Industrial penicillin production with the filamentous fungus Penicillium chrysogenum is based on an unprecedented effort in microbial strain improvement. To gain more insight into penicillin synthesis, we sequenced the 32.19 Mb genome of P. chrysogenum Wisconsin54-1255 and identified numerous genes responsible for key steps in penicillin production. DNA microarrays were used to compare the transcriptomes of the sequenced strain and a penicillinG high-producing strain, grown in the presence and absence of the side-chain precursor phenylacetic acid. Transcription of genes involved in biosynthesis of valine, cysteine and alpha-aminoadipic acid-precursors for penicillin biosynthesis-as well as of genes encoding microbody proteins, was increased in the high-producing strain. Some gene products were shown to be directly controlling beta-lactam output. Many key cellular transport processes involving penicillins and intermediates remain to be characterized at the molecular level. Genes predicted to encode transporters were strongly overrepresented among the genes transcriptionally upregulated under conditions that stimulate penicillinG production, illustrating potential for future genomics-driven metabolic engineering.

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Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum

Peptide antibiotics are known to contain non-protein amino acids, D-amino acids, hydroxy acids, and other unusual constituents. In addition they may be modified by N-methylation and cyclization reactions. Their biosynthetic origin has been connected in many cases to an enzymatic system referred to as the 'thiotemplate multienzymic mechanism'. This mechanism includes the activation of the constituent residues as adenylates on the enzymic template, the acylation of specific template thiol groups, epimerization or N-methylation at this thioester stage, and polymerization in the sequence directed by the multienzymic structure with the aid of 4'-phosphopantetheine as a cofactor, including possible cyclization or terminal modification reactions. The reaction sequences leading to gramicidin S, tyrocidine, cyclosporine, bacitracin, polymyxin, actinomycin, enniatin, beauvericin, delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and linear gramicidin are discussed. The structures of the multienzymes, their genetic organization, the biological functions of these peptides and results on related systems are discussed.

https://febs.onlinelibrary.wiley.com/doi/epdf/10.1111/j.1432-1033.1990.tb19188.x

Nonribosomal Biosynthesis of Peptide Antibiotics

Penicillins and cephalosporins are produced by a wide variety of microorganisms, including some filamentous fungi, many gram-positive streptomycetes, and a few gram-negative unicellular bacteria. All produce these beta-lactam antibiotics by essentially the same biosynthetic pathway. Recently, most of the penicillin and cephalosporin biosynthetic genes have been cloned, sequenced, and expressed. The biosynthetic genes code for enzymes that possess multifunctional peptide synthetase, cyclase, epimerase, expandase, hydroxylase, lysine aminotransferase, and acetyltransferase activities and are organized in chromosomal gene clusters and coordinately expressed. DNA hybridization screens of streptomycetes demonstrate that beta-lactam biosynthetic genes may be more widespread in nature than is indicated by conventional antibiotic screens. They offer the possibility of expanding the search for organisms with potential to make new beta-lactam antibiotics. Attempts to improve current yields of beta-lactams in production strains by introducing into them additional copies of biosynthetic genes have been partially successful. Comparative sequence analysis of bacterial and fungal beta-lactam biosynthetic genes show they share very high sequence identity. A model that explains the similarity of biosynthetic genes from an evolutionary standpoint assumes horizontal gene-transfer between the two groups of organisms. Indirect evidence suggests the transfer occurred from the bacteria to the fungi.

Penicillin and cephalosporin biosynthetic genes: structure, organization, regulation, and evolution.

The most commonly used β-lactam antibiotics for the therapy of infectious diseases are penicillin and cephalosporin. Penicillin is produced as an end product by some fungi, most notably by Aspergillus (Emericella) nidulans and Penicillium chrysogenum. Cephalosporins are synthesized by both bacteria and fungi, e.g., by the fungus Acremonium chrysogenum (Cephalosporium acremonium). The biosynthetic pathways leading to both secondary metabolites start from the same three amino acid precursors and have the first two enzymatic reactions in common. Penicillin biosynthesis is catalyzed by three enzymes encoded by acvA (pcbAB), ipnA (pcbC), and aatA (penDE). The genes are organized into a cluster. In A. chrysogenum, in addition to acvA and ipnA, a second cluster contains the genes encoding enzymes that catalyze the reactions of the later steps of the cephalosporin pathway (cefEF and cefG). Within the last few years, several studies have indicated that the fungal β-lactam biosynthesis genes are controlled by a complex regulatory network, e.g., by the ambient pH, carbon source, and amino acids. A comparison with the regulatory mechanisms (regulatory proteins and DNA elements) involved in the regulation of genes of primary metabolism in lower eukaryotes is thus of great interest. This has already led to the elucidation of new regulatory mechanisms. Furthermore, such investigations have contributed to the elucidation of signals leading to the production of β-lactams and their physiological meaning for the producing fungi, and they can be expected to have a major impact on rational strain improvement programs. The knowledge of biosynthesis genes has already been used to produce new compounds.

Molecular Regulation of β-Lactam Biosynthesis in Filamentous Fungi

The cluster of penicillin biosynthetic genes. Identification and characterization of the pcbAB gene encoding the alpha-aminoadipyl-cysteinyl-valine synthetase and linkage to the pcbC and penDE genes.

Cloning and characterization of the acyl-coenzyme A: 6-aminopenicillanic-aid-acyltransferase gene of Penicillium chrysogenum

Two genes, pcbC and penDE (also named ips and aat, respectively) encoding the enzymes isopenicillin N synthase and acyl-CoA:6-amino penicillanic acid (6-APA) acyltransferase, which are involved in the penicillin biosynthetic pathway in Penicillium chrysogenum, were cloned. Both genes are clustered together in a 5.1 kb SalI DNA fragment and are separated by a nontranscribed intergenic region of 1.5 kb. These genes are transcribed from different promoters in two separate transcripts of about 1.15 kb each. The penDE gene complements mutants of P. chrysogenum deficient in acyltransferase and the pcbC gene increases the level of isopenicillin N synthase in strains containing low levels of this enzyme. The clustering of penicillin biosynthetic genes is of great interest in the light of previous claims of horizontal transfer of the pcbC gene from β-lactam producing Streptomyces to filamentous fungi.

Martinus Antonius Math Groenen

Papers

Cloning and characterization of the acyl-coenzyme A: 6-aminopenicillanic-aid-acyltransferase gene of Penicillium chrysogenum

Two genes involved in penicillin biosynthesis are linked in a 5.1 kb SalI fragment in the genome of Penicillium chrysogenum.

Méthode pour modifier la production des métabolites secondaires en utilisant des gènes biosynthétiques assemblés