多糖型微生物絮凝剂合成途径解析及群体调控机制研究
发布时间：2018-12-17 04:11
【摘要】：作为新兴绿色水处理剂,微生物絮凝剂研发及应用越来越受到各国科学家的重视,多年来一直是环境生物技术领域的研究热点。然而,微生物絮凝剂的生物合成途径、代谢途径及相关基因尚不清晰,致使无法确定野生菌株产絮性状不稳定和产量低的根本原因,无法从根本上解决微生物絮凝剂存在的问题。基于基因组/转录组分析,本文开展了多糖型微生物絮凝剂合成途径及其群体调控机制的研究,为填补微生物絮凝剂合成途径研究的不足,为解决微生物絮凝剂产量低和产絮性状不稳定问题提供理论支持。基于Agrobacterium tumefaciens F2的全基因组测序数据,采用生物信息学和比较基因组方法,分析菌株F2基因组中主要代谢途径与调节机制相关的功能基因;产絮菌F2所产微生物絮凝剂主要有效成分为多糖,因此将多糖型微生物絮凝剂合成基因集中在多糖合成基因上。在菌株F2基因组中发现了84个多糖型微生物絮凝剂合成基因,其中参与琥珀酰聚糖合成exo基因最可能指导多糖型微生物絮凝剂合成;此外,菌株F2质粒上发现了与Rhizobium和Agrobacterium中tra R/tra I群体感应系统具有高度相似的基因簇;完成了多糖型微生物絮凝剂合成基因及其调控基因的解析,为多糖型微生物絮凝剂合成基因和群体感应系统调控基因的功能解析提供大量基因组信息。为了确定多糖型微生物絮凝剂合成基因的功能和合成途径,对产絮菌F2发酵过程中高通量基因转录组和基因表达差异进行分析;从基因组解析的84个多糖型微生物絮凝剂合成基因中确定了65个在发酵过程中发生显著差异的基因,通过比对A.tumefaciens C58和F2合成胞外多糖的单糖组分和合成基因的异同,构建多糖型微生物絮凝剂exo基因簇,并确定了多糖型微生物絮凝剂合成基因的功能和合成途径,揭示引起多糖型微生物絮凝剂产量和效能差异的合成基因类型,为多糖型微生物絮凝剂合成途径的研究提供崭新的思路和方法,为强效表达合成途径的关键基因从而实现多糖型微生物絮凝剂定向合成方法提供理论依据。细菌通过群体感应现象协调群体基因同步表达从而调控胞外物合成的群体行为,而提高调控基因的表达可以实现定向调控多糖型微生物絮凝剂的合成这种群体行为;基因组/转录组分析发现产絮菌F2中具有群体感应系统调控基因,利用UPLC-MS/MS在菌株F2发酵液中检测出七种N-acyl-homoserine lactones(AHLs);同时,研究投加C6-HSL和3-oxo-C8-HSL下对多糖型微生物絮凝剂合成的影响,研究结果表明,投加一定浓度的C6-HSL和3-oxo-C8-HSL时,微生物絮凝剂的糖型微生物絮凝剂效能得到了显著提高,在投加C6-HSL和3-oxo-C8-HSL最优投加量0.4μM和0.2μM下,多糖浓度分别提高1.6倍和1.4倍。此外,对投加C6-HSL和3-oxo-C8-HSL下温度和初始p H值对多糖型微生物絮凝剂合成的影响进行研究,并通过响应面优化得到了C6-HSL和3-oxo-C8HSL投加下微生物絮凝剂发酵最优条件,在此条件下合成的多糖型微生物絮凝剂与未投加相比,多糖浓度分别提高1.75倍和1.55倍,絮凝率分别提高了10%和10.96%。为了进一步探究信号分子AHLs对多糖型微生物絮凝剂合成途径的调控,基于基因转录组测序分析,对C6-HSL投加下菌株F2群体感应系统和多糖型微生物絮凝剂合成基因表达差异进行分析;从基因层面证实了菌株F2基因组中存在与Rhizobium高度相似的traR/tra I群体感应系统,而C6-HSL也可以作为自诱导物与Agau_P200438形成复合物,参与产絮菌F2多糖型微生物絮凝剂合成的调控作用,诱导参与多糖转移和转运基因的表达。综合以上分析,从宏观和微观层面揭示了信号分子AHLs对多糖型微生物絮凝剂合成的群体调控机制,为理解微生物絮凝剂合成的调控机制,为提高调控基因的表达从而实现多糖型微生物絮凝剂定向调控的方法提供理论依据。
[Abstract]:As an emerging green water treatment agent, the research and application of microbial flocculant is becoming more and more important to national scientists, and has been a hot topic in the field of environmental biotechnology for many years. However, the biosynthetic pathway, metabolic pathway and related gene of microbial flocculant are not clear, so that the root cause of the instability and low yield of the wild strain can not be determined, and the problem of the existence of the microbial flocculant can not be fundamentally solved. Based on the analysis of the genome/ transcription group, this paper has carried out the research of the synthetic route of the polysaccharide-type microbial flocculant and its population control mechanism, and provides the theoretical support for solving the problem of the low yield of the microbial flocculant and the instability of the cotton-producing character. Based on the whole genome sequencing data of the Agrobacterium tumefacens F2, the functional genes related to the main metabolic pathway and the regulation mechanism in the strain F2 genome are analyzed by using the bioinformatics and the comparative genomic method, and the main active components of the microbial flocculant produced by the cotton-producing bacteria F2 are polysaccharides, Thus, the polysaccharide-type microbial flocculant synthesis gene is concentrated on the polysaccharide synthesis gene. in that genome of the strain F2, 84 polysaccharide-type microbial flocculant synthesis genes are found, wherein the synthesis of the exo gene involved in the synthesis of the amber-type microbial flocculant is most likely to guide the synthesis of the polysaccharide-type microbial flocculant; in addition, The strain F2 plasmid was found to have a highly similar gene cluster with the tra R/ tra I group induction system in the Rhizobium and the Agrobacterium, and the synthesis gene of the polysaccharide-type microbial flocculant and the analysis of the regulatory gene thereof were completed. and provides a large number of genome information for functional analysis of a polysaccharide-type microbial flocculant synthetic gene and a population induction system regulation gene. in ord to determine that function and the synthetic route of the synthetic gene of the polysaccharide-type microbial flocculant, the difference between the high-throughput gene transcription group and the gene expression in the fermentation process of the cotton-producing strain F2 is analyzed; 65 of the 84 polysaccharide-type microbial flocculant synthetic genes which were analyzed from the genome were identified and 65 genes which had significant difference in the fermentation process were identified, and the polysaccharide-type microbial flocculant exo gene cluster was constructed by the similarity and difference of the monosaccharide components and the synthetic genes of the extracellular polysaccharide synthesized with A. tumefacens C58 and F2. The function and the synthetic route of the synthetic gene of the polysaccharide-type microbial flocculant are determined, the synthetic gene type which causes the difference of the yield and the efficiency of the polysaccharide-type microbial flocculant is disclosed, and a brand-new method and a method are provided for the research of the synthetic route of the polysaccharide-type microbial flocculant, and provides a theoretical basis for realizing the directional synthesis method of the polysaccharide-type microbial flocculant for a key gene of a strong expression and synthesis route. the bacteria are synchronously expressed by the group induction phenomenon to regulate the group behavior of the extracellular substance synthesis, and the expression of the regulation gene can be improved to realize the synthesis of the directional control polysaccharide-type microbial flocculant; In the genome/ transcriptome analysis, a group induction system control gene was found in F2, and seven N-acyl-homoserine lactuones (AHLs) were detected in the strain F2 fermentation broth by UPLC-MS/ MS, and the effects of the addition of C6-HSL and 3-oxo-C8-HSL on the synthesis of polysaccharide-type microbial flocculant were studied. The results showed that When the concentration of C6-HSL and 3-oxo-C8-HSL was added, the efficiency of the microbial flocculant of the microbial flocculant was significantly improved, and the polysaccharide concentration was increased by 1. 6-fold and 1. 4-fold, respectively, with the optimal dosing of C6-HSL and 3-oxo-C8-HSL. In addition, the effects of the temperature and initial p H on the synthesis of the polysaccharide-type microbial flocculant were studied under the conditions of the temperature and the initial p-H value of the addition of C6-HSL and 3-oxo-C8-HSL, and the optimal conditions for the fermentation of the microbial flocculant under the addition of C6-HSL and 3-oxo-C8HSL were obtained through the response surface optimization. The polysaccharide-type microbial flocculant, which was synthesized under this condition, was increased by 1. 75-fold and 1.55-fold, respectively, and the flocculation rate was increased by 10% and 10.96%, respectively, compared with that of the undosed. in ord to further explore that regulation of the synthetic pathway of the polysaccharide-type microbial flocculant by the signal molecule AHLs, the difference of the expression of the synthetic gene of the strain F2 population induction system and the polysaccharide-type microbial flocculant is analyzed based on the gene transcription group sequencing analysis; A TRAR/ tra I population induction system similar to that of Rhizobium in the genome of the strain F2 was confirmed from the genetic level, and the C6-HSL could also be used as the self-inducer and the Adau _ P200438 to form a complex to participate in the regulation and control of the synthesis of the F2 polysaccharide-type microbial flocculant. The expression of the genes involved in the transfer and transport of the polysaccharides was induced. In the light of the above analysis, the group regulation mechanism of the synthesis of the polysaccharide-type microbial flocculant from the signal molecule AHLs is disclosed from the macro and micro level, and the regulation mechanism for the synthesis of the microbial flocculant is provided. and provides a theoretical basis for improving the expression of the regulation gene so as to realize the directional control of the polysaccharide-type microbial flocculant.
【学位授予单位】：哈尔滨工业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：X703.5;TU991.2
[Abstract]:As an emerging green water treatment agent, the research and application of microbial flocculant is becoming more and more important to national scientists, and has been a hot topic in the field of environmental biotechnology for many years. However, the biosynthetic pathway, metabolic pathway and related gene of microbial flocculant are not clear, so that the root cause of the instability and low yield of the wild strain can not be determined, and the problem of the existence of the microbial flocculant can not be fundamentally solved. Based on the analysis of the genome/ transcription group, this paper has carried out the research of the synthetic route of the polysaccharide-type microbial flocculant and its population control mechanism, and provides the theoretical support for solving the problem of the low yield of the microbial flocculant and the instability of the cotton-producing character. Based on the whole genome sequencing data of the Agrobacterium tumefacens F2, the functional genes related to the main metabolic pathway and the regulation mechanism in the strain F2 genome are analyzed by using the bioinformatics and the comparative genomic method, and the main active components of the microbial flocculant produced by the cotton-producing bacteria F2 are polysaccharides, Thus, the polysaccharide-type microbial flocculant synthesis gene is concentrated on the polysaccharide synthesis gene. in that genome of the strain F2, 84 polysaccharide-type microbial flocculant synthesis genes are found, wherein the synthesis of the exo gene involved in the synthesis of the amber-type microbial flocculant is most likely to guide the synthesis of the polysaccharide-type microbial flocculant; in addition, The strain F2 plasmid was found to have a highly similar gene cluster with the tra R/ tra I group induction system in the Rhizobium and the Agrobacterium, and the synthesis gene of the polysaccharide-type microbial flocculant and the analysis of the regulatory gene thereof were completed. and provides a large number of genome information for functional analysis of a polysaccharide-type microbial flocculant synthetic gene and a population induction system regulation gene. in ord to determine that function and the synthetic route of the synthetic gene of the polysaccharide-type microbial flocculant, the difference between the high-throughput gene transcription group and the gene expression in the fermentation process of the cotton-producing strain F2 is analyzed; 65 of the 84 polysaccharide-type microbial flocculant synthetic genes which were analyzed from the genome were identified and 65 genes which had significant difference in the fermentation process were identified, and the polysaccharide-type microbial flocculant exo gene cluster was constructed by the similarity and difference of the monosaccharide components and the synthetic genes of the extracellular polysaccharide synthesized with A. tumefacens C58 and F2. The function and the synthetic route of the synthetic gene of the polysaccharide-type microbial flocculant are determined, the synthetic gene type which causes the difference of the yield and the efficiency of the polysaccharide-type microbial flocculant is disclosed, and a brand-new method and a method are provided for the research of the synthetic route of the polysaccharide-type microbial flocculant, and provides a theoretical basis for realizing the directional synthesis method of the polysaccharide-type microbial flocculant for a key gene of a strong expression and synthesis route. the bacteria are synchronously expressed by the group induction phenomenon to regulate the group behavior of the extracellular substance synthesis, and the expression of the regulation gene can be improved to realize the synthesis of the directional control polysaccharide-type microbial flocculant; In the genome/ transcriptome analysis, a group induction system control gene was found in F2, and seven N-acyl-homoserine lactuones (AHLs) were detected in the strain F2 fermentation broth by UPLC-MS/ MS, and the effects of the addition of C6-HSL and 3-oxo-C8-HSL on the synthesis of polysaccharide-type microbial flocculant were studied. The results showed that When the concentration of C6-HSL and 3-oxo-C8-HSL was added, the efficiency of the microbial flocculant of the microbial flocculant was significantly improved, and the polysaccharide concentration was increased by 1. 6-fold and 1. 4-fold, respectively, with the optimal dosing of C6-HSL and 3-oxo-C8-HSL. In addition, the effects of the temperature and initial p H on the synthesis of the polysaccharide-type microbial flocculant were studied under the conditions of the temperature and the initial p-H value of the addition of C6-HSL and 3-oxo-C8-HSL, and the optimal conditions for the fermentation of the microbial flocculant under the addition of C6-HSL and 3-oxo-C8HSL were obtained through the response surface optimization. The polysaccharide-type microbial flocculant, which was synthesized under this condition, was increased by 1. 75-fold and 1.55-fold, respectively, and the flocculation rate was increased by 10% and 10.96%, respectively, compared with that of the undosed. in ord to further explore that regulation of the synthetic pathway of the polysaccharide-type microbial flocculant by the signal molecule AHLs, the difference of the expression of the synthetic gene of the strain F2 population induction system and the polysaccharide-type microbial flocculant is analyzed based on the gene transcription group sequencing analysis; A TRAR/ tra I population induction system similar to that of Rhizobium in the genome of the strain F2 was confirmed from the genetic level, and the C6-HSL could also be used as the self-inducer and the Adau _ P200438 to form a complex to participate in the regulation and control of the synthesis of the F2 polysaccharide-type microbial flocculant. The expression of the genes involved in the transfer and transport of the polysaccharides was induced. In the light of the above analysis, the group regulation mechanism of the synthesis of the polysaccharide-type microbial flocculant from the signal molecule AHLs is disclosed from the macro and micro level, and the regulation mechanism for the synthesis of the microbial flocculant is provided. and provides a theoretical basis for improving the expression of the regulation gene so as to realize the directional control of the polysaccharide-type microbial flocculant.
【学位授予单位】：哈尔滨工业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：X703.5;TU991.2
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