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Applied Microbiology and Biotechnology May 2022Plant growth-promoting rhizobacteria (PGPR) are a group of microorganisms of utmost interest in agricultural biotechnology for their stimulatory and protective effects... (Review)
Review
Plant growth-promoting rhizobacteria (PGPR) are a group of microorganisms of utmost interest in agricultural biotechnology for their stimulatory and protective effects on plants. Among the various PGPR species, some Pseudomonas putida strains combine outstanding traits such as phytohormone synthesis, nutrient solubilization, adaptation to different stress conditions, and excellent root colonization ability. In this review, we summarize the state of the art and the most relevant findings related to P. putida and its close relatives as PGPR, and we have compiled a detailed list of P. putida sensu stricto, sensu lato, and close relative strains that have been studied for their plant growth-promoting characteristics. However, the mere in vitro analysis of these characteristics does not guarantee correct plant performance under in vivo or field conditions. Therefore, the importance of studying adhesion and survival in the rhizosphere, as well as responses to environmental factors, is emphasized. Although numerous strains of this species have shown good performance in field trials, their use in commercial products is still very limited. Thus, we also analyze the opportunities and challenges related to the formulation and application of bioproducts based on these bacteria. KEY POINTS: •The mini-review updates the knowledge on Pseudomonas putida as a PGPR. • Some rhizosphere strains are able to improve plant growth under stress conditions. • The metabolic versatility of this species encourages the development of a bioproduct.
Topics: Plant Development; Plant Growth Regulators; Plant Roots; Plants; Pseudomonas putida; Rhizosphere; Soil Microbiology
PubMed: 35488932
DOI: 10.1007/s00253-022-11881-7 -
Applied Microbiology and Biotechnology Sep 2020Pseudomonas putida is a Gram-negative, rod-shaped bacterium that can be encountered in diverse ecological habitats. This ubiquity is traced to its remarkably versatile... (Review)
Review
Pseudomonas putida is a Gram-negative, rod-shaped bacterium that can be encountered in diverse ecological habitats. This ubiquity is traced to its remarkably versatile metabolism, adapted to withstand physicochemical stress, and the capacity to thrive in harsh environments. Owing to these characteristics, there is a growing interest in this microbe for industrial use, and the corresponding research has made rapid progress in recent years. Hereby, strong drivers are the exploitation of cheap renewable feedstocks and waste streams to produce value-added chemicals and the steady progress in genetic strain engineering and systems biology understanding of this bacterium. Here, we summarize the recent advances and prospects in genetic engineering, systems and synthetic biology, and applications of P. putida as a cell factory. KEY POINTS: • Pseudomonas putida advances to a global industrial cell factory. • Novel tools enable system-wide understanding and streamlined genomic engineering. • Applications of P. putida range from bioeconomy chemicals to biosynthetic drugs.
Topics: Biotechnology; Genomics; Pseudomonas putida; Synthetic Biology; Systems Biology
PubMed: 32789744
DOI: 10.1007/s00253-020-10811-9 -
ACS Synthetic Biology Nov 2022KT2440 is an emerging microbial chassis for biobased chemical production from renewable feedstocks and environmental bioremediation. However, tools for studying,...
KT2440 is an emerging microbial chassis for biobased chemical production from renewable feedstocks and environmental bioremediation. However, tools for studying, engineering, and modulating protein complexes and biosynthetic enzymes in this organism are largely underdeveloped. Genetic code expansion for the incorporation of unnatural amino acids (unAAs) into proteins can advance such efforts and, furthermore, enable additional controls of biological processes of the strain. In this work, we established the orthogonality of two widely used archaeal tRNA synthetase and tRNA pairs in KT2440. Following the optimization of decoding systems, four unAAs were incorporated into proteins in response to a UAG stop codon at 34.6-78% efficiency. In addition, we demonstrated the utility of genetic code expansion through the incorporation of a photocross-linking amino acid, -benzoyl-l-phenylalanine (pBpa), into glutathione -transferase (GstA) and a chemosensory response regulator (CheY) for protein-protein interaction studies in KT2440. This work reported the successful genetic code expansion in KT2440 for the first time. Given the diverse structure and functions of unAAs that have been added to protein syntheses using the archaeal systems, our research lays down a solid foundation for future work to study and enhance the biological functions of KT2440.
Topics: Pseudomonas putida; Genetic Code; Amino Acyl-tRNA Synthetases; RNA, Transfer; Amino Acids
PubMed: 36287825
DOI: 10.1021/acssynbio.2c00325 -
Journal of Oleo Science Apr 2021A total of 100 environmental samples were investigated for their ability to degrade 1 g/L surfactin as a substrate. Among them, two enrichment cultures, which exhibited...
A total of 100 environmental samples were investigated for their ability to degrade 1 g/L surfactin as a substrate. Among them, two enrichment cultures, which exhibited microbial growth as well as surfactin degradation, were selected and further investigated. After several successive cultivations, nanopore sequencing of full-length 16S rRNA genes with MinION was used to analyze the bacterial species in the enrichment cultures. Variovorax spp., Caulobacter spp., Sphingopyxis spp., and Pseudomonas spp. were found to be dominant in these surfactin-degrading mixed cultures. Finally, one strain of Pseudomonas putida was isolated as a surfactin-degrading bacterium. This strain degraded 1 g/L surfactin below a detectable level within 14 days, and C surfactin was degraded faster than C surfactin.
Topics: Biodegradation, Environmental; Caulobacter; Comamonadaceae; Lipopeptides; Peptides, Cyclic; Pseudomonas putida; Sphingomonadaceae; Surface-Active Agents
PubMed: 33692244
DOI: 10.5650/jos.ess20331 -
Biotechnology Journal Mar 2021Growing environmental concern sparks renewed interest in the sustainable production of (bio)materials that can replace oil-derived goods. Polyhydroxyalkanoates (PHAs)... (Review)
Review
Growing environmental concern sparks renewed interest in the sustainable production of (bio)materials that can replace oil-derived goods. Polyhydroxyalkanoates (PHAs) are isotactic polymers that play a critical role in the central metabolism of producer bacteria, as they act as dynamic reservoirs of carbon and reducing equivalents. PHAs continue to attract industrial attention as a starting point toward renewable, biodegradable, biocompatible, and versatile thermoplastic and elastomeric materials. Pseudomonas species have been known for long as efficient biopolymer producers, especially for medium-chain-length PHAs. The surge of synthetic biology and metabolic engineering approaches in recent years offers the possibility of exploiting the untapped potential of Pseudomonas cell factories for the production of tailored PHAs. In this article, an overview of the metabolic and regulatory circuits that rule PHA accumulation in Pseudomonas putida is provided, and approaches leading to the biosynthesis of novel polymers (e.g., PHAs including nonbiological chemical elements in their structures) are discussed. The potential of novel PHAs to disrupt existing and future market segments is closer to realization than ever before. The review is concluded by pinpointing challenges that currently hinder the wide adoption of bio-based PHAs, and strategies toward programmable polymer biosynthesis from alternative substrates in engineered P. putida strains are proposed.
Topics: Carbon; Metabolic Engineering; Polyhydroxyalkanoates; Pseudomonas; Pseudomonas putida
PubMed: 33085217
DOI: 10.1002/biot.202000165 -
Microbial Cell Factories May 2023Aromatic α-hydroxy ketones, such as S-2-hydroxypropiophenone (2-HPP), are highly valuable chiral building blocks useful for the synthesis of various pharmaceuticals and...
BACKGROUND
Aromatic α-hydroxy ketones, such as S-2-hydroxypropiophenone (2-HPP), are highly valuable chiral building blocks useful for the synthesis of various pharmaceuticals and natural products. In the present study, enantioselective synthesis of 2-HPP was investigated by free and immobilized whole cells of Pseudomonas putida ATCC 12633 starting from readily-available aldehyde substrates. Whole resting cells of P. putida, previously grown in a culture medium containing ammonium mandelate, are a source of native benzoylformate decarboxylase (BFD) activity. BFD produced by induced P. putida resting cells is a highly active biocatalyst without any further treatment in comparison with partially purified enzyme preparations. These cells can convert benzaldehyde and acetaldehyde into the acyloin compound 2-HPP by BFD-catalyzed enantioselective cross-coupling reaction.
RESULTS
The reaction was carried out in the presence of exogenous benzaldehyde (20 mM) and acetaldehyde (600 mM) as substrates in 6 mL of 200 mM phosphate buffer (pH 7) for 3 h. The optimal biomass concentration was assessed to be 0.006 g dry cell weight (DCW) mL. 2-HPP titer, yield and productivity using the free cells were 1.2 g L, 0.56 g 2-HPP/g benzaldehyde (0.4 mol 2-HPP/mol benzaldehyde), 0.067 g 2-HPP g DCW h, respectively, under optimized biotransformation conditions (30 °C, 200 rpm). Calcium alginate (CA)-polyvinyl alcohol (PVA)-boric acid (BA)-beads were used for cell entrapment. Encapsulated whole-cells were successfully employed in four consecutive cycles for 2-HPP production under aerobic conditions without any noticeable beads degradation. Moreover, there was no production of benzyl alcohol as an unwanted by-product.
CONCLUSIONS
Bioconversion by whole P. putida resting cells is an efficient strategy for the production of 2-HPP and other α-hydroxyketones.
Topics: Pseudomonas putida; Carboxy-Lyases; Benzaldehydes; Hydroxypropiophenone; Stereoisomerism; Ketones; Acetaldehyde
PubMed: 37131175
DOI: 10.1186/s12934-023-02073-7 -
Communications Biology Dec 2022Despite advances in understanding the metabolism of Pseudomonas putida KT2440, a promising bacterial host for producing valuable chemicals from plant-derived feedstocks,...
Despite advances in understanding the metabolism of Pseudomonas putida KT2440, a promising bacterial host for producing valuable chemicals from plant-derived feedstocks, a strain capable of producing free fatty acid-derived chemicals has not been developed. Guided by functional genomics, we engineered P. putida to produce medium- and long-chain free fatty acids (FFAs) to titers of up to 670 mg/L. Additionally, by taking advantage of the varying substrate preferences of paralogous native fatty acyl-CoA ligases, we employed a strategy to control FFA chain length that resulted in a P. putida strain specialized in producing medium-chain FFAs. Finally, we demonstrate the production of oleochemicals in these strains by synthesizing medium-chain fatty acid methyl esters, compounds useful as biodiesel blending agents, in various media including sorghum hydrolysate at titers greater than 300 mg/L. This work paves the road to produce high-value oleochemicals and biofuels from cheap feedstocks, such as plant biomass, using this host.
Topics: Pseudomonas putida; Fatty Acids, Nonesterified; Biofuels; Biomass; Fatty Acids
PubMed: 36509863
DOI: 10.1038/s42003-022-04336-2 -
Microbes and Environments 2023Pseudomonas putida is a major species belonging to the genus Pseudomonas. Although several hundred strains of P. putida have been deposited in culture collections, they...
Pseudomonas putida is a major species belonging to the genus Pseudomonas. Although several hundred strains of P. putida have been deposited in culture collections, they potentially differ from the genetically defined "true Pseudomonas putida" because many were classified as P. putida based on their phenotypic and metabolic characteristics. A phylogenetic ana-lysis based on the concatenated sequences of the 16S rRNA and rpoD genes revealed that 46 strains of P. putida deposited in Japanese culture collections were classified into nine operational taxonomic units (OTUs) and eleven singletons. The OTU7 strain produces N-acylhomoserine lactone as a quorum-sensing signal. One of the OTU7 strains, JCM 20066, exhibited a ppuI-rsaL-ppuR quorum-sensing system that controls biofilm formation and motility. The P. putida type strain JCM 13063 and six other strains were classified as OTU4. Classification based on the calculation of whole-genome similarity revealed that three OTU4 strains, JCM 20005, 21368, and 13061, were regarded as the same species as JCM 13063 and defined as true P. putida. When orthologous genes in the whole-genome sequences of true P. putida strains were screened, PP4_28660 from P. putida NBRC 14164 (=JCM 13063) was present in all true P. putida genome sequences. The internal region of PP4_28660 was successfully amplified from all true P. putida strains using the specific primers designed in this study.
Topics: Bacterial Typing Techniques; DNA, Bacterial; Fatty Acids; Genomics; Phylogeny; Pseudomonas putida; RNA, Ribosomal, 16S; Sequence Analysis, DNA
PubMed: 37286511
DOI: 10.1264/jsme2.ME23019 -
Scientific Reports Sep 2021Inducible and tunable expression systems are essential for the microbial production of biochemicals. Five different carbon source- and substrate-inducible promoter...
Inducible and tunable expression systems are essential for the microbial production of biochemicals. Five different carbon source- and substrate-inducible promoter systems were developed and further evaluated in Pseudomonas putida KT2440 by analyzing the expression of green fluorescent protein (GFP) as a reporter protein. These systems can be induced by low-cost compounds such as glucose, 3-hydroxypropionic acid (3HP), levulinic acid (LA), and xylose. 3HP-inducible HpdR/P was also efficiently induced by LA. LvaR/P and XutR/P systems were induced even at low concentrations of LA (0.1 mM) and xylose (0.5 mM), respectively. Glucose-inducible HexR/P showed weak GFP expression. These inducer agents can be used as potent starting materials for both cell growth and the production of a wide range of biochemicals. The efficiency of the reported systems was comparable to that of conventional chemical-inducible systems. Hence, the newly investigated promoter systems are highly useful for the expression of target genes in the widely used synthetic biology chassis P. putida KT2440 for industrial and medical applications.
Topics: Flow Cytometry; Gene Expression; Gene Expression Regulation, Bacterial; Genes, Reporter; Genetic Engineering; Genetic Vectors; Glucose; Lactic Acid; Promoter Regions, Genetic; Pseudomonas putida; Recombinant Proteins
PubMed: 34508142
DOI: 10.1038/s41598-021-97550-7 -
Microbiology (Reading, England) Jan 2023The type VI secretion system (T6SS) is an antimicrobial molecular weapon that is widespread in Proteobacteria and offers competitive advantages to T6SS-positive...
The type VI secretion system (T6SS) is an antimicrobial molecular weapon that is widespread in Proteobacteria and offers competitive advantages to T6SS-positive micro-organisms. Three T6SSs have recently been described in KT2440 and it has been shown that one, K1-T6SS, is used to outcompete a wide range of phytopathogens, protecting plants from pathogen infections. Given the relevance of this system as a powerful and innovative mechanism of biological control, it is critical to understand the processes that govern its expression. Here, we experimentally defined two transcriptional units in the K1-T6SS cluster. One encodes the structural components of the system and is transcribed from two adjacent promoters. The other encodes two hypothetical proteins, the tip of the system and the associated adapters, and effectors and cognate immunity proteins, and it is also transcribed from two adjacent promoters. The four identified promoters contain the typical features of σ-dependent promoters. We have studied the expression of the system under different conditions and in a number of mutants lacking global regulators. K1-T6SS expression is induced in the stationary phase, but its transcription does not depend on the stationary σ factor RpoS. In fact, the expression of the system is indirectly repressed by RpoS. Furthermore, it is also repressed by RpoN and the transcriptional regulator FleQ, an enhancer-binding protein typically acting in conjunction with RpoN. Importantly, expression of the K1-T6SS gene cluster is positively regulated by the GacS-GacA two-component regulatory system (TCS) and repressed by the RetS sensor kinase, which inhibits this TCS. Our findings identified a complex regulatory network that governs T6SS expression in general and K1-T6SS in particular, with implications for controlling and manipulating a bacterial agent that is highly relevant in biological control.
Topics: Type VI Secretion Systems; Bacterial Proteins; Pseudomonas putida; Sigma Factor; Multigene Family; Gene Expression Regulation, Bacterial
PubMed: 36748579
DOI: 10.1099/mic.0.001295