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 (P_GAL1-S), which is stronger than any constitutive or inducible promoter commonly used in yeast. Combining OptoINVRT7 with P_GAL1-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 P_GAL1-S open the door to applying process control tools to engineered metabolisms to improve robustness and yields in microbial fermentations for chemical production.

• PMID: 33232598
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Journal Article | Research Support, N.I.H., Extramural | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, Non-P.H.S.

Authors

Zhao EM, Lalwani MA, Lovelett RJ, García-Echauri SA, Hoffman SM, Gonzalez CL, Toettcher JE, Kevrekidis IG, Avalos JL

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