| 1. |
- Guzman-Chavez, F., et al.
(författare)
-
Mechanism and regulation of sorbicillin biosynthesis by Penicillium chrysogenum
- 2017
-
Ingår i: Microbial Biotechnology. - : Wiley. - 1751-7907 .- 1751-7915. ; 10:4, s. 958-968
-
Tidskriftsartikel (refereegranskat)abstract
- Penicillium chrysogenum is a filamentous fungus that is used to produce -lactams at an industrial scale. At an early stage of classical strain improvement, the ability to produce the yellow-coloured sorbicillinoids was lost through mutation. Sorbicillinoids are highly bioactive of great pharmaceutical interest. By repair of a critical mutation in one of the two polyketide synthases in an industrial P.chrysogenum strain, sorbicillinoid production was restored at high levels. Using this strain, the sorbicillin biosynthesis pathway was elucidated through gene deletion, overexpression and metabolite profiling. The polyketide synthase enzymes SorA and SorB are required to generate the key intermediates sorbicillin and dihydrosorbicillin, which are subsequently converted to (dihydro)sorbillinol by the FAD-dependent monooxygenase SorC and into the final product oxosorbicillinol by the oxidoreductase SorD. Deletion of either of the two pks genes not only impacted the overall production but also strongly reduce the expression of the pathway genes. Expression is regulated through the interplay of two transcriptional regulators: SorR1 and SorR2. SorR1 acts as a transcriptional activator, while SorR2 controls the expression of sorR1. Furthermore, the sorbicillinoid pathway is regulated through a novel autoinduction mechanism where sorbicillinoids activate transcription.
|
|
| 2. |
- Mózsik, László, et al.
(författare)
-
CRISPR-based transcriptional activation tool for silent genes in filamentous fungi
- 2021
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 11:1
-
Tidskriftsartikel (refereegranskat)abstract
- Filamentous fungi are historically known to be a rich reservoir of bioactive compounds that are applied in a myriad of fields ranging from crop protection to medicine. The surge of genomic data available shows that fungi remain an excellent source for new pharmaceuticals. However, most of the responsible biosynthetic gene clusters are transcriptionally silent under laboratory growth conditions. Therefore, generic strategies for activation of these clusters are required. Here, we present a genome-editing-free, transcriptional regulation tool for filamentous fungi, based on the CRISPR activation (CRISPRa) methodology. Herein, a nuclease-defective mutant of Cas9 (dCas9) was fused to a highly active tripartite activator VP64-p65-Rta (VPR) to allow for sgRNA directed targeted gene regulation. dCas9-VPR was introduced, together with an easy to use sgRNA “plug-and-play” module, into a non-integrative AMA1-vector, which is compatible with several filamentous fungal species. To demonstrate its potential, this vector was used to transcriptionally activate a fluorescent reporter gene under the control of the penDE core promoter in Penicillium rubens. Subsequently, we activated the transcriptionally silent, native P. rubens macrophorin biosynthetic gene cluster by targeting dCas9-VPR to the promoter region of the transcription factor macR. This resulted in the production of antimicrobial macrophorins. This CRISPRa technology can be used for the rapid and convenient activation of silent fungal biosynthetic gene clusters, and thereby aid in the identification of novel compounds such as antimicrobials.
|
|
| 3. |
- Mózsik, László, et al.
(författare)
-
Modular synthetic biology toolkit for filamentous fungi
- 2021
-
Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 10:11, s. 2850-2861
-
Tidskriftsartikel (refereegranskat)abstract
- Filamentous fungi are highly productive cell factories, often used in industry for the production of enzymes and small bioactive compounds. Recent years have seen an increasing number of synthetic-biology-based applications in fungi, emphasizing the need for a synthetic biology toolkit for these organisms. Here we present a collection of 96 genetic parts, characterized in Penicillium or Aspergillus species, that are compatible and interchangeable with the Modular Cloning system. The toolkit contains natural and synthetic promoters (constitutive and inducible), terminators, fluorescent reporters, and selection markers. Furthermore, there are regulatory and DNA-binding domains of transcriptional regulators and components for implementing different CRISPR-based technologies. Genetic parts can be assembled into complex multipartite assemblies and delivered through genomic integration or expressed from an AMA1-sequence-based, fungal-replicating shuttle vector. With this toolkit, synthetic transcription units with established promoters, fusion proteins, or synthetic transcriptional regulation devices can be more rapidly assembled in a standardized and modular manner for novel fungal cell factories.
|
|
| 4. |
- Mózsik, László, et al.
(författare)
-
Synthetic control devices for gene regulation in Penicillium chrysogenum
- 2019
-
Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 18:1, s. 203-
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Orthogonal, synthetic control devices were developed for Penicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory. In the synthetic transcription factor, the QF DNA-binding domain of the transcription factor of the quinic acid gene cluster of Neurospora crassa is fused to the VP16 activation domain. This synthetic transcription factor controls the expression of genes under a synthetic promoter containing quinic acid upstream activating sequence (QUAS) elements, where it binds. A gene cluster may demand an expression tuned individually for each gene, which is a great advantage provided by this system. RESULTS: The control devices were characterized with respect to three of their main components: expression of the synthetic transcription factors, upstream activating sequences, and the affinity of the DNA binding domain of the transcription factor to the upstream activating domain. This resulted in synthetic expression devices, with an expression ranging from hardly detectable to a level similar to that of highest expressed native genes. The versatility of the control device was demonstrated by fluorescent reporters and its application was confirmed by synthetically controlling the production of penicillin. CONCLUSIONS: The characterization of the control devices in microbioreactors, proved to give excellent indications for how the devices function in production strains and conditions. We anticipate that these well-characterized and robustly performing control devices can be widely applied for the production of secondary metabolites and other compounds in filamentous fungi.
|
|
| 5. |
- Mózsik, László, et al.
(författare)
-
Synthetic control devices for gene regulation in Penicillium chrysogenum
- 2020
-
Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Synthetic biology aims at controlled gene regulation that can lead to increased production of chemicals and pharmaceuticals. In this work synthetic control devices were developed for Penicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory.
|
|
| 6. |
- Pohl, Carsten, et al.
(författare)
-
Genome editing in penicillium chrysogenum using cas9 ribonucleoprotein particles
- 2018
-
Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1940-6029 .- 1064-3745. ; 1772, s. 213-232
-
Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Several CRISPR/Cas9 tools have been recently established for precise genome editing in a wide range of filamentous fungi. This genome editing platform offers high flexibility in target selection and the possibility of introducing genetic deletions without the introduction of transgenic sequences. This chapter describes an approach for the transformation of Penicillium chrysogenum protoplasts with preassembled ribonucleoprotein particles (RNPs) consisting of purified Cas9 protein and in vitro transcribed single guide RNA (sgRNA) for the deletion of genome sequences or their replacement with alternative sequences. This method is potentially transferable to all fungal strains where protoplasts can be obtained from.
|
|
| 7. |
- Viggiano, Annarita, et al.
(författare)
-
Pathway for the biosynthesis of the pigment chrysogine by Penicillium chrysogenum
- 2018
-
Ingår i: Applied and Environmental Microbiology. - 1098-5336 .- 0099-2240. ; 84:4
-
Tidskriftsartikel (refereegranskat)abstract
- Chrysogine is a yellow pigment produced by Penicillium chrysogenum and other filamentous fungi. Although the pigment was first isolated in 1973, its biosynthetic pathway has so far not been resolved. Here, we show that deletion of the highly expressed nonribosomal peptide synthetase (NRPS) gene Pc21g12630 (chyA) resulted in a decrease in the production of chrysogine and 13 related compounds in the culture broth of P. chrysogenum. Each of the genes of the chyAcontaining gene cluster was individually deleted, and corresponding mutants were examined by metabolic profiling in order to elucidate their function. The data suggest that the NRPS ChyA mediates the condensation of anthranilic acid and alanine into the intermediate 2-(2-aminopropanamido)benzoic acid, which was verified by feeding experiments of a ΔchyA strain with the chemically synthesized product. The remainder of the pathway is highly branched, yielding at least 13 chrysogine-related compounds.
|
|
