Download In vivo analysis of straight-chain and branched

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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
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+ 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
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