Biosensor-driven evolution and metabolic engineering of an Escherichia coli

Biosensor-driven evolution and metabolic engineering of an 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 Biosensor-driven evolution and metabolic engineering of an Escherichia coli Rongshuai Zhu, Zhenqiang Zhao, Weijun Shao, Jin Han, Jiajia You, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9241029/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract L-Tryptophan is an important aromatic amino acid with wide applications across the food, pharmaceutical, and feed industries. However, its efficient microbial production remains challenging due to complex metabolic networks and multi-level feedback regulation. In this study, we constructed a highly efficient Escherichia coli cell factory for L-tryptophan biosynthesis by combining systematic metabolic engineering with high-throughput screening. Initially, a tnaC -based biosensor was developed and coupled with atmospheric and room temperature plasma (ARTP) mutagenesis to isolate high-performance chassis strains. Central carbon metabolism was subsequently reprogrammed to minimize carbon loss and channel metabolic fluxes toward essential precursors, phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P). To further alleviate pathway bottlenecks, promoter engineering was utilized to balance the intracellular supplies of L-glutamine, L-serine, and phosphoribosyl pyrophosphate (PRPP). This targeted intervention yielded a 21.61% increase in L-tryptophan accumulation. Product transport systems were then engineered to enhance extracellular secretion and mitigate intracellular toxicity. Following the optimization of inoculum size and feeding strategies in a 5 L bioreactor, the final engineered strain (W-24) produced 50.83 g/L of L-tryptophan within 40 hours, achieving a yield of 0.185 g/g glucose. This multi-modular engineering framework establishes a robust platform for L-tryptophan biosynthesis and provides a scalable strategy for the industrial production of other valuable aromatic compounds. Biosensor L-Tryptophan Escherichia coli Precursor balance Multi-module engineering Fermentation optimization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. INTRODUCTION L-Tryptophan is an essential aromatic amino acid that serves not only as a fundamental building block for protein biosynthesis but also as a vital precursor for several biologically active molecules [ 1 ], including serotonin, melatonin, and niacin [ 2 ]. In animal nutrition, L-tryptophan is generally regarded as the third limiting amino acid in diets for monogastric animals and poultry [ 3 ]. It plays critical roles in regulating circadian rhythms, neuroimmune responses, and intestinal barrier function, deeply influencing both stress tolerance and overall growth performance [ 4 ]. As the livestock industry continues to expand, the global demand for L-tryptophan has surged. Simultaneously, conventional extraction and chemical synthesis methods are increasingly constrained by high energy consumption, heavy environmental burdens, and limited production efficiency [ 5 ]. Consequently, the development of sustainable, high-efficiency microbial cell factories has emerged as a compelling alternative. However, the complex endogenous regulatory networks in microorganisms persistently restrict the excessive accumulation of target products [ 6 ], making the construction of highly robust production strains a formidable challenge. In microorganisms, particularly in the widely used industrial host Escherichia coli , L-tryptophan biosynthesis originates from glucose and involves several interconnected pathways, including central carbon metabolism, the shikimate pathway, and the branched aromatic amino acid pathway (Fig. 1 ). Central carbon metabolism supplies the key precursors phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) through glycolysis and the pentose phosphate pathway, respectively. These two precursors enter the shikimate pathway via the key enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHP synthase), forming common intermediates required for aromatic amino acid biosynthesis and subsequently directing flux toward the L-tryptophan branch. Previous studies have shown that random mutagenesis is an effective approach for obtaining improved chassis strains for L-tryptophan production [ 7 ]. In addition, enhancing the supply of PEP and E4P [ 8 , 9 ], relieving feedback inhibition of key enzymes [ 10 ], and reinforcing rate-limiting steps in the shikimate pathway have been shown to improve L-tryptophan biosynthesis to varying extents [ 11 ]. From the perspective of metabolic pathway requirements, L-tryptophan biosynthesis depends not only on carbon precursors but also on several donor metabolites, including L-glutamine, L-serine, and phosphoribosyl pyrophosphate (PRPP). These metabolites are simultaneously involved in multiple essential cellular processes, such as amino acid and nucleotide biosynthesis [ 12 – 14 ], Variations in their intracellular availability can trigger homeostatic responses [ 15 ], thereby affecting flux distribution toward the tryptophan pathway and limiting the theoretical yield. Therefore, achieving coordinated regulation among precursor supply, donor metabolism, and downstream flux while maintaining cell growth represents one of the key challenges in further improving L-tryptophan titer and yield. Based on these considerations, systematic metabolic engineering aimed at coordinated regulation of multiple metabolic modules has emerged as an effective strategy to enhance aromatic amino acid biosynthesis [ 16 ]. In this study, a biosensor-assisted high-throughput screening system was established and combined with ARTP mutagenesis to obtain an improved E. coli chassis strain for L-tryptophan production. Central carbon metabolism was reconfigured and key enzymatic steps were reinforced to increase the availability of PEP and E4P and to enhance carbon flux toward the shikimate pathway. In addition, promoter engineering was applied to regulate the biosynthesis of donor metabolites, including L-glutamine, L-serine, and PRPP, thereby balancing their intracellular supply and improving L-tryptophan production. Product secretion was further enhanced through modification of transport systems. After optimization of fermentation conditions, the engineered strain achieved high-level L-tryptophan production. These results demonstrate that coordinated regulation of multiple metabolic modules can effectively alleviate metabolic flux imbalance and improve aromatic amino acid biosynthesis, providing useful insights for the development of efficient microbial production systems. 2. MATERIALS AND METHODS 2.1. Strains and plasmids In this study, E. coli DH5α was used as the host strain for plasmid cloning, and plasmid pTrc99A was used as the expression vector. E. coli W3110 was selected as the starting strain for the construction of L-tryptophan-producing strains, and genome knock-in and knockout manipulations were performed using the CRISPR/Cas12a system [ 17 ]. The strains, plasmids, and primers used in this study are listed in the Tables S1, S2, S3. 2.2. DNA manipulation In this study, all expression cassettes of the reinforced genes were driven by the P trc promoter unless otherwise specified. The CRISPR/Cas12a-mediated genome editing procedure is illustrated using the integration of the tktA gene at the yncK locus as an example. The CRISPR/Cas12a system consisted of the plasmids pEcCpf1 and pcrEG. Using the E. coli genome as the template, approximately 500 bp upstream and downstream homologous arms were amplified with the primer pairs yncK-UP-F/yncK-UP-R and yncK-DO-F/yncK-DO-R, respectively. The target gene tktA driven by the P trc promoter was amplified using primers tktA (Ptrc) -F and tktA-R. The homologous arms and the target gene fragment were then assembled by Gibson assembly to generate the donor DNA. In addition, specific CRISPR RNAs (crRNAs) were designed using the CRISPR-DT tool and constructed into the helper plasmid pcrEG to generate the guide plasmid [ 18 ]. Gene knock-in was performed as described previously [ 17 ]. 2.3. Atmospheric and room temperature plasma (ARTP) mutagenesis The biosensor-containing model strain was cultivated to the exponential growth phase (~ 6–8 h), and then cells were harvested, resuspended in phosphate-buffered saline (PBS), and 10 µL of the cell suspension was spotted onto the center of a sterile metal slide. ARTP mutagenesis was then performed using an ARTP-III mutation system (Tsingyuan Biotechnology Co., Ltd., Beijing, China) with a radio-frequency power of 120 W and a helium gas flow rate of 10 standard liters per minute (SLM) [ 16 ]. The treatment durations were set to 0, 10, 20, 30, 40, 50, 60, 70, and 80 s. After treatment, the cells were immediately collected and subjected to recovery cultivation, followed by plating on LB agar plates. The plates were incubated at 37 ℃ until colonies appeared, generating a mutant library. The lethality rate was calculated as described previously [ 19 ]. 2.4. Shake-flask culture conditions Single colonies of engineered strains grown on agar plates were inoculated into 50 mL shake flasks containing 10 mL of LB medium and cultivated at 37 ℃ with shaking at 220 rpm. After overnight cultivation, 3 mL of the seed culture was transferred into a 500 mL shake flask containing 27 mL of fresh fermentation medium. The flasks were incubated at 37 ℃ and 220 rpm for 36 h. During fermentation, the pH was manually maintained by the addition of 25% (v/v) NH 3 ·H 2 O, and a 60% (w/v) glucose solution was intermittently supplied to replenish the carbon source in the fermentation medium [ 20 ]. The fermentation medium consisted of (per liter): 2.0 g yeast extract, 4.0 g (NH 4 ) 2 SO 4 , 2.0 g sodium citrate, 6.0 g K 2 HPO 4 , 2.5 g KH 2 PO 4 , 2.0 g MgSO 4 ·7H 2 O, 30.0 mg FeSO 4 ·7H 2 O, 5.0 mg MnSO 4 ·H 2 O, 0.5 g sodium glutamate, 0.1 mg biotin, 0.2 mg vitamins B 1 , B 3 , B 5 , and B 12 , and 30 g glucose. 2.5. Fed-batch fermentation in a bioreactor For scale-up cultivation of the engineered strains, fresh colonies were scraped from agar plates using a sterile inoculating loop and inoculated into 500 mL shake flasks containing 120 mL of LB medium. The cultures were incubated at 37 ℃ and 220 rpm for 6–8 h and subsequently transferred into a 5 L bioreactor containing 1.88 L of seed medium for further cultivation for 10–12 h. The resulting seed culture was then inoculated at an appropriate inoculum size into a 5 L bioreactor containing fresh fermentation medium, with the working volume maintained at 2.0 L. When the initial glucose was nearly depleted, an 80% (w/v) glucose solution was rapidly fed to maintain the residual glucose concentration in the broth at 0.05–0.1%. During cultivation, the DO level was controlled at 25–30% by real-time adjustment of agitation speed and aeration rate. The pH was maintained at 7.0 ± 0.02 by automatic addition of 25% (v/v) NH 3 ·H 2 O using a peristaltic pump. The seed medium consisted of (per liter): 1.5 g yeast extract, 4.0 g (NH 4 ) 2 SO 4 , 1.6 g sodium citrate, 6.0 g K 2 HPO 4 , 2.5 g KH 2 PO 4 , 1.5 g MgSO 4 ·7H 2 O, 10.0 mg FeSO 4 ·7H 2 O, 5.0 mg MnSO 4 ·H 2 O, 0.1 mg biotin, 0.2 mg vitamin B 1 , B 3 , B 5 , and B 12 , and 40 g glucose. The fermentation medium consisted of (per liter): 2.0 g yeast extract, 4.0 g (NH 4 ) 2 SO 4 , 2.0 g sodium citrate, 6.0 g K 2 HPO 4 , 2.5 g KH₂PO 4 , 2.0 g MgSO 4 ·7H 2 O, 30.0 mg FeSO 4 ·7H 2 O, 5.0 mg MnSO 4 ·H 2 O, 0.5 g sodium glutamate, 0.5 g/L L-isoleucine, 0.5 g/L betaine, 0.1 mg biotin, 0.2 mg vitamin B 1 , B 3 , B 5 , and B 12 , and 10 g glucose. 2.6. Analytical methods Cell growth was monitored by measuring the optical density at 600 nm (OD 600 ) using a UV-visible spectrophotometer after appropriate dilution of the samples. Glucose concentration was determined using an SBA biosensor analyzer (SBA-40C, Shandong, China) after sample dilution and centrifugation to obtain the supernatant. The fluorescence characteristics of the strains were analyzed using a BD flow cytometer (Becton Dickinson, USA). For the mutant library, L-tryptophan concentration in 96-well plates was quantified using a colorimetric spectrophotometric method. Briefly, under sulfuric acid conditions, L-tryptophan reacts with p -dimethylaminobenzaldehyde to form a blue-colored compound. After incubation in boiling water for 3 min, a specific sodium nitrite solution was added, followed by further incubation for 3 min. The absorbance was then measured at 590 nm (A 590 ) [ 16 ]. For samples obtained from shake-flask and bioreactor cultivations, cultures were diluted to appropriate concentrations, centrifuged, and the supernatants were collected. The clarified supernatants were filtered through 0.22 µm membrane filters to remove cells and impurities. Amino acids and organic acids in the fermentation broth were subsequently quantified by high-performance liquid chromatography (HPLC), using pre-column derivatization with o-phthalaldehyde for amino acid analysis and 5 mM H 2 SO 4 as the mobile phase for organic acid analysis [ 6 , 20 ]. All experimental data are presented as the mean ± standard deviation from at least three independent experiments. 3. RESULTS AND DISCUSSION 3.1 Biosensor-assisted screening of chassis strain for L-tryptophan production Biosensors have become powerful tools for microbial strain development [ 21 ], enabling intracellular sensing of target metabolites and high-throughput screening of high-producing strains, as demonstrated for cadaverine[ 22 ], lycopene [ 23 ], and human milk oligosaccharide [ 24 ]. In E. coli , the transcriptional leader region of tnaA contains a 72-bp sequence, tnaC , which encodes a 24-amino-acid leader peptide (TnaC) and serves as a key regulatory element of the tna operon [ 25 ]. When intracellular tryptophan reaches a threshold concentration, TnaC modulates Rho-dependent transcription termination, thereby enhancing the expression of the downstream tnaAB genes [ 26 ]. Based on this regulatory mechanism, a biosensor-assisted screening platform for tryptophan-producing strains was constructed and designated E. coli W3110 + pUC-trpSensor (Fig. 2 A). Specifically, the regulatory region containing the native tnaC leader sequence and upstream promoter was fused to green fluorescent protein (GFP) via a flexible linker (GGGGS) and cloned into the pUC plasmid, allowing fluorescence output to reflect intracellular L-tryptophan levels. To improve the sensitivity and expand the detection range of the biosensor, the native tnaC was replaced with the hypersensitive variant tnaC R23F [ 25 ], yielding the modified construct pUC-trp*Sensor. The constructed biosensors were characterized in media supplemented with different concentrations of L-tryptophan. As shown in Fig. 2 B, pUC-trp*Sensor exhibited an expanded detection range of 0.05-1.00 g/L, whereas the unmodified biosensor displayed a narrower range of 0.05–0.50 g/L. Compared with pUC-trpSensor, the engineered biosensor displayed an increased upper detection limit and enhanced sensitivity toward L-tryptophan, thereby facilitating the discrimination of strains with elevated intracellular L-tryptophan levels during high-throughput screening. To generate genetic diversity for strain improvement, E. coli W3110 harboring pUC-trp*Sensor was subjected to ARTP mutagenesis under different exposure durations (0–80 s). As the treatment time increased, the lethality rate increased accordingly (Fig. S1 ). Considering both lethality and mutant recovery efficiency [ 7 , 27 ], a treatment duration of 60 s was selected as the optimal mutagenesis condition, corresponding to a lethality rate of approximately 91.04%. Under the optimized ARTP conditions, a mutant library of E. coli W3110 pUC-trp*Sensor was constructed and subjected to high-throughput screening using fluorescence-activated cell sorting (FACS). The sorting gate was defined based on the fluorescence distribution of the parental strain, and cells within the high-fluorescence fraction were collected for subsequent cultivation. Mutants were collected and cultivated in deep-well plates, and the top-performing mutant showing the highest fluorescence intensity was selected as the starting strain for the next round of mutagenesis. After three consecutive rounds of ARTP mutagenesis and screening, 40 preliminary candidates with the highest average fluorescence intensity were selected from approximately 5×10 3 mutants (Fig. 2 C). The top ten candidates were subsequently re-evaluated by shake-flask cultivation. Among them, the best-performing mutant exhibited an L-tryptophan titer of 2.03 g/L and was designated W-1 for subsequent strain engineering aimed at enhanced L-tryptophan production (Fig. 2 D). Notably, the top-ranked mutants selected based on fluorescence intensity consistently exhibited increased L-tryptophan production during shake-flask validation, indicating a strong correlation between biosensor output and intracellular L-tryptophan levels, and thereby supporting the reliability of the biosensor-guided screening strategy. 3.2 Genomic analysis and reinforcement of key enzymes for L-tryptophan biosynthesis To elucidate the genetic basis underlying the enhanced L-tryptophan titer of the mutant strain, whole-genome de novo sequencing was performed for W-1 and the wild-type parental strain E. coli W3110. Comparative genome analysis revealed a total of 47 single-nucleotide variations (SNVs) and insertions/deletions (InDels) in the mutant genome relative to the wild type (Data S1). Notably, multiple mutations were detected in metabolic pathways closely associated with L-tryptophan biosynthesis. Among these, the regulatory gene trpR , which encodes the tryptophan operon repressor, harbored a frameshift insertion. In addition, deletion mutations were observed in tnaA , encoding tryptophanase involved in tryptophan degradation, and tnaB , encoding a tryptophan permease responsible for intracellular transport. Together, these genetic changes are consistent with the enhanced intracellular accumulation of L-tryptophan in strain W-1, which may be associated with the release of transcriptional repression and the attenuation of tryptophan degradation and transport processes, as suggested by previous studies [ 28 , 29 ]. In addition, a frameshift mutation was detected in tyrA , which encodes chorismate mutase/prephenate dehydrogenase and plays a central role in the aromatic amino acid biosynthetic pathway. This alteration is predicted to reduce carbon flux toward L-tyrosine and L-phenylalanine biosynthesis, potentially increasing the availability of shared precursors for L-tryptophan formation [ 8 , 16 , 30 ]. In addition, a nonsynonymous single-nucleotide polymorphism was identified in the key L-tryptophan biosynthetic gene aroG , resulting in an amino acid substitution from lysine to threonine at position 194 (K194T). This mutation was considered a potential contributor to enhanced L-tryptophan biosynthesis and was selected for further functional evaluation. To assess the functional role of the aroG K194T mutation to L-tryptophan biosynthesis, a reverse metabolic engineering strategy was employed. Specifically, the mutated aroG in strain W-1 was replaced with its wild-type sequence, generating strain W-2. Shake-flask fermentation results showed that L-tryptophan titer in W-2 decreased by 10.84% compared with W-1 (Fig. 2 D), indicating that DAHP synthase activity is indeed a critical determinant of L-tryptophan biosynthesis. Similar effects have been reported in previous studies [ 31 ]. To further enhance carbon flux through the aromatic pathway, a well-characterized feedback-resistant DAHP synthase variant, aroG S180F , was introduced to replace the native aroG in W-1, yielding strain W-3, in which the L-tryptophan titer increased to 2.96 g/L. Furthermore, to alleviate feedback inhibition at an important control step within the tryptophan biosynthetic pathway, an S40F mutation was introduced into trpE to relieve feedback inhibition of anthranilate synthase. The engineered trpE S40F DCBA operon was subsequently integrated into the chromosome of W-3 under the control of the strong P trc promoter, yielding strain W-4, which achieved an L-tryptophan titer of 4.29 g/L in shake-flask fermentation (Fig. 2 D). Collectively, these results suggest that relieving feedback inhibition of key enzymes and reinforcing their expression can enhance carbon flux toward the L-tryptophan biosynthetic branch, thereby improving production capacity. 3.3 Enhancing PEP and E4P supply for L-tryptophan biosynthesis After alleviating feedback inhibition at several key enzymatic steps in the L-tryptophan biosynthetic pathway, further enhancement of precursor availability became a prerequisite for increasing product titer. PEP from glycolysis and E4P from the pentose phosphate pathway are the two essential precursors for L-tryptophan biosynthesis, and their intracellular availability is a key factor influencing L-tryptophan biosynthesis and final titer [ 1 ]. However, both PEP and E4P are distributed among multiple competing pathways, and inefficient channeling into the shikimate pathway limits L-tryptophan biosynthesis. Building upon the aforementioned engineering strategies, the precursor supply toward the shikimate pathway was further reinforced to enhance L-tryptophan biosynthesis through three complementary approaches: PEP regeneration, reduction of carbon loss, and enhancement of E4P supply (Fig. 3 A). Intracellular PEP availability was first increased by overexpressing pps , which promoted the conversion of PYR back to PEP. This modification is expected to increase the metabolic capacity for PEP regeneration and thereby support aromatic amino acid biosynthesis. As a result, strain W-5 exhibited a 26.34% increase in L-tryptophan titer compared with W-4. In addition, to reduce the consumption of PEP by non-target metabolic pathways, the biosynthetic routes leading to organic acid byproducts, including lactate, acetate, and formate, were deleted to reduce carbon flux toward competing branches. Specifically, ldhA , poxB , and pflB were deleted, generating strain W-6. This strain produced 5.74 g/L L-tryptophan with a yield of 9.11%. These results suggest that suppressing overflow metabolism may help preserve carbon flux toward the shikimate pathway and support increased L-tryptophan biosynthesis. To balance E4P availability with the expanded PEP pool, zwf and tktA were overexpressed under the control of the P trc promoter, thereby potentially increasing carbon flux through the pentose phosphate pathway. As a result, strain W-8 achieved an L-tryptophan titer of 7.08 g/L in shake-flask fermentation, representing a 65.03% increase relative to W-4 (Fig. 3 B), while the yield increased from 8.57% to 9.61%. These results show that coordinated, multi-level engineering strategies aimed at improving PEP and E4P availability effectively enhanced L-tryptophan biosynthesis and significantly improved production performance. 3.4 Reprogramming central carbon metabolism to reduce carbon loss The citric acid (TCA) cycle serves as the central hub of cellular energy metabolism and provides essential energy to support cell growth [ 32 ]. However, excessive TCA cycle activity is frequently accompanied by the accumulation of organic acids, such as citrate, succinate, and fumarate [ 33 ], which reduces intracellular pyruvate availability [ 34 ], and consequently limit L-tryptophan biosynthesis. To regulate TCA flux and mitigate carbon loss, gltA , which catalyzes the entry step of carbon into the TCA cycle, was dynamically regulated. In shake-flask fermentation, strain W-9 produced 6.17 g/L L-tryptophan when gltA was expressed under the self-regulating promoter P fliC . Compared with strain W-8, biomass accumulation was reduced, and the L-tryptophan titer decreased by 12.85%; however, the glucose-to-L-tryptophan conversion yield increased to 15.27% (Fig. 4 B). This result suggests that targeted intervention in the TCA cycle can improve carbon utilization efficiency toward L-tryptophan biosynthesis. Based on this observation, two alternative engineering routes were designed to optimize central carbon metabolism (Fig. 4 A). Route I targeted the glyoxylate cycle in strain W-8 as a carbon flux redistribution strategy. As a bypass of the TCA cycle, the glyoxylate pathway avoids decarboxylation reactions, thereby preserving carbon skeletons, reducing carbon loss (Fig. 4 A), and improving carbon recovery efficiency, which facilitates the redirection of carbon flux toward biosynthetic pathways [ 35 ]. In E. coli , the transcriptional regulator IclR represses the glyoxylate cycle, and the aceBAK operon encoding key enzymes of this pathway is normally activated only in the presence of acetate. Deletion of iclR in strain W-10 likely enhanced flux through the glyoxylate pathway and increased the L-tryptophan titer to 7.39 g/L in shake-flask fermentation. Consistent with previous studies, deletion of iclR has been shown to reduce excessive carbon flux through the TCA cycle [ 36 ]. Building on this result, further enhancement of glyoxylate cycle flux was achieved by overexpressing aceAB and deleting ycdW , which is involved in glyoxylate degradation. The resulting strain W-11 achieved an L-tryptophan titer of 7.88 g/L and a yield of 12.46% in shake-flask fermentation (Fig. 4 C), suggesting that activation and reinforcement of the glyoxylate cycle help reduce carbon loss and optimize carbon distribution for L-tryptophan biosynthesis. However, the activation of the glyoxylate cycle primarily alleviates carbon flux loss through restructuring the TCA cycle, without directly enhancing the recovery of precursors. Therefore, Route II was designed to improve carbon recovery efficiency by constructing a PYR-OAA-PEP recycling pathway. Specifically, the mutated pyc P458S gene from Corynebacterium glutamicum was integrated into strain W-8, enabling partial conversion of PYR to OAA while bypassing the TCA cycle. The gene pck was then overexpressed to promote the conversion of OAA to PEP, thereby establishing the PYR–OAA–PEP cycle. By comparison, strain W-13 constructed using this strategy achieved an L-tryptophan titer of 7.36 g/L with a yield of 10.62% in shake-flask fermentation (Fig. 4 C). Although lower than that obtained via glyoxylate cycle enhancement strategy, these results indicate that the construction of the PYR-OAA-PEP cycle also improves carbon utilization efficiency. Considering that the enhancement of the glyoxylate cycle might lead to an increased accumulation of OAA, pck was additionally integrated into strain W-11. The resulting strain W-14 achieved an L-tryptophan titer of 8.65 g/L and a yield of 13.07% (Fig. 4 B). Analysis of TCA-related metabolites showed that, compared with strain W-8, glyoxylate accumulation increased by 28.93%, whereas citrate, succinate, malate, oxaloacetate, and pyruvate levels decreased by 49.01%, 25.44%, 45.09%, 47.58%, and 37.93%, respectively (Fig. 4 D). These results indicate that coordinated regulation of the glyoxylate cycle and OAA-PEP recycling effectively alleviates carbon drainage through the TCA cycle and enhances carbon channeling toward L-tryptophan biosynthesis. 3.5 Coordinated supply of donor molecules for L-tryptophan biosynthesis L-tryptophan biosynthesis is a typical multi-precursor-coupled process in terms of both the synthetic pathway and molecular structure. Specifically, PEP and E4P jointly form the aromatic ring backbone, while L-glutamine provides the amide group for indole ring formation. The carbon skeleton of the indole ring is derived from PRPP, and L-serine contributes the side chain of L-tryptophan (Fig. 5 A). Therefore, in addition to PEP and E4P, the intracellular availability of donor molecules, including L-glutamine, L-serine, and PRPP, is also critical for efficient L-tryptophan biosynthesis. In E. coli , L-glutamine is synthesized from L-glutamate by glutamine synthetase (GlnA), which is subject to feedback inhibition by NH 4 + . In contrast, glutamine synthetase from Bacillus subtilis is resistant to this inhibition [ 37 ]. Accordingly, the feedback-resistant glnA L159I,E304A from B. subtilis was overexpressed in strain W-15 to enhance the biosynthetic capacity for L-glutamine, resulting in a 4.16% increase in L-tryptophan titer compared with W-14 (Fig. 5 B). In addition, L-serine, which provides the side chain of L-tryptophan, is synthesized via a pathway whose rate-limiting enzyme SerA is also subject to feedback inhibition. Previous studies reported that introducing the feedback-resistant serA fbr significantly increased intracellular serine levels by 18-fold [ 38 ]. In this study, the feedback-resistant serA H344A,N346A,N364A variant was introduced into strain W-14. In addition, 0.5 g/L L-isoleucine was supplemented to alleviate the metabolic burden associated with L-serine accumulation, as previous studies have shown that L-isoleucine supplementation can mitigate growth inhibition caused by serine overaccumulation [ 30 ]. The resulting strain W-16 exhibited a 2.08% increase in L-tryptophan titer relative to W-14 (Fig. 5 B). On the other hand, PRPP is synthesized from ribose-5-phosphate in the pentose phosphate pathway by phosphoribosylpyrophosphate synthase (Prs), whose expression is negatively regulated by PurR [ 39 ]. To enhance PRPP supply, purR was deleted in strain W-17, followed by heterologous expression of prs genes from E. coli , B. subtilis , C. glutamicum , and Serratia marcescens using the pTrc99A plasmid, generating strains W-17 + Ec, W-17 + Bs, W-17 + Cg, and W-17 + Sm, respectively. As shown in Fig. 5 D, strain W-17 + Sm exhibited a 11.16% increase in L-tryptophan titer compared to the control, suggesting that the prs gene from S. marcescens improves PRPP biosynthetic capacity and supports L-tryptophan production. We also observed that the high-level expression of prs from different sources resulted in varying degrees of growth inhibition (Fig. 5 D). Biomass accumulation in strains W-17 + Ec, W-17 + Bs, and W-17 + Cg decreased by 5.25%, 6.41%, and 2.34%, respectively, whereas W-17 + Sm exhibited the most significant reduction (10.44%). Despite this growth inhibition, W-17 + Sm achieved the highest L-tryptophan accumulation (9.46 g/L), indicating that increased PRPP biosynthetic capacity may favor carbon allocation toward L-tryptophan production at the expense of biomass formation. From an intracellular metabolic pathway perspective, the prs -catalyzed reaction utilizes ribose-5-phosphate and ATP as substrates. Overexpression of prs likely increases competition for ribose-5-phosphate and ATP in the pentose phosphate pathway, which may disrupts intracellular carbon and energy allocation and impairs cell growth [ 39 ]. To assess the balance of donor molecules required for L-tryptophan biosynthesis in strain W-17, expression cassettes for L-glutamine, L-serine, and PRPP were constructed using constitutive promoters of different strengths. Specifically, three promoters—P J23101 (strong), P J23105 (medium), and P J23114 (weak)—were used to drive glnA L159I,E304A ( B. subtilis ), serA H344A,N346A,N364A ( E. coli ), and prs ( S. marcescens ), generating a total of 27 engineered strains (Table S1 ). As shown in Fig. 5 E, screening in 24-well plates identified the strain harboring the M + H+M promoter combination (P J23105 BsglnA L159I,E304A +P J23101 EcserA H344A,N346A,N364A +P J23105 Smprs ) as the best performer, achieving an L-tryptophan titer of 1.59 g/L and a yield of 3.85%. This strain was designated W-22. Shake-flask fermentation further confirmed that W-22 achieved an L-tryptophan titer of 10.58 g/L, representing a 21.61% increase relative to strain W-17, with the yield increasing to 15.13%. These results show that efficient L-tryptophan biosynthesis not only relies on the sufficient supply of key precursors such as PEP and E4P, but also significantly depends on the optimal balance between other donor molecules. Systematic optimization of L-glutamine, L-serine, and PRPP supply modules enabled fine-tuning of precursor metabolism and biosynthetic flux, thereby further improving both L-tryptophan titer and yield. 3.6 Engineering transport systems to promote L-tryptophan export Previous studies have shown that enhancing intracellular overproduction of metabolites through systematic metabolic engineering often leads to negative effects such as intracellular osmotic pressure imbalances and growth inhibition [ 8 , 40 ], thereby reducing the sustained production capacity of engineered strains. By engineering transport channels to alleviate endogenous pressure, further improvement in product titer can be achieved [ 10 , 20 ]. In E. coli , multiple transport proteins cooperate in the permeation of aromatic amino acids. Among them, L-tryptophan transport is subject to complex regulation [ 41 ]. Three transporters—AroP, Mtr, and TnaB—play distinct roles in intracellular L-tryptophan accumulation (Fig. 6 A). Specifically, TnaB functions as a low-affinity transporter, Mtr exhibits high affinity for L-tryptophan, and AroP mediates the transport of multiple aromatic amino acids. Genomic analysis revealed that the starting strain W-1 lacked tnaB , which encodes a low-affinity L-tryptophan transporter. To evaluate the role of TnaB in high-titer tryptophan strains, the tnaB expression cassette was reintroduced into strain W-22 at the ilvG locus via reverse metabolic engineering. Restoration of tnaB expression resulted in a 7.09% decrease in the L-tryptophan titer, suggesting that enhanced L-tryptophan uptake may lead to elevated intracellular concentrations, which could impose additional physiological stress and negatively affect L-tryptophan biosynthesis. On the other hand, overexpression of the L-tryptophan efflux protein YddG in strain W-22 resulted in the creation of strain W-24, which achieved an L-tryptophan titer of 11.76 g/L (Fig. 6 B). These results suggest that engineering L-tryptophan transport pathways to promote product efflux can support higher L-tryptophan titers, likely by reducing intracellular accumulation. 3.7 Optimization of fed-batch fermentation in a 5 L bioreactor During scale-up production of engineered strains, inoculum size is considered an important parameter affecting the adaptability, growth kinetics, and product synthesis of engineered strains [ 42 ]. To investigate the effect of inoculum size on the fermentation performance of strain W-24 under glucose-limited fed-batch conditions, fermentations were conducted in a 5 L bioreactor with controlled dissolved oxygen (DO) and pH. Fresh seed cultures were inoculated at 300 mL (15%), 350 mL (17.5%), 400 mL (20%), and 450 mL (22.5%), while maintaining an initial fermentation working volume of 2.0 L. At an inoculum size of 15%, strain W-24 exhibited slow growth recovery and a prolonged lag phase. Both the specific growth rate and L-tryptophan synthesis rate throughout the fermentation cycle were lower than those observed at higher inoculum sizes (Fig. 7 B, 7 D), resulting in lower final biomass (OD 600 = 68.2) and L-tryptophan titer (43.65 g/L) (Fig. 7 A, 7 C). In contrast, at higher inoculum sizes (20% and 22.5%), the strain rapidly entered the exponential growth phase and maintained higher specific growth rates during the early fermentation stage. Consequently, peak biomass levels of OD 600 78.0 and 81.4 were achieved, corresponding to increases of 10.37% and 19.64%, respectively, relative to the 17.5% inoculum group. However, as fermentation proceeded, the specific growth rates of the 20% and 22.5% inoculum groups gradually declined and became negative, indicating growth cessation and possible decline in cell viability at later stages (Fig. 7 B). Moreover, the rapid early growth at high inoculum sizes may have accelerated nutrient consumption and increased metabolic burden during the early fermentation stage, thereby affecting the later-stage production capacity. Correspondingly, after 24 hours of fermentation, the L-tryptophan synthesis rate significantly decreased (Fig. 7 D), and the final titer (46.08 g/L, 45.16 g/L, respectively) was lower than that of the 17.5% inoculum group (50.83 g/L) (Fig. 7 C). Considering both cell growth and product generation data, W-24 exhibited optimal overall fermentation performance at an inoculum size of 17.5%. During the 40-hour fed-batch fermentation, the maximum biomass reached 74.9, and at the end of fermentation, the L-tryptophan titer was 50.83 g/L, with a yield of 18.5% (Fig. S2 ). The acetic acid concentration at the end of fermentation was 2.30 g/L (Fig. S3). These results show that an appropriate inoculum size facilitates a balanced coordination between cell growth and product biosynthesis, thereby enabling improved fermentation performance of the engineered strain under scaled-up conditions. Overall, these results indicate that the coordinated supplementation and balancing of multiple intracellular precursors is an effective strategy to enhance L-tryptophan biosynthesis. However, the production performance of the engineered strain remains below the theoretical level [ 43 ], indicating that further reduction of carbon flux loss and improvement of precursor utilization efficiency are still required. Future efforts focusing on uncovering potential regulatory mechanisms—such as the identification of novel transcriptional regulators—as well as on the development of more refined metabolic regulation models may provide additional opportunities to further improve amino acid production. 4.CONCLUSION This work describes the construction of an efficient microbial cell factory for L-tryptophan production, starting from a model strain and combining ARTP mutagenesis with systematic metabolic engineering strategies. The main strategies employed include: (i) development of an L-tryptophan-specific biosensor combined with ARTP mutagenesis to obtain an L-tryptophan-producing chassis strain; (ii) reorganization of the central carbon metabolism pathway to enhance the carbon flux of PEP and E4P while reducing carbon loss; (iii) optimization of the supplementation and balance of multiple L-tryptophan donor molecules through promoter engineering; (iv) engineering of L-tryptophan transport pathways; and (v) optimization of inoculum size during fermentation. Ultimately, the engineered strain W-24, without inducers or antibiotics, achieved an L-tryptophan titer of 50.83 g/L in a 5 L bioreactor, with a yield of 0.185 g/g. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the National Key Research and Development Program of China (2024YFA0918000), the Frontier Technology Research and Development Plan of Jiangsu Province (BF2024012), the National Natural Science Foundation of China (32471530), the Major Scientific and Technological Project for “unveiling and commanding” of Hohhot (2023-unveiling and commanding-He-1), the Independent Research Project of the State Key Laboratory of Food Science and Resources of Jiangnan University (SKLF-ZZB-202408), the Project for “unveiling and commanding” of Urumqi (B241011002), the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX24_2594), the National Natural Science Foundation of China (22508213) and the Shandong Postdoctoral Science Foundation (SDCX-ZG-202502064). Author Contribution #R.Z. and Z.Z. contributed equally. R.Z. conceived the study, designed the experiments, analyzed the data, and wrote and revised the manuscript. Z.Z. conducted background investigation and assisted in experimental design. W.S. performed the experiments. J.H. and J.Y. analyzed the data. X.P. and M.S. provided conceptual guidance and supervised the research. All authors have given approval to the final version of the manuscript. Acknowledgements Not applicable. Availability of data and materials All data involved in this study are included in this published article and its additional files. References Niu H, Li R, Liang Q, Qi Q, Li Q, Gu P. Metabolic engineering for improving L-tryptophan production in Escherichia coli . J Ind Microbiol Biotechnol. 2019;46:55–65. Friedman M. Analysis, nutrition, and health benefits of tryptophan. Int J Tryptophan Res. 2018;11:1178646918802282. Lu C, Deng Y, Ma W, Wang W, Li P, Shi P, Yan T, Yin Y, Huang P. Tryptophan nutrition in poultry and ruminant animals. In Tryptophan in Animal Nutrition and Human Health. Edited by Yin Y, Kim SW, Tang X. Singapore: Springer Nature Singapore; 2024: 127–157. 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Supplementary Files Supplementarymaterial1.docx Supplementarymaterial2Data1.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 09 Apr, 2026 Reviews received at journal 09 Apr, 2026 Reviews received at journal 08 Apr, 2026 Reviews received at journal 05 Apr, 2026 Reviewers agreed at journal 03 Apr, 2026 Reviewers agreed at journal 30 Mar, 2026 Reviewers agreed at journal 29 Mar, 2026 Reviewers invited by journal 29 Mar, 2026 Editor assigned by journal 28 Mar, 2026 Submission checks completed at journal 28 Mar, 2026 First submitted to journal 27 Mar, 2026 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-9241029","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":614391712,"identity":"e468c51b-b306-4735-91cf-a771ce8ab52e","order_by":0,"name":"Rongshuai Zhu","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Rongshuai","middleName":"","lastName":"Zhu","suffix":""},{"id":614391713,"identity":"27aaecef-0dda-42cf-af26-b6e38c60164b","order_by":1,"name":"Zhenqiang Zhao","email":"","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhenqiang","middleName":"","lastName":"Zhao","suffix":""},{"id":614391714,"identity":"a4020387-d059-4938-ad94-11d11c40757c","order_by":2,"name":"Weijun Shao","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Weijun","middleName":"","lastName":"Shao","suffix":""},{"id":614391715,"identity":"c999a70d-7e57-4419-8157-93f8d00dfccd","order_by":3,"name":"Jin Han","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Jin","middleName":"","lastName":"Han","suffix":""},{"id":614391716,"identity":"9a71ddd1-ac98-4d34-a104-35018c316dca","order_by":4,"name":"Jiajia You","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Jiajia","middleName":"","lastName":"You","suffix":""},{"id":614391717,"identity":"89cc0ea9-4ddf-4aed-8e09-5e42a9cc764d","order_by":5,"name":"Xuewei Pan","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Xuewei","middleName":"","lastName":"Pan","suffix":""},{"id":614391718,"identity":"f05a0315-17e4-47ab-b04e-b97593203dbe","order_by":6,"name":"Minglong Shao","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Minglong","middleName":"","lastName":"Shao","suffix":""},{"id":614391719,"identity":"38335d42-6f00-4cf6-b8b2-0ae7d8ccac7d","order_by":7,"name":"Zhiming Rao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYJACZgYGG2Y2BD+BKC1ppGs5jMwnoMW8/fDhz4U559n52M8efl1Qc4eBnz3HgOHnDtxaZM6kJRjP3HabmY0nL816xrFnDJI9bwwYe8/g1iLBkGOQzAvSwpBjZszDdpjB4EaOATNjGx4t/G8MDvNuO8fMxv8GqOXfYQZ7glokcgybebcdYGaTyDF+zNsGtEWCoJZnycy825KBWt6YMfP2HeaROPOs4GAvXoclH/7Mu80uWb4/x/gzz7fDcvztyRsf/MSjBQaSgZhNAkjwgHgHCGtgYLADYuYPxKgcBaNgFIyCkQcAZ1JHYMBN2woAAAAASUVORK5CYII=","orcid":"","institution":"Jiangnan University","correspondingAuthor":true,"prefix":"","firstName":"Zhiming","middleName":"","lastName":"Rao","suffix":""}],"badges":[],"createdAt":"2026-03-27 06:40:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9241029/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9241029/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105954099,"identity":"70120f4c-3391-4cb2-bd72-125299a8ae5d","added_by":"auto","created_at":"2026-04-01 19:35:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1610727,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMetabolic engineering strategies for L-tryptophan production in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e“×” indicates knockout of the corresponding gene, whereas “*” represents feedback inhibition-resistant variants. Abbreviations: G6P, glucose-6-phosphate; G3P, Glyceraldehyde-3-phosphate; PEP, phosphoenolpyruvate; PYR, pyruvate; AcoA, acetyl-CoA; CIT, citrate; AKG, α-ketoglutarate; SUC, succinate; MAL, malate; OAA, oxaloacetate; Glo, glyoxylate; Glyo, glycolate; LAC, lactate; FOM, formate; ACE, acetate; Ru5P, ribulose‐5‐phosphate; PRPP, phosphoribosyl pyrophosphate; E4P, erythrose-4-phosphate; DAHP, 3‐deoxy-D‐arabinoheptulosonate‐7‐phosphate; SHIK, shikimate; CHA, chorismate; PPA, prephenate; TFs, transcription factors; L-Ser, L-serine; L-Glu, L-glutamate; L-Gln, L-glutamine; L-Tyr, L-tyrosine; L-Phe, L- phenylalanine; L-Trp, L-tryptophan;\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9241029/v1/e044d809c247cfd92d021465.png"},{"id":106093526,"identity":"6ac012df-3151-4b2d-83b9-8e41f7a4eb94","added_by":"auto","created_at":"2026-04-03 11:37:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":934568,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction of an L-tryptophan biosensor and its application in L-tryptophan-producing strains. \u003c/strong\u003e(A) Schematic illustration of the mechanism for screening an L-tryptophan chassis strain by combining biosensor construction with ARTP mutagenesis. (B) Comparison of L-tryptophan concentration–dependent fluorescence responses of the native and engineered biosensors. (C) L-tryptophan titers of 40 mutant strains obtained by ARTP mutagenesis and screened in 96-deep-well plates. (D) L-tryptophan titers and biomass of strains W1–W4 during shake-flask fermentation.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9241029/v1/389c0b55a06594e2016e282f.png"},{"id":106093160,"identity":"57729be8-d054-4d95-a6e1-e0cfa88229a8","added_by":"auto","created_at":"2026-04-03 11:35:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":494113,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEngineering strategies for enhancing intracellular PEP and E4P supply. \u003c/strong\u003e(A) Schematic overview of multi-step strategies for increasing intracellular PEP and E4P availability. (B) L-tryptophan titer and biomass of strains constructed through the three approaches during shake-flask fermentation.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9241029/v1/c6af0263f8e71b9377be96a0.png"},{"id":106093735,"identity":"8ded6cc5-b40e-441c-8bda-db2d5fbe44d9","added_by":"auto","created_at":"2026-04-03 11:38:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1561326,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSystematic reconstruction of central carbon metabolism for enhanced L-tryptophan biosynthesis.\u003c/strong\u003e (A) Schematic overview of central metabolic pathway reconstruction using two alternative engineering routes. Green indicates pathway reinforcement, and red indicates gene deletion. (B) L-tryptophan titer, biomass and yield of strains W-8, W-9, and W-14 during shake-flask fermentation. (C) L-tryptophan titer and biomass of strains constructed via the two different engineering routes during shake-flask fermentation. (D) Comparison of major organic acid titers in strains W-8 and W-14 during shake-flask fermentation.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9241029/v1/f02b8e920a1bdd12de3d51c4.png"},{"id":105954104,"identity":"d820ff3f-4f6f-4c19-afa0-5d8709cda3e8","added_by":"auto","created_at":"2026-04-01 19:35:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3168112,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCoordinated supply of the donor molecules L-glutamine, L-serine, and PRPP enhances L-tryptophan production. \u003c/strong\u003e(A) Sources of the molecular backbones contributing to L-tryptophan biosynthesis. (B) L-tryptophan titer and biomass of strains W-14–W-17 during shake-flask fermentation. (C) L-tryptophan titers of strains expressing \u003cem\u003eprs\u003c/em\u003e genes from different sources via plasmids, with strain W-17 harboring the empty pTrc99A plasmid as the control. The * symbol represents 0.01 \u0026lt; P \u0026lt; 0.05. (D) Growth curves of strains expressing\u003cem\u003e prs\u003c/em\u003e genes from different sources via plasmids during shake-flask fermentation (12–30 h). (E) Optimization of the intracellular balance of L-glutamine, L-serine, and PRPP through promoter engineering of\u003cem\u003e BsglnA\u003c/em\u003e, \u003cem\u003eEcserA\u003c/em\u003e, and\u003cem\u003e Smprs\u003c/em\u003e, resulting in enhanced L-tryptophan titer. High, medium, and low expression levels were driven by the P\u003csub\u003eJ23101\u003c/sub\u003e, P\u003csub\u003eJ23105\u003c/sub\u003e, and P\u003csub\u003eJ23114\u003c/sub\u003e promoters, respectively.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9241029/v1/1fde87fc7b4a2cbd6425bcf9.png"},{"id":106093566,"identity":"a9dc1360-8f04-4a9d-a546-7086162abecb","added_by":"auto","created_at":"2026-04-03 11:38:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1107618,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEngineering of L-tryptophan transport channels. \u003c/strong\u003e(A) Model of L-tryptophan transport by transport proteins. (B) Comparison of L-tryptophan titer and biomass in strains W-22–W-24 during shake-flask fermentation, constructed by modifying the transport channels.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-9241029/v1/3bdd310b979afd74f99bc52c.png"},{"id":105954107,"identity":"3c6647e9-82d6-4a76-94fc-4a2a27619248","added_by":"auto","created_at":"2026-04-01 19:35:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":849309,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of inoculum size on L-tryptophan production during fed-batch fermentation in a 5 L bioreactor. \u003c/strong\u003e(A) Growth curves of strain W-24 at different inoculum sizes in a 5 L bioreactor. (B) Specific growth rates of strain W-24 at different inoculum sizes in a 5 L bioreactor. (C) L-tryptophan titer profiles of strain W-24 at different inoculum sizes in a 5 L bioreactor. (D) L-tryptophan synthesis rates of strain W-24 at different inoculum sizes during fed-batch fermentation in a 5 L bioreactor.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-9241029/v1/a7512c3880ecbb1b1c7ad49d.png"},{"id":107704943,"identity":"18511ca1-b1b5-43e7-873d-3b0be66ec615","added_by":"auto","created_at":"2026-04-24 09:04:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10117146,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9241029/v1/d4d43ddd-f8d0-419f-9b2c-6d178397f19f.pdf"},{"id":106093496,"identity":"6acea45e-495f-4687-a5ad-4e9ca3d6b605","added_by":"auto","created_at":"2026-04-03 11:37:35","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":289572,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9241029/v1/5eea0182caf28513cd797393.docx"},{"id":105954102,"identity":"6fe16f82-ea48-4d9f-8a81-4c046261e795","added_by":"auto","created_at":"2026-04-01 19:35:34","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13408,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial2Data1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9241029/v1/50e63dd417e8e221f680e4ba.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biosensor-driven evolution and metabolic engineering of an Escherichia coli","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eL-Tryptophan is an essential aromatic amino acid that serves not only as a fundamental building block for protein biosynthesis but also as a vital precursor for several biologically active molecules [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], including serotonin, melatonin, and niacin [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In animal nutrition, L-tryptophan is generally regarded as the third limiting amino acid in diets for monogastric animals and poultry [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. It plays critical roles in regulating circadian rhythms, neuroimmune responses, and intestinal barrier function, deeply influencing both stress tolerance and overall growth performance [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. As the livestock industry continues to expand, the global demand for L-tryptophan has surged. Simultaneously, conventional extraction and chemical synthesis methods are increasingly constrained by high energy consumption, heavy environmental burdens, and limited production efficiency [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Consequently, the development of sustainable, high-efficiency microbial cell factories has emerged as a compelling alternative. However, the complex endogenous regulatory networks in microorganisms persistently restrict the excessive accumulation of target products [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], making the construction of highly robust production strains a formidable challenge.\u003c/p\u003e \u003cp\u003eIn microorganisms, particularly in the widely used industrial host \u003cem\u003eEscherichia coli\u003c/em\u003e, L-tryptophan biosynthesis originates from glucose and involves several interconnected pathways, including central carbon metabolism, the shikimate pathway, and the branched aromatic amino acid pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Central carbon metabolism supplies the key precursors phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) through glycolysis and the pentose phosphate pathway, respectively. These two precursors enter the shikimate pathway via the key enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHP synthase), forming common intermediates required for aromatic amino acid biosynthesis and subsequently directing flux toward the L-tryptophan branch. Previous studies have shown that random mutagenesis is an effective approach for obtaining improved chassis strains for L-tryptophan production [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In addition, enhancing the supply of PEP and E4P [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], relieving feedback inhibition of key enzymes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and reinforcing rate-limiting steps in the shikimate pathway have been shown to improve L-tryptophan biosynthesis to varying extents [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFrom the perspective of metabolic pathway requirements, L-tryptophan biosynthesis depends not only on carbon precursors but also on several donor metabolites, including L-glutamine, L-serine, and phosphoribosyl pyrophosphate (PRPP). These metabolites are simultaneously involved in multiple essential cellular processes, such as amino acid and nucleotide biosynthesis [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], Variations in their intracellular availability can trigger homeostatic responses [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], thereby affecting flux distribution toward the tryptophan pathway and limiting the theoretical yield. Therefore, achieving coordinated regulation among precursor supply, donor metabolism, and downstream flux while maintaining cell growth represents one of the key challenges in further improving L-tryptophan titer and yield.\u003c/p\u003e \u003cp\u003eBased on these considerations, systematic metabolic engineering aimed at coordinated regulation of multiple metabolic modules has emerged as an effective strategy to enhance aromatic amino acid biosynthesis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In this study, a biosensor-assisted high-throughput screening system was established and combined with ARTP mutagenesis to obtain an improved \u003cem\u003eE. coli\u003c/em\u003e chassis strain for L-tryptophan production. Central carbon metabolism was reconfigured and key enzymatic steps were reinforced to increase the availability of PEP and E4P and to enhance carbon flux toward the shikimate pathway. In addition, promoter engineering was applied to regulate the biosynthesis of donor metabolites, including L-glutamine, L-serine, and PRPP, thereby balancing their intracellular supply and improving L-tryptophan production. Product secretion was further enhanced through modification of transport systems. After optimization of fermentation conditions, the engineered strain achieved high-level L-tryptophan production. These results demonstrate that coordinated regulation of multiple metabolic modules can effectively alleviate metabolic flux imbalance and improve aromatic amino acid biosynthesis, providing useful insights for the development of efficient microbial production systems.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.1. Strains and plasmids\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eIn this study, \u003cem\u003eE. coli\u003c/em\u003e DH5α was used as the host strain for plasmid cloning, and plasmid pTrc99A was used as the expression vector. \u003cem\u003eE. coli\u003c/em\u003e W3110 was selected as the starting strain for the construction of L-tryptophan-producing strains, and genome knock-in and knockout manipulations were performed using the CRISPR/Cas12a system [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The strains, plasmids, and primers used in this study are listed in the Tables S1, S2, S3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.2. DNA manipulation\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eIn this study, all expression cassettes of the reinforced genes were driven by the P\u003csub\u003etrc\u003c/sub\u003e promoter unless otherwise specified. The CRISPR/Cas12a-mediated genome editing procedure is illustrated using the integration of the \u003cem\u003etktA\u003c/em\u003e gene at the \u003cem\u003eyncK\u003c/em\u003e locus as an example. The CRISPR/Cas12a system consisted of the plasmids pEcCpf1 and pcrEG. Using the \u003cem\u003eE. coli\u003c/em\u003e genome as the template, approximately 500 bp upstream and downstream homologous arms were amplified with the primer pairs yncK-UP-F/yncK-UP-R and yncK-DO-F/yncK-DO-R, respectively. The target gene \u003cem\u003etktA\u003c/em\u003e driven by the P\u003csub\u003etrc\u003c/sub\u003e promoter was amplified using primers tktA (Ptrc) -F and tktA-R. The homologous arms and the target gene fragment were then assembled by Gibson assembly to generate the donor DNA. In addition, specific CRISPR RNAs (crRNAs) were designed using the CRISPR-DT tool and constructed into the helper plasmid pcrEG to generate the guide plasmid [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Gene knock-in was performed as described previously [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.3. Atmospheric and room temperature plasma (ARTP) mutagenesis\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe biosensor-containing model strain was cultivated to the exponential growth phase (~\u0026thinsp;6\u0026ndash;8 h), and then cells were harvested, resuspended in phosphate-buffered saline (PBS), and 10 \u0026micro;L of the cell suspension was spotted onto the center of a sterile metal slide. ARTP mutagenesis was then performed using an ARTP-III mutation system (Tsingyuan Biotechnology Co., Ltd., Beijing, China) with a radio-frequency power of 120 W and a helium gas flow rate of 10 standard liters per minute (SLM) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The treatment durations were set to 0, 10, 20, 30, 40, 50, 60, 70, and 80 s. After treatment, the cells were immediately collected and subjected to recovery cultivation, followed by plating on LB agar plates. The plates were incubated at 37 ℃ until colonies appeared, generating a mutant library. The lethality rate was calculated as described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.4. Shake-flask culture conditions\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eSingle colonies of engineered strains grown on agar plates were inoculated into 50 mL shake flasks containing 10 mL of LB medium and cultivated at 37 ℃ with shaking at 220 rpm. After overnight cultivation, 3 mL of the seed culture was transferred into a 500 mL shake flask containing 27 mL of fresh fermentation medium. The flasks were incubated at 37 ℃ and 220 rpm for 36 h. During fermentation, the pH was manually maintained by the addition of 25% (v/v) NH\u003csub\u003e3\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO, and a 60% (w/v) glucose solution was intermittently supplied to replenish the carbon source in the fermentation medium [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The fermentation medium consisted of (per liter): 2.0 g yeast extract, 4.0 g (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 2.0 g sodium citrate, 6.0 g K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 2.5 g KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 2.0 g MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 30.0 mg FeSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 5.0 mg MnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO, 0.5 g sodium glutamate, 0.1 mg biotin, 0.2 mg vitamins B\u003csub\u003e1\u003c/sub\u003e, B\u003csub\u003e3\u003c/sub\u003e, B\u003csub\u003e5\u003c/sub\u003e, and B\u003csub\u003e12\u003c/sub\u003e, and 30 g glucose.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.5. Fed-batch fermentation in a bioreactor\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eFor scale-up cultivation of the engineered strains, fresh colonies were scraped from agar plates using a sterile inoculating loop and inoculated into 500 mL shake flasks containing 120 mL of LB medium. The cultures were incubated at 37 ℃ and 220 rpm for 6\u0026ndash;8 h and subsequently transferred into a 5 L bioreactor containing 1.88 L of seed medium for further cultivation for 10\u0026ndash;12 h. The resulting seed culture was then inoculated at an appropriate inoculum size into a 5 L bioreactor containing fresh fermentation medium, with the working volume maintained at 2.0 L.\u003c/p\u003e \u003cp\u003eWhen the initial glucose was nearly depleted, an 80% (w/v) glucose solution was rapidly fed to maintain the residual glucose concentration in the broth at 0.05\u0026ndash;0.1%. During cultivation, the DO level was controlled at 25\u0026ndash;30% by real-time adjustment of agitation speed and aeration rate. The pH was maintained at 7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 by automatic addition of 25% (v/v) NH\u003csub\u003e3\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO using a peristaltic pump. The seed medium consisted of (per liter): 1.5 g yeast extract, 4.0 g (NH\u003csub\u003e4\u003c/sub\u003e) \u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 1.6 g sodium citrate, 6.0 g K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 2.5 g KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 1.5 g MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 10.0 mg FeSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 5.0 mg MnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO, 0.1 mg biotin, 0.2 mg vitamin B\u003csub\u003e1\u003c/sub\u003e, B\u003csub\u003e3\u003c/sub\u003e, B\u003csub\u003e5\u003c/sub\u003e, and B\u003csub\u003e12\u003c/sub\u003e, and 40 g glucose. The fermentation medium consisted of (per liter): 2.0 g yeast extract, 4.0 g (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 2.0 g sodium citrate, 6.0 g K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 2.5 g KH₂PO\u003csub\u003e4\u003c/sub\u003e, 2.0 g MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 30.0 mg FeSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 5.0 mg MnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO, 0.5 g sodium glutamate, 0.5 g/L L-isoleucine, 0.5 g/L betaine, 0.1 mg biotin, 0.2 mg vitamin B\u003csub\u003e1\u003c/sub\u003e, B\u003csub\u003e3\u003c/sub\u003e, B\u003csub\u003e5\u003c/sub\u003e, and B\u003csub\u003e12\u003c/sub\u003e, and 10 g glucose.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.6. Analytical methods\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eCell growth was monitored by measuring the optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) using a UV-visible spectrophotometer after appropriate dilution of the samples. Glucose concentration was determined using an SBA biosensor analyzer (SBA-40C, Shandong, China) after sample dilution and centrifugation to obtain the supernatant. The fluorescence characteristics of the strains were analyzed using a BD flow cytometer (Becton Dickinson, USA). For the mutant library, L-tryptophan concentration in 96-well plates was quantified using a colorimetric spectrophotometric method. Briefly, under sulfuric acid conditions, L-tryptophan reacts with \u003cem\u003ep\u003c/em\u003e-dimethylaminobenzaldehyde to form a blue-colored compound. After incubation in boiling water for 3 min, a specific sodium nitrite solution was added, followed by further incubation for 3 min. The absorbance was then measured at 590 nm (A\u003csub\u003e590\u003c/sub\u003e) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. For samples obtained from shake-flask and bioreactor cultivations, cultures were diluted to appropriate concentrations, centrifuged, and the supernatants were collected. The clarified supernatants were filtered through 0.22 \u0026micro;m membrane filters to remove cells and impurities. Amino acids and organic acids in the fermentation broth were subsequently quantified by high-performance liquid chromatography (HPLC), using pre-column derivatization with o-phthalaldehyde for amino acid analysis and 5 mM H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e as the mobile phase for organic acid analysis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. All experimental data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation from at least three independent experiments.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS AND DISCUSSION","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.1 Biosensor-assisted screening of chassis strain for L-tryptophan production\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eBiosensors have become powerful tools for microbial strain development [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], enabling intracellular sensing of target metabolites and high-throughput screening of high-producing strains, as demonstrated for cadaverine[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], lycopene [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and human milk oligosaccharide [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In \u003cem\u003eE. coli\u003c/em\u003e, the transcriptional leader region of \u003cem\u003etnaA\u003c/em\u003e contains a 72-bp sequence, \u003cem\u003etnaC\u003c/em\u003e, which encodes a 24-amino-acid leader peptide (TnaC) and serves as a key regulatory element of the \u003cem\u003etna\u003c/em\u003e operon [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. When intracellular tryptophan reaches a threshold concentration, TnaC modulates Rho-dependent transcription termination, thereby enhancing the expression of the downstream \u003cem\u003etnaAB\u003c/em\u003e genes [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Based on this regulatory mechanism, a biosensor-assisted screening platform for tryptophan-producing strains was constructed and designated \u003cem\u003eE. coli\u003c/em\u003e W3110\u0026thinsp;+\u0026thinsp;pUC-trpSensor (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Specifically, the regulatory region containing the native \u003cem\u003etnaC\u003c/em\u003e leader sequence and upstream promoter was fused to green fluorescent protein (GFP) via a flexible linker (GGGGS) and cloned into the pUC plasmid, allowing fluorescence output to reflect intracellular L-tryptophan levels. To improve the sensitivity and expand the detection range of the biosensor, the native \u003cem\u003etnaC\u003c/em\u003e was replaced with the hypersensitive variant \u003cem\u003etnaC\u003c/em\u003e\u003csup\u003e\u003cem\u003eR23F\u003c/em\u003e\u003c/sup\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], yielding the modified construct pUC-trp*Sensor. The constructed biosensors were characterized in media supplemented with different concentrations of L-tryptophan. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, pUC-trp*Sensor exhibited an expanded detection range of 0.05-1.00 g/L, whereas the unmodified biosensor displayed a narrower range of 0.05\u0026ndash;0.50 g/L. Compared with pUC-trpSensor, the engineered biosensor displayed an increased upper detection limit and enhanced sensitivity toward L-tryptophan, thereby facilitating the discrimination of strains with elevated intracellular L-tryptophan levels during high-throughput screening.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo generate genetic diversity for strain improvement, \u003cem\u003eE. coli\u003c/em\u003e W3110 harboring pUC-trp*Sensor was subjected to ARTP mutagenesis under different exposure durations (0\u0026ndash;80 s). As the treatment time increased, the lethality rate increased accordingly (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Considering both lethality and mutant recovery efficiency [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], a treatment duration of 60 s was selected as the optimal mutagenesis condition, corresponding to a lethality rate of approximately 91.04%. Under the optimized ARTP conditions, a mutant library of \u003cem\u003eE. coli\u003c/em\u003e W3110 pUC-trp*Sensor was constructed and subjected to high-throughput screening using fluorescence-activated cell sorting (FACS). The sorting gate was defined based on the fluorescence distribution of the parental strain, and cells within the high-fluorescence fraction were collected for subsequent cultivation. Mutants were collected and cultivated in deep-well plates, and the top-performing mutant showing the highest fluorescence intensity was selected as the starting strain for the next round of mutagenesis. After three consecutive rounds of ARTP mutagenesis and screening, 40 preliminary candidates with the highest average fluorescence intensity were selected from approximately 5\u0026times;10\u003csup\u003e3\u003c/sup\u003e mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The top ten candidates were subsequently re-evaluated by shake-flask cultivation. Among them, the best-performing mutant exhibited an L-tryptophan titer of 2.03 g/L and was designated W-1 for subsequent strain engineering aimed at enhanced L-tryptophan production (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Notably, the top-ranked mutants selected based on fluorescence intensity consistently exhibited increased L-tryptophan production during shake-flask validation, indicating a strong correlation between biosensor output and intracellular L-tryptophan levels, and thereby supporting the reliability of the biosensor-guided screening strategy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Genomic analysis and reinforcement of key enzymes for L-tryptophan biosynthesis\u003c/h2\u003e \u003cp\u003eTo elucidate the genetic basis underlying the enhanced L-tryptophan titer of the mutant strain, whole-genome \u003cem\u003ede novo\u003c/em\u003e sequencing was performed for W-1 and the wild-type parental strain \u003cem\u003eE. coli\u003c/em\u003e W3110. Comparative genome analysis revealed a total of 47 single-nucleotide variations (SNVs) and insertions/deletions (InDels) in the mutant genome relative to the wild type (Data S1). Notably, multiple mutations were detected in metabolic pathways closely associated with L-tryptophan biosynthesis. Among these, the regulatory gene \u003cem\u003etrpR\u003c/em\u003e, which encodes the tryptophan operon repressor, harbored a frameshift insertion. In addition, deletion mutations were observed in \u003cem\u003etnaA\u003c/em\u003e, encoding tryptophanase involved in tryptophan degradation, and \u003cem\u003etnaB\u003c/em\u003e, encoding a tryptophan permease responsible for intracellular transport. Together, these genetic changes are consistent with the enhanced intracellular accumulation of L-tryptophan in strain W-1, which may be associated with the release of transcriptional repression and the attenuation of tryptophan degradation and transport processes, as suggested by previous studies [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In addition, a frameshift mutation was detected in \u003cem\u003etyrA\u003c/em\u003e, which encodes chorismate mutase/prephenate dehydrogenase and plays a central role in the aromatic amino acid biosynthetic pathway. This alteration is predicted to reduce carbon flux toward L-tyrosine and L-phenylalanine biosynthesis, potentially increasing the availability of shared precursors for L-tryptophan formation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In addition, a nonsynonymous single-nucleotide polymorphism was identified in the key L-tryptophan biosynthetic gene \u003cem\u003earoG\u003c/em\u003e, resulting in an amino acid substitution from lysine to threonine at position 194 (K194T). This mutation was considered a potential contributor to enhanced L-tryptophan biosynthesis and was selected for further functional evaluation.\u003c/p\u003e \u003cp\u003eTo assess the functional role of the \u003cem\u003earoG\u003c/em\u003e\u003csup\u003e\u003cem\u003eK194T\u003c/em\u003e\u003c/sup\u003e mutation to L-tryptophan biosynthesis, a reverse metabolic engineering strategy was employed. Specifically, the mutated \u003cem\u003earoG\u003c/em\u003e in strain W-1 was replaced with its wild-type sequence, generating strain W-2. Shake-flask fermentation results showed that L-tryptophan titer in W-2 decreased by 10.84% compared with W-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), indicating that DAHP synthase activity is indeed a critical determinant of L-tryptophan biosynthesis. Similar effects have been reported in previous studies [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. To further enhance carbon flux through the aromatic pathway, a well-characterized feedback-resistant DAHP synthase variant, \u003cem\u003earoG\u003c/em\u003e\u003csup\u003e\u003cem\u003eS180F\u003c/em\u003e\u003c/sup\u003e, was introduced to replace the native \u003cem\u003earoG\u003c/em\u003e in W-1, yielding strain W-3, in which the L-tryptophan titer increased to 2.96 g/L. Furthermore, to alleviate feedback inhibition at an important control step within the tryptophan biosynthetic pathway, an S40F mutation was introduced into \u003cem\u003etrpE\u003c/em\u003e to relieve feedback inhibition of anthranilate synthase. The engineered \u003cem\u003etrpE\u003c/em\u003e\u003csup\u003e\u003cem\u003eS40F\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eDCBA\u003c/em\u003e operon was subsequently integrated into the chromosome of W-3 under the control of the strong P\u003csub\u003etrc\u003c/sub\u003e promoter, yielding strain W-4, which achieved an L-tryptophan titer of 4.29 g/L in shake-flask fermentation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Collectively, these results suggest that relieving feedback inhibition of key enzymes and reinforcing their expression can enhance carbon flux toward the L-tryptophan biosynthetic branch, thereby improving production capacity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Enhancing PEP and E4P supply for L-tryptophan biosynthesis\u003c/h2\u003e \u003cp\u003eAfter alleviating feedback inhibition at several key enzymatic steps in the L-tryptophan biosynthetic pathway, further enhancement of precursor availability became a prerequisite for increasing product titer. PEP from glycolysis and E4P from the pentose phosphate pathway are the two essential precursors for L-tryptophan biosynthesis, and their intracellular availability is a key factor influencing L-tryptophan biosynthesis and final titer [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, both PEP and E4P are distributed among multiple competing pathways, and inefficient channeling into the shikimate pathway limits L-tryptophan biosynthesis.\u003c/p\u003e \u003cp\u003eBuilding upon the aforementioned engineering strategies, the precursor supply toward the shikimate pathway was further reinforced to enhance L-tryptophan biosynthesis through three complementary approaches: PEP regeneration, reduction of carbon loss, and enhancement of E4P supply (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Intracellular PEP availability was first increased by overexpressing \u003cem\u003epps\u003c/em\u003e, which promoted the conversion of PYR back to PEP. This modification is expected to increase the metabolic capacity for PEP regeneration and thereby support aromatic amino acid biosynthesis. As a result, strain W-5 exhibited a 26.34% increase in L-tryptophan titer compared with W-4. In addition, to reduce the consumption of PEP by non-target metabolic pathways, the biosynthetic routes leading to organic acid byproducts, including lactate, acetate, and formate, were deleted to reduce carbon flux toward competing branches. Specifically, \u003cem\u003eldhA\u003c/em\u003e, \u003cem\u003epoxB\u003c/em\u003e, and \u003cem\u003epflB\u003c/em\u003e were deleted, generating strain W-6. This strain produced 5.74 g/L L-tryptophan with a yield of 9.11%. These results suggest that suppressing overflow metabolism may help preserve carbon flux toward the shikimate pathway and support increased L-tryptophan biosynthesis. To balance E4P availability with the expanded PEP pool, \u003cem\u003ezwf\u003c/em\u003e and \u003cem\u003etktA\u003c/em\u003e were overexpressed under the control of the P\u003csub\u003etrc\u003c/sub\u003e promoter, thereby potentially increasing carbon flux through the pentose phosphate pathway. As a result, strain W-8 achieved an L-tryptophan titer of 7.08 g/L in shake-flask fermentation, representing a 65.03% increase relative to W-4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), while the yield increased from 8.57% to 9.61%. These results show that coordinated, multi-level engineering strategies aimed at improving PEP and E4P availability effectively enhanced L-tryptophan biosynthesis and significantly improved production performance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Reprogramming central carbon metabolism to reduce carbon loss\u003c/h2\u003e \u003cp\u003eThe citric acid (TCA) cycle serves as the central hub of cellular energy metabolism and provides essential energy to support cell growth [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. However, excessive TCA cycle activity is frequently accompanied by the accumulation of organic acids, such as citrate, succinate, and fumarate [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], which reduces intracellular pyruvate availability [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], and consequently limit L-tryptophan biosynthesis. To regulate TCA flux and mitigate carbon loss, \u003cem\u003egltA\u003c/em\u003e, which catalyzes the entry step of carbon into the TCA cycle, was dynamically regulated. In shake-flask fermentation, strain W-9 produced 6.17 g/L L-tryptophan when \u003cem\u003egltA\u003c/em\u003e was expressed under the self-regulating promoter P\u003csub\u003efliC\u003c/sub\u003e. Compared with strain W-8, biomass accumulation was reduced, and the L-tryptophan titer decreased by 12.85%; however, the glucose-to-L-tryptophan conversion yield increased to 15.27% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). This result suggests that targeted intervention in the TCA cycle can improve carbon utilization efficiency toward L-tryptophan biosynthesis. Based on this observation, two alternative engineering routes were designed to optimize central carbon metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRoute I targeted the glyoxylate cycle in strain W-8 as a carbon flux redistribution strategy. As a bypass of the TCA cycle, the glyoxylate pathway avoids decarboxylation reactions, thereby preserving carbon skeletons, reducing carbon loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), and improving carbon recovery efficiency, which facilitates the redirection of carbon flux toward biosynthetic pathways [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In \u003cem\u003eE. coli\u003c/em\u003e, the transcriptional regulator IclR represses the glyoxylate cycle, and the \u003cem\u003eaceBAK\u003c/em\u003e operon encoding key enzymes of this pathway is normally activated only in the presence of acetate. Deletion of \u003cem\u003eiclR\u003c/em\u003e in strain W-10 likely enhanced flux through the glyoxylate pathway and increased the L-tryptophan titer to 7.39 g/L in shake-flask fermentation. Consistent with previous studies, deletion of \u003cem\u003eiclR\u003c/em\u003e has been shown to reduce excessive carbon flux through the TCA cycle [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Building on this result, further enhancement of glyoxylate cycle flux was achieved by overexpressing \u003cem\u003eaceAB\u003c/em\u003e and deleting \u003cem\u003eycdW\u003c/em\u003e, which is involved in glyoxylate degradation. The resulting strain W-11 achieved an L-tryptophan titer of 7.88 g/L and a yield of 12.46% in shake-flask fermentation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), suggesting that activation and reinforcement of the glyoxylate cycle help reduce carbon loss and optimize carbon distribution for L-tryptophan biosynthesis.\u003c/p\u003e \u003cp\u003eHowever, the activation of the glyoxylate cycle primarily alleviates carbon flux loss through restructuring the TCA cycle, without directly enhancing the recovery of precursors. Therefore, Route II was designed to improve carbon recovery efficiency by constructing a PYR-OAA-PEP recycling pathway. Specifically, the mutated \u003cem\u003epyc\u003c/em\u003e\u003csup\u003e\u003cem\u003eP458S\u003c/em\u003e\u003c/sup\u003e gene from \u003cem\u003eCorynebacterium glutamicum\u003c/em\u003e was integrated into strain W-8, enabling partial conversion of PYR to OAA while bypassing the TCA cycle. The gene \u003cem\u003epck\u003c/em\u003e was then overexpressed to promote the conversion of OAA to PEP, thereby establishing the PYR\u0026ndash;OAA\u0026ndash;PEP cycle. By comparison, strain W-13 constructed using this strategy achieved an L-tryptophan titer of 7.36 g/L with a yield of 10.62% in shake-flask fermentation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Although lower than that obtained via glyoxylate cycle enhancement strategy, these results indicate that the construction of the PYR-OAA-PEP cycle also improves carbon utilization efficiency.\u003c/p\u003e \u003cp\u003eConsidering that the enhancement of the glyoxylate cycle might lead to an increased accumulation of OAA, \u003cem\u003epck\u003c/em\u003e was additionally integrated into strain W-11. The resulting strain W-14 achieved an L-tryptophan titer of 8.65 g/L and a yield of 13.07% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Analysis of TCA-related metabolites showed that, compared with strain W-8, glyoxylate accumulation increased by 28.93%, whereas citrate, succinate, malate, oxaloacetate, and pyruvate levels decreased by 49.01%, 25.44%, 45.09%, 47.58%, and 37.93%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results indicate that coordinated regulation of the glyoxylate cycle and OAA-PEP recycling effectively alleviates carbon drainage through the TCA cycle and enhances carbon channeling toward L-tryptophan biosynthesis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Coordinated supply of donor molecules for L-tryptophan biosynthesis\u003c/h2\u003e \u003cp\u003eL-tryptophan biosynthesis is a typical multi-precursor-coupled process in terms of both the synthetic pathway and molecular structure. Specifically, PEP and E4P jointly form the aromatic ring backbone, while L-glutamine provides the amide group for indole ring formation. The carbon skeleton of the indole ring is derived from PRPP, and L-serine contributes the side chain of L-tryptophan (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Therefore, in addition to PEP and E4P, the intracellular availability of donor molecules, including L-glutamine, L-serine, and PRPP, is also critical for efficient L-tryptophan biosynthesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn \u003cem\u003eE. coli\u003c/em\u003e, L-glutamine is synthesized from L-glutamate by glutamine synthetase (GlnA), which is subject to feedback inhibition by NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e. In contrast, glutamine synthetase from \u003cem\u003eBacillus subtilis\u003c/em\u003e is resistant to this inhibition [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Accordingly, the feedback-resistant \u003cem\u003eglnA\u003c/em\u003e\u003csup\u003e\u003cem\u003eL159I,E304A\u003c/em\u003e\u003c/sup\u003e from \u003cem\u003eB. subtilis\u003c/em\u003e was overexpressed in strain W-15 to enhance the biosynthetic capacity for L-glutamine, resulting in a 4.16% increase in L-tryptophan titer compared with W-14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In addition, L-serine, which provides the side chain of L-tryptophan, is synthesized via a pathway whose rate-limiting enzyme \u003cem\u003eSerA\u003c/em\u003e is also subject to feedback inhibition. Previous studies reported that introducing the feedback-resistant \u003cem\u003eserA\u003c/em\u003e\u003csup\u003e\u003cem\u003efbr\u003c/em\u003e\u003c/sup\u003e significantly increased intracellular serine levels by 18-fold [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In this study, the feedback-resistant \u003cem\u003eserA\u003c/em\u003e\u003csup\u003e\u003cem\u003eH344A,N346A,N364A\u003c/em\u003e\u003c/sup\u003e variant was introduced into strain W-14. In addition, 0.5 g/L L-isoleucine was supplemented to alleviate the metabolic burden associated with L-serine accumulation, as previous studies have shown that L-isoleucine supplementation can mitigate growth inhibition caused by serine overaccumulation [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The resulting strain W-16 exhibited a 2.08% increase in L-tryptophan titer relative to W-14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eOn the other hand, PRPP is synthesized from ribose-5-phosphate in the pentose phosphate pathway by phosphoribosylpyrophosphate synthase (Prs), whose expression is negatively regulated by PurR [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. To enhance PRPP supply, \u003cem\u003epurR\u003c/em\u003e was deleted in strain W-17, followed by heterologous expression of \u003cem\u003eprs\u003c/em\u003e genes from \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eB. subtilis\u003c/em\u003e, \u003cem\u003eC. glutamicum\u003c/em\u003e, and \u003cem\u003eSerratia marcescens\u003c/em\u003e using the pTrc99A plasmid, generating strains W-17\u0026thinsp;+\u0026thinsp;Ec, W-17\u0026thinsp;+\u0026thinsp;Bs, W-17\u0026thinsp;+\u0026thinsp;Cg, and W-17\u0026thinsp;+\u0026thinsp;Sm, respectively. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, strain W-17\u0026thinsp;+\u0026thinsp;Sm exhibited a 11.16% increase in L-tryptophan titer compared to the control, suggesting that the \u003cem\u003eprs\u003c/em\u003e gene from \u003cem\u003eS. marcescens\u003c/em\u003e improves PRPP biosynthetic capacity and supports L-tryptophan production.\u003c/p\u003e \u003cp\u003eWe also observed that the high-level expression of \u003cem\u003eprs\u003c/em\u003e from different sources resulted in varying degrees of growth inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Biomass accumulation in strains W-17\u0026thinsp;+\u0026thinsp;Ec, W-17\u0026thinsp;+\u0026thinsp;Bs, and W-17\u0026thinsp;+\u0026thinsp;Cg decreased by 5.25%, 6.41%, and 2.34%, respectively, whereas W-17\u0026thinsp;+\u0026thinsp;Sm exhibited the most significant reduction (10.44%). Despite this growth inhibition, W-17\u0026thinsp;+\u0026thinsp;Sm achieved the highest L-tryptophan accumulation (9.46 g/L), indicating that increased PRPP biosynthetic capacity may favor carbon allocation toward L-tryptophan production at the expense of biomass formation. From an intracellular metabolic pathway perspective, the \u003cem\u003eprs\u003c/em\u003e-catalyzed reaction utilizes ribose-5-phosphate and ATP as substrates. Overexpression of \u003cem\u003eprs\u003c/em\u003e likely increases competition for ribose-5-phosphate and ATP in the pentose phosphate pathway, which may disrupts intracellular carbon and energy allocation and impairs cell growth [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo assess the balance of donor molecules required for L-tryptophan biosynthesis in strain W-17, expression cassettes for L-glutamine, L-serine, and PRPP were constructed using constitutive promoters of different strengths. Specifically, three promoters\u0026mdash;P\u003csub\u003eJ23101\u003c/sub\u003e (strong), P\u003csub\u003eJ23105\u003c/sub\u003e (medium), and P\u003csub\u003eJ23114\u003c/sub\u003e (weak)\u0026mdash;were used to drive \u003cem\u003eglnA\u003c/em\u003e\u003csup\u003e\u003cem\u003eL159I,E304A\u003c/em\u003e\u003c/sup\u003e (\u003cem\u003eB. subtilis\u003c/em\u003e), \u003cem\u003eserA\u003c/em\u003e\u003csup\u003e\u003cem\u003eH344A,N346A,N364A\u003c/em\u003e\u003c/sup\u003e (\u003cem\u003eE. coli\u003c/em\u003e), and \u003cem\u003eprs\u003c/em\u003e (\u003cem\u003eS. marcescens\u003c/em\u003e), generating a total of 27 engineered strains (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, screening in 24-well plates identified the strain harboring the M\u0026thinsp;+\u0026thinsp;H+M promoter combination (P\u003csub\u003eJ23105\u003c/sub\u003e \u003cem\u003eBsglnA\u003c/em\u003e\u003csup\u003e\u003cem\u003eL159I,E304A\u003c/em\u003e\u003c/sup\u003e+P\u003csub\u003eJ23101\u003c/sub\u003e \u003cem\u003eEcserA\u003c/em\u003e\u003csup\u003e\u003cem\u003eH344A,N346A,N364A\u003c/em\u003e\u003c/sup\u003e+P\u003csub\u003eJ23105\u003c/sub\u003e \u003cem\u003eSmprs\u003c/em\u003e) as the best performer, achieving an L-tryptophan titer of 1.59 g/L and a yield of 3.85%. This strain was designated W-22. Shake-flask fermentation further confirmed that W-22 achieved an L-tryptophan titer of 10.58 g/L, representing a 21.61% increase relative to strain W-17, with the yield increasing to 15.13%. These results show that efficient L-tryptophan biosynthesis not only relies on the sufficient supply of key precursors such as PEP and E4P, but also significantly depends on the optimal balance between other donor molecules. Systematic optimization of L-glutamine, L-serine, and PRPP supply modules enabled fine-tuning of precursor metabolism and biosynthetic flux, thereby further improving both L-tryptophan titer and yield.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Engineering transport systems to promote L-tryptophan export\u003c/h2\u003e \u003cp\u003ePrevious studies have shown that enhancing intracellular overproduction of metabolites through systematic metabolic engineering often leads to negative effects such as intracellular osmotic pressure imbalances and growth inhibition [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], thereby reducing the sustained production capacity of engineered strains. By engineering transport channels to alleviate endogenous pressure, further improvement in product titer can be achieved [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In \u003cem\u003eE. coli\u003c/em\u003e, multiple transport proteins cooperate in the permeation of aromatic amino acids. Among them, L-tryptophan transport is subject to complex regulation [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Three transporters\u0026mdash;AroP, Mtr, and TnaB\u0026mdash;play distinct roles in intracellular L-tryptophan accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Specifically, TnaB functions as a low-affinity transporter, Mtr exhibits high affinity for L-tryptophan, and AroP mediates the transport of multiple aromatic amino acids. Genomic analysis revealed that the starting strain W-1 lacked \u003cem\u003etnaB\u003c/em\u003e, which encodes a low-affinity L-tryptophan transporter. To evaluate the role of TnaB in high-titer tryptophan strains, the \u003cem\u003etnaB\u003c/em\u003e expression cassette was reintroduced into strain W-22 at the \u003cem\u003eilvG\u003c/em\u003e locus via reverse metabolic engineering. Restoration of \u003cem\u003etnaB\u003c/em\u003e expression resulted in a 7.09% decrease in the L-tryptophan titer, suggesting that enhanced L-tryptophan uptake may lead to elevated intracellular concentrations, which could impose additional physiological stress and negatively affect L-tryptophan biosynthesis. On the other hand, overexpression of the L-tryptophan efflux protein YddG in strain W-22 resulted in the creation of strain W-24, which achieved an L-tryptophan titer of 11.76 g/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). These results suggest that engineering L-tryptophan transport pathways to promote product efflux can support higher L-tryptophan titers, likely by reducing intracellular accumulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Optimization of fed-batch fermentation in a 5 L bioreactor\u003c/h2\u003e \u003cp\u003eDuring scale-up production of engineered strains, inoculum size is considered an important parameter affecting the adaptability, growth kinetics, and product synthesis of engineered strains [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. To investigate the effect of inoculum size on the fermentation performance of strain W-24 under glucose-limited fed-batch conditions, fermentations were conducted in a 5 L bioreactor with controlled dissolved oxygen (DO) and pH. Fresh seed cultures were inoculated at 300 mL (15%), 350 mL (17.5%), 400 mL (20%), and 450 mL (22.5%), while maintaining an initial fermentation working volume of 2.0 L.\u003c/p\u003e \u003cp\u003eAt an inoculum size of 15%, strain W-24 exhibited slow growth recovery and a prolonged lag phase. Both the specific growth rate and L-tryptophan synthesis rate throughout the fermentation cycle were lower than those observed at higher inoculum sizes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD), resulting in lower final biomass (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;68.2) and L-tryptophan titer (43.65 g/L) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). In contrast, at higher inoculum sizes (20% and 22.5%), the strain rapidly entered the exponential growth phase and maintained higher specific growth rates during the early fermentation stage. Consequently, peak biomass levels of OD\u003csub\u003e600\u003c/sub\u003e 78.0 and 81.4 were achieved, corresponding to increases of 10.37% and 19.64%, respectively, relative to the 17.5% inoculum group. However, as fermentation proceeded, the specific growth rates of the 20% and 22.5% inoculum groups gradually declined and became negative, indicating growth cessation and possible decline in cell viability at later stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Moreover, the rapid early growth at high inoculum sizes may have accelerated nutrient consumption and increased metabolic burden during the early fermentation stage, thereby affecting the later-stage production capacity. Correspondingly, after 24 hours of fermentation, the L-tryptophan synthesis rate significantly decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD), and the final titer (46.08 g/L, 45.16 g/L, respectively) was lower than that of the 17.5% inoculum group (50.83 g/L) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConsidering both cell growth and product generation data, W-24 exhibited optimal overall fermentation performance at an inoculum size of 17.5%. During the 40-hour fed-batch fermentation, the maximum biomass reached 74.9, and at the end of fermentation, the L-tryptophan titer was 50.83 g/L, with a yield of 18.5% (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The acetic acid concentration at the end of fermentation was 2.30 g/L (Fig. S3). These results show that an appropriate inoculum size facilitates a balanced coordination between cell growth and product biosynthesis, thereby enabling improved fermentation performance of the engineered strain under scaled-up conditions.\u003c/p\u003e \u003cp\u003eOverall, these results indicate that the coordinated supplementation and balancing of multiple intracellular precursors is an effective strategy to enhance L-tryptophan biosynthesis. However, the production performance of the engineered strain remains below the theoretical level [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], indicating that further reduction of carbon flux loss and improvement of precursor utilization efficiency are still required. Future efforts focusing on uncovering potential regulatory mechanisms\u0026mdash;such as the identification of novel transcriptional regulators\u0026mdash;as well as on the development of more refined metabolic regulation models may provide additional opportunities to further improve amino acid production.\u003c/p\u003e \u003c/div\u003e"},{"header":"4.CONCLUSION","content":"\u003cp\u003eThis work describes the construction of an efficient microbial cell factory for L-tryptophan production, starting from a model strain and combining ARTP mutagenesis with systematic metabolic engineering strategies. The main strategies employed include: (i) development of an L-tryptophan-specific biosensor combined with ARTP mutagenesis to obtain an L-tryptophan-producing chassis strain; (ii) reorganization of the central carbon metabolism pathway to enhance the carbon flux of PEP and E4P while reducing carbon loss; (iii) optimization of the supplementation and balance of multiple L-tryptophan donor molecules through promoter engineering; (iv) engineering of L-tryptophan transport pathways; and (v) optimization of inoculum size during fermentation. Ultimately, the engineered strain W-24, without inducers or antibiotics, achieved an L-tryptophan titer of 50.83 g/L in a 5 L bioreactor, with a yield of 0.185 g/g.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Key Research and Development Program of China (2024YFA0918000), the Frontier Technology Research and Development Plan of Jiangsu Province (BF2024012), the National Natural Science Foundation of China (32471530), the Major Scientific and Technological Project for \u0026ldquo;unveiling and commanding\u0026rdquo; of Hohhot (2023-unveiling and commanding-He-1), the Independent Research Project of the State Key Laboratory of Food Science and Resources of Jiangnan University (SKLF-ZZB-202408), the Project for \u0026ldquo;unveiling and commanding\u0026rdquo; of Urumqi (B241011002), the Postgraduate Research \u0026amp; Practice Innovation Program of Jiangsu Province (KYCX24_2594), the National Natural Science Foundation of China (22508213) and the Shandong Postdoctoral Science Foundation (SDCX-ZG-202502064).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e#R.Z. and Z.Z. contributed equally. R.Z. conceived the study, designed the experiments, analyzed the data, and wrote and revised the manuscript. Z.Z. conducted background investigation and assisted in experimental design. W.S. performed the experiments. J.H. and J.Y. analyzed the data. X.P. and M.S. provided conceptual guidance and supervised the research. All authors have given approval to the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e \u003cp\u003eAll data involved in this study are included in this published article and its additional files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNiu H, Li R, Liang Q, Qi Q, Li Q, Gu P. Metabolic engineering for improving L-tryptophan production in \u003cem\u003eEscherichia coli\u003c/em\u003e. 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Metab Eng Commun. 2021;12:e00167.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Biosensor, L-Tryptophan, Escherichia coli, Precursor balance, Multi-module engineering, Fermentation optimization","lastPublishedDoi":"10.21203/rs.3.rs-9241029/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9241029/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eL-Tryptophan is an important aromatic amino acid with wide applications across the food, pharmaceutical, and feed industries. However, its efficient microbial production remains challenging due to complex metabolic networks and multi-level feedback regulation. In this study, we constructed a highly efficient \u003cem\u003eEscherichia coli\u003c/em\u003e cell factory for L-tryptophan biosynthesis by combining systematic metabolic engineering with high-throughput screening. Initially, a \u003cem\u003etnaC\u003c/em\u003e-based biosensor was developed and coupled with atmospheric and room temperature plasma (ARTP) mutagenesis to isolate high-performance chassis strains. Central carbon metabolism was subsequently reprogrammed to minimize carbon loss and channel metabolic fluxes toward essential precursors, phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P). To further alleviate pathway bottlenecks, promoter engineering was utilized to balance the intracellular supplies of L-glutamine, L-serine, and phosphoribosyl pyrophosphate (PRPP). This targeted intervention yielded a 21.61% increase in L-tryptophan accumulation. Product transport systems were then engineered to enhance extracellular secretion and mitigate intracellular toxicity. Following the optimization of inoculum size and feeding strategies in a 5 L bioreactor, the final engineered strain (W-24) produced 50.83 g/L of L-tryptophan within 40 hours, achieving a yield of 0.185 g/g glucose. This multi-modular engineering framework establishes a robust platform for L-tryptophan biosynthesis and provides a scalable strategy for the industrial production of other valuable aromatic compounds.\u003c/p\u003e","manuscriptTitle":"Biosensor-driven evolution and metabolic engineering of an Escherichia coli","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-01 19:35:29","doi":"10.21203/rs.3.rs-9241029/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-09T10:52:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-09T09:48:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-08T05:49:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-06T00:04:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"67604977217938004626982112754394873080","date":"2026-04-04T02:21:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"320711332765496535824947160759849535963","date":"2026-03-30T08:22:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"326832428273520147932013833936002329497","date":"2026-03-30T00:42:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-29T22:57:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-28T04:24:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-28T04:24:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbial Cell Factories","date":"2026-03-27T06:24:14+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":"bb5c01cd-4da8-4cea-b935-d5f7e3d60b31","owner":[],"postedDate":"April 1st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T05:25:04+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-01 19:35:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9241029","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9241029","identity":"rs-9241029","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}