Conversion of Sphingobium chlorophenolicum ATCC 39723 to a Hexachlorobenzene Degrader by Metabolic Engineering

The gene cassette (camA⁺ camB⁺ camC) encoding a cytochrome P-450_camvariant was integrated into the nonessential gene pcpM of thepentachlorophenol degrader Sphingobium chlorophenolicum ATCC39723 by homologous recombination. The recombinant strain coulddegrade hexachlorobenzene at a rate of 0.67 nmol · mg(dry weight)^–1 · h^–1, and intermediate pentachlorophenolwas also identified.

INTRODUCTION

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Introduction

Hexachlorobenzene (C₆Cl₆; HCB) was listed as 1 of the 12 persistentorganic pollutants in the Stockholm Convention for its tendencyto accumulate along the food chain and its recalcitrance todegradation, together with its harmful effects on human beingsand the environment (1). Microbial degradation is a promisingeffective way to bioremediate environmental pollutants, includingpersistent organic pollutants. However, a pure culture capableof completely catabolizing HCB has not yet been isolated. Therefore,constructing an HCB-degrading strain via metabolic engineeringmay be a practical alternative to eliminate HCB in the environment.

It had been demonstrated that the F87W/Y96F/L244A/V247L mutantof cytochrome P-450_cam (CYP101) can oxidize HCB to pentachlorophenol(PCP) (4), which can be completely degraded by many microorganismsin the environment. In particular, the catabolic pathway ofPCP in Sphingobium chlorophenolicum ATCC 39723 has been characterizedthoroughly both biochemically and genetically (2).

In this study, we report the conversion of the PCP utilizerATCC 39723 to an HCB degrader by introducing a genetic segment(camA⁺ camB⁺ camC), which codes for the mutant of CYP101, intothe target gene pcpM through homologous recombination.

Construction of recombinant plasmids and strains.

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Construction of recombinant...

The plasmids, strains, and the primers used are listed in Table1. DNA isolations and manipulations were performed accordingto established methods (11). The construction of the targetingvector for disruption of the genomic pcpM gene is illustratedin Fig. 1A. pcpM was amplified from strain ATCC 39723 with primersMF-1 and MR-1. The resulting 916-bp PCR product was then digestedwith XbaI and HindIII before being ligated into pBluescriptII SK(+) to create pZWY001. The functional expression cassette,including the Ptac-tac-camA⁺B⁺C transcriptional terminator,was PCR amplified from plasmid pCWSGB-camC using primers A5and A6. The amplified 3,386-bp fragment was then digested withEcoRI and SacI before being ligated into pBluescript II SK(+)to generate pZWY002. The neomycin phosphotransferase II gene(nptII) was PCR amplified from pTnMod-OKm (5) with primers KF-1(containing SacI and EcoRI sites in tandem) and KR-1 (containinga SacI site). The amplified 1,179-bp fragment was digested withSacI before being ligated into pBluescript II SK(+) to generatepZWY003. The nptII gene cassette was then excised from pZWY003with SacI and cloned into pZWY002 to generate pZWY004. The insertfrom pZWY004 was released with EcoRI and cloned into pZWY001to generate pZWY005. All constructs were confirmed by DNA sequencing(GiKang, Shanghai, China).

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TABLE 1. Strains, plasmids, and primers used in this study

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FIG. 1. Illustration of construction of plasmids and the engineered strain. (A) Construction of the gene targeting vector pZWY005 for allelic exchange in Sphingobium chlorophenolicum ATCC 39723. (B) Homologous recombination between the flanking sequences of pcpM in the targeting vector pZWY005 and the pcpM sequence in chromosomal DNA in Sphingobium chlorophenolicum ATCC 39723. camA⁺, camB⁺, and camC denote the genes encoding putidaredoxin reductase (PdR), putidaredoxin (Pd), and mutant cytochrome P450_cam, respectively. nptII, neomycin phosphotransferase II gene; Ap^r, ampicillin resistance gene (bla); Ptac, tac promoter; tt, transcriptional terminator; M_L and M_R, the left part and the right part of pcpM, respectively. Arrows indicate the direction of transcription for the respective genes. Relevant restriction sites are indicated.

The plasmid pZWY005 was introduced into strain ATCC 39723 byelectroporation as previously described (8). Positive clonescontaining a pcpM-disrupted allele were screened by their kanamycinresistance (15 µg/ml) and ampicillin sensitivity (15 µg/ml).One hundred kanamycin-resistant colonies were identified, ofwhich 10 were ampicillin sensitive, implying that the disruptionsresulted from a double-crossover recombination rather than deliveryplasmid cointegration (single crossover). The homologous recombination(double crossover) is illustrated in Fig. 1B.

Further identification of the recombinants was performed byPCR with primers A3 and A4 designed from the sequences flankingpcpM. A 5.8-kb fragment was amplified using genomic DNA of therecombinants as template. This clearly indicated that allelicexchange had occurred via homologous recombination between theflanking sequences of pcpM in the targeting vector and the pcpMsequence in the host genome, resulting in an interrupted pcpMallele in the genome. In contrast, a 1.3-kb fragment was producedin a control experiment using wild-type ATCC 39723 genomic DNAas template. One such recombinant was named ZWY005, which stillgrows on PCP as well as the wild type.

HCB degradation by whole cells of engineered strain ZWY005.

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Hcb degradation by whole...

Strains ZWY005 and ATCC 39723 were grown in glutamate mineralsalts medium (10) at 30°C with shaking. When the A₆₀₀ ofa cell culture reached 0.3 to 0.4 (early exponential phase),1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) wasadded and the culture was incubated for an additional 8 h. Cellswere harvested by centrifugation at 3,500 x g for 8 min andwashed twice with MS (glutamate mineral salts medium withoutmonosodium L-glutamate). The cells were then resuspended in300 ml MS with an A₆₀₀ of 3 (1 g [dry weight] of cells per liter)and were shaken at 30°C for HCB degradation. HCB was addedas a 1 mM stock in ethanol to a final concentration of 4.3 µM.For the purpose of analysis of the degradation of HCB and productionof PCP, two 20-ml aliquots of the cultures were removed periodicallyto 50-ml centrifuge tubes.

An internal standard, 1,3,5-tribromobenzene, was added to thesamples for quantitative analysis of HCB to standardize boththe extraction and analysis steps. The sample was centrifugedat 6,000 x g for 10 min. Organics in the supernatant collectedwere adsorbed onto an Argonaut/IST ISOLUTE C₁₈ (EC) column,and the bound organics were eluted with n-hexane. On the otherhand, organics adsorbed on the cell pellet were extracted accordingto methods described previously (9, 12). Control experimentsshowed that the efficiency of this solid-phase extraction combinedwith vortex mixer extraction method was pretty high (>96%),and the results were highly reproducible. The n-hexane layersfrom all above extractions were pooled and dried over anhydroussodium sulfate for quantitation by gas chromatography (GC).

GC analysis was carried out on an Agilent 6820 series instrumentequipped with a ⁶³Ni electron capture detector on a DB-5 fusedsilica capillary column (0.32-mm inner diameter by 30 m by 0.25µm). The column temperature was held at 40°C for 1min and then increased to 100°C at 15°C/min, from 100°Cto 240°C at 20°C/min, and from 240°C to 280°Cat 10°C/min. The injector and detector temperatures were280°C and 300°C, respectively. Nitrogen at a flow rateof 40 ml/min was used as the carrier gas. The injection volumewas 1 µl, and splitless injection was used. The retentiontimes of HCB and 1,3,5-tribromobenzene were 12.43 min and 10.36min, respectively.

Degradation studies demonstrated that the recombinant cellscould degrade 4 µM HCB within 6 h with 1 g (dry weight)of cells per liter (or at a rate of 0.67 nmol · mg [dryweight]^–1 · h^–1). No degradation of HCB wasobserved in the control with ATCC 39723.

Identification of PCP as an intermediate metabolite produced by oxidation of HCB.

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Identification of pcp as...

In the wild-type ATCC 39723, transcription of a gene cluster(pcpB pcpA pcpE) encoding PCP-degrading enzymes has been shownto be inducible by PCP, which takes about 1 to 2 h (13). PCPtransformed from HCB by the engineered strain should then bedetected in the recombinant culture within the early hours afterthe addition of HCB, since it is not further degraded withinthis period.

For qualitative and quantitative analysis of PCP, an internalstandard, 2,4,6-tribromophenol, was added to the samples afterbeing acidified to pH 2.0 with concentrated sulfuric acid. Theextraction procedures were the same as described above, exceptthat the samples were extracted with n-hexane-acetone (1:1)instead of n-hexane only (9). Assays of the trapped intermediatePCP were performed by derivatization with acetic anhydride topentachlorophenyl acetate prior to measurements (9).

The results of GC analysis indicated that PCP had been indeedproduced during the HCB degradation by the recombinants. Theretention time of the derivative from the samples correspondedto that of the derivative of authentic PCP, while the samplesfrom the wild-type ATCC 39723 control or bacteria-free controldid not have the corresponding peak of the PCP derivative. Theretention times were as follows: pentachlorophenyl acetate,13.06 min; 2,4,6-tribromophenyl acetate, 12.49 min. The timecourse analysis showed that the stoichiometric amount of PCPwas formed concomitantly within the first hour of incubationduring the degradation of HCB, apparently before the PCP degradationpathway was induced (Fig. 2).

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FIG. 2. HCB degradation by whole cells of engineered strain ZWY005. ${square}$ , HCB concentration; ${blacktriangleup}$ , PCP concentration. Extracted samples were analyzed with a gas chromatograph equipped with an electron capture detector.

The identity of intermediate PCP was further confirmed by GC-massspectrometry (GC-MS), in which the scanning model was adopted.GC-MS analysis was carried out on an Agilent 6890N/5973 Inertquadrupole instrument. An HP-5 MS column (0.25-mm inner diameterby 30 m by 0.25 µm) was used with temperature programmingfrom 40°C to 280°C at 20°C/min and held at thistemperature for 5 min. Injection volume was 1 µl, andsplitless injection was used. Helium was used as the carriergas at a flow rate of 1 ml/min. The MS ion source temperaturewas 230°C, and the quadrupole temperature was 150°C.Electron energy was 70 eV. Scan range was from 50 to 400 atomicmass units. The possibility that the metabolic intermediateof HCB is PCP was confirmed by comparison of the mass fragmentationpatterns of intermediate and authentic PCP in the NIST02 massspectral database. Molecular ion peaks were observed at m/z312 (M + 6), 310 (M + 4), 308 (M + 2), and 306 (M⁺), whose massesand relative intensities are characteristic of a molecule containingfive chlorine atoms. The relative intensities are consistentwith a natural abundance of 76% for ³⁵Cl and 24% for ³⁷Cl. Majorfragments were observed at m/z 270, 268, 266 (base peak), and264 (loss of CH₂CO from 312, 310, 308, and 306) and m/z 241, 239,237, and 235 (loss of HCO from 270, 268, 266, and 264).

As a leaky tac-tac promoter existed in the expression cassettewithin the engineered strain, HCB degradation and PCP formationwere also observed in the resting cells without induction ofIPTG, but at a lower rate.

In conclusion, an engineered strain with the ability to completelydegrade HCB has been successfully constructed by metabolic engineeringfor the first time, albeit with a low degradation activity.This is in contrast to previous studies on the reductive dechlorinationof HCB, which led to accumulation of toxic intermediates (3,6, 7, 14).

ACKNOWLEDGMENTS

This work was supported by Knowledge Innovation Project grantsfrom the Chinese Academy of Sciences (KSCX2-SW-128) and WuhanInstitute of Virology, Chinese Academy of Sciences (LYQY-020405).

We thank Luet-Lok Wong for the gift of plasmid pCWSGB-camC andLuying Xun for providing Sphingobium chlorophenolicum ATCC 39723.

FOOTNOTES

* Corresponding author. Mailing address: Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China. Phone: 86-27-87197655. Fax: 86-27-87197655. E-mail: n.zhou{at}pentium.whiov.ac.cn.

REFERENCES

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