Unlocking the function promiscuity of old yellow enzyme to catalyze asymmetric Morita-Baylis-Hillman reaction.

Exploring the promiscuity of native enzymes presents a promising strategy for expanding their synthetic applications, particularly for catalyzing challenging reactions in non-native contexts. In this study, we explore the promiscuous potential of old yellow enzymes (OYEs) to facilitate the Morita-Baylis-Hillman reaction (MBH reaction), leveraging substrate similarities between MBH reaction and reduction reaction. Using mass spectrometry and spectroscopic techniques, we confirm promiscuity of GkOYE in both MBH and reduction reactions. By blocking H- and H+ transfer pathways, we engineer GkOYE.8, which loses its reduction ability but enhances its MBH activity. The structural basis of MBH reaction catalyzed by GkOYE.8 is obtained through mutation studies and kinetic simulations. Furthermore, enantiocomplementary mutants GkOYE.11 and GkOYE.13 are obtained by directed evolution, exhibiting the ability to accept various aromatic aldehydes and alkenes as substrates. This study demonstrates the potential of leveraging substrate similarities to unlock enzyme functionalities, enabling the catalysis of new-to-nature reactions.

Anoikis resistance regulates immune infiltration and drug sensitivity in clear-cell renal cell carcinoma: insights from multi omics, single cell analysis and in vitro experiment.

Background: Anoikis is a form of programmed cell death essential for preventing cancer metastasis. In some solid cancer, anoikis resistance can facilitate tumor progression. However, this phenomenon is underexplored in clear-cell renal cell carcinoma (ccRCC). Methods: Using SVM machine learning, we identified core anoikis-related genes (ARGs) from ccRCC patient transcriptomic data. A LASSO Cox regression model stratified patients into risk groups, informing a prognostic model. GSVA and ssGSEA assessed immune infiltration, and single-cell analysis examined ARG expression across immune cells. Quantitative PCR and immunohistochemistry validated ARG expression differences between immune therapy responders and non-responders in ccRCC. Results: ARGs such as CCND1, CDKN3, PLK1, and BID were key in predicting ccRCC outcomes, linking higher risk with increased Treg infiltration and reduced M1 macrophage presence, indicating an immunosuppressive environment facilitated by anoikis resistance. Single-cell insights showed ARG enrichment in Tregs and dendritic cells, affecting immune checkpoints. Immunohistochemical analysis reveals that ARGs protein expression is markedly elevated in ccRCC tissues responsive to immunotherapy. Conclusion: This study establishes a novel anoikis resistance gene signature that predicts survival and immunotherapy response in ccRCC, suggesting that manipulating the immune environment through these ARGs could improve therapeutic strategies and prognostication in ccRCC.

Mechanism-Guided Computational Design Drives meso-Diaminopimelate Dehydrogenase to Efficient Synthesis of Aromatic d-amino Acids.

Aromatic d-amino acids (d-AAs) play a pivotal role as important chiral building blocks and key intermediates in fine chemical and drug synthesis. Meso-diaminopimelate dehydrogenase (DAPDH) serves as an excellent biocatalyst in the synthesis of d-AAs and their derivatives. However, its strict substrate specificity and the lack of efficient engineering methods have hindered its widespread application. Therefore, this study aims to elucidate the catalytic mechanism underlying DAPDH from Proteus vulgaris (PvDAPDH) through the examination of its crystallographic structure, computational simulations of potential energies and molecular dynamics simulations, and site-directed mutagenesis. Mechanism-guided computational design showed that the optimal mutant PvDAPDH-M3 increased specific activity and catalytic efficiency (kcat/Km) for aromatic keto acids up to 124-fold and 92.4-fold, respectively, compared to that of the wild type. Additionally, it expanded the substrate scope to 10 aromatic keto acid substrates. Finally, six high-value-added aromatic d-AAs and their derivatives were synthesized using a one-pot three-enzyme cascade reaction, exhibiting a good conversion rate ranging from 32 to 84% and excellent stereoselectivity (enantiomeric excess >99%). These findings provide a potential synthetic pathway for the green industrial production of aromatic d-AAs.

[Enzymatic production of 1,4-cyclohexanedimethylamine].

1,4-cyclohexanedimethylamine (1,4-BAC) is an important monomer for bio-based materials, it finds wide applications in various fields including organic synthesis, medicine, chemical industry, and materials. At present, its synthesis primarily relies on chemical method, which suffer from issues such as expensive metal catalyst, harsh reaction conditions, and safety risks. Therefore, it is necessary to explore greener alternatives for its synthesis. In this study, a two-bacterium three-enzyme cascade conversion pathway was successfully developed to convert 1,4-cyclohexanedicarboxaldehyde to 1,4-cyclohexanedimethylamine. This pathway used Escherichia coli derived aminotransferase (EcTA), Saccharomyces cerevisiae derived glutamate dehydrogenase (ScGlu-DH), and Candida boidinii derived formate dehydrogenase (CbFDH). Through structure-guided protein engineering, a beneficial mutant, EcTAF91Y, was obtained, exhibiting a 2.2-fold increase in specific activity and a 1.9-fold increase in kcat/Km compared to that of the wild type. By constructing recombinant strains and optimizing reaction conditions, it was found that under the optimal conditions, a substrate concentration of 40 g/L could produce (27.4±0.9) g/L of the product, corresponding to a molar conversion rate of 67.5%±2.1%.

Systems metabolic engineering of Escherichia coli for high-yield production of Para-hydroxybenzoic acid.

Para-hydroxybenzoic acid (PHBA) is extensively used as an additive in the food and cosmetics industries, significantly enhancing product shelf life and stability. While microbial fermentation offers an environment-friendly and sustainable method for producing PHBA, the titer and productivity are limited due to product toxicity and complex metabolic flux distributions. Here, we initially redesigned a L-phenylalanine-producing Escherichia coli by employing rational metabolic engineering strategies, resulting in the production of PHBA reached the highest reported level of 14.17 g/L. Subsequently, a novel accelerated evolution system was devised comprising deaminase, the alpha subunit of RNA polymerase, an uracil-DNA glycosylase inhibitor, and the PHBA-responsive promoter PyhcN. This system enabled us to obtain a mutant strain exhibiting a 47% increase in the half-inhibitory concentration (IC50) for PHBA within 15 days. Finally, the evolved strain achieved a production of 21.35 g/L PHBA in a 5-L fermenter, with a yield of 0.19 g/g glucose and a productivity rate of 0.44 g/L/h. This engineered strain emerges as a promising candidate for industrial production of PHBA through an eco-friendly approach.

Rational Design of the Spatial Effect in a Fe(II)/α-Ketoglutarate-Dependent Dioxygenase Reverses the Regioselectivity of C(sp3)-H Bond Hydroxylation in Aliphatic Amino Acids.

The hydroxylation of remote C(sp3)-H bonds in aliphatic amino acids yields crucial precursors for the synthesis of high-value compounds. However, accurate regulation of the regioselectivity of remote C(sp3)-H bonds hydroxylation in aliphatic amino acids continues to be a common challenge in chemosynthesis and biosynthesis. In this study, the Fe(II)/α-ketoglutarate-dependent dioxygenase from Bacillus subtilis (BlAH) was mined and found to catalyze hydroxylation at the γ and δ sites of aliphatic amino acids. Crystal structure analysis, molecular dynamics simulations, and quantum chemical calculations revealed that regioselectivity was regulated by the spatial effect of BlAH. Based on these results, the spatial effect of BlAH was reconstructed to stabilize the transition state at the δ site of aliphatic amino acids, thereby successfully reversing the γ site regioselectivity to the δ site. For example, the regioselectivity of L-Homoleucine (5 a) was reversed from the γ site (1 : 12) to the δ site (>99 : 1). The present study not only expands the toolbox of biocatalysts for the regioselective functionalization of remote C(sp3)-H bonds, but also provides a theoretical guidance for the precision-driven modification of similarly remote C(sp3)-H bonds in complex molecules.

Fine-Tuning Pyridoxal 5'-Phosphate Synthesis in Escherichia coli for Cadaverine Production in Minimal Culture Media.

Cadaverine is a critical C5 monomer for the production of polyamides. Pyridoxal 5'-phosphate (PLP), as a crucial cofactor for the key enzyme lysine decarboxylase in the cadaverine biosynthesis pathway, has seen a persistent shortage, leading to limitations in cadaverine production. To address this issue, a dual-pathway strategy was implemented, synergistically enhancing both endogenous and heterologous PLP synthesis modules and resulting in improved PLP synthesis. Subsequently, a growth-stage-dependent molecular switch was introduced to balance the precursor competition between PLP synthesis and cell growth. Additionally, a PLP sensor-based negative feedback circuit was constructed by integrating a newly identified PLP-responsive promoter PygjH and an arabinose-regulated system, dynamically regulating the expression of the PLP synthetic genes and preventing excessive intracellular PLP accumulation. The optimal strain, L18, cultivated in the minimal medium AM1, demonstrated cadaverine production with a titer, yield, and productivity of 64.03 g/L, 0.23 g/g glucose, and 1.33 g/L/h, respectively. This represents the highest titer reported to date in engineered Escherichia coli by fed-batch fermentation in a minimal medium.

Metabolic Engineering of Escherichia coli for High-Level Production of l-Phenylalanine.

l-Phenylalanine (l-Phe) is widely used in the food and pharmaceutical industries. However, the biosynthesis of l-Phe using Escherichia coli remains challenging due to its lower tolerance to high concentration of l-Phe. In this study, to efficiently synthesize l-Phe, the l-Phe biosynthetic pathway was reconstructed by expressing the heterologous genes aroK1, aroL1, and pheA1, along with the native genes aroA, aroC, and tyrB in the shikimate-producing strain E. coli SA09, resulting in the engineered strain E. coli PHE03. Subsequently, adaptive evolution was conducted on E. coli PHE03 to enhance its tolerance to high concentrations of l-Phe, resulting in the strain E. coli PHE04, which reduced the cell mortality to 36.2% after 48 h of fermentation. To elucidate the potential mechanisms, transcriptional profiling was conducted, revealing MarA, a DNA-binding transcriptional dual regulator, as playing a crucial role in enhancing cell membrane integrity and fluidity for improving cell tolerance to high concentrations of l-Phe. Finally, the titer, yield, and productivity of l-Phe with E. coli PHE05 overexpressing marA were increased to 80.48 g/L, 0.27 g/g glucose, and 1.68 g/L/h in a 5-L fed-batch fermentation, respectively.

Production of 1,4-Butanediol from Succinic Acid Using Escherichia Coli Whole-Cell Catalysis.

The widespread attention towards 1,4-butanediol (BDO) as a key chemical raw material stems from its potential in producing biodegradable plastics. However, the efficiency of its biosynthesis via current bioprocesses is limited. In this study, a dual-pathway approach for 1,4-BDO production from succinic acid was developed. Specifically, a double-enzyme catalytic pathway involving carboxylic acid reductase and ethanol dehydrogenase was proposed. Optimization of the expression levels of the pathway enzymes led to a significant 318 % increase in 1,4-BDO titer. Additionally, the rate-limiting enzyme MmCAR was engineered to enhance the kcat/KM values by 50 % and increase 1,4-BDO titer by 46.7 %. To address cofactor supply limitations, an NADPH and ATP cycling system was established, resulting in a 48.9 % increase in 1,4-BDO production. Ultimately, after 48 hours, 1,4-BDO titers reached 201 mg/L and 1555 mg/L in shake flask and 5 L fermenter, respectively. This work represents a significant advancement in 1,4-BDO synthesis from succinic acid, with potential applications in the organic chemical and food industries.

Rational Design of Salmonella typhi Acid Phosphatase for Efficient Production of Pyridoxal 5'-Phosphate.

Pyridoxal 5'-phosphate (PLP) is highly valuable in food and medicine. However, achieving the efficient biosynthesis of PLP remains challenging. Here, a salvage pathway using acid phosphatase from Salmonella typhi (StAPase) and pyridoxine oxidase from Escherichia coli (EcPNPO) as pathway enzymes was established for the first time to synthesize PLP from pyridoxine (PN) and pyrophosphate (PPi). StAPase was identified as a rate-limiting enzyme. Two protein modification strategies were developed based on the PN phosphorylation mechanism: (1) improving the binding of PN into StAPase and (2) enhancing the hydrophobicity of StAPase's substrate binding pocket. The kcat/Km of optimal mutant M7 was 4.9 times higher than that of the wild type. The detailed mechanism of performance improvement was analyzed. Under the catalysis of M7 and EcPNPO, a PLP high-yielding strain of 14.5 ± 0.55 g/L was engineered with a productivity of 1.0 ± 0.02 g/(L h) (the highest to date). The study suggests a promising method for industrial-scale PLP production.

Systems engineering Escherichia coli for efficient production p-coumaric acid from glucose.

P-coumaric acid (p-CA), a pant metabolite with antioxidant and anti-inflammatory activity, is extensively utilized in biomedicine, food, and cosmetics industry. In this study, a synthetic pathway (PAL) for p-CA was designed, integrating three enzymes (AtPAL2, AtC4H, AtATR2) into a higher l-phenylalanine-producing strain Escherichia coli PHE05. However, the lower soluble expression and activity of AtC4H in the PAL pathway was a bottleneck for increasing p-CA titers. To overcome this limitation, the soluble expression of AtC4H was enhanced through N-terminal modifications. And an optimal mutant, AtC4HL373T/G211H, which exhibited a 4.3-fold higher kcat/Km value compared to the wild type, was developed. In addition, metabolic engineering strategies were employed to increase the intracellular NADPH pool. Overexpression of ppnk in engineered E. coli PHCA20 led to a 13.9-folds, 1.3-folds, and 29.1% in NADPH content, the NADPH/NADP+ ratio and p-CA titer, respectively. These optimizations significantly enhance p-CA production, in a 5-L fermenter using fed-batch fermentation, the p-CA titer, yield and productivity of engineered strain E. coli PHCA20 were 3.09 g/L, 20.01 mg/g glucose, and 49.05 mg/L/h, respectively. The results presented here provide a novel way to efficiently produce the plant metabolites using an industrial strain.

Rational design improves both thermostability and activity of a new D-tagatose 3-epimerase from Kroppenstedtia eburnean to produce D-allulose.

D-allulose is a naturally occurring rare sugar and beneficial to human health. However, the efficient biosynthesis of D-allulose remains a challenge. Here, we mined a new D-tagatose 3-epimerase from Kroppenstedtia eburnean (KeDt3e) with high catalytic efficiency. Initially, crucial factors contributing to the low conversion of KeDt3e were identified through crystal structure analysis, density functional theory calculations (DFT), and molecular dynamics (MD) simulations. Subsequently, based on the mechanism, combining restructuring the flexible region, proline substitution based onconsensus sequence analysis, introducing disulfide bonds, and grafting properties, and reshaping the active center, the optimal mutant M5 of KeDt3e was obtained with enhanced thermostability and activity. The optimal mutant M5 exhibited an enzyme activity of 130.8 U/mg, representing a 1.2-fold increase; Tm value increased from 52.7 °C to 71.2 °C; and half-life at 55 °C extended to 273.7 min, representing a 58.2-fold improvement, and the detailed mechanism of performance improvement was analyzed. Finally, by screening the ribosome-binding site (RBS) of the optimal mutant M5 recombinant bacterium (G01), the engineered strain G08 with higher expression levels was obtained. The engineered strain G08 catalyzed 500 g/L D-fructose to produce 172.4 g/L D-allulose, with a conversion of 34.4% in 0.5 h and productivity of 344.8 g/L/h on a 1 L scale. This study presents a promising approach for industrial-scale production of D-allulose.

Reprogramming the Transition States to Enhance C-N Cleavage Efficiency of Rhodococcus opacusl-Amino Acid Oxidase.

l-Amino acid oxidase (LAAO) is an important biocatalyst used for synthesizing α-keto acids. LAAO from Rhodococcus opacus (RoLAAO) has a broad substrate spectrum; however, its low total turnover number limits its industrial use. To overcome this, we aimed to employ crystal structure-guided density functional theory calculations and molecular dynamic simulations to investigate the catalytic mechanism. Two key steps were identified: S → [TS1] in step 1 and Int1 → [TS2] in step 2. We reprogrammed the transition states [TS1] and [TS2] to reduce the identified energy barrier and obtain a RoLAAO variant capable of catalyzing 19 kinds of l-amino acids to the corresponding high-value α-keto acids with a high total turnover number, yield (≥95.1 g/L), conversion rate (≥95%), and space-time yields ≥142.7 g/L/d in 12-24 h, in a 5 L reactor. Our results indicated the promising potential of the developed RoLAAO variant for use in the industrial production of α-keto acids while providing a potential catalytic-mechanism-guided protein design strategy to achieve the desired physical and catalytic properties of enzymes.

Chemoenzymatic Installation of Site-Specific Chemical Groups on DNA Enhances the Catalytic Activity.

Functional DNAs are valuable molecular tools in chemical biology and analytical chemistry but suffer from low activities due to their limited chemical functionalities. Here, we present a chemoenzymatic method for site-specific installation of diverse functional groups on DNA, and showcase the application of this method to enhance the catalytic activity of a DNA catalyst. Through chemoenzymatic introduction of distinct chemical groups, such as hydroxyl, carboxyl, and benzyl, at specific positions, we achieve significant enhancements in the catalytic activity of the RNA-cleaving deoxyribozyme 10-23. A single carboxyl modification results in a 100-fold increase, while dual modifications (carboxyl and benzyl) yield an approximately 700-fold increase in activity when an RNA cleavage reaction is catalyzed on a DNA-RNA chimeric substrate. The resulting dually modified DNA catalyst, CaBn, exhibits a kobs of 3.76 min-1 in the presence of 1 mM Mg2+ and can be employed for fluorescent imaging of intracellular magnesium ions. Molecular dynamics simulations reveal the superior capability of CaBn to recruit magnesium ions to metal-ion-binding site 2 and adopt a catalytically competent conformation. Our work provides a broadly accessible strategy for DNA functionalization with diverse chemical modifications, and CaBn offers a highly active DNA catalyst with immense potential in chemistry and biotechnology.

Systems engineering of Escherichia coli for high-level glutarate production from glucose.

Glutarate is a key monomer in polyester and polyamide production. The low efficiency of the current biosynthetic pathways hampers its production by microbial cell factories. Herein, through metabolic simulation, a lysine-overproducing E. coli strain Lys5 is engineered, achieving titer, yield, and productivity of 195.9 g/L, 0.67 g/g glucose, and 5.4 g/L·h, respectively. Subsequently, the pathway involving aromatic aldehyde synthase, monoamine oxidase, and aldehyde dehydrogenase (AMA pathway) is introduced into E. coli Lys5 to produce glutarate from glucose. To enhance the pathway's efficiency, rational mutagenesis on the aldehyde dehydrogenase is performed, resulting in the development of variant Mu5 with a 50-fold increase in catalytic efficiency. Finally, a glutarate tolerance gene cbpA is identified and genomically overexpressed to enhance glutarate productivity. With enzyme expression optimization, the glutarate titer, yield, and productivity of E. coli AMA06 reach 88.4 g/L, 0.42 g/g glucose, and 1.8 g/L·h, respectively. These findings hold implications for improving glutarate biosynthesis efficiency in microbial cell factories.

Efficient production of protocatechuic acid using systems engineering of Escherichia coli.

Protocatechuic acid (3, 4-dihydroxybenzoic acid, PCA) is widely used in the pharmaceuticals, health food, and cosmetics industries owing to its diverse biological activities. However, the inhibition of 3-dehydroshikimate dehydratase (AroZ) by PCA and its toxicity to cells limit the efficient production of PCA in Escherichia coli. In this study, a high-level strain of 3-dehydroshikimate, E. coli DHS01, was developed by blocking the carbon flow from the shikimate-overproducing strain E. coli SA09. Additionally, the PCA biosynthetic pathway was established in DHS01 by introducing the high-activity ApAroZ. Subsequently, the protein structure and catalytic mechanism of 3-dehydroshikimate dehydratase from Acinetobacter pittii PHEA-2 (ApAroZ) were clarified. The variant ApAroZR363A, achieved by modulating the conformational dynamics of ApAroZ, effectively relieved product inhibition. Additionally, the tolerance of the strain E. coli PCA04 to PCA was enhanced by adaptive laboratory evolution, and a biosensor-assisted high-throughput screening method was designed and implemented to expedite the identification of high-performance PCA-producing strains. Finally, in a 5 L bioreactor, the final strain PCA05 achieved the highest PCA titer of 46.65 g/L, a yield of 0.23 g/g, and a productivity of 1.46 g/L/h for PCA synthesis from glucose using normal fed-batch fermentation. The strategies described herein serve as valuable guidelines for the production of other high-value and toxic products.

The association between methylmalonic acid, a biomarker of mitochondrial dysfunction, and risk of prostate cancer.

BACKGROUND: The aim of the study was to investigate the association between methylmalonic acid (MMA), a biomarker of mitochondrial dysfunction, and the risk of prostate cancer (PCa). METHODS AND MATERIALS: The relevant data were collected from the National Health and Nutrition Examination Survey (NHANES). Weighted univariable and multivariable logistic regression analyses were performed to investigate the association between MMA and risk of PCa. A stratified analysis was also carried out. The dose-response relationship was elucidated by conducting a restricted cubic spline function. RESULTS: A total of 2451 participants were included, of which 95 were PCa participants. The fully-adjusted model 2 constructed by weighted multivariable logistic regression analysis showed that the risk of PCa decreased by 53% when every MMA unit was added [OR: 0.47 (0.22-1.00), P = 0.049]. And a decrease in PCa risk was associated with a higher MMA level in MMA subgroups [OR: 0.34 (0.15-0.82), P = 0.02]. The results from a stratified analysis showed that participants in subgroups of other race, BMI (> 30 kg/m2), smoking (former and now), and hypertension (yes), an increase in every MMA unit was linked to a decrease in PCa risk. MMA and the risk of PCa were negatively correlated in a linear manner. CONCLUSION: It was discovered in the study that an increase in MMA level is connected to a decrease in PCa risk. The serum MMA level may be helpful in assessing PCa risk.

Relationship between oxidative balance score and kidney stone prevalence in US adults.

BACKGROUND: The aim of the study was to assess the relationship between prevalence of kidney stones (KS) and the oxidative balance score (OBS). METHODS AND MATERIALS: Participants who participated in the KS questionnaire was extracted from the National Health and Nutrition Examination Survey (NHANES) from 2007 to 2018. A series of covariates were also obtained. Weighted adjusted logistic regression was performed to investigate the association of KS with OBS. Dose-response relationship between KS and OBS was assessed by restricted cubic spline. RESULTS: In the fully adjusted model, we discovered that the risk of KS decreased by 3% with each OBS unit raised (OR = 0.97, 95% CI: 0.95-0.99, P = 0.01). In the OBS subgroups, in contrast to the lowest quartile OBS, the higher quartile OBS was correlated to the decreased risk of KS prevalence (Q3 vs Q1: OR = 0.7, 95% CI: 0.49-0.99, P = 0.04; Q4 vs Q1: OR = 0.66, 95% CI: 0.44-0.99, P = 0.04), and the results maintained relative stability across three models. We also found that the risk of population with KS was negatively linked with each unit increase in dietary OBS (OR = 0.97, 95% CI: 0.95-0.99, P = 0.005). Finally, we detected that there was a linear association between OBS and the risk of KS prevalence (P non-linear > 0.05). CONCLUSION: The study discovered that OBS that comprehensively reflects an individual's overall burden of oxidative stress was negatively related to the risk of KS, and can be utilized as an important indicator for assessing the risk of KS.

Engineering status of protein for improving microbial cell factories.

With the development of metabolic engineering and synthetic biology, microbial cell factories (MCFs) have provided an efficient and sustainable method to synthesize a series of chemicals from renewable feedstocks. However, the efficiency of MCFs is usually limited by the inappropriate status of protein. Thus, engineering status of protein is essential to achieve efficient bioproduction with high titer, yield and productivity. In this review, we summarize the engineering strategies for metabolic protein status, including protein engineering for boosting microbial catalytic efficiency, protein modification for regulating microbial metabolic capacity, and protein assembly for enhancing microbial synthetic capacity. Finally, we highlight future challenges and prospects of improving microbial cell factories by engineering status of protein.

Reprogramming Metabolic Flux in Escherichia Coli to Enhance Chondroitin Production.

Reprogramming metabolic flux is a promising approach for constructing efficient microbial cell factories (MCFs) to produce chemicals. However, how to boost the transmission efficiency of metabolic flux is still challenging in complex metabolic pathways. In this study, metabolic flux is systematically reprogrammed by regulating flux size, flux direction, and flux rate to build an efficient MCF for chondroitin production. The ammoniation pool for UDP-GalNAc synthesis and the carbonization pool for UDP-GlcA synthesis are first enlarged to increase flux size for providing enough precursors for chondroitin biosynthesis. Then, the ammoniation pool and the carbonization pool are rematched using molecular valves to shift flux direction from cell growth to chondroitin biosynthesis. Next, the adaptability of polymerization pool with the ammoniation and carbonization pools is fine-tuned by dynamic and static valve-based adapters to accelerate flux rate for polymerizing UDP-GalNAc and UDP-GlcA to produce chondroitin. Finally, the engineered strain E. coli F51 is able to produce 9.2 g L-1 chondroitin in a 5-L bioreactor. This strategy shown here provides a systematical approach for regulating metabolic flux in complex metabolic pathways for efficient biosynthesis of chemicals.