Stress response regulation to extracellular polymeric substances biosynthesis in Bacillus licheniformis

Stress response regulation to extracellular polymeric substances biosynthesis in Bacillus licheniformis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Stress response regulation to extracellular polymeric substances biosynthesis in Bacillus licheniformis Xiaoyu Wei, Ziwei Pan, Zhen Chen, Ning He This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7275147/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Oct, 2025 Read the published version in Microbial Cell Factories → Version 1 posted 9 You are reading this latest preprint version Abstract Background: The diverse metabolic mechanisms underlying bacterial extracellular polymeric substances give rise to a wide array of components with distinct functionalities, including exopolysaccharides (EPS) and poly-γ-glutamic acid (γ-PGA). The coordinated synthesis of various types of extracellular polymeric substances necessitates comprehensive investigation from a global regulatory perspective. Results: In this study, we examined the impact of multiple environmental stressors on Bacillus species, revealing that the EPS and γ-PGA produced respond to stress through metabolic and cellular process reorganization. The expression of global transcriptional regulators influenced the production of EPS and γ-PGA differently. Specifically, quorum sensing-related global regulators such as rsbRA , rapA , and the carbon utilization regulator ccpA -2 were found to enhance EPS synthesis. Conversely, positive global transcriptional regulators associated with γ-PGA synthesis included carbon and nitrogen utilization-related regulators ccpA -2, cggR , and nrgB . Notably, the global regulators nrgB and cggR increased γ-PGA production by 33.64% and 44.14%, respectively, while this enhancement was accompanied by a concomitant reduction in EPS production. In B. licheniformis , omics analyses have elucidated critical pathways and metabolites implicated in stress response mechanisms that induce alterations in amino acid metabolism, carbon source utilization, alongside the activation of global regulatory elements. These studies indicated that nrgB predominantly governs downstream genes associated with carbon metabolism, energy metabolism, signal transduction, and membrane transport processes. Conclusions: This work combines stress induction strategies and global transcription machinery engineering for investigating the coordinated synthesis of various types of extracellular polymeric substances, which has not been explored before. The insights gained from our research contribute to a deeper understanding of the regulatory networks governing the competition between γ-PGA and EPS, thereby providing a theoretical basis for the engineered modification of Bacillus licheniformis aimed at optimizing the production of extracellular polymeric substances. Bacillus licheniformis γ-PGA EPS Stress response Global transcriptional regulators Omics studies Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Background Extracellular polymeric substances are large molecular polymers that are synthesized and secreted by bacteria in response to the environmental stimuli, which have complex chemical components, encompassing polysaccharides, proteins, nucleic acids, among others [ 1 , 2 ]. Bacillus species are capable of concurrently synthesizing two kinds of extracellular polymeric substances. For instance Bacillus licheniformis CGMCC 2876 produces both EPS and γ-PGA [ 3 , 4 ], B. licheniformis WX-02 synthesizes lichenysin and γ-PGA [ 5 ], and B. amyloliquefaciens NK-1 generates levan and γ-PGA [ 6 ]. Recent research efforts have employed various strategies to elucidate the competitive relationships between different types of extracellular polymeric substances, such as the knockout of EPS operon epsA-O cluster[ 7 ] or pgsBCA [ 8 ], and the cell wall DL-endopeptidase cwlO [ 9 ] in B. amyloliquefaciens . However, investigations into the specific metabolic interactions and regulatory mechanisms governing the synthesis of γ-PGA and EPS from a global regulatory perspective remain limited, thereby impeding targeted metabolic engineering efforts. Additionally, while thermal treatment may enhance EPS yield, it can also lead to a reduction in molecular weight and intrinsic viscosity, resulting in altered antioxidant properties, as evidenced by studies on polysaccharides derived from Inonotus obliquus [ 15 ]. The bioprocess induced by abiotic stress represents a novel approach to augment the production of specific metabolites, with relevant stress conditions encompassing osmotic stress, heat shock, pH fluctuations, and oxidative stress [ 10 ]. Prior research has demonstrated that the production of γ-PGA and validamycin A can be enhanced by 133% and 27.43%, respectively, through the the alkaline pH shock [ 11 , 12 ]. Sandhya et al. reported that salt stress significantly affects the monomer composition of EPS [ 13 ]. Furthermore, EPS production by Rhodotorula sp. CAH2 exhibited a consistent increase with rising concentrations of NaCl [ 14 ]. Additionally, while thermal treatment may enhance EPS yield, it can also lead to a reduction in molecular weight and intrinsic viscosity, resulting in altered antioxidant properties, as evidenced by studies on EPS derived from Inonotus obliquus [ 15 ]. Subsequently, environmental stress signals are detected by sensor proteins, which initiate a signaling cascade that activates the sigma factor, thereby regulating the expression of transcription factor genes and suggesting that these regulators enhance bacterial fitness in response to stressors. Previous works have demonstrated that certain regulatory proteins, such as CcpA and CcpN for carbon metabolism, and NrgB for nitrogen metabolism, play a pivotal role in the stress response mechanisms involved in the synthesis of EPS and γ-PGA in B. licheniformis [ 16 ]. In B. subtilis , the expression of pgsBCA is regulated by the DegS-DegU two-component system and the ComP-ComA quorum sensing (QS) system, which activate pgsBCA transcription in response to environmental osmotic stress and developmental phase transitions [ 17 ]. Through the engineering of the PhrQ-RapQ-DegU QS system, the yield of γ-PGA was remarkably increased by 6.53-fold, achieving a balance between cell growth and high-efficiency γ-PGA production [ 18 ]. Nonetheless, the role of variations in extracellular polymeric substance composition in B. licheniformis in mitigating environmental stress, as well as the underlying metabolic and regulatory mechanisms, remains inadequately understood. This study investigates the regulatory effects of three types of physicochemical stresses—heat shock, osmotic stress, and pH stress—on the production of EPS and γ-PGA production in B. licheniformis . The analysis of intracellular metabolites was conducted to explore potential mechanisms under combined stress conditions that enhance biosynthetic capabilities. Additionally, the research examines the influence of stress-responsive transcriptional regulators on the components of extracellular polymeric substances. The objective of this study is to dissect the competitive interactions by integrating environmental perturbations with global transcriptional engineering, elucidate the underlying molecular mechanisms, and ultimately inform the metabolic engineering of B. licheniformis for the efficient and targeted production of γ-PGA and EPS. 2. Methods 2.1 Plasmids, strains, and culture conditions B. licheniformis CGMCC 2876 was used as the host for extracellular polymeric substances and served as the wild-type strain. Escherichia coli DH5α was employed for plasmid construction and cultured in LB broth at 37°C with 10 mg/L tetracycline as needed. The plasmid pHY300PLK-PamyL-TtamyL was applied for constructing gene overexpression in B. licheniformis CGMCC 2876 (Table S1 and S2). For nrgB gene overexpression, the gene was amplified from B. licheniformis CGMCC 2876 by corresponding primers (Table S3), inserted into the plasmid using ClonExpress One Step Cloning Kit (Vazyme Biotech Co., Ltd., Nanjing, China), resulting in pHY- nrgB . This plasmid was then transformed into DH5α and selected on 10mg/L tetracycline plates, with transformants verified by PCR. This method was also used to construct other regulator expression vectors, which were transferred into B. licheniformis via electroporation (25 kV/cm, 4 ms), resulting in overexpression strain OE- nrgB . The OE- nrgB strain was cultured in LB medium with 10 mg/L tetracycline at 37°C, 220rpm. The scale-up fermentation process was performed within a six-parallel bioreactor system. The compositions and culture conditions of seed and EPS fermentation medium for γ-PGA production were the same as in Wei's previous work [ 19 ]. To determine the effects of independent and multiple stresses on extracellular polymeric substance production, three conditions of osmotic pressure, pH stress, and temperature stress were selected. For osmotic stress, five levels of NaCl (0, 2, 4, 6, and 8 g/L) were added to the EPS fermentation medium at the beginning of cultivation. For pH stress, the medium initial pH was adjusted to 3.0, 5.0, 7.2, 9.0, and 11.0 by the addition of HCl (2 mol/L) or NaOH (2 mol/L). For temperature stress, the strains were inoculated into EPS medium by stimulation with 0, 37, 50, 55, 60, and 65°C for 30min in a water bath, then quickly cooled to 37°C. For combined stress treatment, different stress conditions were applied simultaneously as follows, based on the optimized stress conditions. 2.2 Analysis Methods After the fermentation process, the crude yields of extracellular polymeric substances and the biomass was extracted and purified, based on our previous research [ 20 ]. The yields of γ-PGA were detected by high-performance liquid chromatography (HPLC) with a C18 column (ZORBAX SB-C18). The content of total EPS in the biopolymer was detected using the phenol–sulfuric acid method. The cell density ­ (OD 600 ) was measured using a UV-1800 spectrophotometer (Shimadzu Global Laboratory Consumables Co., Ltd., Shanghai, China). High-performance gel permeation chromatography (HPGPC) was employed to analyze the molecular weight (Mw) of γ-PGA. 2.3 Quantitative realtime PCR Strains were collected after 24 h of incubation for RNA extraction and gene expression analysis. Total RNA was extracted using a Bacterial RNA Kit (Omega Bio-Tek, Guangzhou, China) and quantified by a NanoDrop 2000 (Thermo, CA, USA). First-strand cDNA was synthesized using HiScript III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme, Nanjing, China), and qRT-PCR was performed with a qTOWER3 instrument (Analytik Jena AG, Jena, Germany) using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). For gene transcriptional levels, a SYBR® Premix Ex Taq™ II (RR820A, Takara) and a Bio-Rad iQ5 real-time PCR system were used following the manufacturer’s instructions. Relative quantification relates the PCR signal of the target transcript in a sample to control based on the 2 −ΔΔCt method [ 21 ]. The 16S rDNA served as the reference. Primers used for related genes are shown in Table S4. 2.4 LC-MS performance and metabolomics data analysis Pathway metabolites analysis was performed by liquid chromatography- mass spectrometer (LC-MS AB Sciex TripleTOF 5600 +). The sample pretreatment and metabonomics data analysis methods were as described previously [ 19 ]. In brief, when the cells were grown for 24 h, the culture medium was removed by centrifugation. Two groups of samples, each with three replicates, including the non-stressed fermentation control group (Control) and the combined-stress fermentation experimental group (Stress), were tested for metabolites in both positive and negative ion modes. The cell suspension was washed three times with 10 mM PBS. Cells were resuspended in 1 mL PBS and centrifuged at 4°C. Cells were precipitated in liquid nitrogen for 1 min. Then, 1 mL of MeOH: ACN: H2O (V: V: V, 2:2:1) was vortexed for 30 s for the following 10 min sonication (4°C water bath). The samples were placed in liquid nitrogen for 1 min. This procedure was repeated 3 times to burst the cells in total. Then, the samples were incubated at − 20°C for 1 h to facilitate protein precipitation. Finally, the samples were freeze-centrifuged (1300 rpm, 15 min), and the supernatant was collected for freeze-drying by an FDU-1200 freeze-drying machine. Three replicates were used in this experiment. Metabolomics data have been deposited in the iProX database (www.iprox cn) with accession number IPX0006376000. After processing the data through convolution, internal standard removal, and false-positive elimination, the obtained metabolite information was aligned for subsequent metabolite clustering, screening and identification of differential metabolites, and related bioinformatics analysis. 2.5 Transcriptomic analysis Strains were also primarily inoculated in seed medium and cultured for 20 h to the late logarithmic growth (OD ~ 2), then transferred to EPS medium and further incubated for 24 h. The cells were collected by centrifugation at 8000 rpm for 10 min at 4 ◦C for total RNA purification and cDNA library construction. Sequencing was performed using the Illumina HiSeqTM 2500 platform with paired-end 150 base pair reads by Gene Denovo Biotechnology Co. Raw data were filtered before the quality-trimmed reads were mapped to the genome of B. licheniformis CGMCC 2876 using Bowtie2 [ 22 ]. The gene expression level was analyzed according to FPKM (fragments per kilobase of transcript per million). The edgeR package ( http://www.r-project.org/ ) was used to identify DEGs with fold changes (FC) ≥ 2 and FDR < 0.05. 2.6 Statistical Analyses All the fermentation experiments were repeated at least three times. Data were presented as the mean ± standard deviation for each sample point. All data were collected to analyze the variance at p < 0.05, and the mean values were compared by applying a T-test. 3. Results 3.1 Effect of multiple stresses on extracellular polymeric substances production Environmental stresses effectively enhance target product synthesis. B. licheniformis CGMCC 2876 was fermented under various stress conditions. Adding 16 g/L NaCl increased extracellular polymeric substances yield to 14.53 g/L, a 25.86% rise from the control (11.55 g/L) (Fig. 1 A). Moreover, EPS yield decreased as NaCl concentration increased. Conversely, γ-PGA content rose with higher NaCl levels, reaching 5.22 g/L at 16 g/L NaCl, a 54.33% increase, suggesting γ-PGA production is a stress response to hyperosmotic conditions (Fig. 1 B). Under strong cellular dehydration pressure caused by hyperosmotic stress, γ-PGA, with its high water-absorbing capacity, may help maintain cellular water activity and enhance cell survival [ 23 ]. This indicates that high NaCl-induced osmotic stress boosts γ-PGA synthesis while inhibiting EPS production. Under extreme alkaline stress (pH = 11), B. licheniformis CGMCC 2876 produced 6.57 g/L of extracellular polymeric substances, a 23.73% decrease from the control (8.61 g/L at pH 7.2). At pH = 9, biomass doubled to 1.21 g/L compared to the control (0.63 g/L) (Fig. 1 C) and γ-PGA production rose by 20.88% to 4.68 g/L. EPS production increased to 340 mg/L at pH 9 and 570 mg/L at pH 11. Under acidic conditions (pH = 3 and pH = 5), extracellular polymeric substances decreased to 7.05 g/L and 6.7 g/L, while EPS yields increased by 22.22% and 89.98% to 340 mg/L and 520 mg/L, respectively (Fig. 1 D). γ-PGA yields dropped by 64.44% and 7% under acidic conditions, showing that acidity hinders γ-PGA synthesis. These findings demonstrate that strong alkaline stress increased the yield of γ-PGA, whereas EPS synthesis was more responsive to strong acidic stress. The yield of extracellular polymers showed no significant difference with increasing fermentation temperature, maintaining a stable output of 8.1 g/L (Fig. 1 E). And the EPS production at 55°C reached 380 mg/L, a 49.26% increase over the 37°C condition (0.26 g/L). Similarly, EPS yield rose to 340 mg/L at 65°C (35.6% higher than at 37°C), demonstrating that elevated EPS synthesis is a stress response to thermal challenge. However, beyond 55°C, γ-PGA synthesis declined sharply, dropping to 1.41 g/L at 65°C (Fig. 1 F). These results demonstrate that high-temperature stress at 55°C promoted EPS synthesis, but excessively high temperatures were detrimental to γ-PGA synthesis. Based on the above results, alkaline stress (pH = 9) and hyperosmotic stress (16 g/L NaCl) boost γ-PGA production but reduce EPS synthesis, while high temperature (55°C) has the opposite effect. Under the three stresses, biomass increased from 0.36 g/L to 1.46 g/L, indicating enhanced bacterial growth and extracellular polymer production (Fig. 1 G). EPS production decreased by 12.77% to 200.92 mg/L, while γ-PGA production increased by 18.96% to 3.79 g/L compared to the control (Fig. 1 H). Notably, the Mw of γ-PGA at pH 9 rose by 5.96% to 4.84×10⁵ kDa (Fig. 1 I). This phenomenon may be attributed to H⁺ and OH⁻ ions altering the ionic balance across cell membranes, thereby modifying the transmembrane potential difference and affecting molecular synthesis and transport [ 24 ]. Alkaline stress can lead to a reduction in extracellular enzyme activity, thereby limiting the degradation of extracellular macromolecules and consequently influencing the degree of γ-PGA polymerization. 3.2 Metabolite profile analysis of multiple stresses To elucidate the mechanisms underlying the effects of the three stresses on the metabolic level of B. licheniformis , untargeted metabolomics was employed to analyze changes in intracellular metabolites under these conditions (Table S5). A distinct differentiation is evident between stressed and non-stressed intracellular metabolite samples as demonstrated by PCA and OPLS-DA analyses (Figure S1). To evaluate the accuracy of the models, a 200-response permutation test was performed, which confirmed that the OPLS-DA models, in both ionization modes, exhibit robust discriminative and predictive capabilities. The OPLS-DA model was utilized to screen and identify differential metabolites, employing Variable Importance in the Projection (VIP) and Fold Change (FC) as selection criteria (Figure S2). Based on log 2 FC > 1 or <-1, were identified in positive ion mode, including 11 upregulated metabolites, such as notably coproporphyrin, cyclic GMP, ATP, citrulline, glycerol 3-phosphate, glutamine, and 13 significantly downregulated metabolites, notably hypoxanthine, acetoin, isobutyrate, dimethylglycine, DL-2-aminobutyric acid (Fig. 2 A). In negative ion mode, 97 metabolites were detected, comprising 17 upregulated metabolites, including proline, FAD, pentadecanoic acid, flavin mononucleotide, citrulline, glutamine, phosphoenolpyruvate, and 12 significantly downregulated metabolites, primarily cytidine monophosphate, phthalic acid (Fig. 2 C). Seventeen differential metabolites were identified and visualized in a heatmap by employing a combination of screening criteria: log 2 FC > 1 or 1, and p < 0.05 (Figure S2). In positive ion mode, metabolites such as proline, FAD, citrulline, glutamine, and glycerol 3-phosphate were upregulated, whereas N-acetylglucosamine, N-acetylmannosamine, and N-acetylgalactosamine were downregulated (Fig. 2 B). Conversely, in the negative ion mode, upregulated metabolites included cyclic GMP and citrulline, while 2-aminoisobutyric acid was downregulated (Fig. 2 D). Under stress conditions, glycerol 3-phosphate and phosphoenolpyruvate exhibited upregulation by 3.1-fold and 2.6-fold, respectively, thereby providing additional precursor substances for carbon source utilization within the tricarboxylic acid (TCA) cycle (Fig. 2 E). Within the γ-PGA biosynthetic pathway, both glutamate and glutamine were upregulated by 2.35-fold and 3.28-fold, respectively. Notably, glutamine production slightly surpassed that of glutamate, likely due to the utilization of a portion of glutamate in the downstream porphyrin metabolism pathway for the synthesis of coproporphyrin I. Similarly, the production of 2-aminoisobutyric acid within the downstream pyrimidine metabolism of glutamine exhibited a 0.44-fold decrease, suggesting that elevated levels of glutamine promote glutamate synthesis. I Within amino acid metabolic pathways, intracellular concentrations of proline and citrulline increased significantly, by 14.27-fold and 4.55-fold, respectively. As osmotic regulators, citrulline and glutamine have the capacity to interconvert, while proline can be converted into glutamate, thereby supplying precursor materials for γ-PGA synthesis. Conversely, within the EPS synthesis pathway, the production of N-acetylglucosamine, N-acetylmannosamine, and N-acetylgalactosamine was downregulated, resulting in a reduction of EPS synthesis unit pathways and accompanied by decreased expression of the glycosyltransferase epsG. Collectively, these findings indicate that stress conditions stimulate metabolic pathways associated with γ-PGA synthesis while concurrently attenuating EPS metabolic pathways, thereby highlighting a competitive relationship between EPS and γ-PGA biosynthesis. 3.3 Increasing global regulators related to quorum sensing expression promotes extracellular polymeric substances synthesis Environmental stresses approaches can occasionally disrupt the equilibrium of carbon/nitrogen metabolism and cofactor networks within engineered strains [ 25 ]. To mitigate these issues, global transcription machinery engineering has emerged as a promising strategy [ 26 ]. Notably, eight global transcriptional regulators were significantly upregulated under combined stress conditions (Figure S3). Extracellular polymeric substances constitute a major component of Bacillus biofilms and are also modulated by quorum sensing (QS) [ 27 ]. In the quorum sensing-related global regulator rsbRA mutant strain, EPS production reached 8.76 g/L, representing a 5.12% increase (Fig. 3 A). The proportion of EPS rose to 8.34%, with production levels attaining 730.58 mg/L, marking a twofold increase, while the proportion of γ-polyglutamic acid (γ-PGA) also increased to 37.12%. Similarly, in the quorum sensing-related global regulator rapA mutant strain, EPS and γ-PGA production increased by 43.89% (518 mg/L) and 31.47% (2.77 g/L), respectively. Conversely, in the quorum sensing-related global regulator codY mutant strain, the EPS production decreased by 19.72% and γ-PGA decreased by 20.87%. The quorum sensing-related global regulator nadR mutant strain showed no significant increase in the proportions of EPS and γ-PGA, with total extracellular polymeric substances production slightly decreasing to 7.67 g/L. These results indicate that rsbRA and rapA regulators promote EPS and γ-PGA synthesis, while nadR and codY serve as negative regulators for EPS and γ-PGA production. A protein-protein interaction (PPI) network centered on rsbRA and rapA was constructed utilizing the STRING database and differential gene expression analysis was conducted via qRT-PCR (Fig. 3 B-E). Among the six downstream genes regulated by rsbRA , the expression levels of phosphoserine phosphatase rsbU , serine protein kinase rsbT , and antagonist protein rsbS , all of which are involved in serine metabolism regulation, were found to be downregulated. Conversely, the expression of fliG , which encodes flagellar rotation protein was upregulated and may interact with epsE to inhibit flagellar movement [ 28 ]. The expression of sigB increased 3.45-fold, suggesting that rsbRA enhances signal transduction to sigB , thereby upregulating its transcription. SigB is a well-established regulator of stress response [ 29 ]. Among the critical EPS biosynthetic genes, the expression of epsG , which encodes a glycosyltransferase involved in polysaccharide unit transport, increased by 1.5-fold. This finding indicates an activation of glycosyltransferases within the EPS synthesis pathway. The observed increase in EPS content is likely attributable to enhanced rsbRA -mediated signaling, which upregulates sigB transcription. This, in turn, modulates the transcription of eps genes, thereby promoting EPS biosynthesis. Moreover, in the mutant strain of the global regulator rapA , the competence-damage inducible protein cinA exhibited downregulation, whereas other genes, such as RNA polymerase sigB , sporulation initiation phosphotransferase spo0F , and anti-sigma F factor spoIIAB were upregulated. Genes associated with nucleotide sugar precursor synthesis pathways ( glmU , tuaD , gtaB ) demonstrated varying levels of upregulation, as did genes related to EPS transport and polymerization ( epsH , epsO ). These differential gene expressions collectively contribute to the facilitation of EPS biosynthesis. 3.4 Increasing global regulators related to carbon and nitrogen utilization expression promotes extracellular polymeric substances synthesis The metabolic engineering of carbon and nitrogen pathways is crucial for enhancing the production of various bioproducts [ 30 ]. The global regulator cggR , associated with carbon utilization, was found to increase γ-PGA production by 20.56%, achieving a concentration of 3.87 g/L. This suggests that cggR redirects metabolic flux towards γ-PGA synthesis, while reducing EPS production by 4.72% (Fig. 4 A). In a similar vein, the global regulator nrgB , related to nitrogen utilization, elevated the γ-PGA yield to 4.29 g/L, representing a 33.64% increase and constituting 53.17% of total extracellular polymeric substances. Conversely, EPS yield decreased to 270.47 mg/L, with its proportion diminishing to 3.35%. Furthermore, the ccpA -2 mutant strain, another carbon utilization-related global regulator, produced 3.83 g/L of γ-PGA, marking a 22.42% increase, while EPS production rose to 680.33 mg/L, 1.8 times higher than the wild-type. In contrast, the tnrA mutant strain, associated with nitrogen utilization, exhibited reductions in γ-PGA and EPS content by 40.78% and 47.5%, respectively. These findings underscore that the global regulators cggR and nrgB positively influence γ-PGA synthesis, while negatively impacting EPS production. Protein interaction and qRT-PCR analysis showed that genes regulated by cggR within the glycolysis pathway were generally upregulated (Fig. 4 B and 4 C). Notably, the glyceraldehyde-3-phosphate dehydrogenase-encoding gene, gapA , was downregulated, whereas other genes, including the triose-phosphate isomerase-encoding gene tpiA , the phosphoglycerate kinase-encoding gene pgk , and the enolase-encoding gene eno , exhibited upregulation. The gapA gene is co-transcribed with the downstream genes pgk , tpiA , and eno , as well as with the upstream gene cggR . The observed decrease in gapA expression may be attributed to the inhibitory effect of cggR [ 31 ]. Among the cggR -regulated genes involved in glycolysis, the phosphotransferase gene crr and phosphoglucose isomerase gene pgi were found to be upregulated. The carbon flux diverges at fructose-6-phosphate within glycolysis, leading to downregulation of the glucosamine-1-phosphate acetyltransferase gene glmU in the EPS synthesis pathway. Conversely, in the γ-PGA synthesis pathway via the TCA cycle, pyruvate kinase gene pyk and pyruvate dehydrogenase gene pdhD are upregulated in the OE-cggR strain, suggesting a shift in metabolic flux towards γ-PGA synthesis. The scale-up fermentation process was performed in bioreactors utilizing both the wild-type strain and the global regulator cggR mutant strain within a six-parallel bioreactor system. The wild-type strain functioned as the control (Figure S4). The cggR mutant strain demonstrated a brief adaptation phase, subsequently entering the logarithmic growth phase rapidly, which was characterized by a significant decrease in dissolved oxygen (DO) levels, reaching 30% by 8 hours (Fig. 4 D). To sustain DO at 30%, the agitation speed was dynamically modulated. The biomass growth curve indicated vigorous cell proliferation commencing at 4 hours, with cell density peaking at 6.24 (OD 600 ) by 12 hours, signifying the transition to the stationary phase. Glucose consumption was rapid, approaching depletion by 12 hours, which reflects robust metabolic activity and efficient nutrient utilization. Notably, product accumulation increased significantly after 24 hours (Fig. 4 E). By 50 hours, the OE- cggR strain produced 384.5 mg/L of EPS, while the γ-PGA yield reached 3.98 g/L, representing a 16.4% increase. 3.5 Transcriptomic analysis of the global regulator nrgB Transcriptomic analysis was employed to elucidate the mechanisms by which the nitrogen utilization-related global regulator nrgB positively influences γ-PGA synthesis while negatively regulating EPS production. Utilizing thresholds of log 2 FC greater than 1 for upregulation and less than − 1 for downregulation, alongside a false discovery rate (FDR) of ≤ 0.05 as screening criteria, a total of 52 significantly differentially expressed genes were identified, comprising 29 upregulated and 23 downregulated genes (Figure S5 and Table S6). The Gene Ontology (GO) functional distribution of these differentially expressed genes indicated that they were primarily enriched in cellular metabolic processes, cellular physiological processes, and single-organism processes within the biological processes category (Fig. 5 A). In terms of molecular functions, the genes were predominantly associated with binding and catalytic activities, while cellular components were largely localized to membranes and membrane parts. Furthermore, the KEGG pathway classification of the screened differential genes was also analyzed (Fig. 5 B). Most metabolic pathways showed significant upregulation, particularly in metabolism, with the global and overview maps leading with 8 differential genes. Carbohydrate metabolism followed with 7 genes, while energy and lipid metabolism each had 2, suggesting that nrgB gene overexpression boosts energy metabolism in B. licheniformis . Three pathways related to cellular processes, including cellular community-prokaryotes, cell motility, and cell growth and death, were enriched, with cellular community showing 7 differential genes, indicating changes in cellular communities due to nrgB overexpression. Additionally, environmental information processing pathways were enriched, with signal transduction having 7 differential genes and membrane transport 2, highlighting enhanced signal transduction and membrane transport activities. The study identified 33 differential genes between the wild-type and nrgB mutant strains (Fig. 6 A). In amino and nucleotide sugar metabolism, the expression of wbpA , which includes wbpA 1 and wbpA 2 encoding dehydrogenases for UDP-N-acetylglucosamine and UDP-N-acetylmannosamine, was downregulated. These enzymes produce essential acids for polysaccharide synthesis. Additionally, five genes related to starch and sucrose metabolic pathways and secondary metabolite pathways— glgA (starch synthase), glgB (glycogen branching enzyme), glgC (glucose-1-phosphate adenylyltransferase), glgD (glycogen biosynthesis protein), and glgP (glycogen phosphorylase)—also showed reduced expression. In the glycan pathway, glgA is converted by glgB into essential branched glucans [ 32 ]. Furthermore, in glycan biosynthesis, the N-acetylglucosaminidase lytD , part of peptidoglycan hydrolases, is upregulated, crucial for early bacterial growth and division, suggesting enhanced glycan degradation [ 33 ] Thus, nrgB suppresses EPS synthesis genes while boosting glycan degradation genes. The differential genes were further mapped to the major metabolic pathways (Fig. 6 B). In the glycolytic pathway, the expression of hxlB , encoding 6-phosphohexulose isomerase, was downregulated. While, in the branched pathway of lipid metabolism within the pentose phosphate pathway, egsA , encoding glycerol dehydrogenase, showed the most significant transcriptional upregulation, converting dihydroxyacetone to glycerol-1-phosphate via egsA , indicating that nrgB overexpression stimulates the production of other extracellular polymer components. In amino acid metabolism, fadA , encoding an acetyl-CoA acyltransferase family protein, was markedly downregulated. As a transferase involved in the degradation pathways of valine and other amino acids, which facilitates the conversion of methyl acetyl-CoA to acetyl-CoA, the suppression of fadA indicates a diminished supply of acetyl-CoA for the tricarboxylic acid (TCA) cycle. In the context of nitrogen metabolism pathways, the gene narI , which encodes the gamma subunit of nitrate reductase, was significantly upregulated. Nitrate reductase NarI catalyzes the conversion of nitrite to ammonia, subsequently facilitating the synthesis of glutamine for glutamate production, thereby providing precursors for γ-polyglutamic acid (γ-PGA) synthesis. Bacterial chemotaxis influences the preference for a planktonic state, and motility status impacts biofilm formation through extracellular polymers [ 34 ]. The upregulation of the methyl-accepting chemotaxis protein mcpA modulates signal transduction via the chemotaxis response regulators cheA and cheY , leading to the upregulation of motB , which encodes another chemotaxis protein involved in bacterial flagellar synthesis. This indicates that the overexpression of nrgB enhances bacterial chemotactic motility and flagellar biosynthesis. 4. Discussion Most γ-PGA-producing strains are glutamate-dependent, including B. subtilis C10, B. subtilis chungkookjang, B. licheniformis ATCC 9945a, and B. licheniformis WX-02. In contrast, relatively few strains are glutamate-independent, such as B. subtilis TAM-4, B. licheniformis A35, and B. amyloliquefaciens LL3 [ 23 ]. B. licheniformis CGMCC 2876, a facultative glutamate-dependent γ-PGA producer, is capable of synthesizing γ-PGA both with exogenous glutamate supplementation and through de novo synthesis without glutamate. Our study demonstrated that B. licheniformis CGMCC 2876 cultivated in the presence of 16 g/L NaCl produced 5.22 g/L γ-PGA, marking a 54.33% increase, although a slight reduction in the molecular weight (Mw) of γ-PGA was observed. Similar findings have been reported for halotolerant B. licheniformis WX-02, where salt-induced γ-PGA production achieved a maximal yield of 13.86 g/L under 8% NaCl, despite decreased in Mw with increasing salinity [ 35 ]. Furthermore, NaCl concentration has been shown to modulate the Mw of γ-PGA in B. licheniformis 9945a, B. subtilis natto, B. megaterium WH320, and B. subtilis chungkookjang [ 36 ]. This correlation suggests that γ-PGA serves a protective function for cells under stress conditions, with cells synthesizing higher Mw γ-PGA in response to elevated concentrations of osmotic-disrupting toxic compounds [ 37 ]. While most γ-PGA-producing organisms demonstrate optimal growth at pH 7, some studies have reported that optimal γ-PGA biosynthesis occurs at pH 6.5, coinciding with peak uptake of precursors such as glutamate and citrate [ 38 ]. Under acidic stress conditions (pH 5), there is an 89.98% increase in EPS production, suggesting an acid-stress response mechanism where increased membrane permeability enhances glucose uptake, thereby augmenting the supply and transport of EPS precursors [ 39 ]. Similarly, Alteromonas australica QD synthesizes high-molecular-weight EPS at acidic pH levels as an adaptation to pH stress [ 40 ]. In contrast, under alkaline stress (pH 9), the γ-PGA production increased 20.88%, likely due to the ionization of -COOH groups at elevated pH, which extends γ-PGA chains through enhanced water-polymer interactions, thereby increasing solution viscosity [ 41 ]. Conversely, mildly acidic pH conditions favors the biosynthesis of more free γ-PGA [ 38 ]. Notably, among the metabolites responsive to alkaline pH, hyperosmolarity, and elevated temperature stressors, proline content exhibited the most significant increase, functioning as both an osmolyte and a protective agent in B. subtilis [ 42 ]. In response to osmotic adaptation pathways, B. subtilis enhances its intracellular proline concentration from 20 mM to 500 mM [ 43 ]. We hypothesize that hyperosmotic stress stimulates proline biosynthesis, which subsequently converts to glutamate via arginine, thereby augmenting the supply of γ-PGA precursors. Additionally, there was a notable increase in citrulline accumulation, which may facilitate the conversion of glutamate. Nevertheless, excessive accumulation of citrulline under hyperosmotic conditions could potentially disrupt the cytoplasmic ion balance [ 44 ]. Citrulline serves as a non-proteinogenic amino acid that protects plants under drought stress [ 45 , 46 ] and serves as a novel osmotic protectant in B. subtilis [ 47 ]. Among the regulators promoting EPS and γ-PGA production, the quorum-sensing global regulator rsbRA increased the EPS proportion to 8.34%, with production reaching 730.58 mg/L, representing a two-fold increase. A similar regulatory mechanism is observed in Xanthomonads , where xanthan gum is produced as an exopolysaccharide during the growth phase, and this phase influences protein phosphorylation, specifically the phosphorylation of rsbRA during the transition from the late exponential to the stationary phase [ 48 ]. As a component of the stressosome, rsbRA facilitates responses to environmental stress and starvation signals in B. subtilis by transmitting stress signals to sigB, which is crucial for transcriptional activation necessary for bacterial survival [ 49 ]. Additionally, the quorum-sensing global regulator rapA facilitates the transfer of phosphate groups to the transcriptional regulator Spo0A via the phosphotransfer proteins Spo0F and Spo0B [ 50 ]. The phosphorylated Spo0A ~ P subsequently regulates the transcription of over 500 genes in Bacillus species, including those involved in biofilm formation, sporulation, and secondary metabolite biosynthesis [ 51 ]. The global regulator CcpA recognizes and binds to numerous target genes across various metabolic pathways, modulating glucose metabolic flux by regulating key genes in the pentose phosphate pathway while repressing genes involved in acetate metabolism [ 52 ]. Nonetheless, the quorum-sensing global regulator s, nadR and codY , along with the nitrogen utilization-related global regulator tnrA , are function as negative regulators of EPS and γ-PGA production. CodY exerts its repressive influence on lipopeptide synthesis by directly binding to the srfA promoter, thereby inhibiting its expression [ 53 ]. NadR is capable of repressing key NAD biosynthetic genes, including nadB , nadA - pnuC , and pncB , while simultaneously modulating cellular NAD levels through pnuC-mediated transport [ 54 ]. In B. subtilis , tnrA activates nitrogen reductase genes within the nar and nas operons under conditions of nitrogen limitation [ 55 ]. Among these global regulators, nrgB and cggR demonstrated the most significant differential effects on EPS and γ-PGA production. The central glycolytic gene repressor, cggR , is a member of SorC family of bacterial transcriptional regulators and governs genes and operons involved in carbohydrate catabolism. It acts as a transcriptional repressor of the gapA operon, which encodes central glycolytic enzymes [ 56 , 57 ]. The downstream gene expression changes regulated by nrgB include the downregulation of wbpA (nucleotide sugar dehydrogenase) and the upregulation of lytD (peptidoglycan hydrolase), collectively leading to the suppression of EPS synthesis. 5. Conclusion This study demonstrates that environmental stresses (pH 9 and 16 g/L NaCl) significantly increase γ-PGA production in B. licheniformis while suppressing EPS synthesis. B. licheniformis responds to these stresses by activating precursor synthesis, carbon source utilization, and amino acid metabolism. Key global regulators including rsbRA , rapA , enhance EPS synthesis, whereas ccpA -2 have positive effects on the improvement of both polymers. Notably, cggR and nrgB have pronounced differential effects on EPS and γ-PGA production. Specifically, nrgB primarily regulates genes related to carbon metabolism, energy metabolism, signal transduction, and membrane transport. This study posits that identifying key targets of global transcriptional regulators is essential for improving the production of two types of extracellular polymeric substances in engineered microorganisms. Further research is necessary to achieve comprehensive, large-scale identification of advantageous gene targets involved in the production process of these extracellular polymeric substances. Abbreviations Exopolysaccharides EPS poly-γ-glutamic acid γ-PGA Declarations Ethical approval and consent to participate Not applicable. Consent for publication All authors have given their consent for the publication of this research paper and its accompanying materials. Competing interests The authors declare no competing interests. Supplementary information Supplementary data associated with this article can be found in the online version of the paper. Funding This work was financially supported by the National Natural Science Foundation of China (22208276 and 32170061), the Natural Science Foundation of Xiamen (3502Z202572042), and the Third Institute of Oceanography, Ministry of Natural Resources, Basic Scientific Research Operating Expenses Project (2025016). Author Contribution Xiaoyu Wei: methodology, investigation, formal analysis, the original draft. Zhen Chen, Ning He: data curation, project administration. Xiaoyu Wei, Ziwei Pan, Zhen Chen and Ning He :Writing—review & editing. All authors read and approved the final version of the submitted manuscript. Acknowledgement The authors are grateful for funding support by Natural Science Foundation of Xiamen and the National Natural Science Foundation of China. Data Availability All the data of this study contained in the manuscript and further data can be obtained related to this study from Corresponding authors. References Wei XY, Chen Z, Liu AL, Yang LJ, Xu YY, Cao MF, He N. Advanced strategies for metabolic engineering of Bacillus to produce extracellular polymeric substances. Biotechnol Adv. 2023;67:251–62. Flemming HC, van Hullebusch ED, Little BJ, Neu TR, Nielsen PH, Seviour T, Stoodley P, Wingender J, Wuertz S. Microbial extracellular polymeric substances in the environment, technology and medicine. Nat Rev Microbiol. 2024;23:87–105. Wei XY, Yang LJ, Chen Z, Xia WH, Chen YB, Cao MF, He N. Molecular weight control of poly-γ-glutamic acid reveals novel insights into extracellular polymeric substance synthesis in Bacillus licheniformis . Biotechnol Biofuels Bioprod. 2024;17:60. Chen Z, Liu P, Li Z, Yu W, Wang Z, Yao H, Wang Y, Li Q, Deng X, He N. Identification of key genes involved in polysaccharide bioflocculant synthesis in Bacillus licheniformis . Biotechnol Bioeng. 2017;114:645–55. Hu SY, He PH, Zhang YJ, Jiang M, Wang Q, Yang SH, Chen SW. Transcription factor DegU-mediated multi-pathway regulation on lichenysin biosynthesis in Bacillus licheniformis. Metab Eng. 2022;74:108–20. Feng J, Quan Y, Gu Y, Liu F, Huang X, Shen H, Dang Y, Cao M, Gao W, Lu X, et al. Enhancing poly-gamma-glutamic acid production in Bacillus amyloliquefaciens by introducing the glutamate synthesis features from Corynebacterium glutamicum. Microb Cell Fact. 2017;16:88. Feng J, Gu Y, Quan Y, Cao M, Gao W, Zhang W, Wang S, Yang C, Song C. Improved poly-gamma-glutamic acid production in Bacillus amyloliquefaciens by modular pathway engineering. Metab Eng. 2015;32:106–15. Feng J, Gu YY, Quan YF, Zhang W, Cao MF, Gao WX, Song CJ, Yang C, Wang SF. Recruiting a new strategy to improve levan production in Bacillus amyloliquefaciens. Sci Rep 2015, 5. Qiu YB, Zhang YT, Zhu YF, Sha YY, Xu ZQ, Feng XH, Li S, Xu H. Improving poly-(gamma-glutamic acid) production from a glutamic acid-independent strain from inulin substrate by consolidated bioprocessing. Bioprocess Biosyst Eng. 2019;42:1711–20. Wei X, Tian G, Ji Z, Chen S. A new strategy for enhancement of poly-γ-glutamic acid production by multiple physicochemical stresses in Bacillus licheniformis . J Chem Technol Biotechnol. 2015;90:709–13. Wei XT, Tian GM, Ji ZX, Chen SW. A new strategy for enhancement of poly-γ-glutamic acid production by multiple physicochemical stresses in Bacillus licheniformis. J Chem Technol Biotechnol. 2015;90:709–13. Jiang J, Sun YF, Tang X, He CN, Shao YL, Tang YJ, Zhou WW. Alkaline pH shock enhanced production of validamycin A in fermentation of Streptomyces hygroscopicus. Bioresour Technol. 2018;249:234–40. Sandhya V, Ali SZ. The production of exopolysaccharide by Pseudomonas putida GAP-P45 under various abiotic stress conditions and its role in soil aggregation. Microbiology. 2015;84:512–9. Silambarasan S, Logeswari P, Cornejo P, Kannan VR. Evaluation of the production of exopolysaccharide by plant growth promoting yeast Rhodotorula sp. strain CAH2 under abiotic stress conditions. Int J Biol Macromol. 2019;121:55–62. Nachtigall C, Rohm H, Jaros D. Degradation of Exopolysaccharides from Lactic Acid Bacteria by Thermal, Chemical, Enzymatic and Ultrasound Stresses. Foods 2021, 10. Yu W, Chen Z, Shen L, Wang Y, Li Q, Yan S, Zhong CJ, He N. Proteomic profiling of Bacillus licheniformis reveals a stress response mechanism in the synthesis of extracellular polymeric flocculants. Biotechnol Bioeng. 2016;113:797–806. Hsueh YH, Huang KY, Kunene SC, Lee TY. Poly-γ-glutamic acid synthesis, gene regulation, phylogenetic relationships, and role in fermentation. Int J Mol Sci. 2017;18:2644. Hu LX, Zhao M, Hu WS, Zhou MJ, Huang JB, Huang XL, Gao XL, Luo YN, Li C, Liu K, et al. Poly-γ-glutamic acid production by engineering a DegU quorum-sensing circuit in Bacillus subtilis . ACS Synth Biol. 2022;11:4156–70. Wei XY, Yang LJ, Wang HY, Chen Z, Xu YY, Weng Y, Cao MF, Li QB, He N. Genomic and metabolomic analysis of Bacillus licheniformis with enhanced poly-gamma-glutamic acid production through atmospheric and room temperature plasma mutagenesis. Front Chem Sci Eng. Xu YY, Yang LJ, Wang HY, Wei XY, Shi YY, Liang DF, Cao MF, He N. Putative functions of EpsK in teichuronic acid synthesis and phosphate starvation in Bacillus licheniformis . Synth Syst Biotechnol. 2022;7:815–23. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C) method. Methods. 2001;25:402–8. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9:357–U354. Li DF, Hou LZ, Gao YX, Tian ZL, Fan B, Wang FZ, Li SY. Recent advances in microbial synthesis of poly-gamma-glutamic acid: A review. Foods 2022, 11. Guan NZ, Liu L. Microbial response to acid stress: mechanisms and applications. Appl Microbiol Biotechnol. 2020;104:51–65. Cao HJ, Villatoro-Hernandez J, Weme RDO, Frenzel E, Kuipers OP. Boosting heterologous protein production yield by adjusting global nitrogen and carbon metabolic regulatory networks in Bacillus subtilis . Metab Eng. 2018;49:143–52. Tan FR, Wu B, Dai LC, Qin H, Shui ZX, Wang JL, Zhu QL, Hu GQ, He MX. Using global transcription machinery engineering (gTME) to improve ethanol tolerance of Zymomonas mobilis . Microb Cell Fact 2016, 15. Deo R, Lakra U, Ojha M, Nigam VK, Sharma SR. Exopolysaccharides in microbial interactions: signalling, quorum sensing, and community dynamics. Nat Prod Res 2024:1–16. Guttenplan SB, Blair KM, Kearns DB. The EpsE Flagellar Clutch Is Bifunctional and Synergizes with EPS Biosynthesis to Promote Bacillus subtilis Biofilm Formation. PLos Genet 2010, 6. Patek M, Nesvera J. Sigma factors and promoters in Corynebacterium glutamicum . J Biotechnol. 2011;154:101–13. Wang J, Li X, Wang JF, Wei W, Jin WJ, Zhou LB. Comparative proteomics reveals energy and carbon metabolism changes in Scenedesmus quadricauda mutants induced by heavy-ion beam irradiation. Bioresour Technol 2024, 406. Koberstein JN, Stewart ML, Smith CB, Tarasov AI, Ashcroft FM, Stork PJS, Goodman RH. Monitoring glycolytic dynamics in single cells using a fluorescent biosensor for fructose 1,6-bisphosphate. Proc Natl Acad Sci USA 2022, 119. Agrawal P, Gupta P, Swaminathan K, Parkesh R. alpha-Glucan pathway as a novel Mtb drug target: Structural insights and cues for polypharmcological targeting of GlgB and GlgE. Curr Med Chem. 2014;21:4074–84. Zhang JF, Xu XJ, Li XY, Chen XJ, Zhou CX, Liu YH, Li Y, Lu FP. Reducing the cell lysis to enhance yield of acid-stable alpha amylase by deletion of multiple peptidoglycan hydrolase-related genes in Bacillus amyloliquefaciens. Int J Biol Macromol. 2021;167:777–86. Cremer J, Honda T, Tang Y, Wong-Ng J, Vergassola M, Hwa T. Chemotaxis as a navigation strategy to boost range expansion. Nature. 2019;575:658–. Wei XT, Ji ZX, Chen SW. Isolation of halotolerant Bacillus licheniformis WX-02 and regulatory effects of sodium chloride on yield and molecular sizes of poly-gamma-glutamic acid. Appl Biochem Biotechnol. 2010;160:1332–40. Bhat AR, Irorere VU, Bartlett T, Hill D, Kedia G, Charalampopoulos D, Nualkaekul S, Radecka I. Improving survival of probiotic bacteria using bacterial poly-gamma-glutamic acid. Int J Food Microbiol. 2015;196:24–31. Quach NT, Vu THN, Nguyen TTA, Ha H, Ho PH, Chu-Ky S, Nguyen LH, Nguyen HV, Thanh TTT, Nguyen NA et al. Structural and genetic insights into a poly-gamma-glutamic acid with in vitro antioxidant activity of Bacillus velezensis VCN56. World J Microbiol Biotechnol 2022, 38. Parati M, Khalil I, Tchuenbou-Magaia F, Adamus G, Mendrek B, Hill R, Radecka I. Building a circular economy around poly (D/L-gamma-glutamic acid)- a smart microbial biopolymer. Biotechnol Adv. 2022;61:495–507. Yang FR, Chen JL, Ye SH, Liu ZF, Ding Y. Characterization of antioxidant activity of exopolysaccharides from endophytic Lysinibacillus sphaericus Ya6 under osmotic stress conditions. Process Biochem. 2022;113:87–96. Li FS, Hu X, Sun XJ, Li HS, Lu JR, Li YM, Bao MT. Effect of fermentation pH on the structure, rheological properties, and antioxidant activities of exopolysaccharides produced by Alteromonas australica QD. Glycoconj J. 2022;39:773–87. Wang LL, Chen JT, Wang LF, Wu S, Zhang GZ, Yu HQ, Ye XD, Shi QS. Conformations and molecular interactions of poly-gamma-glutamic acid as a soluble microbial product in aqueous solutions. Sci Rep 2017, 7. Bashir A, Hoffmann T, Kempf B, Xie XL, Smits SHJ, Bremer E. Plant-derived compatible solutes proline betaine and betonicine confer enhanced osmotic and temperature stress tolerance to Bacillus subtilis . Microbiology-Sgm. 2014;160:2283–94. Hoffmann T, Wensing A, Brosius M, Steil L, Volker U, Bremer E. Osmotic control of opuA expression in Bacillus subtilis and its modulation in response to intracellular glycine betaine and proline pools. J Bacteriol. 2013;195:510–22. Morawska LP, Weme R, Frenzel E, Dirkzwager M, Hoffmann T, Bremer E, Kuipers OP. Stress-induced activation of the proline biosynthetic pathway in Bacillus subtilis : a population-wide and single-cell study of the osmotically controlled proHJ promoter. Microb Biotechnol. 2022;15:2411–25. Song QS, Joshi M, DiPiazza J, Joshi V. Functional relevance of citrulline in the vegetative tissues of watermelon during abiotic stresses. Front Plant Sci 2020, 11. Joshi V, Fernie AR. Citrulline metabolism in plants. Amino Acids. 2017;49:1543–59. Stecker D, Hoffmann T, Link H, Commichau FM, Bremer E. L-proline synthesis mutants of Bacillus subtilis overcome osmotic sensitivity by genetically adapting L-arginine metabolism. Front Microbiol 2022, 13. Musa YR, Bäsell K, Schatschneider S, Vorhölter FJ, Becher D, Niehaus K. Dynamic protein phosphorylation during the growth of Xanthomonas campestris pv. campestris B100 revealed by a gel-based proteomics approach. J Biotechnol. 2013;167:111–22. He K, Xin YP, Shan Y, Zhang X, Song HH, Fang WH. Phosphorylation residue T175 in RsbR protein is required for efficient induction of sigma B factor and survival of Listeria monocytogenes under acidic stress. J Zhejiang University-Science B. 2019;20:660–9. Jeon J, Kim D, Jeon TJ. Opposite functions of RapA and RapC in cell adhesion and migration in Dictyostelium. Anim Cells Syst. 2021;25:203–10. Liu N, Sun HW, Tang ZY, Zheng YQ, Qi GF, Zhao XY. Transcription Factor Spo0A Regulates the Biosynthesis of Difficidin in Bacillus amyloliquefaciens. Microbiol Spectr 2023, 11. Zhang Y, Xiao F, Zhang L, Ding Z, Shi G, Li Y. A new mechanism of carbon metabolism and acetic acid balance regulated by CcpA. Microorganisms 2023, 11. Dhali D, Coutte F, Arias AA, Auger S, Bidnenko V, Chataigné G, Lalk M, Niehren J, de Sousa J, Versari C, Jacques P. Genetic engineering of the branched fatty acid metabolic pathway of Bacillus subtilis for the overproduction of surfactin C14 isoform. Biotechnol J 2017, 12. Jaehme M, Slotboom DJ. Structure, function, evolution, and application of bacterial Pnu-type vitamin transporters. Biol Chem. 2015;396:955–66. You JJ, Yang C, Pan XW, Hu MK, Du YX, Osire T, Yang TW, Rao ZM. Metabolic engineering of Bacillus subtilis for enhancing riboflavin production by alleviating dissolved oxygen limitation. Bioresour Technol 2021, 333. Chaix D, Ferguson ML, Atmanene C, Van Dorsselaer A, Sanglier-Cianférani S, Royer CA, Declerck N. Physical basis of the inducer-dependent cooperativity of the Central glycolytic genes Repressor/DNA complex. Nucleic Acids Res. 2010;38:5944–57. Zorrilla S, Chaix D, Ortega A, Alfonso C, Doan T, Margeat E, Rivas G, Aymerich S, Declerckt N, Royer CA. Fructose-1,6-bisphosphate acts both as an inducer and as a structural cofactor of the central glycolytic genes repressor (CggR). Biochemistry. 2007;46:14996–5008. Additional Declarations No competing interests reported. Supplementary Files GA.jpg Graphical abstract Cite Share Download PDF Status: Published Journal Publication published 14 Oct, 2025 Read the published version in Microbial Cell Factories → Version 1 posted Editorial decision: Revision requested 08 Sep, 2025 Reviews received at journal 26 Aug, 2025 Reviews received at journal 14 Aug, 2025 Reviewers agreed at journal 12 Aug, 2025 Reviewers agreed at journal 10 Aug, 2025 Reviewers invited by journal 08 Aug, 2025 Editor assigned by journal 06 Aug, 2025 Submission checks completed at journal 06 Aug, 2025 First submitted to journal 01 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7275147","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":499245734,"identity":"cd2cbcd0-412f-4f0d-b169-f0e4cdca61f5","order_by":0,"name":"Xiaoyu Wei","email":"","orcid":"","institution":"Third Institute of Oceanography, Ministry of Natural Resources","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyu","middleName":"","lastName":"Wei","suffix":""},{"id":499245735,"identity":"b195c1b2-337a-4db2-a737-2ac3f65a33ca","order_by":1,"name":"Ziwei Pan","email":"","orcid":"","institution":"Xiamen University","correspondingAuthor":false,"prefix":"","firstName":"Ziwei","middleName":"","lastName":"Pan","suffix":""},{"id":499245736,"identity":"89ece2c7-6b3c-4438-8fcf-c154960ea3ae","order_by":2,"name":"Zhen Chen","email":"","orcid":"","institution":"Xinyang Normal University","correspondingAuthor":false,"prefix":"","firstName":"Zhen","middleName":"","lastName":"Chen","suffix":""},{"id":499245737,"identity":"cbb8bda6-c2a2-4bcf-968f-aa8d707dc754","order_by":3,"name":"Ning He","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYBACAyA+wMBgA+HxkKAljUQtQHCYBC3m7L0HDxf8Op+4dkYC44O3bQzy5oS0WPacSzg8s++2sdmNBGbDuW0MhjsbCDnsRo7BYd6e23JALWzSvG0MCQYHCGm5/wak5RwPUAv7b+K03OAxOMzz4wDYFmbitJwBOawh2djszMNmyTnnJAw3ENRy/IzxZ54/donbjicf/PCmzEaeoC1gwNgGJhuAhAQx6kHgD7EKR8EoGAWjYEQCAALBQ1guTfPqAAAAAElFTkSuQmCC","orcid":"","institution":"Xiamen University","correspondingAuthor":true,"prefix":"","firstName":"Ning","middleName":"","lastName":"He","suffix":""}],"badges":[],"createdAt":"2025-08-02 03:23:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7275147/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7275147/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12934-025-02846-2","type":"published","date":"2025-10-14T15:57:18+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89008061,"identity":"2a92be9c-270c-4627-a77c-aeec7523d927","added_by":"auto","created_at":"2025-08-13 16:44:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":824141,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of independent and combined stresses on extracellular polymeric substances in \u003cem\u003eB. licheniformis\u003c/em\u003e. (A, B) Different NaCl concentrations; (C, D) Different pH levels; (E, F) Different heating temperatures; (G, H) Combined stress. (I) The molecular weight of γ-PGA.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7275147/v1/17c8850a47ef4b8e2bca2ffe.png"},{"id":89007369,"identity":"264b3695-3325-422a-a180-81b0106acfbb","added_by":"auto","created_at":"2025-08-13 16:36:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2224696,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolite analysis of combined stress group versus the control group. (A) Volcanic map in positive ion mode; (B) Heat map in positive ion mode; (C) Volcanic map in negative ion mode; (D) Heat map in negative ion mode. (E) Schematic diagram of metabolic pathways involved in the synthesis of extracellular polymeric substances under combined stresses.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7275147/v1/fa0756d8cb40c60a5b101482.png"},{"id":89007365,"identity":"6e676f79-a3f1-46b0-88f5-78f04d0423dd","added_by":"auto","created_at":"2025-08-13 16:36:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2052951,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of global regulators related to quorum sensing on extracellular polymeric substances production. (A) Analysis of extracellular polymeric substances produced by global regulator mutant strains; (B) The predicted PPIs of RsbRA; (C) The expression level analysis of key synthesis genes in global regulator RsbRA mutant strains rsbRA. (D) The predicted PPIs of \u003cem\u003erapA\u003c/em\u003e; (E) The expression level analysis of key synthesis genes in global regulator \u003cem\u003erapA\u003c/em\u003emutant strain.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7275147/v1/32583f300b0fde5ae1e71c2a.png"},{"id":89008063,"identity":"7058edec-60f5-4deb-9c9e-3310bd383b53","added_by":"auto","created_at":"2025-08-13 16:44:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1066691,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of regulators related to carbon and nitrogen utilization on extracellular polymeric substances production. (A) Analysis of extracellular polymeric substances produced by global regulator mutant\u003cem\u003e \u003c/em\u003estrains. (B) The predicted PPIs of \u003cem\u003ecggR\u003c/em\u003e; (C) The expression level analysis of key synthesis genes by the global regulator \u003cem\u003ecggR\u003c/em\u003e mutants\u003cem\u003e \u003c/em\u003estrain. (D, E) The fermentation process curve of the global regulator \u003cem\u003ecggR\u003c/em\u003e mutant\u003cem\u003e \u003c/em\u003estrain in a 1.5 L fermenter.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7275147/v1/69120a05a59e940ff6fc23ec.png"},{"id":89007377,"identity":"0178322f-c140-447e-8694-432e9887206e","added_by":"auto","created_at":"2025-08-13 16:36:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2542937,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional enrichment of DEGs in the wild-type strain and the global regulator \u003cem\u003enrgB\u003c/em\u003e mutant strain. (A) The functional categories of DEGs based on GO. (B) The significance analysis of the top 20 Q-value pathways are based on KEGG.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7275147/v1/e954cd03f1c4f462a831ef5a.png"},{"id":89008066,"identity":"06a164b7-5dd1-4214-a622-4490c389b572","added_by":"auto","created_at":"2025-08-13 16:44:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4725952,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Heatmap of significant genes and their enriched pathways of the wild-type strain and the global regulator\u003cem\u003e nrgB\u003c/em\u003e mutant strain. (B) Schematic diagram of metabolic pathways involved in the synthesis of extracellular polymeric substances in the global regulator \u003cem\u003enrgB\u003c/em\u003e mutant strain.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7275147/v1/f3e8db48bb3d6c96968f6c89.png"},{"id":93955967,"identity":"b019042f-f403-4df2-960a-153e6cd065ae","added_by":"auto","created_at":"2025-10-20 16:08:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13527786,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7275147/v1/e0e07efb-bbe9-467f-aba2-5e5a7af5dcc6.pdf"},{"id":89007368,"identity":"e8584b05-b1cb-4e07-8147-8455268f8448","added_by":"auto","created_at":"2025-08-13 16:36:54","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":185049,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"GA.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7275147/v1/3e5d79c051bf4f43b164ef9d.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Stress response regulation to extracellular polymeric substances biosynthesis in Bacillus licheniformis","fulltext":[{"header":"1. Background","content":"\u003cp\u003eExtracellular polymeric substances are large molecular polymers that are synthesized and secreted by bacteria in response to the environmental stimuli, which have complex chemical components, encompassing polysaccharides, proteins, nucleic acids, among others [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. \u003cem\u003eBacillus\u003c/em\u003e species are capable of concurrently synthesizing two kinds of extracellular polymeric substances. For instance \u003cem\u003eBacillus licheniformis\u003c/em\u003e CGMCC 2876 produces both EPS and γ-PGA [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], \u003cem\u003eB. licheniformis\u003c/em\u003e WX-02 synthesizes lichenysin and γ-PGA [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e NK-1 generates levan and γ-PGA [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Recent research efforts have employed various strategies to elucidate the competitive relationships between different types of extracellular polymeric substances, such as the knockout of EPS operon \u003cem\u003eepsA-O\u003c/em\u003e cluster[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] or \u003cem\u003epgsBCA\u003c/em\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and the cell wall DL-endopeptidase \u003cem\u003ecwlO\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] in \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e. However, investigations into the specific metabolic interactions and regulatory mechanisms governing the synthesis of γ-PGA and EPS from a global regulatory perspective remain limited, thereby impeding targeted metabolic engineering efforts. Additionally, while thermal treatment may enhance EPS yield, it can also lead to a reduction in molecular weight and intrinsic viscosity, resulting in altered antioxidant properties, as evidenced by studies on polysaccharides derived from \u003cem\u003eInonotus obliquus\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe bioprocess induced by abiotic stress represents a novel approach to augment the production of specific metabolites, with relevant stress conditions encompassing osmotic stress, heat shock, pH fluctuations, and oxidative stress [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Prior research has demonstrated that the production of γ-PGA and validamycin A can be enhanced by 133% and 27.43%, respectively, through the the alkaline pH shock [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Sandhya et al. reported that salt stress significantly affects the monomer composition of EPS [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, EPS production by \u003cem\u003eRhodotorula\u003c/em\u003e sp. CAH2 exhibited a consistent increase with rising concentrations of NaCl [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Additionally, while thermal treatment may enhance EPS yield, it can also lead to a reduction in molecular weight and intrinsic viscosity, resulting in altered antioxidant properties, as evidenced by studies on EPS derived from \u003cem\u003eInonotus obliquus\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSubsequently, environmental stress signals are detected by sensor proteins, which initiate a signaling cascade that activates the sigma factor, thereby regulating the expression of transcription factor genes and suggesting that these regulators enhance bacterial fitness in response to stressors. Previous works have demonstrated that certain regulatory proteins, such as CcpA and CcpN for carbon metabolism, and NrgB for nitrogen metabolism, play a pivotal role in the stress response mechanisms involved in the synthesis of EPS and γ-PGA in \u003cem\u003eB. licheniformis\u003c/em\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In \u003cem\u003eB. subtilis\u003c/em\u003e, the expression of pgsBCA is regulated by the DegS-DegU two-component system and the ComP-ComA quorum sensing (QS) system, which activate pgsBCA transcription in response to environmental osmotic stress and developmental phase transitions [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Through the engineering of the PhrQ-RapQ-DegU QS system, the yield of γ-PGA was remarkably increased by 6.53-fold, achieving a balance between cell growth and high-efficiency γ-PGA production [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Nonetheless, the role of variations in extracellular polymeric substance composition \u003cem\u003ein B. licheniformis\u003c/em\u003e in mitigating environmental stress, as well as the underlying metabolic and regulatory mechanisms, remains inadequately understood.\u003c/p\u003e\u003cp\u003eThis study investigates the regulatory effects of three types of physicochemical stresses\u0026mdash;heat shock, osmotic stress, and pH stress\u0026mdash;on the production of EPS and γ-PGA production in \u003cem\u003eB. licheniformis\u003c/em\u003e. The analysis of intracellular metabolites was conducted to explore potential mechanisms under combined stress conditions that enhance biosynthetic capabilities. Additionally, the research examines the influence of stress-responsive transcriptional regulators on the components of extracellular polymeric substances. The objective of this study is to dissect the competitive interactions by integrating environmental perturbations with global transcriptional engineering, elucidate the underlying molecular mechanisms, and ultimately inform the metabolic engineering of \u003cem\u003eB. licheniformis\u003c/em\u003e for the efficient and targeted production of γ-PGA and EPS.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Plasmids, strains, and culture conditions\u003c/h2\u003e\u003cp\u003e\u003cem\u003eB. licheniformis\u003c/em\u003e CGMCC 2876 was used as the host for extracellular polymeric substances and served as the wild-type strain. \u003cem\u003eEscherichia coli\u003c/em\u003e DH5α was employed for plasmid construction and cultured in LB broth at 37\u0026deg;C with 10 mg/L tetracycline as needed. The plasmid pHY300PLK-PamyL-TtamyL was applied for constructing gene overexpression in \u003cem\u003eB. licheniformis\u003c/em\u003e CGMCC 2876 (Table S1 and S2). For \u003cem\u003enrgB\u003c/em\u003e gene overexpression, the gene was amplified from \u003cem\u003eB. licheniformis\u003c/em\u003e CGMCC 2876 by corresponding primers (Table S3), inserted into the plasmid using ClonExpress One Step Cloning Kit (Vazyme Biotech Co., Ltd., Nanjing, China), resulting in pHY-\u003cem\u003enrgB\u003c/em\u003e. This plasmid was then transformed into DH5α and selected on 10mg/L tetracycline plates, with transformants verified by PCR. This method was also used to construct other regulator expression vectors, which were transferred into \u003cem\u003eB. licheniformis\u003c/em\u003e via electroporation (25 kV/cm, 4 ms), resulting in overexpression strain OE-\u003cem\u003enrgB\u003c/em\u003e. The OE-\u003cem\u003enrgB\u003c/em\u003e strain was cultured in LB medium with 10 mg/L tetracycline at 37\u0026deg;C, 220rpm. The scale-up fermentation process was performed within a six-parallel bioreactor system.\u003c/p\u003e\u003cp\u003eThe compositions and culture conditions of seed and EPS fermentation medium for γ-PGA production were the same as in Wei's previous work [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. To determine the effects of independent and multiple stresses on extracellular polymeric substance production, three conditions of osmotic pressure, pH stress, and temperature stress were selected. For osmotic stress, five levels of NaCl (0, 2, 4, 6, and 8 g/L) were added to the EPS fermentation medium at the beginning of cultivation. For pH stress, the medium initial pH was adjusted to 3.0, 5.0, 7.2, 9.0, and 11.0 by the addition of HCl (2 mol/L) or NaOH (2 mol/L). For temperature stress, the strains were inoculated into EPS medium by stimulation with 0, 37, 50, 55, 60, and 65\u0026deg;C for 30min in a water bath, then quickly cooled to 37\u0026deg;C. For combined stress treatment, different stress conditions were applied simultaneously as follows, based on the optimized stress conditions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Analysis Methods\u003c/h2\u003e\u003cp\u003eAfter the fermentation process, the crude yields of extracellular polymeric substances and the biomass was extracted and purified, based on our previous research [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The yields of γ-PGA were detected by high-performance liquid chromatography (HPLC) with a C18 column (ZORBAX SB-C18). The content of total EPS in the biopolymer was detected using the phenol\u0026ndash;sulfuric acid method. The cell density \u0026shy; (OD\u003csub\u003e600\u003c/sub\u003e) was measured using a UV-1800 spectrophotometer (Shimadzu Global Laboratory Consumables Co., Ltd., Shanghai, China). High-performance gel permeation chromatography (HPGPC) was employed to analyze the molecular weight (Mw) of γ-PGA.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Quantitative realtime PCR\u003c/h2\u003e\u003cp\u003eStrains were collected after 24 h of incubation for RNA extraction and gene expression analysis. Total RNA was extracted using a Bacterial RNA Kit (Omega Bio-Tek, Guangzhou, China) and quantified by a NanoDrop 2000 (Thermo, CA, USA). First-strand cDNA was synthesized using HiScript III RT SuperMix for qPCR (+\u0026thinsp;gDNA wiper) (Vazyme, Nanjing, China), and qRT-PCR was performed with a qTOWER3 instrument (Analytik Jena AG, Jena, Germany) using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). For gene transcriptional levels, a SYBR\u0026reg; Premix Ex Taq\u0026trade; II (RR820A, Takara) and a Bio-Rad iQ5 real-time PCR system were used following the manufacturer\u0026rsquo;s instructions. Relative quantification relates the PCR signal of the target transcript in a sample to control based on the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The 16S rDNA served as the reference. Primers used for related genes are shown in Table S4.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 LC-MS performance and metabolomics data analysis\u003c/h2\u003e\u003cp\u003ePathway metabolites analysis was performed by liquid chromatography- mass spectrometer (LC-MS AB Sciex TripleTOF 5600 +). The sample pretreatment and metabonomics data analysis methods were as described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In brief, when the cells were grown for 24 h, the culture medium was removed by centrifugation. Two groups of samples, each with three replicates, including the non-stressed fermentation control group (Control) and the combined-stress fermentation experimental group (Stress), were tested for metabolites in both positive and negative ion modes. The cell suspension was washed three times with 10 mM PBS. Cells were resuspended in 1 mL PBS and centrifuged at 4\u0026deg;C. Cells were precipitated in liquid nitrogen for 1 min. Then, 1 mL of MeOH: ACN: H2O (V: V: V, 2:2:1) was vortexed for 30 s for the following 10 min sonication (4\u0026deg;C water bath). The samples were placed in liquid nitrogen for 1 min. This procedure was repeated 3 times to burst the cells in total. Then, the samples were incubated at \u0026minus;\u0026thinsp;20\u0026deg;C for 1 h to facilitate protein precipitation. Finally, the samples were freeze-centrifuged (1300 rpm, 15 min), and the supernatant was collected for freeze-drying by an FDU-1200 freeze-drying machine. Three replicates were used in this experiment. Metabolomics data have been deposited in the iProX database (www.iprox cn) with accession number IPX0006376000. After processing the data through convolution, internal standard removal, and false-positive elimination, the obtained metabolite information was aligned for subsequent metabolite clustering, screening and identification of differential metabolites, and related bioinformatics analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Transcriptomic analysis\u003c/h2\u003e\u003cp\u003eStrains were also primarily inoculated in seed medium and cultured for 20 h to the late logarithmic growth (OD\u0026thinsp;~\u0026thinsp;2), then transferred to EPS medium and further incubated for 24 h. The cells were collected by centrifugation at 8000 rpm for 10 min at 4 ◦C for total RNA purification and cDNA library construction. Sequencing was performed using the Illumina HiSeqTM 2500 platform with paired-end 150 base pair reads by Gene Denovo Biotechnology Co. Raw data were filtered before the quality-trimmed reads were mapped to the genome of \u003cem\u003eB. licheniformis\u003c/em\u003e CGMCC 2876 using Bowtie2 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The gene expression level was analyzed according to FPKM (fragments per kilobase of transcript per million). The edgeR package (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.r-project.org/\u003c/span\u003e\u003cspan address=\"http://www.r-project.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to identify DEGs with fold changes (FC)\u0026thinsp;\u0026ge;\u0026thinsp;2 and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Statistical Analyses\u003c/h2\u003e\u003cp\u003eAll the fermentation experiments were repeated at least three times. Data were presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation for each sample point. All data were collected to analyze the variance at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and the mean values were compared by applying a T-test.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Effect of multiple stresses on extracellular polymeric substances production\u003c/h2\u003e\u003cp\u003eEnvironmental stresses effectively enhance target product synthesis. B. licheniformis CGMCC 2876 was fermented under various stress conditions. Adding 16 g/L NaCl increased extracellular polymeric substances yield to 14.53 g/L, a 25.86% rise from the control (11.55 g/L) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Moreover, EPS yield decreased as NaCl concentration increased. Conversely, γ-PGA content rose with higher NaCl levels, reaching 5.22 g/L at 16 g/L NaCl, a 54.33% increase, suggesting γ-PGA production is a stress response to hyperosmotic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Under strong cellular dehydration pressure caused by hyperosmotic stress, γ-PGA, with its high water-absorbing capacity, may help maintain cellular water activity and enhance cell survival [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This indicates that high NaCl-induced osmotic stress boosts γ-PGA synthesis while inhibiting EPS production.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eUnder extreme alkaline stress (pH\u0026thinsp;=\u0026thinsp;11), \u003cem\u003eB. licheniformis\u003c/em\u003e CGMCC 2876 produced 6.57 g/L of extracellular polymeric substances, a 23.73% decrease from the control (8.61 g/L at pH 7.2). At pH\u0026thinsp;=\u0026thinsp;9, biomass doubled to 1.21 g/L compared to the control (0.63 g/L) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) and γ-PGA production rose by 20.88% to 4.68 g/L. EPS production increased to 340 mg/L at pH 9 and 570 mg/L at pH 11. Under acidic conditions (pH\u0026thinsp;=\u0026thinsp;3 and pH\u0026thinsp;=\u0026thinsp;5), extracellular polymeric substances decreased to 7.05 g/L and 6.7 g/L, while EPS yields increased by 22.22% and 89.98% to 340 mg/L and 520 mg/L, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). γ-PGA yields dropped by 64.44% and 7% under acidic conditions, showing that acidity hinders γ-PGA synthesis. These findings demonstrate that strong alkaline stress increased the yield of γ-PGA, whereas EPS synthesis was more responsive to strong acidic stress.\u003c/p\u003e\u003cp\u003eThe yield of extracellular polymers showed no significant difference with increasing fermentation temperature, maintaining a stable output of 8.1 g/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). And the EPS production at 55\u0026deg;C reached 380 mg/L, a 49.26% increase over the 37\u0026deg;C condition (0.26 g/L). Similarly, EPS yield rose to 340 mg/L at 65\u0026deg;C (35.6% higher than at 37\u0026deg;C), demonstrating that elevated EPS synthesis is a stress response to thermal challenge. However, beyond 55\u0026deg;C, γ-PGA synthesis declined sharply, dropping to 1.41 g/L at 65\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). These results demonstrate that high-temperature stress at 55\u0026deg;C promoted EPS synthesis, but excessively high temperatures were detrimental to γ-PGA synthesis.\u003c/p\u003e\u003cp\u003eBased on the above results, alkaline stress (pH\u0026thinsp;=\u0026thinsp;9) and hyperosmotic stress (16 g/L NaCl) boost γ-PGA production but reduce EPS synthesis, while high temperature (55\u0026deg;C) has the opposite effect. Under the three stresses, biomass increased from 0.36 g/L to 1.46 g/L, indicating enhanced bacterial growth and extracellular polymer production (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). EPS production decreased by 12.77% to 200.92 mg/L, while γ-PGA production increased by 18.96% to 3.79 g/L compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Notably, the Mw of γ-PGA at pH 9 rose by 5.96% to 4.84\u0026times;10⁵ kDa (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). This phenomenon may be attributed to H⁺ and OH⁻ ions altering the ionic balance across cell membranes, thereby modifying the transmembrane potential difference and affecting molecular synthesis and transport [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Alkaline stress can lead to a reduction in extracellular enzyme activity, thereby limiting the degradation of extracellular macromolecules and consequently influencing the degree of γ-PGA polymerization.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Metabolite profile analysis of multiple stresses\u003c/h2\u003e\u003cp\u003eTo elucidate the mechanisms underlying the effects of the three stresses on the metabolic level of \u003cem\u003eB. licheniformis\u003c/em\u003e, untargeted metabolomics was employed to analyze changes in intracellular metabolites under these conditions (Table S5). A distinct differentiation is evident between stressed and non-stressed intracellular metabolite samples as demonstrated by PCA and OPLS-DA analyses (Figure S1). To evaluate the accuracy of the models, a 200-response permutation test was performed, which confirmed that the OPLS-DA models, in both ionization modes, exhibit robust discriminative and predictive capabilities. The OPLS-DA model was utilized to screen and identify differential metabolites, employing Variable Importance in the Projection (VIP) and Fold Change (FC) as selection criteria (Figure S2). Based on log\u003csub\u003e2\u003c/sub\u003eFC\u0026thinsp;\u0026gt;\u0026thinsp;1 or \u0026lt;-1, were identified in positive ion mode, including 11 upregulated metabolites, such as notably coproporphyrin, cyclic GMP, ATP, citrulline, glycerol 3-phosphate, glutamine, and 13 significantly downregulated metabolites, notably hypoxanthine, acetoin, isobutyrate, dimethylglycine, DL-2-aminobutyric acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In negative ion mode, 97 metabolites were detected, comprising 17 upregulated metabolites, including proline, FAD, pentadecanoic acid, flavin mononucleotide, citrulline, glutamine, phosphoenolpyruvate, and 12 significantly downregulated metabolites, primarily cytidine monophosphate, phthalic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSeventeen differential metabolites were identified and visualized in a heatmap by employing a combination of screening criteria: log\u003csub\u003e2\u003c/sub\u003eFC\u0026thinsp;\u0026gt;\u0026thinsp;1 or \u0026lt; -1, VIP\u0026thinsp;\u0026gt;\u0026thinsp;1, and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (Figure S2). In positive ion mode, metabolites such as proline, FAD, citrulline, glutamine, and glycerol 3-phosphate were upregulated, whereas N-acetylglucosamine, N-acetylmannosamine, and N-acetylgalactosamine were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Conversely, in the negative ion mode, upregulated metabolites included cyclic GMP and citrulline, while 2-aminoisobutyric acid was downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Under stress conditions, glycerol 3-phosphate and phosphoenolpyruvate exhibited upregulation by 3.1-fold and 2.6-fold, respectively, thereby providing additional precursor substances for carbon source utilization within the tricarboxylic acid (TCA) cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Within the γ-PGA biosynthetic pathway, both glutamate and glutamine were upregulated by 2.35-fold and 3.28-fold, respectively. Notably, glutamine production slightly surpassed that of glutamate, likely due to the utilization of a portion of glutamate in the downstream porphyrin metabolism pathway for the synthesis of coproporphyrin I. Similarly, the production of 2-aminoisobutyric acid within the downstream pyrimidine metabolism of glutamine exhibited a 0.44-fold decrease, suggesting that elevated levels of glutamine promote glutamate synthesis. I Within amino acid metabolic pathways, intracellular concentrations of proline and citrulline increased significantly, by 14.27-fold and 4.55-fold, respectively. As osmotic regulators, citrulline and glutamine have the capacity to interconvert, while proline can be converted into glutamate, thereby supplying precursor materials for γ-PGA synthesis. Conversely, within the EPS synthesis pathway, the production of N-acetylglucosamine, N-acetylmannosamine, and N-acetylgalactosamine was downregulated, resulting in a reduction of EPS synthesis unit pathways and accompanied by decreased expression of the glycosyltransferase epsG. Collectively, these findings indicate that stress conditions stimulate metabolic pathways associated with γ-PGA synthesis while concurrently attenuating EPS metabolic pathways, thereby highlighting a competitive relationship between EPS and γ-PGA biosynthesis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e3.3 Increasing global regulators related to quorum sensing expression promotes extracellular polymeric substances synthesis\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eEnvironmental stresses approaches can occasionally disrupt the equilibrium of carbon/nitrogen metabolism and cofactor networks within engineered strains [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. To mitigate these issues, global transcription machinery engineering has emerged as a promising strategy [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Notably, eight global transcriptional regulators were significantly upregulated under combined stress conditions (Figure S3). Extracellular polymeric substances constitute a major component of \u003cem\u003eBacillus\u003c/em\u003e biofilms and are also modulated by quorum sensing (QS) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn the quorum sensing-related global regulator \u003cem\u003ersbRA\u003c/em\u003e mutant strain, EPS production reached 8.76 g/L, representing a 5.12% increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The proportion of EPS rose to 8.34%, with production levels attaining 730.58 mg/L, marking a twofold increase, while the proportion of γ-polyglutamic acid (γ-PGA) also increased to 37.12%. Similarly, in the quorum sensing-related global regulator \u003cem\u003erapA\u003c/em\u003e mutant strain, EPS and γ-PGA production increased by 43.89% (518 mg/L) and 31.47% (2.77 g/L), respectively. Conversely, in the quorum sensing-related global regulator \u003cem\u003ecodY\u003c/em\u003e mutant strain, the EPS production decreased by 19.72% and γ-PGA decreased by 20.87%. The quorum sensing-related global regulator \u003cem\u003enadR\u003c/em\u003e mutant strain showed no significant increase in the proportions of EPS and γ-PGA, with total extracellular polymeric substances production slightly decreasing to 7.67 g/L. These results indicate that \u003cem\u003ersbRA\u003c/em\u003e and rapA regulators promote EPS and γ-PGA synthesis, while \u003cem\u003enadR\u003c/em\u003e and \u003cem\u003ecodY\u003c/em\u003e serve as negative regulators for EPS and γ-PGA production.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA protein-protein interaction (PPI) network centered on \u003cem\u003ersbRA\u003c/em\u003e and \u003cem\u003erapA\u003c/em\u003e was constructed utilizing the STRING database and differential gene expression analysis was conducted via qRT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-E). Among the six downstream genes regulated by \u003cem\u003ersbRA\u003c/em\u003e, the expression levels of phosphoserine phosphatase \u003cem\u003ersbU\u003c/em\u003e, serine protein kinase \u003cem\u003ersbT\u003c/em\u003e, and antagonist protein \u003cem\u003ersbS\u003c/em\u003e, all of which are involved in serine metabolism regulation, were found to be downregulated. Conversely, the expression of \u003cem\u003efliG\u003c/em\u003e, which encodes flagellar rotation protein was upregulated and may interact with \u003cem\u003eepsE\u003c/em\u003e to inhibit flagellar movement [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The expression of \u003cem\u003esigB\u003c/em\u003e increased 3.45-fold, suggesting that \u003cem\u003ersbRA\u003c/em\u003e enhances signal transduction to \u003cem\u003esigB\u003c/em\u003e, thereby upregulating its transcription. \u003cem\u003eSigB\u003c/em\u003e is a well-established regulator of stress response [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Among the critical EPS biosynthetic genes, the expression of \u003cem\u003eepsG\u003c/em\u003e, which encodes a glycosyltransferase involved in polysaccharide unit transport, increased by 1.5-fold. This finding indicates an activation of glycosyltransferases within the EPS synthesis pathway. The observed increase in EPS content is likely attributable to enhanced \u003cem\u003ersbRA\u003c/em\u003e-mediated signaling, which upregulates \u003cem\u003esigB\u003c/em\u003e transcription. This, in turn, modulates the transcription of eps genes, thereby promoting EPS biosynthesis. Moreover, in the mutant strain of the global regulator \u003cem\u003erapA\u003c/em\u003e, the competence-damage inducible protein cinA exhibited downregulation, whereas other genes, such as RNA polymerase \u003cem\u003esigB\u003c/em\u003e, sporulation initiation phosphotransferase \u003cem\u003espo0F\u003c/em\u003e, and anti-sigma F factor \u003cem\u003espoIIAB\u003c/em\u003e were upregulated. Genes associated with nucleotide sugar precursor synthesis pathways (\u003cem\u003eglmU\u003c/em\u003e, \u003cem\u003etuaD\u003c/em\u003e, \u003cem\u003egtaB\u003c/em\u003e) demonstrated varying levels of upregulation, as did genes related to EPS transport and polymerization (\u003cem\u003eepsH\u003c/em\u003e, \u003cem\u003eepsO\u003c/em\u003e). These differential gene expressions collectively contribute to the facilitation of EPS biosynthesis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e3.4 Increasing global regulators related to carbon and nitrogen utilization expression promotes extracellular polymeric substances synthesis\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eThe metabolic engineering of carbon and nitrogen pathways is crucial for enhancing the production of various bioproducts [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The global regulator \u003cem\u003ecggR\u003c/em\u003e, associated with carbon utilization, was found to increase γ-PGA production by 20.56%, achieving a concentration of 3.87 g/L. This suggests that \u003cem\u003ecggR\u003c/em\u003e redirects metabolic flux towards γ-PGA synthesis, while reducing EPS production by 4.72% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In a similar vein, the global regulator \u003cem\u003enrgB\u003c/em\u003e, related to nitrogen utilization, elevated the γ-PGA yield to 4.29 g/L, representing a 33.64% increase and constituting 53.17% of total extracellular polymeric substances. Conversely, EPS yield decreased to 270.47 mg/L, with its proportion diminishing to 3.35%. Furthermore, the \u003cem\u003eccpA\u003c/em\u003e-2 mutant strain, another carbon utilization-related global regulator, produced 3.83 g/L of γ-PGA, marking a 22.42% increase, while EPS production rose to 680.33 mg/L, 1.8 times higher than the wild-type. In contrast, the \u003cem\u003etnrA\u003c/em\u003e mutant strain, associated with nitrogen utilization, exhibited reductions in γ-PGA and EPS content by 40.78% and 47.5%, respectively. These findings underscore that the global regulators \u003cem\u003ecggR\u003c/em\u003e and \u003cem\u003enrgB\u003c/em\u003e positively influence γ-PGA synthesis, while negatively impacting EPS production.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eProtein interaction and qRT-PCR analysis showed that genes regulated by \u003cem\u003ecggR\u003c/em\u003e within the glycolysis pathway were generally upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Notably, the glyceraldehyde-3-phosphate dehydrogenase-encoding gene, \u003cem\u003egapA\u003c/em\u003e, was downregulated, whereas other genes, including the triose-phosphate isomerase-encoding gene \u003cem\u003etpiA\u003c/em\u003e, the phosphoglycerate kinase-encoding gene \u003cem\u003epgk\u003c/em\u003e, and the enolase-encoding gene \u003cem\u003eeno\u003c/em\u003e, exhibited upregulation. The \u003cem\u003egapA\u003c/em\u003e gene is co-transcribed with the downstream genes \u003cem\u003epgk\u003c/em\u003e, \u003cem\u003etpiA\u003c/em\u003e, and \u003cem\u003eeno\u003c/em\u003e, as well as with the upstream gene \u003cem\u003ecggR\u003c/em\u003e. The observed decrease in gapA expression may be attributed to the inhibitory effect of cggR [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Among the \u003cem\u003ecggR\u003c/em\u003e-regulated genes involved in glycolysis, the phosphotransferase gene \u003cem\u003ecrr\u003c/em\u003e and phosphoglucose isomerase gene \u003cem\u003epgi\u003c/em\u003e were found to be upregulated. The carbon flux diverges at fructose-6-phosphate within glycolysis, leading to downregulation of the glucosamine-1-phosphate acetyltransferase gene \u003cem\u003eglmU\u003c/em\u003e in the EPS synthesis pathway. Conversely, in the γ-PGA synthesis pathway via the TCA cycle, pyruvate kinase gene \u003cem\u003epyk\u003c/em\u003e and pyruvate dehydrogenase gene \u003cem\u003epdhD\u003c/em\u003e are upregulated in the OE-cggR strain, suggesting a shift in metabolic flux towards γ-PGA synthesis.\u003c/p\u003e\u003cp\u003eThe scale-up fermentation process was performed in bioreactors utilizing both the wild-type strain and the global regulator \u003cem\u003ecggR\u003c/em\u003e mutant strain within a six-parallel bioreactor system. The wild-type strain functioned as the control (Figure S4). The \u003cem\u003ecggR\u003c/em\u003e mutant strain demonstrated a brief adaptation phase, subsequently entering the logarithmic growth phase rapidly, which was characterized by a significant decrease in dissolved oxygen (DO) levels, reaching 30% by 8 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). To sustain DO at 30%, the agitation speed was dynamically modulated. The biomass growth curve indicated vigorous cell proliferation commencing at 4 hours, with cell density peaking at 6.24 (OD\u003csub\u003e600\u003c/sub\u003e) by 12 hours, signifying the transition to the stationary phase. Glucose consumption was rapid, approaching depletion by 12 hours, which reflects robust metabolic activity and efficient nutrient utilization. Notably, product accumulation increased significantly after 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). By 50 hours, the OE-\u003cem\u003ecggR\u003c/em\u003e strain produced 384.5 mg/L of EPS, while the γ-PGA yield reached 3.98 g/L, representing a 16.4% increase.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Transcriptomic analysis of the global regulator \u003cem\u003enrgB\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eTranscriptomic analysis was employed to elucidate the mechanisms by which the nitrogen utilization-related global regulator \u003cem\u003enrgB\u003c/em\u003e positively influences γ-PGA synthesis while negatively regulating EPS production. Utilizing thresholds of log\u003csub\u003e2\u003c/sub\u003eFC greater than 1 for upregulation and less than \u0026minus;\u0026thinsp;1 for downregulation, alongside a false discovery rate (FDR) of \u0026le;\u0026thinsp;0.05 as screening criteria, a total of 52 significantly differentially expressed genes were identified, comprising 29 upregulated and 23 downregulated genes (Figure S5 and Table S6).\u003c/p\u003e\u003cp\u003eThe Gene Ontology (GO) functional distribution of these differentially expressed genes indicated that they were primarily enriched in cellular metabolic processes, cellular physiological processes, and single-organism processes within the biological processes category (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In terms of molecular functions, the genes were predominantly associated with binding and catalytic activities, while cellular components were largely localized to membranes and membrane parts. Furthermore, the KEGG pathway classification of the screened differential genes was also analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Most metabolic pathways showed significant upregulation, particularly in metabolism, with the global and overview maps leading with 8 differential genes. Carbohydrate metabolism followed with 7 genes, while energy and lipid metabolism each had 2, suggesting that \u003cem\u003enrgB\u003c/em\u003e gene overexpression boosts energy metabolism in \u003cem\u003eB. licheniformis\u003c/em\u003e. Three pathways related to cellular processes, including cellular community-prokaryotes, cell motility, and cell growth and death, were enriched, with cellular community showing 7 differential genes, indicating changes in cellular communities due to \u003cem\u003enrgB\u003c/em\u003e overexpression. Additionally, environmental information processing pathways were enriched, with signal transduction having 7 differential genes and membrane transport 2, highlighting enhanced signal transduction and membrane transport activities.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe study identified 33 differential genes between the wild-type and \u003cem\u003enrgB\u003c/em\u003e mutant strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). In amino and nucleotide sugar metabolism, the expression of \u003cem\u003ewbpA\u003c/em\u003e, which includes \u003cem\u003ewbpA\u003c/em\u003e1 and \u003cem\u003ewbpA\u003c/em\u003e2 encoding dehydrogenases for UDP-N-acetylglucosamine and UDP-N-acetylmannosamine, was downregulated. These enzymes produce essential acids for polysaccharide synthesis. Additionally, five genes related to starch and sucrose metabolic pathways and secondary metabolite pathways\u0026mdash;\u003cem\u003eglgA\u003c/em\u003e (starch synthase), \u003cem\u003eglgB\u003c/em\u003e (glycogen branching enzyme), \u003cem\u003eglgC\u003c/em\u003e (glucose-1-phosphate adenylyltransferase), \u003cem\u003eglgD\u003c/em\u003e (glycogen biosynthesis protein), and \u003cem\u003eglgP\u003c/em\u003e (glycogen phosphorylase)\u0026mdash;also showed reduced expression. In the glycan pathway, \u003cem\u003eglgA\u003c/em\u003e is converted by \u003cem\u003eglgB\u003c/em\u003e into essential branched glucans [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Furthermore, in glycan biosynthesis, the N-acetylglucosaminidase \u003cem\u003elytD\u003c/em\u003e, part of peptidoglycan hydrolases, is upregulated, crucial for early bacterial growth and division, suggesting enhanced glycan degradation [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] Thus, nrgB suppresses EPS synthesis genes while boosting glycan degradation genes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe differential genes were further mapped to the major metabolic pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). In the glycolytic pathway, the expression of \u003cem\u003ehxlB\u003c/em\u003e, encoding 6-phosphohexulose isomerase, was downregulated. While, in the branched pathway of lipid metabolism within the pentose phosphate pathway, \u003cem\u003eegsA\u003c/em\u003e, encoding glycerol dehydrogenase, showed the most significant transcriptional upregulation, converting dihydroxyacetone to glycerol-1-phosphate via \u003cem\u003eegsA\u003c/em\u003e, indicating that \u003cem\u003enrgB\u003c/em\u003e overexpression stimulates the production of other extracellular polymer components. In amino acid metabolism, \u003cem\u003efadA\u003c/em\u003e, encoding an acetyl-CoA acyltransferase family protein, was markedly downregulated. As a transferase involved in the degradation pathways of valine and other amino acids, which facilitates the conversion of methyl acetyl-CoA to acetyl-CoA, the suppression of fadA indicates a diminished supply of acetyl-CoA for the tricarboxylic acid (TCA) cycle. In the context of nitrogen metabolism pathways, the gene \u003cem\u003enarI\u003c/em\u003e, which encodes the gamma subunit of nitrate reductase, was significantly upregulated. Nitrate reductase NarI catalyzes the conversion of nitrite to ammonia, subsequently facilitating the synthesis of glutamine for glutamate production, thereby providing precursors for γ-polyglutamic acid (γ-PGA) synthesis.\u003c/p\u003e\u003cp\u003eBacterial chemotaxis influences the preference for a planktonic state, and motility status impacts biofilm formation through extracellular polymers [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The upregulation of the methyl-accepting chemotaxis protein mcpA modulates signal transduction via the chemotaxis response regulators \u003cem\u003echeA\u003c/em\u003e and \u003cem\u003echeY\u003c/em\u003e, leading to the upregulation of \u003cem\u003emotB\u003c/em\u003e, which encodes another chemotaxis protein involved in bacterial flagellar synthesis. This indicates that the overexpression of \u003cem\u003enrgB\u003c/em\u003e enhances bacterial chemotactic motility and flagellar biosynthesis.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eMost γ-PGA-producing strains are glutamate-dependent, including \u003cem\u003eB. subtilis\u003c/em\u003e C10, \u003cem\u003eB. subtilis\u003c/em\u003e chungkookjang, \u003cem\u003eB. licheniformis\u003c/em\u003e ATCC 9945a, and \u003cem\u003eB. licheniformis\u003c/em\u003e WX-02. In contrast, relatively few strains are glutamate-independent, such as \u003cem\u003eB. subtilis\u003c/em\u003e TAM-4, \u003cem\u003eB. licheniformis\u003c/em\u003e A35, and \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e LL3 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. \u003cem\u003eB. licheniformis\u003c/em\u003e CGMCC 2876, a facultative glutamate-dependent γ-PGA producer, is capable of synthesizing γ-PGA both with exogenous glutamate supplementation and through de novo synthesis without glutamate. Our study demonstrated that \u003cem\u003eB. licheniformis\u003c/em\u003e CGMCC 2876 cultivated in the presence of 16 g/L NaCl produced 5.22 g/L γ-PGA, marking a 54.33% increase, although a slight reduction in the molecular weight (Mw) of γ-PGA was observed. Similar findings have been reported for halotolerant \u003cem\u003eB. licheniformis\u003c/em\u003e WX-02, where salt-induced γ-PGA production achieved a maximal yield of 13.86 g/L under 8% NaCl, despite decreased in Mw with increasing salinity [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Furthermore, NaCl concentration has been shown to modulate the Mw of γ-PGA in \u003cem\u003eB. licheniformis\u003c/em\u003e 9945a, \u003cem\u003eB. subtilis\u003c/em\u003e natto, \u003cem\u003eB. megaterium\u003c/em\u003e WH320, and \u003cem\u003eB. subtilis\u003c/em\u003e chungkookjang [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. This correlation suggests that γ-PGA serves a protective function for cells under stress conditions, with cells synthesizing higher Mw γ-PGA in response to elevated concentrations of osmotic-disrupting toxic compounds [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. While most γ-PGA-producing organisms demonstrate optimal growth at pH 7, some studies have reported that optimal γ-PGA biosynthesis occurs at pH 6.5, coinciding with peak uptake of precursors such as glutamate and citrate [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Under acidic stress conditions (pH 5), there is an 89.98% increase in EPS production, suggesting an acid-stress response mechanism where increased membrane permeability enhances glucose uptake, thereby augmenting the supply and transport of EPS precursors [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Similarly, \u003cem\u003eAlteromonas australica\u003c/em\u003e QD synthesizes high-molecular-weight EPS at acidic pH levels as an adaptation to pH stress [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In contrast, under alkaline stress (pH 9), the γ-PGA production increased 20.88%, likely due to the ionization of -COOH groups at elevated pH, which extends γ-PGA chains through enhanced water-polymer interactions, thereby increasing solution viscosity [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Conversely, mildly acidic pH conditions favors the biosynthesis of more free γ-PGA [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNotably, among the metabolites responsive to alkaline pH, hyperosmolarity, and elevated temperature stressors, proline content exhibited the most significant increase, functioning as both an osmolyte and a protective agent in \u003cem\u003eB. subtilis\u003c/em\u003e [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In response to osmotic adaptation pathways, \u003cem\u003eB. subtilis\u003c/em\u003e enhances its intracellular proline concentration from 20 mM to 500 mM [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. We hypothesize that hyperosmotic stress stimulates proline biosynthesis, which subsequently converts to glutamate via arginine, thereby augmenting the supply of γ-PGA precursors. Additionally, there was a notable increase in citrulline accumulation, which may facilitate the conversion of glutamate.\u003c/p\u003e\u003cp\u003eNevertheless, excessive accumulation of citrulline under hyperosmotic conditions could potentially disrupt the cytoplasmic ion balance [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Citrulline serves as a non-proteinogenic amino acid that protects plants under drought stress [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] and serves as a novel osmotic protectant in \u003cem\u003eB. subtilis\u003c/em\u003e [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAmong the regulators promoting EPS and γ-PGA production, the quorum-sensing global regulator \u003cem\u003ersbRA\u003c/em\u003e increased the EPS proportion to 8.34%, with production reaching 730.58 mg/L, representing a two-fold increase. A similar regulatory mechanism is observed in \u003cem\u003eXanthomonads\u003c/em\u003e, where xanthan gum is produced as an exopolysaccharide during the growth phase, and this phase influences protein phosphorylation, specifically the phosphorylation of rsbRA during the transition from the late exponential to the stationary phase [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. As a component of the stressosome, \u003cem\u003ersbRA\u003c/em\u003e facilitates responses to environmental stress and starvation signals in \u003cem\u003eB. subtilis\u003c/em\u003e by transmitting stress signals to sigB, which is crucial for transcriptional activation necessary for bacterial survival [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Additionally, the quorum-sensing global regulator rapA facilitates the transfer of phosphate groups to the transcriptional regulator Spo0A via the phosphotransfer proteins Spo0F and Spo0B [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The phosphorylated Spo0A\u0026thinsp;~\u0026thinsp;P subsequently regulates the transcription of over 500 genes in \u003cem\u003eBacillus\u003c/em\u003e species, including those involved in biofilm formation, sporulation, and secondary metabolite biosynthesis [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The global regulator CcpA recognizes and binds to numerous target genes across various metabolic pathways, modulating glucose metabolic flux by regulating key genes in the pentose phosphate pathway while repressing genes involved in acetate metabolism [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Nonetheless, the quorum-sensing global regulator\u003cem\u003es, nadR\u003c/em\u003e and \u003cem\u003ecodY\u003c/em\u003e, along with the nitrogen utilization-related global regulator \u003cem\u003etnrA\u003c/em\u003e, are function as negative regulators of EPS and γ-PGA production. CodY exerts its repressive influence on lipopeptide synthesis by directly binding to the srfA promoter, thereby inhibiting its expression [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. NadR is capable of repressing key NAD biosynthetic genes, including \u003cem\u003enadB\u003c/em\u003e, \u003cem\u003enadA\u003c/em\u003e-\u003cem\u003epnuC\u003c/em\u003e, and \u003cem\u003epncB\u003c/em\u003e, while simultaneously modulating cellular NAD levels through pnuC-mediated transport [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In \u003cem\u003eB. subtilis\u003c/em\u003e, \u003cem\u003etnrA\u003c/em\u003e activates nitrogen reductase genes within the \u003cem\u003enar\u003c/em\u003e and \u003cem\u003enas\u003c/em\u003e operons under conditions of nitrogen limitation [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAmong these global regulators, \u003cem\u003enrgB\u003c/em\u003e and \u003cem\u003ecggR\u003c/em\u003e demonstrated the most significant differential effects on EPS and γ-PGA production. The central glycolytic gene repressor, \u003cem\u003ecggR\u003c/em\u003e, is a member of SorC family of bacterial transcriptional regulators and governs genes and operons involved in carbohydrate catabolism. It acts as a transcriptional repressor of the \u003cem\u003egapA\u003c/em\u003e operon, which encodes central glycolytic enzymes [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. The downstream gene expression changes regulated by \u003cem\u003enrgB\u003c/em\u003e include the downregulation of \u003cem\u003ewbpA\u003c/em\u003e (nucleotide sugar dehydrogenase) and the upregulation of \u003cem\u003elytD\u003c/em\u003e (peptidoglycan hydrolase), collectively leading to the suppression of EPS synthesis.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study demonstrates that environmental stresses (pH 9 and 16 g/L NaCl) significantly increase γ-PGA production in \u003cem\u003eB. licheniformis\u003c/em\u003e while suppressing EPS synthesis. \u003cem\u003eB. licheniformis\u003c/em\u003e responds to these stresses by activating precursor synthesis, carbon source utilization, and amino acid metabolism. Key global regulators including \u003cem\u003ersbRA\u003c/em\u003e, \u003cem\u003erapA\u003c/em\u003e, enhance EPS synthesis, whereas \u003cem\u003eccpA\u003c/em\u003e-2 have positive effects on the improvement of both polymers. Notably, \u003cem\u003ecggR\u003c/em\u003e and \u003cem\u003enrgB\u003c/em\u003e have pronounced differential effects on EPS and γ-PGA production. Specifically, nrgB primarily regulates genes related to carbon metabolism, energy metabolism, signal transduction, and membrane transport. This study posits that identifying key targets of global transcriptional regulators is essential for improving the production of two types of extracellular polymeric substances in engineered microorganisms. Further research is necessary to achieve comprehensive, large-scale identification of advantageous gene targets involved in the production process of these extracellular polymeric substances.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eExopolysaccharides\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEPS\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003epoly-γ-glutamic acid\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eγ-PGA\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eAll authors have given their consent for the publication of this research paper and its accompanying materials.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eSupplementary information\u003c/h2\u003e\u003cp\u003eSupplementary data associated with this article can be found in the online version of the paper.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was financially supported by the National Natural Science Foundation of China (22208276 and 32170061), the Natural Science Foundation of Xiamen (3502Z202572042), and the Third Institute of Oceanography, Ministry of Natural Resources, Basic Scientific Research Operating Expenses Project (2025016).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eXiaoyu Wei: methodology, investigation, formal analysis, the original draft. Zhen Chen, Ning He: data curation, project administration. Xiaoyu Wei, Ziwei Pan, Zhen Chen and Ning He :Writing\u0026mdash;review \u0026amp; editing. All authors read and approved the final version of the submitted manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors are grateful for funding support by Natural Science Foundation of Xiamen and the National Natural Science Foundation of China.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll the data of this study contained in the manuscript and further data can be obtained related to this study from Corresponding authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWei XY, Chen Z, Liu AL, Yang LJ, Xu YY, Cao MF, He N. Advanced strategies for metabolic engineering of \u003cem\u003eBacillus\u003c/em\u003e to produce extracellular polymeric substances. Biotechnol Adv. 2023;67:251\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFlemming HC, van Hullebusch ED, Little BJ, Neu TR, Nielsen PH, Seviour T, Stoodley P, Wingender J, Wuertz S. Microbial extracellular polymeric substances in the environment, technology and medicine. Nat Rev Microbiol. 2024;23:87\u0026ndash;105.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWei XY, Yang LJ, Chen Z, Xia WH, Chen YB, Cao MF, He N. Molecular weight control of poly-γ-glutamic acid reveals novel insights into extracellular polymeric substance synthesis in \u003cem\u003eBacillus licheniformis\u003c/em\u003e. Biotechnol Biofuels Bioprod. 2024;17:60.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen Z, Liu P, Li Z, Yu W, Wang Z, Yao H, Wang Y, Li Q, Deng X, He N. Identification of key genes involved in polysaccharide bioflocculant synthesis in \u003cem\u003eBacillus licheniformis\u003c/em\u003e. Biotechnol Bioeng. 2017;114:645\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHu SY, He PH, Zhang YJ, Jiang M, Wang Q, Yang SH, Chen SW. Transcription factor DegU-mediated multi-pathway regulation on lichenysin biosynthesis in Bacillus licheniformis. Metab Eng. 2022;74:108\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFeng J, Quan Y, Gu Y, Liu F, Huang X, Shen H, Dang Y, Cao M, Gao W, Lu X, et al. Enhancing poly-gamma-glutamic acid production in Bacillus amyloliquefaciens by introducing the glutamate synthesis features from Corynebacterium glutamicum. Microb Cell Fact. 2017;16:88.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFeng J, Gu Y, Quan Y, Cao M, Gao W, Zhang W, Wang S, Yang C, Song C. Improved poly-gamma-glutamic acid production in \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e by modular pathway engineering. Metab Eng. 2015;32:106\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFeng J, Gu YY, Quan YF, Zhang W, Cao MF, Gao WX, Song CJ, Yang C, Wang SF. Recruiting a new strategy to improve levan production in Bacillus amyloliquefaciens. Sci Rep 2015, 5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eQiu YB, Zhang YT, Zhu YF, Sha YY, Xu ZQ, Feng XH, Li S, Xu H. Improving poly-(gamma-glutamic acid) production from a glutamic acid-independent strain from inulin substrate by consolidated bioprocessing. Bioprocess Biosyst Eng. 2019;42:1711\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWei X, Tian G, Ji Z, Chen S. A new strategy for enhancement of poly-γ-glutamic acid production by multiple physicochemical stresses in \u003cem\u003eBacillus licheniformis\u003c/em\u003e. J Chem Technol Biotechnol. 2015;90:709\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWei XT, Tian GM, Ji ZX, Chen SW. A new strategy for enhancement of poly-γ-glutamic acid production by multiple physicochemical stresses in Bacillus licheniformis. J Chem Technol Biotechnol. 2015;90:709\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJiang J, Sun YF, Tang X, He CN, Shao YL, Tang YJ, Zhou WW. Alkaline pH shock enhanced production of validamycin A in fermentation of Streptomyces hygroscopicus. Bioresour Technol. 2018;249:234\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSandhya V, Ali SZ. The production of exopolysaccharide by Pseudomonas putida GAP-P45 under various abiotic stress conditions and its role in soil aggregation. Microbiology. 2015;84:512\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSilambarasan S, Logeswari P, Cornejo P, Kannan VR. Evaluation of the production of exopolysaccharide by plant growth promoting yeast Rhodotorula sp. strain CAH2 under abiotic stress conditions. Int J Biol Macromol. 2019;121:55\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNachtigall C, Rohm H, Jaros D. Degradation of Exopolysaccharides from Lactic Acid Bacteria by Thermal, Chemical, Enzymatic and Ultrasound Stresses. \u003cem\u003eFoods\u003c/em\u003e 2021, 10.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu W, Chen Z, Shen L, Wang Y, Li Q, Yan S, Zhong CJ, He N. Proteomic profiling of \u003cem\u003eBacillus licheniformis\u003c/em\u003e reveals a stress response mechanism in the synthesis of extracellular polymeric flocculants. Biotechnol Bioeng. 2016;113:797\u0026ndash;806.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHsueh YH, Huang KY, Kunene SC, Lee TY. Poly-γ-glutamic acid synthesis, gene regulation, phylogenetic relationships, and role in fermentation. Int J Mol Sci. 2017;18:2644.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHu LX, Zhao M, Hu WS, Zhou MJ, Huang JB, Huang XL, Gao XL, Luo YN, Li C, Liu K, et al. Poly-γ-glutamic acid production by engineering a DegU quorum-sensing circuit in \u003cem\u003eBacillus subtilis\u003c/em\u003e. ACS Synth Biol. 2022;11:4156\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWei XY, Yang LJ, Wang HY, Chen Z, Xu YY, Weng Y, Cao MF, Li QB, He N. Genomic and metabolomic analysis of Bacillus licheniformis with enhanced poly-gamma-glutamic acid production through atmospheric and room temperature plasma mutagenesis. Front Chem Sci Eng.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu YY, Yang LJ, Wang HY, Wei XY, Shi YY, Liang DF, Cao MF, He N. Putative functions of EpsK in teichuronic acid synthesis and phosphate starvation in \u003cem\u003eBacillus licheniformis\u003c/em\u003e. Synth Syst Biotechnol. 2022;7:815\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLivak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C) method. Methods. 2001;25:402\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLangmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9:357\u0026ndash;U354.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi DF, Hou LZ, Gao YX, Tian ZL, Fan B, Wang FZ, Li SY. Recent advances in microbial synthesis of poly-gamma-glutamic acid: A review. Foods 2022, 11.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGuan NZ, Liu L. Microbial response to acid stress: mechanisms and applications. Appl Microbiol Biotechnol. 2020;104:51\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCao HJ, Villatoro-Hernandez J, Weme RDO, Frenzel E, Kuipers OP. Boosting heterologous protein production yield by adjusting global nitrogen and carbon metabolic regulatory networks in \u003cem\u003eBacillus subtilis\u003c/em\u003e. Metab Eng. 2018;49:143\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTan FR, Wu B, Dai LC, Qin H, Shui ZX, Wang JL, Zhu QL, Hu GQ, He MX. Using global transcription machinery engineering (gTME) to improve ethanol tolerance of \u003cem\u003eZymomonas mobilis\u003c/em\u003e. Microb Cell Fact 2016, 15.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDeo R, Lakra U, Ojha M, Nigam VK, Sharma SR. Exopolysaccharides in microbial interactions: signalling, quorum sensing, and community dynamics. Nat Prod Res 2024:1\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGuttenplan SB, Blair KM, Kearns DB. The EpsE Flagellar Clutch Is Bifunctional and Synergizes with EPS Biosynthesis to Promote Bacillus subtilis Biofilm Formation. PLos Genet 2010, 6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePatek M, Nesvera J. Sigma factors and promoters in \u003cem\u003eCorynebacterium glutamicum\u003c/em\u003e. J Biotechnol. 2011;154:101\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang J, Li X, Wang JF, Wei W, Jin WJ, Zhou LB. Comparative proteomics reveals energy and carbon metabolism changes in Scenedesmus quadricauda mutants induced by heavy-ion beam irradiation. Bioresour Technol 2024, 406.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKoberstein JN, Stewart ML, Smith CB, Tarasov AI, Ashcroft FM, Stork PJS, Goodman RH. Monitoring glycolytic dynamics in single cells using a fluorescent biosensor for fructose 1,6-bisphosphate. Proc Natl Acad Sci USA 2022, 119.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAgrawal P, Gupta P, Swaminathan K, Parkesh R. alpha-Glucan pathway as a novel Mtb drug target: Structural insights and cues for polypharmcological targeting of GlgB and GlgE. Curr Med Chem. 2014;21:4074\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang JF, Xu XJ, Li XY, Chen XJ, Zhou CX, Liu YH, Li Y, Lu FP. Reducing the cell lysis to enhance yield of acid-stable alpha amylase by deletion of multiple peptidoglycan hydrolase-related genes in Bacillus amyloliquefaciens. Int J Biol Macromol. 2021;167:777\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCremer J, Honda T, Tang Y, Wong-Ng J, Vergassola M, Hwa T. Chemotaxis as a navigation strategy to boost range expansion. Nature. 2019;575:658\u0026ndash;.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWei XT, Ji ZX, Chen SW. Isolation of halotolerant \u003cem\u003eBacillus licheniformis\u003c/em\u003e WX-02 and regulatory effects of sodium chloride on yield and molecular sizes of poly-gamma-glutamic acid. Appl Biochem Biotechnol. 2010;160:1332\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBhat AR, Irorere VU, Bartlett T, Hill D, Kedia G, Charalampopoulos D, Nualkaekul S, Radecka I. Improving survival of probiotic bacteria using bacterial poly-gamma-glutamic acid. Int J Food Microbiol. 2015;196:24\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eQuach NT, Vu THN, Nguyen TTA, Ha H, Ho PH, Chu-Ky S, Nguyen LH, Nguyen HV, Thanh TTT, Nguyen NA et al. Structural and genetic insights into a poly-gamma-glutamic acid with in vitro antioxidant activity of \u003cem\u003eBacillus velezensis\u003c/em\u003e VCN56. World J Microbiol Biotechnol 2022, 38.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eParati M, Khalil I, Tchuenbou-Magaia F, Adamus G, Mendrek B, Hill R, Radecka I. Building a circular economy around poly (D/L-gamma-glutamic acid)- a smart microbial biopolymer. Biotechnol Adv. 2022;61:495\u0026ndash;507.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang FR, Chen JL, Ye SH, Liu ZF, Ding Y. Characterization of antioxidant activity of exopolysaccharides from endophytic \u003cem\u003eLysinibacillus sphaericus\u003c/em\u003e Ya6 under osmotic stress conditions. Process Biochem. 2022;113:87\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi FS, Hu X, Sun XJ, Li HS, Lu JR, Li YM, Bao MT. Effect of fermentation pH on the structure, rheological properties, and antioxidant activities of exopolysaccharides produced by \u003cem\u003eAlteromonas australica\u003c/em\u003e QD. Glycoconj J. 2022;39:773\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang LL, Chen JT, Wang LF, Wu S, Zhang GZ, Yu HQ, Ye XD, Shi QS. Conformations and molecular interactions of poly-gamma-glutamic acid as a soluble microbial product in aqueous solutions. Sci Rep 2017, 7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBashir A, Hoffmann T, Kempf B, Xie XL, Smits SHJ, Bremer E. Plant-derived compatible solutes proline betaine and betonicine confer enhanced osmotic and temperature stress tolerance to \u003cem\u003eBacillus subtilis\u003c/em\u003e. Microbiology-Sgm. 2014;160:2283\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHoffmann T, Wensing A, Brosius M, Steil L, Volker U, Bremer E. Osmotic control of \u003cem\u003eopuA\u003c/em\u003e expression in \u003cem\u003eBacillus subtilis\u003c/em\u003e and its modulation in response to intracellular glycine betaine and proline pools. J Bacteriol. 2013;195:510\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMorawska LP, Weme R, Frenzel E, Dirkzwager M, Hoffmann T, Bremer E, Kuipers OP. Stress-induced activation of the proline biosynthetic pathway in \u003cem\u003eBacillus subtilis\u003c/em\u003e: a population-wide and single-cell study of the osmotically controlled proHJ promoter. Microb Biotechnol. 2022;15:2411\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSong QS, Joshi M, DiPiazza J, Joshi V. Functional relevance of citrulline in the vegetative tissues of watermelon during abiotic stresses. Front Plant Sci 2020, 11.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJoshi V, Fernie AR. Citrulline metabolism in plants. Amino Acids. 2017;49:1543\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStecker D, Hoffmann T, Link H, Commichau FM, Bremer E. L-proline synthesis mutants of \u003cem\u003eBacillus subtilis\u003c/em\u003e overcome osmotic sensitivity by genetically adapting L-arginine metabolism. Front Microbiol 2022, 13.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMusa YR, B\u0026auml;sell K, Schatschneider S, Vorh\u0026ouml;lter FJ, Becher D, Niehaus K. Dynamic protein phosphorylation during the growth of Xanthomonas campestris pv. campestris B100 revealed by a gel-based proteomics approach. J Biotechnol. 2013;167:111\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHe K, Xin YP, Shan Y, Zhang X, Song HH, Fang WH. Phosphorylation residue T175 in RsbR protein is required for efficient induction of sigma B factor and survival of \u003cem\u003eListeria monocytogenes\u003c/em\u003e under acidic stress. J Zhejiang University-Science B. 2019;20:660\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJeon J, Kim D, Jeon TJ. Opposite functions of RapA and RapC in cell adhesion and migration in Dictyostelium. Anim Cells Syst. 2021;25:203\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu N, Sun HW, Tang ZY, Zheng YQ, Qi GF, Zhao XY. Transcription Factor Spo0A Regulates the Biosynthesis of Difficidin in Bacillus amyloliquefaciens. Microbiol Spectr 2023, 11.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang Y, Xiao F, Zhang L, Ding Z, Shi G, Li Y. A new mechanism of carbon metabolism and acetic acid balance regulated by CcpA. Microorganisms 2023, 11.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDhali D, Coutte F, Arias AA, Auger S, Bidnenko V, Chataign\u0026eacute; G, Lalk M, Niehren J, de Sousa J, Versari C, Jacques P. Genetic engineering of the branched fatty acid metabolic pathway of \u003cem\u003eBacillus subtilis\u003c/em\u003e for the overproduction of surfactin C14 isoform. Biotechnol J 2017, 12.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJaehme M, Slotboom DJ. Structure, function, evolution, and application of bacterial Pnu-type vitamin transporters. Biol Chem. 2015;396:955\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYou JJ, Yang C, Pan XW, Hu MK, Du YX, Osire T, Yang TW, Rao ZM. Metabolic engineering of Bacillus subtilis for enhancing riboflavin production by alleviating dissolved oxygen limitation. Bioresour Technol 2021, 333.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChaix D, Ferguson ML, Atmanene C, Van Dorsselaer A, Sanglier-Cianf\u0026eacute;rani S, Royer CA, Declerck N. Physical basis of the inducer-dependent cooperativity of the Central glycolytic genes Repressor/DNA complex. Nucleic Acids Res. 2010;38:5944\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZorrilla S, Chaix D, Ortega A, Alfonso C, Doan T, Margeat E, Rivas G, Aymerich S, Declerckt N, Royer CA. Fructose-1,6-bisphosphate acts both as an inducer and as a structural cofactor of the central glycolytic genes repressor (CggR). Biochemistry. 2007;46:14996\u0026ndash;5008.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bacillus licheniformis, γ-PGA, EPS, Stress response, Global transcriptional regulators, Omics studies","lastPublishedDoi":"10.21203/rs.3.rs-7275147/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7275147/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e The diverse metabolic mechanisms underlying bacterial extracellular polymeric substances give rise to a wide array of components with distinct functionalities, including exopolysaccharides (EPS) and poly-γ-glutamic acid (γ-PGA). The coordinated synthesis of various types of extracellular polymeric substances necessitates comprehensive investigation from a global regulatory perspective.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e In this study, we examined the impact of multiple environmental stressors on \u003cem\u003eBacillus\u003c/em\u003especies, revealing that the EPS and γ-PGA produced respond to stress through metabolic and cellular process reorganization. The expression of global transcriptional regulators influenced the production of EPS and γ-PGA differently. Specifically, quorum sensing-related global regulators such as \u003cem\u003ersbRA\u003c/em\u003e, \u003cem\u003erapA\u003c/em\u003e, and the carbon utilization regulator \u003cem\u003eccpA\u003c/em\u003e-2 were found to enhance EPS synthesis. Conversely, positive global transcriptional regulators associated with γ-PGA synthesis included carbon and nitrogen utilization-related regulators \u003cem\u003eccpA\u003c/em\u003e-2, \u003cem\u003ecggR\u003c/em\u003e, and \u003cem\u003enrgB\u003c/em\u003e. Notably, the global regulators \u003cem\u003enrgB\u003c/em\u003e and \u003cem\u003ecggR\u003c/em\u003e increased γ-PGA production by 33.64% and 44.14%, respectively, while this enhancement was accompanied by a concomitant reduction in EPS production. In \u003cem\u003eB. licheniformis\u003c/em\u003e, omics analyses have elucidated critical pathways and metabolites implicated in stress response mechanisms that induce alterations in amino acid metabolism, carbon source utilization, alongside the activation of global regulatory elements. These studies indicated that \u003cem\u003enrgB\u003c/em\u003e predominantly governs downstream genes associated with carbon metabolism, energy metabolism, signal transduction, and membrane transport processes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e This work combines stress induction strategies and global transcription machinery engineering for investigating the coordinated synthesis of various types of extracellular polymeric substances, which has not been explored before. The insights gained from our research contribute to a deeper understanding of the regulatory networks governing the competition between γ-PGA and EPS, thereby providing a theoretical basis for the engineered modification of \u003cem\u003eBacillus licheniformis\u003c/em\u003e aimed at optimizing the production of extracellular polymeric substances.\u003c/p\u003e","manuscriptTitle":"Stress response regulation to extracellular polymeric substances biosynthesis in Bacillus licheniformis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-13 16:36:50","doi":"10.21203/rs.3.rs-7275147/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-08T09:02:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-26T11:39:18+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-14T22:34:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"208264295316882280821947420448586358757","date":"2025-08-12T08:20:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"211501729632349649647283301186253906787","date":"2025-08-10T13:49:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-08T06:47:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-06T08:53:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-06T08:53:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbial Cell Factories","date":"2025-08-02T03:13:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8a21abcc-192c-48c5-9066-d81696c927dd","owner":[],"postedDate":"August 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-20T16:00:37+00:00","versionOfRecord":{"articleIdentity":"rs-7275147","link":"https://doi.org/10.1186/s12934-025-02846-2","journal":{"identity":"microbial-cell-factories","isVorOnly":false,"title":"Microbial Cell Factories"},"publishedOn":"2025-10-14 15:57:18","publishedOnDateReadable":"October 14th, 2025"},"versionCreatedAt":"2025-08-13 16:36:50","video":"","vorDoi":"10.1186/s12934-025-02846-2","vorDoiUrl":"https://doi.org/10.1186/s12934-025-02846-2","workflowStages":[]},"version":"v1","identity":"rs-7275147","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7275147","identity":"rs-7275147","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}