Systematic Metabolic Engineering Enables 2′-Fucosyllactose Biosynthesis from Glucose as the Sole Carbon Source in Escherichia coli

Systematic Metabolic Engineering Enables 2′-Fucosyllactose Biosynthesis from Glucose as the Sole Carbon Source in Escherichia coli | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Systematic Metabolic Engineering Enables 2′-Fucosyllactose Biosynthesis from Glucose as the Sole Carbon Source in Escherichia coli Wentai Wu, Luyao Wang, Yuxuan Li, Tongle Liu, Jiaren Cao, Sheng Chen, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9278536/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 2′-Fucosyllactose (2′-FL) is the most abundant human milk oligosaccharide (HMO), playing vital roles in promoting infant gut health, enhancing immunity, and defending against pathogens. However, conventional 2′-FL biosynthesis typically relies on exogenous supplementation of lactose or fucose precursors, leading to high costs and process complexity. In this study, we report a streamlined de novo biosynthesis process for 2′-FL using glucose as the sole carbon source, achieved through systematic engineering of endogenous precursor pathways in Escherichia coli . First, the complete biosynthetic route from glucose to lactose and then to 2′-FL was reconstructed in a chassis strain capable of producing fucose. Unlike previous studies that primarily focused on the GDP-fucose supply module, this work systematically balanced the metabolic flux between phosphorylated and non-phosphorylated glucose pools to coordinate the supply of precursors for both lactose and GDP-fucose synthesis. This balance was achieved by reconstructing the glucose uptake system, modulating the expression of key enzymes at critical metabolic nodes, and eliminating competing pathways. Subsequently, the endogenous lactose synthesis pathway was further enhanced through coordinated overexpression of pgm , galU , and galE , and the pentose phosphate pathway and purine salvage pathway were optimized to enhance the supply of NADPH and GTP. Using this strategy, an engineered E. coli strain for efficient 2′-FL production was successfully constructed. The engineered strain produced 7.11 g/L 2′-FL in shake-flask fermentation and achieved a titer of 43.2 g/L in a 3-L bioreactor after 58 h, representing the highest reported yield for de novo 2′-FL synthesis from glucose as the sole carbon source. This work provides a promising and simplified strategy for the cost-effective industrial production of 2′-FL. 2′-fucosyllactose sole carbon source de novo synthesis metabolic engineering fermentation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Human milk oligosaccharides (HMOs) are the third-largest solid component in human milk, exhibiting significant prebiotic activity while also modulating the infant immune system, helping infants resist pathogen colonization, and promoting neurodevelopment in infants [ 1 – 4 ]. Among various HMOs, 2′-fucosyllactose (2′-FL) is the most abundant fucosylated oligosaccharide and has been approved as a nutritional additive in infant formula by regulatory bodies including China’s National Health Commission, the U.S. FDA, and the European Union [ 5 – 8 ]. Consequently, its efficient biomanufacturing has become a research hotspot at the intersection of synthetic biology and food science. Currently, three primary routes exist for 2′-FL synthesis: chemical [ 9 ], enzymatic [ 10 ], and microbial fermentation [ 11 – 13 ]. Chemical synthesis involves complex steps and potential safety concerns. The enzymatic method relies on expensive GDP-L-fucose and lactose precursors, limiting its economic viability for scale-up. Microbial fermentation offers advantages such as short cycles, low cost, and readily available feedstocks, making it the most promising strategy [ 13 – 15 ]. In traditional microbial fermentation for 2′-FL, two main technical routes prevail based on the supply mode of the key precursor GDP-L-fucose, both heavily dependent on exogenous lactose addition [ 16 – 18 ]. The first is the salvage pathway, in which engineered bacteria expressing fucosyltransferase catalyze the reaction using exogenously supplied lactose and GDP-L-fucose (or its direct precursor, L-fucose) [ 19 – 22 ]. Although straightforward, this route is hampered by the high cost of both substrates. The more common strategy is the de novo pathway, which involves systematic metabolic engineering to construct an endogenous GDP-L-fucose supply module from cheap carbon sources such as glycerol or glucose, achieved by introducing or enhancing enzymes including mannose mutase and GDP-L-mannose dehydratase, and by expressing efficient fucosyltransferases [ 23 – 25 ]. In this system, exogenously added lactose serves as the essential fucosyl acceptor, accepting the fucosyl group transferred from endogenously synthesized GDP-L-fucose to generate 2′-FL. Although this de novo strategy reduces the cost of the fucose moiety, the obligate dependence on exogenous lactose—coupled with challenges in uptake efficiency, metabolic flux diversion, and additive cost—remains a critical bottleneck that restricts the scalability and overall efficiency of 2′-FL production. To overcome this bottleneck, recent research has focused on developing de novo biosynthetic routes independent of exogenous lactose [ 26 ]. For instance, Mu’s team engineered E. coli BL21(DE3) for 2′-FL production using co-cultivation with glucose and xylose, yielding 6.53 g/L and 27.53 g/L of 2′-FL in shake-flask and 3-L fed-batch bioreactor fermentations, respectively [ 27 ]. Liu’s team successfully developed a Bacillus subtilis platform for 2′-FL production from glucose as the sole carbon source [ 28 ]. Through construction of a lactose biosynthesis module, introduction of a non-phosphotransferase system, and development of a lactose-responsive biosensor for dynamic metabolic regulation, they achieved a 2′-FL titer of 30.1 g/L in a 3-L bioreactor, providing an important technical reference for exogenous lactose-independent de novo 2′-FL biosynthesis. Building upon this foundation, the present study employed low-cost glucose as the sole carbon source for 2′-FL production. Unlike previous approaches that primarily focused on engineering the GDP-L-fucose supply module, this work systematically balanced the metabolic flux between phosphorylated and non-phosphorylated glucose pools to coordinate the supply of precursors for both lactose and GDP-L-fucose synthesis. This balance was achieved by reconstructing the glucose uptake system—specifically, by disrupting the phosphotransferase system (PTS) and introducing a glucose facilitator to provide non-phosphorylated glucose, while modulating glucokinase expression to maintain an adequate pool of glucose-6-phosphate. Additionally, agp was knocked out to eliminate the futile cycling of glucose-1-phosphate back to glucose, further preserving phosphorylated precursors. Subsequently, the carbon flux was further optimized by eliminating other competing pathways (e.g., gcd , udg ) and modulating key node enzymes such as phosphoglucose isomerase to redirect carbon from glycolysis toward the pentose phosphate pathway, thereby enhancing NADPH and ribose-5-phosphate supply. In parallel, the endogenous lactose synthesis pathway was strengthened through coordinated overexpression of pgm , galU , and galE , along with ppa to alleviate pyrophosphate inhibition, while the purine salvage pathway was reinforced via gsk overexpression to boost GTP availability for GDP-L-fucose synthesis. Through this multi-module engineering strategy, a 2′-FL titer of 7.11 g/L was achieved in shake-flask cultivation, which was further scaled to 43.2 g/L in a 3-L bioreactor system. This titer exceeds the previously reported highest levels for de novo 2′-FL synthesis from glucose as the sole carbon source. To our knowledge, this work represents the first demonstration of high-efficiency 2′-FL biosynthesis using glucose as the exclusive carbon source in E. coli , achieving the highest reported yield for exogenous lactose-independent systems. These results validate the feasibility of a fully endogenous 2′-FL synthesis route and offer a scalable technological platform for cost-effective industrial production. Materials and Methods Recombinant plasmid construction and related experimental materials . Table S1 details the overexpression plasmids used in this study. The recombinant plasmids were constructed using E. coli JM109 as the cloning host. Three plasmid vectors (pETDuet-1, pRSFDuet-1, and pCDFDuet-1) with distinct expression strengths were selected to drive the expression of key genes in the 2′-FL biosynthetic pathway. The nucleotide sequences of GalTpm1141 (AEC04686) and glf (AEC04687) were codon-optimized for E. coli and synthesized by GENEWIZ (Suzhou, China). Genes related to the metabolic pathway—including pgm , galU , galE , zwf , glk , pgi , gsk , and ppa —were amplified from the genome of E. coli K12 MG1655. Specifically, BKHT was cloned into the pETDuet-1 vector using the Clone Expression II one-step cloning technique (primer information is provided in Table S2) [ 29 ]. All target genes were inserted into plasmids with different expression levels via the MEGAWHOP method to construct the recombinant plasmids. These constructs were subsequently verified by Sanger sequencing performed by GENEWIZ Biotechnology Co., Ltd. (Suzhou, China). All reagents employed in this study were sourced from Vazyme Biotechnology Co., Ltd. (Nanjing, China). Construction of engineered strains . A dual-plasmid gene editing system based on CRISPR/Cas9 was employed for homologous target gene knockout, using pTargetF (Addgene, #62226) and pCas9 (Addgene, #62225) as tools. The pTargetF plasmid contains an sgRNA sequence paired with a genomic homologous fragment. The N20-specific sequence of the gene to be knocked out was identified using CHOPCHOP ( http://chopchop.cbu.uib.no/ ), obtained via PCR amplification, and then integrated into the sgRNA region at the 5′ end of the pTargetF plasmid. The pCas9 plasmid encodes λ-Red and Cas9 proteins. The Cas9-mediated cleavage of the target site, combined with arabinose-induced (50 mM) λ-Red recombinase activity, enabled efficient homologous recombination. The verified knockout plasmids were introduced into E. coli BL21(DE3) BL-5 via heat shock transformation. Colony PCR validation confirmed successful modification, after which a series of derivative strains were constructed (for full genotype details, see Table S1 ). Using this approach, targeted deletions were accomplished for key metabolic genes including lacZ , lacA , ugd , gcd , ptsG , glk , and agp . Culture conditions of recombinant strains in shake flask. This study employed Luria-Bertani (LB) medium for seed culture and a glucose-defined medium (GDM) for shake-flask fermentation. The LB medium contained (per liter): 5.00 g yeast extract, 10.00 g tryptone, and 10.00 g NaCl. The GDM consisted of (per liter): 10.00 g glucose, 1.70 g citric acid monohydrate, 5.00 g yeast extract, 4.00 g (NH₄)₂HPO₄, 13.50 g KH₂PO₄, 1.40 g MgSO₄·7H₂O, 0.50 g tryptone, and 10 mL of a trace element solution. The medium pH was adjusted to 7.2 using ammonia. All media were supplemented with antibiotics at final concentrations of ampicillin (100 µg/mL), kanamycin (30 µg/mL), and spectinomycin (50 µg/mL) [ 30 ]. The recombinant strain was first inoculated into LB medium and incubated at 37°C with shaking at 200 rpm for 10–12 h. Subsequently, 0.5 mL of this seed culture was transferred into a 250 mL shake flask containing 50 mL of GDM. When the optical density at 600 nm (OD₆₀₀) reached 2.0, protein expression was induced by adding 0.05 mM isopropyl β -D-1-thiogalactopyranoside (IPTG). 3-L Bioreactor Fermentation of Recombinant Strains . The initial medium for the 3-L bioreactor fermentation contained the following components (per liter): 30.00 g glucose, 4.00 g (NH₄)₂SO₄, 6.00 g tryptone, 9.20 g K₂HPO₄, 8.20 g KH₂PO₄, 0.30 g citric acid monohydrate, 0.02 g CaCl₂, 2.00 g yeast extract, and 10 mL of the trace element solution (composition as described above). The pH was adjusted to 7.2 with ammonium hydroxide. The feed medium contained 600.00 g/L glucose, 2.50 g/L tryptone, 22.20 g/L MgSO₄·7H₂O, and 5.00 g/L yeast extract. All media were supplemented with antibiotics at final concentrations of ampicillin (100 µg/mL), kanamycin (30 µg/mL), and spectinomycin (50 µg/mL). The recombinant strain was first inoculated into 10 mL of LB medium as a primary seed culture and incubated at 37°C with shaking at 200 rpm for 10–12 h. Then, 0.1 mL of this culture was aseptically transferred into 50 mL of fresh LB medium in a 250 mL shake flask (secondary seed culture) and grown under the same conditions for another 10–12 h. Subsequently, 100 mL of the secondary culture (10% v/v inoculum) was used to inoculate 1 L of fermentation medium in a 3-L bioreactor (Applikon). During the initial fermentation phase, the key operating parameters were controlled as follows: temperature was maintained at 37°C using a thermostatic system, dissolved oxygen (DO) was kept at 30%, and pH was automatically maintained at 6.8 via an alkali feeding system. When the residual glucose concentration fell below 5 g/L, the feed medium was supplied to maintain glucose levels between 5 and 10 g/L. Once the OD₆₀₀ reached 30, the temperature was shifted from 37°C to 25°C, and protein expression was induced by adding 0.1 mM isopropyl β -D-1-thiogalactopyranoside (IPTG). Analytical Methods. Bacterial growth was monitored by measuring the optical density at 600 nm (OD 600 ) using a UV-1800 spectrophotometer (MeiXi, Shanghai, China). For product analysis, samples were prepared following a standardized three-step protocol: heat inactivation, centrifugation, and filtration. First, fermentation broth was heat-inactivated in a 100°C water bath for 15 min. Subsequently, the samples were centrifuged at 12,000 rpm for 15 min to separate the solid and liquid phases. The clarified supernatant was then filtered through a 0.22 µm membrane, aliquoted, and stored at − 80°C for subsequent analysis. The concentrations of 2′-FL, lactose, and glucose were quantified using a Waters Alliance iS high-performance liquid chromatography (HPLC) system (Waters, Milford, MA, USA) equipped with a refractive index (RI) detector. Chromatographic separation was performed on an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA). The column temperature and detector temperature were maintained at 60°C and 50°C, respectively. The mobile phase was 5 mM sulfuric acid, applied under isocratic elution at a flow rate of 0.5 mL/min. Results and Discussion Construction of an Engineered Chassis Strain for 2′-FL Synthesis Based on a Lactose Self-Synthesis System . Achieving de novo biosynthesis of 2′-FL from glucose as the sole carbon source requires the construction of a complete biosynthetic pathway in E. coli , including a GDP-L-fucose supply module, a lactose self-synthesis module, and a 2′-FL synthesis module. In this study, an E. coli chassis strain (BE0) previously constructed in our laboratory with the ability to synthesize GDP-L-fucose was used as the starting strain, into which exogenous glycosyltransferases were introduced to establish the complete pathway [ 31 ]. The gene encoding α -1,2-fucosyltransferase (BKHT) from Helicobacter pylori was cloned into pETDuet-1, and the gene encoding β -1,4-galactosyltransferase ( GalTpm1141 ) from Neisseria meningitidis was cloned into pRSFDuet-1. Co-transformation of these two recombinant plasmids into the chassis strain yielded the initial strain BE1. Theoretically, this strain could utilize glucose as the sole carbon source to autonomously synthesize lactose and further produce 2′-FL. Shake-flask fermentation results showed that after 72 h of cultivation with glucose as the sole carbon source, BE1 produced only 0.04 g/L of 2′-FL, with 7.4 g/L of glucose remaining, and no lactose accumulation was detected. These results indicated that despite the introduction of the core enzymatic machinery, the intracellular lactose synthesis capacity was severely insufficient to provide an adequate acceptor for 2′-FL synthesis, while glucose uptake and utilization efficiency were low, and carbon flux was not effectively channeled toward the target pathway. Thus, lactose self-synthesis and glucose transport/distribution constituted the main bottlenecks limiting 2′-FL production. Reconstruction and Enhancement of the Glucose Uptake Pathway. In E. coli , glucose uptake is primarily mediated by the phosphotransferase system (PTS), in which the EIICB^Glc enzyme encoded by ptsG simultaneously transports and phosphorylates glucose to glucose-6-phosphate (G-6-P) upon entry, while repressing the expression of non-PTS genes [ 32 ]. When lactose synthesis requires non-phosphorylated glucose as a precursor, this coupled transport-phosphorylation mechanism becomes a metabolic bottleneck. To address this limitation, we first disrupted the PTS pathway by knocking out ptsG in strain BE1 using CRISPR/Cas9, generating strain BE2. After co-transforming the two recombinant plasmids carried by BE1 (pETDuet-1-BKHT and pRSFDuet-1- galTpm1141 ) into this knockout strain, BE2 produced 0.44 g/L of 2′-FL after 72 h of fermentation, a significant increase from the 0.04 g/L produced by BE1, with residual glucose decreasing to 3.1 g/L. These observations indicated that disrupting the dominant PTS pathway partially relieved the bottleneck by redirecting carbon flux toward the non-PTS system, which supplies non-phosphorylated glucose—a key precursor for lactose synthesis. However, the absence of detectable lactose suggested that the endogenous non-phosphorylated glucose supply remained insufficient to support efficient lactose biosynthesis. To further enhance the supply of non-phosphorylated glucose, we introduced the glf gene from Zymomonas mobilis , which encodes a facilitator that mediates passive glucose transport along its concentration gradient without ATP consumption or concomitant phosphorylation. The glf gene was cloned into the low-copy-number plasmid pCDFDuet, yielding pCDFDuet- glf . Additionally, because G-6-P serves as a key precursor for both lactose and GDP-L-fucose, we also aimed to increase the phosphorylated glucose pool within the NPTS framework by cloning glk (encoding glucokinase) into pETDuet and pCDFDuet vectors. Various combinations of these plasmids were transformed into BE2, generating strains BE2-1 through BE2-4. Given that plasmid copy number can affect gene expression levels and metabolic burden, we tested combinations with different expression strengths.Among these, BE2-3 (harboring pETDuet-BKHT, pRSFDuet- GalTpm1141 , and pCDFDuet- glf - glk ) achieved the highest 2′-FL titer of 1.71 g/L after 72 h of shake-flask fermentation (Fig. 1 ). The superior performance of BE2-3 suggested that balanced co-expression of glf and glk from the same low-copy plasmid (pCDFDuet), together with high-copy expression of the fucosyltransferase BKHT (from pETDuet)—which catalyzes the final condensation of GDP-L-fucose and lactose to form 2′-FL—provided an optimal supply of both non-phosphorylated and phosphorylated glucose without imposing excessive metabolic burden. This result demonstrated that optimizing both non-phosphorylated glucose import and its subsequent phosphorylation significantly enhanced lactose production, thereby promoting efficient 2′-FL synthesis. These findings also highlighted that the interplay between glucose uptake, phosphorylation, and downstream lactose synthesis must be finely tuned to maximize product titer, guiding further optimization of the central carbon metabolism in subsequent steps. Reconstruction and Optimization of the Intracellular Phosphorylated Glucose Metabolic Network. As the first key intermediate formed after glucose uptake, D-glucose-6-phosphate (G-6-P) occupies a decisive metabolic branch point. It serves as the primary substrate for glycolysis to generate energy and carbon skeletons, while also being an indispensable precursor for UDP-galactose and GDP-L-fucose synthesis. Therefore, diverting G-6-P from central metabolism toward the 2′-FL biosynthetic pathway is critical for high-yield production. To minimize unnecessary carbon loss from the precursor pool, we first eliminated a non-essential competitive diversion at the glucose-1-phosphate (G-1-P) node. G-1-P, derived from G-6-P via phosphoglucomutase, can be dephosphorylated back to non-phosphorylated glucose by the phosphatase encoded by agp . We knocked out agp in strain BE2 using CRISPR/Cas9, generating strain BE3. This deletion was expected to prevent futile cycling between G-6-P and glucose, thereby preserving phosphorylated glucose intermediates for downstream lactose and GDP-L–fucose synthesis [ 33 ]. After this modification in the engineered E. coli, BE3 was used as the base strain for further optimization of carbon flux distribution. The activity of phosphoglucose isomerase, which interconverts G-6-P and fructose-6-phosphate (F-6-P), acts as a key valve regulating the partition of carbon between glycolysis and the pentose phosphate pathway (PPP). To balance the supply of precursors for both the lactose and GDPL–fucose modules, we overexpressed pgi by cloning it into either pETDuet-BKHT or pCDFDuet- glf - glk vectors. Various combinations of these plasmids were transformed into BE3, generating strains BE3-1 through BE3-6. After 72 h of shake-flask fermentation, strain BE3-2 (harboring pETDuet- pgi -BKHT, pRSFDuet-GalTpm1141, and pCDFDuet- glf - glk ) achieved the highest 2′-FL titer of 3.73 g/L, representing a substantial increase from the 1.71 g/L produced by BE2-3 (Fig. 2 ). The superior performance of BE3-2 suggested that moderate overexpression of pgi from a high-copy plasmid (pETDuet) effectively balanced the carbon flux distribution between glycolysis and the PPP [ 34 ]. This increased the supply of NADPH and ribose-5-phosphate—key precursors for GDP-L-fucose synthesis—while still maintaining sufficient G-6-P flux for lactose production. The marked improvement in 2′-FL titer provided indirect evidence that GDP-L-fucose availability was enhanced. Notably, strains with pgi overexpressed from the low-copy plasmid (pCDFDuet) showed lower titers, which is attributed to insufficient flux balancing or suboptimal expression balance between pgi and other pathway genes. These results demonstrated that enhancing pgi expression served a dual purpose: it balanced the flux between the PPP and glycolysis to drive GDP-L-fucose synthesis, and this balanced redistribution helped overcome the yield bottleneck caused by imbalanced precursor supply between the two modules in the parental strain. Enhancement and Modification of the Lactose Synthesis Pathway . In the absence of exogenous lactose, E. coli must rely on endogenous lactose synthesis to supply the glycosyl acceptor for 2′-FL production. However, the native metabolic network is primarily oriented toward lactose catabolism rather than anabolism, resulting in severely constrained endogenous lactose synthesis capacity. Previous studies have shown that simply introducing a heterologous β -1,4-galactosyltransferase, though sufficient to establish a basic synthetic route, often fails to support high-level 2′-FL production. Therefore, systematic enhancement of the lactose synthesis branch is essential for improving 2′-FL productivity. To minimize precursor loss, we first knocked out setA , which encodes a sugar efflux transporter involved in the export of saccharide precursors, generating strain BE4. This modification was expected to reduce wasteful loss of key small sugar intermediates in the lactose biosynthesis pathway, lower the cellular energy burden associated with active transport, and redirect metabolic resources toward growth and the core synthesis pathway [ 35 , 36 ]. To evaluate the effect of setA deletion, we analyzed the metabolites in the fermentation broth. After 48 h of fermentation, trace amounts of lactose (0.1–0.5 g/L) were detected in whole-cell samples of the setA knockout strain BE4 by HPLC, whereas no lactose was detectable in the setA -proficient parental strain (BE3-2). This observation suggests that SetA likely transports certain sugar intermediates involved in lactose biosynthesis (e.g., glucose or lactose precursors). Its deletion reduces the efflux of these intermediates, allowing their intracellular accumulation and thereby enhancing the availability of precursors for lactose synthesis. The increased lactose pool, in turn, provides more substrate for the downstream fucosylation reaction. Consistent with these findings, BE4 achieved a 2′-FL titer of 4.11 g/L after 72 h of shake-flask cultivation with glucose as the sole carbon source, representing an increase from the 3.73 g/L produced by BE3-2 (Fig. 3 ). To further enhance endogenous lactose synthesis, we next reinforced the UDP-galactose supply pathway. Key genes in this pathway— pgm (encoding phosphoglucomutase), galE (encoding UDP-galactose-4-epimerase), and galU (encoding UTP-glucose-1-phosphate uridylyltransferase)—were individually cloned and overexpressed. A series of recombinant plasmids were constructed to systematically evaluate their synergistic effects. Among strains BE4-1 through BE4-6, the combination of pRSFDuet- GalTpm1141 - pgm - galE - galU , pETDuet- pgi -BKHT, and pCDFDuet- glf - glk yielded the highest 2′-FL titer of 4.61 g/L. This increase suggested that coordinated overexpression of these three genes effectively enhanced the conversion of G-1-P to UDP-galactose, thereby improving lactose precursor availability. Given that the galU -catalyzed reaction (Glc-1-P + UTP → UDP-Glc + PPi) produces inorganic pyrophosphate (PPi) as an equimolar byproduct, we hypothesized that PPi accumulation might limit flux through this step [ 37 ]. To test this, we overexpressed ppa (encoding inorganic pyrophosphatase), which hydrolyzes PPi to 2Pi, thereby shifting the reaction equilibrium toward UDP-Glc formation. The ppa gene was cloned into pETDuet- pgi -BKHT and pCDFDuet- glf - glk vectors, and various plasmid combinations were introduced into BE4, generating strains BE4-7 through BE4-9. Among these, BE4-8 (harboring pCDFDuet- glf - glk - ppa , pRSFDuet- GalTpm1141 - pgm - galE - galU , and pETDuet- pgi -BKHT) achieved the highest 2′-FL titer of 4.95 g/L (Fig. 3 ). However, when ppa was expressed from the high-copy plasmid pETDuet, 2′-FL production decreased. This may be attributed to excessive Pi accumulation disrupting cellular energy homeostasis (e.g., ATP/ADP/Pi balance) or to imbalanced expression between ppa and galU , highlighting the importance of fine-tuning pathway expression levels. To further redirect carbon flux toward lactose synthesis, we disrupted two competing pathways using CRISPR/Cas9: gcd (encoding glucose dehydrogenase, which channels glucose into a direct oxidation pathway) and udg (encoding uracil-DNA glycosylase, potentially involved in futile nucleotide cycling). The resulting strain, BE5, achieved a 2′-FL titer of 5.22 g/L, representing a cumulative improvement from the parental strain and confirming that minimizing precursor diversion is critical for maximizing 2′-FL production. Synergistic Optimization and Balancing of the PPP and Purine Salvage Modules. In the GDP-L-fucose synthesis pathway, the reactions catalyzed by Gmd and WcaG require NADPH as a cofactor. Additionally, the guanosine diphosphate moiety of GDP-L-fucose is derived from 5-phosphoribose, a core precursor generated via the pentose phosphate pathway (PPP). Therefore, both the PPP and purine salvage pathways play critical roles in supporting GDP-L-fucose availability. To enhance NADPH supply and redirect carbon flux toward the PPP, we overexpressed zwf , which encodes glucose-6-phosphate dehydrogenase—the rate-limiting enzyme of the PPP [ 38 ]. Concurrently, to increase the GTP pool required for GDP-L-fucose synthesis, we overexpressed gsk , encoding guanosine kinase, which reinforces the purine salvage pathway [ 39 ]. Based on strain BE5, the zwf and gsk genes were cloned and overexpressed in both pETDuet- pgi -BKHT and pACYC- glf - glk - ppa vectors. A series of recombinant plasmids were constructed to systematically evaluate their synergistic effects, generating strains BE5-1 through BE5-9. After 72 h of shake-flask fermentation, strain BE5-7 (harboring pACYC- glf - glk - zwf - ppa - gsk , pRSFDuet- GalTpm1141 - pgm - galE - galU , and pETDuet- pgi -BKHT) achieved the highest 2′-FL titer, reaching 7.11 g/L (Fig. 4 ). This represented a substantial increase from the 5.22 g/L produced by BE5, suggesting that the coordinated overexpression of zwf and gsk effectively enhanced both NADPH supply and GTP precursor availability. Notably, strains with zwf and gsk expressed from different plasmid combinations showed varying titers, indicating that the expression levels of these genes must be finely balanced to achieve optimal flux distribution without imposing excessive metabolic burden. The significant improvement in 2′-FL titer indicated that the coordinated modulation of these two pathways successfully alleviated precursor limitations in GDP-L-fucose synthesis. Production of 2′-FL by Batch Fermentation in a 3-L Bioreactor. To evaluate the scalability of 2′-FL production, fed-batch fermentation was performed using strain BE5-7 in a 3-L bioreactor (the complete biosynthetic pathway is shown in Fig. 6 ). During the fermentation, temperature was maintained at 37°C, dissolved oxygen (DO) was controlled at 30%, and pH was maintained at 6.8. When the OD₆₀₀ reached 30 at approximately 12 h post-inoculation, the temperature was shifted to 25°C, and protein expression was induced by adding 0.1 mM IPTG. Glucose was fed intermittently to maintain residual glucose concentrations between 5 and 10 g/L throughout the fermentation. As shown in Fig. 5 , BE5-7 achieved a maximum 2′-FL titer of 43.2 g/L after 58 h of fed-batch fermentation. This titer significantly exceeds the highest levels previously reported for de novo 2′-FL synthesis from glucose as the sole carbon source. Analysis of the fermentation kinetics revealed an interesting pattern: no lactose accumulation was detected during the active 2′-FL production phase. Notably, lactose began to accumulate only after the 2′-FL titer reached its maximum and started to decline. This observation suggests that during the active production phase, lactose was efficiently consumed as a substrate for fucosylation, maintaining a low intracellular pool. The accumulation of lactose in the later stage likely reflects the gradual depletion of GDP-L-fucose or a decline in fucosyltransferase activity, leading to an imbalance between lactose supply and its utilization. Throughout the fermentation process, efficient nutrient utilization supported rapid cell growth, and no other detectable byproducts (such as 3-FL or DFL) were observed in the culture supernatant, indicating that the engineered metabolic network effectively channeled carbon flux toward the target product with minimal side reactions. In conclusion this study we successfully established a de novo biosynthetic pathway for 2′-FL in E. coli by introducing a heterologous β -1,4-galactosyltransferase, enabling production from glucose as the sole carbon source. Unlike previous strategies that rely on exogenous lactose supplementation or co-substrate systems, this work demonstrates, to our knowledge, the first efficient synthesis of 2′-FL from glucose as the exclusive carbon source in E. coli . Through systematic metabolic engineering—including optimization of glucose uptake via overexpression of a glucose facilitator and knockout of PTS components, enhancement of endogenous lactose synthesis by modulating key genes ( pgm , galU , galE ), and balancing of cofactor and precursor supply through coordinated overexpression of zwf and gsk —we achieved a 2′-FL titer of 43.2 g/L in a 3-L bioreactor. This represents the highest reported yield for de novo 2′-FL synthesis from glucose as the sole carbon source to date. This study not only provides an innovative platform for efficient 2′-FL biosynthesis but also offers a valuable reference for the microbial synthesis of other high-value lactose-derived oligosaccharides. Conclusion In this study, we successfully constructed an engineered E. coli strain capable of de novo biosynthesis of 2′-fucosyllactose from glucose as the sole carbon source. Unlike previous strategies that relied on exogenous lactose supplementation or co-substrate systems, our approach centered on systematically balancing the metabolic flux between phosphorylated and non-phosphorylated glucose pools to coordinate the supply of precursors for both lactose and GDP-L-fucose synthesis. This balance was achieved through a multi-module engineering strategy. First, the glucose uptake system was reconstructed by disrupting the phosphotransferase system, introducing a glucose facilitator to provide non-phosphorylated glucose, and modulating glucokinase expression to maintain an adequate pool of glucose-6-phosphate. Additionally, agp was knocked out to eliminate futile cycling of phosphorylated precursors. Subsequently, the carbon flux was further optimized by eliminating competing pathways and balancing carbon flux between glycolysis and the pentose phosphate pathway via overexpression of pgi , thereby enhancing NADPH and ribose-5-phosphate supply. The endogenous lactose synthesis pathway was strengthened through coordinated overexpression of pgm , galU , and galE , along with ppa to alleviate pyrophosphate inhibition. Finally, the pentose phosphate pathway and purine salvage pathway were synergistically optimized via overexpression of zwf and gsk to enhance NADPH and GTP availability. Through this systematic engineering, the final engineered strain BE5-7 achieved a 2′-FL titer of 7.11 g/L in shake-flask fermentation and 43.2 g/L in a 3-L bioreactor after 58 h of fed-batch fermentation. This titer represents the highest reported yield for de novo 2′-FL synthesis from glucose as the sole carbon source to date. Collectively, this work not only provides a scalable and cost-effective platform for industrial 2′-FL production but also offers a generalizable engineering framework for the microbial synthesis of other high-value lactose-derived oligosaccharides. Declarations AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] Funding This work was financially supported by the Key Research and Development Program of China (2024YFA0918300), and the National Natural Science Foundation of China (22478155). Notes The authors declare no competing financial interest. References Liu Y, Zhu Y, Wang H, Wan L, Zhang W, Mu W. Strategies for Enhancing Microbial Production of 2′-Fucosyllactose, the Most Abundant Human Milk Oligosaccharide. J Agric Food Chem. 2022; 70:11481-99. https://doi.org/10.1021/acs.jafc.2c04539. Wiciński M, Sawicka E, Gębalski J, Kubiak K, Malinowski B. Human milk oligosaccharides: health benefits, potential applications in infant formulas, and pharmacology. 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Yao R, Hirose Y, Sarkar D, Nakahigashi K, Ye Q, Shimizu K. Catabolic regulation analysis of Escherichia coli and its crp, mlc, mgsA, pgi and ptsG mutants. Microb Cell Fact. 2011; 10:67. https://doi.org/10.1186/1475-2859-10-67. Xia T, Han Q, Costanzo WV, Zhu Y, Urbauer JL, Eiteman MA. Accumulation of d-glucose from pentoses by metabolically engineered Escherichia coli. Appl Environ Microbiol. 2015; 81:3387-94. https://doi.org/10.1128/AEM.04058-14. Boteva E, Doychev K, Kirilov K, Handzhiyski Y, Tsekovska R, Gatev E, et al. Deglycation activity of the Escherichia coli glycolytic enzyme phosphoglucose isomerase. Int J Biol Macromol. 2024; 257:128541. https://doi.org/10.1016/j.ijbiomac.2023.128541. Sugita T, Koketsu K. Transporter engineering enables the efficient production of lacto-N-triose II and lacto-N-tetraose in Escherichia coli. J Agric Food Chem. 2022; 70:5106-14. https://doi.org/10.1021/acs.jafc.2c01369. Liu JY, Miller PF, Willard J, Olson ER. Functional and biochemical characterization of Escherichia coli sugar efflux transporters. J Biol Chem. 1999; 274:22977-84. https://doi.org/10.1074/jbc.274.33.22977. Malykh EA, Golubeva LI, Kovaleva ES, Shupletsov MS, Rodina EV, Mashko SV, et al. H+-translocating membrane-bound pyrophosphatase from Rhodospirillum rubrum fuels Escherichia coli cells via an alternative pathway for energy generation. Microorganisms. 2023; 11:294. https://doi.org/10.3390/microorganisms11020294. Zhao J, Baba T, Mori H, Shimizu K. Effect of zwf gene knockout on the metabolism of Escherichia coli grown on glucose or acetate. Metab Eng. 2004; 6:164-74. https://doi.org/10.1016/j.ymben.2004.02.004. Yu S, Liu JJ, Yun EJ, Kwak S, Kim KH, Jin YS. Production of a human milk oligosaccharide 2′-fucosyllactose by metabolically engineered Saccharomyces cerevisiae. Microb Cell Fact. 2018; 17:101. https://doi.org/10.1186/s12934-018-0947-2. Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.docx Supporting information The strains, plasmids, and primers used in this study, along with colony-PCR-verified gene knockout results. TOC.png Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9278536","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":616227837,"identity":"991e7290-2a87-474e-a4d8-d400987f90cf","order_by":0,"name":"Wentai Wu","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Wentai","middleName":"","lastName":"Wu","suffix":""},{"id":616227838,"identity":"6b85aca2-3c6f-4fee-a99b-1ec8c5d2da9b","order_by":1,"name":"Luyao Wang","email":"","orcid":"","institution":"Jiangnan 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10:39:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9278536/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9278536/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106868988,"identity":"2fef5d9d-5d3b-431c-aee8-bf8273ae3486","added_by":"auto","created_at":"2026-04-14 09:36:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":101414,"visible":true,"origin":"","legend":"\u003cp\u003e2′-FLproduction levels of the strainsBE2-1, BE2-2, BE2-3 and BE2-4.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9278536/v1/f1aa71092b2c9631e11ed302.png"},{"id":106960732,"identity":"970f8bff-7559-4823-a57f-73d56d1382b5","added_by":"auto","created_at":"2026-04-15 09:22:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":100914,"visible":true,"origin":"","legend":"\u003cp\u003e2′-FLproduction levels of the strainsBE3-1, BE3-2, BE3-3, BE3-4, BE3-5 and BE3-6.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9278536/v1/046e9ea9cf48785c18cd16a0.png"},{"id":106868992,"identity":"a82a320a-6b67-46c6-b556-c5191e187642","added_by":"auto","created_at":"2026-04-14 09:36:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":122027,"visible":true,"origin":"","legend":"\u003cp\u003e2′-FL production levels of the strainsBE4-1, BE4-2, BE4-3, BE4-4, BE4-5, BE4-6, BE4-7, BE4-8 and BE4-9.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9278536/v1/483c772906b12666ae0c5830.png"},{"id":106868995,"identity":"f0d71c11-e8fc-40de-8e6e-6a35ba7ba8f1","added_by":"auto","created_at":"2026-04-14 09:36:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":69073,"visible":true,"origin":"","legend":"\u003cp\u003e2′-FL production levels of the strains BE5-1, BE5-2, BE5-3, BE5-4, BE5-5, BE5-6, BE5-7, BE5-8 and BE5-9.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9278536/v1/1ea25977e51d4274f14ec292.png"},{"id":106868993,"identity":"eadddcf3-25f5-4f73-9cc7-86b921e371ce","added_by":"auto","created_at":"2026-04-14 09:36:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":254079,"visible":true,"origin":"","legend":"\u003cp\u003e2′-FL production level of the strain BE5-7 in a 3-L bioreactor.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9278536/v1/96f6426402fa75846c228b7c.png"},{"id":106961202,"identity":"706aff95-2903-4348-8c7d-7f881930d21b","added_by":"auto","created_at":"2026-04-15 09:24:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":171345,"visible":true,"origin":"","legend":"\u003cp\u003eReconstruction of the 2′-FL biosynthetic metabolic pathway.\u003cstrong\u003e \u003c/strong\u003eAbbreviations used for metabolites: G-6-P, Glucose-6-phosphate; G-1-P, Glucose-1-phosphate; UDP-Glc, Uridine 5′-diphospho-glucose; UDP-Gal, Uridine 5′-diphospho-galactose; UDP-Glca, Uridine 5′-diphospho-glucuronate; F-6-P, Fructose-6-phosphate; M-6-P, Mannose-6-phosphate; M-1-P, Mannose-1-phosphate; GDP-Man, Guanosine 5′-diphospho-mannose; GDP-4-keto, GDP-4-keto-6-deoxymannose; GDP-Fuc, Guanosine 5′-diphospho-fucose.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9278536/v1/2b1b72bda611a396d4590596.png"},{"id":106963111,"identity":"23762e95-8b35-4887-8e92-c89b2c553f45","added_by":"auto","created_at":"2026-04-15 09:42:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1503343,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9278536/v1/2fb462d9-770c-4c33-b073-90506ed7b2c4.pdf"},{"id":106960932,"identity":"9718e6cb-25ff-4031-94f5-030383d09226","added_by":"auto","created_at":"2026-04-15 09:23:41","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":42386,"visible":true,"origin":"","legend":"\u003cp\u003eSupporting information\u003c/p\u003e\n\u003cp\u003eThe strains, plasmids, and primers used in this study, along with colony-PCR-verified gene knockout results.\u003c/p\u003e","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-9278536/v1/136f86a740485b640778ad17.docx"},{"id":106868990,"identity":"1f23f48c-b5a2-46a3-a4ba-5cfcc9626af4","added_by":"auto","created_at":"2026-04-14 09:36:25","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":153245,"visible":true,"origin":"","legend":"","description":"","filename":"TOC.png","url":"https://assets-eu.researchsquare.com/files/rs-9278536/v1/892a6c9b166d66c48a583e49.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Systematic Metabolic Engineering Enables 2′-Fucosyllactose Biosynthesis from Glucose as the Sole Carbon Source in Escherichia coli","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHuman milk oligosaccharides (HMOs) are the third-largest solid component in human milk, exhibiting significant prebiotic activity while also modulating the infant immune system, helping infants resist pathogen colonization, and promoting neurodevelopment in infants [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Among various HMOs, 2\u0026prime;-fucosyllactose (2\u0026prime;-FL) is the most abundant fucosylated oligosaccharide and has been approved as a nutritional additive in infant formula by regulatory bodies including China\u0026rsquo;s National Health Commission, the U.S. FDA, and the European Union [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Consequently, its efficient biomanufacturing has become a research hotspot at the intersection of synthetic biology and food science.\u003c/p\u003e \u003cp\u003eCurrently, three primary routes exist for 2\u0026prime;-FL synthesis: chemical [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], enzymatic [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and microbial fermentation [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Chemical synthesis involves complex steps and potential safety concerns. The enzymatic method relies on expensive GDP-L-fucose and lactose precursors, limiting its economic viability for scale-up. Microbial fermentation offers advantages such as short cycles, low cost, and readily available feedstocks, making it the most promising strategy [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn traditional microbial fermentation for 2\u0026prime;-FL, two main technical routes prevail based on the supply mode of the key precursor GDP-L-fucose, both heavily dependent on exogenous lactose addition [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The first is the salvage pathway, in which engineered bacteria expressing fucosyltransferase catalyze the reaction using exogenously supplied lactose and GDP-L-fucose (or its direct precursor, L-fucose) [\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Although straightforward, this route is hampered by the high cost of both substrates. The more common strategy is the \u003cem\u003ede novo\u003c/em\u003e pathway, which involves systematic metabolic engineering to construct an endogenous GDP-L-fucose supply module from cheap carbon sources such as glycerol or glucose, achieved by introducing or enhancing enzymes including mannose mutase and GDP-L-mannose dehydratase, and by expressing efficient fucosyltransferases [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In this system, exogenously added lactose serves as the essential fucosyl acceptor, accepting the fucosyl group transferred from endogenously synthesized GDP-L-fucose to generate 2\u0026prime;-FL.\u003c/p\u003e \u003cp\u003eAlthough this \u003cem\u003ede novo\u003c/em\u003e strategy reduces the cost of the fucose moiety, the obligate dependence on exogenous lactose\u0026mdash;coupled with challenges in uptake efficiency, metabolic flux diversion, and additive cost\u0026mdash;remains a critical bottleneck that restricts the scalability and overall efficiency of 2\u0026prime;-FL production.\u003c/p\u003e \u003cp\u003eTo overcome this bottleneck, recent research has focused on developing \u003cem\u003ede novo\u003c/em\u003e biosynthetic routes independent of exogenous lactose [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. For instance, Mu\u0026rsquo;s team engineered \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) for 2\u0026prime;-FL production using co-cultivation with glucose and xylose, yielding 6.53 g/L and 27.53 g/L of 2\u0026prime;-FL in shake-flask and 3-L fed-batch bioreactor fermentations, respectively [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Liu\u0026rsquo;s team successfully developed a \u003cem\u003eBacillus subtilis\u003c/em\u003e platform for 2\u0026prime;-FL production from glucose as the sole carbon source [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Through construction of a lactose biosynthesis module, introduction of a non-phosphotransferase system, and development of a lactose-responsive biosensor for dynamic metabolic regulation, they achieved a 2\u0026prime;-FL titer of 30.1 g/L in a 3-L bioreactor, providing an important technical reference for exogenous lactose-independent \u003cem\u003ede novo\u003c/em\u003e 2\u0026prime;-FL biosynthesis.\u003c/p\u003e \u003cp\u003eBuilding upon this foundation, the present study employed low-cost glucose as the sole carbon source for 2\u0026prime;-FL production. Unlike previous approaches that primarily focused on engineering the GDP-L-fucose supply module, this work systematically balanced the metabolic flux between phosphorylated and non-phosphorylated glucose pools to coordinate the supply of precursors for both lactose and GDP-L-fucose synthesis. This balance was achieved by reconstructing the glucose uptake system\u0026mdash;specifically, by disrupting the phosphotransferase system (PTS) and introducing a glucose facilitator to provide non-phosphorylated glucose, while modulating glucokinase expression to maintain an adequate pool of glucose-6-phosphate. Additionally, \u003cem\u003eagp\u003c/em\u003e was knocked out to eliminate the futile cycling of glucose-1-phosphate back to glucose, further preserving phosphorylated precursors. Subsequently, the carbon flux was further optimized by eliminating other competing pathways (e.g., \u003cem\u003egcd\u003c/em\u003e, \u003cem\u003eudg\u003c/em\u003e) and modulating key node enzymes such as phosphoglucose isomerase to redirect carbon from glycolysis toward the pentose phosphate pathway, thereby enhancing NADPH and ribose-5-phosphate supply. In parallel, the endogenous lactose synthesis pathway was strengthened through coordinated overexpression of \u003cem\u003epgm\u003c/em\u003e, \u003cem\u003egalU\u003c/em\u003e, and \u003cem\u003egalE\u003c/em\u003e, along with \u003cem\u003eppa\u003c/em\u003e to alleviate pyrophosphate inhibition, while the purine salvage pathway was reinforced via \u003cem\u003egsk\u003c/em\u003e overexpression to boost GTP availability for GDP-L-fucose synthesis. Through this multi-module engineering strategy, a 2\u0026prime;-FL titer of 7.11 g/L was achieved in shake-flask cultivation, which was further scaled to 43.2 g/L in a 3-L bioreactor system. This titer exceeds the previously reported highest levels for \u003cem\u003ede novo\u003c/em\u003e 2\u0026prime;-FL synthesis from glucose as the sole carbon source. To our knowledge, this work represents the first demonstration of high-efficiency 2\u0026prime;-FL biosynthesis using glucose as the exclusive carbon source in \u003cem\u003eE. coli\u003c/em\u003e, achieving the highest reported yield for exogenous lactose-independent systems. These results validate the feasibility of a fully endogenous 2\u0026prime;-FL synthesis route and offer a scalable technological platform for cost-effective industrial production.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eRecombinant plasmid construction and related experimental materials\u003c/b\u003e. Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e details the overexpression plasmids used in this study. The recombinant plasmids were constructed using \u003cem\u003eE. coli\u003c/em\u003e JM109 as the cloning host. Three plasmid vectors (pETDuet-1, pRSFDuet-1, and pCDFDuet-1) with distinct expression strengths were selected to drive the expression of key genes in the 2\u0026prime;-FL biosynthetic pathway.\u003c/p\u003e \u003cp\u003eThe nucleotide sequences of \u003cem\u003eGalTpm1141\u003c/em\u003e (AEC04686) and \u003cem\u003eglf\u003c/em\u003e (AEC04687) were codon-optimized for \u003cem\u003eE. coli\u003c/em\u003e and synthesized by GENEWIZ (Suzhou, China). Genes related to the metabolic pathway\u0026mdash;including \u003cem\u003epgm\u003c/em\u003e, \u003cem\u003egalU\u003c/em\u003e, \u003cem\u003egalE\u003c/em\u003e, \u003cem\u003ezwf\u003c/em\u003e, \u003cem\u003eglk\u003c/em\u003e, \u003cem\u003epgi\u003c/em\u003e, \u003cem\u003egsk\u003c/em\u003e, and \u003cem\u003eppa\u003c/em\u003e\u0026mdash;were amplified from the genome of \u003cem\u003eE. coli\u003c/em\u003e K12 MG1655. Specifically, \u003cem\u003eBKHT\u003c/em\u003e was cloned into the pETDuet-1 vector using the Clone Expression II one-step cloning technique (primer information is provided in Table S2) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAll target genes were inserted into plasmids with different expression levels via the MEGAWHOP method to construct the recombinant plasmids. These constructs were subsequently verified by Sanger sequencing performed by GENEWIZ Biotechnology Co., Ltd. (Suzhou, China). All reagents employed in this study were sourced from Vazyme Biotechnology Co., Ltd. (Nanjing, China).\u003c/p\u003e \u003cp\u003e \u003cb\u003eConstruction of engineered strains\u003c/b\u003e. A dual-plasmid gene editing system based on CRISPR/Cas9 was employed for homologous target gene knockout, using pTargetF (Addgene, #62226) and pCas9 (Addgene, #62225) as tools. The pTargetF plasmid contains an sgRNA sequence paired with a genomic homologous fragment. The N20-specific sequence of the gene to be knocked out was identified using CHOPCHOP (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://chopchop.cbu.uib.no/\u003c/span\u003e\u003cspan address=\"http://chopchop.cbu.uib.no/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), obtained via PCR amplification, and then integrated into the sgRNA region at the 5\u0026prime; end of the pTargetF plasmid. The pCas9 plasmid encodes λ-Red and Cas9 proteins. The Cas9-mediated cleavage of the target site, combined with arabinose-induced (50 mM) λ-Red recombinase activity, enabled efficient homologous recombination. The verified knockout plasmids were introduced into \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) BL-5 via heat shock transformation. Colony PCR validation confirmed successful modification, after which a series of derivative strains were constructed (for full genotype details, see Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Using this approach, targeted deletions were accomplished for key metabolic genes including \u003cem\u003elacZ\u003c/em\u003e, \u003cem\u003elacA\u003c/em\u003e, \u003cem\u003eugd\u003c/em\u003e, \u003cem\u003egcd\u003c/em\u003e, \u003cem\u003eptsG\u003c/em\u003e, \u003cem\u003eglk\u003c/em\u003e, and \u003cem\u003eagp\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCulture conditions of recombinant strains in shake flask.\u003c/b\u003e This study employed Luria-Bertani (LB) medium for seed culture and a glucose-defined medium (GDM) for shake-flask fermentation. The LB medium contained (per liter): 5.00 g yeast extract, 10.00 g tryptone, and 10.00 g NaCl. The GDM consisted of (per liter): 10.00 g glucose, 1.70 g citric acid monohydrate, 5.00 g yeast extract, 4.00 g (NH₄)₂HPO₄, 13.50 g KH₂PO₄, 1.40 g MgSO₄\u0026middot;7H₂O, 0.50 g tryptone, and 10 mL of a trace element solution. The medium pH was adjusted to 7.2 using ammonia. All media were supplemented with antibiotics at final concentrations of ampicillin (100 \u0026micro;g/mL), kanamycin (30 \u0026micro;g/mL), and spectinomycin (50 \u0026micro;g/mL) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe recombinant strain was first inoculated into LB medium and incubated at 37\u0026deg;C with shaking at 200 rpm for 10\u0026ndash;12 h. Subsequently, 0.5 mL of this seed culture was transferred into a 250 mL shake flask containing 50 mL of GDM. When the optical density at 600 nm (OD₆₀₀) reached 2.0, protein expression was induced by adding 0.05 mM isopropyl \u003cem\u003eβ\u003c/em\u003e-D-1-thiogalactopyranoside (IPTG).\u003c/p\u003e \u003cp\u003e \u003cb\u003e3-L Bioreactor Fermentation of Recombinant Strains\u003c/b\u003e. The initial medium for the 3-L bioreactor fermentation contained the following components (per liter): 30.00 g glucose, 4.00 g (NH₄)₂SO₄, 6.00 g tryptone, 9.20 g K₂HPO₄, 8.20 g KH₂PO₄, 0.30 g citric acid monohydrate, 0.02 g CaCl₂, 2.00 g yeast extract, and 10 mL of the trace element solution (composition as described above). The pH was adjusted to 7.2 with ammonium hydroxide. The feed medium contained 600.00 g/L glucose, 2.50 g/L tryptone, 22.20 g/L MgSO₄\u0026middot;7H₂O, and 5.00 g/L yeast extract. All media were supplemented with antibiotics at final concentrations of ampicillin (100 \u0026micro;g/mL), kanamycin (30 \u0026micro;g/mL), and spectinomycin (50 \u0026micro;g/mL).\u003c/p\u003e \u003cp\u003eThe recombinant strain was first inoculated into 10 mL of LB medium as a primary seed culture and incubated at 37\u0026deg;C with shaking at 200 rpm for 10\u0026ndash;12 h. Then, 0.1 mL of this culture was aseptically transferred into 50 mL of fresh LB medium in a 250 mL shake flask (secondary seed culture) and grown under the same conditions for another 10\u0026ndash;12 h. Subsequently, 100 mL of the secondary culture (10% v/v inoculum) was used to inoculate 1 L of fermentation medium in a 3-L bioreactor (Applikon).\u003c/p\u003e \u003cp\u003eDuring the initial fermentation phase, the key operating parameters were controlled as follows: temperature was maintained at 37\u0026deg;C using a thermostatic system, dissolved oxygen (DO) was kept at 30%, and pH was automatically maintained at 6.8 via an alkali feeding system. When the residual glucose concentration fell below 5 g/L, the feed medium was supplied to maintain glucose levels between 5 and 10 g/L. Once the OD₆₀₀ reached 30, the temperature was shifted from 37\u0026deg;C to 25\u0026deg;C, and protein expression was induced by adding 0.1 mM isopropyl \u003cem\u003eβ\u003c/em\u003e-D-1-thiogalactopyranoside (IPTG).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalytical Methods.\u003c/b\u003e Bacterial growth was monitored by measuring the optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) using a UV-1800 spectrophotometer (MeiXi, Shanghai, China). For product analysis, samples were prepared following a standardized three-step protocol: heat inactivation, centrifugation, and filtration. First, fermentation broth was heat-inactivated in a 100\u0026deg;C water bath for 15 min. Subsequently, the samples were centrifuged at 12,000 rpm for 15 min to separate the solid and liquid phases. The clarified supernatant was then filtered through a 0.22 \u0026micro;m membrane, aliquoted, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent analysis.\u003c/p\u003e \u003cp\u003eThe concentrations of 2\u0026prime;-FL, lactose, and glucose were quantified using a Waters Alliance iS high-performance liquid chromatography (HPLC) system (Waters, Milford, MA, USA) equipped with a refractive index (RI) detector. Chromatographic separation was performed on an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA). The column temperature and detector temperature were maintained at 60\u0026deg;C and 50\u0026deg;C, respectively. The mobile phase was 5 mM sulfuric acid, applied under isocratic elution at a flow rate of 0.5 mL/min.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e \u003cb\u003eConstruction of an Engineered Chassis Strain for 2\u0026prime;-FL Synthesis Based on a Lactose Self-Synthesis System\u003c/b\u003e. Achieving \u003cem\u003ede novo\u003c/em\u003e biosynthesis of 2\u0026prime;-FL from glucose as the sole carbon source requires the construction of a complete biosynthetic pathway in \u003cem\u003eE. coli\u003c/em\u003e, including a GDP-L-fucose supply module, a lactose self-synthesis module, and a 2\u0026prime;-FL synthesis module. In this study, an \u003cem\u003eE. coli\u003c/em\u003e chassis strain (BE0) previously constructed in our laboratory with the ability to synthesize GDP-L-fucose was used as the starting strain, into which exogenous glycosyltransferases were introduced to establish the complete pathway [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe gene encoding \u003cem\u003eα\u003c/em\u003e-1,2-fucosyltransferase (BKHT) from \u003cem\u003eHelicobacter pylori\u003c/em\u003e was cloned into pETDuet-1, and the gene encoding \u003cem\u003eβ\u003c/em\u003e-1,4-galactosyltransferase (\u003cem\u003eGalTpm1141\u003c/em\u003e) from \u003cem\u003eNeisseria meningitidis\u003c/em\u003e was cloned into pRSFDuet-1. Co-transformation of these two recombinant plasmids into the chassis strain yielded the initial strain BE1. Theoretically, this strain could utilize glucose as the sole carbon source to autonomously synthesize lactose and further produce 2\u0026prime;-FL.\u003c/p\u003e \u003cp\u003eShake-flask fermentation results showed that after 72 h of cultivation with glucose as the sole carbon source, BE1 produced only 0.04 g/L of 2\u0026prime;-FL, with 7.4 g/L of glucose remaining, and no lactose accumulation was detected. These results indicated that despite the introduction of the core enzymatic machinery, the intracellular lactose synthesis capacity was severely insufficient to provide an adequate acceptor for 2\u0026prime;-FL synthesis, while glucose uptake and utilization efficiency were low, and carbon flux was not effectively channeled toward the target pathway. Thus, lactose self-synthesis and glucose transport/distribution constituted the main bottlenecks limiting 2\u0026prime;-FL production.\u003c/p\u003e \u003cp\u003e \u003cb\u003eReconstruction and Enhancement of the Glucose Uptake Pathway.\u003c/b\u003e In \u003cem\u003eE. coli\u003c/em\u003e, glucose uptake is primarily mediated by the phosphotransferase system (PTS), in which the EIICB^Glc enzyme encoded by \u003cem\u003eptsG\u003c/em\u003e simultaneously transports and phosphorylates glucose to glucose-6-phosphate (G-6-P) upon entry, while repressing the expression of non-PTS genes [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhen lactose synthesis requires non-phosphorylated glucose as a precursor, this coupled transport-phosphorylation mechanism becomes a metabolic bottleneck. To address this limitation, we first disrupted the PTS pathway by knocking out \u003cem\u003eptsG\u003c/em\u003e in strain BE1 using CRISPR/Cas9, generating strain BE2.\u003c/p\u003e \u003cp\u003eAfter co-transforming the two recombinant plasmids carried by BE1 (pETDuet-1-BKHT and pRSFDuet-1-\u003cem\u003egalTpm1141\u003c/em\u003e) into this knockout strain, BE2 produced 0.44 g/L of 2\u0026prime;-FL after 72 h of fermentation, a significant increase from the 0.04 g/L produced by BE1, with residual glucose decreasing to 3.1 g/L. These observations indicated that disrupting the dominant PTS pathway partially relieved the bottleneck by redirecting carbon flux toward the non-PTS system, which supplies non-phosphorylated glucose\u0026mdash;a key precursor for lactose synthesis. However, the absence of detectable lactose suggested that the endogenous non-phosphorylated glucose supply remained insufficient to support efficient lactose biosynthesis.\u003c/p\u003e \u003cp\u003eTo further enhance the supply of non-phosphorylated glucose, we introduced the \u003cem\u003eglf\u003c/em\u003e gene from \u003cem\u003eZymomonas mobilis\u003c/em\u003e, which encodes a facilitator that mediates passive glucose transport along its concentration gradient without ATP consumption or concomitant phosphorylation. The \u003cem\u003eglf\u003c/em\u003e gene was cloned into the low-copy-number plasmid pCDFDuet, yielding pCDFDuet-\u003cem\u003eglf\u003c/em\u003e. Additionally, because G-6-P serves as a key precursor for both lactose and GDP-L-fucose, we also aimed to increase the phosphorylated glucose pool within the NPTS framework by cloning \u003cem\u003eglk\u003c/em\u003e (encoding glucokinase) into pETDuet and pCDFDuet vectors. Various combinations of these plasmids were transformed into BE2, generating strains BE2-1 through BE2-4. Given that plasmid copy number can affect gene expression levels and metabolic burden, we tested combinations with different expression strengths.Among these, BE2-3 (harboring pETDuet-BKHT, pRSFDuet-\u003cem\u003eGalTpm1141\u003c/em\u003e, and pCDFDuet-\u003cem\u003eglf\u003c/em\u003e-\u003cem\u003eglk\u003c/em\u003e) achieved the highest 2\u0026prime;-FL titer of 1.71 g/L after 72 h of shake-flask fermentation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The superior performance of BE2-3 suggested that balanced co-expression of \u003cem\u003eglf\u003c/em\u003e and \u003cem\u003eglk\u003c/em\u003e from the same low-copy plasmid (pCDFDuet), together with high-copy expression of the fucosyltransferase BKHT (from pETDuet)\u0026mdash;which catalyzes the final condensation of GDP-L-fucose and lactose to form 2\u0026prime;-FL\u0026mdash;provided an optimal supply of both non-phosphorylated and phosphorylated glucose without imposing excessive metabolic burden. This result demonstrated that optimizing both non-phosphorylated glucose import and its subsequent phosphorylation significantly enhanced lactose production, thereby promoting efficient 2\u0026prime;-FL synthesis. These findings also highlighted that the interplay between glucose uptake, phosphorylation, and downstream lactose synthesis must be finely tuned to maximize product titer, guiding further optimization of the central carbon metabolism in subsequent steps.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eReconstruction and Optimization of the Intracellular Phosphorylated Glucose Metabolic Network.\u003c/b\u003e As the first key intermediate formed after glucose uptake, D-glucose-6-phosphate (G-6-P) occupies a decisive metabolic branch point. It serves as the primary substrate for glycolysis to generate energy and carbon skeletons, while also being an indispensable precursor for UDP-galactose and GDP-L-fucose synthesis. Therefore, diverting G-6-P from central metabolism toward the 2\u0026prime;-FL biosynthetic pathway is critical for high-yield production.\u003c/p\u003e \u003cp\u003eTo minimize unnecessary carbon loss from the precursor pool, we first eliminated a non-essential competitive diversion at the glucose-1-phosphate (G-1-P) node. G-1-P, derived from G-6-P via phosphoglucomutase, can be dephosphorylated back to non-phosphorylated glucose by the phosphatase encoded by \u003cem\u003eagp\u003c/em\u003e. We knocked out \u003cem\u003eagp\u003c/em\u003e in strain BE2 using CRISPR/Cas9, generating strain BE3. This deletion was expected to prevent futile cycling between G-6-P and glucose, thereby preserving phosphorylated glucose intermediates for downstream lactose and GDP-L\u0026ndash;fucose synthesis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAfter this modification in the engineered \u003cem\u003eE.\u003c/em\u003e coli, BE3 was used as the base strain for further optimization of carbon flux distribution. The activity of phosphoglucose isomerase, which interconverts G-6-P and fructose-6-phosphate (F-6-P), acts as a key valve regulating the partition of carbon between glycolysis and the pentose phosphate pathway (PPP). To balance the supply of precursors for both the lactose and GDPL\u0026ndash;fucose modules, we overexpressed \u003cem\u003epgi\u003c/em\u003e by cloning it into either pETDuet-BKHT or pCDFDuet-\u003cem\u003eglf\u003c/em\u003e-\u003cem\u003eglk\u003c/em\u003e vectors. Various combinations of these plasmids were transformed into BE3, generating strains BE3-1 through BE3-6.\u003c/p\u003e \u003cp\u003eAfter 72 h of shake-flask fermentation, strain BE3-2 (harboring pETDuet-\u003cem\u003epgi\u003c/em\u003e-BKHT, pRSFDuet-GalTpm1141, and pCDFDuet-\u003cem\u003eglf\u003c/em\u003e-\u003cem\u003eglk\u003c/em\u003e) achieved the highest 2\u0026prime;-FL titer of 3.73 g/L, representing a substantial increase from the 1.71 g/L produced by BE2-3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The superior performance of BE3-2 suggested that moderate overexpression of \u003cem\u003epgi\u003c/em\u003e from a high-copy plasmid (pETDuet) effectively balanced the carbon flux distribution between glycolysis and the PPP [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThis increased the supply of NADPH and ribose-5-phosphate\u0026mdash;key precursors for GDP-L-fucose synthesis\u0026mdash;while still maintaining sufficient G-6-P flux for lactose production. The marked improvement in 2\u0026prime;-FL titer provided indirect evidence that GDP-L-fucose availability was enhanced. Notably, strains with \u003cem\u003epgi\u003c/em\u003e overexpressed from the low-copy plasmid (pCDFDuet) showed lower titers, which is attributed to insufficient flux balancing or suboptimal expression balance between \u003cem\u003epgi\u003c/em\u003e and other pathway genes.\u003c/p\u003e \u003cp\u003eThese results demonstrated that enhancing \u003cem\u003epgi\u003c/em\u003e expression served a dual purpose: it balanced the flux between the PPP and glycolysis to drive GDP-L-fucose synthesis, and this balanced redistribution helped overcome the yield bottleneck caused by imbalanced precursor supply between the two modules in the parental strain.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEnhancement and Modification of the Lactose Synthesis Pathway\u003c/b\u003e. In the absence of exogenous lactose, \u003cem\u003eE. coli\u003c/em\u003e must rely on endogenous lactose synthesis to supply the glycosyl acceptor for 2\u0026prime;-FL production. However, the native metabolic network is primarily oriented toward lactose catabolism rather than anabolism, resulting in severely constrained endogenous lactose synthesis capacity. Previous studies have shown that simply introducing a heterologous \u003cem\u003eβ\u003c/em\u003e-1,4-galactosyltransferase, though sufficient to establish a basic synthetic route, often fails to support high-level 2\u0026prime;-FL production. Therefore, systematic enhancement of the lactose synthesis branch is essential for improving 2\u0026prime;-FL productivity.\u003c/p\u003e \u003cp\u003eTo minimize precursor loss, we first knocked out \u003cem\u003esetA\u003c/em\u003e, which encodes a sugar efflux transporter involved in the export of saccharide precursors, generating strain BE4. This modification was expected to reduce wasteful loss of key small sugar intermediates in the lactose biosynthesis pathway, lower the cellular energy burden associated with active transport, and redirect metabolic resources toward growth and the core synthesis pathway [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo evaluate the effect of \u003cem\u003esetA\u003c/em\u003e deletion, we analyzed the metabolites in the fermentation broth. After 48 h of fermentation, trace amounts of lactose (0.1\u0026ndash;0.5 g/L) were detected in whole-cell samples of the \u003cem\u003esetA\u003c/em\u003e knockout strain BE4 by HPLC, whereas no lactose was detectable in the \u003cem\u003esetA\u003c/em\u003e-proficient parental strain (BE3-2). This observation suggests that SetA likely transports certain sugar intermediates involved in lactose biosynthesis (e.g., glucose or lactose precursors). Its deletion reduces the efflux of these intermediates, allowing their intracellular accumulation and thereby enhancing the availability of precursors for lactose synthesis. The increased lactose pool, in turn, provides more substrate for the downstream fucosylation reaction. Consistent with these findings, BE4 achieved a 2\u0026prime;-FL titer of 4.11 g/L after 72 h of shake-flask cultivation with glucose as the sole carbon source, representing an increase from the 3.73 g/L produced by BE3-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further enhance endogenous lactose synthesis, we next reinforced the UDP-galactose supply pathway. Key genes in this pathway\u0026mdash;\u003cem\u003epgm\u003c/em\u003e (encoding phosphoglucomutase), \u003cem\u003egalE\u003c/em\u003e (encoding UDP-galactose-4-epimerase), and \u003cem\u003egalU\u003c/em\u003e (encoding UTP-glucose-1-phosphate uridylyltransferase)\u0026mdash;were individually cloned and overexpressed. A series of recombinant plasmids were constructed to systematically evaluate their synergistic effects. Among strains BE4-1 through BE4-6, the combination of pRSFDuet-\u003cem\u003eGalTpm1141\u003c/em\u003e-\u003cem\u003epgm\u003c/em\u003e-\u003cem\u003egalE\u003c/em\u003e-\u003cem\u003egalU\u003c/em\u003e, pETDuet-\u003cem\u003epgi\u003c/em\u003e-BKHT, and pCDFDuet-\u003cem\u003eglf\u003c/em\u003e-\u003cem\u003eglk\u003c/em\u003e yielded the highest 2\u0026prime;-FL titer of 4.61 g/L. This increase suggested that coordinated overexpression of these three genes effectively enhanced the conversion of G-1-P to UDP-galactose, thereby improving lactose precursor availability.\u003c/p\u003e \u003cp\u003eGiven that the \u003cem\u003egalU\u003c/em\u003e-catalyzed reaction (Glc-1-P\u0026thinsp;+\u0026thinsp;UTP \u0026rarr; UDP-Glc\u0026thinsp;+\u0026thinsp;PPi) produces inorganic pyrophosphate (PPi) as an equimolar byproduct, we hypothesized that PPi accumulation might limit flux through this step [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo test this, we overexpressed \u003cem\u003eppa\u003c/em\u003e (encoding inorganic pyrophosphatase), which hydrolyzes PPi to 2Pi, thereby shifting the reaction equilibrium toward UDP-Glc formation. The \u003cem\u003eppa\u003c/em\u003e gene was cloned into pETDuet-\u003cem\u003epgi\u003c/em\u003e-BKHT and pCDFDuet-\u003cem\u003eglf\u003c/em\u003e-\u003cem\u003eglk\u003c/em\u003e vectors, and various plasmid combinations were introduced into BE4, generating strains BE4-7 through BE4-9. Among these, BE4-8 (harboring pCDFDuet-\u003cem\u003eglf\u003c/em\u003e-\u003cem\u003eglk\u003c/em\u003e-\u003cem\u003eppa\u003c/em\u003e, pRSFDuet-\u003cem\u003eGalTpm1141\u003c/em\u003e-\u003cem\u003epgm\u003c/em\u003e-\u003cem\u003egalE\u003c/em\u003e-\u003cem\u003egalU\u003c/em\u003e, and pETDuet-\u003cem\u003epgi\u003c/em\u003e-BKHT) achieved the highest 2\u0026prime;-FL titer of 4.95 g/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). However, when \u003cem\u003eppa\u003c/em\u003e was expressed from the high-copy plasmid pETDuet, 2\u0026prime;-FL production decreased. This may be attributed to excessive Pi accumulation disrupting cellular energy homeostasis (e.g., ATP/ADP/Pi balance) or to imbalanced expression between \u003cem\u003eppa\u003c/em\u003e and \u003cem\u003egalU\u003c/em\u003e, highlighting the importance of fine-tuning pathway expression levels.\u003c/p\u003e \u003cp\u003eTo further redirect carbon flux toward lactose synthesis, we disrupted two competing pathways using CRISPR/Cas9: \u003cem\u003egcd\u003c/em\u003e (encoding glucose dehydrogenase, which channels glucose into a direct oxidation pathway) and \u003cem\u003eudg\u003c/em\u003e (encoding uracil-DNA glycosylase, potentially involved in futile nucleotide cycling). The resulting strain, BE5, achieved a 2\u0026prime;-FL titer of 5.22 g/L, representing a cumulative improvement from the parental strain and confirming that minimizing precursor diversion is critical for maximizing 2\u0026prime;-FL production.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSynergistic Optimization and Balancing of the PPP and Purine Salvage Modules.\u003c/b\u003e In the GDP-L-fucose synthesis pathway, the reactions catalyzed by Gmd and WcaG require NADPH as a cofactor. Additionally, the guanosine diphosphate moiety of GDP-L-fucose is derived from 5-phosphoribose, a core precursor generated via the pentose phosphate pathway (PPP). Therefore, both the PPP and purine salvage pathways play critical roles in supporting GDP-L-fucose availability.\u003c/p\u003e \u003cp\u003eTo enhance NADPH supply and redirect carbon flux toward the PPP, we overexpressed \u003cem\u003ezwf\u003c/em\u003e, which encodes glucose-6-phosphate dehydrogenase\u0026mdash;the rate-limiting enzyme of the PPP [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Concurrently, to increase the GTP pool required for GDP-L-fucose synthesis, we overexpressed \u003cem\u003egsk\u003c/em\u003e, encoding guanosine kinase, which reinforces the purine salvage pathway [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBased on strain BE5, the \u003cem\u003ezwf\u003c/em\u003e and \u003cem\u003egsk\u003c/em\u003e genes were cloned and overexpressed in both pETDuet-\u003cem\u003epgi\u003c/em\u003e-BKHT and pACYC-\u003cem\u003eglf\u003c/em\u003e-\u003cem\u003eglk\u003c/em\u003e-\u003cem\u003eppa\u003c/em\u003e vectors. A series of recombinant plasmids were constructed to systematically evaluate their synergistic effects, generating strains BE5-1 through BE5-9.\u003c/p\u003e \u003cp\u003eAfter 72 h of shake-flask fermentation, strain BE5-7 (harboring pACYC-\u003cem\u003eglf\u003c/em\u003e-\u003cem\u003eglk\u003c/em\u003e-\u003cem\u003ezwf\u003c/em\u003e-\u003cem\u003eppa\u003c/em\u003e-\u003cem\u003egsk\u003c/em\u003e, pRSFDuet-\u003cem\u003eGalTpm1141\u003c/em\u003e-\u003cem\u003epgm\u003c/em\u003e-\u003cem\u003egalE\u003c/em\u003e-\u003cem\u003egalU\u003c/em\u003e, and pETDuet-\u003cem\u003epgi\u003c/em\u003e-BKHT) achieved the highest 2\u0026prime;-FL titer, reaching 7.11 g/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This represented a substantial increase from the 5.22 g/L produced by BE5, suggesting that the coordinated overexpression of \u003cem\u003ezwf\u003c/em\u003e and \u003cem\u003egsk\u003c/em\u003e effectively enhanced both NADPH supply and GTP precursor availability. Notably, strains with \u003cem\u003ezwf\u003c/em\u003e and \u003cem\u003egsk\u003c/em\u003e expressed from different plasmid combinations showed varying titers, indicating that the expression levels of these genes must be finely balanced to achieve optimal flux distribution without imposing excessive metabolic burden. The significant improvement in 2\u0026prime;-FL titer indicated that the coordinated modulation of these two pathways successfully alleviated precursor limitations in GDP-L-fucose synthesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eProduction of 2\u0026prime;-FL by Batch Fermentation in a 3-L Bioreactor.\u003c/b\u003e To evaluate the scalability of 2\u0026prime;-FL production, fed-batch fermentation was performed using strain BE5-7 in a 3-L bioreactor (the complete biosynthetic pathway is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDuring the fermentation, temperature was maintained at 37\u0026deg;C, dissolved oxygen (DO) was controlled at 30%, and pH was maintained at 6.8. When the OD₆₀₀ reached 30 at approximately 12 h post-inoculation, the temperature was shifted to 25\u0026deg;C, and protein expression was induced by adding 0.1 mM IPTG. Glucose was fed intermittently to maintain residual glucose concentrations between 5 and 10 g/L throughout the fermentation.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e, BE5-7 achieved a maximum 2\u0026prime;-FL titer of 43.2 g/L after 58 h of fed-batch fermentation. This titer significantly exceeds the highest levels previously reported for \u003cem\u003ede novo\u003c/em\u003e 2\u0026prime;-FL synthesis from glucose as the sole carbon source. Analysis of the fermentation kinetics revealed an interesting pattern: no lactose accumulation was detected during the active 2\u0026prime;-FL production phase. Notably, lactose began to accumulate only after the 2\u0026prime;-FL titer reached its maximum and started to decline. This observation suggests that during the active production phase, lactose was efficiently consumed as a substrate for fucosylation, maintaining a low intracellular pool. The accumulation of lactose in the later stage likely reflects the gradual depletion of GDP-L-fucose or a decline in fucosyltransferase activity, leading to an imbalance between lactose supply and its utilization. Throughout the fermentation process, efficient nutrient utilization supported rapid cell growth, and no other detectable byproducts (such as 3-FL or DFL) were observed in the culture supernatant, indicating that the engineered metabolic network effectively channeled carbon flux toward the target product with minimal side reactions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn conclusion this study we successfully established a \u003cem\u003ede novo\u003c/em\u003e biosynthetic pathway for 2\u0026prime;-FL in \u003cem\u003eE. coli\u003c/em\u003e by introducing a heterologous \u003cem\u003eβ\u003c/em\u003e-1,4-galactosyltransferase, enabling production from glucose as the sole carbon source. Unlike previous strategies that rely on exogenous lactose supplementation or co-substrate systems, this work demonstrates, to our knowledge, the first efficient synthesis of 2\u0026prime;-FL from glucose as the exclusive carbon source in \u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThrough systematic metabolic engineering\u0026mdash;including optimization of glucose uptake via overexpression of a glucose facilitator and knockout of PTS components, enhancement of endogenous lactose synthesis by modulating key genes (\u003cem\u003epgm\u003c/em\u003e, \u003cem\u003egalU\u003c/em\u003e, \u003cem\u003egalE\u003c/em\u003e), and balancing of cofactor and precursor supply through coordinated overexpression of \u003cem\u003ezwf\u003c/em\u003e and \u003cem\u003egsk\u003c/em\u003e\u0026mdash;we achieved a 2\u0026prime;-FL titer of 43.2 g/L in a 3-L bioreactor. This represents the highest reported yield for \u003cem\u003ede novo\u003c/em\u003e 2\u0026prime;-FL synthesis from glucose as the sole carbon source to date.\u003c/p\u003e \u003cp\u003eThis study not only provides an innovative platform for efficient 2\u0026prime;-FL biosynthesis but also offers a valuable reference for the microbial synthesis of other high-value lactose-derived oligosaccharides.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we successfully constructed an engineered \u003cem\u003eE. coli\u003c/em\u003e strain capable of \u003cem\u003ede novo\u003c/em\u003e biosynthesis of 2\u0026prime;-fucosyllactose from glucose as the sole carbon source. Unlike previous strategies that relied on exogenous lactose supplementation or co-substrate systems, our approach centered on systematically balancing the metabolic flux between phosphorylated and non-phosphorylated glucose pools to coordinate the supply of precursors for both lactose and GDP-L-fucose synthesis.\u003c/p\u003e \u003cp\u003eThis balance was achieved through a multi-module engineering strategy. First, the glucose uptake system was reconstructed by disrupting the phosphotransferase system, introducing a glucose facilitator to provide non-phosphorylated glucose, and modulating glucokinase expression to maintain an adequate pool of glucose-6-phosphate. Additionally, \u003cem\u003eagp\u003c/em\u003e was knocked out to eliminate futile cycling of phosphorylated precursors. Subsequently, the carbon flux was further optimized by eliminating competing pathways and balancing carbon flux between glycolysis and the pentose phosphate pathway via overexpression of \u003cem\u003epgi\u003c/em\u003e, thereby enhancing NADPH and ribose-5-phosphate supply. The endogenous lactose synthesis pathway was strengthened through coordinated overexpression of \u003cem\u003epgm\u003c/em\u003e, \u003cem\u003egalU\u003c/em\u003e, and \u003cem\u003egalE\u003c/em\u003e, along with \u003cem\u003eppa\u003c/em\u003e to alleviate pyrophosphate inhibition. Finally, the pentose phosphate pathway and purine salvage pathway were synergistically optimized via overexpression of \u003cem\u003ezwf\u003c/em\u003e and \u003cem\u003egsk\u003c/em\u003e to enhance NADPH and GTP availability.\u003c/p\u003e \u003cp\u003eThrough this systematic engineering, the final engineered strain BE5-7 achieved a 2\u0026prime;-FL titer of 7.11 g/L in shake-flask fermentation and 43.2 g/L in a 3-L bioreactor after 58 h of fed-batch fermentation. This titer represents the highest reported yield for \u003cem\u003ede novo\u003c/em\u003e 2\u0026prime;-FL synthesis from glucose as the sole carbon source to date.\u003c/p\u003e \u003cp\u003eCollectively, this work not only provides a scalable and cost-effective platform for industrial 2\u0026prime;-FL production but also offers a generalizable engineering framework for the microbial synthesis of other high-value lactose-derived oligosaccharides.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAUTHOR INFORMATION\u003c/p\u003e\n\u003cp\u003eCorresponding Authors\u003c/p\u003e\n\u003cp\u003e*E-mail: [email protected]\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by the Key Research and Development Program of China (2024YFA0918300), and the National Natural Science Foundation of China (22478155).\u003c/p\u003e\n\u003cp\u003eNotes\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eLiu Y, Zhu Y, Wang H, Wan L, Zhang W, Mu W. 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Microb Cell Fact.\u003cem\u003e\u0026nbsp;\u003c/em\u003e2018; 17:101. https://doi.org/10.1186/s12934-018-0947-2.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"2′-fucosyllactose, sole carbon source, de novo synthesis, metabolic engineering, fermentation","lastPublishedDoi":"10.21203/rs.3.rs-9278536/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9278536/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e2′-Fucosyllactose (2′-FL) is the most abundant human milk oligosaccharide (HMO), playing vital roles in promoting infant gut health, enhancing immunity, and defending against pathogens. However, conventional 2′-FL biosynthesis typically relies on exogenous supplementation of lactose or fucose precursors, leading to high costs and process complexity. In this study, we report a streamlined \u003cem\u003ede novo\u003c/em\u003e biosynthesis process for 2′-FL using glucose as the sole carbon source, achieved through systematic engineering of endogenous precursor pathways in \u003cem\u003eEscherichia coli\u003c/em\u003e. First, the complete biosynthetic route from glucose to lactose and then to 2′-FL was reconstructed in a chassis strain capable of producing fucose. Unlike previous studies that primarily focused on the GDP-fucose supply module, this work systematically balanced the metabolic flux between phosphorylated and non-phosphorylated glucose pools to coordinate the supply of precursors for both lactose and GDP-fucose synthesis. This balance was achieved by reconstructing the glucose uptake system, modulating the expression of key enzymes at critical metabolic nodes, and eliminating competing pathways. Subsequently, the endogenous lactose synthesis pathway was further enhanced through coordinated overexpression of \u003cem\u003epgm\u003c/em\u003e, \u003cem\u003egalU\u003c/em\u003e, and \u003cem\u003egalE\u003c/em\u003e, and the pentose phosphate pathway and purine salvage pathway were optimized to enhance the supply of NADPH and GTP. Using this strategy, an engineered \u003cem\u003eE. coli\u003c/em\u003e strain for efficient 2′-FL production was successfully constructed. The engineered strain produced 7.11 g/L 2′-FL in shake-flask fermentation and achieved a titer of 43.2 g/L in a 3-L bioreactor after 58 h, representing the highest reported yield for \u003cem\u003ede novo\u003c/em\u003e 2′-FL synthesis from glucose as the sole carbon source. This work provides a promising and simplified strategy for the cost-effective industrial production of 2′-FL.\u003c/p\u003e","manuscriptTitle":"Systematic Metabolic Engineering Enables 2′-Fucosyllactose Biosynthesis from Glucose as the Sole Carbon Source in Escherichia coli","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-14 09:36:20","doi":"10.21203/rs.3.rs-9278536/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"225425f0-8a67-404a-b119-2985033c05dd","owner":[],"postedDate":"April 14th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-14T09:53:22+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-14 09:36:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9278536","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9278536","identity":"rs-9278536","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}