Mechanism-guided control of intracellular alkalinization and process pH for production of recombinant human B-type natriuretic peptide under the cSAT scheme in Escherichia coli

Mechanism-guided control of intracellular alkalinization and process pH for production of recombinant human B-type natriuretic peptide under the cSAT scheme in Escherichia coli | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mechanism-guided control of intracellular alkalinization and process pH for production of recombinant human B-type natriuretic peptide under the cSAT scheme in Escherichia coli Hang Nie, Yajun Feng, Guanghui Sun, Haijie Li, Jingwen Kang, Yanqing Zhang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8252127/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Feb, 2026 Read the published version in Microbial Cell Factories → Version 1 posted 10 You are reading this latest preprint version Abstract Background : Tag-based high-load expression in Escherichia coli often causes uncontrolled pH drift, but links between intracellular alkalinization, metabolism and process levers remain unclear. Results : Using a cleavable self-aggregating tag (cSAT) to produce recombinant human B-type natriuretic peptide (rhBNP), we combine broth pH tracking, AF-C ratiometric intracellular pH imaging and multi-omics to show that ammoniagenic amino-acid catabolism, glyoxylate-centered carbon rerouting and respiratory shifts drive alkalinization. Guided by this proton-economy model, stepwise medium engineering (complex nitrogen, phosphate, glucose and ammonium sulfate with tuned carbon to nitrogen ratio) lowers shake-flask broth pH from values above 8.5 to about 7.0, increases titers 2.9-fold over LB medium to 115.4 mg/L and, in 3-L fed-batch, yields 662.1 mg/L rhBNP while maintaining biomass. Conclusions : Proton-economy-based process design stabilizes pH and productivity during cSAT scheme production of rhBNP in E. coli . Intracellular alkalinization Medium engineering Recombinant human B-type natriuretic peptide cSAT scheme Proton economy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Bioactive peptides with defined intramolecular disulfide bonds are increasingly produced in microbial hosts, not only because they can be obtained rapidly and at relatively low cost, but also because they offer tractable chassis to study how cells handle small, cysteine-rich cargos at production-relevant levels [ 1 , 2 ]. In such microbial cell factories, the host must simultaneously support high peptide synthesis rates, correct disulfide formation and folding, and the maintenance of intracellular homeostasis. Recombinant human B-type natriuretic peptide (rhBNP) is a 32-residue hormone whose biological activity depends on a single disulfide, making it a practical model for probing how E. coli manages small disulfide-containing targets under the high loads required for biotechnological production[ 3 , 4 ]. However, even in E. coli , a long-standing workhorse host, such peptides are prone to proteolytic degradation, limited solubility and aggregation, so the recoverable yield often falls short of the apparent expression level and can be highly sensitive to the physiological state of the chassis [ 5 ]. To increase usable yield and mitigate these burdens, a wide range of fusion and tag strategies has been developed to enhance solubility, shield the product from proteases and facilitate downstream purification [ 6 , 7 ]. Among them, the cSAT scheme couples an intein with a self-assembling module and enables release of the target peptide under mild, near-physiological conditions in E. coli , and has been applied to several disulfide-bonded peptides including rhBNP [ 8 – 11 ]. While such schemes efficiently decouple expression and downstream purification, the high intracellular loads and aggregation they impose can strongly perturb cytosolic pH and broader proton homeostasis. How cSAT scheme production of rhBNP reshapes intracellular alkalinization and, in turn, constrains medium formulation and process control in E. coli has not been systematically characterized, even though these links are likely to set practical limits on robust peptide production. Intracellular pH (pHi) in neutralophilic E. coli used as a microbial cell factory is normally maintained between 7.4 and 7.8 over an external pH range of 5 to 9 by the combined action of respiratory proton pumping and Na⁺/H⁺ and K⁺/H⁺ antiport [ 12 ]. This tight control is critical because pHi integrates ATP turnover, ion transport and carbon-nitrogen partitioning, and feeds directly into the proton motive force [ 13 ]. Under high-load heterologous expression, particularly when strong tags or self-aggregating modules are used, several processes can bias pHi upward: reduced net acid formation when carbon is diverted from the full tricarboxylic acid cycle into glyoxylate and related bypass routes [ 14 ], increased proton extrusion through F 1 F 0 -ATP synthase operating in the pumping direction or through secondary antiporters [ 15 ], and deamination with NH 3 release under nitrogen-rich conditions [ 16 ]. Conventional fermentation readouts such as optical density, broth pH and product titer cannot resolve the timing or relative contributions of these routes, and omics-level enrichments remain largely associative unless they are anchored to time-resolved pHi measurements [ 17 ]. As a result, pHi and the underlying proton balance are seldom treated as explicit design variables when optimizing tag-based high-load expression processes. Ratiometric fluorescent probes offer a practical route to make bacterial pHi a measurable state variable in such processes, because the ratio between two emission channels compensates for variations in probe loading, photobleaching and moderate nonspecific binding [ 18 ]. This strategy has been validated for single-cell pHi determination in bacteria using carboxyfluorescein, GFP variants and related dual-excitation reporters [ 19 , 20 ]. Recent reviews on ESIPT and ICT-type 8-hydroxyquinoline probes have summarized red and near-infrared ratiometric scaffolds that are compatible with live-cell imaging and provide large Stokes shifts suitable for dense cultures [ 21 ]. Here, we adapt an HQO-type ratiometric probe (AF-C), calibrated in situ by a standard high-potassium/nigericin protocol [ 22 ], to obtain quantitative, time-resolved single-cell pHi trajectories for E. coli during cSAT scheme production of rhBNP [ 23 , 24 ]. These trajectories are then integrated with transcriptomics, metabolomics and fermentation outputs to link intracellular alkalinization to proton-economy rewiring and to medium and process control levers. At the process level, we first established a baseline for cSAT scheme production of rhBNP in E. coli by reproducing reported conditions in LB medium with induction at 18°C and an optical density at 600 nm (OD 600 ) of 0.4–0.6 [ 25 ]. Increasing the induction temperature to 21°C and shifting induction to an OD 600 of 0.6–0.8 improved volumetric titer and space-time yield but markedly intensified pH drift in both broth and intracellular, revealing a trade-off in which conditions that favor productivity amplify the host pH burden. To resolve and control this behavior, we combined AF-C-based ratiometric intracellular pH imaging with time-resolved transcriptomics and metabolomics to obtain an induction-resolved view of the E. coli response under the cSAT scheme [ 26 ]. The resulting profiles indicate that high anabolic and processing load is accompanied by respiratory and glyoxylate reprogramming and altered ion-transport and energy-related functions, consistent with metabolism-driven intracellular alkalinization [ 13 , 14 , 27 ]. Guided by this mechanistic framework, we then used phosphate buffering, complex nitrogen composition, carbon supply and the carbon to nitrogen ratio as simple process levers to restore near-neutral broth and intracellular pH while increasing shake-flask titers over LB medium. Finally, we validated this medium in a 3-L fed-batch process, demonstrating that mechanism-guided control of intracellular alkalinization and process pH can stabilize rhBNP productivity during cSAT scheme expression in E. coli . 2. Materials and methods 2.1 Strains and plasmid construction The production host was E. coli BL21(DE3) (Sangon Biotech, Shanghai, China) carrying an expression plasmid derived from the pET-30a(+) backbone (Novagen, USA). The DNA sequence encoding rhBNP was codon-optimized for E. coli and synthesized commercially (Tsingke, Xi’an, China). The recombinant plasmid was assembled by standard restriction-ligation cloning and verified by Sanger sequencing. The amino acid sequence of the cSAT-rhBNP fusion and the corresponding coding sequence are provided in Table S1. 2.2 Protein expression coli BL21(DE3) harboring the target plasmid was grown overnight in LB medium supplemented with kanamycin (50 µg/mL) at 37 °C and 220 rpm. The seed culture was diluted 1:50 into fresh LB medium and grown to an OD 600 of 0.6–0.8, after which the temperature was lowered to 21 °C. Following a 15–30 min equilibration, IPTG was added to a final concentration of 0.2 mM to initiate expression. Cultures were incubated for a further 24 h at 21 °C, and cells were harvested by centrifugation (5,000 rpm, 15 min, 4 °C). 2.3 Absorption and emission spectra at different pH values of AF-C A stock of AF-C (1 mM) was diluted to 10 µM in HEPES buffer containing 10 mM SDS. Solution pH was adjusted by incremental addition of NaOH or HCl and verified with a calibrated pH meter. UV-vis spectra were collected on a spectrophotometer; fluorescence emission spectra were recorded on a steady-state/kinetics fluorimeter. All measurements were acquired after 1 h dark equilibration at room temperature. 2.4 Evaluation of AF-C cytotoxicity in E. coli Overnight cultures of E. coli BL21(DE3) were diluted 1:50 into fresh LB medium and grown at 37 °C, 220 rpm until OD 600 reached 0.6–0.8. IPTG (0.2 mM) was added to mimic expression conditions. AF-C (DMSO stock) was then added to final concentrations of 0, 5, 10, 20, 30, or 40 µM, keeping DMSO at 0.5% (v/v) in all groups. After 6 h exposure, cultures were mixed gently and 100–200 µL aliquots transferred to clear 96-well plates. CCK-8 reagent (HY-K0301, MCE) was added at 10% (v/v), mixed gently, and incubated for 30 min at 21 °C protected from light. Absorbance at 450 nm was read on a microplate reader. Reagent and matrix blanks were included for background correction [28]. Background-corrected A 450 nm values were used to calculate cell viability (%) relative to the vehicle control (0 µM AF-C, 0.5% DMSO, set to 100%): In parallel, OD 600 was recorded hourly at each AF-C concentration. For microplate monitoring, 200 µL per well was dispensed into flat-bottom clear plates sealed with breathable film, with three technical wells per biological replicate. Growth curves (OD 600 vs time) were plotted and, where indicated, fitted with a logistic model. 2.5 Real-time confocal imaging of living E. coli cells E. coli cultures were sampled at 0, 12, and 24 h after IPTG induction. At each time point, 2 mL of culture was incubated with AF-C (20 µM, 0.5% DMSO, v/v) at 21 °C. Prior to formal imaging, co-incubation of AF-C with E. coli for 15, 30, 45, 60, or 75 min was screened using cells harvested 12 h after induction; 45 min was selected as the staining period based on maximal ratiometric signal and signal-to-background, low cell-to-cell variability, and unchanged viability (Fig. S4). After incubation, cells were washed three times with PBS, resuspended in 1 mL saline, and imaged on a Leica TCS SP5 spectral confocal microscope. AF-C was excited at 561 and 633 nm, and emissions were collected at 579–668 nm and 674–767 nm, respectively. All images were acquired using identical detector settings across time points. 2.6 Transcriptomic and metabolomic analysis For transcriptomic and metabolomic profiling, E. coli BL21(DE3) cultures in LB medium were induced with IPTG as described above. Samples were collected at 0, 12, and 24 h post-induction from both experimental and control groups. Cell pellets were rapidly frozen in liquid nitrogen and stored at -80 °C until analysis. Total RNA extraction, library preparation and sequencing, metabolite extraction, and LC-MS/MS-based metabolomics were commissioned to and performed by Tsingke (Xi’an, China) according to their standard protocols. 2.7 Quantitative real-time PCR (qRT-PCR) Total RNA was extracted from E. coli BL21(DE3) cells using RNAiso Plus (Takara, Japan) according to the manufacturer’s instructions. RNA quality and concentration were assessed with a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). One microgram of total RNA was reverse-transcribed using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Japan). Quantitative PCR was performed with TB Green® Premix Ex Taq™ II (Takara, Japan) on a CFX96 real-time PCR detection system (Bio-Rad, USA). Gene-specific primers were designed using Primer-BLAST (NCBI); sequences are listed in Table S2. The gyrA gene served as the internal reference, and relative expression levels were calculated using the 2 ⁻ΔΔCt method. All reactions were run in triplicate. 2.8 Stepwise medium optimization in shake flasks Shake-flask medium optimization was carried out in baffled flasks using the same inoculum preparation, cultivation temperature and sampling and analytical procedures as described above. A four-step design was adopted, in which the best-performing composition from each step was carried forward. Step one varied the amount and ratio of tryptone and yeast extract; step two adjusted total phosphate with a KH 2 PO 4 /Na 2 HPO 4 buffer; step three varied the initial glucose concentration; and step four varied ammonium sulfate to tune inorganic nitrogen supply and the overall carbon-to-nitrogen ratio. For all conditions, cultures were induced with IPTG until OD 600 reached 0.8–1.0, and rhBNP titer, optical density and terminal broth pH were measured in biological triplicate to identify the formulation that best balanced near-neutral terminal pH and high rhBNP titer. 2.9 Fed-batch fermentation Fed-batch fermentations were performed in a 3-L stirred-tank using the optimized shake-flask medium supplemented per litre with 1.5 g citric acid, 0.6 g MgSO 4 and 1 mL trace metal solution, which contained (per liter of 250 μL H 2 SO 4 ): 17.6 g FeC 6 H 5 O 7 ·5H 2 O, 2 g ZnSO 4 ·7H 2 O, 0.2 g CuSO 4 ·5H 2 O, 1.6 g MnSO 4 , 0.3g H 3 BO 3 , 0.2g (NH 4 ) 6 Mo 7 O 24 ·4H 2 O, 0.3g CoCl 2 ·6H 2 O. The aeration was set at 3 L/min (2 vvm), and the agitation was set between 200 and 1,000 rpm to maintain the dissolved oxygen (DO) levels at >20% of saturation. pH was controlled at 7.2 by automatic addition of 7% (v/v) NH 3 ·H 2 O. The culture was grown at 37 °C until the OD 600 reached about 35, after which the temperature was reduced to 21 °C and IPTG was added to a final concentration of 1.0 mM to induce protein expression. The cells were harvested after 20–22 h post induction. 2.10 Protein purification by intein-mediated cleavage Intein-mediated cleavage was performed as described previously [25]. Cell pellets were resuspended in disruption buffer (20 mM Tris-HCl, 500 mM NaCl, 1 mM EDTA·2Na, pH 8.5) and sonicated on ice for 20 min (JY92-IIN, Scientz, Ningbo, China). Lysates were clarified by centrifugation (12,000 rpm, 15 min, 4 °C). Pellets were washed twice with disruption buffer; an aliquot was reserved for SDS-PAGE. The remaining pellet was resuspended in an equal volume of intein-cleavage buffer (PBS containing 40 mM Bis-Tris and 2 mM EDTA·2Na, pH 6.2) and incubated at 25 °C for 24 h, after which the soluble fraction was collected by centrifugation (12,000 rpm, 15 min, 4 °C) for downstream analysis. Proteins were analyzed on FastPAGE™ 4–20% Bis-Tris SDS-PAGE gels (Tsingke, China) and stained with Coomassie Brilliant Blue R-250. Band intensities were quantified densitometrically using ImageJ (NIH, USA). Bovine serum albumin (BSA) and aprotinin (APR) were used as standards to assign band identities and determine protein quantities. 3. Results 3.1 rhBNP expression by cSAT scheme and broth alkalinization System setup and expression validation. To establish a process-relevant baseline for rhBNP production under the cSAT scheme in E. coli , we retained the L 6 KD-Mtu ΔI-CM tag (Fig. 1A) and used a slightly intensified induction regime relative to the original report: cultures were grown in LB medium and induced until OD 600 reached 0.6–0.8 at 21 °C with 0.2 mM IPTG [25]. Under these conditions, shake-flask cultures reached high biomass and accumulated the cSAT-rhBNP fusion as a prominent band on SDS-PAGE (Fig. 1C). Intein-mediated self-cleavage released an approximately 3.5 kDa band in the soluble fraction consistent with rhBNP, as confirmed by enzymatic digestion and mass spectrometry (Figs. S2 and S3). The 18 °C condition described previously is used only as a contextual reference and was not pursued further as a benchmark in this study [25]. Growth and broth pH trajectories . During shake-flask cultivations, cultures expressing rhBNP under the cSAT scheme showed a pronounced rise in broth pH. Terminal values exceeded 8.5 both under the process-oriented induction regime used in this study (Fig. 1E) and when the original 18 °C protocol was reproduced (Fig. S1). In contrast, the empty-vector control maintained a terminal pH close to neutrality (Fig. 1D). These data indicate that strong broth alkalinization is a reproducible feature of rhBNP expression under the cSAT scheme across the induction regimes tested and cannot be attributed simply to changes in induction temperature or cell density. Working hypotheses for alkaline drift . To interpret this alkaline drift, we formulated four non-exclusive, testable working hypotheses, each linked to specific experimental readouts. First, self-aggregation-driven proteostasis and energy demand were expected to increase ATP and cofactor turnover and perturb the NADH/NAD + balance [29], providing a route to a modest alkaline shift via respiration-linked proton handling [17]. Second, ammoniagenic amino-acid and one-carbon rewiring could reduce net acid generation from central carbon metabolism [30, 31]. Third, PPi/Pi cycling and tricarboxylic-acid/glyoxylate branch adjustments might limit acid accumulation [32, 33]. Fourth, higher-throughput expression conditions were expected to amplify whichever mechanisms were active. These hypotheses guided the choice of readouts and the design of the omics and medium-engineering experiments described below [34]. 3.2 Spectral pH response and biocompatibility of AF-C Spectral design and operating principle . To follow intracellular pH dynamics during rhBNP production under the cSAT scheme, we implemented an assay based on the HQO-type ratiometric probe AF-C. AF-C interconverts between protonated and deprotonated forms that modulate an intramolecular charge-transfer pathway, yielding anti-correlated spectral signals: the visible absorption band at 548 nm increases with pH, whereas the near-infrared band at 720 nm increases at lower pH. To normalize for probe loading, optical pathlength and illumination, we used the absorbance ratio A 720 nm /A 548 nm and the fluorescence ratio I 740 nm /I 630 nm (excitation at 561 and 640 nm, emission at 630 and 740 nm). All in vitro characterizations were performed in HEPES buffer containing dimethyl sulfoxide and, where indicated, 10 mmol/L SDS to provide a membrane-mimetic microenvironment and stabilise the spectra (Fig. 2A). UV-visible characteristics and band selection . In 10 mM HEPES containing 10 mM SDS and DMSO, AF-C displayed two diagnostic absorbance bands with maxima at 548 and 720 nm (Fig. 2B). Spectra recorded at pH 6.8 and 7.4 showed inverse changes in band intensity: the 548 nm band was stronger at pH 7.4, whereas the 720 nm band was stronger at pH 6.8. These measurements establish an inverse ratiometric relationship for the absorbance ratio A 720 nm /A 548 nm in this cell-mimetic matrix and support pairing one visible and one near-infrared band for downstream imaging. Interference tolerance in a cell-mimetic matrix . Selectivity was assessed at pH 7.4 by spiking the AF-C matrix (10 mM HEPES, 10 mM SDS and DMSO) with representative intracellular modulators, including IPTG, low-molecular-weight thiols (reduced glutathione and cysteine), mono and divalent salts, phosphate species and additional SDS. As summarized in Fig. 2C, the absorbance ratio A 720 nm /A 548 nm remained within experimental variation across these challenges, indicating that under our assay conditions AF-C primarily reports proton activity rather than these coexisting factors. The fluorescence intensity ratio I 740 nm /I 630 nm showed the same qualitative robustness (Fig. 2D). Ratiometric pH calibration for imaging (I 740 nm /I 630 nm , pH 6.6–8.6). The same matrix used for in vitro characterization (DMSO with 10 mM SDS and 10 mM HEPES) was titrated from pH 6.6 to 8.6, and AF-C fluorescence ratios (I 740 nm /I 630 nm ) were measured in ≥3 replicates per point. After background subtraction and channel registration, ratios were summarized as mean ± SEM and plotted to generate the empirical calibration curve (Fig. 2D), which was monotonic over the working range. The data were then fitted to a four-parameter Henderson-Hasselbalch-type model (4PL-HH) by unweighted nonlinear least squares (Levenberg-Marquardt): valid for pH 6.6–8.6. Together, Figs. 2A–D show that AF-C provides anti-correlated visible/NIR bands, robust selectivity at pH 7.4, and a well-behaved ratiometric calibration suitable for cell-to-cell comparable pH mapping during induction. Biocompatibility across working concentrations . AF-C was added to cultures immediately after IPTG induction. Stock solutions were prepared in DMSO and dosed so that the final DMSO content was 0.5% by volume in all cultures, with the control group receiving vehicle only. After 6 hours of co-incubation, CCK-8 measurements indicated viabilities exceeding 85 percent at all AF-C concentrations (Fig. 2E). Over the same 6-hour period, the OD 600 was recorded hourly, and growth trajectories were indistinguishable from the vehicle control (Fig. 2F), indicating no detectable growth penalty attributable to AF-C under imaging-relevant conditions. These results support the use of AF-C in subsequent live-cell intracellular pH measurements without additional correction for probe-induced cytotoxicity or growth inhibition. 3.3 AF-C ratiometric imaging of intracellular pH dynamics under the cSAT scheme Single-cell ratiometric imaging and conversion to intracellular pH . Two-channel confocal images (emission at 630 and 740 nm) were acquired from AF-C-loaded cultures expressing rhBNP under the cSAT scheme and from empty-vector controls at 0, 12 and 24 h after induction. Single-cell regions of interest were segmented in ImageJ, background was subtracted, the two channels were co-registered and a ratiometric signal R=I 740 nm /I 630 nm was calculated for each cell. Ratios were converted to pHi using the four-parameter logistic Henderson-Hasselbalch calibration obtained in vitro (Eqs. 2 and 3). As expected from the spectral characterization, higher pH produced decreased 740 nm emission and increased 630 nm emission, whereas lower pH produced the opposite trend, supporting the reliability of the ratio-to-pHi conversion used in subsequent analyses. Quantitative summary, pHi-pHe relationships and the 12 h separation . At the induction time point (0 h), both cultures maintained pHi slightly higher than the corresponding extracellular pH (pHe), consistent with near-neutral pHi homeostasis in mildly acidic broth (culture expressing rhBNP under the cSAT scheme: pHe 6.81, pHi 7.15; empty-vector control: pHe 6.82, pHi 7.06; Figs. 3E and 3H). Divergence emerged 12 h after induction: the cSAT scheme culture reached a median pHi of about 8.25, whereas the control remained near pHi 7.5 while growth and biomass were still increasing. By 24 h after induction, the cSAT scheme culture remained more alkaline overall (pHe 8.57, pHi 8.53), with intracellular and extracellular pH closely matched, whereas the control lagged behind (pHe 7.76, pHi 7.61) and still showed pHi slightly below pHe. Group differences in pHi and pHe over 12 to 24 h were statistically significant (biological replicates; per-cell medians analysed with a mixed-effects model; P < 0.01). Throughout the time course, red and near-infrared channel trends in cells mirrored the in vitro AF-C fingerprints, supporting the robustness of the ratiometric readout. 3.4 Process-linked integration of transcriptomics and metabolomics Sample grouping and replicates . For RNA-seq and metabolomics, cultures expressing rhBNP under the cSAT scheme and empty-vector controls were sampled at 0, 12 and 24 h after induction, each with three biological replicates (labels cS-0/1/2 and a30-0/1/2). These identifiers are used consistently in the correlation analysis and principal component analysis. Global structure: correlation and principal component analysis . Correlation analysis and principal component analysis (PCA) were used to assess data quality and overall structure (Figs. 4A and 4B). Biological replicates clustered tightly with pairwise correlation coefficients above 0.95, and no outlier libraries were detected, so all samples were retained for downstream analyses. PCA separated samples primarily by time (principal component 1; 0, 12 and 24 h) and secondarily by condition (principal component 2; cSAT scheme rhBNP expression versus control). From 12 h after induction onward, cultures expressing rhBNP under the cSAT scheme were clearly separated from time-matched controls, consistent with the higher intracellular pH observed by AF-C imaging. Time and condition effects on principal component scores were significant by two-way analysis of variance (P<0.01), and technical covariates such as library size and rRNA fraction did not dominate the leading components, indicating that the separation is biologically driven. Differential genes and metabolites across induction . Using thresholds of an absolute log 2 fold change of at least one (corresponding to a fold change of at least two) and a false discovery rate below 0.05, we detected 792, 2,042 and 1,381 differentially expressed genes at 0, 12 and 24 h after induction, respectively (12h volcano plot in Fig. 4C; 0 and 24h results in Fig. S5). Metabolomics showed a similar trend, with 215, 520 and 567 differentially abundant metabolites at the same time points (Fig. S8). Thus, both omics layers identify 12 h after induction as a major inflection point, coinciding with the AF-C imaging evidence for induction-phase intracellular alkalinization and indicating a period of elevated folding and clearance demand and biosynthetic load accompanied by reorganization of organic-acid and nitrogen-related metabolism. Functional enrichment across induction and link to pH control . At 12 h after induction, Gene Ontology (GO) enrichment was dominated by metabolic processes together with ATP-dependent chaperone and folding functions and transporter activity (Fig. 4D), consistent with increased proteostasis demand, ATP turnover and membrane transport under the cSAT scheme. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis at the same time point (Fig. 4E) highlighted pathways with direct relevance to the alkaline phenotype, including central-carbon modules (glycolysis and gluconeogenesis, pyruvate metabolism, glyoxylate and dicarboxylate metabolism), ammoniagenic amino-acid routes (glycine, serine and threonine metabolism; alanine, aspartate and glutamate metabolism) and respiratory and transport modules (oxidative phosphorylation, ATP-binding cassette transporters, the phosphotransferase system and two-component and quorum-sensing systems). Enrichment patterns at 0 and 24 h (Figs. S6–S9) frame this 12 h program as an induction-phase peak between the pre-induction baseline and later adaptations. Overall, the GO and KEGG results support a mechanistic view in which central-carbon rerouting and nitrogen handling, together with transport and respiratory reprogramming, form a core module underlying intracellular alkalinization under the cSAT scheme. 3.5 Mechanism of intracellular alkalinization under the cSAT scheme Heatmap analysis of pH-linked genes and metabolites under the cSAT scheme . The pathway-focused gene heatmaps (Figs. 5A–G) highlight amino-acid, central-carbon, respiratory and phosphate-ion modules that change in parallel with intracellular alkalinization in cultures expressing rhBNP under the cSAT scheme. In the amino-acid block (Fig. 5A), gcvP , gcvT and gcvH (glycine-cleavage system), aspA (aspartate to fumarate), dadA , dadX , sdaB and the ast genes are strongly induced at 12 h after induction, consistent with enhanced conversion of glycine, aspartate and other amino acids into tricarboxylic-acid cycle entry points with ammonia release. In the nitrogen-assimilation block (Fig. 5B), coordinated changes in glnA , gltB , gltD and glmS indicate that glutamate and glutamine pools are actively rebalanced under the cSAT scheme. The glyoxylate-associated block (Fig. 5C) shows increased glcD , glcE , glcF , aceB , aceA , katG , ahpC , ahpF , aldA , aldB and garK at 12 h after induction, pointing to activation of the glyoxylate shunt and the glycolate-glyoxylate-glycerate branch when the alkaline phenotype is established. Tricarboxylic-acid cycle genes ( fumB , fumC , fumD , sdhA to sdhD , mdh , sucA , sucC and sucD ; Fig. 5D), respiratory-chain components ( nuo genes, ndh , cyo and cyd ; Fig. 5E) and the succinate dehydrogenase versus fumarate reductase pair ( sdhA and sdhB versus frdA to frdD ; Fig. 5F) also show their largest separation at 12 h after induction, indicating that electron transport and succinate-fumarate routing are adjusted in the same induction window. Finally, ppa and ppk1 in the phosphate-energy block, together with modest time-dependent changes in nhaA , nhaB and trkA (Fig. 5G), link phosphate turnover and Na⁺/H⁺ antiport and K⁺ handling to the alkaline state, rather than acting as isolated responses. Metabolite heatmaps under the cSAT scheme . The metabolite heatmaps (Figs. 5I–L) mirror these gene-level patterns. Pyrophosphate, inorganic phosphate and ADP all increased at 12 h after induction, consistent with elevated ATP turnover under the cSAT scheme. Glutamate, α-ketoglutarate, oxaloacetate, arginine and alanine rose in the same window, supporting engagement of ammoniagenic amino-acid pathways. γ-Aminobutyric acid and downstream TCA intermediates showed coordinated increases, in line with activation of the glutamate decarboxylase-GABA shunt as a cytosolic proton sink [35]. Glyoxylate-branch metabolites shifted together with aceA , aceB and glycolate-glyoxylate-glycerate genes, indicating glyoxylate-centred rerouting that tempers acid accumulation while maintaining anaplerosis [14]. Taken together, these heatmaps identify a compact set of pathways that co-vary with intracellular alkalinization under the cSAT scheme and provide the basis for the qRT-PCR validation and mechanistic mapping presented next. qRT-PCR validation of alkalinization-linked modules under the cSAT scheme . Relative transcript levels were quantified by the ΔΔCt method using gyrA as the reference gene, with the empty-vector culture serving as the calibrator at each time point [36]. As shown in Fig. 6, the glycine-cleavage genes gcvP , gcvT and gcvH were already elevated at the induction time point in the cSAT scheme culture and remained clearly higher than in the time-matched control at 12 h after induction. Their expression partially declined by 24 h after induction but stayed above control levels, indicating that one-carbon and ammoniagenic fluxes are engaged early in the induction phase and persist through the period in which intracellular alkalinization is maximal. A similar time course was observed for dadA , gdhA , glcD , sucA , aceB , garK and dnaK : these transcripts increased in the cSAT scheme culture at or shortly after induction, peaked at 12 h after induction and then decreased toward 24 h after induction while remaining above the control. In contrast, aspA was slightly lower than the control at the start of induction but clearly higher at 12 h after induction and still elevated at 24 h after induction, consistent with time-dependent activation of the aspartate-to-fumarate node. Taken together, these patterns validate the engagement of ammonia-releasing deamination ( dadA , gdhA ), glyoxylate and glycerate routing ( glcD , aceB , garK ), tricarboxylic-acid cycle entry ( sucA ) and proteostasis demand ( dnaK ) alongside the glycine-cleavage system, and support a mechanistic link between the cSAT scheme, the multi-omic signatures and the intracellular alkalinization quantified by AF-C imaging. Amino acid and one-carbon remodelling under the cSAT scheme . The integrated pathway map (Fig. 7A) indicates that the cSAT scheme couples peptide and amino acid uptake to a set of ammoniagenic and proton-consuming reactions that favour intracellular alkalinization while reshaping central-carbon flow. Oligopeptide and dipeptide ATP-binding cassette transporters (Opp and Dpp) import peptides into the cytosol, where they are hydrolysed and channelled into deamination and lyase routes, linking peptide utilisation directly to nitrogen metabolism [37, 38]. Serine is converted to pyruvate by the pyridoxal 5′-phosphate-dependent dehydratase sdaB [39]; alanine enters the pyruvate node through alaA and avtA , with dadA and dadX oxidising the resulting amino acids; aspartate is transformed into fumarate by aspA [40]; glycine is degraded by the glycine-cleavage system ( gcvP , gcvT and gcvH ) to 5, 10-methylenetetrahydrofolate and NH 3 [30]; glutamate is dehydrogenated to α-ketoglutarate by gdhA with NH 3 release; and arginine proceeds through the ast pathway toward succinate [35, 41, 42]. Together, these reactions increase ammoniagenic pressure and reduce net proton retention, providing a plausible mechanistic explanation for the observed intracellular alkalinization under the cSAT scheme. Glutamate decarboxylation and proton sinks . In parallel, the glutamate decarboxylase system ( gadA , gadB and the antiporter gadC ) consumes cytosolic protons to form γ-aminobutyric acid (GABA), which is converted by gabT and gabD to succinate, establishing a cytosolic proton sink at the periphery of the tricarboxylic-acid cycle [43, 44]. In our data, gcvP , gcvT , gcvH , aspA , dadA , gdhA , the ast genes, gadA , gadB , gabT and gabD are coordinately induced in cultures expressing rhBNP under the cSAT scheme, and 12 h metabolite profiles show increased glutamate, α-ketoglutarate, oxaloacetate, arginine, alanine, GABA, succinate semialdehyde and succinate. Together, these nitrogen and one-carbon circuits provide a mechanistic basis for the early intracellular alkalinization and rising broth pH observed by AF-C imaging and identify amino acid pools (glycine, serine, alanine, aspartate, glutamate and arginine) and NH 3 /NH 4 ⁺ as process-level markers. Central carbon adjusts in concert . Activation of the glyoxylate shunt via aceA and aceB bypasses decarboxylating tricarboxylic-acid cycle steps and tempers net acid generation from oxidative TCA fluxes while maintaining anaplerosis, a classical strategy in E. coli under carbon and redox stress [45, 46]. Upstream, the glycolate-glyoxylate-glycerate branch, which includes glcD , glcE , glcF , aldA and garK , redistributes flux between anaplerotic and gluconeogenic routes [47]. The succinate node receives convergent input from the glutamate decarboxylase-GABA route and from tricarboxylic-acid cycle branches [48]. Increased succinate together with glyoxylate-branch metabolites (hydroxypyruvate, glycerate and glycolaldehyde) and citrate in cultures expressing rhBNP under the cSAT scheme is consistent with this glyoxylate-centred rerouting [45, 49]. Overall, the pathway map supports a process in which ammoniagenesis and proton-consuming decarboxylation reduce net acid load, while glyoxylate-based central-carbon remodelling limits acid accumulation and maintains redox balance and ATP supply for high-load expression under the cSAT scheme. Respiratory-chain and phosphate modules supporting proton balance under the cSAT scheme . The lower part of the pathway schematic (Fig. 7B) links these metabolic shifts to respiratory routing and phosphate-energy management. In E. coli , NADH dehydrogenase I (NDH-1, nuo ) and cytochrome bo 3 ( cyo ) are proton-pumping components of the respiratory chain, whereas NADH dehydrogenase II (NDH-2, ndh ) and cytochrome bd ( cyd ) oxidise quinols without vectorial proton pumping [50-53]. Preferential routing of electrons through an NDH-1/cytochrome bo 3 pathway rather than an NDH-2/cytochrome bd pathway increases net proton extrusion, supports ATP synthesis via the F 1 F 0 -ATP synthase and keeps protons predominantly outside the cytosol, thereby contributing to the rise in intracellular pH [15]. Succinate dehydrogenase ( sdhA to sdhD ) feeds electrons from succinate into the quinone pool, whereas fumarate reductase ( frdA to frdD ) catalyses the reverse reaction and decouples the tricarboxylic-acid cycle from oxidative phosphorylation [54]. In our data, increased expression of nuo and cyo together with enhanced sdh and reduced frd transcripts in cSAT scheme cultures at 12 h after induction is consistent with a more proton-pumping-competent electron-transport configuration supported by elevated succinate and may reinforce the central-carbon and nitrogen remodelling associated with intracellular alkalinization. Phosphate-energy metabolism adjusts in parallel . Polyphosphate kinase ( ppk1 ) and inorganic pyrophosphatase ( ppa ) form a polyphosphate-pyrophosphate-phosphate cascade that buffers high-energy phosphate equivalents and supplies inorganic phosphate for ADP phosphorylation [33, 55]. In cultures expressing rhBNP under the cSAT scheme, increased pyrophosphate, inorganic phosphate and ADP at 12 h after induction, together with induction of ppk1 and ppa , are consistent with intensified ATP turnover coupled to PPi hydrolysis and recycling. Modest time-dependent changes in Na⁺/H⁺ antiporters ( nhaA and nhaB ) and in K⁺ uptake control ( trkA ) suggest envelope-level tuning of ion homeostasis rather than a primary pH-control system. Overall, the respiratory and phosphate modules provide a parsimonious complement to the amino-acid/one-carbon and glyoxylate mechanisms: increased ATP demand from proteostasis and transport is met by PPi-linked phosphate cycling and a more proton-pumping respiratory chain, while protons are kept predominantly outside the cytosol. This integrated picture helps to explain the sustained alkaline intracellular pH trajectory under the cSAT scheme and nominates nuo, ndh , cyo , cyd , sdh , frd , ppk1 and ppa , together with the NADH/NAD⁺ ratio, succinate, PPi, Pi and the ATP/ADP balance, as practical markers for future process monitoring and engineering. 3.6 Stepwise medium optimization and scale-up fermentation Stepwise medium optimization and endpoints . Medium composition was optimized in four sequential steps in shake flasks: adjustment of the complex nitrogen source, addition of phosphate buffer, glucose supplementation and tuning of ammonium supply and the carbon-to-nitrogen ratio. The objective was to define an operating window that maintained high rhBNP productivity while keeping the final broth pH close to neutrality. At each step, the condition providing the best compromise between rhBNP titer, biomass and terminal broth pH was carried forward to the next step. Fusion-protein expression was monitored by SDS-PAGE with densitometric analysis (Fig. S10), and endpoint biomass (OD 600 ), rhBNP titer and terminal broth pH for each condition are summarized in Fig. S11. Tryptone to yeast extract ratio optimization (step 1) . In the first step, the ratio of tryptone to yeast extract in the complex nitrogen source was varied from the LB baseline of 2:1 to 1:1, 1:1.5, 1:2 and 1.5:1. As shown in Fig. 8A and Fig. S12A and summarized in Table S3, a ratio of 1:1.5 gave the highest rhBNP titer (70.8 mg/L versus 39.5 mg/L in LB medium) without loss of biomass. However, the harvest pH remained above 8.5 at all ratios and reached 8.87 at 1:1.5, indicating a pronounced alkaline liability. This behaviour is consistent with our intracellular model, in which richer complex nitrogen is associated with increased ammonia-releasing amino-acid catabolism and proton-consuming reactions, supporting higher expression at the cost of stronger alkalinization [29]. The 1:1.5 ratio was therefore carried forward to subsequent optimization steps, where phosphate buffering and carbon-to-nitrogen ratio control were introduced to retain the yield gain while moderating the pH rise. Phosphate buffer screening (step 2) . Using the 1:1.5 tryptone to yeast extract ratio as the baseline, we evaluated a KH 2 PO 4 /Na 2 HPO 4 buffer at total phosphate concentrations of 0, 10, 20, 50 and 100 mM. The corresponding rhBNP titers were 70.4, 115.6, 98.8, 83.4 and 66.7 mg/L, respectively (Figs. 8B and Fig. S12B; Table S4). The harvest pH remained above 8.3 from 0 to 50 mM and decreased to 7.61 only at 100 mM, where expression was reduced. Thus, moderate phosphate increased titer but only partially dampened the alkaline shift, whereas high phosphate suppressed alkalinity at the expense of titer [56]. Because subsequent steps introduce glucose and ammonium sulfate, we selected 50 mM total phosphate as a compromise that preserves most of the yield increase while providing sufficient buffer capacity. Glucose concentration screening (step 3) . Starting from the step-2 medium, glucose was supplied at 0, 10, 30, 50 and 100 mM. The corresponding rhBNP titers were 84.5, 92.5, 99.1, 103.4 and 42.2 mg/L, with harvest pH values of 8.44, 7.86, 7.49, 7.33 and 5.39, respectively (Fig. 8C and Fig. S12C; Table S5). Moderate glucose (30 to 50 mM) therefore increased titer (maximum 103.4 mg/L at 50 mM) while partially offsetting the alkaline drift from the complex nitrogen source and bringing the terminal pH toward neutrality. At 100 mM glucose, rhBNP production dropped sharply and premature in vivo self-cleavage of the intein fusion was observed, consistent with the final pH of 5.39 falling below the intein induction pH of approximately 6.2, where folding, cleavage kinetics and redox balance are expected to deteriorate. Ammonium sulfate screening (step 4) . Using the step-3 medium (50 mM glucose, 50 mM phosphate, tryptone:yeast extract 1:1.5), we varied ammonium sulfate at 0, 1.70, 3.40, 5.67 and 17.0 g/L to adjust the designed carbon-to-nitrogen (C/N) ratio, defined as the mass of carbon supplied by glucose divided by the mass of nitrogen supplied by ammonium sulfate. With 50 mM glucose supplying 3.603 g carbon per litre, these loadings correspond to nominal C/N ratios of 10:1, 5:1, 3:1 and 1:1. rhBNP titers at 0, 1.70, 3.40, 5.67 and 17.0 g/L were 106.5, 115.4, 77.2, 0 and 0 mg/L, with corresponding final broth pH values of 7.33, 7.04, 6.65, 5.55 and 5.33, respectively (Fig. 8D and Fig. S12D; Table S6). Thus, low to moderate ammonium loadings (0 to 3.40 g/L) maintained high rhBNP production, and 1.70 g/L ammonium sulfate slightly increased titer relative to the ammonium-free baseline while shifting the endpoint pH closer to neutrality. Higher ammonium levels caused progressive acidification and complete loss of recoverable rhBNP despite robust biomass and fusion expression, consistent with the final broth pH falling well below the intended cleavage window around pH 6.2, where folding, cleavage kinetics and redox balance are expected to deteriorate. Overall C/N context of the optimized medium . In the final formulation (10 g/L tryptone, 15 g/L yeast extract, 50 mM glucose, 1.70 g/L (NH 4 ) 2 SO 4 and a nitrogen-free phosphate buffer), the elemental carbon-to-nitrogen ratio calculated from glucose carbon and all nitrogen sources is approximately 1.20 to 1.34 mol per mol, and additional organic carbon from the complex nitrogen sources places the culture in a moderately nitrogen-rich regime. At 1.70 g/L ammonium sulfate (designed C/N 10:1), this regime gave the best compromise, with high fusion and rhBNP titers and a final broth pH close to 7.0, whereas higher ammonium loadings drove acidification, extensive in vivo self-cleavage and loss of recoverable rhBNP. Functionally, the moderately nitrogen-rich background favours ammonia-releasing amino-acid turnover and proton-consuming reactions that tend to raise pH, while 50 mM glucose provides acid-generating flux that counterbalances this tendency. The final medium therefore places expression under the cSAT scheme in a buffered window that sustains rhBNP yield while keeping broth pH within the operational range for intein self-cleavage. Scale-up fermentation in a 3-L stirred-tank fermenter . To evaluate the scale-up potential of the optimized cSAT scheme, we ran a glucose-limited fed-batch fermentation in a 3-L stirred-tank bioreactor using the step-4 medium formulation as the batch phase. After batch growth on the optimized medium, feeding with a concentrated glucose-tryptone-magnesium solution was initiated when residual glucose from the batch phase was depleted and then supplied continuously for the remainder of the fermentation. The culture was grown in batch mode until the OD 600 reached approximately 35, at which point 1.0 mM IPTG was added to induce expression, and fermentation was then continued under fed-batch conditions without interruption until the planned end of the 29.5 h run (Fig. 9A). For SDS-PAGE and densitometric quantification, fermentation samples were diluted before loading (ten-fold for the cSAT-rhBNP fusion suspension and four-fold for rhBNP; Fig. S13A and Fig. S13B), and band intensities were corrected for these dilution factors and broth volume. Under these conditions, the cSAT-rhBNP fusion protein titer in the fermenter was 12.7 g/L and the final rhBNP titer was 662.1 mg/L at a terminal broth pH close to 7.0 (Figs. 9A and 9B). This corresponds to an approximately 16.8-fold increase in rhBNP titer compared with LB medium shake flasks (39.5 mg/L) and about a 5.7-fold increase over the optimized shake-flask medium (115.4 mg/L), while maintaining the near-neutral broth pH anticipated from the proton-economy-based medium design. 4. Discussion Mechanistic summary and process implications . This study helps to explain why high-load rhBNP expression under the cSAT scheme is associated with broth and intracellular alkalinization and how this can be managed at the process level [ 25 ]. AF-C-based single-cell intracellular pH imaging [ 22 – 24 ], together with time-resolved qRT-PCR, transcriptomics and metabolomics, points to ammonia-releasing amino-acid pathways, proton-consuming glutamate decarboxylation [ 31 , 33 , 46 ], glyoxylate-centred carbon rerouting and a shift towards more proton-pumping respiratory branches as key contributors to the alkaline state [ 57 , 58 ]. Treating medium and process design as a proton-economy problem, we used this framework to stabilise the harvest pH near neutrality and increase rhBNP titer 2.9-fold in shake flasks relative to LB medium (from 39.5 to 115.4 mg/L), and in a 3-L fed-batch fermentation to reach 12.7 g/L cSAT-rhBNP fusion protein and 662.1 mg/L rhBNP at a terminal broth pH close to 7.0. Chassis-level interventions suggested by the pathway map . The pathway map points to several chassis-level strategies that could reduce alkaline drift without sacrificing throughput. First, moderating ammoniagenic flux through selected glycine-cleavage, dehydrogenase and deaminase genes (for example gcvP , gcvT , gcvH , gdhA , dadA and aspA ) would weaken a major alkaline drive [ 30 ]. Second, limiting the level or duration of glutamate decarboxylase and GABA-branch activity ( gadA , gadB , gabT and gabD ) could prevent excessive cytosolic proton consumption [ 46 ]. Controlled expression of aceA and aceB would allow glyoxylate shunt activity to support anaplerosis without over-reducing organic-acid pools [ 32 ]. In parallel, tuning respiratory composition ( nuo and cyo relative to ndh and cyd ) and phosphate-energy buffering ( ppk1 and ppa ) offers additional levers to coordinate proton pumping and ATP supply with the desired pH range [ 59 ]. Together, these interventions outline a chassis in which proton economy, redox balance and tag-based high-load expression are engineered in a coordinated rather than sequential manner. Scale-up guidance from the proton-economy mechanism . The 3-L fed-batch runs show that the proton-economy framework can be applied at stirred-tank scale: with the optimized medium, pH-stat control and a glucose-limited feed, the culture maintained a near-neutral broth pH while accumulating 12.7 g/L cSAT-rhBNP fusion protein and 662.1 mg/L rhBNP. These results suggest that further scale-up should retain moderate phosphate buffering with gentle acid and base addition [ 60 ], carbon feeds that keep residual glucose low and avoid strong pH swings [ 61 ], and dissolved-oxygen set points that prevent deep microaeration while avoiding excessive aeration and carbon dioxide stripping [ 62 ]. Balancing yield and proton economy under the cSAT scheme . Even in this controlled window, some in vivo self-cleavage is expected as the average intracellular pH approaches the intein induction pH of about 6.2 [ 63 ], and larger reactors will increase local variation in oxygen, pH and redox state [ 64 , 65 ]. Our data therefore argue that the cSAT scheme expression should be engineered jointly for yield and proton economy: coordinated control of pathway timing, respiratory bias and feed and buffer profiles can turn alkalinity from a liability into a tunable handle for robust fermentation-type production of disulfide-bonded bioactive peptides, with rhBNP as a stringent model. Declarations Acknowledgements We thank the School of Chemical Engineering at Northwest University for kindly providing the facilities for this research. Author contributions M. and H.N. conceived the ideas and designed the experiments. H.N. performed the majority of the experiments. Y.F., G.S. and H.L. assisted in conducting the confocal experiments. 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Supplementary Files supplementary.docx Cite Share Download PDF Status: Published Journal Publication published 01 Feb, 2026 Read the published version in Microbial Cell Factories → Version 1 posted Editorial decision: Revision requested 15 Dec, 2025 Reviews received at journal 11 Dec, 2025 Reviewers agreed at journal 11 Dec, 2025 Reviews received at journal 10 Dec, 2025 Reviewers agreed at journal 09 Dec, 2025 Reviewers agreed at journal 09 Dec, 2025 Reviewers invited by journal 09 Dec, 2025 Editor assigned by journal 06 Dec, 2025 Submission checks completed at journal 06 Dec, 2025 First submitted to journal 01 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8252127","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":559030125,"identity":"1d3183f0-f9fe-445e-b564-63d2508a597f","order_by":0,"name":"Hang Nie","email":"","orcid":"","institution":"Northwest University","correspondingAuthor":false,"prefix":"","firstName":"Hang","middleName":"","lastName":"Nie","suffix":""},{"id":559030129,"identity":"fbd7fa4f-dafa-45f2-9403-e5b6ed749bf8","order_by":1,"name":"Yajun Feng","email":"","orcid":"","institution":"Northwest 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1","display":"","copyAsset":false,"role":"figure","size":123747,"visible":true,"origin":"","legend":"\u003cp\u003eExpression and physiological characterization of rhBNP under cSAT scheme in \u003cem\u003eE. coli\u003c/em\u003e. (A) Schematic diagram of the rhBNP expression by cSAT scheme. (B) Vector map of recombinant pET-30a(+)/cSAT-rhBNP plasmid. (C) SDS-PAGE results of the protein expression in small shake flask experiments by LB medium. EP: precipitate of the cell lysate after expression; ES: supernatant of the cell lysate after expression, CP: precipitate of intein-mediated cleavage; CS: supernatant of intein-mediated cleavage; MI, MII: protein marker. (D) Growth curve and pH profile of strain harboring the pET-30a(+) (empty vector control). (E) Growth curve and pH profile of strain harboring the pET-30a(+)/cSAT-rhBNP plasmid.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8252127/v1/d4fbd3362451a632ab302aa6.png"},{"id":98433639,"identity":"9ae990a5-ffd1-40dd-8efb-97f5294a376c","added_by":"auto","created_at":"2025-12-17 16:50:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":91391,"visible":true,"origin":"","legend":"\u003cp\u003eIn-vitro characterization and biocompatibility of AF-C under a cell-mimetic matrix (A) Operating principle of AF-C. (B) Absorption spectra of AF-C in a HEPES buffer solution (10 mM) containing SDS (10 mM) at different pH 6.8 and pH 7.4. (C) Absorption intensity ratio (A\u003csub\u003e720 nm\u003c/sub\u003e/A\u003csub\u003e548 nm\u003c/sub\u003e) of AF-C (10 μM) in the presence of various analytes (1 mM) in HEPES buffer solution (pH 6.8 for 1 and pH 7.4 for 2–12). 1. blank (pH 6.8); 2. blank (pH 7.4); 3. IPTG; 4.GSH; 5. ZnCl\u003csub\u003e2\u003c/sub\u003e; 6. Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e; 7. NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e; 8. KCl; 9. MgCl\u003csub\u003e2\u003c/sub\u003e; 10. Cys; 11. CaCl\u003csub\u003e2\u003c/sub\u003e; 12. SDS (D) Fluorescence intensity ratio (I\u003csub\u003e740 nm\u003c/sub\u003e/I\u003csub\u003e630 nm\u003c/sub\u003e) versus the pH values in a HEPES buffer solution (10 mM) containing SDS (10 mM). (E) Cytotoxicity across AF-C doses after 6 h cultivation (0–40 μM). (F) Co-culture growth curves under different AF-C doses (0–40 μM).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8252127/v1/f964f96825017a06ca1ed784.png"},{"id":98432497,"identity":"01fba4e8-6162-4ccd-86a3-5d77fd820a5d","added_by":"auto","created_at":"2025-12-17 16:49:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":113051,"visible":true,"origin":"","legend":"\u003cp\u003eReal-time live cell confocal image and two-channel fluorescence quantification. (A, B) Representative confocal fields (Red, 630 nm; NIR, 740 nm) at 0, 12, 24 h (control vs cSAT-rhBNP group). (C, F) Quantification of relative fluorescence intensity in the near-infrared channel and red channel of control and cSAT-rhBNP. (D, G) Quantification of fluorescence intensity ratios (I\u003csub\u003eNIR\u003c/sub\u003e/I\u003csub\u003eRed\u003c/sub\u003e) in control and cSAT-rhBNP group. (E, H) Intracellular and extracellular pH comparison between control and cSAT-rhBNP group. Significance: n.s., not significant; * q \u0026lt; 0.05; ** q \u0026lt; 0.01; *** q \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8252127/v1/aea13a57c151f8a944b78e18.png"},{"id":98218076,"identity":"9eed9e80-43bd-49a5-8881-6f44a6be9387","added_by":"auto","created_at":"2025-12-15 10:59:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":135746,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptomic response post-induction. (A) Heat map of correlation analysis between samples. (B) PCA score plot between samples. (C) Volcano plot of differential gene expression at 12 h post-induction.(D) GO enrichment of DEGs at 12 h post-induction. (E) KEGG enrichment of DEGs at 12 h post-induction.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8252127/v1/c9d6f4a9915cc9e8adcf2a9d.png"},{"id":98218077,"identity":"e7934ede-b95d-4caa-a2af-0debc61dcb01","added_by":"auto","created_at":"2025-12-15 10:59:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":83167,"visible":true,"origin":"","legend":"\u003cp\u003ePathway-focused heatmaps of pH-linked genes and metabolites under the cSAT scheme. (A-G) Time-resolved heatmaps about key genes of alkalinization-relevant pathways (ammonia-releasing amino-acid pathways; glutamate-GABA module; glyoxylate shunt and C\u003csub\u003e2\u003c/sub\u003e conservation; TCA backbone and organic-acid management; respiratory routing and proton-motive balance; transport and envelope regulation; pH/ion homeostasis; energy and phosphate transactions). (H) log\u003csub\u003e2\u003c/sub\u003e fold change of transcriptome. (I-L) Time-resolved heatmaps about key metabolites of alkalinization-relevant pathways. (L) log\u003csub\u003e2\u003c/sub\u003e fold change of metabolome.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8252127/v1/332ed06fe5b6ff3639512ace.png"},{"id":98433564,"identity":"d7730ebe-bce8-4d71-8d60-56d35f9ccd26","added_by":"auto","created_at":"2025-12-17 16:50:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":138152,"visible":true,"origin":"","legend":"\u003cp\u003eqRT-PCR validation of alkalinization-linked gene modules under the cSAT scheme.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8252127/v1/0e3b0b26ce3e5f7f9080e82a.png"},{"id":98218088,"identity":"3757bdad-4b0f-4801-b759-eba7700c59dd","added_by":"auto","created_at":"2025-12-15 10:59:46","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":192049,"visible":true,"origin":"","legend":"\u003cp\u003eMechanistic drivers of intracellular alkalinization under cSAT scheme. (A) Amino-acid catabolism and TCA-glyoxylate remodeling linked to alkalinization. (B) Respiratory-chain routing and proton bookkeeping.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8252127/v1/e224a3a277c8e4db0c9d328e.png"},{"id":98218081,"identity":"71bb5a78-ea31-45a9-b10d-22c728e40ac8","added_by":"auto","created_at":"2025-12-15 10:59:46","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":79066,"visible":true,"origin":"","legend":"\u003cp\u003eYield of rhBNP after stepwise medium optimization. (A) Yield of fusion rhBNP and terminal pH of broth under different tryptone:yeast extract ratios. (B) Yield of fusion rhBNP and terminal pH of broth under different phosphate buffer concentration. (C) Yield of fusion rhBNP and terminal pH of broth under different glucose concentration. (D) Yield of fusion rhBNP and terminal pH of broth under different ammonium sulfate concentration.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8252127/v1/6274285eaa3cf9136573e003.png"},{"id":98431972,"identity":"0f32910a-74a8-4001-84e0-b7c73e4b8d6c","added_by":"auto","created_at":"2025-12-17 16:48:43","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":50676,"visible":true,"origin":"","legend":"\u003cp\u003eFed-batch scale-up of rhBNP production under the cSAT scheme. (A) Time profiles of OD\u003csub\u003e600\u003c/sub\u003e, broth pH and residual glucose during induction and fed-batch operation in the optimized medium. (B) Comparison of the yields and purity of rhBNP for different scales.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8252127/v1/71b7523da5066d03f3ef61ba.png"},{"id":101691314,"identity":"c32749c3-c226-4021-99b2-d89361cb7145","added_by":"auto","created_at":"2026-02-02 16:13:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5419344,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8252127/v1/8dc70177-1bd3-4106-b441-079c3fcede31.pdf"},{"id":98433650,"identity":"1dba8c78-b527-44e6-8b57-fb78eadcaee0","added_by":"auto","created_at":"2025-12-17 16:51:00","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":8006540,"visible":true,"origin":"","legend":"","description":"","filename":"supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-8252127/v1/fb690a5a2c29087b96210256.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mechanism-guided control of intracellular alkalinization and process pH for production of recombinant human B-type natriuretic peptide under the cSAT scheme in Escherichia coli","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eBioactive peptides with defined intramolecular disulfide bonds are increasingly produced in microbial hosts, not only because they can be obtained rapidly and at relatively low cost, but also because they offer tractable chassis to study how cells handle small, cysteine-rich cargos at production-relevant levels [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In such microbial cell factories, the host must simultaneously support high peptide synthesis rates, correct disulfide formation and folding, and the maintenance of intracellular homeostasis. Recombinant human B-type natriuretic peptide (rhBNP) is a 32-residue hormone whose biological activity depends on a single disulfide, making it a practical model for probing how \u003cem\u003eE. coli\u003c/em\u003e manages small disulfide-containing targets under the high loads required for biotechnological production[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, even in \u003cem\u003eE. coli\u003c/em\u003e, a long-standing workhorse host, such peptides are prone to proteolytic degradation, limited solubility and aggregation, so the recoverable yield often falls short of the apparent expression level and can be highly sensitive to the physiological state of the chassis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo increase usable yield and mitigate these burdens, a wide range of fusion and tag strategies has been developed to enhance solubility, shield the product from proteases and facilitate downstream purification [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Among them, the cSAT scheme couples an intein with a self-assembling module and enables release of the target peptide under mild, near-physiological conditions in \u003cem\u003eE. coli\u003c/em\u003e, and has been applied to several disulfide-bonded peptides including rhBNP [\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. While such schemes efficiently decouple expression and downstream purification, the high intracellular loads and aggregation they impose can strongly perturb cytosolic pH and broader proton homeostasis. How cSAT scheme production of rhBNP reshapes intracellular alkalinization and, in turn, constrains medium formulation and process control in \u003cem\u003eE. coli\u003c/em\u003e has not been systematically characterized, even though these links are likely to set practical limits on robust peptide production.\u003c/p\u003e\u003cp\u003eIntracellular pH (pHi) in neutralophilic \u003cem\u003eE. coli\u003c/em\u003e used as a microbial cell factory is normally maintained between 7.4 and 7.8 over an external pH range of 5 to 9 by the combined action of respiratory proton pumping and Na⁺/H⁺ and K⁺/H⁺ antiport [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This tight control is critical because pHi integrates ATP turnover, ion transport and carbon-nitrogen partitioning, and feeds directly into the proton motive force [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Under high-load heterologous expression, particularly when strong tags or self-aggregating modules are used, several processes can bias pHi upward: reduced net acid formation when carbon is diverted from the full tricarboxylic acid cycle into glyoxylate and related bypass routes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], increased proton extrusion through F\u003csub\u003e1\u003c/sub\u003eF\u003csub\u003e0\u003c/sub\u003e-ATP synthase operating in the pumping direction or through secondary antiporters [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and deamination with NH\u003csub\u003e3\u003c/sub\u003e release under nitrogen-rich conditions [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Conventional fermentation readouts such as optical density, broth pH and product titer cannot resolve the timing or relative contributions of these routes, and omics-level enrichments remain largely associative unless they are anchored to time-resolved pHi measurements [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. As a result, pHi and the underlying proton balance are seldom treated as explicit design variables when optimizing tag-based high-load expression processes.\u003c/p\u003e\u003cp\u003eRatiometric fluorescent probes offer a practical route to make bacterial pHi a measurable state variable in such processes, because the ratio between two emission channels compensates for variations in probe loading, photobleaching and moderate nonspecific binding [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This strategy has been validated for single-cell pHi determination in bacteria using carboxyfluorescein, GFP variants and related dual-excitation reporters [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Recent reviews on ESIPT and ICT-type 8-hydroxyquinoline probes have summarized red and near-infrared ratiometric scaffolds that are compatible with live-cell imaging and provide large Stokes shifts suitable for dense cultures [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Here, we adapt an HQO-type ratiometric probe (AF-C), calibrated in situ by a standard high-potassium/nigericin protocol [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], to obtain quantitative, time-resolved single-cell pHi trajectories for \u003cem\u003eE. coli\u003c/em\u003e during cSAT scheme production of rhBNP [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These trajectories are then integrated with transcriptomics, metabolomics and fermentation outputs to link intracellular alkalinization to proton-economy rewiring and to medium and process control levers.\u003c/p\u003e\u003cp\u003eAt the process level, we first established a baseline for cSAT scheme production of rhBNP in \u003cem\u003eE. coli\u003c/em\u003e by reproducing reported conditions in LB medium with induction at 18\u0026deg;C and an optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) of 0.4\u0026ndash;0.6 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Increasing the induction temperature to 21\u0026deg;C and shifting induction to an OD\u003csub\u003e600\u003c/sub\u003e of 0.6\u0026ndash;0.8 improved volumetric titer and space-time yield but markedly intensified pH drift in both broth and intracellular, revealing a trade-off in which conditions that favor productivity amplify the host pH burden. To resolve and control this behavior, we combined AF-C-based ratiometric intracellular pH imaging with time-resolved transcriptomics and metabolomics to obtain an induction-resolved view of the \u003cem\u003eE. coli\u003c/em\u003e response under the cSAT scheme [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The resulting profiles indicate that high anabolic and processing load is accompanied by respiratory and glyoxylate reprogramming and altered ion-transport and energy-related functions, consistent with metabolism-driven intracellular alkalinization [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Guided by this mechanistic framework, we then used phosphate buffering, complex nitrogen composition, carbon supply and the carbon to nitrogen ratio as simple process levers to restore near-neutral broth and intracellular pH while increasing shake-flask titers over LB medium. Finally, we validated this medium in a 3-L fed-batch process, demonstrating that mechanism-guided control of intracellular alkalinization and process pH can stabilize rhBNP productivity during cSAT scheme expression in \u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e2.1 Strains and plasmid construction\u003c/p\u003e\n\u003cp\u003eThe production host was \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) (Sangon Biotech, Shanghai, China) carrying an expression plasmid derived from the pET-30a(+) backbone (Novagen, USA). The DNA sequence encoding rhBNP was codon-optimized for \u003cem\u003eE. coli\u003c/em\u003e and synthesized commercially (Tsingke, Xi\u0026rsquo;an, China). The recombinant plasmid was assembled by standard restriction-ligation cloning and verified by Sanger sequencing. The amino acid sequence of the cSAT-rhBNP fusion and the corresponding coding sequence are provided in Table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Protein expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ecoli\u003c/em\u003e BL21(DE3) harboring the target plasmid was grown overnight in LB medium supplemented with kanamycin (50 \u0026micro;g/mL) at 37 \u0026deg;C and 220 rpm. The seed culture was diluted 1:50 into fresh LB medium and grown to an OD\u003csub\u003e600\u003c/sub\u003e of 0.6\u0026ndash;0.8, after which the temperature was lowered to 21 \u0026deg;C. Following a 15\u0026ndash;30 min equilibration, IPTG was added to a final concentration of 0.2 mM to initiate expression. Cultures were incubated for a further 24 h at 21 \u0026deg;C, and cells were harvested by centrifugation (5,000 rpm, 15 min, 4 \u0026deg;C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Absorption and emission spectra at different pH values of AF-C\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA stock of AF-C (1 mM) was diluted to 10 \u0026micro;M in HEPES buffer containing 10 mM SDS. Solution pH was adjusted by incremental addition of NaOH or HCl and verified with a calibrated pH meter. UV-vis spectra were collected on a spectrophotometer; fluorescence emission spectra were recorded on a steady-state/kinetics fluorimeter. All measurements were acquired after 1 h dark equilibration at room temperature.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Evaluation of AF-C cytotoxicity in \u003cem\u003eE. coli\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOvernight cultures of \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) were diluted 1:50 into fresh LB medium and grown at 37 \u0026deg;C, 220 rpm until OD\u003csub\u003e600\u003c/sub\u003e reached 0.6\u0026ndash;0.8. IPTG (0.2 mM) was added to mimic expression conditions. AF-C (DMSO stock) was then added to final concentrations of 0, 5, 10, 20, 30, or 40 \u0026micro;M, keeping DMSO at 0.5% (v/v) in all groups. After 6 h exposure, cultures were mixed gently and 100\u0026ndash;200 \u0026micro;L aliquots transferred to clear 96-well plates. CCK-8 reagent (HY-K0301, MCE) was added at 10% (v/v), mixed gently, and incubated for 30 min at 21 \u0026deg;C protected from light. Absorbance at 450 nm was read on a microplate reader. Reagent and matrix blanks were included for background correction [28]. Background-corrected A\u003csub\u003e450 nm\u003c/sub\u003e values were used to calculate cell viability (%) relative to the vehicle control (0 \u0026micro;M AF-C, 0.5% DMSO, set to 100%):\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n\u003cp\u003eIn parallel, OD\u003csub\u003e600\u003c/sub\u003e was recorded hourly at each AF-C concentration. For microplate monitoring, 200 \u0026micro;L per well was dispensed into flat-bottom clear plates sealed with breathable film, with three technical wells per biological replicate. Growth curves (OD\u003csub\u003e600\u003c/sub\u003e vs time) were plotted and, where indicated, fitted with a logistic model.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Real-time confocal imaging of living \u003cem\u003eE. coli\u003c/em\u003e cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e cultures were sampled at 0, 12, and 24 h after IPTG induction. At each time point, 2 mL of culture was incubated with AF-C (20 \u0026micro;M, 0.5% DMSO, v/v) at 21 \u0026deg;C. Prior to formal imaging, co-incubation of AF-C with \u003cem\u003eE. coli\u003c/em\u003e for 15, 30, 45, 60, or 75 min was screened using cells harvested 12 h after induction; 45 min was selected as the staining period based on maximal ratiometric signal and signal-to-background, low cell-to-cell variability, and unchanged viability (Fig. S4). After incubation, cells were washed three times with PBS, resuspended in 1 mL saline, and imaged on a Leica TCS SP5 spectral confocal microscope. AF-C was excited at 561 and 633 nm, and emissions were collected at 579\u0026ndash;668 nm and 674\u0026ndash;767 nm, respectively. All images were acquired using identical detector settings across time points.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Transcriptomic and metabolomic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor transcriptomic and metabolomic profiling, \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) cultures in LB medium were induced with IPTG as described above. Samples were collected at 0, 12, and 24 h post-induction from both experimental and control groups. Cell pellets were rapidly frozen in liquid nitrogen and stored at -80 \u0026deg;C until analysis. Total RNA extraction, library preparation and sequencing, metabolite extraction, and LC-MS/MS-based metabolomics were commissioned to and performed by Tsingke (Xi\u0026rsquo;an, China) according to their standard protocols.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Quantitative real-time PCR (qRT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) cells using RNAiso Plus (Takara, Japan) according to the manufacturer\u0026rsquo;s instructions. RNA quality and concentration were assessed with a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). One microgram of total RNA was reverse-transcribed using the PrimeScript\u0026trade; RT reagent Kit with gDNA Eraser (Takara, Japan). Quantitative PCR was performed with TB Green\u0026reg; Premix Ex Taq\u0026trade; II (Takara, Japan) on a CFX96 real-time PCR detection system (Bio-Rad, USA). Gene-specific primers were designed using Primer-BLAST (NCBI); sequences are listed in Table S2. The \u003cem\u003egyrA\u003c/em\u003e gene served as the internal reference, and relative expression levels were calculated using the 2\u003csup\u003e⁻\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method. All reactions were run in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8 Stepwise medium optimization in shake flasks\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShake-flask medium optimization was carried out in baffled flasks using the same inoculum preparation, cultivation temperature and sampling and analytical procedures as described above. A four-step design was adopted, in which the best-performing composition from each step was carried forward. Step one varied the amount and ratio of tryptone and yeast extract; step two adjusted total phosphate with a KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e/Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e buffer; step three varied the initial glucose concentration; and step four varied ammonium sulfate to tune inorganic nitrogen supply and the overall carbon-to-nitrogen ratio. For all conditions, cultures were induced with IPTG until OD\u003csub\u003e600\u003c/sub\u003e reached 0.8\u0026ndash;1.0, and rhBNP titer, optical density and terminal broth pH were measured in biological triplicate to identify the formulation that best balanced near-neutral terminal pH and high rhBNP titer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9 Fed-batch fermentation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFed-batch fermentations were performed in a 3-L stirred-tank using the optimized shake-flask medium supplemented per litre with 1.5 g citric acid, 0.6 g MgSO\u003csub\u003e4\u003c/sub\u003e and 1 mL trace metal solution, which contained (per liter of 250 \u0026mu;L H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e): 17.6 g FeC\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u0026middot;5H\u003csub\u003e2\u003c/sub\u003eO, 2 g ZnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 0.2 g CuSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;5H\u003csub\u003e2\u003c/sub\u003eO, 1.6 g MnSO\u003csub\u003e4\u003c/sub\u003e, 0.3g H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e, 0.2g\u0026nbsp;(NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e6\u003c/sub\u003eMo\u003csub\u003e7\u003c/sub\u003eO\u003csub\u003e24\u003c/sub\u003e\u0026middot;4H\u003csub\u003e2\u003c/sub\u003eO, 0.3g CoCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO. The aeration was set at 3 L/min (2 vvm), and the agitation was set between 200 and 1,000 rpm to maintain the dissolved oxygen (DO) levels at \u0026gt;20% of saturation. pH was controlled at 7.2 by automatic addition of 7% (v/v) NH\u003csub\u003e3\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO. The culture was grown at 37 \u0026deg;C until the OD\u003csub\u003e600\u003c/sub\u003e reached about 35, after which the temperature was reduced to 21 \u0026deg;C and IPTG was added to a final concentration of 1.0 mM to induce protein expression. The cells were harvested after 20\u0026ndash;22 h post induction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10 Protein purification by intein-mediated cleavage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntein-mediated cleavage was performed as described previously [25]. Cell pellets were resuspended in disruption buffer (20 mM Tris-HCl, 500 mM NaCl, 1 mM EDTA\u0026middot;2Na, pH 8.5) and sonicated on ice for 20 min (JY92-IIN, Scientz, Ningbo, China). Lysates were clarified by centrifugation (12,000 rpm, 15 min, 4 \u0026deg;C). Pellets were washed twice with disruption buffer; an aliquot was reserved for SDS-PAGE. The remaining pellet was resuspended in an equal volume of intein-cleavage buffer (PBS containing 40 mM Bis-Tris and 2 mM EDTA\u0026middot;2Na, pH 6.2) and incubated at 25 \u0026deg;C for 24 h, after which the soluble fraction was collected by centrifugation (12,000 rpm, 15 min, 4 \u0026deg;C) for downstream analysis.\u003c/p\u003e\n\u003cp\u003eProteins were analyzed on FastPAGE\u0026trade; 4\u0026ndash;20% Bis-Tris SDS-PAGE gels (Tsingke, China) and stained with Coomassie Brilliant Blue R-250. Band intensities were quantified densitometrically using ImageJ (NIH, USA). Bovine serum albumin (BSA) and aprotinin (APR) were used as standards to assign band identities and determine protein quantities.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 rhBNP expression by cSAT scheme and broth alkalinization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSystem setup and expression validation.\u0026nbsp;\u003c/strong\u003eTo establish a process-relevant baseline for rhBNP production under the cSAT scheme in \u003cem\u003eE. coli\u003c/em\u003e, we retained the L\u003csub\u003e6\u003c/sub\u003eKD-Mtu \u0026Delta;I-CM tag (Fig. 1A) and used a slightly intensified induction regime relative to the original report: cultures were grown in LB medium and induced until OD\u003csub\u003e600\u003c/sub\u003e reached 0.6\u0026ndash;0.8 at 21 \u0026deg;C with 0.2 mM IPTG [25]. Under these conditions, shake-flask cultures reached high biomass and accumulated the cSAT-rhBNP fusion as a prominent band on SDS-PAGE (Fig. 1C). Intein-mediated self-cleavage released an approximately 3.5 kDa band in the soluble fraction consistent with rhBNP, as confirmed by enzymatic digestion and mass spectrometry (Figs. S2 and S3). The 18 \u0026deg;C condition described previously is used only as a contextual reference and was not pursued further as a benchmark in this study [25].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGrowth and broth pH trajectories\u003c/strong\u003e. During shake-flask cultivations, cultures expressing rhBNP under the cSAT scheme showed a pronounced rise in broth pH. Terminal values exceeded 8.5 both under the process-oriented induction regime used in this study (Fig. 1E) and when the original 18 \u0026deg;C protocol was reproduced (Fig. S1). In contrast, the empty-vector control maintained a terminal pH close to neutrality (Fig. 1D). These data indicate that strong broth alkalinization is a reproducible feature of rhBNP expression under the cSAT scheme across the induction regimes tested and cannot be attributed simply to changes in induction temperature or cell density.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWorking hypotheses for alkaline drift\u003c/strong\u003e. To interpret this alkaline drift, we formulated four non-exclusive, testable working hypotheses, each linked to specific experimental readouts. First, self-aggregation-driven proteostasis and energy demand were expected to increase ATP and cofactor turnover and perturb the NADH/NAD\u003csup\u003e+\u003c/sup\u003e balance [29], providing a route to a modest alkaline shift via respiration-linked proton handling [17]. Second, ammoniagenic amino-acid and one-carbon rewiring could reduce net acid generation from central carbon metabolism [30, 31]. Third, PPi/Pi cycling and tricarboxylic-acid/glyoxylate branch adjustments might limit acid accumulation [32, 33]. Fourth, higher-throughput expression conditions were expected to amplify whichever mechanisms were active. These hypotheses guided the choice of readouts and the design of the omics and medium-engineering experiments described below [34].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Spectral pH response and biocompatibility of AF-C\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpectral design and operating principle\u003c/strong\u003e. To follow intracellular pH dynamics during rhBNP production under the cSAT scheme, we implemented an assay based on the HQO-type ratiometric probe AF-C. AF-C interconverts between protonated and deprotonated forms that modulate an intramolecular charge-transfer pathway, yielding anti-correlated spectral signals: the visible absorption band at 548 nm increases with pH, whereas the near-infrared band at 720 nm increases at lower pH. To normalize for probe loading, optical pathlength and illumination, we used the absorbance ratio A\u003csub\u003e720 nm\u003c/sub\u003e/A\u003csub\u003e548 nm\u003c/sub\u003e and the fluorescence ratio I\u003csub\u003e740 nm\u003c/sub\u003e/I\u003csub\u003e630 nm\u003c/sub\u003e (excitation at 561 and 640 nm, emission at 630 and 740 nm). All in vitro characterizations were performed in HEPES buffer containing dimethyl sulfoxide and, where indicated, 10 mmol/L SDS to provide a membrane-mimetic microenvironment and stabilise the spectra (Fig. 2A).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUV-visible characteristics and band selection\u003c/strong\u003e. In 10 mM HEPES containing 10 mM SDS and DMSO, AF-C displayed two diagnostic absorbance bands with maxima at 548 and 720 nm (Fig. 2B). Spectra recorded at pH 6.8 and 7.4 showed inverse changes in band intensity: the 548 nm band was stronger at pH 7.4, whereas the 720 nm band was stronger at pH 6.8. These measurements establish an inverse ratiometric relationship for the absorbance ratio A\u003csub\u003e720 nm\u003c/sub\u003e/A\u003csub\u003e548 nm\u003c/sub\u003e in this cell-mimetic matrix and support pairing one visible and one near-infrared band for downstream imaging.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInterference tolerance in a cell-mimetic matrix\u003c/strong\u003e. Selectivity was assessed at pH 7.4 by spiking the AF-C matrix (10 mM HEPES, 10 mM SDS and DMSO) with representative intracellular modulators, including IPTG, low-molecular-weight thiols (reduced glutathione and cysteine), mono and divalent salts, phosphate species and additional SDS. As summarized in Fig. 2C, the absorbance ratio A\u003csub\u003e720 nm\u003c/sub\u003e/A\u003csub\u003e548 nm\u003c/sub\u003e remained within experimental variation across these challenges, indicating that under our assay conditions AF-C primarily reports proton activity rather than these coexisting factors. The fluorescence intensity ratio I\u003csub\u003e740 nm\u003c/sub\u003e/I\u003csub\u003e630 nm\u003c/sub\u003e showed the same qualitative robustness (Fig. 2D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRatiometric pH calibration for imaging (I\u003csub\u003e740 nm\u003c/sub\u003e/I\u003csub\u003e630 nm\u003c/sub\u003e, pH 6.6\u0026ndash;8.6).\u003c/strong\u003e The same matrix used for in vitro characterization (DMSO with 10 mM SDS and 10 mM HEPES) was titrated from pH 6.6 to 8.6, and AF-C fluorescence ratios (I\u003csub\u003e740 nm\u003c/sub\u003e/I\u003csub\u003e630 nm\u003c/sub\u003e) were measured in \u0026ge;3 replicates per point. After background subtraction and channel registration, ratios were summarized as mean \u0026plusmn; SEM and plotted to generate the empirical calibration curve (Fig. 2D), which was monotonic over the working range. The data were then fitted to a four-parameter Henderson-Hasselbalch-type model (4PL-HH) by unweighted nonlinear least squares (Levenberg-Marquardt):\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cimg 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\"\u003e\u003c/p\u003e\n\u003cp\u003evalid for pH 6.6\u0026ndash;8.6. Together, Figs. 2A\u0026ndash;D show that AF-C provides anti-correlated visible/NIR bands, robust selectivity at pH 7.4, and a well-behaved ratiometric calibration suitable for cell-to-cell comparable pH mapping during induction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiocompatibility across working concentrations\u003c/strong\u003e. AF-C was added to cultures immediately after IPTG induction. Stock solutions were prepared in DMSO and dosed so that the final DMSO content was 0.5% by volume in all cultures, with the control group receiving vehicle only. After 6 hours of co-incubation, CCK-8 measurements indicated viabilities exceeding 85 percent at all AF-C concentrations (Fig. 2E). Over the same 6-hour period, the OD\u003csub\u003e600\u003c/sub\u003e was recorded hourly, and growth trajectories were indistinguishable from the vehicle control (Fig. 2F), indicating no detectable growth penalty attributable to AF-C under imaging-relevant conditions. These results support the use of AF-C in subsequent live-cell intracellular pH measurements without additional correction for probe-induced cytotoxicity or growth inhibition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 AF-C ratiometric imaging of intracellular pH dynamics under the cSAT scheme\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSingle-cell ratiometric imaging and conversion to intracellular pH\u003c/strong\u003e. Two-channel confocal images (emission at 630 and 740 nm) were acquired from AF-C-loaded cultures expressing rhBNP under the cSAT scheme and from empty-vector controls at 0, 12 and 24 h after induction. Single-cell regions of interest were segmented in ImageJ, background was subtracted, the two channels were co-registered and a ratiometric signal \u003cem\u003eR=I\u003csub\u003e740 nm\u003c/sub\u003e/I\u003csub\u003e630 nm\u003c/sub\u003e\u003c/em\u003e was calculated for each cell. Ratios were converted to pHi using the four-parameter logistic Henderson-Hasselbalch calibration obtained in vitro (Eqs. 2 and 3). As expected from the spectral characterization, higher pH produced decreased 740 nm emission and increased 630 nm emission, whereas lower pH produced the opposite trend, supporting the reliability of the ratio-to-pHi conversion used in subsequent analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative summary, pHi-pHe relationships and the 12 h separation\u003c/strong\u003e. At the induction time point (0 h), both cultures maintained pHi slightly higher than the corresponding extracellular pH (pHe), consistent with near-neutral pHi homeostasis in mildly acidic broth (culture expressing rhBNP under the cSAT scheme: pHe 6.81, pHi 7.15; empty-vector control: pHe 6.82, pHi 7.06; Figs. 3E and 3H). Divergence emerged 12 h after induction: the cSAT scheme culture reached a median pHi of about 8.25, whereas the control remained near pHi 7.5 while growth and biomass were still increasing. By 24 h after induction, the cSAT scheme culture remained more alkaline overall (pHe 8.57, pHi 8.53), with intracellular and extracellular pH closely matched, whereas the control lagged behind (pHe 7.76, pHi 7.61) and still showed pHi slightly below pHe. Group differences in pHi and pHe over 12 to 24 h were statistically significant (biological replicates; per-cell medians analysed with a mixed-effects model; P \u0026lt; 0.01). Throughout the time course, red and near-infrared channel trends in cells mirrored the in vitro AF-C fingerprints, supporting the robustness of the ratiometric readout.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eProcess-linked integration of transcriptomics and metabolomics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample grouping and replicates\u003c/strong\u003e. For RNA-seq and metabolomics, cultures expressing rhBNP under the cSAT scheme and empty-vector controls were sampled at 0, 12 and 24 h after induction, each with three biological replicates (labels cS-0/1/2 and a30-0/1/2). These identifiers are used consistently in the correlation analysis and principal component analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGlobal structure: correlation and principal component analysis\u003c/strong\u003e. Correlation analysis and principal component analysis (PCA) were used to assess data quality and overall structure (Figs. 4A and 4B). Biological replicates clustered tightly with pairwise correlation coefficients above 0.95, and no outlier libraries were detected, so all samples were retained for downstream analyses. PCA separated samples primarily by time (principal component 1; 0, 12 and 24 h) and secondarily by condition (principal component 2; cSAT scheme rhBNP expression versus control). From 12 h after induction onward, cultures expressing rhBNP under the cSAT scheme were clearly separated from time-matched controls, consistent with the higher intracellular pH observed by AF-C imaging. Time and condition effects on principal component scores were significant by two-way analysis of variance (P\u0026lt;0.01), and technical covariates such as library size and rRNA fraction did not dominate the leading components, indicating that the separation is biologically driven.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDifferential genes and metabolites across induction\u003c/strong\u003e. Using thresholds of an absolute log\u003csub\u003e2\u003c/sub\u003e fold change of at least one (corresponding to a fold change of at least two) and a false discovery rate below 0.05, we detected 792, 2,042 and 1,381 differentially expressed genes at 0, 12 and 24 h after induction, respectively (12h volcano plot in Fig. 4C; 0 and 24h results in Fig. S5). Metabolomics showed a similar trend, with 215, 520 and 567 differentially abundant metabolites at the same time points (Fig. S8). Thus, both omics layers identify 12 h after induction as a major inflection point, coinciding with the AF-C imaging evidence for induction-phase intracellular alkalinization and indicating a period of elevated folding and clearance demand and biosynthetic load accompanied by reorganization of organic-acid and nitrogen-related metabolism.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional enrichment across induction and link to pH control\u003c/strong\u003e. At 12 h after induction, Gene Ontology (GO) enrichment was dominated by metabolic processes together with ATP-dependent chaperone and folding functions and transporter activity (Fig. 4D), consistent with increased proteostasis demand, ATP turnover and membrane transport under the cSAT scheme. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis at the same time point (Fig. 4E) highlighted pathways with direct relevance to the alkaline phenotype, including central-carbon modules (glycolysis and gluconeogenesis, pyruvate metabolism, glyoxylate and dicarboxylate metabolism), ammoniagenic amino-acid routes (glycine, serine and threonine metabolism; alanine, aspartate and glutamate metabolism) and respiratory and transport modules (oxidative phosphorylation, ATP-binding cassette transporters, the phosphotransferase system and two-component and quorum-sensing systems). Enrichment patterns at 0 and 24 h (Figs. S6\u0026ndash;S9) frame this 12 h program as an induction-phase peak between the pre-induction baseline and later adaptations. Overall, the GO and KEGG results support a mechanistic view in which central-carbon rerouting and nitrogen handling, together with transport and respiratory reprogramming, form a core module underlying intracellular alkalinization under the cSAT scheme.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eMechanism of intracellular alkalinization under the cSAT scheme\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHeatmap analysis of pH-linked genes and metabolites under the cSAT scheme\u003c/strong\u003e. The pathway-focused gene heatmaps (Figs. 5A\u0026ndash;G) highlight amino-acid, central-carbon, respiratory and phosphate-ion modules that change in parallel with intracellular alkalinization in cultures expressing rhBNP under the cSAT scheme. In the amino-acid block (Fig. 5A), \u003cem\u003egcvP\u003c/em\u003e, \u003cem\u003egcvT\u003c/em\u003e and \u003cem\u003egcvH\u003c/em\u003e (glycine-cleavage system), \u003cem\u003easpA\u003c/em\u003e (aspartate to fumarate), \u003cem\u003edadA\u003c/em\u003e, \u003cem\u003edadX\u003c/em\u003e, \u003cem\u003esdaB\u003c/em\u003e and the \u003cem\u003east\u003c/em\u003e genes are strongly induced at 12 h after induction, consistent with enhanced conversion of glycine, aspartate and other amino acids into tricarboxylic-acid cycle entry points with ammonia release. In the nitrogen-assimilation block (Fig. 5B), coordinated changes in \u003cem\u003eglnA\u003c/em\u003e, \u003cem\u003egltB\u003c/em\u003e, \u003cem\u003egltD\u003c/em\u003e and \u003cem\u003eglmS\u003c/em\u003e indicate that glutamate and glutamine pools are actively rebalanced under the cSAT scheme.\u003c/p\u003e\n\u003cp\u003eThe glyoxylate-associated block (Fig. 5C) shows increased \u003cem\u003eglcD\u003c/em\u003e, \u003cem\u003eglcE\u003c/em\u003e, \u003cem\u003eglcF\u003c/em\u003e, \u003cem\u003eaceB\u003c/em\u003e, \u003cem\u003eaceA\u003c/em\u003e, \u003cem\u003ekatG\u003c/em\u003e, \u003cem\u003eahpC\u003c/em\u003e, \u003cem\u003eahpF\u003c/em\u003e, \u003cem\u003ealdA\u003c/em\u003e, \u003cem\u003ealdB\u003c/em\u003e and \u003cem\u003egarK\u003c/em\u003e at 12 h after induction, pointing to activation of the glyoxylate shunt and the glycolate-glyoxylate-glycerate branch when the alkaline phenotype is established. Tricarboxylic-acid cycle genes (\u003cem\u003efumB\u003c/em\u003e, \u003cem\u003efumC\u003c/em\u003e, \u003cem\u003efumD\u003c/em\u003e, \u003cem\u003esdhA\u003c/em\u003e to \u003cem\u003esdhD\u003c/em\u003e, \u003cem\u003emdh\u003c/em\u003e, \u003cem\u003esucA\u003c/em\u003e, \u003cem\u003esucC\u003c/em\u003e and \u003cem\u003esucD\u003c/em\u003e; Fig. 5D), respiratory-chain components (\u003cem\u003enuo\u003c/em\u003e genes, \u003cem\u003endh\u003c/em\u003e, \u003cem\u003ecyo\u003c/em\u003e and \u003cem\u003ecyd\u003c/em\u003e; Fig. 5E) and the succinate dehydrogenase versus fumarate reductase pair (\u003cem\u003esdhA\u003c/em\u003e and \u003cem\u003esdhB\u003c/em\u003e versus \u003cem\u003efrdA\u003c/em\u003e to \u003cem\u003efrdD\u003c/em\u003e; Fig. 5F) also show their largest separation at 12 h after induction, indicating that electron transport and succinate-fumarate routing are adjusted in the same induction window. Finally, \u003cem\u003eppa\u003c/em\u003e and \u003cem\u003eppk1\u003c/em\u003e in the phosphate-energy block, together with modest time-dependent changes in \u003cem\u003enhaA\u003c/em\u003e, \u003cem\u003enhaB\u003c/em\u003e and \u003cem\u003etrkA\u003c/em\u003e (Fig. 5G), link phosphate turnover and Na⁺/H⁺ antiport and K⁺ handling to the alkaline state, rather than acting as isolated responses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetabolite heatmaps under the cSAT scheme\u003c/strong\u003e. The metabolite heatmaps (Figs. 5I\u0026ndash;L) mirror these gene-level patterns. Pyrophosphate, inorganic phosphate and ADP all increased at 12 h after induction, consistent with elevated ATP turnover under the cSAT scheme. Glutamate, \u0026alpha;-ketoglutarate, oxaloacetate, arginine and alanine rose in the same window, supporting engagement of ammoniagenic amino-acid pathways. \u0026gamma;-Aminobutyric acid and downstream TCA intermediates showed coordinated increases, in line with activation of the glutamate decarboxylase-GABA shunt as a cytosolic proton sink [35]. Glyoxylate-branch metabolites shifted together with \u003cem\u003eaceA\u003c/em\u003e, \u003cem\u003eaceB\u003c/em\u003e and glycolate-glyoxylate-glycerate genes, indicating glyoxylate-centred rerouting that tempers acid accumulation while maintaining anaplerosis [14]. Taken together, these heatmaps identify a compact set of pathways that co-vary with intracellular alkalinization under the cSAT scheme and provide the basis for the qRT-PCR validation and mechanistic mapping presented next.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqRT-PCR validation of alkalinization-linked modules under the cSAT scheme\u003c/strong\u003e. Relative transcript levels were quantified by the \u0026Delta;\u0026Delta;Ct method using \u003cem\u003egyrA\u003c/em\u003e as the reference gene, with the empty-vector culture serving as the calibrator at each time point [36]. As shown in Fig. 6, the glycine-cleavage genes \u003cem\u003egcvP\u003c/em\u003e, \u003cem\u003egcvT\u003c/em\u003e and \u003cem\u003egcvH\u003c/em\u003e were already elevated at the induction time point in the cSAT scheme culture and remained clearly higher than in the time-matched control at 12 h after induction. Their expression partially declined by 24 h after induction but stayed above control levels, indicating that one-carbon and ammoniagenic fluxes are engaged early in the induction phase and persist through the period in which intracellular alkalinization is maximal.\u003c/p\u003e\n\u003cp\u003eA similar time course was observed for \u003cem\u003edadA\u003c/em\u003e, \u003cem\u003egdhA\u003c/em\u003e, \u003cem\u003eglcD\u003c/em\u003e, \u003cem\u003esucA\u003c/em\u003e, \u003cem\u003eaceB\u003c/em\u003e, \u003cem\u003egarK\u003c/em\u003e and \u003cem\u003ednaK\u003c/em\u003e: these transcripts increased in the cSAT scheme culture at or shortly after induction, peaked at 12 h after induction and then decreased toward 24 h after induction while remaining above the control. In contrast, \u003cem\u003easpA\u003c/em\u003e was slightly lower than the control at the start of induction but clearly higher at 12 h after induction and still elevated at 24 h after induction, consistent with time-dependent activation of the aspartate-to-fumarate node. Taken together, these patterns validate the engagement of ammonia-releasing deamination (\u003cem\u003edadA\u003c/em\u003e, \u003cem\u003egdhA\u003c/em\u003e), glyoxylate and glycerate routing (\u003cem\u003eglcD\u003c/em\u003e, \u003cem\u003eaceB\u003c/em\u003e, \u003cem\u003egarK\u003c/em\u003e), tricarboxylic-acid cycle entry (\u003cem\u003esucA\u003c/em\u003e) and proteostasis demand (\u003cem\u003ednaK\u003c/em\u003e) alongside the glycine-cleavage system, and support a mechanistic link between the cSAT scheme, the multi-omic signatures and the intracellular alkalinization quantified by AF-C imaging.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAmino acid and one-carbon remodelling under the cSAT scheme\u003c/strong\u003e. The integrated pathway map (Fig. 7A) indicates that the cSAT scheme couples peptide and amino acid uptake to a set of ammoniagenic and proton-consuming reactions that favour intracellular alkalinization while reshaping central-carbon flow. Oligopeptide and dipeptide ATP-binding cassette transporters (Opp and Dpp) import peptides into the cytosol, where they are hydrolysed and channelled into deamination and lyase routes, linking peptide utilisation directly to nitrogen metabolism [37, 38]. Serine is converted to pyruvate by the pyridoxal 5\u0026prime;-phosphate-dependent dehydratase \u003cem\u003esdaB\u003c/em\u003e [39]; alanine enters the pyruvate node through \u003cem\u003ealaA\u003c/em\u003e and \u003cem\u003eavtA\u003c/em\u003e, with \u003cem\u003edadA\u003c/em\u003e and \u003cem\u003edadX\u003c/em\u003e oxidising the resulting amino acids; aspartate is transformed into fumarate by \u003cem\u003easpA\u003c/em\u003e [40]; glycine is degraded by the glycine-cleavage system (\u003cem\u003egcvP\u003c/em\u003e, \u003cem\u003egcvT\u003c/em\u003e and \u003cem\u003egcvH\u003c/em\u003e) to 5, 10-methylenetetrahydrofolate and NH\u003csub\u003e3\u003c/sub\u003e [30]; glutamate is dehydrogenated to \u0026alpha;-ketoglutarate by \u003cem\u003egdhA\u003c/em\u003e with NH\u003csub\u003e3\u003c/sub\u003e release; and arginine proceeds through the \u003cem\u003east\u003c/em\u003e pathway toward succinate [35, 41, 42]. Together, these reactions increase ammoniagenic pressure and reduce net proton retention, providing a plausible mechanistic explanation for the observed intracellular alkalinization under the cSAT scheme.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGlutamate decarboxylation and proton sinks\u003c/strong\u003e. In parallel, the glutamate decarboxylase system (\u003cem\u003egadA\u003c/em\u003e, \u003cem\u003egadB\u003c/em\u003e and the antiporter \u003cem\u003egadC\u003c/em\u003e) consumes cytosolic protons to form \u0026gamma;-aminobutyric acid (GABA), which is converted by \u003cem\u003egabT\u003c/em\u003e and \u003cem\u003egabD\u003c/em\u003e to succinate, establishing a cytosolic proton sink at the periphery of the tricarboxylic-acid cycle [43, 44]. In our data, \u003cem\u003egcvP\u003c/em\u003e, \u003cem\u003egcvT\u003c/em\u003e, \u003cem\u003egcvH\u003c/em\u003e, \u003cem\u003easpA\u003c/em\u003e, \u003cem\u003edadA\u003c/em\u003e, \u003cem\u003egdhA\u003c/em\u003e, the \u003cem\u003east\u003c/em\u003e genes, \u003cem\u003egadA\u003c/em\u003e, \u003cem\u003egadB\u003c/em\u003e, \u003cem\u003egabT\u003c/em\u003e and \u003cem\u003egabD\u003c/em\u003e are coordinately induced in cultures expressing rhBNP under the cSAT scheme, and 12 h metabolite profiles show increased glutamate, \u0026alpha;-ketoglutarate, oxaloacetate, arginine, alanine, GABA, succinate semialdehyde and succinate. Together, these nitrogen and one-carbon circuits provide a mechanistic basis for the early intracellular alkalinization and rising broth pH observed by AF-C imaging and identify amino acid pools (glycine, serine, alanine, aspartate, glutamate and arginine) and NH\u003csub\u003e3\u003c/sub\u003e/NH\u003csub\u003e4\u003c/sub\u003e⁺ as process-level markers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCentral carbon adjusts in concert\u003c/strong\u003e. Activation of the glyoxylate shunt via \u003cem\u003eaceA\u003c/em\u003e and \u003cem\u003eaceB\u003c/em\u003e bypasses decarboxylating tricarboxylic-acid cycle steps and tempers net acid generation from oxidative TCA fluxes while maintaining anaplerosis, a classical strategy in \u003cem\u003eE. coli\u003c/em\u003e under carbon and redox stress [45, 46]. Upstream, the glycolate-glyoxylate-glycerate branch, which includes \u003cem\u003eglcD\u003c/em\u003e, \u003cem\u003eglcE\u003c/em\u003e, \u003cem\u003eglcF\u003c/em\u003e, \u003cem\u003ealdA\u003c/em\u003e and \u003cem\u003egarK\u003c/em\u003e, redistributes flux between anaplerotic and gluconeogenic routes [47]. The succinate node receives convergent input from the glutamate decarboxylase-GABA route and from tricarboxylic-acid cycle branches [48]. Increased succinate together with glyoxylate-branch metabolites (hydroxypyruvate, glycerate and glycolaldehyde) and citrate in cultures expressing rhBNP under the cSAT scheme is consistent with this glyoxylate-centred rerouting [45, 49]. Overall, the pathway map supports a process in which ammoniagenesis and proton-consuming decarboxylation reduce net acid load, while glyoxylate-based central-carbon remodelling limits acid accumulation and maintains redox balance and ATP supply for high-load expression under the cSAT scheme.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRespiratory-chain and phosphate modules supporting proton balance under the cSAT scheme\u003c/strong\u003e. The lower part of the pathway schematic (Fig. 7B) links these metabolic shifts to respiratory routing and phosphate-energy management. In \u003cem\u003eE. coli\u003c/em\u003e, NADH dehydrogenase I (NDH-1, \u003cem\u003enuo\u003c/em\u003e) and cytochrome bo\u003csub\u003e3\u003c/sub\u003e (\u003cem\u003ecyo\u003c/em\u003e) are proton-pumping components of the respiratory chain, whereas NADH dehydrogenase II (NDH-2, \u003cem\u003endh\u003c/em\u003e) and cytochrome bd (\u003cem\u003ecyd\u003c/em\u003e) oxidise quinols without vectorial proton pumping [50-53]. Preferential routing of electrons through an NDH-1/cytochrome bo\u003csub\u003e3\u003c/sub\u003e pathway rather than an NDH-2/cytochrome bd pathway increases net proton extrusion, supports ATP synthesis via the F\u003csub\u003e1\u003c/sub\u003eF\u003csub\u003e0\u003c/sub\u003e-ATP synthase and keeps protons predominantly outside the cytosol, thereby contributing to the rise in intracellular pH [15]. Succinate dehydrogenase (\u003cem\u003esdhA\u003c/em\u003e to \u003cem\u003esdhD\u003c/em\u003e) feeds electrons from succinate into the quinone pool, whereas fumarate reductase (\u003cem\u003efrdA\u003c/em\u003e to \u003cem\u003efrdD\u003c/em\u003e) catalyses the reverse reaction and decouples the tricarboxylic-acid cycle from oxidative phosphorylation [54]. In our data, increased expression of \u003cem\u003enuo\u003c/em\u003e and \u003cem\u003ecyo\u003c/em\u003e together with enhanced \u003cem\u003esdh\u003c/em\u003e and reduced frd transcripts in cSAT scheme cultures at 12 h after induction is consistent with a more proton-pumping-competent electron-transport configuration supported by elevated succinate and may reinforce the central-carbon and nitrogen remodelling associated with intracellular alkalinization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhosphate-energy metabolism adjusts in parallel\u003c/strong\u003e. Polyphosphate kinase (\u003cem\u003eppk1\u003c/em\u003e) and inorganic pyrophosphatase (\u003cem\u003eppa\u003c/em\u003e) form a polyphosphate-pyrophosphate-phosphate cascade that buffers high-energy phosphate equivalents and supplies inorganic phosphate for ADP phosphorylation [33, 55]. In cultures expressing rhBNP under the cSAT scheme, increased pyrophosphate, inorganic phosphate and ADP at 12 h after induction, together with induction of \u003cem\u003eppk1\u003c/em\u003e and \u003cem\u003eppa\u003c/em\u003e, are consistent with intensified ATP turnover coupled to PPi hydrolysis and recycling. Modest time-dependent changes in Na⁺/H⁺ antiporters (\u003cem\u003enhaA\u003c/em\u003e and \u003cem\u003enhaB\u003c/em\u003e) and in K⁺ uptake control (\u003cem\u003etrkA\u003c/em\u003e) suggest envelope-level tuning of ion homeostasis rather than a primary pH-control system. Overall, the respiratory and phosphate modules provide a parsimonious complement to the amino-acid/one-carbon and glyoxylate mechanisms: increased ATP demand from proteostasis and transport is met by PPi-linked phosphate cycling and a more proton-pumping respiratory chain, while protons are kept predominantly outside the cytosol. This integrated picture helps to explain the sustained alkaline intracellular pH trajectory under the cSAT scheme and nominates nuo, \u003cem\u003endh\u003c/em\u003e, \u003cem\u003ecyo\u003c/em\u003e, \u003cem\u003ecyd\u003c/em\u003e, \u003cem\u003esdh\u003c/em\u003e, \u003cem\u003efrd\u003c/em\u003e, \u003cem\u003eppk1\u003c/em\u003e and \u003cem\u003eppa\u003c/em\u003e, together with the NADH/NAD⁺ ratio, succinate, PPi, Pi and the ATP/ADP balance, as practical markers for future process monitoring and engineering.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6\u003c/strong\u003e \u003cstrong\u003eStepwise medium optimization and scale-up fermentation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStepwise medium optimization and endpoints\u003c/strong\u003e. Medium composition was optimized in four sequential steps in shake flasks: adjustment of the complex nitrogen source, addition of phosphate buffer, glucose supplementation and tuning of ammonium supply and the carbon-to-nitrogen ratio. The objective was to define an operating window that maintained high rhBNP productivity while keeping the final broth pH close to neutrality. At each step, the condition providing the best compromise between rhBNP titer, biomass and terminal broth pH was carried forward to the next step. Fusion-protein expression was monitored by SDS-PAGE with densitometric analysis (Fig. S10), and endpoint biomass (OD\u003csub\u003e600\u003c/sub\u003e), rhBNP titer and terminal broth pH for each condition are summarized in Fig. S11.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTryptone to yeast extract ratio optimization (step 1)\u003c/strong\u003e. In the first step, the ratio of tryptone to yeast extract in the complex nitrogen source was varied from the LB baseline of 2:1 to 1:1, 1:1.5, 1:2 and 1.5:1. As shown in Fig. 8A and Fig. S12A and summarized in Table S3, a ratio of 1:1.5 gave the highest rhBNP titer (70.8 mg/L versus 39.5 mg/L in LB medium) without loss of biomass. However, the harvest pH remained above 8.5 at all ratios and reached 8.87 at 1:1.5, indicating a pronounced alkaline liability. This behaviour is consistent with our intracellular model, in which richer complex nitrogen is associated with increased ammonia-releasing amino-acid catabolism and proton-consuming reactions, supporting higher expression at the cost of stronger alkalinization [29]. The 1:1.5 ratio was therefore carried forward to subsequent optimization steps, where phosphate buffering and carbon-to-nitrogen ratio control were introduced to retain the yield gain while moderating the pH rise.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhosphate buffer screening (step 2)\u003c/strong\u003e. Using the 1:1.5 tryptone to yeast extract ratio as the baseline, we evaluated a KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e/Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e buffer at total phosphate concentrations of 0, 10, 20, 50 and 100 mM. The corresponding rhBNP titers were 70.4, 115.6, 98.8, 83.4 and 66.7 mg/L, respectively (Figs. 8B and Fig. S12B; Table S4). The harvest pH remained above 8.3 from 0 to 50 mM and decreased to 7.61 only at 100 mM, where expression was reduced. Thus, moderate phosphate increased titer but only partially dampened the alkaline shift, whereas high phosphate suppressed alkalinity at the expense of titer [56]. Because subsequent steps introduce glucose and ammonium sulfate, we selected 50 mM total phosphate as a compromise that preserves most of the yield increase while providing sufficient buffer capacity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGlucose concentration screening (step 3)\u003c/strong\u003e. Starting from the step-2 medium, glucose was supplied at 0, 10, 30, 50 and 100 mM. The corresponding rhBNP titers were 84.5, 92.5, 99.1, 103.4 and 42.2 mg/L, with harvest pH values of 8.44, 7.86, 7.49, 7.33 and 5.39, respectively (Fig. 8C and Fig. S12C; Table S5). Moderate glucose (30 to 50 mM) therefore increased titer (maximum 103.4 mg/L at 50 mM) while partially offsetting the alkaline drift from the complex nitrogen source and bringing the terminal pH toward neutrality. At 100 mM glucose, rhBNP production dropped sharply and premature in vivo self-cleavage of the intein fusion was observed, consistent with the final pH of 5.39 falling below the intein induction pH of approximately 6.2, where folding, cleavage kinetics and redox balance are expected to deteriorate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAmmonium sulfate screening (step 4)\u003c/strong\u003e. Using the step-3 medium (50 mM glucose, 50 mM phosphate, tryptone:yeast extract 1:1.5), we varied ammonium sulfate at 0, 1.70, 3.40, 5.67 and 17.0 g/L to adjust the designed carbon-to-nitrogen (C/N) ratio, defined as the mass of carbon supplied by glucose divided by the mass of nitrogen supplied by ammonium sulfate. With 50 mM glucose supplying 3.603 g carbon per litre, these loadings correspond to nominal C/N ratios of 10:1, 5:1, 3:1 and 1:1. rhBNP titers at 0, 1.70, 3.40, 5.67 and 17.0 g/L were 106.5, 115.4, 77.2, 0 and 0 mg/L, with corresponding final broth pH values of 7.33, 7.04, 6.65, 5.55 and 5.33, respectively (Fig. 8D and Fig. S12D; Table S6). Thus, low to moderate ammonium loadings (0 to 3.40 g/L) maintained high rhBNP production, and 1.70 g/L ammonium sulfate slightly increased titer relative to the ammonium-free baseline while shifting the endpoint pH closer to neutrality. Higher ammonium levels caused progressive acidification and complete loss of recoverable rhBNP despite robust biomass and fusion expression, consistent with the final broth pH falling well below the intended cleavage window around pH 6.2, where folding, cleavage kinetics and redox balance are expected to deteriorate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOverall C/N context of the optimized medium\u003c/strong\u003e. In the final formulation (10 g/L tryptone, 15 g/L yeast extract, 50 mM glucose, 1.70 g/L (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and a nitrogen-free phosphate buffer), the elemental carbon-to-nitrogen ratio calculated from glucose carbon and all nitrogen sources is approximately 1.20 to 1.34 mol per mol, and additional organic carbon from the complex nitrogen sources places the culture in a moderately nitrogen-rich regime. At 1.70 g/L ammonium sulfate (designed C/N 10:1), this regime gave the best compromise, with high fusion and rhBNP titers and a final broth pH close to 7.0, whereas higher ammonium loadings drove acidification, extensive in vivo self-cleavage and loss of recoverable rhBNP. Functionally, the moderately nitrogen-rich background favours ammonia-releasing amino-acid turnover and proton-consuming reactions that tend to raise pH, while 50 mM glucose provides acid-generating flux that counterbalances this tendency. The final medium therefore places expression under the cSAT scheme in a buffered window that sustains rhBNP yield while keeping broth pH within the operational range for intein self-cleavage.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eScale-up fermentation in a 3-L stirred-tank fermenter\u003c/strong\u003e. To evaluate the scale-up potential of the optimized cSAT scheme, we ran a glucose-limited fed-batch fermentation in a 3-L stirred-tank bioreactor using the step-4 medium formulation as the batch phase. After batch growth on the optimized medium, feeding with a concentrated glucose-tryptone-magnesium solution was initiated when residual glucose from the batch phase was depleted and then supplied continuously for the remainder of the fermentation. The culture was grown in batch mode until the OD\u003csub\u003e600\u003c/sub\u003e reached approximately 35, at which point 1.0 mM IPTG was added to induce expression, and fermentation was then continued under fed-batch conditions without interruption until the planned end of the 29.5 h run (Fig. 9A). For SDS-PAGE and densitometric quantification, fermentation samples were diluted before loading (ten-fold for the cSAT-rhBNP fusion suspension and four-fold for rhBNP; Fig. S13A and Fig. S13B), and band intensities were corrected for these dilution factors and broth volume. Under these conditions, the cSAT-rhBNP fusion protein titer in the fermenter was 12.7 g/L and the final rhBNP titer was 662.1 mg/L at a terminal broth pH close to 7.0 (Figs. 9A and 9B). This corresponds to an approximately 16.8-fold increase in rhBNP titer compared with LB medium shake flasks (39.5 mg/L) and about a 5.7-fold increase over the optimized shake-flask medium (115.4 mg/L), while maintaining the near-neutral broth pH anticipated from the proton-economy-based medium design.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e\u003cb\u003eMechanistic summary and process implications\u003c/b\u003e. This study helps to explain why high-load rhBNP expression under the cSAT scheme is associated with broth and intracellular alkalinization and how this can be managed at the process level [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. AF-C-based single-cell intracellular pH imaging [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], together with time-resolved qRT-PCR, transcriptomics and metabolomics, points to ammonia-releasing amino-acid pathways, proton-consuming glutamate decarboxylation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], glyoxylate-centred carbon rerouting and a shift towards more proton-pumping respiratory branches as key contributors to the alkaline state [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Treating medium and process design as a proton-economy problem, we used this framework to stabilise the harvest pH near neutrality and increase rhBNP titer 2.9-fold in shake flasks relative to LB medium (from 39.5 to 115.4 mg/L), and in a 3-L fed-batch fermentation to reach 12.7 g/L cSAT-rhBNP fusion protein and 662.1 mg/L rhBNP at a terminal broth pH close to 7.0.\u003c/p\u003e\u003cp\u003e\u003cb\u003eChassis-level interventions suggested by the pathway map\u003c/b\u003e. The pathway map points to several chassis-level strategies that could reduce alkaline drift without sacrificing throughput. First, moderating ammoniagenic flux through selected glycine-cleavage, dehydrogenase and deaminase genes (for example \u003cem\u003egcvP\u003c/em\u003e, \u003cem\u003egcvT\u003c/em\u003e, \u003cem\u003egcvH\u003c/em\u003e, \u003cem\u003egdhA\u003c/em\u003e, \u003cem\u003edadA\u003c/em\u003e and \u003cem\u003easpA\u003c/em\u003e) would weaken a major alkaline drive [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Second, limiting the level or duration of glutamate decarboxylase and GABA-branch activity (\u003cem\u003egadA\u003c/em\u003e, \u003cem\u003egadB\u003c/em\u003e, \u003cem\u003egabT\u003c/em\u003e and \u003cem\u003egabD\u003c/em\u003e) could prevent excessive cytosolic proton consumption [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Controlled expression of \u003cem\u003eaceA\u003c/em\u003e and \u003cem\u003eaceB\u003c/em\u003e would allow glyoxylate shunt activity to support anaplerosis without over-reducing organic-acid pools [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In parallel, tuning respiratory composition (\u003cem\u003enuo\u003c/em\u003e and \u003cem\u003ecyo\u003c/em\u003e relative to \u003cem\u003endh\u003c/em\u003e and \u003cem\u003ecyd\u003c/em\u003e) and phosphate-energy buffering (\u003cem\u003eppk1\u003c/em\u003e and \u003cem\u003eppa\u003c/em\u003e) offers additional levers to coordinate proton pumping and ATP supply with the desired pH range [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Together, these interventions outline a chassis in which proton economy, redox balance and tag-based high-load expression are engineered in a coordinated rather than sequential manner.\u003c/p\u003e\u003cp\u003e\u003cb\u003eScale-up guidance from the proton-economy mechanism\u003c/b\u003e. The 3-L fed-batch runs show that the proton-economy framework can be applied at stirred-tank scale: with the optimized medium, pH-stat control and a glucose-limited feed, the culture maintained a near-neutral broth pH while accumulating 12.7 g/L cSAT-rhBNP fusion protein and 662.1 mg/L rhBNP. These results suggest that further scale-up should retain moderate phosphate buffering with gentle acid and base addition [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], carbon feeds that keep residual glucose low and avoid strong pH swings [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], and dissolved-oxygen set points that prevent deep microaeration while avoiding excessive aeration and carbon dioxide stripping [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eBalancing yield and proton economy under the cSAT scheme\u003c/b\u003e. Even in this controlled window, some in vivo self-cleavage is expected as the average intracellular pH approaches the intein induction pH of about 6.2 [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], and larger reactors will increase local variation in oxygen, pH and redox state [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Our data therefore argue that the cSAT scheme expression should be engineered jointly for yield and proton economy: coordinated control of pathway timing, respiratory bias and feed and buffer profiles can turn alkalinity from a liability into a tunable handle for robust fermentation-type production of disulfide-bonded bioactive peptides, with rhBNP as a stringent model.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the School of Chemical Engineering at Northwest University for kindly providing the facilities for this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM. and H.N. conceived the ideas and designed the experiments. H.N. performed the majority of the experiments. Y.F., G.S. and H.L. assisted in conducting the confocal experiments. J.K. and Y.Z. assisted in conducting the scale-up fermentation experiments. H.N. and P.M. analyzed the data, and all authors discussed the results. H.N. prepared the initial draft of the manuscript. P.M., C.Z., Y.M. and Y.G. revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key R\u0026amp;D Program of China (2021YFC2103900).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAppendix A. Supplementary data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary data to this article can be found online.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBaneyx F, Mujacic M: \u003cstrong\u003eRecombinant protein folding and misfolding in Escherichia coli.\u003c/strong\u003e \u003cem\u003eNature biotechnology\u0026nbsp;\u003c/em\u003e2004, \u003cstrong\u003e22:\u003c/strong\u003e1399\u0026ndash;1408.\u003c/li\u003e\n \u003cli\u003eChen R: \u003cstrong\u003eBacterial expression systems for recombinant protein production: E. coli and beyond.\u003c/strong\u003e \u003cem\u003eBiotechnology advances\u0026nbsp;\u003c/em\u003e2012, \u003cstrong\u003e30:\u003c/strong\u003e1102\u0026ndash;1107.\u003c/li\u003e\n \u003cli\u003eSemenov AG, Seferian KR: \u003cstrong\u003eBiochemistry of the human 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\u003cem\u003eScience Advances\u0026nbsp;\u003c/em\u003e2024, \u003cstrong\u003e10:\u003c/strong\u003eeadl5849.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Intracellular alkalinization, Medium engineering, Recombinant human B-type natriuretic peptide, cSAT scheme, Proton economy","lastPublishedDoi":"10.21203/rs.3.rs-8252127/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8252127/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Tag-based high-load expression in \u003cem\u003eEscherichia coli\u003c/em\u003e often causes uncontrolled pH drift, but links between intracellular alkalinization, metabolism and process levers remain unclear.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Using a cleavable self-aggregating tag (cSAT) to produce recombinant human B-type natriuretic peptide (rhBNP), we combine broth pH tracking, AF-C ratiometric intracellular pH imaging and multi-omics to show that ammoniagenic amino-acid catabolism, glyoxylate-centered carbon rerouting and respiratory shifts drive alkalinization. Guided by this proton-economy model, stepwise medium engineering (complex nitrogen, phosphate, glucose and ammonium sulfate with tuned carbon to nitrogen ratio) lowers shake-flask broth pH from values above 8.5 to about 7.0, increases titers 2.9-fold over LB medium to 115.4 mg/L and, in 3-L fed-batch, yields 662.1 mg/L rhBNP while maintaining biomass.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: Proton-economy-based process design stabilizes pH and productivity during cSAT scheme production of rhBNP in \u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Mechanism-guided control of intracellular alkalinization and process pH for production of recombinant human B-type natriuretic peptide under the cSAT scheme in Escherichia coli","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-15 10:59:41","doi":"10.21203/rs.3.rs-8252127/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-15T16:47:16+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-11T20:11:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"15282748374689842631166431947651594436","date":"2025-12-11T15:23:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-10T14:23:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"330949580126564354669738344528140536305","date":"2025-12-10T01:26:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"83221278919336657058050213417181216664","date":"2025-12-09T19:50:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-09T16:55:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-06T23:51:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-06T12:47:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbial Cell Factories","date":"2025-12-01T15:27:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1d5523b9-00b7-42c0-af1c-d71c8a43f946","owner":[],"postedDate":"December 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-02T16:09:06+00:00","versionOfRecord":{"articleIdentity":"rs-8252127","link":"https://doi.org/10.1186/s12934-026-02938-7","journal":{"identity":"microbial-cell-factories","isVorOnly":false,"title":"Microbial Cell Factories"},"publishedOn":"2026-02-01 15:59:04","publishedOnDateReadable":"February 1st, 2026"},"versionCreatedAt":"2025-12-15 10:59:41","video":"","vorDoi":"10.1186/s12934-026-02938-7","vorDoiUrl":"https://doi.org/10.1186/s12934-026-02938-7","workflowStages":[]},"version":"v1","identity":"rs-8252127","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8252127","identity":"rs-8252127","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}