SummaryThe soluble, diffusible red-brown pigment produced by a Saccharopolyspora erythraea 'red variant' has been shown to contain glycosylated and polymerized derivatives of 2,5,7-trihydroxy-1,4-naphthoquinone (flaviolin). Flaviolin is a spontaneous oxidation product of 1,3,6,8-tetrahydroxynaphthalene (THN), which is biosynthesized in bacteria by a chalcone synthase-like (CS-like) type III polyketide synthase (PKS). A fragment of the gene responsible for THN biosynthesis in S. erythraea E_8-7 was amplified by polymerase chain reaction (PCR) using degenerate primers based on conserved regions of known plant CS and bacterial CS-like genes. From the isolated fragment, a suicide vector was prepared, which was subsequently used to disrupt the red-brown pigmentproducing (rpp) locus in S. erythraea, generating a mutant that displayed an albino phenotype. Chromosomal DNA from the albino mutant was subsequently used in a vector-recapture protocol to isolate a plasmid that contained an insert spanning the entire rpp locus. Sequencing of the insert revealed that the disrupted open reading frame (ORF) encodes a CSlike protein displaying 69% sequence identity to the rppA gene of Streptomyces griseus. The S. griseus rppA gene encodes RppA, the first characterized bacterial CS-like protein, which is sufficient in vitro for the synthesis of THN from malonyl-CoA. The rppA disruption mutant and rppA sequence provided a means by which to address the mechanism of diffusible pigment biosynthesis, as well as to investi-

The gene (cefG) encoding the acetyl coenzyme A.deacetylcephalosporin C acetyltransferase of Cephalosporium acremonium (synonym Acremonium chrysogenum) C10 has been cloned. It contains two introns and encodes a protein of 444 amino acids with an Mr of 49,269 that correlates well with the Mr deduced by gel filtration. The cefG gene is linked to the ceJEF gene (encoding the bifunctional deacetoxycephalosporin C synthase/hydroxylase), but it is expressed in an orientation opposite that of the ceJEF gene. Two transcripts of 1.2 and 1.4 kb were found in C. acremonium that correspond to the cefEF and cefG genes, respectively; the degree of expression of the ceiG gene was clearly lower than that of the ceJEF gene in 48-h cultures. The cloned cefG complemented the deficiency of deacetylcephalosporin acetyltransferase in the nonproducer mutant C.acremonium ATCC 20371 and restored cephalosporin biosynthesis in this strain. Heterologous expression of the cefG genes took place in Penicilium chrysogenum. The deacetylcephalosporin acetyltransferase showed a much higher degree of homology with the O-acetylhomoserine acetyltransferases of Saccharomyces cerevisiae and Ascobolus immersus than with other O-acetyltransferases. The cefEF-ceIG cluster of genes encodes the enzymes that carry out the three late steps of the cephalosporin biosynthetic pathway and is not linked to the pcbAB-pcbC gene cluster that encodes the first two steps of the pathway.Cephalosporin is a P-lactam antibiotic formed in Cephalosporium acremonium (synonym Acremonium chrysogenum) and other filamentous fungi by condensation of the three precursor amino acids L-a-aminoadipic acid, L-cysteine, and D-valine to form the tripeptide b-L-a-aminoadipyl-L-cysteinyl-

A DNA vector for expressing an oxygen-binding heme protein (Vitreoscilla hemoglobin, or VHb) in filamentous fungi was constructed and introduced into a cephalosporin C-producing strain of Acremonium chrysogenum. Expression of VHb in transformants was demonstrated by Western immunoblot analysis and by increased carbon monoxide binding activity of cell extracts. Several VHb-expressing transformants produced significantly higher yields of cephalosporin C than control strains in batch culture experiments. Using the same vector system, VHb was also expressed in the related fungus Penicillium 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.

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.

Transcription of the pcbAB, pc6C and penDE genes of Penicilliurn chrysogenum AS-P-78 is repressed by glucose and the repression is not reversed by alkaline pHs Glucose repressed transcription of the penicillin biosynthesis genes pcbAB, pcbC and penDE when added a t inoculation time to cultures of Penici//ium chrysogenum AS-P-78 but it had little repressive effect when added a t 12 h and no effect when added a t 24 or 36 h. A slight increase in the expression of pcbC and penDE (and to a smaller extent of pcbAB) was observed in glucose-grown cultures a t pH 68,7-4 and 8 0 as compared with pH 62, but alkaline pHs did not override the strong repression exerted by glucose. Transcription of the actin gene used as control was not significantly affected by glucose or alkaline pHs. Repression by glucose of the three penicillin biosynthetic genes was also observed using the lacZ reporter gene coupled to each of the three promoters in monocopy transformants with the constructions integrated a t the pyrG locus. Glucose repression of the three genes encoding enzymes of penicillin biosynthesis therefore appears to be exerted by a regulatory mechanism independent from pH regulation.

A DNA fragment containing a gene homologous to LYS2 gene of Saccharomyces cerevisiae was cloned from a genomic DNA library of Penicillium chrysogenum AS-P-78. It encodes a protein of 1409 amino acids (Mr 154859) with strong similarity to the S. cerevisiae (49.9% identity) Schizosaccharomyces pombe (51.3% identity) and Candida albicans (48.12% identity) alpha-aminoadipate reductases and a lesser degree of identity to the amino acid-activating domains of the non-ribosomal peptide synthetases, including the alpha-aminoadipate-activating domain of the alpha-aminoadipyl-cysteinyl-valine synthetase of P. chrysogenum (12.4% identical amino acids). The lys2 gene contained one intron in the 5'-region and other in the 3'-region, as shown by comparing the nucleotide sequences of the cDNA and genomic DNA, and was transcribed as a 4.7-kb monocistronic mRNA. The lys2 gene was localized on chromosome III (7.5 Mb) in P. chrysogenum AS-P-78 and on chromosome IV (5.6 Mb) in strain P2, whereas the penicillin gene cluster is known to be located in chromosome I in both strains. The lys2-encoded protein is a member of the aminoacyladenylate-forming enzyme family with a reductase domain in its C-terminal region.

Methionine stimulated cephalosporin production in cultures of three different strains of Acremonium chrysogenum when added either at inoculation time or at 72 h to cells grown previously in the absence of methionine. When methionine was added at 72 h, the stimulation of cephalosporin biosynthesis was observed only 12 h later and required de novo protein synthesis. Methionine increased the levels of enzymes (isopenicillin N synthase and deacetylcephalosporin C acetyltransferase) expressed from genes (pcbC and cefG, respectively) located in the two clusters of cephalosporin biosynthesis genes in the wild-type A. chrysogenum strain and also in the two improved strains, CW19 and C10. Methionine-supplemented cells showed higher levels of transcripts of the four known genes (pcbAB, pcbC, cefEF and, to a slight extent, cefG) of the cephalosporin biosynthetic pathway than cells grown in the absence of methionine. The levels of the cefG transcript were much lower than those of the pcbAB, pcbC, and cefEF transcripts. The induction by methionine of transcription of the four cephalosporin biosynthesis genes and the known effect of this amino acid on the differentiation of A. chrysogenum indicate that methionine exerts a pleiotropic effect that coordinately regulates cephalosporin biosynthesis and differentiation.