ACS Sustainable Chemistry & Engineering
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2025
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IF 7.3
Weiwei Liu,Zichen Yu,Zhichao Chen,Ling Ma,Yanghao Liu,Zhen Zhang,Qingyang Xu
5-Hydroxytryptamine (5-HT), also known as serotonin, plays a critical role in mood regulation, insomnia treatment, and free radical scavenging. Currently, 5-HT production primarily relies on plant extraction, which is challenged by limited raw materials and environmental concerns, emphasizing the need for innovative green production strategies. In this study, we explored a high-performance producer for biosynthesizing 5-HT in Escherichia coli. First, we constructed and optimized the 5-HT biosynthetic pathway to convert L-tryptophan (l-Trp) to 5-HT, avoiding the accumulation of tryptamine and 5-hydroxytryptophan. Moreover, precursor enrichment eliminated the need for l-Trp supplementation and dynamic downregulation of pyruvate kinase balanced growth and production. Further, the metabolic flux between the tetrahydrobiopterin (BH4) module and the hydroxylation-decarboxylation module was optimized, reducing l-Trp accumulation. A novel “GXAA” system was implemented to enhance intracellular NAD(P)H supply, including (1) expression of gdhbsu (glucose dehydrogenase) from Bacillus subtilis, gapXfbr (mutated glyceraldehyde-3-phosphate dehydrogenase) from Corynebacterium glutamicum, and pntAB (pyridine nucleotide transhydrogenase); (2) knockout of ldhA (lactate dehydrogenase). Finally, transcriptomic analysis under high-5-HT stress revealed a potential 5-HT transporter, YhjX. The resulting strain WW15 produced 15.5 g/L 5-HT and a yield of 0.063 g/g glucose through a two-stage fed-batch fermentation, representing the highest titer and yield reported to date.
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BIOTECHNOLOGY AND BIOENGINEERING
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2014
·Vol. 111
·期 12
·2580-2586
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IF 4.164
Jia-Le Yu,Xiao-Xia Xia,Jian-Jiang Zhong,Zhi-Gang Qian
The C6 dicarboxylic acid, adipic acid, is an important platform chemical in industry. Biobased production of adipic acid is a promising alternative to the current petrochemical route. Here, we report biosynthesis of adipic acid using an artificial pathway inspired by the reversal of beta-oxidation of dicarboxylic acids. The biosynthetic pathway comprises condensation of acetyl-CoA and succinyl-CoA to form the C6 backbone and subsequent reduction, dehydration, hydrogenation, and release of adipic acid from its thioester. The pathway was first tested in vitro with reconstituted pathway enzymes and then functionally introduced into Escherichia coli for the biosynthesis and excretion of adipic acid into the culture medium. The production titer was increased by approximately 20-fold through the combination of recruiting enzymes that were more suitable to catalyze the synthetic reactions and increasing availability of the condensation substrates. This work demonstrates direct biosynthesis of adipic acid via non-natural synthetic pathway, which may enable its renewable production. Biotechnol. Bioeng. 2014;111: 2580–2586. © 2014 Wiley Periodicals, Inc.
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SPECTROCHIMICA ACTA PART A-MOLECULAR AND BIOMOLECULAR SPECTROSCOPY
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2025
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IF 4.6
Mengmeng Wang,Li Zheng,Fan Sun,Qingdan Ye,Pei Liang,Kun Pang,Zihong Ye,Yufeng Wang
Aptamer sensors based on surface-enhanced Raman scattering (SERS) technology have demonstrated great potential in the ultrasensitive and rapid detection of Escherichia coli ( E. coli ). Herein, this paper presents a digital SERS aptamer sensor. This sensor integrates ordered nanoscale array synthesis technology and digital analysis technology, enabling highly sensitive and rapid bacterial quantification. The ordered monolayer gold nanosphere arrays (Au NS) can form uniform and dense “hot spots” on the silicon wafer due to their uniform spherical structures and narrow gaps. Moreover, digital SERS is adopted to further optimize the signal uniformity so as to achieve precise quantification. The sensor modules are combined together through base pairing. The aptamers labeled with Raman tags are detached from the complementary DNA due to the competition of the target substance, thus realizing the detection of E. coli . The digital SERS aptamer sensor has been verified to possess excellent selectivity and reproducibility. It has a wide dynamic linear detection range from 1.0 * 10 1 to 1.0 * 10 9 CFU/ml and a detection limit of 0.657 CFU/ml, maintaining excellent specificity even in the presence of mixed bacterial interference. The spiked recoveries in actual samples range from 98.80 % to 99.81 %. Leveraging different aptamers and digital analysis, the sensor holds promise for food safety and environmental monitoring applications.
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BIOTECHNOLOGY LETTERS
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2016
·Vol. 38
·期 6
·1009-1013
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IF 1.639
Kang Le,Bai Yajun,Cai Yujie,Zheng Xiaohui
Objectives To characterize a novel feruloyl esterase from Escherichia coli BL21 DE3. Results The gene encoding BioH was cloned and overexpressed in E. coli . The protein was purified and its catalytic activity was assessed. BioH exhibited feruloyl esterase activity toward a broad range of substrates, and the corresponding kinetic constants for the methyl ferulate, ethyl ferulate, and methyl p -coumarate substrates were: K
m
values of 0.48, 6.3, and 1.9 mM, respectively, and k
cat
/ K
m
values of 9.3, 3.8, and 3.8 mM −1 s −1 , respectively. Conclusions Feruloyl esterase from E. coli was expressed for the first time. BioH was confirmed to be a feruloyl esterase.
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Current Protocols
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2025
·Vol. 5
·期 12
·e70284
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IF 2.2
Jinwei Zhai,Wansen Tan,Lei Ji,Jingjun Hong
This article mainly describes a fermentation and purification method for expressing recombinant collagen protein in Escherichia coli . The method comprises constructing engineered bacteria expressing human type III collagen and adopting a strategy of feeding in batches for high-density fermentation. The rapid proliferation of bacterial cells is promoted at 37°C, and then the culture is inoculated into a fermentation tank with different carbon sources for growth. When glycerol is used as the main carbon source, the yield of recombinant collagen protein can reach 0.25 to 0.40 g/L. The method allows exploration of the differences in recombinant collagen production with different carbon sources in order to identify the most suitable fermentation medium component. The human type III collagen produced by the method has the typical structure of collagen, with high cell adhesion and the stability of tissue structure. Therefore, it can be used as the raw material for various collagen products, especially facial fillers, dressings, freeze-dried fibers, and gels. © 2025 Wiley Periodicals LLC. Basic Protocol : Fermentation and purification of recombinant human type III collagen expressed in Escherichia coli
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MOLECULES
·
2026
·Vol. 31
·期 15
·2721
·
IF 5.1
Yajia Wang,Yue Chen,Meiling Qiu,Shuqin Xu
Three sulfated derivatives were synthesized using microbially derived defructosylated K4 (DK4) polysaccharide as a chondroitin-like polysaccharide backbone. The obtained derivatives were structurally characterized by nuclear magnetic resonance spectroscopy, Fourier-transform infrared spectroscopy, elemental analysis, and disaccharide composition analysis. The results demonstrated that sulfate groups were successfully introduced into the DK4 structure, while the main chondroitin-like polysaccharide backbone of DK4 was largely preserved after sulfation. Further biological evaluation indicated that the sulfated DK4 derivatives differentially altered the expression levels of Ras homolog family member A/Rho-associated coiled-coil-containing protein kinase 1 (RhoA/ROCK1) and myelin basic protein (MBP). This work provides an experimental basis for further elucidating the structure–activity relationship between sulfation patterns and glial cell functions.
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INTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS
·
2026
·
IF 5
Lvyuan Fan,Jingyun Wu,Ziyi Zhang,Liting Cai,Yubin Su,Jiao Fei,Guozhu Ye
Sodium oleate reduces E. coli susceptibility to gentamicin • Sodium oleate enhances fatty acid degradation and elevates acetyl-CoA levels • Fatty acid metabolism-related substances influence antibiotic sensitivity • Reduced membrane potential and porin permeability limit drug uptake The World Health Organization has listed antibiotic resistance as one of the top ten health threats. There is an urgent need to analyze the resistance mechanism to develop novel anti-infection strategies. Fatty acids have been reported to promote antibiotic resistance; however, the underlying mechanism is complex and requires further exploration. To address this, we investigated the effect of sodium oleate-mediated resistance by proteomics. The present study corroborated that sodium oleate decreased the sensitivity of E. coli to gentamicin being visualized by fluorescence imaging, bacterial survival curve and minimum inhibitory concentration (MIC) assays. Proteomic profiles after treatment with gentamicin or sodium oleate were significantly different, with a number of proteins significantly changing. Pathway enrichment analysis showed that fatty acid biosynthesis and the stress response were upregulated under gentamicin treatment, whereas sodium oleate upregulated fatty acid degradation, amino acids metabolism, siderophore transmembrane transporter activity and enterobactin biosynthetic process. The addition of sodium oleate increased the expression levels of genes involved in the fatty acid degradation pathway by qPCR. Additionally, sodium linoleate and sodium palmitate also reduced bacterial sensitivity to gentamicin. The higher level of acetyl-CoA was found, and acetic acid/sodium acetate also reduced bacterial sensitivity to gentamicin, which was associated with protein acetylation. Moreover, sodium oleate caused a decrease in membrane potential and porin permeability, thereby affecting gentamicin uptake. This study provides new insights of fatty acid-mediated resistance, which may contribute to the development of novel strategies for addressing antibiotic resistance. Download: Download high-res image (238KB) Download: Download full-size image
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ACS Synthetic Biology
·
2025
·
IF 3.9
Zhen Zong,Lianghua Xie,Jiaqi Fu,Zhongyang Liu,Wen-Wen Zhou,Wei Chen
Cyanidin-3-O-galactoside (C3Ga), a natural pigment, has various beneficial biological activities and is widely used as a food colorant. However, traditional plant extraction methods are time-consuming and unsustainable. Rapid and sustainable synthesis of C3Ga by engineered microorganisms offers a promising alternative to traditional plant-based methods and deserves to be explored. In this study, the bioproduction of C3Ga by Escherichia coli was achieved for the first time. The biosynthetic pathway of C3Ga from (+)-catechin was constructed by introducing anthocyanidin synthase (ANS) and UDP-galactose:cyanidin galactosyltransferase. Some strategies, including enhancement of the UDP-galactose biosynthesis pathway, identification of efficient ANS, overexpression of the C3Ga transporter, and modulation of multigene expression, were subsequently used to drive the metabolic flux toward C3Ga production. Next, the two-stage process for C3Ga production was optimized to mitigate limitations for further metabolic engineering. Combined with the knockout of β-phosphoglucomutase (ycjU), a newly identified competitive pathway for UDP-galactose, the production of C3Ga finally reached 217.9 mg/L. The strategies used in this study could be applied to the biosynthesis of other anthocyanins and galactosylated natural products.
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JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2026
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IF 6.7
Weiying Zhao,Yanzhe Shang,Meiyu Pan,Peng Fei,Yuying Wang,Yuanchan Luo,Hui Wu
Limonene is a high-value monoterpenoid used in food, pharmaceutical, and fragrance applications. To overcome limitations of conventional production, this study developed a biosynthesis platform using acetate as the sole carbon source. Acetate is an ideal nonfood substrate that can be sustainably derived from industrial waste streams, syngas, and CO2 reduction. Initially, the heterologous synthetic pathway was constructed to yield 0.13 mg/L limonene from acetate. By introducing the mutated EfMVASA110G to enhance catalytic efficiency, along with promoter engineering and substitution of the synthesis substrate, the production titer was enhanced to 49.24 mg/L. Subsequent host optimization through NADPH regeneration and pathway knockout, combined with fermentation optimization, increased the titer to 167.45 mg/L. Ultimately, whole-cell biocatalysis achieved 450.72 mg/L, establishing the highest reported acetate-based limonene titer. Overall, this study establishes a framework for biosynthesis of high-value terpenoids from nonfood feedstocks and demonstrates acetate as a promising carbon source for advanced bioproduction systems.
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mSystems
·
2022
·
IF 7.324
Zhe Zhao,Ying Xu,Bo Jiang,Qingsheng Qi,Ya-Jie Tang,Mo Xian,Jichao Wang,Guang Zhao
The two-component system CpxRA can sense environmental stresses and regulate transcription of a wide range of genes for the purpose of adaptation. Despite extensive research on this system, the identification of the CpxR regulon is not systematic or comprehensive. Herein, genome-wide screening was performed using a position-specific scoring matrix, resulting in the discovery of more than 10,000 putative CpxR binding sites, which provides an extensive and selective set of targets based on sequence. More than half of the candidate genes ultimately selected (73/97) were experimentally confirmed to be CpxR-regulated genes through experimental analysis. These genes are involved in various physiological functions, indicating that the CpxRA system regulates complex cellular processes. The study also found for the first time that the CpxR-regulated genes ydeE, xylE, alx, and galP contribute to Escherichia coli resistance to acid stress, whereas prlF, alx, casA, yacH, ydeE, sbmA, and ampH contribute to E. coli resistance to cationic antimicrobial peptide stress. Among these CpxR-regulated genes, ydeE and alx responded to both stressors. In a similar way, a cationic antimicrobial peptide is capable of directly activating the periplasmic domain of CpxA kinase in vitro, which is consistent with the CpxA response to acid stress. These results greatly expand our understanding of the CpxRA-dependent stress response network in E. coli.IMPORTANCE CpxRA system is found in many pathogens and plays an essential role in sensing environmental signals and transducing information inside cells for adaptation. It usually regulates expression of specific genes in response to different environmental stresses and is important for bacterial pathogenesis. However, systematically identifying CpxRA-regulated genes and elucidating the regulative role of CpxRA in bacteria responding to environmental stress remains challenging. This study discovered more than 10,000 putative CpxR binding sites based on sequence. This bioinformatics approach, combined with experimental assays, allowed the identification of many previously unknown CpxR-regulated genes. Among the novel 73 CpxRA-regulated genes identified in this study, the role of nine of them in contributing to E. coli resistance to acid or cationic antimicrobial peptide stress was studied. The potential correlation between these two environmental stress responses provides insight into the CpxRA-dependent stress response network. This also improves our understanding of environment-bacterium interaction and Gram-negative pathogenesis.
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BIORESOURCE TECHNOLOGY
·
2022
·
IF 11.889
Lian Wang,Ning Li,Shiqin Yu,Jingwen Zhou
Caffeic acid is a phenylpropanoid which is widely used in medical industry. Microbial fermentation provides a green strategy for producing caffeic acid. To improve the capacity for caffeic acid production in Escherichia coli , the competing pathways for l -tyrosine synthesis were knocked out. The biosynthesis pathway of the cofactor FAD and the expression of previously reported polyphenol transporters were enhanced to promote the production of caffeic acid. Transcriptomics analysis was conducted to mine potential transporters that could further enhance the titer of caffeic acid in engineered E. coli . Transcriptomics data of E. coli under caffeic acid and ferulic acid stress showed that 19 transporters were upregulated. Among them, overexpression of ycjP , which was previously identified as a sugar ABC transporter permease, improved the caffeic acid titer to 775.7 mg/L. The caffeic acid titer was further improved to 7922.0 mg/L in a 5-L fermenter, the highest titer achieved by microorganisms.
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Frontiers in Bioengineering and Biotechnology
·
2022
·
IF 6.064
Wang Jing,Chen Qiaoyu,Wang Xin,Chen Kequan,Ouyang Pingkai
D-1,2,4-Butanetriol (BT) has attracted much attention for its various applications in energetic materials and the pharmaceutical industry. Here, a synthetic pathway for the biosynthesis of BT from d-arabinose was constructed and optimized in Escherichia coli. First, E. coli Trans1-T1 was selected for the synthesis of BT. Considering the different performance of the enzymes from different organisms when expressed in E. coli, the synthetic pathway was optimized. After screening two d-arabinose dehydrogenases (ARAs), two d-arabinonate dehydratases (ADs), four 2-keto acid decarboxylases (ADXs), and three aldehyde reductases (ALRs), ADG from Burkholderia sp., AraD from Sulfolobus solfataricus, KivD from Lactococcus lactis IFPL730, and AdhP from E. coli were selected for the bio-production of BT. After 48 h of catalysis, 0.88 g/L BT was produced by the recombinant strain BT5. Once the enzymes were selected for the pathway, metabolic engineering strategy was conducted for further improvement. The final strain BT5ΔyiaEΔycdWΔyagE produced 1.13 g/L BT after catalyzing for 48 h. Finally, the fermentation conditions and characteristics of BT5ΔyiaEΔycdWΔyagE were also evaluated, and then 2.24 g/L BT was obtained after 48 h of catalysis under the optimized conditions. Our work was the first report on the biosynthesis of BT from d-arabinose which provided a potential for the large-scale production of d-glucose-based BT.
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