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

Abstract

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.This article is part of a Special Collection dedicated to the Biotrans 2021 conference. Please see our homepage for more articles in the collection.

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

“…These findings could be explained by an improved diffusion of the caged IPTG derivatives over the bacterial cell membrane in comparison to IPTG. Similar enhancement of photocaged inducer mediated induction of gene expression could also be observed in other bacterial expression hosts (Hogenkamp et al, 2021;Hogenkamp et al, 2022).…”

“…In the field of optogenetics, the application of light offers various advantageous properties such as non-invasive control with high spatiotemporal resolution (Deiters, 2009;Drepper et al, 2011;Brieke et al, 2012;Gardner and Deiters, 2012; Bardhan and Deiters, 2019;Baumschlager and Khammash, 2021). In this context, photo-labile protecting groups are useful for controlling a multitude of cellular processes including cell signaling (Liu et al, 2017;Bardhan and Deiters, 2019;Kolarski et al, 2019) or gene expression (Young and Deiters, 2007;Gardner et al, 2011;Binder et al, 2014;Bier et al, 2016;Kusen et al, 2016;Hogenkamp et al, 2022). For light-regulated gene expression, especially photocaged isopropyl-β-D-1-thiogalactopyranoside (cIPTG) was applied, e.g., for automated optimization of heterologous gene expression in Escherichia coli using a high-throughput screening system (Wandrey et al, 2016).…”

Recent Synthetic Biology Approaches for Temperature- and Light-Controlled Gene Expression in Bacterial Hosts

“…The activity of the caged inducers is initially inhibited and is regained upon light irradiation at the appropriate wavelength ( Figure 3 A). Small-molecule inducers such as isopropyl β - d -1-thiogalactopyranoside (IPTG) [ 34 , 35 , 36 , 38 , 39 , 42 , 43 ] and l -arabinose [ 34 , 40 ] are usually targeted for caging. Among the collection of many photocleavable protecting groups (PPGs) [ 62 ], limited caging groups including ortho -nitrobenzyl (ONB) derivatives and coumarin derivatives have been employed to develop photocaged small-molecule inducers.…”

“…At the molecular level, researchers have utilized the heat shock response mechanism to construct temperature-sensitive gene expression systems such as the temperature-sensitive dimeric repressor [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 ], RNA polymerase (RNAP) [ 14 , 15 , 16 , 17 ], replication machinery [ 18 , 19 , 20 , 21 , 22 ], and oligonucleotide topology [ 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 ]. Photocaged [ 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 ] or photoswitchable [ 44 , 45 ] small-molecule inducers and photocaged transcription factors [ 46 ] have been used to develop light-driven gene expression systems. Additionally, light-sensitive proteins have been widely used as essential modules to control diverse cellular functions in optogenetics [ 5 ] ( Figure 1 ).…”

Discovery of a Novel Photocaged PI3K Inhibitor Capable of Real-Time Reporting of Drug Release

“…Photocaged compounds, in which a photoactivatable protecting group (PPG) is usually introduced to the binding moiety of bioactive substances, have been extensively studied for manipulating a broad range of biochemical processes, including gene expression, receptor activation, protein binding interactions, and signaling pathway dynamics. − Upon illumination with light at the appropriate wavelength, photocaged compounds restore their activities by cleaving PPGs. To date, various types of exogenous and endogenous substances are designed to be in their photocaged forms for use in chemistry and biology, such as nucleotides and nucleosides, DNA/RNA, , amino acids, , peptides, proteins, neutrotransmitters, , receptor angonists/antagonists, − small-molecule drugs, etc.…”