(1) Field of the Invention
The present invention relates to a modified enzyme of a non-heme iron (II) dependent family of oxygenases and oxidases which renders the enzyme dependent on bicarbonate for activity. In a preferred embodiment, the modification is an arginine, lysine, or other amino acid that is two amino acid residues upstream of a histidine residue that is an iron ligand in the enzyme and is one of the histidine residues of the 2-histidine-1-aspartic acid trifacial motif. In particular, the present invention relates to isopenicillin N synthetase (IPNS), deacetoxycephalosporin C synthetase (DAOCS), and deacetoxycephalosporin C synthetase/deacetylcephalosporin C synthetase (DAOCS/DACS) which are modified to be dependent on bicarbonate for activity. The present invention further relates to organisms that express IPNS and/or DAOCS or DAOCS/DACS having activity dependent on bicarbonate. Finally, the present invention relates to in vivo and in vitro methods for producing antibiotics wherein production is dependent on bicarbonate.
(2) Description of Related Art
β-lactam antibiotics are the largest group of secondary metabolites produced by microorganisms. The penicillins (penam) and cephalosporins (cepham) are the most important of these β-lactams from both a clinical and an economic standpoint. The biosynthesis of these β-lactams occurs by a complex series of enzymatic steps with the first two steps being key for both the biosynthesis of penams and cephams. Afterwards, the pathway for the biosynthesis of penicillins and cephalosporins diverge. The key steps in the biosynthesis of penicillins and cephalosporins are shown in FIG. 1.
The first step in the formation of penams and cephams is the condensation of L-α-amino adipic acid (A), L-cysteine (C), and L-valine (V) by ACV synthetase to form the tripeptide δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (ACV). In the second step, the tripeptide is cyclized in a four electron oxidation of ACV by molecular oxygen catalyzed by isopenicillin synthetase (IPNS) to produce isopenicillin N. IPNS requires dioxygen to form the ferryl form of the enzyme which catalyzes the cyclization reaction. Isopenicillin N contains a β-lactam and thiazolidine ring structure and possess antibacterial activity. To form penicillin G or V, the α-aminoadipic acid side chain of isopenicillin N is exchanged for phenyacetic acid to yield penicillin G or phenoxyacetic acid to yield penicillin V. This reaction is catalyzed by acetyltransferase.
To make the cephams, the A chain of isopenicillin N is racemized in a reaction catalyzed by an epimerase or racemase to form penicillin N. Then the five-member thiazolidine ring in penicillin N is expanded into the six-member dihydrothiazine ring of the cephalosporin nucleus by deacetoxycephalosporin C synthetase/deacetylcephalosporin C synthetase (DAOCS/DACS) or expandase. The reaction requires dioxygen and 2-oxoglutarate (α-ketogluterate) to produce the ferryl form of the enzyme. The same enzyme also catalyzes the subsequent reaction in the pathway which is the hydroxylation of the methyl group at the 3′-position of the ring to form deacetylcephalosporin C. In prokaryotes such as Streptomyces, the deacetoxycephalosporin C synthetase (DAOCS) and deacetylcephalosporin C synthetase (DACS) are encoded by separate genes; however, the prokaryote and eukaryote enzymes are closely related by amino acid sequence. Cephalosporin C is formed from deacetylcephalosporin C by acetylation of the 3′-position. The cephamycins are then formed in several subsequent enzymatic steps.
In the commercial production of antibiotics, a primary concern has been directed towards improving the yield of penams and cephams. Improving the yields in vivo have been achieved primarily by increasing the copy number of IPNS, DAOCS, or DAOCS/DACS or other ancillary enzymes which appear to improve the yield of the penam or cepham in the organism. Other methods for improving yields has been directed towards development of in vitro systems based on the enzymes involved in the biosynthesis of penams and cephams.
The following U.S. Patents disclose methods for improving yields of penams and cephams, both in vivo and in vitro.
U.S. Pat. No. 4,885,251 to Ingolia et al. discloses DNAs encoding IPNS and its flanking regulatory sequences from Cephalosporin acremonium. The isolated gene was used to make novel E. coli expression vectors that drive production of isopenicillin N synthetase in E. coli. 
U.S. Pat. No. 4,885,252 to Ingolia et al. discloses DNAs encoding IPNS from Aspergillus nidulans and its use in the production of β-lactams. In particular, the gene encoding the synthetase can be isolated from plasmid pOGOO4, available from the Northern Regional Research Center under accession number NRRL B-18171.
U.S. Pat. No. 4,892,819 to Carr et al. discloses DNAs containing a gene encoding IPNS and its flanking regulatory sequences from Penicillin chrysogenum. The gene was used to make novel E. coli expression vectors that drive production of IPNS in E. coli. 
U.S. Pat. No. 4,950,603 to Ingolia et al. discloses DNAs containing a gene encoding IPNS from Streptomyces lipmanii, a plasmid pOGO239 containing the gene as NRRL B-18250, and methods for using the gene encoding the synthetase in the production of antibiotics.
U.S. Pat. No. 5,070,020 to Ignolia et al. discloses DNAs containing genes encoding DAOCS activity, recombinant DNA vectors containing the synthetase for expression in a wide variety of host organisms, including E. coli, Penicillium, Streptomyces, Aspergillus, and Cephalosporin.
U.S. Pat. No. 5,462,862 to Groenen et al. discloses DNAs containing genes encoding IPNS, acetyltransferase, and ACV synthetase and transforming host organisms with these DNAs to improve production of an antibiotic. Improvement of production is by increased copy number of the gene in the transformed organism.
U.S. Pat. No. 5,753,435 to Aharonowitz et al. discloses DNAs containing a gene encoding a new oxido reductase activity obtainable from Penicillium chrysogenum involved in the production of β-lactams and provides methods for using the oxido reductase to improve production of β-lactams.
U.S. Pat. No. 5,882,879 to Veenstra et al. discloses DNAs containing genes encoding the ACV synthetase genes from Penicillium chrysogenum and Acremonium chrysogenum, and methods for using clones of the genes to improve antibiotic biosynthesis.
U.S. Pat. No. 5,882,883 to Egel-Mitani et al. provides an improved process for making an antibiotic in a fermentation process by placing a gene encoding a key enzyme in the biosynthesis process under the control of a heterologous promoter which enables the transcription of the gene to be regulatable. This improvement avoids inhibition of transcription of the enzyme by secondary metabolites formed during the synthesis of the antibiotic.
U.S. Pat. No. 5,942,411 to Kaasgaard et al. discloses a method for improving the production of various β-lactam antibiotics in vivo and in vitro by inducing the host organism to express an increased ligase activity, in particular an acetyl-coenzyme A synthetase (ligase).
Other efforts have been directed towards modifying the substrate specificity of expandase to enable biosynthesis of intermediates such as 7-aminodesacetoxycephalosporanic acid (7-ADCA) that can be used in subsequent reactions to make semi-synthetic cephalosporins. For example, U.S. Pat. No. 5,919,680 to Sutherland et al. discloses a process for preparation and recovery of 7-ADCA using a Penicillium chrysogenum transformant expressing a modified expandase that is able to use penicillin G as its substrate.
While the prior art provides methods for improving production of antibiotics by a variety of genetic engineering methods, the prior art has not provided or suggested any means which would enable the activity of an antibiotic biosynthesis enzyme such as IPNS to be dependent on the presence of an activating compound.