PadR对须糖多孢菌丁烯基多杀菌素生物合成及转运相关蛋白表达的影响
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摘要
为研究padR基因表达对须糖多孢菌(Saccharopolyspora pogona)次级代谢产物生物合成及相关蛋白表达的影响,在对须糖多孢菌基因组测序结果的基础上,从基因组中克隆了padR基因上、下游同源臂,利用融合PCR将硫链丝菌素抗性基因插入上、下游同源臂之间,将融合片段与穿梭载体pOJ260连接,构建敲除载体pOJ260-UHA-tsr-DHA。通过接合转移将其导入野生型须糖多孢菌中,通过同源臂双交换获得须糖多孢菌敲除菌株S.pogona-ΔpadR。经HPLC检测分析,敲除菌株次级代谢产物丁烯基多杀菌素产量与原始菌株相比提高了27.3%,且有7个转运相关蛋白出现表达上调。这一研究表明padR基因的敲除能够有效促进须糖多孢菌丁烯基多杀菌素的生物合成。该研究对优化须糖多孢菌丁烯基多杀菌素的生物合成途径具有一定的指导意义,为研究padR基因表达在链霉菌次级代谢产物合成中的作用打下了基础。
In order to investigate the impact of padR gene expression on the biosynthesis of secondary metabolites and the expression of related proteins in Saccharopolyspora pogona, in this work, the thiostrepton resistance gene, amplified by pKCcas9 dO-tsr, was inserted into the upstream and downstream homologous arms of the padR gene by fusion PCR, which were cloned from the genome of S. pogona. Based on the sequencing results, the fusion fragment was ligated to the shuttle vector pOJ260, yielding pOJ260-UHA-tsr-DHA. The constructed vector pOJ260-UHA-tsr-DHA was transformed into S. pogona through conjugation, resulting in S. pogona-ΔpadR by double-crossover. HPLC analysis results showed that the yield of butenyl-spinosyn of the mutant was increased by 27.3% compared with the wide type. Mass spectrometry identified 7 transporters that were up-regulated, suggesting that the knockout of padR could effectively promote the biosynthesis of butenyl-spinosyn in S. pogona. This study is of great significance for optimizing the biosynthesis pathway of S. pogona butenyl-spinosyn, providing an important basis for studying the impacts of padR gene expression on the synthesis of secondary metabolites of Streptomyces.
In order to investigate the impact of padR gene expression on the biosynthesis of secondary metabolites and the expression of related proteins in Saccharopolyspora pogona, in this work, the thiostrepton resistance gene, amplified by pKCcas9 dO-tsr, was inserted into the upstream and downstream homologous arms of the padR gene by fusion PCR, which were cloned from the genome of S. pogona. Based on the sequencing results, the fusion fragment was ligated to the shuttle vector pOJ260, yielding pOJ260-UHA-tsr-DHA. The constructed vector pOJ260-UHA-tsr-DHA was transformed into S. pogona through conjugation, resulting in S. pogona-ΔpadR by double-crossover. HPLC analysis results showed that the yield of butenyl-spinosyn of the mutant was increased by 27.3% compared with the wide type. Mass spectrometry identified 7 transporters that were up-regulated, suggesting that the knockout of padR could effectively promote the biosynthesis of butenyl-spinosyn in S. pogona. This study is of great significance for optimizing the biosynthesis pathway of S. pogona butenyl-spinosyn, providing an important basis for studying the impacts of padR gene expression on the synthesis of secondary metabolites of Streptomyces.
引文
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[11] 林龙镇,万云凤,洪文荣.金霉素生物合成基因ctcK的研究[J].湖南师范大学自然科学学报,2017,40(1):37-43.
[12] 钟贝芬,杜磊,李众,等.基于细胞色素P450单加氧酶介导的4-甲酚氧化降解途径的天麻素生物合成[J].湖南师范大学自然科学学报,2018,41(4):33-40.
[13] OLANO C,LOMBO F,MENDEZ C,et al.Improving production of bioactive secondary metabolites in actinomycetes by metabolic engineering[J].Metab Eng,2008,10(5):281-92.
[14] MARTíN J F,SANTOS-BENEIT F,RODRIGUEZ-GARCIA A,et al.Transcriptomic studies of phosphate control of primary and secondary metabolism in Streptomyces coelicolor[J].Appl Microbiol Biotechnol,2012,95(1):61-75.
[15] LU Y H,WANG W H,SHU D,et al.Characterization of a novel two-component regulatory system involved in the regulation of bothactinorhodin and a type I polyketide in Streptomyces coelicolor[J].Appl Microbiol Biotechnol,2007,77(3):625-635.
[16] QI Y,DING X,LIU X,et al.Differential proteomic profiling reveals regulatory proteins and novel links between primary metabolism and spinosad production in Saccharopolyspora spinosa[J].Microb Cell Fact,2014,13(1):27.
[2] 罗林根,杨燕,魏慧,等.须糖多孢菌Saccharopolyspora pogona的核糖体工程改造对丁烯基多杀菌素合成的影响[J].生物工程学报,2016,32(2):259-263.
[3] CHAUDHARY A K,SINGH B,MAHARJAN S,et al.Switching antibiotics production on and off in actinomycetes by an IclR family transcriptional regulator from Streptomyces peucetius ATCC 27952[J].J Microbiol Biotechnol,2014,24(8):1065-1072.
[4] SHEN J,KONG L,LI Y,et al.A LuxR family transcriptional regulator AniF promotes the production of anisomycin and its derivatives in Streptomyces hygrospinosus var.beijingensis[J].Synth Syst Biotechnol,2019,4(1):40-48.
[5] FLOREZ A B,ALVAREZ S,ZABALA D,et al.Transcriptional regulation of mithramycin biosynthesis in Streptomyces argillaceus:dual role as activator and repressor of the PadR-like regulator MtrY[J].Microbiology-Sgm,2015,161:272-284.
[6] HUILLET E,VELGE P,VALLAEYS T,et al.LadR,a new PadR-related transcriptional regulator from Listeria monocytogenes,negatively regulates the expression of the multidrug efflux pump MdrL[J].FEMS Microbiol Lett,2006,254(1):87-94.
[7] BRINKROLF K,BRUNE I,TAUCH A.Transcriptional regulation of catabolic pathways for aromatic compounds in Corynebacterium glutamicum[J].Genet Mol Res,2006,5(4):773-789.
[8] SWIERCZ J P,NANJI T,GLOYD M,et al.A novel nucleoid-associated protein specific to the actinobacteria[J].Nucleic Acids Res,2013,41(7):4171-4184.
[9] RAPPSILBER J,RYDER U,LAMOND A I,et al.Large-scale proteomic analysis of the human spliceosome[J].Genome Res,2002,12(8):1231-1245.
[10] 张峰,吴柳娟,李躺,等.应用遗传算法和神经网络优化多杀菌素发酵培养基[J].湖南师范大学自然科学学报,2017,40(5):36-43.
[11] 林龙镇,万云凤,洪文荣.金霉素生物合成基因ctcK的研究[J].湖南师范大学自然科学学报,2017,40(1):37-43.
[12] 钟贝芬,杜磊,李众,等.基于细胞色素P450单加氧酶介导的4-甲酚氧化降解途径的天麻素生物合成[J].湖南师范大学自然科学学报,2018,41(4):33-40.
[13] OLANO C,LOMBO F,MENDEZ C,et al.Improving production of bioactive secondary metabolites in actinomycetes by metabolic engineering[J].Metab Eng,2008,10(5):281-92.
[14] MARTíN J F,SANTOS-BENEIT F,RODRIGUEZ-GARCIA A,et al.Transcriptomic studies of phosphate control of primary and secondary metabolism in Streptomyces coelicolor[J].Appl Microbiol Biotechnol,2012,95(1):61-75.
[15] LU Y H,WANG W H,SHU D,et al.Characterization of a novel two-component regulatory system involved in the regulation of bothactinorhodin and a type I polyketide in Streptomyces coelicolor[J].Appl Microbiol Biotechnol,2007,77(3):625-635.
[16] QI Y,DING X,LIU X,et al.Differential proteomic profiling reveals regulatory proteins and novel links between primary metabolism and spinosad production in Saccharopolyspora spinosa[J].Microb Cell Fact,2014,13(1):27.
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