INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2024
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IF 7.7
Ting Wu,Huashan Sun,Wenyao Wang,Bin Xie,Zhengjie Wang,Jianqi Lu,Anming Xu,Weiliang Dong,Jie Zhou,Min Jiang
Enzymatic PET recycling has emerged as a promising green solution in addition to mechanical recycling, but low soluble expression levels of the inherently hydrophobic PET hydrolases hinder large-scale applications. Here, we propose a novel strategy for enhanced production of FastPETase in Escherichia coli using co-expression of molecular chaperones from Ideonella sakaiensis . Co-expression of cognate DnaK and DnaJ chaperones significantly increased soluble FastPETase expression (up to 2.5-fold), surpassing commercial chaperone plasmids. Furthermore, a combinatorial approach employing co-expression of DnaK/DnaJ chaperones and fusion of FastPETase with the VNp6-tag significantly boosted FastPETase secretion, yielding over 2 g/L of target protein in a 5-l bioreactor. Notably, the crude FastPETase in fermentation broth displayed comparable PET hydrolysis effects to the purified enzyme. This work not only provides new insights into the process of chaperones in protein folding but also suggests a novel and efficient strategy for producing recombinant proteins.
研究领域:
生物化学
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环境科学
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蛋白质工程
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PubMed
高影响因子
JOURNAL OF BIOTECHNOLOGY
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2015
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IF 2.871
Xiaolin Pei,Qiuyan Wang,Lijun Meng,Jing Li,Zhengfen Yang,Xiaopu Yin,Lirong Yang,Shaoyun Chen,Jianping Wu
Nitrile hydratase (NHase) is an important industrial enzyme that biosynthesizes high-value amides. However, most of NHases expressed in Escherichia coli easily aggregate to inactive inclusion bodies unless the induction temperature is reduced to approximately 20 °C. The NHase from Aurantimonas manganoxydans has been functionally expressed in E. coli , and exhibits considerable potential for the production of nicotinamide in industrial application. In this study, the effects of chaperones including GroEL/ES, Dnak/J-GrpE and trigger factor on the expression of the recombinant Co-type NHase were investigated. The results indicate that three chaperones can significantly promote the active expression of the recombinant NHase at 30 °C. The total NHase activities reached to 263 and 155 U/ml in shake flasks when the NHase was co-expressed with GroEL/ES and DnaK/J-GrpE, which were 52- and 31-fold higher than the observed activities without chaperones, respectively. This increase is possibly due to the soluble expression of the recombinant NHase assisted by molecular chaperones. Furthermore, GroEL/ES and DnaK/J-GrpE were determined to promote the maturation of the Co-type NHase in E. coli under the absence of the parental activator gene. These knowledge regarding the chaperones effect on the NHase expression are useful for understanding the biosynthesis of Co-type NHase.
研究领域:
生物化学
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工业微生物学
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PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2024
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IF 5.7
Yao Lu,Di Liu,Long Wang,Yongai Ma,Tai-Ping Fan,Huaxiang Deng,Yujie Cai
(−)-α-Bisabolol exhibits analgesic, anti-inflammatory, and skin-soothing properties and is widely applied in the cosmetic and pharmaceutical industries. The use of plant essential oil distillation or chemical synthesis to produce (−)-α-bisabolol is both inefficient and unsustainable. Currently, the microbial production of (−)-α-bisabolol mainly relies on Escherichia coli and Saccharomyces cerevisiae as chassis strains; however, high concentrations of (−)-α-bisabolol have certain toxicity to the strain. This study uses synthetic biology and metabolic engineering strategies to redesign a solvent-tolerant Serratia marcescens for the efficient production of (−)-α-bisabolol. By introducing the Haloarchaea-type mevalonate (MVA) pathway and the (−)-α-bisabolol biosynthesis pathway, we successfully constructed a strain capable of producing (−)-α-bisabolol. The coexpression of the chaperone protein DnaK/J significantly enhanced the soluble expression of the (−)-α-bisabolol synthase, resulting in a 10% increase in (−)-α-bisabolol titer. Furthermore, knockout of the PhoA gene, which reduced the formation of the byproduct farnesol (FOH), further increased the (−)-α-bisabolol titer to 3.21 g/L. In a 5 L bioreactor, S. marcescens achieved a final (−)-α-bisabolol titer of 30.2 g/L, representing the highest titer reported to date. This research provides guidance for the production of (−)-α-bisabolol in nonmodel microorganisms without the requirement for induction.
研究领域:
合成生物学
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代谢工程
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微生物学
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PubMed
高影响因子
BIOPROCESS AND BIOSYSTEMS ENGINEERING
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2025
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IF 3.6
Jin Li-Qun,Liu Meng-Dan,Guan Zi-Yu,Li Yi-Xin,Xue Ya-Ping,Liu Zhi-Qiang,Zheng Yu-Guo
DAAO is applied as a potential catalyst in the biosynthesis of L-PPT. However, its low solubility expression constrains its broader industrial application. Herein, a novel DAAO derived from Cladophialophora carrionii ( Cc DAAO) was identified, which demonstrated superior catalytic performance toward D-Ala (specific activity: 106.38 ± 1.21 U/mg, K m : 1.56 ± 0.06 mM), along with remarkable thermostability and broad substrate spectrum. Under optimal culture conditions, the soluble expression level of Cc DAAO was enhanced through a co-expression strategy with molecular chaperones, and the enzyme activity increased by 36.3% compared with the initial level. Subsequently, Cc DAAO was constructed as a fusion protein (CGD) with catalase from Geobacillus sp. CHB1 ( Gb CAT) and applied in a D-amino acid aminotransferase (DAAT)-mediated cascade system. In a 2 L reaction system, this cascade system achieved complete conversion (> 99%) of 1 M D,L-PPT within 8 h, exhibiting a yield of 11.26 g/L/h for PPO, which represents a significant improvement over existing reports. This study presents a promising practical approach for the industrial production of optically pure L-PPT.
研究领域:
生物催化
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酶工程
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药物合成
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工业微生物
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蛋白质工程
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PubMed
高影响因子
PROCESS BIOCHEMISTRY
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2017
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IF 2.497
Hui Zhang,Mengya Li,Jie Li,Guangli Wang,Feng Li,Minghua Xiong
Nitrile hydratase (NHase) has attracted substantial attention for industrial applications to produce large-scale amides. Several NHases have been investigated for functional expression in Escherichia coli ( E. coli ). A Fe-type NHase was obtained from an acetamiprid-degrading bacterium, Pseudoxanthomonas sp. AAP-7 and functionally expressed in E. coli BL21 (DE3). No significant NHase activity was detected from the E. coli expressing either the NHase gene alone or NHase and P46K genes transcribed as one unit. Purified recombinant NHase, co-expressed with P46K on two separate plasmids, exhibited the maximal enzyme activity. Furthermore, a GST tag attached to the N-terminus of α subunit resulted in a slight increase in the solubility and stability of NHase compared with a His tag at the C-terminus of β subunit. When co-expressed with the chaperones GroEL-GroES, the yield of the soluble recombinant NHase was improved substantially, while a small decrease in NHase activity was observed. The putative activator P46K was strictly required for production of the recombinant NHase for full enzyme activity, although the chaperones GroEL-GroES appeared to assist NHase to fold properly. This study of the expression of a fully active Fe-type NHase would provide another example to enhance our understanding of NHase biosynthesis.
研究领域:
生物化学
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微生物学
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酶学
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工业生物技术
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DOI
高影响因子
Autophagy
·
2024
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IF 14.6
Tian Zhang,Ke-Gang Linghu,Jia Tan,Mingming Wang,Diao Chen,Yan Shen,Junchao Wu,Mingjun Shi,Yuxia Zhou,Lei Tang,Lirong Liu,Zheng-Hong Qin,Bing Guo
Obesity is one of the most common metabolic diseases around the world, which is distinguished by the abnormal buildup of triglycerides within adipose cells. Recent research has revealed that autophagy regulates lipid mobilization to maintain energy balance. TIGAR (Trp53 induced glycolysis regulatory phosphatase) has been identified as a glycolysis inhibitor, whether it plays a role in the metabolism of lipids is unknown. Here, we found that TIGAR transgenic (TIGAR+/+) mice exhibited increased fat mass and trended to obesity phenotype. Non-target metabolomics showed that TIGAR caused the dysregulation of the metabolism profile. The quantitative transcriptome sequencing identified an increased levels of LRRK2 and RAB7B in the adipose tissue of TIGAR+/+ mice. It was confirmed in vitro that TIGAR overexpression increased the levels of LRRK2 by inhibiting polyubiquitination degradation, thereby suppressing macroautophagy and chaperone-mediated autophagy (CMA) while increasing lipid accumulation which were reversed by the LRRK2 inhibitor DNL201. Furthermore, TIGAR drove LRRK2 to interact with RAB7B for suppressing lysosomal degradation of lipid droplets, while the increased lipid droplets in adipocytes were blocked by the RAB7B inhibitor ML282. Additionally, fat-specific TIGAR knockdown of TIGAR+/+ mice alleviated the symptoms of obesity, and adipose tissues-targeting superiority DNL201 nano-emulsion counteracted the obesity phenotype in TIGAR+/+ mice. In summary, the current results indicated that TIGAR performed a vital function in the lipid metabolism through LRRK2-mediated negative regulation of macroautophagy and CMA in adipocyte. The findings suggest that TIGAR has the potential to serve as a viable therapeutic target for treating obesity and its associated metabolic dysfunction.
研究领域:
代谢
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分子生物学
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细胞生物学
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药理学
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PubMed
高影响因子
JOURNAL OF VIROLOGY
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2025
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IF 3.8
Qisheng Qian,Shuang-shuang Zhao,Lei Yang,Guangxu Xing,Yumei Chen,Chao Liang,Haili Wang,Rui Li,Songlin Qiao,Aiping Wang,Gaiping Zhang
Porcine epidemic diarrhea (PED) is a highly pathogenic and infectious intestinal disease caused by the PED virus (PEDV) and has inflicted substantial economic losses on the global swine industry. Therefore, it is imperative to explore appropriate targets to restrain PEDV infection. PEDV spike (S) protein is crucial for viral infection and is regarded as an ideal target for the development of vaccines and antiviral therapeutics. Palmitoylation is a significant post-translational modification implicated in multiple viral replication cycles. Despite the fact that palmitoylation of certain coronavirus S proteins has been reported, the specific biological significance and underlying molecular mechanisms of PEDV S protein palmitoylation have not been fully defined. In the present study, we uncover that palmitoylation enhances the stability of PEDV S protein to promote viral proliferation. Mechanistically, we identify that a cysteine-rich region within the cytoplasmic tail of PEDV S protein is palmitoylated by the zinc finger Asp-His-His-Cys domain palmitoyltransferase 5 (ZDHHC5). We further illustrate that palmitoylation prevents the recognition of Lys-Phe-Glu-Arg-Gln (KFERQ)-like motif in PEDV S protein by heat shock cognate protein of 70 kDa (HSC70), thereby antagonizing its degradation via chaperone-mediated autophagy (CMA). Collectively, our findings underscore the importance of palmitoylation for PEDV pathogenesis and provide prospective targets for the development of antiviral interventions.
研究领域:
病毒学
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分子生物学
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PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2022
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IF 8.025
Xinyi Wang,Xinxin Feng,Rui Xue,Hong Xu,Rui Wang,Lujia Zhang,Sha Li
Mussel foot proteins (Mfps) display application potential with strong adhesion, enabling mussels to adhere firmly to various surfaces. Mytilus galloprovincialis foot protein 3B (Mgfp-3B) exhibits this characteristic remarkably. However, it remains a challenge for further research due to the low soluble expression of heterologous production. In this study, a small ubiquitin-related modifier (SUMO) and thioredoxin A (TrxA), which catalyzed the proper folding of disulfide bridges, were selected to increase the soluble expression of mfps. An additional ribosome binding site was introduced between the molecular chaperones and Mgfp-3B (fp-3) to form a bicistronic translation-coupled expression vector for co-expression. The results revealed that the combination of SUMO-TrxA increased the soluble expression of fp-3 by 18.07 %. Furthermore, the SUMO-TrxA also boosted the soluble expression of hybrid mfps Mgfp-3B-Mfp-1 (fp-3-1) by 11.29 %, Mgfp-3B-Mgfp-3B (fp-3-3) by 19.91 %, and Mgfp-3B-Mgfp-5 (fp-3-5) by 14.03 %. Ultimately, by high cell density cultivation in a 5 L bioreactor, the yields of fp-3, fp-3-3, and fp-3-5 co-expressed with SUMO-TrxA reached 217.75 mg/L, 127.2 mg/L, and 97.28 mg/L, respectively. Consequently, soluble production of mfps holds great potential for the sustainable supply of protein adhesive materials.
研究领域:
海洋生物学
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生物技术
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蛋白质工程
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DOI
PubMed
高影响因子
Stem Cell Research & Therapy
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2021
·Vol. 12
·期 1
·1-13
·
IF 6.832
He Qiting,Qin Ruixi,Glowacki Julie,Zhou Shuanhu,Shi Jie,Wang Shaoyi,Gao Yuan,Cheng Lei
Background Vitamin D is important for the mineralization of bones by stimulating osteoblast differentiation of bone marrow mesenchymal stem cells (BMMSCs). BMMSCs are a target of vitamin D action, and the metabolism of 25(OH)D3 to biologically active 1α,25(OH)2D3 in BMMSCs promotes osteoblastogenesis in an autocrine/paracrine manner. Our previous study with human BMMSCs showed that megalin is required for the 25(OH)D3-DBP complex to enter cells and for 25(OH)D3 to stimulate osteoblast differentiation in BMMSCs. Furthermore, we reported that leptin up-regulates megalin in those cells. Leptin is a known inhibitor of PI3K/AKT-dependent chaperone-mediated autophagy (CMA). In this study, we tested the hypothesis that leptin acts synergistically with 25(OH)D3 to promote osteoblastogenesis in rat BMMSCs by a mechanism that entails inhibition of PI3K/AKT-dependent CMA.Methods BMMSCs were isolated from rat bone marrow (4-week-old male SD rats); qRT-PCR and western immunoblots or immunofluorescence were used to evaluate the expression of megalin, ALP, COL1A1, RUNX2, OSX, OSP, and CMA in rBMMSCs. The osteoblast differentiation was evaluated by ALP activity, ALP staining, and calcium deposition. The viability of rBMMSCs was assessed with the CCK-8 kit. Biosynthesis of 1α,25(OH)2D3 was measured by a Rat 1α,25(OH)2D3 ELISA Kit.Results The combination of leptin and 25(OH)D3 treatment significantly enhanced osteoblast differentiation as shown by ALP activity, ALP staining, and calcium deposition, the expression of osteogenic genes ALP, COL1A1, RUNX2, OSX, and OSP by qRT-PCR and western immunoblots in rBMMSCs. Leptin enhanced the expression of megalin and synthesis of 1α,25(OH)2D3 in rBMMSCs. Our data showed that leptin inhibited CMA activity of rBMMSCs by activating PI3K/AKT signal pathway; the ability of leptin to enhance 25(OH)D3 promoted osteoblast differentiation of rBMMSCs was weakened by the PI3K/AKT signal pathway inhibitor.Conclusion sOur data reveal the mechanism by which leptin and 25(OH)D3 promote osteoblast differentiation in rBMMSCs. Leptin promoted the expression of megalin by inhibiting CMA, increased the utilization of 25(OH)D3 by rBMMSCs, and enhanced the ability of 25(OH)D3 to induce osteoblast differentiation of rBMMSCs. PI3K/AKT is at least partially involved in the regulation of CMA. These data indicate the importance of megalin in BMMSCs for vitamin D’s role in skeletal health.
研究领域:
细胞生物学
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分子生物学
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内分泌学
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骨骼生物学
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DOI
PubMed
高影响因子
Science Advances
·
2025
·Vol. 11
·期 38
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IF 12.5
Wenjing Wang,Xue Xia,Ziqiang Zhang,Qingya Zhu,Yu Chen,Yao Zhang,Lin Chen,Chun Mao,Mimi Wan
Aggregation of α-synuclein (α-syn) represents a pathogenic hallmark of Parkinson’s disease (PD). Using exogenous molecular chaperone systems has the potential to stabilize native conformations and inhibit the aberrant aggregation of α-syn, yet inefficient blood-brain barrier (BBB) penetration and insufficient accumulation at PD sites limit their application. Herein, we developed chemotactic chaperone nanomotors (CNMs) that exploit the unique pathological microenvironment of PD lesions, characterized by elevated inducible nitric oxide synthase (iNOS) levels. The CNMs consist of a chemotactic-targeting module capable of sensing an iNOS concentration gradient, an α-syn–specific recognition module with conformation-sensitive binding domains, and a protein regulatory module with a hydrophobic microdomain that can stabilize α-syn and prevent misfolding. Results demonstrate that CNMs can achieve active chemotactic navigation across BBB and dual modulation of α-syn proteostasis through aggregate dissolution and prevention of misfolding. CNMs not only supplement exogenous chaperone activity but also restore endogenous protein stabilization mechanisms, concurrently reducing neuroinflammatory markers, promoting the application of nanochaperones for PD treatment.
研究领域:
神经生物学
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纳米医学
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分子生物学
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转化医学
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神经退行性疾病
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药物递送系统
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蛋白质折叠
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PubMed
高影响因子
JOURNAL OF ALZHEIMERS DISEASE
·
2015
·Vol. 49
·期 3
·829-844
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IF 4.151
Shan Ye,Wang Dan-Dan,Xu Yu-Xia,Wang Chu,Cao Lan,Liu Yun-Sheng,Zhu Cui-Qing
Stress is an important risk factor of Alzheimer’s disease (AD). It has been evidenced that stress could induce tau phosphorylation and increase tau insolubility in brain; however, little is known about the interactional effect of stress with aging on tauopathy. Therefore, we explored the effects of aging on stress-induced tauopathy and the potential mechanism in mouse model of chronic restraint stress (CRS). Here we found that in general, the level of phosphorylated tau (P-tau) was higher in brain of middle-aged mice than that in adult mice under physiological conditions. CRS-induced tau phosphorylation and its insolubility were more prominent in middle-aged mice. The increase of AT8-labeled insoluble P-tau was dramatic in middle-aged mice, which was highly ubiquitinated but did not form PHF structures. The levels of chaperones were relatively lower in middle-aged mice brain; CRS further reduced the expression, especially for HDJ2/HSP40. CRS also suppressed the expression of Pin1, the peptidylprolyl cis/trans isomerase, in middle-aged mice but not in adult mice. Downregulation of HSP40 or Pin1 caused an increase of transfected extraneous tau in 293 cells. Rosmarinic acid (RA) could effectively suppress the elevation of P-tau and insoluble P-tau formation induced by CRS, and reversed the abnormal changes of chaperones and Pin1 particularly in middle-aged mice. Taken together, our findings provided evidence that aging could be a promoting factor in stress-induced tauopathy, which was relevant with malregulation of chaperones and Pin1, and RA might be a promising beneficial agent for stress-induced tauopathy.
研究领域:
神经科学
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药理学
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老年学
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分子生物学
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DOI
PubMed
高影响因子
Polymers
·
2024
·Vol. 16
·期 15
·2179
·
IF 4.7
Yazhen Liao,Xiaoxue Yin,Wenqian Liu,Zhenrui Du,Jie Du
Adenosine is an endogenous molecule that plays a vital role in biological processes. Research indicates that abnormal adenosine levels are associated with a range of diseases. The development of sensors capable of detecting adenosine is pivotal for early diagnosis of disease. For example, elevated adenosine levels are closely associated with the onset and progression of cancer. In this study, we designed a novel DNA biosensor utilizing chaperone copolymer-assisted catalytic hairpin assembly for highly sensitive detection of adenosine. The functional probe comprises streptavidin magnetic beads, an aptamer, and a catalytic chain. In the presence of adenosine, it selectively binds to the aptamer, displacing the catalytic chain into the solution. The cyclic portion of H1 hybridizes with the catalytic strand, while H2 hybridizes with the exposed H1 fragment to form an H1/H2 complex containing a G-quadruplex. Thioflavin T binds specifically to the G-quadruplex, generating a fluorescent signal. As a nucleic acid chaperone, PLL-g-Dex expedites the strand exchange reaction, enhancing the efficiency of catalytic hairpin assembly, thus amplifying the signal and reducing detection time. The optimal detection conditions were determined to be a temperature of 25 °C and a reaction time of 10 min. Demonstrating remarkable sensitivity and selectivity, the sensor achieved a lowest limit of detection of 9.82 nM. Furthermore, it exhibited resilience to interference in complex environments such as serum, presenting an effective approach for rapid and sensitive adenosine detection.
研究领域:
生物化学
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分析化学
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生物技术
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分子生物学
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产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）