant. The recent isolation of both <em>cre</em> A and <em>are</em> A mutants in <em>A.</em> niger combined <em></em> with the construction of a <em>pac</em> 2C <sup><font size="-2">c</font></SUP>disruption strain enabled a detailed analysis of protease expression at the level of mRNA content (chapter XII). Northern analyses using the wide domain regulatory mutants and disruptant proved that the observed regulatory effects for extracellular proteases (carbon catabolite repression, nitrogen metabolite repression and pH regulation) were indeed mediated by the <em>cre</em> A, <em>are</em> A and <em>pac</em> C genes, respectively. Apart from identification of severe <em>cre</em> A and <em>are</em> A alleles and the expected derepression phenomena for carbon catabolite repression and nitrogen metabolite repession of extracellular protease, also elevated mRNA levels for the vacuolar proteases were observed. Possibly, the derepressed strains need higher degradation capacities in order to remove the increased levels of damaged or unwanted protein. Furthermore, in the <em>pac</em> 2C <sup><font size="-2">c</font></SUP>disruption strain the expression of the extracellular acid proteases is increased upon transfer to alkaline pH. As the current model for pH regulation predicts a strong reduced expression for acid target genes in <em>pac</em> C <sup><font size="-2">c</font></SUP>strains, this observation could imply that for acid pH regulation not all the components involved have been identified.<p><em>A. nidulans</em> has a very different protease spectrum compared to <em>A. niger</em> (chapter III). However, although the pH optimum for degradation of 'a-specific' proteins is much higher than for <em>A. niger</em> , <em>A.</em><em>nidulans</em> can <em></em> also express an acid protease gene, <em>pep</em> A, which is probably regulated in a similar complex way as described for the <em>A.</em><em>niger</em> extracellular proteases (chapter XIII). Cloning and characterization of two novel metallo protease genes, <em>pep</em> I <em></em> and <em>pep</em> J <em>,</em> from <em>A.</em><em>nidulans</em> is <em></em> described in chapter XIV. These two metallo proteases appear (together with the <em>A.</em><em>oryzae</em> neutral protease II, the <em>A.</em><em>fumigatus</em> and <em>A.</em><em>flavus</em> mp20 and the <em>P. citrinum</em> neutral metallo protease) to belong to a novel class of metallo proteases, as no significant homologies with any of the known metallo protease families can be detected. As the metallo protease genes are expressed in <em>A.</em><em>nidulans</em> and metallo proteases in general have neutral pH optima, these metallo proteases could well be involved in the overall degradation of expressed heterologous proteins, which is also observed in <em>A.</em><em>nidulans.</em> Disruption of these metallo proteases together with the major extracellular serine protease could be a starting point for construction of <em>A.</em><em>nidulans</em> expression strains with reduced proteolytic activities.
|Qualification||Doctor of Philosophy|
|Award date||11 Jun 1996|
|Place of Publication||S.l.|
|Publication status||Published - 1996|
- aspergillus niger
- cum laude