安莎类抗生素产生菌分子生物学筛选及其化学结构早期鉴别的研究
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摘要
3-氨基-5-羟基苯甲酸(AHBA)是微生物产生安莎类抗生素及其相关抗生素的必需前体。通过对这类抗生素已知的生物合成基因簇的比较,发现AHBA生物合成基因簇中的AHBA合酶基因高度保守。AHBA合酶基因的保守性为筛选这类化合物的产生菌提供了新的思路。本论文拟通过分子生物学手段,建立一种基于检测AHBA合酶基因来筛选安莎类化合物及相关抗生素产生菌的方法。
本文首先针对从约2000株土壤放线菌分离得到的33株具有可能产生安莎类抗生素潜力的放线菌,初步研究了它们的发酵条件及发酵产物的生物活性(抗菌、抗病毒和抗肿瘤活性),确定了每株菌对可用于选择性标记的不同抗生素的抗性。综合以上实验结果选择了六株具研究价值(有较好的抗菌、抗病毒或抗肿瘤活性且具有相应的抗药性或进化树分析属于不同从属分枝)的菌株:8-21、2209、22-24、4088、3-27、4353进行AHBA合酶基因的阻断研究,进一步筛选安莎类及相关抗生素产生菌。
研究并建立上述菌株PEG-介导的原生质体转化及接合转移的转化系统,最后通过接合转移方法获得了22-24、4088、3-27、4353等四株菌基因单交换阻断AHBA合酶基因阻断变株。采用4353 AHBA合酶部分基因,构建了其在4353菌株的阻断变株、采用22-24 AHBA合酶部分基因,构建了其在22-24、4088、3-27菌株的阻断变株。基因阻断实验结果显示:AHBA合酶基因的阻断使菌株4353不再产生一种黄色的活性化合物;尽管目前菌株22-24、4088中AHBA合酶基因的阻断尚没有在基因水平上得到完全的验证,但4阻断使菌株22-2变株产生的一种活性化合物明显减少,而菌株4088变株则产生了一种原株中不产生的新的具有抗菌活性的物质;菌株3-27 AHBA阻断株的发酵产物与原株相比没有明显变化。菌株4353和4088及22-24有继续深入研究价值。
此外,我们又针对安莎类化合物结构中含有酰胺键的特点,根据碱性处理能使其安莎链与苯环连接的酰胺键开裂,形成的化合物呈紫色的原理,建立了发酵液粗提品TLC和NaOH溶液喷涂显色的早期鉴别苯安莎类抗生素(格尔德霉素类)方法,实验表明此种方法在硅胶板上的检测灵敏度为4μg,用此方法对两株确证为新的格尔德霉素产生菌Streptomyces sp.4-4以及Streptomyces sp.3-57进行了佐证。
为了更进一步研究AHBA合酶基因在菌株4353发酵活性物质的产生过程中的作用,我们获得了菌株4353AHBA合酶的全基因序列及其下游氧化还原酶基因序列和部分磷酸酶基因序列,并成功构建了两种带有不同标记序列的表达载体,在大肠杆菌中得到表达,并鉴定了重组蛋白在大肠杆菌中以包涵体形式存在,为以后的研究奠定了基础。
The compound,3-amino-5-hydroxybenzoic acid(AHBA),is the specific and indispensible starter unit for the biosynthesis of ansamycins in microbes.AHBA synthase is the terminal enzyme catalyzing the final formation of AHBA in the aminoshikimate pathway for AHBA biosynthesis. Multi-alignment of known AHBA synthases revealed that they are highly similar.Therefore, degenerate oligonucleotides could be designed to amplify AHBA synthase genes from unkown microbial strains.The existence of AHBA synthase gene in a microbial strain could also be used as an indicator that the strain possess the potential for AHBA,and furtherly,ansamycin biosynthesis. Under such belief,a PCR screening method for potential ansamycin-producing actinomycete strains had been established and 33 strains from 2000 soil-isolates had been obtained as AHBA-synthase gene positive strains in our lab.
In this paper,the anti-bacterial/anti-virus/anti-cancer cell activities of the fermentation products of 33 AHBA synthase gene positive strains obtained above were assayed.Six strains,that is,Streptomyces sp.8-21,2209,22-24,4088,3-27 and 4353,showed good biological activities in their fermentation products,which are worthy to study furtherly.
Several methods including PEG-mediated protoplasts transformation and conjugal transfer were investigated for each of the six strians.Conjugal transfer was accomplished successfully in four of the six strains,that is Streptomyces sp.22-24,4088,3-27 and 4353.AHBA synthase gene disruption mutants for each of the four strains,by single cross-over,were obtained by conjugal transfer of pKC 1139,inserted with the coresponding AHBA synthase gene fragment in its multiple cloning sites,from E.coli ET12567(pUZ8002)to the Streptomyces sp..
Mutants of Streptomyces sp.4353 produced no further a yellow compound,which was identified later as actinomycin D.Mutants of Streptomyces sp.3-27 showed no observed changes in its fermentation products.Mutants of Streptomyces sp.22-24 produced less amounts of an active compound in its fermentation production.Mutants of Streptomyces sp.4088 produced a new active compound in its fermentation production.
In this paper,we established also a simple method for early preliminary discrimination of benzenic ansamycins and their derivatives.With the treatment of alkali such as NaOH,which break the amide bond formed between the benzenic amino-group and the ansa-bridge's terminal carboxy-group,benzenic ansamycins such as geldanamycin changed its color from yellow to purple. The minimal detection limit of this method on the silica gel plate for geldanamycin was 4μg if NaOH(2.0mol/L)was sprayed on the TLC plate.This method was used successfully for early preliminary discrimination of geldanamycin produced by two soil-isolated novel strains, Streptomyces sp.4-4 and Streptomyces sp.3-57,which were strains with ansamycin producing potential(s).
To investigate the AHBA synthase gene and some other genes associated with AHBA biosynthesis in Streptomyces sp.4353,the AHBA synthase gene and its flanking regions were amplified by combinations of PCR,then cloned and sequenced.In the 2872bp DNA we obtained, the ORFs of AHBA synthase and AHBA oxidoreductase and AHBA phosphotase(partial)were discovered.The AHBA synthase gene from Streptomyces sp.4353 was expressed sucessfully in E. coli as inclusion bodies in our study.The AHBA synthase gene,AHBA oxidoreductase gene and, possibly,the AHBA phosphotase gene(partial)could be useful candidates for re-constitution of hybrid AHBA biosynthetic pathways heterologously(such as in E.coli),therefore providing a good starting point for combinatorial biosynthesis of ansamycins.
本文首先针对从约2000株土壤放线菌分离得到的33株具有可能产生安莎类抗生素潜力的放线菌,初步研究了它们的发酵条件及发酵产物的生物活性(抗菌、抗病毒和抗肿瘤活性),确定了每株菌对可用于选择性标记的不同抗生素的抗性。综合以上实验结果选择了六株具研究价值(有较好的抗菌、抗病毒或抗肿瘤活性且具有相应的抗药性或进化树分析属于不同从属分枝)的菌株:8-21、2209、22-24、4088、3-27、4353进行AHBA合酶基因的阻断研究,进一步筛选安莎类及相关抗生素产生菌。
研究并建立上述菌株PEG-介导的原生质体转化及接合转移的转化系统,最后通过接合转移方法获得了22-24、4088、3-27、4353等四株菌基因单交换阻断AHBA合酶基因阻断变株。采用4353 AHBA合酶部分基因,构建了其在4353菌株的阻断变株、采用22-24 AHBA合酶部分基因,构建了其在22-24、4088、3-27菌株的阻断变株。基因阻断实验结果显示:AHBA合酶基因的阻断使菌株4353不再产生一种黄色的活性化合物;尽管目前菌株22-24、4088中AHBA合酶基因的阻断尚没有在基因水平上得到完全的验证,但4阻断使菌株22-2变株产生的一种活性化合物明显减少,而菌株4088变株则产生了一种原株中不产生的新的具有抗菌活性的物质;菌株3-27 AHBA阻断株的发酵产物与原株相比没有明显变化。菌株4353和4088及22-24有继续深入研究价值。
此外,我们又针对安莎类化合物结构中含有酰胺键的特点,根据碱性处理能使其安莎链与苯环连接的酰胺键开裂,形成的化合物呈紫色的原理,建立了发酵液粗提品TLC和NaOH溶液喷涂显色的早期鉴别苯安莎类抗生素(格尔德霉素类)方法,实验表明此种方法在硅胶板上的检测灵敏度为4μg,用此方法对两株确证为新的格尔德霉素产生菌Streptomyces sp.4-4以及Streptomyces sp.3-57进行了佐证。
为了更进一步研究AHBA合酶基因在菌株4353发酵活性物质的产生过程中的作用,我们获得了菌株4353AHBA合酶的全基因序列及其下游氧化还原酶基因序列和部分磷酸酶基因序列,并成功构建了两种带有不同标记序列的表达载体,在大肠杆菌中得到表达,并鉴定了重组蛋白在大肠杆菌中以包涵体形式存在,为以后的研究奠定了基础。
The compound,3-amino-5-hydroxybenzoic acid(AHBA),is the specific and indispensible starter unit for the biosynthesis of ansamycins in microbes.AHBA synthase is the terminal enzyme catalyzing the final formation of AHBA in the aminoshikimate pathway for AHBA biosynthesis. Multi-alignment of known AHBA synthases revealed that they are highly similar.Therefore, degenerate oligonucleotides could be designed to amplify AHBA synthase genes from unkown microbial strains.The existence of AHBA synthase gene in a microbial strain could also be used as an indicator that the strain possess the potential for AHBA,and furtherly,ansamycin biosynthesis. Under such belief,a PCR screening method for potential ansamycin-producing actinomycete strains had been established and 33 strains from 2000 soil-isolates had been obtained as AHBA-synthase gene positive strains in our lab.
In this paper,the anti-bacterial/anti-virus/anti-cancer cell activities of the fermentation products of 33 AHBA synthase gene positive strains obtained above were assayed.Six strains,that is,Streptomyces sp.8-21,2209,22-24,4088,3-27 and 4353,showed good biological activities in their fermentation products,which are worthy to study furtherly.
Several methods including PEG-mediated protoplasts transformation and conjugal transfer were investigated for each of the six strians.Conjugal transfer was accomplished successfully in four of the six strains,that is Streptomyces sp.22-24,4088,3-27 and 4353.AHBA synthase gene disruption mutants for each of the four strains,by single cross-over,were obtained by conjugal transfer of pKC 1139,inserted with the coresponding AHBA synthase gene fragment in its multiple cloning sites,from E.coli ET12567(pUZ8002)to the Streptomyces sp..
Mutants of Streptomyces sp.4353 produced no further a yellow compound,which was identified later as actinomycin D.Mutants of Streptomyces sp.3-27 showed no observed changes in its fermentation products.Mutants of Streptomyces sp.22-24 produced less amounts of an active compound in its fermentation production.Mutants of Streptomyces sp.4088 produced a new active compound in its fermentation production.
In this paper,we established also a simple method for early preliminary discrimination of benzenic ansamycins and their derivatives.With the treatment of alkali such as NaOH,which break the amide bond formed between the benzenic amino-group and the ansa-bridge's terminal carboxy-group,benzenic ansamycins such as geldanamycin changed its color from yellow to purple. The minimal detection limit of this method on the silica gel plate for geldanamycin was 4μg if NaOH(2.0mol/L)was sprayed on the TLC plate.This method was used successfully for early preliminary discrimination of geldanamycin produced by two soil-isolated novel strains, Streptomyces sp.4-4 and Streptomyces sp.3-57,which were strains with ansamycin producing potential(s).
To investigate the AHBA synthase gene and some other genes associated with AHBA biosynthesis in Streptomyces sp.4353,the AHBA synthase gene and its flanking regions were amplified by combinations of PCR,then cloned and sequenced.In the 2872bp DNA we obtained, the ORFs of AHBA synthase and AHBA oxidoreductase and AHBA phosphotase(partial)were discovered.The AHBA synthase gene from Streptomyces sp.4353 was expressed sucessfully in E. coli as inclusion bodies in our study.The AHBA synthase gene,AHBA oxidoreductase gene and, possibly,the AHBA phosphotase gene(partial)could be useful candidates for re-constitution of hybrid AHBA biosynthetic pathways heterologously(such as in E.coli),therefore providing a good starting point for combinatorial biosynthesis of ansamycins.
引文
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