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Journal of Bacteriology, December 2002, p. 6690-6699, Vol. 184, No. 23
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.23.6690-6699.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Evolution of the C30 Carotenoid Synthase CrtM for Function in a C40 Pathway

Daisuke Umeno,* Alexander V. Tobias, and Frances H. Arnold*

Department of Chemical Engineering, California Institute of Technology 210-41, Pasadena, California 91125

Received 6 June 2002/ Accepted 28 August 2002

The C30 carotene synthase CrtM from Staphylococcus aureus and the C40 carotene synthase CrtB from Erwinia uredovora were swapped into their respective foreign C40 and C30 biosynthetic pathways (heterologously expressed in Escherichia coli) and evaluated for function. Each displayed negligible ability to synthesize the natural carotenoid product of the other. After one round of mutagenesis and screening, we isolated 116 variants of CrtM able to synthesize C40 carotenoids. In contrast, we failed to find a single variant of CrtB with detectable C30 activity. Subsequent analysis revealed that the best CrtM mutants performed comparably to CrtB in an in vivo C40 pathway. These mutants showed significant variation in performance in their original C30 pathway, indicating the emergence of enzymes with broadened substrate specificity as well as those with shifted specificity. We discovered that Phe 26 alone determines the specificity of CrtM. The plasticity of CrtM with respect to its substrate and product range highlights the potential for creating further new carotenoid backbone structures.

* Corresponding author. Mailing address: Department of Chemical Engineering, California Institute of Technology 210-41, Pasadena, CA 91125. Phone: (626) 395-4162. Fax: (626) 568-8743. E-mail for Frances H. Arnold: frances{at}cheme.caltech.edu. E-mail for Daisuke Umeno: umeno{at}cheme.caltech.edu.

Journal of Bacteriology, December 2002, p. 6690-6699, Vol. 184, No. 23
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.23.6690-6699.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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  • Umeno, D., Tobias, A. V., Arnold, F. H. (2005). Diversifying Carotenoid Biosynthetic Pathways by Directed Evolution. Microbiol. Mol. Biol. Rev. 69: 51-78 [Abstract] [Full Text]  
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