Abstract
Functional evolution of an arsenic resistance operon has been accomplished by DNA shuffling, involving multiple rounds of in vitro recombination and mutation of a pool of related sequences, followed by selection for increased resistance in vivo. Homologous recombination is achieved by random fragmentation of the PCR templates and reassembly by primerless PCR. Plasmid-determined arsenate resistance from plasmid pI258 encoded by genes arsR, arsE, and arsC was evolved in Escherichia coli. Three rounds of shuffling and selection resulted in cells that grew in up to 0.5 M arsenate, a 40-fold increase in resistance. Whereas the native plasmid remained episomal, the evolved operon reproducibly integrated into the bacterial chromosome. In the absence of shuffling, no increase in resistance was observed after four selection cycles, and the control plasmid remained episomal. The integrated ars operon had 13 mutations. Ten mutations were located in arsB, encoding the arsenite membrane pump, resulting in a fourfold to sixfold increase in arsenite resistance. While arsC, the arsenate reductase gene, contained no mutations, its expression level was increased, and the rate of arsenate reduction was increased 12–fold. These results show that DNA shuffling can improve the function of pathways by complex and unexpected mutational mechanisms that may be activated by point mutation. These mechanisms may be difficult to explain and are likely to be overlooked by rational design.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Stemmer, W.P.C. 1994. DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA 91: 10747–10751.
Stemmer, W.P.C. 1994. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370: 389–391.
Stemmer, W.P.C. 1995. Searching sequence space. Bio/Technology 13: 549–553.
Crameri, A., Cwirla, S. and Stemmer, W.P.C. 1996. Construction and evolution of antibody-phage libraries by DNA shuffling. Nature Medicine 2: 100–103.
Crameri, A., Whitehorn, E.A., Tate, E. and Stemmer, W.P.C. 1996. Improved green fluorescent protein by molecular evolution using DNA shuffling. Nature Biotechnology 14: 315–319.
Zhang, J., Dawes, G. and Stemmer, W.P.C. . 1997. Evolution of a fucosidase from a galactosidase by DNA shuffling. Proc. Natl. Acad. Sci. USA. In press.
Cervantes, C., Ji, G., Ramirez, J.L. and Silver, S. .1994. Resistance to arsenic compounds in microorganisms. FEMS Microbiol. Rev. 15: 355–367.
Rawlings, D.E. and Silver, S. .1995. Mining with microbes.Bio/Technology 13: 33–778.
Ji, G. and Silver, S. .1992. Regulation and expression of the arsenic resistance operon from Staphylococcus aureus plasmid pl258.J. Bacteriol. 174: 3684–3694.
Wu, J., Tisa, L.S. and Rosen, B.R. 1992. Membrane topology of the ArsB protein, the membrane subunit of an anion-translocating ATPase. J. Biol. Chem. 267: 12570–12576.
Ji, G. and Silver, S. 1992. Reduction of arsenate to arsenite by the ArsC protein of the arsenic resistance operon of Staphylococcus aureus plasmid pl258.Proc. Natl. Acad. Sci. USA 89: 9474–9478.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Crameri, A., Dawes, G., Rodriguez Jr., E. et al. Molecular evolution of an arsenate detoxification pathway by DNA shuffling. Nat Biotechnol 15, 436–438 (1997). https://doi.org/10.1038/nbt0597-436
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/nbt0597-436
This article is cited by
-
In vivo Protein Evolution, Next Generation Protein Engineering Strategy: from Random Approach to Target-specific Approach
Biotechnology and Bioprocess Engineering (2019)
-
Directed evolution of a cellobiose utilization pathway in Saccharomyces cerevisiae by simultaneously engineering multiple proteins
Microbial Cell Factories (2013)
-
Revealing biases inherent in recombination protocols
BMC Biotechnology (2007)