利用CRISPR-Cas9系统构建新型异戊酰螺旋霉素Ⅰ产生菌
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摘要
异戊酰螺旋霉素(Isovalerylspiramycin,ISP)Ⅰ是必特螺旋霉素(Bitespiramycin,BT)的一种主组分,其抗菌活性与BT相似,而且作为单一组分在质控和剂型上更具优势,目前正在进行临床前研究。原有的 ISPⅠ工程菌株经过3次基因改造,已经具有2种抗性基因,很难再进行遗传操作。前期研究利用经典同源重组的方法无法构建无抗性的ISPⅠ产生菌,文中利用CRISPR-Cas9基因编辑系统在螺旋霉素(Spiramycin,SP)产生菌中成功将3位酰基化酶的基因bsm4替换为组成型强启动子ermEp~*控制下的异戊酰基转移酶基因(Isovaleryltansferase gene,ist)。删除bsm4后突变株只能产生SPⅠ组分,外源基因ist的表达产物催化SPⅠ在其4″位进行异戊酰化修饰形成ISPⅠ。经过HPLC和质谱鉴定,阳性菌株ΔEI的发酵产物中只有ISPⅠ一种ISP组分,证实新的ISPⅠ工程菌株构建成功。ΔEI菌株不带有抗性基因,可重复利用CRISPR-Cas9系统进行基因操作来获得新的改良菌株。
Isovalerylspiramycin(ISP)Ⅰ, as a major component of bitespiramycin(BT), exhibits similar antimicrobial activities with BT and has advantages in quality control and dosage forms. It has been under preclinical studies. The existing ISPⅠ producing strain, undergoing three genetic modifications, carries two resistant gene markers. Thus, it is hard for further genetic manipulation. It is a time-consuming and unsuccessful work to construct a new ISPⅠ strain without resistant gene marker by means of the classical homologous recombination in our preliminary experiments. Fortunately, construction of the markerless ISPⅠ strain, in which the bsm4(responsible for acylation at 3 of spiramycin) gene was replaced by the Isovaleryltansferase gene(ist) under control of the constitutive promoter ermEp~*, was efficiently achieved by using the CRISPR-Cas9 gene editing system. The mutant of bsm4 deletion can only produce SPⅠ. Isovaleryltransferase coded by ist catalyzes the isovalerylation of the SPⅠat C-4" hydroxyl group to produce ISPⅠ. As anticipated, ISPⅠ was the sole ISP component of the resultant strain(ΔEI) when detected by HPLC and mass spectrometry. The ΔEI mutant is suitable for further genetic engineering to obtain improved strains by reusing CRISPR-Cas9 system.
Isovalerylspiramycin(ISP)Ⅰ, as a major component of bitespiramycin(BT), exhibits similar antimicrobial activities with BT and has advantages in quality control and dosage forms. It has been under preclinical studies. The existing ISPⅠ producing strain, undergoing three genetic modifications, carries two resistant gene markers. Thus, it is hard for further genetic manipulation. It is a time-consuming and unsuccessful work to construct a new ISPⅠ strain without resistant gene marker by means of the classical homologous recombination in our preliminary experiments. Fortunately, construction of the markerless ISPⅠ strain, in which the bsm4(responsible for acylation at 3 of spiramycin) gene was replaced by the Isovaleryltansferase gene(ist) under control of the constitutive promoter ermEp~*, was efficiently achieved by using the CRISPR-Cas9 gene editing system. The mutant of bsm4 deletion can only produce SPⅠ. Isovaleryltransferase coded by ist catalyzes the isovalerylation of the SPⅠat C-4" hydroxyl group to produce ISPⅠ. As anticipated, ISPⅠ was the sole ISP component of the resultant strain(ΔEI) when detected by HPLC and mass spectrometry. The ΔEI mutant is suitable for further genetic engineering to obtain improved strains by reusing CRISPR-Cas9 system.
引文
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[2]Wu LZ,Ma CY,Wang YG,et al.Deletion of spiramycin 3-O-acyltransferase gene from Streptomyces spiramyceticus F21 resulting in the production of spiramycinⅠas major component.Chin J Biotech,2007,23(4):612-617(in Chinese).武临专,马春燕,王以光,等.螺旋霉素3-O-酰基转移酶基因的删除和主要产生螺旋霉素组分Ⅰ菌株的获得.生物工程学报,2007,23(4):612-617.
[3]Wang YG,He WQ,Dai JL,et al.Gene series technology for increasing main component content of gene engineering isovalerylspiramycin CN,200910148767.8.2011-09-07(in Chinese).王以光,赫卫清,戴剑漉,等.一种提高基因工程异戊酰螺旋霉素主组分含量的基因串连技术:中国,200910148767.8.2011-09-07.
[4]Ma CY,Zhou HX,Li JY,et al.Construction of4"-isovalerylspiramycin-I-producing strain by in-frame partial deletion of 3-O-acyltransferase gene in Streptomyces spiramyceticus WSJ-1,the bitespiramycin producer.Curr Microbiol,2011,62(1):16-20.
[5]Wang YG,Wu LZ,Jiang Y,et al.Isovalerylspiramycin I component high-content high-yield genetic engineering bacteria:CN,201010237595.4.2012-07-04(in Chinese).王以光,武临专,姜洋,等.一株异戊酰螺旋霉素I组分高含量、高产量基因工程菌:中国,201010237595.4.2012-07-04.
[6]Dai JL,Lu ZL,Lin L,et al.Construction of the isomycin I high-yield strain by introducing a heterologous positive regulatory gene acyB2.China Biotechnol,2017,37(3):65-72(in Chinese).戴剑漉,卢智黎,林灵,等.利用异源正调控基因acyB2构建埃莎霉素I高产菌株.中国生物工程杂志,2017,37(3):65-72.
[7]Dai JL,Zhang XT,Lu ZL,et al.Breeding of high isomycin-I-producing strain by MPMS composite mutagenesis with plasma and UV.Chin J Antibiot,2018,43(2):182-188(in Chinese).戴剑漉,张晓婷,卢智黎,等.新型常压室温等离子体-紫外复合诱变选育埃莎霉素I高产菌株.中国抗生素杂志,2018,43(2):182-188.
[8]Ran FA,Hsu PD,Wright J,et al.Genome engineering using the CRISPR-Cas9 system.Nat Protoc,2013,8(11):2281-2308.
[9]Ran FA,Hsu PD,Lin CY,et al.Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.Cell,2013,154(6):1380-1389.
[10]Bierman M,Logan R,O’Brien K,et al.Plasmid cloning vectors for the conjugal transfer of DNAfrom Escherichia coli to Streptomyces spp.Gene,1992,116(1):43-49.
[11]Huang H,Zheng GS,Jiang WH,et al.One-step high-efficiency CRISPR/Cas9-mediated genome editing in Streptomyces.Acta Biochim Biophys Sin,2015,47(4):231-243.
[12]Kieser T,Bibb MJ,Buttner MJ,et al.Practical Streptomyces genetics.Norwich:The John Innes Foundation,2000.
[13]Wang YG,Jin LF,Jin WZ,et al.Cloning of midecamycin 4?-acyltransferase and expression spiramycin producing strains.Chin J Biotech,1992,8(1):1-14(in Chinese).王以光,金莲舫,金文藻,等.麦迪霉素4″酰化酶基因的克隆及在螺旋霉产生菌中的表达.生物工程学报,1992,8(1):1-14.
[14]Xu P,Li WJ,Xu LH,et al.A microwave-based method for genomic DNA extraction from Actinomycetes.Microbiology China,2003,30(4):82-84(in Chinese).徐平,李文均,徐丽华,等.微波法快速提取放线菌基因组DNA.微生物学通报,2003,30(4):82-84.
[15]Sambrook J,Fritsch EF,Maniatis T.Molecular Cloning:A Laboratory Manual.2nd ed.New York:Cold Spring Harbor Laboratory Press,1989:20-25.
[16]Yang YL,Yang JN,Hu M,et al.Determination of the components of bitespiramycin by HPLC.Acta Pharmac Sin,2009,44(10):1183-1186(in Chinese).杨亚莉,杨剑宁,胡敏,等.HPLC法分析可利霉素的组分.药学学报,2009,44(10):1183-1186.
[17]Bi YW,Sun L,Gao DD,et al.High-efficiency targeted editing of large viral genomes by RNA-guided nucleases.PLoS Pathog,2014,10(5):e1004090.
[18]Gratz SJ,Wildonger J,Harrison MM,et al.CRISPR/Cas9-mediated genome engineering and the promise of designer flies on demand.Fly,2013,7(4):249-255.
[19]Jiang WZ,Yang B,Weeks DP.Efficient CRISPR/Cas9-mediated gene editing in Arabidopsis thaliana and inheritance of modified genes in the T2and T3 generations.PLoS ONE,2014,9(6):e99225.
[20]Jiang WZ,Zhou HB,Bi HH,et al.Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis,tobacco,sorghum and rice.Nucleic Acids Res,2013,41(20):e188.
[21]Hruscha A,Krawitz P,Rechenberg A,et al.Efficient CRISPR/Cas9 genome editing with low off-target effects in zebrafish.Development,2013,140(24):4982-4987.
[22]Bibb MJ,Janssen GR,Ward JM.Cloning and analysis of the promoter region of the erythromycin resistance gene(ermE)of Streptomyces erythraeus.Gene,1985,38(1/3):215-226.
[23]Villsen ID,Vester B,Douthwaite S.ErmEmethyltransferase recognizes features of the primary and secondary structure in a motif within domain Vof 23S rRNA.J Mol Biol,1999,286(2):365-374.
[24]Jin ZH,Jin X,Jin QC.Conjugal transferring of resistance gene ptr for improvement of pristinamycin-producing Streptomyces pristinaespiralis.Appl Biochem Biotechnol,2010,160(6):1853-1864.
[25]Stojkovi?V,Noda-Garcia L,Tawfik DS,et al.Antibiotic resistance evolved via inactivation of a ribosomal RNA methylating enzyme.Nucleic Acids Res,2016,44(18):8897-8907.
[26]Zhang JH,Zhong JJ,Dai JL,et al.Expression of4?-O-isovaleryltransferase gene from Streptomyces thermotolerans in Streptomyces lividans TK24.Chin J Biotech,2014,30(9):1390-1400(in Chinese).张家瑚,钟晶晶,戴剑漉,等.耐热链霉菌4″-O-戊酰基转移酶基因在变铅青链霉菌TK24中的表达.生物工程学报,2014,30(9):1390-1400.