Cloning of FAD synthetase gene from Corynebacterium ammoniagenes and its application to FAD and FMN production

The cloning of a bifunctional FAD synthetase gene, which shows flavokinase and FMN adenylyltransferase activities, from Corynebacterium ammoniagenes was tried by hybridization with synthetic DNAs corresponding to the N-terminal amino acid sequence. The cloned PstI-digested 4.4 × 10³-base (4.4-kb) fragment could not express the FAD synthetase activity in E. coli, but could increase the two activities by the same factor of about 20 in C. amminoagenes. The FAD-synthetase-gene-amplified C. amminoagenes cells were applied to the production of FAD from FMN or riboflavin. The productivity of FAD from FMN was increased four to five times compared with the parent strain, and reached a 90% molar yield. The productivity of FAD from riboflavin was increased about eight times, with a 50% molar yield. The addition of Zn²⁺ to the reaction mixtures for the conversion from riboflavin to FAD brought about the specific inhibition of adenylyltransferase activity and resulted in the accumulation of FMN.     

Characterization of salt-tolerant glutaminase from Stenotrophomonas maltophilia NYW-81 and its application in Japanese soy sauce fermentation

Glutaminase from Stenotrophomonas maltophilia NYW-81 was purified to homogeneity with a final specific activity of 325 U/mg. The molecular mass of the native enzyme was estimated to be 41 kDa by gel filtration. A subunit molecular mass of 36 kDa was measured with SDS-PAGE, thus indicating that the native enzyme is a monomer. The N-terminal amino acid sequence of the enzyme was determined to be KEAETQQKLANVVILATGGTIA. Besides l-glutamine, which was hydrolyzed with the highest specific activity (100%), l-asparagine (74%), d-glutamine (75%), and d-asparagine (67%) were also hydrolyzed. The pH and temperature optima were 9.0 and approximately 60°C, respectively. The enzyme was most stable at pH 8.0 and was highly stable (relative activities from 60 to 80%) over a wide pH range (5.0–10.0). About 70 and 50% of enzyme activity was retained even after treatment at 60 and 70°C, respectively, for 10 min. The enzyme showed high activity (86% of the original activity) in the presence of 16% NaCl. These results indicate that this enzyme has a higher salt tolerance and thermal stability than bacterial glutaminases that have been reported so far. In a model reaction of Japanese soy sauce fermentation, glutaminase from S. maltophilia exhibited high ability in the production of glutamic acid compared with glutaminases from Aspergillus oryzae, Escherichia coli, Pseudomonas citronellolis, and Micrococcus luteus, indicating that this enzyme is suitable for application in Japanese soy sauce fermentation.     

Bioprocess engineering to produce 10-hydroxystearic acid from oleic acid by recombinant Escherichia coli expressing the oleate hydratase gene of Stenotrophomonas maltophilia

Microbial hydroxylation of long chain fatty acids has been extensively investigated. However, biotransformation productivity remains below ca. 1.0 g/g cell dry weight (CDW)/h under process conditions. In the present study, a highly efficient microbial hydroxylation process to convert oleic acid into 10-hydroxystearic acid was developed. A recombinant Escherichia coli expressing ohyA, the gene encoding oleate hydratase of Stenotrophomonas maltophilia, was used as the biocatalyst. Investigation of the ohyA expression and biotransformation conditions (e.g., inducer concentration, gene expression period before initiating biotransformation, mixing condition of reaction medium) enabled 10-hydroxystearic acid to accumulate to a final concentration of approximately 46 g/L in the culture medium. The specific product formation rate and product yield reached approximately 2.0 g/g CDW/h (i.e., 110 U/g CDW) and 91%, respectively. The specific product formation rate was more than 3-fold higher than those of a bioprocess using wild type Stenotrophomonas sp. cells. Additionally, the product of the whole-cell biotransformation was recovered at a yield of 70.9% and a purity of 99.7% via solvent fraction crystallization at low temperature. These results will contribute to developing a biological process for hydroxylation of oleic acid.     

Cloning and overexpression in Escherichia coli of the gene encoding dihydroxyacetone kinase isoenzyme I from Schizosaccharomyces pombe, and its application to dihydroxyacetone phosphate production

The gene dak1 encoding a dihydroxyacetone kinase (DHAK) isoenzyme I, one of two isoenzymes in the Schizosaccharomyces pombe IFO 0354 strain, was cloned and sequenced. The dak1 gene comprises 1743 bp and encodes a protein of 62 245 Da. The deduced amino acid sequence showed a similarity to a putative DHAK of Saccharomyces cerevisiae and DHAK of Citrobacter freundii. The dak1 gene was expressed at a high level in Escherichia coli, and the recombinant enzyme was purified to homogeneity and characterized. The acetone powder of recombinant E. coli cells was used to produce dihydroxyacetone phosphate. Received: 25 August 1998 / Received revision: 22 September 1998 / Accepted: 11 October 1998     

嗜麦芽寡养单胞菌D2株单加氧酶基因的克隆与表达

构建嗜麦芽寡养单胞菌D2株荧光素样单加氧酶基因表达克隆载体,并进行融合表达。PCR扩增出嗜麦芽寡养单胞菌D2菌株基因组DNA中包含单加氧酶基因约1300bp的核酸片段,将其克隆到T载体pMD-18中进行序列测定,所得序列申请并获得GenBank登记号(GQ122330)。DNA star软件分析发现该基因片段中含有一个996bp的完整开放读码框架(ORF),与GenBank中收录的S.maltophilia R551-3(CP001111/GenomeProject17107)和K279a(AM743169)的MO基因核酸序列同源性分别为90%和89%,氨基酸序列同源性分别为93%和90%。根据该ORF序列设计分别含有BamHⅠ和HindⅢ酶切位点的表达克隆扩增引物,PCR扩增、双酶切后将产物亚克隆到pET32a载体中,经过双酶切验证,证实成功获得了表达重组载体pET32a/MO;将其转化宿主菌E.coli BL21,IPTG诱导后成功表达出54.2ku的MO融合蛋白,为该酶进一步的功能研究和开发奠定了基础。     

Characterization of a chitinase gene from Stenotrophomonas maltophilia strain 34S1 and its involvement in biological control

A chitinase gene was cloned on a 2.8-kb DNA fragment from Stenotrophomonas maltophilia strain 34S1 by heterologous expression in Burkholderia cepacia. Sequence analysis of this fragment identified an open reading frame encoding a deduced protein of 700 amino acids. Removal of the signal peptide sequence resulted in a predicted protein that was 68 kDa in size. Analysis of the sequence indicated that the chitinase contained a catalytic domain belonging to family 18 of glycosyl hydrolases. Three putative binding domains, a chitin binding domain, a novel polycystic kidney disease (PKD) domain, and a fibronectin type III domain, were also identified within the sequence. Pairwise comparisons of each domain to the most closely related sequences found in database searches clearly demonstrated variation in gene sources and the species from which related sequences originated. A 51-kDa protein with chitinolytic activity was purified from culture filtrates of S. maltophilia strain 34S1 by hydrophobic interaction chromatography. Although the protein was significantly smaller than the size predicted from the sequence, the N-terminal sequence verified that the first 15 amino acids were identical to the deduced sequence of the mature protein encoded by chiA. Marker exchange mutagenesis of chiA resulted in mutant strain C5, which was devoid of chitinolytic activity and lacked the 51-kDa protein in culture filtrates. Strain C5 was also reduced in the ability to suppress summer patch disease on Kentucky bluegrass, supporting a role for the enzyme in the biocontrol activity of S. maltophilia.     

Cloning and overexpression of the old yellow enzyme gene of Candida macedoniensis, and its application to the production of a chiral compound

The gene encoding old yellow enzyme (OYE), which catalyzes the conversion of ketoisophorone (KIP; 2,6,6-trimethyl-2-cyclohexen-1,4-dione) to (6R)-levodione (2,2,6-trimethylcyclohexane-1,4-dione), of Candida macedoniensis was cloned and sequenced. A 1212bp nucleotide fragment (oye) was confirmed to be the gene encoding OYE based on the agreement of internal amino acid sequences. Oye encodes a total 403 amino acid residues, and the deduced amino acid sequence shows a high degree of similarity to those of other microbial OYE family proteins. An expression vector, pETOYE, that contains the full length of oye was constructed. Escherichia coli harboring pETOYE exhibited an about six-fold increase in specific KIP-reducing activity under the control of the T7 promoter as compared with that of C. macedoniensis. (6R)-Levodione formed with washed cells of the transformant and a cofactor regeneration system amounted to 638 mM (98.2 mg ml(-1)), the a molar yield being 96.9%. The asymmetric reduction of KIP to (6R)-levodione with E. coli cells, which co-expressed both oye and the glucose dehydrogenase gene (gdh), as a catalyst was investigated. The (6R)-levodione formed amounted to 627 mM (96.6 mg ml(-1)), the a molar yield being 95.4%. Since the use of E. coli BL21 (DE3) cells co-expressing oye and gdh as a catalyst is simple and does not require the addition of glucose dehydrogenase, it is highly advantageous for the practical synthesis of (6R)-levodione.     

Cloning,heterologous expression and characterization of two keratinases from Stenotrophomonas maltophilia BBE11-1

The keratin-degrading strain Stenotrophomonas maltophilia BBE11-1 secretes two keratinolytic proteases, KerSMD and KerSMF. However, the genes encoding these proteases remain unknown. Here, we have isolated these two genes with a modified TAIL-PCR (thermal asymmetric interlaced PCR) method based on the N-terminal amino acid sequences of mature keratinases. These two keratinase genes encode serine proteases with PPC (bacterial pre-peptidase C-terminal) domain, which are successfully expressed with the help of pelB leader in Escherichia coli cells. Recombinant KerSMD (48 kDa) shows a better activity in feather degradation, higher thermostability and substrate specificity than KerSMF (40 kDa). KerSMD has a t_1/2 of 90 min at 50 °C and 64 min at 60 °C, and a better tolerance to surfactants SDS and triton X-100. The predicted model of KerSMD helps to explain the phenomenon of auto-catalytic C-terminal propeptide truncation, the special function of PPC domain, and the molecular weight of the C-terminal-processed mature keratinase KerSMD. This work not only provides a new way to overproduce keratinases but also helps to explore keratinases folding mechanism.     

2005年至2008年嗜麦芽窄食单胞菌的耐药性变迁

调查2005年至2008年嗜麦芽窄食单胞菌的耐药情况,为临床有效治疗嗜麦芽窄食单胞菌感染提供依据。从各类标本中分离出嗜麦芽窄食单胞菌株并利用API系统鉴定。采用Kirby-Bauer法和微量肉汤稀释法进行药敏试验。2005年至2008年共分离出嗜麦芽窄食单胞菌株337株。嗜麦芽窄食单胞菌对碳氢酶烯类药物、氨基糖苷类、三代头孢菌素、氨曲南和β-内酰胺酶抑制剂复方制剂高度耐药,复方新诺明和米诺环素对嗜麦芽窄食单胞菌的敏感率在90%以上。嗜麦芽窄食单胞菌对临床上常用的大多数抗菌药物高度耐药,可首选复方新诺明和米诺环素治疗嗜麦芽窄食单胞菌感染。     

2008-2010年嗜麦芽窄食单胞菌医院感染的临床分布及耐药性分析

目的了解医院感染嗜麦芽窄食单胞菌（Stenotrophomonasmaltophilia,Sm）的临床分布及耐药性情况,为临床诊治提供依据。方法采用回顾性资料,对中山大学附属第三医院2008—2010年间住院患者的各种临床标本中分离到的Sm及其药敏结果进行统计分析。结果Sm主要来源于呼吸道标本（痰及咽拭子）,占82．97％,临床分布以肝胆外科最多,占25．27％,其次为感染科（21．43％）,ICU和神经外科均占8．79％;40岁以上的中老年患者占75．82％;Sm对复方新诺明的敏感率最高,达84．40％,其次为左氧氟沙星（81．21％）,对头孢他啶和替卡西kS／克拉维酸的敏感性均较低（〈40％）。结论该院感染Sm的易感人群主要是以中老年患者为主,Sm对CLSI推荐的抗菌药物已有一定的耐药性,临床应高度重视,控制感染。     

A molecular biological protocol to distinguish potentially human pathogenic Stenotrophomonas maltophilia from plant-associated Stenotrophomonas rhizophila

In recent years, the importance of the Gram-negative bacterium Stenotrophomonas as an opportunistic pathogen as well as in biotechnology has increased. The aim of the present study was to develop new methods for distinguishing between strains closely related to the potentially human pathogenic Stenotrophomonas maltophilia and those closely related to the plant-associated Stenotrophomonas rhizophila. To accomplish this, 58 strains were characterized by 16S rDNA sequencing and amplified ribosomal DNA restriction analysis (ARDRA), and the occurrence of specific functional genes. Based on 16S rDNA sequences, an ARDRA protocol was developed which allowed differentiation between strains of the S. maltophilia and the S. rhizophila group. As it was known that only salt-treated cells of S. rhizophila were able to synthesize the compatible solute glucosylglycerol (GG), the ggpS gene responsible for GG synthesis was used for differentiation between both species and it was confirmed that it only occurred in S. rhizophila strains. As a further genetic marker the smeD gene, which is part of the genes coding for the multidrug efflux pump SmeDEF from S. maltophilia, was used. Based on the results we propose a combination of fingerprinting techniques using the 16S rDNA and the functional genes ggpS and smeD to distinguish both Stenotrophomonas species.     

产气肠杆菌fhlA基因克隆、表达及重组菌株产氢量

【目的】本研究以产氢细菌产气肠杆菌Enterobacter aerogenes ATCC13408为研究对象,克隆甲酸-氢裂解酶(formate hydrogen lyase,FHL)系统的转录激活蛋白FHL activator(fhlA)基因,构建过表达重组菌株,以提高菌株产氢效率。【方法】利用简并引物和Genome walking技术,克隆fhlA的全长基因,将该基因连接到改造质粒pGEX-4T-2-Cat中,电击转化得到重组菌株,用厌氧发酵方法测定重组细菌的产氢量。【结果】E.aerogenes ATCC13408fhlA ORF全长2073bp,编码一个含690个氨基酸残基的蛋白(GenBank accessionGU188474)。SDS-PAGE和Western blot分析证明fhlA基因在重组菌中得到了融合表达。对重组后菌株的产氢量进行了测定,结果表明:底物产氢潜力由原来的1.23±0.08mol H2/mol葡萄糖提高到了1.48±0.04mol H2/mol葡萄糖,提高了20.36%。【结论】本研究首次克隆了E.aerogenes ATCC13408的fhlA基因,并将该基因在原菌中过量表达。重组后菌株的产氢量得到显著提高,为进一步研究和开发利用E.aerogenes ATCC13408的fhlA基因提供了基础。     

Functional characterization of an NADPH dependent 2-alkyl-3-ketoalkanoic acid reductase involved in olefin biosynthesis in Stenotrophomonas maltophilia

OleD is shown to play a key reductive role in the generation of alkenes (olefins) from acyl thioesters in Stenotrophomonas maltophilia. The gene coding for OleD clusters with three other genes, oleABC, and all appear to be transcribed in the same direction as an operon in various olefin producing bacteria. In this study, a series of substrates varying in chain length and stereochemistry were synthesized and used to elucidate the functional role and substrate specificity of OleD. We demonstrated that OleD, which is an NADP(H) dependent reductase, is a homodimer which catalyzes the reversible stereospecific reduction of 2-alkyl-3-ketoalkanoic acids. Maximal catalytic efficiency was observed with syn-2-decyl-3-hydroxytetradecanoic acid, with a k(cat)/K(m) 5- and 8-fold higher than for syn-2-octyl-3-hydroxydodecanoic acid and syn-2-hexyl-3-hydroxydecanoic acid, respectively. OleD activity was not observed with syn-2-butyl-3-hydroxyoctanoic acid and compounds lacking a 2-alkyl group such as 3-ketodecanoic and 3-hydroxydecanoic acids, suggesting the necessity of the 2-alkyl chain for enzyme recognition and catalysis. Using diastereomeric pairs of substrates and 4 enantiopure isomers of 2-hexyl-3-hydroxydecanoic acid of known stereochemistry, OleD was shown to have a marked stereochemical preference for the (2R,3S)-isomer. Finally, experiments involving OleA and OleD demonstrate the first 3 steps and stereochemical course in olefin formation from acyl thioesters; condensation to form a 2-alkyl-3-ketoacyl thioester, subsequent thioester hydrolysis, and ketone reduction.     

DNA profiling of Stenotrophomonas maltophilia by PCR targeted to its species-specific repetitive palindromic sequences

Aims: The aim of this study was to develop a simple protocol for a PCR‐based fingerprinting of Stenotrophomonas maltophilia (SmrepPCR) that utilizes primers complementary to repetitive extragenic palindromic elements (REPs) of this micro‐organism. Methods and Results: The relatedness of 34 isolates of environmental and clinical origin was investigated by two SmrepPCRs with two different primers, gyrB sequencing and XbaI macrorestriction followed by pulsed‐field gel electrophoresis. While SmrepPCR (with primer DIR) results matched data obtained from the analysis of gyrB nucleotide sequences and identified several clonal complexes, XbaI macrorestriction showed high level of heterogeneity between isolates. The macrorestriction‐based clustering of isolates did not correspond to both gyrB and DIR‐SmrepPCR grouping. Conclusions: Our results show that SmrepPCR‐inferred relationship of isolates is in a good agreement with sequence‐based methods. The combined information from all methods used suggests that rapid evolution of S. maltophilia genomes might be predominantly due to high rate of rearrangements caused by mobile genetic elements. Significance and Impact of the Study: The presented method is an inexpensive and easy to perform alternative to genotype S. maltophilia isolates and to study their population genetics. SmrepPCR demonstrates the usefulness of species‐specific repetitive elements in genomic analyses.     

High activity catechol 1,2-dioxygenase from Stenotrophomonas maltophilia strain KB2 as a useful tool in cis,cis-muconic acid production

This is the first report of a catechol 1,2-dioxygenase from Stenotrophomonas maltophilia strain KB2 with high activity against catechol and its methyl derivatives. This enzyme was maximally active at pH 8.0 and 40 °C and the half-life of the enzyme at this temperature was 3 h. Kinetic studies showed that the value of K _m and V _max was 12.8 μM and 1,218.8 U/mg of protein, respectively. During our studies on kinetic properties of the catechol 1,2-dioxygenase we observed substrate inhibition at >80 μM. The nucleotide sequence of the gene encoding the S. maltophilia strain KB2 catechol 1,2-dioxygenase has high identity with other catA genes from members of the genus Pseudomonas. The deduced 314-residue sequence of the enzyme corresponds to a protein of molecular mass 34.5 kDa. This enzyme was inhibited by competitive inhibitors (phenol derivatives) only by ca. 30 %. High tolerance against condition changes is desirable in industrial processes. Our data suggest that this enzyme could be of use as a tool in production of cis,cis-muconic acid and its derivatives.     

Intra-hospital dissemination of clinical and environmental isolates of Stenotrophomonas maltophilia from Tehran

Stenotrophomonas maltophilia isolates are responsible for various hospital-acquired infections and are particularly increasing in the immunocompromised patients. The aim of this study was to determine the clonal relatedness between S. maltophilia isolates originating from the clinic and environment. A total of 150 S. maltophilia isolates from patients and 1108 environmental samples obtained in three hospitals from Tehran. Following molecular identification targeting 23S rRNA gene, the clonal relatedness of the environmental and clinical isolates was determined using pulsed field gel electrophoresis (PFGE). Of the 150 clinical and 18 environmental isolates identified using phenotypic tests, the speciation of 120 and 15 was confirmed by targeting the 23S rRNA gene. The 24 common pulsotypes (PTs) and 32 single PTs were identified by PFGE. Only a small cluster was shared among the clinic and environment within a hospital; therefore, the intra-hospital dissemination of certain isolates of S. maltophilia among the clinic and environment was demonstrated.     

Production of 10-hydroxystearic acid from oleic acid by whole cells of recombinant Escherichia coli containing oleate hydratase from Stenotrophomonas maltophilia

A putative fatty acid hydratase from Stenotrophomonas maltophilia was cloned and expressed in Escherichia coli. The recombinant enzyme showed the highest hydration activity for oleic acid among the fatty acids tested, indicating that the enzyme is an oleate hydratase. The optimal conditions for the production of 10-hydroxystearic acid from oleic acid using whole cells of recombinant E. coli containing the oleate hydratase were pH 6.5, 35°C, 0.05% (w/v) Tween 40, 10 g l(-1) cells, and 50 g l(-1) oleic acid. Under these conditions, whole recombinant cells produced 49 g l(-1) 10-hydroxystearic acid for 4 h, with a conversion yield of 98% (w/w), a volumetric productivity of 12.3 g l(-1) h(-1), and a specific productivity of 1.23 g g-cells(-1) h(-1), which were 18%, 2.5-, and 2.5-fold higher than those of whole wild-type S. maltophilia cells, respectively. This is the first report of 10-hydroxystearic acid production using recombinant cells and the concentration and productivity are the highest reported thus far among cells.     

Stenotrophomonas maltophilia Gd2: A potential and novel isolate for fibrinolytic enzyme production

The bacterium with an ability to produce extracellular fibrinolytic protease was isolated and identified as Stenotrophomonas maltophilia Gd2 based on ribotyping. The in-vitro fibrinolytic profile of this enzyme depicted 73% of fibrin clot dissolution within 4 h. Fibrinolytic enzyme yield influenced by different physiological (incubation time, temperature, agitation and pH), nutritional (macronutrients such as carbon and nitrogen sources) and biological (inoculums age and inoculums concentration) parameters of fermentation which were optimized based on one-factor-at-a-time (OFAT) approach. The enzyme yield improved from 886 to 1795 FU ml⁻¹ upon OFAT; optimized conditions include temperature – 33 °C, pH – 8.0, incubation time – 36 h, agitation – 150 RPM, 3% v/v inoculums and age of inoculum – 18 h. Further optimization of enzyme production was achieved with implementation of Plackett-Burman media designing where the production levels increased to 3411 FU ml⁻¹ and noticed that peptone, pH, dextrose and K₂HPO₄ was found to be significant factor. This ms reports the highest fibrinolytic enzyme yield with S. maltophilia to that of literature reports.     

嗜麦芽寡养单胞菌(DR-929)纤溶酶的发酵条件优化及其分离纯化

本试验研究了嗜麦芽寡养单胞菌（DR-929）纤溶酶的液体发酵条件及其分离纯化。最佳发酵条件为：可溶性淀粉2.0%,黄豆粉1.0%,酵母膏0.5%,NaCl 1.0%, CaCl2 0.02%,MgSO4 0.05%,种龄36h,发酵时间4d,初始pH 8.0或9.0,温度25℃,装样量30mL,接种量5%或6%。采用发酵液离心除菌,25%～70%饱和度的硫酸铵沉淀,Phenyl FF（high sub）疏水层析,Q-Sepharose FF离子交换层析,Superdex 75凝胶过滤层析对活性成分分离纯化。用SDS-PAGE电泳对纯化效果进行检验,结果表明在SDS-PAGE中得到单一条带,分子量28.3KD。最终纯化倍数和酶活回收率分别为271.5和24.5%。     

Fimbriae and adherence of Stenotrophomonas maltophilia to epithelial cells and to abiotic surfaces

Stenotrophomonas maltophilia is an emerging nosocomial bacterial pathogen associated with several infectious diseases and opportunistic infections, especially in immunocompromised patients. These bacteria adhere avidly to medical implants and catheters forming a biofilm that confers natural protection against host immune defences and different antimicrobial agents. The nature of the bacterial surface factors involved in biofilm formation on inert surfaces and in adherence of S. maltophilia to epithelial cells is largely unknown. In this study, we identified and characterized fimbrial structures produced by S. maltophilia grown at 37 degrees C. The S. maltophilia fimbriae 1 (SMF-1) are composed of a 17 kDa fimbrin subunit which shares significant similarities with the N-terminal amino acid sequences of several fimbrial adhesins (G, F17, K99 and 20K) found in Escherichia coli pathogenic strains and the CupA fimbriae of Pseudomonas aeruginosa. All of the clinical S. maltophilia isolates tested produced the 17 kDa fimbrin. Antibodies raised against SMF-1 fimbriae inhibited the agglutination of animal erythrocytes, adherence to HEp-2 cells and biofilm formation by S. maltophilia. High resolution electron microscopy provided evidence of the presence of fimbriae acting as bridges between bacteria adhering to inert surfaces or to cultured epithelial cells. This is the first characterization of fimbriae in this genus. We provide compelling data suggesting that the SMF-1 fimbriae are involved in haemagglutination, biofilm formation and adherence to cultured mammalian cells.