Prodiginine Biosynthesis Research Articles

PpGpp is a dual-role regulator involved in balancing iron absorption and prodiginine biosynthesis in Pseudoalteromonas.

Iron is an essential element for microbial survival and secondary metabolism. However, excess iron availability and overloaded secondary metabolites can hinder microbial growth and survival. Microorganisms must tightly control iron homeostasis and secondary metabolism. Our previous studies have found that the stringent starvation protein A (SspA) positively regulates prodiginine biosynthesis by activating iron uptake in Pseudoalteromonas sp. strain R3. It is believed that the interaction between SspA and the small nucleotide ppGpp is important for iron to exert regulation functions. However, the roles of ppGpp in iron absorption and prodiginine biosynthesis, and the underlying relationship between ppGpp and SspA in strain R3 remain unclear. In this study, we found that ppGpp accumulation in strain R3 could be induced by limiting iron. In addition, ppGpp not only positively regulated iron uptake and prodiginine biosynthesis via increasing the SspA level but also directly repressed iron uptake and prodiginine biosynthesis independent of SspA, highlighting the finding that ppGpp can stabilize both iron levels and prodiginine production. Notably, the abolishment of ppGpp significantly increased prodiginine production, thus providing a theoretical basis for manipulating prodiginine production in the future. This dynamic ppGpp-mediated interaction between iron uptake and prodiginine biosynthesis has significant implications for understanding the roles of nutrient uptake and secondary metabolism for the survival of bacteria in unfavorable environments.

Stringent Starvation Protein SspA and Iron Starvation Sigma Factor PvdS Coordinately Regulate Iron Uptake and Prodiginine Biosynthesis in Pseudoalteromonas sp. R3.

Organisms need sufficient intracellular iron to maintain biological processes. However, cells can be damaged by excessive iron-induced oxidation stress. Therefore, iron homeostasis must be strictly regulated. In general, bacteria have evolved complex mechanisms to maintain iron homeostasis. In this study, we showed that Pseudoalteromonas sp. R3 has four sets of iron uptake systems. Among these, the siderophore pyoverdine-dependent iron uptake system and the ferrous iron transporter Feo system are more important for iron uptake and prodiginine biosynthesis. Stringent starvation protein SspA positively controls iron uptake and iron-dependent prodiginine biosynthesis by regulating the expression of all iron uptake systems. In turn, the expression of SspA can be induced and repressed by extracellular iron deficiency and excess, respectively. Interestingly, extracytoplasmic function sigma factor PvdS also regulates iron uptake and prodiginine production and responds to extracellular iron levels, exhibiting a similar phenomenon as SspA. Notably, not only do SspA and PvdS function independently, but they can also compensate for each other, and their expression can be affected by the other. All of these findings demonstrate that SspA and PvdS coordinate iron homeostasis and prodiginine biosynthesis in strain R3. More importantly, our results also showed that SspA and PvdS homologs in Pseudomonas aeruginosa PAO1 have similar functions in iron uptake to their counterparts in Pseudoalteromonas, suggesting that coordination between SspA and PvdS on iron homeostasis could be conserved in typical Gram-negative bacteria. Since master regulation of iron homeostasis is extremely important for cell survival, this cross talk between SspA and PvdS may be environmentally significant. IMPORTANCE Both deficiency and excess of intracellular iron can be harmful, and thus, the iron homeostasis needs to be tightly regulated in organisms. At present, the ferric uptake regulator (Fur) is the best-characterized regulator involved in bacterial iron homeostasis, while other regulators of iron homeostasis remain to be further explored. Here, we demonstrated that the stringent starvation protein SspA and the extracytoplasmic function sigma factor PvdS coordinate iron uptake and iron-dependent prodiginine biosynthesis in Pseudoalteromonas sp. R3. These two regulators work independently, but their functions can compensate for the other and their expression can be affected by the other. Moreover, their expression can be activated and repressed by extracellular iron deficiency and excess, respectively. Notably, SspA and PvdS homologs in Pseudomonas aeruginosa PAO1 exhibit similar functions in iron uptake to their counterparts in Pseudoalteromonas, suggesting that this novel fine-tuned mode of iron homeostasis could be conserved in typical Gram-negative bacteria.

Stringent Starvation Protein Regulates Prodiginine Biosynthesis via Affecting Siderophore Production in Pseudoalteromonas sp. Strain R3.

Prodiginines are a family of red-pigmented secondary metabolites with multiple biological activities. The biosynthesis of prodiginines is affected by various physiological and environmental factors. Thus, prodiginine biosynthesis regulation is highly complex and multifaceted. Although the regulatory mechanism for prodiginine biosynthesis has been extensively studied in Serratia and Streptomyces species, little is known about that in the marine betaproteobacterium Pseudoalteromonas In this study, we report that stringent starvation protein A (SspA), an RNA polymerase-associated regulatory protein, is required for the biosynthesis of prodiginine in Pseudoalteromonas sp. strain R3. The strain lacking sspA (ΔsspA) fails to produce prodiginine, which resulted from the downregulation of the prodiginine biosynthetic gene (pig) cluster. The effect of SspA on prodiginine biosynthesis is independent of histone-like nucleoid structuring protein (H-NS) and RpoS (σS). Further analysis demonstrates that the ΔsspA strain has a significant decrease in the transcription of the siderophore biosynthesis gene (pvd) cluster, leading to the inhibition of siderophore production and iron uptake. The ΔsspA strain regains the ability to synthesize prodiginine by cocultivation with siderophore producers or the addition of iron. Therefore, we conclude that SspA-regulated prodiginine biosynthesis is due to decreased siderophore levels and iron deficiency. We further show that the iron homeostasis master regulator Fur is also essential for pig transcription and prodiginine biosynthesis. Overall, our results suggest that SspA indirectly regulates the biosynthesis of prodiginine, which is mediated by the siderophore-dependent iron uptake pathway.IMPORTANCE The red-pigmented prodiginines are attracting increasing interest due to their broad biological activities. As with many secondary metabolites, the biosynthesis of prodiginines is regulated by both environmental and physiological factors. At present, studies on the regulation of prodiginine biosynthesis are mainly restricted to Serratia and Streptomyces species. This work focused on the regulatory mechanism of prodiginine biosynthesis in Pseudoalteromonas sp. R3. We found that stringent starvation protein A (SspA) positively regulates prodiginine biosynthesis via affecting the siderophore-dependent iron uptake pathway. The connections among SspA, iron homeostasis, and prodiginine biosynthesis were investigated. These findings uncover a novel regulatory mechanism for prodigiosin biosynthesis.

Biosynthesis and Bioactivity of Prodiginine Analogs in Marine Bacteria, Pseudoalteromonas: A Mini Review.

The Prodiginine family consists of primarily red-pigmented tripyrrole secondary metabolites that were first characterized in the Gram-negative bacterial species Serratia marcescens and demonstrates a wide array of biological activities and applications. Derivatives of prodiginine have since been characterized in the marine γ-proteobacterium, Pseudoalteromonas. Although biosynthetic gene clusters involved in prodiginine synthesis display homology among genera, there is an evident structural difference in the resulting metabolites. This review will summarize prodiginine biosynthesis, bioactivity, and gene regulation in Pseudoalteromonas in comparison to the previously characterized species of Serratia, discuss the ecological contributions of Pseudoalteromonas in the marine microbiome and their eukaryotic hosts, and consider the importance of modern functional genomics and classic DNA manipulation to understand the overall prodiginine biosynthesis pathway.

Condensing Enzymes from Pseudoalteromonadaceae for Prodiginine Synthesis

AbstractProdiginines are natural products that are produced by various microorganisms. As the biological activity of prodiginines varies depending on their structure, prodiginine derivatives are of interest. In order to extend the prodiginine structural diversity, new condensing enzyme homologs of PigC were analysed. The condensing enzyme PigC from S. marcescens catalyses the last step in prodiginine biosynthesis and accepts not only its natural substrate, but also synthetically produced monopyrroles. Still, the substrate spectrum of PigC is partially limited. In order to enable the biocatalytic production of new prodiginine derivatives, PigC homologs supplementing the substrate spectra of PigC are of interest. Two condensing enzymes homologs from two tambjamine producing Pseudoalteromonadaceae strains were analysed towards their catalytic ability to produce the structurally related prodiginines. Besides the investigation of their substrate spectrum and enzyme kinetic data, a preparative scale biocatalysis approach for the production of prodiginine derivatives was established, comparing different condensing enzymes.magnified image

Efficient recombinant production of prodigiosin in Pseudomonas putida.

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.

Use of red autofluorescence for monitoring prodiginine biosynthesis

Prodigiosin-like pigments or prodiginines (PdGs) are promising drugs owing to their reported antitumor, antibiotic, and immunosuppressive activities. These natural compounds are produced by several bacteria, including Streptomyces coelicolor and Serratia marcescens as most commonly studied models. The bright red color of these tripyrrole pigments made them excellent reporter molecules for studies aimed at understanding the molecular mechanisms that control secondary metabolite production in microorganisms. However, the natural red fluorescence of PdGs has only been rarely used as a biophysical parameter for detection and assessment of PdG biosynthesis. In this work, we used S. coelicolor in order to exemplify how intrinsic red fluorescence could be utilized for rapid, low-cost, sensitive, specific and accurate semi-quantitative analyses of PdG biosynthesis. Additionally, and contrary to the colorimetric-based approach, the fluorescence-based method allows in situ spatio-temporal visualization of PdG synthesis throughout a solid culture of S. coelicolor. As PdG production is related to cell differentiation, their red autofluorescence could be exploited, by means of confocal microscopy, as a natural marker of the entrance into a crucial developmental stage in the course of the S. coelicolor life cycle.

Structure and Function of the RedJ Protein, a Thioesterase from the Prodiginine Biosynthetic Pathway in Streptomyces coelicolor

Prodiginines are a class of red-pigmented natural products with immunosuppressant, anticancer, and antimalarial activities. Recent studies on prodiginine biosynthesis in Streptomyces coelicolor have elucidated the function of many enzymes within the pathway. However, the function of RedJ, which was predicted to be an editing thioesterase based on sequence similarity, is unknown. We report here the genetic, biochemical, and structural characterization of the redJ gene product. Deletion of redJ in S. coelicolor leads to a 75% decrease in prodiginine production, demonstrating its importance for prodiginine biosynthesis. RedJ exhibits thioesterase activity with selectivity for substrates having long acyl chains and lacking a β-carboxyl substituent. The thioesterase has 1000-fold greater catalytic efficiency with substrates linked to an acyl carrier protein (ACP) than with the corresponding CoA thioester substrates. Also, RedJ strongly discriminates against the streptomycete ACP of fatty acid biosynthesis in preference to RedQ, an ACP of the prodiginine pathway. The 2.12 Å resolution crystal structure of RedJ provides insights into the molecular basis for the observed substrate selectivity. A hydrophobic pocket in the active site chamber is positioned to bind long acyl chains, as suggested by a long-chain ligand from the crystallization solution bound in this pocket. The accessibility of the active site is controlled by the position of a highly flexible entrance flap. These data combined with previous studies of prodiginine biosynthesis in S. coelicolor support a novel role for RedJ in facilitating transfer of a dodecanoyl chain from one acyl carrier protein to another en route to the key biosynthetic intermediate 2-undecylpyrrole.

Elucidation of the Streptomyces coelicolor Pathway to 2-Undecylpyrrole, a Key Intermediate in Undecylprodiginine and Streptorubin B Biosynthesis

The red gene cluster of Streptomyces coelicolor directs production of undecylprodiginine. Here we report that this gene cluster also directs production of streptorubin B and show that 2-undecylpyrrole (UP) is an intermediate in the biosynthesis of undecylprodiginine and streptorubin B. The redPQRKL genes are involved in UP biosynthesis. RedL and RedK are proposed to generate UP from dodecanoic acid or a derivative. A redK(-) mutant produces a hydroxylated undecylprodiginine derivative, whereas redL(-) and redK(-) mutants require addition of chemically synthesized UP for production of undecylprodiginine and streptorubin B. Fatty acid biosynthetic enzymes can provide dodecanoic acid, but efficient and selective prodiginine biosynthesis requires RedPQR. Deletion of redP, redQ, or redR leads to an 80%-95% decrease in production of undecylprodiginine and an array of prodiginine analogs with varying alkyl chains. In a redR(-) mutant, the ratio of these can be altered in a logical manner by feeding various fatty acids.

Anticancer and Immunosuppressive Properties of Bacterial Prodiginines

Bacterial prodiginines are a family of red-pigmented, tripyrrolic compounds that display numerous biological activities, including antibacterial, antifungal, antiprotozoal, antimalarial, immunosuppressive and anticancer properties. Recently, significant progress has been made in understanding the biosynthesis and regulation of bacterial prodiginines. An understanding of the biosynthesis of prodiginines will allow engineering of bacterial strains capable of synthesizing novel prodiginines through rational design and mutasynthesis experiments. Bacterial prodiginines and synthetic derivatives are effective proapoptotic agents with multiple cellular targets, and they are active against numerous cancer cell lines, including multidrug-resistant cells, with little or no toxicity towards normal cell lines. A synthetic derivative, GX15-070 (Obatoclax), developed through structure-activity relationship studies of the pyrrolic ring A of GX15, is in multiple Phase I and II clinical trials in both single and dual-agent studies to treat different types of cancer. Therefore, prodiginines have real therapeutic potential in the clinic.

The biosynthesis and regulation of bacterial prodiginines

The red-pigmented prodiginines are bioactive secondary metabolites produced by both Gram-negative and Gram-positive bacteria. Recently, these tripyrrole molecules have received renewed attention owing to reported immunosuppressive and anticancer properties. The enzymes involved in the biosynthetic pathways for the production of two of these molecules, prodigiosin and undecylprodigiosin, are now known. However, the biochemistry of some of the reactions is still poorly understood. The physiology and regulation of prodiginine production in Serratia and Streptomyces are now well understood, although the biological role of these pigments in the producer organisms remains unclear. However, research into the biology of pigment production will stimulate interest in the bioengineering of strains to synthesize useful prodiginine derivatives.

Biosynthesis of antibiotic prodiginines in the marine bacterium Hahella chejuensis KCTC 2396

Hahella chejuensis KCTC 2396 produces red pigments, showing antibacterial and algicidal activities. The main red-coloured metabolite of the pigments was identified as antibiotic prodigiosin. With the expectation that the red pigments are a mixture of a series of close relatives, the aim of the present study is to detect new antibiotic prodigiosin analogues and to analyse the biosynthetic pattern for prodiginines in KCTC 2396. Except prodigiosin, the other constituents in the red pigments were confirmed as well-known dipyrrolyldipyrromethene prodigiosin, norprodigiosin, and undecylprodiginine. Additionally, four new prodigiosin analogues, each of which was distinguished from prodigiosin (C(5)), according to differences in alkyl chain length (C(3)-C(7)), were detected in small quantities by liquid chromatography mass spectrometry/mass spectrometry spectroscopy. Owing to the presence of a cytotoxic methoxy group, it is expected that all the new prodigiosin analogues are bioactive. Four characterized prodiginines, including prodigiosin and four new prodigiosin analogues are produced in different ratio in KCTC 2396. All of the prodiginines possess a common linear tripyrrolyl structure and a cytotoxic methoxy group. This study shows for the first time that KCTC 2396 is able to produce antibiotic prodigiosin, undecylprodiginine and new prodigiosin analogues in a mixture of pigments. It is also shown that KCTC 2396 possesses a novel system for the simultaneous production of multiple prodiginines in a single micro-organism.

Elucidation of the Streptomyces coelicolor pathway to 4-methoxy-2,2′-bipyrrole-5-carboxaldehyde, an intermediate in prodiginine biosynthesis

The biosynthetic pathway to 4-methoxy-2,2'-bipyrrole-5-carboxaldehyde (MBC), a key intermediate in the biosynthesis of prodiginine antibiotics in Streptomyces coelicolor, has been elucidated using a combination of gene replacements and feeding experiments with chemically synthesised MBC and a synthetic analogue of a pathway intermediate.

Analysis of the prodiginine biosynthesis gene cluster of Streptomyces coelicolor A3(2): new mechanisms for chain initiation and termination in modular multienzymes

Prodiginines are a large family of pigmented oligopyrrole antibiotics with medicinal potential as immunosuppressants and antitumour agents that are produced by several actinomycetes and other eubacteria. Recently, a gene cluster in Streptomyces coelicolor encoding the biosynthesis of undecylprodiginine and butyl-meta-cycloheptylprodiginine has been sequenced. Using sequence comparisons, functions have been assigned to the majority of the genes in the cluster, several of which encode homologues of enzymes involved in polyketide, non-ribosomal peptide, and fatty acid biosynthesis. Based on these assignments, a complete pathway for undecylprodiginine and butyl-meta-cycloheptylprodiginine biosynthesis in S. coelicolor has been deduced. Gene knockout experiments have confirmed the deduced roles of some of the genes in the cluster. The analysis presented provides a framework for a general understanding of the genetics and biochemistry of prodiginine biosynthesis, which should stimulate rational approaches to the engineered biosynthesis of novel prodiginines with improved immunosuppressant or antitumour activities. In addition, new mechanisms for chain initiation and termination catalysed by hitherto unobserved domains in modular multienzyme systems have been deduced.

Biosynthesis of prodiginines. 13C resonance assignments and enrichment patterns in nonyl-, cyclononyl-, methylcyclodecyl-, and butylcycloheptylprodiginine produced by actinomycete cultures supplemented with 13C-labeled acetate and 15N-labeled nitrate

Resonances in the 13C nmr spectra of nonylprodiginine, cyclononylprodiginine, methylcyclodecylprodiginine, and butylcycloheptylprodiginine were assigned using information from the literature, from high resolution spectra and double irradiation experiments, and from 13C–15N couplings observed in samples of nonylprodiginine enriched with 15N. Supplementation of cultures with sodium [1-13C]- and [1,2-13C]acetate gave 13C-enriched prodiginines and the labeling patterns were established from 13C nmr spectra. These indicated that rings A and B were formed in a similar manner to rings A and B in prodigiosin and other prodiginine pigments. Ring C and the C2-alkyl substituent were derived in part from acetate units that were condensed in a linear 12- or 14-carbon chain. The same pattern of acetate assembly has been found in ail actinomycete prodiginines examined.