Synthetic biology aims at controlled gene regulation that can lead to increased production of chemicals andpharmaceuticals. In this work synthetic control devices were developed for Penicillium chrysogenum, a modelfilamentous fungus and industrially relevant cell factory

B\ufcttel, Zs\uf3fia

Bovenberg, R. A. L.

Driessen, Arnold J.M.

M\uf3zsik, L\ue1szl\uf3

Nyg\ue5rd, Yvonne

English

Chalmers Research

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/339313738Synthetic control devices for gene regulation in filamentous fungusPenicillium chrysogenum (STF/CRISPRa)Poster · February 2020CITATIONS0READS165 authors, including:Some of the authors of this publication are also working on these related projects:(Multi)drug resistance View projectOMNIYEAST View projectLaszlo MozsikUniversity of Groningen3 PUBLICATIONS   9 CITATIONS   SEE PROFILERoel A L BovenbergRoyal DSM104 PUBLICATIONS   2,834 CITATIONS   SEE PROFILEArnold J.M. DriessenUniversity of Groningen546 PUBLICATIONS   24,582 CITATIONS   SEE PROFILEAll content following this page was uploaded by Laszlo Mozsik on 17 February 2020.The user has requested enhancement of the downloaded file.Synthetic biology aims at controlled gene regulation that can lead to increased production of chemicals andpharmaceuticals. In this work synthetic control devices were developed for Penicillium chrysogenum, a modelfilamentous fungus and industrially relevant cell factory.In the synthetic transcription factor (STF) the QF DNA-binding domain of the transcription factor of the quinic acid genecluster of Neurospora crassa2 is fused to the VP16 activation domain. This synthetic transcription factor controls theexpression of genes under a synthetic promoter containing quinic acid upstream activating sequence (QUAS) elements,where it binds prior to a core promoter (CP) (Fig.1.).Day 3Day 5Day 70 .00 .20 .40 .6T im e  (d a y s )Extracellular Penicillin V [mg/mL]D S 5 4 4 6 85 x Q U A S  C P1 1 x Q U A S  C P5xQ U A S1 1xQ U A Smolecular microbiology                and synthetic biologySynthetic control devices for gene regulationin Penicillium chrysogenumConclusions/Outlook• Modular, synthetic control devices were developed for P. chrysogenum and their function was demonstrated with fluorescent reporters and Penicillin V production.• The strength of the control devices can be altered by altering the expression of the STF, the core promoter upstream the QUAS or the amount of QUAS elements, leadingto expression ranging from barely detectable to similar the highest expressed native genes.• We anticipate that these well-characterized and robustly performing control devices are highly useful tools in the development of filamentous fungi as production hosts.• Silent promoter activation was envisioned with the CRISPRa system. This genome editing free transcriptional regulatory tool could be further expand the fungal toolboxof synthetic regulatory systems and could be used to interrogate transcriptionally silent fungal gene clusters.László Mózsik1*, Zsofia Buttel1, Roel A.L. Bovenberg2,3, Arnold J. M. Driessen1, Yvonne Nygård 41 Department of Molecular Microbiology, GBB, University of Groningen, Groningen, NL;  2 DSM Biotechnology Center, DSM, Delft, NL; 3 Synthetic Biology and Cell Engineering, GBB,University of Groningen, Groningen, NL;  4 Department of Biology and Biological Engineering, Division of Industrial Biotechnology, Chalmers University of Technology, Göteborg, SE*E-mail: L.Mozsik@rug.nl  This research was supported by the QuantFung Project (Seventh Framework No. 607332) and ALERT Project (Marie Skłodowska-Curie No. 713482).References:1) Mózsik, L., Büttel, Z., Bovenberg, R.A.L., Driessen, A. J. M., & Nygård, Y. I.; Synthetic control devices for gene regulation in Penicillium chrysogenum. Microb Cell Fact 18, 203. (2019)2) Potter, C. J., Tasic, B., Russler, E. V., Liang, L., and Luo, L. ; The Q System: A Repressible Binary System for Transgene Expression, Lineage Tracing and Mosaic Analysis, Cell 141, 536-548. (2010)3) Polli, F., Meijrink, B., Bovenberg, R. A. L., and Driessen, A. J. M.; New promoters for strain engineering of Penicillium chrysogenum, Fungal Genet Biol 89. (2016)4) Pohl, C., Mózsik, L., Driessen, A. J. M., Bovenberg, R. A. L., & Nygård, Y. I.; Genome Editing in Penicillium chrysogenum Using Cas9 Ribonucleoprotein Particles. Synthetic biology: Methods and Protocols (pp. 213-232). (2018)Fig.1. Schematic representation of the control devices.IntroductionFig.5.a) Schematic representation of CRISPR/Cas9 based integration4 and co-transformation of the synthetic control deviceand QUAS elements into the penicillin cluster of DS54468. Designed sgRNA targets boundary between the promoter ofpcbAB and the core promoter of pcbC. b) Extracellular Penicillin V production of strains where the penicillin biosynthesiscluster is under control of the control device and parental strain DS54468 in shake flask cultivation in penicillinproducing medium. Data shows 3 independent cultures, measured in replicates.Validation by Penicillin V productiona bc dca bba baResultsTunability of the synthetic control device Fig.2. Development of biomass (a), GFP fluorescence (b), GFPfluorescence/biomass (c) and RFP (DsRed) fluorescence (d) overtime of selected P. chrysogenum strains in BioLector micro-bioreactor system3. Promoter driving STF p40S (when not indicateddifferently). Parental strain DS68530 (Wt) shown in dark green.Fig.4. Development of DsRed/GFP fluorescence over time duringgrowth of P. chrysogenum strains a) Effect of different CPs drivingDsRed with 5xQUAS. b) Strains expressing DsRed under a syntheticpromoter containing 0, 1, 5 or 11 QUAS elements upstream thepcbC CP.The strength of the control device can be altered by altering the expression of the transcription factor, the corepromoter upstream the QUAS or the number of QUAS elements (Fig.2.-4.).The versatility of the control device was demonstrated by fluorescent reporters (eGFP-NLS, DsRed-SKL) and itsapplication was confirmed by synthetically controlling the production of Penicillin V (Fig.5.).Validation by fluorescent reporters Validation of synthetic promotersFig.3. Fluorescence microscopy images of STF(-eGFP-NLS)expressing strains with (a) and without (b) 5xQUAS bindingsites prior core promoter driving DsRed. c) Ranking of theexpression of 6 control devices. All devices contain 5xQUAS;promoters driving the STF (on top) and CPs (on bottom).Envisioned silent promoter activation with CRISPRaa b1b2Fig.6.a) Schematic representation of plasmid delivery of the CRISPRa components(dCas9m4-AD and sgRNA) where AD stands for transcriptional activator domain.b1) Representation of synthetic silent promoter: no transcription from the geneof interest (GOI) without the integrated QUAS sequences (Figure 1).b2) sgRNA guided dCas9-AD binding upstream the synthetic silent promoter,promoting transcriptional activation.View publication stats

Synthetic control devices for gene regulation in Penicillium chrysogenum

https://research.chalmers.se/publication/515625/file/515625_Fulltext.pdf

Synthetic control devices for gene regulation in Penicillium chrysogenum

Abstract

Similar works

Full text

Available Versions

Chalmers Research