Downloaded from orbit.dtu.dk on: Sep 23, 2021 Regulatory processes in Aspergillus niger Poulsen, Lars Publication date: 2012 Document Version Early version, also known as pre-print Link back to DTU Orbit Citation (APA): Poulsen, L. (2012). Regulatory processes in Aspergillus niger. Department of Systems Biology, Technical University of Denmark. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Regulatory processes in Aspergillus niger Lars Poulsen Ph. D. Thesis October 2012 i ii Regulatory processes in Aspergillus niger Ph. D. Thesis Lars Poulsen Department of Systems Biology Technical university of university of Denmark Supervisors Assoc. Prof. Jette Thykær Assoc. Prof. Anna E. Lantz iii iv “The first step towards getting somewhere is to decide that you are not going to stay where you are.” - John Pierpont Morgan v i Summary Filamentous fungi are extensively used in the fermentation industry for synthesis of numerous products. One of the most important, is the fungus Aspergillus niger, used industrially for production of organic acids, and homologous as well as heterologous enzymes. This fungus has numerous of advantages, including tolerance for low pH, which is important for acid production. Furthermore, it has the capability of metabolizing a wide variety of carbon sources, possesses an exceptional efficient protein secretion capacity, and three genome sequences are publicly available. However, A. niger have some disadvantages as well, those are byproduct formation, secretion of proteolytic enzymes and formation of mycotoxins. The aim of this project was to reduce these disadvantages, though investigating the regulatory processes. The first objective was to study the regulatory events leading to A. niger’s citric acid overflow metabolism. This was done with analysis of both transcriptome and proteome profiles, from cultivations in manganese limitation and manganese excess conditions. Beside already described responses, that were used to verify the experimental setup, identification of novel events was done. The most interesting was the strong down regulation of phosphoenolpyruvate carboxykinase (PEPCK) at manganese limited conditions that could be one of the main initiators for the citrate overflow metabolism. To gain further insight into A. niger’s metabolism, a new metabolic engineering tool, termed transcription factor modulation was developed. Using this approach, two novel mutants were isolated and formed the basis for the following studies. Through knock out studies of putative trans-acting pH responding transcription factors, a mutant exhibiting an oxalate overproducing phenotype was identified and entitled Oxalic Acid repression Factor, OafA. This mutant was physiological characterized in details, using continues cultivation (chemostats), followed by transcriptional analysis. Two phosphoketolases were found to be down-regulated in the ΔoafA mutant and it was argued, that this was the main cause, for the increase oxalate formation. From similar knock out studies, another mutant was identified and this strain was shown to be a protease mutant and the responsible transcription factor was entitled Protease Regulator B, PrtB. This was compared against the already described, protease deficient strain, ΔprtT. The physiological batch characterization showed that the ΔprtT strain had the lowest protease activity (fivefold reduced), but also featured excessive CO2 yield, reduced growth rate and lower biomass yields. The ΔprtB strain had a close to twofold reduced levels of secreted proteases but with additional beneficial characteristics, as a lower oxalic i acid formation and wild type growth performance; it was therefore argued that this strain could be an attractive alternative to ΔprtT. Finally, in order to characterize the formation of the carcinogenic mycotoxin fumonisin, a reporter strain of A. niger was constructed, where the promoter from the fumonisin synthase was fused to the green fluorescent protein. This strain was used together with the commercial large-scale nutrient profiling platform, Biolog Phenotype MicroArrays. Out of the 476 conditions tested, six compounds significantly induce fumonisin production, identified. These formed the basis for the subsequent examinations, which resulted in the identification of azelaic acid, a plant hormone and a very potent fumonisin inducer. ii Dansk Resumé Skimmelsvampe anvendes i stort omfang i fermenteringindustrien, til at producere en række forskellige produkter. En af de vigtigste er svampen Aspergillus niger, som anvendes til industriel produktion af organiske syrer og homologe samt heterologe proteiner. Denne svamp har en række fordele, heriblandt tolerance for lav pH, hvilket er vigtigt når den anvendes til produktion af organiske syrer. Ydermere, kan den metabolisere en bred vifte af kulstofsklider, udskille proteiner i store mængder samt der er tre offentlig tilgænglige genom sekvenser. Men A. niger har også ulemper, heribland dannelse af biprodukter, udskillelse af proteolytiske enzymer og dannelse af mykotoxiner. Målet med dette projekt er at reducere disse ulemper, via studier af de regulatoriske processer. Det første mål var at undersøge de regulatoriske hændelser der forårsager citron syre overflow i A. niger’s metabolisme. Dette blev udført via analyser af både transkriptom- og proteomprofiler, fra mangan begrænsede og mangan overskuds kultiveringer. Udover identifikation af allerede beskrevne processer, som blev anvendt til at verifice den eksperimentelle setup, blev nye og ukendte respons også identificeret. Det mest interessante af disse var en kraftige nedregulering af phosphoenolpyruvate carboxykinase (PEPCK) i de mangan begrænsede kulturer, som kunne være en af de primære igangsættere af citronsyre produktionen. For at få en udvidet forståelse af A. niger’s metabolisme, blev et nyt ”metabolic engineering” værktøj udviklet, betegnet transkriptionsfaktor modulering. Med anvendelse af denne fremgangsmåde, blev to nye mutanter identificeret og disse dannede grundlaget for de efterfølgende undersøgelser. Med ”knock out” studier af formodet trans bindende pH reagerende transkriptionsfaktore, blev en transkriptionsmutant, der udviste en oxalsyre overproducerende fænotype, identificeret og efterfølgende navngivet Oxalic Acid repression Factor, OafA. Denne mutant blev fysiologisk karakteriseret vha. Kontinuerte gæringer (kemostater), efterfulgt af transkriptionsanalyse. To phophoketolaser blev fundet opreguleret i ΔoafA mutanten og derfor ansås dette respons at være den primære årsag, for den forøgede oxalsyre produktion. Fra lignende “knock out” studier, blev en yderligere mutant identificeret og denne viste sig at være en protease deficient mutant. Den ansvarlige transkriptionsfaktor blev navngivet Protease Regulator B, PrtB. Denne blev sammenlignet med en allerede beskrevet protease deficient stamme, ΔprtT. Fysiologiske batch karakteriseringer, viste at ΔprtT stammen, havde den laveste protease aktivitet (femfold reduceret), men udviste også en forøget CO2 produktion, reduceret vækst rate samt lavere biomasse udbytte. ΔprtB iii stammens protease aktivitet var reduceret tofold, i forhold til vildtypen men udtrykte ydereligere fordelagtige karakteristika, så som lavere oxalsyre produktion samt vækst på vildtype niveau. Det blev derfor argumenteret for at denne stamme kunne være et attraktivt alternativ til ΔprtT. Endeligt, for at karakterisere dannelsen af det kræftfremkaldende mykotoxin fumonisin, blev en rapportørstamme af A. niger konstrueret, hvor promotoren fra fumonisinsyntasen var fusioneret med det grønne fluorescerende protein. Denne stamme blev anvendt sammen med den kommercielle stor skala platform, ”Biolog Phenotype MicroArrays”. Ud af de 476 forhold undersøgt, fandtes seks stoffer der signifikant inducerer fumonisin produktionen. Disse dannede grundlaget for de efterfølgende undersøgelser, som resulterede i opdagelsen af azelaic syre, et plantehormer og en yderst potent fumonisin inducer. iv Preface The work presented in this thesis was performed between October 2009 and October 2012 at the Center for Microbial Biotechnology, Department of Systems Biology, Technical university of Denmark (DTU). The PhD study was a part of CMB’s IVC (Ingeniør-Videnskabelige centre) supported by the Danish Resarch Council for Technology and Production. The work was supervised by associate professor Jette Thykær and associate professor Anna E. Lantz. I wish to express my sincere gratitude to my supervisor Jette Thykær, I am thankful for the excellent supervision, guidance and daily scientific support. I will also like to thank Anna E. Lantz, my co-supervisor, for giving me the opportunity to carry out my Ph.D study at CMB and for her support and for the valuable scientific discussions. Through Jette I came in touch with Scott E. Baker of the Pacific