Design of a synthetic miniR1 plasmid and its production by engineered Escherichia coli

• Published in: Bioprocess and biosystems engineering 2019-08, Vol.42 (8), p.1391-1397
• Main Authors: Lara, Alvaro R., Velázquez, Daniela, Penella, Inés, Islas, Fabiola, González-De la Rosa, Claudia H., Sigala, Juan-Carlos
• Format: Article
• Language: English
• Subjects: Bacteria; Bacterial Proteins - biosynthesis; Bacterial Proteins - genetics; Biotechnology; Chemistry; Chemistry and Materials Science; Chromosomes, Bacterial - genetics; Chromosomes, Bacterial - metabolism; Deoxyribonucleic acid; DNA; E coli; Environmental Engineering/Biotechnology; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Food Science; Hemoglobin; Industrial and Production Engineering; Industrial Chemistry/Chemical Engineering; Kanamycin; Microorganisms, Genetically-Modified - genetics; Microorganisms, Genetically-Modified - metabolism; Plasmids; Plasmids - biosynthesis; Plasmids - genetics; Rapid Communication; RecA protein; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Truncated Hemoglobins - biosynthesis; Truncated Hemoglobins - genetics
• ISSN: 1615-7591; 1615-7605
• DOI: 10.1007/s00449-019-02129-2

A synthetic plasmid consisting of the minimal elements for replication control of the R1 replicon and kanamycin resistance marker, which was named pminiR1, was developed. pminiR1 production was tested at 30 °C under aerobic and microaerobic conditions in Escherichia coli W3110 recA − (W1). The plasmid DNA yields from biomass ( Y pDNA/X ) were only 0.06 ± 0.02 and 0.22 ± 0.11 mg/g under aerobic and microaerobic conditions, respectively. As an option to increase Y pDNA/X values, pminiR1 was introduced in an engineered E. coli strain expressing the Vitreoscilla hemoglobin inserted in chromosome (W12). The Y pDNA/X values using strain W12 increased to 0.85 ± 0.05 and 1.53 ± 0.14 mg/g under aerobic and microaerobic conditions, respectively. pminiR1 production in both strains was compared with that of pUC57Kan at 37 °C under aerobic and microaerobic conditions. The Y pDNA/X values for pminiR1 using strain W12 were 6.25 ± 0.16 and 9.27 ± 0.95 mg/g under aerobic and microaerobic conditions, respectively. Such yields were similar to those obtained for plasmid pUC57Kan using strain W12 (6.9 ± 0.64 and 10.85 ± 1.06 mg/g for aerobic and microaerobic cultures, respectively). Therefore, the synthetic minimal plasmid based on the R1 replicon is a valuable alternative to pUC plasmids for biotechnological applications.