Optogenetic Control by Pulsed Illumination

Hennemann, Julia; Iwasaki, Roman S.; Grund, Tamara N.; Diensthuber, Ralph P.; Richter, Florian; Möglich, Andreas

doi:10.1002/cbic.201800030

Cited by 37 publications

(56 citation statements)

References 58 publications

(99 reference statements)

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“…Besides the tracking of optical density, the cultivation volume of 750 µL was sufficient to allow endpoint measurement of intracellular hydrocarbons. Originating from a custom-made LED-device [15], adapters for LED-matrix and 24-well plates, as well as a universal plate-holder for incubators were fabricated (Fig. 3a, b).…”

Section: Resultsmentioning

confidence: 99%

“…CAD for the custom labware was performed using OpenSCAD version 2015.03-1. The basic design of the 24-well plates was kindly provided by the Möglich lab and is based on their previously published 96-well plates [15]. This design was modified in tinkercad, to fit into our microwell holders as well as to accommodate an easier electronic setup by using a FadeCandy (Adafruit Part Number: 1689) driver for the LED-matrix (Adafruit NeoPixel, Adafruit industries, New York, USA).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Drop-in biofuel production using fatty acid photodecarboxylase from Chlorella variabilis in the oleaginous yeast Yarrowia lipolytica

Bruder

Moldenhauer

Lemke

et al. 2019

Biotechnol Biofuels

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Background Oleaginous yeasts are potent hosts for the renewable production of lipids and harbor great potential for derived products, such as biofuels. Several promising processes have been described that produce hydrocarbon drop-in biofuels based on fatty acid decarboxylation and fatty aldehyde decarbonylation. Unfortunately, besides fatty aldehyde toxicity and high reactivity, the most investigated enzyme, aldehyde-deformylating oxygenase, shows unfavorable catalytic properties which hindered high yields in previous metabolic engineering approaches. Results To demonstrate an alternative alkane production pathway for oleaginous yeasts, we describe the production of diesel-like, odd-chain alkanes and alkenes, by heterologously expressing a recently discovered light-driven oxidase from Chlorella variabilis (CvFAP) in Yarrowia lipolytica. Initial experiments showed that only strains engineered to have an increased pool of free fatty acids were susceptible to sufficient decarboxylation. Providing these strains with glucose and light in a synthetic medium resulted in titers of 10.9 mg/L of hydrocarbons. Using custom 3D printed labware for lighting bioreactors, and an automated pulsed glycerol fed-batch strategy, intracellular titers of 58.7 mg/L were achieved. The production of odd-numbered alkanes and alkenes with a length of 17 and 15 carbons shown in previous studies could be confirmed. Conclusions Oleaginous yeasts such as Yarrowia lipolytica can transform renewable resources such as glycerol into fatty acids and lipids. By heterologously expressing a fatty acid photodecarboxylase from the algae Chlorella variabilis hydrocarbons were produced in several scales from microwell plate to 400 mL bioreactors. The lighting turned out to be a crucial factor in terms of growth and hydrocarbon production, therefore, the evaluation of different conditions was an important step towards a tailor-made process. In general, the developed bioprocess shows a route to the renewable production of hydrocarbons for a variety of applications ranging from being substrates for further enzymatic or chemical modification or as a drop-in biofuel blend. Electronic supplementary material The online version of this article (10.1186/s13068-019-1542-4) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Drop-in biofuel production using fatty acid photodecarboxylase from Chlorella variabilis in the oleaginous yeast Yarrowia lipolytica

Bruder

Moldenhauer

Lemke

et al. 2019

Biotechnol Biofuels

View full text Add to dashboard Cite

show abstract

“…Additionally, the influence of a fourth light intensity (1 mW cm −2 ) was investigated. To reach the intensity of 1 mW cm −2 a programmable matrix of light-emitting diodes has been assembled with the assistance of Prof. Dr. Andreas Möglich as previously described [65]. For this approach, 1000 µL of the E. coli cells, which have been adjusted to a final OD 580 nm of 0.1 were transferred into a black colored 96-deepwell plate (Riplate ® RW, 43001-0216, black, Ritter GmbH, Schwabmünchen, Germany) and illuminated with the programmable LED matrix which has been directly placed on top of the deepwell plate.…”

Section: Methodsmentioning

confidence: 99%

Genetically Encoded Photosensitizers as Light-Triggered Antimicrobial Agents

Hilgers

Bitzenhofer

Ackermann

et al. 2019

IJMS

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Diseases caused by multi-drug resistant pathogens have become a global concern. Therefore, new approaches suitable for treating these bacteria are urgently needed. In this study, we analyzed genetically encoded photosensitizers (PS) related to the green fluorescent protein (GFP) or light-oxygen-voltage (LOV) photoreceptors for their exogenous applicability as light-triggered antimicrobial agents. Depending on their specific photophysical properties and photochemistry, these PSs can produce different toxic ROS (reactive oxygen species) such as O2•− and H2O2 via type-I, as well as 1O2 via type-II reaction in response to light. By using cell viability assays and microfluidics, we could demonstrate differences in the intracellular and extracellular phototoxicity of the applied PS. While intracellular expression and exogenous supply of GFP-related PSs resulted in a slow inactivation of E. coli and pathogenic Gram-negative and Gram-positive bacteria, illumination of LOV-based PSs such as the singlet oxygen photosensitizing protein SOPP3 resulted in a fast and homogeneous killing of these microbes. Furthermore, our data indicate that the ROS type and yield as well as the localization of the applied PS protein can strongly influence the antibacterial spectrum and efficacy. These findings open up new opportunities for photodynamic inactivation of pathogenic bacteria.

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“…67 Knowledge of the recovery kinetics of a given optogenetic circuit can be exploited for the optimization of illumination protocols and for the parallel deployment of actuators that respond to the same light color but differ in their sensitivity. 161…”

Section: Photoreceptor Engineeringmentioning

confidence: 99%

Blue-Light Receptors for Optogenetics

2018

Self Cite

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Sensory photoreceptors underpin light-dependent adaptations of organismal physiology, development, and behavior in nature. Adapted for optogenetics, sensory photoreceptors become genetically encoded actuators and reporters to enable the noninvasive, spatiotemporally accurate and reversible control by light of cellular processes. Rooted in a mechanistic understanding of natural photoreceptors, artificial photoreceptors with customized light-gated function have been engineered that greatly expand the scope of optogenetics beyond the original application of light-controlled ion flow. As we survey presently, UV/blue-light-sensitive photoreceptors have particularly allowed optogenetics to transcend its initial neuroscience applications by unlocking numerous additional cellular processes and parameters for optogenetic intervention, including gene expression, DNA recombination, subcellular localization, cytoskeleton dynamics, intracellular protein stability, signal transduction cascades, apoptosis, and enzyme activity. The engineering of novel photoreceptors benefits from powerful and reusable design strategies, most importantly light-dependent protein association and (un)folding reactions. Additionally, modified versions of these same sensory photoreceptors serve as fluorescent proteins and generators of singlet oxygen, thereby further enriching the optogenetic toolkit. The available and upcoming UV/blue-light-sensitive actuators and reporters enable the detailed and quantitative interrogation of cellular signal networks and processes in increasingly more precise and illuminating manners.

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Optogenetic Control by Pulsed Illumination

Cited by 37 publications

References 58 publications

Drop-in biofuel production using fatty acid photodecarboxylase from Chlorella variabilis in the oleaginous yeast Yarrowia lipolytica

Drop-in biofuel production using fatty acid photodecarboxylase from Chlorella variabilis in the oleaginous yeast Yarrowia lipolytica

Genetically Encoded Photosensitizers as Light-Triggered Antimicrobial Agents

Blue-Light Receptors for Optogenetics

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