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Identification of a cluster-situated activator of oxytetracycline biosynthesis and manipulation of its expression for improved oxytetracycline production in Streptomyces rimosus.

ABSTRACT: Oxytetracycline (OTC) is a broad-spectrum antibiotic commercially produced by Streptomyces rimosus. Despite its importance, little is known about the regulation of OTC biosynthesis, which hampered any effort to improve OTC production via engineering regulatory genes.A gene encoding a Streptomyces antibiotic regulatory protein (SARP) was discovered immediately adjacent to the otrB gene of oxy cluster in S. rimosus and designated otcR. Deletion and complementation of otcR abolished or restored OTC production, respectively, indicating that otcR encodes an essential activator of OTC biosynthesis. Then, the predicted consensus SARP-binding sequences were extracted from the promoter regions of oxy cluster. Transcriptional analysis in a heterologous GFP reporter system demonstrated that OtcR directly activated the transcription of five oxy promoters in E. coli, further mutational analysis of a SARP-binding sequence of oxyI promoter proved that OtcR directly interacted with the consensus repeats. Therefore, otcR was chosen as an engineering target, OTC production was significantly increased by overexpression of otcR as tandem copies each under the control of strong SF14 promoter.A SARP activator, OtcR, was identified in oxy cluster of S. rimosus; it was shown to directly activate five promoters from oxy cluster. Overexpression of otcR at an appropriate level dramatically increased OTC production by 6.49 times compared to the parental strain, thus demonstrating the great potential of manipulating OtcR to improve the yield of OTC production.

SUBMITTER: Yin S

PROVIDER: S-EPMC4393881 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Identification of a cluster-situated activator of oxytetracycline biosynthesis and manipulation of its expression for improved oxytetracycline production in Streptomyces rimosus.

Yin Shouliang S Wang Weishan W Wang Xuefeng X Zhu Yaxin Y Jia Xiaole X Li Shanshan S Yuan Fang F Zhang Yuxiu Y Yang Keqian K

Microbial cell factories 20150402

<h4>Background</h4>Oxytetracycline (OTC) is a broad-spectrum antibiotic commercially produced by Streptomyces rimosus. Despite its importance, little is known about the regulation of OTC biosynthesis, which hampered any effort to improve OTC production via engineering regulatory genes.<h4>Results</h4>A gene encoding a Streptomyces antibiotic regulatory protein (SARP) was discovered immediately adjacent to the otrB gene of oxy cluster in S. rimosus and designated otcR. Deletion and complementatio ...[more]

PMID: 25886456

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Project description:BackgroundNatural products are a valuable source of biologically active compounds that have applications in medicine and agriculture. One disadvantage with natural products is the slow, time-consuming strain improvement regimes that are necessary to ensure sufficient quantities of target compounds for commercial production. Although great efforts have been invested in strain selection methods, many of these technologies have not been improved in decades, which might pose a serious threat to the economic and industrial viability of such important bioprocesses.ResultsIn recent years, introduction of extra copies of an entire biosynthetic pathway that encodes a target product in a single microbial host has become a technically feasible approach. However, this often results in minor to moderate increases in target titers. Strain stability and process reproducibility are the other critical factors in the industrial setting. Industrial Streptomyces rimosus strains for production of oxytetracycline are one of the most economically efficient strains ever developed, and thus these represent a very good industrial case. To evaluate the applicability of amplification of an entire gene cluster in a single host strain, we developed and evaluated various gene tools to introduce multiple copies of the entire oxytetracycline gene cluster into three different Streptomyces rimosus strains: wild-type, and medium and high oxytetracycline-producing strains. We evaluated the production levels of these engineered S. rimosus strains with extra copies of the oxytetracycline gene cluster and their stability, and the oxytetracycline gene cluster expression profiles; we also identified the chromosomal integration sites.ConclusionsThis study shows that stable and reproducible increases in target secondary metabolite titers can be achieved in wild-type and in high oxytetracycline-producing strains, which always reflects the metabolic background of each independent S. rimosus strain. Although this approach is technically very demanding and requires systematic effort, when combined with modern strain selection methods, it might constitute a very valuable approach in industrial process development.

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.

Project description:Streptomyces rimosus is an industrial streptomycete, best known as a producer of oxytetracycline, one of the most widely used antibiotics. Despite the significant contribution of Streptomyces species to the pharmaceutical industry, most omics analyses have only been conducted on the model organism Streptomyces coelicolor. In recent years, protein phosphorylation on serine, threonine, and tyrosine (Ser, Thr, and Tyr, respectively) has been shown to play a crucial role in the regulation of numerous cellular processes, including metabolic changes leading to antibiotic production and morphological changes. In this study, we performed a comprehensive quantitative (phospho)proteomic analysis during the growth of S. rimosus under conditions of oxytetracycline production and pellet fragmentation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis combined with phosphopeptide enrichment detected a total of 3,725 proteins, corresponding to 45.6% of the proteome and 417 phosphorylation sites from 230 phosphoproteins. Significant changes in abundance during three distinct growth phases were determined for 494 proteins and 98 phosphorylation sites. Functional analysis revealed changes in phosphorylation events of proteins involved in important cellular processes, including regulatory mechanisms, primary and secondary metabolism, cell division, and stress response. About 80% of the phosphoproteins detected during submerged growth of S. rimosus have not yet been reported in streptomycetes, and 55 phosphoproteins were not reported in any prokaryote studied so far. This enabled the creation of a unique resource that provides novel insights into the dynamics of (phospho)proteins and reveals many potential regulatory events during antibiotic production in liquid culture of an industrially important bacterium. IMPORTANCE Streptomyces rimosus is best known as a primary source of oxytetracycline (OTC). The significant global market value of OTC highlights the need for a better understanding of the regulatory mechanisms that lead to production of this antibiotic. Our study provides, for the first time, a detailed insight into the dynamics of (phospho)proteomic profiles during growth and antibiotic production in liquid culture of S. rimosus. Significant changes in protein synthesis and phosphorylation have been revealed for a number of important cellular proteins during the growth stages that coincide with OTC production and morphological changes of this industrially important bacterium. Most of these proteins have not been detected in previous studies. Therefore, our results significantly expand the insight into phosphorylation events associated with important cellular processes and antibiotic production; they also greatly increase the phosphoproteome of streptomycetes and contribute with newly discovered phosphoproteins to the database of prokaryotic phosphoproteomes. This can consequently lead to the design of novel research directions in elucidation of the complex regulatory network in Streptomyces.

Dataset Information

Identification of a cluster-situated activator of oxytetracycline biosynthesis and manipulation of its expression for improved oxytetracycline production in Streptomyces rimosus.

Publications

Identification of a cluster-situated activator of oxytetracycline biosynthesis and manipulation of its expression for improved oxytetracycline production in Streptomyces rimosus.

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