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Review
, 62 (3), 547-85

Molecular Regulation of Beta-Lactam Biosynthesis in Filamentous Fungi

Affiliations
Review

Molecular Regulation of Beta-Lactam Biosynthesis in Filamentous Fungi

A A Brakhage. Microbiol Mol Biol Rev.

Abstract

The most commonly used beta-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 beta-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 beta-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.

Figures

FIG. 1
FIG. 1
Naturally occurring classes of β-lactam antibiotics essentially as compiled by O’Sullivan and Sykes (249). Modified from reference with permission of the publisher.
FIG. 2
FIG. 2
Biosynthesis of penicillin, cephalosporin C, and cephamycin C. Gene and organism names are in italics, names of enzymes are in capital letters. l-α-AAA is an intermediate of the l-lysine biosynthetic pathway but can also be provided by catabolic degradation of l-lysine. The question mark indicates that the contribution of the latter to β-lactam production has not been clarified yet. The penicillin biosynthesis occurs in fungi only, whereas cephalosporins are synthesized in both fungi (e.g., cephalosporin C by A. chrysogenum) and bacteria (e.g., cephamycin C by S. clavuligerus) (Fig. 1). See the text for details.
FIG. 3
FIG. 3
β-Lactam biosynthesis gene cluster in fungi and bacteria. References to each fungal gene are listed in Tables 1 to 5 or in the text. The A. chrysogenum cefD gene has not been identified yet. The whole gene cluster of N. lactamdurans has been characterized (–79). The organization of the L. lactamgenus gene cluster was taken from references and . Roman numerals indicate the chromosomes (in fungi) on which the genes are localized (115, 201, 235, 300). The intergenic regions between acvA and ipnA of P. chrysogenum, A. nidulans, and A. chrysogenum are 1,107 bp (91, 307), 872 bp (203) and 1,233 bp (230), respectively. Bacterial genes with fungal homologs are boxed. The transcriptional orientation and the transcript units (Bacteria), as far as it has been determined, are indicated by arrows above or below the boxes. Arrows between boxes (Bacteria) and arrows with broken lines below boxes mark the orientation of genes. The function of ORF is unknown. Abbreviations (–79, 258): cmcT, transmembrane protein; pbp, penicillin binding protein; bla, β-lactamase; blp, showing similarity to the extracellular β-lactamase inhibitory protein BLIP.
FIG. 4
FIG. 4
Compartmentalization of penicillin biosynthesis gene products. Enzyme names are boxed. Reactions which are hypothetical are labelled by two question marks. Most of the transport processes (indicated by a single question mark) which seem likely to exist because of the compartmentalization of the different enzymes have not been elucidated yet. The ACVS seems to be located within or bound to the vacuolar membrane. IPNS and IAT are located in the cytoplasm and in microbodies, respectively. See the text for details. The stage at which the processing of IAT occurs remains to be determined. Abbreviations: Aa, amino acids; cA, cyclized l-α-AAA (6-oxo-piperdine-2-carboxylic acid [Fig. 6]); CV, l-cysteinyl–d-valine; Mito, mitochondrion.
FIG. 5
FIG. 5
Regulatory circuits affecting the expression of the penicillin biosynthesis genes acvA and ipnA in A. nidulans and P. chrysogenum. Large and small arrows indicate major and minor transcription start sites, respectively. Transcript start sites of acvA and aatA in P. chrysogenum have not been reported yet. Until now, in P. chrysogenum the effect of alkaline pH has been shown for the ipnA gene (316). The addition of certain amino acids to the medium results in the indicated effects on gene expression. Some of these effects are mediated by the ambient pH. See the text for details. Regulatory circuits affecting the expression of the aatA gene in both fungi have not been identified yet.
FIG. 6
FIG. 6
Lysine biosynthetic pathway (aminoadipate pathway) with the intermediate l-α-AAA. It has not been clarified whether the reaction from homocitric acid to cis-homoaconitic acid is catalyzed by homoaconitase, as is the following reaction, or by a specific enzyme designated homocitrate dehydrase (38). Therefore, this reaction is labelled with a question mark. OPC is cyclized l-α-AAA and is found as by-product in the culture broth of penicillin fermentations. The minus signs indicate negative effects of l-lysine on the indicated reactions which were identified from either P. chrysogenum or A. nidulans. These might be mediated at the transcriptional level or posttranscriptionally. See the text for details.
FIG. 7
FIG. 7
Pathways leading to the synthesis of l-cysteine. (A) Assimilation of sulfate. For reduction of sulfate to sulfide, several enzymes are required. Sulfate is transported into the cytoplasm by a specific permease, encoded by the sB gene in A. nidulans. See the text for details. (B) Starting from sulfide, three different routes leading to the formation of cysteine are used by the various β-lactam-producing fungi. They are designated transsulfuration, direct sulfhydration (grey background), and reverse transsulfuration (grey background bounded by broken lines). Although all the pathways seem to exist in A. chrysogenum, the fungus prefers to generate cysteine for optimal cephalosporin C synthesis by conversion of methionine to cysteine via reverse transsulfuration. In contrast, P. chrysogenum and A. nidulans synthesize cysteine mainly by direct sulfhydrylation starting from serine. See the text for details. Abbreviations: SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine.
FIG. 8
FIG. 8
Biosynthetic pathway of l-valine. The minus sign indicates a negative effect of l-valine on the indicated reaction, as determined in P. chrysogenum and A. chrysogenum. See the text for details.
FIG. 9
FIG. 9
Diagrammatic representation of possible homologous recombination events of a plasmid (pAXB4A) carrying acvA-uidA and ipnA-lacZ gene fusions integrated in single or double copy at the chromosomal argB gene locus of A. nidulans. The restriction map around the argB locus with and without integrated plasmid is shown. By using an appropriate probe, transformants carrying the plasmid integrated in double copy were identified by Southern blot analysis (46, 50). Essentially the same system with an ipnA-lacZ gene fusion only was independently used by Pérez-Esteban et al. (256) to identify the npeE1 mutation. Abbreviations: argB*, mutated argB allele which can complement the chromosomal argB2 mutation by homologous recombination; B, BamHI restriction site; lacZ, E. coli β-galactosidase gene; PacvA, promoter region of the acvA gene; PipnA, promoter region of the ipnA gene; uidA, E. coli β-glucuronidase gene. Modified from reference with permission of the publisher.
FIG. 10
FIG. 10
Regulatory genes involved in the regulation of the penicillin biosynthesis genes of A. nidulans. Addition of the indicated amino acids to the medium results in the indicated effects on gene expression (318). Some of these effects are mediated by the ambient pH, most probably via pal genes and the central regulatory protein PACC encoded by the pacC gene (318). PACCi symbolizes the inactive form, which is proteolytically cleaved upon the pH signal (alkalinity) into PACCa, i.e., the active shorter version of PACC (246, 322). The two identified PENR1 binding sites containing CCAAT sequences are marked with black boxes and roman numerals (I and II). One of the protein components of the PENR1 complex is HAPC (in parentheses below PENR1). The four PACC binding sites bound in vitro by PACC (322) are designated 1 (ipnA1), 2 (ipnA2), 3 (ipnA3), and 4AB (ipnA4AB) (102) and are boxed in the intergenic region between acvA and ipnA. PACC sites that were found to be functional in vivo for expression of ipnA-lacZ and/or acvA-uidA are indicated by hatched boxes. Site 3 seems to be of major importance for both ipnA-lacZ (102) and acvA-uidA (318) expression. Large and small arrows indicate major and minor transcription start sites, respectively. Names printed in italics indicate mutations which formally represent trans-acting genes with positive effects on the expression of the indicated genes. The existence of the repressor is hypothetical. Only two putative regulatory proteins have been described so far in P. chrysogenum. PACC most probably mediates the effect of pH on ipnA transcription (316), and NRE mediates the nitrogen regulation of acvA and ipnA (109, 132) (Fig. 5).
FIG. 11
FIG. 11
Production of cephalosporin intermediates via production of adipyl-cephalosporins by using recombinant strains of P. chrysogenum (82). The single arrows indicate reactions of the recombinant P. chrysogenum strains, and the double arrows indicate contact of the culture filtrate with immobilized Pseudomonas amidase. Modified from reference with permission of the publisher.

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