Biosurfactants are amphiphilic secondary metabolites produced by microorganisms. Marine bacteria have recently emerged as a rich source for these natural products which exhibit surface-active ...properties, making them useful for diverse applications such as detergents, wetting and foaming agents, solubilisers, emulsifiers and dispersants. Although precise structural data are often lacking, the already available information deduced from biochemical analyses and genome sequences of marine microbes indicates a high structural diversity including a broad spectrum of fatty acid derivatives, lipoamino acids, lipopeptides and glycolipids. This review aims to summarise biosyntheses and structures with an emphasis on low molecular weight biosurfactants produced by marine microorganisms and describes various biotechnological applications with special emphasis on their role in the bioremediation of oil-contaminated environments. Furthermore, novel exploitation strategies are suggested in an attempt to extend the existing biosurfactant portfolio.
Τhe morphology, physiology and immunology, of solid tumors exhibit spatial heterogeneity which complicates our understanding of cancer progression and therapy response. Understanding spatial ...heterogeneity necessitates high resolution in vivo imaging of anatomical and pathophysiological tumor information. We introduce Rhodobacter as bacterial reporter for multispectral optoacoustic (photoacoustic) tomography (MSOT). We show that endogenous bacteriochlorophyll a in Rhodobacter gives rise to strong optoacoustic signals >800 nm away from interfering endogenous absorbers. Importantly, our results suggest that changes in the spectral signature of Rhodobacter which depend on macrophage activity inside the tumor can be used to reveal heterogeneity of the tumor microenvironment. Employing non-invasive high resolution MSOT in longitudinal studies we show spatiotemporal changes of Rhodobacter spectral profiles in mice bearing 4T1 and CT26.WT tumor models. Accessibility of Rhodobacter to genetic modification and thus to sensory and therapeutic functions suggests potential for a theranostic platform organism.
Summary
Hydrolases acting on polyesters like cutin, polycaprolactone or polyethylene terephthalate (PET) are of interest for several biotechnological applications like waste treatment, biocatalysis ...and sustainable polymer modifications. Recent studies suggest that a large variety of such enzymes are still to be identified and explored in a variety of microorganisms, including bacteria of the genus Pseudomonas. For activity‐based screening, methods have been established using agar plates which contain nanoparticles of polycaprolactone or PET prepared by solvent precipitation and evaporation. In this protocol article, we describe a straightforward agar plate‐based method using emulsifiable artificial polyesters as substrates, namely Impranil® DLN and liquid polycaprolactone diol (PLD). Thereby, the currently quite narrow set of screening substrates is expanded. We also suggest optional pre‐screening with short‐chain and middle‐chain‐length triglycerides as substrates to identify enzymes with lipolytic activity to be further tested for polyesterase activity. We applied these assays to experimentally demonstrate polyesterase activity in bacteria from the P. pertucinogena lineage originating from contaminated soils and diverse marine habitats.
Hydrolases acting on polyesters like cutin, polycaprolactone or polyethylene terephthalate (PET) are of interest for several biotechnological applications like waste treatment, biocatalysis, and sustainable polymer modifications. In this protocol article, we describe a straightforward agar plate based method using emulsifiable artificial polyesters as substrates. We applied these assays to experimentally demonstrate polyesterase activity in bacteria from the Pseudomonas pertucinogena lineage originating from contaminated soils and diverse marine habitats.
The biosynthesis of natural products by heterologous expression of biosynthetic pathways in amenable production strains enables biotechnological access to a variety of valuable compounds by ...conversion of renewable resources.
Pseudomonas putida
has emerged as a microbial laboratory work horse, with elaborated techniques for cultivation and genetic manipulation available. Beyond that, this bacterium offers several particular advantages with regard to natural product biosynthesis, notably a versatile intrinsic metabolism with diverse enzymatic capacities as well as an outstanding tolerance to xenobiotics. Therefore, it has been applied for recombinant biosynthesis of several valuable natural products. This review provides an overview of applications of
P. putida
as a host organism for the recombinant biosynthesis of such natural products, including rhamnolipids, terpenoids, polyketides and non-ribosomal peptides, and other amino acid-derived compounds. The focus is on de novo natural product synthesis from intrinsic building blocks by means of heterologous gene expression and strain engineering. Finally, the future potential of the bacterium as a chassis organism for synthetic microbiology is pointed out.
Recombinant protein production is mostly realized with large-scale cultivations and monitored at the level of the entire population. Detailed knowledge of cell-to-cell variations with respect to ...cellular growth and product formation is limited, even though phenotypic heterogeneity may distinctly hamper overall production yields, especially for toxic or difficult-to-express proteins. Unraveling phenotypic heterogeneity is thus a key aspect in understanding and optimizing recombinant protein production in biotechnology and synthetic biology. Here, microfluidic single-cell analysis serves as the method of choice to investigate and unmask population heterogeneities in a dynamic and spatiotemporal fashion. In this study, we report on comparative microfluidic single-cell analyses of commonly used E. coli expression systems to uncover system-inherent specifications in the synthetic M9CA growth medium. To this end, the PT7lac/LacI, the PBAD/AraC and the Pm/XylS system were systematically analyzed in order to gain detailed insights into variations of growth behavior and expression phenotypes and thus to uncover individual strengths and deficiencies at the single-cell level. Specifically, we evaluated the impact of different system-specific inducers, inducer concentrations as well as genetic modifications that affect inducer-uptake and regulation of target gene expression on responsiveness and phenotypic heterogeneity. Interestingly, the most frequently applied expression system based on E. coli strain BL21(DE3) clearly fell behind with respect to expression homogeneity and robustness of growth. Moreover, both the choice of inducer and the presence of inducer uptake systems proved crucial for phenotypic heterogeneity. Conclusively, microfluidic evaluation of different inducible E. coli expression systems and setups identified the modified lacY-deficient PT7lac/LacI as well as the Pm/XylS system with conventional m-toluic acid induction as key players for precise and robust triggering of bacterial gene expression in E. coli in a homogeneous fashion.
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.
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•Valuable natural products can be accessed via engineered microbes.•The soil bacterium Pseudomonas putida represents an effective production host.•Products are rhamnolipids, ...terpenoids, polyketides, and amino acid-related metabolites.•Novel effective tools for genetic engineering and gene expression are available.
Organisms from all kingdoms of life represent a rich source for valuable natural products with various applications. Microbial systems are useful to implement the expression of respective biosynthetic genes and establish the production and effective access to natural products, or to elucidate the biochemistry of the underlying pathways. Pseudomonas putida, a Gram-negative soil bacterium, appears to be particularly suitable for natural product biosynthesis because of an advantageous intrinsic metabolism and a remarkable tolerance toward various xenobiotics. This article presents the current state of engineering P. putida for the biosynthesis of rhamnolipids, terpenoids, polyketides, non-ribosomal peptides, and products from amino acid metabolism. Technological advances facilitating chromosomal integration and expression of biosynthetic genes, and smart metabolic engineering are key to success.
Cyclic triterpenes constitute one of the most diverse groups of plant natural products. Besides the intriguing biochemistry of their biosynthetic pathways, plant triterpenes exhibit versatile ...bioactivities, including antimicrobial effects against plant and human pathogens. While prokaryotes have been extensively used for the heterologous production of other classes of terpenes, the synthesis of cyclic triterpenes, which inherently includes the two-step catalytic formation of the universal linear precursor 2,3-oxidosqualene, is still a major challenge. We thus explored the suitability of the metabolically versatile photosynthetic α-proteobacterium Rhodobacter capsulatus SB1003 and cyanobacterium Synechocystis sp. PCC 6803 as alternative hosts for biosynthesis of cyclic plant triterpenes. Therefore, 2,3-oxidosqualene production was implemented and subsequently combined with different cyclization reactions catalyzed by the representative oxidosqualene cyclases CAS1 (cycloartenol synthase), LUP1 (lupeol synthase), THAS1 (thalianol synthase) and MRN1 (marneral synthase) derived from model plant Arabidopsis thaliana. While successful accumulation of 2,3-oxidosqualene could be detected by LC-MS analysis in both hosts, cyclase expression resulted in differential production profiles. CAS1 catalyzed conversion to only cycloartenol, but expression of LUP1 yielded lupeol and a triterpenoid matching an oxidation product of lupeol, in both hosts. In contrast, THAS1 expression did not lead to cyclic product formation in either host, whereas MRN1-dependent production of marnerol and hydroxymarnerol was observed in Synechocystis but not in R. capsulatus. Our findings thus indicate that 2,3-oxidosqualene cyclization in heterologous phototrophic bacteria is basically feasible but efficient conversion depends on both the respective cyclase enzyme and individual host properties. Therefore, photosynthetic α-proteo- and cyanobacteria are promising alternative candidates for providing new bacterial access to the broad class of triterpenes for biotechnological applications.
Plant parasitic nematodes, including the beet cyst nematode
, constitute a devastating problem for crops worldwide. The limited availability of sustainable management options illustrates the need for ...new eco-friendly control means. Plant metabolites represent an invaluable source of active compounds for the discovery of such novel antagonistic agents. Here, we evaluated the impact of eight plant terpenoids on the
parasitism of
. None of the metabolites affected the plant development (5 or 10 ppm). Nootkatone decreased the number of adult nematodes on
to 50%, with the female nematodes being smaller compared to the control. In contrast, three other terpenoids increased the parasitism and/or female size. We discovered that nootkatone considerably decreased the number of nematodes that penetrated
roots, but neither affected the nematode viability or attraction to plant roots, nor triggered the production of plant reactive oxygen species or changed the plant's sesquiterpene profile. However, we demonstrated that nootkatone led to a significant upregulation of defense-related genes involved in salicylic and jasmonic acid pathways. Our results indicate that nootkatone is a promising candidate to be developed into a novel plant protection agent acting as a stimulator of plant immunity against parasitic nematodes.
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