Environmentally safe production of 7-aminodeacetoxycephalosporanic acid (7-ADCA) using recombinant strains of Acremonium chrysogenum

Velasco, Javier; Adrio, José L.; Moreno, M.; Dı́ez, Bruno; Soler, Gloria; Barredo, José Luis

doi:10.1038/78467

Cited by 87 publications

(58 citation statements)

References 22 publications

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“…Deacylation of DAOC is carried out by two final enzymatic bioconversions catalyzed by D-amino acid oxidase and glutaryl acylase yielding 7-aminocephalosporanic acid, precursor of all clinically important semi-synthetic cephalosporin derivatives. 197 In S. clavuligerus, expandase was modified by site-directed mutagenesis to promote the conversion of penicillin G to 7-phenylacetylamino-deacetoxycephalosporonic acid, precursor of 7-aminodeacetoxycephalosporonic acid. The resulting mutants showed increased k cat /K m values and 13-fold increase in relative activity toward penicillin G. 198 Addition of different side chains to 7-aminodeacetoxycephalosporonic acid converts this compound into the oral cephalosporins: cephradine, cephalexin and cephadroxil.…”

Section: Strain Improvement By Avoiding or Removing Ccrmentioning

confidence: 99%

Carbon source regulation of antibiotic production

et al. 2010

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Section: Strain Improvement By Avoiding or Removing Ccrmentioning

confidence: 99%

Carbon source regulation of antibiotic production

et al. 2010

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“…To date, A. chrysogenum propagation has been accepted to be strictly asexual, by the formation of conidiospores and arthrospores (27,42), and no associated teleomorph showing sexual propagation has been found. Thus, strains for improved industrial cephalosporin production can be obtained only by conventional mutagenesis and selection procedures or by molecular genetic techniques (29,60,72). However, the ability to mate fungi under controlled laboratory conditions would be a valuable tool for genetic analysis, as well as for conventional strain improvement programs (52).…”

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Asexual Cephalosporin C Producer Acremonium chrysogenum Carries a Functional Mating Type Locus

Pöggeler

Hoff

Kück

2008

Appl Environ Microbiol

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Acremonium chrysogenum, the fungal producer of the pharmaceutically relevant ␤-lactam antibiotic cephalosporin C, is classified as asexual because no direct observation of mating or meiosis has yet been reported. To assess the potential of A. chrysogenum for sexual reproduction, we screened an expressed sequence tag library from A. chrysogenum for the expression of mating type (MAT) genes, which are the key regulators of sexual reproduction. We identified two putative mating type genes that are homologues of the ␣-box domain gene, MAT1-1-1 and MAT1-1-2, encoding an HPG domain protein defined by the presence of the three invariant amino acids histidine, proline, and glycine. In addition, cDNAs encoding a putative pheromone receptor and pheromone-processing enzymes, as well as components of a pheromone response pathway, were found. Moreover, the entire A. chrysogenum MAT1-1 (AcMAT1-1) gene and regions flanking the MAT region were obtained from a genomic cosmid library, and sequence analysis revealed that in addition to AcMAT1-1-1 and AcMAT1-1-2, the AcMAT1-1 locus comprises a third mating type gene, AcMAT1-1-3, encoding a high-mobility-group domain protein. The ␣-box domain sequence of AcMAT1-1-1 was used to determine the phylogenetic relationships of A. chrysogenum to other ascomycetes. To determine the functionality of the AcMAT1-1 locus, the entire MAT locus was transferred into a MAT deletion strain of the heterothallic ascomycete Podospora anserina (the Pa⌬MAT strain). After fertilization with a P. anserina MAT1-2 (MAT ؉ ) strain, the corresponding transformants developed fruiting bodies with mature ascospores. Thus, the results of our functional analysis of the AcMAT1-1 locus provide strong evidence to hypothesize a sexual cycle in A. chrysogenum.

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“…Due to potential pollution by the toxic chemicals used for the ring expansion, Penicillium chrysogenum and A. chrysogenum have been engineered metabolically to produce 7-ADCA derivatives (7,27). However, the derivatives from these strains still need further complicated enzymatic processing.…”

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Family Shuffling of Expandase Genes To Enhance Substrate Specificity for Penicillin G

Hsu¹,

Yang²,

Deng³

et al. 2004

Appl Environ Microbiol

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Deacetoxycephalosporin C synthase (expandase) from Streptomyces clavuligerus, encoded by cefE, is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid from penicillin G. To improve the substrate specificity for penicillin G, eight cefE-homologous genes were directly evolved by using the DNA shuffling technique. After the first round of shuffling and screening, using an Escherichia coli ESS bioassay, four chimeras with higher activity were subjected to a second round. Subsequently, 20 clones were found with significantly enhanced activity. The kinetic parameters of two isolates that lack substrate inhibition showed 8.5-and 118-fold increases in the k cat /K m ratio compared to the S. clavuligerus expandase. The evolved enzyme with the 118-fold increase is the most active obtained to date anywhere. Our shuffling results also indicate the remarkable plasticity of the expandase, suggesting that more-active chimeras might be achievable with further rounds.Cephalosporins, which constitute one of the major ␤-lactam antibiotic classes, are produced by several microorganisms, such as filamentous fungi, actinomycetes, and gram-negative bacteria. The biosynthesis of cephalosporins in Streptomyces clavuligerus and Acremonium chrysogenum has been studied extensively (19). In S. clavuligerus, deacetoxycephalosporin C synthase (DAOCS) (expandase), encoded by cefE, catalyzes the oxidative expansion of the five-membered thiazolidine ring of penicillin N into the six-membered dihydrothiazine ring of deacetoxycephalosporin C (DAOC) (13) (Fig. 1). DAOC is sequentially converted to deacetylcephalosporin C (DAC) by deacetylcephalosporin C synthase (DACS) (hydroxylase), encoded by cefF (12). Both expandase and hydroxylase are ␣-ketoglutarate and Fe(II) dependent oxygenases and share 55 to 60% amino acid identity. Interestingly, the enzyme from A. chrysogenum can catalyze the direct conversion of penicillin N to DAC due to possession of both expandase and hydroxylase activity; hence the encoded gene is termed cefEF (24).7-Aminodeacetoxycephalosporanic acid (7-ADCA) is one of the most important semisynthetic cephalosporins, and its current production includes the ring expansion of penicillin G into phenylacetyl-7-ADCA and the well-documented enzymatic removal of the phenylacetyl group. Due to potential pollution by the toxic chemicals used for the ring expansion, Penicillium chrysogenum and A. chrysogenum have been engineered metabolically to produce 7-ADCA derivatives (7, 27). However, the derivatives from these strains still need further complicated enzymatic processing. Therefore, the production of a penicillin G-specific expandase would be a more straightforward solution to the problems of 7-ADCA production.Several expandases have been screened from actinomycetes, and the S. clavuligerus enzyme has been well characterized. These enzymes have broad substrate specificity and can catalyze ring expansion in penicillins with various side chains. However, they prefer their physiological substrate, peni...

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Environmentally safe production of 7-aminodeacetoxycephalosporanic acid (7-ADCA) using recombinant strains of Acremonium chrysogenum

Cited by 87 publications

References 22 publications

Carbon source regulation of antibiotic production

Carbon source regulation of antibiotic production

Asexual Cephalosporin C Producer Acremonium chrysogenum Carries a Functional Mating Type Locus

Family Shuffling of Expandase Genes To Enhance Substrate Specificity for Penicillin G

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