Use of directed enzyme evolution to create novel biosynthetic pathways for production of rare or non-natural carotenoids

Maiko Furubayashi*, Daisuke Umeno

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Citation (Scopus)


In recent years, advances in bioengineering and synthetic biology techniques have been used to create carotenoid diversity in the laboratory. In this chapter, we describe the step-by-step method to perform directed evolution of carotenoid biosynthetic enzymes. We first explain how to establish an efficient Escherichia coli colony-based screening, including a detailed description of plasmid DNA construction design as well as tips and tricks to handle and manipulate cells to produce stable colonies. As an example for the directed evolution experiment, we engineer a bacterial phytoene desaturase CrtI to obtain a C50-phytoene desaturase, which catalyzes formation of a non-natural long-chain carotenoid. The method described in this chapter can be applied to many carotenoid biosynthetic enzymes, whose numbers have been rapidly expanding with recent advances in genomics. The use of directed evolution for carotenoid enzymes will contribute not only to the discovery of novel carotenoids but also to a deeper understanding of the creation and evolution of carotenoid biosynthetic pathways in nature.

Original languageEnglish
Title of host publicationCarotenoids
Subtitle of host publicationCarotenoid and apocarotenoid biosynthesis metabolic engineering and synthetic biology
EditorsEleanore T. Wurtzel, Eleanore T. Wurtzel
PublisherAcademic Press Inc.
Number of pages32
ISBN (Print)9780323913539
Publication statusPublished - 2022 Jan

Publication series

NameMethods in Enzymology
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988


  • Carotenoids
  • Directed evolution
  • Non-natural
  • Pathway engineering

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology


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