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National Key Laboratory of Plant Molecular Genetics

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National Center for Gene Research, CAS

Key Laboratory of Synthetic Biology, CAS

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Molecular Genetics of Actinomycetes and Synthetic Microbiology

My laboratory is currently interested in functions of actinomycetes linear and circular replicons, genomicengineering of antibiotic industrial strains, development ofgenetic and genomic tools, and synthetic biology of Escherichia coli genome.

Principal Investigator: Dr. Zhong-Jun Qin, Professor. Email: qin@sibs.ac.cn
Staff: Li Zhong, Xiao-Li Xue, Hai-Yang Xia, Tao Wang, Peng Jiang, Fu-Jun Dang, Qing-Yu Xu
Graduate Students: Jian-Ting Zhou, Yang-Yang Shao, Min Zhou, Rong-Hai Wu

Recent research progress

1. Characterization of a replication locus and formation of a higher-order complex between RepA protein and two inverted-repeats in Streptomyces plasmid pSV1
We identified the minimal locus of 163-kb plasmid pSV1 of S. violaceoruber for replication in S. lividans. This locus comprised a repA gene and an upstream 407-bp sequence containing two inverted repeats (IR-III and IR-IV) within an iteron, an AT-rich region and a 300-bp non-coding sequence (NCS). RepA protein bound specifically to a 94-bp sequence covering the intact IR-III and IR-IV to form multimers of DNA/protein complexes, but was unable to bind specifically to the NCS and the promoter ofrepAgene. Interestingly, this “bound” regionalso leaves eight 1-bp “unbound” spacers at 7-11-9-11-9-11-9-11-8 bp intervals. RepA protein-protein interaction could form dimers or trimers in vitro. These results suggest that a high-order complex between pSV1 RepA protein and the long inverted-repeats may be formed during initiation of plasmid replication.

2. Three functional replication origins of the linear and artificially-circularized plasmid SCP1 of Streptomyces coelicolor
Previous reports showed that the large linear plasmid SCP1 of Streptomyces coelicolorA3(2) contains a 5.4-kb centrally-located replication locus. We report here that SCP1 actually contains three internal replication loci. Sub-cloning of the 5.4-kb sequence identified a 3.2-kb minimal locus (rep1A/repB/iteron) that determined propagation in S. lividans. The two newly identified replication genes, rep2A and rep3A, resemble the rep gene of Streptomyces circular plasmid pZL12. Transcription start points of the three replication genes were determined. The three replication loci could independently determine propagation in linear mode in S. lividans. Individual and sequential deletions of the rep1A and rep3Agenes were successful. The SCP1-derived linear plasmids with deletions of the rep1A and/or rep3Agenes still propagated in similar copy numbers, were inheritedlargely stable and transferred efficiently by conjugation in S. coelicolor. Interestingly, SCP1 can be artificially circularized to yield a 280-kb circular plasmid, C-SCP1, which contains the three replication loci. Strikingly, the copy numbers, inheritance and transfer of C-SCP1 resembled that of the linear SCP1 plasmids.Transcriptions of the rep1A,rep2A and rep3Agenes in linear or artificially-circularizedSCP1 were detectedat all the time-points of strain growth.

3. Recombinational cloning the antibiotic biosynthetic gene clusters in linear plasmid SCP1 of Streptomyces coelicolor
The model organism Streptomyces coelicolorA3(2) harbors a 356-kb linear plasmid, SCP1. We report here development of a recombinational cloning method for deleting large segment from one telomere of SCP1 followed by replacing with the telomere of pSLA2 and sequentially inserting with the overlapping cosmids in vivo. The procedure depends on homologous recombination coupled with cleavage at telomere termini by telomere terminal protein. Using this procedure we cloned the 81-kb avermectin and the 76-kb spinosad biosynthetic gene clusters into SCP1. Heterologous expression of avermectin production in S. coelicolor was detected. These results demonstrate the utility of SCP1 for cloning large DNA segments such as antibiotic biosynthetic gene clusters.

4. Genome sequencing and proteomics analysis of the fast-growing and moderately thermophilicStreptomyces sp. 4F
We have developed a gene cloning and expression system in a fast-growing and moderately thermophilicStreptomyces species. The actinorhodin and anthramycin biosynthetic gene clusters from mesophilicS. coelicolor and thermophilicS. refuineus were heterologously expressed in strain 4F.The complete nucleotide sequence of the 4F genome consists of 8,047,771 bp with long terminal inverted-repeats of 334,071 bp. About 22 genes or gene clusters for biosynthesis of secondary metabolites are predicted. Comparison to these mesophilicStreptomyces species, 4F proteins contain abundant IVYWREL motif possible for its thermo-tolerance. Two-dimensional gel electrophoresis reveals that proteins involved in heat-shock and defense of oxidative stress etc are highly expressed at 45oC than at 30oC.

5. Sequential deletion of all the PKS and NRPS biosynthetic gene clusters and a 900-kb subtelomeric sequence of the linear chromosome of Streptomycescoelicolor
Streptomyces coelicolor, with its 8,667,507-bp linear chromosome,is the genetically most studied Streptomyces species and is an excellent model for studying antibiotic production and cell differentiation. Here we report construction ofS. coelicolorderivatives containing sequential deletions of all the ten polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) biosynthetic gene clusters and a 900-kb subtelomeric sequence (total ca. 1.22 Mb, 14% of the genome). No obvious differencesin growth rates and sporulation of the strains werefound. An artificially circularized S. coelicolorgenome with deletions of total ~1.6 Mb segments (840-kb for the left and 761-kbfor the right arm of the linear chromosome) was obtained. The actinorhodin biosynthetic gene cluster could be over-expressed in some of the constructed strains.

6. The MEGA (Multiple essential gene assembling)–deletion and replacement method for long genomic deletion in Escherichia coli
Top-down reduction of bacterial genome to construct desired chassis cells is important for synthetic biology. Life essential genes interspersed on the chromosome greatly hinder the genomic deletion due to their indispensability. Here we described a new method designated ‘MEGA–deletion and replacement’ by first cloning and assembly of multiple essential genes in the E. coli-yeast shuttle vector, and then deleting the corresponding DNA sequences including the cloned essential genes along with their adjacent dispensable genes on the original chromosome. As a proof of concept, we generated 6 long deletions (>100 kbp) at different positions of E. coli chromosome. We also demonstrated that with slight modifications, the cloned multiple essential genes could be integrated into chromosome, thus allowed serial deletions. Our results strongly supported that multiple essential genes, regardless of their locations on the chromosome, could simultaneously acting in trans. We suggested that the MEGA-deletion and replacement method has the potential to become widely used in large scale genomic deletions in E. coli.

Major Publications

  1. Yalan Chen, Huarong Tan, Zhongjun Qin* (2013) Characterization of a replication locus and formation of a higher-order complex between RepA protein and two inverted-repeats in Streptomyces plasmid pSV1. FEMS Microbiol Lett. 349(2): 144-152.
  2. Shiyuan Peng, Ana Zeng, Li Zhong, Ran Zhang, Min Zhou, Qiuxiang Cheng, Liqian Zhao, Tao Wang, Huarong Tan, Zhongjun Qin* (2013) Three functional replication origins of the linear and artificially-circularized plasmid SCP1 of Streptomyces coelicolor. Microbiology. 159: 2127-2140.
  3. Ran Zhang, Haiyang Xia, Qingyu Xu, Fujun Dang, Zhongjun Qin* (2013) Recombinational cloning of the antibiotic biosynthetic gene clusters in linear plasmid SCP1 of Streptomyces coelicolor A3(2). FEMS Microbiol Lett. 345(1): 39-48.
  4. Pengfei Xie, Ana Zeng, Zhongjun Qin* (2013) A putative transglycosylase encoded by SCO4132 influences morphological differentiation and actinorhodin production in Streptomyces coelicolor. Act Bioch Bioph Sin. 45(4):296-302.
  5. Tao Wang, Zhenhua Chen, Qiuxiang Cheng, Min Zhou, Xinli Tian, Pengfei Xie, Li Zhong, Meijuan Shen and Zhongjun Qin* (2012) Characterization of replication and conjugation of plasmid pWTY27 from a widely distributed Streptomyces species. BMC Microbiol. 12:253.
  6. Zhenhua Chen, Li Zhong, Meijuan Shen, Ping Fang*, Zhongjun Qin* (2012) Characterization of Streptomyces plasmid-phage pFP4 and its evolutionary implications. Plasmid. 68(3): 170-178.
  7. Min Zhou, Xinyun Jing, Pengfei Xie, Weihua Chen, Tao Wang, Haiyang Xia, Zhongjun Qin* (2012) Sequential deletion of all the PKS and NRPS biosynthetic gene clusters and a 900-kb subtelomeric sequence of the linear chromosome of Streptomyces coelicolor. FEMS Microbiol Lett. 333(2):169-179.
  8. Yun Fan, Yumei Dai, Qiuxiang Cheng, Guangjun Zhang, Dongshu Zhang, Ping Fang*, Hang Wu, Linquan Bai*, Zixin Deng, Zhongjun Qin* (2012) A self-ligation method for PCR-sequencing the telomeres of Streptomyces and Mycobacterium linear replicons. J Microbiol Meth. 90(2):105-107.
  9. Weihua Chen and Zhongjun Qin* (2011) Development of a gene cloning system in a fast-growing and moderately thermophilic Streptomyces species and heterologous expression of Streptomyces antibiotic biosynthetic gene clusters. BMC Microbiol. 11(1): 243.
  10. Ana Zeng , Tao Wang ,Haiyang Xia, Shiyuan Peng, Weihua Chen, Chaneglin Jiang, Lihua Xu, Li Zhong, Meijuan Shen*, and Zhongjun Qin* (2011) Development of a vector and host system and characterization of replication of plasmid pSQ10 in moderately halophilic Nocardiopsis. Acta Biochim Biophys Sin (Shanghai) 43(9): 738-743.
  11. Peng Guo, Qiuxiang Cheng, Pegnfei Xie, Yun Fan, Weihong Jiang, and Zhongjun Qin* (2011) Characterization of the multiple CRISPR loci on Streptomyces linear plasmid pSHK1. Acta Biochim Biophys Sin (Shanghai). 43(8): 630-639.
  12. Li Zhong, Qiuxiang Cheng, Xinli Tian, Liqian Zhao, and Zhongjun Qin* (2010) Characterization of the Replication, Transfer and Plasmid/Lytic Phage Cycle of the Streptomyces Plasmid-Phage pZL12. J Bacteriol. 192(14):3747-54.
  13. Ran Zhang, Shiyuan Peng, and Zhongjun Qin* (2010)Two Internal Origins of Replication on Streptomyces Linear Plasmid pFRL1. Appl Environ Microb. 76(17): 5676-5683.
  14. Zhiqun Lu, Pengfei Xie, and Zhongjun Qin* (2010) Promotion of markerless deletion of the actinorhodin biosynthetic gene cluster in Streptomyces coelicolor. Acta Biochim Biophys Sin (Shanghai). 42(10): 717-721.
  15. Pengfei Xie, Ana Zeng and Zhongjun Qin* (2009) cmdABCDEF, a cluster of genes encoding membrane proteins for differentiation and antibiotic production in Streptoymyces coelicolor A3(2). BMC Microbiol. 9: 157.
  16. Ran Zhang, Haiyang Xia, Peng Guo and Zhongjun Qin* (2009) Variation in the replication loci of Streptomyces linear plasmids. FEMS Microbiol Lett. 290(2): 209-216.
  17. Yongqiang Tian, Weihong Jiang, Guoping Zhao* and Zhongjun Qin* (2009) In vivo conjugation-coupled recombinational cloning of a Streptomyces lividans chromosomal telomeric DNA using a linear plasmid. Biotechnol Lett. 31(8): 1253-1258.
  18. Ran Zhang, Ana Zeng, Ping Fang and Zhongjun Qin* (2008) Characterization of the replication and conjugation loci of Streptomyces circular plasmids pFP11 and pFP1 and their ability to propagate in linear mode with artificially attached telomeres. Appl Environl Microb. 74(11):3368-3376.
  19. Jingyi Zhao, Li Zhong, Meijuan Shen, Zhijie Xia, Qiuxiang Cheng, Xia Sun, Guoping Zhao, Yuezhong Li* and Zhongjun Qin* (2008) Discovery of an autonomously-replicating plasmid pMF1 from Myxococcusfulvus and the development of a gene cloning system in Myxococcus Xanthus. Appl Environl Microb. 74(7):1980-1987.
  20. Mingxuan Xu, Yingmin Zhu, Ran Zhang, Meijuan Shen, Weihong Jiang, Guoping Zhao* and Zhongjun Qin* (2006) Characterization of the genetic components of Streptomyces lividans linear plasmid SLP2 for replication in circular and linear modes. J Bacteriol. 188:6851-6857.
  21. Ran Zhang, YongYang, Ping Fang, Chenglin Jiang, Lihua Xu, Yingmin Zhu, Meijuan Shen, Haiyang Xia, Jianfu Zhao, Tongbing Chen and Zhongjun Qin* (2006) Diversity of telomere palindromic sequences and replication genes among Streptomyces linear plasmids. Appl Environl Microb. 72(9):5728-5733.
  22. Meijuan Shen, Ping Fang, Deqiang Xu, Yalei Zhang, Weihuan Cao, Yingmin Zhu, JianfuZhao and Zhongjun Qin* (2006) Replication and Inheritance of Nocardia Plasmid pC1. FEMS Microbiol Lett. 261:47-52.
  23. Haiyang Xia, YongqiangTian, Ran Zhang, Kaichun Lin and Zhongjun Qin* (2006) Characterization ofNocardia Plasmid pXT107. Acta Biochim Biophys Sin. 38(9):620-624.
  24. Mingxuan Xu, Yingmin Zhu, Meijuan Shen, Weihong Jiang, Guoping Zhao* and Zhongjun Qin* (2006) Characterization of the essential genetic components for conjugal transfer of Streptomyces lividans linear plasmid SLP2. Prog Biochem and Biophy. 33(10):1-8.
  25. Yongqiang Tian, Pei Hao, Guoping Zhao* and Zhongjun Qin* (2005) Cloning and characterization of the chromosomal replication origin region of Amycolatopsismediterranei U32. Biochem Bioph Res Co. 333(1):14-20.
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