The role of P-oxidation of short-chain alkanoates in polyhydroxyalkanoate copolymer synthesis in Azotobacter

William J. Page and Janet Manchak

Abstract: Valerate and other short-chain, uneven-length fatty acids promoted the formation of the polyhydroxyalkanoate (PHA) copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in Azotobacter vinelandii UWD growing in glucose medium. The uptake of valerate was inducible, being repressed by acetate but not by glucose. A likely route that would direct valerate into PHA synthesis involved the P-oxidation pathway. The short-chain fatty acids butyrate, valerate, trans-2-pentenoate, crotonate, hexanoate, heptanoate, and octanoate induced the coordinate production of the P-oxidation enzymes enoyl-CoA hydratase (ECH) and L-(+)-3- hydroxybutyryl-CoA dehydrogenase (HAD). trans-3-Pentenoate was the best inducer of these activities, which suggested that the isomerase of the P-oxidation complex also was present. However, 3-hydroxyacyl-CoA epimerase activity of the P-oxidation complex was not detected. 3-Ketoacyl-CoA thiolase activity was constitutive in A. vinelandii and appeared to associate only loosely with the 73 000 Da ECH-HAD complex. Thus, 3-ketoacyl- CoA, the end product of HAD activity, could be directed into PHA synthesis through acetoacetyl-CoA reductase generating the 3-hydroxyvalerate subunit of the polymer. When valerate was the sole carbon source, the incorpora- tion of valerate into the polymer was normal, but most of the valerate was directed into metabolism and very little PHA was formed. When glucose also was present, the P-oxidation of short-chain alkanoates inhibited the specific activity of acetoacetyl-CoA reductase and 3-ketothiolase and the PHA yield. A model for PHA synthesis was developed that suggests that the use of fatty acids to promote PHA copolymer formation in A. vinelandii will inevitably result in decreased PHA yield.

Key words: P-oxidation, poly(P-hydroxyalkanoate) synthesis, short chain fatty acids, regulation.

Resume : Le valerate et d'autres acides gras a chaines courtes de longueurs inkgales ont favoris6 la formation d'un polyhydroxyalcanoate (PHA), le copolymkre poly(3-hydroxybutyrate-co-3-hydroxyval6rate) par 1'Azotobacter vinelandii UWD cultive dans un milieu contenant du glucose. Le captage de valerate fut inductible, Ctant reprim6 par de l'acetate mais pas par du glucose. Le sentier de la P-oxydation constituerait une route probable qui dirigerait le valkrate vers la synthkse de PHA. Les acides gras 5 chaines courtes butyrate, valerate, trans-2-penthoate, crotonate, hexanoate, heptanoate et octonoate ont induit la production coordonnke des enzymes de P-oxydation knoyl-CoA hydratase (ECH) et L-(+)-3-hydroxybutyryl-CoA dkshydrogenase (HAD). Le trans-3-penthoate a kt6 le meilleur inducteur de ces activites, ce qui suggbre que l'isomerase du complexe de P-oxydation ktait aussi pksente. Cependant, l'activite 3-hydroxyacyl-CoA epimkrase du complexe de P-oxydation n'a pas kt6 dktectee. L'activite 3-cktoacyl-CoA thiolase fut constitutive chez 1'A. vinelandii et semblait s'associer seulement vaguement avec le complexe ECH-HAD de 73 000 Da. h s i , le 3-cktoacyl-CoA, le produit final de l'activit6 HAD, a pu Ctre dirigk vers la synthbse de PHA, par l'intermedmire de l'ac6toacetyl-CoA rductase, generant la sous-unit6 3-hydroxyvalerate du polymkre. Lorsque le valkrate fut la seule source de carbone, l'incorporation de valerate dans le polymkre a kt6 normale, mais la majoritk du valerate a kt6 orient& vers le metabolisme et tr&s peu de PHA a Ctk forme. Lorsque le glucose aussi fut present, la P-oxydation d'alcanoates a chaines courtes a inhibe l'activit6 specifique de I'adtoacCtyl-CoA Muctase et de la 3-~Ctothiolase, ainsi que le rendement de PHA. Les auteurs ont developpe un modble pour la synthbse de PHA selon lequel l'utilisation d'acides gras pour favoriser la formation de copolymkre de PHA chez 1'A. vinelandii mknera inevitablement B un amoindrissement du rendement de PHA.

Mots cle's : P-oxydation, synthkse de poly(P-hydroxyalcanoate), acides gras B chaines courtes, regulation. [Traduit par la Redaction]

Received July 25, 1994. Revision received December 23, 1994. Introduction Accepted January 4, 1995.

I Poly(P-hydroxyalkanoates) (PHAs) are natural polyesters W. I. Page1 and I. Manchak. Department of Biological Sciences, formed by a wide variety of bacte~a. This material is University of Alberta, Edmonton, AB T6G 2E9, Canada.

represented most commonly by poly(P-hydroxybutyrate) Author to whom all correspondence should be addressed. (PHB), a well-known intracellular storage polymer essential

Can. J. Microbiol. 41(Suppl. 1): 106-114 (1995). Printed in Canada / ImprimC au Canada

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for bacterial viability and differentiation during the stationary phase of growth (Dawes and Senior 1973). While the ability of a cell to produce PHB has some value in taxonomy and PHB is of obvious value to the cell, a new use for PHB and related polymers has been identified. These polymers can be used to form natural, biocompatible, and biodegradable plastics (Howells 1982; King 1982). The controlled, large-scale production of PHAs by bacteria has become a subject of increasing interest (Anderson and Dawes 1990; Doi 1990; Luzier 1992; Steinbiichel 1991), with the hope that the use of biodegradable polymers to replace some petrochemically based polymers will help to reduce the highly visible pollution of land and sea environments by plastic products. In addition, PHA plastics are part of the natural carbon cycle and, hence, a renewable resource (Schlegel 1992).

An important consideration in the exploitation of PHB as a biodegradable plastic is that it shares characteristics with polypropylene, but forms a very brittle plastic (Howells 1982). The random incorporation of 8-20% 3-hydroxyvalerate (HV) into the polymer is necessary to generate a copolymer with increased flexibility (Luzier 1992). Copolymers are formed by a variety of methods and any one PHA-producing organism will usually have only one or two routes for copolymer formation (Anderson et al. 1992; Doi 1990; Haywood et al. 1989; Steinbiichel and Pieper 1992).

The focus of our research has been the development of strains of Azotobacter vinelandii for the commercial production of the bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(HI3- co-HV), Page and Knosp 1989,1991,1992). Use of the mutant strain UWD allows rapid production of polymer during growth on inexpensive, unrefined carbon sources (Page 1992). Supplementation of the simple mineral salts medium with fish peptone also generates large, fragile pleomorphic cells that are easily broken by alkaline extraction with aqueous NH3 (Page and Cornish 1993). Recently we examined the control of PHA biosynthesis in strain UWD (Manchak and Page 1994). The strain UWD mutation limits the respiratory oxidation of NADH (Fig. 1, step 1). This results in an excess of reducing power in the cell (Fig. 1, step 2), which will feed back on the tricarboxylic acid cycle (Fig. 1, step 3) to generate a surplus of acetyl-CoA (AcCoA) relative to CoA. This in turn relieves the negative regulation of 3-ketothiolase and promotes the condensation of AcCoA (Fig. 1, step 4). The acetoacetyl-CoA (AcAcCoA) reductase (Fig. 1, step 5) becomes an alternative electron sink, allowing PHB synthesis during active growth on glucose (Page and Knosp 1989).

Copolymer formation in strain UWD is promoted by feeding odd-chain fatty acids to the microbial culture during growth on a primary carbon source, which is usually a sugar (Page et al. 1992). Thus, strain UWD will form P(HB-co-HV) from valeric, heptanoic, or nonanoic acid. Strain UWD forms only trace amounts of copolymer from propionic acid by the condensation (Fig. 1, step 4) of AcCoA and propionyl-CoA (Page et al. 1992). The results to date suggest that P-oxidation of odd-chain n-alkanoates is the primary source of HV subunits for copolymer synthesis in A. vinelandii (Page et al. 1992; Manchak and Page 1994). However, nothing is known about P-oxidation in Azotobacter species and this pathway has been studied in only a few bacteria.

The P-oxidation pathway of Escherichia coli and Pseudomonas fragi is very similar to the P-oxidation pathway

in eukaryotes (Pawar and Schulz 1981; Sato et al. 1992~). P-Oxidation starts with the ATP-dependent transport and activation of the fatty acid by acyl-CoA synthetase (Lynen and Ochoa 1953). The acyl-CoA is oxidized by an FAD-dependent dehydrogenase before it is passed to a multienzyme complex that channels the intermediates through cyclic P-oxidations to final products. Thus, P-oxidation of even-chain alkanoates will generate AcCoA and odd-chain alkanoates will generate AcCoA and propionyl-CoA. The multienzyme complex is comprised of two &-subunits that have multiple enzyme activities (enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, cis-A3-trans-A2 enoyl-CoA isomerase, 3-hydroxyacyl-CoA epimerase), and two P-subunits that have 3-ketoacyl-CoA thiolase activity. The multienzyme complex HDT (named for the three key activities, hydratase, dehydrogenase, and thiolase) is encoded by two genes, fadAB in E. coli and faoAB in P. fragi, which are transcribed coordinately as an operon (Yang and Schulz 1983; Sato et al. 1994). P-Oxidation is not constitutive, but must be induced by the presence of long-chain (>Clo) fatty acids as the exogenous carbon source (DiRusso et al. 1993). Short-chain (<Cs) fatty acids do not induce P-oxidation in E. coli (Weeks et al. 1969) or P. fragi (Sato et al. 19926) and mutants of E. coli that do oxidize short-chain fatty acids have derepressed P-oxidation enzymes and a derepressed ability to take up short-chain fatty acids (Salinitro and Wegener 1971).

The presence of a single constitutive 3-ketoacyl-CoA thiolase in strain UWD (Manchak and Page 1994) suggested that the P-oxidation complex could also be constitutive in A. vinelandii. Complete P-oxidation of odd-chain fatty acids channelled to AcCoA and propionyl-CoA would appear to be a futile cycle, because the thiolase would then be used in the reverse direction to reconstruct 3-ketovaleryl-CoA for PHA synthesis. Also, the inhibition of 3-ketothiolase during the catabolism of valerate has increased HV content in PHA (Page et al. 1992). This suggested that P-oxidation was not channelled to produce simple products, but 3-ketoacyl-CoA could be removed at an intermediate step (Fig. 1). These observations and questions, therefore, prompted a further investigation of the role of P-oxidation in PHA synthesis in A. vinelandii UWD.

Materials and methods

Bacterial strain and growth conditions Azotobacter vinelandii UWD (ATCC 53799; Page and Knosp 1989) was grown in glucose medium composed of 0.81 mM MgS04, 0.58 mM CaS04, 50 pM femc citrate, and 1 pM Na2Mo04 in 5 mM potassium phosphate buffer, pH 7.2, containing 3% glucose, 15 mM ammonium acetate, and 0.1% fish peptone (product No. HOlOOBT from Protan A/S, Drammen, Norway). Burk's buffer contained only the mineral salts and phosphate buffer components of this medium. The cultures were inoculated with a 4% (v/v) inoculum and incubated at 28-30°C with vigorous aeration (50 mL culture in each 500-mL flask, shaken at 225 rpm on a New Brunswick Scientific Co. model G-10 platform shaker) for up to 24 h.

Copolymers were formed by the addition of 10 mM (final concentration) fatty acid (as a neutral sodium salt) to a culture pregrown in glucose medium for 12 h (Page et al. 1992). After 10- 12 h additional incubation the cultures were harvested by

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Fig. 1. Control of PHA synthesis in Azotobacter vinelandii UWD. Points of inhibitory (-) or stimulatory (+) control by effector compounds (boxed) are shown with open arrows. The pathway steps are (1) UWD mutation in respiratory NADH oxidase, (2) pyridine nucleotide transhydrogenase, (3) citrate synthase and isocitrate dehydrogenase, (4) 3-ketothiolase condensation reaction, (5 and 6) AcAcCoA reductase, (7) L-(+)-P-hydroxyacyl-CoA dehydrogenase, (8) PHA synthase, (9) PHA depolymerase, (10) P-hydroxybutyrate dehydrogenase, (11) succinyl-CoA transferase, and (12) 3-ketothiolase thiolysis reaction. Figure reproduced from Manchak and Page (1994).

Entner-Doudoroff pathway

co, 1 Pyruvate

TCA cycle

3

Acetoacetate

NADH /

p - Oxidation

A \ A mutation

centrifugation for the determination of total cell dry mass, the PHAformed by A. vinelandii was determined after polymer polymer dry mass, and residual dry mass (RM) (total mass minus purification, hydrolysis, and methyl-ester formation (Page polymer mass). Polymer mass was determined after digestion et al. 1992). Fatty acid methyl esters also were separated on the of RM in commercial bleach (Law and Slepecky 1961). Nukol column and quantitated (Budwill et al. 1992).

Chemical analysis of cells, polymer, and culture fluid Cell growth was estimated as the amount of cell protein and the glucose remaining in the medium was measured spectro- photometrically (Page et al. 1992). The uptake of valerate or acetate from the culture medium was measured by gas chromatography (Hewlett Packard model 5890A fitted with an HP7673 autoinjector) using a Nukol column (Supelco) described previously (Budwill et al. 1992). The culture fluid (300 pL) was acidified with 10 p,L of 1 M HCl and 1 p,L of the acidified mixture was injected on the column. The mol% HV content of

Cell extract preparation and enzyme activity determination

After about 24 h growth the cells were harvested by centrifugation (10 000 x g for 15 min at 4OC), washed once in Burk's buffer, and resuspended in 20 mM potassium phosphate buffer, pH 7.0, containing 1 mM dithiothreitol. The cells were ruptured by sonication (Manchak and Page 1994) and the cell extract was cleared by centrifugation (39 000 x g for 60 min at 4°C). Glycerol was added to about 10% (v/v) and the extracts were stored at -20°C until use.

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Enzyme activities were determined in the clear cell-free extract or other preparations according to the assays listed below. KCN (2 mM final concentration) was added to all enzyme assays containing NADH or NADPH to decrease background oxidation by respiratory particles (Manchak and Page 1994) and the assays were conducted at 30°C. The protein content of cell extracts was determined by the Bio-Rad dye binding assay (Bradford 1976).

3-Ketoacyl-CoA thiolase was assayed in the direction of thiolysis as described previously (Nishimura et al. 1978). One unit of enzyme activity was the amount of enzyme required to catalyse the cleavage of 1 pmol AcAcCoA . m i d .

NADPH-dependent AcAcCoA reductase, an enzyme required for PHA synthesis, was measured as described previously (enzyme A in Haywood et al. (1988)). One unit was the amount of enzyme required to oxidize 1 pmol NADPH . min-I .

L-(+)-3-Hydroxyacyl-CoA dehydrogenase (HAD) was measured in the reverse direction as an NADH-dependent AcAcCoA reductase using the assay for NADPH-dependent AcAcCoA reductase described above.

Enoyl-CoA hydratase (ECH) was followed by measuring the dehydration of crotonyl-CoA as a loss of absorbance at 263 nm caused by the double bond (Binstock and Schulz 1981). The assay contained (final concentration per millilitre) 0.2 M potassium phosphate buffer, pH 8.0, 0.2 mg bovine serum albumin, and 30 pM crotonyl-CoA. One unit was the amount of enzyme required to hydrate 1 pmol crotonyl-CoA . min-l.

3-Hydroxyacyl-CoA epimerase was assayed by following an increase in absorbance at 340 nrn (A340) owing to the formation of NADH in a linked assay containing (final concentration per millilitre) 0.15 M potassium phosphate buffer, pH 8.0,0.3 mg bovine serum albumin, 0.5 mM NADC, 0.1 mM CoA, 60 pM DL-3-hydroxybutyryl-CoA, 2 yg pig heart L-specific HAD (Sigma), and =2 pg Azotobacter 3-ketothiolase. In the first stage of the assay (10 min), increased as the L-3-hydroxybutyryl-CoA was completely oxidized to AcAcCoA by the pig heart HAD. Thiolase was present in the assay to remove this product and prevent feedback inhibition of HAD. In the second stage of the assay, the Azotobacter ECH-HAD enzyme complex partially purified by column chromatography was added to the assay. If epimerase activity was present, the remaining D-3-hydroxybutyryl-CoA would be directed into the ECH-HAD complex and an additional increase in A340 should be observed (Binstock and Schulz 1981).

Column chromatography of cell extracts The cell extract was precipitated with protamine sulfate as described by Haywood et al. (1988) and the supernatant fluid (4 mL, about 3 mg proteinmL-1) was applied to a DEAE Sepharose CL-6B (Pharmacia) column (Manchak and Page 1994). The column was washed with a column volume of 20 mM potassium phosphate buffer, pH 7.0, containing 1 mM dithiothreitol and the enzymes were eluted with a linear 0-0.5 M NaCl gradient (in column buffer). Each fraction tube was loaded with 200 pL glycerol and 2-mL fractions were collected. Fractions from the column were assayed for Azso (data not shown), enzyme activity, and conductivity.

Fig. 2. Uptake of valerate by A. vinelandii UWD. Sodium valerate (10 mM) was added at zero time to glucose medium and the uptake of glucose (o), acetate (o), and valerate (A) was followed. Valerate uptake by cells preincubated for 12 h in glucose medium (0) and in medium in which valerate was the sole carbon source (m) also was measured.

Incubation time (h)

The molecular weight of the HAD-ECH multienzyme complex was estimated on a calibrated DEAE Sepharose CL-6B column as described previously (Manchak and Page 1994).

Results and discussion

Valerate uptake is inducible and subject to acetate repression

When copolymers were synthesized in strain UWD, the culture was pregrown in glucose medium for 12 h, then 10 mM valerate (final concentration) was added and the culture incubated for an additional 10- 12 h (Page et al. 1992). During the first 0-8 h the culture used the 15 mM acetate originally present in the medium and glucose use was delayed until acetate was depleted (Fig. 2). This pattern of diauxic growth has been described previously by George et al. (1985). When valerate was fed to the culture at 0 h, valerate uptake also was inhibited until acetate was depleted, but when valerate was added at 12 h, there was no apparent delay in valerate uptake (Fig. 2). The addition of valerate did not significantly inhibit glucose use, so valerate cannot be considered to be a preferred carbon source like acetate.

When the glucose medium was altered to contain 15 rnM ammonium bicarbonate rather than ammonium acetate and 30 mM valerate (final concentration) was present as the sole carbon source, the uptake of valerate also was delayed by 6 h (Fig. 2). This indicated that valerate use was not constitutive but inducible and that a period of adaptation was needed before fatty acid uptake occurred. Amore precise timing of short chain fatty acid use by cells pregrown for 12 h in glucose medium showed that there was a brief lag before the use of 15 mM propionate, butyrate, or valerate (5 1 h) and a longer lag before the use of 15 mM hexanoate, heptanoate, nonanoate, and

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Fig. 3. Induction of ECH and HAD by short-chain fatty acids. Enzyme specific activity (units. mg protein-') was assayed after 24 h growth of strain UWD in glucose medium (Gluc), or in glucose medium fed 10 rnM propionate (Prop), butyrate (But), valerate (Val), trans-2-pentenoate (T2P), or trans-3-pentenoate (T3P) 12 h before harvesting.

0 Gluc Prop But Val T2 P T3 P

Growth Substrate

Fig. 4. Inhibition of 3-ketothiolase and decreased PHA yield in the presence of P-oxidation inducers. Enzyme specific activity (units . mg protein-', solid bar) and PHA yield (mg . mg protein-1, hatched bar) were measured in cells prepared for Fig. 3.

Gluc Val T2P T3 P

Growth Substrate

octanoate (2-4 h) (data not shown). The C3-5 fatty acids were consumed completely by 20 h whereas the consumption of the other fatty acids was less (C6 > C7 > C8 = C9). There was no noticeable growth of strain UWD on longer chain (C11- 18) fatty

acids in a 24-h period, despite the addition of detergents (0.5% (final concentration) Tween 40 or 80) to make these compounds more soluble and hence available.

P-Oxidation enzymes in A. vinelandii UWD It has been previously shown that 3-ketothiolase is a constitutive enzyme in A. vinelandii (Manchak and Page 1994). However, the other P-oxidation enzymes ECH and HAD were absent from cells grown in glucose medium or in glucose medium supplemented with 10 mM propionate (Fig. 3) or acetate (data not shown). These enzymes were induced coordinately and equally well in glucose medium containing 10 mM butyrate, valerate, trans-Zpentenoate (Fig. 3), trans-2-butenoate (crotonate), hexanoate, heptanoate, or octanoate (data not shown). trans-3-Pentenoate was the most effective inducer of the @-oxidation enzymes (Fig. 3), which also indicated that ~is-A-~-trans-A~ enoyl-CoA isomerase was present to direct the trans-3-pentenoate into the P-oxidation pathway. There was no induction of P-oxidation by L-(+)-P-hydroxybutyrate, which suggested that there was channelling of this intermediate through the complex. Channelling was also suggested by the fact that DL-P-hydroxybutyryl-CoA was not a substrate for HAD in vitro, hence this enzyme was always measured in the reverse direction (i.e., as an NADH-dependent AcAcCoA reductase).

Concomitant with the induction of ECH and HAD (Fig. 3) there was always a reduction in the constitutive 3-ketothiolase activity and a reduction in PHA yield (Fig. 4). Thiolase activity was always highest in cells from glucose medium and was lowest in cells from glucose medium containing trans-3- pentenoate. Intermediate degrees of &oxidation induction resulted in less severe thiolase inhibition, but there was not an absolute or linear relation between these values.

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Fig. 5. Resolution of enzymes involved in P-oxidation and PHA synthesis. Cell extract prepared from strain UWD grown for 24 h in glucose medium alone (A) or in glucose medium with 10 mM butyrate added at 12 h (B) was eluted from a DEAE Sepharose CL-6B column. The NaCl gradient started at about fraction 38 and continued to at least fraction 80 (not shown).

I \ I - HAD I \

I \ - AcAcCoA Red. \ I

3- Ketothiolase

Fraction

4 i \I - HAD - AcAcCoA Red.

Fraction

These studies concerning the induction of ECH and HAD and the reduction in thiolase activity were repeated using strain UW, the parent of strain UWD, and the results were essentially identical to those reported here.

The P-oxidation complex of A. vinelandii Ion exchange column chromatography showed that cells grown in glucose medium contained 3-ketothiolase and the NADPH-dependent AcAcCoA reductase required for PHA synthesis, but were missing HAD andECH activities (Fig. 5A). The small amount of apparent HAD activity coincident with the AcAcCoA reductase was likely due to the small amount of NADPH-dependent AcAcCoA reductase activity that is obtained with NADH (Manchak and Page 1994). Some 3-ketothiolase activity eluted from the column at lower NaCl concentrations ( 3 W 0 mM), but the majority of the activity eluted at about 6&65 mM NaC1. Cells grown in glucose medium containing butyrate, valerate, trans-2-pentenoate, or

octanoate were similarly examined and a representative elution profile is shown in Fig. 5B. The induction of ECH and HAD was clearly demonstrated and these two enzymes eluted together as a complex at about 80 mM NaC1. These enzymes did not associate with the 3-ketothiolase peak that eluted at 60 mM NaC1, but possibly formed an aggregate with the thiolase and eluted at a lower NaCl concentration (35 mM NaCl). No 3-hydroxybutyryl-CoA epimerase activity could be detected in the ECH-HAD (HD) complex. This activity also was missing from the P. fragi HDT complex (Imamura et al. 1990), but was present in the E. coli HDT complex (Pawar and Schulz 1981). Hyperinduction of ECH and HAD by trans-2- pentenoate (Fig. 3) increased the enzyme specific activity but did not alter the elution profile (data not shown).

The A. vinelandii HD complex had an apparent molecular weight of 73 000. The molecular weight of the 3-ketothiolase from A. vinelandii has been previously determined to be 150 000 and this enzyme is likely a dimer (Manchak and Page

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Table 1. Fate of valerate transported into strain UWD.

Can. J. Microbiol. Vol. 41(Suppl. I ) , 1995

Total dry HV content Valerate used in RM Valerate Other mass PHA of polymer Valerate used polymer (% of dry mass (mM) additions (mg/mL) (% dry mass) (mol%) (p,mol/mL) total consumed) (mg/mL)

10 Glucose 7.0 94 18 10 76 1.8 20 Glucose 5.9 64 22 18 38 2.1 30 Glucose 5.6 74 25 22 4 1 1.5 10 - 0.7 67 16 10 6.6 0.2 20 - 1.4 36 21 20 4.5 0.9 30 - 2.0 29 28 30 4.5 1.4

NOTE: The cultures were incubated for 24 h in glucose medium containing valerate added at 12 h, or in medium containing valerate as the sole carbon source.

Fig. 6. B-Oxidation and the initiation of copolymer synthesis in A. vinelandii. Enzymes not measured in this study include (1) acyl-CoA synthetase and (2) acyl-CoA dehydrogenase, although a transport step (1) is inducible. As shown in this study, the enzymes (3) enoyl-CoA hydratase (ECH) and (4) 3-hydroxyacyl- CoA dehydrogenase (HAD) are associated with the a-subunit (A) and form the HD complex. cis-A3-trans-A2-Enoyl-COA isomerase (5) appears to be present, but 3-hydroxyacyl-CoA epimerase is not present. 3-Ketoacyl-CoA is an end product of the HD complex. This may be degraded by the 3-ketothiolase (6) on the loosely associated P-subunit (B) or it may enter PHA biosynthesis through the NADPH-dependent AcAcCoA reductase (7). AcAcCoA also can be generated by the reversed (condensation) reaction of the 3-ketothiolase (6). The AcAcCoA reductase (7) is regulated (open arrows) negatively (-) by 3-ketoacyl-CoA and NADP+ and positively (+) by NADPH (Manchak and Page 1994).

Fatty acid

AMP + ATp$cOA PPi

1994; J. Manchak and W.J. Page, unpublished results). Thus the aggregated P-oxidation complex of A. vinelandii could have a molecular weight of 223 000, similar to the complex with a molecular weight of 240 000 found in P. fragi and E. coli

(Sato et al. 1992a; Pawar and Schulz 1981), assuming an a2P2 configuration as found in these organisms. Greater similarities are found between the molecular weights of the a-subunits of P. fragi and A. vinelandii (73 000) and E. coli (78 OOO), than between those for the 3-ketothiolase dimer, which is about twice as large in A. vinelandii as in E. coli and P. fragi (84 000).

P-Oxidation and control of PHA synthesis in A. vinelandii A major difference between the A. vinelandii P-oxidation complex and the others is that the a- and P-subunits are not tightly associated. The E. coli and P.fragi HDT complexes are held together very tightly and only dissociate during electrophoresis in the presence of SDS (Imamura et al. 1990; Pawar and Schulz 1981). Also, the induction of the 3-ketothiolase, ECH, and HAD is coordinate in E. coli and P. fragi (Saito et al. 1994; Yang and Schulz 1983), but not in A. vinelandii. This must reflect the dual role of the 3-ketothiolase for biosynthesis of PHA and degradation of lipids (including PHA) in A. vinelandii, while the 3-ketothiolase of E. coli and P. fragi performs only a degradative role. The results suggest that 3-ketoacyl-CoAs are end products of P-oxidation in A. vinelandii and are not channelled to complete degradation. These should, therefore, be available for PHA synthesis and according to the control model (Fig. I), excess 3-ketoacyl-CoAs generated by a-oxidation should interfere with AcAcCoA reductase activity especially when NADPH levels are low (Manchak and Page 1994).

This was shown to be the case in growth experiments in which fatty acids were used to promote copolymer formation. Valerate was incorporated equally well into PHA produced by strain UWD in glucose medium and in a medium in which valerate was the sole carbon source (Table 1). When glucose and valerate were both in the medium, valerate was directed largely into polymer synthesis. Increased valerate availability had an overall negative effect on polymer formation, but growth of the cells (measured as RM) was not significantly affected (mean 1.8 ? 0.3 mg . mL-I). However, when valerate was the sole carbon source, increased valerate availability resulted in increased cell growth and greatly decreased polymer production. These data show that valerate metabolism interfered with the quantity of PHA synthesized, but not the efficiency of HV insertion into the polymer. The quantity of

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PHA synthesized should be affected by the availability of substrates and the specific activity of PHA biosynthetic enzymes, while HV content in the polymer (which does not change) should be affected by the enzyme's affinity for HV versus HB as a substrate.

Inhibition of PHA biosynthetic enzyme activities by valerate addition to the medium also was observed. Inhibition of 3-ketothiolase activity has been shown (Fig. 4). The NADPH-dependent AcAcCoA reductase activity was 4-6 units . mg protein-' in glucose medium and was decreased 57% by the addition of 10 mM valerate to the culture. Similar inhibition of these enzymes was seen when valerate was the sole carbon source. Even more severe inhibition of the AcAcCoA reductase and the 3-ketothiolase were observed when 10 mM butyrate or hexanoate was added to glucose medium at 12 h. In these cases, the quantity of polymer decreased by 62% and the specific activites of the two enzymes each decreased by 70-77%.

These effects on AcAcCoA reductase were consistent with PHA synthesis being controlled by 3-ketoacyl-CoA intermediates obtained from the P-oxidation of even- and odd-chain fatty acids (Fig. 6). Thus, AcAcCoA formed from the degradation of butyrate or hexanoate could inhibit the NADPH-dependent AcAcCoA reductase, and 3-ketovaleryl- CoA formed during the P-oxidation of valerate could also inhibit this enzyme, but to a lesser extent. The degree of inhibition is consistent with AcAcCoA being the preferred substrate of the NADPH-dependent AcAcCoA reductase

1 (Manchak and Page 1994). Decreased 3-ketothiolase activity would also promote a buildup of 3-ketoacyl-CoA intermediates, exacerbating the inhibition of AcAcCoA i reductase.

Inhibition of the 3-ketothiolase, however, cannot be explained by a buildup of 3-ketoacyl-CoA, since this enzyme is not subject to substrate inhibition (Manchak and Page 1994). These data suggest that there may be some control feature shared between the inducible enzymes of P-oxidation and the constitutive 3-ketothiolase. A model can be drawn for this, based on the control of P-oxidation in E. coli that is regulated by FadR (DiRusso et al. 1993; Henry and Cronan 1992). FadR is a positive transcriptional regulator of lipid synthesis and a negative regulator of P-oxidation (Henry and Cronan 199 1). When long-chain fatty acids are transported into the cell and activated, the acyl-CoA binds to FadR to prevent DNA binding. Unsaturated fatty acids are more effective than saturated fatty acids in interfering with FadR functions (Henry and Cronan 1992). A similar control system may possibly operate in A. vinelandii. The intriguing parallels, despite the differences in fatty acid chain length, are that an unsaturated fatty acid (T3P) was the best inducer of P-oxidation and that induction of P-oxidation was always accompanied by decreased activity of 3-ketothiolase, the first enzyme involved in lipid (PHA) biosynthesis. If this type of control does exist in A. vinelandii, it would suggest that induction of P-oxidation must inevitably result in decreased polymer synthesis. Thus, further understanding of the molecular genetic control of P-oxidation in A. vinelandii and the search for alternative routes for copolymer synthesis will be worthwhile studies, with potential benefit to the development of this organism as a commercial

I source of PHA.

I

Acknowledgements

This study was supported by the Natural Sciences and Engineering Research Council of Canada operating grants program and the Cooperative Research and Development program and a grant from the University of Alberta Central Research Fund.

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