Using gene shuffling to increase genetic diversity in genes involved in beta-lactam biosynthesis
- Authors: Tarr, Shahida
- Date: 2001
- Subjects: Beta lactam antibiotics , Genes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4014 , http://hdl.handle.net/10962/d1004074 , Beta lactam antibiotics , Genes
- Description: The actinomycetes are gram-positive bacteria that produce more than two-thirds of the known biologically active microbial natural products, including many commercially important antibiotics, anti-cancer agents, other pharmacologically useful agents, animal health products and agrochemicals. The prevailing utilization of antibiotics continues to be the mainstay against microbial infections and a majority ofthe over six thousand antibiotics discovered thus far are from Streptomyces spp. One of the most well-characterized antibiotic biosynthetic pathway is the one involving the biosynthesis of the penicillins, cephalosporins and cephamycins. This pathway involves two initial steps which are common in filamentous fungi, lower eukaryotes and prokaryotes. The penam nucleus of penicillins and the cephem nucleus of both cephamycins andcephalosporins are formed by the condensation of the three precursor amino acids L-a-aminoadipic acid, Lcysteine and L-valine by a mechanism designated as 'non-ribosomal peptide synthesis', which involves activation and condensation of the three component amino acids and epimerization of the L- to D-valine to form a linear acyclic tripeptide called o-(L-a-aminoadipyl)-L-cysteinyl-Dvaline (ACV) by the action of a peptide synthetase. ACV is then cyclized to form isopenicillin N, an intermediate that contains an L-a-aminoadipyl side-chain attached to the penem nucleus (Fig. 1.2) by isopenicilin N synthase (IPNS or Cyclase) and this encompasses the creation of the Beta-lactam and thiazolidine rings. A broad range of ~-lactam producing Streptomyces spp were grown, the DNA extraction procedure optimised and total chromosomal DNA isolated. A bioinformatics analysis of known IPNS gene sequences allowed the synthesis of PCR primers for the iso-penicillin N synthase gene. IPNS genes and lPNS-like genes were successfully amplified from the total DNA of ten strains including two novel thermophilic strains, A. and B. Sequencing was carried out on the genes from S. hygroscopicus, S. tanashiensis and the two thermophiles A and B. This allowed development of the conditions for gene shuffiing of the IPNS gene which was carried out pairwise and resulted in the reconstitution of shuffied genes of the correct size. The resulting mixed gene sequences were cloned into the pTrcHis2-TOPO expression vector and the plasmid DNA screened and assayed for IPNS activity using HPLC which showed ten fold increase in IPNS activity as a result of the shuffiing.
- Full Text:
- Date Issued: 2001
- Authors: Tarr, Shahida
- Date: 2001
- Subjects: Beta lactam antibiotics , Genes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4014 , http://hdl.handle.net/10962/d1004074 , Beta lactam antibiotics , Genes
- Description: The actinomycetes are gram-positive bacteria that produce more than two-thirds of the known biologically active microbial natural products, including many commercially important antibiotics, anti-cancer agents, other pharmacologically useful agents, animal health products and agrochemicals. The prevailing utilization of antibiotics continues to be the mainstay against microbial infections and a majority ofthe over six thousand antibiotics discovered thus far are from Streptomyces spp. One of the most well-characterized antibiotic biosynthetic pathway is the one involving the biosynthesis of the penicillins, cephalosporins and cephamycins. This pathway involves two initial steps which are common in filamentous fungi, lower eukaryotes and prokaryotes. The penam nucleus of penicillins and the cephem nucleus of both cephamycins andcephalosporins are formed by the condensation of the three precursor amino acids L-a-aminoadipic acid, Lcysteine and L-valine by a mechanism designated as 'non-ribosomal peptide synthesis', which involves activation and condensation of the three component amino acids and epimerization of the L- to D-valine to form a linear acyclic tripeptide called o-(L-a-aminoadipyl)-L-cysteinyl-Dvaline (ACV) by the action of a peptide synthetase. ACV is then cyclized to form isopenicillin N, an intermediate that contains an L-a-aminoadipyl side-chain attached to the penem nucleus (Fig. 1.2) by isopenicilin N synthase (IPNS or Cyclase) and this encompasses the creation of the Beta-lactam and thiazolidine rings. A broad range of ~-lactam producing Streptomyces spp were grown, the DNA extraction procedure optimised and total chromosomal DNA isolated. A bioinformatics analysis of known IPNS gene sequences allowed the synthesis of PCR primers for the iso-penicillin N synthase gene. IPNS genes and lPNS-like genes were successfully amplified from the total DNA of ten strains including two novel thermophilic strains, A. and B. Sequencing was carried out on the genes from S. hygroscopicus, S. tanashiensis and the two thermophiles A and B. This allowed development of the conditions for gene shuffiing of the IPNS gene which was carried out pairwise and resulted in the reconstitution of shuffied genes of the correct size. The resulting mixed gene sequences were cloned into the pTrcHis2-TOPO expression vector and the plasmid DNA screened and assayed for IPNS activity using HPLC which showed ten fold increase in IPNS activity as a result of the shuffiing.
- Full Text:
- Date Issued: 2001
Isolation and identification of Beta-Lactam Producing Microorganisms using PCR based methodologies
- Authors: Krallis, Myrsini
- Date: 1997
- Subjects: Polymerase chain reaction , Bacterial genetics , Fungi -- Genetics , Beta lactam antibiotics , Microbial enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4155 , http://hdl.handle.net/10962/d1018237
- Description: The polymerase chain reaction (PCR) was investigated as a potential tool in microbial screening for 13-lactam. producing organisms. Optimization of PCR conditions and the addition of acetamide to the PCR reaction allowed for the successful amplification of the isopenicillin N synthetase (lPNS) gene in S. clavuligerus, S. tanashiensis, S. griseus, S. olivaceus, S. lipmanii, and S. chartreusis. PCR was used to produce a radiolabelled probe from S. clavuligerus that was used to detect analogous genes in bacteria and fungi. Southern blot and dot blot analysis using the lPNS probe revealed the presence of IPNS-like sequences in seventeen organisms. Fourteen of these sequences belonged to known 13-lactam. producing organisms; one unidentified soil isolate; and two non-/3-lactam. producing organisms viz. S. venezuelae ATCC 10712 and S. hygroscopicus ATCC 21703. The lPNS gene was also detected in a 13-lactam producer (S. chartreusis) that had lost its ability to produce antibiotic. It would therefore have been overlooked in a conventional antibiotic screening program. The use of PCR, coupled with Southern hybridization and dot blot analysis, increased the sensitivity and specificity of the antibiotic screening procedures and allowed for the investigation of evolutionary relationships between the eukaryotes and the prokaryotes. A preliminary investigation into the potential use of RAPD PCR and protein fmgerprinting as tools for solving discrepancies in streptomycete identification was conducted. A variety of streptomycete species that were chosen as being representative of a number of numerical taxonomic classes were amplified using various RAPD primers. Streptomycetes appear to be genetically diverse organisms as was reflected by their RAPD and protein profiles. The application of PCR in an antibiotic screening program showed great potential as a specific and sensitive tool in the detection of /3-lactam producers and in the elimination of duplicate strains.
- Full Text:
- Date Issued: 1997
- Authors: Krallis, Myrsini
- Date: 1997
- Subjects: Polymerase chain reaction , Bacterial genetics , Fungi -- Genetics , Beta lactam antibiotics , Microbial enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4155 , http://hdl.handle.net/10962/d1018237
- Description: The polymerase chain reaction (PCR) was investigated as a potential tool in microbial screening for 13-lactam. producing organisms. Optimization of PCR conditions and the addition of acetamide to the PCR reaction allowed for the successful amplification of the isopenicillin N synthetase (lPNS) gene in S. clavuligerus, S. tanashiensis, S. griseus, S. olivaceus, S. lipmanii, and S. chartreusis. PCR was used to produce a radiolabelled probe from S. clavuligerus that was used to detect analogous genes in bacteria and fungi. Southern blot and dot blot analysis using the lPNS probe revealed the presence of IPNS-like sequences in seventeen organisms. Fourteen of these sequences belonged to known 13-lactam. producing organisms; one unidentified soil isolate; and two non-/3-lactam. producing organisms viz. S. venezuelae ATCC 10712 and S. hygroscopicus ATCC 21703. The lPNS gene was also detected in a 13-lactam producer (S. chartreusis) that had lost its ability to produce antibiotic. It would therefore have been overlooked in a conventional antibiotic screening program. The use of PCR, coupled with Southern hybridization and dot blot analysis, increased the sensitivity and specificity of the antibiotic screening procedures and allowed for the investigation of evolutionary relationships between the eukaryotes and the prokaryotes. A preliminary investigation into the potential use of RAPD PCR and protein fmgerprinting as tools for solving discrepancies in streptomycete identification was conducted. A variety of streptomycete species that were chosen as being representative of a number of numerical taxonomic classes were amplified using various RAPD primers. Streptomycetes appear to be genetically diverse organisms as was reflected by their RAPD and protein profiles. The application of PCR in an antibiotic screening program showed great potential as a specific and sensitive tool in the detection of /3-lactam producers and in the elimination of duplicate strains.
- Full Text:
- Date Issued: 1997
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