Cloning and expression in Escherichia coli of isopenicillin N synthetase genes from Streptomyces lipmanii and Aspergillus nidulans

Abstract

,-Lactam antibiotics such as penicillins and cephalosporins are synthesized by a wide variety of microbes, including procaryotes and eucaryotes. Isopenicillin N synthetase catalyzes a key reaction in the biosynthetic pathway of penicillins and cephalosporins. The genes encoding this protein have previously been cloned from the ifiamentous fungi Cephalosporium acremonium and Penicillium chrysogenum and characterized. We have extended our analysis to the isopenicillin N synthetase genes from the fungus Aspergillus nidulans and the gram-positive procaryote Streptomyces lipmanii. The isopenicillin N synthetase genes from these organisms have been cloned and sequenced, and the proteins encoded by the open reading frames were expressed in Escherichia coli. Active isopenicillin N synthetase enzyme was recovered from extracts of E. coli cells prepared from cells containing each of the genes in expression vectors. The four isopenicillin N synthetase genes studied are closely related. Pairwise comparison of the DNA sequences showed between 62.5 and 75.7% identity; comparison of the predicted amino acid sequences showed between 53.9 and 80.6% identity. The close homology of the procaryotic and eucaryotic isopenicillin N synthetase genes suggests horizontal transfer of the genes during evolution.The penicillin, cephalosporin, and cephamycin classes of P-lactam antibiotics share two common steps in their respective biosynthetic pathways: the joining of three amino acids to form 8-(L-a-aminoadipyl)-L-cysteinyl-D-valine (LLD-ACV) and the conversion of LLD-ACV to isopenicillin N. These two reactions are carried out in filamentous fungi and streptomycetes, including Penicillium chrysogenum and Aspergillus nidulans (producers of penicillin), Cephalosporium acremonium (producer of cephalosporin C), and Streptomyces lipmanii and Streptomyces clavuligerus (producers of cephamycins) (16). We have studied the biochemical and genetic basis of the conversion of LLD-ACV to isopenicillin N by cloning and characterizing the gene encoding the isopenicillin N synthetase (IPNS) enzyme from C. acremonium and P. chrysogenum (6, 23). The IPNS genes from these two fungi show a high degree of nucleotide and amino acid sequence identity. The 77% identity of the predicted amino acid sequences of the IPNSs from these fungi is so extensive that it is difficult to identify regions of potential functional importance. We therefore decided to extend the comparison to IPNS genes from other organisms which might be more distant evolutionarily.The IPNS gene from A. nidulans was of particular interest because of the well-developed genetic techniques available for this organism. Also, mutant A. nidulans strains with altered ,-lactam-biosynthetic ability have been described (10) and characterized (20

Linear plasmids and chromosomes in bacteria

“…The organization and nucleotide sequences of some Streptomyces antibiotic synthesis genes are similar to those of certain fungi (Hara et al, 1991). Genetic exchange between soil bacteria and fungi may account for this similarity (Weigel et al, 1988), and Kinashi et ai (1992) hypothesized that S/reptomyces conjugative linear plasmids were likely vehicles for this horizontal exchange.…”

Molecular characterization of the acyl-coenzyme A:isopenicillin N acyltransferase gene (penDE) from Penicillium chrysogenum and Aspergillus nidulans and activity of recombinant enzyme in Escherichia coli

“…Therefore, the fungal pcbC genes also show a similar pattern of relatedness (5,11,25 Barredo et al (3) which amino acid is the N-terminus of the 29-kDa protein. They report that Leu-110 is the seventh amino acid from the N-terminus, yet cleavage into the 29-and 11-kDa species is indicated to occur between Asp-101 and Gly-102.…”

“…Preliminary data indicated that the AT protein from A. nidulans was similar to the enzyme from P. chrysogenum, including the N-terminal sequence of the 29-kDa polypeptide (25a). A HindIII-EcoRI restriction fragment from pXL2 located downstream from pcbC in P. chrysogenum (5) and the oligonucleotide described for Penicillium penDE were used to screen clones containing DNA downstream from pcbC (25). Hybridization and restriction analysis located penDE downstream from pcbC, and DNA blot and sequence analysis of the region downstream of pcbC indicated that the 4.5-kbp HindIII fragment in pOGO4 contained both pcbC and penDE (Fig.…”

DNA homology between Saccharopolyspora strains and other erythromycin-producing actinomycetes

“…The lack of extensive DNA sequence homology among diverse erythromycin-producing strains suggests several evolutionary possibilities : (1) that erythromycin biosynthetic genes evolved before the divergence of actinomycete genera; (2) that these genes were transferred horizontally between certain genera early on the evolutionary time scale; or (3) that all or parts of the erythromycin pathway genes evolved more than once. In the case of p-lactam antibiotics that are produced by several species of Streptomyces and by several genera of fungi, there is evidence to suggest that the genes evolved in a streptomycete and were transferred around 370 million years ago to a fungal progenitor of Aspergillus, Cephalosporium and Penicillium (Weigel et al, 1988). More detailed studies on the structural organization of erythromycin biosynthetic genes in different actinomycetes, and DNA sequence analysis of several specific genes cloned from taxonomically distinct micro-organisms should provide additional insights into the evolution of erythromycin biosynthetic genes.…”