Characterization of Pathway-Specific Regulator NigR for High Yield Production of Nigericin in Streptomyces malaysiensis F913.
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SUBMITTER: Wei J
PROVIDER: S-EPMC9312159 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The genome sequence of Streptomyces ambofaciens, a species known to produce the congocidine and spiramycin antibiotics, has revealed the presence of numerous gene clusters predicted to be involved in the biosynthesis of secondary metabolites. Among them, the type II polyketide synthase-encoding alp cluster was shown to be responsible for the biosynthesis of a compound with antibacterial activity. Here, by means of a deregulation approach, we gained access to workable amounts of the antibiotics for structure elucidation. These compounds, previously designated as alpomycin, were shown to be known members of kinamycin family of antibiotics. Indeed, a mutant lacking AlpW, a member of the TetR regulator family, was shown to constitutively produce kinamycins. Comparative transcriptional analyses showed that expression of alpV, the essential regulator gene required for activation of the biosynthetic genes, is strongly maintained during the stationary growth phase in the alpW mutant, a stage at which alpV transcripts and thereby transcripts of the biosynthetic genes normally drop off. Recombinant AlpW displayed DNA binding activity toward specific motifs in the promoter region of its own gene and that of alpV and alpZ. These recognition sequences are also targets for AlpZ, the γ-butyrolactone-like receptor involved in the regulation of the alp cluster. However, unlike that of AlpZ, the AlpW DNA-binding ability seemed to be insensitive to the signaling molecules controlling antibiotic biosynthesis. Together, the results presented in this study reveal S. ambofaciens to be a new producer of kinamycins and AlpW to be a key late repressor of the cellular control of kinamycin biosynthesis.
| S-EPMC3067597 | biostudies-literature
Project description:Thaxtomins are virulence factors of most plant-pathogenic Streptomyces strains. Due to their potent herbicidal activity, attractive environmental compatibility, and inherent biodegradability, thaxtomins are key active ingredients of bioherbicides approved by the U.S. Environmental Protection Agency. However, the low yield of thaxtomins in native Streptomyces producers limits their wide agricultural applications. Here, we describe the high-yield production of thaxtomins in a heterologous host. The thaxtomin gene cluster from S. scabiei 87.22 was cloned and expressed in S. albus J1074 after chromosomal integration. The production of thaxtomins and nitrotryptophan analogs was observed using liquid chromatography-mass spectrometry (LC-MS) analysis. When the engineered S. albus J1074 was cultured in the minimal medium Thx defined medium supplemented with 1% cellobiose (TDMc), the yield of the most abundant and herbicidal analog, thaxtomin A, was 10 times higher than that in S. scabiei 87.22, and optimization of the medium resulted in the highest yield of thaxtomin analogs at about 222 mg/liter. Further engineering of the thaxtomin biosynthetic gene cluster through gene deletion led to the production of multiple biosynthetic intermediates important to the chemical synthesis of new analogs. Additionally, the versatility of the thaxtomin biosynthetic system in S. albus J1074 was capitalized on to produce one unnatural fluorinated analog, 5-fluoro-thaxtomin A (5-F-thaxtomin A), whose structure was elucidated by a combination of MS and one-dimensional (1D) and 2D nuclear magnetic resonance (NMR) analyses. Natural and unnatural thaxtomins demonstrated potent herbicidal activity in radish seedling assays. These results indicated that S. albus J1074 has the potential to produce thaxtomins and analogs thereof with high yield, fostering their agricultural applications.IMPORTANCE Thaxtomins are agriculturally valuable herbicidal natural products, but the productivity of native producers is limiting. Heterologous expression of the thaxtomin gene cluster in S. albus J1074 resulted in the highest yield of thaxtomins ever reported, representing a significant leap forward in its wide agricultural use. Furthermore, current synthetic routes to thaxtomins and analogs are lengthy, and two thaxtomin biosynthetic intermediates produced at high yields in this work can provide precursors and building blocks to advanced synthetic routes. Importantly, the production of 5-F-thaxtomin A in engineered S. albus J1074 demonstrated a viable alternative to chemical methods in the synthesis of new thaxtomin analogs. Moreover, our work presents an attractive synthetic biology strategy to improve the supply of herbicidal thaxtomins, likely finding general applications in the discovery and production of many other bioactive natural products.
| S-EPMC5960955 | biostudies-literature
Project description:Milbemycins, a group of 16-membered macrolides with potent anthelminthic and insecticidal activity, are produced by several Streptomyces and used widely in agricultural, medical and veterinary fields. Milbemycin A3 and A4, the main components produced by Streptomyces bingchenggensis, have been developed as an acaricide to control mites. The subsequent structural modification of milbemycin A3/A4 led to other commercial products, such as milbemycin oxime, lepimectin and latidectin. Despite its importance, little is known about the regulation of milbemycin biosynthesis, which has hampered efforts to enhance milbemycin production via engineering regulatory genes.milR, a regulatory gene in the milbemycin (mil) biosynthetic gene cluster of S. bingchenggensis, encodes a large ATP-binding regulator of the LuxR family (LAL family), which contains an ATPase domain at its N-terminus and a LuxR-like DNA-binding domain at the C-terminus. Gene disruption and genetic complementation revealed that milR plays an important role in the biosynthesis of milbemycin. ?-glucuronidase assays and transcriptional analysis showed that MilR activates the expression of the milA4-E operon and milF directly, and activates the other mil genes indirectly. Site-directed mutagenesis confirmed that the ATPase domain is indispensable for MilR's function, and particularly mutation of the conserved amino acids K37A, D122A and D123A, led to the loss of MilR function for milbemycin biosynthesis. Overexpression of an extra copy of milR under the control of its native promoter significantly increased production of milbemycin A3/A4 in a high-producing industrial strain S. bingchenggensis BC04.A LAL regulator, MilR, was characterized in the mil gene cluster of S. bingchenggensis BC04. MilR could activate milbemycin biosynthesis through direct interaction with the promoter of the milA4-E operon and that of milF. Overexpression of milR increased milbemycin A3/A4 production by 38 % compared with the parental strain BC04, suggesting that genetic manipulation of this activator gene could enhance the yield of antibiotics.
| S-EPMC5015266 | biostudies-literature
Project description:Lutein is an industrially important carotenoid pigment, which is essential for photoprotection and photosynthesis in plants. Lutein is crucial for maintaining human health due to its protective ability from ocular diseases. However, its pathway engineering research has scarcely been performed for microbial production using heterologous hosts, such as Escherichia coli, since the engineering of multiple genes is required. These genes, which include tricky key carotenoid biosynthesis genes typically derived from plants, encode two sorts of cyclases (lycopene ε- and β-cyclase) and cytochrome P450 CYP97C. In this study, upstream genes effective for the increase in carotenoid amounts, such as isopentenyl diphosphate isomerase (IDI) gene, were integrated into the E. coli JM101 (DE3) genome. The most efficient set of the key genes (MpLCYe, MpLCYb and MpCYP97C) was selected from among the corresponding genes derived from various plant (or bacterial) species using E. coli that had accumulated carotenoid substrates. Furthermore, to optimize the production of lutein in E. coli, we introduced several sorts of plasmids that contained some of the multiple genes into the genome-inserted strain and compared lutein productivity. Finally, we achieved 11 mg/l as lutein yield using a mini jar. Here, the high-yield production of lutein was successfully performed using E. coli through approaches of pathway engineering. The findings obtained here should be a base reference for substantial lutein production with microorganisms in the future.
| S-EPMC8546607 | biostudies-literature
Project description:BackgroundThe macrolide antibiotic avermectin, a natural product derived from Streptomyces avermitilis, finds extensive applications in agriculture, animal husbandry and medicine. The mtrA (sav_5063) gene functions as a transcriptional regulator belonging to the OmpR family. As a pleiotropic regulator, mtrA not only influences the growth, development, and morphological differentiation of strains but also modulates genes associated with primary metabolism. However, the regulatory role of MtrA in avermectin biosynthesis remains to be elucidated.ResultsIn this study, we demonstrated that MtrA, a novel OmpR-family transcriptional regulator in S. avermitilis, exerts global regulator effects by negatively regulating avermectin biosynthesis and cell growth while positively controlling morphological differentiation. The deletion of the mtrA gene resulted in an increase in avermectin production, accompanied by a reduction in biomass and a delay in the formation of aerial hyphae and spores. The Electrophoretic Mobility Shift Assay (EMSA) revealed that MtrA exhibited binding affinity towards the upstream region of aveR, the intergenic region between aveA1 and aveA2 genes, as well as the upstream region of aveBVIII in vitro. These findings suggest that MtrA exerts a negative regulatory effect on avermectin biosynthesis by modulating the expression of avermectin biosynthesis cluster genes. Transcriptome sequencing and fluorescence quantitative PCR analysis showed that mtrA deletion increased the transcript levels of the cluster genes aveR, aveA1, aveA2, aveC, aveE, aveA4 and orf-1, which explains the observed increase in avermectin production in the knockout strain. Furthermore, our findings demonstrate that MtrA positively regulates the cell division and differentiation genes bldM and ssgC, while exerting a negative regulatory effect on bldD, thereby modulating the primary metabolic processes associated with cell division, differentiation and growth in S. avermitilis, consequently impacting avermectin biosynthesis.ConclusionsIn this study, we investigated the negative regulatory effect of the global regulator MtrA on avermectin biosynthesis and its effects on morphological differentiation and cell growth, and elucidated its transcriptional regulatory mechanism. Our findings indicate that MtrA plays crucial roles not only in the biosynthesis of avermectin but also in coordinating intricate physiological processes in S. avermitilis. These findings provide insights into the synthesis of avermectin and shed light on the primary and secondary metabolism of S. avermitilis mediated by OmpR-family regulators.
| S-EPMC11000389 | biostudies-literature
Project description:BackgroundO-Methylated phenylpropanoids, which are generally present in small amounts in plants, have improved or distinct biological activities and pharmacological properties as opposed to their unmethylated counterparts. Although microbial production could be a useful tool for the efficient and environment-friendly production of methylated phenylpropanoids, a high-yield microbial production of neither tri-methylated stilbenes nor di-/tri-methylated flavonoids has been achieved to date.ResultsA methyltransferase from Streptomyces avermitilis (SaOMT2), which has been known to possess 7-O-methylation activity toward several flavonoids, exhibited more diverse regiospecificity and catalyzed mono-, di-, and tri-methylation of stilbene, flavanone, and flavone when it was expressed in Streptomyces venezuelae. For the efficient production of multi-methylated phenylpropanoids, a cocultivation system was developed by employing engineered Escherichia coli strains producing pterostilbene, naringenin, and apigenin, respectively, along with SaOMT2-expressing S. venezuelae mutant. Consequently, high-yield microbial production of tri-methylated stilbenes and di-/tri-methylated flavonoids (including 3,5,4'-trimethoxystilbene, 5-hydroxy-7,4'-dimethoxyflavanone, 4'-hydroxy-5,7-dimethoxyflavanone, 5,7,4'-trimethoxyflavanone, 5-hydroxy-7,4'-dimethoxyflavone, and 5,7,4'-trimethoxyflavone) has been demonstrated for the first time.ConclusionsThis cocultivation system based on the phenylpropanoid-producing E. coli and SaOMT2-expressing S. venezuelae provides an efficient tool for producing scarce and potentially valuable multi-methylated phenylpropanoids and will enable further development of these compounds as pharmaceuticals and nutraceuticals.
| S-EPMC6456975 | biostudies-literature
Project description:BackgroundStreoptomyces rimosus M527 is a producer of the polyene macrolide rimocidin which shows activity against various plant pathogenic fungi. Notably, the regulatory mechanisms underlying rimocidin biosynthesis are yet to be elucidated.ResultsIn this study, using domain structure and amino acid alignment and phylogenetic tree construction, rimR2, which located in the rimocidin biosynthetic gene cluster, was first found and identified as a larger ATP-binding regulators of the LuxR family (LAL) subfamily regulator. The rimR2 deletion and complementation assays were conducted to explore its role. Mutant M527-ΔrimR2 lost its ability to produce rimocidin. Complementation of M527-ΔrimR2 restored rimocidin production. The five recombinant strains, M527-ER, M527-KR, M527-21R, M527-57R, and M527-NR, were constructed by overexpressing rimR2 gene using the promoters permE*, kasOp*, SPL21, SPL57, and its native promoter, respectively, to improve rimocidin production. M527-KR, M527-NR, and M527-ER exhibited 81.8%, 68.1%, and 54.5% more rimocidin production, respectively, than the wild-type (WT) strain, while recombinant strains M527-21R and M527-57R exhibited no obvious differences in rimocidin production compared with the WT strain. RT-PCR assays revealed that the transcriptional levels of the rim genes were consistent with the changes in rimocidin production in the recombinant strains. Using electrophoretic mobility shift assays, we confirmed that RimR2 can bind to the promoter regions of rimA and rimC.ConclusionA LAL regulator RimR2 was identified as a positive specific-pathway regulator of rimocidin biosynthesis in M527. RimR2 regulates the rimocidin biosynthesis by influencing the transcriptional levels of rim genes and binding to the promoter regions of rimA and rimC.
| S-EPMC9942304 | biostudies-literature
Project description:BackgroundIsatropolone A and C, produced by Streptomyces sp. CPCC 204095, belong to an unusual class of non-benzenoid aromatic compounds and contain a rare seven-membered ring structure. Isatropolone A exhibits potent activity against Leishmania donovani, comparable to the only oral drug miltefosine. However, its variably low productivity represents a limitation for this lead compound in the future development of new anti-leishmaniasis drugs to meet unmet clinical needs.ResultsHere we first elucidated the regulatory cascade of biosynthesis of isatropolones, which consists of two SARP family regulators, IsaF and IsaJ. Through a series of in vivo and in vitro experiments, IsaF was identified as a pathway-specific activator that orchestrates the transcription of the gene cluster essential for isatropolone biosynthesis. Interestingly, IsaJ was found to only upregulate the expression of the cytochrome P450 monooxygenase IsaS, which is crucial for the yield and proportion of isatropolone A and C. Through targeted gene deletions of isaJ or isaS, we effectively impeded the conversion of isatropolone A to C. Concurrently, the facilitation of isaF overexpression governed by selected promoters, prompted the comprehensive activation of the production of isatropolone A. Furthermore, meticulous optimization of the fermentation parameters was conducted. These strategies culminated in the attainment of an unprecedented maximum yield-980.8 mg/L of isatropolone A-achieved in small-scale solid-state fermentation utilizing the genetically modified strains, thereby establishing the highest reported titer to date.ConclusionIn Streptomyces sp. CPCC 204095, the production of isatropolone A and C is modulated by the SARP regulators IsaF and IsaJ. IsaF serves as a master pathway-specific regulator for the production of isatropolones. IsaJ, on the other hand, only dictates the transcription of IsaS, the enzyme responsible for the conversion of isatropolone A and C. By engineering the expression of these pivotal genes, we have devised a strategy for genetic modification aimed at the selective and high-yield biosynthesis of isatropolone A. This study not only unveils the unique regulatory mechanisms governing isatropolone biosynthesis for the first time, but also establishes an essential engineering framework for the targeted high-level production of isatropolone A.
| S-EPMC11020959 | biostudies-literature
Project description:BackgroundAurantimycin (ATM), produced by Streptomyces aurantiacus JA 4570, is a potent antimicrobial and antitumor antibiotic. Although the chemical structure of ATM is highly distinctive and features a cyclohexadepsipeptide scaffold attached with a C14 acyl side chain, little is known about its biosynthetic pathway and regulatory mechanism.ResultsIn this work, we report the identification and characterization of the ATM biosynthetic gene cluster from S. aurantiacus JA 4570. Targeted inactivation of artG, coding for a NRPS enzyme, completely abolished ATM production, thereof demonstrating the target gene cluster (art) is responsible for ATM biosynthesis. Moreover, four NRPS adenylation (A) domains including a freestanding enzyme ArtC have been characterized in vitro, whose substrate specificities are consistent with in silico analysis. Further genetic analysis of the two regulatory genes artB and artX unambiguously suggested both of them play positive roles in ATM biosynthesis, and ATM-A production was thus rationally enhanced to about 2.5 fold via tandem overexpression of artB and artX in S. aurantiacus JA 4570.ConclusionsThese results will provide the basis for the understanding of precise mechanisms for ATM biosynthesis, and open the way for both rational construction of high-production ATM producer and orient-directed generation of designer ATM derivatives via synthetic biology strategies.
| S-EPMC5031334 | biostudies-literature