Biosci. Biotechnol. Biochem., 68 (10), 2171–2177, 2004

Biotransformation of Various Alkanes Using the Escherichia coli Expressing an Alkane Hydroxylase System from Gordonia sp. TF6

y Tadashi FUJII,1; Tatsuya NARIKAWA,1 Koji TAKEDA,1 and Junichi KATO2

1Bioresource Laboratories, Mercian Corporation, 1808 Nakaizumi, Iwata, Shizuoka 438-0078, Japan 2Department of Molecular Biotechnology Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan

Received June 8, 2004; Accepted July 20, 2004

Biotransformation using alkane-oxidizing or rubredoxin reductase (AlkT), which act as electron their alkane hydroxylase (AH) systems have been little carriers between NADH and monooxygenase.2) Among studied at the molecular level. We have cloned and gram-positive bacteria, the AH systems of two Rhodo- sequenced genes from Gordonia sp. TF6 encoding an AH coccus strains, Q15 and NRRL B-16531, have been system, alkB2 (alkane 1-monooxygenase), rubA3 (rubre- studied in detail. Both organisms contained at least four doxin), rubA4 (rubredoxin), and rubB (rubredoxin alkane 1-monooxygenase gene homologues (alkB1, reductase). When expressed in Escherichia coli, these alkB2, alkB3, and alkB4).3) The alkB1 and alkB2 genes allowed the construction of biotransformation homologues were part of AH gene clusters, each systems for various alkanes. Normal alkanes with 5 to 13 encoding two rubredoxins (rubA1 and rubA2; rubA3 carbons were good substrates for this biotransforma- and rubA4), and, in the alkB1 cluster, a rubredoxin tion, and oxidized to their corresponding 1-alkanols. reductase (rubB).3) Surprisingly, cycloalkanes with 5 to 8 carbons were Several AH system genes had been expressed hetero- oxidized to their corresponding cycloalkanols as well. logously. A DNA region of about 35-kbp containing AH This is the first study to achieve biotransformation of system genes from P. putida GPo1 was cloned into an alkanes using the E. coli expressing the minimum E. coli strain and into a mutant strain of P. putida, component genes of the AH system. Our biotransfor- unable to grow on alkanes. These transformants metab- mation system has facilitated assays and analysis leading olized n-alkanes as shown by mineralization and growth to improvement of AH systems, and has indicated a assays.2) Heterologous expression of other alkane 1- cycloalkane oxidation pathway in microorganisms for monooxygenase genes from several bacteria such as the first time. Rhodococcus strains,3) borkumensis AP1,4) Prauserella rugosa NRRL B-2295,4) and Mycobacteri- Key words: alkane hydroxylase; biotransformation; um tuberculosis H37Rv4) were confirmed using this Gordonia sp. TF6; cycloalkane oxidation P. putida system. Almost all the AH activities in these previous studies Alkanes are the most abundant family of hydro- were shown not by detection of the products, but by carbons in crude oil and are generated by many plants mineralization and growth assays in vivo. That is, AH and algae.1) Bacterial of normal alkanes (n- activity was detected only indirectly. In only one study alkanes) normally proceeds via sequential oxidation of a using P. putida strain PpS81, which lacks alcohol terminal methyl group to produce alcohol, aldehydes, dehydrogenase activity and carried the plasmids with a and finally fatty acids.1) It is known that many micro- DNA region of about 29-kbp containing AH system organisms are able to metabolize n-alkanes, but rela- genes, was the product of AH activity detected using the tively little is known about the system of alkane whole-cell reaction.5,6) In contrast, only the long DNA metabolism at the molecular level. region responsible for AH activity was characterized in The best-characterized system of n-alkane degrada- this study. tion is that of Pseudomonas putida GPo1.2) In this case, AH assays in vitro were performed with a reconsti- the initial oxidation step is performed by an alkane tuted hydroxylase system consisting of AlkB and AlkG hydroxylase (AH) system composed of a particulate expressed in E. coli, and with spinach ferredoxin nonheme integral-membrane alkane 1-monooxygenase reductase.6) But again, AH activity was detected only (AlkB) and two soluble proteins, rubredoxin (AlkG) and indirectly, this time by measuring cooxidation of

y To whom correspondence should be addressed. Tel: +81-538-21-1134; Fax: +81-538-21-1135; E-mail: [email protected] Abbreviations: AH, alkane hydroxylase; GC–MS, gas chromatography–mass spectrometry; IPTG, isopropyl--D-thiogalactopyranoside; n-alkanes, normal alkanes 2172 T. FUJII et al. Table 1. Primers Used in This Study

Primers Sequence (50!30) Primer 16SF GTGTTTGATCCTGGCTCAG Primer 16SF GTATTACCGCGGCTGCTG Primer TS2S AAYAGAGCTCAYGARYTRGGTCAYAAG Primer Deg1RE GTGGAATTCGCRTGRTGRTCIGARTG Primer Inv1 AAGACCAACACCGGTCGCTACGAACGAT Primer Inv2 TTCGACAGCCAGCGCTCGAGGTCGTCCTT Primer Inv3 TGGCGACCCTGCACCGTGAACGCGGTGT Primer Inv4 TACAGCACTGGACATTAACCGAGACAAT Primer AEx1 TTAGATCTGCATGAGTACCGCACCGACCCC Primer AEx2 GGCTGCAGGCCCTGCTGTCCTGGGCGAAGT Primer MutA3F GCGATGAGCACATTCCGTTGACCGGTCTGCGA Primer MutA3R CAACGGAATGTGCTCATCGCGGCGACACCCC Primer MutA4F AATTTCAAGCTTTTCCGTTGAGAGGTATGCGG Primer MutA4R CAACGGAAAAGCTTGAAATTCATCGTACTGC

R, A+G; Y, C+T; I, inosine.

NADH. The extent of the AH reaction was very limited, previous study.10) Gordonia sp. TF6 was characterized possibly due to the insolubility of alkanes and enzymes and identified by biochemical tests and 16S rRNA gene in water. sequences. The biochemical tests were performed based Recently, much attention has been focused on the on Cowan and Steel’s manual.11) Part of the 16S rRNA importance of regio-selective oxidation of chemicals for gene of 479-bp was cloned and sequenced by PCR using the production of medicinal drugs. These oxidized Primer 16SF and Primer 16SR as shown in Table 1. chemicals can be prepared by chemical or biocatalytic reactions. The advantages of biocatalytic reactions are DNA manipulation. Chromosomal DNA from Gordo- that they are regio-selective, one-step reactions, and can nia sp. TF6 was prepared using Isoplant (Nippon Gene). take place at ambient temperature and pressure. Fur- Plasmids from E. coli strains were prepared using a thermore, biotransformation using living microbial cells GFX Micro Plasmid Prep Kit (Amersham Pharmacia by fermentation is useful, especially when the enzymatic Biotech). DNA ligation was performed using a Ligation reaction requires cofactors that must be regenerated. Kit (TaKaRa). All restriction enzymes were obtained Considerable interest is being devoted to the use of from TaKaRa. All amplifications by PCR or inverse bacterial alkane oxidation systems as biocatalysts for the PCR were performed in a reaction mixture (50 l) production of fine chemicals and pharmaceuticals. Using containing template DNA (50 ng), both primers (25 pM bacteria with alkane-oxidizing ability, biotransformation each), MgCl2 (2.5 mM), dNTPs (0.25 mM each), and LA reactions of various , such as cumene,7) 2, Taq polymerase (1 U) in the buffer for LA Taq polymer- 5-dimethylhexane,8) and N-(2-hexylamino-4-phenylimi- ase (TaKaRa). The primers used in this study were dazole-1-yl)-acetamide9) have been performed. How- synthesized by Sigma Genosys, as shown in Table 1, ever, little study has been conducted on these biotrans- and the plasmids used in this study were constructed as formations at the molecular level. shown in Table 2. Site-directed mutagenesis was per- No AH system of Gordonia sp. has been characterized formed using the GeneTailor Site-Directed Mutagenesis at the molecular level. In this study we isolated a n- System (Invitrogen). DNA sequencing analysis was decane utilizing bacterium, Gordonia sp. TF6, and from done using the BigDye Terminator v3.1 Cycle Sequenc- this strain genes encoding an AH system (alkane 1- ing Kit (Applied Biosystems). monooxygenase, rubredoxins, and rubredoxin reductase) were cloned, sequenced, and expressed in E. coli. This is Cloning of a DNA fragment encoding part of AlkB2. the first study to achieve biotransformation of alkanes In order to clone part of the gene encoding AlkB2, we using the E. coli expressing the minimum component used the method described previously.12) Highly degen- genes of the AH system. erate primers, Primer TS2S and Primer deg1RE, shown in Table 1, were designed on the basis of conserved Materials and Methods sequence motifs. Degenerate PCR was performed for 25 cycles of denaturation (98 C, 20 s), annealing (40 C, Isolation of Gordonia sp. TF6. We isolated the n- 1 min), and extension (68 C, 1 min) with these primers decane utilizing bacterium Gordonia sp. TF6 from the and Gordonia sp. TF6 chromosomal DNA. About 550- soil on a tennis court in Fujisawa, Kanagawa, Japan by bp of PCR product was amplified, ligated into pT7 Blue the methods described previously.10) Growth assay of T-vector (Novagen), and sequenced. Gordonia sp. TF6 was performed using submerged medium with 1% (v/v) n-alkane with 5 to 16 carbons. Inverse PCR for sequences of the DNA region The medium compositions were the same as shown in encoding the AH system. In order to clone the 50- and Biotransformation Using E. coli Expressing an Alkane Hydroxylase System 2173 Table 2. Plasmids Used in This Study

Plasmids Relevant characteristics Genotype pAL526 pTrcHisA (Invitrogen) derivative containing 2825-bp BglII–PstI alkB2, rubA3, rubA4, rubB insert amplified by PCR with Primer AEx1 and Primer AEx2 pAL520 pAL526 derivative digested with PshAI and self-ligated with T4 ligase. alkB2, rubA3, rubA4 pAL501 pAL526 derivative carrying rubA4 whose Cys8-codon (TGC) was exchanged to alkB2, rubA3, rubB a termination codon (TGA)a with Primer MutA4F and Primer MutA4R pAL502 pAL526 derivative carrying rubA3 whose Cys6-codon (TGT) was exchanged to alkB2, rubA4, rubB a termination codon (TGA)a with Primer MutA3F and Primer MutA3R pAL503 pAL502 derivative carrying rubA4 whose Cys8-codon (TGC) was exchanged to alkB2, rubB a termination codon (TGA)a with Primer MutA4F and Primer MutA4R pAL026 pAL526 derivative digested with XhoI and self-ligated with T4 ligase. rubA3, rubA4, rubB

Primers are shown in Table 1. a, Site-directed mutagenesis was performed using the GeneTailor Site-Directed Mutagenesis System (Invitrogen).

30-flanking regions of the sequenced PCR product, (50 g per ml final concentration). After 19 h of inverse PCR was performed.13) As a template, chromo- incubation at 28 C with shaking, 300 lof2NHCl somal DNA from Gordonia sp. TF6 was completely and 500 l of the alkane were added and mixed. We digested with SalIorBamHI and circularized. Ampli- defined the supernatant of this biotransformation reac- fications by inverse PCR were performed for 25 cycles tion mixture as the alkane-phase. We performed qual- of denaturation (98 C, 20 s) and annealing-extension itative analysis of each biotransformation reaction (68 C, 4 min) with Primer Inv1 and Primer Inv2, as product in the alkane-phase by gas chromatography– shown in Table 1. These inverse PCR products were mass spectrometry (GC–MS), and quantitative analysis ligated into pT7 Blue T-vector, and a DNA region of by gas chromatography. In particular, as for the about 2-kbp was sequenced. Further, in order to clone biotransformation of n-octane or cyclohexane, we the 50- and 30-flanking regions of the sequenced region, performed quantitative analysis of each reaction inverse PCR was performed again. As a further product after 0.5 h, 1 h, 3 h, 12 h, and 19 h of incubation template, another chromosomal DNA from Gordonia time. sp. TF6 was completely digested with MluI and circularized. Amplifications by inverse PCR were GC–MS analysis. Qualitative analysis of each bio- performed for 25 cycles of denaturation (98 C, 20 s) transformation reaction product in the alkane-phase was and annealing-extension (68 C, 3 min) with Primer Inv3 performed by GC–MS using the TurboMass Gold and Primer Inv4, as shown in Table 1. This inverse PCR system (PerkinElmer). A CombiPAL system and Cycle product was ligated into pT7 Blue T-vector and Composer software (CTC Analytics AG) were used for sequenced. autosampling. Mass spectra were obtained by the electron ionization method (70 eV), and chromatograms Expression of the AH system in E. coli TOP10. E. coli were measured by total and selected ion monitoring. A TOP10 (Invitrogen) harboring pAL526, pAL520, TC-WAX capillary column (30 m by 0.25 mm by pAL501, pAL502, pAL503, pAL026, or pTrcHisA was 0.25 m, GL Sciences) was used with helium at a flow cultivated at 28 C with shaking in L-broth (polypeptone rate of 20 ml/min as the carrier gas. The temperature 1.0%, yeast extract 0.5%, NaCl 0.5%, glucose 0.1%, program had an initial oven temperature of 120 C, pH 7.2) containing 100 g of ampicillin sodium per ml. which was then increased at rates of 5 C/min to 160 C, Five hundred l of overnight-grown culture was added 10 C/min to 180 C for 7 min, and 10 C/min to 230 C to 50 ml of L-broth containing 100 g ampicillin sodium for 3 min. The injector and detector temperatures were per ml. The culture was grown at 28 C with shaking, 260 C and 250 C respectively. One l of the sample and after 3 h of cultivation, expression of AH genes was was analyzed by injecting 1/20 of this amount in the induced by the addition of isopropyl--D-thiogalacto- split injection mode onto the column. The products were pyranoside (IPTG) (0.1 mM final concentration). After identified by matching the retention times and mass an additional 5 h of cultivation, cells were collected. spectra with authentic standards. The TurboMass NIST/ EPA/NIH Mass Spectral Database was used in achiev- Biotransformation of various alkanes. Fifty ml of ing agreement between mass spectra. E. coli cells expressing the AH system were collected (about 0.2 g of wet cells) and suspended in 5 ml of 0.2 M Gas chromatography analysis. Quantitative analysis sodium phosphate buffer (pH 7.2). Five hundred lof of each biotransformation reaction product in the each alkane was added to 1.5 ml of this cell-suspended alkane-phase was performed by gas chromatography buffer containing glycerol (0.66% final concentration), using a GC-17A system with a flame ionization detector FeSO4.7H2O (100 g per ml final concentration), IPTG (Shimadzu). The analytical conditions were the same as (0.1 mM final concentration), and ampicillin sodium for GC–MS analysis. 2174 T. FUJII et al. Results and Methods, we obtained a PCR product of the expected length of 556-bp. This PCR product was Isolation of Gordonia sp. TF6 sequenced, and a BLAST search indicated that this PCR We isolated a n-decane utilizing bacterium. This product encodes peptides that have a high level of amino strain was able to metabolize and grow on n-alkanes acid sequence identity with the corresponding region of with 5 to 16 carbons, except for n-hexane and n-heptane. alkane 1-monooxygenase homologous proteins. Then, We cloned and sequenced the 16S rRNA gene of 479-bp using the inverse PCR method, the 50- and 30-flanking from this strain (accession no. AB125675). A BLAST regions of the sequenced PCR product were cloned search indicated that this DNA sequence showed great and sequenced. The 3491-bp DNA sequence in this identity with the DNA sequences of 16S rRNA genes region has been deposited in GenBank (accession no. from several strains of Gordonia sp., and we designated AB112870). It was found that there were four consec- this bacterium Gordonia sp. TF6. The results of several utive open reading frames whose products show the biochemical tests are shown in Table 3. greatest sequence identities with the com- plete sequences of AH components from Rhodococcus Cloning of the genes encoding the AH system from strain NRRL B-16531 (Table 4). We have designated Gordonia sp. TF6 these four genes alkB2 (alkane 1-monooxygenase), Sequence alignments of the amino acid sequences of rubA3 (rubredoxin), rubA4 (rubredoxin), and rubB AHs from Pseudomonas putida GPo1 and Acinetobacter (rubredoxin reductase). These sequence data suggested sp. ADP1 indicated that several regions are well that these four genes compose the AH system. conserved, and two degenerate PCR primers, Primer TS2S and Primer deg1RE, had been described previ- Expression of the genes encoding the AH system from ously.12) Using the PCR program described in Materials Gordonia sp. TF6 and biotransformation of n-octane To determine whether the four genes (alkB2, rubA3, rubA4, and rubB) encode the component proteins of the Table 3. Properties of Gordonia sp. TF6 AH system, we tried to express them in E. coli. Plasmid Properties Results pAL526, containing these four genes under the control Gram staining positive of trc promoter, or vector control plasmid pTrcHisA Morphology rod were introduced into E. coli TOP10, and each biotrans- Colony color orange formation of n-octane was performed using these cells. Spore formation About 74.4 g/ml of 1-octanol was produced from n- þ Glossy octane in the alkane-phase by biotransformation using Denitrification Indole production E. coli TOP10 carrying pAL526. On the other hand, Hydrogen sulfide production no 1-octanol was produced by biotransformation using -Galactosidase reaction E. coli TOP10 carrying pTrcHisA. Catalase reaction þ Moreover, several deletion or mutant plasmids were Urease reaction constructed, and biotransformation reactions of n-octane Hydrolysis of starch Acid or gas production from were performed using E. coli TOP10 carrying each arabinose plasmid (Fig. 1). Relative biotransformation activity fructose was estimated as the percentage of the amount of 1- glucose octanol in the alkane-phase produced by E. coli Top10 inositol carrying each plasmid against that by E. coli Top10 mannitol maltose carrying pAL526 (74.4 g/ml). Biotransformation using lactose cells carrying pAL520, which had a deletion of 264-bp sucrose in rubB, showed almost the same, but slightly lower, glycerol level of relative biotransformation activity. Biotransfor- xylose mation using cells carrying pAL502, having a mutation

Table 4. Comparison of Alkane Hydroxylase Systems between Gordonia sp. TF6 and R. erythropolis NRRL B-16531

Number of amino acid residues Gene Locationa (bp) Functions of possible gene products Amino acid identityb TF6 NRRL B-16531 alkB2 597–1832 Alkane 1-monooxygenase 411 408 301/396 (76%) rubA3 1829–1999 Rubredoxin 56 61 39/51 (76%) rubA4 1996–2175 Rubredoxin 59 60 45/56 (80%) rubB 2172–3374 Rubredoxin reductase 400 418 161/390 (41%)

a, Location in reference to the 3,491-bp contig. b, Number of amino acids with identities between TF6 and NRRL B-16531/Number of amino acids (%). Biotransformation Using E. coli Expressing an Alkane Hydroxylase System 2175

Fig. 1. Alkane Hydroxylase System Genes on Various Plasmids and Relative Biotransformation Activity Using E. coli TOP10 Transformed with Each Plasmid. Each arrow shows the corresponding gene belonging to the AH system. Bold bars show the DNA region carried on each plasmid. Crosses show the points of stop codon exchanged from cysteine codon described in Table 2. N.D., not detected.

in rubA3 whose Cys6-codon (TGT) was exchanged to a Table 5. Biotransformation of Various Alkanes Using E. coli TOP10 termination codon (TGA), or biotransformation using Carrying pAL526 cells carrying pAL501, having a mutation in rubA4 Amount of the product whose Cys8-codon (TGC) was exchanged to a termi- Substrate Product nation codon (TGA), showed about 20% of relative (g/ml) biotransformation activity. Moreover, cells carrying n-Pentane 1-Pentanol 28.5 pAL503, which had mutations both in rubA3 and in n-Hexane 1-Hexanol 102.9 rubA4, showed very little relative biotransformation n-Heptane 1-Heptanol 56.1 n-Octane 1-Octanol 74.4 activity. Still more, biotransformation using cells carry- n-Nonane 1-Nonanol 64.9 ing pAL026, which had a deletion of 874-bp in alkB2, n-Decane 1-Decanol 68.1 showed no relative biotransformation activity. n-Undecane 1-Undecanol 49.1 From these results and sequence data, we considered n-Dodecane 1-Dodecanol 35.5 that alkB2, rubA3, rubA4, and rubB are the minimum n-Tridecane 1-Tridecanol 12.1 n-Tetradecane 1-Tetradecanol N.D. component genes of the AH system when expressed in n-Pentadecane 1-Pentadecanol N.D. E. coli, so we used E. coli TOP10 carrying pAL526 for n-Hexadecane 1-Hexadecanol N.D. following biotransformation. Cyclopentane Cyclopentanol 1.13 Cyclohexane Cyclohexanol 26.8 Biotransformation of various alkanes Cycloheptane Cycloheptanol 25.1 Cyclooctane Cyclooctanol 8.7 We have identified the minimum component genes of the AH system when expressed in E. coli, which The Amount of the product in the alkane-phase was measured by gas chromatography analysis. allowed the construction of the biotransformation N.D., not detected. system oxidizing n-octane to 1-octanol. Then we performed biotransformation of various alkanes using E. coli TOP10 carrying pAL526 (Table 5). n-Alkanes only one carbon of each cycloalkane, and no more with 5 to 13 carbons were good substrates for this oxidized products were detected. On the other hand, no biotransformation system, and were oxidized to their alkanols were detected by biotransformation using corresponding 1-alkanols. The greatest amount of 1- E. coli TOP10 carrying pTrcHisA. alkanol (102.9 g/ml of 1-hexanol) was accumulated in The time course of the biotransformation of n-octane the alkane-phase by biotransformation of n-hexane. or cyclohexane using E. coli TOP10 carrying pAL526 is Surprisingly, cycloalkanes with 5 to 8 carbons were shown in Fig. 2. The amount of each reaction product oxidized to their corresponding cycloalkanols as well, (1-octanol or cyclohexanol) increased with time, and and a significant amount of cycloalkanol (26.8 g/ml of reached almost to the highest amount after 12 h of cyclohexanol) was accumulated in the alkane-phase by incubation time. At the same time, we performed biotransformation of cyclohexane. This biotransforma- biotransformation using E. coli TOP10 carrying tion using E. coli TOP10 carrying pAL526 oxidized pTrcHisA, and no oxidized alkanes were detected in only one side of the terminal carbon of each n-alkane or any incubation time. 2176 T. FUJII et al. condition. First, the substrate should always be in plentiful supply in order to produce as much biotrans- formation product (alkanol) as possible. Secondly, the product should be in the substrate-phase during the biotransformation reaction in order to avoid the pro- duct’s toxicity for host cells. In the substrate-limited condition, we might be able to improve the conversion rate of this biotransformation. E. coli expressing the AH system converted n-alkanes with 5 to 13 carbons to their corresponding alcohols. On the other hand, Gordonia sp. TF6 was able to grow on n- alkanes with 5 to 16 carbons, except for n-hexane and n- heptane. These results suggest that Gordonia sp. TF6 has other AH systems to oxidize n-alkanes with more than 14 carbons. In fact, previous studies have reported that some microorganisms have more than two AH systems.3) Further, it is interesting that Gordonia sp. TF6 Fig. 2. Time Course of Biotransformation of n-Octane or Cyclo- was not able to grow on n-hexane or n-heptane. It might hexane. be that n-hexane and n-heptane are more toxic to Biotransformation of n-octane or cyclohexane was performed using E. coli Top10 carrying pAL526. Its reaction product in the microbial cells than other n-alkanes. The superiority of alkane-phase, 1-octanol ( ) or cyclohexanol ( ), was measured biotransformation using E. coli might have overcome after 0.5 h, 1 h, 3 h, 12 h, and 19 h of incubation time. this toxicity. Cycloalkanes are components of . Under- standing the bacterial degradation pathway of cyclo- Discussion alkanes, as well as that of n-alkanes, is important from the standpoint of bioremediation. Although n-alkane Genes encoding an AH system (alkane 1-monooxy- hydroxylase systems have been studied as described genase, rubredoxins, and rubredoxin reductase) from above, the enzyme that oxidizes cycloalkanes has not Gordonia sp. TF6 were cloned, sequenced, and ex- been identified. It has merely been reported that cell pressed in E. coli. This is the first study to achieve extracts of a cyclohexane-growing strain Xanthobacter biotransformation of n-alkanes or cycloalkanes using the sp. oxidized cyclohexane to cyclohexanol depending on E. coli expressing the minimum component genes of the NADPH.14,15) It is known that the bacterial metabolism AH system. of cyclohexanol to adipic acid proceeds via cyclo- Although the AH system has been considered to be a hexanone, "-caprolactone, 6-hydroxyhexanoic acid, and useful method for the regio-selective oxidation of 6-oxohexanoic acid.16) Each gene encoding the enzyme various hydrocarbons, and although many alkane-oxi- involved in this pathway has been identified,17) so that dizing bacteria have been used for the production of the identification of the enzyme oxidizing cyclohexane hydroxyl chemicals, little has been studied about these to cyclohexanol and its coding gene is essential for at the molecular level. As described studies of the cycloalkane degradation pathway. It has above, several heterologous expressions of AH systems been thought that AH catalyzes the terminal oxidation of were studied in vivo based on the long DNA region from n-alkanes. However, previous studies have suggested by P. putida GPo1, and AH activity in vitro was detected detecting NADH cooxidation6) and our biotransforma- only indirectly by measuring cooxidation of NADH. tion system has confirmed that AH oxidizes cyclo- These vague or complicated methods have probably alkanes as well as n-alkanes. This study has indicated a been impediments to the study of AH systems. Our cycloalkane degradation pathway in microorganisms for biotransformation system using the E. coli expressing the first time. the minimum component genes of the AH system has Our biotransformation system using E. coli express- facilitated assays and analysis leading to improvement ing the minimum component genes of the AH system of AH systems. For example, analysis using amino acid oxidized various alkanes to their corresponding alco- exchange methods or error-prone PCR methods can hols, resulting in a considerable accumulation of these provide useful information about the active sites of an products. This probably depends on the characteristics enzyme, the increases in enzyme activity, or the of E. coli, namely, the high level of expression of the extension of substrate specificity. We are in fact trying AH system, the capacity for NADH regeneration, and to create genetically improved AH systems. the lack of a metabolic pathway for the alcohols Our biotransformation reactions in this paper were produced. In the near future, biotransformation using performed under conditions of substrate (alkane) excess, E. coli expressing the genetically improved AH system in which the volume ratio of the substrate to the cell- will perhaps be capable of oxidizing a wider range of suspended buffer was 1:3. There are two reasons for this hydrocarbons. Biotransformation Using E. coli Expressing an Alkane Hydroxylase System 2177 Acknowledgment grown cells. Appl. Microbiol. Biotechnol., 41, 178–182 (1994). This study was carried out as part of the Project 8) Matsui, T., and Furuhashi, K., Asymmetric oxidation of for Development of a Technological Infrastructure for isopropyl moieties of aliphatic and aromatic hydro- Industrial Bioprocesses on R&D of New Industrial carbons by Rhodococcus sp. 11B. Biosci. Biotechnol. Biochem., 59, 1342–1344 (1995). Science and Technology Frontiers conducted by the 9) Mikolasch, A., Hammer, E., and Schauer, F., Synthesis Ministry of Economy, Trade and Industry (METI) of of imidazole-2-yl amino acids by using cells from Japan, and entrusted to us by the New Energy and alkane-oxidizing bacteria. Appl. Environ. Microbiol., 69, Industrial Technology Development Organization 1670–1679 (2003). (NEDO). 10) Vomberg, A., and Klinner, U., Distribution of alkB genes within n-alkane-degrading bacteria. J. Appl. References Microbiol., 89, 339–348 (2000). 11) Barrow, G. I., and Feltham, R. K. A., Cowan and Steel’s 1) Rehm, H. J., and Reiff, I., Mechanisms and occurrence manual for the identification of medical bacteria, 3rd of microbial oxidation of long chain alkanes. Adv. edition., Cambridge University Press (1993). Biochem. Eng., 19, 175–215 (1981). 12) Smits, T. H. M., Rothlissberger, M., Witholt, B., and van 2) Eggink, G., Lageveen, R. G., Altenburg, B., and Witholt, Beilen, J. B., Molecular screening for alkane hydrox- B., Controlled and functional expression of the Pseudo- ylase genes in gram-negative and gram-positive strains. monas oleovorans alkane utilizing system in Pseudomo- Environ. Microbiol., 1, 307–317 (1999). nas putida and Escherichia coli. J. Biol. Chem., 262, 13) Ochman, H., Gerber, A. S., and Hartl, D. L., Genetic 17712–17718 (1987). applications of an inverse polymerase chain reaction. 3) Whyte, L. G., Smits, T. H. M., Labbe, D., Witholt, B., Genetics, 120, 621–623 (1988). Geer, C. W., and van Beilen, J. B., Gene cloning and 14) Trower, M. K., Buckland, R. M., Higgins, R., and characterization of multiple alkane hydroxylase systems Griffin, M., Isolation and characterization of a cyclo- in Rhodococcus strains Q15 and NRRL B-16531. Appl. hexane-metabolizing Xanthobacter sp. Appl. Environ. Environ. Microbiol., 68, 5933–5942 (2002). Microbiol., 49, 1282–1289 (2003). 4) Smits, T. H. M., Balada, S. B., Witholt, B., and van 15) Trickett, J. M., Hammonds, E. J., Worrall, T. L., Trower, Beilen, J. B., Functional analysis of alkane hydroxylase M. K., and Griffin, M., Characterization of cyclohexane from gram-negative and gram-positive bacteria. J. hydroxylase. A three-component enzyme system from a Bacteriol., 184, 1733–1742 (2002). cyclohexane-grown Xanthobacter sp. FEMS Microbiol. 5) Bosetti, A., van Beilen, J. B., Preusting, H., Lageveen, Lett., 82, 329–334 (1991). R. G., and Witholt, B., Production of primary aliphatic 16) Donoghue, N. A., and Trudgill, P. W., The metabolism alcohols with a recombinant Pseudomonas strain. of cyclohexanol by Acinetobacter NCIB 9871. Eur. J. Enzyme Microb. Technol., 14, 702–708 (1992). Biochem., 60, 1–7 (1975). 6) Van Beilen, J. B., Kingma, J., and Witholt, B., Substrate 17) Cheng, Q., Thomas, S. M., Kichka, K., Valentine, J. R., specificity of the alkane hydroxylase of Pseudomonas and Nagarajan, V., Genetic analysis of a gene cluster for oleovorans GPo1. Enzyme Microb. Technol., 16, 904– cyclohexanol oxidation in Acinetobacter sp. strain SE19 911 (1994). by in vitro transposition. J. Bacteriol., 182, 4744–4751 7) Hou, C. T., Jackson, M. A., Bagby, M. O., and Becker, (2000). L. A. M., Microbial oxidation of cumene by octane-