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Updated: Aug 16, 2026
Site-specific Bacterial Chromosome Engineering: ΦC31 Integrase Mediated Cassette Exchange (IMCE)
Published on: March 16, 2012
Characterization of the Micromonospora rosaria pMR2 plasmid and development of a high G+C codon optimized integrase
Thomas J Hosted1, Tim Wang, Ann C Horan
1New Lead Discovery, Schering Plough Research Institute, 2015 Galloping Hill Road, K15-C321-MS3600, Kenilworth, NJ 07033, USA. thomas.hosted@spcorp.com
Abstract:
pMR2, an 11.1 kb plasmid was isolated from Micromonospora rosaria SCC2095, NRRL3718, and its complete nucleotide sequence determined. Analysis revealed 13 ORFs including homologs of a KorSA regulatory protein and TraB plasmid transfer protein found on other actinomycete plasmids. pMR2 contains att/int functions consisting of an integrase, an excisionase, and a putative plasmid attachment site (attP). The integrase gene contained a high frequency of codons rarely used in high G+C actinomycete coding regions. The gene was codon optimized for actinomycete codon usage to create the synthetic gene int-OPT. pSPRX740, containing an rpsL promoter and the att/int-OPT region, was introduced into Micromonospora halophytica var. nigra ATCC33088. Analysis of DNA flanking the pSPRX740 integration site confirmed site-specific integration into a tRNA(Phe) gene in the M. halopytica var. nigra chromosome. The pMR2 attP element and chromosomal attachment (attB) site contain a 63 bp region of sequence identity overlapping the 3' end of the tRNA(Phe) gene. Plasmids comprising the site-specific att/int-OPT functions of pMR2 can be used to integrate genes into the chromosome of actinomycetes with an appropriate tRNA gene. The development of an integrative system for Micromonospora will expand our ability to study antibiotic biosynthesis in this important actinomycete genus.
Insights
Researchers developed a novel gene integration system for Micromonospora using the pMR2 plasmid. This system enables site-specific integration into actinomycete chromosomes, aiding antibiotic biosynthesis research.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Actinomycetes are crucial for antibiotic production.
- Gene integration systems are vital for studying microbial genetics and biosynthesis pathways.
- The Micromonospora genus presents unique challenges for genetic manipulation.
Purpose of the Study:
- To characterize the pMR2 plasmid from Micromonospora rosaria.
- To develop a site-specific integration system for Micromonospora species.
- To facilitate genetic studies and enhance antibiotic biosynthesis research in Micromonospora.
Main Methods:
- Isolation and sequencing of the pMR2 plasmid.
- Identification and analysis of open reading frames (ORFs).
- Codon optimization of the integrase gene (int-OPT).
- Construction of a new plasmid (pSPRX740) for integration.
- Site-specific integration into Micromonospora halophytica var. nigra chromosome.
Main Results:
- The pMR2 plasmid (11.1 kb) was fully sequenced, revealing 13 ORFs.
- A functional att/int system was identified, including integrase and attachment site (attP).
- Codon-optimized integrase (int-OPT) facilitated site-specific integration into a tRNA(Phe) gene in M. halopytica.
- Integration occurred at a 63 bp region of sequence identity between attP and attB.
Conclusions:
- The pMR2 plasmid provides a basis for a novel site-specific gene integration system in actinomycetes.
- This system utilizes the att/int-OPT functions and an appropriate tRNA gene for chromosomal integration.
- The developed system will significantly advance genetic manipulation and antibiotic biosynthesis studies in Micromonospora.
