Gene manipulation of catabolic activities for production of intermediates of various biphenyl compounds

Appl Microbiol Biotechnol (1988) 29:363--369 Applied Microbiology

Biotechnology © Springer-Verlag 1988

Gene manipulation of catabolic activities for production of intermediates of various biphenyl compounds

Kensuke Furukawa and Hideo Suzuki

Fermentation Research Institute, Agency qf Industrial Science and Technology, Tsukuba, Ibaraki 305, Japan

Summary. A bioconversion process was demonstrated by manipulation of catabolic genes. Cata- bolic intermediates of various biphenyl compounds could be efficiently produced by Pseudo- monas aeruginosa carrying recombinant plasmids containing a set of cloned bph genes. A dihydrodiol compound was produced by the strain carrying plasmid pMFB4 containing bphA (encoding biphenyl dioxygenase) gene. A dihydroxy compound was produced from 4-chlorobiphenyl by the strain carrying plasmid pMFB6 containing bphA and bphB (encoding dihydrodiol dehydro- genase) genes. Tetrahydroxybiphenyl was accumulated as the final product via dihydroxybiphenyl from biphenyl by the same pMFB6 carrying strain. Meta-cleavage yellow compounds were produced from biphenyl and its derivatives substituted with methyl, chloro, bromo, or nitro group on one of the biphenyl rings by the strain carrying plasmid pMFB2 containing bphA, bphB and bphC (encoding dihydroxybiphenyl dioxygenase) genes.

Introduction

Soil pseudomonads exhibit a wide range of catabolic activities for many aromatic compounds. Genes of catabolic pathways are often highly clustered. The clustering of catabolic genes in pseudomonads has been found on chromosome and on plasmids such as TOL (specifying xylene/ toluene catabolism) (Franklin et al. 1983; Inouye et al. 1983), NAH (specifying naphthalene catabolism) (Yen and Gunsalus 1982), and OCT (specifying n-alkane catabolism) (Owen et al. 1984). A

Offprint requests to: Kensuke Furukawa

cluster of genes of mandelate and benzoate pathways has been shown in Pseudomonas aeruginosa using the transducing phage Fl l6 (Kemp and Hegeman 1968). It has been demonstrated that the biphenyl catabolic gene cluster bphA, bphB, and bphC in Pseudomonas. pseudoalcaligenes were also assembled on a single operon and placed under the control of regulatory elements so that the genes could be induced simultaneously (Furukawa and Miyazaki 1986). The catabolic intermediates of interest of aromatic hydrocarbons do not accumulate in microbial dissimilation un- less a mutation in structural genes is introduced. Moreover, since such catabolic intermediates are usually hydroxylated and ring-cleaved, it appears to be very difficult to synthesize them by multi- step chemical processes. However, these compounds can be produced enzymatically, if the genes responsible for the catabolism are properly introduced in host cells. The use of genetic manipulation of catabolic pathways for new chemical compounds has become an increased concern in biotechnology.

Biphenyl and its substituted derivatives in- cluding polychlorinated biphenyls (PCB) are ca- tabolized to benzoic acid and its derivatives by many bacterial strains (Amed and Focht 1973; Catelani et al. 1971; Furukawa et al. 1978; Furu- kawa et al. 1983; Furukawa and Miyazaki 1986) through an oxidative route as shown in Fig. 1. Bi- phenyl (compound I in Fig. 1) is oxidized to the dihydrodiol compound (compound II) by biphenyl dioxygenase (encoded by the bphA gene). The dihydrodiol is dehydrogenated to the dihydroxy compound (compound III) by dihydrodiol dehy- drogenases (encoded by the bphB gene). The dihydroxy compound is then meta-cleaved to form 2- hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (compound IV) by 2,3-dihydroxybiphenyl dioxygenase

364 K. Furukawa and H. Suzuki: Production of catabolic intermediates of biphenyls

H OH OOH COOH ~ " ~ , J OH OH H

I II III IV

Xhol - 7.2 kb I pMf82 [ bphA ~ bphB ~ bphC

,,,, V ~Xhol pMFBS x,o[ bp, A ) bp, B

Xhol~ 5.4 kb i,, Smal pMfB4 --~ bphA ' ~

bphD

V

}

Fig. 1. The catabolic pathway for degradation of biphenyl and its derivatives and proposed gene organization of the bph operon in Pseudomonaspseudoalcaligenes KF707 (Furukawa and Miyazaki 1986). (top) Compounds: I, biphenyl; II, 2,3-dihydroxy-4-phenylhexa-4,6-diene (dihydrodiol compound); III, 2,3-dihydroxybiphenyl; IV, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (meta cleavage compound); V, benzoic acid. Enzymes catalyzing the conversion: A, biphenyl dioxygenase; B, dihydrodiol dehydrogen- ase; C, 2,3-dihydroxybiphenyl dioxygenase; D, meta cleavage compound hydrolase. (Bottom) The structures of plasmids pMFB2, pMFB6 and pMFB4 are shown. The bphD gene has not yet been cloned. In vitro mutation at the ClaI site within the bph C gene of pMFB6 is shown by an arrowhead (V). R: H, CI, Br, CH3, NO2, CH2OH

(encoded by the bphC gene) (Furukawa and Ari- mura 1987; Furukawa et al. 1987). The meta- cleavage yellow compound is hydrolyzed to benzoic acid (compound V) by hydrolase (encoded by a putative bphD gene) (Furukawa and Miyazaki 1986). We cloned previously a 7.9 kilobase pair (kb) DNA fragment containing bphABC genes from the chromosomal DNA of biphenyl-utilizing Pseudomonas pseudoalcaligenes strain KF707 (9). Here we report that various catabolic intermediates of biphenyl compounds could be efficiently produced by manipulation of the cloned bph genes.

Materials and methods

Bacterial strains, plasmids and culture conditions

Bacterial strains used in this study are Pseudomonas aeruginosa PA01161 (leu hsdR hsdM) carrying recombinant plasmids which contain various bph genes: strain KF260 carrying pMFB4 containing only the bphA gene; strain KF274 carrying pMFB6 containing the bphA and bphB genes; strain KF258 carrying pMFB2 containing the bphA, bphB and bphC genes. pMFB2 and pMFB4 were previously constructed from pMFB1 (Furukawa and Miyazaki 1986). pMFB6 was obtained as a mutant plasmid of pMFB2 by introducing in vitro mutation in the bphC gene. The unique ClaI site in the bphC gene was cleaved and filled in with Klenow fragment, so that addi- tional 2 base pairs were inserted, causing the frame shift in the bphC gene. Thereby, only bphAB genes are intact in pMFB6. P. aeruginosa cells carrying each recombinant plasmid were grown at 37°C in Luria broth (tryptone, 10g; yeast extract, 5 g; NaC1, 5 g per liter, pH 7.0) in the presence of streptomycin (300 Ixg/ml).

Plasmid isolation and enzymatic treatment of DNA

Plasmid DNA was prepared by either the rapid alkaline extraction method (Birnboim and Doly 1979) or CsCl-ethidium bromide equilibrium density gradient centrifugation. Diges- tion of DNA with restriction endonuclease, filling in with Kle- now fragment and ligation of DNA with T4 DNA ligase were performed as recommended by the manufacturer.

Incubation method and analysis of the metabolites

Biphenyl compounds used in this study were biphenyl, 4- and 4,4'-dimethylbiphenyls, 2-bromobiphenyl, 4-, 4,4'-, 2,3- and 2,4,5-chlorobiphenyls, 2-hydroxybiphenyl, 2,3-dihydroxybiphenyl, 2-nitrobiphenyl, 4-biphenylacetic acid, 4-biphenylcar- boxylic acid, 4-biphenylmethanol, and diphenylmethane. Resting cells of P. aeruginosa carrying each hybrid plasmid containing the corresponding bph genes were used for production of catabolic intermediates of biphenyl compounds. The washed cells were suspended in 10 ml of phosphate buffer (pH 7.5) to which 10 lxmoles of each biphenyl compound dissolved in ethanol were added. The final absorbance of the cell sus- pension was adjusted to 1.0 at 660 nm (1.2 x 10 9 cells per ml). Incubations were carried out with shaking at 37 ° C. The incubation mixtures were then extracted with 10 ml of ethylacetate after acidification to pH 1 with concentrated HC1. The ethylacetate layer was removed and evaporated to dryness under a gentle stream of nitrogen gas, and the residue was dissolved in a small amount of ethylacetate. Bistrimethylsilylacetamide was added to obtain trimethylsilyl (TMS) derivatives of produced compounds. The samples were analyzed with a gas-liquid chromatograph-mass spectrometer (GC-MS) (JEOL Ltd., model JMS D-300) with a coiled glass column (1 m by 4 mm internal diameter) packed with silicon OV1 (at 2% on 80- to 100-mesh chromosorb G). Helium was used as a carrier gas at a flow rate of 20 ml/min. The column temperature was increased from 140 to 250°C at a rate of 8 o C/min. The electron impact spectra were measured at 70 eV ionization potential, 300 IxA trap current, and 200°C ion source temperature.

K. Furukawa and H. Suzuki: Production of catabolic intermediates of biphenyls 365

Results

Production of dihydrodiol compound by the recombinant strain KF260

Strain KF260 carrying plasmid pMFB4 containing the bphA gene was cultured in Luria broth

with streptomycin, and the washed cells were incubated with biphenyl in phosphate buffer (pH 7.5). After 2 hr incubation, the total reaction mixture was extracted with ethylacetate at pH 1. The concentrated ethylacetate extract was subjected to GC-MS (Fig.2). Two major peaks with close re- tention times were detected, and they were both

( a )

1 O0 0 1

3 /

~ 0 , ,

0 1 2 t I I I I

3 4 5 rain

( b )

I000 227

193

:= b I "_" ,,t , ,~,,I,,, I , I, ,, I, -. ,, 100 150 200

M ÷ 242

~OTHS

J,LL, 2~o 300m/z

1000

211 2[7

! o l,.,.IJ,.. ,~. ,J,J.,,~ .... g,L,, 150 200

|* 330 ~ OTNS

OTM$

242 315

, J , , , . . . . . I, !,,li.~ 250 300 350 m,

(c )

A A oH

H ~ ~H

OH OH

acid ~ ac id

Fig. 2. Production of dihydrodiol compounds from biphenyl by P. aeruginosa KF260 carrying pMFB4. (a) The GC-MS total ion monitor of the ethylacetate extract. The metabolites were treated with bistrimethylsilylacetamide to obtain the TMS derivatives. (b) Mass spectra of TMS derivatives of compound 1 (left) and compound 3 (right) in Fig. la. (c) Proposed conversion of biphenyl to dihydrodiol by strain KF260. Enzymes: see the legend of Fig. 1


identified to be monohydroxybiphenyl (M + as a TMS derivative, m/z 242; M + - C H 3 , m/z 227; M ÷ - OTMS, m/z 154). The third minor peak was identified to be dihydroxybiphenyl (M + as a TMS derivative, m/z 330; M + - C H 3 , m/z 315; M + - O T M S , m/z 242), but the fragmentation profile was different from that of 2,3-dihydroxybiphenyl (data not shown). These results indicate that biphenyl was converted to the dihydrodiol compound (2,3-dihydroxy-4-phenylcyclohexa-4,6- diene), which was spontaneously converted to 2- hydroxy- and 3-hydroxybiphenyl in the acid extraction procedure. The minor compound was considered to be a dihydroxy compound in which a single hydroxyl group might be substituted on each biphenyl ring. This can be derived from 1,1'- bis(2,3-dihydroxycyclohexa-4,6-diene) spontaneously in acid extraction procedure as shown in Fig. 2c.

Production of dihydroxy compound and tetrahydroxy compound by the recombinant strain KF274

Strain KF274 carying plasmid pMFB6 containing the bphA and bphB genes were grown as above, and the washed cells were incubated with biphenyl. After 1 h incubation, the incubation mixture turned to dark brown because of the oxidative po- lymerization of hydroxylated biphenyls. Two major metabolites were detected in the GC-MS analysis (Fig. 3). The first peak was identified to be 2,3-dihydroxybiphenyl from the mass spectrum (M + as a TMS derivative, m/z 330; M + - C H 3 , m/z 315; M + - O T M S , m/z 242) which was iden- tical with that of authentic 2,3-dihydroxybiphenyl. The second peak was considered to be 2,3,2',3'-tetrahydroxybiphenyl (M + as a TMS derivative, m/z 506; M + - C H 3 , m/z 481; M + - T M S , m/z 434) (Fig. 3b). After 2 h incubation, tetrahydroxybiphenyl predominantly accumulated in the reaction mixture. These results indicate that 2,3-dihydroxybiphenyl was first produced, then the nonhydroxylated ring of the same compound was further oxidized to form tetrahydroxybiphenyl and accumulated as the final product (Fig. 3c). Tetrahydroxybiphenyl was produced from authentic 2,3-dihydroxybiphenyl by the same strain. On the other hand, when 4-chlorobiphenyl was used as the substrate, only the dihydroxy compound (M + as TMS derivative, m/z 364; M + --CH3, m/z 249) was produced and accumulated after 2 hr incubation (Fig. 4), Thus it was found that the bphA gene product, biphenyi

1000.

Z

1000

~ OH OH

J

2

OH

OH OH

min ~ ~ ~ 1'o

( a )

434 ld + 506

315

"~ 0 300

0°:

403

,k ~ II 481

3~o 4~o ~o slo sso®,,~

( c )

, o, o,

~ O H OH OH

Fig. 3. Production of tetrahydroxybiphenyl from biphenyl by P. aeruginosa KF274 carrying pMFB6. (a) The GC-MS total ion monitor of ethylacetate. The metabolites were treated with bistrimethylsilylacetamide to obtain the TMS derivatives. (b) The mass spectrum of the TMS derivative of compound 2 in Fig. 2a. (e) A proposed converison of biphenyl to tetrahydroxybiphenyl by strain KF274. Enzymes: see the legend of Fig. 1

dioxygenase in P. pseudoalcaligenes KF707 at- tacks only the non-substituted ring of biphenyl compounds.

Production of ring meta-cleavage compounds from various biphenyl-derivatives by the recombinant strain KF258

Cells of strain KF258 carrying plasmid pMFB2 containing bphA, bphB, and bphC genes were prepared and the washed cells were incubated with various biphenyl derivatives such as biphenyl, 4-, 2,3-di-, 4,4'-di, 2,4,5-tri-chlorobiphenyls, 2-bromo-, 4-methyl-, 4,4'-dimethyl-, 2-nitro-, and 2-hydroxy- biphenyls. 4-Bisphenylacetic acid, 4-biphenyl- carboxylic acid, 4-biphenylmethanol and diphe-


(a) ( a ) 1000.

_=

CI

OH

rain

(b) 1000.

/

oL L, ,,

15o

M ÷ 364 CI

~ TldS OTMS

275 249

240 250 300 350 m/z

(c)

cI el el

Fig. 4. Production of dihydroxy compound from 4-chlorobiphenyl by P. aeruginosa KF274 carrying pMFB6. (a) The GC- MS total ion monitor of ethylacetate extract. The metabolites were treated with bistrimethylsilylacetamide to obtain the TMS derivatives. (b) The mass spectrum of The TMS derivative of the product. (e) Proposed conversion of 4-chlorobiphenyl to dihydroxy compound by strain 274. Enzymes: see the legend of Fig. 1

5

Incubation Time (hr)

( b )

A B C

OH OH HH H H

R=H, CI, Br,CH3, NO2, CH20H

Fig. 5. Production of meta-cleavage compounds from various biphenyls and diphenylmethane by P. aeruginosa KF258 carrying pMFB2. (a) Time course of meta-cleavage yellow compounds production. The absorption maximum of each meta-cleavage compound was monitored: biphenyl(l), 434 nm; 4-chlorobiphenyl(2), 430 nm; 3,4-dichlorobiphenyl(3), 436 nm; 2,4,5-trichlorobiphenyl(4), 390 nm; 2-bromobiphenyl(5), 400 nm, 4-methylbiphenyl(6), 436 nm; 2-nitrobiphenyl(7), 385 nm; 4-biphenylmethanol(8), 400 nm; diphenylmethane(9), 390 nm. (b) Proposed conversion of various biphenyls and diphenylmethane by strain KF258. Enzymes: see the legend of Fig. 1

nylmethane were also tested. Since it was previously demonstrated that biphenyl and chlorobiphenyls were converted to the corresponding meta-cleavage yellow compound via dihydrodiols and dihydroxy compounds (Furukawa and Miya- zaki 1986), the formation of catabolic products from various biphenyl derivatives were monitored by measuring absorbance at the corresponding absorption maxima (Fig. 5). Biphenyl, 4-chloro-, 2-bromo-, 4-methyl-, 2-nitro- and 3,4-dichlorobi- phenyls quickly turned to yellow, and the absorp-

tions were reached maxima within 1 hr, then decreased gradually for further incubation in the case of biphenyl, 4-methyl- and 4-chlorobiphenyls. Yellow compounds were also produced from 2,4,5-trichlorobiphenyl, diphenylmethane, and 4- biphenylmethanol, but slowly. Slightly-yellow color was detected for 2-hydoxybiphenyl and 4- biphenylacetic acid after 16 hr incubation. No yellow color was observed from 4,4'-dichloro-, and 4,4'-dimethylbiphenyls and 4-biphenyl carboxylic acid.


Discussion

The applied genetics of soil pseudomonads seems to forcus on two directions; (1) degradation or de- toxification of recalcitrant chemical wastes, and (2) production of fine chemicals by use of catabolic genes. Cloned genes were used to construct Pseudomonas sp. B13 derivatives capable of utilizing 4-chlorobenzoate, 3,5-dichlorobenzoate, salicylate and chlorosalicylates as new growth substrates (Lehrbach et al. 1984). A novel route for bacterial production of indigo via hydroxyla- tion of indol was demonstrated using cells of E. coli containing a cloned fragment of P. putida TOL plasmid pWW0 (Mermod et al. 1986). In the present paper, we have demonstrated one practi- cal application of genetic techniques to the production of specialty chemicals. Using bphABC genes cloned from chromosomal DNA of P. pseu- doatcaligenes, we have constructed recombinant strains that can produce catabolic intermediates from various biphenyl compounds. Bioconversion rates of biphenyl derivatives are dependent on the substituents on the biphenyl molecule. Single sub- stistution of chloro-, bromo-, and methyl group did not significantly affect the susceptibility for the oxidative catabolism, since the conversion rates of these compounds were comparable with that of biphenyl (Fig. 5). Increased substitution with chlorine on one ring such as 3,4-dichloro- and 2,4,5-trichlorobiphenyls decreased the reaction rate accordingly. However, hydroxyl-, car- boxyl-, and carboxymethyl groups made the biphenyl molecule extremely less susceptible. More- over, if both rings are substituted with chloro- or methyl groups as in the case of 4,4'-dichlorobiphenyl and 4,4'-dimethylbiphenyl, initial oxidation did not occur. When biphenyl is used as the substrate for strain KF274 carrying pMFB6(bphAB), 2,3-dihydroxybiphenyl was ini- tially produced and then converted to tetrahydroxybiphenyl, which predominantly accumulated in the reaction mixture as the final product (Fig. 3). On the other hand, when 4-chlorobiphenyl is used as the substrate, only the dihydroxy- compound was produced and accumulated (Fig. 4). Another ring substituted with chlorine could not be attacked for oxidation. Thus, it is clear that the biphenyl dioxygenase in P. pseudoalcaligenes KF707 can attack only non-substituted ring, so that biphenyl compounds with substituents on both rings such as 4,4'-dichloro- and 4,4'-dimethylbiphenyls could not be the substrates. In con- trast to P. pseudoalcaligenes KF707, Acinetobacter sp. P6 could attack various PCBs with chlorine

substitution on both rings such as 2,2'-, 2,4'-, 3,3'-, 4,4'-, 2,5,3'-, 2,5,4'-, 2,4,4'-, 3,4,2'-, and 2,5,2',5'- chlorobiphenyls, in which the less chlorinated ring was always oxidized (Furukawa et al. 1978; Furukawa et al. 1979). Since we constructed the recombinant strains that produce dihydrodiol compounds, dihydroxy compounds and meta- cleavage compounds for various biphenyl derivatives, enzymes involved in the bipheny! catabolism could be further studied using such catabolic intermediates.

References

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Bradford M (1976) A rapid and sensitive method for the quan- tatitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248- 254

Catelani D, Sorlini C, Treccani V (1971) The metabolism of biphenyl by Pseudomonasputida. Experientia 27:1173-1174

Franklin FCH, Bagdasarian M, Bagdasarian MM, Timmis KN (1981) Molecular and functional analysis of the TOL plasmid pWW0 from Pseudomonas putida and cloning of genes for entire regulated aromatic ring meta cleavage pathway. Proc Natl Acad Sci USA 78:7458-7462

Furukawa K, Arimura N (1987) Purification and properties of 2,3-dihydroxybiphenyl dioxygenase from polychlorinated biphenyl-degrading Pseudomonas pseudoalcaligenes and Pseudomonas aeruginosa carrying the cloned bphC gene. J Bacteriol 169:924-927

Furukawa K, Miyazaki T (1986) Cloning of a gene cluster encoding biphenyl and chlorobiphenyl degradation in Pseu- domonas pseudoalcali#enes. J Bacteriol 166:392-398

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Furukawa K, Tomizuka N, Kamibayashi A (1979) Effect of chlorine substitution on the bacterial metabolism of various polychlorinated biphenyls. Appl Environ Microbiol 38:301-310

Furukawa K, Simon JR, Chakrabarty AM (1983) Common in- duction and regulation of biphenyl, xylene/toluene, and salicylate catabolism in Pseudomonaspaucimobilis. J Bacte- riol 154:1356-1362

Furukawa K, Arimura N, Miyazaki T (1987) Nucleotide se- quence of the 2,3-dihydroxybiphenyl dioxygenase gene of Pseudomonas pseudoalcaligenes. J Bacteriol 169:427-429

Inouye S, Nakazawa A, Nakazawa T (1983) Molecular cloning of regulatory gene xylR and operator-promoter regions of the xylABC and xylDEGF operons of the TOL plasmid. J Bacteriol 155:1192-1199


Kemp MB, Hegeman GD (1968) Genetic control of the keto- adipate pathway in Pseudomonas aeruoinosa. J Bacteriol 96:1488-1499

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Mermod N, Harayama S, Timmis KN (1986) New Route to bacterial production of indigo. Biotechnology 4:321-324

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Received February 15, 1988/Accepted May 26, 1988

Documents

Gene manipulation of catabolic activities for production of intermediates of various biphenyl compounds