Design and Characterization of Rapid Optogenetic Circuits for Dynamic Control in Yeast Metabolic Engineering

ACS Synth Biol. 2020 Dec 18;9(12):3254-3266. doi: 10.1021/acssynbio.0c00305. Epub 2020 Nov 24.

Abstract

The use of optogenetics in metabolic engineering for light-controlled microbial chemical production raises the prospect of utilizing control and optimization techniques routinely deployed in traditional chemical manufacturing. However, such mechanisms require well-characterized, customizable tools that respond fast enough to be used as real-time inputs during fermentations. Here, we present OptoINVRT7, a new rapid optogenetic inverter circuit to control gene expression in Saccharomyces cerevisiae. The circuit induces gene expression in only 0.6 h after switching cells from light to darkness, which is at least 6 times faster than previous OptoINVRT optogenetic circuits used for chemical production. In addition, we introduce an engineered inducible GAL1 promoter (PGAL1-S), which is stronger than any constitutive or inducible promoter commonly used in yeast. Combining OptoINVRT7 with PGAL1-S achieves strong and light-tunable levels of gene expression with as much as 132.9 ± 22.6-fold induction in darkness. The high performance of this new optogenetic circuit in controlling metabolic enzymes boosts production of lactic acid and isobutanol by more than 50% and 15%, respectively. The strength and controllability of OptoINVRT7 and PGAL1-S open the door to applying process control tools to engineered metabolisms to improve robustness and yields in microbial fermentations for chemical production.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Butanols / metabolism
  • Galactokinase / genetics
  • Gene Expression Regulation, Fungal / drug effects
  • Lactic Acid / metabolism
  • Light
  • Metabolic Engineering / methods*
  • Optogenetics
  • Plasmids / genetics
  • Plasmids / metabolism
  • Promoter Regions, Genetic
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*

Substances

  • Butanols
  • Lactic Acid
  • isobutyl alcohol
  • Galactokinase