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。     

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。     

氧化葡糖杆菌sndh-sdh基因簇的克隆表达

目的:从氧化葡糖杆菌H763中克隆sndh-sdh基因簇,在大肠杆菌和氧化葡糖杆菌621H中分别表达山梨酮脱氢酶-山梨糖脱氢酶(SNDH-SDH),并检测其活性。方法与结果:以氧化葡糖杆菌H763基因组DNA为模板,PCR扩增包括启动子、结构基因及终止序列在内的sndh-sdh基因簇,回收3533 bp的扩增产物,连入pMD18T载体,转化至大肠杆菌DH5α中表达;以山梨糖或木糖为底物,DCIP法检测菌体裂解液,DCIP检测液颜色由蓝绿色变为黄色,表明大肠杆菌表达产物具有脱氢酶活性。构建pBBR1MCS2-sndh-sdh载体,通过接合转移导入氧化葡糖杆菌621H,重组葡糖杆菌在以山梨醇或山梨糖为底物的培养基中培养,采用薄层层析检测法检测其培养上清中的代谢产物,层析板上显示了2-酮基-L-古龙酸斑点。结论:重组大肠杆菌DH5α和氧化葡糖杆菌621H中均表达了有脱氢酶活性的SNDH-SDH。     

Glucose oxidation and PQQ-dependent dehydrogenases in Gluconobacter oxydans

Gluconobacter oxydans is famous for its rapid and incomplete oxidation of a wide range of sugars and sugar alcohols. The organism is known for its efficient oxidation of D-glucose to D-gluconate, which can be further oxidized to two different keto-D-gluconates, 2-keto-D-gluconate and 5-keto-D-gluconate, as well as 2,5-di-keto-D-gluconate. For this oxidation chain and for further oxidation reactions, G. oxydans possesses a high number of membrane-bound dehydrogenases. In this review, we focus on the dehydrogenases involved in D-glucose oxidation and the products formed during this process. As some of the involved dehydrogenases contain pyrroloquinoline quinone (PQQ) as a cofactor, also PQQ synthesis is reviewed. Finally, we will give an overview of further PQQ-dependent dehydrogenases and discuss their functions in G. oxydans ATCC 621H (DSM 2343).     

Inactivation of pqq genes of Enterobacter intermedium 60-2G reduces antifungal activity and induction of systemic resistance

Enterobacter intermedium 60-2G, a phosphate solubilizing bacterium, has the ability to induce systemic resistance in plants against soft rot pathogen Erwinia carotovora. Glucose dehydrogenase, an enzyme that utilizes pyrroloquinoline quinone (PQQ) as a cofactor, is required for the synthesis of gluconic acid by E. intermedium 60-2G. Here, we report that the pqqA and pqqB genes are required for phosphate solubilization and induced systemic resistance against a soft rot pathogen in tobacco. Mutations in either the pqqA or pqqB gene abolished the production of 2-ketogluconic acid and eliminated the ability of E. intermedium to solubilize hydroxyapatite. Addition of gluconic acid to the growth media restored the ability of the pqqA mutant to produce 2-ketogluconic acid. Interestingly, both pqqA and pqqB mutants of E. intermedium lost their ability to inhibit the growth of the rice pathogen Magnaporthe grisea KI-409. Additionally, induced systemic resistance against the soft rot pathogen was attenuated in the pqq mutants. These functions were restored by complementation with the wild-type pqq gene cluster. Our findings suggest that PQQ plays an important function in beneficial traits including phosphate solubilization, antifungal activity, and induced systemic resistance of E. intermedium, possibly by acting as a cofactor for several enzymes including glucose dehydrogenase.     

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。     

Molecular properties of membrane-bound FAD-containing D-sorbitol dehydrogenase from thermotolerant Gluconobacter frateurii isolated from Thailand

There are two types of membrane-bound D-sorbitol dehydrogenase (SLDH) reported: PQQ-SLDH, having pyrroloquinoline quinone (PQQ), and FAD-SLDH, containing FAD and heme c as the prosthetic groups. FAD-SLDH was purified and characterized from the PQQ-SLDH mutant strain of a thermotolerant Gluconobacter frateurii, having molecular mass of 61.5 kDa, 52 kDa, and 22 kDa. The enzyme properties were quite similar to those of the enzyme from mesophilic G. oxydans IFO 3254. This enzyme was shown to be inducible by D-sorbitol, but not PQQ-SLDH. The oxidation product of FAD-SLDH from D-sorbitol was identified as L-sorbose. The cloned gene of FAD-SLDH had three open reading frames (sldSLC) corresponding to the small, the large, and cytochrome c subunits of FAD-SLDH respectively. The deduced amino acid sequences showed high identity to those from G. oxydans IFO 3254: SldL showed to other FAD-enzymes, and SldC having three heme c binding motives to cytochrome c subunits of other membrane-bound dehydrogenases.     

Construction of expression vectors for protein production in Gluconobacter oxydans

The characteristic ability of Gluconobacter oxydans to incompletely oxidize numerous sugars, sugar acids, polyols, and alcohols has been exploited in several biotechnological processes, for example vitamin C production. The genome sequence of G. oxydans 621H is known but molecular tools are needed for the characterization of putative proteins and for the improvement of industrial strains by heterologous and homologous gene expression. To this end, promoter regions for the genes encoding G. oxydans ribosomal proteins L35 and L13 were introduced into the broad-host-range plasmid pBBR1MCS-2 to construct two new expression vectors for gene expression in Gluconobacter spp. These vectors were named pBBR1p264 and pBBR1p452, respectively, and have many advantages over current vectors for Gluconobacter spp. The uidA gene encoding β-D-glucuronidase was inserted downstream of the promoter regions and these promoter-reporter fusions were used to assess relative promoter strength. The constructs displayed distinct promoter strengths and strong (pBBR1p264), moderate (pBBR1p452) and weak (pBBR1MCS-2 carrying the intrinsic lac promoter) promoters were identified.     

Complete genome sequence of the acetic acid bacterium Gluconobacter oxydans

Gluconobacter oxydans is unsurpassed by other organisms in its ability to incompletely oxidize a great variety of carbohydrates, alcohols and related compounds. Furthermore, the organism is used for several biotechnological processes, such as vitamin C production. To further our understanding of its overall metabolism, we sequenced the complete genome of G. oxydans 621H. The chromosome consists of 2,702,173 base pairs and contains 2,432 open reading frames. In addition, five plasmids were identified that comprised 232 open reading frames. The sequence data can be used for metabolic reconstruction of the pathways leading to industrially important products derived from sugars and alcohols. Although the respiratory chain of G. oxydans was found to be rather simple, the organism contains many membrane-bound dehydrogenases that are critical for the incomplete oxidation of biotechnologically important substrates. Moreover, the genome project revealed the unique biochemistry of G. oxydans with respect to the process of incomplete oxidation.     

Localization of a pyrroloquinoline quinone biosynthesis gene near the methanol dehydrogenase structural gene in Methylobacterium organophilum DSM 760

A partial Sau3AI genomic bank of Methylobacterium organophilum DSM 760 was constructed in the cosmid pSUP106 and moxF, the structural gene for methanol dehydrogenase, was isolated. In M. organophilum, pSUP106 behaves as a suicide plasmid. This property was used to insert Tn5 into the bacterial chromosome, in the vicinity of moxF, by marker exchange. Mobilization of the Tn5-labelled chromosomal region by a broad-host-range plasmid, pJB3J1 (an R68-45 derivative), allowed the selection of several large R' hybrid plasmids in Escherichia coli HB101. Most of them were able to complement both mutants of the moxF region and mutant MTM1, the first mutant of the pyrroloquinoline quinone (PQQ) biosynthesis pathway in M. organophilum. The gene involved, pqqA, was subcloned and localized.     

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.     

Physiological properties of a pyrroloquinoline quinone mutant of Methylobacterium organophilum

Abstract The grwoth of MTMl, a mutant of methylobacterium organophilum) blocked in the use of methanol as a carbon and energy source, was restored by addition of pyrroloquinoline quinone (PQQ) in the culture medium. No PQQ could be detected in crude medium. No PQQ could be of MTMl. Therefore, MTMl can be regarded as a mutant blocked in the biosynthesis of PQQ. Under the conditions of growth employed, growth rates of MTMl on methanol, comparable to those of the wild type, occured at a PQQ concentration of 1 μM. Since lower amounts of methanol dehydrogenase (MDH) wer found in cell-free extracts of PQQ-supplemented MTMl, the wild type strain synthesizes a surplus of MDH under these conditions. Growth of M. organophilum on ethanol proceeds via MDH as a catalyst for the first step, since (NAD(P) -dependent etanol. dehydrogenase was absent in cell-free extracts and growth of MTMl on ethanol only took place in the presence of PQQ. On the hand, growth of MTMl on mthylamine was unimpaired. This is in accordance with the fact that methylamine dehydrogenase was absent and N -methylamine mate dehydrogenase was present in cell-free extracts     

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.     

Sequence analysis of the Gluconobacter oxydans RecA protein and construction of a recA-deficient mutant.

The deduced amino acid sequence of Gluconobacter oxydans RecA protein shows 75.2, 69.4, and 66.2% homology with those from Aquaspirillum magnetotacticum, Escherichia coli, and Pseudomonas aeruginosa, respectively. The amino acid residues essential for function of the recombinase, protease, and ATPase in E. coli recA protein are conserved in G. oxydans. Of 24 amino acid residues believed to be the ATP binding domain of E. coli RecA, 17 are found to be identical in G. oxydans RecA. Interestingly, nucleotide sequence alignment between the SOS box of G. orphans recA gene and those from different microorganisms revealed that all the DNA sequences examined have dyad symmetry that can form a stem-loop structure. A G. oxydans recA-deficient mutant (LCC96) was created by allelic exchange using the cloned recA gene that had been insertionally inactivated by a kanamycin-resistance cassette. Such replacement of the wild-type recA with a kanamycin resistance gene in the chromosome was further verified by Southern hybridization. Phenotypically, the recA-deficient mutant is significantly more sensitive to UV irradiation than the wild-type strain, suggesting that the recA gene of G. oxydans ATCC9324 plays a role in repairing DNA damage caused by UV irradiation. Moreover, the mutant strain is much more plasmid transformable than its parent strain, illustrating that G. oxydans LCC96 could be used as a host to take up the recombinant plasmid for gene manipulation.     

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。     

Incapability of Gluconobacter oxydans to produce tartaric acid

The dependence of tartaric acid production by Gluconobacter oxydans ssp. oxydans ATCC 19357 and G. oxydans ssp. suboxydans ATCC 621 on vanadate was investigated. It was found with both organisms that trataric acid could only be produced in a medium containing vanadate (NH(4)VO(3)). A proposed intermediate of the tartaric acid metabolism in G. oxydans, 5-ketogluconic acid, was tested on its reactivity in the presence of the oxidizing catalyst vanadate. It could be shown that 5-ketogluconic acid and the catalyst vanadate, but not the activity of G. oxydans, were responsible for the formation of tartaric acid. G. oxydans was not able to produce tartaric acid by itself. The stereochemical identity of the formed tartaric acid could be identified as the L-(+)-type. Oxalic acid was formed from 5-ketogluconic acid with vanadate in the absence and in the presence of G. oxydans. The ratio of oxalic acid to tartaric acid was 1:1.