Light-mediated control of gene expression in the anoxygenic phototrophic bacterium Rhodobacter capsulatus using photocaged inducers.

Photocaged inducer molecules, especially photocaged isopropyl-ß-d-1-thiogalactopyranoside (cIPTG), are well-established optochemical tools for light-regulated gene expression and have been intensively applied in Escherichia coli and other bacteria including Corynebacterium glutamicum, Pseudomonas putida or Bacillus subtilis. In this study, we aimed to implement a light-mediated on-switch for target gene expression in the facultative anoxygenic phototroph Rhodobacter capsulatus by using different cIPTG variants under both phototrophic and non-phototrophic cultivation conditions. We could demonstrate that especially 6-nitropiperonyl-(NP)-cIPTG can be applied for light-mediated induction of target gene expression in this facultative phototrophic bacterium. Furthermore, we successfully applied the optochemical approach to induce the intrinsic carotenoid biosynthesis to showcase engineering of a cellular function. Photocaged IPTG thus represents a light-responsive tool, which offers various promising properties suitable for future applications in biology and biotechnology including automated multi-factorial control of cellular functions as well as optimization of production processes.

Optochemical Control of Bacterial Gene Expression: Novel Photocaged Compounds for Different Promoter Systems.

Photocaged compounds are applied for implementing precise, optochemical control of gene expression in bacteria. To broaden the scope of UV-light-responsive inducer molecules, six photocaged carbohydrates were synthesized and photochemically characterized, with the absorption exhibiting a red-shift. Their differing linkage through ether, carbonate, and carbamate bonds revealed that carbonate and carbamate bonds are convenient. Subsequently, those compounds were successfully applied in vivo for controlling gene expression in E. coli via blue light illumination. Furthermore, benzoate-based expression systems were subjected to light control by establishing a novel photocaged salicylic acid derivative. Besides its synthesis and in vitro characterization, we demonstrate the challenging choice of a suitable promoter system for light-controlled gene expression in E. coli. We illustrate various bottlenecks during both photocaged inducer synthesis and in vivo application and possibilities to overcome them. These findings pave the way towards novel caged inducer-dependent systems for wavelength-selective gene expression.

Effect of Photocaged Isopropyl ß-d-1-thiogalactopyranoside Solubility on the Light Responsiveness of LacI-controlled Expression Systems in Different Bacteria.

Photolabile protecting groups play a significant role in controlling biological functions and cellular processes in living cells and tissues, as light offers high spatiotemporal control, is non-invasive as well as easily tuneable. In the recent past, photo-responsive inducer molecules such as 6-nitropiperonyl-caged IPTG (NP-cIPTG) have been used as optochemical tools for Lac repressor-controlled microbial expression systems. To further expand the applicability of the versatile optochemical on-switch, we have investigated whether the modulation of cIPTG water solubility can improve the light responsiveness of appropriate expression systems in bacteria. To this end, we developed two new cIPTG derivatives with different hydrophobicity and demonstrated both an easy applicability for the light-mediated control of gene expression and a simple transferability of this optochemical toolbox to the biotechnologically relevant bacteria Pseudomonas putida and Bacillus subtilis. Notably, the more water-soluble cIPTG derivative proved to be particularly suitable for light-mediated gene expression in these alternative expression hosts.

Light-Controlled Cell Factories: Employing Photocaged Isopropyl-ß-d-Thiogalactopyranoside for Light-Mediated Optimization of lac Promoter-Based Gene Expression and (+)-Valencene Biosynthesis in Corynebacterium glutamicum.

Precise control of microbial gene expression resulting in a defined, fast, and homogeneous response is of utmost importance for synthetic bio(techno)logical applications. However, even broadly applied biotechnological workhorses, such as Corynebacterium glutamicum, for which induction of recombinant gene expression commonly relies on the addition of appropriate inducer molecules, perform moderately in this respect. Light offers an alternative to accurately control gene expression, as it allows for simple triggering in a noninvasive fashion with unprecedented spatiotemporal resolution. Thus, optogenetic switches are promising tools to improve the controllability of existing gene expression systems. In this regard, photocaged inducers, whose activities are initially inhibited by light-removable protection groups, represent one of the most valuable photoswitches for microbial gene expression. Here, we report on the evaluation of photocaged isopropyl-ß-d-thiogalactopyranoside (IPTG) as a light-responsive control element for the frequently applied tac-based expression module in C. glutamicum In contrast to conventional IPTG, the photocaged inducer mediates a tightly controlled, strong, and homogeneous expression response upon short exposure to UV-A light. To further demonstrate the unique potential of photocaged IPTG for the optimization of production processes in C. glutamicum, the optogenetic switch was finally used to improve biosynthesis of the growth-inhibiting sesquiterpene (+)-valencene, a flavoring agent and aroma compound precursor in food industry. The variation in light intensity as well as the time point of light induction proved crucial for efficient production of this toxic compound. IMPORTANCE: Optogenetic tools are light-responsive modules that allow for a simple triggering of cellular functions with unprecedented spatiotemporal resolution and in a noninvasive fashion. Specifically, light-controlled gene expression exhibits an enormous potential for various synthetic bio(techno)logical purposes. Before our study, poor inducibility, together with phenotypic heterogeneity, was reported for the IPTG-mediated induction of lac-based gene expression in Corynebacterium glutamicum By applying photocaged IPTG as a synthetic inducer, however, these drawbacks could be almost completely abolished. Especially for increasing numbers of parallelized expression cultures, noninvasive and spatiotemporal light induction qualifies for a precise, homogeneous, and thus higher-order control to fully automatize or optimize future biotechnological applications.

Light-induced gene expression with photocaged IPTG for induction profiling in a high-throughput screening system.

BACKGROUND: Inducible expression systems are frequently used for the production of heterologous proteins. Achieving maximum product concentrations requires induction profiling, namely the optimization of induction time and inducer concentration. However, the respective experiments can be very laborious and time-consuming. In this work, a new approach for induction profiling is presented where induction in a microtiter plate based cultivation system (BioLector) is achieved by light using photocaged isopropyl ß-D-1-thiogalactopyranoside (cIPTG). RESULTS: A flavin mononucleotide-based fluorescent reporter protein (FbFP) was expressed using a T7-RNA-polymerase dependent E. coli expression system which required IPTG as inducer. High power UV-A irradiation was directed into a microtiter plate by light-emitting diodes placed above each well of a 48-well plate. Upon UV irradiation, IPTG is released (uncaged) and induces product formation. IPTG uncaging, formation of the fluorescent reporter protein and biomass growth were monitored simultaneously in up to four 48-well microtiter plates in parallel with an in-house constructed BioLector screening system. The amount of released IPTG can be gradually and individually controlled for each well by duration of UV-A exposure, irradiance and concentration of photocaged IPTG added at the start of the cultivation. A comparison of experiments with either optical or conventional IPTG induction shows that product formation and growth are equivalent. Detailed induction profiles revealed that for the strain and conditions used maximum product formation is reached for very early induction times and with just 6-8 s of UV-A irradiation or 60-80 µM IPTG. CONCLUSIONS: Optical induction and online monitoring were successfully combined in a high-throughput screening system and the effect of optical induction with photocaged IPTG was shown to be equivalent to conventional induction with IPTG. In contrast to conventional induction, optical induction is less costly to parallelize, easy to automate, non-invasive and without risk of contamination. Therefore, light-induced gene expression with photocaged IPTG is a highly advantageous method for the efficient optimization of heterologous protein production and has the potential to replace conventional induction with IPTG.

Photocaged Arabinose: A Novel Optogenetic Switch for Rapid and Gradual Control of Microbial Gene Expression.

Controlling cellular functions by light allows simple triggering of biological processes in a non-invasive fashion with high spatiotemporal resolution. In this context, light-regulated gene expression has enormous potential for achieving optogenetic control over almost any cellular process. Here, we report on two novel one-step cleavable photocaged arabinose compounds, which were applied as light-sensitive inducers of transcription in bacteria. Exposure of caged arabinose to UV-A light resulted in rapid activation of protein production, as demonstrated for GFP and the complete violacein biosynthetic pathway. Moreover, single-cell analysis revealed that intrinsic heterogeneity of arabinose-mediated induction of gene expression was overcome when using photocaged arabinose. We have thus established a novel phototrigger for synthetic bio(techno)logy applications that enables precise and homogeneous control of bacterial target gene expression.

Light-responsive control of bacterial gene expression: precise triggering of the lac promoter activity using photocaged IPTG.

Light can be used to control numerous cellular processes including protein function and interaction as well as gene expression in a non-invasive fashion and with unprecedented spatiotemporal resolution. However, for chemical phototriggers tight, gradual, and homogeneous light response has never been attained in living cells. Here, we report on a light-responsive bacterial T7 RNA polymerase expression system based on a photocaged derivative of the inducer molecule isopropyl-ß-d-thiogalactopyranoside (IPTG). We have comparatively analyzed different Escherichia coli lac promoter-regulated expression systems in batch and microfluidic single-cell cultivation. The lacY-deficient E. coli strain Tuner(DE3) harboring additional plasmid-born copies of the lacI gene exhibited a sensitive and defined response to increasing IPTG concentrations. Photocaged IPTG served as a synthetic photo-switch to convert the E. coli system into an optogenetic expression module allowing for precise and gradual light-triggering of gene expression as demonstrated at the single cell level.