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JOURNAL OF BACTERIOLOGY, Dec. 2002, p. 66906699 Vol. 184, No. 23 0021-9193/02/$04.00 0 DOI: 10.1128/JB.184.23.66906699.2002
 

Summary: JOURNAL OF BACTERIOLOGY, Dec. 2002, p. 66906699 Vol. 184, No. 23
0021-9193/02/$04.00 0 DOI: 10.1128/JB.184.23.66906699.2002
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 (heterolo-
gously 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.

  

Source: Arnold, Frances H. - Division of Chemistry and Chemical Engineering, California Institute of Technology

 

Collections: Chemistry; Biology and Medicine