Engineered bidirectional promoters enable rapid multi-gene co-expression optimization

Nat Commun. 2018 Sep 4;9(1):3589. doi: 10.1038/s41467-018-05915-w.

Abstract

Numerous synthetic biology endeavors require well-tuned co-expression of functional components for success. Classically, monodirectional promoters (MDPs) have been used for such applications, but MDPs are limited in terms of multi-gene co-expression capabilities. Consequently, there is a pressing need for new tools with improved flexibility in terms of genetic circuit design, metabolic pathway assembly, and optimization. Here, motivated by nature's use of bidirectional promoters (BDPs) as a solution for efficient gene co-expression, we generate a library of 168 synthetic BDPs in the yeast Komagataella phaffii (syn. Pichia pastoris), leveraging naturally occurring BDPs as a parts repository. This library of synthetic BDPs allows for rapid screening of diverse expression profiles and ratios to optimize gene co-expression, including for metabolic pathways (taxadiene, β-carotene). The modular design strategies applied for creating the BDP library could be relevant in other eukaryotic hosts, enabling a myriad of metabolic engineering and synthetic biology applications.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkenes / metabolism
  • Cytochrome P-450 CYP2D6 / genetics
  • Diterpenes / metabolism
  • Farnesyltranstransferase / genetics
  • Gene Expression Regulation, Fungal
  • Genetic Engineering / methods*
  • Histones / genetics
  • Microorganisms, Genetically-Modified
  • Pichia / genetics*
  • Pichia / metabolism
  • Promoter Regions, Genetic*
  • beta Carotene / genetics
  • beta Carotene / metabolism

Substances

  • Alkenes
  • Diterpenes
  • Histones
  • taxa-4(5),11(12)diene
  • beta Carotene
  • Cytochrome P-450 CYP2D6
  • Farnesyltranstransferase