Reconstitution of Active Mycobacterial Binuclear Iron MonooxygenaseComplex in Escherichia coli
Toshiki Furuya, Mika Hayashi, Kuniki Kino
Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo, Japan
Bacterial binuclear iron monooxygenases play numerous physiological roles in oxidative metabolism. Monooxygenases of thistype found in actinomycetes also catalyze various useful reactions and have attracted much attention as oxidation biocatalysts.However, difficulties in expressing these multicomponent monooxygenases in heterologous hosts, particularly in Escherichiacoli, have hampered the development of engineered oxidation biocatalysts. Here, we describe a strategy to functionally expressthe mycobacterial binuclear iron monooxygenase MimABCD in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gelelectrophoretic analysis of the mimABCD gene expression in E. coli revealed that the oxygenase components MimA and MimCwere insoluble. Furthermore, although the reductase MimB was expressed at a low level in the soluble fraction of E. coli cells, aband corresponding to the coupling protein MimD was not evident. This situation rendered the transformed E. coli cells inac-tive. We found that the following factors are important for functional expression of MimABCD in E. coli: coexpression of thespecific chaperonin MimG, which caused MimA and MimC to be soluble in E. coli cells, and the optimization of the mimD nucle-otide sequence, which led to efficient expression of this gene product. These two remedies enabled this multicomponent mono-oxygenase to be actively expressed in E. coli. The strategy described here should be generally applicable to the E. coli expressionof other actinomycetous binuclear iron monooxygenases and related enzymes and will accelerate the development of engineeredoxidation biocatalysts for industrial processes.
Bacterial binuclear iron monooxygenases are a family of pro-teins that contain a binuclear iron center at the active site and
are widely distributed throughout prokaryotes (1–3). These en-zymes play numerous physiological roles in the oxidative metab-olism of organic compounds, including alkanes, alkenes, andaromatics. Monooxygenases of this type found in actinomycetesconstitute a new subfamily within the family of binuclear ironmonooxygenases (1–3). These actinomycetous monooxygenasesconsist of four components, an oxygenase large subunit, an oxy-genase small subunit, a reductase, and a coupling protein. Nota-bly, the oxygenase component in these actinomycetous enzymescomprises two subunits in an �� or an �2�2 quaternary structure,whereas this component in the enzymes of other bacteria, includ-ing methanotrophs and pseudomonads, comprises three subunitsin an �2�2�2 quaternary structure. The oxygenase component ac-tivates molecular oxygen using electrons that are transferred fromNAD(P)H by the reductase component (4, 5). The coupling pro-tein interacts with the oxygenase component and is essential forfull oxidation activity (6, 7). The actinomycetous monooxygenasescatalyze various interesting reactions and have attracted much atten-tion as oxidation biocatalysts. For example, AmoABCD from Nocar-dia corallina (Rhodococcus rhodochrous) strain B-276 catalyzes thestereoselective epoxidation of various aliphatic alkenes (8, 9). Inparticular, this enzyme is able to catalyze the epoxidation of ter-minal alkenes, which is particularly difficult to perform by chem-ical methods. PmoABCD from Mycobacterium sp. strain M156also shows epoxidation activity toward alkenes (10). In addition,propane monooxygenase (PrmABCD) (11, 12) and tetrahydrofu-ran monooxygenase (ThmABCD) (13) from actinomycetousstrains have high catalytic potential for applications in biocatalysisand biodegradation.
The gene clusters encoding the actinomycetous monooxyge-nases described above have been successfully identified andcloned, while attempts to express these gene clusters in heterolo-
gous hosts have encountered difficulties (10, 14). In particular,expression of these gene clusters in Escherichia coli has been un-successful, although this extensively characterized and developedmodel microorganism is an ideal host for biochemical character-ization and biotechnological applications of enzymes. For exam-ple, although functional expression of amoABCD from N. coral-lina B-276 in E. coli cells has been reported (9), experiments toconfirm the reproducibility of the experiment were unsuccessful(14). Similarly, E. coli cells transformed with pmoABCD from My-cobacterium sp. strain M156 were not able to acquire oxidationactivity (10). Chan Kwo Chion et al. suggested that the unsuccess-ful expression could be attributed to overlapping reading framesbetween pmoA and pmoB and between pmoC and pmoD (10). Inaddition to these actinomycetous monooxygenases, it has beenreported that several binuclear iron monooxygenases of otherbacteria, including methanotrophs and a Xanthobacter strain,were not functionally expressed in E. coli cells (15, 16). Thesestudies suggest that the oxygenase components are unstable in E.coli hosts. The fact that these fascinating binuclear iron monooxy-genases are difficult to express in E. coli has hampered the devel-opment of the engineered oxidation biocatalysts and prevents thepractical application of these enzymes (17–19).
More recently, we succeeded in functionally expressing the
mimABCD gene clusters from Mycobacterium smegmatis strainmc2155 and Mycobacterium goodii strain 12523 in the actinomy-cetous strain Rhodococcus opacus B-4 (20). The four genes mimA,mimB, mimC, and mimD encode an oxygenase large subunit, areductase, an oxygenase small subunit, and a coupling protein,respectively (Fig. 1). The mimABCD gene cluster plays essentialroles in propane and acetone metabolism in these mycobacteria(21, 22). Interestingly, MimABCD fortuitously catalyzes the regi-oselective oxidation of phenol to hydroquinone, which is of bio-technological importance (21). We have already found thatMimABCD requires the specific chaperonin-like protein MimG,which is encoded downstream from the mimABCD gene cluster(Fig. 1), for functional expression in the Rhodococcus host; whenthe mimG gene was coexpressed with the mimABCD gene clusterin R. opacus strain B-4, this host successfully acquired oxidationactivity toward phenol (20). Furthermore, we demonstrated thatMimG was involved in the productive folding of the oxygenaselarge subunit MimA (20). We speculated that this chaperonin-likeprotein might also be an important factor in active expression inE. coli.
In this study, we attempted to reconstitute the activeMimABCD complex in E. coli. Based on the findings describedabove, the mimABCD gene cluster was coexpressed with themimG gene in an E. coli host. Furthermore, the nucleotide se-quence of the mimD gene was optimized for an expression systemin E. coli, as we found that the expression level of the MimD pro-tein in E. coli cells was extremely low. These attempts led to thesuccessful expression of the mycobacterial binuclear iron mono-oxygenase in E. coli. The approach described here provides a ro-bust platform applicable for the functional expression of otheractinomycetous binuclear iron monooxygenases.
MATERIALS AND METHODSBacterial strains, plasmids, and cultivation media. The bacterial strainsand plasmids that were used or constructed in this study are listed in Table1. Schematic maps of the plasmids are shown in Fig. S1 in the supplemen-tal material. Bacteria were grown in Luria-Bertani (LB) medium, whichcontained (per liter) Bacto tryptone (10 g), Bacto yeast extract (5 g), andNaCl (10 g), pH 7.0.
Construction of mimABCD, mimG, and groES expression plasmids.The plasmids used for expression of the mimA and mimC genes in E. coli
FIG 1 Scheme showing genetic organization and enzymatic reaction of the mycobacterial binuclear iron monooxygenase.
TABLE 1 Bacterial strains and plasmids used in this study
Strain or plasmid CharacteristicsaReference(s)or source
StrainsE. coli JM109 Host used for cloning TaKaRa BioE. coli Rosetta 2(DE3)pLysS Host used for expression Novagen
PlasmidspETmimABCDgo pET21a containing mimABCDgo 20, 21pTipmimGsm pTip-RT2 containing mimGsm 20pRSFDuet-1 Vector used for cloning, RSF
origin, KnrNovagen
pCDFDuet-1 Vector used for cloning, CDForigin, Smr
Novagen
pETDuet-1 Vector used for cloning, pBR322origin, Apr
Novagen
pRSFDmimA pRSFDuet-1 containing mimA inMCS-1 under the control ofthe T7 promoter
This study
pRSFDmimAC pRSFDuet-1 containing mimA inMCS-1 and mimC in MCS-2under the control of the T7promoters
This study
pCDFDmimG pCDFDuet-1 containing mimGin MCS-1 under the control ofthe T7 promoter
This study
pCDFDmimG-groES pCDFDuet-1 containing mimGin MCS-1 and groES in MCS-2under the control of the T7promoters
This study
pETDmimB pETDuet-1 containing mimB inMCS-1 under the control ofthe T7 promoter
This study
pETDmimBD pETDuet-1 containing mimB inMCS-1 and mimD in MCS-2under the control of the T7promoters
This study
pETDmimDop pETDuet-1 containing mimDop
in MCS-2 under the control ofthe T7 promoter
This study
pETDmimBDop pETDuet-1 containing mimB inMCS-1 and mimDop in MCS-2under the control of the T7promoters
This study
pETDmimB5=opDop pETDuet-1 containing mimB5=op
in MCS-1 and mimDop inMCS-2 under the control ofthe T7 promoters
This study
a Knr, kanamycin resistance; Smr, streptomycin resistance; Apr, ampicillin resistance.
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6034 aem.asm.org Applied and Environmental Microbiology
cells were constructed using the pRSFDuet-1 vector (Table 1). Two oligo-nucleotide primers, mimA-F and mimA-R (see Table S1 in the supple-mental material), were designed to amplify the mimA gene. The regionbetween the two oligonucleotide primers was amplified from thepETmimABCDgo plasmid (Table 1) by PCR. This amplified DNA frag-ment was digested with BspHI and EcoRI and was then inserted intomulticloning site 1 (MCS-1) of pRSFDuet-1. The resulting plasmid,pRSFDmimA (Table 1), was amplified in E. coli JM109 cells (Table 1).Next, the mimC gene was amplified from pETmimABCDgo by PCR usingtwo oligonucleotide primers, mimC-F and mimC-R (see Table S1). This am-plified DNA fragment was digested with NdeI and FseI and was then insertedinto MCS-2 of pRSFDmimA to construct pRSFDmimAC (Table 1).
In a similar technique, the plasmids used for expression of the mimGand groES genes in E. coli cells were constructed using the pCDFDuet-1vector (Table 1). The mimG gene was amplified from the pTipmimGsm
plasmid (Table 1) by PCR using two oligonucleotide primers, mimG-Fand mimG-R (see Table S1 in the supplemental material). This amplifiedDNA fragment was digested with NcoI and EcoRI and was then insertedinto MCS-1 of pCDFDuet-1 to construct pCDFDmimG (Table 1). Next,two oligonucleotide primers, groES-F and groES-R (see Table S1), weredesigned to amplify the groES gene that corresponds to the Msmeg_1582open reading frame (ORF), based on the genome sequence of M. smeg-matis strain mc2155 (GenBank accession number NC_008596). PCR wasused to amplify the region between the two oligonucleotide primers fromgenomic DNA of M. smegmatis strain mc2155. This amplified DNA frag-ment was digested with NdeI and BglII and was then inserted into MCS-2of pCDFDmimG to construct pCDFDmimG-groES (Table 1).
The plasmids used for expression of the mimB and mimD genes in E.coli cells were constructed using the pETDuet-1 vector (Table 1). ThemimB gene was amplified from pETmimABCDgo by PCR using twooligonucleotide primers, mimB-F and mimB-R (see Table S1). This am-plified DNA fragment was digested with NcoI and EcoRI and was theninserted into MCS-1 of pETDuet-1 to construct pETDmimB (Table 1).Next, the mimD gene was amplified from pETmimABCDgo by PCR usingtwo oligonucleotide primers, mimD-F and mimD-R (see Table S1). Thisamplified DNA fragment was digested with NdeI and BglII and was theninserted into MCS-2 of pETDmimB to construct pETDmimBD (Table 1).
These plasmids were introduced into E. coli Rosetta 2(DE3)pLysS cells(Table 1) by heat shock. When three plasmids were introduced into E. colicells, two plasmids, derived from pRSFDuet-1 and pCDFDuet-1, werefirst simultaneously introduced into E. coli cells. After preparation of thecompetent E. coli cells carrying the two plasmids, the third pETDuet-1-derived plasmid was introduced into the cells by electroporation.
Construction of mimDop and mimB5=op expression plasmids. Theoverall nucleotide sequence of the mimD gene and the partial nucleotidesequence of the mimB gene in the translation initiation region were opti-mized for the E. coli expression system based on codon usage using soft-ware by Fasmac Co., Ltd. (Kanagawa, Japan). The nucleotide sequences ofthe optimized genes, mimDop and mimB5=op, are shown in Fig. S2 and S3,respectively, in the supplemental material. The mimDop gene was chemi-cally synthesized by Fasmac Co., Ltd. The mimDop gene was amplifiedfrom the synthetic gene by PCR using two oligonucleotide primers, mim-Dop-F and mimDop-R (see Table S1). This amplified DNA fragment wasdigested with NdeI and BglII and was then inserted into MCS-2 ofpETDuet-1 to construct pETDmimDop (Table 1) or into MCS-2 ofpETDmimB to construct pETDmimBDop (Table 1). The mimB5=op genewas synthesized and amplified from pETmimABCDgo by PCR using twooligonucleotide primers, mimB5=op-F and mimB5=op-R (see Table S1). Theoptimized nucleotide sequence was included in the 5=-terminal primermimB5=op-F. This amplified DNA fragment was digested with NcoI andBamHI and was then inserted into MCS-1 of pETDmimDop to constructpETDmimB5=opDop (Table 1).
Preparation of whole cells. The transformed E. coli Rosetta2(DE3)pLysS cells were cultivated at 30°C in LB medium supplementedwith chloramphenicol (34 �g/ml), kanamycin (15 �g/ml), streptomycin
(50 �g/ml), and/or ampicillin (50 �g/ml). When the cell growth reachedan optical density at 600 nm (OD600) of 0.6 to 0.8, ferric citrate (100�g/ml), ammonium ferric citrate (100 �g/ml), and L-cysteine (0.2 mM)were added to the medium to supply iron and sulfur for an iron protein(i.e., MimA) and an iron-sulfur protein (i.e., MimB) (23, 24). Isopropyl-�-D-thiogalactopyranoside (IPTG, 0.1 mM) was also added to the me-dium, and cultivation was continued at 25°C for an additional 15 h. Cellswere harvested by centrifugation at 15,000 � g for 10 min at 4°C, washedwith potassium phosphate buffer (50 mM, pH 7.5) containing glycerol(10%, vol/vol), and stored at �80°C until use.
SDS-PAGE analysis. The expression levels of Mim-related proteinswere examined by sodium dodecyl sulfate-polyacrylamide gel electropho-resis (SDS-PAGE) analysis. Frozen cells were suspended in potassiumphosphate buffer (50 mM, pH 7.5) containing glycerol (10%, vol/vol) andwere disrupted by sonication. Disrupted cells were used for the prepara-tion of whole-cell samples that included both soluble and insoluble pro-teins. After centrifugation at 15,000 � g for 30 min at 4°C, the resultingsupernatant was used for the preparation of soluble-fraction samples.Protein concentrations were measured using a Coomassie protein assaykit (Pierce, Rockford, IL) with a bovine serum albumin standard (25).Samples (2.5 to 5 �g protein) were treated with sodium dodecyl sulfateand then loaded onto a polyacrylamide gel. The acrylamide concentrationwas adjusted to 7.5% or 15%, depending on the molecular weights of Mimproteins.
Reaction using whole cells. In whole-cell assays using transformed E.coli cells, the reaction mixture (250 �l) contained cells of the E. coli strain(2 g dry cell weight per liter), the substrate phenol (10 mM), ethanol (1%,vol/vol), and aqueous basal medium (20, 26) containing glucose (5 g/li-ter).
Product analysis. High-performance liquid chromatography (HPLC)analysis was performed using an HPLC system (1100 series; Agilent, PaloAlto, CA) with a Wakosil-II 5C18 HG column (4.6 mm by 150 mm, 4.2- to4.7-�m particle size; Wako pure chemicals, Osaka, Japan). Following thereaction of the transformed E. coli cells with phenol, methanol (250 �l)was added to the reaction mixture. The resulting sample (10 �l) was theninjected into the HPLC system. Mobile phase A was composed of a mix-ture of acetonitrile-methanol-potassium phosphate buffer (10 mM, pH2.7) at a ratio of 2.5:2.5:95, and phase B was methanol. Samples wereeluted with 20% B for 30 min at a flow rate of 0.5 ml/min. The reactionproduct hydroquinone was detected spectrophotometrically at a wave-length of 220 nm.
Sequence analysis. The GC contents of the nucleotide sequence werecalculated using Genetyx version 10 (Genetyx Corporation, Tokyo, Ja-pan). The minimum free energy associated with the mRNA secondarystructure was also calculated using Genetyx version 10.
RESULTSConstruction of mimABCD expression system in E. coli. Wehave previously attempted to express the mimABCD gene clusterin E. coli cells using pETmimABCD, in which the tandemmimABCD gene cluster was located downstream from the T7 pro-moter and the ribosome-binding site (RBS) on the pET21a vector(20). In this plasmid, the mimA gene utilizes the vector-derivedRBS, whereas the mimB, mimC, and mimD genes have their nat-ural RBSs. The transformed E. coli cells, however, did not oxidizethe substrate phenol. SDS-PAGE analysis revealed the absence ofany bands corresponding to MimB, MimC, or MimD and thepresence of a band corresponding to MimA in the insoluble frac-tion (20).
In this study, we designed and exploited a new system formimABCD expression using three compatible plasmids to over-come the low-level expression and insolubilization of the Mimcomponents in E. coli cells. Here, the mimA, mimB, mimC, andmimD genes were placed under the respective T7 promoters and
Reconstitution of Mycobacterial Monooxygenase Complex
E. coli RBSs. The mimA and mimC genes, encoding the oxygenaselarge and small subunits, respectively, were cloned into the twoMCSs of the pRSFDuet-1 vector (Table 1). In a similar technique,the mimB and mimD genes, encoding a reductase and a couplingprotein, respectively, were cloned into the two MCSs of thepETDuet-1 vector (Table 1). Furthermore, the mimG gene, whoseGroEL-like product had been required for the productive foldingof MimA in R. opacus cells (20), was cloned into MCS-1 of thepCDFDuet-1 vector (Table 1). In addition, GroELs generally re-quire a cochaperonin, GroES, to assist in the folding of targetproteins. The genome sequence of M. smegmatis strain mc2155 hasonly one copy of the groES gene, Msmeg_1582 (20, 27). This groESgene was cloned into MCS-2 of the pCDFDuet-1 vector (Table 1).
Expression analysis of mimA and mimC in E. coli. To recon-stitute the active MimABCD complex in E. coli cells, we first at-tempted to express the oxygenase large and small subunits, MimAand MimC, in the soluble fraction of E. coli cells. The pRSFDmimACplasmid (Table 1) was introduced into E. coli Rosetta 2(DE3)pLysScells, and the expression of the mimA and mimC genes was in-duced by IPTG. As observed for the expression analysis of MimAin R. opacus cells, SDS-PAGE analysis revealed that the MimAprotein was insoluble in E. coli cells (Fig. 2, mimAC). Further-more, we found that MimC was also insoluble (Fig. 2, mimAC). Incontrast, when the mimG gene was coexpressed with the mimAand mimC genes, these gene products were successfully expressedas their soluble forms (Fig. 2, mimAC � mimG). These resultsindicate that MimG functioned as a chaperonin for the productivefolding of MimA and MimC even in E. coli cells. We also examinedthe effects of mycobacterial cochaperonin GroES on the expres-sion of MimA and MimC. By SDS-PAGE analysis using 15% poly-acrylamide gel, we confirmed that the GroES protein (10.8 kDa)was expressed in the soluble fraction (data not shown). However,
the coexpression of the groES gene with the mimA, mimC, andmimG genes did not further enhance the proportions of thesoluble forms of MimA and MimC compared to their expres-sion without the coexpression of groES (Fig. 2, mimAC �mimG � groES).
Expression analysis of mimB and mimD in E. coli. We nextexamined the expression of the mimB and mimD genes in E. colicells using the pETDmimBD plasmid carrying these genes (Table1). Although SDS-PAGE analysis revealed very low-level expres-sion of the MimB protein in the soluble fraction of E. coli cells, aband corresponding to MimD was not evident (Fig. 3, mimBD).These results indicate the difficulties in expressing native myco-bacterial genes in Gram-negative E. coli, which is phylogeneticallydistant from Gram-positive actinomycetous Mycobacterium spe-cies and possesses transcription and translation systems differentfrom those of mycobacteria.
In order to overcome low-level expression of the MimD pro-tein in E. coli cells, the overall nucleotide sequence of the mimDgene (345 bp) was optimized for the expression system of E. coliusing software (see Materials and Methods). The synthetic genedesignated mimDop (see Fig. S2 in the supplemental material)shares 77% nucleotide identity with the native mimD gene,whereas the encoded protein sequences are identical for the twogenes. The GC content decreased to 51% from 62% through theoptimization. The mimDop gene was cloned into the pETDuet-1vector instead of the native mimD gene, and was then introducedinto the E. coli host. The induction of the expression of this gene byIPTG resulted in markedly high-level expression of MimD in E.coli; SDS-PAGE analysis revealed the presence of a major bandcorresponding to MimD in the soluble fraction of E. coli cells (Fig.3, mimBDop).
We also attempted to express the MimB protein at higher levels
FIG 2 SDS-PAGE analysis of expression of mimA and mimC in E. coli. Samples prepared from E. coli cells carrying pRSFDuet-1 and pCDFDuet-1 (empty vector),pRSFDmimAC and pCDFDuet-1 (mimAC), pRSFDmimAC and pCDFDmimG (mimAC � mimG), or pRSFDmimAC and pCDFDmimG-groES (mimAC �mimG � groES) were loaded onto a polyacrylamide gel (7.5%). S, soluble-fraction sample; W, whole-cell sample. The molecular masses corresponding to MimA,MimC, and MimG are indicated by arrows.
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6036 aem.asm.org Applied and Environmental Microbiology
in E. coli cells. Because MimB is encoded by a relatively long nu-cleotide sequence (1,047 bp), we examined the substitution of apartial nucleotide sequence. Recent studies have suggested thatpoor expression levels of recombinant proteins in E. coli wereoften caused by the formation of excessively stable secondarystructures of mRNA in the translation initiation region (28, 29).Thus, we calculated the minimum free energy associated with themRNA secondary structure of the translation initiation region formimB using Genetyx software (29). The folding energy for theregion from nucleotide �4 to �37 relative to the start site wasfound to be �6.6 to ��6.2 kcal/mol. This value was lower thanthe threshold predicted for inhibition of translation initiation, ca.�6 kcal/mol (29–31). When the partial nucleotide sequence of themimB gene in the translation initiation region was optimized forthe expression system of E. coli, the folding energy increased to�3.9 to ��3.8 kcal/mol. Therefore, we synthesized the mimBgene with an optimized nucleotide sequence from �1 to �37 (seeMaterials and Methods). The synthetic gene designated mimB5=op
(see Fig. S3 in the supplemental material) is different in only threenucleotides from the native mimB gene, whereas the encoded pro-tein sequences are identical for the two genes. The GC contents arealmost the same (64%) for the two genes. The mimB5=op gene wascloned into the pETDuet-1 vector instead of the native mimB geneand then expressed in the E. coli host. Through this optimization,the expression level of MimB was extensively improved; SDS-PAGE analysis, however, revealed the presence of MimB in theinsoluble fraction of E. coli cells (Fig. 3, mimB5=opDop). We alsoconfirmed that induction of mimB5=op gene expression by IPTGunder 15°C instead of 25°C did not affect the solubility of this geneproduct.
Catalytic activity of E. coli cells carrying mimABCD towardphenol. We examined the catalytic activity of E. coli cells carryingthe mimABCD genes toward phenol using the three-recombi-nant-plasmid system. As expected, E. coli cells carrying only themimABCD genes did not oxidize phenol. In contrast, when themimABCD genes were coexpressed with the mimG gene, E. colicells successfully acquired oxidation activity toward phenol, al-
though the activity was low (0.067 �mol per gram dry cell weightper minute) (Fig. 4). When the mimDop gene was used as a sub-stitute for mimD, however, the oxidation activity was extensivelyenhanced; E. coli cells carrying mimABCDop and mimG rapidlyoxidized phenol to hydroquinone (Fig. 4). The initial rate of hy-droquinone production by cells carrying mimABCDop and mimGwas estimated to be 0.71 �mol per gram dry cell weight per min-ute, which was 11 times higher than that by cells carryingmimABCD and mimG. When the mimB gene was optimized inaddition to the mimD gene, E. coli cells carrying mimAB5=opCDop
and mimG showed lower oxidation activities than cells carryingmimABCDop and mimG (Fig. 4).
FIG 3 SDS-PAGE analysis of expression of mimB and mimD in E. coli. Samples prepared from E. coli cells carrying pETDuet-1 (empty vector), pETDmimBD(mimBD), pETDmimBDop (mimBDop), or pETDmimB5=opDop (mimB5=opDop) were loaded onto a polyacrylamide gel (15%). S, soluble-fraction sample; W,whole-cell sample. The molecular masses corresponding to MimB and MimD are indicated by arrows.
FIG 4 Monooxygenase activities of engineered E. coli cells toward phenol.Whole cells of E. coli carrying pRSFDmimAC, pETDmimBD, andpCDFDuet-1 (mimAC � mimBD), pRSFDmimAC, pETDmimBD, andpCDFDmimG (mimAC � mimBD � mimG), pRSFDmimAC, pETDmimBDop,and pCDFDmimG (mimAC � mimBDop � mimG), or pRSFDmimAC,pETDmimB5=opDop, and pCDFDmimG (mimAC � mimB5=opDop � mimG)were reacted with phenol for 6 h, and the monooxygenation product hydro-quinone was quantified using HPLC. Bars represent the means of three inde-pendent experiments, and error bars represent standard deviations of themeans.
Reconstitution of Mycobacterial Monooxygenase Complex
In this study, we succeeded in reconstituting the active MimABCDcomplex in E. coli. We found that the following factors are impor-tant for functional expression of the mycobacterial binuclear ironmonooxygenase in E. coli: the coexpression of MimG, whichcaused MimA and MimC to be soluble in E. coli cells, and theoptimization of the mimD nucleotide sequence, which led to effi-cient expression of this gene product (Fig. 1). To our knowledge,this is the second report regarding the active expression of anactinomycetous binuclear iron monooxygenase in an E. coli host.The first functional expression in E. coli was reported for Amo-ABCD of N. corallina B-276; E. coli cells carrying only the nativeamoABCD gene cluster were able to oxidize 3,3,3-trifluoropro-pene (9). However, it has also been reported that experiments tocarefully confirm the reproducibility of the experiment were un-successful (14).
The GroEL-like protein MimG functioned as a chaperonin forthe productive folding of the oxygenase large subunit MimA in E.coli cells, as well as in R. opacus cells. Notably, in the absence ofMimG, the oxygenase small subunit MimC was also insoluble in E.coli cells (Fig. 2), although this protein had been soluble in R.opacus cells (20). These results suggest that the environment in E.coli cells tended to render the MimC protein unstable. Neverthe-less, MimG was able to support MimC in the maturation processin E. coli cells (Fig. 2). Because the oxygenase large and smallsubunits are known to be a paralogous protein and show similarityin quaternary structure (2), it is unsurprising that MimG plays arole for both MimA and MimC. Coexpression of the mycobacte-rial cochaperonin GroES with MimG did not further enhance theproportions of the soluble forms of MimA and MimC comparedto their proportions without groES coexpression (Fig. 2). It is pos-sible that MimG did not require any cochaperonin for its functionor that a proper cochaperonin was sufficiently supplied to MimGby the host E. coli strain. The genome sequence of E. coliBL21(DE3), the parent strain of Rosetta 2(DE3)pLysS (GenBankaccession number NC_012971), contains one copy of the groESgene, ECD_04012, whose gene product shares ca. 45% amino acididentity with the mycobacterial GroES.
In this study, we succeeded in reconstituting the activeMimABCD complex using protein components whose aminoacid sequences are identical to those of the native Mim compo-nents. The optimization of the mimD nucleotide sequence for theexpression system of E. coli provided efficient expression of thismimDop gene product (Fig. 3), which endowed E. coli cells withhigh oxidation activity (Fig. 4). We also achieved high-level ex-pression of MimB in E. coli by increasing the folding energy asso-ciated with the mRNA secondary structure of the translationinitiation region. However, this mimB5=op gene product was insol-uble in E. coli cells (Fig. 3), and this situation reduced the oxida-tion activity of the transformed E. coli cells (Fig. 4). It was reportedthat the reductase components of binuclear iron monooxygenasesthat contain [Fe-S] centers were amenable to inactivation in E. colicells, particularly under high-level-expression conditions (15, 24,32). For example, the reductase component of toluene 4-mono-oxygenase has been functionally expressed under the control of aweak promoter (32). Similarly, it would be important to restrictthe expression of MimB at low levels to escape inactivation. As aresult, the engineered E. coli cells carrying mimABCDop and mimGacquired the highest oxidation activity (0.71 �mol per gram dry
cell weight per minute) toward phenol. This E. coli biocatalyst hasgreat potential for the industrial production of hydroquinonefrom phenol. To further enhance the oxidation activity of theengineered E. coli cells, it would be important to achieve high-leveland soluble expression of MimB. It has recently been reported thatfusion of the reductase component of tetrahydrofuran monooxy-genase to maltose binding protein markedly increased the solubil-ity of this component (24). Similar to this, fusion of MimB to anappropriate tag sequence might lead to its high-level soluble ex-pression. Also, the genome sequence of M. smegmatis strainmc2155 has two other copies of the groEL gene, Msmeg_0880 andMsmeg_1583, in addition to mimG (Msmeg_1978) (27). Thesemycobacterial chaperonins might be helpful in the solubilizationof MimB.
Finally, the strategy described here should be generally appli-cable to the active expression of other actinomycetous binucleariron monooxygenases in E. coli. As we have reported previously(20), many homologous gene clusters encoding binuclear ironmonooxygenase exist in cloned nucleotide sequences and pub-lished genome sequences of actinomycetes, and these are alwaysaccompanied by mimG homologs. This strongly suggests the highpotential of our strategy for wider application. Furthermore, al-though methane monooxygenase is the archetypal and most in-vestigated member of the binuclear iron monooxygenase family,active expression of this monooxygenase in E. coli has yet to beachieved (18, 19). Because it was found that a specific chaperoninwas involved in the expression system of the methane monooxy-genase (33, 34), our experimental platform may be helpful for thefunctional expression of this monooxygenase in E. coli. The ap-proaches described here accelerate not only the biochemical char-acterization of these binuclear iron monooxygenase systems butalso the development of the engineered oxidation biocatalysts andwill pave the way for industrial application of these enzymes.
ACKNOWLEDGMENT
This work was supported by Japan Society for the Promotion of Science,Grant-in-Aid for Young Scientists (B) 24780083 to T.F.
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Reconstitution of Mycobacterial Monooxygenase Complex
