Gene disruption of the pcbAB gene encoding ACV synthetase in Cephalosporium acremonium

Hoskins, JoAnn; O'callaghan, Niamh M.; Queener, Stephen W.; Cantwell, Cathleen A.; Wood, John S.; Chen, Victor; Skatrud, Paul L.

doi:10.1007/bf00327023

Cited by 43 publications

(14 citation statements)

References 31 publications

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“…The nucleotide sequence in contiguous S. clavuligerus restriction fragments containing DNA immediately downstream from lat encodes amino acid sequences similar to those of the N and C termini of the "core" polypeptide found as three repeated domains in both C. acremonium and P. chrysogenum ACVS (5,10,34). The C. acremonium pebAB sequence is near completion (11; see Addendum below); in the region encoding the N terminus, it contains a sequence for one of the three repeated domains that is similar to the streptomycete ORF reported here, as well as to tyrocidine A and gramicidin S synthetases.…”

Section: Discussionmentioning

confidence: 99%

Localization of the lysine epsilon-aminotransferase (lat) and delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (pcbAB) genes from Streptomyces clavuligerus and production of lysine epsilon-aminotransferase activity in Escherichia coli

et al. 1991

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Lysine e-aminotransferase (LAT) in the ,I-lactam-producing actinomycetes is considered to be the first step in the antibiotic biosynthetic pathway. putative N and C termini of S. clavuligerus pcbAB suggests that the coding region occupies 12 kbp and codes for a polypeptide related in size to the fungal ACV synthetases, the molecular characterization of the I8-lactam biosynthetic cluster between pcbC and cefE (-25 kbp) is nearly complete.Many naturally occurring P-lactam antibiotics (isopenicillin N, cephalosporin C, and cephamycins) contain a side chain derived from a-aminoadipic acid (a-AAA). When the producer is a filamentous fungus (Penicillium chrysogenum, Aspergillus nidulans, Cephalosporium acremonium), this amino acid is formed as an obligate intermediate in the synthesis of lysine. However, in procaroytes, lysine is synthesized via the diaminopimelic acid pathway without the production of a-AAA (for reviews, see references 23 and 36). Whitney et al. (37) established that in the P-lactamproducing Streptomyces spp., ot-AAA is derived from the breakdown of lysine. Although the catabolism of lysine in bacteria is diverse, conversion of lysine to a-AAA by removal of the E-amino group was tentatively linked in Streptomyces lipmanii to an aminotransferase (16) which was later conclusively shown to be present in Nocardia (previously Streptomyces) lactamdurans (15). Recently, Madduri et al. (21) reported that in these actinomycetes, L-lysine-e-aminotransferase (LAT) is specific to P-lactam (cephamycin C) producers and provides the precursor for antibiotic synthesis; the pathway via cadaverine is obligatory for lysine catabolism (Fig. 1). Moreover, a gene governing LAT production was found exclusively in Streptomyces spp. producing P-lactams (22) MATERIALS AND METHODS Bacterial strains, growth conditions, DNA manipulations, and DNA sequence determination. E. coli strains were grown in TY broth (18) supplemented with either 5 ,ug of tetracycline per ml or 80 jig of ampicillin per ml under standard conditions, except as noted.Restriction endonuclease digestion, ligation, and determination of DNA restriction fragments by agarose or acrylamide gel electrophoresis followed established procedures (29). DNA fragments were subcloned into pUC or M13 vectors for further analysis. DNA sequencing was performed by the dideoxy method with TAQ-TRACK (Promega Biotec) and followed the supplier's recommendations. Fragments were sequenced either directly from the plasmid or after subcloning into M13 bacteriophage vectors, using forward and reverse sequencing primers for pUC and M13 templates, as well as several custom DNA oligonucleotide primers. Custom DNA oligonucleotide primers were synthesized by using an Applied Biosystems DNA Synthesizer (model 380A) according to the manufacturer's instructions.Computing. DNA sequences were analyzed with the GCG

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Section: Discussionmentioning

confidence: 99%

Localization of the lysine epsilon-aminotransferase (lat) and delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (pcbAB) genes from Streptomyces clavuligerus and production of lysine epsilon-aminotransferase activity in Escherichia coli

et al. 1991

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“…Transforming DNA is integrated into the genome of A. chrysogenum at a very low efficiency, mainly via heterologous recombination (14,36). The frequency of gene disruption in A. chrysogenum is one event every 200 transformants screened (36).…”

Section: Resultsmentioning

confidence: 99%

“…Some of them are able to efficiently integrate homologous DNA into the genome, as is the case with Aspergillus nidulans (33,37), Ascobolus immersus (11), and Cochiobolus heterostrophus (15). On the other hand, with other filamentous fungi such as A. chrysogenum (14,36), Podospora anserina (2), and Penicillium chrysogenum (5), the frequency of homologous recombination is very low (usually lower than 1 in 100 transformants).…”

Section: Discussionmentioning

confidence: 99%

Targeted Inactivation of the mecB Gene, Encoding Cystathionine-γ-Lyase, Shows that the Reverse Transsulfuration Pathway Is Required for High-Level Cephalosporin Biosynthesis in Acremonium chrysogenum C10 but Not for Methionine Induction of the Cephalosporin Genes

et al. 2001

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Targeted gene disruption efficiency in Acremonium chrysogenum was increased 10-fold by applying the double-marker enrichment technique to this filamentous fungus. Disruption of the mecB gene by the doublemarker technique was achieved in 5% of the transformants screened. Mutants T6 and T24, obtained by gene replacement, showed an inactive mecB gene by Southern blot analysis and no cystathionine-␥-lyase activity. These mutants exhibited lower cephalosporin production than that of the control strain, A. chrysogenum C10, in MDFA medium supplemented with methionine. However, there was no difference in cephalosporin production between parental strain A. chrysogenum C10 and the mutants T6 and T24 in Shen's defined fermentation medium (MDFA) without methionine. These results indicate that the supply of cysteine through the transsulfuration pathway is required for high-level cephalosporin biosynthesis but not for low-level production of this antibiotic in methionine-unsupplemented medium. Therefore, cysteine for cephalosporin biosynthesis in A. chrysogenum derives from the autotrophic (SH 2 ) and the reverse transsulfuration pathways. Levels of methionine induction of the cephalosporin biosynthesis gene pcbC were identical in the parental strain and the mecB mutants, indicating that the induction effect is not mediated by cystathionine-␥-lyase. ␤-Lactam biosynthesis inAcremonium chrysogenum (formerly known as Cephalosporium acremonium) begins with the nonribosomal condensation of the three precursor amino acids L-␣-aminoadipic acid, L-cysteine, and L-valine (1, 21, 23). There are two ways to synthesize cysteine in filamentous fungi (35,26). One pathway, the so-called autotrophic pathway, converts inorganic sulfur to cysteine via serine O-acetyltransferase and O-acetylserine sulfhydrilase (Fig. 1). In the second route, cysteine can be obtained via the reverse transsulfuration pathway, in which the sulfur atom of methionine is transferred to cysteine through S-adenosylmethionine, S-adenosylhomocysteine, homocysteine, and cystathionine as intermediates.The predominant route of the cysteine supply for ␤-lactam biosynthesis depends on the producer microorganism (6). Cysteine for penicillin biosynthesis in Penicillium chrysogenum is obtained mainly from sulfate reduction (31). In this microorganism, the transsulfuration pathway has been considered to have a minor role in penicillin biosynthesis. On the other hand, the sulfur atom for ␤-lactam biosynthesis by A. chrysogenum is believed to derive preferentially from methionine via the reverse transsulfuration pathway (3, 24). However, there is no genetic evidence for, or against, this hypothesis.DL-Methionine is known to stimulate cephalosporin biosynthesis in A. chrysogenum (7, 16). For many years it was unclear whether the stimulatory effect of DL-methionine was due to a precursor effect (one providing cysteine) or to an inducing effect (8,20,23). Sawada and coworkers established that methionine increases isopenicillin N synthase, deacetoxycephalosporin C synthase (30), and ␣-...

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“…The Psy1-encoded sequence also contains a region that matches the thioesterase/ transferase consensus region (GXSXG) often found near the C termini of other peptide synthetases, including the peptide synthetases for ACV, gramicidin S, and surfactin (20). Although the open reading frame fragment encoding 1,609 amino acids is only a partial coding region, as expected from the larger sizes of other peptide synthetases, the cloned fragment is large enough to permit manipulation of the gene through targeted gene disruption (15).…”

Section: Discussionmentioning

confidence: 99%

Peptide Synthetase Gene in Trichoderma virens

Wilhite

Lumsden

Straney

2001

Appl Environ Microbiol

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Trichoderma virens (synonym, Gliocladium virens), a deuteromycete fungus, suppresses soilborne plant diseases caused by a number of fungi and is used as a biocontrol agent. Several traits that may contribute to the antagonistic interactions of T. virens with disease-causing fungi involve the production of peptide metabolites (e.g., the antibiotic gliotoxin and siderophores used for iron acquisition). We cloned a 5,056-bp partial cDNA encoding a putative peptide synthetase (Psy1) from T. virens using conserved motifs found within the adenylate domain of peptide synthetases. Sequence similarities with conserved motifs of the adenylation domain, acyl transfer, and two condensation domains support identification of the Psy1 gene as a gene that encodes a peptide synthetase. Disruption of the native Psy1 gene through gene replacement was used to identify the function of this gene. Psy1 disruptants produced normal amounts of gliotoxin but grew poorly under low-iron conditions, suggesting that Psy1 plays a role in siderophore production. Psy1 disruptants cannot produce the major T. virens siderophore dimerum acid, a dipetide of acylated N ␦ -hydroxyornithine. Biocontrol activity against damping-off diseases caused by Pythium ultimum and Rhizoctonia solani was not reduced by the Psy1 disruption, suggesting that iron competition through dimerum acid production does not contribute significantly to disease suppression activity under the conditions used.

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Gene disruption of the pcbAB gene encoding ACV synthetase in Cephalosporium acremonium

Cited by 43 publications

References 31 publications

Localization of the lysine epsilon-aminotransferase (lat) and delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (pcbAB) genes from Streptomyces clavuligerus and production of lysine epsilon-aminotransferase activity in Escherichia coli

Localization of the lysine epsilon-aminotransferase (lat) and delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (pcbAB) genes from Streptomyces clavuligerus and production of lysine epsilon-aminotransferase activity in Escherichia coli

Targeted Inactivation of the mecB Gene, Encoding Cystathionine-γ-Lyase, Shows that the Reverse Transsulfuration Pathway Is Required for High-Level Cephalosporin Biosynthesis in Acremonium chrysogenum C10 but Not for Methionine Induction of the Cephalosporin Genes

Peptide Synthetase Gene in Trichoderma virens

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