Mitochondrial aspartate regulates TNF biogenesis and autoimmune tissue inflammation.

Misdirected immunity gives rise to the autoimmune tissue inflammation of rheumatoid arthritis, in which excess production of the cytokine tumor necrosis factor (TNF) is a central pathogenic event. Mechanisms underlying the breakdown of self-tolerance are unclear, but T cells in the arthritic joint have a distinctive metabolic signature of ATPlo acetyl-CoAhi proinflammatory effector cells. Here we show that a deficiency in the production of mitochondrial aspartate is an important abnormality in these autoimmune T cells. Shortage of mitochondrial aspartate disrupted the regeneration of the metabolic cofactor nicotinamide adenine dinucleotide, causing ADP deribosylation of the endoplasmic reticulum (ER) sensor GRP78/BiP. As a result, ribosome-rich ER membranes expanded, promoting co-translational translocation and enhanced biogenesis of transmembrane TNF. ERrich T cells were the predominant TNF producers in the arthritic joint. Transfer of intact mitochondria into T cells, as well as supplementation of exogenous aspartate, rescued the mitochondria-instructed expansion of ER membranes and suppressed TNF release and rheumatoid tissue inflammation.

N-myristoyltransferase deficiency impairs activation of kinase AMPK and promotes synovial tissue inflammation.

N-myristoyltransferase (NMT) attaches the fatty acid myristate to the N-terminal glycine of proteins to sort them into soluble and membrane-bound fractions. Function of the energy-sensing AMP-activated protein kinase, AMPK, is myristoylation dependent. In rheumatoid arthritis (RA), pathogenic T cells shift glucose away from adenosine tri-phosphate production toward synthetic and proliferative programs, promoting proliferation, cytokine production, and tissue invasion. We found that RA T cells had a defect in NMT1 function, which prevented AMPK activation and enabled unopposed mTORC1 signaling. Lack of the myristate lipid tail disrupted the lysosomal translocation and activation of AMPK. Instead, myristoylation-incompetent RA T cells hyperactivated the mTORC1 pathway and differentiated into pro-inflammatory TH1 and TH17 helper T cells. In vivo, NMT1 loss caused robust synovial tissue inflammation, whereas forced NMT1 overexpression rescued AMPK activation and suppressed synovitis. Thus, NMT1 has tissue-protective functions by facilitating lysosomal recruitment of AMPK and dampening mTORC1 signaling.

Tumor-derived exosomes antagonize innate antiviral immunity.

Malignancies can compromise innate immunity, but the mechanisms of this are largely unknown. Here we found that, via tumor-derived exosomes (TEXs), cancers were able to transfer activated epidermal growth factor receptor (EGFR) to host macrophages and thereby suppress innate antiviral immunity. Screening of the human kinome identified the kinase MEKK2 in macrophages as an effector of TEX-delivered EGFR that negatively regulated the antiviral immune response. In the context of experimental tumor implantation, MEKK2-deficient mice were more resistant to viral infection than were wild-type mice. Injection of TEXs into mice reduced innate immunity, increased viral load and increased morbidity in an EGFR- and MEKK2-dependent manner. MEKK2 phosphorylated IRF3, a transcription factor crucial for the production of type I interferons; this triggered poly-ubiquitination of IRF3 and blocked its dimerization, translocation to the nucleus and transcriptional activity after viral infection. These findings identify a mechanism by which cancer cells can dampen host innate immunity and potentially cause patients with cancer to become immunocompromised.

OTUB2 Promotes Cancer Metastasis via Hippo-Independent Activation of YAP and TAZ.

The transcriptional regulators YAP and TAZ play important roles in development, physiology, and tumorigenesis and are negatively controlled by the Hippo pathway. It is yet unknown why the YAP/ TAZ proteins are frequently activated in human malignancies in which the Hippo pathway is still active. Here, by a gain-of-function cancer metastasis screen, we discovered OTUB2 as a cancer stemness and metastasis-promoting factor that deubiquitinates and activates YAP/TAZ. We found OTUB2 to be poly-SUMOylated on lysine 233, and this SUMOylation enables it to bind YAP/TAZ. We also identified a yet-unknown SUMO-interacting motif (SIM) in YAP and TAZ required for their association with SUMOylated OTUB2. Importantly, EGF and oncogenic KRAS induce OTUB2 poly-SUMOylation and thereby activate YAP/TAZ. Our results establish OTUB2 as an essential modulator of YAP/TAZ and also reveal a novel mechanism via which YAP/TAZ activity is induced by oncogenic KRAS.

KAT8 is upregulated and recruited to the promoter of Atg8 by FOXO to induce H4 acetylation for autophagy under 20-hydroxyecdysone regulation.

Selective gene expression in cells in physiological or pathological conditions is important for the growth and development of organisms. Acetylation of histone H4 at K16 (H4K16ac) catalyzed by histone acetyltransferase 8 (KAT8) is known to promote gene transcription; however, the regulation of KAT8 transcription and the mechanism by which KAT8 acetylates H4K16ac to promote specific gene expression are unclear. Using the lepidopteran insect Helicoverpa armigera as a model, we reveal that the transcription factor FOXO promotes KAT8 expression and recruits KAT8 to the promoter region of autophagy-related gene 8 (Atg8) to increase H4 acetylation at that location, enabling Atg8 transcription under the steroid hormone 20-hydroxyecdysone (20E) regulation. H4K16ac levels are increased in the midgut during metamorphosis, which is consistent with the expression profiles of KAT8 and ATG8. Knockdown of Kat8 using RNA interference results in delayed pupation and repression of midgut autophagy and decreases H4K16ac levels. Overexpression of KAT8-GFP promotes autophagy and increases H4K16ac levels. FOXO, KAT8, and H4K16ac colocalized at the FOXO-binding region to promote Atg8 transcription under 20E regulation. Acetylated FOXO at K180 and K183 catalyzed by KAT8 promotes gene transcription for autophagy. 20E via FOXO promotes Kat8 transcription. Knockdown or overexpression of FOXO appeared to give similar results as knockdown or overexpression of KAT8. Therefore, FOXO upregulates KAT8 expression and recruits KAT8 to the promoter region of Atg8, where the KAT8 induces H4 acetylation to promote Atg8 transcription for autophagy under 20E regulation. This study reveals the mechanism that KAT8 promotes transcription of a specific gene.

CRISPR-dCas12a-mediated genetic circuit cascades for multiplexed pathway optimization.

The production efficiency of microbial cell factories is sometimes limited by the lack of effective methods to regulate multiple targets in a coordinated manner. Here taking the biosynthesis of glucosamine-6-phosphate (GlcN6P) in Bacillus subtilis as an example, a 'design-build-test-learn' framework was proposed to achieve efficient multiplexed optimization of metabolic pathways. A platform strain was built to carry biosensor signal-amplifying circuits and two genetic regulation circuits. Then, a synthetic CRISPR RNA array blend for boosting and leading (ScrABBLE) device was integrated into the platform strain, which generated 5,184 combinatorial assemblies targeting three genes. The best GlcN6P producer was screened and engineered for the synthesis of valuable pharmaceuticals N-acetylglucosamine and N-acetylmannosamine. The N-acetylglucosamine titer reached 183.9 g liter-1 in a 15-liter bioreactor. In addition, the potential generic application of the ScrABBLE device was also verified using three fluorescent proteins as a case study.

Deficiency of inflammation-sensing protein neuropilin-2 in myeloid-derived macrophages exacerbates colitis via NF-κB activation.

Neuropilin-2 (NRP2) is a multifunctional protein engaged in the regulation of angiogenesis, lymphangiogenesis, axon guidance, and tumor metastasis, but its function in colitis remains unclear. Here, we found that NRP2 was an inflammation-sensing protein rapidly and dramatically induced in myeloid cells, especially in macrophages, under inflammatory contexts. NRP2 deficiency in myeloid cells exacerbated dextran sulfate sodium salt-induced experimental colitis by promoting polarization of M1 macrophages and colon injury. Mechanistically, NRP2 could be induced via NF-κB activation by TNF-α in macrophages, but exerted an inhibitory effect on NF-κB signaling, forming a negative feedback loop with NF-κB to sense and alleviate inflammation. Deletion of NRP2 in macrophages broke this negative feedback circuit, leading to NF-κB overactivation, inflammatory exacerbation, and more severe colitis. Collectively, these findings reveal inflammation restriction as a role for NRP2 in macrophages under inflammation contexts and suggest that NRP2 in macrophages may relieve inflammation in inflammatory bowel disease. © 2023 The Pathological Society of Great Britain and Ireland.

Cortical-cerebellar circuits changes in preschool ASD children by multimodal MRI.

OBJECTIVE: To investigate the alterations in cortical-cerebellar circuits and assess their diagnostic potential in preschool children with autism spectrum disorder using multimodal magnetic resonance imaging. METHODS: We utilized diffusion basis spectrum imaging approaches, namely DBSI_20 and DBSI_combine, alongside 3D structural imaging to examine 31 autism spectrum disorder diagnosed patients and 30 healthy controls. The participants' brains were segmented into 120 anatomical regions for this analysis, and a multimodal strategy was adopted to assess the brain networks using a multi-kernel support vector machine for classification. RESULTS: The results revealed consensus connections in the cortical-cerebellar and subcortical-cerebellar circuits, notably in the thalamus and basal ganglia. These connections were predominantly positive in the frontoparietal and subcortical pathways, whereas negative consensus connections were mainly observed in frontotemporal and subcortical pathways. Among the models tested, DBSI_20 showed the highest accuracy rate of 86.88%. In addition, further analysis indicated that combining the 3 models resulted in the most effective performance. CONCLUSION: The connectivity network analysis of the multimodal brain data identified significant abnormalities in the cortical-cerebellar circuits in autism spectrum disorder patients. The DBSI_20 model not only provided the highest accuracy but also demonstrated efficiency, suggesting its potential for clinical application in autism spectrum disorder diagnosis.

Compensatory thickening of cortical thickness in early stage of schizophrenia.

Brain structural abnormality has been observed in the prodromal and early stages of schizophrenia, but the mechanism behind it is not clear. In this study, to explore the association between cortical abnormalities, metabolite levels, inflammation levels and clinical symptoms of schizophrenia, 51 drug-naive first-episode schizophrenia (FES) patients, 51 ultra-high risk for psychosis (UHR), and 51 healthy controls (HC) were recruited. We estimated gray matter volume (GMV), cortical thickness (CT), concentrations of different metabolites, and inflammatory marks among four groups (UHR converted to psychosis [UHR-C], UHR unconverted to psychosis [UHR-NC], FES, HC). UHR-C group had more CT in the right lateral occipital cortex and the right medial orbito-frontal cortex (rMOF), while a significant reduction in CT of the right fusiform cortex was observed in FES group. UHR-C group had significantly higher concentration of IL-6, while IL-17 could significantly predict CT of the right fusiform and IL-4 and IL-17 were significant predictors of CT in the rMOF. To conclude, it is reasonable to speculate that the increased CT in UHR-C group is related to the inflammatory response, and may participate in some compensatory mechanism, but might become exhaustive with the progress of the disease due to potential neurotoxic effects.

Krüppel-like factor 15 integrated autophagy and gluconeogenesis to maintain glucose homeostasis under 20-hydroxyecdysone regulation.

The regulation of glycometabolism homeostasis is vital to maintain health and development of animal and humans; however, the molecular mechanisms by which organisms regulate the glucose metabolism homeostasis from a feeding state switching to a non-feeding state are not fully understood. Using the holometabolous lepidopteran insect Helicoverpa armigera, cotton bollworm, as a model, we revealed that the steroid hormone 20-hydroxyecdysone (20E) upregulated the expression of transcription factor Krüppel-like factor (identified as Klf15) to promote macroautophagy/autophagy, apoptosis and gluconeogenesis during metamorphosis. 20E via its nuclear receptor EcR upregulated Klf15 transcription in the fat body during metamorphosis. Knockdown of Klf15 using RNA interference delayed pupation and repressed autophagy and apoptosis of larval fat body during metamorphosis. KLF15 promoted autophagic flux and transiting to apoptosis. KLF15 bound to the KLF binding site (KLF bs) in the promoter of Atg8 (autophagy-related gene 8/LC3) to upregulate Atg8 expression. Knockdown Atg8 reduced free fatty acids (FFAs), glycerol, free amino acids (FAAs) and glucose levels. However, knockdown of Klf15 accumulated FFAs, glycerol, and FAAs. Glycolysis was switched to gluconeogenesis, trehalose and glycogen synthesis were changed to degradation during metamorphosis, which were accompanied by the variation of the related genes expression. KLF15 upregulated phosphoenolpyruvate carboxykinase (Pepck) expression by binding to KLF bs in the Pepck promoter for gluconeogenesis, which utilised FFAs, glycerol, and FAAs directly or indirectly to increase glucose in the hemolymph. Taken together, 20E via KLF15 integrated autophagy and gluconeogenesis by promoting autophagy-related and gluconeogenesis-related genes expression.

Tethered Helical Ladder-Type Aromatic Lactams.

Tethered nonplanar aromatics (TNAs) make up an important class of nonplanar aromatic compounds showing unique features. However, the knowledge on the synthesis, structures, and properties of TNAs remains insufficient. In this work, a new type of TNAs, the tethered aromatic lactams, is synthesized via Pd-catalyzed consecutive intramolecular direct arylations. These molecules possess a helical ladder-type conjugated system of up to 13 fused rings. The overall yields ranged from 3.4 to 4.3%. The largest of the tethered aromatic lactams, 6L-Bu-C14, demonstrates a guest-adaptive hosting capability of TNAs for the first time. When binding fullerene guests, the cavity of 6L-Bu-C14 became more circular to better accommodate spherical fullerene molecules. The host-guest interaction is thoroughly studied by X-ray crystallography, theoretical calculations, fluorescence titration, and nuclear magnetic resonance (NMR) titration experiments. 6L-Bu-C14 shows stronger binding with C70 than with C60 due to the better convex-concave π-π interaction. P and M enantiomers of all tethered aromatic lactams show distinct and persistent chiroptical properties and demonstrate the potential of chiral TNAs as circularly polarized luminescence (CPL) emitters.

Superconductivity and Charge-Density-Wave-Like Transition in Th2Cu4As5.

We report the synthesis, crystal structure, and physical properties of a novel ternary compound, Th2Cu4As5. The material crystallizes in a tetragonal structure with lattice parameters a = 4.0639(3) Å and c = 24.8221(17) Å. Its structure can be described as an alternating stacking of fluorite-type Th2As2 layers with antifluorite-type double-layered Cu4As3 slabs. The measurement of electrical resistivity, magnetic susceptibility, and specific heat reveals that Th2Cu4As5 undergoes bulk superconducting transition at 4.2 K. Additionally, all these physical quantities exhibit anomalies at 48 K, accompanied by a sign change in the Hall coefficient, suggesting a charge-density-wave-like (CDW) phase transition. Drawing from both experimental data and band calculations, we propose that the superconducting and CDW-like phase transitions are, respectively, associated with the Cu4As3 slabs and the As plane in the Th2As2 layers.

Compact In Situ Electrochemical NMR with Wireless and Anti-interference Strategy in Multiscenario Applications.

The integration of electrochemistry with nuclear magnetic resonance (NMR) spectroscopy recently offers a powerful approach to understanding oxidative metabolism, detecting reactive intermediates, and predicting biological activities. This combination is particularly effective as electrochemical methods provide excellent mimics of metabolic processes, while NMR spectroscopy offers precise chemical analysis. NMR is already widely utilized in the quality control of pharmaceuticals, foods, and additives and in metabolomic studies. However, the introduction of additional and external connections into the magnet has posed challenges, leading to signal deterioration and limitations in routine measurements. Herein, we report an anti-interference compact in situ electrochemical NMR system (AICISENS). Through a wireless strategy, the compact design allows for the independent and stable operation of electrochemical NMR components with effective interference isolation. Thus, it opens an avenue toward easy integration into in situ platforms, applicable not only to laboratory settings but also to fieldwork. The operability, reliability, and versatility were validated with a series of biomimetic assessments, including measurements of microbial electrochemical systems, functional foods, and simulated drug metabolisms. The robust performance of AICISENS demonstrates its high potential as a powerful analytical tool across diverse applications.

Tailoring planar slip to achieve pure metal-like ductility in body-centred-cubic multi-principal element alloys.

Uniform tensile ductility (UTD) is crucial for the forming/machining capabilities of structural materials. Normally, planar-slip induced narrow deformation bands localize the plastic strains and hence hamper UTD, particularly in body-centred-cubic (bcc) multi-principal element high-entropy alloys (HEAs), which generally exhibit early necking (UTD < 5%). Here we demonstrate a strategy to tailor the planar-slip bands in a Ti-Zr-V-Nb-Al bcc HEA, achieving a 25% UTD together with nearly 50% elongation-to-failure (approaching a ductile elemental metal), while offering gigapascal yield strength. The HEA composition is designed not only to enhance the B2-like local chemical order (LCO), seeding sites to disperse planar slip, but also to generate excess lattice distortion upon deformation-induced LCO destruction, which promotes elastic strains and dislocation debris to cause dynamic hardening. This encourages second-generation planar-slip bands to branch out from first-generation bands, effectively spreading the plastic flow to permeate the sample volume. Moreover, the profuse bands frequently intersect to sustain adequate work-hardening rate (WHR) to large strains. Our strategy showcases the tuning of plastic flow dynamics that turns an otherwise-undesirable deformation mode to our advantage, enabling an unusual synergy of yield strength and UTD for bcc HEAs.

Clinical characteristics and outcomes of COVID-19-associated encephalopathy in children.

Apart from the typical respiratory symptoms, coronavirus disease 2019 (COVID-19) also affects the central nervous system, leading to central disorders such as encephalopathy and encephalitis. However, knowledge of pediatric COVID-19-associated encephalopathy is limited, particularly regarding specific subtypes of encephalopathy. This study aimed to assess the features of COVID-19-associated encephalopathy/encephalitis in children. We retrospectively analyzed a single cohort of 13 hospitalized children with COVID-19-associated encephalopathy. The primary outcome was the descriptive analysis of the clinical characteristics, magnetic resonance imaging and electroencephalography findings, treatment progression, and outcomes. Thirteen children among a total of 275 (5%) children with confirmed COVID-19 developed associated encephalopathy/encephalitis (median age, 35 months; range, 3-138 months). Autoimmune encephalitis was present in six patients, acute necrotizing encephalopathy in three, epilepsy in three, and central nervous system small-vessel vasculitis in one patient. Eight (62%) children presented with seizures. Six (46%) children exhibited elevated blood inflammatory indicators, cerebrospinal fluid inflammatory indicators, or both. Two (15%) critically ill children presented with multi-organ damage. The magnetic resonance imaging findings varied according to the type of encephalopathy/encephalitis. Electroencephalography revealed a slow background rhythm in all 13 children, often accompanied by epileptic discharges. Three (23%) children with acute necrotizing encephalopathy had poor prognoses despite immunotherapy and other treatments. Ten (77%) children demonstrated good functional recovery without relapse. This study highlights COVID-19 as a new trigger of encephalopathy/encephalitis in children. Autoimmune encephalitis is common, while acute necrotizing encephalopathy can induce poor outcomes. These findings provide valuable insights into the impact of COVID-19 on children's brains.

Integrating trans-omics, cellular experiments and clinical validation to identify ILF2 as a diagnostic serum biomarker and therapeutic target in gastric cancer.

BACKGROUND: Gastric cancer (GC) lacks serum biomarkers with clinical diagnostic value. Multi-omics analysis is an important approach to discovering cancer biomarkers. This study aimed to identify and validate serum biomarkers for GC diagnosis by cross-analysis of proteomics and transcriptomics datasets. METHODS: A cross-omics analysis was performed to identify overlapping differentially expressed genes (DEGs) between our previous aptamer-based GC serum proteomics dataset and the GC tissue RNA-Seq dataset in The Cancer Genome Atlas (TCGA) database, followed by lasso regression and random forest analysis to select key overlapping DEGs as candidate biomarkers for GC. The mRNA levels and diagnostic performance of these candidate biomarkers were analyzed in the original and independent GC datasets to select valuable candidate biomarkers. The valuable candidate biomarkers were subjected to bioinformatics analysis to select those closely associated with the biological behaviors of GC as potential biomarkers. The clinical diagnostic value of the potential biomarkers was validated using serum samples, and their expression levels and functions in GC cells were validated using in vitro cell experiments. RESULTS: Four candidate biomarkers (ILF2, PGM2L1, CHD7, and JCHAIN) were selected. Their mRNA levels differed significantly between tumor and normal tissues and showed different diagnostic performances for GC, with areas under the receiver operating characteristic curve (AUROCs) of 0.629-0.950 in the TCGA dataset and 0.736-0.840 in the Gene Expression Omnibus (GEO) dataset. In the bioinformatics analysis, only ILF2 (interleukin enhancer-binding factor 2) gene levels were associated with immune cell infiltration, some checkpoint gene expression, chemotherapy sensitivity, and immunotherapy response. Serum levels of ILF2 were higher in GC patients than in controls, with an AUROC of 0.944 for the diagnosis of GC, and it was also detected in the supernatants of GC cells. Knockdown of ILF2 by siRNA significantly reduced the proliferation and colony formation of GC cells. Overexpression of ILF2 significantly promotes the proliferation and colony formation of gastric cancer cells. CONCLUSIONS: Trans-omics analysis of proteomics and transcriptomics is an efficient approach for discovering serum biomarkers, and ILF2 is a potential diagnostic biomarker and therapeutic target of gastric cancer.

Iodomethane in C1 chemistry: application in palladium-catalyzed [2 + 2 + 1] annulation.

An efficient palladium-catalyzed [2 + 2 + 1] annulation of 3-iodochromones, bridged olefins, and iodomethane is described, affording a range of chromone-containing polycyclic compounds. Additionally, the corresponding deuterated products were smoothly obtained with iodomethane-d3 instead of iodomethane. Moreover, the synthetic utility of this method is further substantiated by gram scale preparation and application to late-stage modification of estrone.

Severe pneumonia with co-infection of H5N1 and SARS-CoV-2: a case report.

BACKGROUND: The H5N1 influenza virus is a cause of severe pneumonia. Co-infection of influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may lead to poor prognosis of patients during the COVID-19 epidemic. However, reports on patients co-infected with avian influenza virus and SARS-CoV-2 are scarce. CASE PRESENTATION: A 52-year-old woman presented with a fever, which has persisted for the past eight days, along with worsening shortness of breath and decreased blood pressure. Computed tomography (CT) revealed an air bronchogram, lung consolidation, and bilateral pleural effusion. The subsequent polymerase chain reaction (PCR) of the bronchoalveolar lavage fluid (BALF) revealed positivity for H5N1 and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). CONCLUSION: The H5N1 influenza virus is a cause of severe pneumonia. The clinical presentation of the patient had a predomination of H5N1 influenza rather than COVID-19. A PCR analysis for the identification of the virus is necessary to reveal the pathogen causing the severe pneumonia. The patient exhibited an excellent prognosis upon the use of the appropriate antiviral medicine.

Arginine Methylation of MDH1 by CARM1 Inhibits Glutamine Metabolism and Suppresses Pancreatic Cancer.

Distinctive from their normal counterparts, cancer cells exhibit unique metabolic dependencies on glutamine to fuel anabolic processes. Specifically, pancreatic ductal adenocarcinoma (PDAC) cells rely on an unconventional metabolic pathway catalyzed by aspartate aminotransferase, malate dehydrogenase 1 (MDH1), and malic enzyme 1 to rewire glutamine metabolism and support nicotinamide adenine dinucleotide phosphate (NADPH) production. Here, we report that methylation on arginine 248 (R248) negatively regulates MDH1. Protein arginine methyltransferase 4 (PRMT4/CARM1) methylates and inhibits MDH1 by disrupting its dimerization. Knockdown of MDH1 represses mitochondria respiration and inhibits glutamine metabolism, which sensitizes PDAC cells to oxidative stress and suppresses cell proliferation. Meanwhile, re-expression of wild-type MDH1, but not its methylation-mimetic mutant, protects cells from oxidative injury and restores cell growth and clonogenic activity. Importantly, MDH1 is hypomethylated at R248 in clinical PDAC samples. Our study reveals that arginine methylation of MDH1 by CARM1 regulates cellular redox homeostasis and suppresses glutamine metabolism of pancreatic cancer.

A pathway independent multi-modular ordered control system based on thermosensors and CRISPRi improves bioproduction in Bacillus subtilis.

Dynamic regulation is an effective strategy for control of gene expression in microbial cell factories. In some pathway contexts, several metabolic modules must be controlled in a time dependent or ordered manner to maximize production, while the creation of genetic circuits with ordered regulation capacity still remains a great challenge. In this work, we develop a pathway independent and programmable system that enables multi-modular ordered control of metabolism in Bacillus subtilis. First, a series of thermosensors were created and engineered to expand their thresholds. Then we designed single-input-multi-output circuits for ordered control based on the use of thermosensors with different transition points. Meanwhile, a repression circuit was constructed by combining CRISPRi-based NOT gates. As a proof-of-concept, these genetic circuits were applied for multi-modular ordered control of 2'-fucosyllactose (2'-FL) biosynthesis, resulting in a production of 1839.7 mg/l in shake flask, which is 5.16-times that of the parental strain. In a 5-l bioreactor, the 2'-FL titer reached 28.2 g/l with down-regulation of autolysis. Taken together, this work provides programmable and versatile thermosensitive genetic toolkits for dynamic regulation in B. subtilis and a multi-modular ordered control framework that can be used to improve metabolic modules in other chassis cells and for other compounds.