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Web End = Biotechnol Lett (2015) 37:24452452 DOI 10.1007/s10529-015-1934-x
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Web End = Improving the electro-transformation efciencyof Corynebacterium glutamicum by weakening its cell wall and increasing the cytoplasmic membrane uidity
Yili Ruan . Linjiang Zhu . Qi Li
Received: 12 June 2015 / Accepted: 4 August 2015 / Published online: 9 September 2015 Springer Science+Business Media Dordrecht 2015
AbstractObjective To improve the transformation efciency of Corynebacterium glutamicum cells with heterogenous plasmid DNA and single-strand DNA (ssDNA) using a methodology based on electro-transformation. Results A semicomplex hypertonic medium was selected with addition of glycine and DL-threonine to weaken cell walls and addition of Tween 80 and isonicotinic acid hydrazide to increase cytoplasmic membrane uidity. Their contents were optimized by response surface methodology. Cell growth, electro-transformation buffer, and transformation protocol were also optimized. Temporary heating inactivation of the host restriction enzyme showed a signicant
effect. Finally, a high transformation efciency of3.57 0.13 9 107 cfu/lg DNA of plasmid and1.05 9 106 StrR cfu per 109 viable cells with a ssDNA was achieved.
Conclusion The results shed light on the application in functional genomics and genome editing ofC. glutamicum.
Keywords Cell-wall weakening Corynebacterium
glutamicum Cytoplasmic membrane uidity
Electro-transformation Single-strand DNA
Introduction
Corynebacterium glutamicum is an industrial producer of various amino acids, vitamins and organic acids. Production strains were initially isolated by the classical method of mutagen-induced mutagenesis. Later, its metabolic capability was rationally improved by genetic tools like constitutive and inducible promoters (Ptek et al. 2013), T7 RNA polymerase-based gene expression system (Kortmann et al. 2014), and sacB-based gene deletion system (Tan et al. 2012). Genome-scale editing in this species has attracted wide interest like oligonucleotide-mediated recombination (Esvelt and Wang 2013). However, this one manipulation, multiple-sites modication genome editing technology is dependent upon a high transformation efciency.
Electronic supplementary material The online version of this article (doi:http://dx.doi.org/10.1007/s10529-015-1934-x
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Y. Ruan L. Zhu Q. Li (&)
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, No...