Juan-Francisco Martín

Engineering of regulatory cascades and networks controlling antibiotic biosynthesis in Streptomyces.

Engineering of regulatory mechanisms that control the biosynthesis of bioactive secondary metabolites is an approach to increase the production of valuable fermentation products. Two types of regulatory mechanisms have been studied in Streptomyces species: (1) pyramidal cascades of regulation that usually involve a butyrolactone and its receptor protein triggering the formation of pathway-associated regulatory proteins (SARP), and (2) global regulators that transduce protein phosphorylation signals responding to stress factors. Global regulators are frequently two-component systems; for example, the PhoR-PhoP system, the AsbA1-AsbA2, the orphan response regulator GlnR and the STAND-family regulator AfsR. Several strategies have been used to obtain overproducer strains, including: (i) obtention of phosphate-deregulated mutants by alteration of phoP, (ii) amplification and/or overexpression of pathway-associated positive regulators, and (iii) modification of butyrolactone receptor proteins. The success of these strategies is hampered by the poor knowledge of interactions between regulatory mechanisms.

Dissociation of cephamycin and clavulanic acid biosynthesis in Streptomyces clavuligerus

SummaryStreptomyces clavuligerus produced simultaneously cephamycin C and clavulanic acid in defined medium in long-term fermentations and in resting-cell cultures. Biosynthesis of cephamycin by phosphate-limited resting cells was dissociated from clavulanic acid formation by removing either glycerol or sulphate from the culture medium. In absence of glycerol no clavulanic acid was formed but cephamycin production occurred, whereas in absence of sulphate no cephamycin was synthesized but clavulanic biosynthesis took place. Sulphate, sulphite and thiosulphate were excellent sulphur sources for cephamycin biosynthesis while l-methionine and l-cysteine were poor precursors of this antibiotic. Increasing concentrations of sulphate also stimulated clavulanic acid formation. The biosynthesis of clavulanic acid was much more sensitive to phosphate (10–100 mM) regulation than that of cephamycin. Therefore, the formation of both metabolites was pertially dissociated at 25 mM phosphate. By contrast, nitrogen regulation by ammonium salts or glutamic acid strongly reduced the biosynthesis of both cephamycin and clavulanic acid.

The cephamycin biosynthetic genes pcbAB, encoding a large multidomain peptide synthetase, and pcbC of Nocardia lactamdurans are clustered together in an organization different from the same genes in Acremonium chrysogenum and Penicillium chrysogenum

A 34 kb fragment of the Nocardia lactamdurans DNA carrying the cluster of early cephamycin biosynthetic genes was cloned in λ EMBL3 by hybridization with probes internal to the pcbAB and pcbC genes of Penicillium chrysogenum and Streptomyces griseus. The pcbAB and pcbC genes were found to be closely linked together in the genome of N. lactamdurans. The pcbAB gene of N. lactamdurans showed the same orientation as the pcbC gene, in contrast to the divergent expression of the genes in the pcbAB‐pcbC cluster of P. chrysogenum and Acremonium chrysogenum. The pcbAB gene encodes a large (3649 amino acids) multidomain δ‐(L‐α‐aminoadipyl)‐L‐cysteinyl‐D‐valine synthetase with a deduced Mr, of 404134. This enzyme contains three repeated domains and a consensus thioesterase active‐site sequence. The pcbC gene encodes a protein of 328 amino acids with a deduced Mr of 37469, which is similar to other isopenicillin N synthases except that it lacks one of two cysteine residues conserved in all other isopenicillin N synthases. The different organization of the pcbAB‐pcbC gene cluster in N. lactamadurans and Streptomyces clavuligerus relative to P. chrysogenum and A. chrysogenum is intriguing in relation to the hypothesis of horizontal transference of these genes from actinomycetes to filamentous fungi by a single transfer event.

New aspects of genes and enzymes for β-lactam antibiotic biosynthesis

Abstract Penicillins, cephalosporins and cephamycins are peptide antibiotics synthesized by condensation of l-α-aminoadipic acid, l-cysteine and l-valine to form the tripeptide δ(l-α-aminoadipyl)-l-cysteinyl-d-valine (Aad-Cys-Val) by a non-ribosomal peptide synthetase. The genes pcbAB and pcbC, common to all penicillin and cephalosporin producers, that encode the Aad-Cys-Val synthetase1 and isopenicillin N (IPN) synthase1 respectively, have been cloned and the encoded enzymes studied in detail. The IPN synthase has been crystallized and its active center identified, providing evidence for the molecular mechanism of cyclization of the tripeptide Aad-Cys-Val to isopenicillin N. The late genes of the penicillin and cephalosporin pathways have also been characterized although some of the molecular mechanisms catalyzed by the encoded enzymes (e.g. IPN acyltransferase) are still obscure. In cephamycin-producing organisms, biosynthesis of the α-aminoadipic acid precursor proceeds in two steps catalyzed by lysine 6-aminotransferase and piperideine-6-carboxylic acid dehydrogenase. The gene lat for the first of these enzymes is located in the cephamycin gene cluster, providing an interesting example of association of genes encoding enzymes for the formation of a precursor with genes involved in assembly of the antibiotics. Novel enzymes involved in methoxylation at C-7 and carbamoylation at C-3′ of the cephem nucleus were isolated from Nocardia lactamdurans and Streptomyces clavuligerus. The methoxylation system is encoded by two linked genes cmcI-cmcJ and their products (proteins P7 and P8) form a complex that is required for hydroxylation at C-7 and for the subsequent methylation of the 7-hydroxycephem derivative to form the methoxyl group. Carbamoylation at the C-3′-hydroxyl group of the cephem nucleus is catalyzed by a specific carbamoyltransferase encoded by the gene cmcH. Finally, genes for a β-lactamase (bla), a penicillin-binding protein (pbp) and a transmembrane protein (cmcT) that appears to be involved in cephamycin exportation, are clustered together with the biosynthetic genes in the cephamycin clusters of S. clavuligerus and N. lactamdurans. Availability of the cloned genes allows metabolic engineering of the β-lactam biosynthetic pathways such as a channelling precursors and directed removal of bottlenecks in the β-lactam biosynthetic pathways. Several new β-lactam antibiotics have been discovered in gram-positive and gram-negative bacteria that will provide new genes for combinatorial synthesis of new molecules.

Mutants blocked in penicillin biosynthesis show a deletion of the entire penicillin gene cluster at a specific site within a conserved hexanucleotide sequence

The organization of the genes of the penicillin cluster has been studied in three different mutants of P. chrysogenum impaired in penicillin biosynthesis. The three blocked mutants (derived from the parental strain P. chrysogenum Bb-1) lacked the genes pcbAB, pcbC and penDE of the penicillin biosynthetic pathway and were unable to form isopenicillin N synthase and isopenicillin N acyltransferase. All strains were identified as P. chrysogenum derivatives by fingerprinting analysis with (GTG)n as a probe. The borders of the deleted region were cloned and sequenced, showing the same junction point in the three mutants. The deleted DNA region was found to be identical to that described in P. chrysogenum npe10. The frequent deletion of the pen gene cluster at this point may indicate that this cluster is located in an unstable genetic region, flanked by hot spots of recombination, that is easily lost by mutagen-induced recombination.

Selection and characterization of pyrG mutants of Penicillium chrysogenum lacking orotidine-5′-phosphate decarboxylase and complementation by the pyr4 gene of Neurospora crassaa

SummaryPyrimidine auxotrophs of Penicillium chrysogenum have been isolated at a high frequency among mutants resistant to 5-fluoroorotic acid (5.2 mM). Some of the pyrimidine auxotrophs (e.g. strain pyrG1) showed no reversion. A radiometric assay based on the conversion of (6-14C)orotidine 5′-monophosphate (OMP) into (6-14C)uridine 5′-monophosphate (UMP) was developed to determine OMP-decarboxylase activity. One of the pyrimidine auxotrophs (P. chrysogenum pyrGl) was studied in detail. It was deficient in OMP-decarboxylase activity, whereas the parental strain (P. chrysogenum Wis. 54-1255) showed a normal enzyme activity. A five-fold higher OMP-decarboxylase activity was found in a P. chrysogenum pyrGI clone transformed with plasmids containing the Neurospora crassa pyr4 gene (which codes for the same enzyme).

The cefT gene of Acremonium chrysogenum C10 encodes a putative multidrug efflux pump protein that significantly increases cephalosporin C production.

Abstract. Transcriptional analysis of the region downstream of the pcbAB gene (which encodes the α-aminoadipyl-cysteinyl-valine synthetase involved in cephalosporin synthesis) of Acremonium chrysogenum revealed the presence of two different transcripts corresponding to two new ORFs. ORF3 encodes a putative D-hydroxyacid dehydrogenase and cefT (for transmembrane protein) encodes a multidrug efflux pump belonging to the Major Faciltator Superfamily (MFS) of membrane proteins. The CefT protein has 12 transmembrane segments (TMS) and contains motifs A, B, C, D2 and G characteristic of the Drug:H+ antiporter 12-TMS group of the major facilitator superfamily. The CefT protein confers resistance to some toxic organic acids, including isovaleric acid and phenylacetic acid. Targeted inactivation of ORF3 and cefT by gene replacement showed that they are not essential for cephalosporin biosynthesis. However, amplification of the cefT gene results in increments of up to 100% in cephalosporin production in the A. chrysogenum C10 strain. Amplification of a truncated form of the cefT insert did not lead to cephalosporin overproduction. It seems that the CefT protein is involved in cephalosporin export from A. chrysogenum or in transmembrane signal transduction, and that there are redundant systems involved in cephalosporin export.

Expression of the cefG gene is limiting for cephalosporin biosynthesis in Acremonium chrysogenum

Abstract The conversion of deacetylcephalosporin C to cephalosporin C is inefficient in most Acremonium chrysogenum strains. The cefG gene, which encodes deacetylcephalosporin C acetyltransferase, is expressed very poorly in A. chrysogenum as compared to other genes of the cephalosporin pathway. Introduction of additional copies of the cefG gene with its native promoter (in two different constructions with upstream regions of 1056 bp and 538 bp respectively) did not produce a significant increase of the steady-state level of the cefG transcript. Expression of the cefG gene from the promoters of (i) the glyceraldehyde-3-phosphate dehydrogenase (gpd ) gene of Aspergillus nidulans, (ii) the glucoamylase (gla) gene of Aspergillus niger, (iii) the glutamate dehydrogenase (gdh) and (iv) the isopenicillin N synthase ( pcbC ) genes of Penicillium chrysogenum, led to very high steady-state levels of cefG transcript and to increased deacetylcephalosporin-C acetyltransferase protein concentration (as shown by immunoblotting) and enzyme activity in the transformants. Southern analysis showed that integration of the new constructions occurred at sites different from that of the endogenous cefG gene. Cephalosporin production was increased two- to threefold in A. chrysogenum C10 transformed with constructions in which the cefG gene was expressed from the gdh or gpd promoters as a result of a more efficient acetylation of deacetylcephalosporin C.

Cloning, expression in Streptomyces lividans and biochemical characterization of a thermostable endo-β-1,4-xylanase of Thermomonospora alba UL JB1 with cellulose-binding ability

Abstract Several thermophilic actinomycetes were isolated from urban solid waste. One of them, Thermomonospora alba ULJB1, showed a broad degradative activity on xylan, cellulose, starch and other polymers. Xylanase and cellulase activities were quantified and compared with those of Thermomonospora fusca. Genes encoding two different endo-β-1,4-xylanases were cloned from T.␣alba ULJB1. One of them, xylA, was sequenced, subcloned and overexpressed in Streptomyces lividans. It encodes a protein of 482 amino acids with a deduced molecular mass of 48 456 Da. The protein contains a 38-amino-acid leader peptide with six Arg+ residues in its amino-terminal end, a catalytic domain and a cellulose-binding domain connected by a linker region rich in proline and glycine. The XylA protein was purified to near homogeneity from S. lividans/xylA cultures. Two forms of the extracellular xylanase, of 48 kDa and 38 kDa, were produced that differed in their cellulose-binding ability. The 48-kDa protein showed a strong binding to cellulose whereas the 38-kDa form did not bind to this polymer, apparently because of the removal during processing of the cellulose-binding domain. Both forms were able to degrade xylans form different origins but not lichenam or carboxymethylcellulose. The major degradation product was xylobiose with traces of xylose. The xylanase activity was thermostable, showing a good activity up to 95 °C, and had broad pH stability in the range from pH 4.0 to pH 10.0.

The Penicillium chrysogenum extracellular proteome. Conversion from a food-rotting strain to a versatile cell factory for white biotechnology

The filamentous fungus Penicillium chrysogenum is well-known by its ability to synthesize β-lactam antibiotics as well as other secondary metabolites. Like other filamentous fungi, this microorganism is an excellent host for secretion of extracellular proteins because of the high capacity of its protein secretion machinery. In this work, we have characterized the extracellular proteome reference map of P. chrysogenum Wisconsin 54–1255 by two-dimensional gel electrophoresis. This method allowed the correct identification of 279 spots by peptide mass fingerprinting and tandem MS. These 279 spots included 328 correctly identified proteins, which corresponded to 131 different proteins and their isoforms. One hundred and two proteins out of 131 were predicted to contain either classical or nonclassical secretion signal peptide sequences, providing evidence of the authentic extracellular location of these proteins. Proteins with higher representation in the extracellular proteome were those involved in plant cell wall degradation (polygalacturonase, pectate lyase, and glucan 1,3-β-glucosidase), utilization of nutrients (extracellular acid phosphatases and 6-hydroxy-d-nicotine oxidase), and stress response (catalase R). This filamentous fungus also secretes enzymes specially relevant for food industry, such as sulfydryl oxidase, dihydroxy-acid dehydratase, or glucoamylase. The identification of several antigens in the extracellular proteome also highlights the importance of this microorganism as one of the main indoor allergens. Comparison of the extracellular proteome among three strains of P. chrysogenum, the wild-type NRRL 1951, the Wis 54–1255 (an improved, moderate penicillin producer), and the AS-P-78 (a penicillin high-producer), provided important insights to consider improved strains of this filamentous fungus as versatile cell-factories of interest, beyond antibiotic production, for other aspects of white biotechnology.