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Related Experiment Videos

A C35 carotenoid biosynthetic pathway.

Daisuke Umeno1, Frances H Arnold

  • 1Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA. umeno@cheme.caltech.edu

Applied and Environmental Microbiology
|June 6, 2003
PubMed
Summary

Researchers engineered a novel C(35) carotenoid biosynthetic pathway in E. coli, producing unique carotenoids from a C(30) synthase and C(40) or C(30) enzymes. Directed evolution further expanded this pathway, creating over 10 new compounds.

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Area of Science:

  • Biochemistry
  • Synthetic Biology
  • Metabolic Engineering

Background:

  • Carotenoids are vital pigments with diverse biological functions.
  • Existing carotenoid biosynthesis pathways primarily focus on C(40) and C(30) backbones.
  • Enzyme engineering offers potential for novel carotenoid structures.

Purpose of the Study:

  • To engineer a novel C(35) carotenoid biosynthetic pathway in Escherichia coli.
  • To explore the substrate flexibility of carotenoid enzymes.
  • To generate structurally diverse carotenoids using combinatorial biosynthesis and directed evolution.

Main Methods:

  • Coexpression of Staphylococcus aureus CrtM (C(30) carotenoid synthase) and Erwinia geranylgeranyldiphosphate (GGDP) synthase in E. coli.
  • Utilizing farnesyldiphosphate and GGDP as precursors for C(35) carotenoid synthesis.

Related Experiment Videos

  • Employing carotene desaturases and cyclases from C(40) or C(30) pathways to process C(35) substrates.
  • Applying directed evolution to desaturase variants and combinatorial expression with lycopene cyclases.

    • Main Results:

    • Successful production of novel C(35) carotenoids with an asymmetrical backbone.
    • C(35) carotenoids constituted 40-60% of the total accumulated carotenoids.
    • Demonstrated acceptance and conversion of C(35) substrates by C(40) and C(30) pathway enzymes.
    • Generated at least 10 previously undescribed carotenoid compounds through directed evolution and combinatorial expression.

    Conclusions:

    • Carotenoid pathways exhibit significant plasticity and expansibility.
    • Combinatorial biosynthesis coupled with directed evolution is an effective strategy for rapid generation of diverse chemical structures.
    • This study establishes a foundation for engineering novel carotenoid biosynthesis pathways with tailored properties.