Frontiers in Bioengineering and Biotechnology
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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.
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JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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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.
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ENVIRONMENTAL MICROBIOLOGY
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2019
·Vol. 21
·期 5
·1847-1863
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IF 5.147
Lu Lin,Xiaopeng Wang,Lanfang Cao,Meiying Xu
Summary Lignin is one of the largest carbon reservoirs in the environment, playing an important role in the global carbon cycle. However, lignin degradation in bacteria, especially non-model organisms, has not been well characterized either enzymatically or genetically. Here, a lignin-degrading bacterial strain, Pseudomonas putida A514, was used as the research model. Genomic and proteomic analyses suggested that two B subfamily dye-decolorizing peroxidases (DypBs) were prominent in lignin depolymerization, while the classic O 2 -dependent ring cleavage strategy was utilized in central pathways to catabolize lignin-derived aromatic compounds that were funnelled by peripheral pathways. These enzymes, together with a range of transporters, sequential and expression-dose dependent regulation and stress response systems coordinated for lignin metabolism. Catalytic assays indicated these DypBs show unique Mn 2+ independent lignin depolymerization activity, while Mn 2+ oxidation activity is absent. Furthermore, a high synergy between DypB enzymes and A514 cells was observed to promote cell growth (5 × 10 12 cfus/ml) and lignin degradation (27%). This suggested DypBs are competitive lignin biocatalysts and pinpointed limited extracellular secretion capacity as the rate-limiting factor in bacterial lignin degradation. DypB production was, therefore, optimized in recombinant strains and a 14,141-fold increase in DypB activity (56,565 U/l) was achieved, providing novel insights for lignin bioconversion.
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WATER RESEARCH
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2017
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IF 6.942
Yuancai Lv,Zhuyu Niu,Yuancai Chen,Yongyou Hu
With the introduction of nano zero valent iron (nZVI) technology into our environment, its potential environmental risk to environmental microorganisms has attracted considerable attention. In this study, Pseudomonas putida was chosen as a typical strain to study the bacterial toxicity of nZVI/Pd. The CFU assay results indicated that nZVI/Pd was toxic to P. putida cells but the toxicity decreased with an increase in DO. The experiments isolated by dialysis bag and flow cytometry analysis suggested that both membrane disruption caused by direct contact and oxidative stress were the main bactericidal mechanisms under the aerobic condition , while membrane disruption resulting from direct contact was the primary bactericidal mechanism in the anaerobic system. Furthermore, according to TEM, SEM, EDS , XRD , FTIR and XPS , it was indicated that in the aerobic system, the reactive oxygen species (ROS) generated by nZVI/Pd could oxidize the amide and hydroxyl groups into carboxyl groups , resulting in a decline in peptides and increase in polysaccharides . In addition, the ROS also accumulated inside the cell and caused cell inactivation via oxidative stress . In the anaerobic system, the adhered nZVI/Pd particles would attack the functional groups such as carboxyl, ester and amide, leading to the decline in proteins and polysaccharides and subsequent damage of the membrane. The findings provide a significant guide for the application of nano-bio combined technology.
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JOURNAL OF HAZARDOUS MATERIALS
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2023
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IF 13.6
Jinfeng Lu,Yuexian Liu,Ruili Zhang,Zhengyi Hu,Kai Xue,Biya Dong
Controversial results are reported whereby biodegradation of polycyclic aromatic hydrocarbons (PAHs) can be promoted or inhibited by biochar amendment of soil. Metabolomics was applied to analyze the metabolic profiles of amendment with biochar (BB) and biochar inoculated with functional bacteria ( Pseudomonas putida ) (BP) involved in phenanthrene (PHE) degradation. Additionally, metagenomic analysis was utilized to assess the impact of different treatments on PHE degradation by soil microorganisms. Results indicated that BB treatment decreased the PHE biodegradation of the soil indigenous bacterial consortium, but BP treatment alleviated this inhibitory effect. Metabolomics revealed the differential metabolite 9-phenanthrol was absent in the BB treatment, but was found in the control group (CK), and in the treatment inoculated with the Pseudomonas putida (Ps) and the BP treatment. Metagenomic analysis showed that biochar decreased the abundance of the cytochrome P450 monooxygenase (CYP116), which was detected in the Pseudomonas putida , thus alleviating the inhibitory effect of biochar on PHE degradation. Moreover, a noticeable delayed increase of functional gene abundance and enzymes abundance in the BB treatment was observed in the PHE degradation pathway. Our findings elucidate the mechanism of inhibition with biochar amendment and the alleviating effect of biochar inoculated with degrading bacteria.
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Molecular Catalysis
·
2021
·
IF 5.062
Yu-Ting Shang,Jiufu Qin,Jin-Song Gong,Zi-Kai Wang,Heng Li,Hui Li,Jin-Song Shi,Zheng-Hong Xu
A nicotinate dehydrogenase producing strain was isolated and identified to be Pseudomonas putida , which could bio-transform nicotinic acid into 6-hydroxynicotinic acid. The activity of nicotinate dehydrogenase towards nicotinic acid could reach up to 1.11 U/mL and the yield of 6-hydroxynicotinic acid accumulated to 176 g/L within 30 h with a space time yield of 1.36 g/(g•h). In order to further improve its catalytic potential, a mutant library was constructed through atmospheric and room temperature plasma (ARTP) and rapidly screened by a high-throughput screening strategy, which obtained a mutant strain mut-5 with activity of 2.68 U/mL. Via developing a nitrilase-nicotinate dehydrogenase enzymatic cascade system, 54.5 g/L 6-hydroxynicotinic acid could be obtained within 585 min with 3-cyanopyridine as the substrate, which would further reduce the production costs. The results indicated that the nicotinate dehydrogenase (separately or combining with nitrilase) could serve as a superior catalyst for 6-hydroxynicotinic acid production. To our knowledge, the nicotinate dehydrogenase activity reported in this study was the highest level in literature to date.
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CHEMOSPHERE
·
2019
·
IF 5.108
Yating Jia,Fan Hu,Yuancai Lv,Yuancai Chen,Yongyou Hu
Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants and challenges for water treatment due to their persistence and toxicity. In this study, the reduction of 2′2′4′4′-tetrabromodiphenyl ether (BDE-47) was investigated in a nano-bio-integrated system. Results showed that the introducing of P. putida could markedly accelerate the demineralization of BDE-47 in nZVI/Pd- P.p system; the continuous generation of acidic metaboliates by P. putida could decrease pH, which could alleviate the surface passivation to some extent, resulting in the releasing of Fe 2+ and high generation of H 2 O 2 , the shift in reactive oxygen species from Fe(IV) to •OH. The BDE-47 was firstly debrominated to the DE by the highly reductive [Pd·2H] generated by nZVI/Pd, then oxidized to bromophenol and phenol, catechol as well as hydroquinone via the P. putida strain and the Fenton-like system. The toxicity assays confirmed the combined system could avert generation of nocuous intermediates, and could be an alternative strategy for complete remediation of recalcitrant POPs.
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JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2024
·
IF 5.7
Huoyong Jiang,Xuexiu Yu,Jingjing Guo,Guangdong Shang,Yijun Dai
The amides 4-trifluoromethylnicotinamide, acrylamide, and benzamide are widely used in agriculture and industry, posing hazards to the environment and animals. Immobilized bacteria are preferred in wastewater treatment, but degradation of these amides by immobilized engineered bacteria has not been explored. Here, engineered Pseudomonas putida KT2440 pLSJ15-amiA was constructed by introducing a new amidase gene expression vector into environmentally safe P. putida KT2440. P. putida KT2440 pLSJ15-amiA had high amidase activity, even at 80 °C. P. putida KT2440 pLSJ15-amiA immobilized with calcium alginate exhibited a greater environmental tolerance than free cells. The amides were rapidly degraded by the immobilized cells, but the activity was inhibited by high concentrations of substrates. The substrate inhibition model revealed that the optimum initial concentrations of 4-trifluoromethylnicotinamide, acrylamide, and benzamide for degradation by immobilized cells were 197.65, 350.76, and 249.40 μmol/L, respectively. This study develops a novel and excellent immobilized biocatalyst for remediation of wastewater containing hazardous amides.
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2016
·Vol. 82
·期 2
·
IF 3.823
Hong-Jun Chao,Yan-Fei Chen,Ti Fang,Ying Xu,Wei E. Huang,Ning-Yi Zhou
In addition to growing on p-cresol, Pseudomonas putida NCIMB 9866 is the only reported strain capable of aerobically growing on 2,4-xylenol, which is listed as a priority pollutant by the U.S. Environmental Protection Agency. Several enzymes involved in the oxidation of the para-methyl group, as well as the corresponding genes, have previously been reported. The enzyme catalyzing oxidation of the catabolic intermediate 4-hydroxyisophthalate to the ring cleavage substrate protocatechuate was also purified from strain NCIMB 9866, but its genetic determinant is still unavailable. In this study, the gene hipH, encoding 4-hydroxyisophthalate hydroxylase, from strain NCIMB 9866 was cloned by transposon mutagenesis. Purified recombinant HipH-His6 was found to be a dimer protein with a molecular mass of approximately 110 kDa. HipH-His6 catalyzed the hydroxylation of 4-hydroxyisophthalate to protocatechuate with a specific activity of 1.54 U mg−1 and showed apparent Km values of 11.40 ± 3.05 μM for 4-hydroxyisophthalate with NADPH and 11.23 ± 2.43 μM with NADH and similar Km values for NADPH and NADH (64.31 ± 13.16 and 72.76 ± 12.06 μM, respectively). The identity of protocatechuate generated from 4-hydroxyisophthalate hydroxylation by HipH-His6 has also been confirmed by high-performance liquid chromatography and mass spectrometry. Gene transcriptional analysis, gene knockout, and complementation indicated that hipH is essential for 2,4-xylenol catabolism but not for p-cresol catabolism in this strain. This fills a gap in our understanding of the gene that encodes a critical step in 2,4-xylenol catabolism and also provides another example of biochemical and genetic diversity of microbial catabolism of structurally similar compounds.
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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.
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Frontiers in Chemistry
·
2020
·
IF 3.693
Zheng Zhaojuan,Xu Qianqian,Tan Huanghong,Zhou Feng,Ouyang Jia
Upgrading of furanic aldehydes to their corresponding furancarboxylic acids has received considerable interest recently. Herein we reported selective oxidation of furfural (FAL) to furoic acid (FA) with quantitative yield using whole-cells of Pseudomonas putida KT2440. The biocatalytic capacity could be substantially promoted through adding 5-hydroxymethylfurfural into media at the middle exponential growth phase. The reaction pH and cell dosage had notable impacts on both FA titer and selectivity. Based on the validation of key factors for FAL conversion, the capacity of P. putida KT2440 to produce FAL was substantially improved. In batch bioconversion, 170 mM FA was produced with selectivity nearly 100% in 2 h, whereas 204 mM FA was produced with selectivity above 97% in 3 h in fed-batch bioconversion. Particularly, the role of molybdate transporter in oxidation of FAL and 5-hydroxymethylfurfural was demonstrated for the first time. The furancarboxylic acids synthesis was repressed markedly by destroying molybdate transporter, which implied Mo-dependent enzyme/molybdoenzyme played pivotal role in such oxidation reactions. This research further highlights the potential of P. putida KT2440 as next generation industrial workhorse and provides a novel understanding of molybdoenzyme in oxidation of furanic aldehydes.
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BIOPROCESS AND BIOSYSTEMS ENGINEERING
·
2019
·Vol. 43
·期 1
·67-73
·
IF 2.371
Xu Qianqian,Zheng Zhaojuan,Zou Lihua,Zhang Chen,Yang Fen,Zhou Kaijie,Ouyang Jia
Currently, biotransformation of 5-hydroxymethylfurfural (HMF) into a series of high-value bio-based platform chemicals is massively studied. In this study, selective biooxidation of HMF to 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) by Pseudomonas putida KT2440 with superior titer, yield, and productivity was reported. The biocatalytic performances of P. putida KT2440 were optimized separately. Under optimal conditions, 100% yield of HMFCA was obtained when HMF concentration was less than 150 mM, while the maximum concentration of 155 mM was achieved from 160 mM HMF in 12 h. P. putida KT2440 was highly tolerate to HMF, up to 190 mM. Besides, it was capable of selective oxidation of other furan aldehydes to the corresponding carboxylic acids with good yield of 100%. This study further demonstrates the potential of P. putida KT2440 as a biocatalyst for biomass conversion, as this strain has been proved the capacity to convert and utilize many kinds of biomass-derived sugars and ligin-derived aromatic compounds.
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