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

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 (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 lighttunable 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.

Modular and Molecular Optimization of a LOV (Light–Oxygen–Voltage)-Based Optogenetic Switch in Yeast

“…Different blue-light optogenetic systems have been implemented in yeast to control gene expression, with remarkable applications in metabolic engineering and yeast biotechnology [8][9][10][11][12]. Among them, the OptoEXP and OptoINVRT optogenetic systems, both based on the blue-light photoreceptor EL222 from Erythrobacter litoralis, have been used to redirect the glycolytic metabolic flux depending on the applied light or dark regimens [10][11][12].…”

Cellulosic biofuel production using emulsified simultaneous saccharification and fermentation (eSSF) with conventional and thermotolerant yeasts

“…Biosynthesis of isobutanol in yeast is challenged by the strong competition with ethanol biosynthesis, a pathway that cannot be easily deleted genetically due to its essential role for growth on glucose [ 54 , 55 ]. We have previously shown that this challenge can be overcome by dynamically controlling the ethanol and the mitochondrial isobutanol biosynthetic pathways with light using optogenetic circuits [ 56 , 57 ]. The two pathways compete for pyruvate, metabolized by either pyruvate decarboxylases (encoded by PDC1, PDC5 and PDC6 ) for ethanol production or by acetolactate synthase (encoded by ILV2 ) for isobutanol.…”

“…6 a). By controlling PDC1 (in a strain with the three native PDC genes knocked out) using a light-activated circuit (OptoEXP) [ 57 ], and ILV2 with a dark-activated circuit (OptoINVRT7) [ 56 ], the engineered yeast can grow only in the light (producing ethanol), and then direct its metabolic flux towards isobutanol production in the dark (Fig. 6 a).…”

“…6 a). This approach has been shown to improve isobutanol titers by allowing biomass to build up before repressing an essential competing pathway and subjecting the cells to the metabolic burden of production [ 56 , 57 ].

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“…For the isobutanol tests, we used the S. cerevisiae YEZ546-2 strain, which is engineered with dynamic optogenetic controls to grow robustly in the light and produce isobutanol in the dark [ 56 ]. In this strain, the native PDC1 , PDC2 , and PDC3 genes are deleted, which are essential for fermentation of glucose into ethanol.…”