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Nonribosomal peptide synthetases (NRPS) are large multimodular enzymes that synthesize a diverse variety of peptides. Many of these are currently used as pharmaceuticals, thanks to their activity as antimicrobials (penicillin, vancomycin, daptomycin, echinocandin), immunosuppressant (cyclosporin) and anticancer compounds (bleomycin). Because of their biotechnological potential, NRPSs have been extensively studied in the past decades. In this review, we provide an overview of the main structural and functional features of these enzymes, and we consider the challenges and prospects of engineering NRPSs for the synthesis of novel compounds. Furthermore, we discuss secondary metabolism and NRP synthesis in the filamentous fungus Penicillium rubens and examine its potential for the production of novel and modified β-lactam antibiotics., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

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.

The l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine synthetase (ACVS) is a trimodular nonribosomal peptide synthetase (NRPS) that provides the peptide precursor for the synthesis of β-lactams. The enzyme has been extensively characterized in terms of tripeptide formation and substrate specificity. The first module is highly specific and is the only NRPS unit known to recruit and activate the substrate l-α-aminoadipic acid, which is coupled to the α-amino group of l-cysteine through an unusual peptide bond, involving its δ-carboxyl group. Here we carried out an in-depth investigation on the architecture of the first module of the ACVS enzymes from the fungus Penicillium rubens and the bacterium Nocardia lactamdurans. Bioinformatic analyses revealed the presence of a previously unidentified domain at the N-terminus which is structurally related to condensation domains, but smaller in size. Deletion variants of both enzymes were generated to investigate the potential impact on penicillin biosynthesis in vivo and in vitro. The data indicate that the N-terminal domain is important for catalysis., (© 2021 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

We present a Penicillium rubens strain with an industrial background in which the four highly expressed biosynthetic gene clusters (BGC) required to produce penicillin, roquefortine, chrysogine and fungisporin were removed. This resulted in a minimal secondary metabolite background. Amino acid pools under steady-state growth conditions showed reduced levels of methionine and increased intracellular aromatic amino acids. Expression profiling of remaining BGC core genes and untargeted mass spectrometry did not identify products from uncharacterized BGCs. This platform strain was repurposed for expression of the recently identified polyketide calbistrin gene cluster and achieved high yields of decumbenone A, B and C. The penicillin BGC could be restored through in vivo assembly with eight DNA segments with short overlaps. Our study paves the way for fast combinatorial assembly and expression of biosynthetic pathways in a fungal strain with low endogenous secondary metabolite burden.