| Oxytetracycline (OTC) is a widely used antibiotic, which has a great market demand for clinic and aquaculture application. The commercial producer of OTC, Streptomyces rimosus, attracts extensive attention of researchers for scientific investigation. The genomic sequencing of S. rimosus type strain M4018 has been accomplished by the cooperation work of our team and Professor Iain Hunter’s team, which brought about a plenty of gene sequence information, while some functional genes have not been identified yet. It provides potential opportunities for deep insight of the regulation mechanisms in S. rimosus by characterization of functional genes, especially the functional genes which associated with OTC biosynthesis, which have great value of investigation and application. In this study, an OTC biosynthesis associated ATP-binding-cassette (ABC) transporter OtrC and a OTC biosynthesis transcript regulator RgC in S. rimosus were firstly functional characterized, and the effects of oxy-encoding type II minimal PKS on carbon flux and OTC biosynthesis was also investigated. Finally, the functional genes were knocked into an industrial OTC overproducer, respectively, with expect of OTC improvement. The results were as follows:1. Functional characterization of an ATP-binding cassette (ABC) OtrC in S. rimosus and investigation of its functional effect on OTC biosynthesisThe otrC gene of S. rimosus type strain M4018 was cloned and the alignment of the otrC sequence showed that the two open reading frames of it encoding a putative ATP-binding subunit and a putative transmembrane subunit, respectively, therefore it could be an ABC transporter. The two open reading frames of the otrC gene were expressed in E. coli BL21(DE3). The exporter function of OtrC was identified by ATPase activity determination and fluorimetric ethidium bromide (EB) efflux assays, which confirms that OtrC is a member of ABC transporter family. OtrC duplication mutant M4018/pSEC and disruption mutant M4018/pKC △ otrC were generated from M4018, respectively. The susceptibilities of OtrC-overexpressing cells to 9 structurally unrelated drugs were compared with those of OtrC-non-expressing cells by minimal inhibitory concentration (MIC) assays. Both OtrC-overexpressing E. coli and S. rimosus exhibited enhanced MICs to ampicillin, OTC, doxorubicin, EB, ofloxacin and vancomycin, it indicates that OtrC is a drug exporter with a broad range of drug specificities. The OTC production was significantly enhanced by 1.6-fold in otrC duplication mutant and while that decreased by 20% in disruption mutants, it demonstrates that OtrC has functional effect on the OTC biosynthesis in S. rimosus. 2. Functional characterization of a AsnC-like regulator RgC and its directly regulation on OTC biosynthesis in S. rimosusRgC contains a conserved AsnC regulator conserved motif which has high similarity that of Lrp/AsnC family proteins (similarity over 70%). The rgC gene adjacents with the multidrug ABC transporter otrC gene on S. rimosus chromosome, which raise a bold hypothesis that rgC might encoding a regulator of OTC biosynthesis pathway. To date, there is no report about functional characterization of Lrp/AsnC regulator in S. rimosus. Thus, in this study, the function regulator RgC from S. rimosus was firstly characterized. The rgC gene was overexpressed in E. coli and Western blotting analysis showed that a certain proportion of RgC exist as a dimmer or tetramer complex. EMSA assay was employed for RgC-binding promoter screening, and the results showed that RgC can bind to the promoters of OTC minimal PKS genes and OTC resistance gene otrB, the addition of asparitic acid and asparagine increased the forming of RgC-DNA complex. The binding sites of RgC was identified using footprinting analyze, and the conserved sequence of RgC binding site is G(C) G(C) _ T C(G) _ A(C/G) A(C/G) T(A) G(C) C(A/G) C(A/G), the conserved sequence was different from that of reported Lrp/AsnC-like protein. Mutation investigation of RgC was also performed and revealed that the mutant in AsnC regulator conserved motif with T17-A、T17 H21-A17 L21、M7-F have no effect on the DNA-binding ability of RgC, while the deletion of three amino acids L34P35A36 outside brought about the abolishing of DNA-binding ability. The regulation function of RgC was further characterized in vivo, quantitative real-time PCR analyze revealed a significantly increase of transcripts of oxyA, oxyB, oxyC, around 7.0- fold in rgC knock-in mutant, and for otrB around 3.6-fold, while the transcripts of all target genes decreased by 20% in rgC knock-out mutant. The mutants were used for OTC fermentation of mutants showed that, the OTC production of rgC knock-in mutant was significantly enhanced around 1.7-fold, and the L-aspartic acid consumption and growth rates were also remarkably increased, for rgC knock-out mutant the OTC production, L-aspartic acid consumption and growth rates were significantly decreased. The results demonstrated that RgC regulates the L-aspartic acid metabolic pathway and also directly regulates the OTC biosynthesis pathway, which has not been discovered in Lrp/AsnC family protein before.3. The effects of minimal PKS genes duplication on cell growth and OTC production in S. rimosusThe type Ⅱ minimal polyketide synthases (minimal PKS) multienzyme system was encoded by oxyA, oxyB and oxyC genes, and it responsible for the biosynthesis of a amidated decaktide backbone, which plays an important role in OTC production. A second copy of the minimal PKS genes was introduced into the chromosome of S. rimosus M4018 to generate the mutant strain MR69, and the overexpression of the minimal PKS genes elicited significant change of phenotype, retardation of cell growth and a significant improvement in OTC (approximately 51.2%). The difference of organic acids production between parental strains and mutants are significant during the cell growth, which suggests that the change of morphology and biomass yield are due to redirection of carbon into the OTC biosynthesis pathway with the result that less carbon is available for cell growth. The RT-qPCR analysis revealed a significantly increased transcriptional level of oxyA, oxyB and oxyC, additionally, the transcriptional level of an OTC resistance gene otrB was also found increased, which suggests that the expression of minimal PKS genes could affect the expression of otrB gene.4. Regulation of OTC biosynthesis in an industrial overproducer S. rimosus SRI 6The genetic manipulation of industrial overproducer S. rimosus SRI 6 were performed based on the investigation results, with respect of OTC production improvement.otrC duplication and disruption mutants were generated from SRI6, respectively. The result showed that the OTC production was significantly enhanced by 1.4-fold in otrC knock-in mutants, while it decreased by 20% in disruption mutants.The regulation mechanism of RgC on OTC biosynthesis in industrial overproducer SRI 6 was investigated by rgC gene knock-in and knock-out. The OTC production of rgC knock-in was significantly enhanced, about 2.1-fold of parental strain, while the OTC production of rgC knock-out was about 80% of parental strain, and the OTC productivity was recovered by rgC gene complementary. Compared with the parental strain, the rgC knock-in mutant is more stable.A remarkable improvement of OTC production about 32.9% in the industrial overproducer SR16 were successfully achieved by introducing a second copy of minimal PKSs genes into their chromosome, however, the overexpression of minimal PKS genes also brought about a retarded cell growth and lower production of organic acids, which suggests that minimal PKS plays an important role in carbon flux distribution. Furthermore, the results of RT-qPCR analysis revealed that the expression of minimal PKS genes in SR16 also has important effect on the expression of otrB gene.This study provides new strategies of genetic manipulation of industrial overproducer, and also provides basic theory and technology for deep insight into regulation mechanism of OTC biosynthesis in S. rimosus. |
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