Optogenetic Amplification Circuits for Light-Induced Metabolic Control

Abstract

Dynamic control of microbial metabolism is an effective strategy to improve chemical production in fermentations. While dynamic control is most often implemented using chemical inducers, optogenetics offers an attractive alternative due to the high tunability and reversibility afforded by light. However, a major concern of applying optogenetics in metabolic engineering is the risk of insufficient light penetration at high cell densities, especially in large bioreactors. Here, we present a new series of optogenetic circuits we call OptoAMP, which amplify the transcriptional response to blue light by as much as 23-fold compared to the basal circuit (OptoEXP). These circuits show as much as a 41-fold induction between dark and light conditions, efficient activation at light duty cycles as low as ∼1%, and strong homogeneous light-induction in bioreactors of at least 5 L, with limited illumination at cell densities above 40 OD 600 . We demonstrate the ability of OptoAMP circuits to control engineered metabolic pathways in novel three-phase fermentations using different light schedules to control enzyme expression and improve production of lactic acid, isobutanol, and naringenin. These circuits expand the applicability of optogenetics to metabolic engineering.

Bifunctional optogenetic switch for improving shikimic acid production in E. coli

“…Product yields in microbial cell factories are currently limited by metabolic flux competition between biomass production and biosynthesis. Therefore, several strategies to decouple growth and production phases have been created, including the application of substrate-dependent promoters [ 45 ], protein degradation based cell growth [ 46 ], CRISPRi governed intermittent gene expression [ 41 ], light controlled production phase [ 47 ], aptamer-based regulatory biosensor [ 48 ], etc. Herein, the potential of optogenetics in decoupling cell growth and shikimic acid production was demonstrated for the first time in shikimic acid production, and a more than twofold higher shikimic acid titer than previous reports was obtained under mineral salt medium.…”

Toward Multiplexed Optogenetic Circuits

“…When the LOV domain absorbs blue light, it changes its conformation and exposes HTH domains to bind to a cognate DNA sequence (C20) ( Rivera-Cancel et al, 2012 ) Beyond that, the small size of EL222 and abundance of cofactors could be beneficial for its portability in different hosts. Several studies have shown that the expression of EL222 and cognate promoters resulted in blue light–mediated transcriptional regulation ( Jayaraman et al, 2016 ; Zhao et al, 2018 ; Ding et al, 2020 ; Zhao et al, 2021a ). Jayaraman and others replaced the lux box in the native luxI promoter with the 18-bp EL222-binding region ( Jayaraman et al, 2016 ).…”

“…Temporal pulses in the light-duty cycle are sufficient to induce the optogenetic system while reducing the light exposure ( Lalwani et al, 2021 ). Light pulsing has been implemented in engineered yeast harboring different metabolic pathways ( Zhao et al, 2021a ). Interestingly, the metabolite ratio is modified in the function of the light pattern, indicating that fine-tuning of the inducer can have a major effect on the actuator, opening room for more sustainable metabolic engineering design: do more and purest final product with less precursor.…”

“…Nevertheless, this approach is less adapted for pulse design as at height cell density, light will be required for the inhibition part. To limit this issue, Zhao and others have recently created an OptoAMP circuit which amplifies the transcriptional response to blue light by as much as 23-fold compared to the basal circuit and allows efficient blue light activation of high–cell density culture in a 5-L bioreactor ( Zhao et al, 2021a ). Whatever the optogenetic design, at height cell density, each cell should receive the correct amount of light, according to the light-duty cycle.…”

Development of Optogenetic Dual-Switch System for Rewiring Metabolic Flux for Polyhydroxybutyrate Production

“…Previously, we developed several dynamic methods for regulating metabolic flux and enhancing PHB production in E. coli ; they include an autoinduced AND gate, a native interspaced short palindromic repeats interference (CRISPRi) system, and quorum sensing-based (QS-based) bifunctional dynamic switches (QS switches) [ 26 , 27 , 28 ]. However, the activation and repression of gene expression in such strategies has proven inconvenient, especially when different sequences and intervals of gene expression must be regulated [ 20 , 29 ].…”