2-萘酸细菌代谢途径的研究

采用质粒消除、转座子Tn10插入突变和回复突变等试验方法证实了2-萘酸细菌代谢途径中邻苯二酸环节的存在,探讨了2-萘酸代谢菌2-NAT菌株对2-萘酸代谢的调控机制。发现该菌至少带有一个质粒,该菌的2-萘酸代谢途径由质粒和染色体DNA共同编码控制。     

2-萘酸加单氧酶基因及NADH:黄素还原酶基因的克隆与分析

伯克霍尔德氏菌(Burkholderiasp.)JT1500对2-萘酸(2-naphthoate)生物降解的关键步骤之一是通过2-萘酸加单氧酶羟化2-萘酸生成1-羟基-2-萘酸(1-hydroxy-2-naphthoate)。在已确定2-萘酸加单氧酶基因及其功能的基础上对含有该基因的一个4.8kb长度的基因簇进行了克隆测序。该序列上含有4个可能的阅读框orfB、orfC、orfD、orfA。序列比对发现,orfA序列与JaponicumUSDA110和RalstoniaeutrophaHF39中的加单氧酶基因同源性较高,orfB序列与BordetllapertussisTohamaI、RalstoniasolanacearumGMI1000和BordetellabronchisepticaRB50等菌中的黄素还原酶基因有一定的同源性。酶活分析发现只含基因orfA的重组大肠杆菌SA细胞提取液有很低的加氧活性,含基因orfB的重组子SB细胞提取液没有加氧活性,但在反应体系中同时加入SA和SB的细胞提取液后,其加氧活性显著增强,包含片段orfB orfA的重组子SB A在黄素(FMN、FAD)存在的情况下也表现出很强的加氧活性;在厌氧条件下,能检测出SB细胞提取液的黄素还原活性。基于以上信息,认为2-萘酸加单氧酶基因簇含有两个重要的组分黄素还原酶基因(nmoB)和加单氧酶基因(nmoA)。2-萘酸加单氧酶Nmo羟化2-萘酸的过程为先由黄素还原酶(NmoB)在NADH存在的条件下将黄素(FMN、FAD)还原为还原型黄素(FMNH2、FADH2),然后加单氧酶(NmoA)利用还原型黄素和O2羟化底物2-萘酸,生成1-羟基-2-萘酸。NmoB是NmoA的偶联蛋白。     

假单胞杆菌2-萘酸加氧酶基因的克隆与表达

采用Cosmid pLARF1构建了完整的2-萘酸代谢菌2-NAT菌株的基因文库.通过分析基因文库中产生蓝色色素的克隆验证了2-萘酸代谢基因在大肠杆菌体内得到表达,并导致重组细菌产生靛蓝.从产生靛蓝的克隆抽提重组的Cosmid DNA进行酶解分析,发现所有能产生靛蓝的克隆均含有一大小相同的DNA片段.用重组细菌进行靛蓝生物合成的试验展示了微生物法生产靛蓝的美好前景.     

伯克霍尔德氏菌（Burkholderia sp.）2-萘酸单加氧酶基因（nmo）的克隆及表达

通过酶切连接将Burkholderia sp. JT1500的一段DNA片段(4.8kb)亚克隆到表达载体pUC18上,得到重组子pEK123。测序后的pEK123重组子48kb插入片段的序列已经登陆欧洲EMBL基因库,序列接受号为AJ566333。对这一DNA片段的序列分析显示,此DNA片段含有3个阅读框,且在这3个阅读框5′端发现一启动子特异序列。再用酶切连接方法得到仅含一个阅读框的重组子pXK3,其阅读框长度为1158bp,编码386个氨基酸,与已报道的Ralstonia eutropha HF39羟化酶（单加氧酶,bec）氨基酸序列有64%的同源性。pEK123对2萘酸代谢途径中4个关键底物的转化实验结果显示,其基因产物仅对2萘酸发生加氧转化反应,而且2萘酸浓度有明显的降低,证实此基因是2萘酸单加氧酶基因（nmo）。同时发现其基因产物也可以转化苯甲酸钠。该酶对苯甲酸的加氧转化途径正在研究中。 SDSPAGE结果表明,pXK3、pEK123两重组子中2萘酸单加氧酶表达量并没明显区别,但加氧酶酶活却存在显著的差别。推测在启动子后,单加氧酶阅读框前的两个阅读框的基因产物,对单加氧酶活有促进作用。     

伯克霍尔德氏菌(Burkholderia sp.)2-萘酸单加氧酶基因(nmo)的克隆及表达

通过酶切连接将Burkholderia sp．JTl500的一段DNA片段(4．8kb)亚克隆到表达载体pUC18上,得到重组子pEKl23。测序后的pEKl23重组子4．8kb插入片段的序列已经登陆欧洲EMBL基因库,序列接受号为AJ566333。对这一DNA片段的序列分析显示,此DNA片段含有3个阅读框,且在这3个阅读框5’端发现一启动子特异序列。再用酶切连接方法得到仅含一个阅读框的重组子pXK3,其阅读框长度为1158bp,编码386个氨基酸,与已报道的Ralstonia eutropha HF39羟化酶(单加氧酶,bec)氨基酸序列有64％的同源性。pEKl23对2-萘酸代谢途径中4个关键底物的转化实验结果显示,其基因产物仅对2-萘酸发生加氧转化反应,而且2-萘酸浓度有明显的降低,证实此基因是2-萘酸单加氧酶基因(nmo)。同时发现其基因产物也可以转化苯甲酸钠。该酶对苯甲酸的加氧转化途径正在研究中。SDS-PAGE结果表明,pXK3、pEKl23两重组子中2-萘酸单加氧酶表达量并没明显区别,但加氧酶酶活却存在显著的差别。推测在启动子后,单加氧酶阅读框前的两个阅读框的基因产物,对单加氧酶活有促进作用。     

好客嗜酸两面菌W1寡营养酸性热泉环境的适应机制

【目的】从基因组水平揭示好客嗜酸两面菌(Acidianus hospitalis)W1对寡营养酸性热泉环境的适应机制。【方法】使用NCBI非冗余蛋白数据库、Uniport蛋白数据库以及Sulfolobus数据库对好客嗜酸两面菌W1基因组序列进行功能注释,使用KEGG数据库对基因组进行In slico代谢途径重构,在此基础之上,采用比较基因组学方法对代谢途径进行鉴定和完善。【结果】好客嗜酸两面菌W1具备多样化的代谢方式以适应寡营养酸性热泉环境。W1菌株通过3-羟基丙酸/4-羟基丁酸和乙二酸-4-羟基丁酸循环进行CO2固定、通过氧化还原型无机硫化物(Reduced inorganic sulfur compounds,RISCs)获取能量营自养型生长。但W1菌株与模式菌株Acidianus ambivalens的硫代谢方式不同,W1基因组中缺少编码亚硫酸-受体氧化还原酶(SAOR)、腺苷硫酸(APS)还原酶、硫酸腺苷酰转移酶(SAT)和腺苷硫酸:磷酸腺苷转移酶(APAT)的基因。此外,W1菌株可以通过非磷酸化的分支的ED途径和TCA循环进行葡萄糖代谢,糖类和氨基酸转运蛋白以及相应水解酶的存在表明W1能够利用部分有机物营兼性自养型生长。与A.ambivalens不同,W1菌株不能利用H2作为电子供体。【结论】多样化的代谢方式为好客嗜酸两面菌W1更好地适应寡营养酸性热泉环境提供了重要保障,对于W1菌株特有代谢方式的揭示也丰富了对于嗜酸两面菌(Acidianus)属物种代谢多样性的认知。     

维生素C生物转化的代谢工程研究

本从代谢工程角度出发,综述维生素C(Vc)生物转化代谢研究进展。分别论述了3条产生Vc重要前体2-酮基-L-古龙酸(2-KLG)反应路线的代谢机制、反应酶系及代谢工程菌构建等方面的问题;并对Vc生物转化应用前景作了展望。     

食品抗氧剂-D-异抗坏血酸的研究——Ⅱ.产酮产碱菌E54产生2-酮基-D-葡萄糖酸的发酵条件

2-酮基-D-葡萄糖酸是合成D-异抗坏血酸(简称异维生素C)的前体。而D-异抗坏血酸及其钠盐是广泛应用于食品工业中的优良抗氧剂。本文通过新种产酮产碱菌使葡萄糖发酵产生2-酮基-D-葡萄糖酸,并对该菌发酵的碳源、氮源、通气量、温度、金属离子等影响作了探讨,通过正交试验确定了产生2-酮基-D-葡萄糖酸最佳种子培养基和发酵培养基。并对该菌的发酵代谢作了初步的观察。     

食品抗氧剂-D-异抗坏血酸的研究——Ⅱ.产酮产碱菌E54产生2-酮基-D-葡萄糖酸的发酵条件*

2-酮基-D-葡萄糖酸是合成D-异抗坏血酸(简称异维生素C)的前体。而D-异抗坏血酸及其钠盐是广泛应用于食品工业中的优良抗氧剂。本文通过新种产酮产碱菌使葡萄糖发酵产生2-酮基-D-葡萄糖酸,并对该菌发酵的碳源、氮源、通气量、温度、金属离子等影响作了探讨,通过正交试验确定了产生2-酮基-D-葡萄糖酸最佳种子培养基和发酵培养基。并对该菌的发酵代谢作了初步的观察。     

氧化葡萄糖酸杆菌酶学和分子生物学研究

对氧化葡萄糖酸杆菌初级代谢途径中的关键酶及分子生物学研究做了系统的评述 ,展望了分子技术改造氧化葡萄糖酸杆菌和优化 2 KGA代谢途径的可能。     

O-phthalic acid, a dead-end product in one of the two pathways of phenanthrene degradation in Pseudomonas sp. strain PP2

Phenanthrene is degraded via either o-phthalic acid or 1, 2-dihydroxynaphthalene in bacteria. A soil isolate Pseudomonas sp. strain PP2 degrades phenanthrene as the sole source of carbon, but failed to utilize naphthalene [Prabhu and Phale (2003) Appl Microbiol Biotechnol 61:342-351]. Analysis of the phenanthrene-grown culture spent media of this strain by gas chromatography-mass spectrometry (GC-MS) showed accumulation of o-phthalic acid. The cell-free extract prepared from this strain showed activity of 1-hydroxy-2-naphthoic acid dioxygenase (1-H-2-NADO). The extract showed conversion of 1-hydroxy-2-naphthoic acid and 2-carboxybenzaldehyde to o-phthalic acid, as analyzed by thin layer chromatography and GC-MS. However, it failed to grow or respire on o-phthalic acid. These results suggest that besides 1, 2-dihydroxynaphthalene pathway, the strain has a truncated o-phthalic acid pathway for phenanthrene metabolism and excretes o-phthalic acid as a dead-end product, indicating the co-existence of two pathways. 1-H-2-NADO, the key enzyme of o-phthalic acid pathway is inducible, has pH optima of 7.5, does not require external addition of Fe(II) as a co-factor and is completely inhibited by 1,10-phenanthroline. Absence of product formation under anaerobic condition and stoichiometric consumption of 0.82 moles of O2 per mole of product formed confirmed the dioxygenase nature of the enzyme.     

Degradation of phenanthrene via meta-cleavage of 2-hydroxy-1-naphthoic acid by Ochrobactrum sp. strain PWTJD

The present study describes the assimilation of phenanthrene by an aerobic bacterium, Ochrobactrum sp. strain PWTJD, isolated from municipal waste-contaminated soil sample utilizing phenanthrene as a sole source of carbon and energy. The isolate was identified as Ochrobactrum sp. based on the morphological, nutritional and biochemical characteristics as well as 16S rRNA gene sequence analysis. A combination of chromatographic analyses, oxygen uptake assay and enzymatic studies confirmed the degradation of phenanthrene by the strain PWTJD via 2-hydroxy-1-naphthoic acid, salicylic acid and catechol. The strain PWTJD could also utilize 2-hydroxy-1-naphthoic acid and salicylic acid, while the former was metabolized by a ferric-dependent meta-cleavage dioxygenase. In the lower pathway, salicylic acid was metabolized to catechol and was further degraded by catechol 2,3-dioxygenase to 2-hydroxymuconoaldehyde acid, ultimately leading to tricarboxylic acid cycle intermediates. This is the first report of the complete degradation of a polycyclic aromatic hydrocarbon molecule by Gram-negative Ochrobactrum sp. describing the involvement of the meta-cleavage pathway of 2-hydroxy-1-naphthoic acid in phenanthrene assimilation.     

Evidence for aromatic ring reduction in the biodegradation pathwayof carboxylated naphthalene by a sulfate reducing consortium

Naphthalene was used as a model compound in order to study the anaerobic pathway of polycyclic aromatic hydrocarbon degradation. Previously we had determined that carboxylation is an initial step for anaerobic metabolism of naphthalene, but no other intermediate metabolites were identified (Zhang & Young 1997). In the present study we further elucidate the pathway with the identification of six novel naphthalene metabolites detected when cultures were fed naphthalene in the presence of its analog 1-fluoronaphthalene. Results from cultures supplemented with either deuterated naphthalene or non-deuterated naphthalene plus [¹³C]bicarbonate confirm that the metabolites originated from naphthalene. Three of these metabolites were identified by comparison with the following standards: 2-naphthoic acid (2-NA), 5,6,7,8-tetrahydro-2-naphthoic acid, and decahydro-2-naphthoic acid. The presence of 5,6,7,8-tetrahydro-2-NA as a metabolite of naphthalene degradation indicates that the first reduction reaction occurs at the unsubstituted ring, rather than the carboxylated ring. The overall results suggest that after the initial carboxylation of naphthalene, 2-NA is sequentially reduced to decahydro-2-naphthoic acid through 5 hydrogenation reactions, each of which eliminated one double bond. Incorporation of deuterium atoms from D₂O into 5,6,7,8-tetrahydro-2-naphthoic acid suggests that water is the proton source for hydrogenation.     

A catabolic pathway for the degradation of chrysene by Pseudoxanthomonas sp. PNK-04

The chrysene-degrading bacterium Pseudoxanthomonas sp. PNK-04 was isolated from a coal sample. Three novel metabolites, hydroxyphenanthroic acid, 1-hydroxy-2-naphthoic acid and salicylic acid, were identified by TLC, HPLC and MS. Key enzyme activities, namely 1-hydroxy-2-naphthoate hydroxylase, 1,2-dihydroxynaphthalene dioxygenase, salicylaldehyde dehydrogenase and catechol-1,2-dioxygenase, were noted in the cell-free extract. These results suggest that chrysene is catabolized via hydroxyphenanthroic acid, 1-hydroxy-2-naphthoic acid, salicylic acid and catechol. The terminal aromatic metabolite, catechol, is then catabolized by catechol-1,2-dioxygenase to cis,cis-muconic acid, ultimately forming TCA cycle intermediates. Based on these studies, the proposed catabolic pathway for chrysene degradation by strain PNK-04 is chrysene → hydroxyphenanthroic acid → 1-hydroxy-2-naphthoic acid → 1,2-dihydroxynaphthalene → salicylic acid → catechol →cis,cis-muconic acid.     

Isolation of phenanthrene-degrading bacteria and characterization of phenanthrene metabolites

Three aerobic bacterial consortia GY2, GS3 and GM2 were enriched from polycyclic aromatic hydrocarbon-contaminated soils with water-silicone oil biphasic systems. An aerobic bacterial strain utilizing phenanthrene as the sole carbon and energy source was isolated from bacterial consortium GY2 and identified as Sphingomonas sp. strain GY2B. Within 48 h and at 30°C the strain metabolized 99.1% of phenanthrene (100 mg/l) added to batch culture in mineral salts medium and the cell number increased by about 40-fold. Three metabolites 1-hydroxy-2-naphthoic acid, 1-naphthol and salicylic acid, were identified by gas chromatographic mass spectrometry and UV–visible spectroscopy analysis. A degradation pathway was proposed based on the identified metabolites. In addition to phenanthrene, strain GY2B could use other aromatic compounds such as naphthalene, 2-naphthol, salicylic acid, catechol, phenol, benzene and toluene as a sole source of carbon and energy.     

Degradation of phenanthrene and anthracene by Nocardia otitidiscaviarum strain TSH1, a moderately thermophilic bacterium

Aims:  The metabolism of phenanthrene and anthracene by a moderate thermophilic Nocardia otitidiscaviarum strain TSH1 was examined.
Methods and Results:  When strain TSH1 was grown in the presence of anthracene, four metabolites were identified as 1,2-dihydroxy-1,2-dihydroanthracene, 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid, 2,3-dihydroxynaphthalene and benzoic acid using gas chromatography-mass spectrometry (GC-MS), reverse phase-high performance liquid chromatography (RP-HPLC) and thin-layer chromatography (TLC). Degradation studies with phenanthrene revealed 2,2'-diphenic acid, phthalic acid, 4-hydroxyphenylacetic acid, o -hydroxyphenylacetic acid, benzoic acid, a phenanthrene dihydrodiol, 4-[1-hydroxy(2-naphthyl)]-2-oxobut-3-enoic acid and 1-hydroxy-2-naphthoic acid (1H2NA), as detectable metabolites.
Conclusions:  Strain TSH1 initiates phenanthrene degradation via dioxygenation at the C-3 and C-4 or at C-9 and C-10 ring positions. Ortho -cleavage of the 9,10-diol leads to formation of 2,2'-diphenic acid. The 3,4-diol ring is cleaved to form 1H2NA which can subsequently be degraded through o -phthalic acid pathway. Benzoate does not fit in the previously published pathways from mesophiles. Anthracene metabolism seems to start with a dioxygenation at the 1 and 2 positions and ortho -cleavage of the resulting diol. The pathway proceeds probably through 2,3-dicarboxynaphthalene and 2,3-dihydroxynaphthalene. Degradation of 2,3-dihydroxynaphthalene to benzoate and transformation of the later to catechol is a possible route for the further degradation of anthracene.
Significance and Impact of the Study:  For the first time, metabolism of phenanthrene and anthracene in a thermophilic Nocardia strain was investigated.     

Novel intermediates of acenaphthylene degradation by Rhizobium sp. strain CU-A1: evidence for naphthalene-1,8-dicarboxylic acid metabolism

The acenaphthylene-degrading bacterium Rhizobium sp. strain CU-A1 was isolated from petroleum-contaminated soil in Thailand. This strain was able to degrade 600 mg/liter acenaphthylene completely within three days. To elucidate the pathway for degradation of acenaphthylene, strain CU-A1 was mutagenized by transposon Tn5 in order to obtain mutant strains deficient in acenaphthylene degradation. Metabolites produced from Tn5-induced mutant strains B1, B5, and A53 were purified by thin-layer chromatography and silica gel column chromatography and characterized by mass spectrometry. The results suggested that this strain cleaved the fused five-membered ring of acenaphthylene to form naphthalene-1,8-dicarboxylic acid via acenaphthenequinone. One carboxyl group of naphthalene-1,8-dicarboxylic acid was removed to form 1-naphthoic acid which was transformed into salicylic acid before metabolization to gentisic acid. This work is the first report of complete acenaphthylene degradation by a bacterial strain.     

Biodegradation of Phenanthrene by a Bacillus Species

A bacterial strain capable of utilizing phenanthrene as sole source of carbon was isolated from soil and identified as a Bacillus sp. The organism also utilized naphthalene, biphenyl, anthracene, and other aromatic compounds as growth substrates. The organism degraded phenanthrene through the intermediate formation of 1-hydroxy-2-naphthoic acid, which was further metabolized via o-phthalate by a protocatechuate pathway, as evidenced by oxygen uptake and enzymatic studies. Received: 1 December 1999 / Accepted: 5 January 2000     

Novel Intermediates of Acenaphthylene Degradation by Rhizobium sp. Strain CU-A1: Evidence for Naphthalene-1,8-Dicarboxylic Acid Metabolism

The acenaphthylene-degrading bacterium Rhizobium sp. strain CU-A1 was isolated from petroleum-contaminated soil in Thailand. This strain was able to degrade 600 mg/liter acenaphthylene completely within three days. To elucidate the pathway for degradation of acenaphthylene, strain CU-A1 was mutagenized by transposon Tn5 in order to obtain mutant strains deficient in acenaphthylene degradation. Metabolites produced from Tn5-induced mutant strains B1, B5, and A53 were purified by thin-layer chromatography and silica gel column chromatography and characterized by mass spectrometry. The results suggested that this strain cleaved the fused five-membered ring of acenaphthylene to form naphthalene-1,8-dicarboxylic acid via acenaphthenequinone. One carboxyl group of naphthalene-1,8-dicarboxylic acid was removed to form 1-naphthoic acid which was transformed into salicylic acid before metabolization to gentisic acid. This work is the first report of complete acenaphthylene degradation by a bacterial strain.     

Identification of novel metabolites in the degradation of phenanthrene by Sphingomonas sp. strain P2

Sphingomonas sp. strain P2, which is capable of utilizing phenanthrene as a sole carbon and energy source, was isolated from petroleum-contaminated soil in Thailand. Gas chromatography-mass spectrometry and (1)H and (13)C nuclear magnetic resonance analyses revealed two novel metabolites from the phenanthrene degradation pathway. One was identified as 5,6-benzocoumarin, which was derived by dioxygenation at the 1- and 2-positions of phenanthrene, and the other was determined to be 1,5-dihydroxy-2-naphthoic acid. Other metabolites from phenanthrene degradation were identified as 7, 8-benzocoumarin, 1-hydroxy-2-naphthoic acid and coumarin. From these results, it is suggested that strain P2 can degrade phenanthrene via dioxygenation at both 1,2- and 3,4-positions followed by meta-cleavage.