Strain Engineering and Pathway Enhancement of Bacillus subtilis for Efficient Hyaluronic Acid Production

Strain Engineering and Pathway Enhancement of Bacillus subtilis for Efficient Hyaluronic Acid Production | 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 Article Strain Engineering and Pathway Enhancement of Bacillus subtilis for Efficient Hyaluronic Acid Production Rouzbeh Almasi Ghale, Reza Faghihi, Marjan Talebi, Mehdi Shamsara, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7842276/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Hyaluronic acid (HA) is a biopolymer with broad biomedical and pharmaceutical uses, yet microbial HA production is limited by strain safety, and process inefficiencies; to address these challenges we engineered a biosafe Bacillus subtilis chassis by integrating catalytic genes identified from the BRENDA database. Two constructs on the pHT01 shuttle vector were prepared, a dual-gene plasmid ( hasA –NX02_04625) and a single gene plasmid ( hasA ), and following transformation and molecular confirmation recombinant colonies were screened in different media to identify optimal production conditions. HA was quantified by the CTAB assay, its structure validated by FTIR, and molecular weight distribution characterized by GPC. Cloning and molecular validation were successful, HA synthesis was detected only in engineered strains and not in wild type controls, and the dual-gene strain produced significantly more HA than the single gene strain, reaching up to 1.2 g/L in shake flask cultures. Preliminary bioreactor cultivation of the dual gene strain in the selected medium yielded 0.62 g/L HA. These results indicate that engineered B. subtilis is a biosafe, scalable platform for HA production, offering an industrially viable alternative to pathogenic hosts and highlighting a promising source for HA synthesis applicable to pharmaceutical, biomedical, and cosmetic industries. Biological sciences/Biological techniques Biological sciences/Biotechnology Biological sciences/Microbiology Hyaluronic acid production Bacillus subtilis 168 Strain development hasA Gene Metabolic Engineering NX02_04625 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Hyaluronic acid (HA) is a high-value, linear glycosaminoglycan renowned for its exceptional biocompatibility, viscoelasticity, and hygroscopic properties. Structurally composed of repeating disaccharide units of D-glucuronic acid (GlcUA) and N-acetyl-D-glucosamine (GlcNAc), HA is a ubiquitous component of the extracellular matrix (ECM) in vertebrates, where it regulates cell proliferation, tissue hydration, inflammation, and wound repair 1 . Due to its shear-thinning behavior and high moisture retention, HA has become indispensable in biomedicine (dermal fillers, viscosupplementation, drug delivery), cosmetics (anti-aging formulations), and regenerative medicine 2 . The unique physicochemical properties of HA, including its molecular weight-dependent biological functions, underpin its clinical versatility. High-molecular-weight HA (> 1 MDa) is anti-inflammatory and immunosuppressive, while lower molecular weights can stimulate angiogenesis and cell migration, making tailored HA products valuable for diverse therapeutic applications 3 , 4 . Despite its widespread applications, conventional HA production methods face critical limitations. Animal-derived extraction (e.g., rooster combs) poses risks of zoonotic contaminants and batch variability, while fermentation using pathogenic Streptococcus strains ( S. zooepidemicu s) raises biosafety concerns and complicates downstream processing 5 , 6 . The relatively low HA yield (~ 6–8 g/L in wild-type strains), when weighed against the high cost of downstream purification, renders conventional production methods economically and industrially unjustifiable. These challenges have accelerated efforts to develop GRAS (Generally Recognized As Safe) microbial platforms for sustainable HA synthesis 7 , 8 . Bacillus subtilis , a non-pathogenic, endotoxin-free, and industrially robust bacterium, has emerged as a versatile chassis for recombinant HA production 9 , 10 . Its well-annotated genome and high metabolic flux make it ideal for synthetic biology and metabolic engineering. However, native Bacillus subtilis lacks the hasA gene (hyaluronan synthase) essential for HA biosynthesis, while it naturally encodes tuaD (UDP-glucose dehydrogenase). Therefore, heterologous expression of hasA is required to reconstruct the HA biosynthetic pathway 8 , 11 , 12 . In this study, we developed a biosafe and genetically engineered Bacillus subtilis strain capable of efficient HA production through heterologous expression of two key genes: hasA , which encodes hyaluronan synthase and is absent in the native genome, and NX02_04625, a functional homolog of tuaD that enhances the supply of UDP-glucuronic acid precursors 1 . We constructed and compared single- and dual-gene expression systems, and evaluated various fermentation media to identify the most productive condition. This approach addresses biosafety concerns associated with pathogenic production strains and provides a promising foundation for scalable and safe HA biosynthesis. 2. Materials and Methods 2.1. Bacterial Strains, Plasmids, and Growth Conditions Escherichia coli DH5α was used for plasmid propagation, and Bacillus subtilis 168 served as the expression host for HA production. Cultures were grown in Luria–Bertani (LB) broth at 37°C with shaking at 180 rpm. Ampicillin (100 µg/mL) and chloramphenicol (10 µg/mL) were used for plasmid selection in E. coli and B. subtilis , respectively. 2.2. Gene Selection and Vector Design To confer HA biosynthetic capability to Bacillus subtilis 168, a key synthase gene ( hasA , EC 2.4.1.212) from Streptococcus dysgalactiae ATCC12394 was selected based on its catalytic performance reported in the BRENDA database. This gene encodes hyaluronan synthase with a turnover number (TN) of approximately 120, comprising 1254 bp and 417 amino acids (~ 47.8 kDa). To further enhance HA production, a second gene (NX02_04625) from Sphingomonas sanxanigenens DSM19645 was co-expressed. This gene encodes UDP-glucose 6-dehydrogenase, a homolog of tuaD , and was selected for its low Km and high TN values. It spans 1365 bp and encodes a 454-amino-acid protein (~ 48 kDa). Both genes were codon-optimized for B. subtilis and designed using SnapGene (version 3.2.1). They were sequentially inserted into the shuttle vector pHT01 under the IPTG-inducible promoter Pgrac01, forming a single transcriptional unit with an intergenic ribosome binding site (RBS) to enable independent translation. The final dual-gene construct (pHT01- hasA - NX02_04625) was 10,782 bp in length. Detailed information regarding reagents, suppliers, cloning sites, sequence validation, and complete gene feature tables is provided in the Supplementary Materials ( Tables S1–S2 ). A schematic representation of the dual-gene construct, designed in SnapGene (version 3.2.1), is shown in Fig. 1 . 2.2.1. Plasmid Dilution and Storage The dual-gene plasmid (pHT01-hasA-NX02_04625), hereafter referred to as PST, was reconstituted and diluted to a working concentration. Aliquots were prepared and stored at − 20°C for downstream applications. Detailed dilution schemes, final concentrations, and storage conditions are provided in Supplementary Protocol S1. 2.2.2. Transformation into E. coli DH5α Chemically competent E. coli DH5α cells were transformed using the standard heat-shock method with minor modifications. Recovery was performed in antibiotic-free LB, followed by plating on LB agar supplemented with ampicillin. Exact volumes, timings, and plating schemes are detailed in Supplementary Protocol S1 13 . 2.3. Cloning Procedures and Construct Validation To enable comparative analysis, two plasmids were utilized: the externally synthesized PST and a single-gene variant (pHT01- hasA ), hereafter referred to as PS, created in-house. Construct sizes were confirmed by gel electrophoresis: PST: 10,782 bp PS: 9,245 bp The ~ 1,537 bp reduction corresponds to the removal of the NX02_04625, including associated intergenic regions and tags. Agarose gels (0.7%) were prepared in TBE buffer and used for construct validation by electrophoresis. Details of gel casting, buffer composition, and run conditions are provided in Supplementary Protocol S3. 2.3.1. Enzymatic Digestion and Backbone Isolation To excise the NX02_04625 region, PST was digested with Xba I, resolved on agarose gel, and the backbone containing the hasA gene was excised and purified. Full reaction compositions, device settings, and gel preparation details are provided in Supplementary Protocol S2 and Table S1 . 2.3.2. Ligation and Control Reactions The purified backbone- hasA was ligated using T4 DNA ligase; PEG was included to enhance ligation efficiency. A ligation-negative control (no ligase) confirmed that colony formation required successful ligation. Ligation products were transformed into E. coli DH5α and plated on ampicillin LB agar. Colonies were analyzed by gel electrophoresis and hasA -specific colony PCR. Detailed ligation recipes, incubation schemes, and control setups are provided in Supplementary Protocol S1. 2.3.3. Plasmid Extraction and Culture Preparation Overnight E. coli DH5α cultures harboring either PS or PST were processed for plasmid isolation using a commercial kit following the manufacturer’s instructions. Media and buffer compositions are listed in Supplementary Table S3 . 2.3.3.1. Verification of Gel-Purified Plasmid Gel-purified PS was transformed into chemically competent E. coli DH5α by heat shock. Transformants were selected on ampicillin plates and screened by colony PCR to confirm the presence of the insert. Full volumes, timings, and recovery steps are in Supplementary Protocol S4. 2.4. Transformation into Bacillus subtilis 168 and Validation The minimum inhibitory concentration (MIC) of chloramphenicol for B. subtilis 168 was determined to be 10 µg/mL. Electrocompetent B. subtilis cells were transformed with PS or PST constructs via electroporation and selected on LB–chloramphenicol plates. Colonies were validated by colony PCR using gene-specific primers. Detailed protocols for cell preparation, electroporation parameters 14 , recovery, plating strategies, and PCR conditions are provided in Supplementary Protocols S5 and S6 , and Supplementary Table S3 , following standard procedures with minor modifications tailored to the host system. 2.4.1. Colony PCR for Construct Integration Colony PCR was performed on B. subtilis transformants to confirm the insertion of recombinant constructs. Two primer pairs were used: OptisehasA-F (“hf”) and OptiNX02-R (“nr”) for PST, yielding a ~ 1,000 bp product; and OptisehasA-F (“hf”) and OptisehasA-R (“hr”) for PS, yielding a ~ 500 bp product. PCR reactions were carried out using a standard Taq Master Mix and thermal cycling parameters as described for plasmid validation. Primer sequences, reaction mixes, and cycling conditions are provided in Supplementary Protocol S6 and Supplementary Table S4 . 2.5. HA Purification, and Quantification 2.5.1. HA Purification Engineered B. subtilis culture supernatants were clarified by centrifugation and precipitated sequentially with trichloroacetic acid and ethanol. The dried HA pellet was redissolved in deionized water. 2.5.2. CTAB Turbidimetric Assay HA concentration was determined by measuring the turbidity of HA–CTAB complexes in a microplate format. A standard curve was generated using commercial HA. Optical density at 540 nm was interpolated against the standard curve. Detailed protocols for HA recovery, ethanol precipitation, pellet solubilization, CTAB reagent preparation, microplate layout, and data analysis are provided in Supplementary Protocols S7–S8 15 . 2.6. Bioreactor Evaluation Batch fermentation was carried out with two different conditions aiming to enhance cell density and HA production. Pre-culture LB medium was prepared and supplemented with chloramphenicol for selective growth. Bacillus subtilis glycerol stock (PST) was used to inoculate tubes (0.5% v/v, Experiment 1, and 1% v/v, Experiment 2), which were incubated at 37°C and 180 rpm for 18 h. Main culture Fermentation was conducted in volume of 1.5 L in a stirred bioreactor (Pierre Guerin Tryton) using a modified medium based on Westbrook et al. 11 . Chloramphenicol was added post-sterilization. The fermenter was inoculated with 6.7% v/v (Experiment 1) and 2% v/v (Experiment 2) pre-culture and operated at two different conditions, which are summarized in Table 2 , to investigate the effect of pH control and temperature on dual gene B. subtilis growth and HA production. To control pH, NaOH 5 M was added to the fermentation broth automatically over a 72 h period of the fermentation process. Fermentation supernatants were processed by sequential TCA and ethanol precipitation. HA pellets were dried and redissolved in deionized water. The CTAB turbidimetric assay was used to quantify HA concentration. Detailed purification steps and assay conditions are provided in Supplementary Protocols S7–S8 . Fermentation medium composition is listed in Supplementary Table S5 . 2.7. Structural and Molecular Weight Characterization 2.7.1. FTIR Analysis Fourier-transform infrared (FTIR) spectroscopy was used to confirm the structural identity of purified hyaluronic acid. Spectra were recorded in the range of 4000–400 cm⁻¹ using standard scanning parameters. Characteristic peaks corresponding to hydroxyl, carboxyl, and amide groups were evaluated. Full instrument settings and spectral interpretation are provided in Supplementary Protocol S9 . 2.7.2. Molecular Weight Determination by GPC Gel permeation chromatography (GPC) was performed to determine the molecular weight distribution of HA samples. Water was used as the mobile phase, and detection was carried out via refractive index (RI). Samples were filtered through 0.45 µm membranes prior to injection. Detailed column specifications, calibration standards, and run conditions are provided in Supplementary Protocol S10. 3. Results 3.1. Gene Selection and Vector Design hasA (1,254 bp) from Streptococcus dysgalactiae and NX02_04625 (1,365 bp) from Sphingomonas sanxanigenens were codon-optimized for B. subtilis and assembled under the IPTG-inducible Pgrac01 promoter with an intergenic RBS in the pHT01 backbone to yield the dual-gene plasmid (PST, 10782 bp), synthesized and sequence-verified by Gene Universal Inc. A single-gene control plasmid (PS, ~ 9 kb) was constructed in-house. 3.2. Validation of Synthetic Constructs via PCR As an additional quality control, PCR was performed on the synthetic plasmid template (PST); single bands at ~ 540 bp ( hasA ) and ~ 1000 bp ( hasA –NX02_04625) were observed with no amplification in no-template controls (see Supplementary Figure S2 ). Primer sequences and PCR conditions are provided in Supplementary Table S4 and Protocol S6 . 3.3. Transformation into E. coli DH5α Successful transformation of PST into E. coli DH5α was confirmed qualitatively by the growth of discrete colonies on LB agar supplemented with 100 µg/mL ampicillin (see Supplementary Figure S1 ). 3.4. Recovery and Quality Control of the Dual-Gene Plasmid Prior to the construction of the single-gene plasmid, the PST was extracted from E. coli DH5α using a commercial miniprep kit (see Supplementary Figure S1 ). Plasmid integrity and size (~ 10.8 kb) were confirmed by agarose gel electrophoresis (see Supplementary Figure S3 ), ensuring suitability for downstream subcloning procedures. 3.5. Restriction Digestion of Dual-Gene Plasmid for Single-Gene Construct Preparation To generate the PS, the PST was digested with the restriction enzyme Xba I to remove the NX02_04625 coding sequence. The digestion reaction yielded two distinct fragments of approximately 9 kb and 1.5 kb, corresponding to the plasmid backbone containing the hasA and the excised NX02_04625 region, respectively. Agarose gel electrophoresis confirmed complete digestion and accurate fragment separation (see Supplementary Figure S4 ), validating the suitability of the backbone for subsequent ligation and transformation. 3.6. Recycling and Validation of the Vector Backbone Containing hasA for Single-Gene Construct Assembly The backbone fragment containing hasA (9,245 bp) was recovered and recycled for single-gene construct assembly (see Supplementary Figure S5 ). 3.7. Transformation of the Single-Gene Construct into E. coli DH5α To evaluate the necessity of ligation for plasmid circularization, chemically competent E. coli DH5α cells were transformed with either linearized or ligated pHT01- hasA . As a negative control, transformation of the linear construct (no T4 DNA ligase) yielded no colonies on LB–ampicillin agar, confirming that ligation is required for replication in the host. In contrast, transformation with the ligated construct produced numerous colonies on LB–ampicillin plates. After overnight incubation, clear colony expansion was observed, demonstrating the stability and viability of E. coli DH5α harboring the single-gene plasmid (see Supplementary Figure S6 ). 3.8. Confirmation of Single-Gene Construct via Plasmid Size Comparison To validate the successful removal of the NX02_04625 and generation of the PS plasmid DNA from both constructs, agarose gel electrophoresis was used. The single-gene plasmid exhibited a distinct band at 9245 bp, while the dual-gene plasmid showed a higher molecular weight band at 10782 bp. The upward shift in the PST relative to the PS variant confirms the expected size difference and supports the accuracy of the cloning procedure (see Fig. 2 ). 3.9. Electroporation of Bacillus subtilis 168 with PS and PST Constructs Electroporation of B. subtilis 168 with the PS and the PST was performed under identical conditions on LB agar supplemented with chloramphenicol (10 µg/mL). In both cases, transformants yielded distinct colonies, whereas the negative control cells electroporated without plasmid DNA showed no growth. Subsequent re-plating of colonies on fresh selective media confirmed stable propagation and plasmid maintenance. Colony formation by the PS construct is shown in Supplementary Figure S7 , and by the PST construct in Supplementary Figure S8 . 3.10. Colony PCR Confirmation of PS and PST Constructs in Bacillus subtilis 168 To verify the integration of both PS and PST constructs into chloramphenicol-resistant Bacillus subtilis 168 colonies, colony PCR was performed under identical conditions (see Supplementary Protocol S6 ). PS transformants produced a clear ~ 540-bp amplicon with hasA -specific primers, with no band in the negative control. PST transformants yielded a distinct ~ 1,000-bp product, with no amplification in the negative control or no-template control (NTC). These data confirm successful uptake, specific integration, and stable maintenance of both constructs in B. subtilis (see Supplementary Table S4 for primer sequences, Supplementary Figure S9 for ~ 540 bp gel, and Supplementary Figure S10 for ~ 1000 bp gel). 3.11. Initial Assessment of HA Yield in Wild-Type Versus PST Recombinant Bacillus subtilis HA production was assessed in two Bacillus subtilis 168 strains, wild-type (electrocompetent cell) and recombinant carrying the PST, at 24 and 48 hours post-induction using the CTAB turbidimetric method (CTM), (n = 3; see Supplementary Protocol S8 ). This pilot experiment was conducted in LB medium to establish a baseline comparison between the native and engineered strains in terms of HA biosynthetic capacity. No biologically meaningful HA production was detected in the wild-type strain at either time point. However, since the CTM relies on turbidity measurements at 540 nm, minor absorbance values may appear in HA-negative samples due to optical noise, suspended particles, or nonspecific interactions with CTAB. These values are considered background and do not reflect actual HA synthesis. In contrast, the recombinant strain produced 0.22 mg/mL HA at 24 hours and 0.31 mg/mL at 48 hours, while the wild-type strain yielded 0.04 mg/mL and 0.09 mg/mL at the corresponding time points. Both comparisons between recombinant and wild-type strains at 24 h and 48 h were statistically significant ( p < 0.05) ( Fig. 3 ) . Replicate values and full statistical analyses are provided in Supplementary Table S7 . HA concentrations were calculated using the standard curve shown in Supplementary Table S8 . 3.12. Comparison of HA Production in PS and PST Bacillus subtilis Strains Multiple media formulations were initially tested for HA production 16 , 17 , but only the modified Westbrook medium supported efficient synthesis and was therefore selected for all subsequent experiments. HA production was quantified at 24, 48, and 72 hours post-induction using the CTM (n = 2 per strain after outlier removal; see Supplementary Protocol S8 ). Cultures were grown in a modified Westbrook medium, adapted from the original formulation described by Westbrook et al., 11 with full composition provided in Supplementary Table S5 . Final HA concentrations for each strain and time point are summarized in Supplementary Table S6 , reported as mean ± SD based on two biological replicates. The calibration curve used for quantification is shown in Supplementary Figure S11 . In the dual-gene strain, HA concentration at 48 hours was 1.20 ± 0.03 mg/mL, and at 72 hours it was 1.14 ± 0.07 mg/mL. Statistical analysis showed no significant difference between these two time points ( p > 0.05 ). In the single-gene strain, HA concentration at 48 hours was 0.49 ± 0.07 mg/mL, and at 72 hours it was 0.50 ± 0.13 mg/mL. This difference was also not statistically significant ( p > 0.05 ). However, at both time points, the HA yield in the PST strain was significantly higher than that of the PS strain ( p < 0.05 ), confirming the superior performance of the dual-gene construct (Fig. 4 ). 3.13. FTIR Analysis of Produced HA To confirm the structural identity of the HA produced by the engineered Bacillus subtilis 168 carrying the PST construct, Fourier-transform infrared spectroscopy (FTIR) was performed. The IR spectrum of the purified sample exhibited characteristic absorption bands corresponding to key functional groups, including: O–H stretch at 3415 cm⁻¹ C–H stretch at 2934 cm⁻¹ Asymmetric COO⁻ stretch at 1683 cm⁻¹ Symmetric COO⁻ stretch at 1459 cm⁻¹ C–O–C stretch at 1097 cm⁻¹ 3.14. Molecular Weight Distribution by GPC Molecular Weight Parameters Quantitative MW data for fractions within the calibration range (1,030–1.18×10⁶ g/mol, PEG standards) are reported in Table 1 . Table 1 . Table 1 Molecular weight distribution of validated HA fractions. Fraction % Area M n (g/mol) M w (g/mol) PDI Fragmented HA 24.69 8.26 × 10 5 9.88 × 10 5 1.20 Oligomers 31.86 4.12 × 10 4 5.54 × 10 4 1.34 M n : Number-average molecular weight; M w : Weight-average molecular weight; PDI: Polydispersity Index (M w / M n ). High-MW HA (Peak 1) exceeded the column’s calibration range; its MW is estimated as > 1.18×10⁶ g/mol but requires absolute quantification. The trimodal distribution indicates significant heterogeneity in the HA sample. The dominant high-MW fraction (43.45%) aligns with native HA’s intrinsic chain length, while the mid- and low-MW fractions (56.55% combined) suggest partial hydrolysis during processing. The narrow PDI (1.20) of fragmented HA implies controlled degradation, whereas the broader PDI (1.34) of oligomers reflects heterogeneous chain cleavage. 3.15. HA Production in Bioreactor Cultures This preliminary study evaluated recombinant Bacillus subtilis 168 (PST) strains under controlled bioreactor conditions (Table 2 ). In Experiment 2 (37°C, pH 6.0–7.0), PST produced 0.62 mg/mL of HA at 72 h, a 2.8-fold increase compared to Experiment 1 (0.22 mg/mL, 30°C, without pH control). As shown in Table 2 , both pH and temperature had a remarkable impact on cell growth and HA production. The recombinant PST B. subtilis 168 strain remained stable and sustained HA synthesis throughout the 72 h fermentation period. Sporulation was observed in Experiment 2 and is considered a potential consideration during scale-up for production. This study highlights the importance of tightly controlling fermentation parameters to achieve optimal HA yield. Table 2 . Table 2 Two batch fermentation conditions for culture and HA production by PST B. subtilis 168 Parameter Experiment 1 Experiment 2 Temp. 30°C 37°C pH Control None 6.0–7.0 (NaOH 5M) Agitation 180 rpm 200 rpm Aeration 2–3 vvm 5–6 vvm HA (72 h) 0.22 ± 0.03 mg/mL 0.62 ± 0.09 mg/mL OD 600 1.32 (56 h) 31.54 ± 1.22 (72h) Sporulation - Observed (48&72h) 4. Discussion The escalating demand for HA across the pharmaceutical, biomedical, and cosmetic industries necessitates a paradigm shift away from traditional, limited production methods. While extraction from animal tissues raises concerns about pathogen contamination and batch-to-batch variability, and fermentation with pathogenic Streptococcus strains introduces significant biosafety and regulatory hurdles, the development of efficient GRAS microbial platforms has become a central focus of metabolic engineering. This study successfully establishes a recombinant Bacillus subtilis 168 as a safe and effective cell factory for HA production by strategically addressing the fundamental metabolic limitations of this non-pathogenic host, achieving a final yield of 1.20 g/L in shake flasks and 0.62 g/L in a bioreactor. The core of our engineering strategy was the rational reconstruction and enhancement of the heterologous HA biosynthetic pathway. Native B. subtilis possesses the genetic machinery for UDP-N-acetylglucosamine (UDP-GlcNAc) synthesis but lacks the hasA gene and has a suboptimal capacity for producing the second precursor, UDP-glucuronic acid (UDP-GlcUA) 11 , 18 . Our approach involved the heterologous co-expression of two key genes: hasA from Streptococcus dysgalactiae and a high-performance tuaD homolog (NX02_04625) from Sphingomonas sanxanigenens , selected for its favorable enzymatic kinetics (low Km, high turnover number) from the BRENDA database. The results unequivocally validate this strategy. The dual-gene construct (pHT01- hasA - NX02_04625) demonstrated a significant advantage over the single-gene construct (pHT01- hasA ), achieving a yield of 1.20 ± 0.03 mg/mL in shake flasks compared to 0.50 ± 0.13 mg/mL at 72 hours, a 2.4-fold increase ( Fig. 4 ) . This stark difference underscores a critical bottleneck in the precursor supply chain, specifically the conversion of UDP-glucose to UDP-GlcUA, which is efficiently alleviated by the expression of NX02_04625. This finding aligns with and reinforces the metabolic engineering principle established by Westbrook et al 11 , who identified the augmentation of the UDP-GlcUA pool via the native tuaD gene as the most impactful intervention, boosting HA titer 5.6-fold in their B. subtilis system. Our use of a heterologous homolog with potentially superior catalytic properties further advances this strategy. Our achieved shake flask titer (~ 1.2 g/L) is notably higher than the 0.5–0.7 g/L often reported in early studies with similar systems 10 , 19 . The transition from shake flasks to a controlled bioreactor environment revealed the profound impact of physicochemical parameters on microbial performance and HA synthesis. Our bioreactor experiments yielded critical insights: The HA titer of 0.62 ± 0.09 mg/mL was achieved in experiment 2 (37°C, pH maintained at 6.0–7.0), which was a 182% increase over the yield in experiment 1 (30°C, no pH control), highlighting that suboptimal conditions severely constrain the engineered strain's potential. The maintained pH likely prevented acid-induced inhibition of enzyme activity and supported robust cell growth, as evidenced by the final OD 600 = 31.54 ± 1.22, which was an order of magnitude higher than in uncontrolled fermenter 17 , 20 . Product characterization confirmed the successful biosynthesis of authentic hyaluronic acid. FTIR spectroscopy provided definitive evidence, with all characteristic functional group peaks (O-H, C-H, COO-, C-O-C) matching the commercial standard. This confirms the functional fidelity of the heterologously expressed hasA synthase, a crucial step for biomedical applicability 21 . Gel Permeation Chromatography analysis revealed a heterogeneous molecular weight distribution, which is typical for microbial HA production due to the presence of endogenous hydrolases or shear stress 22 – 24 . The product profile was promising: a dominant fraction (43.45% of the total area) consisted of high-molecular-weight HA ( > > 1.18 MDa), which is highly valued for its superior rheological and biological properties in medical applications such as viscosupplementation and ophthalmic surgery 25 , 26 . The remaining fractions consisted of fragmented HA (PDI: 1.20) and oligomers (PDI: 1.34). While this heterogeneity may require further purification for specific applications, it also presents an opportunity, as low- and medium-MW HA fractions have their own unique therapeutic applications, such as in angiogenesis and wound healing 2 , 27 . The ability to produce a broad spectrum of MWs in a single fermentation could be advantageous. The weight-average molecular weight of the main fragmented fraction was calculated at 9.88 × 10 5 g/mol, which is within the highly desirable range for many commercial applications 28 . When benchmarked against other engineered non-pathogenic hosts, our platform demonstrates strong potential. Our optimized bioreactor titer (0.62 g/L) is comparable to early reports in Agrobacterium sp. (0.7–0.9 g/L) 29 , and exceeds yields from some Lactococcus lactis systems (0.3 g/L) 30 . While some hyper-producing engineered E. coli or Bacillus subtilis strains have reported titers exceeding 6–8 g/L 11, 18 , it is crucial to note that these often involve extensive multi-gene mutagenesis and pathway engineering over several generations. Our study, focusing on the strategic introduction of two key genes, provides a robust and simplified foundation. The primary advantage of our B. subtilis -based system is its combination of a GRAS status, high secretory capacity, well-known genetics, and absence of endotoxins, which collectively offer a more straightforward and safer path to industrial-scale production and regulatory approval for biomedical uses compared to E. coli 31 . 5. Conclusion In this study, we successfully engineered Bacillus subtilis for efficient and safe HA production by co-expressing hasA , encoding hyaluronan synthase, and NX02_04625, involved in the biosynthesis of the essential precursor UDP-glucuronic acid. The dual-gene recombinant strain demonstrated significantly enhanced HA yield compared to the single-gene and wild-type strains, underscoring the critical role of precursor supply in HA biosynthesis. Optimization of fermentation conditions, particularly pH control, substantially improved HA titers, with the highest yield reaching 0.62 ± 0.09 mg/mL in bioreactor cultures. Characterization of the purified HA by FTIR confirmed its chemical identity, showing all expected functional groups, while gel permeation chromatography analysis revealed a predominantly high molecular weight polymer fraction, suitable for biomedical and cosmetic applications. Compared to traditional extraction and pathogenic bacterial fermentation, recombinant production in B. subtilis offers advantages in biosafety, product purity, scalability, and environmental impact. Our findings establish this engineered B. subtilis platform as a robust candidate for industrial-scale HA biosynthesis, with potential to meet growing market demand for safe and high-quality HA. Future research should focus on minimizing sporulation, refining molecular weight distribution, and further fermentation process optimization to enhance yield and product consistency. Future optimization can focus on implementing a fed-batch strategy with controlled carbon source feeding to prevent potential catabolite repression and support prolonged production phases, as successfully demonstrated by others to achieve titers exceeding 6–8 g/L 11, 18 . The use of sucrose as a carbon source is economically advantageous, and its efficient hydrolysis and utilization is a positive feature of our system 32 . Collectively, this study successfully demonstrates a comprehensive strategy from strain construction to process optimization for HA production in a safe microbial host. We have confirmed that precursor supply is a critical bottleneck and that environmental parameters like pH and temperature are just as important as genetic design for maximizing yield. The engineered B. subtilis strain produces authentic HA with a significant proportion of high molecular weight polymer. Future work will focus on (i) deleting endogenous hydrolase genes to control and increase molecular weight, (ii) implementing fed-batch fermentation strategies with optimized feeding to boost titers to industrially competitive levels (> 2–3 g/L), and (iii) further engineering to decouple production from sporulation for more predictable and scalable processes 20 . This work solidifies B. subtilis as a premier chassis for the safe and efficient biomanufacturing of high-value biopolymers. Declarations Funding: This work is based upon research funded by Iran National Science Foundation (INSF) under project No.4024643. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors declare no competing interests. Acknowledgments: This study was conducted as part of the first author's PhD project, with financial support from the Iran National Science Foundation (INSF) and NIGEB. Data availability : All relevant data are included in the manuscript and its supplementary materials. Author Contributions Rouzbeh Almasi Ghale : Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Validation; Visualization; Writing original draft; Writing review & editing. Reza Faghihi ; Investigation; Formal analysis; Writing original draft; Visualization Marjan Talebi ; Formal analysis; Writing original draft; Writing review & editing; Visualization * Mehdi Shamsara : Conceptualization; Formal analysis; Investigation; Methodology; Supervision; Validation; Writing original draft; Writing review & editing. * Fatemeh Tabandeh : Conceptualization; Formal analysis; Investigation; Methodology; Project administration; Funding acquisition; Supervision; Validation; Writing original draft; Writing review & editing. References Saadati, F. et al. Advances and principles of hyaluronic acid production, extraction, purification, and its applications: a review. Int. J. Biol. Macromol. 143839 (2025). Talebi, M., Ghale, R. A., Asl, R. M. & Tabandeh, F. Advancements in characterization and preclinical applications of hyaluronic acid-based biomaterials for wound healing: a review. Carbohydr. Polym. Technol. Appl. 100706 (2025). Valachová, K. & Šoltés, L. Hyaluronan as a prominent biomolecule with numerous applications in medicine. Int. J. Mol. Sci. 22 , 7077 (2021). Jiang, Y. et al. High-molecular-weight hyaluronic acid can be used as a food additive to improve the symptoms of persistent inflammation, immunosuppression and catabolism syndrome (PICS). Biology 13 , 319 (2024). Shikina, E., Kovalevsky, R., Shirkovskaya, A. & Toukach, P. V. Prospective bacterial and fungal sources of hyaluronic acid: a review. Comput. Struct. Biotechnol. J. 20 , 6214–6236 (2022). Liu, L., Liu, Y., Li, J., Du, G. & Chen, J. Microbial production of hyaluronic acid: current state, challenges, and perspectives. Microb. Cell. Fact. 10 , 99 (2011). Grabowski, M., Gmyrek, D., Żurawska, M. & Trusek, A. Hyaluronic acid: production strategies, gel-forming properties, and advances in drug delivery systems. Gels 11 , 424 (2025). Manfrão-Netto, J. H. et al. Genetic strategies for improving hyaluronic acid production in recombinant bacterial culture. J. Appl. Microbiol. 132 , 822–840 (2022). Su, Y., Liu, C., Fang, H. & Zhang, D. Bacillus subtilis: a universal cell factory for industry, agriculture, biomaterials and medicine. Microb. Cell. Fact. 19 , 173 (2020). Widner, B. et al. Hyaluronic acid production in Bacillus subtilis. Appl. Environ. Microbiol. 71 , 3747–3752 (2005). Westbrook, A. W., Ren, X., Oh, J., Moo-Young, M. & Chou, C. P. Metabolic engineering to enhance heterologous production of hyaluronic acid in Bacillus subtilis. Metab. Eng. 47 , 401–413 (2018). Zhao, X., Chen, X., Xue, Y. & Wang, X. Development of an efficient iterative genome editing method in Bacillus subtilis using the CRISPR-AsCpf1 system. J. Basic. Microbiol. 62 , 824–832 (2022). Froger, A. & Hall, J. E. Transformation of plasmid DNA into E. coli using the heat shock method. J. Vis. Exp. e253 (2007). Harwood, C. R. & Cutting, S. M. Molecular Biological Methods for Bacillus, Vol. 35Wiley Chichester,. (1990). Chen, Y. H. & Wang, Q. Establishment of CTAB turbidimetric method to determine hyaluronic acid content in fermentation broth. Carbohydr. Polym. 78 , 178–181 (2009). Jin, P., Kang, Z., Yuan, P., Du, G. & Chen, J. Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168. Metab. Eng. 35 , 21–30 (2016). Chien, L. J. & Lee, C. K. Enhanced hyaluronic acid production in Bacillus subtilis by coexpressing bacterial hemoglobin. Biotechnol. Prog . 23 , 1017–1022 (2007). Jia, Y. et al. Metabolic engineering of Bacillus subtilis for the efficient biosynthesis of uniform hyaluronic acid with controlled molecular weights. Bioresour Technol. 132 , 427–431 (2013). Kim, J. H. et al. Selection of a Streptococcus equi mutant and optimization of culture conditions for the production of high molecular weight hyaluronic acid. Enzyme Microb. Technol. 19 , 440–445 (1996). Schallmey, M., Singh, A. & Ward, O. P. Developments in the use of Bacillus species for industrial production. Can. J. Microbiol. 50 , 1–17 (2004). Fallacara, A., Baldini, E., Manfredini, S. & Vertuani, S. Hyaluronic acid in the third millennium. Polymers 10 , 701 (2018). Iqbal, Z. et al. Discussion of the AED workshop. Pharm. World Sci. 19 , 246–250 (1997). Gomes, V. A. M., Netto, C. M., Carvalho, L. S. & Parachin, N. S. Heterologous hyaluronic acid production in Kluyveromyces lactis. Microorganisms 7 , 294 (2019). Güngör, G. et al. Bacterial hyaluronic acid production through an alternative extraction method and its characterization. J. Chem. Technol. Biotechnol. 94 , 1843–1852 (2019). Kogan, G., Šoltés, L., Stern, R. & Gemeiner, P. Hyaluronic acid: a natural biopolymer with a broad range of biomedical and industrial applications. Biotechnol. Lett. 29 , 17–25 (2007). Tabasi, A. et al. Improved production of food-grade hyaluronic acid in recombinant Corynebacterium glutamicum by medium optimization and feeding strategy. Appl. Food Biotechnol. 12 , 1–14 (2022). Tammi, R. H. et al. Transcriptional and post-translational regulation of hyaluronan synthesis. FEBS J. 278 , 1419–1428 (2011). La Gatta, A., Schiraldi, C., Papa, A. & De Rosa, M. Comparative analysis of commercial dermal fillers based on crosslinked hyaluronan: physical characterization and in vitro enzymatic degradation. Polym. Degrad. Stab. 96 , 630–636 (2011). Mao, Z. & Chen, R. R. Recombinant synthesis of hyaluronan by Agrobacterium sp. Biotechnol. Prog . 23 , 1038–1042 (2007). Chien, L. J. & Lee, C. K. Hyaluronic acid production by recombinant Lactococcus lactis. Appl. Microbiol. Biotechnol. 77 , 339–346 (2007). van Dijl, J. & Hecker, M. Bacillus subtilis: from soil bacterium to super-secreting cell factory. Microb. Cell. Fact. 12 , 3 (2013). Boels, I. C., van Kranenburg, R., Hugenholtz, J., Kleerebezem, M. & De Vos, W. M. Sugar catabolism and its impact on the biosynthesis and engineering of exopolysaccharide production in lactic acid bacteria. Int. Dairy. J. 11 , 723–732 (2001). Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterialsAlmasietalSciRepOct2025.docx.pdf Cite Share Download PDF Status: Posted Version 1 posted 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. 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17:45:43","extension":"html","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":113557,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7842276/v1/ddb2966b43fa342e42af464e.html"},{"id":94789808,"identity":"fa55e002-06a3-497c-9a3b-74556ba21991","added_by":"auto","created_at":"2025-10-30 17:45:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":136725,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic plasmid map of the dual-gene construct (pHT01-\u003cem\u003ehasA\u003c/em\u003e-\u003cem\u003eNX02_04625\u003c/em\u003e) designed for HA biosynthesis in \u003cem\u003eBacillus subtilis\u003c/em\u003e 168. The construct includes the IPTG-inducible Pgrac01 promoter, lacO operator, RBS, codon-optimized \u003cem\u003ehasA\u003c/em\u003eand \u003cem\u003eNX02_04625\u003c/em\u003e, and transcription terminators. Restriction sites used for cloning and excision (\u003cem\u003eBam\u003c/em\u003eHI, \u003cem\u003eSma\u003c/em\u003eI, \u003cem\u003eXba\u003c/em\u003eI) are indicated. The total plasmid length is 10,782 bp. Primer binding sites used for colony PCR verification are also shown: OptisehasA-F (forward primer for \u003cem\u003ehasA\u003c/em\u003e), OptisehasA-R (reverse primer for \u003cem\u003ehasA\u003c/em\u003e), and OptiNX02-R (reverse primer for \u003cem\u003eNX02_04625\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7842276/v1/02d4519384b24557eb07d1cb.png"},{"id":94825630,"identity":"b23febab-737a-40c2-b19a-64f6ed91a849","added_by":"auto","created_at":"2025-10-31 06:50:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":334622,"visible":true,"origin":"","legend":"\u003cp\u003eAgarose gel electrophoresis comparing plasmid sizes of (A) PS (9245 bp), and (B) PST (10782 bp) constructs. The upward shift of the PST band confirms successful excision of the NX02_04625 segment and integrity of the PS plasmid.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7842276/v1/42be17e57905d02dd0676114.png"},{"id":94789807,"identity":"f6ec1fa5-4e3b-4ede-b5e1-4def34e97bd6","added_by":"auto","created_at":"2025-10-30 17:45:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":89378,"visible":true,"origin":"","legend":"\u003cp\u003eHA production in wild-type (WT) and recombinant \u003cem\u003eBacillus subtilis\u003c/em\u003e 168 strains (PST) at 24 and 48 hours. The x-axis represents time post-induction (hours), and the y-axis indicates HA concentration (mg/mL). Bars represent mean ± SD (n = 3). PST at 24 h (0.22 mg/mL); PST at 48 h (0.31 mg/mL); WT at 24 h (0.04 mg/mL); WT at 48 h (0.09 mg/mL). Asterisks (*) indicate statistical significance (\u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7842276/v1/c48b0e593b3f79d906e93e50.png"},{"id":94789811,"identity":"de01f046-5a7b-4517-823a-75ea9c4eff51","added_by":"auto","created_at":"2025-10-30 17:45:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":82640,"visible":true,"origin":"","legend":"\u003cp\u003eHA production in recombinant \u003cem\u003eBacillus subtilis\u003c/em\u003e 168 strains carrying PS and PST constructs at 24, 48, and 72 hours post-induction. The x-axis represents time post-induction (hours), and the y-axis indicates HA concentration (mg/mL). Bars represent mean ± SD (n = 2). No significant difference was observed between 48 and 72 hours within each strain (\u003cem\u003ep \u0026gt; 0.05\u003c/em\u003e). \u0026nbsp;At both time points, HA production in the PST strain was significantly higher than in the PS strain (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7842276/v1/a28349a04884d352e341748a.png"},{"id":94789812,"identity":"4b4e1871-59dd-4988-9746-0bda3e2e59d5","added_by":"auto","created_at":"2025-10-30 17:45:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":96044,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectrum of HA produced by the engineered \u003cem\u003eBacillus subtilis\u003c/em\u003e 168 carrying the PST construct. The presence of characteristic absorption bands corresponding to O–H, C–H, asymmetric and symmetric COO⁻, and C–O–C functional groups confirms the structural integrity of the biosynthesized polymer.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7842276/v1/ecf826c5376a29886f937457.png"},{"id":94789817,"identity":"ab094a80-0a31-49a9-bff5-d0e95c85b44f","added_by":"auto","created_at":"2025-10-30 17:45:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":38221,"visible":true,"origin":"","legend":"\u003cp\u003eGPC chromatogram of HA produced by the engineered Bacillus subtilis 168 carrying PST construct strain showing (A) high-MW native HA, (B) fragmented HA, and (C) oligomers. Dashed lines denote calibration limits.\u003c/p\u003e\n\u003cp\u003ePeak 1 (RT = 11.884 min): 43.45% of total area, assigned to high-MW native HA (\u0026gt;1.18×10⁶ g/mol).\u003c/p\u003e\n\u003cp\u003ePeaks 2+3 (RT = 14.229–14.956 min): 24.69% of total area, assigned to moderately fragmented HA.\u003c/p\u003e\n\u003cp\u003ePeak 4 (RT = 17.030 min): 31.86% of total area, assigned to low-MW HA oligomers.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7842276/v1/2f442b9a3985e2478af06de5.png"},{"id":96247639,"identity":"4fbd3932-ea8c-4a10-a87f-dc3e99a4f2b3","added_by":"auto","created_at":"2025-11-19 07:27:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2639357,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7842276/v1/eac0f0b5-3679-403c-aa0e-4380296eab2f.pdf"},{"id":94789813,"identity":"08a123cb-fb4e-48ec-b168-f13b325cb863","added_by":"auto","created_at":"2025-10-30 17:45:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":939246,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialsAlmasietalSciRepOct2025.docx.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7842276/v1/e24ce843f7b3c832144573f2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Strain Engineering and Pathway Enhancement of Bacillus subtilis for Efficient Hyaluronic Acid Production","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eHyaluronic acid (HA) is a high-value, linear glycosaminoglycan renowned for its exceptional biocompatibility, viscoelasticity, and hygroscopic properties. Structurally composed of repeating disaccharide units of D-glucuronic acid (GlcUA) and N-acetyl-D-glucosamine (GlcNAc), HA is a ubiquitous component of the extracellular matrix (ECM) in vertebrates, where it regulates cell proliferation, tissue hydration, inflammation, and wound repair \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Due to its shear-thinning behavior and high moisture retention, HA has become indispensable in biomedicine (dermal fillers, viscosupplementation, drug delivery), cosmetics (anti-aging formulations), and regenerative medicine \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe unique physicochemical properties of HA, including its molecular weight-dependent biological functions, underpin its clinical versatility. High-molecular-weight HA (\u0026gt;\u0026thinsp;1 MDa) is anti-inflammatory and immunosuppressive, while lower molecular weights can stimulate angiogenesis and cell migration, making tailored HA products valuable for diverse therapeutic applications \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDespite its widespread applications, conventional HA production methods face critical limitations. Animal-derived extraction (e.g., rooster combs) poses risks of zoonotic contaminants and batch variability, while fermentation using pathogenic \u003cem\u003eStreptococcus\u003c/em\u003e strains (\u003cem\u003eS. zooepidemicu\u003c/em\u003es) raises biosafety concerns and complicates downstream processing \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The relatively low HA yield (~\u0026thinsp;6\u0026ndash;8 g/L in wild-type strains), when weighed against the high cost of downstream purification, renders conventional production methods economically and industrially unjustifiable. These challenges have accelerated efforts to develop GRAS (Generally Recognized As Safe) microbial platforms for sustainable HA synthesis \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e, a non-pathogenic, endotoxin-free, and industrially robust bacterium, has emerged as a versatile chassis for recombinant HA production \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Its well-annotated genome and high metabolic flux make it ideal for synthetic biology and metabolic engineering. However, native \u003cem\u003eBacillus subtilis\u003c/em\u003e lacks the \u003cem\u003ehasA\u003c/em\u003e gene (hyaluronan synthase) essential for HA biosynthesis, while it naturally encodes \u003cem\u003etuaD\u003c/em\u003e (UDP-glucose dehydrogenase). Therefore, heterologous expression of \u003cem\u003ehasA\u003c/em\u003e is required to reconstruct the HA biosynthetic pathway \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In this study, we developed a biosafe and genetically engineered \u003cem\u003eBacillus subtilis\u003c/em\u003e strain capable of efficient HA production through heterologous expression of two key genes: \u003cem\u003ehasA\u003c/em\u003e, which encodes hyaluronan synthase and is absent in the native genome, and NX02_04625, a functional homolog of \u003cem\u003etuaD\u003c/em\u003e that enhances the supply of UDP-glucuronic acid precursors \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. We constructed and compared single- and dual-gene expression systems, and evaluated various fermentation media to identify the most productive condition. This approach addresses biosafety concerns associated with pathogenic production strains and provides a promising foundation for scalable and safe HA biosynthesis.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Bacterial Strains, Plasmids, and Growth Conditions\u003c/h2\u003e\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e DH5α was used for plasmid propagation, and \u003cem\u003eBacillus subtilis\u003c/em\u003e 168 served as the expression host for HA production. Cultures were grown in Luria\u0026ndash;Bertani (LB) broth at 37\u0026deg;C with shaking at 180 rpm. Ampicillin (100 \u0026micro;g/mL) and chloramphenicol (10 \u0026micro;g/mL) were used for plasmid selection in \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eB. subtilis\u003c/em\u003e, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Gene Selection and Vector Design\u003c/h2\u003e\u003cp\u003eTo confer HA biosynthetic capability to \u003cem\u003eBacillus subtilis\u003c/em\u003e 168, a key synthase gene (\u003cem\u003ehasA\u003c/em\u003e, EC 2.4.1.212) from \u003cem\u003eStreptococcus dysgalactiae\u003c/em\u003e ATCC12394 was selected based on its catalytic performance reported in the BRENDA database. This gene encodes hyaluronan synthase with a turnover number (TN) of approximately 120, comprising 1254 bp and 417 amino acids (~\u0026thinsp;47.8 kDa).\u003c/p\u003e\u003cp\u003eTo further enhance HA production, a second gene (NX02_04625) from \u003cem\u003eSphingomonas sanxanigenens\u003c/em\u003e DSM19645 was co-expressed. This gene encodes UDP-glucose 6-dehydrogenase, a homolog of \u003cem\u003etuaD\u003c/em\u003e, and was selected for its low Km and high TN values. It spans 1365 bp and encodes a 454-amino-acid protein (~\u0026thinsp;48 kDa).\u003c/p\u003e\u003cp\u003eBoth genes were codon-optimized for \u003cem\u003eB. subtilis\u003c/em\u003e and designed using SnapGene (version 3.2.1). They were sequentially inserted into the shuttle vector pHT01 under the IPTG-inducible promoter Pgrac01, forming a single transcriptional unit with an intergenic ribosome binding site (RBS) to enable independent translation. The final dual-gene construct (pHT01-\u003cem\u003ehasA\u003c/em\u003e- NX02_04625) was 10,782 bp in length. Detailed information regarding reagents, suppliers, cloning sites, sequence validation, and complete gene feature tables is provided in the Supplementary Materials (\u003cb\u003eTables S1\u0026ndash;S2\u003c/b\u003e). A schematic representation of the dual-gene construct, designed in SnapGene (version 3.2.1), is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.2.1. Plasmid Dilution and Storage\u003c/h2\u003e\u003cp\u003eThe dual-gene plasmid (pHT01-hasA-NX02_04625), hereafter referred to as PST, was reconstituted and diluted to a working concentration. Aliquots were prepared and stored at \u0026minus;\u0026thinsp;20\u0026deg;C for downstream applications. Detailed dilution schemes, final concentrations, and storage conditions are provided in Supplementary \u003cb\u003eProtocol S1.\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.2.2. Transformation into \u003cem\u003eE. coli\u003c/em\u003e DH5α\u003c/h2\u003e\u003cp\u003eChemically competent \u003cem\u003eE. coli\u003c/em\u003e DH5α cells were transformed using the standard heat-shock method with minor modifications. Recovery was performed in antibiotic-free LB, followed by plating on LB agar supplemented with ampicillin. Exact volumes, timings, and plating schemes are detailed in Supplementary \u003cb\u003eProtocol S1\u003c/b\u003e \u003csup\u003e13\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Cloning Procedures and Construct Validation\u003c/h2\u003e\u003cp\u003eTo enable comparative analysis, two plasmids were utilized: the externally synthesized PST and a single-gene variant (pHT01-\u003cem\u003ehasA\u003c/em\u003e), hereafter referred to as PS, created in-house. Construct sizes were confirmed by gel electrophoresis:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003ePST: 10,782 bp\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003ePS: 9,245 bp\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eThe ~\u0026thinsp;1,537 bp reduction corresponds to the removal of the NX02_04625, including associated intergenic regions and tags. Agarose gels (0.7%) were prepared in TBE buffer and used for construct validation by electrophoresis. Details of gel casting, buffer composition, and run conditions are provided in Supplementary \u003cb\u003eProtocol S3.\u003c/b\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1. Enzymatic Digestion and Backbone Isolation\u003c/h2\u003e\u003cp\u003eTo excise the NX02_04625 region, PST was digested with \u003cem\u003eXba\u003c/em\u003eI, resolved on agarose gel, and the backbone containing the \u003cem\u003ehasA\u003c/em\u003e gene was excised and purified. Full reaction compositions, device settings, and gel preparation details are provided in Supplementary \u003cb\u003eProtocol S2\u003c/b\u003e and \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2. Ligation and Control Reactions\u003c/h2\u003e\u003cp\u003eThe purified backbone-\u003cem\u003ehasA\u003c/em\u003e was ligated using T4 DNA ligase; PEG was included to enhance ligation efficiency. A ligation-negative control (no ligase) confirmed that colony formation required successful ligation. Ligation products were transformed into \u003cem\u003eE. coli\u003c/em\u003e DH5α and plated on ampicillin LB agar. Colonies were analyzed by gel electrophoresis and \u003cem\u003ehasA\u003c/em\u003e-specific colony PCR. Detailed ligation recipes, incubation schemes, and control setups are provided in Supplementary \u003cb\u003eProtocol S1.\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.3.3. Plasmid Extraction and Culture Preparation\u003c/h2\u003e\u003cp\u003eOvernight \u003cem\u003eE. coli\u003c/em\u003e DH5α cultures harboring either PS or PST were processed for plasmid isolation using a commercial kit following the manufacturer\u0026rsquo;s instructions. Media and buffer compositions are listed in Supplementary \u003cb\u003eTable S3\u003c/b\u003e.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section4\"\u003e\u003ch2\u003e2.3.3.1. Verification of Gel-Purified Plasmid\u003c/h2\u003e\u003cp\u003eGel-purified PS was transformed into chemically competent \u003cem\u003eE. coli\u003c/em\u003e DH5α by heat shock. Transformants were selected on ampicillin plates and screened by colony PCR to confirm the presence of the insert. Full volumes, timings, and recovery steps are in Supplementary \u003cb\u003eProtocol S4.\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Transformation into \u003cem\u003eBacillus subtilis\u003c/em\u003e 168 and Validation\u003c/h2\u003e\u003cp\u003eThe minimum inhibitory concentration (MIC) of chloramphenicol for \u003cem\u003eB. subtilis\u003c/em\u003e 168 was determined to be 10 \u0026micro;g/mL. Electrocompetent \u003cem\u003eB. subtilis\u003c/em\u003e cells were transformed with PS or PST constructs via electroporation and selected on LB\u0026ndash;chloramphenicol plates. Colonies were validated by colony PCR using gene-specific primers. Detailed protocols for cell preparation, electroporation parameters \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, recovery, plating strategies, and PCR conditions are provided in Supplementary \u003cb\u003eProtocols S5\u003c/b\u003e and \u003cb\u003eS6\u003c/b\u003e, and Supplementary \u003cb\u003eTable S3\u003c/b\u003e, following standard procedures with minor modifications tailored to the host system.\u003c/p\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.4.1. Colony PCR for Construct Integration\u003c/h2\u003e\u003cp\u003eColony PCR was performed on \u003cem\u003eB. subtilis\u003c/em\u003e transformants to confirm the insertion of recombinant constructs. Two primer pairs were used: OptisehasA-F (\u0026ldquo;hf\u0026rdquo;) and OptiNX02-R (\u0026ldquo;nr\u0026rdquo;) for PST, yielding a\u0026thinsp;~\u0026thinsp;1,000 bp product; and OptisehasA-F (\u0026ldquo;hf\u0026rdquo;) and OptisehasA-R (\u0026ldquo;hr\u0026rdquo;) for PS, yielding a\u0026thinsp;~\u0026thinsp;500 bp product.\u003c/p\u003e\u003cp\u003ePCR reactions were carried out using a standard Taq Master Mix and thermal cycling parameters as described for plasmid validation. Primer sequences, reaction mixes, and cycling conditions are provided in Supplementary \u003cb\u003eProtocol S6\u003c/b\u003e and Supplementary \u003cb\u003eTable S4\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.5. HA Purification, and Quantification\u003c/h2\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e2.5.1. HA Purification\u003c/h2\u003e\u003cp\u003eEngineered \u003cem\u003eB. subtilis\u003c/em\u003e culture supernatants were clarified by centrifugation and precipitated sequentially with trichloroacetic acid and ethanol. The dried HA pellet was redissolved in deionized water.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e2.5.2. CTAB Turbidimetric Assay\u003c/h2\u003e\u003cp\u003eHA concentration was determined by measuring the turbidity of HA\u0026ndash;CTAB complexes in a microplate format. A standard curve was generated using commercial HA. Optical density at 540 nm was interpolated against the standard curve.\u003c/p\u003e\u003cp\u003eDetailed protocols for HA recovery, ethanol precipitation, pellet solubilization, CTAB reagent preparation, microplate layout, and data analysis are provided in Supplementary \u003cb\u003eProtocols S7\u0026ndash;S8\u003c/b\u003e \u003csup\u003e15\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Bioreactor Evaluation\u003c/h2\u003e\u003cp\u003e\u003cb\u003eBatch fermentation was carried out with two different conditions aiming to enhance cell density and HA production.\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePre-culture\u003c/p\u003e\u003cp\u003eLB medium was prepared and supplemented with chloramphenicol for selective growth. \u003cem\u003eBacillus subtilis\u003c/em\u003e glycerol stock (PST) was used to inoculate tubes (0.5% v/v, Experiment 1, and 1% v/v, Experiment 2), which were incubated at 37\u0026deg;C and 180 rpm for 18 h.\u003c/p\u003e\u003cp\u003eMain culture\u003c/p\u003e\u003cp\u003eFermentation was conducted in volume of 1.5 L in a stirred bioreactor (Pierre Guerin Tryton) using a modified medium based on Westbrook et al. \u003csup\u003e11\u003c/sup\u003e. Chloramphenicol was added post-sterilization. The fermenter was inoculated with 6.7% v/v (Experiment 1) and 2% v/v (Experiment 2) pre-culture and operated at two different conditions, which are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, to investigate the effect of pH control and temperature on dual gene \u003cem\u003eB. subtilis\u003c/em\u003e growth and HA production. To control pH, NaOH 5 M was added to the fermentation broth automatically over a 72 h period of the fermentation process. Fermentation supernatants were processed by sequential TCA and ethanol precipitation. HA pellets were dried and redissolved in deionized water. The CTAB turbidimetric assay was used to quantify HA concentration. Detailed purification steps and assay conditions are provided in Supplementary \u003cb\u003eProtocols S7\u0026ndash;S8\u003c/b\u003e. Fermentation medium composition is listed in Supplementary \u003cb\u003eTable S5\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Structural and Molecular Weight Characterization\u003c/h2\u003e\u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\u003ch2\u003e2.7.1. FTIR Analysis\u003c/h2\u003e\u003cp\u003eFourier-transform infrared (FTIR) spectroscopy was used to confirm the structural identity of purified hyaluronic acid. Spectra were recorded in the range of 4000\u0026ndash;400 cm⁻\u0026sup1; using standard scanning parameters. Characteristic peaks corresponding to hydroxyl, carboxyl, and amide groups were evaluated. Full instrument settings and spectral interpretation are provided in Supplementary \u003cb\u003eProtocol S9\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\u003ch2\u003e2.7.2. Molecular Weight Determination by GPC\u003c/h2\u003e\u003cp\u003eGel permeation chromatography (GPC) was performed to determine the molecular weight distribution of HA samples. Water was used as the mobile phase, and detection was carried out via refractive index (RI). Samples were filtered through 0.45 \u0026micro;m membranes prior to injection.\u003c/p\u003e\u003cp\u003eDetailed column specifications, calibration standards, and run conditions are provided in Supplementary Protocol S10.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Gene Selection and Vector Design\u003c/h2\u003e\u003cp\u003e\u003cem\u003ehasA\u003c/em\u003e (1,254 bp) from \u003cem\u003eStreptococcus dysgalactiae\u003c/em\u003e and NX02_04625 (1,365 bp) from \u003cem\u003eSphingomonas sanxanigenens\u003c/em\u003e were codon-optimized for \u003cem\u003eB. subtilis\u003c/em\u003e and assembled under the IPTG-inducible Pgrac01 promoter with an intergenic RBS in the pHT01 backbone to yield the dual-gene plasmid (PST, 10782 bp), synthesized and sequence-verified by Gene Universal Inc. A single-gene control plasmid (PS, ~\u0026thinsp;9 kb) was constructed in-house.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Validation of Synthetic Constructs via PCR\u003c/h2\u003e\u003cp\u003eAs an additional quality control, PCR was performed on the synthetic plasmid template (PST); single bands at ~\u0026thinsp;540 bp (\u003cem\u003ehasA\u003c/em\u003e) and ~\u0026thinsp;1000 bp (\u003cem\u003ehasA\u003c/em\u003e\u0026ndash;NX02_04625) were observed with no amplification in no-template controls (see Supplementary \u003cb\u003eFigure S2\u003c/b\u003e). Primer sequences and PCR conditions are provided in Supplementary \u003cb\u003eTable S4\u003c/b\u003e and \u003cb\u003eProtocol S6\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Transformation into \u003cem\u003eE. coli\u003c/em\u003e DH5α\u003c/h2\u003e\u003cp\u003eSuccessful transformation of PST into \u003cem\u003eE. coli\u003c/em\u003e DH5α was confirmed qualitatively by the growth of discrete colonies on LB agar supplemented with 100 \u0026micro;g/mL ampicillin (see Supplementary \u003cb\u003eFigure S1\u003c/b\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Recovery and Quality Control of the Dual-Gene Plasmid\u003c/h2\u003e\u003cp\u003ePrior to the construction of the single-gene plasmid, the PST was extracted from \u003cem\u003eE. coli\u003c/em\u003e DH5α using a commercial miniprep kit (see Supplementary \u003cb\u003eFigure S1\u003c/b\u003e). Plasmid integrity and size (~\u0026thinsp;10.8 kb) were confirmed by agarose gel electrophoresis (see Supplementary \u003cb\u003eFigure S3\u003c/b\u003e), ensuring suitability for downstream subcloning procedures.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Restriction Digestion of Dual-Gene Plasmid for Single-Gene Construct Preparation\u003c/h2\u003e\u003cp\u003eTo generate the PS, the PST was digested with the restriction enzyme \u003cem\u003eXba\u003c/em\u003eI to remove the NX02_04625 coding sequence. The digestion reaction yielded two distinct fragments of approximately 9 kb and 1.5 kb, corresponding to the plasmid backbone containing the \u003cem\u003ehasA\u003c/em\u003e and the excised NX02_04625 region, respectively. Agarose gel electrophoresis confirmed complete digestion and accurate fragment separation (see Supplementary \u003cb\u003eFigure S4\u003c/b\u003e), validating the suitability of the backbone for subsequent ligation and transformation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e3.6. Recycling and Validation of the Vector Backbone Containing \u003cem\u003ehasA\u003c/em\u003e for Single-Gene Construct Assembly\u003c/h2\u003e\u003cp\u003eThe backbone fragment containing \u003cem\u003ehasA\u003c/em\u003e (9,245 bp) was recovered and recycled for single-gene construct assembly (see Supplementary \u003cb\u003eFigure S5\u003c/b\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e3.7. Transformation of the Single-Gene Construct into\u003c/b\u003e \u003cem\u003eE. coli\u003c/em\u003e \u003cb\u003eDH5α\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eTo evaluate the necessity of ligation for plasmid circularization, chemically competent \u003cem\u003eE. coli\u003c/em\u003e DH5α cells were transformed with either linearized or ligated pHT01-\u003cem\u003ehasA\u003c/em\u003e. As a negative control, transformation of the linear construct (no T4 DNA ligase) yielded no colonies on LB\u0026ndash;ampicillin agar, confirming that ligation is required for replication in the host. In contrast, transformation with the ligated construct produced numerous colonies on LB\u0026ndash;ampicillin plates. After overnight incubation, clear colony expansion was observed, demonstrating the stability and viability of \u003cem\u003eE. coli\u003c/em\u003e DH5α harboring the single-gene plasmid (see Supplementary \u003cb\u003eFigure S6\u003c/b\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003e3.8. Confirmation of Single-Gene Construct via Plasmid Size Comparison\u003c/h2\u003e\u003cp\u003eTo validate the successful removal of the NX02_04625 and generation of the PS plasmid DNA from both constructs, agarose gel electrophoresis was used. The single-gene plasmid exhibited a distinct band at 9245 bp, while the dual-gene plasmid showed a higher molecular weight band at 10782 bp. The upward shift in the PST relative to the PS variant confirms the expected size difference and supports the accuracy of the cloning procedure (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e\u003ch2\u003e3.9. Electroporation of \u003cem\u003eBacillus subtilis\u003c/em\u003e 168 with PS and PST Constructs\u003c/h2\u003e\u003cp\u003eElectroporation of \u003cem\u003eB. subtilis\u003c/em\u003e 168 with the PS and the PST was performed under identical conditions on LB agar supplemented with chloramphenicol (10 \u0026micro;g/mL). In both cases, transformants yielded distinct colonies, whereas the negative control cells electroporated without plasmid DNA showed no growth. Subsequent re-plating of colonies on fresh selective media confirmed stable propagation and plasmid maintenance. Colony formation by the PS construct is shown in Supplementary \u003cb\u003eFigure S7\u003c/b\u003e, and by the PST construct in Supplementary \u003cb\u003eFigure S8\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003e3.10. Colony PCR Confirmation of PS and PST Constructs in \u003cem\u003eBacillus subtilis\u003c/em\u003e 168\u003c/h2\u003e\u003cp\u003eTo verify the integration of both PS and PST constructs into chloramphenicol-resistant \u003cem\u003eBacillus subtilis\u003c/em\u003e 168 colonies, colony PCR was performed under identical conditions (see Supplementary \u003cb\u003eProtocol S6\u003c/b\u003e). PS transformants produced a clear\u0026thinsp;~\u0026thinsp;540-bp amplicon with \u003cem\u003ehasA\u003c/em\u003e-specific primers, with no band in the negative control. PST transformants yielded a distinct\u0026thinsp;~\u0026thinsp;1,000-bp product, with no amplification in the negative control or no-template control (NTC). These data confirm successful uptake, specific integration, and stable maintenance of both constructs in \u003cem\u003eB. subtilis\u003c/em\u003e (see Supplementary \u003cb\u003eTable S4\u003c/b\u003e for primer sequences, Supplementary \u003cb\u003eFigure S9\u003c/b\u003e for ~\u0026thinsp;540 bp gel, and Supplementary \u003cb\u003eFigure S10\u003c/b\u003e for ~\u0026thinsp;1000 bp gel).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\u003ch2\u003e3.11. Initial Assessment of HA Yield in Wild-Type Versus PST Recombinant \u003cem\u003eBacillus subtilis\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eHA production was assessed in two \u003cem\u003eBacillus subtilis\u003c/em\u003e 168 strains, wild-type (electrocompetent cell) and recombinant carrying the PST, at 24 and 48 hours post-induction using the CTAB turbidimetric method (CTM), (n\u0026thinsp;=\u0026thinsp;3; see Supplementary \u003cb\u003eProtocol S8\u003c/b\u003e). This pilot experiment was conducted in LB medium to establish a baseline comparison between the native and engineered strains in terms of HA biosynthetic capacity. No biologically meaningful HA production was detected in the wild-type strain at either time point. However, since the CTM relies on turbidity measurements at 540 nm, minor absorbance values may appear in HA-negative samples due to optical noise, suspended particles, or nonspecific interactions with CTAB. These values are considered background and do not reflect actual HA synthesis. In contrast, the recombinant strain produced 0.22 mg/mL HA at 24 hours and 0.31 mg/mL at 48 hours, while the wild-type strain yielded 0.04 mg/mL and 0.09 mg/mL at the corresponding time points. Both comparisons between recombinant and wild-type strains at 24 h and 48 h were statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Replicate values and full statistical analyses are provided in Supplementary \u003cb\u003eTable S7\u003c/b\u003e. HA concentrations were calculated using the standard curve shown in Supplementary \u003cb\u003eTable S8\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec33\" class=\"Section2\"\u003e\u003ch2\u003e3.12. Comparison of HA Production in PS and PST \u003cem\u003eBacillus subtilis\u003c/em\u003e Strains\u003c/h2\u003e\u003cp\u003eMultiple media formulations were initially tested for HA production \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, but only the modified Westbrook medium supported efficient synthesis and was therefore selected for all subsequent experiments. HA production was quantified at 24, 48, and 72 hours post-induction using the CTM (n\u0026thinsp;=\u0026thinsp;2 per strain after outlier removal; see Supplementary \u003cb\u003eProtocol S8\u003c/b\u003e). Cultures were grown in a modified Westbrook medium, adapted from the original formulation described by Westbrook et al.,\u003csup\u003e11\u003c/sup\u003e with full composition provided in Supplementary \u003cb\u003eTable S5\u003c/b\u003e. Final HA concentrations for each strain and time point are summarized in Supplementary \u003cb\u003eTable S6\u003c/b\u003e, reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD based on two biological replicates. The calibration curve used for quantification is shown in Supplementary \u003cb\u003eFigure S11\u003c/b\u003e. In the dual-gene strain, HA concentration at 48 hours was 1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 mg/mL, and at 72 hours it was 1.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 mg/mL. Statistical analysis showed no significant difference between these two time points (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e). In the single-gene strain, HA concentration at 48 hours was 0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 mg/mL, and at 72 hours it was 0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 mg/mL. This difference was also not statistically significant (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e). However, at both time points, the HA yield in the PST strain was significantly higher than that of the PS strain (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), confirming the superior performance of the dual-gene construct (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec34\" class=\"Section2\"\u003e\u003ch2\u003e3.13. FTIR Analysis of Produced HA\u003c/h2\u003e\u003cp\u003eTo confirm the structural identity of the HA produced by the engineered \u003cem\u003eBacillus subtilis\u003c/em\u003e 168 carrying the PST construct, Fourier-transform infrared spectroscopy (FTIR) was performed. The IR spectrum of the purified sample exhibited characteristic absorption bands corresponding to key functional groups, including:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eO\u0026ndash;H stretch\u003c/b\u003e at 3415 cm⁻\u0026sup1;\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eC\u0026ndash;H stretch\u003c/b\u003e at 2934 cm⁻\u0026sup1;\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eAsymmetric COO⁻ stretch\u003c/b\u003e at 1683 cm⁻\u0026sup1;\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eSymmetric COO⁻ stretch\u003c/b\u003e at 1459 cm⁻\u0026sup1;\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eC\u0026ndash;O\u0026ndash;C stretch\u003c/b\u003e at 1097 cm⁻\u0026sup1;\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec35\" class=\"Section2\"\u003e\u003ch2\u003e3.14. Molecular Weight Distribution by GPC\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMolecular Weight Parameters\u003c/b\u003e\u003c/p\u003e\u003cp\u003eQuantitative MW data for fractions within the calibration range (1,030\u0026ndash;1.18\u0026times;10⁶ g/mol, PEG standards) are reported in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMolecular weight distribution of validated HA fractions.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026times;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026times;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFraction\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e% Area\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003csub\u003en\u003c/sub\u003e (g/mol)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eM\u003csub\u003ew\u003c/sub\u003e (g/mol)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePDI\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFragmented HA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e24.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c3\"\u003e\u003cp\u003e8.26 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c4\"\u003e\u003cp\u003e9.88 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOligomers\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e31.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c3\"\u003e\u003cp\u003e4.12 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c4\"\u003e\u003cp\u003e5.54 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.34\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eM\u003csub\u003en\u003c/sub\u003e: Number-average molecular weight; M\u003csub\u003ew\u003c/sub\u003e: Weight-average molecular weight; PDI: Polydispersity Index (M\u003csub\u003ew\u003c/sub\u003e/ M\u003csub\u003en\u003c/sub\u003e).\u003c/p\u003e\u003cp\u003eHigh-MW HA (Peak 1) exceeded the column\u0026rsquo;s calibration range; its MW is estimated as \u0026gt;\u0026thinsp;1.18\u0026times;10⁶ g/mol but requires absolute quantification.\u003c/p\u003e\u003cp\u003eThe trimodal distribution indicates significant heterogeneity in the HA sample. The dominant high-MW fraction (43.45%) aligns with native HA\u0026rsquo;s intrinsic chain length, while the mid- and low-MW fractions (56.55% combined) suggest partial hydrolysis during processing. The narrow PDI (1.20) of fragmented HA implies controlled degradation, whereas the broader PDI (1.34) of oligomers reflects heterogeneous chain cleavage.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec36\" class=\"Section2\"\u003e\u003ch2\u003e3.15. HA Production in Bioreactor Cultures\u003c/h2\u003e\u003cp\u003eThis preliminary study evaluated recombinant \u003cem\u003eBacillus subtilis\u003c/em\u003e 168 (PST) strains under controlled bioreactor conditions (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In Experiment 2 (37\u0026deg;C, pH 6.0\u0026ndash;7.0), PST produced 0.62 mg/mL of HA at 72 h, a 2.8-fold increase compared to Experiment 1 (0.22 mg/mL, 30\u0026deg;C, without pH control). As shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, both pH and temperature had a remarkable impact on cell growth and HA production. The recombinant PST \u003cem\u003eB. subtilis\u003c/em\u003e 168 strain remained stable and sustained HA synthesis throughout the 72 h fermentation period. Sporulation was observed in Experiment 2 and is considered a potential consideration during scale-up for production. This study highlights the importance of tightly controlling fermentation parameters to achieve optimal HA yield.\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTwo batch fermentation conditions for culture and HA production by PST \u003cem\u003eB. subtilis\u003c/em\u003e 168\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExperiment 1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eExperiment 2\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTemp.\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e30\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e37\u0026deg;C\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003epH Control\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e6.0\u0026ndash;7.0 (NaOH 5M)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAgitation\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e180 rpm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e200 rpm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAeration\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2\u0026ndash;3 vvm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026ndash;6 vvm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHA (72 h)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 mg/mL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 mg/mL\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eOD\u003c/b\u003e\u003csub\u003e\u003cb\u003e600\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.32 (56 h)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e31.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22 (72h)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSporulation\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eObserved (48\u0026amp;72h)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe escalating demand for HA across the pharmaceutical, biomedical, and cosmetic industries necessitates a paradigm shift away from traditional, limited production methods. While extraction from animal tissues raises concerns about pathogen contamination and batch-to-batch variability, and fermentation with pathogenic \u003cem\u003eStreptococcus\u003c/em\u003e strains introduces significant biosafety and regulatory hurdles, the development of efficient GRAS microbial platforms has become a central focus of metabolic engineering. This study successfully establishes a recombinant \u003cem\u003eBacillus subtilis\u003c/em\u003e 168 as a safe and effective cell factory for HA production by strategically addressing the fundamental metabolic limitations of this non-pathogenic host, achieving a final yield of 1.20 g/L in shake flasks and 0.62 g/L in a bioreactor.\u003c/p\u003e\u003cp\u003eThe core of our engineering strategy was the rational reconstruction and enhancement of the heterologous HA biosynthetic pathway. Native \u003cem\u003eB. subtilis\u003c/em\u003e possesses the genetic machinery for UDP-N-acetylglucosamine (UDP-GlcNAc) synthesis but lacks the \u003cem\u003ehasA\u003c/em\u003e gene and has a suboptimal capacity for producing the second precursor, UDP-glucuronic acid (UDP-GlcUA) \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Our approach involved the heterologous co-expression of two key genes: \u003cem\u003ehasA\u003c/em\u003e from \u003cem\u003eStreptococcus dysgalactiae\u003c/em\u003e and a high-performance \u003cem\u003etuaD\u003c/em\u003e homolog (NX02_04625) from \u003cem\u003eSphingomonas sanxanigenens\u003c/em\u003e, selected for its favorable enzymatic kinetics (low Km, high turnover number) from the BRENDA database. The results unequivocally validate this strategy. The dual-gene construct (pHT01-\u003cem\u003ehasA\u003c/em\u003e- NX02_04625) demonstrated a significant advantage over the single-gene construct (pHT01-\u003cem\u003ehasA\u003c/em\u003e), achieving a yield of 1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 mg/mL in shake flasks compared to 0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 mg/mL at 72 hours, a 2.4-fold increase \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. This stark difference underscores a critical bottleneck in the precursor supply chain, specifically the conversion of UDP-glucose to UDP-GlcUA, which is efficiently alleviated by the expression of NX02_04625. This finding aligns with and reinforces the metabolic engineering principle established by Westbrook et al \u003csup\u003e11\u003c/sup\u003e, who identified the augmentation of the UDP-GlcUA pool via the native \u003cem\u003etuaD\u003c/em\u003e gene as the most impactful intervention, boosting HA titer 5.6-fold in their \u003cem\u003eB. subtilis\u003c/em\u003e system. Our use of a heterologous homolog with potentially superior catalytic properties further advances this strategy. Our achieved shake flask titer (~\u0026thinsp;1.2 g/L) is notably higher than the 0.5\u0026ndash;0.7 g/L often reported in early studies with similar systems \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe transition from shake flasks to a controlled bioreactor environment revealed the profound impact of physicochemical parameters on microbial performance and HA synthesis. Our bioreactor experiments yielded critical insights:\u003c/p\u003e\u003cp\u003eThe HA titer of 0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 mg/mL was achieved in experiment 2 (37\u0026deg;C, pH maintained at 6.0\u0026ndash;7.0), which was a 182% increase over the yield in experiment 1 (30\u0026deg;C, no pH control), highlighting that suboptimal conditions severely constrain the engineered strain's potential. The maintained pH likely prevented acid-induced inhibition of enzyme activity and supported robust cell growth, as evidenced by the final OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;31.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22, which was an order of magnitude higher than in uncontrolled fermenter \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eProduct characterization confirmed the successful biosynthesis of authentic hyaluronic acid. FTIR spectroscopy provided definitive evidence, with all characteristic functional group peaks (O-H, C-H, COO-, C-O-C) matching the commercial standard. This confirms the functional fidelity of the heterologously expressed \u003cem\u003ehasA\u003c/em\u003e synthase, a crucial step for biomedical applicability \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Gel Permeation Chromatography analysis revealed a heterogeneous molecular weight distribution, which is typical for microbial HA production due to the presence of endogenous hydrolases or shear stress \u003csup\u003e\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The product profile was promising: a dominant fraction (43.45% of the total area) consisted of high-molecular-weight HA (\u0026thinsp;\u0026gt;\u0026thinsp;\u0026gt;\u0026thinsp;1.18 MDa), which is highly valued for its superior rheological and biological properties in medical applications such as viscosupplementation and ophthalmic surgery \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The remaining fractions consisted of fragmented HA (PDI: 1.20) and oligomers (PDI: 1.34). While this heterogeneity may require further purification for specific applications, it also presents an opportunity, as low- and medium-MW HA fractions have their own unique therapeutic applications, such as in angiogenesis and wound healing \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The ability to produce a broad spectrum of MWs in a single fermentation could be advantageous. The weight-average molecular weight of the main fragmented fraction was calculated at 9.88 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e g/mol, which is within the highly desirable range for many commercial applications \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWhen benchmarked against other engineered non-pathogenic hosts, our platform demonstrates strong potential. Our optimized bioreactor titer (0.62 g/L) is comparable to early reports in \u003cem\u003eAgrobacterium\u003c/em\u003e sp. (0.7\u0026ndash;0.9 g/L) \u003csup\u003e29\u003c/sup\u003e, and exceeds yields from some \u003cem\u003eLactococcus lactis\u003c/em\u003e systems (0.3 g/L) \u003csup\u003e30\u003c/sup\u003e. While some hyper-producing engineered \u003cem\u003eE. coli\u003c/em\u003e or \u003cem\u003eBacillus subtilis\u003c/em\u003e strains have reported titers exceeding 6\u0026ndash;8 g/L \u003csup\u003e11, 18\u003c/sup\u003e, it is crucial to note that these often involve extensive multi-gene mutagenesis and pathway engineering over several generations. Our study, focusing on the strategic introduction of two key genes, provides a robust and simplified foundation. The primary advantage of our \u003cem\u003eB. subtilis\u003c/em\u003e-based system is its combination of a GRAS status, high secretory capacity, well-known genetics, and absence of endotoxins, which collectively offer a more straightforward and safer path to industrial-scale production and regulatory approval for biomedical uses compared to \u003cem\u003eE. coli\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this study, we successfully engineered \u003cem\u003eBacillus subtilis\u003c/em\u003e for efficient and safe HA production by co-expressing \u003cem\u003ehasA\u003c/em\u003e, encoding hyaluronan synthase, and NX02_04625, involved in the biosynthesis of the essential precursor UDP-glucuronic acid. The dual-gene recombinant strain demonstrated significantly enhanced HA yield compared to the single-gene and wild-type strains, underscoring the critical role of precursor supply in HA biosynthesis.\u003c/p\u003e\u003cp\u003eOptimization of fermentation conditions, particularly pH control, substantially improved HA titers, with the highest yield reaching 0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 mg/mL in bioreactor cultures. Characterization of the purified HA by FTIR confirmed its chemical identity, showing all expected functional groups, while gel permeation chromatography analysis revealed a predominantly high molecular weight polymer fraction, suitable for biomedical and cosmetic applications.\u003c/p\u003e\u003cp\u003eCompared to traditional extraction and pathogenic bacterial fermentation, recombinant production in \u003cem\u003eB. subtilis\u003c/em\u003e offers advantages in biosafety, product purity, scalability, and environmental impact. Our findings establish this engineered \u003cem\u003eB. subtilis\u003c/em\u003e platform as a robust candidate for industrial-scale HA biosynthesis, with potential to meet growing market demand for safe and high-quality HA.\u003c/p\u003e\u003cp\u003eFuture research should focus on minimizing sporulation, refining molecular weight distribution, and further fermentation process optimization to enhance yield and product consistency.\u003c/p\u003e\u003cp\u003eFuture optimization can focus on implementing a fed-batch strategy with controlled carbon source feeding to prevent potential catabolite repression and support prolonged production phases, as successfully demonstrated by others to achieve titers exceeding 6\u0026ndash;8 g/L \u003csup\u003e11, 18\u003c/sup\u003e. The use of sucrose as a carbon source is economically advantageous, and its efficient hydrolysis and utilization is a positive feature of our system \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eCollectively, this study successfully demonstrates a comprehensive strategy from strain construction to process optimization for HA production in a safe microbial host. We have confirmed that precursor supply is a critical bottleneck and that environmental parameters like pH and temperature are just as important as genetic design for maximizing yield. The engineered \u003cem\u003eB. subtilis\u003c/em\u003e strain produces authentic HA with a significant proportion of high molecular weight polymer. Future work will focus on (i) deleting endogenous hydrolase genes to control and increase molecular weight, (ii) implementing fed-batch fermentation strategies with optimized feeding to boost titers to industrially competitive levels (\u0026gt;\u0026thinsp;2\u0026ndash;3 g/L), and (iii) further engineering to decouple production from sporulation for more predictable and scalable processes \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. This work solidifies \u003cem\u003eB. subtilis\u003c/em\u003e as a premier chassis for the safe and efficient biomanufacturing of high-value biopolymers.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work is based upon research funded by Iran National Science Foundation (INSF) under project No.4024643.\u0026nbsp;The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting\u0026nbsp;interests:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eThis study was conducted as part of the first author's PhD project, with financial support from the Iran National Science Foundation (INSF) and NIGEB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eAll relevant data are included in the manuscript and its supplementary materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRouzbeh Almasi Ghale\u003c/strong\u003e: Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Validation; Visualization; Writing original draft; Writing review \u0026amp; editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReza Faghihi\u003c/strong\u003e; Investigation; Formal analysis; Writing original draft; Visualization\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMarjan Talebi\u003c/strong\u003e; Formal analysis; Writing original draft; Writing review \u0026amp; editing; Visualization\u003c/p\u003e\n\u003cp\u003e*\u003cstrong\u003eMehdi Shamsara\u003c/strong\u003e: Conceptualization; Formal analysis; Investigation; Methodology; Supervision; Validation; Writing original draft; Writing review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e*\u003cstrong\u003eFatemeh Tabandeh\u003c/strong\u003e: Conceptualization; Formal analysis; Investigation; Methodology; Project administration; Funding acquisition; Supervision; Validation; Writing original draft; Writing review \u0026amp; editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSaadati, F. et al. Advances and principles of hyaluronic acid production, extraction, purification, and its applications: a review. \u003cem\u003eInt. J. Biol. Macromol.\u003c/em\u003e 143839 (2025).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTalebi, M., Ghale, R. A., Asl, R. M. \u0026amp; Tabandeh, F. Advancements in characterization and preclinical applications of hyaluronic acid-based biomaterials for wound healing: a review. \u003cem\u003eCarbohydr. Polym. Technol. Appl.\u003c/em\u003e 100706 (2025).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eValachov\u0026aacute;, K. \u0026amp; Šolt\u0026eacute;s, L. Hyaluronan as a prominent biomolecule with numerous applications in medicine. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cb\u003e22\u003c/b\u003e, 7077 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJiang, Y. et al. High-molecular-weight hyaluronic acid can be used as a food additive to improve the symptoms of persistent inflammation, immunosuppression and catabolism syndrome (PICS). \u003cem\u003eBiology\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 319 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShikina, E., Kovalevsky, R., Shirkovskaya, A. \u0026amp; Toukach, P. V. Prospective bacterial and fungal sources of hyaluronic acid: a review. \u003cem\u003eComput. Struct. Biotechnol. J.\u003c/em\u003e \u003cb\u003e20\u003c/b\u003e, 6214\u0026ndash;6236 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu, L., Liu, Y., Li, J., Du, G. \u0026amp; Chen, J. Microbial production of hyaluronic acid: current state, challenges, and perspectives. \u003cem\u003eMicrob. Cell. Fact.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 99 (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGrabowski, M., Gmyrek, D., Żurawska, M. \u0026amp; Trusek, A. Hyaluronic acid: production strategies, gel-forming properties, and advances in drug delivery systems. \u003cem\u003eGels\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 424 (2025).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eManfr\u0026atilde;o-Netto, J. H. et al. Genetic strategies for improving hyaluronic acid production in recombinant bacterial culture. \u003cem\u003eJ. Appl. Microbiol.\u003c/em\u003e \u003cb\u003e132\u003c/b\u003e, 822\u0026ndash;840 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSu, Y., Liu, C., Fang, H. \u0026amp; Zhang, D. Bacillus subtilis: a universal cell factory for industry, agriculture, biomaterials and medicine. \u003cem\u003eMicrob. Cell. Fact.\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e, 173 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWidner, B. et al. Hyaluronic acid production in Bacillus subtilis. \u003cem\u003eAppl. Environ. Microbiol.\u003c/em\u003e \u003cb\u003e71\u003c/b\u003e, 3747\u0026ndash;3752 (2005).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWestbrook, A. W., Ren, X., Oh, J., Moo-Young, M. \u0026amp; Chou, C. P. Metabolic engineering to enhance heterologous production of hyaluronic acid in Bacillus subtilis. \u003cem\u003eMetab. Eng.\u003c/em\u003e \u003cb\u003e47\u003c/b\u003e, 401\u0026ndash;413 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhao, X., Chen, X., Xue, Y. \u0026amp; Wang, X. Development of an efficient iterative genome editing method in Bacillus subtilis using the CRISPR-AsCpf1 system. \u003cem\u003eJ. Basic. Microbiol.\u003c/em\u003e \u003cb\u003e62\u003c/b\u003e, 824\u0026ndash;832 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFroger, A. \u0026amp; Hall, J. E. Transformation of plasmid DNA into E. coli using the heat shock method. \u003cem\u003eJ. Vis. Exp.\u003c/em\u003e e253 (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHarwood, C. R. \u0026amp; Cutting, S. M. Molecular Biological Methods for Bacillus, Vol. 35Wiley Chichester,. (1990).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen, Y. H. \u0026amp; Wang, Q. Establishment of CTAB turbidimetric method to determine hyaluronic acid content in fermentation broth. \u003cem\u003eCarbohydr. Polym.\u003c/em\u003e \u003cb\u003e78\u003c/b\u003e, 178\u0026ndash;181 (2009).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJin, P., Kang, Z., Yuan, P., Du, G. \u0026amp; Chen, J. Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168. \u003cem\u003eMetab. Eng.\u003c/em\u003e \u003cb\u003e35\u003c/b\u003e, 21\u0026ndash;30 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChien, L. J. \u0026amp; Lee, C. K. Enhanced hyaluronic acid production in Bacillus subtilis by coexpressing bacterial hemoglobin. \u003cem\u003eBiotechnol. Prog\u003c/em\u003e. \u003cb\u003e23\u003c/b\u003e, 1017\u0026ndash;1022 (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJia, Y. et al. Metabolic engineering of Bacillus subtilis for the efficient biosynthesis of uniform hyaluronic acid with controlled molecular weights. \u003cem\u003eBioresour Technol.\u003c/em\u003e \u003cb\u003e132\u003c/b\u003e, 427\u0026ndash;431 (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim, J. H. et al. Selection of a Streptococcus equi mutant and optimization of culture conditions for the production of high molecular weight hyaluronic acid. \u003cem\u003eEnzyme Microb. Technol.\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e, 440\u0026ndash;445 (1996).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchallmey, M., Singh, A. \u0026amp; Ward, O. P. Developments in the use of Bacillus species for industrial production. \u003cem\u003eCan. J. Microbiol.\u003c/em\u003e \u003cb\u003e50\u003c/b\u003e, 1\u0026ndash;17 (2004).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFallacara, A., Baldini, E., Manfredini, S. \u0026amp; Vertuani, S. Hyaluronic acid in the third millennium. \u003cem\u003ePolymers\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 701 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIqbal, Z. et al. Discussion of the AED workshop. \u003cem\u003ePharm. World Sci.\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e, 246\u0026ndash;250 (1997).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGomes, V. A. M., Netto, C. M., Carvalho, L. S. \u0026amp; Parachin, N. S. Heterologous hyaluronic acid production in Kluyveromyces lactis. \u003cem\u003eMicroorganisms\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e, 294 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eG\u0026uuml;ng\u0026ouml;r, G. et al. Bacterial hyaluronic acid production through an alternative extraction method and its characterization. \u003cem\u003eJ. Chem. Technol. Biotechnol.\u003c/em\u003e \u003cb\u003e94\u003c/b\u003e, 1843\u0026ndash;1852 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKogan, G., Šolt\u0026eacute;s, L., Stern, R. \u0026amp; Gemeiner, P. Hyaluronic acid: a natural biopolymer with a broad range of biomedical and industrial applications. \u003cem\u003eBiotechnol. Lett.\u003c/em\u003e \u003cb\u003e29\u003c/b\u003e, 17\u0026ndash;25 (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTabasi, A. et al. Improved production of food-grade hyaluronic acid in recombinant Corynebacterium glutamicum by medium optimization and feeding strategy. \u003cem\u003eAppl. Food Biotechnol.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 1\u0026ndash;14 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTammi, R. H. et al. Transcriptional and post-translational regulation of hyaluronan synthesis. \u003cem\u003eFEBS J.\u003c/em\u003e \u003cb\u003e278\u003c/b\u003e, 1419\u0026ndash;1428 (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLa Gatta, A., Schiraldi, C., Papa, A. \u0026amp; De Rosa, M. Comparative analysis of commercial dermal fillers based on crosslinked hyaluronan: physical characterization and in vitro enzymatic degradation. \u003cem\u003ePolym. Degrad. Stab.\u003c/em\u003e \u003cb\u003e96\u003c/b\u003e, 630\u0026ndash;636 (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMao, Z. \u0026amp; Chen, R. R. Recombinant synthesis of hyaluronan by Agrobacterium sp. \u003cem\u003eBiotechnol. Prog\u003c/em\u003e. \u003cb\u003e23\u003c/b\u003e, 1038\u0026ndash;1042 (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChien, L. J. \u0026amp; Lee, C. K. Hyaluronic acid production by recombinant Lactococcus lactis. \u003cem\u003eAppl. Microbiol. Biotechnol.\u003c/em\u003e \u003cb\u003e77\u003c/b\u003e, 339\u0026ndash;346 (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003evan Dijl, J. \u0026amp; Hecker, M. Bacillus subtilis: from soil bacterium to super-secreting cell factory. \u003cem\u003eMicrob. Cell. Fact.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 3 (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBoels, I. C., van Kranenburg, R., Hugenholtz, J., Kleerebezem, M. \u0026amp; De Vos, W. M. Sugar catabolism and its impact on the biosynthesis and engineering of exopolysaccharide production in lactic acid bacteria. \u003cem\u003eInt. Dairy. J.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 723\u0026ndash;732 (2001).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Hyaluronic acid production, Bacillus subtilis 168, Strain development, hasA Gene, Metabolic Engineering, NX02_04625","lastPublishedDoi":"10.21203/rs.3.rs-7842276/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7842276/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHyaluronic acid (HA) is a biopolymer with broad biomedical and pharmaceutical uses, yet microbial HA production is limited by strain safety, and process inefficiencies; to address these challenges we engineered a biosafe \u003cem\u003eBacillus subtilis\u003c/em\u003e chassis by integrating catalytic genes identified from the BRENDA database. Two constructs on the pHT01 shuttle vector were prepared, a dual-gene plasmid (\u003cem\u003ehasA\u003c/em\u003e\u0026ndash;NX02_04625) and a single gene plasmid (\u003cem\u003ehasA\u003c/em\u003e), and following transformation and molecular confirmation recombinant colonies were screened in different media to identify optimal production conditions. HA was quantified by the CTAB assay, its structure validated by FTIR, and molecular weight distribution characterized by GPC. Cloning and molecular validation were successful, HA synthesis was detected only in engineered strains and not in wild type controls, and the dual-gene strain produced significantly more HA than the single gene strain, reaching up to 1.2 g/L in shake flask cultures. Preliminary bioreactor cultivation of the dual gene strain in the selected medium yielded 0.62 g/L HA. These results indicate that engineered \u003cem\u003eB. subtilis\u003c/em\u003e is a biosafe, scalable platform for HA production, offering an industrially viable alternative to pathogenic hosts and highlighting a promising source for HA synthesis applicable to pharmaceutical, biomedical, and cosmetic industries.\u003c/p\u003e","manuscriptTitle":"Strain Engineering and Pathway Enhancement of Bacillus subtilis for Efficient Hyaluronic Acid Production","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-30 17:45:38","doi":"10.21203/rs.3.rs-7842276/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"04577c72-7b17-4aa0-9520-da87382a2366","owner":[],"postedDate":"October 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":56988375,"name":"Biological sciences/Biological techniques"},{"id":56988376,"name":"Biological sciences/Biotechnology"},{"id":56988377,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2025-11-17T08:09:55+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-30 17:45:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7842276","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7842276","identity":"rs-7842276","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}