Serratia marcescens and several other bacteria produce the red-colored pigment prodigiosin which possesses bioactivities as an antimicrobial, anticancer, and immunosuppressive agent. Therefore, there is a great interest to produce this natural compound. Efforts aiming at its biotechnological production have so far largely focused on the original producer and opportunistic human pathogen S. marcescens. Here, we demonstrate efficient prodigiosin production in the heterologous host Pseudomonas putida. Random chromosomal integration of the 21 kb prodigiosin biosynthesis gene cluster of S. marcescens in P. putida KT2440 was employed to construct constitutive prodigiosin production strains. Standard cultivation parameters were optimized such that titers of 94 mg/L culture were obtained upon growth of P. putida at 20°C using rich medium under high aeration conditions. Subsequently, a novel, fast and effective protocol for prodigiosin extraction and purification was established enabling the straightforward isolation of prodigiosin from P. putida growth medium. In summary, we describe here a highly efficient method for the heterologous biosynthetic production of prodigiosin which may serve as a basis to produce large amounts of this bioactive natural compound and may provide a platform for further in-depth studies of prodiginine biosynthesis.
Microbial secondary metabolites represent a rich source of valuable compounds with a variety of applications in medicine or agriculture. Effective exploitation of this wealth of chemicals requires the functional expression of the respective biosynthetic genes in amenable heterologous hosts. We have previously established the TREX system which facilitates the transfer, integration and expression of biosynthetic gene clusters in various bacterial hosts. Here, we describe the yTREX system, a new tool adapted for one-step yeast recombinational cloning of gene clusters. We show that with yTREX, Pseudomonas putida secondary metabolite production strains can rapidly be constructed by random targeting of chromosomal promoters by Tn5 transposition. Feasibility of this approach was corroborated by prodigiosin production after yTREX cloning, transfer and expression of the respective biosynthesis genes from Serratia marcescens. Furthermore, the applicability of the system for effective pathway rerouting by gene cluster adaptation was demonstrated using the violacein biosynthesis gene cluster from Chromobacterium violaceum, producing pathway metabolites violacein, deoxyviolacein, prodeoxyviolacein, and deoxychromoviridans. Clones producing both prodigiosin and violaceins could be readily identified among clones obtained after random chromosomal integration by their strong color-phenotype. Finally, the addition of a promoter-less reporter gene enabled facile detection also of phenazine-producing clones after transfer of the respective phenazine-1-carboxylic acid biosynthesis genes from Pseudomonas aeruginosa. All compounds accumulated to substantial titers in the mg range. We thus corroborate here the suitability of P. putida for the biosynthesis of diverse natural products, and demonstrate that the yTREX system effectively enables the rapid generation of secondary metabolite producing bacteria by activation of heterologous gene clusters, applicable for natural compound discovery and combinatorial biosynthesis.
Since high-value bacterial secondary metabolites, including antibiotics, are often naturally produced in only low amounts, their efficient biosynthesis typically requires the transfer of entire metabolic pathways into suitable bacterial hosts like Pseudomonas putida . Stable maintenance and sufficient expression of heterologous pathway-encoding genes in host microbes, however, still remain key challenges. In this study, the 21 kb prodigiosin gene cluster from Serratia marcescens was used as a reporter to identify genomic sites in P. putida KT2440 especially suitable for maintenance and expression of pathway genes. After generation of a strain library by random Tn5 transposon-based chromosomal integration of the cluster, 50 strains exhibited strong prodigiosin production. Remarkably, chromosomal integration sites were exclusively identified in the seven rRNA-encoding rrn operons of P. putida . We could further demonstrate that prodigiosin production was mainly dependent on (i) the individual rrn operon where the gene cluster was inserted as well as (ii) the distance between the rrn promoter and the inserted prodigiosin biosynthetic genes. In addition, the recombinant strains showed high stability upon subculturing for many generations. Consequently, our findings demonstrate the general applicability of rDNA loci as chromosomal integration sites for gene cluster expression and recombinant pathway implementation in P. putida KT2440.
Bacterial secondary metabolites are naturally produced to prevail amongst competitors in a shared habitat and thus represent a valuable source for antibiotic discovery. The transformation of newly discovered antibiotic compounds into effective drugs often requires additional surfactant components for drug formulation. Nature may also provide blueprints in this respect: A cocktail of two compounds consisting of the antibacterial red pigment prodigiosin and the biosurfactant serrawettin W1 is naturally produced by the bacterium Serratia marcescens, which occurs in highly competitive habitats including soil. We show here a combinatorial antibacterial effect of these compounds, but also of prodigiosin mixed with other (bio)surfactants, against the soil-dwelling bacterium Corynebacterium glutamicum taken as a model target bacterium. Prodigiosin exerted a combinatorial inhibitory effect with all tested surfactants in a disk diffusion assay which was especially pronounced in combination with N-myristoyltyrosine. Minimal inhibitory and bactericidal concentrations (MIC and MBC) of the individual compounds were 2.56 μg/mL prodigiosin and 32 μg/mL N-myristoyltyrosine, and the MIC of prodigiosin was decreased by 3 orders of magnitude to 0.005 μg/mL in the presence of 16 μg/mL N-myristoyltyrosine, indicative of synergistic interaction. Investigation of bacterial survival revealed similar combinatorial effects; moreover, antagonistic effects were observed at higher compound concentrations. Finally, the investigation of microcolony formation under combined application of concentrations just below the MBC revealed heterogeneity of responses with cell death or delayed growth. In summary, this study describes the combinatorial antibacterial effects of microbial biomolecules, which may have ecological relevance by inhibiting cohabiting species, but shall furthermore inspire drug development in the combat of infectious disease.
SummaryBacterial gene clusters, which represent a genetic treasure trove for secondary metabolite pathways, often need to be activated in a heterologous host to access the valuable biosynthetic products. We provide here a detailed protocol for the application of the yTREX ‘gene cluster transplantation tool’: Via yeast recombinational cloning, a gene cluster of interest can be cloned in the yTREX vector, which enables the robust conjugational transfer of the gene cluster to bacteria like Pseudomonas putida, and their subsequent transposon Tn5‐based insertion into the host chromosome. Depending on the gene cluster architecture and chromosomal insertion site, the respective pathway genes can be transcribed effectively from a chromosomal promoter, thereby enabling the biosynthesis of a natural product. We describe workflows for the design of a gene cluster expression cassette, cloning of the cassette in the yTREX vector by yeast recombineering, and subsequent transfer and expression in P. putida. As an example for yTREX‐based transplantation of a natural product biosynthesis, we provide details on the cloning and activation of the phenazine‐1‐carboxylic acid biosynthetic genes from Pseudomonas aeruginosa in P. putida KT2440 as well as the use of β‐galactosidase‐encoding lacZ as a reporter of production levels.
The expression of biosynthetic genes in bacterial hosts can enable access to high-value compounds, for which appropriate molecular genetic tools are essential. Therefore, we developed a toolbox of modular vectors, which facilitate chromosomal gene integration and expression in Pseudomonas putida KT2440. To this end, we designed an integrative sequence, allowing customisation regarding the modus of integration (random, at attTn7, or into the 16S rRNA gene), promoters, antibiotic resistance markers as well as fluorescent proteins and enzymes as transcription reporters. We thus established a toolbox of vectors carrying integrative sequences, designated as pYT series, of which we present 27 ready-to-use variants along with a set of strains equipped with unique ‘landing pads’ for directing a pYT interposon into one specific copy of the 16S rRNA gene. We used genes of the well-described violacein biosynthesis as reporter to showcase random Tn5-based chromosomal integration leading to constitutive expression and production of violacein and deoxyviolacein. Deoxyviolacein was likewise produced after gene integration into the 16S rRNA gene of rrn operons. Integration in the attTn7 site was used to characterise the suitability of different inducible promoters and successive strain development for the metabolically challenging production of mono-rhamnolipids. Finally, to establish arcyriaflavin A production in P. putida for the first time, we compared different integration and expression modes, revealing integration at attTn7 and expression with NagR/PnagAa to be most suitable. In summary, the new toolbox can be utilised for the rapid generation of various types of P. putida expression and production strains.
Waddlia chondrophila is a obligate intracellular bacterium belonging to the Chlamydiales order, a clade that also includes the well-known classical Chlamydia responsible for a number of severe human and animal diseases. Waddlia is an emerging pathogen associated with adverse pregnancy outcomes in humans and abortion in ruminants. Adhesion to the host cell is an essential prerequisite for survival of every strict intracellular bacteria and, in classical Chlamydia, this step is partially mediated by polymorphic outer membrane proteins (Pmps), a family of highly diverse autotransporters that represent about 15% of the bacterial coding capacity. Waddlia chondrophila genome however only encodes one putative Pmp-like protein. Using a proteomic approach, we identified several bacterial proteins potentially implicated in the adhesion process and we characterized their expression during the replication cycle of the bacteria. In addition, we demonstrated that the Waddlia Pmp-like autotransporter as well as OmpA2 and OmpA3, two members of the extended Waddlia OmpA protein family, exhibit adhesive properties on epithelial cells. We hypothesize that the large diversity of the OmpA protein family is linked to the wide host range of these bacteria that are able to enter and multiply in various host cells ranging from protozoa to mammalian and fish cells.
Toxoplasma gondii ( T. gondii ) is an obligate intracellular parasite and belongs to the phylum Apicomplexa. T. gondii is of medical and veterinary importance, because T. gondii causes the parasitic disease toxoplasmosis. In human cells, the interferon-gamma inducible indoleamine 2,3-dioxygenase 1 (IDO1) is an antimicrobial effector mechanism that degrades tryptophan to kynurenine and thus limits pathogen proliferation in vitro . Furthermore, IDO is described to have immunosuppressive properties, e.g., regulatory T cell differentiation and T cell suppression in humans and mice. However, there is only little known about the role of IDO1 in mice during acute toxoplasmosis. To shed further light on the role of mIDO1 in vivo , we have used a specifically adjusted experimental model. Therein, we infected mIDO1-deficient (IDO −/− ) C57BL/6 mice and appropriate wild-type (WT) control mice with a high dose of T. gondii ME49 tachyozoites (type II strain) via the intraperitoneal route and compared the phenotype of IDO −/− and WT mice during acute toxoplasmosis. During murine T. gondii infection, we found mIDO1 mRNA and mIDO1 protein, as well as mIDO1-mediated tryptophan degradation in lungs of WT mice. IDO −/− mice show no tryptophan degradation in the lung during infection. Even though T. gondii is tryptophan auxotroph and rapidly replicates during acute infection, the parasite load was similar in IDO −/− mice compared to WT mice 7 days post-infection. IDO1 is described to have immunosuppressive properties, and since T cell suppression is observed during acute toxoplasmosis, we analyzed the possible involvement of mIDO1. Here, we did not find differences in the intensity of ex vivo mitogen stimulated T cell proliferation between WT and IDO −/− mice. Concomitant nitric oxide synthase inhibition and interleukin-2 supplementation increased the T cell proliferation from both genotypes drastically, but not completely. In sum, we analyzed the involvement of mIDO1 during acute murine toxoplasmosis in our specifically adjusted experimental model and found a definite mIDO1 induction. Nevertheless, mIDO1 seems to be functional redundant as an antiparasitic defense mechanism during acute toxoplasmosis in mice. Furthermore, we suggest that the systemic T cell suppression observed during acute toxoplasmosis is influenced by nitric oxide activity and IL-2 deprivation.
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