[reaction: see text] Enzymatic approaches to prepare sugar nucleotides are gaining in importance and offer several advantages over chemical synthesis including high yields and stereospecificity. We report the cloning, expression, and purification of two new wild-type thymidylyltransferases and observed catalysis with a wide variety of substrates. Significant product inhibition was not observed with the enzymes studied over a 24 h period, enabling the efficient preparation of 15 sugar nucleotides, clearly demonstrating the synthetic utility of these biocatalysts.

Hybridizing fragments in the genomic DNA of Streptomyces venezuelae lSP5230, which produces the jadomycin group of angucycline antibiotics, were detected by probing with act/ DNA from Streptomyces coelicolor A3(2). The hybridizing regions were isolated from a 16.5 kb insert of S. venezuelae DNA recovered from a genomic library cloned in a A replacement vector. Subcloning and sequencing of a 4 8 kb segment of the insert, containing regions hybridizing to act/// as well as act/, identified five open reading frames (ORFs). The deduced polypeptide products of the ORFs closely resemble in sequence the components of streptomycete t y p e 4 polyketide synthases (PKSs) : the ORFI product corresponds to the ketoacyl synthase, and the ORF2 product to a polypeptide closely related to the ketoacyl synthase and involved in determining chain length; the ORF3 product matches the acyl carrier protein; ORF4 encodes a bifunctional cyclase/dehydrase; and ORF5 encodes a ketoreductase. Integration into the chromosomal DNA of a plasmid containing a segment of the ORF2-ORF4 region severely depressed jadomycin B biosynthesis; since the integrant showed no change in growth or spore pigmentation, the cloned PKS genes are presumed to encode enzymes in the pathway for jadomycin biosynthesis.

An approximately 12.5-kbp region of DNA sequence from beyond the end of the previously described clavulanic acid gene cluster was analyzed and found to encode nine possible open reading frames (ORFs). Involvement of these ORFs in clavulanic acid biosynthesis was assessed by creating mutants with defects in each of the ORFs. orf12 and orf14 had been previously reported to be involved in clavulanic acid biosynthesis. Now five additional ORFs are shown to play a role, since their mutation results in a significant decrease or total absence of clavulanic acid production. Most of these newly described ORFs encode proteins with little similarity to others in the databases, and so their roles in clavulanic acid biosynthesis are unclear. Mutation of two of the ORFs, orf15 and orf16, results in the accumulation of a new metabolite, N-acetylglycylclavaminic acid, in place of clavulanic acid. orf18 and orf19 encode apparent penicillin binding proteins, and while mutations in these genes have minimal effects on clavulanic acid production, their normal roles as cell wall biosynthetic enzymes and as targets for ␤-lactam antibiotics, together with their clustered location, suggest that they are part of the clavulanic acid gene cluster.

The Streptomyces clavuligerus clavam gene cluster was examined to identify genes specifically involved in 5S clavam biosynthesis. A reduction/loss of 5S clavam production was seen in cvm2 and cvm5 gene mutants, and a clavam metabolite not previously observed, 2-carboxymethylideneclavam, accumulated in the cvm5 mutant. Disruption of additional genes from the region of the clavam cluster did not have any effect on 5S clavam production. Examination of the paralog gene cluster region for 5S clavam biosynthetic genes led to the identification of cvm6P and cvm7P, which encode a putative aminotransferase and a transcriptional regulator, respectively. Mutants defective in cvm6P and cvm7P were completely blocked in 5S clavam but not clavulanic acid production. The loss of 5S clavam production in cvm7P mutants suggests that this gene encodes a transcriptional regulator specific for 5S clavam metabolite biosynthesis.

No abstract

A 6.5 kb DNA fragment containing a chloramphenicol-resistance gene of Streptomyces venezuelae ISP5230 was cloned in Streptomyces fitlidans M252 using the high-copy-number plasmid vector pIJ702. The gene was located within a 2.4 kb KpnI-SstI fragment of the cloned DNA and encoded an enzyme (chloramphenicol hydrolase) that catalysed removal of the dichloroacetyl moiety from the antibiotic. The deacylated product, p-nitrophenylserinol, was metabolized to p-nitrobenzyl alcohol and other compounds by enzymes present in S. f i v i h s M252. Examination of the genomic DNA from several sources using the cloned 6.5 kb SstI fragment from S. venezuefue ISP5230 as a probe showed a hybridizing region in the DNA from S. venezuelae 13s but none in the DNA from another chloramphenicol producer, Streptomycesphaeochromogenes NRRLB 3559. The resistance phenotype was not expressed when the 6.5 kb SstI fragment or a subfragment was subcloned behind the lac-promoter of plasmid pTZ18R in Escherichia cofi.

Portions of the Streptomyces clavuligerus chromosome flanking cas1, which encodes the clavaminate synthase 1 isoenzyme (CAS1), have been cloned and sequenced. Mutants of S. clavuligerus disrupted in cvm1, the open reading frame located immediately upstream of cas1, were constructed by a gene replacement procedure. Similar techniques were used to generate S. clavuligerus mutants carrying a deletion that encompassed portions of the two open reading frames,cvm4 and cvm5, located directly downstream ofcas1. Both classes of mutants still produced clavulanic acid and cephamycin C but lost the ability to synthesize the antipodal clavam metabolites clavam-2-carboxylate, 2-hydroxymethyl-clavam, and 2-alanylclavam. These results suggested that cas1 is clustered with genes essential and specific for clavam metabolite biosynthesis. When a cas1 mutant of S. clavuligerus was constructed by gene replacement, it produced lower levels of both clavulanic acid and most of the antipodal clavams except for 2-alanylclavam. However, a double mutant of S. clavuligerus disrupted in both cas1 andcas2 produced neither clavulanic acid nor any of the antipodal clavams, including 2-alanylclavam. This outcome was consistent with the contribution of both CAS1 and CAS2 to a common pool of clavaminic acid that is shunted toward clavulanic acid and clavam metabolite biosynthesis.

Cephamycin C production was blocked in wild-type cultures of the clavulanic acid-producing organism Streptomyces clavuligerus by targeted disruption of the gene (lat) encoding lysine -aminotransferase. Specific production of clavulanic acid increased in the lat mutants derived from the wild-type strain by 2-to 2.5-fold. Similar beneficial effects on clavulanic acid production were noted in previous studies when gene disruption was used to block the production of the non-clavulanic acid clavams produced by S. clavuligerus. Therefore, mutations in lat and in cvm1, a gene involved in clavam production, were introduced into a high-titer industrial strain of S. clavuligerus to create a double mutant with defects in production of both cephamycin C and clavams. Production of both cephamycin C and non-clavulanic acid clavams was eliminated in the double mutant, and clavulanic acid titers increased about 10% relative to those of the parental strain. This represents the first report of the successful use of genetic engineering to eliminate undesirable metabolic pathways in an industrial strain used for the production of an antibiotic important in human medicine.