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Microbial Cell Factories May 20202-Acetamidophenol (AAP) is an aromatic compound with the potential for antifungal, anti-inflammatory, antitumor, anti-platelet, and anti-arthritic activities. Due to the...
BACKGROUND
2-Acetamidophenol (AAP) is an aromatic compound with the potential for antifungal, anti-inflammatory, antitumor, anti-platelet, and anti-arthritic activities. Due to the biosynthesis of AAP is not yet fully understood, AAP is mainly produced by chemical synthesis. Currently, metabolic engineering of natural microbial pathway to produce valuable aromatic compound has remarkable advantages and exhibits attractive potential. Thus, it is of paramount importance to develop a dominant strain to produce AAP by elucidating the AAP biosynthesis pathway.
RESULT
In this study, the active aromatic compound AAP was first purified and identified in gene phzB disruption strain HT66ΔphzB, which was derived from Pseudomonas chlororaphis HT66. The titer of AAP in the strain HT66ΔphzB was 236.89 mg/L. Then, the genes involved in AAP biosynthesis were determined. Through the deletion of genes phzF, Nat and trpE, AAP was confirmed to have the same biosynthesis route as phenazine-1-carboxylic (PCA). Moreover, a new arylamine N-acetyltransferases (NATs) was identified and proved to be the key enzyme required for generating AAP by in vitro assay. P. chlororaphis P3, a chemical mutagenesis mutant strain of HT66, has been demonstrated to have a robust ability to produce antimicrobial phenazines. Therefore, genetic engineering, precursor addition, and culture optimization strategies were used to enhance AAP production in P. chlororaphis P3. The inactivation of phzB in P3 increased AAP production by 92.4%. Disrupting the phenazine negative regulatory genes lon and rsmE and blocking the competitive pathway gene pykA in P3 increased AAP production 2.08-fold, which also confirmed that AAP has the same biosynthesis route as PCA. Furthermore, adding 2-amidophenol to the KB medium increased AAP production by 64.6%, which suggested that 2-amidophenol is the precursor of AAP. Finally, by adding 5 mM 2-amidophenol and 2 mM Fe to the KB medium, the production of AAP reached 1209.58 mg/L in the engineered strain P3ΔphzBΔlonΔpykAΔrsmE using a shaking-flask culture. This is the highest microbial-based AAP production achieved to date.
CONCLUSION
In conclusion, this study clarified the biosynthesis process of AAP in Pseudomonas and provided a promising host for industrial-scale biosynthesis of AAP from renewable resources.
Topics: Acetaminophen; Arylamine N-Acetyltransferase; Bacterial Proteins; Biosynthetic Pathways; Genes, Bacterial; Industrial Microbiology; Metabolic Engineering; Pseudomonas chlororaphis
PubMed: 32430011
DOI: 10.1186/s12934-020-01364-7 -
Journal of Agricultural and Food... Feb 20221-Hydroxyphenazine derivatives are phenazine family chemicals with broad-spectrum antibacterial and potential biological activities. However, the lack of variety and low...
1-Hydroxyphenazine derivatives are phenazine family chemicals with broad-spectrum antibacterial and potential biological activities. However, the lack of variety and low titer hinder their applications. In this research, three enzymes PhzS (monooxygenase), NaphzNO1 (-monooxygenase), and LaphzM (methyltransferase) were heterologously expressed in a phenazine-1-carboxylic acid generating strain H18. Four phenazines, 1-hydroxyphenazine, 1-methoxyphenazine, 1-hydroxyphenazine ' 10-oxide, and a novel phenazine derivative 1-methoxyphenazine ' 10-oxide, were isolated, characterized in the genetically modified strains, and exhibited excellent antimicrobial activities. Next, we verified the hydroxyl methylation activity of LaphzM and elucidated the biosynthetic pathway of 1-methoxyphenazine ' 10-oxide . Moreover, the titer of 1-hydroxyphenazine derivatives was engineered. The three compounds 1-methoxyphenazine, 1-hydroxyphenazine ' 10-oxide, and 1-methoxyphenazine ' 10-oxide all reach the highest titer reported to date. This work provides a promising platform for phenazine derivatives' combinatorial biosynthesis and engineering.
Topics: Biosynthetic Pathways; Methyltransferases; Phenazines; Pseudomonas chlororaphis
PubMed: 35057615
DOI: 10.1021/acs.jafc.1c07760 -
New Biotechnology Mar 2020Pseudomonas chlororaphis subsp. aurantiaca DSM 19603 was cultivated using glycerol as the sole carbon source for the simultaneous production of medium-chain length...
Pseudomonas chlororaphis subsp. aurantiaca DSM 19603 was cultivated using glycerol as the sole carbon source for the simultaneous production of medium-chain length polyhydroxyalkanoates (mcl-PHA), extracellular polysaccharide (EPS) and phenazines. A maximum cell dry mass of 11.79 g/L was achieved with a mcl-PHA content of 19 wt%, corresponding to a polymer concentration of 2.23 g/L. A considerably higher EPS production, 6.10 g/L, was attained. Phenazines synthesis was evidenced by the bright orange coloration developed by the culture during the cell growth phase. The mcl-PHA produced by P. chlororaphis was composed mainly of 3-hydroxydecanoate (50 wt%) with lower amounts of 3-hydroxyoctanoate (17 wt%), 3-hydroxytetradecanoate (17 wt%), 3-hydroxydodecanoate (13 wt%) and 3-hydroxyhexanoate (3 wt%). This PHA showed unique thermal features being highly amorphous, with a degree of crystallinity of 27% and a low melting temperature (45.0 °C). The secreted EPS was mostly composed of glucose, glucosamine, rhamnose and mannose, with smaller amounts of three other unidentified monomers. Although the bioprocess can be improved further to define the optimal conditions to produce each bioproduct (mcl-PHA, EPS or phenazines), this study has demonstrated for the first time the ability of P. chlororaphis to simultaneously produce three high-value products from a single substrate.
Topics: Biomass; Biopolymers; Glycerol; Kinetics; Phenazines; Polyhydroxyalkanoates; Polysaccharides, Bacterial; Pseudomonas chlororaphis; X-Ray Diffraction
PubMed: 31605767
DOI: 10.1016/j.nbt.2019.10.002 -
International Journal of Molecular... Feb 2023Although many bacterial lipases and PHA depolymerases have been identified, cloned, and characterized, there is very little information on the potential application of...
Although many bacterial lipases and PHA depolymerases have been identified, cloned, and characterized, there is very little information on the potential application of lipases and PHA depolymerases, especially intracellular enzymes, for the degradation of polyester polymers/plastics. We identified genes encoding an intracellular lipase (LIP3), an extracellular lipase (LIP4), and an intracellular PHA depolymerase (PhaZ) in the genome of the bacterium PA23. We cloned these genes into and then expressed, purified, and characterized the biochemistry and substrate preferences of the enzymes they encode. Our data suggest that the LIP3, LIP4, and PhaZ enzymes differ significantly in their biochemical and biophysical properties, structural-folding characteristics, and the absence or presence of a lid domain. Despite their different properties, the enzymes exhibited broad substrate specificity and were able to hydrolyze both short- and medium-chain length polyhydroxyalkanoates (PHAs), para-nitrophenyl (pNP) alkanoates, and polylactic acid (PLA). Gel Permeation Chromatography (GPC) analyses of the polymers treated with LIP3, LIP4, and PhaZ revealed significant degradation of both the biodegradable as well as the synthetic polymers poly(ε-caprolactone) (PCL) and polyethylene succinate (PES).
Topics: Pseudomonas; Carboxylic Ester Hydrolases; Lipase; Polyesters; Polyhydroxyalkanoates; Pseudomonas chlororaphis; Substrate Specificity
PubMed: 36901931
DOI: 10.3390/ijms24054501 -
Frontiers in Microbiology 2020PCL1606 (PcPCL1606) is a rhizobacterium isolated from avocado roots, which is a favorable niche for its development. This strain extensively interacts with plant roots...
PCL1606 (PcPCL1606) is a rhizobacterium isolated from avocado roots, which is a favorable niche for its development. This strain extensively interacts with plant roots and surrounding microbes and is considered a biocontrol rhizobacterium. Genome sequencing has shown the presence of thirty-one potential methyl-accepting chemotaxis proteins (MCPs). Among these MCPs, two candidates are putative functional aerotaxis receptors, encoded at locus PCL1606_41090 (1-1) and locus PLC1606_20530 (1-2), that are homologous to the Aer receptor of strain PaO1. Single- and double-deletion mutants in one or both genes have led to motility deficiencies in oxygen-rich areas, particularly reduced swimming motility compared with that of wildtype PcPCL1606. No differences in swarming tests were detected, and less adhesion by the double mutant was observed. However, the single and double mutants on avocado plant roots showed delayed biocontrol ability. During the first days of the biocontrol experiment, the -defective mutants also showed delayed root colonization. The current research characterizes the presence of transductors on . Thus, the functions of the PCL1606_41090 and PCL1606_20530 loci, corresponding to genes 1-1 and 1-2, respectively, are elucidated.
PubMed: 32754135
DOI: 10.3389/fmicb.2020.01560 -
Frontiers in Microbiology 2022Currently, plant growth-promoting rhizobacteria (PGPR) microbial inoculants are heavily used in agricultural production among which sp. and sp. are two excellent...
Currently, plant growth-promoting rhizobacteria (PGPR) microbial inoculants are heavily used in agricultural production among which sp. and sp. are two excellent inoculum strains, which are widely used in plant growth promotion and disease control. However, few studies have been conducted on the combined use of the two bacteria. The aim of this study was to investigate the effects of co-inoculation of these two bacteria on soybean [ (L.) ] growth and physiological indexes and further study the effect of microbial inoculants on native soil bacterial communities and plant endophyte microbiota, especially microorganisms in rhizosphere and root. A pot experiment was conducted and four treatments were designed: group without any strain inoculant (CK); group inoculated with H1 inoculant (J); group inoculated with Y1 inoculant (Y) and group inoculated with equal volume of H1 inoculant and Y1 inoculant (H). Compared with CK, the three inoculant groups J, Y, and H exhibited improved soybean growth and physiological indexes, and group H was the most significant ( < 0.05). In terms of rhizosphere bacterial community structure, the relative abundance of native (9.31%) was higher in the H group than in the J (6.07%), Y (3.40%), and CK (5.69%) groups, which has potential value of disease suppression. Besides, compared with bacterial communities of the other three groups in soybean roots, group H increased the abundance of beneficial bacterial community for the contents of , , and significantly increased ( < 0.05). In conclusion, we found that the composite inoculum of H1 and Y1 could effectively promote soybean growth, increase yield and improve the beneficial bacterial community in root and rhizosphere and have certain value for soil improvement.
PubMed: 36699592
DOI: 10.3389/fmicb.2022.1079348 -
Frontiers in Plant Science 2022(Pc) representatives are found as part of the rhizosphere-associated microbiome, and different rhizospheric Pc strains frequently perform beneficial activities for the...
(Pc) representatives are found as part of the rhizosphere-associated microbiome, and different rhizospheric Pc strains frequently perform beneficial activities for the plant. In this study we described the interactions between the rhizospheric Pc strains PCL1601, PCL1606 and PCL1607 with a focus on their effects on root performance. Differences among the three rhizospheric Pc strains selected were first observed in phylogenetic studies and confirmed by genome analysis, which showed variation in the presence of genes related to antifungal compounds or siderophore production, among others. Observation of the interactions among these strains under lab conditions revealed that PCL1606 has a better adaptation to environments rich in nutrients, and forms biofilms. Interaction experiments on plant roots confirmed the role of the different phenotypes in their lifestyle. The PCL1606 strain was the best adapted to the habitat of avocado roots, and PCL1607 was the least, and disappeared from the plant root scenario after a few days of interaction. These results confirm that 2 out 3 rhizospheric Pc strains were fully compatible (PCL1601 and PCL1606), efficiently colonizing avocado roots and showing biocontrol activity against the fungal pathogen . The third strain (PCL1607) has colonizing abilities when it is alone on the root but displayed difficulties under the competition scenario, and did not cause deleterious effects on the other Pc competitors when they were present. These results suggest that strains PCL1601 and PCL1606 are very well adapted to the avocado root environment and could constitute a basis for constructing a more complex beneficial microbial synthetic community associated with avocado plant roots.
PubMed: 36589057
DOI: 10.3389/fpls.2022.1063182 -
ACS Synthetic Biology Mar 2024The advancement of metabolic engineering and synthetic biology has promoted in-depth research on the nonmodel microbial metabolism, and the potential of nonmodel...
The advancement of metabolic engineering and synthetic biology has promoted in-depth research on the nonmodel microbial metabolism, and the potential of nonmodel organisms in industrial biotechnology is becoming increasingly evident. The nonmodel organism is a safe plant growth promoting bacterium for the production of phenazine compounds; however, its application is seriously hindered due to the lack of an effective gene expression precise regulation toolkit. In this study, we constructed a library of 108 promoter-5'-UTR (PUTR) and characterized them through fluorescent protein detection. Then, 6 PUTRs with stable low, intermediate, and high intensities were further characterized by report genes encoding β-galactosidase from K12 and encoding PCA monooxygenase from GP72 and thus developed as a static gene expression regulation system. Furthermore, the stable and high-intensity expressed PUTR was fused with the LacO operator to construct an IPTG-induced plasmid, and a self-induced plasmid was constructed employing the high-intensity PUTR regulated by cell density, resulting in a dynamic gene expression regulation system. In summary, this study established two sets of static and dynamic regulatory systems for , providing an effective toolkit for fine-tuning gene expression and reprograming the metabolism flux.
Topics: Pseudomonas chlororaphis; Metabolic Engineering; Gene Expression Regulation, Bacterial; Promoter Regions, Genetic; Bacterial Proteins
PubMed: 38377538
DOI: 10.1021/acssynbio.3c00714 -
Microbiological Research Oct 2020Three morphological mutants (M71a, M71b, M71c) of the antagonist Pseudomonas chlororaphis M71, naturally arose during a biocontrol trial against the phytopathogenic...
Three morphological mutants (M71a, M71b, M71c) of the antagonist Pseudomonas chlororaphis M71, naturally arose during a biocontrol trial against the phytopathogenic fungus Fusarium oxysporum f.sp. radicis-lycopersisci. In this study, the three mutants were investigated to elucidate their role in the biocontrol of plant pathogens. M71a and M71b phenotypes were generated by a mutation in the two-component system GacS/GacA. The mutation determined an increase in siderophore production and an impaired ability to release proteases, to swarm, to produce phenazine and AHLs and to colonize tomato roots. In vitro antagonistic activity against different plant pathogens was partially reduced in M71a, while M71b resulted effective only against Pythium ultimum. Biocontrol efficacy against Fusarium oxysporum f.sp. radicis-lycopersisci, was partially reduced in M71a and completely lost in M71b. M71c phenotype was impaired in swarming motility, did not produce biofilms and its antagonistic activity was similar to the parental M71 strain. M71c showed an enhanced ability to colonize tomato roots, on which its progeny in part reverted to the M71 parental phenotype. Volatile organic compounds (VOCs) emitted by all four strains, inhibited the growth of Clavibacter michiganensis subsp. michiganensis and Seiridium cardinale in vitro. Real-time screening of VOCs by PTR-MS combined with GC-MS analysis, showed that methanethiol was the main component of the blend produced by all four M71 strains. However, the emissions of hydrogen cyanide, dimethyl disulfide, 1,3-butadiene and acetone were significantly affected by the three different mutations. These findings highlight that the simultaneous presence of different M71 phenotypes may improve, through the integration of different mechanisms, the ecological fitness and biocontrol efficacy of P. chlororaphis M71.
Topics: Bacterial Proteins; Biological Control Agents; Fusarium; Solanum lycopersicum; Mutation; Pest Control, Biological; Phenazines; Phenotype; Plant Diseases; Plant Roots; Pseudomonas chlororaphis; Siderophores; Volatile Organic Compounds
PubMed: 32535393
DOI: 10.1016/j.micres.2020.126517 -
JACS Au Nov 2023Chemical fertilizers have been crucial for sustaining the current global population by supplementing overused farmland to support consistent food production, but their...
Chemical fertilizers have been crucial for sustaining the current global population by supplementing overused farmland to support consistent food production, but their use is unsustainable. is a nitrogen-fixing bacterium that could be used as a fertilizer replacement, but this microbe is delicate. It is sensitive to stressors, such as freeze-drying and high temperatures. Here, we demonstrate protection of from freeze-drying, high temperatures (50 C), and high humidity using self-assembling metal-phenolic network (MPN) coatings. The composition of the MPN is found to significantly impact its protective efficacy, and with optimized compositions, no viability loss is observed for MPN-coated microbes under conditions where uncoated cells do not survive. Further, we demonstrate that MPN-coated microbes improve germination of seeds by 150% as compared to those treated with fresh . Taken together, these results demonstrate the protective capabilities of MPNs against environmental stressors and represent a critical step towards enabling the production and storage of delicate microbes under nonideal conditions.
PubMed: 38034965
DOI: 10.1021/jacsau.3c00426