T P Pirog - Peculiarities of c2 metabolism and intensification of the synthesis of surface-active substances in rhodococcus erythropolis ek-1 grown in ethanol - страница 1
ISSN 0026-2617, Microbiology, 2008, Vol. 77, No. 6, pp. 665-673. © Pleiades Publishing, Ltd., 2008.
Original Russian Text © T.P. Pirog, Yu.V. Korzh, T.A. Shevchuk, D.A. Tarasenko, 2008, published in Mikrobiologiya, 2008, Vol. 77, No. 6, pp. 749-757.
Peculiarities of C2 Metabolism and Intensification of the Synthesis of Surface-Active Substances in Rhodococcus erythropolis EK-1 Grown in Ethanol
T. P. Pirog, Yu. V. Korzh, T. A. Shevchuk, and D. A. Tarasenko
Institute of Microbiology and Virology, National Academy of Sciences of Ukraine, ul. Zabolotnogo 154, Kiev, 03680 Ukraine Received November 12, 2007
Abstract—Oxidation of ethanol, acetaldehyde, and acetate in Rhodococcus erythropolis EK-1, producer of surface-active substances (SAS), is catalyzed by N,N-dimethyl-4-nitrosoaniline (DMNA)-dependent alcohol dehydrogenase, NAD+/NADP+-dependent dehydrogenases (optimum pH 9.5), and acetate kinase/acetyl-CoA-synthetase, respectively. The glyoxylate cycle and complete tricarboxylic acid cycle function in the cells of R. erythropolis EK-1 growing on ethanol; the synthesis of phosphoenolpyruvate (PEP) is provided by the two key enzymes of gluconeogenesis, PEP carboxykinase and PEP synthetase. Introduction of citrate (0.1%) and fumarate (0.2%) into the cultivation medium of R. erythropolis EK-1 containing 2% ethanol resulted in the 1.5- and 3.5-fold increase in the activities of isocitrate lyase and PEP synthetase (the key enzymes of the gly-oxylate cycle and gluconeogenesis branch of metabolism, respectively) and of lipid synthesis, as evidenced by the 1.5-fold decrease of isocitrate dehydrogenase activity. In the presence of fumarate and citrate, the indices of SAS synthesis by strain R. erythropolis EK-1 grown on ethanol increased by 40-100%.
Key words: Rhodococcus erythropolis, ethanol metabolism, glyoxylate cycle, gluconeogenesis, intensification of biosynthesis, surface-active substances.
Strain Rhodococcus erythropolis EK-1 was isolated from petroleum-contaminated soil samples . R. erythropolis EK-1 was previously shown to produce surface-active substances (SAS) both on hydrophobic (hexadecane, liquid paraffins) and hydrophilic (glucose, ethanol) substrates , with much lower values of SAS synthesis on ethanol than on hexadecane. Since ethanol is a cheaper and more producible substrate than water-insoluble, hydrophobic compounds, its application for SAS biosynthesis may significantly enhance the efficiency of the SAS production technology. Although literature provides some data on the ability of Rhodococcus bacteria to assimilate ethanol as a carbon and energy source [3, 4], we did not find any information concerning SAS synthesis by rhodococci growing on this substrate.
One of the ways for intensification in the technologies of microbial synthesis is to reveal the possible sites of metabolic limitation and to develop approaches to their elimination based on the analysis of the peculiarities of energetic and constructive metabolism of producers of practically valuable metabolites. Earlier studies of C2 metabolism regulation in Acinetobacter sp.
IMV B-7005, a producer of the polysaccharide ethapo-lan, resulted in development of the method for its production on a nonbuffered medium with a fourfold decreas in content of salts (up to 2.95 g/l) .
Another approach that increases the efficiency of microbial biotechnologies is introduction of exogenous precursors into the cultivation medium. Previously we have proved the possibility for intensification of ethap-olan synthesis by introduction of C4-dicarboxylic acids, the intermediates of ethanol metabolism involved in gluconeogenesis . Introduction of precursors into the cultivation medium is known to enhance the synthesis of macrolide antibiotics . In the 80-90s of the 20th century, some researchers established the stimulating effect of sodium citrate on SAS formation by microorganisms [8-10]. Our studies showed that after introduction of fumarate (0.2%) and citrate (0.1%) into the medium with hexadecane in the beginning of the stationary growth phase of R. erythropolis EK-1 the values of SAS synthesis increased by 40-70% as compared with the analogous values at cultivation of the strain on the medium without these precursors .
The goal of this work was to study the peculiarities of ethanol metabolism in R. erythropolis EK-1 and to establish the possible mechanisms for enhancement of
SAS synthesis on this substrate in the presence of precursors (fumarate and citrate).
MATERIALS AND METHODS
Object of research. The object of research was strain R. erythropolis EK-1 registered at the Depository of microorganisms of the Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine with accession number IMV Ac-5017.
Medium composition and cultivation conditions for R. erythropolis EK-1. Bacteria were grown in liquid mineral media. Medium 1 contained the following (g/l): KNO3, 1.5; NaCl, 1.0; Na2HPO4 • 12H2O - 0.6; KH2PO4 - 0.14; MgSO4 • 7H2O, 0.1; pH 6.8-7.0. Medium 2 contained the following (g/l): KH2PO4, 6.8; NaOH, 1.0; NH4NO3 - 0.6; MgSO4 • 7H2O - 0.4; CaCl2 • 2H2O - 0.1; FeCl3 • 6H2O, 0.01; pH 6.8-7.0. Ethanol (2%, vol/vol) was used as both a carbon and energy source. Medium 1 is a modified  Muenz medium , which is used for cultivation of hydrocarbon-oxidizing bacteria. Medium 2 was developed by us for the cultivation of biopolymer producers .
The precursors of SAS synthesis, sodium citrate and sodium fumarate (0.1 and 0.2%, respectively), were added to the medium with ethanol in one of the experimental variants. Precursors were introduced as 10% solutions in the beginning of cultivation and in the beginning of the stationary growth phase.
Citrate and fumarate are additional sources of carbon nutrition and their introduction into the medium results in the change of not only carbon concentration but also the C/N ratio. Hence, the content of the main carbon source (ethanol) in the control variants was corrected. The purpose of such a correction was to ensure equimolar amounts of carbon, to provide stability of the optimal carbon/nitrogen ratio in the cultivation medium.
Cultivation was performed in 750-ml flasks with 100 ml of the medium in a shaker (220 rpm) at 30°C for
The daily culture grown on meat-peptone (MPA) or glucose-potato (GPA) agar and the culture in the exponential phase (48-72 h), grown on media 1 or 2 with 0.5% ethanol (vol/vol) in the presence or absence of SAS synthesis precursors, were used as inoculum. If the inoculum was grown in a liquid medium, its concentration was 5% of the volume of the inoculated medium.
Determination of growth and SAS synthesis characteristics. The biomass was assayed by the optical density of cell suspension followed by recalculation per absolutely dry weight according to the calibration diagram.
The ability for SAS synthesis was assessed by the following indices:
(1) Surface tension (as) of cell-free culture liquid measured using a glass plate [1, 2].
(2) Express evaluation of the quantitative content of SAS in the culture liquid was performed using an index of conventional concentration of SAS (SAS*) defined as the degree of dilution of cell-free culture liquid (supernatant) to the MCC point (Micelle formation Critical Concentration). The dependence of surface tension as on the logarithm of supernatant dilution was plotted as a diagram [1, 2]. The abscissa of the inflection point corresponds to the SAS* value. Conditional SAS concentration was expressed in dimensionless units.
(3) Index of culture liquid emulsification (E24, %) was determined as described . Sunflower oil was used as a hydrophobic substrate for emulsification.
(4) The quantity of synthesized SAS was determined by the gravimetric method of Bligh and Dyer  in our modification. Modification was necessary, because R. erythropolis EK-1 synthesizes a complex of polar and nonpolar lipids [1, 2], whereas the method of Bligh and Dyer allows extraction of mainly nonpolar lipids. In view of this fact, we modified the classical system of solvents (Folch mixture) by introducing 1 M HCl (chloroform: methanol: 1 M HCl = 4 : 3 : 2).
Obtaining the cell-free extracts. The bacterial suspension obtained after cultivation of R. erythropolis EK-1 in a liquid, mineral medium was centrifuged (4000 g, 15 min, 4°C). The cell precipitate was washed twice from the medium debris with 0.05 M K+ phosphate buffer (pH 7.0) and centrifuged at 4000 g (15 min, 4°C). Washed cells were resuspended in 0.05 M K+ phosphate buffer (pH 7.0) and sonicated (22 kHz) four times by 6 s at 4°C in an UZDN-1 apparatus. The resulting liquid was centrifuged (12000 g, 30 min, 4°C), the precipitate was removed, and the supernatant was used as a cell-free extract.
Cell-free extracts were obtained from cells in the early, middle, and late exponential growth phases (24, 48, and 72 h of cultivation, respectively).
Enzymatic analyses. The activities of alcohol dehydrogenase (EC 126.96.36.199, EC 188.8.131.52, and EC 184.108.40.206), acetaldehyde dehydrogenase (EC 220.127.116.11, EC 18.104.22.168, EC 22.214.171.124), acetate kinase (EC 126.96.36.199), acetyl-CoA-synthetase (EC 188.8.131.52), and isocitrate lyase (EC 184.108.40.206) were determined as described in . The activities of nicotinoprotein (NAD(P)H-con-taining) alcohol dehydrogenase (EC 1.1.99. -) and ace-taldehyde dehydrogenase (EC 1.2.99. -) were determined spectrophotometrically by N,N-dimethyl-4-nitrosoaniline (DMNA) reduction at 440 nm with etha-nol, methanol, and acetaldehyde as electron donors, respectively . The activity of alcohol oxidase (EC 220.127.116.11) was analyzed by H202 formation or dissolved oxygen consumption as described in . The activity of acetaldehyde dehydrogenase (EC 18.104.22.168) was determined by the reduction of dichlorophenol indophenol in the presence of phenazine methosulfate
at 600 nm .
Table 1. Activity of alcohol and acetaldehyde dehydrogenases in R. erythropolis EK-1 grown on ethanol
Activity (nmol min 1 mg 1 protein) when grown in
NAD+-dependent (pH 9.0)
8.91 ± 0.4
9.41 ± 0.3
NADP+-dependent (pH 9.0)
10.52 ± 0.5
8.57 ± 0.3
7.23 ± 0.3
3.74 ± 0.1
51.45 ± 2.5
40.16 ± 0.21
(85.32 ± 3.9)
(155.92 ± 0.69)
40.16 ± 2.1
10.93 ± 0.5
(83.73 ± 4.0)
(145.89 ± 7.3)
NAD+-dependent (pH 9.5)
96.72 ± 4.8
110.55 ± 5.5
NADP+-dependent (pH 9.5)
44.82 ± 2.4
29.71 ± 1.1
7.22 ± 0.3
3.15 ± 0.1
7.34 ± 0.2
4.38 ± 0.2
Note: The cells from the middle of exponential growth phase (48 h) were sampled. In brackets, the enzymatic activities in the cells from the early exponential growth phase (24 h) are given.
The activities of malate synthase (EC 22.214.171.124), citrate synthase (EC 126.96.36.199), aconitate hydratase (EC 188.8.131.52), isocitrate dehydrogenase (EC 184.108.40.206 and EC 220.127.116.11), 2-oxoglutarate dehyderogenase (EC 18.104.22.168), succinate dehydrogenase (EC 22.214.171.124), fumarate hydratase (EC 126.96.36.199), malate dehydrogenase
(EC 188.8.131.52, EC 184.108.40.206), malate dehydrogenase
(decarboxylating) (EC 220.127.116.11 and EC 18.104.22.168), phos-phoenolpyruvate (PEP) synthetase (EC 22.214.171.124), and PEP carboxykinase (EC 126.96.36.199) were analyzed as described in .
Protein content in cell-free extracts was determined according to Bradford . Enzyme activities were determined at 28-30°C, the temperature optimal for growth of R. erythropolis EK-1.
All experiments were carried out in three repeats; parallel measurements in the experiments were three to five. Statistical processing of experimental data was performed according to Lakin . The experimental results, according to the Student's t-criterion, proved to be statistically reliable at a 5% level of significance.
RESULTS AND DISCUSSION
Oxidation of ethanol and acetaldehyde. Alcohol dehydrogenases oxidizing ethanol to acetaldehyde in microorganisms belong to three groups [20, 21]. The first group includes NAD(P)+-dependent alcohol dehy-drogenases that have been studied thoroughly. Representatives of the second group are NAD(P)+-indepen-dent alcohol dehydrogenases that utilize pyrrole quino-line quinone (PQQ), heme (associated with PQQ), or factor F420 as a cofactor. The third group includes flavin adenine dinucleotide (FAD+)-dependent alcohol oxi-dases catalyzing irreversible oxidation of alcohols.
In the 90s, of the 20th century, some Gram-positive bacteria (Mycobacterium gastri, Rhodococcus rhodo-chrous, R. erythropolis, Rhodococcus sp., and Amyco-latopsis methanolica) were shown to have a new type of nicotinoprotein (NAD(P)H-containing) alcohol dehy-drogenases revealed through the reaction with N,N-dimethyl-4-nitrosoaniline (DMNA) as an artificial electron acceptor . Such enzymes are known as DMNA-dependent alcohol dehydrogenases. As an active site, they contain the bound NAD(P)H which is a cofactor but not a coenzyme of these dehydrogenases.
It is known from the literature that NAD(P)+ and DMNA-dependent alcohol dehydrogenases function in the members of the genus Rhodococcus growing on ethanol [15, 22]. It is interesting to note that DMNA-dependent enzymes of ethanol-grown rhodococci are able to oxidize both ethanol and methanol [15, 22]. It is mentioned in these works that methanol and ethanol oxidation involves two different DMNA-dependent alcohol dehydrogenases, i.e. methanol: N,N-dimethyl-4-nitrosoaniline oxidoreductase (MNO) and alcohol: N,N-dimethyl-4-nitrosoaniline oxidoreductase (DMNA-ADH). The cells of R. erythropolis DSM 1069 growing on the medium with ethanol were shown to have the NAD+-alcohol dehydrogenase activity with the optimum pH 9.0 and the DMNA-ADH and MNO activities .
The NAD+, NADP+, PQQ and DMNA-dependent
enzymes as well as MNO were found in the cells of R. erythropolis EK-1 grown on ethanol (Table 1). The activities of PQQ- and NAD(P)+-dependent alcohol dehydrogenases were not high (4—10 nmol min-1 mg-1 protein) and obviously could not be of essential significance for ethanol metabolism in the studied rhodococ-cus strain. These enzyme activities remained practi
cally on the same level irrespective of the bacterial growth phase. Investigation of the activities of NAD+-and NADP+-dependent enzymes in the pH range from 7.0 to 9.5 showed that the optimal pH value was 9.0, when the activity did not exceed 10 nmol min-1 mg-1 protein (Table 1).
As can be seen from the data presented in Table 1, the cells of R. erythropolis EK-1 growing on ethanol possess both DMNA-ADH and MNO activities with the maximal values in the early exponential phase of bacterial growth. The activities of these alcohol dehy-drogenases significantly decreased in the middle of the exponential phase and were only 4-5 nmol min-1 mg-1 protein by the end of the exponential phase.
It is known from the literature that DMNA-ADH activity determined in the cell-free extract of rhodococci grown on ethanol does not exceed 4-6 nmol min-1 mg-1 protein [15, 22]. It may be explained by the fact that DMNA-ADH activity decreases 2-20 times in the presence of adenylates, acetaldehyde, and many cations that are inhibitors of this enzyme . The content of such inhibitors in cell-free extracts is rather high. The above assumption is supported by the fact that the activity of this enzyme increased by two orders of magnitude after its isolation and preliminary purification .
Thus, ethanol oxidation in R. erythropolis EK-1, like in other rhodococci, involves DMNA-dependent alcohol dehydrogenase.
On medium 1, DMNA-ADH and MNO activities in R. erythropolis EK-1 measured in the beginning of the exponential growth phase were almost two times lower than those obtained on medium 2 (84-85 and 146-156 nmol min-1 mg-1 protein, respectively). At the same time, by the middle of the exponential growth phase these enzymatic activities decreased only twice on medium 1 (to 40-50 nmol min-1 mg-1 protein) and 4-15 times on medium 2 (to 11-40 nmol min-1 mg-1 protein).
Acetaldehyde formed during ethanol oxidation is involved in metabolism with participation of acetalde-hyde dehydrogenases. Most microorganisms have NAD(P)+-dependent enzymes and acylating acetalde-hyde dehydrogenase [23-25]. Recently, reports have appeared on acetaldehyde oxidation in bacterial cells by PQQ- and DMNA-dependent acetaldehyde dehy-drogenases [15, 17]. The cell-free extract of R. erythropolis EK-1 was shown to contain several acetaldehyde dehydrogenases (Table 1). The activity of PQQ- and DMNA-dependent enzymes did not exceed 7-7.5 nmol min-1 mg-1 protein. The data presented in Table 1 demonstrate that acetaldehyde oxidation in R. erythropolis EK-1 is performed by NAD+- and NADP+-dependent acetaldehyde dehydrogenases. The study of the effect of the reaction mixture pH on these enzyme activities revealed the pH optimum at 9.5.
As is known from the literature, NAD+-dependent acetaldehyde dehydrogenase, which can also oxidize formaldehyde, functions in R. erythropolis UPV-1 growing on ethanol . As a result, strain UPV-1 can be used for removal of formaldehyde from industrial wastewater. Our preliminary experiments also demonstrated the possibility of formaldehyde oxidation by the cells of R. erythropolis EK-1. It should be noted that representatives of the genus Rhodococcus, including R. erythropolis strains, are characterized by a wide range of various enzymes including dehydrogenases. Hence, rhodococcus strains and their enzymes may be considered promising for application in different environment-protecting biotechnologies .
Central metabolism. The activity of the enzymes of central metabolism at cultivation of R. erythropolis EK-1 on ethanol is given in Table 2. Acetate formed by the acetaldehyde dehydrogenase reaction is oxidized by acetate kinase and acetyl-CoA-synthetase (280-300 and 45-55 nmol min-1 mg-1 protein, respectively). The role of the glyoxylate cycle as the anaplerotic sequence of reactions completing the pool of C4-dicarboxylic acids in ethanol-grown R. erythropolis EK-1 is confirmed by the high activity of both isocitrate lyase and malate synthase (730-740 and 135-165 nmol min-1 mg-1 protein, respectively).
The cell-free extract of R. erythropolis EK-1 shows high activity of all the enzymes of the tricarboxylic acid cycle, with the exception of 2-oxoglutarate dehydroge-nase: 10-20 nmol min-1 mg-1 protein. High activities of isocitrate dehydrogenase and isocitrate lyase and low activity of 2-oxoglutarate dehydrogenase at cultivation of R. erythropolis EK-1 on ethanol may demonstrate that the tricarboxylic acid cycle in these bacteria plays a mainly, biosynthetic role.
It should be noted that the activity of some enzymes of this cycle was practically the same at cultivation of strain EK-1 on media 1 and 2. At the same time, the activities of fumarate hydratase, NAD+-dependent malate dehydrogenase, and NADP+-dependent malate dehydrogenase (decarboxylating) were nearly 3 times higher and the activity of NADP+-dependent isocitrate dehydrogenase was 1.5 times higher at cultivation of this rhodococcus strain on medium 1 than on medium 2
At growth on ethanol, the synthesis of carbohydrates necessary for the formation of nucleic acids, polysaccharides, and a number of metabolites including surface active substances in R. erythropolis EK-1 is provided by the gluconeogenetic branch of metabolism, as demonstrated by the high activity of the two key enzymes of gluconeogenesis: PEP synthetase and PEP carboxykinase (Table 2).
It is notable that the activity of PEP synthetase at cultivation of R. erythropolis EK-1 on medium 1 was higher (nearly threefold) than the activity of this enzyme at cultivation on medium 2. At the same time, PEP carboxylase activity was practically the same at cultivation of the strain on both media. Since not only the PEP synthetase activity but also the activities of malate synthase, fumarase, NAD+-dependent malate dehydrogenase and NADP+-dependent malate dehy-
PECULIARITIES OF C2 METABOLISM 669
Table 2. Activity of the enzymes of central metabolism in R. erythropolis EK-1 cultivation on ethanol
Activity (nmol min-1 mg-1 protein) when grown in
52.0 ± 2.6
44.6 ± 2.2
287.6 ± 14.3
302.2 ± 15.1
741.1 ± 37.0
733.0 ± 36.5
164.8 ± 8.2
138.5 ± 6.9
216.4 ± 10.2
201.8 ± 10.0
152.4 ± 7.6
168.7 ± 8.4
NAD+-dependent isocitrate dehydrogenase
10.2 ± 0.5
10.5 ± 0.5
NADP+-dependent isocitrate dehydrogenase
608.6 ± 30.4
400.0 ± 20.0
20.4 ± 1.0
10.5 ± 0.5
99.3 ± 4.9
68.7 ± 3.4
776.9 ± 38.4
268.7 ± 13.4
NAD+-dependent malate dehydrogenase
406.4 ± 20.0
148.4 ± 7.3
NADP+ dependent malate dehydrogenase
50.8 ± 2.5
42.2 ± 2.1
NAD+-dependent malate dehydrogenase (decarboxylating)
NAD+-dependent malate dehydrogenase (decarboxylating)
304.8 ± 15.2
105.4 ± 5.2
210.9 ± 10.5
1307.1 ± 63.2
464.3 ± 23.2
Note: Cells from the middle of exponential growth phase (48 h) were sampled.
drogenase (decarboxylating) were higher at cultivation of R. erythropolis EK-1 on medium 1, a higher level of glycolipid synthesis could be expected under these conditions than on medium 2. However, the previous data  demonstrated that the amount of carbohydrate SAS synthesized during the growth of R. erythropolis EK-1 on medium 1 was somewhat less than that on medium 2. Elucidation of the causes of this phenomenon will be the subject of our further work. Nevertheless, the results of enzymatic studies demonstrate the existence of potential reserves for the increase of SAS synthesis on medium 1.
We believe that the differences in the activity of some enzymes at cultivation of R. erythropolis EK-1 on media 1 and 2 (see Tables 1 and 2) may be determined by the different mineral compositions of these media, in particular, different contents of potassium, sodium and ammonium cations, which are potential enzyme activators or inhibitors.
Mechanisms of intensification of SAS synthesis at cultivation of R. erythropolis EK-1 on ethanol in the presence of precursors. The SAS synthesized by R. erythropolis EK-1 in ethanol are a complex of gly-colipids and neutral lipids with polysaccharide-protein compounds . Hence, we have assumed that it is pos-sible,similar to the case of cultivation on hexadecane , to increase the efficiency of SAS biosynthesis on C2 substrates by an introduction into the medium of sodium citrate (the regulator of lipid synthesis) and C4
Table 3. Formation of surface-active substances in the course of cultivation of R. erythropolis EK-1 on the medium with ethanol in the presence of biosynthetic precursors
Indicators of synthesis
3.0 ± 0.15
0.94 ± 0.04
60 ± 3.0
3.0 ± 0.15
0.80 ± 0.03
90 ± 4.0
4.2 ± 0.21
1.4 ± 0.07
75 ± 3.0
Citrate, 0.1% + fuma-
4.8 ± 0.24
1.9 ± 0.09
80 ± 4.0
Note: Cultivation on medium 2; inoculum was grown on ethanol; fumarate and citrate were introduced in the beginning of the stationary growth phase.
900 800 700 600 500 400 300 200 100
Activity of the enzymes providing the involvement of exogenous fumarate and citrate in metabolism of R. erythropolis EK-1 grown on the medium with ethanol. Enzymes: 1, NADP+-dependent isocitrate dehydrogenase; 2, isocitrate lyase; 3, NAD+-dependent malate dehydrogenase; 4, fumarate hydratase. Cultivation conditions: A, without fumarate and citrate (control); B, citrate (0.1%); C, fumarate (0.2%); D, citrate (0.1%) + fumarate (0.2%). Cultivation was carried out on medium 2. The inoculum was grown on medium 2 with ethanol and respective precursors. The precursors were introduced in the beginning of cultivation. Enzyme activities were determined for cells from the middle of exponential growth phase.