FEMS Microbiology Letters 100 (1992) 281-286 © 1992 Federation of European Microbiological Societies 0378-1097/92/$05.00 Published by Elsevier
281
FEMSLE 80013
Growth temperature-dependent activity of glycerol dehydratase in Escherichia coli expressing the Citrobacter freundii dha regulon Rolf Daniel and G e r h a r d Gottschalk lnstitut fiJr Mikrobiologie der Georg-August-Universitiit, G&tingen, FRG Received 28 May 1992 Accepted 16 June 1992
Key words: Citrobacterfreundii; Escherichia coli ECL707; Glycerol dehydratase; 1,3-Propanediol; Glycerol fermentation; dha Regulon 1. SUMMARY Using the cosmid pWE15, a genomic library of Citrobacter freundii DNA in Escherichia coli ECL707 was prepared and screened for glycerol utilization. Six out of approximately 3000 clones were positive. One clone, harboring the recombinant cosmid pRD1, expressed glycerol dehydratase in high activity when grown at 28°C but not at 37°C. The growth temperature had little effect on the activity of the other enzymes encoded by the dha regulon. When the glycerolcontaining medium was supplemented with corrinoids, the recombinant E. coli strain produced 1,3-propanediol in high amounts at 28°C.
2. INTRODUCTION The dha regulon enables microorganisms such as Klebsiella pneumoniae or Citrobacter freundii
Correspondence to: G. Gottschalk, Institut t'fir Mikrobiologie der Georg-August-Universit~it, Grisebachstr. 8, W-3400 G6ttingen, FRG.
to grow anaerobically on glycerol. Four enzymes are encoded by this regulon: glycerol dehydrogenase (dhaD), dihydroxyacetone kinase (dhaK), glycerol dehydratase (dhaB) and 1,3-propanediol dehydrogenase (dhaT). The first two enzymes represent the oxidative branch of glycerol fermentation and the latter two the reductive branch; their activity leads to the formation of 1,3-propanediol. Glycerol dehydratase, a coenzyme B12containing enzyme is of special interest and its activity seems to be rate-limiting when resting cells of C. freundii are employed for the production of 1,3-propanediol from glycerol [1]. The dha regulon of K. pneumoniae was transferred to Escherichia coli but expression of the genes was incomplete. Sprenger et al. [2] found expression of glycerol and 1,3-propanediol dehydrogenases as well as dihydroxyacetone kinase but glycerol dehydratase was undetectable and 1,3-propanediol was not produced. Tong et al. [3] described a recombinant E. coli strain which produced some 1,3-propanediol (about 1 g/l) but the dehydratase activity was extremely low (0.0016 U / m g protein). We have cloned the dha regulon of C. freundii into an E. coli strain and describe here its full expression.
282 3. M A T E R I A L S AND M E T H O D S
3.1. Bacterial strains and cosmids C. freundii DSM 30040 was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen G m b H (Braunschweig, FRG). E. coli ECL707 (F gld::ATnlO g l p K : : A T n l O
ptsD::ATnlO araD139 flbB A(lacU169) ptsF25 relA rpsL thi) [2] was a gift from E.C.C. Lin (Harvard Medical School, Boston, MA). The cosmid pWE15 was obtained from Stratagene (Heidelberg, FRG).
3.4. Preparation of cell extracts Cells of the stationary growth phase from 500ml anaerobic cultures were harvested by centrifugation at 6000 × g for 20 min, washed once with 100 mM potassium phosphate buffer (pH 8.0) and resuspended in 2-3 ml of the same buffer. The cells were disrupted by French pressing (1.38 × l0 n Pa) and the extract was cleared by centrifugation at 32 000 × g for 35 min. All steps were done under anaerobic conditions.
3.5. Enzyme assays
The basal medium for liquid cultures contained per litre: KzHPO4, 14.0 g; KHePO4, 6.0 g; (NH4)2S04, 3.0 g; MgSO4" 7 H 2 0 , 0.2 g; CoCI 2 • 6H20, 0.0119 g; yeast extract, 0.2 g; cysteine-HC1 0.2 g; guanidine-HC1, 0.04 g; thiamin-HC1, 0.001 g; trace element solution SL4 [4], 1 ml; pH 7.5. Further supplements and the incubation temperatures are given in the Tables and Figures. Fermentations were done in Hungate tubes or anaerobic flasks and media were gassed with N 2 for 30 min before sterilization. A modified MacConkey agar (lactose was replaced by 70 mM glycerol) was used to identify glycerol-utilizing recombinant E. coli strains. All growth media for these strains additionally contained 75 /xg/ml ampicillin to maintain the presence of the cosmids.
Glycerol dehydrogenase was assayed by the method of Ruch et al. [7] and dihydroxyacetone kinase by the method of Johnson et al. [8]. Glycerol dehydratase was estimated by the 3-methyl2-benzothiazolinone hydrazone method [9] in 1min assays with glycerol as substrate [10]. The ratios between apoenzyme and holoenzyme were calculated in parallel assays with and without coenzyme B~2 in the assay mixture. The activity of 1,3-propanediol dehydrogenase was determined spectrophotometrically (E36s) at 25°C by the initial rate of substrate-dependent NADHdecrease [1]. The assay mixture contained 28 mM propionaldehyde, 0.37 mM N A D H and 100 mM triethanolamine buffer (pH 7.5) in a 1-ml final volume. Protein concentrations were determined by the method of Bradford [11] with bovine serum albumin as standard. All enzyme activities are expressed in /xmol m i n - l mg protein-~.
3.3. Cloning of the dha regulon from Citrobacter freundii Chromosomal DNA from C. freundii was iso-
3.6. Determination of 1,3-propanediol and glycerol
3.2. Media and growth conditions
lated applying the method of Marmur [5], partially digested with Sau3AI, and ligated into the cosmid pWE15 which had been linearized with BamHI. Ligated DNA was packed in vitro into the bacteriophage A and transduced into the glycerol -mutant, E. coli ECL707. Transductants were screened on MacConkey-glycerol-ampicillin agar for glycerol utilization, which was indicated by a red colour of the colonies. Cosmids from such colonies were isolated and retransduced into E. coli ECL707 to confirm the ability of the transductants to use glycerol. Digestion with restriction endonuclease, ligation, packaging of DNA, transduction, and isolation of cosmids were done according to standard procedures [6].
1,3-Propanediol was determined by gas chromatography using a Chromosorb 101 column of 1.5 m length installed in a Packard Instruments Modell 439 A (Chrompack, Frankfurt, FRG). The column temperature was 200°C, whereas the injector and detector temperature were 220°C. Gas chromatographic data were quantified with a Shimadzu (Kyoto, Japan) C-R2AX integrator. Glycerol was determined as described by Eggstein und Kuhlmann [12].
4. RESULTS Approximately 3000 recombinant E. coli strains, containing a cosmid library of C. freundii
283
DNA, were screened on MacConkey-glycerolampicillin agar. Six of these clones had the ability to utilize glycerol; one (E. coli ECL707/pRD1) containing an approx. 40 kb cosmid (pRD1) was studied further. When strain E. coli ECL707/pRD1 was grown at 37°C in basal medium containing 100 mM glycerol, 6 mM pyruvate and 0.3% CAA growth was poor and 1,3-propanediol was not produced in detectable amounts. The activity of glycerol dehydratase in cell extracts appeared very low in contrast to the high activities of the glycerol dehydrogenase, 1,3-propanediol dehydrogenase and dihydroxyacetone kinase. However, it was observed that the activity of the glycerol dehydratase was growth temperature-dependent. The specific activities of the four enzymes encoded by the dha regulon in extracts of cells grown at 25, 28, 32 and 37°C, respectively are given in Table 1. A slight increase in the activities of the two dehydrogenases and the kinase with growth temperature was recorded but dehydratase activity went through a pronounced maximum at 28°C. At this temperature the specific activities of all four enzymes in E. coli ECL707/pRD1 exceeded those of C. freundii (Table 1). Although glycerol dehydratase activity in E. coli ECL707/pRD1 was considerably increased by changing the growth temperature from 37°C to 28°C, this had little effect on 1,3-propanediol formation (1-3 mM were produced). A reason for this was found in that most of the enzyme was present as the apoenzyme. To enhance the level of holoenzyme and the formation of 1,3-propanediol, corrinoids were added to the growth
3.0'
S
1.0~
0.5: © 0.1 0.03
-60
120 ~,,,~. --
100 pJLYC~
~,~
F
I'3-PD "50
80 -
~
-40 -30
60= 40-
o°
2
~
-20
--
"10
°
0 0
5
10
15
20
25
30
t [h] Fig. 1. Anaerobic growth on glycerol and 1,3-propanediol formation of ~E. coli ECL707/pRD1. The growth experiment was carried out at 28°C in anaerobic flasks with 500 ml basal medium, which was supplemented with 120 mM glycerol and 0.4 mg/l vitamin Bl2. Measurements of glycerol (GLYC) and 1,3-propanediol (1,3-PD) were made as described in MATERIALS A N D M E T H O D S .
medium. In the presence of 0.4 mg/1 vitamin B12 , E. coli ECL707/pRD1 grew well at 28°C in basal medium containing glycerol and produced approx. 0.5 mol 1,3-propanediol per tool of glycerol consumed (Fig. 1). With 120 mM glycerol in the medium the final concentration of 1,3-propanediol was 56 mM. With higher amounts of glycerol, final 1,3-propanediol could be increased to
Table 1 Effect of growth temperature on the level of the enzymes for glycerol fermentation in E. coli ECL707/pRD1. Cultures were grown in anaerobic flasks with 500 ml basal medium, which was supplemented with 100 mM glycerol, 0.3% CAA and 6 mM pyruvate. Cell extracts were prepared and assayed as described in MATERIALSAND METHODS. Growth temperature
Specific activity of dha-encoded enzymes [~zmol min-1 mg protein 1] GLYC dehydrogenase
DHA kinase
GLYC dehydratase
1,3-PD dehydrogenase
25°C 28°C 32°C 37°C C. freundii at 28°C
4.40 5.41 5.81 6.55 4.33
0.115 0.123 0.140 0.196 0.092
1.19 1.71 0.90 0.19 0.84
0.63 0.74 0.77 0.83 0.61
284 Table 2
erich& coil strain two conditions have to be ful-
Effect of various additions to the growth medium on the ability of E. coli ECL707/pRD1 to produce 1,3-propanediol.
filled: (i) the growth temperature has to be decreased to 28°C; and (ii) corrinoids have to be added to the growth medium. In contrast to the related enteric bacteria C. freundii and Salmonella typhimurium [13], E. coli seems not to be able to synthesize corrinoids de novo under anaerobic conditions, but the recombinant strain E C L 7 0 7 / p R D 1 can use the biosynthetic precursor cobinamide for formation of coenzyme B12-containing glycerol dehydratase, which was indicated by the production of 1,3-propanediol, so it must contain the enzyme machinery for the conversion of cobinamide to coenzyme B12. This should include the pathway for 5,6-dimethylbenzimidazole-ribose-5'-phosphate synthesis, the formation of GDP-cobinamide and the final reactions leading to coenzyme B12. All, or part of, the genetic information required could reside on the approx. 32-kb insert and actually originate from C. freundii or could be present on the E. coli genome. Reasons for the growth temperature-dependent formation of glycerol dehydratase in the recombinant strain could be that the expression was temperature-dependent or that the enzyme was rapidly degraded at 37°C. This result indicates that it is advisable to try different growth temperatures if expression of certain genes in recombinant E. coli strains cannot be achieved.
Compound added
Final 1,3-propanediol concentration [mM]
None
1-3 68 64 45 1-3 68 59 50
281
FEMSLE 80013
Growth temperature-dependent activity of glycerol dehydratase in Escherichia coli expressing the Citrobacter freundii dha regulon Rolf Daniel and G e r h a r d Gottschalk lnstitut fiJr Mikrobiologie der Georg-August-Universitiit, G&tingen, FRG Received 28 May 1992 Accepted 16 June 1992
Key words: Citrobacterfreundii; Escherichia coli ECL707; Glycerol dehydratase; 1,3-Propanediol; Glycerol fermentation; dha Regulon 1. SUMMARY Using the cosmid pWE15, a genomic library of Citrobacter freundii DNA in Escherichia coli ECL707 was prepared and screened for glycerol utilization. Six out of approximately 3000 clones were positive. One clone, harboring the recombinant cosmid pRD1, expressed glycerol dehydratase in high activity when grown at 28°C but not at 37°C. The growth temperature had little effect on the activity of the other enzymes encoded by the dha regulon. When the glycerolcontaining medium was supplemented with corrinoids, the recombinant E. coli strain produced 1,3-propanediol in high amounts at 28°C.
2. INTRODUCTION The dha regulon enables microorganisms such as Klebsiella pneumoniae or Citrobacter freundii
Correspondence to: G. Gottschalk, Institut t'fir Mikrobiologie der Georg-August-Universit~it, Grisebachstr. 8, W-3400 G6ttingen, FRG.
to grow anaerobically on glycerol. Four enzymes are encoded by this regulon: glycerol dehydrogenase (dhaD), dihydroxyacetone kinase (dhaK), glycerol dehydratase (dhaB) and 1,3-propanediol dehydrogenase (dhaT). The first two enzymes represent the oxidative branch of glycerol fermentation and the latter two the reductive branch; their activity leads to the formation of 1,3-propanediol. Glycerol dehydratase, a coenzyme B12containing enzyme is of special interest and its activity seems to be rate-limiting when resting cells of C. freundii are employed for the production of 1,3-propanediol from glycerol [1]. The dha regulon of K. pneumoniae was transferred to Escherichia coli but expression of the genes was incomplete. Sprenger et al. [2] found expression of glycerol and 1,3-propanediol dehydrogenases as well as dihydroxyacetone kinase but glycerol dehydratase was undetectable and 1,3-propanediol was not produced. Tong et al. [3] described a recombinant E. coli strain which produced some 1,3-propanediol (about 1 g/l) but the dehydratase activity was extremely low (0.0016 U / m g protein). We have cloned the dha regulon of C. freundii into an E. coli strain and describe here its full expression.
282 3. M A T E R I A L S AND M E T H O D S
3.1. Bacterial strains and cosmids C. freundii DSM 30040 was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen G m b H (Braunschweig, FRG). E. coli ECL707 (F gld::ATnlO g l p K : : A T n l O
ptsD::ATnlO araD139 flbB A(lacU169) ptsF25 relA rpsL thi) [2] was a gift from E.C.C. Lin (Harvard Medical School, Boston, MA). The cosmid pWE15 was obtained from Stratagene (Heidelberg, FRG).
3.4. Preparation of cell extracts Cells of the stationary growth phase from 500ml anaerobic cultures were harvested by centrifugation at 6000 × g for 20 min, washed once with 100 mM potassium phosphate buffer (pH 8.0) and resuspended in 2-3 ml of the same buffer. The cells were disrupted by French pressing (1.38 × l0 n Pa) and the extract was cleared by centrifugation at 32 000 × g for 35 min. All steps were done under anaerobic conditions.
3.5. Enzyme assays
The basal medium for liquid cultures contained per litre: KzHPO4, 14.0 g; KHePO4, 6.0 g; (NH4)2S04, 3.0 g; MgSO4" 7 H 2 0 , 0.2 g; CoCI 2 • 6H20, 0.0119 g; yeast extract, 0.2 g; cysteine-HC1 0.2 g; guanidine-HC1, 0.04 g; thiamin-HC1, 0.001 g; trace element solution SL4 [4], 1 ml; pH 7.5. Further supplements and the incubation temperatures are given in the Tables and Figures. Fermentations were done in Hungate tubes or anaerobic flasks and media were gassed with N 2 for 30 min before sterilization. A modified MacConkey agar (lactose was replaced by 70 mM glycerol) was used to identify glycerol-utilizing recombinant E. coli strains. All growth media for these strains additionally contained 75 /xg/ml ampicillin to maintain the presence of the cosmids.
Glycerol dehydrogenase was assayed by the method of Ruch et al. [7] and dihydroxyacetone kinase by the method of Johnson et al. [8]. Glycerol dehydratase was estimated by the 3-methyl2-benzothiazolinone hydrazone method [9] in 1min assays with glycerol as substrate [10]. The ratios between apoenzyme and holoenzyme were calculated in parallel assays with and without coenzyme B~2 in the assay mixture. The activity of 1,3-propanediol dehydrogenase was determined spectrophotometrically (E36s) at 25°C by the initial rate of substrate-dependent NADHdecrease [1]. The assay mixture contained 28 mM propionaldehyde, 0.37 mM N A D H and 100 mM triethanolamine buffer (pH 7.5) in a 1-ml final volume. Protein concentrations were determined by the method of Bradford [11] with bovine serum albumin as standard. All enzyme activities are expressed in /xmol m i n - l mg protein-~.
3.3. Cloning of the dha regulon from Citrobacter freundii Chromosomal DNA from C. freundii was iso-
3.6. Determination of 1,3-propanediol and glycerol
3.2. Media and growth conditions
lated applying the method of Marmur [5], partially digested with Sau3AI, and ligated into the cosmid pWE15 which had been linearized with BamHI. Ligated DNA was packed in vitro into the bacteriophage A and transduced into the glycerol -mutant, E. coli ECL707. Transductants were screened on MacConkey-glycerol-ampicillin agar for glycerol utilization, which was indicated by a red colour of the colonies. Cosmids from such colonies were isolated and retransduced into E. coli ECL707 to confirm the ability of the transductants to use glycerol. Digestion with restriction endonuclease, ligation, packaging of DNA, transduction, and isolation of cosmids were done according to standard procedures [6].
1,3-Propanediol was determined by gas chromatography using a Chromosorb 101 column of 1.5 m length installed in a Packard Instruments Modell 439 A (Chrompack, Frankfurt, FRG). The column temperature was 200°C, whereas the injector and detector temperature were 220°C. Gas chromatographic data were quantified with a Shimadzu (Kyoto, Japan) C-R2AX integrator. Glycerol was determined as described by Eggstein und Kuhlmann [12].
4. RESULTS Approximately 3000 recombinant E. coli strains, containing a cosmid library of C. freundii
283
DNA, were screened on MacConkey-glycerolampicillin agar. Six of these clones had the ability to utilize glycerol; one (E. coli ECL707/pRD1) containing an approx. 40 kb cosmid (pRD1) was studied further. When strain E. coli ECL707/pRD1 was grown at 37°C in basal medium containing 100 mM glycerol, 6 mM pyruvate and 0.3% CAA growth was poor and 1,3-propanediol was not produced in detectable amounts. The activity of glycerol dehydratase in cell extracts appeared very low in contrast to the high activities of the glycerol dehydrogenase, 1,3-propanediol dehydrogenase and dihydroxyacetone kinase. However, it was observed that the activity of the glycerol dehydratase was growth temperature-dependent. The specific activities of the four enzymes encoded by the dha regulon in extracts of cells grown at 25, 28, 32 and 37°C, respectively are given in Table 1. A slight increase in the activities of the two dehydrogenases and the kinase with growth temperature was recorded but dehydratase activity went through a pronounced maximum at 28°C. At this temperature the specific activities of all four enzymes in E. coli ECL707/pRD1 exceeded those of C. freundii (Table 1). Although glycerol dehydratase activity in E. coli ECL707/pRD1 was considerably increased by changing the growth temperature from 37°C to 28°C, this had little effect on 1,3-propanediol formation (1-3 mM were produced). A reason for this was found in that most of the enzyme was present as the apoenzyme. To enhance the level of holoenzyme and the formation of 1,3-propanediol, corrinoids were added to the growth
3.0'
S
1.0~
0.5: © 0.1 0.03
-60
120 ~,,,~. --
100 pJLYC~
~,~
F
I'3-PD "50
80 -
~
-40 -30
60= 40-
o°
2
~
-20
--
"10
°
0 0
5
10
15
20
25
30
t [h] Fig. 1. Anaerobic growth on glycerol and 1,3-propanediol formation of ~E. coli ECL707/pRD1. The growth experiment was carried out at 28°C in anaerobic flasks with 500 ml basal medium, which was supplemented with 120 mM glycerol and 0.4 mg/l vitamin Bl2. Measurements of glycerol (GLYC) and 1,3-propanediol (1,3-PD) were made as described in MATERIALS A N D M E T H O D S .
medium. In the presence of 0.4 mg/1 vitamin B12 , E. coli ECL707/pRD1 grew well at 28°C in basal medium containing glycerol and produced approx. 0.5 mol 1,3-propanediol per tool of glycerol consumed (Fig. 1). With 120 mM glycerol in the medium the final concentration of 1,3-propanediol was 56 mM. With higher amounts of glycerol, final 1,3-propanediol could be increased to
Table 1 Effect of growth temperature on the level of the enzymes for glycerol fermentation in E. coli ECL707/pRD1. Cultures were grown in anaerobic flasks with 500 ml basal medium, which was supplemented with 100 mM glycerol, 0.3% CAA and 6 mM pyruvate. Cell extracts were prepared and assayed as described in MATERIALSAND METHODS. Growth temperature
Specific activity of dha-encoded enzymes [~zmol min-1 mg protein 1] GLYC dehydrogenase
DHA kinase
GLYC dehydratase
1,3-PD dehydrogenase
25°C 28°C 32°C 37°C C. freundii at 28°C
4.40 5.41 5.81 6.55 4.33
0.115 0.123 0.140 0.196 0.092
1.19 1.71 0.90 0.19 0.84
0.63 0.74 0.77 0.83 0.61
284 Table 2
erich& coil strain two conditions have to be ful-
Effect of various additions to the growth medium on the ability of E. coli ECL707/pRD1 to produce 1,3-propanediol.
filled: (i) the growth temperature has to be decreased to 28°C; and (ii) corrinoids have to be added to the growth medium. In contrast to the related enteric bacteria C. freundii and Salmonella typhimurium [13], E. coli seems not to be able to synthesize corrinoids de novo under anaerobic conditions, but the recombinant strain E C L 7 0 7 / p R D 1 can use the biosynthetic precursor cobinamide for formation of coenzyme B12-containing glycerol dehydratase, which was indicated by the production of 1,3-propanediol, so it must contain the enzyme machinery for the conversion of cobinamide to coenzyme B12. This should include the pathway for 5,6-dimethylbenzimidazole-ribose-5'-phosphate synthesis, the formation of GDP-cobinamide and the final reactions leading to coenzyme B12. All, or part of, the genetic information required could reside on the approx. 32-kb insert and actually originate from C. freundii or could be present on the E. coli genome. Reasons for the growth temperature-dependent formation of glycerol dehydratase in the recombinant strain could be that the expression was temperature-dependent or that the enzyme was rapidly degraded at 37°C. This result indicates that it is advisable to try different growth temperatures if expression of certain genes in recombinant E. coli strains cannot be achieved.
Compound added
Final 1,3-propanediol concentration [mM]
None
1-3 68 64 45 1-3 68 59 50
