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Novel ceftazidime-resistance beta-lactamases generated by a codon-based mutagenesis method and selection
Paul Gaytán1, Joel Osuna, Xavier Soberón
1Instituto de Biotecnología/UNAM, Ap. Postal 510-3, Cuernavaca, Morelos 62250, México. paul@ibt.unam.mx
Abstract:
Four known and nine new ceftazidime-resistance beta-lactamases were generated by a novel, contaminating codon-based mutagenesis approach. In this method, wild-type codons are spiked with a set of mutant codons during oligonucleotide synthesis, generating random combinatorial libraries of primers that contain few codon replacements per variant. Mutant codons are assembled by tandem addition of a diluted mixture of five Fmoc-dimer amidites to the growing oligo and a mixture of four DMTr-monomer amidites to generate 20 trinucleotides that encode a set of 18 amino acids. Wild-type codons are assembled with conventional chemistry and the whole process takes place in only one synthesis column, making its automation feasible. The random and binomial behavior of this approach was tested in the polylinker region of plasmid pUC19 by the synthesis of three oligonucleotide libraries mutagenized at different rates and cloned as mutagenic cassettes. Additionally, the method was biologically assessed by mutating six contiguous codons that encode amino acids 237-243 (ABL numbering) of the TEM(pUC19) beta-lactamase, which is functionally equivalent to the clinically important TEM-1 beta-lactamase. The best ceftazidime-recognizing variant was a triple mutant, R164H:E240K: R241A, displaying a 333-fold higher resistance than the wild-type enzyme.
Insights
A novel codon-based mutagenesis method efficiently generated 13 beta-lactamases, including new ceftazidime-resistant variants. This approach enables rapid enzyme evolution for antibiotic resistance research.
Area of Science:
- Molecular Biology
- Enzyme Engineering
- Drug Discovery
Background:
- Beta-lactamase enzymes confer resistance to antibiotics like ceftazidime.
- Developing novel beta-lactamases is crucial for understanding and combating antibiotic resistance.
- Existing mutagenesis methods can be inefficient for generating diverse enzyme variants.
Purpose of the Study:
- To develop and validate a novel, automated codon-based mutagenesis approach.
- To generate a library of ceftazidime-resistance beta-lactamases.
- To identify enhanced beta-lactamase variants with significant ceftazidime resistance.
Main Methods:
- A contaminating codon-based mutagenesis strategy was employed during oligonucleotide synthesis.
- Mutant and wild-type codons were combined in a single synthesis column for automation.
- The method was tested using plasmid pUC19 and applied to TEM-1 beta-lactamase mutagenesis.
Main Results:
- Four known and nine new beta-lactamase variants were successfully generated.
- The mutagenesis approach demonstrated random and binomial behavior.
- A triple mutant (R164H:E240K:R241A) showed a 333-fold increase in ceftazidime resistance compared to wild-type.
Conclusions:
- The novel codon-based mutagenesis method is efficient and automatable for generating enzyme variants.
- This approach accelerates the discovery of enzymes with enhanced antibiotic resistance properties.
- The identified beta-lactamase variants contribute to understanding ceftazidime resistance mechanisms.