Host strain influences on supercoiled plasmid DNA production in Escherichia coli: Implications for efficient design of large-scale processes.
We set out to investigate if E. coli genotype plays a significant role in host strain selection for optimal processing of plasmid DNA based on both quality and quantity of supercoiling. Firstly 17 E. coli commercial and non-commercial strains were selected and their available genetic backgrounds were researched in the open literature. Growth characteristics of all the strains were considered and made impartial by using a common medium and growth condition platform. By keeping the growth conditions constant for each strain/plasmid combination, we are only looking at one variable which is the host strain. The second step was to attempt to correlate the findings with common genotype characteristics (e.g. mutations such as endA or recA). We found that one can screen the number of strains which are likely to give good productivity early on, before any further optimisation and verification is performed, both for small and large plasmids. Also, it is worth noting that complex plasmid interactions with each strain prevent the use of genotype alone in making an intelligent choice for supercoiling optimisation. This leads to a third optimisation step selecting a few of the potentially high performing strains based on high DNA yield and supercoiling, with a view to identify the factors which need improvement in strain design and bioreactor optimisation. We found that high specific growth rates of some strains did not affect the level of DNA supercoiling but did influence the total plasmid yield, potentially an important aspect in the design of fermentation strategy. Interestingly, a few host/plasmid combinations result in what appears to be runaway plasmid replication.
Yau
Sin Yee
SY
Advanced Centre of Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, United Kingdom.
Keshavarz-Moore
Eli
E
Ward
John
J
D004269:DNA, Bacterial / Q000096:biosynthesis* / Q000302:isolation & purification*; D004278:DNA, Superhelical / Q000096:biosynthesis* / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D010957:Plasmids / Q000096:biosynthesis* / Q000302:isolation & purification*
D004269:DNA, Bacterial; D004278:DNA, Superhelical