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An Analysis of the Proteolytic System Inaspergillus in Order to Improve An analysis of theproteolyti c system inAspergillus in order to improve protein production Promotor: dr. ir. J.A.M. de Bont Hoogleraar in de Industriele Microbiologic Co-promotor: dr. ir. J. Visser Universitair hoofddocent sectie Moleculaire Genetica van Industriele Microorganismen /ucl)08?o 0^ Johannes Petrus Theodorus Wilhelmus van den Hombergh An analysis of theproteolyti c system in Aspergillus in order to improve protein production Proefschrift ter verkrijging van de graad van doctor in de Landbouw- en Milieuwetenschappen op gezag van de rector magnificus dr. CM. Karssen in het openbaar te verdedigen op dinsdag 11jun i 1996 des morgens om 11uu r in de Aula van de Landbouwuniversiteit te Wageningen r Sr\0[£>n~>C\\v The research described in this thesis was financially supported by Ciba Geigy AG (Basel, Switzerland) and by the European Community in the framework of BRIDGE (BIOTCT- 90169) and BIOTECH (BIO-CT9301974), and was performed in the labs of the section Molecular Genetics of Industrial Microorganisms at Wageningen Agricultural University BIBIJOTIU^K' lANDBOUWUNiVK.'s: V. An analysis of the proteolytic system in Aspergillus in order to improve protein producti­ on / Johannes P.T.W. van den Hombergh. - [S.I.: s.n.] Thesis Landbouwuniversiteit Wageningen. - With ref. - With Summary in Dutch. ISBN 90-5485-545-2 Subject headings: Aspergillus niger I proteolytic degradation / protease regulation Cover: Annemaria Bruinslot Gerrit Rietveld Akademie, Amsterdam , pi i0a2o'12 °^ Stellingen 1. Er wordt bij het optimaliseren van eiwitproduktie inAspergillus stammen ten onrechte te weinig aandacht besteed aan de mate waarin een dergelijke stam in staat is tot verzuring van het cultuurmedium in relatie tot proteolyse. 2. De door Caddick (1994) beschreven indeling van mutaties in het bij de ammonium metabolietrepressie betrokken areA gen in Aspergillus is arbitrair omdat een dergelijke indeling in sterke mate afhankelijk is van de keuze van de targetgenen die hiervoor worden gebruikt. Caddick (1994) Progress in Industrial Microbiol. 29:347-353 3. Bij het tijdens autoregulatie negatief moduleren van de functionaliteit van het PacC eiwit is deC-terminal e QED repeat niet betrokken. 4. De extracellulaire serinecarboxypeptidases in Aspergillus niger hebben mogelijk een functie bij de autokatalytisch gei'nduceerde maturatie van het extracellulaire pepstatine-rembare zure protease PEPA. Inouee tal . (1996)Eur .J .Biochem . 237:719-725 5. Bij het verklaren van de verschillen in opbrengsten tussen homologe en heterologe eiwitproduktie in filamenteuze schimmels wordt de vaak afwijkende aminozuursamenstelling van heterologe eiwitten en de invloed van de veranderde aminozuurbehoefte veelal over het hoofd gezien. 6. Omdat de door Tilburn et al. (1995) waargenomen pH regulatie van hetprtA gen in Aspergillus nidulans door Katz et al. (1996) niet gereproduceerd kan worden is het niet uitgsloten dat de xprE mutaties en xpr¥,G suppressoren in Aspergillus nidulans betrokken zijn bij pH regulatie. Tilburn et al. (1995)EMB O J. 14:779-790; Katze t al. (1996)Mol .Gen .Genet .250:715-72 4 7. Niet-functionerende gemeentesecretarissen, procureurs generaal, ABP functionarissen en besturen van toezichthoudende organen blijken bij niet functioneren alleen vergezeld van forse afkooppremies (de gouden handdruk) te kunnen vertrekken. Opmerkelijk hierbij is verder dat de hierover verontwaardigde Tweede-Kamerleden zelf bij vertrek gebruik maken van een zogenaamde 'aanvulling op hun nieuwe inkomen'. 8. Het voorgenomen teruggeven aan denatuu r van grote stukken cultuurgrond in deprovinci e Zeeland vereist een aanpassing van de Zeeuwse lijfspreuk in 'Luctor et Submergo'. 9. De vertaling van het Latijnse woord doctorandus suggereert ten onrechte dat een ingenieur niet zou kunnen promoveren. 10. Het beursale AlO systeem is een stap in de richting van het creeren van een wetenschapsproletariaat. 11. Bij het uit aerodynamisch oogpunt inklapbaar maken van de verlichting van bepaalde sportauto's hebben de constructeurs over het hoofd gezien dat de wetten van de aerodynamica ook 's nachts van toepassing zijn. 12. Het aanbieden van walkman-vrije coupes zal meer mensen overhalen tot het gebruik van de trein dan de uitgebreide 'We gaan ervoor' reclamecampagnes. Stellingen behorende bij het proefschrift An analysis of the proteolytic system in Aspergillus in order to improve protein production Wageningen, 11jun i 1996 J.P.T.W. van den Hombergh aan Caroline en mijn ouders Contents Chapter 1 General introduction; fungal proteases, structure, characteristics, 1 function and regulation Chapter 2 Aim and outline of the thesis 71 Chapter 3 Expression of an Erwinia pectate lyase in three species of Asper- Ti gillus Chapter 4 Cloning and characterization of the pepE gene of Aspergillus 89 niger encoding a new aspartic protease and regulation of the pepE and pepC genes Chapter 5 Cloning, characterization and expression ofpepF, a gene encoding 107 a serine carboxypeptidase from Aspergillus niger Chapter 6 Residual protease spectra in Aspergillus niger prt mutants contain 123 elevated metallo protease activities: identification, cloning, charac­ terization and expression ofpepH, a novel metallo protease Chapter 7 Disruption of three acid proteases in Aspergillus niger: effects on 137 residual protease spectra and degradation of target proteins Chapter 8 New protease mutants in Aspergillus niger result in strongly 157 reduced in vitro degradation of target proteins: genetic and bio­ chemical characterization of seven complementation groups Chapter 9 Production of the homologous pectin lyase B protein in six diffe- 175 rent genetically defined protease deficient Aspergillus niger mutant strains Chapter 10 Identification, cloning and analysis of the Aspergillus niger pacC 193 wide domain pH regulatory gene Chapter 11 Regulation of acid phosphatases in an Aspergillus niger paclQ" 209 disruption strain Chapter 12 Complex regulation of extracellular proteases in Aspergillus niger: 229 an analysis of wide domain regulatory mutants demonstrates CREA, AREA and PACC mediated control Chapter 13 Reduced degradation of Erwinia pectate lyase expressed in Asper- 251 gillus through modulation of pH regulation and cloning of pep A, an Aspergillopepsin from Aspergillus nidulans Chapter 14 Identification, cloning and analyses ofpepl and pep], two metallo 269 proteases from Aspergillus nidulans: indications for an unique acid metallo protease class in filamentous fungi Chapter 15 General discussion; towards further improving protein production 287 Summary 297 Samenvatting (Nederlands) 301 Curriculum vitae 305 List of publications 307 Nawoord 309 -| General Introduction; fungal proteases, structure, characteristics, function and regulation1 J.P.T.W. van den Hombergh and J. Visser 1.1 Introduction Traditionally the proteases have been regarded as degradative enzymes, capable of cleaving proteins into small peptides and/or amino acids, and whose role it ist o digest nutrient protein or to participate in the turnover of cellular proteins. However, although the best characterized proteases have this elementary function, it has also been shown that proteases play key roles in a wide range of cellular processes, via mechanisms of selectivemodificatio n by limited proteolysis,an d thusca nhav e essential regulatory functions (Holzer and Tschensche 1979; Holzer and Heinrich, 1980). There is a number of reasons why proteases of filamentous fungi are of particular interest. The basic process of hydrolytic cleavage of peptide bonds in proteins appears costly and potentially detrimental to an organism if not properly controlled. The desired limits to proteolytic action are achieved through the specificity of proteinases, by compartmentalization of proteases and substrates within the cell, through modification of the substrates allowing recognition by the respective proteases, by regulation via zymogen activation, and the presence or absence of specific inhibitors, and of course also by regulation of protease gene expression. Studying proteases in fungi will help to understand fundamental cellular functions involving proteolysis, including intracellular protein turnover, processing, translocation, sporulation, germination and differentiation. Filamentous fungi provide interesting possibilities to study proteases as it is relatively easy to generate mutants, and powerful genetical, biochemical and molecular biological techniquesar e availablefo r analysis. In fact, Aspergillus nidulans and Neurospora crassaar e used asmode l organisms for analyzing the molecular basis of a range of physiological and developmental processes. Their well-developed genetics enable direct access tobiochemica l and genetical studies,unde r defined nutritionalan d cultivationconditions . Furthermore, a large group of fungi pathogenic to humans, live-stock and crop, have been isolated and proteolysis could play a role in their pathogenicity (host penetration, countering host defense mechanisms and/or nutrition during infection). Proteases are also frequently used in laboratory and clinical practice and in industrial processes; proteases both microbial and non-microbial are in wide­ spread use in the food industry (baking, brewing, cheese manufacturing, meat tenderizing), in the ' Accepted for publication in Applied Molecular and Cellular Biology of Filamentous Fungi. S. Brown and R.M. Berka (Eds.) 2 Chapter 1 tanning industry and in the manufacture of biological detergents (Aunstrup, 1980). The commercial interest in exploiting certain filamentous
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