Lovastatin biosynthesis in Aspergillus terreus: characterization of blocked mutants, enzyme activities and a multifunctional polyketide synthase gene.

BACKGROUND: Lovastatin, an HMG-CoA reductase inhibitor produced by the fungus Aspergillus terreus, is composed of two polyketide chains. One is a nonaketide that undergoes cyclization to a hexahydronaphthalene ring system and the other is a simple diketide, 2-methylbutyrate. Fungal polyketide synthase (PKS) systems are of great interest and their genetic manipulation should lead to novel compounds. RESULTS: An A. terreus mutant (BX102) was isolated that could not synthesize the nonaketide portion of lovastatin and was missing a approximately 250 kDa polypeptide normally present under conditions of lovastatin production. Other mutants produced lovastatin intermediates without the methylbutyryl sidechain and were missing a polypeptide of approximately 220 kDa. The PKS inhibitor cerulenin reacted covalently with both polypeptides. Antiserum raised against the approximately 250 kDa polypeptide was used to isolate the corresponding gene, which complemented the BX102 mutation. The gene encodes a polypeptide of 269 kDa containing catalytic domains typical of vertebrate fatty acid and fungal PKSs, plus two additional domains not previously seen in PKSs: a centrally located methyltransferase domain and a peptide synthetase elongation domain at the carboxyl terminus. CONCLUSIONS: The results show that the nonaketide and diketide portions of lovastatin are synthesized by separate large multifunctional PKSs. Elucidation of the primary structure of the PKS that forms the lovastatin nonaketide, as well as characterization of blocked mutants, provides new details of lovastatin biosynthesis.

Production of cephalosporin intermediates by feeding adipic acid to recombinant Penicillium chrysogenum strains expressing ring expansion activity.

We demonstrate a novel and efficient bioprocess for production of the cephalosporin intermediates, 7-aminocephalosporanic acid (7-ACA) or 7-amino deacetoxycephalosporanic acid (7-ADCA). The Streptomyces clavuligerus expandase gene or the Cephalosporium acremonium expandase-hydroxylase gene, with and without the acetyltransferase gene, were expressed in a penicillin production strain of Penicillium chrysogenum. Growth of these transformants in media containing adipic acid as the side chain precursor resulted in efficient production of cephalosporins having an adipyl side chain, proving that adipyl-6-APA is a substrate for either enzyme in vivo. Strains expressing expandase produced adipyl-7-ADCA, whereas strains expressing expandase-hydroxylase produced both adipyl-7-ADCA and adipyl-7-ADAC (aminodeacetylcephalosporanic acid). Strains expressing expandase-hydroxylase and acetyltransferase produced adipyl-7-ADCA, adipyl-7-ADAC and adipyl-7-ACA. The adipyl side chain of these cephalosporins was easily removed with a Pseudomonas-derived amidase to yield the cephalosporin intermediates.

Sequence of the dengue-1 virus genome in the region encoding the three structural proteins and the major nonstructural protein NS1.

Sequence results are presented for a 3745-nucleotide region at the 5' end of the dengue type-1 virus (DEN-1) genome. The strain characterized is a Western Pacific isolate from Nauru Island. The sequenced region contains the beginning of a continuous open reading frame which specifies the capsid (C), membrane (M), and envelope (E) structural proteins and the nonstructural protein NS1. The sequences are compared with corresponding segments for seven other flaviviruses, including two of the three remaining dengue serotypes, DEN-2 and DEN-4. The results show the DEN-1 genome size and organization to be similar to those of other characterized flaviviruses and that major features of the individual proteins are conserved. It is of special interest that comparisons of the E glycoprotein sequences between the dengue serotypes (DEN-1, -2, -4) reveal only moderately greater sequence relatedness (63-68%) than occurs in comparisons of DEN-1 with five other flaviviruses (46-54%). For the other structural proteins, C and M, the relatedness values are 59-74% for comparisons between DEN-1 and the other dengue serotypes and 31-45% for comparisons between DEN-1 and the five other flaviviruses.

Expression of Japanese encephalitis virus antigens in Escherichia coli.

The expression of Japanese encephalitis virus (JE) cDNA in Escherichia coli has been used to study the functional organization of the viral genome. JE protein coding sequences were expressed in E. coli by subcloning random fragments of cloned cDNA (P.C. McAda, P.W. Mason, C.S. Schmaljohn, J.M. Dalrymple, T.L. Mason, and M.J. Fournier, 1987, Virology 158, 348-360) into the bacteriophage lambda gt11 expression vector. Over 120 lambda gt11 recombinants expressing viral protein sequences as beta-galactosidase fusion proteins were identified immunologically with monoclonal antibodies (MAbs) and polyclonal hyperimmune mouse ascites fluid (HMAF). This expression and immunological detection strategy has been used to (1) map viral protein coding sequences to the JE genome; (2) demonstrate that contiguous viral protein coding regions can be expressed as single polypeptides in E. coli, providing functional confirmation for a long viral open reading frame; (3) localize important antigenic domains within the envelope protein E; and (4) identify in JE-infected cells a form of the glycosylated nonstructural protein NS1 that contains a hydrophobic C-terminal extension encoded by portions of the "ns2a" region of the JE genome.

Partial nucleotide sequence of the Japanese encephalitis virus genome.

Approximately 10 kb of the estimated 10.9-kb genome of the Japanese encephalitis virus (JE; Nakayama strain) has been cloned as cDNA; the uncloned portion includes 430 bases at the 5'-terminus and 450 bases at the 3'-end. A map of the genome has been developed through nucleotide sequencing and in vivo expression with the Escherichia coli expression vector lambda gt11 and immunological identification. Sequence results for 4320 nucleotides suggest the JE genome organization is very similar to those of three other flaviviruses for which sequence information is available. Like the other flaviviruses, the JE proteins are encoded by a single open reading frame that continues uninterrupted throughout the region sequenced. Considerable homology exists between the JE RNA and protein sequences and those of the other characterized flaviviruses. Comparative nucleotide and (amino acid) homology values for the M-E-NS1-ns2 segment of JE are approximately MVE, 70% (80%), WN, 68% (76%), and YF, 50% (45%). Even greater homology is suggested when the protein hydrophobicity profiles are compared. The molecular relationships are consistent with the established serological relationships among JE, MVE, and WN viruses and argue that these flaviviruses may have been derived from a common evolutionary ancestor.

A neutral metallo endoprotease involved in the processing of an F1-ATPase subunit precursor in mitochondria.

A post-translational processing assay of the precursor to the yeast F1-ATPase subunit has been utilized to examine a mitochondrial endoprotease which cleaves this subunit precursor to the size of a mature subunit. The endoprotease is extracted from purified mitochondria as a soluble complex of Mr = 115,000 which is composed of subunits of lower molecular weight when examined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It exhibits a pH optimum of between pH 7 and 8 and is inactive at pH 6.5 and below. The mitochondrial endoprotease is insensitive to serine esterase inhibitors, but is inhibited by EDTA and o-phenanthroline. Restoration of precursor subunit processing activity in the presence of metal chelators is strictly dependent on excess Co2+ and Mn2+ over other heavy metals examined. These and additional data indicate that this soluble metallo endoprotease is involved in the processing of other cytoplasmically synthesized precursor subunits of the ATPase complex in addition to the subunit 2 precursor. The role of this processing enzyme in the assembly of mitochondrial inner membrane complexes is discussed in light of the current model of mitochondrial biogenesis.