Synthetic control devices for gene regulation in Penicillium chrysogenum

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.

CRISPR-Based Transcriptional Activation Tool for Silent Genes in Filamentous Fungi

“…To assess the CRISPRa vector for activation of transcriptionally silent promoter activation, we integrated a penDE core promoter driven DsRed gene into the penicillin-locus of P. rubens DS68530 (∆penicillin-BGC). This penDE-CP was selected because it has been reported previously to be insufficient to express the fluorescent reporter on its own, instead depending on the presence of a synthetic transcription factor 36 . Fluorescence microscopy showed a clear increase in fluorescence with 4/6 sgRNAs tested, compared to a non-sgRNA expressing control (Fig.…”

“…In order to test if expression of dCas9-VPR and the sgRNA from the CRISPRa vector could activate transcription of a silent gene, we targeted dCas9-VPR to the penDE core promoter (penDE-CP). The 200 bp long penDE-CP has previously been shown to be functional, but insufficient to drive expression on its own 36 . For easy visualization of CRISPR based transcriptional activation, the penDE-CP was set to drive DsRed-T1-SKL, a red fluorescent reporter gene with peroxisomal targeting signal (Fig.…”

“…The HH and HDV ribozyme based "Plug-and-Play" sgRNA transcription unit was amplified in three parts, where the gpdA promoter and the trpC terminator together with the HDV self-cleaving sequence were amplified from vector pFC334 (AddGene ID # 87846) 33 and LacZ alpha gene was amplified from the MoClo ToolKit vector pICH41308 (AddGene ID # 47998) 55 . The promoter of 40S ribosomal protein S8 of A. nidulans (AN0465.2, referred to as 40S) and the tif35 terminator of P. rubens Pc22g19890, as well as the transcription unit penDE-CP-DsRed-SKL-TAct, were amplified from pVE2_10 (AddGene ID #154228) 36 . The terbinafine selection marker as Pgpda-ergA-TamdS transcription unit was amplified from pCP1_45 41 .…”

“…The terbinafine selection marker as Pgpda-ergA-TamdS transcription unit was amplified from pCP1_45 41 . The promoter of pcbC (Pc21g21380, IPNS) was amplified from pVE2_19 (AddGene ID #154241) 36 , adding 80 bp flanking regions for homologous recombination. The phleomycin selection marker was amplified from pDSM-JAK-109 56 providing the PpcbC-ble-TCYC1 transcription unit, adding 80 bp flanking regions for homologous recombination ( Supplementary Fig.…”

Identification of a conserved N‐terminal domain in the first module of ACV synthetases

“…The sequence encoding Sp Cas9‐NLS (nuclear localization sequence) was amplified from pYTK036 (yeast MoClo toolkit, AddGene ID # 65143). The gene was placed under control of the p 40S promoter (AN0465, 40S ribosomal protein S8 promoter), amplified from pVE2_10_p40S_QfDBD_VP16AD_cp_penDE_DsRed_0xQUAS (AddGene ID # 154228) (Mózsik et al, 2019). For the sgRNA transcription unit, the hammerhead (HH) and hepatitis delta virus (HDV) ribozyme sequences (self‐cleavage), gpdA (AN8041) promoter, and trpC terminator were amplified from pFC334 (AddGene ID # 87846) (Nødvig et al, 2015).…”