食甲基杆菌J1-1吡咯喹啉醌生物合成突变株的筛选和鉴定

目的:通过Tn5转座诱变筛选食甲基杆菌J1-1吡咯喹啉醌(PQQ)生物合成相关基因。方法:构建食甲基杆菌J1-1 Tn5转座突变体库,筛选PQQ合成水平差异明显的突变株,利用质粒拯救法鉴定突变基因,通过基因敲除、回补及过表达进一步研究该基因与PQQ合成的关系。结果:构建了J1-1的Tn5转座突变体库,筛选得到一株PQQ合成水平显著下降的突变株,经鉴定Tn5插入位点为mpq0056基因,该突变株在以甲醇为惟一碳源的培养基中生长速度略慢;敲除J1-1中mpq0056基因后,PQQ的合成水平下降,与Tn5诱变结果一致;回补该基因后,PQQ产量恢复到野生菌水平。结论:mpq0056基因参与了PQQ的生物合成,该基因可能编码分支酸盐裂合酶,并在PQQ生物合成中起重要作用。     

基于重组氧化葡萄糖酸杆菌生物合成吡咯喹啉醌

吡咯喹啉醌(Pyrroloquinoline quinone,PQQ)是一种重要的氧化还原酶辅基,具有多种生理生化功能,在食品、医药卫生及农业等领域具有广泛的应用。文中采用重组氧化葡萄糖酸杆菌生物合成吡咯喹啉醌。首先构建丙酮酸脱羧酶基因GOX1081敲除的重组菌G. oxydans T1,减少副产物乙酸的形成。然后利用筛选的内源性组成型启动子P0169融合表达pqqABCDE基因簇及tldD基因,构建重组菌G. oxydans T2。最后对发酵培养基添加物和发酵条件进行优化。结果显示重组菌G. oxydans T1、G. oxydans T2生物量较野生菌分别提高43.02%和38.76%,而PQQ的产量分别是野生菌的4.82倍和20.5倍。进一步优化G. oxydans T2碳源及培养条件,最终PQQ产量达(51.3241±0.8997)mg/L,是野生菌的345.62倍。通过基因工程手段,可以有效提高氧化葡萄糖酸杆菌的生物量和合成PQQ的产量,为改善PQQ生物合成效率奠定基础。     

吡咯喹啉醌生物合成研究进展

吡咯喹啉醌(pyrroloquinoline quinone,PQQ)是一种多肽修饰类天然产物,是继烟酰胺和核黄素之后第三类辅酶,具有抗氧化、抗衰老、提高免疫力等重要生理功能,在医药、保健等领域具有重要价值。目前,PQQ的大规模制备仍然存在诸多问题,限制了PQQ的广泛应用。当前迫切需求低成本的合成方式,以充分实现其广阔的应用潜力。综述了近年来PQQ生物合成途径的解析、关键酶的催化反应机理以及高产菌株选育等方面的研究动态及发展趋势,并针对PQQ生物合成微生物细胞工厂构建研究策略提出了建议及展望。     

利用山梨糖脱氢酶活性筛选氧化葡糖杆菌PQQ合成基因簇

【目的】利用山梨糖脱氢酶醌酶活性从氧化葡糖杆菌H24中分离PQQ生物合成基因簇。【方法】利用ptsG位点整合sdh基因的大肠杆菌JM109作为宿主菌构建了氧化葡糖杆菌H24的基因组DNA文库。通过山梨糖脱氢酶活性检测,从文库中筛选具有PQQ合成能力的单菌落并进行亚克隆。【结果】从氧化葡糖杆菌H24的基因组文库中筛选得到一株具有山梨糖脱氢酶活性的单菌落,亚克隆后序列分析显示插入片段全长5400bp,对应5个编码框(pqqABCDE),与其他细菌PQQ生物合成基因簇有很高的序列同源性。【结论】利用山梨糖脱氢酶醌酶活性成功从氧化葡糖杆菌H24中分离克隆得到了PQQ生物合成基因簇pqqABCDE。     

吡咯喹啉醌合成及生产工艺研究进展

吡咯喹啉醌(pyrroloquinoline quinone, PQQ)是继烟酰胺和核黄素之后发现的第三类氧化还原酶辅因子,普遍存在于生物体中参与呼吸链电子传递,具有促进线粒体产生、清除自由基、增强细胞代谢和预防心肌损伤等生理功能,在医药、食品和农业领域具有广泛的应用前景。微生物发酵法是PQQ生产的主要方式,解析PQQ生物合成途径及其调控机制,通过代谢工程选育短周期、高产量的生产菌是PQQ工业化的研究方向之一。本文综述了PQQ的合成途径、高产菌株选育以及微生物发酵生产与分离纯化的研发工作,为深入阐释PQQ的生物合成机制和工业化生产菌株的选育提供参考。     

吡咯喹啉醌研究进展

吡咯喹啉醌（PQQ）是继烟酰胺和黄素核苷酸之后发现的氧化还原酶的第3种辅酶,具有多种生理功能,在食品、医药及农业等行业有广泛的应用前景。我们简要综述了PQQ参与醌酶电子传递、增强微生物对极端环境的适应能力、促进植物生长、刺激神经生长因子生成等生物学功能及相关作用机制,介绍了PQQ生产菌、PQQ合成基因及PQQ生物合成的调控等方面的研究进展。     

甲基营养菌MP688甲醇脱氢酶基因mpq1818的敲除及功能研究

目的:研究甲醇脱氢酶基因mpq1818在甲基营养菌MP688生长代谢中的作用。方法:利用同源重组原理构建中间为庆大霉素抗性基因Gmr、两侧mpq1818基因上下游序列同源的敲除载体pAK0-up-Gmr-down,接合转移导入MP688,通过庆大霉素抗性和组合PCR方法筛选基因敲除菌,并检测其生长、甲醇脱氢酶活性、甲醇利用及吡咯喹啉醌(PQQ)生物合成能力等方面的差异。结果:抗性和PCR验证显示mpq1818缺失株构建成功;与野生菌相比,缺失株的甲醇脱氢酶活力及利用甲醇的能力降低,而且菌株的生长和PQQ产量也有显著下降。结论:基因mpq1818的缺失影响菌株前期生长与PQQ合成。     

高产吡咯喹啉醌扭脱甲基杆菌的高通量选育

吡咯喹啉醌(PQQ)是一种细菌脱氢酶的辅酶,具有促进机体生长、调节机体自由基水平等功能,应用于食品、医药等领域。由于化学合成法成本较高,微生物发酵法生产PQQ受到关注。目前,发酵法生产PQQ产量较低,限制了其工业应用。然而,由于对PQQ菌株的合成与调控机制尚缺乏深入理解,以及对野生型菌株缺乏必要的基因工程改造手段,目前采用代谢工程强化PQQ合成菌株还缺乏相关基础。因此,本研究以扭脱甲基杆菌Methylobacterium extorquens I-F2为研究对象,整合常压室温等离子体诱变、流式细胞术分选和高通量筛选策略,对样品制备和流式分选过程进行优化,最终筛选出一株PQQ高产突变菌株1-C6,PQQ产量比出发菌株I-F2提高98.02%。本文所述的流式细胞术结合高通量筛选方法能简单、快速地获得高产突变菌株,相比于基因工程改造和传统筛选方法,具有提升效果明显且易于实施等优势。     

甲基营养菌甲醇脱氢酶基因的克隆及表达

目的:从甲基营养菌MP681中扩增甲醇脱氢酶(MDH)基因,在大肠杆菌中表达并检测其活性,同时在MP681中考察该基因对吡咯喹啉醌(PQQ)产生的影响。方法:根据MP681基因组序列设计引物,PCR扩增靶基因mdh,构建表达载体,考察活性,利用接合转移转化至MP681,考察PQQ的合成。结果:扩增得到甲基营养菌MP681甲醇脱氢酶基因,在大肠杆菌中的表达产物能够催化甲醇脱氢;将携带mdh基因的质粒转入MP681后,PQQ产量略有提高。结论:获得编码MDH的基因,该基因能够在大肠杆菌中表达,且表达产物具有生物活性;甲醇脱氢酶基因表达对宿主菌的PQQ合成可能有一定影响。     

紫杉醇生物合成途径中相关酶的研究进展

抗癌新药紫杉醇是具有萜类环状结构的一种重要次生代谢产物 ,研究紫杉醇的生物合成对于通过基因工程手段提高紫杉醇的产量 ,解决目前资源紧缺造成的巨大供求矛盾具有重要意义 ,这就需要对紫杉醇生物合成途径中催化各步反应 (尤其是关键步骤 )的酶以及编码这些酶的基因有个全面的了解。对近年来紫杉醇生物合成途径中相关酶的研究进行了综述 ,大部分酶及相关基因已被分离、克隆 ,但还有一些酶及相关基因没有发现 ,有待继续深入研究。     

新辅基吡咯喹啉醌(PQQ)生物合成基因研究进展

吡咯喹啉醌（Ｐｙｒｏｌｏｑｕｉｎｏｌｉｎｅ－Ｑｕｉｎｏｎｅ,ＰＱＱ）是氧化还原酶的新辅基。它在细菌体内是由一组排列成簇的相关基因即ｐｑｑ基因控制合成的。根据不同细菌来源ｐｑｑ基因的同源性和对应关系,可将ｐｑｑ基因归为７类：簇基因１～７。在Ａｃｉｎｅｔｏｂａｃｔｅｒｃａｌｃｏａｃｅｔｉｃｕｓ中存在其中四个,ＫｌｅｂｓｉｅｌａＰｎｅｕｍｏｎｉａｅ和ＭｅｔｈｙｌｏｂａｃｔｅｒｉｕｍＯｒｇａｎｏｐｈｉｌｕｍＤＳＭ７６０中６个,而Ｍｅｔｈｙｌｏｂａｃｔｅｒｉｕｍｅｘ－ｔｏｒｑｕｅｎｓＡＭ１中存在全部７个簇基因。簇基因１编码一个由２２～２９年氨基酸组成的小肽,此小肽可能是ＰＱＱ的前体,簇基因２可能涉及ＰＱＱ跨膜转运,簇基因３可能负责ＰＱＱ合成的最后一步酶催化,簇基因５可能涉及ＰＱＱ合成中某种酶的辅因子合成,簇基因６和７可能负责小肽的加工。簇基因４功能还不清楚,但在Ｍ．ｅｘｔｏｒｑｕｅｎｓＡＭ１中簇基因３和４是以融合基因存在的。     

Research on the metabolic engineering of the direct oxidation pathway for extraction of phosphate from ore has generated preliminary evidence for PQQ biosynthesis in Escherichia coli as well as a possible role for the highly conserved region of quinoprotein dehydrogenases

The ability of some bacteria to dissolve poorly soluble calcium phosphates (CaPs) has been termed 'mineral phosphate solubilizing' (MPS). Since most microorganisms and plants must assimilate P via membrane transport, biotransformation of CaP into soluble phosphate is considered an essential component of the global P cycle. In many Gram-negative bacteria, strong organic acids produced in the periplasm via the direct oxidation pathway have been shown to dissolve CaP in the adjacent environment. Therefore, the quinoprotein glucose dehydrogenase (PQQGDH) may function in the ecophysiology of many soil bacteria. There is interest in using MPS bacteria for industrial bioprocessing of rock phosphate ore (a substituted fluroapatite) or even for direct inoculation of soils as a 'biofertilizer' analogous to nitrogen fixation. Our laboratory has spent 20 years studying superior MPS bacteria. Screening genomic libraries in the appropriate E. coli genetic background can 'trap' PQQ or GDH genes from these bacteria via functional complementation. In setting the 'trap' for PQQ genes, we have identified DNA fragments that apparently induce PQQGDH activity in E. coli with no sequence homology to known PQQ genes. These data suggest that E. coli may have an alternative, inducible PQQ biosynthesis pathway. Finally, a novel protein engineering strategy to increase the catalytic rate of PQQGDH has emerged and will be discussed.     

PQQ and quinoprotein research--the first decade

On the occasion of the first international symposium on pyrroloquinoline quinone (PQQ) and quinoproteins (Delft, September 1988), a review of this novel field in enzymology is presented. Quinoproteins (PQQ-containing enzymes) are widespread, from bacteria to mammalian organisms (including man), and occur in several classes of enzymes. Indications already exist that PQQ is a versatile cofactor, involved not only in oxidation but also in hydroxylation, transamination, decarboxylation and hydration reactions. The current list of quinoproteins shows that it was overlooked in several well-studied enzymes where the presence of a common cofactor had already been established. Up until now, all eukaryotic quinoproteins have covalently bound PQQ (or perhaps pro-PQQ), while free PQQ occurs exclusively in a number of (bacterial) dehydrogenases and in the culture fluid of certain Gram-negative bacteria. Biosynthesis of free PQQ in methylotrophic bacteria starts with tyrosine and glutamic acid as precursors while intermediates in the route have not been detected and the presence of free PQQ is not required for synthesis of the covalently bound form of the cofactor in glutamic acid decarboxylase from Escherichia coli. Therefore, the assembly of covalently bound cofactor might occur in situ, i.e. in the quinoproteins themselves. If the latter also applies to mammalian quinoproteins, this implies that PQQ is not a vitamin. On the other hand, positive effects have been reported upon administration of PQQ to test animals. Methods suited to detach and to detect PQQ with a derivatized o-quinone moiety may answer questions on the uptake and processing of the compound.     

一株新的产吡咯喹啉醌甲基营养菌全基因组测序和比较基因组学分析

【背景】由于甲基营养菌被发现的时间较短,而且可以生产吡咯喹啉醌(pyrroloquinoline quinone,PQQ)的甲基杆菌属细菌只有少数菌株的全基因组序列被公布,增加了该类细菌基因组学和生物代谢途径研究的难度。【目的】将本实验室筛选的PQQ生产菌经多种诱变方式处理,用于提高PQQ的发酵产量。对高产突变菌株进行全基因组解析,以探究甲基杆菌PQQ合成的分子机制,为后续分子育种提供序列背景信息。【方法】将野生型PQQ生产菌株进行紫外诱变、亚硝基胍诱变、甲基磺酸乙酯诱变、硫酸二乙酯诱变和紫外-氯化锂复合诱变。将突变菌株利用PromethION三代测序平台和MGISEQ-2000二代测序平台测序,然后进行组装和功能注释。组装得到的全基因组序列与模式菌株扭脱甲基杆菌AM1 (Methylobacterium extorquens AM1)进行比较基因组学分析。【结果】经11轮诱变获得一株突变菌株NI91,其PQQ产量为19.49 mg/L,相较原始菌株提高44.91%。突变菌株NI91的基因组由一个5 409 262 bp的染色体组成,共编码4 957个蛋白,与模式菌株M. extorquens AM1比较发现其PQQ合成过程中剪切加工相关的基因pqqF和pqqG缺失,但首次在甲基营养菌中发现与基因pqqF具有相似功能的基因pqqL,且基因pqqC/D的序列存在较大差异。【结论】为甲基营养类细菌甲基杆菌的功能基因组学研究及PQQ合成机理研究提供了基础数据支持,NI91与模式菌株M. extorquens AM1的比较基因组学分析为揭示PQQ合成的不同机理提供了分子基础。     

Synthesis of pyrroloquinoline quinone in vivo and in vitro and detection of an intermediate in the biosynthetic pathway.

In Klebsiella pneumoniae, six genes, constituting the pqqABCDEF operon, which are required for the synthesis of the cofactor pyrroloquinoline quinone (PQQ) have been identified. The role of each of these K. pneumoniae Pqq proteins was examined by expression of the cloned pqq genes in Escherichia coli, which cannot synthesize PQQ. All six pqq genes were required for PQQ biosynthesis and excretion into the medium in sufficient amounts to allow growth of E. coli on glucose via the PQQ-dependent glucose dehydrogenase. Mutants lacking the PqqB or PqqF protein synthesized small amounts of PQQ, however. PQQ synthesis was also studied in cell extracts. Extracts made from cells containing all Pqq proteins contained PQQ. Lack of each of the Pqq proteins except PqqB resulted in the absence of PQQ. Extracts lacking PqqB synthesized PQQ slowly. Complementation studies with extracts containing different Pqq proteins showed that an extract lacking PqqC synthesized an intermediate which was also detected in the culture medium of pqqC mutants. It is proposed that PqqC catalyzes the last step in PQQ biosynthesis. Studies with cells lacking PqqB suggest that the same intermediate might be accumulated in these mutants. By using pqq-lacZ protein fusions, it was shown that the expression of the putative precursor of PQQ, the small PqqA polypeptide, was much higher than that of the other Pqq proteins. Synthesis of PQQ most likely requires molecular oxygen, since PQQ was not synthesized under anaerobic conditions, although the pqq genes were expressed.     

A search for intermediates in the bacterial biosynthesis of PQQ

Studies on the biosynthesis of pyrroloquinoline quinone (PQQ) were performed with Acinetobacter calcoaceticus PQQ- -mutants belonging to genetically different complementation groups. All mutants were unable to grow on L-arabinose, the conversion of this substrate by the organism only occurring via membrane-bound quinoprotein (PQQ-containing) glucose dehydrogenase. In general, the same observation and conclusion applied to shikimate and quinate, requiring active quinoprotein quinate dehydrogenase (EC 1.1.99.--), although some mutants appeared to be leaky with respect to PQQ biosynthesis under this condition. A number of mutants were unable to grow on anthranilate and accumulated this compound when the growth medium was supplemented with L-kynurenine. Combined with other observations, it strongly suggests that these are deletion mutants, missing a gene for synthesis of anthranilate hydroxylase (EC 1.14.12.1) as well as nearby located genes for the biosynthesis of PQQ. Supplementation of the growth media with amino acids did not result in stimulation of PQQ biosynthesis. Also cross-feeding experiments, using normal and permeabilized cells with extensive variation in combination and conditions, resulted in neither stimulation nor reconstitution of PQQ synthesis. Under conditions optimal for PQQ production in the wild-type strain, as well as under stress conditions using a limiting amount of added cofactor, excretion of intermediates by PQQ- -mutants could not be detected. Similar results were obtained with PQQ- -mutants from Methylobacterium organophilum and Pseudomonas aureofaciens. A tentative explanation, accounting for the absence of detectable intermediates in the biosynthetic route, is given.     

高浓度2-KLG对氧化葡萄糖酸杆菌WSH-003中2-KLG合成途径关键基因表达的影响

【目的】研究高浓度的2-KLG对其生产菌株氧化葡萄糖酸杆菌生产过程中关键的脱氢酶合成基因、辅因子合成基因及其转运蛋白编码基因的影响。【方法】测定高浓度梯度2-KLG下氧化葡萄糖酸杆菌的生长情况,确定合适的添加浓度对氧化葡萄糖酸杆菌进行胁迫。使用实时定量PCR技术检测2-KLG合成中关键山梨醇脱氢酶基因sld AB、关键辅因子PQQ合成基因pqq ABCDE及5个潜在转运蛋白合成基因的变化。【结果】根据氧化葡萄糖酸杆菌在2-KLG高浓度梯度下生长测定实验结果,选定40、80和120 g/L 2-KLG作为添加浓度。实时定量PCR结果显示,在高浓度的2-KLG压力下,PQQ合成基因pqq ABCDE未受到显著影响,山梨醇脱氢酶基因sld AB以及部分PQQ潜在转运蛋白编码基因的表达均显著下调。【结论】高浓度2-KLG会抑制氧化葡萄糖酸杆菌中山梨醇脱氢酶基因的表达,有可能会影响辅酶PQQ的转运,但不会显著影响辅酶PQQ的合成。     

Pyrroloquinoline quinone biosynthesis in Escherichia coli through expression of the Gluconobacter oxydans pqqABCDE gene cluster

We have expressed the pqqABCDE gene cluster from Gluconobacter oxydans, which is involved in pyrroloquinoline quinone (PQQ) biosynthesis, in Escherichia coli, resulting in PQQ accumulation in the medium. Since the gene cluster does not include the tldD gene needed for PQQ production, this result suggests that the E. coli tldD gene, which shows high homology to the G. oxydans tldD gene, carries out that function. The synthesis of PQQ activated d-glucose dehydrogenase in E. coli and the growth of the recombinant was improved. In an attempt to increase the production of PQQ, which acts as a vitamin or growth factor, we transformed E. coli with various recombinant plasmids, resulting in the overproduction of the PQQ synthesis enzymes and, consequently, PQQ accumulation—up to 6 mM—in the medium. This yield is 21.5-fold higher than that obtained in previous studies.