To promote spinosad biosynthesis by improving the limited oxygen supply during high-density fermentation of Saccharopolyspora spinosa, the open reading frame of the Vitreoscilla hemoglobin gene was placed under the co...To promote spinosad biosynthesis by improving the limited oxygen supply during high-density fermentation of Saccharopolyspora spinosa, the open reading frame of the Vitreoscilla hemoglobin gene was placed under the control of the promoter for the erythromycin resistance gene by splicing using overlapping extension PCR. This was cloned into the integrating vector pSET152, yielding the Vitreoscilla hemoglobin gene expression plasmid pSET152EVHB. This was then introduced into S. spinosa SP06081 by conjugal transfer, and integrated into the chromosome by site-specific recombination at the integration site ФC31 on pSET152EVHB. The resultant conjugant, S. spinosa S078-1101, was genetically stable. The integration was further confirmed by PCR and Southern blotting analysis. A carbon monoxide differential spectrum assay showed that active Vitreoscilla hemoglobin was successfully expressed in S. spinosa S078-1101. Fermentation results revealed that expression of the Vitreoscilla hemoglobin gene significantly promoted spinosad biosynthesis under normal oxygen and moderately oxygen-limiting conditions （P〈0.01）. These findings demonstrate that integrating expression of the Vitreoscilla hemoglobin gene improves oxygen uptake and is an effective means for the genetic improvement of S. spinosa fermentation. Saccharopolyspora spinosa, spinosad, Vitreoscilla hemoglobin, integrating vector, homologous recombination展开更多
Erythromycin A is a widely used antibiotic produced by Saccharopolyspora erythraea;however,its biosynthetic cluster lacks a regulatory gene,limiting the yield enhancement via regulation engineering of S.erythraea.Here...Erythromycin A is a widely used antibiotic produced by Saccharopolyspora erythraea;however,its biosynthetic cluster lacks a regulatory gene,limiting the yield enhancement via regulation engineering of S.erythraea.Herein,six TetR family transcriptional regulators(TFRs)belonging to three genomic context types were individually inactivated in S.erythraea A226,and one of them,SACE_3446,was proved to play a negative role in regulating erythromycin biosynthesis.EMSA and qRT-PCR analysis revealed that SACE_3446 covering intact N-terminal DNA binding domain specifically bound to the promoter regions of erythromycin biosynthetic gene eryAI,the resistant gene ermE and the adjacent gene SACE_3447(encoding a longchain fatty-acid CoA ligase),and repressed their transcription.Furthermore,we explored the interaction relationships of SACE_3446 and previously identified TFRs(SACE_3986 and SACE_7301)associated with erythromycin production.Given demonstrated relatively independent regulation mode of SACE_3446 and SACE_3986 in erythromycin biosynthesis,we individually and concomitantly inactivated them in an industrial S.erythraea WB.Compared with WB,the WBΔ3446 and WBΔ3446Δ3986 mutants respectively displayed 36%and 65%yield enhancement of erythromycin A,following significantly elevated transcription of eryAI and ermE.When cultured in a 5 L fermentor,erythromycin A ofWBΔ3446 and WBΔ3446Δ3986 successively reached 4095 mg/L and 4670 mg/L with 23%and 41%production improvement relative to WB.The strategy reported here will be useful to improve antibiotics production in other industrial actinomycete.展开更多
Objective:To investigate the larvicidal and pupicidal activity of spinosad against Anopheles stephensi Listen.Methods:Spinosad from the actinomycete,Saccharopolyspora spinosa was tested against Anopheles stephensi at ...Objective:To investigate the larvicidal and pupicidal activity of spinosad against Anopheles stephensi Listen.Methods:Spinosad from the actinomycete,Saccharopolyspora spinosa was tested against Anopheles stephensi at different concentrations（0.01,0.02,0.04,0.06 and 0.08 ppm.）, and against first to fourth instar larvae and pupae.Results:The larval mortality ranged from 36.1±1.7 in（0.01 ppm） to 79.3±1.8（0.08 ppm） the first instar larva.The LC<sub>50</sub> and LC<sub>90</sub> values of first, second,third and fourth instar larva were 0.001,0.031,0.034,0.036 and 0.0113,0.102,0.111,0.113, respectively.The pupal mortality ranged from 33.0±2.0（0.01 ppm） to 80.0±0.9（0.08 ppm）.The LC<sub>50</sub> and LC<sub>90</sub> values were 0.028 and 0.1020,respectively.The reduction percentage of Anopheles larvae was 82.7%,91.4%and 96.0%after 24,48,72 hours,respectively,while more than 80% reduction was observed after 3 weeks.Conclusions:In the present study spinosad effectively caused mortality of mosquito larvae in both the laboratory and field trial.It is predicted that spinosad is likely to be an effective larvicide for treatment of mosquito breeding sites.展开更多
采用响应面法对红色糖多孢菌产红霉素发酵培养基进行优化。用Minimum Run Equireolicated Res IV设计对初始发酵培养基添加的6个影响因素的效应进行评价,选择有显著影响的4个因素,即硫酸镁、甜菜碱、硫酸铜和氯化钴。再用最陡爬坡实验...采用响应面法对红色糖多孢菌产红霉素发酵培养基进行优化。用Minimum Run Equireolicated Res IV设计对初始发酵培养基添加的6个影响因素的效应进行评价,选择有显著影响的4个因素,即硫酸镁、甜菜碱、硫酸铜和氯化钴。再用最陡爬坡实验为中心组合实验确定最大响应区间,最后经过响应面分析得到最优化结果,硫酸镁0.106%(w/v),甜菜碱0.0185%(w/v),硫酸铜0.106mmol/L,氯化钴0.0003%(w/v)。优化后红霉素生物效价比优化前提高了30%。展开更多
基金supported by the National Basic Research Program of China (Grant Nos. 2012CB722301 and 2011CB111605)the National High Technology Research and Development Project of China (Grant No. 2011AA10A203)the National Natural Science Foundation of China (Grant No. 31070006)
文摘To promote spinosad biosynthesis by improving the limited oxygen supply during high-density fermentation of Saccharopolyspora spinosa, the open reading frame of the Vitreoscilla hemoglobin gene was placed under the control of the promoter for the erythromycin resistance gene by splicing using overlapping extension PCR. This was cloned into the integrating vector pSET152, yielding the Vitreoscilla hemoglobin gene expression plasmid pSET152EVHB. This was then introduced into S. spinosa SP06081 by conjugal transfer, and integrated into the chromosome by site-specific recombination at the integration site ФC31 on pSET152EVHB. The resultant conjugant, S. spinosa S078-1101, was genetically stable. The integration was further confirmed by PCR and Southern blotting analysis. A carbon monoxide differential spectrum assay showed that active Vitreoscilla hemoglobin was successfully expressed in S. spinosa S078-1101. Fermentation results revealed that expression of the Vitreoscilla hemoglobin gene significantly promoted spinosad biosynthesis under normal oxygen and moderately oxygen-limiting conditions （P〈0.01）. These findings demonstrate that integrating expression of the Vitreoscilla hemoglobin gene improves oxygen uptake and is an effective means for the genetic improvement of S. spinosa fermentation. Saccharopolyspora spinosa, spinosad, Vitreoscilla hemoglobin, integrating vector, homologous recombination
基金the National Program on Key Basic Research Project(973 program,2013CB734000)the National Natural Science Foundation of China(Grant Nos.31300081,31570074)the Initial Foundation of Doctoral Scientific Research in Anhui University(01001904,J01001935).
文摘Erythromycin A is a widely used antibiotic produced by Saccharopolyspora erythraea;however,its biosynthetic cluster lacks a regulatory gene,limiting the yield enhancement via regulation engineering of S.erythraea.Herein,six TetR family transcriptional regulators(TFRs)belonging to three genomic context types were individually inactivated in S.erythraea A226,and one of them,SACE_3446,was proved to play a negative role in regulating erythromycin biosynthesis.EMSA and qRT-PCR analysis revealed that SACE_3446 covering intact N-terminal DNA binding domain specifically bound to the promoter regions of erythromycin biosynthetic gene eryAI,the resistant gene ermE and the adjacent gene SACE_3447(encoding a longchain fatty-acid CoA ligase),and repressed their transcription.Furthermore,we explored the interaction relationships of SACE_3446 and previously identified TFRs(SACE_3986 and SACE_7301)associated with erythromycin production.Given demonstrated relatively independent regulation mode of SACE_3446 and SACE_3986 in erythromycin biosynthesis,we individually and concomitantly inactivated them in an industrial S.erythraea WB.Compared with WB,the WBΔ3446 and WBΔ3446Δ3986 mutants respectively displayed 36%and 65%yield enhancement of erythromycin A,following significantly elevated transcription of eryAI and ermE.When cultured in a 5 L fermentor,erythromycin A ofWBΔ3446 and WBΔ3446Δ3986 successively reached 4095 mg/L and 4670 mg/L with 23%and 41%production improvement relative to WB.The strategy reported here will be useful to improve antibiotics production in other industrial actinomycete.
文摘Objective:To investigate the larvicidal and pupicidal activity of spinosad against Anopheles stephensi Listen.Methods:Spinosad from the actinomycete,Saccharopolyspora spinosa was tested against Anopheles stephensi at different concentrations（0.01,0.02,0.04,0.06 and 0.08 ppm.）, and against first to fourth instar larvae and pupae.Results:The larval mortality ranged from 36.1±1.7 in（0.01 ppm） to 79.3±1.8（0.08 ppm） the first instar larva.The LC<sub>50</sub> and LC<sub>90</sub> values of first, second,third and fourth instar larva were 0.001,0.031,0.034,0.036 and 0.0113,0.102,0.111,0.113, respectively.The pupal mortality ranged from 33.0±2.0（0.01 ppm） to 80.0±0.9（0.08 ppm）.The LC<sub>50</sub> and LC<sub>90</sub> values were 0.028 and 0.1020,respectively.The reduction percentage of Anopheles larvae was 82.7%,91.4%and 96.0%after 24,48,72 hours,respectively,while more than 80% reduction was observed after 3 weeks.Conclusions:In the present study spinosad effectively caused mortality of mosquito larvae in both the laboratory and field trial.It is predicted that spinosad is likely to be an effective larvicide for treatment of mosquito breeding sites.
基金supported by the National Natural Science Foundation of China(No.21076148 and No.31270087)Plan of Tianjin Science and Technology Support(11ZCKFSY0100)
文摘采用响应面法对红色糖多孢菌产红霉素发酵培养基进行优化。用Minimum Run Equireolicated Res IV设计对初始发酵培养基添加的6个影响因素的效应进行评价,选择有显著影响的4个因素,即硫酸镁、甜菜碱、硫酸铜和氯化钴。再用最陡爬坡实验为中心组合实验确定最大响应区间,最后经过响应面分析得到最优化结果,硫酸镁0.106%(w/v),甜菜碱0.0185%(w/v),硫酸铜0.106mmol/L,氯化钴0.0003%(w/v)。优化后红霉素生物效价比优化前提高了30%。
