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In vivo analysis of straight-chain and branched-chain fatty acid biosynthesis in three actinomycetes
ELSEVIER
FEMS Microbiology
Letters 131 (1995) 227-234
In vivo analysis of straight-chain and branched-chain fatty acid biosynthesis in three actinomycetes Kimberlee K. Wallace a, Bitao Zhao a, Hamish A.I. McArthur b, Kevin A. Reynolds aY* a Department of Pharmaceutical Sciences, School of Pharmacy, Universiv of Maryland at Baltimore, Baltimore, MD 21201, USA b Central Research Division, Pfizer Incorporated,
Groton, CT 06340, USA
Received 6 June 1995; revised 4 July 1995; accepted 10 July 1995
Abstract The starter units for branched-chain and straight-chain fatty acid biosynthesis was investigated in vivo in three actinomycetes using stable isotopes. Branched-chain fatty acids, which constitute the majority of the fatty acid pool, were
confirmed to be biosynthesized using the amino acid degradation products methylbutyryl-CoA and isobutyryl-CoA as starter units. Straight-chain fatty acids were shown to be constructed using butyryl-CoA as a starter unit. Isomerization of the valine catabolite isobutyryl-CoA was shown to be only a minor source of this butyryl-CoA. KeywordF: Actinomycete;
Butyrate
metabolism;
Fatty acid biosynthesis;
1. Introduction The cellular fatty acids of streptomycetes are known to consist primarily of branched-chain fatty acids with only minor straight-chain components [l]. A similar combination of branched-chain and straight-chain fatty acids is observed in Bacillus [2]. Research on Bacillus has shown that branched-chain fatty acids are likely produced using the amino acid catabolites, isobutyryl-CoA, 2-methylbutyryl-CoA and 3-methylbutyryl-CoA, as biosynthetic starter units [3]. The same starter units are probably utilized in branched-chain fatty acid biosynthesis in strepto-
Corresponding author. Tel.: + 1 (410) 706 5008; Fax: (410) 706 0346; E-mail: [email protected]. l
037%1097/95/$09.50
Q 1995 Federation
SSDIO378-1097(95)00263-4
of European
+ 1
Microbiological
Amino acid catabolism
mycetes. Incorporation of deuterated isobutyrate into the branched-chain fatty acid isopalmitate, in Streptomyces fiadiae, have suggested that isobutyryl-CoA is indeed a biosynthetic starter unit [4]. The role that 2-methylbutyryl-CoA plays in both avermectin and branched-chain fatty acid biosynthesis in Srrepcomyces aoermitilis has also been described [5]. The preferred starter unit for the biosynthesis of the minor straight-chain fatty acids in streptomycetes is not known. One suggestion is that acetyl-CoA is utilized directly by transacylation to acetyl ACP (acyl carrier protein) [6]. However, the deuterium labeling of the palmitate pool by deuterated isobutyrate in S. fiudiue is consistent with an isomerization of isobutyryl-CoA to n-butyryl-CoA, which is subsequently used in straight-chain fatty acid biosynthesis [4]. This interpretation is consistent with reSocieties. All rights reserved
palmitate,
isopentadecanoate,
and isoheptadecanoate
B
C
A
D
of
pools
to the abundances
labeled Isopalmitate/palmitate pool labeled Isopalmitate/palmitate pool labeled Palmitate pool labeled Isopentadecanoate/isoheptadecanoate by intact incorporation of by incorporation of pool labeled by incorporation by incorporation of methylvalerate/hexanoate as methylvalerate/hexanoate (%) of leucine (%) butyrate (%I isobutyrate/butyrate (%)
into the isopalmitate,
A=[M+7]/~[M]+[M+7]~~100;B=[M+11]/~[M]+[M+7]+[M+lll)X100;C=[M+7]/([M]+[M+7]+[M+Il])X100;D=[M+9]/(IM]+[M+9])~100.[M],abundance of the molecular ion (m/z) corresponding to non-deuterated methyl ester of the fatty acid, whereas [M+7],[M+9], and [M+ 111 correspond molecular ions at 7, 9 and 11 atomic mass units greater than this value and reflect fatty acids labeled with 7, 9, and 11 deuterlums, respectively.
Calculation A
Isopalmitate/palmitate by valine (%l
Table 1 Mass spectral analysis calculations of the percentage incorporation of perdeuterated compounds of Streptomyces cinnamonensis, Streptomyces collinus and Saccharopolyspora erythraea
K.K. Wallace et al. / FEMS Microbiology Letters 131 (1995) 227-234
sults observed in Bacillus where in vitro analysis has shown that butyryl-CoA is the preferred starter unit for straight-chain fatty acid biosynthesis [7,8]. In this work we have utilized an in vivo method for investigating the probable starter units for both straight-chain and branched-chain fatty acid biosynthesis in Streptomyces collinus, Streptomyces cinnamonensis and Saccharopoiyspora (Sp.) erythraea; actinomycetes in which either aspects of butyrate metabolism or fatty acid biosynthesis have previously been studied [9-111.
harvested by centrifugation. The supematant was discarded, and the cell pellet was washed with distilled water. 2.2. Fatty acid analysis
2. Materials and methods 2.1. Culture conditions for fatty acid analysis S. collinus Tu 1892 and Sp. erythraea (ATCC 11635) were maintained on agar slants containing 4 g yeast extract, 10 g malt extract, 4 g glucose and 20 g agar in 1 1 distilled water. S. cinnamonensis (A 3823.5) was maintained on agar slants containing 10 g Bacto-soytone, 10 g glucose, 1 g calcium carbonate and 20 g agar in 1 1 distilled water. A liquid medium (3 g yeast extract, 5 g malt extract, 3 g peptone and 10 g glucose in 1 1 distilled water at pH 7.2) in which all three actinomycetes could grow and which would not give background signals in the subsequent fatty acid analysis was selected. Spores (approx. 109) from one slant were used to inoculate a 250-ml flask containing 50 ml of this medium. After incubation at 30°C with shaking (200 rpm) for 48 h, 1 ml of this inoculum was transferred to a 50-ml flask containing 10 ml of the same medium supplemented with specific concentrations of labeled precursors. After incubation for 24-48 h, cells were
Table 2 Typical fatty acid compositions
of S. collinus, S. cinnamonensis
Strain
C 14
Cl5
Cl,
Cl7
%SCFA
S. collinus S. cinnamonensis Sp. erythraea
1.6 M> 0.3
3.4 3.3 1.3
7.1 11.4 4.8
0.3 4.0 1.3
12.4 18.7 7.8
229
Cell pellets were suspended in 1 ml of methanolic sodium hydroxide (1.2 N NaOH in 50% methanol) and heated in an airtight tube at 100°C for 30 min. Upon cooling to room temperature, 0.5 ml of 6 N HCl and 1 ml of BCl, were added to the suspension. The acidified suspension (pH < 2) was heated at 85°C for 10 min. Upon cooling to room temperature, fatty acid methyl esters were extracted by the addition of 1 ml of hexane/ether (50/50). Fatty acid methyl esters were analysed by injecting a sample (l-2 ~1) of the organic phase onto a Hewlett Packard 5970/5970A series gas chromatograph-mass selective detector equipped with an HP1 methyl silicone gum capillary column (0.33 PM film thickness). Ramp conditions for analysis were 50°C to 250°C at a rate of 20°C min- ‘. General fatty acid profiles were collected by scanning from 50 to 350 atomic mass units. Peaks were assigned based upon comparisons with retention times and mass spectral fragmentation patterns of standards. Fatty acid profiles for stable isotope incorporation studies were collected by scanning between 250 and 300 atomic mass units. The percentage of the fatty acid pool derived from perdeuterated precursors was calculated from the mass spectral analysis in the method shown in Table 1. In the [2-13C]acetate experiment, the average percentage labeling of each acetate-derived position of palmitate was calculated using (1 -Xl X 100; X = N/(Y + N) where N is defined as the number of
and Sp. erythraea i-14
i-15
ai-
i-16
i-17
ai-
%BCFA
13.8 0.7 7.9
15.5 11.0 14.0
31.9 18.0 22.3
19.1 15.2 29.8
2.5 11.0 6.8
4.9 25.7 11.4
87.7 81.6 92.1
Abbreviations: C,,, my&ate; C,,, pentadecanoate; C,,, palmitate; C,,, margarate; i-14, isomyristate; i-15, isopentadecanoate; ai-15, anteisopentadecanoate; i-16, isopalmitate; i-17, isoheptadecanoate; ai-17, anteisoheptadecanoate; SCFA, straight-chain fatty acids; BCFA, branched-chain fatty acids. The sum of %SCFA and %BCFA may exceed 100% due to rounding off the percentages of the individual fatty acids.
230
K.K. Wallace et al./ FEMS Microbiology Letters 131 (1995) 227-234
3. Results and discussion
potential acetate-derived sites (i.e. N = 8 for palmitate) and Y = (A4 + 1)/M. Y is the ratio of palmitate containing one 13C to unlabeled palmitate (after correction for natural abundance 13C). Results reported in the text for the stable isotope incorporation studies represent the findings of a single experiment with a single flask. Reproducibility was confirmed, in S. collinus, using perdeuterated hexanoate which resulted in 20 + 5% labeling of the palmitate pool from butyrate from three separate experiments.
3.1. Biosynthesis of long branched-chain fatty acids Typical fatty acid profiles for S. collinus, S. cinnamonensis and Sp. erythraea show that branched-chain fatty acids with chain lengths ranging from 14 to 17 carbon atoms predominate (Table 2). Like Bacillus, these three microorganisms also produce straight-chain fatty acids which constitute less than 20% of the total fatty acid pool (Table 2).
Leucine Degndation
Valine
-
-penladecanoyl CoA
Methylvalerate (exogenous)
I
I
Degrsdstlln
lsobutytyl CoA
’
Methylvaleryl CoA
I Biosynthesis
[Methylvaleryl ACP] -
Bulyryl CoA crotonyl COA Rsduc.tsse
aiisyntiwrit -
T\
Crotonyl CoA
t (Hydroxybutyryl CoA)
-
lsopalmitoyl CoA
Biosynthesis
[Hexanoyl ACP]
-
--+
Palmitoyl CoA
t
Hexanoyl CoA
f Hexanoate (exogenous)
t
I
(Acetoacetyl CoA) 4
2 Ace+ CoA
Fig. 1. Proposed pathways of fatty acid and amino acid metabolism in S. collinus. Compounds in square brackets represent presumed fatty acid biosynthetic intermediates which may exist as either CoA-or ACP-activated thioesters. Fatty acids are represented as CoA thioesters although they may also exist as ACP thioesters or free fatty acids.
8 10 7 $i 18
pools
of Swepromyces
14 ND 23
Streptomyces
collinus,
and Saccharopolyspora
1.5 5 26
20 37 49
Palmitate pool labeled by Palm&ate pool labeled by b intact incorporation of intact incorporation of hexanoate ’ (o/o) hexaooate ’ as butyrate (o/o)
cirmomonensis,
Isopalmitate pool labeled by incorporation of methylvalerate as isobutyrate (%I
palmitate
a Valine was added at time of inoculation to a final concentration of 200 mM to S. cinnamonensis and Sp. erythraea and 250 mM to S. collinus. b Perdeuterated methylvalerate was added to a final concentration of 4.3 mM at the time of inoculation. ’ Perdeuterated hexanoate was added to a final concentration of 4.3 mM at the time of inoculation. d ND, below detectable limits.
61 66 62
S. cinnamonensis s. collinus So. ervrhraea
and/or
Isopalmitate pool labeled by intact incorporation of methylvalerate b (o/o)
into the isopalmitate
Palm&ate labeled by valine a (o/o)
compounds
Isopalmitate labeled by valioe a (o/o)
of perdeuterated
Organism
Table 3 Incorporation erythraea
232
K.K. Wallace et al. / FEMS Microbiology Letters 131 (1995) 227-234
Long branched-chain fatty acid biosynthesis in these three actinomycetes was investigated by following the incorporation of stable isotope labeled precursors into the corresponding fatty acid pool. Addition of perdeuterated leucine (4.3 mM) to a S. collinus fermentation resulted in approximately 53% labeling of the isopentadecanoate CC,,) pool and 58% labeling of the isoheptadecanoate CC,,> pool with nine deuterium atoms. This result is consistent with the catabolism of leucine to 3-methylbutyrylCoA, which is utilized as a starter unit for the biosynthesis of odd-numbered fatty acids containing a branch at the o-1 carbon (Fig. 1). As shown in Table 3, addition of perdeuterated valine (200-250 mM) resulted in efficient isotopic labeling of the isopalmitate pool, consistent with the catabolism of valine to isobutyryl-CoA and subsequent incorporation into even-numbered branched-chain fatty acids. Under these conditions, a 35% labeling of the isopentadecanoate pool with seven deuteriums was also observed. This result is consistent with the catabolism of valine to a-ketoisovaleryl-CoA by valine dehydrogenase [12] and subsequent biosynthesis of leucine from this intermediate (Fig. 1) [13]. This leucine is then catabolized as described above. These results indicate that the even-numbered branchedchain fatty acids are built from an isobutyryl-CoA starter unit and that the odd-numbered branched chain fatty acids with a methyl branch at ~1 (the iso fatty acids) are built using 3-methylbutyryl-CoA as a starter unit. Presumably, the odd-numbered branched-chain fatty acids and a methyl branch at the ~2 carbon (the anteiso fatty acids) are built from the isoleucine catabolite, 2-methylbutyryl-CoA, as previously suggested in S. avermitilis [5]. In two of the bacterial systems perdeuterated methylvalerate, a putative six-carbon intermediate in branched-chain fatty acid biosynthesis, was incorporated intact with 11 deuterium atoms into the isopalmitate pool (Table 3). The degradation of the methylvalerate to isobutyryl-CoA prior to use in isopalmitoyl-CoA biosynthesis (Fig. 1) was observed by the labeling of 14-23% of the isopalmitate pool with seven deuteriums. This result is again consistent with the utilization of isobutyryl-CoA as a starter unit for branched-chain fatty acid biosynthesis. The lack of incorporation of methylvalerate into the isopalmitate pool in the case of S. collinus may arise
from limited transport of the compound into the cell, although this has not been demonstrated. The observed intact incorporation of the six carbon intermediate into the isopalmitate pool in S. cinnamonensis and Sp. erythraea suggests that these branched-chain fatty acids are made by a Type II fatty acid synthase. Type II fatty acid synthases are commonly observed in prokaryotes and plants [14]. These synthases consist of at least seven distinct and separable enzymes where potential fatty acid biosynthetic intermediates, such as exogenously supplied methylvalerate, can be transacylated and converted through to their products [15]. Like other prokaryotes, Streptomyces are generally thought to contain Type II fatty acid synthases; however, some preliminary evidence for a Type I synthase in Sp. erythraea [11,16] and Streptomyces coelicolor [17] has been reported. In contrast to Type II synthases, Type I synthases are multifunctional enzyme complexes which do not allow for fatty acid biosynthetic intermediates to enter and be converted to their products 1151.Such complexes are predominantly observed in eukaryotic systems [14,15], although their presence has been reported in other actinomycetes [18,19]. 3.2. Biosynthesis
of straight-chain
fatty acids
The formation of straight-chain fatty acids in the three bacterial systems was investigated by monitoring the incorporation of [2- l3Clacetate, perdeuterated butyrate and perdeuterated hexanoate. The experiments with [2-‘3C]acetate (11.9 mM) resulted in approximately 5% labeling of each of the acetate/malonate-derived positions. The low incorporation into the palmitate pool most likely arises due to dilution of the labeled acetate by endogenous acetyl-CoA and by the myriad of biochemical pathways that utilize acetyl-CoA. Addition of perdeuterated butyrate (4.3 mM) resulted in 57% labeling of the palmitate pool with seven deuterium atoms in S. cinnamonensis and 53% in Sp. erythraea. The lower level of intact incorporation of butyrate observed with S. collinus (22%) is consistent with studies with 14C-labeled butyrate which demonstrated that less than 5% of the butyrate is taken up by the cell (unpublished results). If straight-chain fatty acids were assembled in these systems by a Type I fatty acid synthase that used acetyl-CoA as a starter unit,
K.K. Wallace et al. / FEMS Microbiology Letters 131 (1995) 227-234
no significant intact incorporation of butyrate into the palmitate pool would be predicted. Some level of intact butyrate incorporation would be predicted if acetyl-CoA were the preferred or the physiological starter unit for a Type II fatty acid synthase. The very high level of intact butyrate incorporation is, however, more consistent with the direct utilization of butyryl-CoA for fatty acid biosynthesis using either a Type I or Type II fatty acid synthase (Fig. 1). In contrast, a low level of intact hexanoate incorporation into the straight-chain fatty acids was observed (Table 3). This intact incorporation indicates that straight-chain fatty acids, like branched-chain fatty acids, are potentially synthesized by a Type II fatty acid synthase. However, as shown in Table 3, the majority of the hexanoate that was fed was oxidized to butyryl-CoA prior to utilization as a starter unit. This result is consistent with butyryl-CoA being the preferred starter unit for straight-chain fatty acid biosynthesis in these microorganisms. One possible physiological source of butyryl-CoA for palmitoyl-CoA biosynthesis may be an isomerization of the valine-derived catabolite isobutyrylCoA (Fig. 1). This pathway has been observed in the formation of a butyryl-CoA building block in the biosynthesis of secondary metabolites [4,10,20-221 and straight-chain fatty acids in S. frudiae [4], suggesting that this isomerization may be general among streptomycetes. Valine feeding studies at high concentrations (200-250 mM as opposed to 5 mM) demonstrated that in these three actinomycetes a similar isomerization provides a component of the butyryl-CoA pool, putatively used for straight-chain fatty acid biosynthesis. In S. collinus cultures grown in the presence of 250 mM perdeuterated valine, 66% of the isopalmitate pool was labeled with deuterium. If the sole starter unit for isopalmitoyl-CoA biosynthesis is isobutyryl-CoA, 66% of the isobutyryl-CoA pool must, by definition, be perdeuterated. If all of the palmitoyl-CoA is assembled from butyryl-CoA, of which the sole source is the isomerization of isobutyryl-CoA, it would be predicted that 66% of the palmitate pool in the same experiment would be isotopically enriched with seven deuteriurns. However, the results indicate that only 10% of the palmitate pool is labeled with deuterium. Thus, in this experiment, only 14% of the butyryl-CoA pool is derived from an isomerization of isobutyryl-
233
CoA with the remaining 86% of the butyryl-CoA being derived from an alternative source. Similarly, 87% and 88% of the butyryl-CoA utilized for straight-chain fatty acid biosynthesis in the valine feeding studies of S. cinnamonensis and Sp. erythrueu, respectively, must also be obtained from a source other than isobutyryl-CoA (Fig. 1). It has already been demonstrated that butyrate units for secondary metabolism in streptomycetes can be formed from the condensation of two acetate molecules [22]. This process, which may be responsible for providing the majority of butyryl-CoA for palmitoyl-CoA biosynthesis, has not been investigated in streptomycetes. In mammalian mammary glands [23] and Euglena gracilis [a], butyryl-CoA used for fatty acid biosynthesis is thought to be formed from the condensation of two acetyl-CoA molecules (Fig. 11. The final critical step in this pathway is catalysed by an NADPH-specific crotonyl-CoA reductase [23-251. A similar enzyme may also operate in the conversion of acetyl-CoA to butyryl-CoA in the actinomycetes studied herein [9]. In summary, we have utilized an in vivo stable isotope analysis method for rapid, facile studies of both branched-chain amino acid degradation and fatty acid biosynthesis. This assay has been used to study these processes in three actinomycetes and has not only confirmed that branched-chain fatty acids are biosynthesized from isobutyryl-CoA and methylbutyryl-CoA, but shown that straight-chain fatty acids are preferentially biosynthesized from butyryl-CoA. Furthermore, evidence has been obtained that two alternative sources for butyryl-CoA formation are in operation in these organisms.
Acknowledgements This work was supported in part by a National Science Foundation grant (DCB-0104933) and by a National Institutes of Health grant (GM 50541-02) to K.A.R. and by a National Science Foundation Creativity Award in Engineering (EID-9021048) and an American Foundation of Pharmaceutical Education Fellowship (AFPE) to K.K.W. The authors are indebted to Professor A. Zeeck and Professor H. Zlhner for supplying S. collinus and to Eli Lilly for supplying S. cinnamonensis.
234
K.K. Wallace et al. /FEMS Microbiology Letters 131 (1995) 227-234
References [I]
Saddler, G.S., O’Donnel, A.G., Goodfellow. M. and Minnikin, D.E. (1987) SIMCA pattern recognition in the analysis of streptomycete fatty acids. J. Gen. Microbial. 13, 11371147. [2] Kaneda, T. (1963) Biosynthesis of branched-chain fatty acids. I. Isolation and identification of fatty acids from Bacillus subtilts. J. Biol. Chem. 238, 1222-1228. [3] Kaneda, T. (1991) lso-and anteiso-fatty acids in bacteria: biosynthesis, function and taxonomic significance Microbial. Rev. 55, 288-302. 141 Rezanka, T., Reichelova, J. and Kopecky, J. (1991) Isobutyrate as a precursor of n-butyrate in the biosynthesis of tylosine and fatty acids. FEMS Microbial. Lett. 84, 33-36. [S] Rezanka, T., Mikovl, H. and JurkovL, M. (1992) Influence of inhibitors on lipid biosynthesis on the production of avermectins in Streptomyces auermitilis. FEMS Microbial. Lett. 96, 31-36. [6] Suutari, M. and Laasko, S. (1992) Changes in fatty acid branching and unsaturation of Streptomyces griseus and Breuibacterium fermentans as a response to growth temperature. Appl. Environ. Microbial. 58, 2338-2340. [7] Kaneda, T. and Smith, E.J. (1980) Relationship of primer specificity of fatty acid de nouo synthetase to fatty acid composition in 10 species of bacteria and yeasts. Can. J. Microbial. 26, 893-898. IS] Kaneda, T., Smith, E.J. and Naik, D.N. (1983) Fatty acid composition and primer specificity of de novo fatty acid synthetase activity in Bacillus globisporw, Bacillus insolitus and Bacilluspsychrophilus. Can. J. Microbial. 29, 1634-41. [9] Reynolds, K.A. (1993) Comparison of two unusual enoyl COG reductases in Sfreptomyces collinus. J. Nat. Prod. Sk, 175-185. [lOI Reynolds, K.A., O’Hagan, D., Gani, D. and Robinson, J.A. (1988) Butyrate metabolism in Streptomycetes. Characterization of an intramolecular vicinal interchange rearrangement linking isobutyrate and n-butyrate in Streptomyces cinnamonensis. J. Chem. Sot., Perkins Trans. 1, 3195-3207. [Ill Revill, W.P. and Leadlay, P.F. (1991) Cloning, characterization and high-level expression in Escherichia coli of the Saccharopolyspora erythraea gene encoding an acyl carrier protein potentially involved in fatty acid biosynthesis. J. Bacterial. 173, 4379-4385. I121Nguyen, L.T., Nguyen, K.T., Spiiek, J. and Behal, V. (1995) The tylosin producer, Streptomycesfradiae contains a second valine dehydrogenase. Microbiology 141, 1139-1145.
and its I131 Umbarber, H.E. (1978) Amino acid biosynthesis regulation. Annu. Rev. Biochem. 47, 533-606. [I41Volpc, J.J. and Vagelos, P.R. (1976) Mechanisms and regulation of the biosynthesis of saturated fatty acids. Physiol. Rev. 56, 339-417. [I51Lynen, F. (1980) On the structure of fatty acid synthetase of yeast. Eur. J. Biochem. 112, 431-442. [I61 Rossi, A. and Corcoran, J.W. (1973) Identification of a multienzyme complex synthesizing fatty acids in the actinomycete Strepromyces erythraeus. Biochem. Biophys. Res. Commun. 50,597-602. [171Flatman, S. and Packter, N.M. (1983) Partial purification of fatty acid synthetase from Streptomyces coelicolor. Biochem. Sot. Trans. 11, 597. [I81 Wood, WI., Peterson, D.O. and Bloch, K. (1978) Subunit structure of Mycobacterium smegmatis fatty acid synthetase. Evidence for identical multifunctional polypeptide chains. J. Biol. Chem. 253, 2650-2656. [191 Vance, D.E., Mitsuhashi, 0. and Bloch, K. (1973) Purification and properties of the fatty acid synthetase from Mycobacterium phlei. J. Biol. Chem. 248, 2303-2309. DO1Pospisil, S., Sedmera, P., Havranek, M., Krumphanzl, V. and VanEk, Z. (1983) Biosynthesis of monensins A and B. J. Antibiot. 36, 617-619. [21] Gmura, S., Tsuzuki, K., Tanaka, Y., Sakakibara, H., Aizawa, M. and Lukacs, G. (1983) VaIine as a precursor of n-butyrate in the biosynthesis of macrolide aglycone. J. Antibiot. 36, 614-616. [22] Byrne, K.M., Shafiee, A., Nielsen, J.B., Arison, B., Monaghan, R.L. and Kaplan, L. (1993) The biosynthesis and enzymology of an immunosuppressant, immunomycin, produced by Streptomyces hygroscopicus var. ascomyceticus. Dev. Ind. Microbial. 32, 29-44. [23] Maitra, SK. and Kumar, S. (1974) Crotonyl coenzyme A reductase activity in bovine mammary fatty acid synthetase. J. Biol. Chem. 249, 111-117. [24] Inui, H., Miyatake, K., Nakano, Y. and Kitaoka, S. (1986) Purification and some properties of short chain-length specific fruns-2-enoyl-CoA teductase in mitochondria of Euglena gracilis. J. Biochem. 100, 995-1000. [25] Podack, E.R. and Seubert, W. (1972) On the mechanism of malonyl-CoA independent fatty acid synthesis. Il. Isolation, properties and subcellular location of trans-2,3-hexenoylCoA and tranr-2,3-decenoyl-CoA reductase. Biochim. Biophys. Acta 280, 235-247.
FEMS Microbiology
Letters 131 (1995) 227-234
In vivo analysis of straight-chain and branched-chain fatty acid biosynthesis in three actinomycetes Kimberlee K. Wallace a, Bitao Zhao a, Hamish A.I. McArthur b, Kevin A. Reynolds aY* a Department of Pharmaceutical Sciences, School of Pharmacy, Universiv of Maryland at Baltimore, Baltimore, MD 21201, USA b Central Research Division, Pfizer Incorporated,
Groton, CT 06340, USA
Received 6 June 1995; revised 4 July 1995; accepted 10 July 1995
Abstract The starter units for branched-chain and straight-chain fatty acid biosynthesis was investigated in vivo in three actinomycetes using stable isotopes. Branched-chain fatty acids, which constitute the majority of the fatty acid pool, were
confirmed to be biosynthesized using the amino acid degradation products methylbutyryl-CoA and isobutyryl-CoA as starter units. Straight-chain fatty acids were shown to be constructed using butyryl-CoA as a starter unit. Isomerization of the valine catabolite isobutyryl-CoA was shown to be only a minor source of this butyryl-CoA. KeywordF: Actinomycete;
Butyrate
metabolism;
Fatty acid biosynthesis;
1. Introduction The cellular fatty acids of streptomycetes are known to consist primarily of branched-chain fatty acids with only minor straight-chain components [l]. A similar combination of branched-chain and straight-chain fatty acids is observed in Bacillus [2]. Research on Bacillus has shown that branched-chain fatty acids are likely produced using the amino acid catabolites, isobutyryl-CoA, 2-methylbutyryl-CoA and 3-methylbutyryl-CoA, as biosynthetic starter units [3]. The same starter units are probably utilized in branched-chain fatty acid biosynthesis in strepto-
Corresponding author. Tel.: + 1 (410) 706 5008; Fax: (410) 706 0346; E-mail: [email protected]. l
037%1097/95/$09.50
Q 1995 Federation
SSDIO378-1097(95)00263-4
of European
+ 1
Microbiological
Amino acid catabolism
mycetes. Incorporation of deuterated isobutyrate into the branched-chain fatty acid isopalmitate, in Streptomyces fiadiae, have suggested that isobutyryl-CoA is indeed a biosynthetic starter unit [4]. The role that 2-methylbutyryl-CoA plays in both avermectin and branched-chain fatty acid biosynthesis in Srrepcomyces aoermitilis has also been described [5]. The preferred starter unit for the biosynthesis of the minor straight-chain fatty acids in streptomycetes is not known. One suggestion is that acetyl-CoA is utilized directly by transacylation to acetyl ACP (acyl carrier protein) [6]. However, the deuterium labeling of the palmitate pool by deuterated isobutyrate in S. fiudiue is consistent with an isomerization of isobutyryl-CoA to n-butyryl-CoA, which is subsequently used in straight-chain fatty acid biosynthesis [4]. This interpretation is consistent with reSocieties. All rights reserved
palmitate,
isopentadecanoate,
and isoheptadecanoate
B
C
A
D
of
pools
to the abundances
labeled Isopalmitate/palmitate pool labeled Isopalmitate/palmitate pool labeled Palmitate pool labeled Isopentadecanoate/isoheptadecanoate by intact incorporation of by incorporation of pool labeled by incorporation by incorporation of methylvalerate/hexanoate as methylvalerate/hexanoate (%) of leucine (%) butyrate (%I isobutyrate/butyrate (%)
into the isopalmitate,
A=[M+7]/~[M]+[M+7]~~100;B=[M+11]/~[M]+[M+7]+[M+lll)X100;C=[M+7]/([M]+[M+7]+[M+Il])X100;D=[M+9]/(IM]+[M+9])~100.[M],abundance of the molecular ion (m/z) corresponding to non-deuterated methyl ester of the fatty acid, whereas [M+7],[M+9], and [M+ 111 correspond molecular ions at 7, 9 and 11 atomic mass units greater than this value and reflect fatty acids labeled with 7, 9, and 11 deuterlums, respectively.
Calculation A
Isopalmitate/palmitate by valine (%l
Table 1 Mass spectral analysis calculations of the percentage incorporation of perdeuterated compounds of Streptomyces cinnamonensis, Streptomyces collinus and Saccharopolyspora erythraea
K.K. Wallace et al. / FEMS Microbiology Letters 131 (1995) 227-234
sults observed in Bacillus where in vitro analysis has shown that butyryl-CoA is the preferred starter unit for straight-chain fatty acid biosynthesis [7,8]. In this work we have utilized an in vivo method for investigating the probable starter units for both straight-chain and branched-chain fatty acid biosynthesis in Streptomyces collinus, Streptomyces cinnamonensis and Saccharopoiyspora (Sp.) erythraea; actinomycetes in which either aspects of butyrate metabolism or fatty acid biosynthesis have previously been studied [9-111.
harvested by centrifugation. The supematant was discarded, and the cell pellet was washed with distilled water. 2.2. Fatty acid analysis
2. Materials and methods 2.1. Culture conditions for fatty acid analysis S. collinus Tu 1892 and Sp. erythraea (ATCC 11635) were maintained on agar slants containing 4 g yeast extract, 10 g malt extract, 4 g glucose and 20 g agar in 1 1 distilled water. S. cinnamonensis (A 3823.5) was maintained on agar slants containing 10 g Bacto-soytone, 10 g glucose, 1 g calcium carbonate and 20 g agar in 1 1 distilled water. A liquid medium (3 g yeast extract, 5 g malt extract, 3 g peptone and 10 g glucose in 1 1 distilled water at pH 7.2) in which all three actinomycetes could grow and which would not give background signals in the subsequent fatty acid analysis was selected. Spores (approx. 109) from one slant were used to inoculate a 250-ml flask containing 50 ml of this medium. After incubation at 30°C with shaking (200 rpm) for 48 h, 1 ml of this inoculum was transferred to a 50-ml flask containing 10 ml of the same medium supplemented with specific concentrations of labeled precursors. After incubation for 24-48 h, cells were
Table 2 Typical fatty acid compositions
of S. collinus, S. cinnamonensis
Strain
C 14
Cl5
Cl,
Cl7
%SCFA
S. collinus S. cinnamonensis Sp. erythraea
1.6 M> 0.3
3.4 3.3 1.3
7.1 11.4 4.8
0.3 4.0 1.3
12.4 18.7 7.8
229
Cell pellets were suspended in 1 ml of methanolic sodium hydroxide (1.2 N NaOH in 50% methanol) and heated in an airtight tube at 100°C for 30 min. Upon cooling to room temperature, 0.5 ml of 6 N HCl and 1 ml of BCl, were added to the suspension. The acidified suspension (pH < 2) was heated at 85°C for 10 min. Upon cooling to room temperature, fatty acid methyl esters were extracted by the addition of 1 ml of hexane/ether (50/50). Fatty acid methyl esters were analysed by injecting a sample (l-2 ~1) of the organic phase onto a Hewlett Packard 5970/5970A series gas chromatograph-mass selective detector equipped with an HP1 methyl silicone gum capillary column (0.33 PM film thickness). Ramp conditions for analysis were 50°C to 250°C at a rate of 20°C min- ‘. General fatty acid profiles were collected by scanning from 50 to 350 atomic mass units. Peaks were assigned based upon comparisons with retention times and mass spectral fragmentation patterns of standards. Fatty acid profiles for stable isotope incorporation studies were collected by scanning between 250 and 300 atomic mass units. The percentage of the fatty acid pool derived from perdeuterated precursors was calculated from the mass spectral analysis in the method shown in Table 1. In the [2-13C]acetate experiment, the average percentage labeling of each acetate-derived position of palmitate was calculated using (1 -Xl X 100; X = N/(Y + N) where N is defined as the number of
and Sp. erythraea i-14
i-15
ai-
i-16
i-17
ai-
%BCFA
13.8 0.7 7.9
15.5 11.0 14.0
31.9 18.0 22.3
19.1 15.2 29.8
2.5 11.0 6.8
4.9 25.7 11.4
87.7 81.6 92.1
Abbreviations: C,,, my&ate; C,,, pentadecanoate; C,,, palmitate; C,,, margarate; i-14, isomyristate; i-15, isopentadecanoate; ai-15, anteisopentadecanoate; i-16, isopalmitate; i-17, isoheptadecanoate; ai-17, anteisoheptadecanoate; SCFA, straight-chain fatty acids; BCFA, branched-chain fatty acids. The sum of %SCFA and %BCFA may exceed 100% due to rounding off the percentages of the individual fatty acids.
230
K.K. Wallace et al./ FEMS Microbiology Letters 131 (1995) 227-234
3. Results and discussion
potential acetate-derived sites (i.e. N = 8 for palmitate) and Y = (A4 + 1)/M. Y is the ratio of palmitate containing one 13C to unlabeled palmitate (after correction for natural abundance 13C). Results reported in the text for the stable isotope incorporation studies represent the findings of a single experiment with a single flask. Reproducibility was confirmed, in S. collinus, using perdeuterated hexanoate which resulted in 20 + 5% labeling of the palmitate pool from butyrate from three separate experiments.
3.1. Biosynthesis of long branched-chain fatty acids Typical fatty acid profiles for S. collinus, S. cinnamonensis and Sp. erythraea show that branched-chain fatty acids with chain lengths ranging from 14 to 17 carbon atoms predominate (Table 2). Like Bacillus, these three microorganisms also produce straight-chain fatty acids which constitute less than 20% of the total fatty acid pool (Table 2).
Leucine Degndation
Valine
-
-penladecanoyl CoA
Methylvalerate (exogenous)
I
I
Degrsdstlln
lsobutytyl CoA
’
Methylvaleryl CoA
I Biosynthesis
[Methylvaleryl ACP] -
Bulyryl CoA crotonyl COA Rsduc.tsse
aiisyntiwrit -
T\
Crotonyl CoA
t (Hydroxybutyryl CoA)
-
lsopalmitoyl CoA
Biosynthesis
[Hexanoyl ACP]
-
--+
Palmitoyl CoA
t
Hexanoyl CoA
f Hexanoate (exogenous)
t
I
(Acetoacetyl CoA) 4
2 Ace+ CoA
Fig. 1. Proposed pathways of fatty acid and amino acid metabolism in S. collinus. Compounds in square brackets represent presumed fatty acid biosynthetic intermediates which may exist as either CoA-or ACP-activated thioesters. Fatty acids are represented as CoA thioesters although they may also exist as ACP thioesters or free fatty acids.
8 10 7 $i 18
pools
of Swepromyces
14 ND 23
Streptomyces
collinus,
and Saccharopolyspora
1.5 5 26
20 37 49
Palmitate pool labeled by Palm&ate pool labeled by b intact incorporation of intact incorporation of hexanoate ’ (o/o) hexaooate ’ as butyrate (o/o)
cirmomonensis,
Isopalmitate pool labeled by incorporation of methylvalerate as isobutyrate (%I
palmitate
a Valine was added at time of inoculation to a final concentration of 200 mM to S. cinnamonensis and Sp. erythraea and 250 mM to S. collinus. b Perdeuterated methylvalerate was added to a final concentration of 4.3 mM at the time of inoculation. ’ Perdeuterated hexanoate was added to a final concentration of 4.3 mM at the time of inoculation. d ND, below detectable limits.
61 66 62
S. cinnamonensis s. collinus So. ervrhraea
and/or
Isopalmitate pool labeled by intact incorporation of methylvalerate b (o/o)
into the isopalmitate
Palm&ate labeled by valine a (o/o)
compounds
Isopalmitate labeled by valioe a (o/o)
of perdeuterated
Organism
Table 3 Incorporation erythraea
232
K.K. Wallace et al. / FEMS Microbiology Letters 131 (1995) 227-234
Long branched-chain fatty acid biosynthesis in these three actinomycetes was investigated by following the incorporation of stable isotope labeled precursors into the corresponding fatty acid pool. Addition of perdeuterated leucine (4.3 mM) to a S. collinus fermentation resulted in approximately 53% labeling of the isopentadecanoate CC,,) pool and 58% labeling of the isoheptadecanoate CC,,> pool with nine deuterium atoms. This result is consistent with the catabolism of leucine to 3-methylbutyrylCoA, which is utilized as a starter unit for the biosynthesis of odd-numbered fatty acids containing a branch at the o-1 carbon (Fig. 1). As shown in Table 3, addition of perdeuterated valine (200-250 mM) resulted in efficient isotopic labeling of the isopalmitate pool, consistent with the catabolism of valine to isobutyryl-CoA and subsequent incorporation into even-numbered branched-chain fatty acids. Under these conditions, a 35% labeling of the isopentadecanoate pool with seven deuteriums was also observed. This result is consistent with the catabolism of valine to a-ketoisovaleryl-CoA by valine dehydrogenase [12] and subsequent biosynthesis of leucine from this intermediate (Fig. 1) [13]. This leucine is then catabolized as described above. These results indicate that the even-numbered branchedchain fatty acids are built from an isobutyryl-CoA starter unit and that the odd-numbered branched chain fatty acids with a methyl branch at ~1 (the iso fatty acids) are built using 3-methylbutyryl-CoA as a starter unit. Presumably, the odd-numbered branched-chain fatty acids and a methyl branch at the ~2 carbon (the anteiso fatty acids) are built from the isoleucine catabolite, 2-methylbutyryl-CoA, as previously suggested in S. avermitilis [5]. In two of the bacterial systems perdeuterated methylvalerate, a putative six-carbon intermediate in branched-chain fatty acid biosynthesis, was incorporated intact with 11 deuterium atoms into the isopalmitate pool (Table 3). The degradation of the methylvalerate to isobutyryl-CoA prior to use in isopalmitoyl-CoA biosynthesis (Fig. 1) was observed by the labeling of 14-23% of the isopalmitate pool with seven deuteriums. This result is again consistent with the utilization of isobutyryl-CoA as a starter unit for branched-chain fatty acid biosynthesis. The lack of incorporation of methylvalerate into the isopalmitate pool in the case of S. collinus may arise
from limited transport of the compound into the cell, although this has not been demonstrated. The observed intact incorporation of the six carbon intermediate into the isopalmitate pool in S. cinnamonensis and Sp. erythraea suggests that these branched-chain fatty acids are made by a Type II fatty acid synthase. Type II fatty acid synthases are commonly observed in prokaryotes and plants [14]. These synthases consist of at least seven distinct and separable enzymes where potential fatty acid biosynthetic intermediates, such as exogenously supplied methylvalerate, can be transacylated and converted through to their products [15]. Like other prokaryotes, Streptomyces are generally thought to contain Type II fatty acid synthases; however, some preliminary evidence for a Type I synthase in Sp. erythraea [11,16] and Streptomyces coelicolor [17] has been reported. In contrast to Type II synthases, Type I synthases are multifunctional enzyme complexes which do not allow for fatty acid biosynthetic intermediates to enter and be converted to their products 1151.Such complexes are predominantly observed in eukaryotic systems [14,15], although their presence has been reported in other actinomycetes [18,19]. 3.2. Biosynthesis
of straight-chain
fatty acids
The formation of straight-chain fatty acids in the three bacterial systems was investigated by monitoring the incorporation of [2- l3Clacetate, perdeuterated butyrate and perdeuterated hexanoate. The experiments with [2-‘3C]acetate (11.9 mM) resulted in approximately 5% labeling of each of the acetate/malonate-derived positions. The low incorporation into the palmitate pool most likely arises due to dilution of the labeled acetate by endogenous acetyl-CoA and by the myriad of biochemical pathways that utilize acetyl-CoA. Addition of perdeuterated butyrate (4.3 mM) resulted in 57% labeling of the palmitate pool with seven deuterium atoms in S. cinnamonensis and 53% in Sp. erythraea. The lower level of intact incorporation of butyrate observed with S. collinus (22%) is consistent with studies with 14C-labeled butyrate which demonstrated that less than 5% of the butyrate is taken up by the cell (unpublished results). If straight-chain fatty acids were assembled in these systems by a Type I fatty acid synthase that used acetyl-CoA as a starter unit,
K.K. Wallace et al. / FEMS Microbiology Letters 131 (1995) 227-234
no significant intact incorporation of butyrate into the palmitate pool would be predicted. Some level of intact butyrate incorporation would be predicted if acetyl-CoA were the preferred or the physiological starter unit for a Type II fatty acid synthase. The very high level of intact butyrate incorporation is, however, more consistent with the direct utilization of butyryl-CoA for fatty acid biosynthesis using either a Type I or Type II fatty acid synthase (Fig. 1). In contrast, a low level of intact hexanoate incorporation into the straight-chain fatty acids was observed (Table 3). This intact incorporation indicates that straight-chain fatty acids, like branched-chain fatty acids, are potentially synthesized by a Type II fatty acid synthase. However, as shown in Table 3, the majority of the hexanoate that was fed was oxidized to butyryl-CoA prior to utilization as a starter unit. This result is consistent with butyryl-CoA being the preferred starter unit for straight-chain fatty acid biosynthesis in these microorganisms. One possible physiological source of butyryl-CoA for palmitoyl-CoA biosynthesis may be an isomerization of the valine-derived catabolite isobutyrylCoA (Fig. 1). This pathway has been observed in the formation of a butyryl-CoA building block in the biosynthesis of secondary metabolites [4,10,20-221 and straight-chain fatty acids in S. frudiae [4], suggesting that this isomerization may be general among streptomycetes. Valine feeding studies at high concentrations (200-250 mM as opposed to 5 mM) demonstrated that in these three actinomycetes a similar isomerization provides a component of the butyryl-CoA pool, putatively used for straight-chain fatty acid biosynthesis. In S. collinus cultures grown in the presence of 250 mM perdeuterated valine, 66% of the isopalmitate pool was labeled with deuterium. If the sole starter unit for isopalmitoyl-CoA biosynthesis is isobutyryl-CoA, 66% of the isobutyryl-CoA pool must, by definition, be perdeuterated. If all of the palmitoyl-CoA is assembled from butyryl-CoA, of which the sole source is the isomerization of isobutyryl-CoA, it would be predicted that 66% of the palmitate pool in the same experiment would be isotopically enriched with seven deuteriurns. However, the results indicate that only 10% of the palmitate pool is labeled with deuterium. Thus, in this experiment, only 14% of the butyryl-CoA pool is derived from an isomerization of isobutyryl-
233
CoA with the remaining 86% of the butyryl-CoA being derived from an alternative source. Similarly, 87% and 88% of the butyryl-CoA utilized for straight-chain fatty acid biosynthesis in the valine feeding studies of S. cinnamonensis and Sp. erythrueu, respectively, must also be obtained from a source other than isobutyryl-CoA (Fig. 1). It has already been demonstrated that butyrate units for secondary metabolism in streptomycetes can be formed from the condensation of two acetate molecules [22]. This process, which may be responsible for providing the majority of butyryl-CoA for palmitoyl-CoA biosynthesis, has not been investigated in streptomycetes. In mammalian mammary glands [23] and Euglena gracilis [a], butyryl-CoA used for fatty acid biosynthesis is thought to be formed from the condensation of two acetyl-CoA molecules (Fig. 11. The final critical step in this pathway is catalysed by an NADPH-specific crotonyl-CoA reductase [23-251. A similar enzyme may also operate in the conversion of acetyl-CoA to butyryl-CoA in the actinomycetes studied herein [9]. In summary, we have utilized an in vivo stable isotope analysis method for rapid, facile studies of both branched-chain amino acid degradation and fatty acid biosynthesis. This assay has been used to study these processes in three actinomycetes and has not only confirmed that branched-chain fatty acids are biosynthesized from isobutyryl-CoA and methylbutyryl-CoA, but shown that straight-chain fatty acids are preferentially biosynthesized from butyryl-CoA. Furthermore, evidence has been obtained that two alternative sources for butyryl-CoA formation are in operation in these organisms.
Acknowledgements This work was supported in part by a National Science Foundation grant (DCB-0104933) and by a National Institutes of Health grant (GM 50541-02) to K.A.R. and by a National Science Foundation Creativity Award in Engineering (EID-9021048) and an American Foundation of Pharmaceutical Education Fellowship (AFPE) to K.K.W. The authors are indebted to Professor A. Zeeck and Professor H. Zlhner for supplying S. collinus and to Eli Lilly for supplying S. cinnamonensis.
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K.K. Wallace et al. /FEMS Microbiology Letters 131 (1995) 227-234
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