代谢工程改造的链霉菌生物合成TLM H-1和Rapamycin的研究
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摘要
在了解天然产物生物合成途径以及相关基因信息的基础上,人工改造调控基因,或者实现相关基因簇的异源表达,从而获得具有新结构新活性的抗生素,是链霉菌代谢工程改造的两个重要途径。
Tallysomycin(TLM)H-1是由tlmH基因失活后的工程菌S.hindustanus SB8005合成的主要产物。它作为糖肽类抗肿瘤抗生素TLM和BLM的新型类似物,具有与母体相似的DNA切割活性。然而,这种新型抗生素在工程菌中的低产量极大地限制了对其活性和应用的进一步研究。本研究旨在通过采用各种有效的方法来优化TLM H-1的培养基组分,并进行发酵放大提高其产量。文章结合单因素优化,Plackett-Burman设计和响应面实验等方法来进行优化,结果显示CuSO_4,麦芽糖,DGS是培养基中3个最重要的影响因子,统计学分析确定最佳培养基后,相应的上罐放大发酵最高产量为249.9 mg/L,比原始培养基的产量高出26.8倍,比原始菌TLM的产量高出12.9倍。经Amberlite(?) IRC50离子交换树脂和Diaion HP-20大孔树脂上柱分离后,HPLC分析纯度可达到95%以上。
Rapamycin(RAP)是一种由吸水链霉菌合成的大环内酯抗生素。尽管其生物合成途径已得到全面深入的研究,但其详细的合成机制尤其是调控机理并未完全明了,极大地阻碍了RAP产量的提高和新型衍生物的产生,以及其工业化生产和应用。本文在构建三种RAP异源生产系统的基础上,首先用生物分析方法检测各系统中RAP异源生产的情况,结果表明仅S.albus 28-1-1显示出与RAP相似的抗菌活性,而HPLC和LC-MS分析并未检测到RAP,添加重要前体L-赖氨酸也无明显作用。随后的RT-PCR分析结果表明绝大部分的RAP基因在S.albus中未被正常转录,只有下游的rapG,rapF和rapD显示转录信号,5个可能的RAP调节基因中仅有rapG正常转录,因此RAP基因表达调控的不足或不当很可能是限制基因簇正常转录的重要原因。通过这些基因转录表达情况的研究,我们可深入了解RAP生物合成的调控机理,为最终实现高产奠定基础。
Discovery of novel antibiotic analogs by combinatorial biosynthesis via engineering regulatory genes and heterologous expression of gene clusters are the two significant approaches in metabolic engineering based on fully understand the biosynthetic pathways and related gene information.
Tallysomycin(TLM) H-1,a novel TLM and BLM analog displaying similar DNA cleavage activity,is the major product isolated from Streptoalloteichus hindustanus SB8005,a genetically engineered strain from S.hindustanus E465-94 ATCC 31158.The low titer of TLM H-1 in the engineered SB8005 strain has greatly limited its further study.This research aims to improve the titer of TLM H-1 production in the SB8005 strain through optimization of fermentation culture and scaling up the fermentation process.In the present work,single factor optimization, Plackett-Burman design and response surface methodology were introduced.The results indicated that three variables including distiller's grains and solubles,copper sulfate and maltose out of eight parameters could significantly influence the TLM H-1 production.With systematic comparison and evaluation,the final optimized fermentation medium was determined. The optimized yield of TLM H-1 in the bench-top fermentor was 249.9 mg/L,which is 26.8 times higher than reported using the original medium,and 12.9-fold higher than that of the parent compound TLM produced by the wild-type strain.Moreover,an efficient purification procedure was developed to separate TLM H-1 from the fermentation broth by the application of Amberlite(?) IRC50 resin and Diaion HP-20 resin,and the HPLC purity of the product reached over 95%.
Rapamycin(RAP) is a macrolide antibiotic produced by Streptomyces hygroscopicus. Though the biosynthesis of RAP has been extensively and intensively studied,the detailed biosynthetic machinery especially its regulatory mechanism was not fully understood,which greatly obstructed the improvement of the titer and generation of novel RAP analogs,as well as its industrialization and further application.To test the possibility of gaining this important but low yield natural product via novel heterologous production technique and set up a stage for systematically study of its biosynthesis,the RAP biosynthetic gene cluster has been respectively integrated into three Streptomyces mode hosts S.albus,S.ceolicolor M512,and S.lividans K4-114 by BAC-based technology to generate the RAP heterologous production systems.The bioassay results suggested that olny S.albus 28-1-1 from the constructed three heterologous systems has clearly showed the antibiotic activity similar to RAP.But the parallel HPLC and LC-MS analyses did not detect any trace of the production.Supplementation of special precursor L-lysine indeed had no effect on the improvement of RAP production or the antibiotic activity in the bioassay.Subsequent RT-PCR analyses has indicated that the majority of RAP biosynthetic genes were not transcripted,only three downstream genes rapG,rapF and rapD showed transcription signal in S.albus.Thus,insufficient or improper regulation of RAP genes could be a critical reason that limited the normal gene transcription.These heterologous production studies in S.albus not only provide us a simplified stage with the clean background to study the details especially the regulation mechanism of RAP biosynthesis,but also present an alternative way to obtain some high-yielded natural products to be profitable drug leads.
Tallysomycin(TLM)H-1是由tlmH基因失活后的工程菌S.hindustanus SB8005合成的主要产物。它作为糖肽类抗肿瘤抗生素TLM和BLM的新型类似物,具有与母体相似的DNA切割活性。然而,这种新型抗生素在工程菌中的低产量极大地限制了对其活性和应用的进一步研究。本研究旨在通过采用各种有效的方法来优化TLM H-1的培养基组分,并进行发酵放大提高其产量。文章结合单因素优化,Plackett-Burman设计和响应面实验等方法来进行优化,结果显示CuSO_4,麦芽糖,DGS是培养基中3个最重要的影响因子,统计学分析确定最佳培养基后,相应的上罐放大发酵最高产量为249.9 mg/L,比原始培养基的产量高出26.8倍,比原始菌TLM的产量高出12.9倍。经Amberlite(?) IRC50离子交换树脂和Diaion HP-20大孔树脂上柱分离后,HPLC分析纯度可达到95%以上。
Rapamycin(RAP)是一种由吸水链霉菌合成的大环内酯抗生素。尽管其生物合成途径已得到全面深入的研究,但其详细的合成机制尤其是调控机理并未完全明了,极大地阻碍了RAP产量的提高和新型衍生物的产生,以及其工业化生产和应用。本文在构建三种RAP异源生产系统的基础上,首先用生物分析方法检测各系统中RAP异源生产的情况,结果表明仅S.albus 28-1-1显示出与RAP相似的抗菌活性,而HPLC和LC-MS分析并未检测到RAP,添加重要前体L-赖氨酸也无明显作用。随后的RT-PCR分析结果表明绝大部分的RAP基因在S.albus中未被正常转录,只有下游的rapG,rapF和rapD显示转录信号,5个可能的RAP调节基因中仅有rapG正常转录,因此RAP基因表达调控的不足或不当很可能是限制基因簇正常转录的重要原因。通过这些基因转录表达情况的研究,我们可深入了解RAP生物合成的调控机理,为最终实现高产奠定基础。
Discovery of novel antibiotic analogs by combinatorial biosynthesis via engineering regulatory genes and heterologous expression of gene clusters are the two significant approaches in metabolic engineering based on fully understand the biosynthetic pathways and related gene information.
Tallysomycin(TLM) H-1,a novel TLM and BLM analog displaying similar DNA cleavage activity,is the major product isolated from Streptoalloteichus hindustanus SB8005,a genetically engineered strain from S.hindustanus E465-94 ATCC 31158.The low titer of TLM H-1 in the engineered SB8005 strain has greatly limited its further study.This research aims to improve the titer of TLM H-1 production in the SB8005 strain through optimization of fermentation culture and scaling up the fermentation process.In the present work,single factor optimization, Plackett-Burman design and response surface methodology were introduced.The results indicated that three variables including distiller's grains and solubles,copper sulfate and maltose out of eight parameters could significantly influence the TLM H-1 production.With systematic comparison and evaluation,the final optimized fermentation medium was determined. The optimized yield of TLM H-1 in the bench-top fermentor was 249.9 mg/L,which is 26.8 times higher than reported using the original medium,and 12.9-fold higher than that of the parent compound TLM produced by the wild-type strain.Moreover,an efficient purification procedure was developed to separate TLM H-1 from the fermentation broth by the application of Amberlite(?) IRC50 resin and Diaion HP-20 resin,and the HPLC purity of the product reached over 95%.
Rapamycin(RAP) is a macrolide antibiotic produced by Streptomyces hygroscopicus. Though the biosynthesis of RAP has been extensively and intensively studied,the detailed biosynthetic machinery especially its regulatory mechanism was not fully understood,which greatly obstructed the improvement of the titer and generation of novel RAP analogs,as well as its industrialization and further application.To test the possibility of gaining this important but low yield natural product via novel heterologous production technique and set up a stage for systematically study of its biosynthesis,the RAP biosynthetic gene cluster has been respectively integrated into three Streptomyces mode hosts S.albus,S.ceolicolor M512,and S.lividans K4-114 by BAC-based technology to generate the RAP heterologous production systems.The bioassay results suggested that olny S.albus 28-1-1 from the constructed three heterologous systems has clearly showed the antibiotic activity similar to RAP.But the parallel HPLC and LC-MS analyses did not detect any trace of the production.Supplementation of special precursor L-lysine indeed had no effect on the improvement of RAP production or the antibiotic activity in the bioassay.Subsequent RT-PCR analyses has indicated that the majority of RAP biosynthetic genes were not transcripted,only three downstream genes rapG,rapF and rapD showed transcription signal in S.albus.Thus,insufficient or improper regulation of RAP genes could be a critical reason that limited the normal gene transcription.These heterologous production studies in S.albus not only provide us a simplified stage with the clean background to study the details especially the regulation mechanism of RAP biosynthesis,but also present an alternative way to obtain some high-yielded natural products to be profitable drug leads.
引文
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