A SURF4-to-proteoglycan relay mechanism that mediates the sorting and secretion of a tagged variant of sonic hedgehog.

SignificanceSonic Hedgehog (Shh) is a key signaling molecule that plays important roles in embryonic patterning, cell differentiation, and organ development. Although fundamentally important, the molecular mechanisms that regulate secretion of newly synthesized Shh are still unclear. Our study reveals a role for the cargo receptor, SURF4, in facilitating export of Shh from the endoplasmic reticulum (ER) via a ER export signal. In addition, our study provides evidence suggesting that proteoglycans promote the dissociation of SURF4 from Shh at the Golgi, suggesting a SURF4-to-proteoglycan relay mechanism. These analyses provide insight into an important question in cell biology: how do cargo receptors capture their clients in one compartment, then disengage at their destination?

Red-Shifting B12-Dependent Photoreceptor Protein via Optical Coupling for Inducible Living Materials.

Cobalamin (B12)-dependent photoreceptors are gaining traction in materials synthetic biology, especially for optically controlling cell-to-cell adhesion in living materials. However, these proteins are mostly responsive to green light, limiting their deep-tissue applications. Here, we present a general strategy for shifting photoresponse of B12-dependent photoreceptor CarHC from green to red/far-red light via optical coupling. Using thiol-maleimide click chemistry, we labeled cysteine-containing CarHC mutants with SulfoCyanine5 (Cy5), a red light-capturing fluorophore. The resulting photoreceptors not only retained the ability to tetramerize in the presence of adenosylcobalamin (AdoB12), but also gained sensitivity to red light; labeled tetramers disassembled on red light exposure. Using genetically encoded click chemistry, we assembled the red-shifted proteins into hydrogels that degraded rapidly in response to red light. Furthermore, Saccharomyces cerevisiae cells were genetically engineered to display CarHC variants, which, alongside in situ Cy5 labeling, led to living materials that could assemble and disassemble in response to AdoB12 and red light, respectively. These results illustrate the CarHC spectrally tuned by optical coupling as a versatile motif for dynamically controlling cell-to-cell interactions within engineered living materials. Given their prevalence and ecological diversity in nature, this spectral tuning method will expand the use of B12-dependent photoreceptors in optogenetics and living materials.

Correction: Highly reusable nanoporous silver sheet for sensitive SERS detection of pesticides.

Correction for 'Highly reusable nanoporous silver sheet for sensitive SERS detection of pesticides' by Huanyu Chi et al., Analyst, 2020, 145, 5158-5165, https://doi.org/10.1039/D0AN00999G.

Tunable lipid-coated nanoporous silver sheet for characterization of protein-membrane interactions by surface-enhanced Raman scattering (SERS).

Membrane environments affect protein structures and functions through protein-membrane interactions in a wide range of important biological processes. To better study the effects from the lipid's hydrophilic and hydrophobic interaction with protein on different membrane regions, we developed the lipid-coated nanoporous silver sheets to provide tunable supported lipid monolayer/bilayer environments for in situ surface-enhanced Raman vibrational spectroscopy (SERS) characterizations. Under the controllable surface pressure, lipid monolayer/bilayer was coated along the microscopic curved surface of nanoporous silver sheets to serve as a cell membrane mimic as well as a barrier to avoid protein denaturation while empowering the high SERS enhancements from the underlying metallic bases allowing detection sensitivity at low physiological concentrations. Moreover, we fine-tuned the lipid packing density and controlled the orientation of the deposited lipid bilayers and monolayers to directly monitor the protein structures upon interactions with various membrane parts/positions. Our results indicate that lysozyme adopted the α-helical structure in both hydrophilic and hydrophobic interaction with lipid membrane. Interestingly, alpha-synuclein folded into the α-helical structure on the negatively charged lipid heads, whereas the hydrophobic lipid tails induced the ß-sheet structural conversion of alpha-synuclein originated from its unstructured monomers. These direct observations on protein hydrophilic and hydrophobic interaction with lipid membrane might provide profound insights into the formation of the ß-sheet-containing alpha-synuclein oligomers for further membrane disruptions and amyloid genesis associated with Parkinson's disease. Hence, with the controllability and tunability of lipid environments, our platform holds great promise for more general applications in investigating the influences from membranes and the correlative structures of proteins under both hydrophilic and hydrophobic effects.

Comparison of nutritional value, bioactivity, and volatile compounds of soybean meal-corn bran mixed substrates fermented by different microorganisms.

To evaluate the impact of fermentation with different microorganisms on the nutritional quality and bioactivity of soybean meal-corn bran mixed substrates (MS), five lactic acid bacteria (LAB) strains, two Bacillus, and two yeast strains with excellent probiotics were selected for solid-state fermentation of soybean meal and corn bran MS. The fermented mixed substrate (FMS) inoculated with Lacticaseibacillus casei, Lactobacillus fermentum, Lactiplantibacillus plantarum, and Lactobacillus acidophilus presents lower risk of infection with pathogenic bacteria, probably due to their low pH and high lactate content. Compared to the FMS with LAB and yeast, Bacillus subtilis and B. pumilus showed significant improvements in nutritional quality and bioactivity, including TCA-SP, small peptide, free amino acids, total phenol, and protein digestibility. More than 300 volatile compounds were identified in FMS, including alcohols, ketones, aldehydes, esters, acids, ethers, furans, pyrazines, benzene, phenols, amines, alkanes, and others. FMS with Bacillus was characterized as containing a greater number of compounds such as ketones, aldehydes, and pyrazines. This study showed that microbial fermented feeds differed with various microorganism, and fermentation was an effective way to improve the quality of soybean meal-corn bran mixed feeds. This study might be the basis for excellent strains screening for multi-microbial combined fermentation in the future.

Dynamics of bacterial community, metabolites profile and physicochemical characteristics during solid-state fermentation of soybean meal and corn mixed substrates inoculated with Bacillus pumilus and Limosilactobacillus fermentum.

BACKGROUND: Solid-state fermentation (SSF) is a general approach for preparing food and feed, which not only improves nutrition but also provides prebiotics and metabolites. Although many studies have been conducted on the effects of fermentation on feed substrate, the dynamics of microbiota and metabolites in SSF remain unclear. Here, high-throughput sequencing combined with gas chromatography-quadrupole time-of-flight mass spectrometry was used to evaluate the dynamic changes of solid fermented soybean meal and corn mixed matrix inoculated with Bacillus pumilus and Limosilactobacillus fermentum. RESULTS: Generally, inoculated bacteria rapidly proliferated, accompanied by the degradation of macromolecular proteins and an increase in the content of small peptides, trichloroacetic acid-soluble protein, free amino acids and organic acids. Bacillus, Lactobacillus and Enterococcus dominated the whole fermentation process. 389 non-volatile metabolites and 182 volatile metabolites were identified, including amino acids, organic acids, ketones, aldehydes, furans and pyrazine. Typical non-volatile metabolites such as lactic acid, 4-aminobutanoic acid, l-glutamic acid, d-arabinose and volatile metabolites such as 4-ethyl-2-methoxyphenol, 4-penten-2-ol, 2-pentanone, 2-ethylfuran, 2-methylhexanoic acid and butanoic acid-ethyl ester were significantly increased in two-stage solid fermentation. However, some adverse metabolites were also produced, such as oxalic acid, acetic acid, tyramine and n-butylamine, which may affect the quality of fermented feed. Sixteen genera were significantly correlated with differential non-volatile metabolites, while 11 genera were significantly correlated with differential volatile metabolites. CONCLUSION: These results characterized the dynamic changes in the process of two-stage solid-state fermentation with Bacillus pumilus and Limosilactobacillus fermentum and provided a potential reference for additional intervention on improving the effectiveness and efficiency of solid-state fermentation of feed in the future. © 2023 Society of Chemical Industry.

FOXO1 represses MCL1 transcription to regulate the function of vascular smooth muscle cells in intracranial aneurysm.

Intracranial aneurysm (IA) is a pathological dilation of the cerebral arteries. Vascular smooth muscle cell (VSMC) dysfunction assumes a role in IA development. In this context, this study probed the role of FOXO1 in human brain VSMC (HBVSMC) function via MCL1. FOXO1 and MCL1 expression in arterial wall tissues from IA patients and inflammatory cytokines (IL-1ß, TNF-α, and IL-6) levels in the serum of IA patients were, respectively, detected with qRT-PCR and ELISA. Pearson's correlation analysis was utilized to analyze the correlation between FOXO1 and MCL1. After FOXO1 and/or MCL1 were overexpressed in HBVSMCs, caspase-3 and Cyt-c protein expression were examined by western blot, cell proliferation by CCK-8 and EdU assays, and cell apoptosis by flow cytometry. IL-1ß, TNF-α, and IL-6 levels were assessed in the supernatant of HBVSMCs with ELISA. Dual-luciferase gene reporter and ChIP assays were conducted to evaluate the binding of FOXO1 to MCL1. FOXO1 expression was high and MCL expression was low in arterial wall tissues from IA patients, and IL-1ß, TNF-α, and IL-6 levels were high in the serum of IA patients. There was an inverse correlation between FOXO1 and MCL1 mRNA levels. Moreover, FOXO1 bound to the MCL1 promoter to decrease MCL1 transcription. In addition, FOXO1 overexpression augmented cell apoptosis, caspase-3 and Cyt-c protein expression, and IL-1ß, TNF-α, and IL-6 secretion, while reducing cell proliferation in HBVSMCs, which was abrogated by further MCL1 overexpression. FOXO1 impeded MCL1 transcription to curb HBVSMC proliferation and facilitate their apoptosis and inflammation.

Composition and functional diversity of fecal bacterial community of wild boar, commercial pig and domestic native pig as revealed by 16S rRNA gene sequencing.

The bacterial community in mammalian gastrointestinal tract is abundant and complex. To date, little is known about the gut microbiota of wild boar. This study aimed to investigate the fecal bacterial diversity of wild boar and compare with commercial pig and domestic native pig. The diet composition showed that the diets of wild boar, commercial pig and domestic native pig were different from each other. More than 1,760,000 quality-filtered sequences were obtained, and the results revealed distinct compositions and diversity of fecal microbiota in three groups. PCoA and NMDS analyses showed that fecal bacterial communities of wild boar, commercial pig and domestic native pig formed distinctly different clusters. Although the three groups shared a large size of OTUs comprising a core microbiota community, a strong distinction existed at family and genus levels. Ruminococcaceae, Prevotellaceae and Christensenellaceae were more abundant in the feces of wild boar than in domestic native pig and commercial pig. At the genus level, the proportion of unidentified Christensenellaceae was remarkably higher in wild boar group, while commercial pig and domestic native pig group had a higher abundance of Streptococcus and Lactobacillus. Tax4Fun predictions of metagenome function showed statistically significant differences in the functions of fecal microbiota in three groups. There were more bacteria genes with amino acid metabolism, cell growth and death, cell motility, energy metabolism, immune system and environmental adaptation observed in wild boar feces, while commercial pig feces contained more bacteria genes with carbohydrate metabolism, drug resistance, aging, infectious diseases, lipid metabolism, endocrine and metabolic diseases. These results indicated that the fecal microbial ecosystem of the wild boar is significantly different from that of domestic native pig and commercial pig, suggesting that diet is an important factor leading to differences in bacterial abundance and diversity in feces.

Highly reusable nanoporous silver sheet for sensitive SERS detection of pesticides.

Surface-enhanced Raman spectroscopy (SERS) enables pesticide detection at the point-of-need, but its practical application is limited by expensive and disposable SERS substrates. Here, we report a reusable nanoporous silver (NPAg) sheet for the SERS detection of organochlorine pesticides, aiming to maximize the cost-efficiency of substrate regeneration. The NPAg sheet is prepared by a reduction-induced decomposition method without chemical induced random aggregations. This SERS substrate is sensitive to various analytes regardless of their affinity to a metal surface such as rhodamine B, dichlorodiphenyl-trichloroethane (DDT), and lindane due to its large surface area and the coral rock-like morphology. The SERS signal of lindane, a typical organochlorine pesticide, is identified and quantified with a minimum detectable concentration of 3 × 10-7 M (87 ppb), which is below the maximum residue limits in various foods set by the regulators across the world. More importantly, after a few minutes of ultrasonic cleaning in water, the NPAg sheet can be reused at least 20 times with a reproducible SERS activity. Furthermore, the NPAg sheet remains stable in terms of its sensitivity and reusability after several months of bare strorage. Therefore, the NPAg sheet as a SERS substrate holds great promise for mass production and convenient applications in low-cost pesticide analysis.

Oreonectes guilinensis (Teleostei, Cypriniformes, Nemacheilidae), a new loach species from Guangxi, China.

A new loach Oreonectes guilinensis sp. nov. is described from Guangxi Zhuang Autonomous Region, China. It is the second surface-dwelling fish identified in the genus Oreonectes after the type species Oreonectes platycephalus. This new species can be distinguished from other species within the genus by the combination of the following features: a round caudal fin, 13-14 branched rays, an incomplete lateral line with 4-6 pores, a short pelvic fin located some distance from the anus, a scale-covered body, a stout build (body width 14.0%-16.7% of standard length), a posterior chamber of well-developed airbladder and a yellowish-brown body with a lateral dark brown strip. The maximum likelihood phylogenetic analysis based on cytb gene recovered this new species in a well-supported clade with the type species O. platycephalus, sister to the Lefua clade. Morphological comparisons as well as our phylogenetic tree support Oreonectes shuilongensis, Oreonectes daqikongensis, Oreonectes jiarongensis as species of Troglonectes. Based on our results there are 6 valid species in the genus Oreonectes.

A spectroscopic study of the excited state proton transfer processes of (8-bromo-7-hydroxyquinolin-2-yl)methyl-protected phenol in aqueous solutions.

A combination of spectroscopic methods and density functional theory (DFT) computations was used to study the excited state proton transfer (ESPT) processes of (8-bromo-7-hydroxyquinolin-2-yl)methyl-protected phenol (BHQ-OPh). Characterization of the prototropic forms of BHQ-OPh in different solvent environments revealed that the neutral form predominates in acetonitrile and in 1 : 1 acetonitrile/water (pH 5.0), whereas the anionic form predominates in 1 : 1 acetonitrile/PBS (pH 7.4). Both the neutral and anionic forms were significantly populated in 1 : 1 acetonitrile/water. Upon irradiation in acetonitrile the triplet neutral form was observed, whereas the triplet anionic form was detected in 1 : 1 acetonitrile/PBS (pH 7.4). The existence of the triplet tautomeric form of BHQ-OPh in both 1 : 1 acetonitrile/water and 1 : 1 acetonitrile/water (pH 5.0), and the ESPT processes from the neutral to the anionic to the tautomeric forms in the excited state were observed using time-resolved spectroscopy. A reaction mechanism in 1 : 1 acetonitrile/water and 1 : 1 acetonitrile/water (pH 5.0) was proposed based on the spectroscopic and DFT computational results. A comparison of the results for BHQ-OPh with those of BHQ-OAc reveals that the initial prototropic states and photochemical processes are similar. The understanding gained of the initial photo-induced processes of BHQ-based photoremovable protecting groups (PPGs) is useful for the design of new quinolinyl-based PPGs for specialized applications.

Octave-spanning mid-infrared pulses by plasma generation in air pumped with an Yb:KGW source.

Femtosecond mid-infrared (IR) supercontinuum generation in gas media provides a broadband source suited for time-domain spectroscopies and microscopies. This technology has largely utilized <100 fs Ti:sapphire pump lasers. In this Letter, we describe the first plasma generation mid-IR source based on a 1030 nm, 171 fs Yb:KGW laser system; when its first three harmonics are focused in air, a conical mode supercontinuum is generated that spans <1000 to 2700 cm-1 with a 190 pJ pulse energy and 0.5% RMS stability.

How Does the C-Halogen Bond Break in the Photosubstitution Reaction of 3-Fluorobenzophenone in Acidic Aqueous Solutions?

The efficient photosubstitution reaction of m-fluorobenzophenone and the related photohydration reactions were systematically investigated in acidic aqueous solutions. The mechanisms and intermediates were directly characterized by femtosecond transient absorption spectroscopy and nanosecond time-resolved resonance Raman spectroscopy, which is supported by density functional theory calculations. This photosubstitution was found to be a two-step process, based on the observation of a meta-hydration intermediate. The protonation of the ketone was confirmed as a crucial precursor step for further photochemical reactions as indicated by the observation of the absorption spectrum of an excited triplet protonated species. More interestingly, the efficient photosubstitution reaction could selectively occur under specific conditions. Control experiments on a series of halogen-substituted benzophenones were conducted to study the influence of the solution acidity, substituent positions, and the kind of substituted halogens on the efficiency in forming the corresponding hydroxyl photosubstitution product. Some practical conditions in predicting the efficiency of the photosubstitution reaction of interest are summarized, and they were successfully used to predict when the photosubstitution reaction takes place for some other halogen-substituted benzophenone derivatives. The driving force of this photosubstitution reaction may provide insights into several possible applications which are also briefly discussed.

Direct time-resolved spectroscopic observation of arylnitrenium ion reactions with guanine-containing DNA oligomers.

The metabolic activation of a number of aromatic amine compounds to arylnitrenium ions that can react with DNA to form covalent adducts has been linked to carcinogenesis. Guanine in DNA has been shown to be the main target of N-containing carcinogens, and many monomeric guanine derivatives have been utilized as models for product analysis and spectroscopic investigations to attempt to better understand the reaction mechanisms of DNA with arylnitrenium ions. However, there are still important unresolved issues regarding how arylnitrenium ions attack guanine residues in DNA oligomers. In this article, we employed ns-TA and ns-TR(3) spectroscopies to directly observe the reaction of the 2-fluorenylnitrenium ion with selected DNA oligomers, and we detected an intermediate possessing a similar C8 structure as the intermediates produced from the reaction of monomeric guanosine derivatives with arylnitrenium ions. Our results suggest that the oligomeric structure can lead to a faster reaction rate of arylnitrenium ions with guanine residues in DNA oligomers and the reaction of arylnitrenium ions take place in a manner similar to reactions with monomeric guanosine derivatives.

A computational chemistry investigation of the mechanism of the water-assisted decomposition of trichloroethylene oxide.

Trichloroethylene oxide is a downstream product in the oxidative metabolism of trichloroethylene (TCE) and it may be involved in cytochrome P450 inactivation, protein function destruction, and nucleic acid base alkalization. To explore the hydrolysis mechanism of the decomposition of TCE oxide, an investigation using Second-order Møller-Plesset perturbation theory in conjunction with density functional theory has been conducted to analyze the effect of the water solvation shell on probable reaction steps. The decomposition of TCE oxide is accelerated by coordinated water molecules (up to seven), which reveals that water molecules can help to solvate the TCE oxide molecule and activate the release of the Cl(-) leaving group. After the opening of the epoxide ring, several pathways are proposed to account for the dehalogenation step along with the formation of CO as well as three carboxylic acids (formic acid, glyoxylic acid, and dichloroacetic acid). The predominant pathways were examined by comparing the computed activation energies for the formation of the products to each other for the possible reaction steps examined in this work. After rationally analyzing the computational results, the ring-opening reaction has been identified as the rate-determining step. The rate constant estimated for the TCE oxide decomposition from the calculations performed here was found to be reasonably consistent with previous experimental observations reported in the literature.

The dynamic changes and correlations between biochemical properties, flavor and microbial community during fermentation of asparagus by-products.

Asparagus by-products are the promising resource that urgently need to be re-valorized. This study investigated the dynamic changes in physicochemical properties, organic acids, free amino acids, volatile flavor compounds, microbial succession, and their correlations during 7-day spontaneous fermentation of asparagus by-products. Dominant organic acids (lactic acid and acetic acid) and free amino acids (Ser, Glu, and Ala) increased with fermentation time, with lactic acid reaching 7.73 ± 0.05 mg/mL and Ser increasing 56-fold after 7 days. A total of 58 volatile flavor compounds were identified using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPEM/GC-MS), with esters, alcohols and acids as the main volatile flavor compounds. Fourteen volatile flavor compounds had odor activity value >1. High-throughput sequencing showed Firmicutes and Proteobacteria as the main bacterial phyla, dominated by lactic acid bacteria (Levilactobacillus, Lactiplantibacillus, Weissella). Correlation analysis revealed that five bacterial genera (Levilactobacillus, Lactiplantibacillus, Enterobacter, Pediococcus and Acetobacter) were highly correlated with organic acids, free amino acids, and volatile flavor compounds, indicating their pivotal role in forming the characteristic flavor of fermented asparagus by-products (FAPS). This study provides new insights into the flavor and microbial profile of FAPS, offering a strategy for value-added processing and industrial production.

Two-stage fermentation of corn and soybean meal mixture by Bacillus subtilis and Lactobacillus acidophilus to improve feeding value: optimization, physicochemical property, and microbial community.

Microbial treatment can reduce the antinutritional factors and allergenic proteins in corn-soybean meal mixture (CSMM), but the role of the microbial community in hypoallergenicity and digestibility during the fermentation process remains unclear. Therefore, the fermentation strains of Bacillus and LAB were determined, and the compatibility and fermentation process of two-stage solid fermentation composite bacteria were optimized, and the dynamic changes in physicochemical property and microbial community during two-stage fermentation were investigated. Results showed that Bacillus subtilis NCUBSL003 and Lactobacillus acidophilus NCUA065016 were the best fermentation combinations. The optimal fermentation conditions were inoculum 7.14%, solid-liquid ratio of 1:0.88 and fermentation time of 74.30 h. The contents of TI, ß-conglycinin and glycinin decreased significantly after fermentation. Besides, TCA-SP, small peptides and FAA increased. Bacillus and Lactobacillus were the main genera. Pathogenic bacteria genera were inhibited effectively. This study suggests the feasibility of two-stage fermentation in improving the nutrient values and safety of the CSMM. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01426-7.

ZBP1 condensate formation synergizes Z-NAs recognition and signal transduction.

Z-DNA binding protein 1 (ZBP1) is a crucial player in the intracellular recognition of Z-form nucleic acids (Z-NAs) through its Zαß domain, initiating downstream interactions with RIPK1 and RIPK3 via RHIM domains. This engagement leads to the assembly of PANoptosomes, ultimately inducing programmed cell death to curb pathogen dissemination. How Zαß and RHIM domain cooperate to trigger Z-NAs recognition and signal transduction remains unclear. Here, we show that ZBP1 condensate formation facilitates Z-NAs binding and antiviral signal transduction. The ZBP1 Zαß dimerizes in a concentration-dependent manner, forming characteristic condensates in solutions evidenced by DLS and SAXS methods. ZBP1 exhibits a binding preference for 10-bp length CG (10CG) DNA and Z-RNA ligand, which in turn enhanced Zαß dimerization, expediting the formation of droplet condensates in vitro and amyloid-like puncta in cells. Subsequent investigations reveal that Zαß could form condensates with liquid-liquid phase separation property upon HSV and IAV infections, while full-length ZBP1 forms amyloid-like puncta with or without infections. Furthermore, ZBP1 RHIM domains show typical amyloidal fibril characterizations and cross-polymerize with RIPK1 depending on the core motif of 206IQIG209, while mutated ZBP1 could impede necroptosis and antiviral immunity in HT-29 cells. Thus, ZBP1 condensate formation facilitates the recognition of viral Z-NAs and activation of downstream signal transduction via synergic action of different domains, revealing its elaborated mechanism in innate immunity.

Brain region changes following a spinal cord injury.

Brain-related complications are common in clinical practice after spinal cord injury (SCI); however, the molecular mechanisms of these complications are still unclear. Here, we reviewed the changes in the brain regions caused by SCI from three perspectives: imaging, molecular analysis, and electrophysiology. Imaging studies revealed abnormal functional connectivity, gray matter volume atrophy, and metabolic abnormalities in brain regions after SCI, leading to changes in the structure and function of brain regions. At the molecular level, chemokines, inflammatory factors, and damage-associated molecular patterns produced in the injured area were retrogradely transmitted through the corticospinal tract, cerebrospinal fluid, or blood circulation to the specific brain area to cause pathologic changes. Electrophysiologic recordings also suggested abnormal changes in brain electrical activity after SCI. Transcranial magnetic stimulation, transcranial direct current stimulation, and deep brain stimulation alleviated pain and improved motor function in patients with SCI; therefore, transcranial therapy may be a new strategy for the treatment of patients with SCI.

Integrated metatranscriptomics and metabolomics reveal microbial succession and flavor formation mechanisms during the spontaneous fermentation of Laotan Suancai.

Laotan Suancai, a Chinese traditional fermented vegetable, possesses a unique flavor that depends on the fermentative microbiota. However, the drivers of microbial succession and the correlation between flavor and active microbiota remain unclear. A total of 21 characteristic flavor metabolites were identified in Laotan Suancai by metabolomics, including 8 sulfides, 6 terpenes, 3 organic acids, 2 isothiocyanates, 1 ester, and 1 pyrazine. Metatranscriptome analysis revealed variations in the active microbiota at different stages of fermentation, and further analysis indicated that organic acids were the primary drivers of microbial succession. Additionally, we reconstructed the metabolic network responsible for the formation of characteristic flavor compounds and identified Companilactobacillus alimentarius, Weissella cibaria, Lactiplantibacillus plantarum, and Loigolactobacillus coryniformis as the core functional microbes involved in flavor development. This study contributed to profoundly understanding the relationship between the active microbiota and flavor quality formation, as well as the targeted selection of starters with flavor regulation abilities.