【作者】 柯崇榕；

【导师】 黄建忠；

【作者基本信息】 福建师范大学 ， 微生物学， 2016， 博士

【摘要】 吡咯喹啉醌(PQQ)是一种具有氧化还原活性的芳香三环邻位醌,参与脱氢酶的氧化还原过程,协同COQ完成呼吸链的电子传递,是继核黄素和吡啶核苷酸之后发现的第三种氧化还原酶辅因子。PQQ广泛存在于生物体内,具有促进机体生长、防护肝损伤、维护线粒体功能、促进神经生长因子合成、调节机体自由基水平、防止皮肤衰老、防治帕金森病和老年痴呆症等功能,是一种独特的生理活性物质,在医药、食品和化妆品领域具有广阔的应用前景。因此,选育PQQ高产菌株并建立相应的发酵控制及纯化制备工艺具有重要的意义。1、定向驯化选育高产PQQ的甲基营养菌:根据PQQ的吸收光谱特性建立了光谱法结合液相色谱法的PQQ产生菌快速筛选体系,从化工污水中筛选获得蛋白分泌量低而PQQ合成量超过20 mg/L的甲基营养菌FJNU-6,经形态学观察、生理生化测定及系统发生分析命名为脱氮生丝微菌(Hyphomicrobium denitrificans);利用紫外线和亚硝基胍轮流间隔诱变提高突变率,以高浓度甲醇为拮抗因子进行8轮定向驯化,选育获得高产突变株FJNU-R8;突变株FJNU-R8在不同甲醇浓度下pqq和moxF基因簇的表达量较高且差异较小,但生长速度较慢,甲醇消耗速度快,PQQ产量达到 65.73±1.65 mg/L(132 h),比出发株FJNU-6(30.61±3.69 mg/L,160h)提高了 1.17倍。2、建立DO-stat结合pH-stat的甲醇分段控制补料分批发酵工艺:通过单因素试验设计、析因设计及中心组合设计对H.denitrificans FJNU-R8产PQQ的摇瓶培养体系进行优化,PQQ产量提高至135.67±2.34 mg/L;5L发酵罐的分批培养数据发现培养液中较低的溶氧和pH有利于PQQ的合成,动力学模型表明FJNU-R8产PQQ属于生长部分偶联型发酵,甲醇是PQQ合成的限制性底物;建立DO-stat和pH-stat相结合的分段甲醇浓度控制补料分批发酵工艺,5L发酵罐中PQQ产量达到1.18 g/L,生产效率为52.17 mg/g DCW;按照单位体积培养液所消耗的搅拌功率相同为准则进行5L-50L-1T发酵参数的逐级放大,1T发酵罐的PQQ产量高达2.05 g/L,生产效率达到77.82 mg/g DCW,比5L发酵罐提高49.17%。3、建立离心结合膜过滤、层析分离耦合色谱纯化的结晶制备工艺:利用离心和膜过滤技术结合添加15%的硫酸铵完全去除发酵液中的菌体和95%的蛋白多肽,防止发酵液变绿,提高了 PQQ的稳定性;通过静态吸附试验,确定大孔树脂DK-6和缓冲液为PQQ富集纯化的分离介质和解析剂;动力学研究表明,树脂DK-6对PQQ的吸附属于优惠型化学吸附,该吸附过程是一个放热过程,吸附速率由内外扩散共同控制;动态吸附试验确定柱层析条件为:样液浓度1.5 g/L(pH 3.0),以5 BV/h的流速上样6 BV,按体积倍数进行线性放大,PQQ最高可浓缩3.3倍,回收率达到90%;析因设计确定浓度≥3.5 g/L的PQQ母液(pH≤3.0)在4℃条件下结晶5 h,PQQ残留量≤100 mg/L,结晶得率>90%,经过两次结晶,PQQ产品的纯度从90.36%提高至99.32%;根据分离纯化制备参数进行工艺放大,PQQ的最终得率大于70%,纯度高于99%。4、完成H.denitrificans FJNU-R8的基因组测序分析:FJNU-R8的基因组全长3.54M bp,GC含量为60.78%,含有4个CRISPR结构以及1个完整的前噬菌体区域,编码3498个基因,3个rRNA和sRNA以及46个tRNA,与ATCC 51888具有较好的共线性。FJNU-R8基因组只含有I型限制性修饰系统,含有完整的甲醇氧化代谢、硝酸盐还原以及Fe-S簇合成基因,以及丰富的无机盐转运与代谢、信号转导、细胞膜及膜生物合成和转录的相关基因。基因组中还包含由4个独立moxFα基因和1个moxFαβ基因簇组成甲醇脱氢酶体系以及4个独立pqqA基因、1个pqqDE和pqqABCDE基因簇组合的PQQ合成系统。pqq基因簇中各个基因是单独转录的,pqq基因中pqqA3表达量最高,moxF基因中moxFβ表达量最高。5、强化Fe-S的合成和pqqA的表达提高PQQ的合成量:以大肠杆菌为基盘细胞,采用携带阿拉伯糖启动子的表达载体实现pqqABCDE基因簇的异源表达,PQQ产量达到4.09±0.21 mg/L,生产效率为2.16±0.06 mg/g DCW。通过添加30 μM的Fe2+或以IscR缺失菌为宿主,促进铁硫簇的合成,PQQ产量提高到3.13±0.07mg/g DCW;FJNU-R8含有的5个pqqA基因中pqqA3最有利于PQQ的产生,单独表达时PQQ产量达到4.52±0.05 mg/g DCW。当串联表达5个不同pqqA或pqqA3时,PQQ 产量分别为 5.28±0.12 mg/g DCW 和 5.48±0.09 mg/g DCW,而融合 5 个不同的pqqA或pqqA3 时,PQQ 产量仅为 4.89±0.1 mg/g DCW 和 4.78±0.05 mg/g DCW,表明PqqA加工形成PQQ既需要保守结构域还需形成一定的空间结构。综上所述,本论文通过定向驯化选育获得高产PQQ的脱氮生丝微菌,优化了培养体系,建立了分批流加补料生产工艺及色谱分离纯化结晶制备工艺。在此基础上,对高产突变株进行基因组测序分析,以大肠杆菌为基盘细胞分析了铁硫簇的合成和pqqA的表达对PQQ合成量的影响。更多还原

【Abstract】 Pyrroloquinoline quinone(PQQ)is a redox active aromatic,tricyclic o-quinone that invloves in redox reactions by serving as a cofactor for dehydrogenases,and electron transport in respiratory chain by collaborating coenzyme Q.In general,ortho-quinone cofactors is the third family of cofactors following pyridine nucleotide-and flavin-dependent cofactors.PQQ is widely distributed in biological organisms,and it is an unique physiologically active substance with the function of supporting body growth,protection against liver injury,maintenance of mitochondrial function,promotion of nerve growth factor synthesis,regulation of free radicals level,prevention of skin aging,and neuroprotective function.Because of the broad application in cosmetics,food and pharmaceutical industry,it is of great significance to breed high-yield PQQ strain and establish efficient fermentation control and purification process.1、Directional breeding of methylotrophic bacteria with high-yield PQQ production:Rapid screening system for PQQ producing bacteria was established by combining spectroscopy with liquid chromatography according to the absorption spectrum properties of PQQ.The methylotrophic strain Hyphomicrobium denitrificans FJNU-6 with high-yield PQQ and low protein secretion was isolated from chemical sewage,which was identified by morphological,physiological,biochemical and phylogenetic analysis.Ahigh-yield PQQ mutant FJNU-R8 was acquired through eight-times’ directional domestication with a high concentration of methanol as the antagonistic factor by alternate UV and NTG mutagenesis according to the secretion mechanism.PQQ production of mutant FJNU-R8 reached 65.73±1.65 mg/L.Compared with the original strain FJNU-6,PQQ production impoved 1.17-fold with the methanol consumption accelerating albeit at a rate reduced in the growth.2、The establishment of DO-stat and pH-stat fed-batch fermentation process with methanol segmented control:Firstly,optimization of PQQ production from FJNU-R8 in shake flask culture system by single factor experimental design,factorial and central composite design,PQQ production increased to 135.67 mg/L after optimization.Secondly,PQQ production from FJNU-R8 was proved to be growth-partial ly coupled fermentation based on the kinetic model,which implied lower methanol concentration in favor of PQQ generation.Then PQQ production reached 1.18 g/L with 52.17 mg/g DCW efficiency by applying methanol segmented control,DO-stat and pH-stat fed-batch fermentation process in 5 L fermenter batch culture.Furthmore,fermentation scale-up(5L-50L-1T)by applying the criterion "the same power was consumed per liquid volume" was conducted.As a result,2.05 g/L PQQ with 77.82 mg/g DCW efficiency was achieved in 1 T fermenter,which increased 49.17%than in 5 L fermenter.3、The establishment of crystalline preparation process by the combination of centrifugation,membrane filtration with adsorption chromatography coupled chromatographic separation:Firstly,all cells and 95%peptides PQQ fermentation broth was removed from fermentation broth by utilizing 15%ammonium sulfate together with centrifugation and membrane filtration technology,which could prevent the fermentation broth from turning green,and improve the stability of PQQ.Secondly,the macroporous resin DK-6 and appropriate buffers were selected as the separation medium and parsing agent for the purification and enrichment of PQQ by static adsorption experiments.Thirdly,the dynamical study showed that the adsorption using resin DK-6 belonged favorable adsorption,so the process was an exothermic process,and the adsorption rate is controlled by internal/external diffusion.Furthermore,the dynamic adsorption experiments determined the conditions of the chromatography:sample concentration of 1.5 g/L(pH 3.0),6 BV sample with the flow rate of 5 BV/h,multiple linear amplification by volume.As a result,PQQ prodution was concentrated up to 3.3 times with the recovery rate above 90%.After the factorial design,PQQ crystallization yield within 3h was more than 90%when the PQQ initial concentration was more than or equal to 3.5 g/L(pH<3.0)at 4℃,meanwhile,PQQ residues was no more than 100 mg/L.After the second crystallization,the purity of PQQ crystal increased from 90.36 to 99.32%.Finally,more than 70%PQQ produciton yield with 99%purity was achieved according to the parameters of the separation and purification process4、The analysis of genome sequencing data from the high-yield PQQ mutant FJNU-R8:The genome of Hyphomicrobium denitrificans FJNU-R8 consists of a single circular chromosome of 3.54 M bp with a G+C content of 60.78%.There are 3498 putative open reading frames by Glimmer,and 46 tRNA-encoding genes and 3 rRNA-encoding operons were identified.The genes related to the methanol oxidation,serine pathway,tricarboxylic acid pathway,glyoxylate regeneration pathway and nitrate reduction are invovled in the genome.In addition,the genome contains six methanol dehydrogenase encoding genes and 11 genes associated with PQQ synthesis.Each gene involved in PQQ biosynthetic gene cluster was transcribed separately,and the expression of pqqA genes was significant difference with the highest expression level from pqqA3 gene.5、Improvement of PQQ synthesis by enhanced PqqA and Fe-S cluster expression in Escherichia coli:The E.coli BW25113 harboring pqqABCDE gene cluster under arabinose-inducible araBAD promoter was constructed for PQQ production.Therefore,4.09 ± 0.21 mg/L PQQ with 2.16±0.06 mg/g DCW efficiency was produced by the recombinant E.coli.When IscR(iron-sulfur cluster regulator)knockout strain was used as a host,3.13±0.07 mg/g DCW productivity was acquired.The pqqA3 gene was the most efficient for PQQ production among the five pqqA genes.The over-expression of pqqA3 alone resulted in 4.52±0.05 mg/g DCW efficiency in the recombinant △IscR strain.When five concatenate pqqA3 genes was expressed,PQQ productivity reached 5.48 ±0.09 mg/g DCW,on the contrary,only 4.78±0.05 mg/g DCW efficiency was achieved by fusion expression of five pqqA3 gene.The results implied that the spatial structure was as important as the conserved domain for transfomation of PqqA to PQQ.In summary,a high-yield PQQ production strain Hyphomicrobium denitrificans FJNU-R8 was acquired by directional breeding.After optimization of the culture system,DO-stat and pH-stat fed-batch fermentation process with methanol segmented control as well as crystalline preparation process by chromatographic purification were established to achieve PQQ with high purity.Furthermore,genome sequencing of FJNU-R8 and profound analysis of metabolic pathway,especially for PQQ synthetic pathway,were conducted.Finally,PQQ productivity was improved by over-expression of a variety of pqqA genes in recombinant E.coli.更多还原