Multimodal Control of Bacterial Gene Expression by Red and Blue Light

Meier, Stefanie S. M.; Multamäki, Elina; Ranzani, Américo T.; Takala, Heikki; Möglich, Andreas

doi:10.1007/978-1-0716-3658-9_26

Cited by 3 publications

(1 citation statement)

References 34 publications

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“…We next addressed whether these systems support multiplexing with optogenetic circuits that drive gene expression in response to other light colors. We previously combined Dr REDusk and Dr REDawn with the blue-light-responsive pCrepusculo and pAurora circuits 28 , 44 , 45 (Suppl. Fig.…”

Section: Resultsmentioning

confidence: 99%

Leveraging the histidine kinase-phosphatase duality to sculpt two-component signaling

Meier,

Multamäki,

Ranzani

et al. 2024

Nat Commun

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Bacteria must constantly probe their environment for rapid adaptation, a crucial need most frequently served by two-component systems (TCS). As one component, sensor histidine kinases (SHK) control the phosphorylation of the second component, the response regulator (RR). Downstream responses hinge on RR phosphorylation and can be highly stringent, acute, and sensitive because SHKs commonly exert both kinase and phosphatase activity. With a bacteriophytochrome TCS as a paradigm, we here interrogate how this catalytic duality underlies signal responses. Derivative systems exhibit tenfold higher red-light sensitivity, owing to an altered kinase-phosphatase balance. Modifications of the linker intervening the SHK sensor and catalytic entities likewise tilt this balance and provide TCSs with inverted output that increases under red light. These TCSs expand synthetic biology and showcase how deliberate perturbations of the kinase-phosphatase duality unlock altered signal-response regimes. Arguably, these aspects equally pertain to the engineering and the natural evolution of TCSs.

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Section: Resultsmentioning

confidence: 99%

Leveraging the histidine kinase-phosphatase duality to sculpt two-component signaling

Meier,

Multamäki,

Ranzani

et al. 2024

Nat Commun

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Light-directed evolution of dynamic, multi-state, and computational protein functionalities

Gligorovski,

Labagnara,

Rahi

2024

Preprint

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Directed evolution is a powerful method in biological engineering. Current approaches were devised for evolving steady-state properties such as enzymatic activity or fluorescence intensity. A fundamental problem remains how to evolve dynamic, multi-state, or computational functionalities, e.g., folding times, on-off kinetics, state-specific activity, stimulus-responsiveness, or switching and logic capabilities. These require applying selection pressure on all of the states of a protein of interest (POI) and the transitions between them. We realized that optogenetics and cell cycle oscillations could be leveraged for a novel directed evolution paradigm (‘optovolution’) that is germane for this need: We designed a signaling cascade in budding yeast where optogenetic input switches the POI between off (0) and on (1) states. In turn, the POI controls a Cdk1 cyclin, which in the re-engineered cell cycle system is essential for one cell cycle stage but poisonous for another. Thus, the cyclin must oscillate (1-0-1-0…) for cell proliferation. In this system, evolution can act efficiently on the dynamics, transient states, and input-output relations of the POI in every cell cycle. Further, controlling the pacemaker, light, directs and tunes selection pressures. Optovolution isin vivo, continuous, self-selecting, and genetically robust. We first evolved two optogenetic systems, which relay 0/1 input to 0/1 output: We obtained 25 new variants of the widely used LOV transcription factor El222. These mutants were stronger, less leaky, or green- and red-responsive. The latter was conjectured to be impossible for LOV domains but is needed for multiplexing and lowering phototoxicity. Evolving the PhyB-Pif3 optogenetic system, we discovered that loss ofYOR1makes supplementing the expensive and unstable chromophore phycocyanobilin (PCB) unnecessary. Finally, we demonstrate the generality of the method by creating and evolving a destabilized rtTA transcription factor, which performs an AND operation between transcriptional and doxycycline input. Optovolution makes coveted, difficult-to-change protein functionalities evolvable.

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Potent photoswitch for expression of biotherapeutics in mammalian cells by light

Gebel,

Ciglieri,

Stahl

et al. 2024

Preprint

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Precise temporal and spatial control of gene expression is of great benefit for the study of specific cellular circuits and activities. Compared to chemical inducers, light-dependent control of gene expression by optogenetics achieves a higher spatial and temporal resolution. This could also prove decisive beyond basic research for manufacturing difficult-to-express proteins in pharmaceutical bioproduction. However, current optogenetic gene-expression systems limit this application in mammalian cells as expression levels and fold induction upon light stimulation are not sufficient. To overcome this limitation, we designed a photoswitch by fusing the blue light-activated light-oxygen-voltage receptor EL222 from Erythrobacter litoralis to the three tandem transcriptional activator domains VP64, p65, and Rta. The resultant photoswitch, dubbed DEL-VPR, allows an up to 400-fold induction of target gene expression by blue light, achieving expression levels that surpass those for strong constitutive promoters. Here, we utilized DEL-VPR to enable light-induced expression of complex monoclonal and bispecific antibodies with reduced byproduct expression, increasing the yield of functional protein complexes. Our approach offers temporally controlled yet strong gene expression and applies to both academic and industrial settings.

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Multimodal Control of Bacterial Gene Expression by Red and Blue Light

Cited by 3 publications

References 34 publications

Leveraging the histidine kinase-phosphatase duality to sculpt two-component signaling

Leveraging the histidine kinase-phosphatase duality to sculpt two-component signaling

Light-directed evolution of dynamic, multi-state, and computational protein functionalities

Potent photoswitch for expression of biotherapeutics in mammalian cells by light

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