Environmentally safe production of 7-aminodeacetoxycephalosporanic acid (7-ADCA) using recombinant strains of Acremonium chrysogenum.

Medically useful semisynthetic cephalosporins are made from 7-aminodeacetoxycephalosporanic acid (7-ADCA) or 7-aminocephalosporanic acid (7-ACA). Here we describe a new industrially amenable bioprocess for the production of the important intermediate 7-ADCA that can replace the expensive and environmentally unfriendly chemical method classically used. The method is based on the disruption and one-step replacement of the cefEF gene, encoding the bifunctional expandase/hydroxylase activity, of an actual industrial cephalosporin C production strain of Acremonium chrysogenum. Subsequent cloning and expression of the cefE gene from Streptomyces clavuligerus in A. chrysogenum yield recombinant strains producing high titers of deacetoxycephalosporin C (DAOC). Production level of DAOC is nearly equivalent (75-80%) to the total beta-lactams biosynthesized by the parental overproducing strain. DAOC deacylation is carried out by two final enzymatic bioconversions catalyzed by D-amino acid oxidase (DAO) and glutaryl acylase (GLA) yielding 7-ADCA. In contrast to the data reported for recombinant strains of Penicillium chrysogenum expressing ring expansion activity, no detectable contamination with other cephalosporin intermediates occurred.

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

A gene, aat, encoding acyl-CoA: 6-aminopenicillanic acid acyltransferase (AAT), the last enzyme of the penicillin (Pn) biosynthetic pathway, has been cloned from the genome of Penicillium chrysogenum AS-P-78. The gene contains three introns in the 5'-region and encodes a protein of 357 amino acids with an Mr of 39,943. It complements mutants of P. chrysogenum deficient in AAT activity. The aat gene is expressed as a 1.15-kb transcript and the encoded protein appears to be processed post-translationally into two nonidentical polypeptides of 102 and 255 aa, with Mrs of 11,498 and 28,461, respectively. Three proteins of 40, 11, and 29 kDa (the last one corresponding to the previously purified AAT), were identified in extracts of P. chrysogenum. The aa sequence of the N-terminal end of the 11-kDa polypeptide matched the nucleotide (nt) sequence of the 5'-region of aat. The N-terminal end of the 29-kDa polypeptide corresponded to the sequence beginning at nt position 916 of the sequenced DNA fragment (nt 441 of aat gene). The aat gene of P. chrysogenum resembles the genes encoding Pn acylases of Escherichia coli, Proteus rettgeri and Pseudomonas sp., all of which encode two nonidentical subunits derived from a common precursor, encoded by a single open reading frame.

Conjugation and transformation of Streptomyces species by tylosin resistance.

The tlrB gene from Streptomyces fradiae has been cloned and used to construct bifunctional Streptomyces-Escherichia coli shuttle vectors carrying the antibiotic resistance genes to kanamycin-neomycin, thiostrepton and tylosin as selection markers. In the same way, the tlrB gene was subcloned in plasmids including the apramycin resistance gene and the oriT sequence from the plasmid pSET152 to facilitate conjugation of Streptomyces spores. The usefulness of the tlrB gene as tylosin resistance marker was ascertained in Streptomyces lividans, Streptomyces parvulus and Streptomyces coelicolor, but not in Streptomyces clavuligerus. The tlrB gene constitutes a useful selection marker when high-frequency of conjugation/transformation is not required or as secondary marker in recombinant Streptomyces species where thiostrepton and kanamycin have been utilized for primary selection.

The ddcA gene from Streptomyces fradiae encodes an extracellular beta-lactamase with penicillinase and cephalosporinase activities.

The ddcA gene from Streptomyces fradiae, which is located adjacent to the left edge of the tylosin biosynthetic cluster, has been cloned and sequenced. DNA sequence analysis revealed an ORF of 1194 bp that encodes a product of 42.6 kDa. This protein showed significant similarity to the extracellular endopeptidase with beta-lactamase activity encoded by the adp gene from Bacillus cereus and to PBPs (DD-carboxypeptidases and DD-endopeptidases) and beta-lactamases. Moreover, it contains three characteristic motifs conserved in PBPs and beta-lactamases, including an essential serine residue in the active centre and a putative leader peptide. Heterologous expression of the ddcA gene in Streptomyces lividans demonstrated the presence in the transformants of an extracellular beta-lactamase active against penicillin G, ampicillin and the chromogenic cephalosporin nitrocefin.

Selective adsorption of poly-His tagged glutaryl acylase on tailor-made metal chelate supports.

A poly-His tag was fused in the glutaryl acylase (GA) from Acinetobacter sp. strain YS114 cloned in E. coli yielding a fully active enzyme. Biochemical analyses showed that the tag did not alter the maturation of the chimeric GA (poly-His GA) that undergoes a complex post-translational processing from an inactive monomeric precursor to the active heterodimeric enzyme. This enzyme has been used as a model to develop a novel and very simple procedure for one-step purification of poly-His proteins via immobilized metal-ion affinity chromatography on tailor-made supports. It was intended to improve the selectivity of adsorption of the target protein on tailor-made chelate supports instead of performing a selective desorption. The rate and extent of the adsorption of proteins from a crude extract from E. coli and of pure poly-His tagged GA on different metal chelate supports was studied. Up to 90% of proteins from E. coli were adsorbed on commercial chelate supports having a high density of ligands attached to the support through long spacer arms, while this adsorption becomes almost negligible when using low ligand densities, short spacer arms and Zn2+ or Co2+ as cations. On the contrary, poly-His GA adsorbs strongly enough on all supports. A strong affinity interaction between the poly-His tail and a single chelate moiety seems to be the responsible for the adsorption of poly-His GA. By contrast, multipoint weak interactions involving a number of chelate moieties seem to be mainly responsible for adsorption of natural proteins. By using tailor-made affinity supports, a very simple procedure for one-step purification of GA with minimal adsorption of host proteins could be performed. Up to 20 mg of GA were adsorbed on each ml of chelate support while most of accompanying proteins were hardly adsorbed on such supports. Following few washing steps, the target enzyme was finally recovered (80% yield) by elution with 50 mM imidazole with a very high increment of specific activity (up to a 120 purification factor).

Genes directly involved in the biosynthesis of beta-lactam antibiotics.

Several genes encoding enzymatic activities involved in penicillin and cephalosporin biosynthesis have been identified. The first two steps in the biosynthesis of both antibiotics are common in penicillin, cephalosporin and cephamycin producers: condensation of the three precursor amino acids to form the tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine, and oxidative cyclization of the tripeptide to form isopenicillin N. The genes involved in the two steps are pcbAB and pcbC respectively. The conversion of isopenicillin N to penicillin G is carried out by the enzyme isopenicillin N:6-APA acyltransferase encoded by the gene penDE. The biosynthesis of cephalosporin diverges from that of penicillin G at the isopenicillin N level. The isopenicillin N is first isomerized to penicillin N by an epimerase that is encoded by the gene cefD. The penicillin N is converted in deacetoxycephalosporin C by an expansion of the five-membered thiazolidine ring to the six-membered dihydrothiazine ring. The deacetoxycephalosporin C is finally converted into cephalosporin C by a hydroxylation and O-acetylation. The enzymes which catalyze these last three steps are encoded by the genes cefE, cefF and cefG. The penicillin, cephalosporin and cephamycin biosynthetic genes are organized in clusters (and subclusters) of genes.

Expression of the penDE gene of Penicillium chrysogenum encoding isopenicillin N acyltransferase in Cephalosporium acremonium: production of benzylpenicillin by the transformants.

No DNA sequence homologous to the penDE gene of Penicillium chrysogenum was found in the genome of three different strains of Cephalosporium acremonium. The pcbC-penDE gene cluster of P. chrysogenum complemented the isopenicillin N synthase deficiency of C. acremonium mutant N2 and resulted in the production of penicillin, in addition to cephalosporin, in cultures supplemented with phenylacetic acid. The penicillin formed was identified as benzylpenicillin by HPLC and NMR studies. The penDE gene of P. chrysogenum is expressed in C. acremonium forming a transcript of 1.15 kb. The transcript is processed and translated in C. acremonium resulting in the formation of acyl CoA: isopenicillin N acyl transferase. When the penDE gene was introduced into a cephalosporin producing strain, the total titre of beta-lactam antibiotics comprised distinct proportions of penicillin and cephalosporin in different transformants. Analysis of the hybridization patterns of the DNA of C. acremonium transformed with the pcbC or penDE genes indicated that integration occurs by non-homologous recombination.

The NADP-dependent glutamate dehydrogenase gene from Penicillium chrysogenum and the construction of expression vectors for filamentous fungi.

The gdhA gene encoding the NADP-dependent glutamate dehydrogenase activity from Penicillium chrysogenum has been isolated and characterized for its use in gene expression. The nucleotide sequence of a 2816-bp genomic fragment was determined, showing an open reading frame of 1600 bp interrupted by two introns, of 160 bp and 57 bp respectively, with fungal consensus splice-site junctions. The predicted amino acid sequence revealed a high degree of identity to glutamate dehydrogenase enzymes, especially to those from the fungi Aspergillus nidulans (82%) and Neurospora crassa (78%). The gdhA gene was found to be present in a single copy in the genome of several P. chrysogenum strains with different penicillin productivity. The use of the gdhA promoter for homologous and heterologous gene expression in fungi and Escherichia coli was analyzed. Heterologous gene expression was ascertained by the construction of gene fusions with the lacZ gene from E. coli and the bleomycin-resistance determinant (bleR) from Streptoalloteichus hindustanus. Homologous gene expression was shown through the use of the penicillin-biosynthetic genes pchC and penDE from P. chrysogenum and the cephalosporin biosynthetic genes cefEF and cefG from Acremonium chrysogenum.

Purification to homogeneity and characterization of acyl coenzyme A:6-aminopenicillanic acid acyltransferase of Penicillium chrysogenum.

The acyl coenzyme A (CoA):6-aminopenicillanic acid (6-APA) acyltransferase of Penicillium chrysogenum AS-P-78 was purified to homogeneity, as concluded by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The enzyme is a monomer with a molecular weight of 30,000 +/- 1,000 and a pI of about 5.5. The optimal pH and temperature were 8.0 and 25 degrees C, respectively. This enzyme converts 6-APA into penicillin by using phenylacetyl CoA or phenoxyacetyl CoA as acyl donors. The pure enzyme showed a high specificity and affinity for 6-APA and did not accept benzylpenicillin, 7-aminocephalosporanic acid, cephalosporin C, or isocephalosporin C as substrates. The enzyme converted isopenicillin N into penicillin G, although with a lower efficiency than when 6-APA was used as the substrate. It did not show penicillin G acylase activity. The acyl CoA:6-APA acyltransferase required dithiothreitol or other thiol-containing compounds, and it was protected by thiol-containing reagents against thermal inactivation. The acyltransferase was inhibited by several divalent and trivalent cations and by p-chloromercuribenzoate and N-ethylmaleimide. The activity was absent in four different mutants that were blocked in penicillin biosynthesis.

Recombinant Acremonium chrysogenum strains for the industrial production of cephalosporin.

Conventional strain improvement programs based on random mutagenesis and rational screening have meant valuable results to the antibiotic producing companies. The development of recombinant DNA techniques and their applications to the industrially-used cephalosporin-producing fungus Acremonium chrysogenum has provided a new tool, complementary to classical mutation, promoting the design of alternative biosynthetic pathways making it possible to obtain new antibiotics and to improve cephalosporin production. Yield increases have been achieved by increasing the dosage of the biosynthetic genes cefEF (deacetoxycephalosporin C expandase/hydroxylase) and cefG (deacetylcephalosporin C acetyltransferase) or enhancing the oxygen uptake by expressing a bacterial oxygen-binding heme protein (Vitreoscilla hemoglobin). New biosynthetic capacities such as the production of 7-aminocephalosporanic acid (7-ACA) or penicillin G have been achieved through the expression of the foreign genes dao (D-amino acid oxidase) coupled with cephalosporin acylase or penDE(acyl-CoA:6-APA acyltransferase) respectively. Confined manipulation of the above-mentioned recombinant strains must be performed according to standing rules.

Recombinant microorganisms for industrial production of antibiotics.

The enhancement of industrial antibiotic yield has been achieved through technological innovations and traditional strain improvement programs based on random mutation and screening. The development of recombinant DNA techniques and their application to antibiotic producing microorganisms has allowed yield increments and the design of biosynthetic pathways giving rise to new antibiotics. Genetic manipulations of the cephalosporin producing fungus Cephalosporium acremonium have included yield improvements, accomplished increasing biosynthetic gene dosage or enhancing oxygen uptake, and new biosynthetic capacities as 7-aminocephalosporanic acid (7-ACA) or penicillin G production. Similarly, in Penicillium chrysogenum, the industrial penicillin producing fungus, heterologous expression of cephalosporin biosynthetic genes has led to the biosynthesis of adipyl-7-aminodeacetoxycephalosporanic acid (adipyl-7-ADCA) and adipyl-7-ACA, compounds that can be transformed into the economically relevant 7-ADCA and 7-ACA intermediates. Escherichia coli expression of the genes encoding D-amino acid oxidase and cephalosporin acylase activities has simplified the bioconversion of cephalosporin C into 7-ACA, eliminating the use of organic solvents. The genetic manipulation of antibiotic producing actinomycetes has allowed productivity increments and the development of new hybrid antibiotics. A legal framework has been developed for the confined manipulation of genetically modified organisms.

The manganese superoxide dismutase from the penicillin producer Penicillium chrysogenum.

The antioxidant enzyme superoxide dismutase has been studied in order to define mechanisms for the influence of oxygen on penicillin production. Manganese-containing SOD activity was purified from penicillin-producing cultures of the filamentous fungus Penicillium chrysogenum and reverse genetics was used to identify full-length cDNA and genomic clones. Sequence analysis revealed a 630-bp ORF containing three exons and two introns with fungal consensus splice-site junctions. The deduced amino-acid sequence (210 amino acids; 23.13 kDa) includes conserved residues required for enzymatic activity and metal binding, and shares significant similarity with Mn- and Fe-containing superoxide dismutases. The sod gene is present as a single copy in the genome of different P. chrysogenum strains and its expression level is not correlated with penicillin-G productivity.

Glucokinase-deficient mutant of Penicillium chrysogenum is derepressed in glucose catabolite regulation of both beta-galactosidase and penicillin biosynthesis.

One glucokinase-deficient mutant (glk1) of Penicillium chrysogenum AS-P-78 was isolated after germ tube-emitting spores were mutated with nitrosoguanidine and selected for growth on lactose-containing medium in the presence of inhibitory concentrations of D-2-deoxyglucose (3 mM). Penicillin biosynthesis was greatly reduced (55%) in D-glucose-grown cultures of the parental strain, but this sugar had no repressive effect on the rate of penicillin biosynthesis in the mutant glk1. This mutant was deficient in ATP-dependent glucokinase and showed a greatly reduced uptake of D-glucose. The parental strain P. chrysogenum AS-P-78 showed in vitro ATP-dependent phosphorylating activities of D-glucose, D-2-deoxyglucose, and D-galactose. The glk1 mutant was deficient in the in vitro phosphorylation of D-glucose and D-2-deoxyglucose but retained a normal D-galactose-phosphorylating activity. D-Glucose repressed both beta-galactosidase and isopenicillin-N-synthase but not acyl coenzyme A:6-aminopenicillanic acid acyltransferase in the parental strain. The glucokinase-deficient mutant was simultaneously derepressed in carbon catabolite regulation of beta-galactosidase and isopenicillin-N-synthase, suggesting that a common regulatory mechanism is involved in carbon catabolite regulation of both sugar utilization and penicillin biosynthesis.

Large amplification of a 35-kb DNA fragment carrying two penicillin biosynthetic genes in high penicillin producing strains of Penicillium chrysogenum.

The isopenicillin N synthase (pcbC) and acyl-CoA:6-APA acyltransferase (penDE) genes of Penicillium chrysogenum were located in a 19.5-kb DNA fragment that had been previously cloned in phage vector EMBL3. This 19.5-kb DNA fragment was mapped with several endonucleases, and the pcbC and penDE genes were located by hybridization with probes corresponding to internal fragments of each gene. A low penicillin producing strain (P. chrysogenum Wis 54-1255) and two high producing strains (AS-P-78 and P2) showed hybridizing fragments of identical sizes in their chromosomes. Dot-blot hybridization of serial dilutions of the total DNA of the three strains showed that the intensity of all the hybridizing bands was much higher in strains AS-P-78 and P2 than in Wis 54-1255. Hybridization of total DNA digestions with probes corresponding to fragments which mapped upstream or downstream of the pcbC-penDE region revealed that a fragment of at least 35 kb DNA has been amplified 9 to 14 fold in the high penicillin producing strains. The amplified region did not include the previously cloned pyrG gene that encodes OMP-decarboxylase, an enzyme involved in pyrimidine biosynthesis.

Cloning, characterization of the acyl-CoA:6-amino penicillanic acid acyltransferase gene of Aspergillus nidulans and linkage to the isopenicillin N synthase gene.

The penDE gene encoding acyl-CoA:6-amino penicillanic acid acyltransferase (AAT), the last enzyme of the penicillin biosynthetic pathway, has been cloned from the DNA of Aspergillus nidulans. The gene contains three introns which are located in the 5' region of the open reading frame. It encodes a protein of 357 amino acids with a molecular weight of 39,240 Da. The penDE gene of A. nidulans shows 73% similarity at the nucleotide level with the penDE gene of Penicillium chrysogenum. The A. nidulans gene was expressed in P. chrysogenum and complemented the AAT deficiency of the non-producer mutants of P. chrysogenum, npe6 and npe8. The penDE gene of A. nidulans is linked to the pcbC gene, which encodes the isopenicillin N synthase, as also occurs in P. chrysogenum. Both genes show the same orientation and are separated by an intergenic region of 822 nucleotides.

Cloning, sequence analysis and transcriptional study of the isopenicillin N synthase of Penicillium chrysogenum AS-P-78.

A gene (ips) encoding the isopenicillin N synthase of Penicillium chrysogenum AS-P-78 was cloned in a 3.9 kb SalI fragment using a probe corresponding to the amino-terminal end of the enzyme. The SalI fragment was trimmed down to a 1.3 kb NcoI-BglII fragment that contained an open reading frame of 996 nucleotides encoding a polypeptide of 331 amino acids with an Mr of 38012 dalton. The predicted polypeptide encoded by the ips gene of strain AS-P-78 contains a tyrosine at position 195, whereas the gene of the high penicillin producing strain 23X-80-269-37-2 shows an isoleucine at the same position. The ips gene is expressed in Escherichia coli minicells using the lambda phage PL promoter. Some similar sequence motifs were found in the upstream region of the ips gene of P. chrysogenum when compared with the upstream sequences of the ips genes of Cephalosporium acremonium and Aspergillus nidulans. Primer extension studies indicated that the start of the mRNA coincides with a T in position -11 which is located in a conserved pyrimidine-rich sequence, near two CAAG boxes. Clones of P. chrysogenum Wis 54-1255 transformed with the ips gene showed a five-fold higher isopenicillin N synthase activity than the untransformed cultures.

The isopenicillin-N acyltransferase of Penicillium chrysogenum has isopenicillin-N amidohydrolase, 6-aminopenicillanic acid acyltransferase and penicillin amidase activities, all of which are encoded by the single penDE gene.

The isopenicillin-N acyltransferase of Penicillium chrysogenum catalyzes the conversion of the biosynthetic intermediate isopenicillin N to the hydrophobic penicillins. The isopenicillin-N acyltransferase copurified with the acyl-CoA:6-aminopenicillanic acid (6-APA) acyltransferase activity which transfers an acyl residue from acyl-CoA derivatives (e.g. phenylacetyl-CoA, phenoxyacetyl-CoA) to 6-APA. Other thioesters of phenylacetic acid were also used as substrates. An amino acid sequence similar to that of the active site of thioesterases was found in the isopenicillin-N acyltransferase, suggesting that this site is involved in the transfer of phenylacetyl residues from phenylacetyl thioesters. Purified isopenicillin-N acyltransferase also showed isopenicillin-N amidohydrolase, penicillin transacylase and penicillin amidase activities. The isopenicillin-N amidohydrolase (releasing 6-APA) showed a much lower specific activity than the isopenicillin-N acyltransferase of the same enzyme preparation, suggesting that in the isopenicillin-N acyltransferase reaction the 6-APA is not released and is directly converted into benzylpenicillin. Penicillin transacylase exchanged side chains between two hydrophobic penicillin molecules; or between one penicillin molecule and 6-APA. The penicillin amidase activity is probably the reverse of the biosynthetic acyl-CoA:6-APA acyltransferase. Four P. chrysogenum mutants deficient in acyl-CoA:6-APA acyltransferase lacked the other four related activities. Transformation of these mutants with the penDE gene restored all five enzyme activities.

The penicillin gene cluster is amplified in tandem repeats linked by conserved hexanucleotide sequences.

The penicillin biosynthetic genes (pcbAB, pcbC, penDE) of Penicillium chrysogenum AS-P-78 were located in a 106.5-kb DNA region that is amplified in tandem repeats (five or six copies) linked by conserved TTTACA sequences. The wild-type strains P. chrysogenum NRRL 1951 and Penicillium notatum ATCC 9478 (Fleming's isolate) contain a single copy of the 106.5-kb region. This region was bordered by the same TTTACA hexanucleotide found between tandem repeats in strain AS-P-78. A penicillin overproducer strain, P. chrysogenum E1, contains a large number of copies in tandem of a 57.9-kb DNA fragment, linked by the same hexanucleotide or its reverse complementary TGTAAA sequence. The deletion mutant P. chrysogenum npe10 showed a deletion of 57.9 kb that corresponds exactly to the DNA fragment that is amplified in E1. The conserved hexanucleotide sequence was reconstituted at the deletion site. The amplification has occurred within a single chromosome (chromosome I). The tandem reiteration and deletion appear to arise by mutation-induced site-specific recombination at the conserved hexanucleotide sequences.

Extracellular production of biologically active deacetoxycephalosporin C synthase from Streptomyces clavuligerus in Pichia pastoris.

We have successfully expressed and observed secretion of the Streptomyces clavuligerus deacetoxycephalosporin C synthase (DAOCS) using the Pichia pastoris expression system. Two clones having multiple copies of the expression cassette were selected and used for protein-expression analysis. SDS-PAGE showed efficient expression and secretion of the bacterial recombinant DAOCS. The highest yield (120 microg/mL) was obtained when expression was induced with 2% methanol. Free and immobilized protein were assayed for biological activity and found to expand penicillin N (its natural substrate) and penicillin G to deacetoxycephalosporin C (DAOC) and deacetoxycephalosporin G (DAOG), respectively.

Reduced function of a phenylacetate-oxidizing cytochrome p450 caused strong genetic improvement in early phylogeny of penicillin-producing strains.

The single-copy pahA gene from Penicillium chrysogenum encodes a phenylacetate 2-hydroxylase that catalyzes the first step of phenylacetate catabolism, an oxidative route that decreases the precursor availability for penicillin G biosynthesis. PahA protein is homologous to cytochrome P450 monooxygenases involved in the detoxification of xenobiotic compounds, with 84% identity to the Aspergillus nidulans homologue PhacA. Expression level of pahA displays an inverse correlation with the penicillin productivity of the strain and is subject to induction by phenylacetic acid. Gene expression studies have revealed a reduced oxidative activity of the protein encoded by pahA genes from penicillin-overproducing strains of P. chrysogenum compared to the activity conferred by phacA of A. nidulans. Sequencing and expression of wild-type pahA from P. chrysogenum NRRL 1951 revealed that an L181F mutation was responsible for the reduced function in present industrial strains. The mutation has been tracked down to Wisconsin 49-133, a mutant obtained at the Department of Botany of the University of Wisconsin in 1949, at the beginning of the development of the Wisconsin family of strains.