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Microbial Cell Factories Mar 2018The soil bacterium Pseudomonas putida KT2440 is a "generally recognized as safe"-certified strain with robust property and versatile metabolism. Thus, it is an ideal...
BACKGROUND
The soil bacterium Pseudomonas putida KT2440 is a "generally recognized as safe"-certified strain with robust property and versatile metabolism. Thus, it is an ideal candidate for synthetic biology, biodegradation, and other biotechnology applications. The known genome editing approaches of Pseudomonas are suboptimal; thus, it is necessary to develop a high efficiency genome editing tool.
RESULTS
In this study, we established a fast and convenient CRISPR-Cas9 method in P. putida KT2440. Gene deletion, gene insertion and gene replacement could be achieved within 5 days, and the mutation efficiency reached > 70%. Single nucleotide replacement could be realized, overcoming the limitations of protospacer adjacent motif sequences. We also applied nuclease-deficient Cas9 binding at three locations upstream of enhanced green fluorescent protein (eGFP) for transcriptional inhibition, and the expression intensity of eGFP reduced to 28.5, 29.4, and 72.1% of the control level, respectively. Furthermore, based on this CRISPR-Cas9 system, we also constructed a CRISPR-Cpf1 system, which we validated for genome editing in P. putida KT2440.
CONCLUSIONS
In this research, we established CRISPR based genome editing and regulation control systems in P. putida KT2440. These fast and efficient approaches will greatly facilitate the application of P. putida KT2440.
Topics: CRISPR-Cas Systems; Endonucleases; Gene Deletion; Gene Editing; Gene Expression; Green Fluorescent Proteins; Mutagenesis, Insertional; Pseudomonas putida
PubMed: 29534717
DOI: 10.1186/s12934-018-0887-x -
Molecules (Basel, Switzerland) Sep 2022Natural coumarins contribute to the aroma of licorice, and they are often used as a flavoring and stabilizing agents. However, coumarins usage in food has been banned by...
Natural coumarins contribute to the aroma of licorice, and they are often used as a flavoring and stabilizing agents. However, coumarins usage in food has been banned by various countries due to its toxic effect. In this study, a strain of HSM-C2 that can biodegrade coumarin with high efficiency was isolated from soil and identified as through performing 16S rDNA sequence analysis. The HSM-C2 catalyzed the biodegradation up to 99.83% of 1 mg/mL coumarin within 24 h under optimal culture conditions, such as 30 °C and pH 7, which highlights the strong coumarin biodegrading potential of this strain. The product, such as dihydrocoumarin, generated after the biodegradation of coumarin was identified by performing GC-MS analysis. The present study provides a theoretical basis and microbial resource for further research on coumarin biodegradation.
Topics: Biodegradation, Environmental; Coumarins; DNA, Ribosomal; Excipients; Pseudomonas putida; Soil; Soil Microbiology
PubMed: 36144743
DOI: 10.3390/molecules27186007 -
Current Opinion in Biotechnology Feb 2024The soil bacterium Pseudomonas putida, especially the KT2440 strain, is increasingly being utilized as a host for biotransformations of both industrial and environmental... (Review)
Review
The soil bacterium Pseudomonas putida, especially the KT2440 strain, is increasingly being utilized as a host for biotransformations of both industrial and environmental interest. The foundations of such performance include its robust redox metabolism, ability to tolerate a wide range of physicochemical stresses, rapid growth, versatile metabolism, nonpathogenic nature, and the availability of molecular tools for advanced genetic programming. These attributes have been leveraged for hosting engineered pathways for production of valuable chemicals or degradation/valorization of environmental pollutants. This has in turn pushed the boundaries of conventional enzymology toward previously unexplored reactions in nature. Furthermore, modifications to the physical properties of the cells have been made to enhance their catalytic performance. These advancements establish P. putida as bona fide chassis for synthetic biology, on par with more traditional metabolic engineering platforms.
Topics: Metabolic Engineering; Pseudomonas putida; Synthetic Biology; Biotransformation; Oxidation-Reduction
PubMed: 38061264
DOI: 10.1016/j.copbio.2023.103025 -
Microbial Biotechnology Jan 2016Pseudomonas putida BIRD-1 has the potential to be used for the industrial production of butanol due to its solvent tolerance and ability to metabolize low-cost...
Pseudomonas putida BIRD-1 has the potential to be used for the industrial production of butanol due to its solvent tolerance and ability to metabolize low-cost compounds. However, the strain has two major limitations: it assimilates butanol as sole carbon source and butanol concentrations above 1% (v/v) are toxic. With the aim of facilitating BIRD-1 strain design for industrial use, a genome-wide mini-Tn5 transposon mutant library was screened for clones exhibiting increased butanol sensitivity or deficiency in butanol assimilation. Twenty-one mutants were selected that were affected in one or both of the processes. These mutants exhibited insertions in various genes, including those involved in the TCA cycle, fatty acid metabolism, transcription, cofactor synthesis and membrane integrity. An omics-based analysis revealed key genes involved in the butanol response. Transcriptomic and proteomic studies were carried out to compare short and long-term tolerance and assimilation traits. Pseudomonas putida initiates various butanol assimilation pathways via alcohol and aldehyde dehydrogenases that channel the compound to central metabolism through the glyoxylate shunt pathway. Accordingly, isocitrate lyase - a key enzyme of the pathway - was the most abundant protein when butanol was used as the sole carbon source. Upregulation of two genes encoding proteins PPUBIRD1_2240 and PPUBIRD1_2241 (acyl-CoA dehydrogenase and acyl-CoA synthetase respectively) linked butanol assimilation with acyl-CoA metabolism. Butanol tolerance was found to be primarily linked to classic solvent defense mechanisms, such as efflux pumps, membrane modifications and control of redox state. Our results also highlight the intensive energy requirements for butanol production and tolerance; thus, enhancing TCA cycle operation may represent a promising strategy for enhanced butanol production.
Topics: Acetate-CoA Ligase; Acyl-CoA Dehydrogenases; Bacterial Proteins; Butanols; Proteomics; Pseudomonas putida
PubMed: 26986205
DOI: 10.1111/1751-7915.12328 -
Microbiology (Reading, England) Nov 2020Microbial bioproduction of the aromatic acid anthranilate (-aminobenzoate) has the potential to replace its current, environmentally demanding production process. The...
Microbial bioproduction of the aromatic acid anthranilate (-aminobenzoate) has the potential to replace its current, environmentally demanding production process. The host organism employed for such a process needs to fulfil certain demands to achieve industrially relevant product levels. As anthranilate is toxic for microorganisms, the use of particularly robust production hosts can overcome issues from product inhibition. The microorganisms and are known for high tolerance towards a variety of chemicals and could serve as promising platform strains. In this study, the resistance of both wild-type strains towards anthranilate was assessed. To further enhance their native tolerance, adaptive laboratory evolution (ALE) was applied. Sequential batch fermentation processes were developed, adapted to the cultivation demands for and to enable long-term cultivation in the presence of anthranilate. Isolation and analysis of single mutants revealed phenotypes with improved growth behaviour in the presence of anthranilate for both strains. The characterization and improvement of both potential hosts provide an important basis for further process optimization and will aid the establishment of an industrially competitive method for microbial synthesis of anthranilate.
Topics: Adaptation, Physiological; Bioreactors; Corynebacterium glutamicum; Directed Molecular Evolution; Industrial Microbiology; Mutation; Pseudomonas putida; ortho-Aminobenzoates
PubMed: 33095135
DOI: 10.1099/mic.0.000982 -
Journal of Microbiology and... Feb 2021Many bacteria metabolize aromatic compounds via catechol as a catabolic intermediate, and possess multiple genes or clusters encoding catechol-cleavage enzymes. The...
Many bacteria metabolize aromatic compounds via catechol as a catabolic intermediate, and possess multiple genes or clusters encoding catechol-cleavage enzymes. The presence of multiple isozyme-encoding genes is a widespread phenomenon that seems to give the carrying strains a selective advantage in the natural environment over those with only a single copy. In the naphthalene-degrading strain ND6, catechol can be converted into intermediates of the tricarboxylic acid cycle via either the - or -cleavage pathways. In this study, we demonstrated that the catechol ortho-cleavage pathway genes ( and ) on the chromosome play an important role. The and operons are co-transcribed, whereas and are under independent transcriptional regulation. We examined the binding of regulatory proteins to promoters. In the presence of -muconate, a well-studied inducer of the cat gene cluster, CatR and CatR occupy an additional downstream site, designated as the activation binding site. Notably, CatR binds to both the and promoters with high affinity, while CatR binds weakly. This is likely caused by a T to G mutation in the G/T-N-A motif. Specifically, we found that CatR and CatR regulate and in a cooperative manner, which provides new insights into naphthalene degradation.
Topics: Bacterial Proteins; Catechols; Gene Expression Regulation, Bacterial; Multigene Family; Operon; Promoter Regions, Genetic; Pseudomonas putida
PubMed: 33323670
DOI: 10.4014/jmb.2009.09026 -
Microbiology Spectrum Jun 2023Biotransformation of plastics or their depolymerization monomers as raw materials would offer a better end-of-life solutions to the plastic waste dilemma. 1,4-butanediol...
Biotransformation of plastics or their depolymerization monomers as raw materials would offer a better end-of-life solutions to the plastic waste dilemma. 1,4-butanediol (BDO) is one of the major depolymerization monomers of many plastics polymers. BDO valorization presents great significance for waste plastic up-recycling and fermenting feedstock exploitation. In the present study, atmospheric pressure room temperature plasma (ARTP)-induced mutation combined with adaptive laboratory evolution (ALE) was used to improve the BDO utilization capability of Pseudomonas putida KT2440. The excellent mutant P. putida NB10 was isolated and stored in the China Typical Culture Preservation Center (CCTCC) with the deposit number M 2021482. Whole-genome resequencing and transcriptome analysis revealed that the BDO degradation process consists of β-oxidation, glyoxylate carboligase (GCL) pathway, glyoxylate cycle and gluconeogenesis pathway. The imbalance between the two key intermediates (acetyl-CoA and glycolyl-CoA) and the accumulation of cytotoxic aldehydes resulted in the weak metabolism performance of KT2440 in the utilization of BDO. The balance of the carbon flux and enhanced tolerance to cytotoxic intermediates endow NB10 with great BDO degradation capability. This study deeply revealed the metabolic mechanism behind BDO degradation and provided an excellent chassis cell for BDO further up-cycling to high-value chemicals. Plastic waste represents not only a global pollution problem but also a carbon-rich, low-cost, globally renewable feedstock for industrial biotechnology. BDO is the basic material for polybutylene terephthalate (PBT), poly butylene adipate-co-terephthalate (PBAT), poly (butylene succinate) (PBS), etc. Herein, the construction of BDO valorization cell factory presents great significance for waste plastic up-recycling and novel fermentation feedstock exploitation. However, BDO is hard to be metabolized and its metabolic pathway is unclear. This study presents a P. putida mutant NB10, obtained through the integration of ARTP and ALE, displaying significant growth improvement with BDO as the sole carbon source. Further genome resequencing, transcriptome analysis and genetic engineering deeply revealed the metabolic mechanism behind BDO degradation in P. putida, this study offers an excellent microbial chassis and modification strategy for plastic waste up-cycling.
Topics: Pseudomonas putida; Mutation; Carbon; Plastics
PubMed: 37067433
DOI: 10.1128/spectrum.04988-22 -
Microbial Cell Factories Jun 2022Biocatalysis offers a promising path for plastic waste management and valorization, especially for hydrolysable plastics such as polyethylene terephthalate (PET)....
BACKGROUND
Biocatalysis offers a promising path for plastic waste management and valorization, especially for hydrolysable plastics such as polyethylene terephthalate (PET). Microbial whole-cell biocatalysts for simultaneous PET degradation and growth on PET monomers would offer a one-step solution toward PET recycling or upcycling. We set out to engineer the industry-proven bacterium Pseudomonas putida for (i) metabolism of PET monomers as sole carbon sources, and (ii) efficient extracellular expression of PET hydrolases. We pursued this approach for both PET and the related polyester polybutylene adipate co-terephthalate (PBAT), aiming to learn about the determinants and potential applications of bacterial polyester-degrading biocatalysts.
RESULTS
P. putida was engineered to metabolize the PET and PBAT monomer terephthalic acid (TA) through genomic integration of four tphII operon genes from Comamonas sp. E6. Efficient cellular TA uptake was enabled by a point mutation in the native P. putida membrane transporter MhpT. Metabolism of the PET and PBAT monomers ethylene glycol and 1,4-butanediol was achieved through adaptive laboratory evolution. We then used fast design-build-test-learn cycles to engineer extracellular PET hydrolase expression, including tests of (i) the three PET hydrolases LCC, HiC, and IsPETase; (ii) genomic versus plasmid-based expression, using expression plasmids with high, medium, and low cellular copy number; (iii) three different promoter systems; (iv) three membrane anchor proteins for PET hydrolase cell surface display; and (v) a 30-mer signal peptide library for PET hydrolase secretion. PET hydrolase surface display and secretion was successfully engineered but often resulted in host cell fitness costs, which could be mitigated by promoter choice and altering construct copy number. Plastic biodegradation assays with the best PET hydrolase expression constructs genomically integrated into our monomer-metabolizing P. putida strains resulted in various degrees of plastic depolymerization, although self-sustaining bacterial growth remained elusive.
CONCLUSION
Our results show that balancing extracellular PET hydrolase expression with cellular fitness under nutrient-limiting conditions is a challenge. The precise knowledge of such bottlenecks, together with the vast array of PET hydrolase expression tools generated and tested here, may serve as a baseline for future efforts to engineer P. putida or other bacterial hosts towards becoming efficient whole-cell polyester-degrading biocatalysts.
Topics: Biocatalysis; Hydrolases; Plastics; Polyethylene Terephthalates; Pseudomonas putida
PubMed: 35717313
DOI: 10.1186/s12934-022-01849-7 -
PloS One 2019Pseudomonas putida is one of 13 major groups of Pseudomonas spp. and contains numerous species occupying diverse niches and performing many functions such as plant...
Pseudomonas putida is one of 13 major groups of Pseudomonas spp. and contains numerous species occupying diverse niches and performing many functions such as plant growth promotion and bioremediation. Here we compared a set of 19 P. putida isolates obtained from sugarcane rhizosphere or bulk soil using a population genomics approach aiming to assess genomic and metabolic differences between populations from these habitats. Phylogenomics placed rhizosphere versus bulk soil strains in separate clades clustering with different type strains of the P. putida group. Multivariate analyses indicated that the rhizosphere and bulk soil isolates form distinct populations. Comparative genomics identified several genetic functions (GO-terms) significantly different between populations, including some exclusively present in the rhizosphere or bulk soil strains, such as D-galactonic acid catabolism and cellulose biosynthesis, respectively. The metabolic profiles of rhizosphere and bulk soil populations analyzed by Biolog Ecoplates also differ significantly, most notably by the higher oxidation of D-galactonic/D-galacturonic acid by the rhizosphere population. Accordingly, D-galactonate catabolism operon (dgo) was present in all rhizosphere isolates and absent in the bulk soil population. This study showed that sugarcane rhizosphere and bulk soil harbor different populations of P. putida and identified genes and functions potentially associated with their soil niches.
Topics: Antibiosis; Genetics, Population; Genome, Bacterial; Genomics; Metabolomics; Phylogeny; Pseudomonas putida; Rhizosphere; Saccharum; Soil Microbiology
PubMed: 31581220
DOI: 10.1371/journal.pone.0223269 -
Metabolic Engineering Nov 2018The itinerary followed by Pseudomonas putida from being a soil-dweller and plant colonizer bacterium to become a flexible and engineer-able platform for metabolic... (Review)
Review
The itinerary followed by Pseudomonas putida from being a soil-dweller and plant colonizer bacterium to become a flexible and engineer-able platform for metabolic engineering stems from its natural lifestyle, which is adapted to harsh environmental conditions and all sorts of physicochemical stresses. Over the years, these properties have been capitalized biotechnologically owing to the expanding wealth of genetic tools designed for deep-editing the P. putida genome. A suite of dedicated vectors inspired in the core tenets of synthetic biology have enabled to suppress many of the naturally-occurring undesirable traits native to this species while enhancing its many appealing properties, and also to import catalytic activities and attributes from other biological systems. Much of the biotechnological interest on P. putida stems from the distinct architecture of its central carbon metabolism. The native biochemistry is naturally geared to generate reductive currency [i.e., NAD(P)H] that makes this bacterium a phenomenal host for redox-intensive reactions. In some cases, genetic editing of the indigenous biochemical network of P. putida (cis-metabolism) has sufficed to obtain target compounds of industrial interest. Yet, the main value and promise of this species (in particular, strain KT2440) resides not only in its capacity to host heterologous pathways from other microorganisms, but also altogether artificial routes (trans-metabolism) for making complex, new-to-Nature molecules. A number of examples are presented for substantiating the worth of P. putida as one of the favorite workhorses for sustainable manufacturing of fine and bulk chemicals in the current times of the 4th Industrial Revolution. The potential of P. putida to extend its rich native biochemistry beyond existing boundaries is discussed and research bottlenecks to this end are also identified. These aspects include not just the innovative genetic design of new strains but also the incorporation of novel chemical elements into the extant biochemistry, as well as genomic stability and scaling-up issues.
Topics: Biocatalysis; Metabolic Engineering; Metabolic Networks and Pathways; Oxidation-Reduction; Pseudomonas putida
PubMed: 29758287
DOI: 10.1016/j.ymben.2018.05.005