Inducible Promoter Research Articles

Metabolic engineering of Pichia pastoris for myo-inositol production by dynamic regulation of central metabolism

BackgroundThe methylotrophic budding yeast Pichia pastoris GS115 is a powerful expression system and hundreds of heterologous proteins have been successfully expressed in this strain. Recently, P. pastoris has also been exploited as an attractive cell factory for the production of high-value biochemicals due to Generally Recognized as Safe (GRAS) status and high growth rate of this yeast strain. However, appropriate regulation of metabolic flux distribution between cell growth and product biosynthesis is still a cumbersome task for achieving efficient biochemical production.ResultsIn this study, P. pastoris was exploited for high inositol production using an effective dynamic regulation strategy. Through enhancing native inositol biosynthesis pathway, knocking out inositol transporters, and slowing down carbon flux of glycolysis, an inositol-producing mutant was successfully developed and low inositol production of 0.71 g/L was obtained. The inositol production was further improved by 12.7% through introduction of heterologous inositol-3-phosphate synthase (IPS) and inositol monophosphatase (IMP) which catalyzed the rate-limiting steps for inositol biosynthesis. To control metabolic flux distribution between cell growth and inositol production, the promoters of glucose-6-phosphate dehydrogenase (ZWF), glucose-6-phosphate isomerase (PGI) and 6-phosphofructokinase (PFK1) genes were replaced with a glycerol inducible promoter. Consequently, the mutant strain could be switched from growth mode to production mode by supplementing glycerol and glucose sequentially, leading to an increase of about 4.9-fold in inositol formation. Ultimately, the dissolved oxygen condition in high-cell-density fermentation was optimized, resulting in a high production of 30.71 g/L inositol (~ 40-fold higher than the baseline strain).ConclusionsThe GRAS P. pastoris was engineered as an efficient inositol producer for the first time. Dynamic regulation of cell growth and inositol production was achieved via substrate-dependent modulation of glycolysis and pentose phosphate pathways and the highest inositol titer reported to date by a yeast cell factory was obtained. Results from this study provide valuable guidance for engineering of P. pastoris for the production of other high-value bioproducts.

A next generation inducible armored CAR to overcome the immunosuppressive tumor microenvironment and enhances cytotoxicity of CAR-T and TILs.

e14517 Background: The full potential of CAR-T cells in clinic is limited by the immunosuppressive tumour microenvironment (TME), which induces expression of exhaustion markers and limits the activity of tumour infiltrating lymphocytes (TILs) including CAR-T cells. We developed a next generation inducible Armored CAR platform (aCAR) that releases an anti-PD-1 checkpoint inhibitor upon CAR-T cell activation, limiting payload release exclusively to the TME, thereby reducing the risk of systemic exposure. This differentiated strategy facilitates activation of CAR-T cells and TILs within the TME and has the potential for lower toxicity. Methods: A ROR1-targeting second-generation CAR containing a 41BB-CD3ζ intracellular domain was cloned into a lentivirus transfer vector alongside a prototypic anti-PD1 antibody. The CAR was under the control of a constitutively active promoter, and the PD1 targeting payload was controlled by an inducible promoter that was activated upon engagement of CAR with ROR1 on tumour cells. CAR-T cells were subject to in vitro co-culture assays with target ROR1+ tumour cells and their cytotoxic responses evaluated by flow cytometry. Levels and binding functionality of released payload were analyzed by ELISA and flow cytometry. In vivo xenograft models were performed in NSG mice with tumor growth assessed by bioluminescent imaging (BLI) and caliper measurement of the tumour volume. Results: Non-Armored CAR cells displayed potent and specific cytotoxic responses directed towards ROR1+ TNBC and NSCLC cells lines at different effector to target (E:T) ratios. The results were equivalent or superior to CARs generated with comparator anti-ROR1 antibodies, which may be due to the ROR1 CAR targeting a membrane proximal epitope within the ROR1 frizzled domain. The aCAR cells released measurable levels of anti-PD1 payload within 5 hours of binding to ROR1 on tumors and enhanced the cytotoxic effects at challenging 1:10 E:T ratios. Established PDL1+ TNBC xenograft models using the aCAR cells and comparing with non-Armored CAR cells displayed a qualitative abrogation in tumor growth by BLI, which was confirmed and shown to be significant by caliper measurement of the tumor volume. Continuing the experiment out to 3 months showed a significant survival advantage for the animals receiving aCAR. All other cohorts were terminated by day 70, however 20% of the aCAR cohort survived at day 95. Conclusions: Our next generation inducible aCAR platform enables the release of an immune stimulating payload only in the presence of target tumor cells, enhancing the therapeutic activity of the CAR-T cells and limiting payload exposure to the site of action. This technology provided a significant survival advantage in challenging in vivo xenograft models. This coupled with its potential safety attributes merits further clinical evaluation of this approach.

Binding of the HSF-1 DNA-binding domain to multimeric C. elegans consensus HSEs is guided by cooperative interactions

The protein HSF-1 is the controlling transcription factor of the heat-shock response (HSR). Its binding to the heat-shock elements (HSEs) induces the strong upregulation of conserved heat-shock proteins, including Hsp70s, Hsp40s and small HSPs. Next to these commonly known HSPs, more than 4000 other HSEs are found in the promoter regions of C. elegans genes. In microarray experiments, few of the HSE-containing genes are specifically upregulated during the heat-shock response. Most of the 4000 HSE-containing genes instead are unaffected by elevated temperatures and coexpress with genes unrelated to the HSR. This is also the case for several genes related to the HSP chaperone system, like dnj-12, dnj-13, and hsp-1. Interestingly, several promoters of the dedicated HSR-genes, like F44E5.4p, hsp-16.48p or hsp-16.2p, contain extended HSEs in their promoter region, composed of four or five HSE-elements instead of the common trimeric HSEs. We here aim at understanding how HSF-1 interacts with the different promoter regions. To this end we purify the nematode HSF-1 DBD and investigate the interaction with DNA sequences containing these regions. EMSA assays suggest that the HSF-1 DBD interacts with most of these HSE-containing dsDNAs, but with different characteristics. We employ sedimentation analytical ultracentrifugation (SV-AUC) to determine stoichiometry, affinity, and cooperativity of HSF-1 DBD binding to these HSEs. Interestingly, most HSEs show cooperative binding of the HSF-1 DBD with up to five DBDs being bound. In most cases binding to the HSEs of inducible promoters is stronger, even though the consensus scores are not always higher. The observed high affinity of HSF-1 DBD to the non-inducible HSEs of dnj-12, suggests that constitutive expression may be supported from some promoter regions, a fact that is evident for this transcription factor, that is essential also under non-stress conditions.

Efficient Synthesis of Phycocyanobilin by Combinatorial Metabolic Engineering in Escherichia coli.

Phycocyanobilin (PCB) is a kind of light-harvesting pigment which naturally exists in algae and plays important roles in absorbing and transferring energy. Based on its antioxidant and optical properties, PCB has been applied in food, medicine, and cosmetics. Currently, PCB is mainly extracted from Spirulina through complicated steps; thus, the biosynthesis of PCB in Escherichia coli has attracted more attention. However, due to the lower catalytic efficiency of synthetic enzymes and the deficiency of precursors and cofactors, the titer of PCB remains at a low level. Here, we report the efficient synthesis of PCB by the expression of heme oxygenase-1 from Thermosynechococcus elongatus and PCB: ferredoxin oxidoreductase (PcyA) from Synechocystis sp. using a high-copy number plasmid with an inducible T7lac promoter and the assembly of these two enzymes at a suitable ratio of 2:1 with DNA scaffolds. Additionally, the synthesis of PCB was further enhanced by direct supplementation of 5-aminolevulinic acid (ALA), moderate overexpression of key enzymes in the heme biosynthetic pathway (hemB and hemH), and accelerated cycle of cofactors (NADPH) through the expression of NAD+ kinase and the addition of a reducing agent. Finally, based on the optimal conditions (Modified R medium with 200 mg/L ALA, 20 mg/L FeSO4·7H2O, and 5 g/L vitamin C induced by 0.8 mM isopropylthio-β-galactoside at 30 °C), the highest reported titer of PCB (28.32 mg/L) was obtained at the fermenter level by feeding glucose and FeSO4·7H2O. The strategies applied in this study will be useful for the synthesis of other natural pigments and PCB or heme derivatives in E. coli.

A CRISPR/Cas9-Based System with Controllable Auto-Excision Feature Serving Cisgenic Plant Breeding and Beyond.

Transgenic or genetically modified crops have great potential in modern agriculture but still suffer from heavy regulations worldwide due to biosafety concerns. As a promising alternative route, cisgenic crops have received higher public acceptance and better reviews by governing authorities. To serve the purpose of cisgenic plant breeding, we have developed a CRISPR/Cas9-based vector system, which is capable of delivering target gene-of-interest (GOI) into recipient plants while removing undesired genetic traces in the plants. The new system features a controllable auto-excision feature, which is realized by a core design of embedded multi-clonal sequence and the use of inducible promoters controlling the expression of Cas9 nuclease. In the current proof-of-concept study in Arabidopsis thaliana (L.) Heynh., we have successfully incorporated a GOI into the plant and removed the selection marker and CRISPR/Cas9 components from the final product. Following the designed workflow, we have demonstrated that novel cisgenic plant germplasms with desired traits could be developed within one to two generations. Further characterizations of the vector system have shown that heat treatment at 37 °C could significantly improve the editing efficiency (up to 100%), and no off-target mutations were identified in the Arabidopsis background. This novel vector system is the first CRISPR/Cas9-based genome editing tool for cisgenic plant breeding and should prove powerful for other similar applications in the bright future of precision molecular breeding.

Agar Art as an Instructional Tool to Teach Inducible Promoters via Fluorescent Protein Expression

Agar Art as an Instructional Tool to Teach Inducible Promoters via Fluorescent Protein Expression

High throughput screen for the improvement of inducible promoters for tumor microenvironment cues

Cancer immunotherapies are highly potent and are gaining wide clinical usage. However, severe side effects require focusing effector immune cell activities on the tumor microenvironment (TME). We recently developed a chimeric antigen receptor tumor-induced vector (CARTIV), a synthetic promoter activated by TME factors. To improve CARTIV functions including background, activation levels, and synergism, we screened a library of promoters with variations in key positions. Here, we present a screening method involving turning ON/OFF stimulating TNFα and IFNγ cytokines, followed by sequential cell sorting. Sequencing of enriched promoters identified seventeen candidates, which were cloned and whose activities were then validated, leading to the identification of two CARTIVs with lower background and higher induction. We further combined a third hypoxia element with the two-factor CARTIV, demonstrating additional modular improvement. Our study presents a method of fine-tuning synthetic promoters for desired immunotherapy needs.

Agrobacterium mediated transformation of ABA biosynthetic pathway coding genes for enhanced drought tolerance in Nicotiana tabacum

Abscisic acid (ABA) is an important hormone that regulates stomata closure, developmental stages and drought tolerance in plants. NCED (EC 1.13.11.51) is a key enzyme involved in the biosynthesis of abscisic acid. Transgenic tobacco plants with the integration of nced and rpk genes under the control of stress inducible promoters viz., sal T and lea P were developed through Agrobacterium mediated transformation. Over expression of integrated genes were confirmed by leaf disc assay method by imposing artificial stress using methyl Viologen (5 μMmethyl Viologen for 36 hrs).On exposure of stress, transgenic plants showed high content of chlorophyll pigments and low level of electrolyte leakage and MDA were observed than the wild type plants. Over expression of nced and rpk enhanced the abscisic acid content and increased the tolerance to drought stress.

An Application of Mycobacteriophage Genome Engineering using Bacteriophage Recombineering with Electroporated DNA (BRED) and CRISPR Cas‐9 Systems

The increasing occurrence of antibiotic‐resistant pathogens remains a global threat to public health, spurring research to develop alternative treatments for infectious bacteria. Phage therapy, the targeted use of bacteria‐specific viral particles called bacteriophages, has emerged as a promising antibiotic alternative. Our lab studies mycobacteriophages – phages specific to bacteria of the genus Mycobacterium,which includes infamous, antibiotic‐resistant pathogens such as M. tuberculosisand M. abscessus.For the use of many mycobacteriophages in phage therapy treatments, gene deletions are often necessary to ensure host lethality. Previous methods for mutant generation used a technique called bacteriophage recombination with electroporated DNA (BRED), which used electrocompetent host cells equipped with recombination machinery, genomic phage DNA, and a synthetic DNA g‐block to generate mutants. This process proved inefficient with recalcitrant recovery of some deletions, requiring extensive screening of plaques. To improve the process of genetic modifications with mycobacteriophages, greater precision and improved screening has recently been achieved through the combined use of BRED with a mycobacteria‐efficient Streptococcus thermophilus CRISPR Cas‐9 gene knockdown system for selection against the parent phage (CRISPY‐BRED). Here we present an application of the CRISPY‐BRED technique for the deletion of the immunity repressor gene in the mycobacteriophage known as Mufasa. The Cas‐9 system involves a plasmid containing the modified, nuclease‐dead Cas9 gene, a sgRNA fused to an anhydrotetracycline inducible promoter, a kanamycin resistance gene, an E. colireplication origin, and an attPsite for chromosomal integration in mycobacteria. The identification of a Protospacer Adjacent Motif (PAM) within the mycobacteriophage immunity repressor gene allowed for the design of a 20 bp sgRNA targeting sequence from the phage DNA. With host cell transformation of this CRISPR Cas9 plasmid programmed to target the parent phage immunity repressor and inhibit overall parent phage gene expression, straightforward mutant recovery should be achievable.

Expanding the promoter toolbox for metabolic engineering of methylotrophic yeasts.

Methylotrophic yeasts have been widely recognized as a promising host for production of recombinant proteins and value-added chemicals. Promoters for controlled gene expression are critical for construction of efficient methylotrophic yeasts cell factories. Here, we summarized recent advances in characterizing and engineering promoters in methylotrophic yeasts, such as Komagataella phaffii and Ogataea polymorpha. Constitutive and inducible promoters controlled by methanol or other inducers/repressors were introduced to demonstrate their applications in production of proteins and chemicals. Furthermore, efforts of promoter engineering, including site-directed mutagenesis, hybrid promoter, and transcription factor regulation to expand the promoter toolbox were also summarized. This mini-review also provides useful information on promoters for the application of metabolic engineering in methylotrophic yeasts. KEY POINTS: • The characteristics of six methylotrophic yeasts and their promoters are described. • The applications of Komagataella phaffii and Ogataea polymorpha inmetabolic engineeringare expounded. • Threepromoter engineering strategies are introduced in order to expand the promoter toolbox.

Cohesin couples transcriptional bursting probabilities of inducible enhancers and promoters. Figure plotting code. Version v1.0.0 Chocolate.

Cohesin couples transcriptional bursting probabilities of inducible enhancers and promoters. Figure plotting code. Version v1.0.0 Chocolate.

Spatiotemporal Regulation of CRISPR/Cas9 Enables Efficient, Precise, and Heritable Edits in Plant Genomes.

CRISPR/Cas-mediated editing has revolutionized crop engineering. Due to the broad scope and potential of this technology, many studies have been carried out in the past decade towards optimizing genome editing constructs. Clearly, the choice of the promoter used to drive gRNA and Cas9 expression is critical to achieving high editing efficiency, precision, and heritability. While some important considerations for choosing a promoter include the number and nature of targets, host organism, mode of transformation and goal of the experiment, spatiotemporal regulation of Cas9 expression using tissue-specific or inducible promoters enables higher heritability and efficiency of targeted mutagenesis with reduced off-target effects. In this review, we discuss specific studies that highlight the prospects and trade-offs associated with the choice of promoters on genome editing and emphasize the need for inductive exploration and discovery to further advance this area of research in crop plants.

Specific Bacterial Pathogen Phytosensing Is Enabled by a Synthetic Promoter-Transcription Factor System in Potato.

Phytosensors are genetically engineered plant-based sensors that feature synthetic promoters fused to reporter genes to sense and report the presence of specific biotic and abiotic stressors on plants. However, when induced reporter gene output is below detectable limits, owing to relatively weak promoters, the phytosensor may not function as intended. Here, we show modifications to the system to amplify reporter gene signal by using a synthetic transcription factor gene driven by a plant pathogen-inducible synthetic promoter. The output signal was unambiguous green fluorescence when plants were infected by pathogenic bacteria. We produced and characterized a phytosensor with improved sensing to specific bacterial pathogens with targeted detection using spectral wavelengths specific to a fluorescence reporter at 3 m standoff detection. Previous attempts to create phytosensors revealed limitations in using innate plant promoters with low-inducible activity since they are not sufficient to produce a strong detectable fluorescence signal for standoff detection. To address this, we designed a pathogen-specific phytosensor using a synthetic promoter-transcription factor system: the S-Box cis-regulatory element which has low-inducible activity as a synthetic 4xS-Box promoter, and the Q-system transcription factor as an amplifier of reporter gene expression. This promoter-transcription factor system resulted in 6-fold amplification of the fluorescence after infection with a potato pathogen, which was detectable as early as 24 h post-bacterial infection. This novel bacterial pathogen-specific phytosensor potato plant demonstrates that the Q-system may be leveraged as a powerful orthogonal tool to amplify a relatively weak synthetic inducible promoter, enabling standoff detection of a previously undetectable fluorescence signal. Pathogen-specific phytosensors would be an important asset for real-time early detection of plant pathogens prior to the display of disease symptoms on crop plants.

Examination of Genetic Control Elements in the Phototrophic Firmicute Heliomicrobium modesticaldum.

Heliomicrobium modesticaldum has been used as a model organism for the Heliobacteria, the only phototrophic family in the Firmicutes. It is a moderately thermophilic anoxygenic phototrophic bacterium that is capable of fermentative growth in the dark. The genetic manipulation of H. modesticaldum is still in its infancy. Methods to introduce genes through the use of exogenous plasmids and to delete genes from the chromosome through the use of the native CRISPR/Cas system have been developed in the last several years. To expand our genetic toolkit, it was necessary to control gene expression. In this study, we analyzed constitutive and inducible promoters developed for clostridia for their use in H. modesticaldum and further tested two reporters, adhB and lacZ, as indicators of promoter strength. Alcohol dehydrogenase (AdhB) was unsuitable as a reporter in this species due to high endogenous activity and/or low activity of the reporter, but a thermostable LacZ worked well as a reporter. A set of constitutive promoters previously reported to work in Clostridium thermocellum was found to be reliable for controlling the expression of the lacZ reporter gene in H. modesticaldum at a range of activities spanning an order of magnitude. An anhydrotetracycline-inducible promoter was created by inserting tetO operators into a strong constitutive promoter, but it was not fully repressible. The implementation of a xylose-inducible promoter resulted in complete repression of β-gal in the absence of xylose, and reliable expression tunable through the concentration of xylose added to the culture.

Transcriptional Interference in Toehold Switch-Based RNA Circuits

Gene regulation based on regulatory RNA is an important mechanism in cells and is increasingly used for regulatory circuits in synthetic biology. Toehold switches are rationally designed post-transcriptional riboregulators placed in the 5' untranslated region of mRNA molecules. In the inactive state of a toehold switch, the ribosome-binding site is inaccessible to the ribosome. In the presence of a trigger RNA molecule, protein production is turned on. Using antisense RNA against trigger molecules (antitrigger RNA), gene expression can also be switched off again. We here study the utility of antisense transcription in this context, which enables a particularly compact circuit design. Our circuits utilize two inducible promoters that separately regulate trigger and antitrigger transcription, whereas their cognate toehold switch, regulating the expression of a reporter protein, is transcribed from a constitutive promoter. We explore various design options for the arrangement of the promoters and demonstrate that the resulting dynamic behavior is influenced by transcriptional interference (TI) effects depending on the promoter distance. Our experimental results are consistent with previous findings that enhanced local RNA polymerase concentrations due to active promoters in close proximity lead to an increase in transcriptional activity of the strongest promoter in the circuits. We observed that the range of this effect is larger when the participating promoters are stronger. Based on this insight, we combined two promoter arrangements for the realization of a genetic circuit comprised of two toehold switches, two triggers, and two antitriggers that function as a two-input two-output logic gate.

The roles of grouper TAK1 in regulating the infection of Singapore grouper iridovirus

Transforming growth factor-β activated kinase 1 (TAK1) is a member of the mitogen-activated protein kinase family. It is an upstream factor of the IκB kinase, which activates IKKα and IKKβ. TAK1 is a key factor in the induction of nuclear factor κB (NF-κB) and plays a crucial role in the activation of inflammatory responses. However, the roles of TAK1 during viral infection in teleost fish are largely unknown. In this study, we cloned a TAK1 homolog (HgTAK1) from the hybrid grouper (Epinephelus fuscoguttatus♂ × Epinephelus lanceolatus♀). The open reading frame of HgTAK1 consists of 1728 nucleotides encoding 575 amino acids, and the predicted molecular weight is 64.32 kDa HgTAK1 has an S_TKc domain, which consists of a serine/threonine protein kinase and a catalytic domain. Expression pattern analysis showed that HgTAK1 was distributed in all tested tissues, with abundant contents in the heart, head kidney, and blood. Additionally, HgTAK1 was distributed in the cytoplasm of grouper spleen (GS) cells. After Singapore grouper iridovirus (SGIV) infection, the expression of HgTAK1 increased in GS cells. Overexpression of HgTAK1 could promote the replication of SGIV in GS cells and inhibit the activation of NF-κB and IFN stimulated response elements (ISRE) in reporter assay. When co-expressed with IRF3 or HgIRF7 in GS cells, HgTAK1 obviously down-regulated IRF3- or IRF7-mediated the NF-κB and ISRE promoter induction. The interaction between HgTAK1 and IRF3 or IRF7 has been identified by co-immunoprecipitation assay. These findings provide a basis for understanding the innate immune mechanism of the grouper response to viral infection.

Exploitation of strong constitutive and stress-driven promoters from Acetobacter pasteurianus for improving acetic acid tolerance

Acetobacter pasteurianus is an excellent cell factory for production of highly-strength acetic acid, and attracts an increasing attention in metabolic engineering. However, the available well-characterized constitutive and inducible promoters are rather limited to adjust metabolic fluxes in A. pasteurianus. In this study, we screened a panel of constitutive and acid stress-driven promoters based on time-series of RNA-seq data and characterized in A. pasteurianus and Escherichia coli. Nine constitutive promoters ranged in strength from 1.7-fold to 100-fold that of the well-known strong promoter Padh under non-acetic acid environment. Subsequently, an acetic acid-stable red fluorescent visual reporting system was established and applied to evaluate acid stress-driven promoter in A. pasteurianus during highly-acidic fermentation environment. PgroES was identified as acid stress-driven strong promoters, with expression outputs varied from 100% to 200% when acetic acid treatment. To assess their application potential, ultra-strong constitutive promoter Ptuf and acid stress-driven strong promoter PgroES were selected to overexpress acetyl-CoA synthase and greatly improved acetic acid tolerance. Notably, the acid stress-driven promoter displayed more favorable for regulating strain robustness against acid stress by overexpressing tolerance gene. In summary, this is the first well-characterized constitutive and acid stress-driven promoter library from A. pasteurianus, which could be used as a promising toolbox for metabolic engineering in acetic acid bacteria and other gram-negative bacteria.

Characterizing and designing promoter of gene \(\textit{ZmDREB2.7}\) into plant expression vector

In the regulation of gene expression, particularly abiotic-stress tolerance characteristics in plants, inducible promoters play an essential role in ensuring the endurance and productivity of crops. In several previous studies, the ZmDREB2.7 gene and some DNA polymorphisms in its promoter have been shown to enhance drought tolerance traits in maize (Zea mays L.). With the goal of creating a strong drought-tolerant promoter region derived from maize, in this study, the 1 kilobase 5’-upstream region of the ZmDREB2.7 coding sequence from the drought resistant maize line Tevang1 was isolated and characterized. Essential putative cis-regulatory elements (as TATA box or CAAT box) and other elements that regulate drought tolerant properties (as RY/G box, ABRE element or MYB core) were investigated. After that, we designed the transgenic plant vector containing the isolated promoter and the ZmDREB2.7 gene which were isolated in our earlier study. The promoter segment was removed 20 nucleotides ahead of the transcription start site and transferred into the recombinant pZY vector containing the ZmDREB2.7 gene. In conclusion, we created a recombinant vector containing the modified ZmDREB2.7 promoter and the ZmDREB2.7 gene segment derived from maize. This is a promising resource for the creation of a transgenic cultivar with effective resistance to dry stress in the future. Besides, we will keep on examining other potential genetic regions and promoters to diversify sources of genetic materials as well as understand deeper the stress-resistant transgenic plants.

Some novel features of strong promoters discovered in Cytophaga hutchinsonii.

Cytophaga hutchinsonii is an important Gram-negative bacterium belonging to the Bacteroides phylum that can efficiently degrade cellulose. But the promoter that mediates the initiation of gene transcription has been unknown for a long time. In this study, we determined the transcription start site (TSS) of C. hutchinsonii by 5' rapid amplification of cDNA ends (5'RACE). The promoter structure was first identified as TAAT and TATTG which are located -5 and -31bp upstream of TSS, respectively. The function of -5 and -31 regions and the spacer length of the promoter Pchu_1284 were explored by site directed ligase-independent mutagenesis (SLIM). The results showed that the promoter activities were sharply decreased when the TTG motif was mutated into guanine (G) or cytosine (C). Interestingly, we found that the strong promoter was accompanied with many TTTG motifs which could enhance the promoter activities within certain copies. These characteristics were different from other promoters of Bacteriodes species. Furthermore, we carried out genome scanning analysis for C. hutchinsonii and another Bacteroides species by Perl6.0. The results indicated that the promoter structure of C. hutchinsonii possessed more unique features than other species. Also, the screened inducible promoter Pchu_2268 was used to overexpress protein CHU_2196 with a molecular weight of 120kDa in C. hutchinsonii. The present study enriched the promoter structure of Bacteroidetes species and also provided a novel method for the highly expressed large protein (cellulase) in vivo, which was helpful to elucidate the unique cellulose degradation mechanism of C. hutchinsonii.Key points• The conserved structure of strong promoter of C. hutchinsonii was elucidated.• Two novel regulation motifs of TTTG and AATTATG in the promoter were discovered.• A new method for induced expression of cellulase in vivo was established.• Helpful for explained the unique cellulose degradation mechanism of C. hutchinsonii.

Sense-and-Respond Payload Delivery Using a Novel Antigen-Inducible Promoter Improves Suboptimal CAR-T Activation

Chimeric antigen receptor (CAR)-T cell therapies demonstrate the clinical potential of lymphocytes engineered with synthetic properties. However, CAR-T cells are ineffective in most solid tumors, partly due to inadequate activation of the infused lymphocytes at the site of malignancy. To selectively enhance antitumor efficacy without exacerbating off-target toxicities, CAR-T cells can be engineered to preferentially deliver immunostimulatory payloads in tumors. Here, we report a novel antigen-inducible promoter for conditional payload expression in primary human T cells. In therapeutic T cell models, the novel NR4A-based promoter induced higher reporter gene expression than the conventional NFAT-based promoter under weakly immunogenic conditions, where payload expression is most needed. Minimal activity was detected from the inducible promoters in the absence of antigen and after withdrawal of stimulation. As a functional proof-of-concept, we used the NR4A-based promoter to express cytokines in an antimesothelin CAR-T model with suboptimal stimulation and observed improved proliferation compared to T cells engineered with the conventional NFAT promoter or CAR alone. Our system achieves CAR-directed payload expression under weakly immunogenic conditions and could enable the next generation of cell therapies with enhanced antitumor efficacy.