Difucosyllactose (DFL), a fucosylated human milk oligosaccharide, exhibits significant application potential in the food and pharmaceutical industries. However, the microbial production of DFL faces challenges such as the accumulation of the intermediate 2'-fucosyllactose (2'-FL) and the high costs associated with the use of antibiotics and inducers. In this study, we constructed a plasmid- and inducer-free Escherichia coli MG1655 strain to enhance the biosynthesis of DFL while minimizing 2'-FL accumulation. Initially, the de novo DFL biosynthetic pathway was established by introducing different α-1,3-fucosyltransferases (α-1,3-FucT) into a 2'-FL-producing strain. Combinatorial metabolic engineering strategies were then employed to improve DFL accumulation. Subsequently, guided by the in silico multienzyme assembling by reshaping space (iMARS) framework, a fusion enzyme, FucTaY218K-L60-FutC, was rationally designed to effectively alleviate 2'-FL accumulation. Furthermore, modulation of the hydrophobic microenvironment in the substrate-binding pocket of FucTaY218K yielded the beneficial mutant FucTaY218K/W31R. Finally, modifications to the lactose operon significantly improved strain growth while concurrently enhancing DFL production. The best-performing strain achieved a DFL titer of 74 g/L in a 5-L bioreactor, corresponding to a productivity of 0.96 g/L/h, representing the highest productivity reported to date. This study establishes a robust and environmentally friendly platform for the industrial-scale production of DFL and provides a strategy for the microbial synthesis of other high-value human milk oligosaccharides.
3’-Sialyllactose (3’-SL), the simplest sialylated human milk oligosaccharide (HMO), is widely incorporated into infant formula due to its roles in shaping the gut microbiota, inhibiting pathogen adhesion, and supporting neurodevelopment. In this study, we developed an efficient microbial cell factory for 3’-SL production by enhancing carbon utilization and CTP regeneration. Following the identification of a highly active α-2,3-sialyltransferase, metabolic flux was redirected through pfkA deletion and fbaA overexpression. The precursor pool was strengthened by optimizing the expression of key enzymes involved in UDP-GlcNAc and CMP-Neu5Ac biosynthesis. To further improve pathway balance and stability, promoter engineering was applied to optimize the expression of four key genes (neuA, neuB, neuC and ST). The optimized multigene cassette was subsequently integrated into the chromosome using a one-step multicopy integration strategy, generating a plasmid-free and antibiotic marker-free production strain. The resulting strain achieved 3’-SL titers of 7.75 g L−1 in shake flasks and 102.18 g L−1 in a 5-L fed-batch bioreactor, representing, to the best of our knowledge, the highest reported level to date. This work demonstrates a robust strategy combining metabolic rewiring, adaptive gene expression tuning and multicopy genome integration, providing a versatile platform for the sustainable production of 3’-SL and other high-value HMOs.
Yan Wu, Tao-Ling Min, Zhenfeng Ma et al.· Bioresources and Bioprocessi...· 0 citations
1,4-Butanediamine is an important raw material for the synthesis of engineering plastics such as Polyamide 46 with excellent performance, which is widely used in automotive, electronics and machinery manufacturing industries. In this study, Escherichia coli BL21(DE3) was used as the starting strain to systematically modify the 1,4-butanediamine biosynthesis pathway using a modular strategy. 1,4-Butanediamine biosynthesis was re-divided into two modules: 1,4-butanediamine production module and α-ketoglutaric acid production module. By controlling the expression intensity of genes pykF, ppc, aceEF, gltA, icdA and gdhA, argD, argCB, argJ, ODC10, a recombinant producing 1,4-butanediamine strain PKT was obtained, achieving a yield of 862.82 mg/L of 1,4-butanediamine, which increased 8.62-fold compared to that of Escherichia coli BL21(DE3). Then, the fermentation medium of PKT strain was optimized. After 24 h of fermentation, the yield of 1,4-butanediamine reached 1843.60 mg/L, representing a 16.23-fold increase over the original strain. Furthermore, non-target metabolomics analysis was used to analyze the changes of metabolites during the efficient synthesis of 1,4-butanediamine by the recombinant strain PKT. The results showed that the total number of differential metabolites detected was 653. Differential metabolite pathway analysis showed that there were 9 differential metabolites in the 1,4-butanediamine synthesis pathway, with 5 involved in its synthesis pathway and 4 in its degradation branch. These differences in metabolites provide a theoretical basis for the modification of recombinant strains. The green production of 1,4- butanediamine by microbial cell factories through fermentation is the future direction. This research provides a theoretical guidance of building high yield 1,4-butanediamine engineering strains.
Xiangxiang Sun, Zongda Li, Yan-Ling Sun et al.· Journal of Genetic Engineeri...· 0 citations
Efficient biosynthesis of 1,3-PDO, a key bio-based chemical, depends on precise regulation of the host metabolic network. In this study, a heterologous CRISPR-Cas12a genome editing system was established and systematically optimized in Klebsiella pneumoniae, enabling efficient and stable genome editing (75-100% efficiency). Using on this platform, by-product pathways were reduced through multi-gene deletions (frdA, poxB, adhE, ldhA, glpK, ptsG and dhaM) to enhance the yield of 1,3-PDO from glycerol. By heterologously expressing GPD1/GPP2 and optimizing promoter, a metabolic network for the "glucose-glycerol-1,3-PDO" pathways was reconstructed. Cometabolism studies indicated that low concentrations of xylose and arabinose as co-substrates enhanced conversion efficiency of glycerol, whereas glucose alleviated metabolic competition. Under cometabolism of glucose and glycerol, the engineered strain K. pneumoniae S2 ΔABEAKGM-1-2 produced 1003.7 mmol/L (76.4 g/L) of 1,3-PDO with a yield of 0.83 mol/mol glycerol, an overall molar yield of 0.78 mol/mol based on total substrate consumption, and a productivity of 27.9 mmol/L/h. When lignocellulosic hydrolysate was used as co-substrate, 981.1 mmol/L (74.7 g/L) of 1,3-PDO was produced with a yield of 0.77 mol/mol glycerol and a productivity of 27.3 mmol/L/h. This study achieved efficient redirection of carbon flux toward 1,3-PDO through systematic metabolic engineering, providing valuable strain resources and technical guidance for the sustainable and cost-effective biomanufacturing of bio-based 1,3-PDO.
Li Wang, Ming-Yang Zhao, Yuan-Ming Ye et al.· Bioresource Technology· 0 citations
O-Succinyl-L-homoserine (OSH) plays a pivotal role in L-methionine biosynthesis. Microbial cell factories for high-yield OSH production have been progressively optimized, achieving substantial improvements in fermentation titers. In this study, a multi-step progressive optimization strategy was adopted to construct a high-yield OSH-producing strain. First, the feedback inhibition of the key enzyme HST was relieved, and the key genes involved in the byproduct metabolic pathways were knocked out. This modification enabled engineered strain to produce 9.77 ± 0.27g/L OSH in shake-flask fermentation. Second, ribosome binding site (RBS) engineering, promoter engineering, and dynamic metabolic regulation were integrated to strengthen and balance the intracellular supply of the two core precursors, L-homoserine and succinyl-CoA. These strategies greatly increased the OSH titer to 18.54 ± 0.03g/L. Finally, global optimization of cofactor and energy optimization was carried out to further enhance strain performance, and the engineered strain OSHM40 achieved the OSH titer of 20.15 ± 0.21g/L via shake-flask cultivation, and 104.09 ± 2.06g/L in a 5-L bioreactor under fed-batch fermentation, with a sugar-acid conversion rate of 64.99% and a volumetric productivity of 1.43g/L/h. Notably, the OSH titer and sugar-acid conversion rate of this strain represent the highest levels reported to date among all plasmid-free OSH-producing strains. The plasmid-free system constructed in this study effectively avoids the plasmid-induced metabolic burden and genetic instability. This work demonstrates the prominent advantages and great application potential of plasmid-free modular engineering for the efficient biosynthesis of OSH and other high-value amino acids.
Si-Min Huang, Xu-Yue He, Ruo-Nan Wang et al.· Journal of Biotechnology· 0 citations
Chemical synthesis of indigo relies on aniline and formaldehyde feedstocks, inevitably generating carcinogenic byproducts and imposing severe environmental burdens, driving the urgent demand for sustainable bio-manufacturing alternatives. As a water-soluble glycoside precursor of indigo, indican circumvents the cytotoxicity and product accumulation issues caused by hydrophobic indigo precipitation, emerging as an ideal intermediate for green indigo production. Herein, we developed an antibiotic- and inducer-free biosynthesis platform using the probiotic Escherichia coli Nissle 1917 (EcN). By harnessing its native high-copy plasmids, constitutive expression of the flavin-containing monooxygenase FMOK223R and glycosyltransferase PtUGT1 was achieved to enable the de novo synthesis of indican. To redirect metabolic flux toward indican accumulation, competing pathways were blocked via inactivation of pheA, tyrA, pykA and ppc, while disruption of the trpR gene alleviated feedback inhibition to the tryptophan biosynthetic pathway. Combined overexpression of rate-limiting genes tnaA and the feedback-resistant trpES40F synergistically enhanced indican accumulation. Further semi-rational mutagenesis of PtUGT1(S112A), coupled with fusion expression optimization, substantially elevated indican titers to 449.3 mg/L in shake-flask cultivation, and crude glycerol-fed-batch fermentation reached a peak titer of 1923.2 mg/L at 32 h. This study demonstrates that the EcN endogenous plasmid engineering eliminates dependence on antibiotics and inducers, establishing a robust and eco-friendly alternative to traditional chemical synthesis.
Li-Jing Mao, Jia-Qi Su, Yu Mo et al.· Bioresources and Bioprocessi...· 0 citations
Vanillin is an important flavor compound widely used in the food, fragrance, and pharmaceutical industries. Current biotransformation processes from ferulic acid or eugenol are limited by high substrate cost and low carbon efficiency, motivating de novo biosynthesis from glucose. This study employed Escherichia coli as the chassis organism to establish a modular vanillin biosynthesis system based on the phenylpropanoid metabolic pathway. Heterologous expression of sam8, sam5, and comt established a biosynthetic module for the sequential conversion of l-tyrosine to p-coumaric acid, then to caffeic acid, and finally to ferulic acid. This module enabled the production of 15.86 mg/L ferulic acid from glucose. Two ferulic acid-to-vanillin modules were compared: a CoA-dependent deacetylation pathway (fcs/ech) and an oxidative decarboxylation pathway (fdc/cso2). With ferulic acid feeding, the deacetylation route produced 445.78 mg/L vanillin, far exceeding the 3.49 mg/L obtained via oxidative decarboxylation. When integrated with the upstream module, the deacetylation pathway enabled de novo vanillin production from glucose at 4.46 mg/L, whereas the oxidative decarboxylation route yielded only 0.46 mg/L, indicating better performance of the former under the tested conditions. Metabolite profiling indicated accumulation of caffeic acid and limited ferulic acid levels, identifying O-methylation and S-adenosyl-L-methionine (SAM) supply as major bottlenecks. Implementation of SAM regeneration modules revealed that mtn overexpression enhanced the vanillin titer by about 3-fold, to 12.36 mg/L, while luxS overexpression had a negligible effect. In summary, this study establishes a functional de novo phenylpropanoid pathway for vanillin in E. coli, underscores the critical role of terminal‑pathway selection, and demonstrates that SAM regeneration effectively improves vanillin production from glucose.
Yue Wang, Tian-Jie Han, Yan-Xiang Bao et al.· Biotechnology and applied bi...· 0 citations
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