Conditional Control of Universal CAR T Cells by Cleavable OFF-Switch Adaptors.

As living drugs, engineered T cell therapies are revolutionizing disease treatment with their unique functional capabilities. However, they suffer from limitations of potentially unpredictable behavior, toxicities, and nontraditional pharmacokinetics. Engineering conditional control mechanisms responsive to tractable stimuli such as small molecules or light is thus highly desirable. We and others previously developed "universal" chimeric antigen receptors (CARs) that interact with coadministered antibody adaptors to direct target cell killing and T cell activation. Universal CARs are of high therapeutic interest due to their ability to simultaneously target multiple antigens on the same disease or different diseases by combining with adaptors to different antigens. Here, we further enhance the programmability and potential safety of universal CAR T cells by engineering OFF-switch adaptors that can conditionally control CAR activity, including T cell activation, target cell lysis, and transgene expression, in response to a small molecule or light stimulus. Moreover, in adaptor combination assays, OFF-switch adaptors were capable of orthogonal conditional targeting of multiple antigens simultaneously, following Boolean logic. OFF-switch adaptors represent a robust new approach for the precision targeting of universal CAR T cells with potential for enhanced safety.

Balancing Redox Homeostasis to Improve l-Cysteine Production in Corynebacterium glutamicum.

l-Cysteine is a valuable sulfur-containing amino acid with applications across a wide range of fields. Recently, microbial fermentation has emerged as a method to produce l-cysteine. However, cellular redox stress from high levels of l-cysteine is a bottleneck for achieving efficient production. In this study, we aimed to facilitate l-cysteine biosynthesis by modulating cellular redox homeostasis through the introduction of the natural antioxidant astaxanthin in Corynebacterium glutamicum. To achieve this, we first introduced an exogenous astaxanthin synthesis module in C. glutamicum. Then, an l-cysteine-dependent autonomous bifunctional genetic switch was developed to dynamically regulate the l-cysteine and astaxanthin biosynthesis pathway to maintain cellular redox homeostasis. This regulation system achieved high biosynthesis of astaxanthin, which significantly facilitated l-cysteine production. Finally, engineered strain Cg-10 produced 8.45 g/L l-cysteine and 95 mg/L astaxanthin in a 5 L bioreactor, both of which are the highest reported levels in C. glutamicum.

Development of a "turn off-on" whole-cell biosensor for sulforaphane detection based on the ultrasensitive activator HrpRS.

Sulforaphane (SFN), a defense secondary metabolite, can be used to predict the health status of plants and also has pharmacological effects, including anticancer, antioxidant, and anti-inflammatory properties. The detection of SFN is therefore of great significance for the prevention and treatment of diseases. In this study, a "turn off" whole-cell biosensor that can rapidly and robustly respond to the presence of SFN was constructed based on the orthogonal genetic components (hrpR, hrpS, and PhrpL ) of Pseudomonas syringae (PS). The final optimized biosensor, p114(30R-30S), was able to inhibit 91.7% of the fluorescence intensity in the presence of 100-µM SFN. Subsequently, a HrpRS-regulated OFF-ON genetic switch was designed by reconstituting a reverse σ70 promoter on the σ54 -PhrpL promoter sequence; this was coupled with dual-color reporter genes to construct a "turn off-on" whole-cell SFN biosensor. The PhrpLB variant increased the expression of green fluorescence a factor of 11.9 and reduced the expression of red fluorescence by 85.8% compared with the system in the absence of SFN. Thus, a robust switching of signal output from "turn off" to "turn on" was realized. In addition, the biosensor showed good linearity in the SFN concentration ranges of 0.1-10 µM (R2  = 0.99429) and 10-100 µM (R2  = 0.99465) and a detection limit of ~0.1 µM.

Construction of the genetic switches in response to mannitol based on artificial MtlR box.

Synthetic biology has rapidly advanced from the setup of native genetic devices to the design of artificial elements able to provide organisms with highly controllable functions. In particular, genetic switches are crucial for deploying new layers of regulation into the engineered organisms. While the assembly and mutagenesis of native elements have been extensively studied, limited progress has been made in rational design of genetic switches due to a lack of understanding of the molecular mechanism by which a specific transcription factor interacts with its target gene. Here, a reliable workflow is presented for designing two categories of genetic elements, one is the switch element-MtlR box and the other is the transcriptional regulatory element- catabolite control protein A (CcpA) box. The MtlR box was designed for ON/OFF-state selection and is controlled by mannitol. The rational design of MtlR box-based molecular structures can flexibly tuned the selection of both ON and OFF states with different output switchability in response to varied kind effectors. Different types of CcpA boxes made the switches with more markedly inducer sensitivities. Ultimately, the OFF-state value was reduced by 90.69%, and the maximum change range in the presence of two boxes was 15.31-fold. This study presents a specific design of the switch, in a plug-and-play manner, which has great potential for controlling the flow of the metabolic pathway in synthetic biology.

Engineering of Synthetic Transcriptional Switches in Yeast.

Transcriptional switches can be utilized for many purposes in synthetic biology, including the assembly of complex genetic circuits to achieve sophisticated cellular systems and the construction of biosensors for real-time monitoring of intracellular metabolite concentrations. Although to date such switches have mainly been developed in prokaryotes, those for eukaryotes are increasingly being reported as both rational and random engineering technologies mature. In this review, we describe yeast transcriptional switches with different modes of action and how to alter their properties. We also discuss directed evolution technologies for the rapid and robust construction of yeast transcriptional switches.

Engineering of Corynebacterium glutamicum for high-level γ-aminobutyric acid production from glycerol by dynamic metabolic control.

Synthetic biology seeks to reprogram microbial cells for efficient production of value-added compounds from low-cost renewable substrates. A great challenge of chemicals biosynthesis is the competition between cell metabolism and target product synthesis for limited cellular resource. Dynamic regulation provides an effective strategy for fine-tuning metabolic flux to maximize chemicals production. In this work, we created a tunable growth phase-dependent autonomous bifunctional genetic switch (GABS) by coupling growth phase responsive promoters and degrons to dynamically redirect the carbon flux for metabolic state switching from cell growth mode to production mode, and achieved high-level GABA production from low-value glycerol in Corynebacterium glutamicum. A ribosome binding sites (RBS)-library-based pathway optimization strategy was firstly developed to reconstruct and optimize the glycerol utilization pathway in C. glutamicum, and the resulting strain CgGly2 displayed excellent glycerol utilization ability. Then, the initial GABA-producing strain was constructed by deleting the GABA degradation pathway and introducing an exogenous GABA synthetic pathway, which led to 5.26 g/L of GABA production from glycerol. In order to resolve the conflicts of carbon flux between cell growth and GABA production, we used the GABS to reconstruct the GABA synthetic metabolic network, in which the competitive modules of GABA biosynthesis, including the tricarboxylic acid (TCA) cycle module and the arginine biosynthesis module, were dynamically down-regulated while the synthetic modules were dynamically up-regulated after sufficient biomass accumulation. Finally, the resulting strain G7-1 accumulated 45.6 g/L of GABA with a yield of 0.4 g/g glycerol, which was the highest titer of GABA ever reported from low-value glycerol. Therefore, these results provide a promising technology to dynamically balance the metabolic flux for the efficient production of other high value-added chemicals from a low-value substrate in C. glutamicum.

Flexible linker modulates the binding affinity of the TP901-1 CI phage repressor to DNA.

Temperate bacteriophages can switch between two life cycles following infection of a host bacterium: the lytic or lysogenic life cycle. The choice between these is controlled by a bistable genetic switch. We investigated the genetic switch of the lactococcal temperate bacteriophage, TP901-1, which is controlled by two regulatory proteins, the Clear 1 (CI) repressor and modulator of repression (MOR) antirepressor. CI consists of a DNA-binding N-terminal domain and a C-terminal domain responsible for oligomerization, connected by a flexible interdomain linker. Full-length CI is hexameric, whereas the truncated version CI with 58 C-terminal residues truncated (CIΔ58), missing the second C-terminal subdomain, is dimeric, but binds with the same affinity as full-length CI to the OL operator site, responsible for lytic genes transcription repression. Three variants of CIΔ58 with shorter, longer, and PP substituted linkers were produced and confirmed by circular dichroism spectroscopy and nanodifferential scanning fluorimetry to be well folded. With small-angle X-ray scattering, we delineated the conformational space sampled by the variants and wild-type in solution and found that shortening and lengthening the linker decrease and increase this, respectively, as also substantiated by molecular dynamics and as intended. Isoelectric focusing electrophoresis confirmed that all variants are able to bind to the MOR antirepressor. However, using electrophoretic mobility shift assays, we showed that shortening and lengthening the linker lead to a 94 and 17 times decrease in affinity to OL , respectively. Thus, an appropriate linker length appears to be crucial for appropriate DNA-binding and subsequent TP901-1 genetic switch function.

Context-Dependent Stability and Robustness of Genetic Toggle Switches with Leaky Promoters.

Multistable switches are ubiquitous building blocks in both systems and synthetic biology. Given their central role, it is thus imperative to understand how their fundamental properties depend not only on the tunable biophysical properties of the switches themselves, but also on their genetic context. To this end, we reveal in this article how these factors shape the essential characteristics of toggle switches implemented using leaky promoters such as their stability and robustness to noise, both at single-cell and population levels. In particular, our results expose the roles that competition for scarce transcriptional and translational resources, promoter leakiness, and cell-to-cell heterogeneity collectively play. For instance, the interplay between protein expression from leaky promoters and the associated cost of relying on shared cellular resources can give rise to tristable dynamics even in the absence of positive feedback. Similarly, we demonstrate that while promoter leakiness always acts against multistability, resource competition can be leveraged to counteract this undesirable phenomenon. Underpinned by a mechanistic model, our results thus enable the context-aware rational design of multistable genetic switches that are directly translatable to experimental considerations, and can be further leveraged during the synthesis of large-scale genetic systems using computer-aided biodesign automation platforms.

CRISPR-Based Genetic Switches and Other Complex Circuits: Research and Application.

CRISPR-based enzymes have offered a unique capability to the design of genetic switches, with advantages in designability, modularity and orthogonality. CRISPR-based genetic switches operate on multiple levels of life, including transcription and translation. In both prokaryotic and eukaryotic cells, deactivated CRISPR endonuclease and endoribonuclease have served in genetic switches for activating or repressing gene expression, at both transcriptional and translational levels. With these genetic switches, more complex circuits have been assembled to achieve sophisticated functions including inducible switches, non-linear response and logical biocomputation. As more CRISPR enzymes continue to be excavated, CRISPR-based genetic switches will be used in a much wider range of applications.

Characterization of the genetic switch from phage ɸ13 important for Staphylococcus aureus colonization in humans.

Temperate phages are bacterial viruses that after infection either reside integrated into a bacterial genome as prophages forming lysogens or multiply in a lytic lifecycle. The decision between lifestyles is determined by a switch involving a phage-encoded repressor, CI, and a promoter region from which lytic and lysogenic genes are divergently transcribed. Here, we investigate the switch of phage ɸ13 from the human pathogen Staphylococcus aureus. ɸ13 encodes several virulence factors and is prevalent in S. aureus strains colonizing humans. We show that the ɸ13 switch harbors a cI gene, a predicted mor (modulator of repression) gene, and three high-affinity operator sites binding CI. To quantify the decision between lytic and lysogenic lifestyle, we introduced reporter plasmids that carry the 1.3 kb switch region from ɸ13 with the lytic promoter fused to lacZ into S. aureus and Bacillus subtilis. Analysis of ß-galactosidase expression indicated that decision frequency is independent of host factors. The white "lysogenic" phenotype, which relies on the expression of cI, could be switched to a stable blue "lytic" phenotype by DNA damaging agents. We have characterized lifestyle decisions of phage ɸ13, and our approach may be applied to other temperate phages encoding virulence factors in S. aureus.

Bacteriophage λ RexA and RexB functions assist the transition from lysogeny to lytic growth.

The CI and Cro repressors of bacteriophage λ create a bistable switch between lysogenic and lytic growth. In λ lysogens, CI repressor expressed from the PRM promoter blocks expression of the lytic promoters PL and PR to allow stable maintenance of the lysogenic state. When lysogens are induced, CI repressor is inactivated and Cro repressor is expressed from the lytic PR promoter. Cro repressor blocks PRM transcription and CI repressor synthesis to ensure that the lytic state proceeds. RexA and RexB proteins, like CI, are expressed from the PRM promoter in λ lysogens; RexB is also expressed from a second promoter, PLIT , embedded in rexA. Here we show that RexA binds CI repressor and assists the transition from lysogenic to lytic growth, using both intact lysogens and defective prophages with reporter genes under the control of the lytic PL and PR promoters. Once lytic growth begins, if the bistable switch does return to the immune state, RexA expression lessens the probability that it will remain there, thus stabilizing the lytic state and activation of the lytic PL  and PR  promoters. RexB modulates the effect of RexA and may also help establish phage DNA replication as lytic growth ensues.

Molecular Basis of Lysis-Lysogeny Decisions in Gram-Positive Phages.

Temperate bacteriophages (phages) are viruses of bacteria. Upon infection of a susceptible host, a temperate phage can establish either a lytic cycle that kills the host or a lysogenic cycle as a stable prophage. The life cycle pursued by an infecting temperate phage can have a significant impact not only on the individual host bacterium at the cellular level but also on bacterial communities and evolution in the ecosystem. Thus, understanding the decision processes of temperate phages is crucial. This review delves into the molecular mechanisms behind lysis-lysogeny decision-making in Gram-positive phages. We discuss a variety of molecular mechanisms and the genetic organization of these well-understood systems. By elucidating the strategies used by phages to make lysis-lysogeny decisions, we can improve our understanding of phage-host interactions, which is crucial for a variety of studies including bacterial evolution, community and ecosystem diversification, and phage therapeutics.

Engineering genetic devices for in vivo control of therapeutic T cell activity triggered by the dietary molecule resveratrol.

Chimeric antigen receptor (CAR)-engineered T cell therapies have been recognized as powerful strategies in cancer immunotherapy; however, the clinical application of CAR-T is currently constrained by severe adverse effects in patients, caused by excessive cytotoxic activity and poor T cell control. Herein, we harnessed a dietary molecule resveratrol (RES)-responsive transactivator and a transrepressor to develop a repressible transgene expression (RESrep) device and an inducible transgene expression (RESind) device, respectively. After optimization, these tools enabled the control of CAR expression and CAR-mediated antitumor function in engineered human cells. We demonstrated that a resveratrol-repressible CAR expression (RESrep-CAR) device can effectively inhibit T cell activation upon resveratrol administration in primary T cells and a xenograft tumor mouse model. Additionally, we exhibit how a resveratrol-inducible CAR expression (RESind-CAR) device can achieve fine-tuned and reversible control over T cell activation via a resveratrol-titratable mechanism. Furthermore, our results revealed that the presence of RES can activate RESind-CAR T cells with strong anticancer cytotoxicity against cells in vitro and in vivo. Our study demonstrates the utility of RESrep and RESind devices as effective tools for transgene expression and illustrates the potential of RESrep-CAR and RESind-CAR devices to enhance patient safety in precision cancer immunotherapies.

Evaluation of Ogataea (Hansenula) polymorpha for Hyaluronic Acid Production.

Hyaluronic acid (HA) is a biopolymer formed by UDP-glucuronic acid and UDP-N-acetyl-glucosamine disaccharide units linked by ß-1,4 and ß-1,3 glycosidic bonds. It is widely employed in medical and cosmetic procedures. HA is synthesized by hyaluronan synthase (HAS), which catalyzes the precursors' ligation in the cytosol, elongates the polymer chain, and exports it to the extracellular space. Here, we engineer Ogataea (Hansenula) polymorpha for HA production by inserting the genes encoding UDP-glucose 6-dehydrogenase, for UDP-glucuronic acid production, and HAS. Two microbial HAS, from Streptococcus zooepidemicus (hasAs) and Pasteurella multocida (hasAp), were evaluated separately. Additionally, we assessed a genetic switch using integrases in O. polymorpha to uncouple HA production from growth. Four strains were constructed containing both has genes under the control of different promoters. In the strain containing the genetic switch, HA production was verified by a capsule-like layer around the cells by scanning electron microscopy in the first 24 h of cultivation. For the other strains, the HA was quantified only after 48 h and in an optimized medium, indicating that HA production in O. polymorpha is limited by cultivation conditions. Nevertheless, these results provide a proof-of-principle that O. polymorpha is a suitable host for HA production.

Construction of an IS-Free Corynebacterium glutamicum ATCC 13 032 Chassis Strain and Random Mutagenesis Using the Endogenous ISCg1 Transposase.

Mobile genetic elements (MGEs) contribute to instability of the host genome and plasmids. Previously, removal of the prophages in the industrial amino acid producer Corynebacterium glutamicum ATCC 13 032 resulted in strain MB001 which showed better survival under stress conditions and increased transformability. Still, eight families of Insertion Sequence (IS) elements with 27 potentially active members remain in MB001, two of which were demonstrated to be detrimental in biotechnological processes. In this study, systematical deletion of all complete IS elements in MB001 resulted in the MGE-free strain CR101. CR101 shows growth characteristics identical to the wildtype and the increased transformability of MB001. Due to its improved genome stability, we consider this strain to be an optimal host for basic research and biotechnology. As a "zero-background" host, it is also an ideal basis to study C. glutamicum IS elements. Re-sequencing of CR101 revealed that only five spontaneous point mutations had occurred during the construction process, highlighting the low mutation rate of C. glutamicum on the nucleotide level. In a second step, we developed an easily applicable ISCg1-based transposon mutagenesis system to randomly transpose a selectable marker. For optimal plasmid stability during cloning in Escherichia coli, the system utilizes a genetic switch based on the phage integrase Bxb1. Use of this integrase revealed the presence of a functional attB site in the C. glutamicum genome. To avoid cross-talk with our system and increase ease-of-use, we removed the attB site and also inserted the Bxb1 encoding gene into the chromosome of CR101. Successful insertion of single markers was verified by sequencing randomly selected mutants. Sequencing pooled mutant libraries revealed only a weak target site specificity, seemingly random distribution of insertion sites and no general strand bias. The resulting strain, ML103, together with plasmid pML10 provides a easily customizable system for random mutagenesis in an otherwise genomically stable C. glutamicum. Taken together, the MGE-free C. glutamicum strain CR101, the derivative ML103, and the plasmid pML10 provide a useful set of tools to study C. glutamicum in the future.

Directed evolution of transcriptional switches using dual-selector systems.

Genetic switches provide core components of gene expression induction systems, genetic circuits, and metabolite sensors, including those for high-throughput screening and selection of enzyme functions. However, it is rare that natural transcription factors meet the required specifications for each application. Fortunately, the directed evolution of transcription switches is a straightforward process, given that the two states of genetic switches (on and off) are both selectable, using a wide range of positive and negative selection tools developed in the field of molecular genetics. On/off-state selections based on bactericidal mechanism allow greatly accelerate the entire process. The key to success is finding the selection conditions that minimize false-positive and false-negative clones as with any directed evolution experiment. We introduce a reliable and automatable directed evolution platform to rapidly evolve genetic switches with desired specifications in this chapter. Highlighting the importance and ease of screening for selection conditions, we demonstrate how selection conditions influence the resultant population of the selected pools.

Revealing the mechanism of repressor inactivation during switching of a temperate bacteriophage.

Temperate bacteriophages can enter one of two life cycles following infection of a sensitive host: the lysogenic or the lytic life cycle. The choice between the two alternative life cycles is dependent upon a tight regulation of promoters and their cognate regulatory proteins within the phage genome. We investigated the genetic switch of TP901-1, a bacteriophage of Lactococcus lactis, controlled by the CI repressor and the modulator of repression (MOR) antirepressor and their interactions with DNA. We determined the solution structure of MOR, and we solved the crystal structure of MOR in complex with the N-terminal domain of CI, revealing the structural basis of MOR inhibition of CI binding to the DNA operator sites. 15N NMR Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion and rotating frame R1ρ measurements demonstrate that MOR displays molecular recognition dynamics on two different time scales involving a repacking of aromatic residues at the interface with CI. Mutations in the CI:MOR binding interface impair complex formation in vitro, and when introduced in vivo, the bacteriophage switch is unable to choose the lytic life cycle showing that the CI:MOR complex is essential for proper functioning of the genetic switch. On the basis of sequence alignments, we show that the structural features of the MOR:CI complex are likely conserved among a larger family of bacteriophages from human pathogens implicated in transfer of antibiotic resistance.

Direct Readout of Neural Stem Cell Transgenesis with an Integration-Coupled Gene Expression Switch.

Stable genomic integration of exogenous transgenes is essential in neurodevelopmental and stem cell studies. Despite tools driving increasingly efficient genomic insertion with DNA vectors, transgenesis remains fundamentally hindered by the impossibility of distinguishing integrated from episomal transgenes. Here, we introduce an integration-coupled On genetic switch, iOn, which triggers gene expression upon incorporation into the host genome through transposition, thus enabling rapid and accurate identification of integration events following transfection with naked plasmids. In vitro, iOn permits rapid drug-free stable transgenesis of mouse and human pluripotent stem cells with multiple vectors. In vivo, we demonstrate faithful cell lineage tracing, assessment of regulatory elements, and mosaic analysis of gene function in somatic transgenesis experiments that reveal neural progenitor potentialities and interaction. These results establish iOn as a universally applicable strategy to accelerate and simplify genetic engineering in cultured systems and model organisms by conditioning transgene activation to genomic integration.

The thermo-regulated genetic switch of deep-sea filamentous phage SW1 and its distribution in the Pacific Ocean.

Viruses, especially bacteriophages, are thought to have important functions in the deep-sea ecosystem, but little is known about the induction mechanism of benthic phages in response to environmental change. Our prior work characterized a cold-active filamentous phage SW1 that infects the deep-sea bacterium Shewanella piezotolerans WP3; however, the underlying mechanism of the putative thermo-regulated genetic switch of SW1 is still unclear. In this study, the DNA copy number and mRNA abundance of the deep-sea phage SW1 were quantified in the whole life cycle of its host S. piezotolerans WP3 at different temperatures. Our results demonstrated that the induction of SW1 is dependent on a threshold temperature (4°C), but this dependency is not proportional to temperature gradient. RNA-Seq analyses revealed two highly transcribed regions at 4°C and verified the presence of a long 3' untranslated region (UTR) in the SW1 genome. Interestingly, recruitment analysis showed that SW1-like inoviruses prevail in deep sea (depth >1000 m) and photic epipelagic and mesopelagic zones (depth <1000 m), which suggested that the thermo-regulated genetic switch revealed in SW1 may be widely distributed in the ocean.

Construction of Inducible Genetic Switch for the Global Regulator WblA To Sustain Both Overproduction of Tiancimycins and On-Demand Sporulation in Streptomyces sp. CB03234.

The complex life cycle of streptomycetes is closely related to their secondary metabolisms, all controlled by cascade regulations. Tiancimycins (TNMs) are ten-membered enediynes possessing great potential for antitumor drug development. However, their low yields in Streptomyces sp. CB03234 have greatly limited subsequent studies. Through transcriptome analysis and genetic characterization, we proved that WblA is one pivotal global regulator to repress the biosynthesis of TNMs. The deletion of wblA could significantly enhance the production of TNMs, but also abolish the sporulation in CB03234. By constructing the NitR-ε-caprolactam inducible genetic switch, the expression of wblA was governed in CB03234-NRW, thereby sustaining the overproduction of TNMs and recovering the normal sporulation upon induction, which were practical for the scaled-up production of TNMs. Considering the prevalence and conserved regulatory roles of WblA in streptomycetes, our developed strategy shall provide an effective and practical approach to facilitate titer improvement and discovery of natural products.