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Structure of the aglycone core of pimaricin. Mycosamine (M) is attached to the hydroxyl at C-15 in the pimaricin molecule. The 12 C-C bonds formed by the polyketide synthase are boxed and numbered in italics. Bold lines indicate the building units. Lactonization of the acyl chain between C-1 and C-25 results in the formation of the pimaricinolide ring.
Source publication
The biosynthetic gene cluster for the 26-membered
ring of the polyene macrolide pimaricin extends for
about 110 kilobase pairs of contiguous DNA in the genome
of Streptomyces natalensis. Two sets of polyketide
synthase (PKS) genes are separated by a group of small
polyketide-functionalizing genes. Two of the polyketide
synthase genes, pimS0 and pim...
Citations
... Most countries approve natamycin on cheese as surface treatment, and its addition into other foods depends on legislation (de Boer and Stolk-Horsthuis, 1977;Aparicio et al., 1999). Natamycin's antifungal activity against diverse pathogenic fungal strains has reported that natamycin blocks fungal growth by binding to ergosterol specifically without permeabilising the membrane (Te Welscher et al., 2008;Delattin et al., 2014a). ...
In this present study, a highly stable gum acacia -gold nanocomposite fabricated with food preservative agent natamycin (GA-AuNC–NT) was prepared via green science principles under in vitro conditions. Various characterisation techniques reveal highly stable structural, functional properties of the synthesised nanocomposite with marked antifungal activity and adsorption efficacy against congo red dye. The antifungal activity was investigated against the fungal strain Aspergillus ochraceopealiformis isolated from spoiled, expired bread. The well diffusion assay, fungal hyphae fragmentation assay and spore germination inhibition assay were used to determine the antifungal activity of the synthesised nanocomposite. Potential antifungal activity of the synthesised nanocomposite was confirmed by recording zone of inhibition, high rate of hyphae fragmentation and marked spore germination inhibition against the tested fungal strain. The molecular mechanism of antifungal activity was studied by measuring oxidative stress marker genes like catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) induction adopting quantitative real-time polymerase chain reaction (q RT-PCR). Among the various treatment, a notable reduction in all the tested marker genes expression was recorded in the nanocomposite treated fungal strain. Release profile studies using different solvents reveals sustained or controlled release of natamycin at the increasing periods. The synthesised nanocomposite's high safety or biocompatibility was evaluated with the Wistar animal model by determining notable changes in behavioural, biochemical, haematological and histopathological parameters. The synthesised nanocomposite did not exhibit any undesirable changes in all the tested parameters confirming the marked biosafety or biocompatibility. The nanocomposite was coated on the bread packaging material. The effect of packaging on the proximate composition, antioxidative enzymes status, and fungal growth of bread samples incubated under the incubation period were studied. Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) studies reveal that the nanocomposite was effectively coated on the packaging material without changing size, shape, and functional groups. No changes in the proximate composition and antioxidative enzymes of the packaged bread samples incubated under different incubation periods reveal the nanocomposite's marked safety. The complete absence of the fungal growth also indicates the uniqueness of the nanocomposite. Further, the sorption studies revealed the utilisation of Langmuir mechanism and pseudo II order model successfully The present finding implies that the synthesised nanocomposite can be used as an effective, safe food preservative agent and adsorbent of toxic chemicals.
... 16 Meanwhile, formal fermentation is also greatly influenced by the cell status and activity in seed preculture, which depends on the spore's distribution level, inoculum age, environmental pH and inoculum concentration. 17,18 Therefore, parameter optimization and process regulation in seed preculture are necessary for biochemical fermentations. ...
... Natamycin-producing strains are mainly composed of Streptomyces species, such as S. natalensis, 11,18 S. gilvosporeus, 8,10 S. lydicus 19 and S. chattanoogensis 7 . These filamentous microorganisms usually exhibit various morphologies of mycelial pellets under different culture conditions. ...
BACKGROUND
Natamycin, an antifungal agent, has been widely used as a food preservative and medicine for fungal disease therapy. Seed preculture plays a crucial role in natamycin production, while studies on the effects of seed morphology on natamycin biosynthesis and corresponding regulation approaches are still absent.
RESULTS
Correlation analyses among spore age, seed morphology and natamycin production showed that old spores tended to form larger mycelial pellets, which gave rise to a significant reduction of natamycin biosynthesis. To solve this problem, microparticle talc was added to regulate the mycelial morphology in seed preculture. Optimal talc addition led to small mycelial pellets with hairy superficial mycelia and loose structure, resulting in higher glucose‐6‐phosphate dehydrogenase activity and energy charge. The seed morphology regulation effectively improved the natamycin titer and decreased the culture time, especially for old spores (age 28 days), with a 1.7‐fold higher natamycin titer and 16.9% culture time reduction. Unfortunately, direct talc addition proved to be infeasible for natamycin fermentation in a 5 L fermentor, which was attributed to physical damage from rapid agitation and excessive talc crashing. Therefore, the morphology engineering strategy was preferred in the seed preculture process rather than formal fermentation for natamycin production.
CONCLUSION
The new strategy proposed in this study could not only improve natamycin production, but also reduced the culture time. Furthermore, this work could provide references for other biochemicals production by filamentous bacteria or fungi, which would be of great significance in industrial antibiotic fermentation. © 2021 Society of Chemical Industry
... The biosynthesis of natamycin was first described by Aparicio et al. (1999) who identified and cloned a large Streptomyces natalensis gene cluster involved in the biosynthesis of the polyketide backbone that formed the 26-membered tetraene macrolide ring of natamycin. Natamycin is synthesized by polyketide synthases that sequentially assemble acyl precursors in a mechanism that was described as resembling fatty acid biosynthesis (Aparicio et al. 2003). ...
... Over the last several decades, complete polyene biosynthetic gene clusters from nystatin, amphotericin, pimaricin, and candicidin have been isolated and characterized (Aparicio et al., 1999(Aparicio et al., , 2003Zotchev et al., 2000;Caffrey et al., 2001). Polyene compounds are biosynthesized typically by a giant enzyme complex called polyketide synthase (PKS), followed by further modification of the core macrolide ring by post-PKS modification enzymes, including P450 hydroxylases and glycosyltransferases (Kim et al., 2015). ...
Polyene macrolides, such as nystatin A1, amphotericin B, and NPP A1, belong to a large family of valuable antifungal polyketide compounds that are typically produced by soil actinomycetes. Previously, NPP B1, a novel NPP A1 derivative harboring a heptaene core structure, was generated by introducing two amino acid substitutions in the putative NADPH-binding motif of the enoyl reductase domain in module 5 of the NPP A1 polyketide synthase in Pseudonocardia autotrophica. This derivative showed superior antifungal activity to NPP A1. In this study, another novel derivative called NPP B2 was developed, which lacks a hydroxyl group at the C10 position by site-specific gene disruption of the P450 hydroxylase NppL. To stimulate the extremely low expression of the NPP B2 biosynthetic pathway genes, the 32-kb NPP-specific regulatory gene cluster was overexpressed via site-specific chromosomal integration. The extra copy of the six NPP-specific regulatory genes led to a significant increase in the NPP B2 yield from 0.19 to 7.67 mg/L, which is the highest level of NPP B2 production ever achieved by the P. autotrophica strain. Subsequent in vitro antifungal activity and toxicity studies indicated that NPP B2 exhibited similar antifungal activity but significantly lower hemolytic toxicity than NPP B1. These results suggest that an NPP biosynthetic pathway refactoring and overexpression of its pathway-specific regulatory genes is an efficient approach to stimulating the production of an extremely low-level metabolite, such as NPP B2 in a pathway-engineered rare actinomycete strain.
... Natamycin not only exhibits strong inhibition and wide antifungal spectrum against most yeasts and moulds, but also prevents fungal toxin biosynthesis. 1 Confirmed as a GRAS (generally regarded as safe) compound by Food and Drug Administration (FDA), natamycin has been used widely as a green preservative in the food industry, an antifungal agent in disease therapy and an antifungal pesticide in plant disease control for several decades. 2 Currently, submerged batch and fed-batch cultures with free suspended cells are the main approaches to commercial natamycin production. [3][4][5][6][7] However, these traditional strategies have considerable drawbacks. ...
BACKGROUND
Natamycin is an antibiotic which shows strong inhibition and wide spectrum against most fungi. It is produced mainly through batch and fed‐batch fermentations, which are characterized by long culture time, low productivity and enormous discharge of waste biomass. In the present study, a new strategy using immobilized cells was proposed for continuous natamycin production via repeated batch culture to address these shortcomings.
RESULTS
A combination of cell immobilization and repeated batch culture was employed to continuously produce natamycin in both flasks and a 5‐L fermenter. The immobilized cells served as seeds and producing strains, and exhibited high vitality of cell growth and natamycin biosynthesis during six cycles of repeated batch culture. The application of the new strategy led to remarkable decrease of lag phase and removal of time intervals among traditional multiple batch cultures, resulting in a 32.10% reduction of total culture time. In addition, the new strategy showed 1.8‐fold higher natamycin titre and 195.24% productivity improvement relative to traditional strategies, and decreased waste biomass discharge, thus consituting a high‐efficiency, environmentally friendly strategy for natamycin production.
CONCLUSION
This study proposes a new approach to continuous natamycin production, which not only promoted natamycin productivity, but also reduced the fermentation time and the biomass residue discharge. Moreover, it provides reference points for the continuous production of other biochemicals by filamentous bacteria or funguses, which could be of great significance in industrial antibiotics production. © 2019 Society of Chemical Industry
... The biosynthetic mechanisms for several polyene macrolide antibiotics, including candicidin (FR-008) [4,5], amphotericin [6], pimaricin [7], nystatin [8], tetramycin [9], and NPP [10] have been widely studied. The formation of the macrolactone ring is typically catalyzed by a modular type I polyketide synthetase (PKS), after which two tailoring steps usually occur to furnish the exocyclic carboxyl group and the mycosamine sugar(s) [11]. ...
... The occurrence of cis-double bonds in RDM B-E (2-5) might be due to spontaneous trans-cis isomerization [11]. In comparison to other polyene macrolide gene clusters, such as pimaricin [7], amphotericin [6], candicidin [4,5], nystatin [8], NPP [10] and tetramycin [9] biosynthetic gene clusters, the rdm gene cluster is devoid of mycosamine biosynthetic genes and cytochrome P450 genes, which are involved in tailoring steps after macrocyclization. Many studies have demonstrated that mycosaminyltransferases tolerate structural changes in their aglycones and show moderately strict specificity for NDP-sugar donors [11,29]. ...
Background
Polyene antibiotics are important as antifungal medicines albeit with serious side effects such as nephrotoxicity. Reedsmycin (RDM) A (1), produced by marine-derived Streptomyces youssoufiensis OUC6819, is a non-glycosylated polyene macrolide antibiotic with antifungal activity comparable to that of clinically used nystatin. To elucidate its biosynthetic machinery, herein, the rdm biosynthetic gene cluster was cloned and characterized. ResultsThe rdm cluster is located within a 104 kb DNA region harboring 21 open reading frames (ORFs), among which 15 ORFs were designated as rdm genes. The assembly line for RDM A is proposed on the basis of module and domain analysis of the polyketide synthetases (PKSs) RdmGHIJ, which catalyze 16 rounds of decarboxylative condensation using malonyl-CoA as the starter unit (loading module), two methylmalonyl-CoA (module 1 and 2), and fourteen malonyl-CoA (module 3–16) as extender units successively. However, the predicted substrate specificity of AT0 in the loading module is methylmalonyl-CoA instead of malonyl-CoA. Interestingly, the rdm cluster contains a five-gene regulation system RdmACDEF, which is different from other reported polyene gene clusters. In vivo experiments demonstrated the XRE family regulator RdmA and the PAS/LuxR family regulator RdmF function in negative and positive manner, respectively. Notably, inactivation of rdmA and overexpression of rdmF led to increased production of RDM A by ~ 2.0-fold and ~ 2.5-fold, reaching yields of 155.3 ± 1.89 and 184.8 ± 9.93 mg/L, respectively. Conclusions
Biosynthesis of RDM A is accomplished on a linear assembly line catalyzed by Rdm PKSs harboring a unique AT0 under the control of a complex regulatory system. These findings enable generation of new biologically active RDM derivatives at high yield and with improved properties by engineered biosynthesis.
... It inhibits the growth of fungi via the immediate inhibition of amino acid and glucose transport across the plasma membrane. Due to its broad spectrum of antifungal activity and naturally occurring fungal resistance to natamycin being exceptionally rare, natamycin is widely used in the treatment of fungal keratitis, as a food preservative and as an antifungal agent in agriculture 1,3,4 . In addition to S. lydicus, three other species of Streptomyces are known to produce natamycin, i.e., S. natalensis, S. gilvosporeus, and S. chattanoogensis 3,5,6 . ...
... Due to its broad spectrum of antifungal activity and naturally occurring fungal resistance to natamycin being exceptionally rare, natamycin is widely used in the treatment of fungal keratitis, as a food preservative and as an antifungal agent in agriculture 1,3,4 . In addition to S. lydicus, three other species of Streptomyces are known to produce natamycin, i.e., S. natalensis, S. gilvosporeus, and S. chattanoogensis 3,5,6 . Despite extensive studies on their natamycin biosynthetic pathways, the underlying mechanisms of natamycin production and the regulatory role at the genomic level in these Streptomyces species remain unclear. ...
... Genomic DNA was isolated from A02 with TIANamp Bacteria DNA Kit (Tiangen, Beijing, China) and dissolved in DNAse-free double-distilled water. For RNA extraction, the wild-type A02 and recombinant strains of S. lydicus were inoculated into YEME medium without sucrose 3 Genome sequencing and assembly. The genomic DNA of A02 was sequenced by combining next-generation sequencing platforms (Illumina paired end, 2*90-bp, and 500-bp insert size) and SMRT sequencing (Pacific Biosystems RS) by the Wuhan Institute of Biotechnology (Wuhan, China). ...
Streptomyces lydicus A02 is used by industry because it has a higher natamycin-producing capacity than the reference strain S. natalensis ATCC 27448. We sequenced the complete genome of A02 using next-generation sequencing platforms, and to achieve better sequence coverage and genome assembly, we utilized single-molecule real-time (SMRT) sequencing. The assembled genome comprises a 9,307,519-bp linear chromosome with a GC content of 70.67%, and contained 8,888 predicted genes. Comparative genomics and natamycin biosynthetic gene cluster (BGC) analysis showed that BGC are highly conserved among evolutionarily diverse strains, and they also shared closer genome evolution compared with other Streptomyces species. Forty gene clusters were predicted to involve in the secondary metabolism of A02, and it was richly displayed in two-component signal transduction systems (TCS) in the genome, indicating a complex regulatory systems and high diversity of metabolites. Disruption of the phoP gene of the phoR-phoP TCS and nsdA gene confirmed phosphate sensitivity and global negative regulation of natamycin production. The genome sequence and analyses presented in this study provide an important molecular basis for research on natamycin production in Streptomyces, which could facilitate rational genome modification to improve the industrial use of A02.
... This polyene antibiotic (also known as pimaricin) is used as an antifungal agent with a broad spectrum of activity, and is widely applied in the food industry. Natamycin is a 26-membered ring macrocyclic polyketide that is synthesized by a common pathway in which units originated from acetate, propionate or butyrate are condensed onto the growing chain by a polyketide synthase (PKS) in a process that resembles the long-chain fatty acid biosynthesis [19, 20]. It is produced by Streptomyces natalensis, S. chattanoogensis, S. gilvosporeus and S. lydicusin and it binds to ergosterol in fungal and yeast membranes, presumably impeding the sterol of properly distributing in the membrane, and killing the cell. ...
Processed foods depend on conservation methods to ensure biological
stability of the product until consumption. Foods in general are a rich source of
nutrients, and therefore may support the proliferation of opportunistic microorganisms.
Most of these microorganisms only reduce the nutritional value and sensorial quality of
the product; but in some cases, pathogenic contaminants may also grow. Traditional
antimicrobial additives are decreasing in use due to some disadvantages related to
physicochemical and sensory aspects, such as undesirable interactions with the food
matrix, accumulation into consumer organism and even possible allergic reactions.
Besides food traditional methods for preservation such as acidification or reduction of
water activity, a possible strategy for increasing food shelf life is the use of natural
antimicrobial compounds. Nowadays, the great challenge of the food industry is to
make better use of these additives ensuring product integrity and, at the same time,
generate minimal residual effects, avoiding undesirable physicochemical and sensorial
modifications. In this context, there is an increasing preference of antimicrobial
compounds from natural sources (microbial, animal or plant) targeting a wide use in the
production of technologically advanced foods which, besides high quality and healthy,
must be biologically friendly, meeting the demands of 21st century consumers.
... Current industrial concerns include a significant improvement of its production to meet increasing commercial demand. Recently, the polyene macrolide antibiotic biosynthetic gene cluster was characterized [3]. Consequently, increasing attention has been given to genetic manipulation as a good approach to significantly improve the production of polyene antibiotics. ...
Natamycin is a widely used antifungal antibiotic. For natamycin biosynthesis, the gene pimE encodes cholesterol oxidase, which acts as a signalling protein. To confirm the positive effect of the gene pimE on natamycin biosynthesis, an additional copy of the gene pimE was inserted into the genome of Streptomyces gilvosporeus 712 under the control of ermE* promoter (permE*) using intergeneric conjugation. Overexpression of the target protein engendered 72% and 81% increases in the natamycin production and cell productivity, respectively, compared to the control strain. Further improvement in the antibiotic production was achieved in a 1-l fermenter to 7.0 g/l, which was a 153% improvement after 120 h cultivation. Exconjugants highly expressing pimE and pimM were constructed to investigate the effects of both genes on the increase of natamycin production. However, co-effect of pimE and pimM did not enhance the antibiotic production obviously, compared to the exconjugants highly expressing pimE alone. These results suggest not only a new application of cholesterol oxidase but also a useful strategy to genetically engineer natamycin production.
... These are b-type cytochromes that carry out oxygenation reactions on an enormous array of substrates [1], among them the polyketide precursors of macrolide antibiotics. Examples include reactions involved in the biosynthesis of erythromycin [2,3], tylosin [4], oleandomycin [5], narbomycin [6,7], epothilone [8,9] , ampho- tericin [10], nystatin [11], pimaricin [12,13] and rapamycin [14], among others. These reactions typically occur during the late stages of the biosynthesis, once the macrolide ring has been constructed by the polyketide synthase, and constitute an important contribution for biological activity [6,15]. ...
... cured meat, sausages, ham etc.). The biosynthetic gene cluster for pimaricin has been characterized [12,13,15,20]. Insertional inactivation of one of the genes of the pimaricin cluster, pimD, generated a mutant that accumulated a glycosylated pimaricin precursor, 4,5- de-epoxypimaricin [15], thus suggesting that this gene codes for a mono-oxygenase that is responsible for the introduction of the epoxy group at C-4–C-5 of the pimaricin molecule. ...
The biosynthesis of the antifungal agent pimaricin by Streptomyces natalensis has been proposed to involve a cytochrome P450 encoded by the gene pimD. Pimaricin is derived from its immediate precursor de-epoxypimaricin by epoxidation of the C-4-C-5 double bond on the macrolactone ring. We have overproduced PimD with a N-terminal His6 affinity tag in Escherichia coli and purified the enzyme for kinetic analysis. The protein showed a reduced CO-difference spectrum with a Soret maximum at 450 nm, indicating that it is a cytochrome P450. Purified PimD was shown to catalyse the in vitro C-4-C-5 epoxidation of 4,5-de-epoxypimaricin to pimaricin. The enzyme was dependent on NADPH for activity with optimal pH at 7.5, and the temperature optimum was 30 degrees C. The kcat value for the epoxidation of de-epoxypimaricin was similar to the values reported for other macrolide oxidases. Enzyme activity was inhibited at high substrate concentration. This is the first time that a polyene macrolide P450 mono-oxygenase has been expressed heterologously and studied. The unique specificity of this epoxidase should be useful for the oxidative modification of novel polyene macrolide antibiotics.
