Nickel-Catalyzed Reductive Cross-Coupling of Propargylic Acetates with Chloro(vinyl)silanes: Access to Silylallenes.

Because of their various reactivities, propargyl acetates are refined chemical intermediates that are extensively applied in pharmaceutical synthesis. Currently, reactions between propargyl acetates and chlorosilanes may be the most effective method for synthesizing silylallenes. Nevertheless, owing to the adaptability and selectivity of substrates, transition metal catalysis is difficult to achieve. Herein, nickel-catalyzed reductive cross-coupling reactions between propargyl acetates and substituted vinyl chlorosilanes for the synthesis of tetrasubstituted silylallenes are described. Therein, metallic zinc is a crucial reductant that effectively enables two electrophilic reagents to selectively construct C(sp2)-Si bonds. Additionally, a Ni-catalyzed reductive mechanism involving a radical process is proposed on the basis of deuteration-labeled experiments.

The ldh Gene Plays a Crucial Role in Mediating the Pathogen Control of Lactiplantibacillus plantarum AR113.

Effectively managing foodborne pathogens is imperative in food processing, where probiotics play a crucial role in pathogen control. This study focuses on the Lactiplantibacillus plantarum AR113 and its gene knockout strains, exploring their antimicrobial properties against Escherichia coli O157:H7 and Staphylococcus aureus. Antimicrobial assays revealed that the inhibitory effect of AR113 increases with its growth and the potential bacteriostatic substance is acidic. AR113Δldh, surpassed AR113Δ0273&2024, exhibited a complete absence of bacteriostatic properties, which indicates that lactic acid is more essential than acetic acid in the bacteriostatic effect of AR113. However, the exogenous acid validation test affirmed the equivalent superior bacteriostatic effect of lactic acid and acetic acid. Notably, AR113 has high lactate production and deletion of the ldh gene not only lacks lactate production but also affects acetic production. This underscores the ldh gene's pivotal role in the antimicrobial activity of AR113. In addition, among all the selected knockout strains, AR113ΔtagO and ΔccpA also had lower antimicrobial effects, suggesting the importance of tagO and ccpA genes of AR113 in pathogen control. This study contributes insights into the antimicrobial potential of AR113 and stands as the pioneering effort to use knockout strains for comprehensive bacteriostatic investigations.

Genome-scale reconstruction of the metabolic network in Streptococcus thermophilus S-3 and assess urea metabolism.

BACKGROUND: Streptococcus thermophilus is an important strain widely used in dairy fermentation, with distinct urea metabolism characteristics compared to other lactic acid bacteria. The conversion of urea by S. thermophilus has been shown to affect the flavor and acidification characteristics of milk. Additionally, urea metabolism has been found to significantly increase the number of cells and reduce cell damage under acidic pH conditions, resulting in higher activity. However, the physiological role of urea metabolism in S. thermophilus has not been fully evaluated. A deep understanding of this metabolic feature is of great significance for its production and application. Genome-scale metabolic network models (GEMs) are effective tools for investigating the metabolic network of organisms using computational biology methods. Constructing an organism-specific GEM can assist us in comprehending its characteristic metabolism at a systemic level. RESULTS: In the present study, we reconstructed a high-quality GEM of S. thermophilus S-3 (iCH492), which contains 492 genes, 608 metabolites and 642 reactions. Growth phenotyping experiments were employed to validate the model both qualitatively and quantitatively, yielding satisfactory predictive accuracy (95.83%), sensitivity (93.33%) and specificity (100%). Subsequently, a systematic evaluation of urea metabolism in S. thermophilus was performed using iCH492. The results showed that urea metabolism reduces intracellular hydrogen ions and creates membrane potential by producing and transporting ammonium ions. This activation of glycolytic fluxes and ATP synthase produces more ATP for biomass synthesis. The regulation of fluxes of reactions involving NAD(P)H by urea metabolism improves redox balance. CONCLUSION: Model iCH492 represents the most comprehensive knowledge-base of S. thermophilus to date, serving as a potent tool. The evaluation of urea metabolism led to novel insights regarding the role of urease. © 2023 Society of Chemical Industry.

Software-based screening for efficient sgRNAs in Lactococcus lactis.

BACKGROUND: The two essential editing elements in the clustered regularly interspaced short palindromic repeats (CRISPR) editing system are promoter and single-guide RNA (sgRNA), the latter of which determines whether Cas protein can precisely target a specific location to edit the targeted gene. Therefore, the selection of sgRNA is crucial to the efficiency of the CRISPR editing system. Various online prediction tools for sgRNA are currently available. These tools can predict all possible sgRNAs of the targeted gene and rank sgRNAs according to certain scoring criteria according to the demands of the user. RESULTS: We designed sgRNAs for Lactococcus lactis NZ9000 LLNZ_RS02020 (ldh) and LLNZ_RS10925 (upp) individually using online prediction software - CRISPOR - and successfully constructed a series of knockout strains to allow comparison of the knockout efficiency of each sgRNA and analyze the differences between software predictions and actual experimental results. CONCLUSION: Our experimental results showed that the actual editing efficiency of the screened sgRNAs did not match the predicted results - a phenomenon that suggests that established findings from eukaryotic studies are not universally applicable to prokaryotes. Software prediction can still be used as a tool for the initial screening of sgRNAs before further selection of suitable sgRNAs through experimental experience. © 2023 Society of Chemical Industry.

Establishment of CRISPR-Cas9 system in Bifidobacteria animalis AR668.

Bifidobacteria are representative intestinal probiotics that have extremely high application value in the food and medical fields. However, the lack of molecular biology tools limits the research on functional genes and mechanisms of bifidobacteria. The application of an accurate and efficient CRISPR system to genome engineering can fill the gap in efficient genetic tools for bifidobacteria. In this study, CRISPR system of B. animalis AR668 was established, which successfully knocked out gene 0348 and gene 0208. The influence of different homology arms and fragments on the knockout effect of the system was explored. In addition, the inducible plasmid curing system of bifidobacteria was innovatively established. This study contributes to the genetic modification and functional mechanism analysis of bifidobacteria.

Chemodivergent Synthesis of Benzothiadiazin-3-one 1-Oxides and Benzisothiazol-3-ones via Visible Light-Promoted Intramolecular N-S Bond Formation.

We reported a versatile protocol to chemodivergently construct significant heterocyclic scaffolds of benzothiadiazin-3-one 1-oxides and benzisothiazol-3-ones by visible light-promoted photocatalysis. This substrate-dependent chemoselective strategy enables N-(2-mercaptophenyl)-N'-substituted ureas through the N-S bond coupling/oxidation cascade to selectively produce benzothiadiazin-3-one 1-oxides; however, the transformation of 2-mercaptobenzamides only occurs via N-S bond coupling to access benzisothiazol-3-ones with moderate to good yields. This strategy features mild conditions, excellent chemoselectivity, and functional group compatibility, which has potential applications in organic and medicinal chemistry.

Recent Advances in the Biological Responses to Nano-black Phosphorus: Understanding the Importance of Intrinsic Properties and Cell Types.

The production scalability and increasing demand for nano-black phosphorus materials (nano-BPs) inevitably lead to their environmental leakage, thereby raising the risk of human exposure through inhalation, ingestion, dermal, and even intravenous pathways. Consequently, a systematic evaluation of their potential impacts on human health is necessary. This Review outlines recent progress in the understanding of various biological responses to nano-BPs. Attention is particularly given to the inconsistent toxicological findings caused by a wide variation of nano-BPs' physicochemical properties, toxicological testing methods, and cell types examined in each study. Additionally, cellular uptake and intracellular trafficking, cell death modes, immunological effects, and other biologically relevant processes are discussed in detail, providing evidence for the potential health implications of nano-BPs. Finally, we address the remaining challenges related to the health risk evaluation of nano-BPs and propose a broader range of applications for these promising nanomaterials.

Circulating microRNAs as diagnostic biomarkers for ischemic stroke: evidence from comprehensive analysis and real-world validation.

Ischemic stroke (IS) is the majority of strokes which remain the second leading cause of deaths in the last two decades. Circulating microRNAs (miRNAs) have been suggested as potential diagnostic and therapeutic tools for IS by previous studies analyzing their differential expression. However, inconclusive and controversial conclusions of these results have to be addressed. In this study, comprehensive analysis and real-world validation were performed to assess the associations between circulating miRNAs and IS. 29 studies with 112 miRNAs were extracted after manual selection and filtering, 12 differentially expressed miRNAs were obtained from our results of meta-analysis. These miRNAs were evaluated in 20 IS patients, compared to 20 healthy subjects. 4 miRNAs (hsa-let-7e-5p, hsa-miR-124-3p, hsa-miR-17-5p, hsa-miR-185-5p) exhibited the significant expression level in IS patient plasma samples. Pathway and biological process enrichment analysis for the target genes of the 4 validated miRNAs identified cellular senescence and neuroinflammation as key post-IS response pathways. The results of our analyses closely correlated with the pathogenesis and implicated pathways observed in IS subjects suggested by the literature, which may provide aid in the development of circulating diagnostic or therapeutic targets for IS patients.

Identification of lncRNAs associated with uterine corpus endometrial cancer prognosis based on the competing endogenous RNA network.

Uterine Corpus Endometrial Carcinoma (UCEC) is one of the major malignant tumors of the female reproductive system. However, there are limitations in the currently available diagnostic approaches for UCEC. Long non-coding RNAs (lncRNAs) play important roles in regulating biological processes as competitive endogenous RNA (ceRNA) in tumors. To study the potential of lncRNAs as non-invasive diagnostic tumor markers, RNA-sequencing dataset of UCEC patients from The Cancer Genome Atlas was used to identify differentially expressed genes. A lncRNA-miRNA-mRNA ceRNA network was constructed by differentially expressed lncRNAs, miRNAs and miRNAs. Pathway enrichment and functional analysis for the mRNAs in the constructed ceRNA network provide the direction of future research for UCEC by demonstrating the most affected processes and pathways. Seven potential lncRNA biomarkers (C20orf56, LOC100144604, LOC100190940, LOC151534, LOC727677, FLJ35390, LOC158572) were validated in UCEC patients by quantitative real-time PCR. Notably, LOC100190940 and LOC158572 were identified as novel RNA molecules with unknown functions. Receiver operating characteristic (ROC) curve analysis demonstrated that the combined 7 lncRNAs had a high diagnostic value for UCEC patients with area under curve (AUC) of 0.941 (95% CI: 0.875-0.947). Our study highlights the potential of the validated 7 lncRNAs panel as diagnostic biomarkers in UCEC, providing new insights into the UCEC pathogenesis.

Identification of new reference genes for colony counting by reverse-transcription quantitative PCR in Bifidobacterium animalis.

Bifidobacterium animalis, one of the predominant bacteria in the intestines of humans and other mammals, is widely added to dairy products. We employed RNA sequencing to analyze gene expression variance on a genome-wide scale and found stable reference genes (RG) in B. animalis. A total of 1,665 genes were identified by analyzing the data from the transcriptome under 4 different conditions, and 13 probable candidate RG with variation coefficient values <0.1 were validated using reverse-transcription quantitative PCR (RT-qPCR). The amplification efficiency of candidate RG were ranging from 94.16% to 126.25%. We integrated the analysis results of BestKeeper, geNorm, NormFinder, and RefFinder algorithms and revealed that rplD and atpA comprehensive ranked 1.68 and 2.82, respectively, which were more stable than traditional RG. Compared with plate count (1.58 × 106 cfu/mL), the concentrations of B. animalis AR668 by RT-qPCR using rplD, atpA, and 16S rRNA as RG were 2.27 × 106, 2.24 × 106, and 6.66 × 106 cfu/mL, respectively, after 10 h of fermentation in fermented skim milk. It suggested that rplD and atpA as RG can be accurate for colony counting of B. animalis. Our study provides the foundation for more accurate analysis of colony counting by RT-qPCR of B. animalis in dairy foods.

Evaluating Urban Building Damage of 2023 Kahramanmaras, Turkey Earthquake Sequence Using SAR Change Detection.

On February 6, 2023 (local time), two earthquakes (Mw7.8 and Mw7.7) struck central and southern Turkey, causing extensive damage to several cities and claiming a toll of 40,000 lives. In this study, we propose a method for seismic building damage assessment and analysis by combining SAR amplitude and phase coherence change detection. We determined building damage in five severely impacted urban areas and calculated the damage ratio by measuring the urban area and the damaged area. The largest damage ratio of 18.93% is observed in Nurdagi, and the smallest ratio of 7.59% is found in Islahiye. We verified the results by comparing them with high-resolution optical images and AI recognition results from the Microsoft team. We also used pixel offset tracking (POT) technology and D-InSAR technology to obtain surface deformation using Sentinel-1A images and analyzed the relationship between surface deformation and post-earthquake urban building damage. The results show that Nurdagi has the largest urban average surface deformation of 0.48 m and Antakya has the smallest deformation of 0.09 m. We found that buildings in the areas with steeper slopes or closer to earthquake faults have higher risk of collapse. We also discussed the influence of SAR image parameters on building change recognition. Image resolution and observation geometry have a great influence on the change detection results, and the resolution can be improved by various means to raise the recognition accuracy. Our research findings can guide earthquake disaster assessment and analysis and identify influential factors of earthquake damage.

Heterologous expression of C30 carotenoid biosynthetic gene crtNM from Lactiplantibacillus plantarum.

BACKGROUND: Probiotic lactic acid bacterium Lactiplantibacillus plantarum is widely used in the dairy and other fermented food industries. L. plantarum AR113 harbors a C30 carotenoid operon crtNM based on genomic analysis, but the yield of C30 carotenoid is only 8.1 µg g-1 DCW. RESULTS: To improve the productivity of C30 carotenoid, crtNM from L. plantarum AR113 was cloned and reconstructed in Escherichia coli BL21(DE3). The proteins crtN and crtM were successfully expressed based on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and the carotenoid was detected using high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). In comparison with the constitutive promoter P44 , the use of the inducible T7 promoter significantly increased the carotenoid content in E. coli. The fermentation conditions were also optimized with induction by 0.5 mmol/L IPTG at 20 °C for 7 h. The yield of C30 carotenoid reached 154.5 µg g-1 DCW, which was 18-fold higher than that of L. plantarum AR113. The 2,2-diphenyl-1-picryl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6sulfonic acid (ABTS) radical scavenging capacity of C30 carotenoids synthesized by heterologous expression in E. coli was also higher than that of the antioxidant food additive butylated hydroxytoluene. CONCLUSIONS: Our findings suggest that E. coli has strong potential as a basic chassis for the production of C30 carotenoids from Lactiplantibacillus with high antioxidant activity. © 2022 Society of Chemical Industry.

Effects of tamarind seed polysaccharides on physicochemical characteristics of frozen dough: structure-function relationship.

BACKGROUND: Recently, frozen dough has become more popular because of its ability to be quickly transformed into freshly baked foods. During the storage and transport process, frozen dough can suffer some degree of damage caused by ice crystallization and recrystallization. Adding polysaccharides to frozen dough is a good way to solve this problem. Tamarind seed polysaccharide (TSP) has excellent ice crystal steady ability and has also been widely used in frozen foods. However, there is no study on the use of TSP in frozen dough. RESULTS: TSP can stabilize the bound water content, inhibit the freezable water content, and increase elasticity. However, the dough with different structures of TSP added was less firm after 30 days of freezing compared to the dough without TSP, and the porosity and stomatal density of the prepared steamed bread gradually decreased. The addition of TSP reduced gluten deterioration during the freezing process, thus decreasing the collapse and uneven porosity of the steamed bread. CONCLUSIONS: The results could provide new insights into the structure of TSP and its effect on the quality characteristics of frozen dough. © 2023 Society of Chemical Industry.

Intermolecular 1,2-Difunctionalization of Alkenes Enabled by Fluoroamide-Directed Remote Benzyl C(sp3)-H Functionalization.

A copper-catalyzed remote benzylic C-H functionalization strategy enabling 1,2-difunctionalization of alkenes with 2-methylbenzeneamides and nucleophiles, including alcohols, indoles, pyrroles, and the intrinsic amino groups, is reported, which is characterized by its redox-neutral conditions, exquisite site-selectivity, broad substrate scope, and wide utilizations of late-stage modifying bioactive molecules. This reaction proceeds through nitrogen-centered radical generation, hydrogen atom transfer, benzylic radical addition across the alkenes, single-electron oxidation, and carbocation electrophilic course cascades. While using external nucleophiles manipulates three-component alkene alkylalkoxylation and alkyl-heteroarylation with 2-methylbenzeneamides to access dialkyl ethers, 3-alkylindoles, and 3-alkylpyrroles, omitting the external nucleophiles results in two-component alkylamidation ([5+2] annulation) of alkenes with 2-methylbenzeneamides to benzo-[f][1,2]thiazepine 1,1-dioxides.

Functional characterization of a Colchicum autumnale L. double-bond reductase (CaDBR1) in colchicine biosynthesis.

MAIN CONCLUSION: An alkenal double-bond reductase enzyme (CaDBR1) was cloned from Colchicum autumnale L. The encoded enzyme catalysed 4-coumaraldehyde to 4-hydroxydihydrocinnamaldehyde (4-HDCA). Its functional characterization increased the understanding of colchicine biosynthesis. As a traditional medical plant, Colchicum autumnale L. is famous for producing colchicine, a widely used drug for alleviating gout attacks. The biosynthetic pathway of colchicine was revealed most recently, and 4-hydroxydihydrocinnamaldehyde (4-HDCA) has been verified as a crucial intermediate derived from L-phenylalanine. However, the functional gene that catalyses the formation of 4-HDCA remains controversial. In this study, the alkenal double-bond reductase (DBR) gene member CaDBR1 was cloned and characterized from C. autumnale. Bioinformatics analysis predicted and characterized the basic physicochemical properties of CaDBR1. Recombinant CaDBR1 protein was heterologously expressed in Escherichia coli and purified by a Ni-NTA column. In vitro enzyme assays indicated that CaDBR1 could catalyse 4-coumaraldehyde to form 4-HDCA but could not generate 4-HDCA by taking cinnamaldehyde as a substrate. Stable transformation into tobacco BY-2 cells revealed that CaDBR1 localized in the cytoplasm, and tissue-specific expression results showed that CaDBR1 had the highest expression in bulbs. All these results verify and confirm the participation and contribution of CaDBR1 in the biosynthesis pathway of 4-HDCA and colchicine alkaloids in C. autumnale.

Determination of the regulatory network and function of the lysR-type transcriptional regulator of Lactiplantibacillus plantarum, LpLttR.

BACKGROUND: Lactiplantibacillus plantarum has various healthcare functions including the regulation of immunity and inflammation, reduction of serum cholesterol levels, anti-tumor activity, and maintenance of the balance of intestinal flora. However, the underlying metabolic and regulatory mechanisms of these processes remain unclear. Our previous studies have shown that the LysR type transcriptional regulator of L. plantarum (LpLttR) regulates the biotransformation of conjugated linoleic acids (CLAs) through the transcriptional activation of cla-dh (coding gene for CLA short-chain dehydrogenase) and cla-dc (coding gene for CLA acetoacetate decarboxylase). However, the regulatory network and function of LpLttR have not yet been characterized in L. plantarum. RESULTS: In this study, the regulatory role of LpLttR in various cellular processes was assessed using transcriptome analysis. The deletion of LpLttR had no evident influence on the bacterial growth. The transcriptome data showed that the expression of nine genes were positively regulated by LpLttR, and the expression of only two genes were negatively regulated. Through binding motif analysis and molecular interaction, we demonstrated that the regulatory region of the directly regulated genes contained a highly conserved sequence, consisting of a 15-base long box and rich in AT. CONCLUSION: This study revealed that LpLttR of L. plantarum did not play a global regulatory role similar to that of the other transcriptional regulators in this family. This study broadens our knowledge of LpLttR and provides a theoretical basis for the utilization of L. plantarum.

Enhancement of triterpene production via in situ extractive fermentation of Sanghuangporus vaninii YC-1.

There have been many studies on the activities and polysaccharide production of Sanghuangporus vaninii. However, few studies have looked at triterpene production from S. vaninii using liquid-state fermentation. A method for enhancing the production of triterpenes by in situ extractive fermentation (ISEF) was studied. Eight solvents were investigated as extractants for triterpene production in the ISEF system. The results showed that using vegetable oil as an extractant significantly increased the yield of total triterpenes and biomass of S. vaninii YC-1, reaching 18.98 ± 0.71 and 44.67 ± 2.21 g/L, respectively. In 5 L fermenter experiments, the added vegetable oil improved the dissolved oxygen condition of the fermentation broth and promoted the growth of S. vaninii YC-1. Furthermore, adding vegetable oil increased the expression of fatty acid synthesis-related genes such as FAD2 and SCD, thereby increasing the synthesis of unsaturated fatty acids in the cell membrane of S. vaninii YC-1. Therefore, the cell membrane permeability of S. vaninii YC-1 increased by 19%. Our results indicated that vegetable oil increased the permeability of S. vaninii YC-1 cell membranes to promote the production of total triterpenes. The use of vegetable oil as an extractant was thus effective in increasing the yield of triterpenes in the ISEF system.

Complete Genome Sequence of Lactobacillus salivarius AR809, a Probiotic Strain with Oropharyngeal Tract Resistance and Adhesion to the Oral Epithelial Cells.

Lactobacillus salivarius AR809 was isolated from a healthy adult oral cavity with multiple probiotic properties, such as high antimicrobial activity, adhesion to the oral epithelium, resistance to acidic pH, bile, lysozyme, and H2O2. In this study, to investigate the genetic basis on probiotic potential and identify the functional genes in the strain, the complete genome of strain AR809 was sequenced by Illumina and PacBio platforms. Then comparative genome analysis on 11 strains of Lactobacillus salivarius was performed. The complete genome of AR809 consisted of a circular 1,747,224 bp chromosome with 33.00% GC content and four circular plasmids [pA (247,948 bp), pB (27,292 bp), pC (3349 bp), and pD (2898 bp), respectively]. From among the 1866 protein-coding genes, 130 carbohydrate metabolism-related genes, 18 bacteriocin biosynthesis-related genes, 74 environmental stress-related genes, and a series of adhesion-related genes were identified via clusters of orthologous genes, Koyto Encyclopedia of Genes and Genomes, and carbohydrate-active enzymes annotation. The comparative genome analysis indicated that genomic homology between AR809 and CICC23174 was the highest. In conclusion, the present work provided valuable insights into the gene's function prediction and understanding the genetic basis on adapting to host oropharyngeal-gastrointestinal tract in strain AR809.

Genetic evidence for the requirements of antroquinonol biosynthesis by Antrodia camphorata during liquid-state fermentation.

The solid-state fermentation of Antrodia camphorata could produce a variety of ubiquinone compounds, such as antroquinonol (AQ). However, AQ is hardly synthesized during liquid-state fermentation (LSF). To investigates the mechanism of AQ synthesis, three precursors (ubiquinone 0 UQ0, farnesol and farnesyl diphosphate FPP) were added in LSF. The results showed that UQ0 successfully induced AQ production; however, farnesol and FPP could not induce AQ synthesis. The precursor that restricts the synthesis of AQ is the quinone ring, not the isoprene side chain. Then, the Agrobacterium-mediated transformation system of A. camphorata was established and the genes for quinone ring modification (coq2-6) and isoprene synthesis (HMGR, fps) were overexpressed. The results showed that overexpression of genes for isoprene side chain synthesis could not increase the yield of AQ, but overexpression of coq2 and coq5 could significantly increase AQ production. This is consistent with the results of the experiment of precursors. It indicated that the A. camphorata lack the ability to modify the quinone ring of AQ during LSF. Of the modification steps, prenylation of UQ0 is the key step of AQ biosynthesis. The result will help us to understand the genetic evidence for the requirements of AQ biosynthesis in A. camphorata.

CRISPR/dCas9-based metabolic pathway engineering for the systematic optimization of exopolysaccharide biosynthesis in Streptococcus thermophilus.

Streptococcus thermophilus is used extensively in the dairy industry and has shown great promise as a chassis cell for the biosynthesis of high-value metabolites. However, metabolic engineering in S. thermophilus lacks effective genetic modification tools to modulate gene expression to relieve metabolic burden and maximize the production of desired compounds. Here, we developed a clustered regularly interspaced short palindromic repeats interference (CRISPRi) system for efficient gene transcriptional modulation in S. thermophilus. Our CRISPRi system typically achieved 66 to 98% knockdown of single or multiple gene expression. We used CRISPRi for the biosynthesis of a new exopolysaccharide (EPS) as a paradigm model. Repression of galK at module of uridine diphosphate glucose sugar metabolism and overexpression of epsA and epsE at EPS synthesis module resulted in an approximately 2-fold increase in EPS titer (277 mg/L) when compared with a control strain. This study demonstrated the effectiveness of CRISPRi as a powerful metabolic engineering tool and synthetic biology strategy for S. thermophilus.