纤维堆囊菌遗传操作体系建立及其在埃博霉素生物合成改造中的应用

【作者】 夏志洁；

【导师】 李越中；

【作者基本信息】 山东大学 ， 微生物学， 2007， 博士

【摘要】 粘细菌是革兰氏阴性的滑动细菌,它以复杂的多细胞社会学行为及产生多种生物活性物质而著称。至今研究者已经从粘细菌次级代谢物中鉴定了100种以上的基本化学结构和500多种结构衍生物,约占微生物来源活性物质总数的3.5%。在粘细菌的研究中,急需适宜的遗传操作工具。但是到目前为止,转导和电穿孔方法只能在模式菌粘球菌中有效的应用。在其他的粘细菌类群中,甚至是不同的种或菌株,遗传操作都很难获得结果或效率很低。堆囊菌属是粘细菌中唯一能够降解纤维素的生理类群,尤其令人感兴趣的是该属菌株能够产生丰富的次级代谢产物。菌株次级代谢物产生率接近100%,而且在粘细菌共17个属所产生的次级代谢产物种类中,堆囊菌属的次级代谢产物可达到48.4%。菌株So0157-2是本实验室从盐水湖岸边土样品中分离鉴定的一株纤维堆囊菌。实验室前期生物学活性分析结果证实So0157-2可以产生包括埃博霉素（epothilones）在内的多种生物活性物质,是重要的药源菌。从天然药物的结构出发,进行结构修饰、类似物的合成及系统的活性研究,以作为设计新药目标化合物的基础,是国际上研究天然活性成分的主要思路和方法。埃博霉素作为极具潜力的抗癌药物,其分子结构与生物合成特点激发了科研人员和制药公司的极大热情,并已经成为化学界、生物界和临床医学研究的热点。埃博霉素属于聚酮类化合物,由大型多酶复合体催化羧酸单体连续缩合而形成,该酶复合体称为聚酮合酶即PKSs（Polyketide synthases）。模块PKSs是聚酮合酶家族中的独特类群,表现出极其丰富的结构和功能,该类模块PKS的逻辑组织结构形式,为人工创造各种非天然“天然”化合物提供了灵感,是组合生物合成的良好模式材料。作为粘细菌产生的次级代谢产物中最具有应用前景的埃博霉素,其组合生物学改造的重要前提之一是建立良好的遗传操作手段。目前,纤维堆囊菌中主要的遗传操作方法是接合转移,该方法在1992年由Jaoua S等人建立。随着纤维堆囊菌次级代谢研究的展开,研究人员发现由于不同的纤维堆囊菌菌株生理生化性能存在显著差异,已建立的遗传操作系统缺乏通用性,如在纤维堆囊菌So ce26或So ce56菌株中建立的遗传操作系统并不能有效地应用于纤维堆囊菌的其它菌株。而且,遗憾的是文献所报道的接合转移方法同样不适用于本实验室分离的埃博霉素产生菌株So0157-2的遗传操作。所以,纤维堆囊菌遗传操作手段的匮乏极大地阻碍了埃博霉素以及其他次级代谢产物的研究。为了避开纤维堆囊菌难于操作的问题,研究者将目光转到了发酵友好的异源宿主中,然而异源表达面临的难题是:epothilones对于非纤维堆囊菌宿主具有强烈的细胞毒活性,而epothilones的抗性机理尚不明确,使得异源表达产量较低,给后续的产物分析及工业化制备带来很多困难。纤维堆囊菌So0157-2是一株具有优良发酵性状的埃博霉素产生菌,具有非常广阔的应用前景。为了实现埃博霉素在产生菌中的定向结构改造及代谢调控改造,探索和建立其高效的遗传操作方法势在必行。因此本论文从接合转移和转化两个方面对纤维堆囊菌的遗传体系进行了研究。在建立起高效广泛适用于不同纤维堆囊菌的抗生素添加接合转移方法的同时,还发现纤维堆囊菌子实体形成过程中存在自然转化的现象。利用本文所建立的抗生素添加的接合转移方法,在epothilones产生菌So0157-2中进行了epothilone的定向改造及代谢调控改造,并检测到了epothilones结构类似物的合成。论文的相关工作为进行纤维堆囊菌的组合生物合成建立了技术平台,希望产生大量的新化合物,丰富药物筛选原料库。此外,本论文还在纤维堆囊菌中发现了自然转化并对其实现条件进行了初步探索,相关结果为纤维堆囊菌遗传操作提供了新的思路。实验通过在氯霉素抗性基因cat前端加入纤维堆囊菌能够识别的启动子aphⅡ,构建了氯霉素抗性基因盒,然后将其插入诱动载体pCVD442中,解决了纤维堆囊菌So0157-2接合转移中无可用筛选抗生素的问题。这是首次将抗性基因序列短、不易产生耐药性的氯霉素引入到纤维堆囊菌的遗传操作系统中,使氯霉素继腐草霉素与潮霉素后成为第三个纤维堆囊菌遗传操作筛选标记。通过对纤维堆囊菌接合转移条件的摸索,最终确定了低剂量双筛选抗生素添加的接合转移方法。该方法与现有的纤维堆囊菌So ce56遗传操作方法相比,接合转移效率提高了10倍以上,阳性率几乎达到100%。并且对于So0157-2等难于操作的纤维堆囊菌来说,抗生素添加的接合转移方法使其遗传操作的效率提高了100倍以上,为次级代谢改造在纤维堆囊菌中的直接进行铺平了道路。进一步实验证明,该方法不仅在纤维堆囊菌中具有广泛的适用性,并且对大肠杆菌的接合转移同样具有促进作用。通过质粒的连续添加实验,首次实现了纤维堆囊菌So0003-3的自然转化,而且基本确定了自然感受态出现的时间阶段,即子实体形成的早期。实验还确定了CaCl₂对感受态的形成有较好的促进作用,并发现纤维堆囊菌对外界DNA的吸收重组可能具有序列识别能力。通过自然转化的方法将含有埃博酶素epoF基因片段的质粒pCCK700转入纤维堆囊菌So0003-3中,并对转化子进行了验证。此外,通过纤维堆囊菌So0003-3对外源DNA的吸附及降解情况以及子实体形成中释放出大量DNA的实验现象,证明了在自然环境中纤维堆囊菌发生自然转化的可能性。目前,纤维堆囊菌转化的研究还处于起步的阶段,随着研究的深入该方法极有可能成为纤维堆囊菌遗传操作又一有力工具。与此同时,自然转化在粘细菌中的发现还为其如此庞大的基因组可能是通过基因水平转移进化而来提供了实验支持。以本论文所建立的抗生素添加的纤维堆囊菌高效接合转移体系为基础,利用聚酮合酶的模块特性,设计了单一结构域失活以及模块缺失的结构改造路线。实现了基因epoD中模块7的结构域失活,并将epoE中的TE结构域提前到了epoD中,对埃博霉素的合成进行了提前终止,并对所获得的突变株进了产物分析。目前尚未见对埃博霉素合成酶基因簇中这两个模块进行研究的报道。相关实验结果证明,模块7中KR结构域是没有功能的,这与氨基酸序列以及产物结构分析的结果相吻合;模块7中MT结构域的失活使产物epthiloneA1、A2的发酵量有所提高。另外,TE结构域提前使埃博霉素合成提前终止,但其产物的鉴定仍需要更多的结构化学分析。相关的实验内容对埃博霉素定向改造进行了初步探索,相关结果可以为筛选新的埃博霉素类似物提供备选化合物,并为聚酮类化合物的组合生物合成奠定基础和提供理论指导。纤维堆囊菌所产生的次级代谢产物中,聚酮类化合物是主要的产物,其合成酶基因通常表现出独特的序列组成和组织结构的多样性。美国基因组学研究所（TIGR）对粘细菌DK1622的全基因组序列分析发现,PKSs基因约占其基因组的8.6%,因此在堆囊菌中仍可能有大量的次级代谢物有待我们去研究。实验室的前期工作从So0157-2的cosmid粘粒文库中筛选到了一个非埃博霉素聚酮合酶基因,该聚酮合酶与埃博霉素合酶的组织结构有一定的相似性。本论文对该粘粒cosmid6进行了测序,共获得了31777 bp的序列,其中包括一个完整的新的聚酮合酶基因dx1,全长14673 bp,初步判断其含有3个模块。还获得了dx1下游部分dx2基因。对dx1、dx2基因进行了结构域的预测,证实该聚酮合酶为一类结构较为新颖的聚酮合酶基因簇——反式AT型模块PKS。同时确定了该基因簇中没有NRPS模块的存在,对前期的cosmid6所含聚酮合酶的特征预测进行了修正。使用本论文所建立的抗生素添加的接合转移方法对野生型纤维堆囊菌So0157-2的dx1基因进行了插入失活。对突变子的产物分析表明,埃博霉素产量明显提高。相关研究不仅可以帮助我们认识纤维堆囊菌So0157-2中聚酮合酶的多样性和特殊性,认识纤维堆囊菌聚酮类化合物合成酶基因的表达及代谢关联,帮助我们构建高效合成目标化合物的工程菌,并且为发现和利用更多的粘细菌次级代谢产物,发掘粘细菌巨大的次级代谢潜力提供可能。更多还原

【Abstract】 Myxobacteria are Gram-negative gliding bacteria that are noted for the complicated multicellular social behavior and the excellent ability to produce various bioactive compounds.In the past two decades,about 100 basic structures and 500 structural variants have been discovered in myxobacteria and have been fully characterized chemically,which account for about 3.5%of the presently known secondary metabolites of microbial origin.Nearly 100%of the Sorangium strains produce some kinds of compounds with diverse biological activities.The genetic tools are urgently needed in myxobacteria but to date,transduction and electroporation have been well described only for the model species Myxococcus xanthus.In other myxobacterial taxa and even different Myxococcus species or strains,the genetic performance is usually difficult to achieve or suffers from low efficiency.In Sorangium,the only cellulose-degrader among all of the 17 myxobacterial genera but the producer of almost half of the discovered secondary metabolites from myxobacteria,there are many barriers for genetic performances.The sorangial cells grow slowly,possess multiple antibiotics-resistances,produce abundant extracellular polysaccharides,and tend to aggregate.In Sorangium,conjugation,which was first reported by Jaoua et al.in 1992,is the major genetic tool.In 2002,Pradella et al. adapted the conjugation conditions for S.cellulosum So ce56.However,the conjugation protocol developed for S.cellulosum So ce56 or So ce26 is probably not applicable for other Sorangium strains,due to the discrepant physiological characteristics of Sorangium strains.The Sorangium So0157-2 is a facultative alkaliphile strain,which was isolated from a soil sample collected on ChengHai lake bank.Our previous bioactivity screeninganalysis confirmed that So0157-2 is a promising strain for the production of bioactive secondary metabolites.But using the normal conjugation protocol,there was no visible conjugant on the selection plates.At present,structure modification and analogue synthesis of natural products are the main methods for screening new drugs.The polyketide compounds-epothilones are produced by Sorangium strains,and has generated substantial interests over the last few years in the areas of chemistry,biology,and medicine due to their interesting structure and,more importantly,their activity against numerous cancer cell lines. Polyketides are one important large family of complex natural products.They are synthesized from simple carboxylic acids by sequential reactions catalyzed by the large multienzyme protein complexes called polyketide synthases（PKSs）.Modular PKSs constitute a unique class of type I polyketide synthases,exhibiting remarkable diversity in terms of their structures and functions.In resent years,the combinatorial genetic protocols have been applied and confirmed to expand vastly the molecular diversity of these pharmacologically important metabolites,taking advantage of the natural modularity of polyketides.However the most serious problem is that the Sorangium strains have poorly developed the conjugation system and thus lacks flexible genetic tools.The studies of epothilones and other secondary metabolites are lagging.To bypass the serious limitations of Sorangium genetics manipulation, researchers integrated by homologous recombination the entire epothilones gene clusters to heterologous hosts,in which the background genetic methodology is already more highly developed,for the production of value metabolites.But epothilones had great toxicity to heterologous hosts,and the yields were quite low.Sorangium cellulosum So0157-2 is an epothilone producer with good fermentation characteristics.Efficient genetic manipulation methods are needed to establish for epothilone structure modification and regulation controlling in Sorangium cellulosum So0157-2.In this work we discovered two important protocols.One is that we established an efficient conjugation method by the presence of the dual selective antibiotics,which fit for different Sorangium strains even different microorganisms. The other is that we discovered the natural transformation during the development of fruiting bodies.Using the improved conjugation protocol,we achieved in epothilones structure modification in its producing strain.The efficient and flexible conjugation toolbox established in this work meet the major requirements for practical exploitations of combinatorial biosynthesis.The natural transformation was discovered for the first time in myxobacteria,which can afford new ideas and potential means for developing new genetic performance techniques in myxobacteria.Before this work,the selection markers used for Sorangium conjugation protocols was only phlenmycin and hygromycin.However,many Sorangium strains were found to be able to tolerate phleomycin and/or hygromycin,such as So0157-2 and So02007-3.To select a competent marker for Sorangium conjugation,we screened different Sorangium strains on a large scale for their antibiotics-resistances,which revealed that chloramphenicol was one of the few antibiotics to which all of the screened Sorangium strains were sensitive.Thus,a chloramphenicol resistance cassette containing an aphⅡpromoter element and a followed chloramphenicol resistant gene was constructed into the mobilizable plasmid pCVD442.Aider insertion of the fragments of epothilone biosynthetic genes,the resulting plasmids were used for transfer from DH5α（λpir）to S.cellulosum strains with the help of pRK2013.Using the previously described procedures,there was no visible transconjugant from the mating of S.cellulosum So0157-2 cells and the E.coil cells,even adjusting the ratio of the donor and recipient cells,conjugative time,and culture stages. However,when low dosages of the dual selective antibiotics were added into the mating medium,many clones appeared on the selection plates.The mating efficiency for transfer of pCCMT61 containing a 3 kb-insert of epothilone gene fragment was 1-3×10^-6.For So0157-2,the genetic manipulation efficiency was improved 100 times, and the background growth was smoothed away.The improved protocol is able to circumvent the barriers for the Sorangium conjugation and is potentially applicable for different Sorangium strains,either sensitive or less sensitive to living E.coli cells.The effect of the dual selective antibiotics was also confirmed in the mating for transfer of the self-transmissible plasmid pRK2013 from E.coli strain DH5αto HB101.The finding is significant for those less-efficient or unachievable conjugation performances, especially in those slowly growing bacteria.By the addition of plasmid pCCK700 to So0003-3,the natural genetic transformation appeared.Our experimental results indicate that competence for the uptake of exogenous DNA materials is able to be transiently developed during the fruiting body formation.We also found that CaCl₂ was helpful for the forming of competence stage,and sequence-specific DNA uptake in transformation of Sorangium. Developmental lysis of cells occurs during the morphogenesis and provides a DNA pool,which is suggested to be available for the natural transformation if competence is developed.The discovery of competence of cells in fruiting bodies and natural transformation provides not only a reasonable explanation for the expansion means of myxobacterial genomes,but also new ideas and potential means for developing new genetic performance techniques in myxobacteria.Based on the conjugation method with low dosages of dual selecting antibiotics established by this work,module 7 and module 8 of epothilones synthases gene were used for structure modification.β-ketoacyl-ACP reductase（KR）domain and methyltransferase（MT）domain of module 7 were inactivated by the insertion of chloramphenicol resistance cassette.It was turned out that KR domain in module 7 was inactive and MT domain inactivation in module 7 could lead to production increase of epothiloneA1 and epothiloneA2.Meanwhile the thioesterase（TE）domain was repositioned to module 7,which promote the epothilones specific cleavage in forward,but the products need to be confirmed further.The results were helpful to the combinatorial manipulation of the structures of polyketides,and the resulting products can help to enrich the compounds pool for the selection of drugs.Genome sequencing indicated that 5-10%genome was constituted by PKSs,and it was about twice the capacity for producing polyketides of either Streptomyces coelicolor or Streptomyces avermitilis.Our previous construction of So0157-2 genome library got a cosmid 6 which had high organized similar with epothilones synthases genes.We got 31777 bp sequences by sequencing,in which there was a new polyketide synthase gene,named dx1.There were three modules in dx1,but no acyl-transferase（AT）was found.We did not found NRPS module in dx1.So the dx1 gene was trans-AT module PKS.The inactivation of dx1 make the epothilones product of So0157-2 increased apparently.The exploration of regulatory alteration in So0157-2 can help us not only reconstruct efficient industrial strain,but also find and utilize more secondary metabolites.更多还原