Vol. 176, No. 6JOURNAL OF BACTERIOLOGY, Mar. 1994, P. 1661-16660021-9193/94/$04.00+0Copyright © 1994, American Society for Microbiology

13C Nuclear Magnetic Resonance Studies of Pseudomonasputida Fatty Acid Metabolic Routes Involved in

Poly(3-Hydroxyalkanoate) SynthesisGERN N. M. HUIJBERTS, THEO C. DE RIJK, PIETER DE WAARD, AND GERRIT EGGINK*

Agrotechnological Research Institute (ATO-DLO), 6700 AA Wageningen, The Netherlands

Received 27 October 1993/Accepted 7 January 1994

The formation of poly(3-hydroxyalkanoates) (PHAs) in Pseudomonas putida KT2442 from various carbonsources was studied by '3C nuclear magnetic resonance spectroscopy, gas chromatography, and gaschromatography-mass spectroscopy. By using [1-_3C]decanoate, the relation between beta-oxidation and PHAformation was confirmed. The labeling pattern in PHAs synthesized from [1-_3C]acetate corresponded to theformation of PHAs via de novo fatty acid biosynthesis. Studies with specific inhibitors of the fatty acidmetabolic pathways demonstrated that beta-oxidation and de novo fatty acid biosynthesis function indepen-dently in PHA formation. Analysis of PHAs derived from [1_-3C]hexanoate showed that both fatty acidmetabolic routes can function simultaneously in the synthesis of PHA. Furthermore, evidence is presented thatduring growth on medium-chain-length fatty acids, PHA precursors can be generated by elongation of thesefatty acids with an acetyl coenzyme A molecule, presumably by a reverse action of 3-ketothiolase.

Fluorescent Pseudomonas strains belonging to RNA homol-ogy group I are able to accumulate poly(3-hydroxyalkanoates)(PHAs), which consist of 3-hydroxy fatty acids, as a carbon andenergy reserve (11, 18). The monomer compositions of thesepolyesters are variable and are determined by the specificity ofthe PHA polymerase system, the nature of the substrate, andthe metabolic routes leading to PHA formation (5). On thebasis of a structural analysis of PHAs synthesized by Pseudo-monas oleovorans growing on medium-chain-length saturatedand unsaturated alkanes, it has been proposed that 3-hydroxy-acyl coenzyme A (acyl-CoA) intermediates of the beta-oxida-tion route are channeled to PHA synthesis (13). In line withthese results, de Waard et al. (3) showed that the structure ofthe monomer units in PHAs formed by Pseudomonas putidaKT2442 during growth on (poly)unsaturated long-chain fattyacids matches the structures that can be predicted from thebeta-oxidation pathway for unsaturated fatty acids (16).

Detailed one- and two-dimensional nuclear magnetic reso-nance (NMR) studies on PHA synthesized from carbohydratesrevealed the presence of both saturated and unsaturated3-hydroxy fatty acids. The chemical structures of these constit-uents are identical to the structures of the acyl moieties of the3-hydroxy-acyl acyl carrier protein (ACP) intermediates of thefatty acid biosynthetic pathway (10). In addition, it was dem-onstrated that the degree of unsaturation of PHA and mem-brane lipids is similarly influenced by shifts in cultivationtemperature. On the basis of these results, we proposed thatduring growth on carbohydrates, intermediates of the de novofatty acid biosynthetic pathway are diverted to PHA synthesis.It remained, however, to be determined whether PHA precur-sors are (in part) generated by beta-oxidation of newly synthe-sized long-chain fatty acids.

In order to establish in more detail the relation betweenPHA synthesis and fatty acid metabolism in P. putida, westudied PHA formation by using '3C-labeled substrates andspecific inhibitors of the fatty acid metabolic pathways. This

* Corresponding author. Mailing address: Agrotechnological Re-search Institute (ATO-DLO), P.O. Box 17,6700 AA Wageningen, TheNetherlands. Phone: 31-8370-75112. Fax: 31-8370-12260.

approach allowed us to determine that in P. putida, bothbeta-oxidation and de novo fatty acid biosynthesis can functionindependently and simultaneously in generating precursors forPHA synthesis.

MATERIALS AND METHODS

Media and culture conditions. P. putida KT2442 (1) wasused in all experiments. P. putida KT2442 was cultivated in1-liter Erlenmeyer shake flasks containing 250 ml of 0.5 x E2medium (10) at 30°C in an orbital incubator at 150 rpm(Gallenkamp) with various carbon sources.

13C-labeled fatty acids were added to the cultures at the endof the exponential growth phase. The substrate concentrationsused were as follows: 10 mM decanoate plus 5 mM [1-13C]de-canoate and 20 mM hexanoate plus 5 mM [1-13C]hexanoate.[1-13C]acetate (0.6 g/liter) was added to cultures growing on15-g/liter glucose at the end of the exponential growth phase.Cells were harvested by centrifugation (10 min at 5,000 x g),washed, and lyophilized. "3C-labeled substrates were obtainedfrom Isotec, Miamisburg, Ohio. Hexanoate and decanoatewere obtained from Merck, Darmstadt, Federal Republic ofGermany.Polymer isolation and analysis. Isolation of PHA was per-

formed by chloroform extraction of lyophilized cells followedby methanol precipitation of PHA. Gas chromatography (GC)analyses of PHA were performed as described previously (10).GC-mass spectroscopy (GC-MS) analyses were performedwith an HRGC/MS GC attached by direct interface to a QMD1000 ms mass spectrometer (both from Carlo-Erba, Milan,Italy). Spectra were obtained as electron impact (EI) spectra(70 eV) or as chemical ionization spectra, with isobutane as thereacting agent. The scan rate was 1 s-l.

13C NMR analysis was performed on PHA samples dis-solved in deuterated chloroform. 13C NMR spectra wererecorded on a Bruker AC200 NMR spectrometer at a probetemperature of 27°C.

Inhibition of fatty acid metabolism. Inhibitors of beta-oxidation and fatty acid synthesis were acrylic acid (17)(Merck) and cerulenin (6, 12) (Sigma, St. Louis, Mo.), respec-

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tively. In E2 medium with octanoate (10 mM) as the solesource of carbon and energy, the growth of P. putida KT2442was inhibited by acrylic acid at a concentration of 0.1 mg/ml.Cerulenin inhibited the growth of P. putida KT2442 on mineralE2 medium at a concentration of 0.3 mg/ml.

RESULTS

PHA synthesis from [1-13C]acetate. The relation betweenPHA formation and de novo fatty acid biosynthesis was studiedby "3C NMR and GC-MS analyses of PHAs synthesized from13C-labeled substrates. For this purpose, (1- 3C]glucose and[6-'3C]glucose are not suitable because the 3C-labeled carbonatom is released as CO2 in the reaction steps leading toacetyl-CoA. Therefore, we used [1-13C]acetate, which afterconversion to acetyl-CoA is a precursor for de novo fatty acidbiosynthesis. Because P. putida KT2442 grows poorly onacetate as the sole carbon and energy source, we used glucoseas the substrate with 13C-labeled acetate as a cosubstrate. Thisresults in a dilution of 13C label so only a fraction of the carbonatoms in PHA are labeled. When P. putida KT2442 wascultivated on 1.5% glucose and 0.15% 13C-labeled acetate,PHA formation was observed. GC analysis of the 3-hydroxyfatty acid methyl esters derived from purified polyester re-vealed that the monomeric composition did not differ fromthat of PHA isolated from P. putida KT2442 cultivated withglucose as the sole carbon source.El GC-MS showed that all 3-hydroxy fatty acid methyl esters

derived from PHA synthesized from glucose-[1-_3C]acetatewere enriched with 13C. The 13C NMR spectrum of PHApurified from P. putida KT2442 cultivated on glucose-[1-3C]acetate is shown in Fig. 1, and the carbon chemical shiftdata are summarized in Table 1. The assignment of the signalsin this spectrum is in agreement with earlier NMR studies (3,4, 7, 9, 15). Compared with the signals of the even-numberedcarbon atoms, all signals corresponding to odd-numberedcarbon atoms are enhanced, indicating a'C enrichment of theodd-numbered carbon atoms.

Synthesis of 13C-labeled PHA from [1-13C]decanoate. Toinvestigate the relation between fatty acid degradation andPHA synthesis, PHA formed from 1-13C-fatty acids was ana-lyzed by GC-MS and 13C NMR. The compositions of PHAsisolated from P. putida KT2442 grown on [1-13C]decanoateand on unlabeled decanoate are shown in Table 2. The 13CNMR spectrum of PHA purified from P. putida KT2442cultivated on [1-13C]decanoate is depicted in Fig. 1. From theintensities of the signals corresponding to carbon atoms 1 and2 in PHA derived from [1-'3C]decanoate and unlabeled de-canoate, we estimate a 10-fold 13C enrichment of the C-1carbon atom in PHA from [1-13C]decanoate.

El GC-MS analysis of the 3-hydroxy fatty acid methyl estersderived from PHA formed on [1-13C]decanoate showed thatonly the 3-hydroxydecanoate methyl esters were enriched. Onthe basis of the El GC-MS spectra (Fig. 2), it was alsodetermined that enrichment was exclusively located in a frag-ment with an mie of 103 corresponding to carbon atoms C-1,C-2, and C-3 of the monomers (7).

Synthesis of "3C-labeled PHA from [1-_3C]hexanoate. Thecomposition of PHA derived from [1-13C]hexanoate and un-labeled hexanoate as determined by GC is shown in Table 2. Inaddition to the major constituent, 3-hydroxyhexanoate, sixother monomer units were identified: 3-hydroxyoctanoate,3-hydroxydecanoate, 3-hydroxydodecanoate, 3-hydroxydode-cenoate, 3-hydroxytetradecanoate, and 3-hydroxytetradeceno-ate. These monomers are normally found in PHA isolatedfrom P. putida KT2442 cells cultivated on non-PHA-related

Acl1

C3 C0+C7C2 C4C81 C5C9 C10

1LLLLLU,

170 70 40 30 20 10

03 (ppm)

0 0

1 1CH3-CH2-CH2-CH2- CH2 CH2- CH2 CH-CH2-C-0-

10 9 8 7 6 5 4 3 2 1

C5-

B 07'06I

C3-

c1-

C12:1,&5 C3'-_ C1-C12:1A5

~~~~-C6 C8 C7' C5'

,i- \ \\1 ILI

170 140 130 120

2C (ppm)

0114:1A7 C9~-

12:1A5 C7-

C8C2 C4

LL1.

C9-

C10

70 40 30 20 10

FIG. 1. 13C NMR spectra of PHA isolated from P. putida KT2442cultivated on [1-'3C]decanoate (A) and on glucose and [1-_3C]acetate(B). The assignments of the carbon atoms of the 3-hydroxydecanoatemonomers (A) and of the major constituent 3-hydroxydecanoate (B)are indicated. 13C-enriched carbon atoms are marked (*). The signalscorresponding to carbon atoms in the double bonds of the unsaturatedmonomers are also assigned. The chemical shifts of all constituents canbe found in Table 1.

substrates (10). The presence of these monomers was furtherconfirmed by 13C NMR spectroscopy (Fig. 32.

Chemical ionization GC-MS analyses of 3C-labeled PHArevealed that the 3-hydroxyhexanoate methyl esters containedone 13C-enriched carbon atom. EI GC-MS data showed thatthe label was located in a fragment with an mie of 103corresponding to the carbon atoms C-1, C-2, and C-3 of the3-hydroxy fatty acids. The 13C NMR spectrum of PHA isolatedfrom cells cultivated on [1-13C]hexanoate is depicted in Fig. 3together with the assignment. It can be seen that the intensity

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TABLE 1. 13C chemical shifts of PHA monomers isolated from P.putida KT2442 cultivated on glucose

Chemical shift (ppm) of 3-hydroxyalkanoate monomersCarbon

C6 C8 Clo C12 C12:1 C14 C14:11 169.40 169.40 169.40 169.40 169.40 169.40 169.402 39.14 39.14 39.14 39.14 38.47 39.14 39.143 70.63 70.88 70.88 70.88 70.51 70.88 70.884 35.92 33.78 33.84 33.84 31.47 33.84 33.445 18.34 24.74 25.10 25.10 122.98 25.10 25.106 13.82 31.55 29.38 29.38 133.88 29.38 26.857 22.52 29.21 29.54 27.43 29.54 128.808 13.99 31.81 29.38 29.54 29.54 130.739 22.65 29.21 29.02 29.54 27.2910 14.09 31.81 31.92 29.38 29.5411 22.65 22.65 29.21 29.0212 14.09 14.09 31.81 31.9213 22.65 22.6514 14.09 14.09a Downfield from internal tetramethylsilane. C6, 3-hydroxyhexanoate; C8,

3-hydroxyoctanoate; C1O, 3-hydroxydecanoate; C12:1, 3-hydroxy-cis-5-dodeceno-ate; C12, 3-hydroxydodecanoate; C14:1, 3-hydroxy-cis-7-tetradecenoate; C14, 3-hy-droxytetradecanoate.

of the signal assigned to the C-1 carbon atom is enhanced incomparison with the same signal in the 13C NMR spectrum ofPHA derived from unlabeled hexanoate. Comparing the inten-sities of the signals assigned to carbon atoms C-1 and C-2 inPHA synthesized from [1-13C]hexanoate with those of thesignals from unlabeled hexanoate, we estimate a 70-fold 13Cenrichment of carbon atom C-1 in the PHA derived from[1-13C]hexanoate.The El and chemical ionization GC-MS analyses revealed

that the 3-hydroxyoctanoate methyl esters derived from thisPHA contain two 13C-enriched carbon atoms and that thesetwo carbon atoms are located in the fragment with an mie of103. From the 13C NMR spectrum, it is evident that theodd-numbered carbon atoms are 13C enriched, and thereforewe conclude that '3C enrichment in the 3-hydroxyoctanoatemethyl esters has occurred at positions C-1 and C-3. Inaddition, a fragment with an mie of 74 containing carbon atomsC-1 and C-2, which is the result of a McLafferty rearrangement(14) in 3-hydroxy fatty acid methyl esters, shows 13C enrich-ment of only one carbon atom, thus excluding the possibilitythat carbon atom C-2 is 13C enriched.GC-MS analyses of the 3-hydroxydecanoate methyl esters

TABLE 2. Composition of PHA isolated from P. putida KT2442cultivated on decanoate, hexanoate, [1-13C]decanoate, and

[1-l3C]hexanoateaRelative amount of monomers in purified PHA

Substrate (%, Wt/Wt)bC6 C8 C1O C12:1 C12 C14:1 C14

Decanoate 6 53 41[1-13C]decanoate 5 52 43

Hexanoate 72 14 10 1 1 1 1[1-13C]hexanoate 78 8 10 1 1 1 1

a Cells were cultivated as described in Materials and Methods and wereharvested after 48 to 72 h. 20 mM hexanoate was added to the cultures in twoportions of 10 mM with a time interval of 16 h to prevent toxicity.

b C6, 3-hydroxyhexanoate; C8, 3-hydroxyoctanoate; C10, 3-hydroxydecanoate;C12:1, 3-hydroxy-cis-5-dodecenoate; C12, 3-hydroxydodecanoate; C14:1, 3-hy-droxy-cis-7-tetradecenoate; C14, 3-hydroxytetradecanoate.

show that three or more carbon positions are 13C enriched. 13CNMR analysis reveals a specific 13C labeling pattern for allmonomers with a carbon chain length of 10 or more (Fig. 3).Compared with the signals belonging to the even-numberedcarbon atoms, the signals corresponding to the odd-numberedcarbon atoms are enhanced, indicating a 13C enrichment of theodd-numbered carbon atoms in these monomers.

Inhibition of PHA synthesis. The fatty acid metabolic path-ways, beta-oxidation and fatty acid synthesis, can be inhibitedby acrylic acid and cerulenin, respectively. Acrylic acid inhibitsthe enzymes acyl-CoA synthase and 3-ketoacyl-CoA thiolasepresumably via an interaction of acrylic acid with CoA (17).Cerulenin binds to the active sites of keto acyl synthases I and11 (6, 12). We have determined the inhibitory concentrations ofthese compounds, and it was found that acrylic acid effectivelyinhibits beta-oxidation in P. putida KT2442 at 0.1 mg/ml.Cerulenin inhibits fatty acid synthesis in P. putida KT2442 at0.3 mg/ml.

In order to study the effects of these inhibitors on thesynthesis and composition of PHA, an exponentially growingculture of P. putida was harvested, washed, and resuspended innitrogen-free medium containing either 20 g of glucose perliter or 10 mM octanoate. Under these conditions, PHAformation is observed within 24 h. The effects of the inhibitionof fatty acid metabolism on PHA formation were examined byadding the appropriate amount of inhibitor to the cell suspen-sions.

In cell suspensions containing 10 mM octanoate as thesubstrate, no PHA formation was observed in the presence of0.1 mg of acrylic acid per ml. Synthesis ofPHA from octanoate,however, was affected by the presence of 0.3 mg of ceruleninper ml, but the composition of this PHA was identical to PHAsynthesized from octanoate in the absence of the inhibitor(Table 3).

In cell suspensions with glucose as the substrate, PHAformation was inhibited by 0.3 mg of cerulenin per ml, whereasPHA formation was not affected by the presence of 0.1 mg ofacrylic acid per ml. The composition of the PHA was notinfluenced by the presence of acrylic acid (Table 3).

DISCUSSION

PHA synthesis and de novo fatty acid synthesis. Previously,we proposed that during growth on non-PHA-related sub-strates, PHA precursors are generated by de novo fatty acidsynthesis. In this study, the relation between PHA synthesisand the fatty acid metabolic pathways was further establishedby 13C NMR analysis of PHAs purified from P. putida KT2442cultivated on a combination of glucose and [1-13C]acetate.From the GC analyses, it is clear that the addition of 13C-labeled acetate to cultures does not result in aberrant PHAcompositions, and so we conclude that this is a reliable way ofstudying the formation of PHA from non-PHA-related sub-strates.The labeling pattern of P1-A synthesized from [1-13C]

acetate can be understood from the reactions in the fatty acidbiosynthetic pathway. First, acetyl-CoA molecules are carbox-ylated to malonyl-CoA and then converted to malonyl-ACP.Acetyl-CoA labeled at the C-1 position yields malonyl-ACPlabeled at the C-1 position. Malonyl-ACP is coupled to the acylchain with the release of C02, which results in the formation ofacyl chains with "3C label at odd-numbered carbon positions.The 13C NMR spectra and the GC-MS data show that in PHAderived from [1-13C]acetate the '3C label is found at odd-

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100

90

80

4 70cm(ac 604)C.)

@ 50

c 40

- 30

20

10

0127 152I.. 11

5 5 9 1. 1 135 55 75 95 115 135 155 175 195 215

Mass unitsFIG. 2. El GC-MS spectrum of 3-hydroxydecanoate methyl ester derived from PHA isolated from P. putida KT2442 cultivated on decanoate.

The structure of the fragment with an mie of 103, characteristic of 3-hydroxyalkanoate methyl esters, is shown. From the ratio of signals 103, 104,105, and 106, the number of 13C-enriched carbon atoms in the fragment can be calculated.

numbered carbon positions. In a similar way, the 13C label isfound at even-numbered positions when [2-13C]acetate is used(5). These data confirm the role of fatty acid biosynthesis inPHA formation. However, the possibility that PHA precursorsare derived (in part) from the beta-oxidation of newly synthe-sized fatty acids cannot be excluded. Therefore, we investi-gated the formation of PHA in the presence of the beta-oxidation inhibitor acrylic acid. At a concentration of 0.1mg/ml, acrylic acid completely inhibited beta-oxidation,whereas the amount and the composition of PHAs were notaffected (Table 3). From this, we conclude that the precursorsfor PHA polymerization can be directly and exclusively derivedfrom the de novo fatty acid biosynthesis route.PHA synthesis from 1-'3C-labeled fatty acids. PHA synthe-

sized from [1-13C]decanoate exhibits a specific 13C enrichmentof the C-1 carbon atoms. The GC-MS spectra revealed that theincrease of label was present only in the 3-hydroxydecanoatemethyl esters. No 13C enrichment was found in the 3-hy-droxyoctanoate and 3-hydroxyhexanoate methyl esters. Theseresults support the hypothesis that intermediates for PHApolymerization are generated via beta-oxidation of fatty acids.The C2 fragment containing the label is removed in the firstcycle of beta-oxidation, yielding unlabeled intermediates with acarbon chain length of eight.From 13C NMR, GC, and GC-MS analyses of PHA formed

by P. putida KT2442 growing on [1-13C]hexanoate, it can bededuced that in this case PHA precursors are generated bythree different routes. In Fig. 4, a schematic representation ofthe labeling patterns, as determined by 13C NMR and GC-MS,is shown. Partial beta-oxidation of [1-13C]hexanoate yields[1-13C]3-hydroxyhexanoate monomers which account for ap-proximately 72% of the PHA monomers. Degradation of[1-13C]hexanoate via beta-oxidation yields three molecules ofacetyl-CoA of which one is [1-13C]acetyl-CoA. The presence ofunsaturated monomers indicates that there is also PHA for-mation via de novo fatty acid synthesis. This is also clear from

the labeling pattern of the medium-chain-length monomers(C10, C12:0, C12:1, C14:0, and C14:1). The '3C label is found at allodd-numbered carbon positions in these monomers. Appar-ently, the_1-13C]acetyl-CoA which is formed in the degrada-tion of [1- 3C]hexanoate is used as a substrate for the forma-tion of PHA. The ratio in which these medium-chain-lengthmonomers are present in PHA formed from hexanoate corre-sponds to the composition of PHA synthesized from carbohy-drates. From these results, we estimate that 13% of the PHAmonomers synthesized from hexanoate are derived from thede novo fatty acid biosynthetic pathway.However, on the basis of the composition of PHA synthe-

sized from carbohydrates, the de novo fatty acid biosyntheticroute can account for only approximately 15% of the 3-hy-droxyoctanoate monomers. The remaining 85% of the 3-hy-droxyoctanoate monomers must be the result of anotherbiosynthetic reaction. The 13C NMR spectra and GC-MS datashow that in the 3-hydroxyoctanoate monomers carbon atoms1 and 3 are more frequently labeled. Therefore, we concludethat most of the 3-hydroxyoctanoate monomers in PHA syn-thesized from hexanoate are generated via elongation ofhexanoate with, presumably, acetyl-CoA.

Relationship between fatty acid metabolism and PHA for-mation. In this report, we have provided further evidence thatintermediates for PHA formation can be generated via de novofatty acid synthesis, beta-oxidation, and elongation of fattyacids. An overview of the pathways and the structures of theintermediates formed in the synthesis ofPHA from [1- 3CJhex-anoate is shown in Fig. 4. The experiments with inhibitors offatty acid metabolism have demonstrated that, depending onthe nature of the substrate, precursors for PHA synthesis canbe derived from either beta-oxidation or fatty acid biosynthe-sis. Both routes can operate independently and simultaneously.The PHA polymerizing enzyme system appears to have a

preference for monomers with 8 or 10 carbon atoms, whilelarger and smaller monomers are incorporated less efficiently

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o

11CH3-CH2-CH2-CH- CH2-c -

6 5 4 3 2 1

ci

0

oxidaon - SCoA

OH I0

SCoA

C3

C12:1A5 C5C14:1A7 C7

_ 1,,-

170 140 130 120 70 40 30 20 10'3C (pp)

FIG. 3. 13C NMR spectrum of PHA isolated from P. putidaKT2442 cultivated on [1-_3C]hexanoate. The assignment of the signalscorresponding to carbon atoms in the major compound 3-hydroxyhex-anoate is indicated. The signals in the 120- to 140-ppm part of thespectrum correspond to carbon atoms in the unsaturated monomers3-hydroxy-cis-5-dodecenoate and 3-hydroxy-cis-7-tetradecenoate. Theunassigned signals in the 10- to 40-ppm part correspond to carbonatoms in the medium-chain-length monomers 3-hydroxyoctanoate,3-hydroxydecanoate, 3-hydroxydodecanoate, and 3-hydroxytetrade-canoate. The exact chemical shifts of the carbon atoms of thesecompounds are listed in Table 1.

(11). This might explain why, during growth on hexanoate,PHA monomers are synthesized by three different routes. Bothelongation and de novo fatty acid synthesis result in thegeneration of the more preferable C8 and ClO monomers. Thepresence ofPHA monomers with'a larger chain length than the

0 0

A SCoA )fi SCoA

fatty acid

synthesisOH 0

SCoA

OH 1oR ACP

OH 0

ACPOH 0

ACP

FIG. 4. Overview of reactions yielding precursors for PHA synthe-sis from hexanoate. 3-Hydroxyhexanoate monomers are synthesizedvia beta-oxidation, and the concomitantly generated acetyl-CoA mol-ecules can be incorporated into PHA via de novo fatty acid biosynthe-sis. R represents the acyl groups of the saturated 3-hydroxyacyl-ACPintermediates. "3C-labeled carbon atoms are indicated (*), and lessfrequently labeled carbon atoms are also indicated (*). 3-Hy-droxyoctanoate monomers can also be generated via elongation ofhexanoate by an acetyl-CoA molecule.

substrate has been observed before (2, 7). Haywood et al. (8)have suggested that these monomers are formed via a conden-sation reaction of acyl-CoA molecules with acetyl-CoA cata-lyzed by the beta-oxidation enzyme 3-ketothiolase. It cannot beruled out, however, that elongation of exogenous fatty acids inP. putida proceeds via de novo fatty acid biosynthesis. In P.putida, 3-hydroxydecanoate is the major constituent of PHAsynthesized via de novo fatty acid biosynthesis. If elongation ofhexanoate in P. putida were the result of de novo fatty acidbiosynthesis, we would expect 3-hydroxydecanoate to be thepredominant product and only a minor amount of 3-hy-droxyoctanoate to be present. Since this is not the case, weconclude that elongation of hexanoate via a reverse action ofthe 3-ketothiolase is more likely than elongation via de novofatty acid biosynthesis.

TABLE 3. Inhibition of P. putida KT2442 fatty acid metabolic routes and effects on PHA synthesisa

Substrate%

bRelative amount of monomers in PHA (%, wt/wt)c

uHA C6 C8 CIO C12:1 C12 C14:1 C14

1.5% glucose 8.5 1 11 66 11 8 2 11.5 glucose + 8.3 1 12 65 12 8 1 1

acrylic acid1.5% glucose + _d

cerulenin10 mM octanoate 22.3 6 92 210 mM octanoate+ acrylic acid

10 mM octanoate 7.0 5 94 1+ cerulenin

aCell suspensions of P. putida KT2442 were incubated with substrates and inhibitors as described in Materials and Methods.b Percent PHA was determined by GC analysis and calculated as a percentage of cell dry weight.C6, 3-hydroxyhexanoate; C8, 3-hydroxyoctanoate; CIO, 3-hydroxydecanoate; C12:1, 3-hydroxy-cis-5-dodecenoate; C12, 3-hydroxydodecanoate; C14.1, 3-hydroxy-cis-

7-tetradecenoate; C14, 3-hydroxytetradecanoate.d, not detected (<0.1%).

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ACKNOWLEDGMENT

We thank B. Witholt for critically reading the manuscript and forstimulating discussions.

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