Evolution and function of cis-/trans elements of fungal secondary metabolism with emphasis on the penicillin biosynthesis

AXEL A. BRAKHAGE
Abteilung für Molekulare und Angewandte Mikrobiologie
Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie
Hans-Knöll-Institut und Lehrstuhl für Mikrobiologie und Molekularbiologie
Friedrich-Schiller-Universität Jena
Beutenbergstraße 11a
D-07745 Jena
Axel.Brakhage@hki-jena.de
www.hki-jena.de

References
Brakhage AA, Spröte P, Al-Abdallah Q, Gehrke A, Plattner H, Tüncher A. Regulation of penicillin biosynthesis in filamentous fungi. Adv Biochem Eng Biotechnol. 2004;88:45-90. Review.
The beta-lactam antibiotic penicillin is one of the mainly used antibiotics for the therapy of infectious diseases. It is produced as end product by some filamentous fungi only, most notably by Aspergillus (Emericella) nidulans and Penicillium chrysogenum. The penicillin biosynthesis is catalysed by three enzymes which are encoded by the following three genes: acvA (pcbAB), ipnA (pcbC) and aatA (penDE). The genes are organised into a gene cluster. Although the production of secondary metabolites as penicillin is not essential for the direct survival of the producing organisms, several studies indicated that the penicillin biosynthesis genes are controlled by a complex regulatory network, e.g. by the ambient pH, carbon source, amino acids, nitrogen etc. 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. Positively acting regulators have been identified such as the pH dependent transcriptional regulator PACC, the CCAAT-binding complex AnCF and seem also to be represented by recessive trans-acting mutations of A. nidulans (prgA1, prgB1, npeE1) and P. chrysogenum (carried by mutants Npe2 and Npe3). In addition, repressors like AnBH1 and VeA are involved in the regulation. Furthermore, such investigations have contributed to the elucidation of signals leading to the production of penicillin and can be expected to have a major impact on rational strain improvement programs.

Brakhage AA, Al-Abdallah Q, Tüncher A, Spröte P. Evolution of beta-lactam biosynthesis genes and recruitment of trans-acting factors. Phytochemistry. 2005 Jun;66(11):1200-10. Review.
Penicillins and cephalosporins belong chemically to the group of beta-lactam antibiotics. The formation of hydrophobic penicillins has been reported in fungi only, notably Penicillium chrysogenum and Emericella nidulans, whereas the hydrophilic cephalosporins are produced by both fungi, e.g., Acremonium chrysogenum (cephalosporin C), and bacteria. The producing bacteria include Gram-negatives and Gram-positives, e.g. Lysobacter lactamdurans (cephabacins) and Streptomyces clavuligerus (cephamycin C), respectively. For a long time the evolutionary origin of beta-lactam biosynthesis genes in fungi has been discussed. As often, there are arguments for both hypotheses, i.e., horizontal gene transfer from bacteria to fungi versus vertical descent. There were strong arguments in favour of horizontal gene transfer, e.g., fungal genes were clustered or some genes lack introns. The recent identification and characterisation of cis-/trans-elements involved in the regulation of the beta-lactam biosynthesis genes has provided new arguments in favour of horizontal gene transfer. In contrast to the bacterium S. clavuligerus, all regulators of fungal beta-lactam biosynthesis genes represent wide-domain regulators which were recruited to also regulate the beta-lactam biosynthesis genes. Moreover, the fungal regulatory genes are not part of the gene cluster. If bacterial regulators were co-transferred with the gene cluster from bacteria to fungi, most likely they would have been non-functional in eukaryotes and lost during evolution. Alternatively, it is conceivable that only a part of the beta-lactam biosynthesis gene cluster was transferred to some fungi, e.g., the acvA and ipnA gene without a regulatory gene.

Tüncher A, Spröte P, Gehrke A, Brakhage AA. The CCAAT-binding complex of eukaryotes: evolution of a second NLS in the HapB subunit of the filamentous fungus Aspergillus nidulans despite functional conservation at the molecular level between yeast, A. nidulans and human. J Mol Biol. 2005 Sep 23;352(3):517-33.
The heterotrimeric CCAAT-binding complex is evolutionarily conserved in eukaryotic organisms, including fungi, plants and mammals. In the filamentous fungus Aspergillus nidulans, the corresponding complex was designated AnCF (A. nidulans CCAAT-binding factor). AnCF consists of the subunits HapB, HapC and HapE. All three subunits are necessary for DNA binding. HapB contains two putative nuclear localisation signal sequences (NLSs) designated NLS1 and NLS2. Previously, it was shown that only NLS2 was required for nuclear localisation of HapB. Furthermore, HapC and HapE are transported to the nucleus only in complex with HapB via a piggy back mechanism. Here, by using various GFP constructs and by establishing a novel marker gene for transformation of A. nidulans, i.e. the pabaA gene encoding p-aminobenzoic acid synthase, it was shown that the HapB homologous proteins of both Saccharomyces cerevisiae (Hap2p) and human (NF-YA) use an NLS homologous to HapB NLS1 for nuclear localisation in S. cerevisiae. Interestingly, for A. nidulans HapB, NLS1 was sufficient for nuclear localisation in S. cerevisiae. In A. nidulans, HapB NLS1 was also functional when present in a different protein context. However, in A. nidulans, both S. cerevisiae Hap2p and human NF-YA entered the nucleus only when HapB NLS2 was present in the respective proteins. In that case, both proteins Hap2p and NF-YA complemented, at least in part, the hap phenotype of A. nidulans with respect to lack of growth on acetamide. Similarly, A. nidulans HapB and human NF-YA complemented a hap2 mutant of S. cerevisiae. In summary, HapB, Hap2p and NF-YA are interchangeable. Because the A. nidulans hapB mutant was complemented, at least in part, by both the human NF-YA and S. cerevisiae Hap2p this finding suggests that the piggy-back mechanism of nuclear transport found for A. nidulans is conserved in yeast and human.

Herrmann M, Spröte P, Brakhage AA. Protein kinase C (PkcA) of Aspergillus nidulans is involved in penicillin production. Appl Environ Microbiol. 2006 Apr;72(4):2957-70.
The biosynthesis of the beta-lactam antibiotic penicillin in the filamentous fungus Aspergillus nidulans is catalyzed by three enzymes that are encoded by the acvA, ipnA, and aatA genes. A variety of cis-acting DNA elements and regulatory factors form a complex regulatory network controlling these beta-lactam biosynthesis genes. Regulators involved include the CCAAT-binding complex AnCF and AnBH1. AnBH1 acts as a repressor of the penicillin biosynthesis gene aatA. Until now, however, little information has been available on the signal transduction cascades leading to the transcription factors. Here we show that inhibition of protein kinase C (Pkc) activity in A. nidulans led to cytoplasmic localization of an AnBH1-enhanced green fluorescent protein (EGFP) fusion protein. Computer analysis of the genome and screening of an A. nidulans gene library revealed that the fungus possesses two putative Pkc-encoding genes, which we designated pkcA and pkcB. Only PkcA showed all the characteristic features of fungal Pkc's. Production of pkcA antisense RNA in A. nidulans led to reduced growth and conidiation in Aspergillus minimal medium, while in fermentation medium it led to enhanced expression of an aatAp-lacZ gene fusion, reduced pencillin production, and predominantly cytoplasmic localization of AnBH1. These data agree with the finding that inhibition of Pkc activity prevented nuclear localization of AnBH1-EGFP. As a result, repression of aatA expression was relieved. The involvement of Pkc in penicillin biosynthesis is also interesting in light of the fact that in the yeast Saccharomyces cerevisiae, Pkc plays a major role in maintaining cell integrity.

Spröte P, Brakhage AA. The light-dependent regulator velvet A of Aspergillus nidulans acts as a repressor of the penicillin biosynthesis. Arch Microbiol. 2007 Jul;188(1):69-79.
The biosynthesis of the beta-lactam antibiotic penicillin in Aspergillus nidulans is catalysed by three enzymes that are encoded by the genes acvA, ipnA and aatA. Several studies have indicated that these genes are controlled by a complex regulatory network, including a variety of cis-acting DNA elements and regulatory factors. Until now, however, relatively little information is available on external signals and their transmission influencing the expression of the structural genes. Here, we show that the light-dependent regulator velvet A (VeA) acts as a repressor on the penicillin biosynthesis, mainly via repression of the acvA gene. Expression of a regulatable alcAp-veA gene fusion in an A. nidulans strain carrying, in addition, acvAp-uidA and ipnAp-lacZ gene fusions indicated that under alcAp-inducing conditions, penicillin titres and expression of acvAp-uidA were drastically reduced compared with untransformed wild-type strains. The same level of repression was found irrespective of whether the alcAp-veA gene fusion was expressed in a veA1 or DveA background, with or without light. The expression of the ipnAp-lacZ gene fusion was only moderately affected indicating a less prominent effect. These findings were confirmed by the analysis of a regulatable niiAp-veA gene fusion. Under niiAp-inducing conditions, penicillin titres and acvAp-uidA expression were much lower than in untransformed wild-type strains.

Bergmann S, Schümann J, Scherlach K, Lange C, Brakhage AA, Hertweck C. Genomics-driven discovery of PKS-NRPS hybrid metabolites from Aspergillus nidulans. Nat Chem Biol. 2007 Apr;3(4):213-7. Epub 2007 Mar 18.
In the postgenomic era it has become increasingly apparent that the vast number of predicted biosynthesis genes of microorganisms is not reflected by the metabolic profile observed under standard fermentation conditions. In the absence of a particular (in most cases unknown) trigger these gene loci remain silent. Because these cryptic gene clusters may code for the biosynthesis of important virulence factors, toxins, or even drug candidates, new strategies for their activation are urgently needed to make use of this largely untapped reservoir of potentially bioactive compounds. The discovery of new microbial metabolites through genome mining has proven to be a very promising approach. Even so, the investigation of silent gene clusters is still a substantial challenge, particularly in fungi. Here we report a new strategy for the successful induction of a silent metabolic pathway in the important model organism Aspergillus nidulans, which led to the discovery of novel PKS-NRPS hybrid
metabolites.

Hortschansky P, Eisendle M, Al-Abdallah Q, Schmidt AD, Bergmann S, Thön M, Kniemeyer O, Abt B, Seeber B, Werner ER, Kato M, Brakhage AA, Haas, H. Interaction of HapX with the CCAAT-binding complex-a novel mechanism of gene regulation by iron. EMBO J. 2007 Jun 14; [Epub ahead of print]
Iron homeostasis requires subtle control systems, as iron is both essential and toxic. In Aspergillus nidulans, iron represses iron acquisition via the GATA factor SreA, and induces iron-dependent pathways at the transcriptional level, by a so far unknown mechanism. Here, we demonstrate that iron-dependent pathways (e.g., heme biosynthesis) are repressed during iron-depleted conditions by physical interaction of HapX with the CCAAT-binding core complex (CBC). Proteome analysis identified putative HapX targets. Mutual transcriptional control between hapX and sreA and synthetic lethality resulting from deletion of both regulatory genes indicate a tight interplay of these control systems. Expression of genes encoding CBC subunits was not influenced by iron availability, and their deletion was deleterious during iron-depleted and iron-replete conditions. Expression of hapX was repressed by iron and its deletion was deleterious during iron- depleted conditions only. These data indicate that the CBC has a general role and that HapX function is confined to iron-depleted conditions. Remarkably, CBC-mediated regulation has an inverse impact on the expression of the same gene set in A. nidulans, compared with Saccharomyces cerevisae.

Thön M, Al-Abdallah Q, Hortschansky P, Brakhage AA. The thioredoxin system of Aspergillus nidulans: Impact on development and oxidative stress response. J Biol Chem 2007 (in revision)
Redox regulation has been shown to be of increasing importance for many cellular processes. Aspergillus nidulans is an important model organism to address fundamental biological questions such as development, gene regulation or the regulation of the production of secondary metabolites. Here, we describe the characterization of a thioredoxin system from the filamentous fungus A. nidulans. The A. nidulans thioredoxin A (AnTrxA) is an 11.6 kDa protein with a characteristic thioredoxin active site motif (WCGPC) encoded by the trxA gene. The corresponding thioredoxin reductase (AnTrxR) encoded by the trxR gene, represents a homodimeric flavoprotein with a native molecular weight of 72.2 kDa. When combined in vitro, the in Escherichia coli overproduced recombinant proteins AnTrxA and AnTrxR were able to reduce insulin and oxidized glutathione (GSSG) in an NADPH-dependent manner indicating that this in vitro redox system is functional. Moreover, we have created a thioredoxin A deletion strain which shows decreased growth, an increased catalase activity and the inability to form reproductive structures like conidiophores or cleistothecia when cultivated under standard conditions. However, addition of reduced glutathione (GSH) at low concentrations led to the development of sexual cleistothecia whereas high GSH levels resulted in the formation of asexual conidiophores. Furthermore, by applying the principle of thioredoxin-affinity chromatography we identified several novel putative targets of thioredoxin A, including a hypothetical protein with peroxidase activity and an aldehyde dehydrogenase.

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