Optogenetic control of the lac operon for bacterial chemical and protein production

Lalwani, Makoto A.; Ip, Samantha S.; Carrasco-López, César; Day, Catherine; Zhao, Evan M.; Kawabe, Hinako; Avalos, José L.

doi:10.1038/s41589-020-0639-1

Cited by 93 publications

(102 citation statements)

References 58 publications

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“…IPTG, the gold standard inducer in E. coli , binds the LacI repressor and thus induces the expression of genes containing a lac operator in their promoter by preventing LacI from shielding DNA from RNA polymerase. Lalwani et al [ 26 ] ( Figure 1 a) placed LacI under the control of the pDawn system, so that blue light induces LacI expression and therefore represses the genes of interest. In contrast, the absence of light represses LacI expression and therefore activates the various IPTG-inducible promoters.…”

Section: Merging Optogenetics and Bioproductionmentioning

confidence: 99%

“…This system was optimized to reduce leakiness, and although the expression dynamics are slower than those of IPTG induction systems (2 h delay), the final induction levels are higher and production exceeds that of the IPTG induction systems. Thus, the pDawn system is a successful alternative to IPTG induction, and has already been tested and applied to bioproduction and scaled-up to 2 L [ 26 ]. The pDusk and pDawn systems also highlight the possibility of activating or repressing a system by illumination or darkness, depending on how the optogenetic system is connected to the bioproduction system.…”

Section: Merging Optogenetics and Bioproductionmentioning

confidence: 99%

“…Both studies demonstrated enrichment in their respective competing pathways, as expected. To obtain more insight into induction timing, Raghavan et al [ 28 ] used the PhyB-PIF system ( Figure 1 b) and Lalwani et al [ 26 ] used FixJ ( Figure 1 a) to explore light induction at different optical densities during growth. Raghavan et al found that the illumination pulse was most efficient during the late exponential phase of growth, and Lalwani et al found that constant illumination at an OD of about 1 was optimal.…”

Section: Control Strategiesmentioning

confidence: 99%

“…Given the ease with which such strategies can be implemented using optogenetics, dynamic control is starting to appear in bioproduction studies. For instance, Lalwani et al [ 26 ] tested the ability of illumination duty cycles to control protein expression levels over a period of about 17 min. They managed to recapitulate a full range of induction strengths (similarly to [ 33 ]), which can be very difficult to obtain using chemical inducers (such as IPTG) that induce high expression levels regardless of their concentration, i.e., in a more switch-like manner.…”

Section: Control Strategiesmentioning

confidence: 99%

“…Another solution to the light penetration issue could be the use of “inverted” optogenetic systems, where genes for the production phase are activated in the dark, when light becomes scarce, i.e., when a certain cell density is reached [ 26 ]; however, this would hardly allow for dynamic control.…”

Section: Scale-up Instrumentsmentioning

confidence: 99%

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The Promise of Optogenetics for Bioproduction: Dynamic Control Strategies and Scale-Up Instruments

et al. 2020

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Progress in metabolic engineering and synthetic and systems biology has made bioproduction an increasingly attractive and competitive strategy for synthesizing biomolecules, recombinant proteins and biofuels from renewable feedstocks. Yet, due to poor productivity, it remains difficult to make a bioproduction process economically viable at large scale. Achieving dynamic control of cellular processes could lead to even better yields by balancing the two characteristic phases of bioproduction, namely, growth versus production, which lie at the heart of a trade-off that substantially impacts productivity. The versatility and controllability offered by light will be a key element in attaining the level of control desired. The popularity of light-mediated control is increasing, with an expanding repertoire of optogenetic systems for novel applications, and many optogenetic devices have been designed to test optogenetic strains at various culture scales for bioproduction objectives. In this review, we aim to highlight the most important advances in this direction. We discuss how optogenetics is currently applied to control metabolism in the context of bioproduction, describe the optogenetic instruments and devices used at the laboratory scale for strain development, and explore how current industrial-scale bioproduction processes could be adapted for optogenetics or could benefit from existing photobioreactor designs. We then draw attention to the steps that must be undertaken to further optimize the control of biological systems in order to take full advantage of the potential offered by microbial factories.

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Section: Merging Optogenetics and Bioproductionmentioning

confidence: 99%

Section: Merging Optogenetics and Bioproductionmentioning

confidence: 99%

Section: Control Strategiesmentioning

confidence: 99%

Section: Control Strategiesmentioning

confidence: 99%

Section: Scale-up Instrumentsmentioning

confidence: 99%

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The Promise of Optogenetics for Bioproduction: Dynamic Control Strategies and Scale-Up Instruments

et al. 2020

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Extracellularly Detectable Electrochemical Signals of Living Cells Originate from Metabolic Reactions

Koo

Kim

et al. 2023

Advanced Science

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Direct detection of cellular redox signals has shown immense potential as a novel living cell analysis tool. However, the origin of such signals remains unknown, which hinders the widespread use of electrochemical methods for cellular research. In this study, the authors found that intracellular metabolic pathways that generate adenosine triphosphate (ATP) are the main contributors to extracellularly detectable electrochemical signals. This is achieved through the detection of living cells (4,706 cells/chip, linearity: 0.985) at a linear range of 7,466–48,866. Based on this discovery, the authors demonstrated that the cellular signals detected by differential pulse voltammetry (DPV) can be rapidly amplified with a developed medium containing metabolic activator cocktails (MACs). The DPV approach combined with MAC treatment shows a remarkable performance to detect the effects of the anticancer drug CPI‐613 on cervical cancer both at a low drug concentration (2 µm) and an extremely short treatment time (1 hour). Furthermore, the senescence of mesenchymal stem cells could also be sensitively quantified using the DPV+MAC method even at a low passage number (P6). Collectively, their findings unveiled the origin of redox signals in living cells, which has important implications for the characterization of various cellular functions and behaviors using electrochemical approaches.

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Light‐induced fermenter production of derivatives of the sweet protein monellin is maximized in prestationary Saccharomyces cerevisiae cultures

Gramazio

Trauth

Bezold

et al. 2022

Biotechnology Journal

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Optogenetics has great potential for biotechnology and metabolic engineering due to the cost-effective control of cellular activities. The usage of optogenetics techniques for the biosynthesis of bioactive molecules ensures reduced costs and enhanced regulatory possibilities. This requires development of efficient methods for light-delivery during a production process in a fermenter. Here, we benchmarked the fermenter production of a low-caloric sweetener in Saccharomyces cerevisiae with optogenetic tools against the production in small scale cell culture flasks. An expression system based on the light-controlled interaction between Cry2 and Cib1 was used for sweet-protein production. Optimization of the fermenter process was achieved by increasing the light-flux during the production phase to circumvent shading by yeast cells at high densities. Maximal amounts of the sweet-protein were produced in a pre-stationary growth phase, whereas at later stages, a decay in protein abundance was observable.Our investigation showcases the upscaling of an optogenetic production process from small flasks to a bioreactor. Optogenetic-controlled production in a fermenter is highly cost-effective due to the cheap inducer and therefore a viable alternative to chemicals for a process that requires an induction step.

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Optogenetic control of the lac operon for bacterial chemical and protein production

Cited by 93 publications

References 58 publications

The Promise of Optogenetics for Bioproduction: Dynamic Control Strategies and Scale-Up Instruments

The Promise of Optogenetics for Bioproduction: Dynamic Control Strategies and Scale-Up Instruments

Extracellularly Detectable Electrochemical Signals of Living Cells Originate from Metabolic Reactions

Light‐induced fermenter production of derivatives of the sweet protein monellin is maximized in prestationary Saccharomyces cerevisiae cultures

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