Genetic analysis of a gene cluster for cyclohexanol oxidation in Acinetobacter sp. Strain SE19 by in vitro transposition

Biological oxidation of cyclic alcohols normally results in formation of the corresponding dicarboxylic acids, which are further metabolized and enter the central carbon metabolism in the cell. We isolated an Acinetobacter sp. from an industrial wastewater bioreactor that utilized cyclohexanol as a sole carbon source. A cosmid library was constructed from Acinetobacter sp. strain SE19, and oxidation of cyclohexanol to adipic acid was demonstrated in recombinant Escherichia coli carrying a SE19 DNA segment. A region that was essential for cyclohexanol oxidation was localized to a 14-kb fragment on the cosmid DNA. Several putative open reading frames (ORFs) that were expected to encode enzymes catalyzing the conversion of cyclohexanol to adipic acid were identified. Whereas one ORF showed high homology to cyclohexanone monooxygenase from Acinetobacter sp. strain NCIB 9871, most of the ORFs showed only moderate homology to proteins in GenBank. In order to assign functions of the various ORFs, in vitro transposon mutagenesis was performed using the cosmid DNA as a target. A set of transposon mutants with a single insertion in each of the ORFs was screened for cyclohexanol oxidation in E. coli. Several of the transposon mutants accumulated a variety of cyclohexanol oxidation intermediates. The in vitro transposon mutagenesis technique was shown to be a powerful tool for rapidly assigning gene functions to all ORFs in the pathway.     

Cloning and characterization of a gene cluster for cyclododecanone oxidation in Rhodococcus ruber SC1

Biological oxidation of cyclic ketones normally results in formation of the corresponding dicarboxylic acids, which are further metabolized in the cell. Rhodococcus ruber strain SC1 was isolated from an industrial wastewater bioreactor that was able to utilize cyclododecanone as the sole carbon source. A reverse genetic approach was used to isolate a 10-kb gene cluster containing all genes required for oxidative conversion of cyclododecanone to 1,12-dodecanedioic acid (DDDA). The genes required for cyclododecanone oxidation were only marginally similar to the analogous genes for cyclohexanone oxidation. The biochemical function of the enzymes encoded on the 10-kb gene cluster, the flavin monooxygenase, the lactone hydrolase, the alcohol dehydrogenase, and the aldehyde dehydrogenase, was determined in Escherichia coli based on the ability to convert cyclododecanone. Recombinant E. coli strains grown in the presence of cyclododecanone accumulated lauryl lactone, 12-hydroxylauric acid, and/or DDDA depending on the genes cloned. The cyclododecanone monooxygenase is a type 1 Baeyer-Villiger flavin monooxygenase (FAD as cofactor) and exhibited substrate specificity towards long-chain cyclic ketones (C₁₁ to C₁₅), which is different from the specificity of cyclohexanone monooxygenase favoring short-chain cyclic compounds (C₅ to C₇).     

一株太平洋深海环己酮降解菌的筛选与功能研究

从太平洋深海菌株中筛选到一株能以环己酮为唯一碳源生长的微球菌(CN1),其最适生长温度为25℃~37℃,最适生长pH8,最适生长盐度6%。该菌可耐受高浓度环己酮(>44% V/V),并且在16.7%(V/V)的环己酮中生长最好。CN1可转化环己醇成环己酮,环己酮又可被快速降解、矿化。这表明该菌含有环己醇脱氢酶并且很可能还含有环己酮单加氧酶。通过兼并PCR克隆到450bp环己酮单加氧酶基因片段,其编码的氨基酸序列不仅具有Baeyer-Villiger单加氧酶家族的保守序列,而且与节杆菌(Arthrobacter BP2)的环己酮单加氧酶同源性最高(80%),而与研究较深入的不动杆菌(Acinetobacter sp.NCIMB 9871)单加氧酶的同源性仅为53%。由于目前报道的环己醇和环己酮的降解都是通过环己酮单加氧酶进行的,所以CN1的环己酮单加氧酶应该负责环己酮的降解。目前报道的环己酮降解菌都可以降解环戊酮,而CN1不可降解环戊酮,暗示了CN1的环己酮单加氧酶比较特别。另外,我们还首次发现在CN1中环己醇对环己酮的降解有一定的抑制作用。     

mRNA differential display in a microbial enrichment culture: simultaneous identification of three cyclohexanone monooxygenases from three species

mRNA differential display has been used to identify cyclohexanone oxidation genes in a mixed microbial community derived from a wastewater bioreactor. Thirteen DNA fragments randomly amplified from the total RNA of an enrichment subculture exposed to cyclohexanone corresponded to genes predicted to be involved in the degradation of cyclohexanone. Nine of these DNA fragments are part of genes encoding three distinct Baeyer-Villiger cyclohexanone monooxygenases from three different bacterial species present in the enrichment culture. In Arthrobacter sp. strain BP2 and Rhodococcus sp. strain Phi2, the monooxygenase is part of a gene cluster that includes all the genes required for the degradation of cyclohexanone, while in Rhodococcus sp. strain Phi1 the genes surrounding the monooxygenase are not predicted to be involved in this degradation pathway but rather seem to belong to a biosynthetic pathway. Furthermore, in the case of Arthrobacter strain BP2, three other genes flanking the monooxygenase were identified by differential display, demonstrating that the repeated sampling of bacterial operons shown earlier for a pure culture (D. M. Walters, R. Russ, H. Knackmuss, and P. E. Rouvière, Gene 273:305-315, 2001) is also possible for microbial communities. The activity of the three cyclohexanone monooxygenases was confirmed and characterized following their expression in Escherichia coli.     

The metabolism of cyclohexanol by Acinetobacter NCIB 9871.

Acinetobacter NCIB 9871 was isolated by elective culture on cyclohexanol and grows with this compound as sole source of carbon. It displays a restricted growth spectrum, being unable to grow on a wide range of alternative alicyclic alcohols and ketones. Cyclohexanol-grown cells oxidize the growth substrate at a rate of 230 mul of O2/h per mg dry wt with the consumption of 5.65 mumol of O2/mumol substrate. Cyclohexanone is oxidized at a similar rate with the consumption of 4.85 mumol of O2/mumol. 1-Oxa-2-oxocycloheptane and 6-hydroxyhexanoate are both oxidized at the same slow rate of 44 mul of O2/h per mg dry wt and adipate is not oxidized. Studies with cell extracts reveal the presence of inducible dehydrogenases for cyclohexanol, 6-hydroxyhexanoate and 6-oxohexanoate and a monooxygenase, that in conjunction with a lactonase converts cyclohexanone to 6-hydroxyhexanoate. The monooxygenase is therefore presumed to be of the lactone-forming type and the pathway for conversion of cyclohexanol to adipate; cyclohexanol leads to cyclohexanone leads to 1-oxa-2-oxocycloheptane leads to 6-hydroxyhexanoate leads to 6-oxohexanoate leads to adipate; for which key intermediates have been identified chromatographically, is identical with the route for the oxidation of cyclohexanol by Nocardia globerula CL1.     

mRNA Differential Display in a Microbial Enrichment Culture: Simultaneous Identification of Three Cyclohexanone Monooxygenases from Three Species

mRNA differential display has been used to identify cyclohexanone oxidation genes in a mixed microbial community derived from a wastewater bioreactor. Thirteen DNA fragments randomly amplified from the total RNA of an enrichment subculture exposed to cyclohexanone corresponded to genes predicted to be involved in the degradation of cyclohexanone. Nine of these DNA fragments are part of genes encoding three distinct Baeyer-Villiger cyclohexanone monooxygenases from three different bacterial species present in the enrichment culture. In Arthrobacter sp. strain BP2 and Rhodococcus sp. strain Phi2, the monooxygenase is part of a gene cluster that includes all the genes required for the degradation of cyclohexanone, while in Rhodococcus sp. strain Phi1 the genes surrounding the monooxygenase are not predicted to be involved in this degradation pathway but rather seem to belong to a biosynthetic pathway. Furthermore, in the case of Arthrobacter strain BP2, three other genes flanking the monooxygenase were identified by differential display, demonstrating that the repeated sampling of bacterial operons shown earlier for a pure culture (D. M. Walters, R. Russ, H. Knackmuss, and P. E. Rouvière, Gene 273:305-315, 2001) is also possible for microbial communities. The activity of the three cyclohexanone monooxygenases was confirmed and characterized following their expression in Escherichia coli.     

Cloning of Baeyer-Villiger monooxygenases from Comamonas,Xanthobacter and Rhodococcus using polymerase chain reaction with highly degenerate primers

To clone novel type 1 Baeyer-Villiger monooxygenase (BVMO) genes, we isolated or collected 25 bacterial strains able to grow on alicyclic compounds. Twelve of the bacterial strains yielded polymerase chain reaction (PCR) fragments with highly degenerate primers based on the sequences of known and putative BVMOs. All these fragments were found to encode peptides homologous to published BVMO sequences. The complete BVMO genes and flanking DNA were cloned from a Comamonas, a Xanthobacter and a Rhodococcus strain using the PCR fragments as probes. BVMO genes cloned from the first two strains could be expressed to high levels in Escherichia coli using standard expression vectors, and the recombinants converted cyclopentanone and cyclohexanone to the corresponding lactones. The Rhodococcus BVMO, a putative steroid monooxygenase, could be expressed after modification of the N-terminal sequence. However, recombinants expressing this protein did not show activity towards progesterone. An esterase homologue located directly upstream of the Xanthobacter BVMO gene and a dehydrogenase homologue encoded directly downstream of the Comamonas sp. NCIMB 9872 BVMO gene were also expressed in E. coli and shown to specify lactone hydrolase and cyclohexanol dehydrogenase activity respectively.     

A Method for Screening Baeyer-Villiger Monooxygenase Activity Against Monocyclic Ketones

The assay for Baeyer-Villiger monooxygenase (BVMO) enzyme activity has relied to date on the spectrophotometric change observed on the oxidation of the nicotinamide cofactor during the enzymatic reaction. By analogy to the cyclohexanol catabolic pathway of Acinetobacter calcoaceticus NCIMB 9871, we have developed a specific colorimetric screening method that utilises an esterase to cleave the lactone that is formed in the BVMO reaction. When carried out in a non-buffered or weakly buffered system the resultant change in pH can be visually detected. This allows the rapid assaying and screening of BVMO enzymes. This has been demonstrated with cyclohexanone monooxygenase from A. calcoaceticus. The resultant colour change has been visualised with washed cell suspensions, individual bacterial colonies on Petri dishes and with semi-purified recombinant enzyme utilising Linbro dishes.     

Purification and characterization of hexahistidine-tagged cyclohexanone monooxygenase expressed in Saccharomyces cerevisiae and Escherichia coli

Cyclohexanone monooxygenase (CMO) is a soluble flavoenzyme originally isolated from Acinetobacter spp. which carries out Baeyer-Villiger reactions with cyclic ketone substrates. In the present study we cloned the Acinetobacter CMO gene and modified it for facile purification from heterologous expression systems by incorporation of a His(6)-tag at its C-terminus. A single purification step employing metal (Ni(2+))-affinity column chromatography provided essentially homogeneous enzyme in yields of 69-72%. The properties of the purified, recombinant enzymes (rCMO) were compared with that of native CMO (nCMO) isolated from Acinetobacter cultures grown in the presence of cyclohexanone. The specific activities of His(6)-tagged rCMO and nCMO toward their index substrate, cyclohexanone, were similar and ranged from 14 to 20 micromol/min/mg. nCMO and rCMO from the Escherichia coli expression system exhibited molecular masses, determined by electrospray mass spectrometry, of 60,800 and 61,615 Da, respectively, an increase for the recombinant enzyme equivalent to the mass of the His(6)-tag. However, rCMO expressed in Saccharomyces cerevisiae consistently exhibited a mass some 50 Da larger than rCMO expressed in bacteria. Edman degradation confirmed that rCMO purified from the E. coli system and nCMO shared the same N-terminal sequence, whereas no sequence information could be obtained for rCMO expressed in yeast. Therefore, the yeast-expressed enzyme possesses an additional posttranslational modification(s), possibly acylation, at the N-terminus. Expression in E. coli is the preferred system for future site-directed mutagenesis studies and crystallization efforts.     

Processing of the Hepatitis C virus precursor protein expressed in the methylotrophic yeast Pichia pastoris

In this paper, the base frequency at the second codon position of the 3839 open reading frames (ORFs) in the Vibrio cholerae genome is analyzed. It is shown that according to the base content at this codon site, the ORFs can be divided into two clusters, each containing 673 and 3166 ORFs, respectively. ORFs in the smaller cluster usually have significantly higher T frequency than that of A at the second codon position. For the two clusters of ORFs, there are significant differences in the frequencies for 18 of the 20 amino acids in the encoding proteins. The two clusters of ORFs are also significantly different in their functions. More than half of the known genes involved in transport and binding are included in the smaller cluster, while few genes involved in amino acid biosynthesis, protein synthesis, and so on are included in this cluster.     

Identification of the monooxygenase gene clusters responsible for the regioselective oxidation of phenol to hydroquinone in mycobacteria

Mycobacterium goodii strain 12523 is an actinomycete that is able to oxidize phenol regioselectively at the para position to produce hydroquinone. In this study, we investigated the genes responsible for this unique regioselective oxidation. On the basis of the fact that the oxidation activity of M. goodii strain 12523 toward phenol is induced in the presence of acetone, we first identified acetone-induced proteins in this microorganism by two-dimensional electrophoretic analysis. The N-terminal amino acid sequence of one of these acetone-induced proteins shares 100% identity with that of the protein encoded by the open reading frame Msmeg_1971 in Mycobacterium smegmatis strain mc(2)155, whose genome sequence has been determined. Since Msmeg_1971, Msmeg_1972, Msmeg_1973, and Msmeg_1974 constitute a putative binuclear iron monooxygenase gene cluster, we cloned this gene cluster of M. smegmatis strain mc(2)155 and its homologous gene cluster found in M. goodii strain 12523. Sequence analysis of these binuclear iron monooxygenase gene clusters revealed the presence of four genes designated mimABCD, which encode an oxygenase large subunit, a reductase, an oxygenase small subunit, and a coupling protein, respectively. When the mimA gene (Msmeg_1971) of M. smegmatis strain mc(2)155, which was also found to be able to oxidize phenol to hydroquinone, was deleted, this mutant lost the oxidation ability. This ability was restored by introduction of the mimA gene of M. smegmatis strain mc(2)155 or of M. goodii strain 12523 into this mutant. Interestingly, we found that these gene clusters also play essential roles in propane and acetone metabolism in these mycobacteria.     

Survey of microbial oxygenases: trichloroethylene degradation by propane-oxidizing bacteria

Microorganisms that biosynthesize broad-specificity oxygenases to initiate metabolism of linear and branched-chain alkanes, nitroalkanes, cyclic ketones, alkenoic acids, and chromenes were surveyed for the ability to biodegrade trichloroethylene (TCE). The results indicated that TCE oxidation is not a common property of broad-specificity microbial oxygenases. Bacteria that contained nitropropane dioxygenase, cyclohexanone monooxygenase, cytochrome P-450 monooxygenases, 4-methoxybenzoate monooxygenase, and hexane monooxygenase did not degrade TCE. However, one new unique class of microorganisms removed TCE from incubation mixtures. Five Mycobacterium strains that were grown on propane as the sole source of carbon and energy degraded TCE. Mycobacterium vaccae JOB5 degraded TCE more rapidly and to a greater extent than the four other propane-oxidizing bacteria. At a starting concentration of 20 microM, it removed up to 99% of the TCE in 24 h. M. vaccae JOB5 also biodegraded 1,1-dichloroethylene, trans-1,2-dichloroethylene, cis-1,2-dichloroethylene, and vinyl chloride.