Modular pathway engineering of diterpenoid synthases and the mevalonic acid pathway for miltiradiene production

J Am Chem Soc. 2012 Feb 15;134(6):3234-41. doi: 10.1021/ja2114486. Epub 2012 Feb 6.

Abstract

Microbial production can be advantageous over the extraction of phytoterpenoids from natural plant sources, but it remains challenging to rationally and rapidly access efficient pathway variants. Previous engineering attempts mainly focused on the mevalonic acid (MVA) or methyl-d-erythritol phosphate (MEP) pathways responsible for the generation of precursors for terpenoids biosynthesis, and potential interactions between diterpenoids synthases were unexplored. Miltiradiene, the product of the stepwise conversion of (E,E,E)-geranylgeranyl diphosphate (GGPP) catalyzed by diterpene synthases SmCPS and SmKSL, has recently been identified as the precursor to tanshionones, a group of abietane-type norditerpenoids rich in the Chinese medicinal herb Salvia miltiorrhiza . Here, we present the modular pathway engineering (MOPE) strategy and its application for rapid assembling synthetic miltiradiene pathways in the yeast Saccharomyces cerevisiae . We predicted and analyzed the molecular interactions between SmCPS and SmKSL, and engineered their active sites into close proximity for enhanced metabolic flux channeling to miltiradiene biosynthesis by constructing protein fusions. We show that the fusion of SmCPS and SmKSL, as well as the fusion of BTS1 (GGPP synthase) and ERG20 (farnesyl diphosphate synthase), led to significantly improved miltiradiene production and reduced byproduct accumulation. The MOPE strategy facilitated a comprehensive evaluation of pathway variants involving multiple genes, and, as a result, our best pathway with the diploid strain YJ2X reached miltiradiene titer of 365 mg/L in a 15-L bioreactor culture. These results suggest that terpenoids synthases and the precursor supplying enzymes should be engineered systematically to enable an efficient microbial production of phytoterpenoids.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • DNA / chemistry
  • DNA Primers / chemistry
  • Diterpenes / chemistry*
  • Diterpenes / pharmacology*
  • Drug Design
  • Humans
  • Mevalonic Acid / chemistry*
  • Models, Chemical
  • Phytotherapy / methods
  • Plant Extracts / metabolism
  • Protein Structure, Tertiary
  • Saccharomyces cerevisiae / metabolism
  • Salvia / metabolism
  • Sequence Analysis, DNA
  • Sequence Homology, Amino Acid

Substances

  • DNA Primers
  • Diterpenes
  • Plant Extracts
  • miltiradiene
  • DNA
  • Mevalonic Acid