Metabolic engineering of ketocarotenoids biosynthetic pathway in Chlamydomonas reinhardtii strain CC-4102

Sci Rep. 2020 Jul 1;10(1):10688. doi: 10.1038/s41598-020-67756-2.

Abstract

In Chlamydomonas reinhardtii, ketocarotenoid biosynthesis is limited to the diploid zygospore stage. In this study, we attempted to engineer the ketocarotenoid pathway into Chlamydomonas haploid vegetative green cells by overexpressing the key enzyme ß-carotene ketolase (CrBKT). We chose strain CC-4102 for the approach; competitive pathways, α-carotene biosynthesis and xanthophyll cycle are silenced in this strain. Driven by the strong constitutive HSP70/RBCS2 promoter CrBKT overexpression resulted in the production of canthaxanthin, the ketolation product from ß-carotene as well as a drastic reduction in the chlorophyll concentration. Intriguingly, these phenotypes could only be detected from lines transformed and grown heterotrophically in the dark. Once exposed to light, these transformants lost the aforementioned phenotypes as well as their antibiotic resistance. This phenomenon is in agreement with the fact that we were unable to recover any canthaxanthin-producing line among light-selected transformants.

MeSH terms

  • Biosynthetic Pathways / genetics
  • Biosynthetic Pathways / physiology
  • Biotechnology / methods
  • Canthaxanthin / biosynthesis*
  • Carotenoids / metabolism*
  • Cell Line
  • Chlamydomonas reinhardtii / genetics*
  • Chlamydomonas reinhardtii / metabolism*
  • Chlorophyll / analysis
  • DNA, Plant / genetics
  • Gene Expression Regulation, Plant / genetics
  • HSP70 Heat-Shock Proteins / genetics
  • Metabolic Engineering / methods*
  • Oxygenases / genetics
  • Oxygenases / metabolism
  • Plants, Genetically Modified / genetics
  • Promoter Regions, Genetic / genetics
  • Secondary Metabolism / genetics
  • beta Carotene / metabolism

Substances

  • DNA, Plant
  • HSP70 Heat-Shock Proteins
  • beta Carotene
  • Chlorophyll
  • Carotenoids
  • Canthaxanthin
  • Oxygenases
  • beta-carotene ketolase