Development of a strain for efficient degradation of polychlorinated biphenyls by patchwork assembly of degradation pathways

Rhodococcus jostii RHA1 accumulates chlorobenzoates (CBA) during the degradation of polychlorinated biphenyls (PCBs). CBA degradation is considered one of the rate-limiting steps in the complete degradation of PCBs. To reduce the accumulation of CBAs, the upper pathway enzyme genes for PCB degradation of RHA1 were introduced into a CBA-degrading bacterium, Burkholderia sp. NK8. The resulting recombinant strain exhibited no biphenyl 2,3-dioxygenase (BphA) activity encoded by bphAaAbAcAd genes, which encode the large and small subunits of the terminal oxygenase component and the ferredoxin and reductase subunits responsible for electron transfer from NADH to the large subunit. The remaining enzyme genes involved in the transformation of biphenyl to benzoate, bphB2C1D1, which encode dehydrogenase, ring-cleavage dioxygenase and hydrolase, conferred activities to NK8. To obtain the BphA activity of RHA1 in NK8, sets of BphA genes were constructed by combining the bphAaAbAcAd genes of RHA1 and bphA3A4 of Pseudomonas pseudoalcaligenes KF707, encoding the ferredoxin and reductase subunits. Hybrid derivatives of BphA containing the KF707 bphA3 conferred BphA activity to NK8, and a derivative containing the RHA1 bphAaAb and KF707 bphA3A4 genes exhibited the highest BphA activity. A plasmid containing the RHA1 bphAaAb and KF707 bphA3A4 genes plus the RHA1 bphB2C1D1 genes was constructed and introduced into NK8. The resulting recombinant strain efficiently degraded 2-, 3- and 4-chlorobiphenyls with an apparent reduction in CBA accumulation in comparison to the recombinant mutant strain, which had an insertion in the cbeA gene to inactivate CBA dioxygenase.     

Nucleotide sequences and characterization of genes encoding naphthalene upper pathway of pseudomonas aeruginosa PaK1 and Pseudomonas putida OUS82

A 12,808-nucleotide containing DNA fragment cloned from naphthalene-utilizing (Nah+) Pseudomonas aeruginosa PaK1 was analyzed and compared with the genes (pah(OUS)) of a 14,462-nucleotide DNA fragment from Pseudomonas putida OUS82. The DNA sequence analyses demonstrated that the naphthalene upper-pathway genes and their deduced enzymes were very similar between the two bacteria: nucleotide similarities, 83-93%; amino acid similarities, 79-95%. These genes were also similar to those of the nah operon of plasmid NAH7; in particular, the OUS82 genes were similar to the nah genes, whereas the PaK1 genes were almost identical to the dox genes of Pseudomonas sp. C18. A region homologous with the 84-bp repeated sequence that Eaton (J. Bacteriol., 176, 7757-7762, 1994) has found at a site upstream of he nah operon was found only in a region downstream of the pah(PaK) gene cluster in PaK1 and on both sides of the pah(OUS) gene cluster in OUS82. A PaK1 gene, corresponding to an unknown gene (nahQ) in the nah operon, is located between the 1,2-dihydroxynaphthalene dioxygenase gene and the trans-o-hydroxybenzylindenepyruvate (tHBP A) hydratase-aldolase gene (nahE), and was suggested to be involved in the conversion of naphthalene to salicylate. Just downstream of the pah(PaK) gene cluster, a portion of a region was identical to one-third of the transposase gene (tnpA) in a phenol-catabolic plasmid pEST1226.     

Molecular cloning, identification and characterization of 2-methyl-3-hydroxypyridine-5-carboxylic-acid-dioxygenase-coding gene from the nitrogen-fixing symbiotic bacterium Mesorhizobium loti

The gene (mlr6788) of a nitrogen-fixing symbiotic bacterium Mesorhizobium loti MAFF303099 has been identified as a gene coding for 2-methyl-3-hydroxypyridine-5-carboxylic acid dioxygenase (MHPCO), the seventh enzyme in degradation pathway I for pyridoxine, a free form of vitamin B(6). The gene was cloned and overexpressed in Escherichia coli cells co-transformed with chaperonin genes. The homogeneous recombinant enzyme showed similar enzymatic properties to the enzyme from Pseudomonas sp. MA-1. MHPCO was essential for the assimilation of pyridoxine in M. loti, but not for its growth in a nutrient-rich medium. From the infection experiment of a symbiotic plant Lotus japonicus with an M. loti mlr6788 gene disruptant, MHPCO was demonstrated to be dispensable for at least nodule formation on roots of seedlings in symbiosis.     

Trichloroethylene degradation by Ralstonia sp. KN1-10A constitutively expressing phenol hydroxylase: transformation products, NADH limitation, and product toxicity

Ralstonia sp. KN1-10A, which was constructed by inserting the tac promoter upstream of the phenol hydroxylase (PH) gene in the chromosomal DNA of the wild-type strain, Ralstonia sp. KN1, is a useful recombinant strain for eliminating trichloroethylene (TCE) from contaminated sites because it exhibits constitutive TCE oxidation activity. During TCE degradation by Ralstonia sp. KN1-10A, noxious chlorinated compounds, such as dichloroacetic acid, trichloroacetic acid, 2,2,2-trichloroethanol, and chloral, were not detected, and more than 95% of chlorine in TCE was released as chloride ions. Among the possible TCE transformation products, only carbon monoxide was detected, and its conversion percentage was 7 mol%. The addition of formate, which Ralstonia sp. KN1-10A could use as an exogenous electron donor, did not enhance the TCE degradation performance, suggesting that NADH depletion did not limit the degradation. The phenol degradation activity of Ralstonia sp. KN1-10A that previously degraded TCE was not markedly lower than that of cells not exposed to TCE, suggesting that Ralstonia sp. KN1-10A was not susceptible to product toxicity associated with TCE degradation. Furthermore, to clarify the mechanisms underlying TCE degradation by PH from Ralstonia sp. KN1, this enzyme was compared with another enzyme, a hybrid aromatic ring dioxygenase exhibiting a high TCE degradation activity in Escherichia coli and Pseudomonas sp. The initial TCE degradation rate of Ralstonia sp. KN1 (pKTP100), which produced PH, was 1 50 lower than that of Ralstonia sp. KN1 (pKTF200), which produced the hybrid aromatic ring dioxygenase. However, because of its lower product toxicity, the strain producing PH could degrade 2.3 times more TCE than that generated by the strain producing the hybrid aromatic ring dioxygenase.     

Isolation of genes coding for chitin-degrading enzymes in the novel chitinolytic bacterium, Chitiniphilus shinanonensis, and characterization of a gene coding for a family 19 chitinase

Chitiniphilus shinanonensis type strain SAY3(T) is a strongly chitinolytic bacterium, originally isolated from the moat water in Ueda, Japan. To elucidate the chitinolytic activity of this strain, 15 genes (chiA-chiO) coding for putative chitin-degrading enzymes were isolated from a genomic library. Sequence analysis revealed the genes comprised 12 family 18 chitinases, a family 19 chitinase, a family 20 β-N-acetylglucosaminidase, and a polypeptide with a chitin-binding domain but devoid of a catalytic domain. Two operons were detected among the sequences: chiCDEFG and chiLM. The gene coding for the polypeptide (chiN) showed sequence similarity to family 19 chitinases and was successfully expressed in Escherichia coli. ChiN demonstrated a multi-domain structure, composed of the N-terminal, two chitin-binding domains connected by a Pro- and Thr-rich linker, and a family 19 catalytic domain located at the C-terminus. The recombinant protein rChiN catalyzed an endo-type cleavage of N-acetyl-d-glucosamine oligomers, and also degraded insoluble chitin and soluble chitosan (degree of deacetylation of 80%). rChiN exhibited an inhibitory effect on hyphal growth of the fungus Trichoderma reesei. The chitin-binding domains of ChiN likely play an important role in the degradation of insoluble chitin, and are responsible for a growth inhibitory effect on fungi.     

Identification and characterization of another 4-nitrophenol degradation gene cluster, nps, in Rhodococcus sp. strain PN1

4-Nitrophenol (4-NP) is a toxic compound formed in soil by the hydrolysis of organophosphorous pesticides, such as parathion. We previously reported the presence of the 4-NP degradation gene cluster (nphRA1A2) in Rhodococcus sp. strain PN1, which encodes a two-component 4-NP hydroxylase system that oxidizes 4-NP into 4-nitrocatechol. In the current study, another gene cluster (npsC and npsRA2A1B) encoding a similar 4-NP hydroxylase system was cloned from strain PN1. The enzymes from this 4-NP hydroxylase system (NpsA1 and NpsA2) were purified as histidine-tagged (His-) proteins and then characterized. His-NpsA2 showed NADH/FAD oxidoreductase activity, and His-NpsA1 showed 4-NP oxidizing activity in the presence of His-NpsA2. In the 4-NP oxidation using the reconstituted enzyme system (His-NpsA1 and His-NpsA2), hydroquinone (35% of 4-NP disappeared) and hydroxyquinol (59% of 4-NP disappeared) were detected in the presence of ascorbic acid as a reducing reagent, suggesting that, without the reducing reagent, 4-NP was converted into their oxidized forms, 1,4-benzoquinone and 2-hydroxy-1,4-benzoquinone. In addition, in the cell extract of recombinant Escherichia coli expressing npsB, a typical spectral change showing conversion of hydroxyquinol into maleylacetate was observed. These results indicate that this nps gene cluster, in addition to the nph gene cluster, is also involved in 4-NP degradation in strain PN1.     

Engineering a hybrid pseudomonad to acquire 3,4-dioxygenase activity for polychlorinated biphenyls

We constructed a hybrid strain that acquired 3,4-dioxygenase activity for polychlorinated biphenyls (PCBs). This strain, KF707-D34, possessed a chimeric biphenyl dioxygenase gene, of which a portion of bphA1 (coding for a large subunit of biphenyl dioxygenase) of Pseudomonas pseudoalcaligenes KF707 was replaced with that of a PCB-degrader, Burkholderia cepacia LB400 by homologous recombination. KF707-D34 retained the ability to degrade 4,4'-dichlorobiphenyl via 2,3-dioxygenation in a fashion identical to that of KF707 and gained novel capability to degrade 2,5,4'-trichlorobiphenyl and 2,5,2',5'-tetrachlorobiphenyl via 3,4-dioxygenation in a fashion identical to that of LB400. Sequence analysis of bphA1 from KF707-D34 revealed that three nucleotides in the 3'-terminal region of KF707 bphA1 were changed to correspond to those in LB400 bphA1. The resulting BphA1 protein in KF707-D34 was changed at position 376 from threonine (Thr) to asparagine (Asn). The results demonstrate that a minor alteration of the amino acid sequence in BphA1 improved the PCB degradation capability in biphenyl-utilizing bacteria.     

Characterization of alkylphenol degradation gene cluster in Pseudomonas putida MT4 and evidence of oxidation of alkylphenols and alkylcatechols with medium-length alkyl chain

Alkylphenols (APs) are ubiquitous contaminants in aquatic environments and have endocrine disrupting and toxic effects on aquatic organisms. To investigate biodegradation mechanisms of APs, an AP degradation gene cluster was cloned from a butylphenol (BP)-degrading bacterium, Pseudomonas putida MT4. The gene cluster consisted of 13 genes named bupBA1A2A3A4A5A6CEHIFG. From the nucleotide sequences, bupA1A2A3A4A5A6 were predicted to encode a multicomponent phenol hydroxylase (PH), whereas bupBCEHIFG were expected to encode meta-cleavage pathway enzymes. A partial sequence of a putative NtrC-type regulatory gene, bupR, was also found upstream of the gene bupB. This result indicates that APs can be initially oxidized into alkylcatechols (ACs), followed by the meta-cleavage of the aromatic rings. To confirm this pathway, AP degradation tests were carried out using the recombinant P. putida KT2440 harboring the PH genes (bupA1A2A3A4A5A6). The recombinant strain oxidized 4-n-APs with an alkyl chain of up to C7 (< or = C7) efficiently and also several BPs including those with an alkyl chain with some degree of branching. Therefore, it was found that PH had a broad substrate specificity for APs with a medium-length alkyl chain (C3-C7). Moreover, the cell extract of a recombinant Escherichia coli harboring bupB (a catechol 2,3-dioxygenase gene) converted 4-n-ACs with an alkyl chain of < or = C9 into yellow meta-cleavage products with a maximum absorbance at 379 nm, indicating that the second step enzyme in this pathway is also responsible for the degradation of ACs with a medium-length alkyl chain. These results suggest that MT4 is a very useful strain in the biodegradation of a wide range of APs with a medium-length alkyl chain, which known nonylphenol-degrading Sphingomonas strains have never degraded.     

Characterization of methylhydroquinone-metabolizing oxygenase genes encoded on plasmid in Burkholderia sp. NF100

Methylhydroquinone is an intermediate in the degradation of fenitrothion by Burkholderia sp. NF100. The catabolic gene (mhq) for methylhydroquinone degradation encoded on the plasmid pNF1 in the strain was cloned and sequenced. The mhq clone contained two ORFs, mhqA and mhqB, of which the deduced amino acid sequence shared significant homology with NAD(P)H-dependent flavoprotein monooxygenases and extradiol dioxygenases, respectively. Parts of the consensus sequences of the monooxygenase gene and dioxygenase gene have been identified in MhqA and MhqB from strain NF100, respectively. MhqA was overexpressed in Escherichia coli, and partially purified MhqA catalyzed the NADPH-dependent hydroxylation of methylhydroquinone. MhqB was also overexpressed in E. coli, and the purified enzyme showed an extradiol ring cleavage activity toward 3-methylcatechol but a very low activity was observed toward 4-methylcatechol.     

Organization of the capsule biosynthesis gene locus of the oral streptococcus Streptococcus anginosus

The capsular polysaccharide (CPS) of the important oral streptococcus Streptococcus anginosus, which causes endocarditis, and the genes for its synthesis have not been clarified. In this study, we investigated the gene locus required for CPS synthesis in S. anginosus. Southern hybridization using the cpsE gene of the well-characterized bacterium S. agalactiae revealed that there is a similar gene in the genome of S. anginosus. By using the colony hybridization technique and inverse PCR, we isolated the CPS synthesis (cps) genes of S. anginosus. This gene cluster consisted of genes containing typical regulatory genes, cpsA-D, and glycosyltransferase genes coding for glucose, rhamnose, N-acetylgalactosamine, and galactofuranose transferases. Furthermore, we confirmed that the cps locus is required for CPS synthesis using a mutant strain with a defective cpsE gene. The cps cluster was found to be located downstream the nrdG gene, which encodes ribonucleoside triphosphate reductase activator, as is the case in other oral streptococci such as S. gordonii and S. sanguinis. However, the location of the gene cluster was different from those of S. pneumonia and S. agalactiae.     

组成型过表达ido基因对4-羟基异亮氨酸合成的影响及其发酵培养基的响应面优化

为考察组成型过表达异亮氨酸羟化酶（isoleucine dioxygenase,IDO）基因ido对4-羟基异亮氨酸合成的影响,构建ido组成型表达质粒pXM01-ido及菌株HIL017,其ido转录量及IDO活性较诱导型过表达菌株HIL016显著提升,4-羟基异亮氨酸产量较HIL016提高19.4%。为进一步提高4-羟基异亮氨酸产量,通过Plackett-Burman试验确定HIL017发酵培养基中玉米浆、谷氨酸和FeSO4·7H2O用量为主要影响因素,利用最陡爬坡试验和响应面法确定其最优用量为玉米浆34.1 mL/L、谷氨酸2.98 g/L、FeSO4·7H2O 0.016 7 g/L,此时4-羟基异亮氨酸理论产量为5.57 g/L。验证实验结果表明,最佳条件下4-羟基异亮氨酸产量为5.53 g/L,较优化前提高19.7%。     

Over-expression of GSH1 gene and disruption of PEP4 gene in self-cloning industrial brewer's yeast

Foam stability is often influenced by proteinase A, and flavor stability is often affected by oxidation during beer storage. In this study, PEP4, the gene coding for proteinase A, was disrupted in industrial brewing yeast. In the meantime, one copy of GSH1 gene increased in the same strain. GSH1 is responsible for gamma-glutamylcysteine synthetase, a rate-limiting enzyme for synthesis of glutathione which is one kind of important antioxidant and beneficial to beer flavor stability. In order to improve the brewer's yeast, plasmid pYPEP, pPC and pPCG1 were firstly constructed, which were recombined plasmids with PEP4 gene, PEP4's disruption and PEP4's disruption+GSH1 gene respectively. These plasmids were verified to be correct by restriction enzymes' assay. By digesting pPCG1 with AatII and PstI, the DNA fragment for homologous recombination was obtained carrying PEP4 sequence in the flank and GSH1 gene internal to the fragment. Since self-cloning technique was applied in the study and the modified genes were from industrial brewing yeast itself, the improved strains, self-cloning strains, were safe to public. The genetic stability of the improved strains was 100%. The results of PCR analysis of genome DNA showed that coding sequence of PEP4 gene had been deleted and GSH1 gene had been inserted into the locus of PEP4 gene in self-cloning strains. The fermentation ability of self-cloning strain, SZ-1, was similar to that of the host. Proteinase A could not be detected in beer brewed with SZ-1, and GSH content in the beer increased 35% compared to that of the host, Z-1.     

Isolation and characterization of benzene-tolerant Rhodococcus opacus strains

Twenty-two benzene-utilizing bacteria were isolated from soil samples. Among them, three isolates were highly tolerant to benzene. They grew on benzene when liquid benzene was added to the basal salt medium at 10--90% (v/v). Taxonomical analysis identified the benzene-tolerant isolates as Rhodococcus opacus. One of the benzene-tolerant isolates, designated B-4, could utilize many aromatic and aliphatic hydrocarbons including benzene, toluene, styrene, xylene, ethylbenzene, propylbenzene, n-octane and n-decane as sole sources of carbon and energy. Strain B-4 grew well in the presence of 10% (v/v) organic solvents that it was capable of using as growth substrates. Genetic analysis revealed the benzene dioxygenase pathway is involved in benzene catabolism in strain B-4. A deletion-insertion mutant defective in the benzene dioxygenase large and small subunits genes (bnz A 1 and bnz A 2) was as tolerant to organic solvents as the wild-type strain B-4, suggesting that utilization or degradation of organic solvents is not essential for the organic solvent tolerance of R. opacus B-4.     

Relationship between some Staphylococcus aureus pathogenic factors and growth rates and somatic cell counts

Staphylococcus aureus isolates produce several pathogenic factors. The combination of these products influences the pathogenic role of different isolates, but their specific effects are well known in the pathogenesis of udder infections. This study focused on the association of polymorphism of the coagulase gene, protein A gene, collagen-binding protein gene, and of fibrinogen-binding protein gene on somatic cell count (SCC) and on Staph. aureus growth rate. Fifty Staph. aureus isolates from 13 dairy cow herds, located in seven different provinces, were considered. The results showed a low frequency of cna gene, similar to the one observed in human isolates. Meanwhile, the high frequency of efb gene indirectly confirmed the role of this factor in bacterial pathogenesis, being associated with adhesion to epithelia. The association of these two single genes with SCC and growth rate showed to be not significant. The polymorphism of spa gene was confirmed to be significantly associated with inflammatory response and growth rate, albeit with a pattern different from the one suggested for human isolates. Sorting of isolates based on the clusters obtained by combining polymorphisms of spa and coa genes and the presence of cna and efb genes, showed that a single cluster (cluster V) was prevalent in the different herds and provinces, while the other six clusters identified were widely spread among the remaining 60% of the isolates. Results showed that clusters VI and VII had significantly higher growth rates at 3, 4, and 6 h in comparison with the other clusters. Meanwhile, quarters infected with these strains showed significantly lower SCC levels. The frequency of isolates from cluster V, suggested that they should possess pathogenic factors increasing their invasiveness, even if in the presence of a stronger inflammatory response. These results indirectly confirm previous findings on the different interactions between isolates and the udder immune system. They also suggest that isolates with higher growth rates and inducing a lower inflammatory response have better chances to spread among the herd. The relatively simple genomic method proposed in this study could be applied by an increasing number of diagnostic laboratories and could be useful in studying the epidemiology of Staph. aureus intra-mammary infections in dairy herds when collecting data from the field.     

The Characterization of Two New Clusters of Duplicated Genes Suggests a ‘Lego’ Organization of the Yeast Saccharomyces cerevisiae Chromosomes

The systematic sequencing of 42 485 bp of yeast chromosome VII (nucleotides 377948 to 420432) has revealed the presence of 27 putative open reading frames (ORFs) coding for proteins of at least 100 amino acids. The degree of redundancy observed is elevated since five of the 27 ORFs are duplications of a previously identified gene. These duplicated copies may be classified in two types of cluster organization. The first type includes genes sharing a significant level of identity in the amino acid sequences of their predicted protein product. They are recovered on two different chromosomes, transcribed in the same orientation and the distance between them is conserved. The second type of cluster is based on one gene unit tandemly repeated. This duplication is itself repeated elsewhere in the genome. The level of nucleic acid identity is high within the coding sequence and the non-coding region between the two repeats. In addition, the basic gene unit is recovered many times in the genome and is a component of a multigene family of unknown function. These organizations in clusters of genes suggest a ‘Lego organization’ of the yeast chromosomes, as recently proposed for the genome of plants (Moore, 1995). The sequence is deposited in the Yeast Genome Databank under Accession Number from Z72562 to Z72586. © 1997 John Wiley & Sons, Ltd.     

17beta-estradiol-degrading bacteria isolated from activated sludge

Fourteen phylogenetically diverse 17beta-estradiol-degrading bacteria (strains KC1-14) were isolated from activated sludge of a wastewater treatment plant. These isolates widely distributed among eight different genera--Aminobacter (strains KC6 and KC7), Brevundimonas (strain KC12), Escherichia (strain KC13), Flavobacterium (strain KC1), Microbacterium (strain KC5), Nocardioides (strain KC3), Rhodococcus (strain KC4), and Sphingomonas (strains KC8-KC11 and KC14)--of three Phyla: Proteobacteria, Actinobacteria, and Bacteroidetes. All 14 isolates were capable of converting 17beta-estradiol to estrone, but only three strains (strains KC6, KC7, and KC8) showed the ability to degrade estrone. Only strain KC8 could use 17beta-estradiol as a sole carbon source. Based on the degree of estrogens being transformed and the estrogenicity of metabolites and/ or end products of estrogen degradation, three different degradation patterns (patterns A-C) were observed from degradation tests using resting cells. Eleven out of 14 isolates showed degradation pattern A, where 17beta-estradiol was stoichiometrically converted to estrone. Estrone was confirmed to be a degradation product of 17beta-estradiol; however, estrone was not further degraded during the course of experiments. Strains KC6 and KC7 exhibited degradation pattern B, where both 17beta-estradiol and estrone were degraded, with slower 17beta-estradiol degradation rates than those observed in pattern A. Strain KC8 was the only strain exhibited degradation pattern C, where 17beta-estradiol and estrone were rapidly degraded within 3 days. No residual 17beta-estradiol and estrone or estrogenic activity was detected after 5 days, suggesting that strain KC8 could degrade 17beta-estradiol into nonestrogenic metabolites/end products. Strains KC6-8 exhibited nonspecific monooxygenase activity but not nonspecific dioxygenase activity. However, the relationship between nonspecific monooxygenase activity and its estrogen degradation ability was unclear.     

Genomic differences between Candida glabrata and Saccharomyces cerevisiae around the MRPL28 and GCN3 loci

We report the sequences of two genomic regions from the pathogenic yeast Candida glabrata and their comparison to Saccharomyces cerevisiae. A 3 kb region from C. glabrata was sequenced that contains homologues of the S. cerevisiae genes TFB3, MRPL28 and STP1. The equivalent region in S. cerevisiae includes a fourth gene, MFA1, coding for mating factor a. The absence of MFA1 is consistent with C. glabrata's asexual life cycle, although we cannot exclude the possibility that a-factor gene(s) are located somewhere else in its genome. We also report the sequence of a 16 kb region from C. glabrata that contains a five-gene cluster similar to S. cerevisiae chromosome XI (including GCN3) followed by a four-gene cluster similar to chromosome XV (including HIS3). A small-scale rearrangement of gene order has occurred in the chromosome XI-like section.     

DNA sequence of the beta-isopropylmalate dehydrogenase gene and phylogenetic analysis of the yeast Saccharomyces exiguus Yp74L-3

The beta-isopropylmalate dehydrogenase (LEU2) gene from a homothallic wild-type yeast, Saccharomyces exiguus Yp74L-3, was analyzed to estimate the phylogenetic position of this strain in yeasts. The beta-isopropylmalate dehydrogenase gene of Yp74L-3 was first isolated as a clone complementing the leu2 mutation of Saccharomyces cerevisiae, and then confirmed to complement the haploid leu2 mutant derived from strain Yp74L-3 through genetic transformation. The nucleotide sequence of the cloned DNA revealed an open reading frame (ORF) encoding the beta-isopropylmalate dehydrogenase composed of 365 amino acids. The beta-isopropylmalate dehydrogenase coding sequence from the Yp74L-3 strain displayed 76.7% similarity to that of S. cerevisiae. Candidates for a UAS and a TATA-box in the 5'-upstream region and for a poly-A attachment site in the 3'-downstream region were found. A phylogenetic tree constructed from the nucleotide sequences of the beta-isopropylmalate dehydrogenase coding regions revealed that Yp74L-3 is located between S. cerevisiae and the Kluyveromyces yeasts. The LEU2 gene cloned from Yp74L-3 will serve as an effective genetic marker for constructing the transformation system in S. exiguus Yp74L-3.     

霍氏肠杆菌β-胡萝卜素9,10'双加氧酶的制备及其应用

以霍氏肠杆菌（Enterobacter hormaechei）基因组DNA为模板，通过聚合酶链式反应（PCR）法扩增β-胡萝卜素9,10'双加氧酶基因，构建重组质粒，在大肠杆菌（Escherichia coli）BL21（DE3）中表达，并采用高效液相色谱（HPLC）法检测9,10'双加氧酶活性。结果表明，通过镍柱亲和层析和分子筛Sephacryl TM S-200，得到纯化重组β-胡萝卜素9,10'双加氧酶，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）结果表明，该酶分子质量约57 kDa，最适反应温度为40 ℃，最适反应pH值为8.5；当底物β-胡萝卜素质量浓度为500 mg/L，β-胡萝卜素9,10'双加氧酶酶活为0.4 U/mL时，β-紫罗兰酮产量为142.3 mg/L，产率达到79.4%。