APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
·
2016
·Vol. 181
·期 3
·972-985
·
IF 1.606
Wu Hua-Lei,Zhang Jian-Dong,Zhang Chao-Feng,Fan Xiao-Jun,Chang Hong-Hong,Wei Wen-Long
Four uncharacterized ω-transaminases (ωTAs) from Pseudomonas putida NBRC 14164 have been identified and cloned from the pool of fully sequenced genomes. The genes were functionally expressed in Escherichia coli BL21, and the enzymes were purified and characterized. Four TAs showed highly ( S )-selective ωTA activity and converted ( S )-α-methylbenzylamine and pyruvate to acetophenone and l -Ala. The maximum activity of cloned enzymes was in the pH range of 8.0– 8.5 (Pp36420), 8.5 –9. 5 (Pp21050), 9 .0– 9.5 (PpspuC), and 9.5 –1 0 . 5 (PpbauA), and the optimal temperatures were at 35 °C (Pp36420, Pp21050, and PpspuC) and 50 °C (PpbauA), respectively, with K
M of 161.3 mM (Pp21050), 136.7 mM (PpbauA), 398.5 mM (Pp36420), and 130.9 mM (PpspuC) and yielding a catalytic efficiency k
cat / K
M of 0.015, 0.003, 0.012, and 0.023 mM −1 s −1 . Several racemic amines and amino alcohols were resolved by the cloned ωTAs; perfect conversions (48–50 %) were obtained by at least one enzyme, and the residual substrates were left with 97–99 % ee. Kinetic resolution of racemic phenylglycinol was done with PpspuC in a 100-mL scale. Enaniomeric excess of ( S )-phenylglycinol reached 99 % with 45 % isolated yield. The high enantioselectivity and large substrate spectra of the cloned PpTAs showed an attractive potency for biotechnology application in production of chiral amines and amino alcohols.
研究领域:
生物化学
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酶工程
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生物技术
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PubMed
高影响因子
Journal of Environmental Chemical Engineering
·
2022
·
IF 7.968
Xiaoyan Qi,Huimin Wang,Xinyu Gao,Lei Zhang,Sen wang,Xia Wang,Ping Xu
Environmental pollution caused by aromatic compounds (ACs) has long stimulated the emergence of creative bioremediation strategies. Pseudomonas putida B6–2, an aerobic ACs degrading bacterium, successfully coupled ACs degradation with power generation in microbial fuel cells (MFCs) when a nickel-coated sponge with a three-dimensional macroporous structure served as the anode. The simultaneous removal of coexisting ACs pollutants including biphenyl (BP), carbazole (CA), and dibenzothiophene (DBT) were achieved in MFCs, accompanied by the efficient power densities of 469 mW m −2 , 499 mW m −2 , 541 mW m −2 , and 587 mW m −2 for single (BP), binary (BP+DBT or BP+CA), and ternary (BP+DBT+CA) ACs fueled MFCs, respectively. Degradation dynamics analysis indicated that the degradation of BP, CA, and DBT was consistent with the pseudo-first order kinetic model in the ternary ACs fueled MFC, and BP had a higher degradation rate ( k obs =0.0337 h –1 ) than DBT ( k obs =0.0201 h –1 ) and CA ( k obs =0.0149 h –1 ). It is the first time that P. putida B6–2 was discovered as an electroactive bacterium with high extracellular electron transfer (EET) capability. The EET preliminary analysis showed that P. putida B6–2 might transfer extracellular electrons by redox mediators and microbial nanowires . The mediators may play a key role in indirect EET at the start-up period of the MFCs, while the nanowires may involve in direct EET during the whole discharge process of the MFCs. The interesting findings may lay a foundation for further exploration of the EET of P. putida B6–2 and bioremediation of ACs pollutants.
研究领域:
环境科学
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微生物学
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高影响因子
INTERNATIONAL MICROBIOLOGY
·
2024
·
IF 2.3
Zhang Mingle,He Zhimin,Xu Xiaoyi,Ji Fan,Wang Bin
This study investigates the advantages of combined microbial degradation of polycyclic aromatic hydrocarbons (PAHs) in reducing the inhibitory effects of high-concentration eluents commonly used in soil washing. A microbial synergistic strategy was proposed using Arthrobacter sp. SZ-3 and Pseudomonas putida B6-2 as the key bacteria in the presence of Tween 80. The results show that in systems with Tween 80, the SZ-3 strain exhibits a strong capacity to degrade three types of PAH compounds, while the B6-2 strain follows multiple degradation pathways. Mixed bacteria achieved degradation rates 60.70% higher than single bacteria at varying concentrations of Tween 80. Additionally, the average growth rates of mixed bacteria increased by 1.17–1.37 times, aligning with the changes in the functional group. Protein activity detection within each degradation system corresponded with growth quantity and the cyclic variation characteristics of ETS enzyme activity. Notably, the ETS activity of mixed bacteria was 150% higher than that of single bacteria. At a Tween 80 concentration of 500 mg/L, the degradation rates of PAHs (Phe, Flu, Pyr) by mixed bacteria were significantly higher than those by single bacteria. The catechol 1,2-dioxygenase activity of mixed bacteria was 2.30 times higher than that of single bacteria. While Tween 80 did not alter the PAH degradation pathways, it significantly influenced the accumulation amount and duration of the characteristic intermediate product. This provides a reference for the remediation of recalcitrant pollutants under conditions involving high-concentration surfactants.
研究领域:
环境微生物学
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污染修复
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PubMed
高影响因子
ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH
·
2022
·Vol. 30
·期 10
·28227-28237
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IF 5.19
Liu Huan-Huan,Yang Lei,Guo Lin-Kai,Tu Li-Xin,Li Xiao-Tong,Wang Jia,Ren Yong-Xiang
The occurrence of metal oxide nanoparticles (NPs) in wastewater treatment plants (WWTPs) has raised great concerns about their adverse impacts on nitrification performance. In this study, a heterotrophic nitrifying bacterium Pseudomonas putida strain NP5 showed strong resistance against TiO 2 and NiO NPs. Under 5–50 mg/L NP stress, cell viability was still normal, and the final nutrient removal rates, always higher than 80%, were slightly inhibited. Correspondingly, the PO 4 3− -P removal rates were almost the same as those observed in the control test. Although the enzyme assay demonstrated ammonia monooxygenase and hydroxylamine oxidoreductase activities markedly decreased caused by increased reactive oxygen species (ROS) level under 50 mg/L NPs stress. The total antioxidant capability of NP5 could eliminate excess ROS to maintain a balance between oxidants and antioxidants. Besides, in response to the escalating burden of NPs, strain NP5 tended to secrete more extracellular polymeric substances (EPS), which could protect cell from being damaged by binding to ions and coating. Thus, the strong NP resistance of NP5 would help to overcome the vulnerability of the nitrification process in WWTPs.
研究领域:
环境微生物学
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纳米毒理学
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污水处理
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微生物生理学
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DOI
高影响因子
BIORESOURCE TECHNOLOGY
·
2019
·
IF 6.669
Lei Yang,Xu-Hui Wang,Shen Cui,Yong-Xiang Ren,Jie Yu,Ning Chen,Qian Xiao,Lin-Kai Guo,Rui-Hua Wang
A novel strain NP5 with efficient heterotrophic nitrification, aerobic denitrification and phosphorus accumulation ability was isolated and identified as Pseudomonas putida strain NP5. The removed ammonium and phosphate were mainly converted into intracellular components by assimilation, and negligible nitrification intermediates and N 2 O were accumulated during heterotrophic nitrification. In addition, the optimal conditions for nutrient removal were: succinate as carbon source, C/N 10, P/N 0.2, temperature 30 °C, salinity 0% and shaking speed 160 rpm. Besides, strain NP5 possessed an exceptional heavy metal and nanoparticles resistance. Cr 6+ was found to be the most toxic among the tested metals, and it could be removed simultaneously. Moreover, an obvious phosphorus release was observed under anaerobic condition, and repeated exposure to the anaerobic/aerobic conditions could significantly improve the nutrient removal. Furthermore, the successful expression of key enzymes for nitrogen and phosphorous removal provided additional evidence for possibility of simultaneous nitrification, denitrification and phosphorus removal.
研究领域:
环境微生物学
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废水处理
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生物修复
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PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2022
·
IF 5.895
Ya’nan Shi,Rongshan Li,Jie Zheng,Yubin Xue,Yong Tao,Bo Yu
Bio-based propionate attracts increasing attention owing to its green nature and specific food additive market. To date, the time-consuming and costly fermentation process by strict anaerobes makes propionate production not ideal. In this study, we designed a new route for propionate production, in which 1,2-propanediol was first dehydrated to propionaldehyde and then to propionate by taking advantage of the robust oxidization capacity of the Pseudomonas putida KT2440 strain. The high atom economy (0.97 g/g) in this proposed pathway is more advantageous than the previous l-threonine-derived route (0.62 g/g). The molecular mechanism of the extraordinary oxidation capacity of P. putida KT2440 was first deciphered. The propionate production was realized in P. putida KT2440 by screening suitable glycerol dehydratases and optimizing the expression to eliminate the formation of 1-propanol and the accumulation of the intermediate propionaldehyde. The engineered strain produced propionate with a molar conversion rate of >99% from 1,2-propanediol. A high titer of 46.5 g/L pure propionic acid with a productivity of 1.55 g/L/h and a mass yield of 0.96 g/g was achieved in fed-batch biotransformation. Thus, this study provides another idea for the production of high-purity bio-based propionate from renewable materials with high atom economy.
研究领域:
生物技术
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微生物学
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化学工程
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代谢工程
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PubMed
高影响因子
JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC
·
2013
·
IF 2.823
Xiaolin Pei,Lirong Yang,Gang Xu,Qiuyan Wang,Jianping Wu
Microbial nitrile hydratases (NHases) are important industrial catalysts to produce valuable amides. However, only some NHase genes have been reported and studied at the molecular level. In this study, we developed a genome mining method to discover Fe-type NHases from GenBank. The putative NHase gene from Pseudomonas putida F1 was cloned and functionally expressed in Escherichia coli BL21 (DE3) by assisting of a putative activator gene adjacent to β-subunit region. Three recombinant plasmids containing NHase gene or the activator gene were designed and constructed. Maximal enzyme activity was obtained when the structural and activator genes were transcribed as one unit in plasmid pCDFDuet-1 at 18 °C. However, the expressed product did not show any NHase activity when the downstream activator gene was ignored, and the product completely existed in insoluble inclusion body. The activator gene might be involved in protein folding of the α- and β-subunits of NHase. In addition, the Fe-type NHase exhibited broad substrate specificity. The enzyme can efficiently hydrate aromatic nitriles, such as 3-cyanopyridine, 4-cyanopyridine, and benzonitrile, asides from aliphatic nitriles preferentially. Therefore, the recombinant NHase shows potential applications in some amides preparation.
研究领域:
生物化学
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微生物学
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工业酶学
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高影响因子
SCIENCE OF THE TOTAL ENVIRONMENT
·
2023
·
IF 9.8
Kaiyue Huo,Siqi Wang,Wanwan Zhao,Hongfu Guo,Weini Xiong,Ruihua Liu,Chao Yang
Currently, 1,2-dichloroethane (DCA) is frequently detected in groundwater and has been listed as a potential human carcinogen by the U.S. EPA. Owing to its toxicity and recalcitrant nature, inefficient DCA mineralization has become a bottleneck of DCA bioremediation. In this study, the first engineered DCA-mineralizing strain KTU-P8DCA was constructed by functional assembly of DCA degradation pathway and enhancing pathway expression with a strong promoter P8 in the biosafety strain Pseudomonas putida KT2440. Strain KTU-P8DCA can metabolize DCA to produce CO 2 and utilize DCA as the sole carbon source for cell growth by quantifying 13 C stable isotope ratios in collected CO 2 and in lyophilized cells. Strain KTU-P8DCA exhibited superior tolerance to high concentrations of DCA. Excellent genetic stability was also observed in continuous passage culture. Therefore, strain KTU-P8DCA has enormous potential for use in bioremediation of sites heavily contaminated with DCA. In the future, our strategy for pathway construction and optimization is expected to be developed as a standard pipeline for creating a wide variety of new contaminants-mineralizing microorganisms. The present study also highlights the power of synthetic biology in creating novel degraders for environmental remediation.
研究领域:
环境科学
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合成生物学
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微生物学
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PubMed
高影响因子
Frontiers in Bioengineering and Biotechnology
·
2020
·
IF 3.644
Sun Jun,Lu Li-Bing,Liang Tian-Xin,Yang Li-Rong,Wu Jian-Ping
Pseudomonas putida (P. putida) KT2440 is a paradigmatic environmental-bacterium that possesses significant potential in synthetic biology, metabolic engineering and biodegradation applications. However, most genome editing methods of P. putida KT2440 depend on heterologous repair proteins and the provision of donor DNA templates, which is laborious and inefficient. In this report, an efficient cytosine base editing system was established by using cytidine deaminase (APOBEC1), enhanced specificity Cas9 nickase (eSpCas9ppD10A) and the uracil DNA glycosylase inhibitor (UGI). This constructed base editor converts C-G into T-A in the absence of DNA strands breaks and donor DNA templates. By introducing a premature stop codon in target spacers, we successfully applied this system for gene inactivation with an efficiency of 25–100% in various Pseudomonas species, including P. putida KT2440, P. aeruginosa PAO1, P. fluorescens Pf-5 and P. entomophila L48. We engineered an eSpCas9ppD10A-NG variant with a NG protospacer adjacent motif to expand base editing candidate sites. By modifying the APOBEC1 domain, we successfully narrowed the editable window to increase gene inactivation efficiency in cytidine-rich spacers. Additionally, multiplex base editing in double and triple loci was achieved with mutation efficiencies of 90–100% and 25–35%, respectively. Taken together, the establishment of a fast, convenient and universal base editing system will accelerate the pace of future research undertaken with P. putida KT2440 and other Pseudomonas species.
研究领域:
合成生物学
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代谢工程
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微生物遗传学
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生物技术
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DOI
PubMed
高影响因子
JOURNAL OF ENVIRONMENTAL MANAGEMENT
·
2025
·
IF 8.4
Qing Qiu,Xuejian Sun,Han Li,Fenglin Zhang,Dandan Zhou,Kejian Tian,Xinwen Zhang,Hongliang Huo
Polystyrene (PS), being resistant to biodegradation, poses a significant environmental challenge. This study isolated highly effective lignin-degrading microbial consortia from samples collected at six sites rich in lignin-degrading bacteria. After 360 days of enrichment, a stable lignin-degrading microbial consortium, LQX-03, was successfully established. LQX-03 demonstrated notable degradation efficiency not only for lignin (21-day degradation rate of 54.6 %) but also for PS (21-day degradation rate of 13.1 %). Importantly, PS-induced LQX-03 communities overlapped with the original lignin communities in 13 genera, revealing a close relationship between the degrading microbial compositions of the two substrates.Additionally, Pseudomonas putida Q1, isolated from LQX-03, exhibited significant capability in simultaneously degrading lignin and PS, achieving degradation rates of 36.1 % and 4.4 %, respectively. The strain was also able to alter the functional groups of PS, increasing its hydrophilicity. Gene and enzyme expression analyses revealed that key lignin-degrading enzymes, such as laccase (CopA) and DyP peroxidase, were significantly upregulated when PS was the sole substrate. Laccase CopA expression increased by 1.76-fold and 1.41-fold, while DyP expression increased by 1.24-fold. These results indicate that these enzymes likely play a crucial role in PS depolymerization and biodegradation. Further molecular docking analysis confirmed that laccase CopA could bind to PS. In summary, this study provides preliminary insights into the potential links between lignin-degrading and plastic-degrading microorganisms and their enzymes. It suggests that the biodegradation of synthetic plastics may rely on ancient natural lignin-degrading enzymes. These findings offer a new perspective and valuable data for developing efficient plastic biodegradation strategies.
研究领域:
环境科学
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微生物学
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分子生物学
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PubMed
高影响因子
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
·
2018
·Vol. 102
·期 10
·4425-4433
·
IF 3.34
Yin Xinjian,Wu Jianping,Yang Lirong
The objective of this study was to identify and exploit a robust biocatalyst that can be applied in reductive amination for enantioselective synthesis of the competitive herbicide L-phosphinothricin. Applying a genome mining-based library construction strategy, eight NADPH-specific glutamate dehydrogenases (GluDHs) were identified for reductively aminating 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO) to L-phosphinothricin. Among them, the glutamate dehydrogenase cloned from Pseudomonas putida ( Pp GluDH) exhibited relatively high catalytic activity and favorable soluble expression. This enzyme was purified to homogeneity for further characterization. The specific activity of Pp GluDH was 296.1 U/g-protein, which is significantly higher than the reported value for a GluDH. To the best of our knowledge, there has not been any report on protein engineering of GluDH for PPO-oriented activity. Taking full advantage of the available information and the diverse characteristics of the enzymes in the enzyme library, Pp GluDH was engineered by site-directed mutation based on multiple sequence alignment. The mutant I170M, which had 2.1-fold enhanced activity, was successfully produced. When the I170M mutant was applied in the batch production of L-phosphinothricin, it showed markedly improved catalytic efficiency compared with the wild type enzyme. The conversion reached 99% (0.1 M PPO) with an L-phosphinothricin productivity of 1.35 g/h·L, which far surpassed the previously reported level. These results show that Pp GluDH I170M is a promising biocatalyst for highly enantioselective synthesis of L-phosphinothricin by reductive amination.
研究领域:
生物化学
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酶工程
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合成生物学
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PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2024
·
IF 5.7
Yihan Wang,Jie Zheng,Yubin Xue,Bo Yu
Dipicolinic acid (DPA), a cyclic diacid, has garnered significant interest due to its potential applications in antimicrobial agents, antioxidants, chelating reagents, and polymer precursors. However, its natural bioproduction is limited since DPA is only accumulated in Bacillus and Clostridium species during sporulation. Thus, heterologous production by engineered strains is of paramount importance for developing a sustainable biological route for DPA production. Pseudomonas putida KT2440 has emerged as a promising host for the production of various chemicals thanks to its robustness, metabolic versatility, and genetic tractability. The dominant Entner–Doudoroff (ED) pathway for glucose metabolism in this strain offers an ideal route for DPA production due to the advantage of NADPH generation and the naturally balanced flux between glyceraldehyde-3-phosphate and pyruvate, which are both precursors for DPA synthesis. In this study, DPA production via the ED pathway was in silico designed in P. putida KT2440. The systematically engineered strain produced dipicolinate with a titer of 11.72 g/L from glucose in a 5 L fermentor. This approach not only provides a sustainable green route for DPA production but also expands our understanding of the metabolic potential of the ED pathway in P. putida KT2440.
研究领域:
微生物学
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生物化学
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代谢工程
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PubMed
高影响因子
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