Optimization of Electroporation Conditions for Introducing Heterologous DNA into Rhodobacter sphaeroides
0
조회
0
다운로드
0
인용
0
참고문헌
키워드
초록
Rhodobacter sphaeroides is a strain capable of both photoautotrophic and chemoautotrophic growth, with various metabolic pathways that make it highly suitable for converting carbon dioxide into high value-added products. However, its low transformation efficiency has posed challenges for genetic and metabolic engineering of this strain. In this study, we aimed to increase the transformation efficiency of R. sphaeroides by deleting the rshI gene coding for an endogenous DNA restriction enzyme that inhibits. We evaluated the effects of growth conditions for making electrocompetent cells and optimized electroporation parameters to be a cuvette width of 0.1 cm, an electric field strength of 30 kV/cm, a resistance of 200 Ω, and a plasmid DNA amount of 0.5 μg, followed by a 24-h recovery period. As a result, we observed over 7,000 transformants per μg of DNA under the optimized electroporation conditions using the R. sphaeroides ΔrshI strain, which is approximately 10 times higher than that of wild-type R. sphaeroides under standard bacterial electroporation conditions. These findings are expected to enhance the application of R. sphaeroides in various industrial fields in the future.
Abstract
Rhodobacter sphaeroides is a strain capable of both photoautotrophic and chemoautotrophic growth, with various metabolic pathways that make it highly suitable for converting carbon dioxide into high value-added products. However, its low transformation efficiency has posed challenges for genetic and metabolic engineering of this strain. In this study, we aimed to increase the transformation efficiency of R. sphaeroides by deleting the rshI gene coding for an endogenous DNA restriction enzyme that inhibits. We evaluated the effects of growth conditions for making electrocompetent cells and optimized electroporation parameters to be a cuvette width of 0.1 cm, an electric field strength of 30 kV/cm, a resistance of 200 Ω, and a plasmid DNA amount of 0.5 μg, followed by a 24-h recovery period. As a result, we observed over 7,000 transformants per μg of DNA under the optimized electroporation conditions using the R. sphaeroides ΔrshI strain, which is approximately 10 times higher than that of wild-type R. sphaeroides under standard bacterial electroporation conditions. These findings are expected to enhance the application of R. sphaeroides in various industrial fields in the future.
참고문헌 (28)
- Lu S, "A critical combination of operating parameters can significantly increase the electrotransformation efficiency of a gram-positive Dietzia strain", J. Microbiol. Methods, 103, pp.144~151 (2014)
- Wang C, "Optimization of electrotransformation(ETF)conditions in lactic acid bacteria(LAB)", J. Microbiol Methods, 174, pp.105944~ (2020)
- Lorenz MG, "Bacterial gene transfer by natural genetic transformation in the environment", Microbiol. Rev, 58, pp.563~602 (1994)
- Fiedler S, "Transformation of bacteria with plasmid DNA by electroporation", Anal. Biochem, 170, pp.38~44 (1988)
- Nybo SE, "Metabolic engineering in chemolithoautotrophic hosts for the production of fuels and chemicals", Metab. Eng, 30, pp.105~120 (2015)
- Tee KL, "An efficient transformation method for the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16", Biotechnol. J, 12 (2017)[DOI]
- Dower WJ, "High efficiency transformation of E. coli by high voltage electroporation", Nucleic Acids Res, 16, pp.6127~6145 (1988)
- Taketo A, "DNA transfection of Escherichia coli by electroporation", Biochimica et Biophysica Acta(BBA)-Gene Structure and Expression, 949, pp.318~324 (1988)
- Kim YH, "Optimization of technical conditions for the transformation of Lactobacillus acidophilus strains by electroporation", J. Appl. Microbiol, 99, pp.167~174 (2005)
- Jun D, "Use of new strains of Rhodobacter sphaeroides and a modified simple culture medium to increase yield and facilitate purification of the reaction centre", Photosynth. Res, 120, pp.197~205 (2014)
- Jaschke P, "Modification of the genome of Rhodobacter sphaeroides and construction of synthetic operons", Methods Enzymol, 497, pp.519~538 (2011)
- Sistrom WR, "The Kinetics of the synthesis of photopigments in Rhodopseudomonas spheroides", Microbiology, 28, pp.607~616 (1962)
- Fischer HM, "One member of a gro-ESL-like chaperonin multigene family in Bradyrhizobium japonicum is co-regulated with symbiotic nitrogen fixation genes", EMBO J, 12, pp.2901~2912 (1993)
- Thomas S, "DNA library construction using Gibson Assembly®", Nat. Methods, 12, pp.i~ii (2015)
- Marx CJ, "Broad-host-range cre-lox system for antibiotic marker recycling in Gram-negative bacteria", BioTechniques, 33, pp.1062~1067 (2002)
- Jun D, "Use of new strains of Rhodobacter sphaeroides and a modified simple culture medium to increase yield and facilitate purification of the reaction centre", Photosynth. Res, 120, pp.197~205 (2014)
- Kovach ME, "Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes", Gene, 166, pp.175~176 (1995)
- Sadykov MR, "Restriction-modification systems as a barrier for genetic manipulation of Staphylococcus aureus", Methods Mol. Biol, 1373, pp.9~23 (2016)
- Lee IH, "Reductive effect of H2 uptake and poly-beta-hydroxybutyrate formation on nitrogenase-mediated H2 accumulation of Rhodobacter sphaeroides according to light intensity", Appl. Microbiol. Biotechnol, 60, pp.147~153 (2002)
- Su A, "Metabolic redesign of Rhodobacter sphaeroides for lycopene production", J. Agric. Food Chem, 66, pp.5879~5885 (2018)
- Zhang H, "Optimization of electroporation conditions for Arthrobacter with plasmid PART2", J. Microbiol. Methods, 84, pp.114~120 (2011)
- Lofblom J, "Optimization of electroporation-mediated transformation : Staphylococcus carnosus as model organism", J. Appl. Microbiol, 102, pp.736~747 (2007)
- Thompson K, "Improvement in electroporation efficiency for Lactobacillus plantarum by the inclusion of high concentrations of glycine in the growth medium", J. Microbiol. Methods, 26, pp.73~79 (1996)
- Holo H, "High-frequency transformation, by electroporation, of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media", Appl. Environ. Microbiol, 55, pp.3119~3123 (1989)
- Xue D, "Study on the construction of recombined plasmid pMG36e-lacc1 and the electroporation of Lactobacillus buchneri", Bio-Medical Mater. Eng, 24, pp.3855~3861 (2014)
- Dallaire-Dufresne S, "Optimization of a plasmid electroporation protocol for Aeromonas salmonicida subsp. salmonicida", J. Microbiol. Methods, 98, pp.44~49 (2014)
- Turgeon N, "Elaboration of an electroporation protocol for Bacillus cereus ATCC 14579", J. Microbiol. Methods, 67, pp.543~548 (2006)
- Mougiakos I, "Efficient Cas9-based genome editing of Rhodobacter sphaeroides for metabolic engineering", Microb. Cell Fact, 18, pp.204~ (2019)