Abstract
Genetic manipulations including chromosome engineering are essential techniques used to restructure cell metabolism. Lambda/Red (λ/Red)-mediated recombination is the most commonly applied approach for chromosomal modulation in Escherichia coli. However, the efficiency of this method is significantly hampered by the laborious removal of the selectable markers. To overcome the problem, the integration helper plasmid was constructed, pSBC1a-CtR, which contains Red recombinase, Cre recombinase, and exogenous orthogonal aminoacyl-transfer RNA (tRNA) synthetase/tRNA pairs, allows an unnatural amino acid (UAA) to be genetically encoded at the defined site of the antibiotic resistance gene-encoded protein. When UAAs are not in the culture medium, there was no expression in the antibiotic resistance gene-encoded protein. Accordingly, the next procedure of antibiotic gene excising is not needed. To verify this method, poxB gene was knocked out successfully. Furthermore, sequential deletion of three target genes (galR, ptsG, and pgi) was able to generate neurosporene-producing strain marked by high growth rate. Thus, the site-specific incorporation UAA mutagenesis system were used to control and expand the use of conditional selectable marker, and the technique is used to facilitate a rapid continuous genome editing in Escherichia coli.
Highlights
Synthetic biology methods have greatly facilitated the optimization of processes in metabolic engineering
This study describes the novel unnatural amino acid (UAA) mutagenesis system that utilizes a pair of exogenous M. jannaschii tyrosyl-transfer RNA (tRNA) and tyrosyl-tRNA synthetase (TyrRS) construct
To reduce the false-positive rate due to plasmid incompatibility, the vector pSBC1a-CtR and the template vector including pSLKM, pSLC, and pSLK2272 were constructed with lowcopy number replication origin pSC101
Summary
Synthetic biology methods have greatly facilitated the optimization of processes in metabolic engineering. Ribosome binding sites, and gene circuits were designed using synthetic technologies and assembled into metabolic pathways to improve the metabolic flux and gene regulation efficiency. By these ways, the production of medicinal chemicals such as artemisinin (Chang et al, 2007) and taxol (Li et al, 2019) has been successfully optimized. The λ/Red-based system was developed to mediate chromosomal homologous recombination, and resistance gene markers were used for genetic selection (Wang et al, 2016). The pKD46 vectors include three genes: γ, β, and exo, whose products make chromosomal recombination. The PCR products were generated using pairs of primers that included homology extension
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