cis-benzeneglycol production using a mutant Pseudomonas strain

[J. Ferment. Technol., Vol. 66, No. 3, 305-312. 1988]

cis-Benzeneglycol Production Using a Mutant Pseudomonas Strain JEAN-JACqUES YARMOFF, YASUSHI KAWAKAMI*, TAKESHI YAGO, HIROYUKI MARUO, a n d HAJIME NISHIMURA

Departmentof ChemicalEngineering, Universityof Tokyo, Tokyo 113, Japan The biotransformation of commodity aromatic chemicals into dihydroxy derivatives was studied. A strain isolated from the environment, PseudomonasJIl04, used benzene, toluene, and other hydrocarbons as sole carbon and energy sources. We selected mutants unable to grow with benzene, and among these, screened for strains with deficient ¢is-benzeneglycol dehydrogenase able to stably produce cis-benzeneglycol when another carbon source was co-metabolized. We examined the possibility of cis-benzeneglycol production by growing the mutant strain in the presence of benzene vapor. Ethanol was the carbon and energy source most adapted to the eis-benzeneglycol production phase, and lactate or propanol could also be used. Glucose inhibited the production of the metabolite. The growth rates were barely affected by the presence of benzene at a reduced partial pressure (less than 20% of saturation), showing that continuous culture is possible. In a batch process, 0.54 g.1-1 of a cell suspension produced 5.1 mmol.1-1 eis-benzeneglycol in 27 h, using ethanol as the energy source.

T h e possibility o f using m i c r o o r g a n i s m s for the p r o d u c t i o n o f fine c h e m i c a l s using c h e a p c o m m o d i t y c h e m i c a l s as substrates has attracted considerable attention. Mild prod u c t i o n conditions, e n a n t i o m e r specificity, a n d the p o s s i b i l i t y to p r o d u c e molecules w h i c h a r e difficult to synthesize b y classical m e t h o d s a r e a m o n g the m a i n a d v a n t a g e s o f b i o l o g i c a l conversions. T h e r e is c u r r e n t i n d u s t r i a l interest in t h e use o f a r o m a t i c dioxygenases for the p r o d u c t i o n o f d i h y d r o d i o l s a n d access to n e w classes o f s u b s t i t u t e d catechols. T h e simplest o f these c o m p o u n d s , the d i h y d r o d i o l d e r i v e d from b e n z e n e , cis-l,2-dihydroxy-cyclohexa3,5-diene (cis-benzeneglycol, cBG) has b e e n shown to be a p r e c u r s o r to p o l y p a r a p h e n y l e n e (ppp).l) The exceptional physical properties o f ppp,2) t o g e t h e r w i t h its p r o m i s e s as a c o n d u c t i v e polymerS) f u r t h e r increase t h e interest i n cis-benzeneglycol. However, * Corresponding author

t h o u g h cis-benzeneglycol has b e e n identified as a n i n t e r m e d i a t e in b a c t e r i a l b e n z e n e degradation,4) a n d the use o f b a c t e r i a as catalysts in a p r o d u c t i o n process has b e e n described,5) the possibility o f c o n v e n t i o n a l f e r m e n t a t i o n techniques to p r o d u c e ¢isb e n z e n e g l y c o l has b e e n little investigated. I n this work, we r e p o r t the isolation o f a s t r a i n a b l e to g r o w w i t h b e n z e n e as t h e sole c a r b o n source, w i t h a v e r y stable d e g r a d a t i o n a b i l i t y . M u t a n t s u n a b l e to g r o w w i t h benzene after c h e m i c a l m u t a g e n e s i s were p i c k e d up, a n d cis-benzeneglycol p r o d u c i n g strains were screened for. W e show t h a t p r o d u c t i o n b y c o - m e t a b o l i s m o f e t h a n o l i n presence o f b e n z e n e v a p o r is feasible, a n d discuss the b a s i c c o n d i t i o n s o f the b i o t r a n s f o r m a t i o n . Materials and Methods

Bacterial strains The benzene degrading strain used in this study, named JI104, was isolated from soil which had been polluted by aromatic hydrocarbons, near the coke oven attached to a gasworks, by enrich-

306

YARMOFFet al.

ment culture with benzene as the sole carbon source. E. coli containing some conjugative plasmids unable to replicate in Pseudomonas and carrying Tn 5 (marker for kanamycin resistance), were used for transposon mutagenesis (suicide vectors). The E. coli strains were E. coli S17-1 (pSUP2021), ~) ED2196 (pLG221), 7) and J53 (pJB4JI). s) The highest transposition/conjugation frequency (1.8 × 10-5 per recipient) was observed with strain S17-I. Enrichment method Aromatic hydrocarbons are often toxic at high concentration, s) Therefore, benzene was supplied into the gas phase of the culture, at a partial pressure below saturation. This was achieved by allowing some benzene vapor to diffuse through a tight cotton plug into the air flow. The air was then bubbled through the culture. Isolation of individual benzene degrading strains was done by streaking repeatedly on nutrient agar, and growing single colonies in minimum liquid medium with benzene as the carbon source. Growth conditions Non-selective Nutrient Broth (NB medium), TM pH 7.2, was used as the standard culture medium for all bacterial strains. The benzene degrading strain, JI104, was routinely grown with M56 minimum medium containing the following (per liter): 11.7g of Na~HPO4'12H20, 2.7g of KH2PO4, 1.0g of (NH4)~SO4, 100mg of MgSO4. 7H,O, 5 mg of Ca(NOs)~, and 0.25 mg of FeSO47H,O, adjusted to pH 7.2. This medium was used both for liquid cultures, and for growth in Petri dishes after the addition of 1.5% agar. The minimum medium M9 I°) was also used. Benzene was supplied from granular activated charcoal, which had first been equilibrated with air containing a low concentration of benzene, usually between 20 and 30% of saturation. (30°C). This was accomplished by circulating air over cold benzene at a temperature sucb, that saturation pressure of benzene at the low temperature was equal to 30% of that at 30°C. The benzene air mixture was then percolated through a bed of charcoal pellets for several days. The pellets were kept in a tightly closed vessel before use; for plate cultures, a spoonful of pellets was added to the lid of the Petri dish. In the case of liquid cultures, a paper plug was inserted inside the mouth of a 200 × 20 mm test tube, and charcoal pellets were placed over it. The benzene vapor was supplied to the medium through the paper plug, the mouth of the tube being sealed with aluminium foil. While the concentration of benzene might have changed slightly from batch to batch, experiments were always done using the same batch to ensure the comparability of the results. The strain could not grow when liquid benzene was directly introduced into the test tubes. Incubations were

[J. Ferment. Technol.,

conducted in a controlled temperature room (30°G). All chemicals were obtained from commercial sources, and were used without further purification. Mutagenesis N-Methyl-N'-nitro-N-nitrosoguanidine (NTG) was used for chemical mutagenesis. The procedure was essentially based on the method for producing auxotrophs in E. coli. TM Treated JI104 cells were exposed to benzene in the presence of antibiotics (Penicillin-G, 10000units m1-1 and D-cycloserine, 100/~g.m1-1) for 8 h for mutant enrichment. Transposon mutagenesis was done by filter mating the suicide vector donor strains with JI104. Cultures of JI104 and of the donor strain were allowed to rest, were then mixed and filtered, and the filter carrying the bacteria was incubated overnight on a fresh NB plate. Ceils were then scraped off, suspended in M56, and plated on a minimum plate containing benzoate as carbon source (2 g./-1) and kanamycin (200 ,ug.ml-l). Only JI104 strains has,ing integrated Tn5 in their chromosome could form colonies on this selective medium. The number of colonies growing allowed us to measure the conjugation/transposition frequency. Production of metabolltes The screening of eis-benzeneglycol producing strains, or the analyses of some production conditions were done by growing the strains in test tubes with an appropriate carbon source, in the presence of benzene vapor. The substrate was added to 10 ml of M56 medium before autoclaving (e.g. succinate) or after the sterilization (e.g. glucose or ethanol). The benzene vapor was provided from charcoal pellets which were placed over a previously sterilized paper plug. The incubation period was usually 24 or 36 h.

Measurement of els-benzeneglyeol accumulation The product accumulation was measured by spectrophotometry (Hitachi 340 recording spectrophotometer), or by HPLC analysis of the liquid culture supernatant. A sample of the culture was spun for 5rain (15k rpm); 500#1 were then filtered using a Millipore UFC3 TGC membrane (maximum molecular weight of the solute, 10,000) adapted to a microfuge tube. After dilution, 100/A were analyzed without further purification. The apparatus used was a TOSO HPLC system with a TSK gel column ODS807M coupled to a UV analyzer (260nm). The absorption coefficient of !og(~202)=3.49 was used to calculate the concentration ofcis-benzeneglycol. TM

Results and Discussion Isolation of benzene degrading

strains

S e v e r a l s t r a i n s g r o w i n g w i t h b e n z e n e as t h e sole c a r b o n s o u r c e w e r e i s o l a t e d b y e n r i c h ment culture. Axenic cultures were obtained

Vol. 66, 1988]

307

cis-Benzeneglycol Producing Mutant

Table I. Aromatic degradation ability of the strain JI104. Good growth o n : benzene biphenyl m-toluate o-cresol catechol Slow growth on: m-xylene o-phthalate p-phthalate No growth on: naphthalene

toluene benzoate p-toluate m-cresol protocatechuate

ethylbenzene o-toluate phenol p-cresol 4-methylcatechol

anthracene m-phthalate 2,3-dimethyl naphthalene

decahydronaphthalene 1,2,4-trimethylbenzene

o-xylene

by repeatedly streaking single colonies on NB plates and transferring isolated colonies to M56 m i n i m u m m e d i u m supplied with benzene vapor. All the isolated bacteria were G r a m negative, unicellular short rods, motile, obligately aerobic, and non-fermentative. T h e y were tentatively identified as Pseudomonas. T h e strain showing the best growth, n a m e d J I l 0 4 , was chosen for further studies. T h e colonies were smooth and glistening. Besides benzene, this strain could degrade a number of aromatic compounds, as shown in T a b l e 1. Strategy of mutant isolation The only well characterized benzene degradation pathwayXZ) in bacteria is shown in Fig. 1. I t involves the action of a three-enzyme dioxygenase system,18,14) and the subsequent dehydrogenation of the cis-benzeneglycol Benzene c~-Benzene glycol H

Catechol meta

Pathway

ortho Pothwoy

Fe +~BFea+

"~

X

Fp2~Fp hY~D÷

NADH + H+

Fig. 1. Bacterial benzene degradation pathway. A2, Flavoprotein A2. A1 and B, iron-sulfur proteins.

produced. 15) I n contrast, the degradation of benzoate by Pseudornonas putida mt-2 leads to catechol through different sets of enzymes, xs,iT) As we were primarily interested in finding mutants having lesions either in the benzene dioxygenase system or in the cBG dehydrogenase, we screened for mutants unable to grow with benzene, but able to use benzoate. T h e strains would have an intact degradation pathway downstream of catechol, limiting the n u m b e r of unrelated mutants recovered. Strains accumulating cis-benzeneglycol were then chosen from this collection of mutants. Mutagenesis Both chemical and transposon mutagenesis were used. Cells treated with N T G were incubated in minimal mediu m in the presence of benzene, penicillin-G, and D-cycloserine for mutant enrichment. Comparison of the growth of potential mutants with benzene and with benzoate allowed us to pick up 156 clones having some lesion in the benzene degradation pathway out of 60,000 colonies. By screening an additional 25,000 clones having incorporated Tn5 during transposon mutagenesis, 8 more strains unable to grow with benzene as the sole carbon source were recovered. T o screen for mutants producing cisbenzeneglycol, the strains were separately grown on m i n i m u m medium supplemented with either succinate or ethanol in the presence of benzene vapor Although transposon mutants in the benzene genes were

YARIVlOFFet al.

308

l --[ V-262nm

[J. Ferment. Technol.,

b e n z e n e g l y c o [ - p r o d u c i n g mutants. As shown in T a b l e 2, the p r o d u c t i o n v a r i e d from s t r a i n to strain. T h e best p r o d u c e r seemed to be m u t a n t J I 1 0 4 - 1 6 , a n d this s t r a i n was used for all s u b s e q u e n t e x p e r i m e n t s . T h e e x c l u d e d strains e i t h e r d i d not a c c u m u l a t e a n y byp r o d u c t s , were leaky, or were u n s t a b l e a n d r e v e r t e d to the o r i g i n a l p h e n o t y p e after several subcultures.

g JI104-16

Confirmation

of

cis-benzeneglycol p r o -

T h e m u t a n t strains were culI I t u r e d in test tubes on M 5 6 m i n i m u m m e d i u m 200 300 400 200 300 40O(nm) W0velength w i t h 2 ml./-1 e t h a n o l as the c a r b o n source, Fig. 2. UV spectra of the culture supernatants of a n d in the presence o f a low level o f benzene strain JI104-16, and JI104-20, mutants unable to v a p o r s u p p l i e d f r o m c h a r c o a l pellets at grow with benzene alone. The strains were a c o n c e n t r a t i o n below 3 0 % o f s a t u r a t i o n , as grown in test tubes on M56 medium supplemented d e s c r i b e d before. T h e c u l t u r e s u p e r n a t a n t with ethanol. Benzene was supplied from char- was a n a l y z e d b y l i q u i d c h r o m a t o g r a p h y coal pellets as described in MaterialsandMethods. ( H P L C ) (Fig. 3a). T o g e t h e r w i t h cis-benzeneglycol, p h e n o l (the p r o d u c t ofcis-benzeneglycol d e h y d r a t i o n ) o b t a i n e d a t a f r e q u e n c y o f 3 . 2 × 10 -4, n o n e o f these strains seemed to a c c u m u l a t e a pro- was also d e t e c t e d . T h e t r a n s f o r m a t i o n o f d u c t w i t h a n a b s o r p t i o n a t 260 nm. F i g u r e cis-benzeneglycol to p h e n o l was v e r y slow at 2 shows the U V a b s o r p t i o n s p e c t r a o f the the p h y s i o l o g i c a l p H used (a few p e r cent s u p e r n a t a n t s o f two N T G m u t a n t s w h i c h p e r d a y a t r o o m t e m p e r a t u r e ) . H o w e v e r , were b o t h u n a b l e to g r o w w i t h benzene, w h e n the c u l t u r e m e d i u m was acidified with one, JI104-16, a c c u m u l a t i n g a p r o d u c t w i t h sulfuric a c i d to p H 1 a n d i n c u b a t e d at 37°C a U V a b s o r p t i o n m a x i m u m a r o u n d 262 nm, for one hour, analysis showed t h a t the p e a k w h i l e a n o t h e r J I 1 0 4 - 2 0 , d i d not. T h i s of cis-benzeneglycol was g r e a t l y r e d u c e d , p r o d u c t was identified as cis-benzeneglycol w h i l e the p h e n o l c o n c e n t r a t i o n increased b y H P L C analysis (see below). conversely (Fig. 3b). is) I n this assay, it was F i n a l l y 9 strains were selected as cis- found t h a t w h i l e 120.5 # m o l . l - x cis-benzeneduction

Table 2. cis-Benzeneglycol production by various JI104 strains, grown on succinate in the presence of benzene vapor (incubation time 48 It). Strain

Growth O D s e 0 nm

JIl04 (wild type) JIl04- 16

1.36 0. 87

[cBG] rag.l-1

Efficiency nag cBG/g dry cell 5.6 70 25 49 9. 8 67.7 45 7.8 13.2 56

- 43

1.09

4.0 32.2 14. 5

- 88

0.83

21, 7

-137 -143 -145 -146 -148 -156

1.28 0. 89 1.15 1.32 1.08 1.08

6.7 32.0 27.5 5.5 7.5 32. 0

cis-Benzeneglycol Producing M u t a n t

Vol. 66, 1988]

a)

b)

Table 3. Correlation between the growth on toluene a n d that on benzene of the mutant strains. T h e figures give the n u m b e r of strains able to grow with benzene or with toluene, as counted in Petri dishes.

J g

o

*~

g

Growth with benzene:

ri

ill ~ L _ i

2,5

5 Time

(rain)

2'.5

309

5 Time

(rain)

Fig. 3. Chromatograrn showing the quantitative transformation of ¢is-benzeneglycol into phenol. A culture supernatant was a) analysed directly, b) was acidified to pI-I 1 then neutralized before analysis. Because of the sample dilution during these steps, the concentration of the sample b) is half that of a). T h e U V absorption at 260 n m of the eluent was monitored. Note the presence of a small quantity of phenol (dehydration of cisbenzeneglycol) before the medium acidification.

glycol disappeared, 123.3pmol.l-t phenol were formed, cis-Benzeneglycol was measured using the valve from the literature, TM log(~s2)=3.49, while phenol was measured by calibration with a commercial sample. This unambiguously confirms that the accumulated metabolite is 1,2-dihydroxycyclohexa-3,5-diene. Relationship between hydrocarbon degradation p a t h w a y s When the collection of 156 mutants (and revertants) was tested for the ability to grow on aromatics, they all showed a growth similar to that of the wild type when supplied with m-toluate, p-toluate, or biphenyl. It is possible that all these compounds are degraded by a single pathwayt~,z0) that has no step in common with the benzene degradation. In contrast, mutants unable (revertants able) to grow with benzene were also unable (able) to use toluene with few exceptions (Table 3). Since the mutations generated by N T G are essentially random, this striking correlation between benzene and toluene degradation strongly suggests that both substrates are oxidized and metabolized by the same pathway. Another organism which can degrade

Growth with toluene: Good Poor No growth

Good

Poor

No growth

57 5

5 6

0 0

1

13

69

benzene and toluene by the same pathway has been described by Gibson et al.4,TM However, it is not a general property since bacteria that can degrade benzene but cannot use toluene have also been described by Hogn and Jaenicke. 22) The somewhat relaxed specificity of the benzene degradative pathway might have important implications. I f a production protess for cis-benzeneglycol can be devised, it is also possible that by switching the substrate from benzene to toluene, or to other benzene derivatives, the corresponding diols will be produced. Choice of the carbon source for the production process The conversion of benzene to cis-benzeneglycol requires reducing power in the form of NADH.15, 22~ T o maintain the production of cis-benzeneglycol, the bacteria will have to compensate for this energy depletion by the co-metabolism of another carbon source. In addition to the aromatic compounds that the wild type PseudomonasJIl04 could degrade, we observed growth with a wide range of hydrocarbons. The effects of some of these compounds on cell growth and cis-benzeneglycol production by the mutant JI104-16 are summarized in Table 4. T h e different carbon sources were added to 10 rnl of M56 minimal medium, so as to reach a 0.2% final concentration. Cells grown overnight in liquid medium were washed several times in M56 buffer before inoculation (initial O . D . = 0 . 1 ) . The test

YARMOFret al.

310

Table 4. Effectsof various carbon sources on growth of strain JI104-16, and on cis-benzeneglycol production. Test tubes were inoculated to an initial O.D. of 0.1. Carbon sources

Growth ODse0 am

[cBG] mg'1-1

0. 787 0. 580 0. 763 0. 692 0. 570 0. 761 0. 787 0. 708 0. 479 0. 667 0. 593 0. 737 0. 150

0. 3 1.1 1.9 0. 4 5.4 7.0 4. 9 0. 8 0. 8 0. 05 0. 0 3.5 1.7

Glucose Fructose Citrate Acetate Lactate Ethanol Propanol Butanol 2-Propanol Glycerol Catechol Benzoate Blank

....... tubes were t h e n i n c u b a t e d for 48 h at 30°C. A l t h o u g h glucose was one of the best substrates for cell growth, the level of cisbenzeneglycol a c c u m u l a t i o n was very low, w h a t e v e r the glucose c o n c e n t r a t i o n (Fig. 4a). O t h e r substrates, like fructose or glycerol, also prevented the f o r m a t i o n of cis-benzeneglycol. Propanol, ethanol, or lactate, o n the other h a n d , allowed a significant a m o u n t of cis-benzeneglycol to be produced. W h e n e t h a n o l was the c a r b o n source, while at low c o n c e n t r a t i o n s the p r o d u c t f o r m a t i o n seemed

x/

Z~ -g

x'--'------

x --------/,~--.-.-_~ 0 5

E

-IoJ 2 -~ 4o

o = o

® 20 g, .~ 0 _o-•, I

o

I

2

P

I

J

3 4 5 Glucose ( g . / - I )

// cf

l0

Fig. 4(a). Effects of various glucose concentrations on the cis-benzeneglycol production. Symbols: ( O ) , cis-benzeneglycol; (×), cell growth.

[J. Ferment. Technol., ff )/

I x....

g,

40 I

x- - - - - - - - - - - × ~

•

ID

:

m !/ .~ 0 r

I

0.3 o

~0.1 o -io £ [ o , ~'

i2og o

i

~ 2

o-----4)-__~ ,

5 4 5 Ethanol ( m l . / - ~ )

io

Fig. 4(b). Effects of various ethanol concentrations on cis-benzeneglycol production. Same symbols as (a). p r o p o r t i o n a l to the a m o u n t of c a r b o n source introduced, a n o p t i m u m was reached for a n i n i t i a l c o n c e n t r a t i o n of 1 m l . / - k F u r t h e r increase of the e t h a n o l c o n c e n t r a t i o n did not i m p r o v e the p r o d u c t i o n (Fig. 4b). T h e a b i l i t y of our s t r a i n to produce cisbenzeneglycol w h e n the energy is derived from lactate is i n contrast w i t h the c a r b o n source r e q u i r e m e n t of a c a t e c h o l - p r o d u c i n g s t r a i n isolated b y Shirai, zS) where lactate was n o t found to support a good p r o d u c t i o n . Since use of cheap fructose-glucose syrup as a c a r b o n source would be a d v a n t a g e o u s for a n i n d u s t r i a l process, it m i g h t be necessary to isolate a derepressed m u t a n t . Growth rates of JI104-16 with different carbon sources T h e efficient growth of the m u t a n t strain is the first step i n dsbenzeneglycol p r o d u c t i o n . Therefore, we m e a s u r e d the growth rate of the p r o d u c i n g s t r a i n w i t h different c a r b o n sources: glucose or e t h a n o l i n M56 m i n i m u m m e d i u m or N u t r i e n t Broth. T h e highest specific growth rate was observed w i t h the complex m e d i u m NB (0.94 h -x) w h e t h e r b e n z e n e v a p o r was present or not. At high cell densities (O.D. over 5) the s t r a i n h a d a t e n d e n c y to flocculate. T h e strain g r o w n o n glucose (5 g./-x) h a d a slightly higher growth rate t h a n the strain utilizing ethanol (5 ml.l-x). However, a c o m b i n a t i o n of the c a r b o n sources allowed the s t r a i n to grow faster; w h e n glucose was a d d e d to a final c o n c e n t r a t i o n of 1 g.l-1 to the m e d i u m

cis-Benzeneglycol Producing Mutant

Vol. 66, 1988]

311

...--o

-~ 5 .-] kD ~JO (3

2

.c

I

.J

o

o

#,

-

J

0.5

..

0.2

I

I

T

T

r

4

8

12

16

20

J

1

l

I

J

Ethanol I

(h

r

i

T

i

4

8

12

16

20

l

J

~

[

]

(h}

2 /

.e.l - e--e-''l

I

0.5 I

-

Incubation t i m e

F/

00o..

0.2 Incubation time

Fig. 5. Growth rate of the strain JI104-16, with different carbon sources, in presence or absence of benzene vapor. Five hundred-ml Erlenmeyer flasks with 200 ml liquid phase were used. Benzene vapor was provided from charcoal pellets placed over a frltted glass funnel fitting in the mouth of the flask. Samples (1 ml) were taken at regular intervals to measure the optical density of the culture. ©, growth without benzene; e , growth with benzene.

containing 0.5% ethanol, the specific maximal growth rate was increased to 0.30 h -1 (compared with growth rates of 0.17 and 0.18 h -x on ethanol and glucose as sole carbon sources, respectively). I f the use of glucose alone shows no obvious advantage, the growth with mixed substrates or with a complex m e d i u m will minimize the time required for the production of the biomass. When the strain was grown in the presence of benzene, the growth rates were not appreciably affected. However, the final cell concentrations were in all cases lower than those without benzene (Fig. 5). T h e results show the possibility of a continuous production process, since the growth rate of the producing strain is not m u c h affected by the presence of benzene at a low concentration (in this case not higher than

20%). Since ethanol allows both high production of c/s-benzeneglycol and satisfactory growth of the strain, it seems to be the carbon source best suited for a production process. P r o d u c t i o n o f cis-benzeneglycol in batch culture T h e strain JT104-16 was incubated in M9 medium, with 0.2% ethanol as the carbon source, and in the presence of charcoal pellets containing benzene. Cells were collected by centrifugation and resuspended in 200 ml of M9 medium, with I ml-/-x of ethanol, in a 500-ml Erlenmeyer flask, at an initial concentration of 0.54 g.l-1 ( O . D . = I ) . Charcoal pellets provided the benzene. T h e results are shown in Fig. 6. Ethanol (0.2 ml) was added to the culture after 6 h of incubation. After 2 7 h of incubation, the cis-benzeneglycol concen-

312

YARMOFFet al. Ji5

- 8~

o

] 41"

-~

-Io

._J/

i

--

o5.

/"

2 ~

._---.

i ~a 0 "'; /°

0

5

I0

~----~

15 20 Time (h)

7'5

~ 0 30

Fig. 6. Production of cis-benzeneglycol in batch culture. The system described in Fig. 5 was used for benzene delivery. During the growth a small quantity of phenol was also produced. Symbols: liD,cis-benzeneglycol concentration; x, cell growth (ODes0 nm); [:3, phenol concentration.

tration was 5.1 mmol./-1. These figures are particularly promising for an industrial production process; under the conditions described, we could produce more than 5 mmol./-1 cis-benzeneglycol in 27 h, or 560/~g ml-1. Translated to a large scale production process, a 2 td reactor could produce more than 1 kg of cis-benzeneglycol per day. These results were obtained with an unsophisticated mutant, in a culture medium designed primarily for E. coli. I f the level reached can be considered too low to support an economical production process, optimization of the parameters best suited for our strain will undoubtedly allow us to increase the production of cis-benzeneglycol. Acknowledgments

Systems for transposon mutagenesis are kind gifts from Professor R. Simon, Universitat Bielefeld. This work was supported by Grants from The Asahi Glass Foundation for Industrial Technology and from Nippon Steel Corporation. References

1) Ballard, D . G . H . , Courtis, A., Shirley, I. ML, Taylor, S.C.: ,Jr. Chem. Sot., Chem. Comm., 954 (1983).

2) Ballard, D . G . H . , Courtis, A., Shirley, I.M., Taylor, S.C.: Proceedings of the 1st SPSJ International Polymer Conference, p. 173 (1984). 3) Wegner, G.: Makromol. Chem., Macromol. Symp., 1, 151 (1986). 4) Gibson, D.T., Cardini, G.E., Maseles, F.C., Kallio, R.E.: Biochemistry,9, 1631 (1970). 5) Taylor, S.C., Brown, S.: Performance Chemicals, 11, 18 (1986). 6) Simon, R., Priefer, U., Puhler, A.: Biotechnology, I1, 137 (1983). 7) Boulnois, G.J., Vaxley, J . M . , Sharpe, G.S., Franlin, F . C . H . : Mol. Gen. Genet., 200, 65 (1985). 8) Beringer, J. E., Beynon, J. L., Buchanan-Wollaston, A.V., Johnston, A.W.B.: Nature, 276, 633 (1978). 9) Playne, M.J., Smith, B.R.: Biotechnol. Bioeng., 25, 1251 (1983). 10) Gerhardt, P.: Manual of Methods for General Bacteriology, American Society for Microbiology (1981). 1l) Nakajima, M., Tomida, I., Hashizume, A., Takei, S.: Chem.Ber., 89, 2224 (1956). 12) Gibson, D.T., Koch, J.R., Kallio, R.E.: Biochemistry, 7, 2653 (1968). 13) Axcell, B. C., Geary, P.J.: Biochem. J., 146, 173 (1975). 14) Crutcher, S.E., Geary, P.J.: Biochem. J., 177, 393 (1979). 15) Axcell, B. C., Geary, P.J.: Biachem. J., 136, 927 (1973). 16) Harayama, S., Lehrbach, P. R., Timmis, K.N.: J. Bacteriol., 160, 251 (1984). 17) Nakazawa, T., Yokota, T.: J. Bacteriol., 115, 262 (1973). 18) Whited, G. M., McCombie, W. R., Kwart, L. D., Gibson, D.T.: J. Bacteriol., 166(3), 1028 (1986). 19) Kunz, D.A., Chapman, P. J. : J. Bacteriol., 146, 179 (1981). 20) Gibson, D.T., Roberts, R.L., Wells, M.C., Kobal, U . M . : Biachem. Biophys. Res. Commun., 50, 211 (1973). 21) Gibson, D. T., Hensley, M., Yoshioka, H., Mabry, T.J.: Biochemistry,9(7), 1626 (1970). 22) Hog"n, T., Jaenicke, L.: Eur. J. Biochem., 30, 369 (1972). 23) Shirai, K.: Agric. Biol. Chem., 51, 121 (1987). (Received October 30, 1987)