The initial metabolic conversion of levulinic acid in Cupriavidus necator

Journal of Biotechnology 155 (2011) 293–298

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The initial metabolic conversion of levulinic acid in Cupriavidus necator Matt Jaremko ∗ , Jian Yu ¯ Hawai‘i Natural Energy Institute, University of Hawai‘i at Manoa, 1680 East West Road, POST 109, Honolulu, HI 96822, USA

a r t i c l e

i n f o

Article history: Received 6 May 2011 Received in revised form 16 July 2011 Accepted 22 July 2011 Available online 30 July 2011 Keywords: Levulinic acid 4-Oxopatenoic acid Polyhydroxyalkanoate Ralstonia eutropha Biopolyester

a b s t r a c t Levulinic acid or 4-ketovaleric acid is a potential renewable substrate for production of polyhydroxyalkanoates. In this work, the initial reactions of LA metabolism by Cupriavidus necator were examined in vitro. The organic acid was converted by membrane-bound crude enzymes obtained from the cells pre-grown on LA, while no LA activity was detected from cells pre-grown on acetic acid. Acetyl-CoA and propionyl-CoA were two major intermediates in the initial reactions of LA conversion. A mass balance on propionyl-CoA accounts for 84 mol% of LA added in vitro. It explains an interesting phenomenon that 3-hydroxbutyrate and 3-hydroxyvalerate are two major monomers of the biopolyester formed from LA, instead of 4-hydroxvalerate that has the similar chemical structure of LA as the precursor. A Monod model was used to describe the kinetics of LA utilization as a sole carbon source or a co-substrate of glucose and fructose. The max and Km of LA alone were 0.26 h−1 and 0.01 g/L, respectively. The content and composition of PHA are also dependent on the culture conditions such as carbon to nitrogen ratio. The in vitro observation is supported by the high utilization rate of LA and the high molar percentage of 3HB and 3HV in the PHA derived from LA. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Polyhydroxyalkanoates (PHAs) are a family of biopolyesters formed and accumulated in microbial cells as carbon and energy reserve (Du et al., 2001; Naik et al., 2008; Yu et al., 2005). After recovery and purification from the cells, the biopolyesters exhibit the thermal and mechanical properties of petrochemical plastics, but are biocompatible and biodegradable in the environment (Gross and Kalra, 2002; Luzier, 1992; Volova et al., 2006). PHA bioplastics have a small carbon footprint because they can be produced from various renewable feedstocks such as starch and molasses (Bengtsson et al., 2010; Harding et al., 2007; Quillaguamán et al., 2010). Glucose and fructose are the common carbon sources from which poly-(3-hydroxybutyrate) (P3HB) is usually formed by many microbial species (Du et al., 2001; Page et al., 1992; Raberg et al., 2011; Yezza et al., 2007). P3HB is a rigid plastic, exhibiting

Abbreviations: , specific growth rate (h−1 ); max , maximum specific growth rate (h−1 ); 3HB, 3-hydroxybutyrate; 3HV, 3-hydroxyvalerate; 4HV, 4hydroxyvalerate; AcCoA, acetyl-CoA; ATP, adenosine tri-phosphate; C/N, carbon to nitrogen; CoA, coenzyme A; Km , substrate saturation constant (g/L); LA, levulinic acid; P3HB, poly-(3-hydroxybutyrate); P3HB3HV, poly-(3-hydroxybutyrate-co-3hydroxyvalerate); P3HB3HV4HV, poly-(3-hydroxybutyrate-co-3-hydroxyvalerateco-4-hydroxyvalerate); PHAs, polyhydroxyalkanoates; PrpCoA, propionyl-CoA; S, substrate concentration (g/L); V, specific consumption rate (h−1 ); Vmax , maximum specific consumption rate (h−1 ); YX/S , overall cell yield. ∗ Corresponding author. Tel.: +1 808 956 4207; fax: +1 808 956 2336. E-mail address: [email protected] (M. Jaremko). 0168-1656/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2011.07.027

high tensile strength and low ductility (Lee, 1996). The material properties of PHA bioplastics can be altered and controlled by introducing other monomers into the P3HB backbone. Poly-(3hydroxybutyrate-co-3-hydroxyvalerate) (P3HB3HV), for instance, has a good balance of tensile strength, ductility and melting behavior (Kulkarni et al., 2011; Lee, 1996). In order to produce co-polyesters, microbial cells are fed with co-substrates, including one primary carbon source such as glucose and one precursor substrate for alternative monomers. Propionic acid or valeric acid, for instance, is often used with glucose to produce P3HB3HV (Page et al., 1992; Pereira et al., 2008; Ramsay et al., 1990; Yu et al., 2005). It has been known that one acetyl-CoA (AcCoA) from glucose and one propionyl-CoA (PrpCoA) from propionic acid are condensed by ␤-ketothiolase to form a 3-ketovaleryl-CoA that is further reduced to 3-hydroxyvaleryl-CoA (Bramer and Steinbuchel, 2001; Doi et al., 1987; Yu and Si, 2004). The precursor of 3-hydroxyvalerate (3HV) is integrated into a growing polyester backbone by PHA synthase (Slater et al., 1998; Yu et al., 2002). In the presence of valeric acid, the organic acid is first activated to form valeryl-CoA, which is then converted into 3-hydroxyvaleryl-CoA directly via the betaoxidation pathway (Page et al., 1992; Yu et al., 2009). Levulinic acid (LA) or 4-ketovaleric acid is a platform chemical that can be derived from simple carbohydrates, starch, and cellulosic biomass (Assary et al., 2010; Bozell et al., 2000). It is an inexpensive renewable precursor substrate for production of PHA co-polyesters. A few studies have shown that LA can be used by Cupriavidus necator as a sole carbon source or cosubstrates for cell growth and PHA formation (Chung et al., 2001;

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Growth and Maintenance

O

Acyl-CoA Synthetase COOH [1]

O

CoA, ATP

O

[2]

CoA COSCoA ATP

O SCoA

+

SCoA

[3]

[4]

PhaA

BktB

(2 Acetyl-CoAs) O

O

O

O

SCoA

SCoA

[5]

[7] PhaB NADPH

PhaB NADPH OH

O

O

OH

O

PhaC SCoA [6]

O

PhaC O

O 3HB

3HV

O n

SCoA [8]

PHA Co-polymer Fig. 1. A suggested metabolic pathway of PHA synthesis from levulinic acid. [1] Levulinic acid; [2] levulinyl-CoA; [3] acetyl-CoA; [4] propionyl-CoA; [5] acetoacetyl-CoA; [6] 3-hydroxybutryl-CoA; [7] 3-ketovaleryl-CoA; [8] 3-hydroxyvaleryl-CoA; CoA, coenzyme-A; ATP, adenosine triphosphate; PhaA, ␤-ketothiolase A; BktB, ␤-ketothiolase B; PhaB, NADPH-dependent acetoacetyl-CoA reductase; NADPH, nicotinamide adenine dinucleotide phosphate; PhaC, PHA synthase.

Lee et al., 2009; Yu et al., 2009). It is interesting to notice that the monomeric composition of co-polyesters formed on LA is quite different from the chemical structure of the precursor (Yu et al., 2009). For instance, a terpolyester, poly-(3-hydroxybutyrateco-3-hydroxyvalerate-co-4-hydroxyvalerate) or P3HB3HV4HV, is formed in C. necator from LA as the sole carbon source. The two primary monomers are 3HB and 3HV, 55 mol% and 43 mol%, respectively, and 4HV is only a minor monomer (1–3 mol%) even though 4-ketovaleric acid seems a natural precursor of 4-hydroxyvalerate (Yu et al., 2009). The small difference of 3HV and 4HV in chemical structure may have a great effect on the material properties of biopolyesters (Kulkarni et al., 2011; Sudesh et al., 2000; Yu et al., 2005). Little knowledge, however, exists regarding the formation of 3HV from 4-ketovaleric acid or LA. One possible mechanism is based on the initial reactions of LA metabolism: the organic acid is first activated to form 4-ketovaleryl-CoA and then split into PrpCoA and AcCoA (Fig. 1). The two intermediates are either used via the main metabolism pathway for cell growth or condensed into 3-ketovaleryl-CoA for 3HV via the well-established PHA biosynthesis pathway in C. necator. This hypothesis is supported by the up-regulation of a probable acyl-CoA synthetase in C. necator when exposed to LA (Lee et al., 2009). Also, C. necator HF39 exhibits poor growth on LA when the metabolic reactions of PrpCoA utilization are blocked (Bramer and Steinbuchel, 2001; Horswill and Escalante-Semerena, 1997). Another possible mechanism of LA conversion is beta-oxidation. Beta-oxidation involves formation of a ketone group or hydroxyl group on carbon 3 of an organic acid. For example, valeryl-CoA is converted into 3-ketovaleryl-CoA and then split into acetyl-CoA and propionyl-CoA via beta-oxidation (Slater et al., 1998). However, levulinic acid contains a ketone group on carbon 4. It is reasonable to assume that multiple reactions with cofactors must occur on C4 of LA for the ketone group to be reduced if beta-oxidation is the primary reactions. Identification of the intermediates in the initial reactions of LA metabolism in vitro may reveal the pathway. Organic acids, such as LA, often inhibit cell growth at moderate concentrations (Yu et al., 2002). In small amounts, however, the

acids can be utilized for cell growth and PHA formation (Page et al., 1992; Yu et al., 2009). Nitrogen limitation is a common method used to induce PHA formation because nitrogen limitation directs the carbon flux to PHA synthesis rather than cellular components (Budde et al., 2011; Du et al., 2001; Yu et al., 2005). The change in carbon to nitrogen ratio may not only alter the accumulation of PHA or PHA content, but also the monomeric composition because the formation of 3HB and 3HV may be affected to a different extent. C. necator (formerly Ralstonia eutropha) is a representative bacterium for PHA biosynthesis (Budde et al., 2011; Khanna and Srivastava, 2005; Pereira et al., 2008; Reinecke and Steinbüchel, 2008). In this work, we investigated the kinetics of cell growth and utilization on levulinic acid, glucose and fructose. The kinetic parameters were examined by using a Monod model. We further investigated the initial metabolic reactions of levulinic acid with the crude enzymes of C. necator pre-grown on the organic acid. The monomeric composition of PHA biopolyesters formed under different culture conditions were measured and analyzed. 2. Materials and methods 2.1. Chemicals, media, and cultures Glucose, fructose, levulinic acid, coenzyme A (CoA), AcCoA, and PrpCoA were purchased from Sigma Aldrich. Acetic acid was purchased from Fisher Scientific. A laboratory strain of Cupriavidus necator was maintained on slant cultures containing 2 g/L NaH2 PO4 , 0.25 g/L MgSO4 ·7H2 O, 2 g/L (NH4 )2 SO4 , 3.67 g/L K2 HPO4 , 1 g/L yeast extract, 15 g/L agar, 6 g/L glucose, and 1 mL/L trace solution. For inoculum preparation, cells were transferred from slant culture to a 20 mL test tube containing 5 mL solution of 5 g/L yeast extract, 5 g/L peptone, 2.5 g/L meat extract, and 2 g/L (NH4 )2 SO4 . The test tube culture was incubated at 30 ◦ C for 48 h. The 5 mL test tube culture was then transferred to a 250 mL baffled flask containing 100 mL medium of 10 g/L substrate, 94% v/v mineral solution,

M. Jaremko, J. Yu / Journal of Biotechnology 155 (2011) 293–298

and 0.1% v/v trace solution. The 100 mL seed flask culture was shaken at 30 ◦ C and 200 rpm in an orbital rotary incubator for 24 h. Depending on the experiment, the substrates were levulinic acid, glucose, fructose, and acetic acid. For example, if an experiment involved glucose and fructose in culture, the 100 mL seed flask culture would contain 5 g/L glucose and 5 g/L fructose to a total substrate concentration of 10 g/L. In the case of levulinic acid and acetic acid, the 100 mL seed flask culture contained 9 g/L glucose and 1 g/L organic acid to avoid growth inhibition from organic acids. Unless otherwise stated, the mineral solution contained 2 g/L NaH2 PO4 , 0.5 g/L MgSO4 ·7H2 O2, 2 g/L (NH4 )2 SO4 , and 7.34 g/L K2 HPO4 ·3H2 O. When noted, the (NH4 )2 SO4 concentration was changed to control the C/N ratio. Trace mineral solution contained 700 ␮M Fe(NH4 )SO4 , 17 ␮M ZnSO4 ·7H2 O, 25 ␮M MnCl2 ·4H2 O, 8 ␮M CuSO4 ·5H2 O, 7.2 ␮M NaB4 O2 ·10H2 O, and 8.3 ␮M NaMoO4 ·2H2 O. The 100 mL seed flask culture was then used as inoculum for experimental cultures. Unless stated otherwise, in shake flask experiments, cultures were grown in 500 mL baffled flask containing a 200 mL medium composed of 8–10 mL C. necator inoculum (from 100 mL seed flask culture), a specified substrate concentration, 94% v/v mineral solution, and 0.1% v/v trace solution. Cultures were shaken at 30 ◦ C and 200 rpm in an orbital rotary incubator. Aliquots of 50 mL culture medium were collected for analysis. Crude enzyme solutions were prepared from the shake flask cultures on levulinic or acetic acid. The individual organic acid was equally fed five times to culture at 0, 12, 24, 36, and 48 h to a total of 10 g/L to avoid growth inhibition. Cells were harvested after 50-h cultivation with centrifugation for later use. Batch fermentations were performed in a 3 L bioreactor (BioFlo 110, New Brunswick Scientific Co. Inc., NJ, USA). The temperature, pH, and dissolved oxygen probes were used to monitor and control the fermentations. Culture conditions were maintained at 30 ◦ C and pH 6.8. Agitation speeds were 200 RPM or above to control the dissolved oxygen at 10% of air saturation. Cultures were inoculated with a 100 mL seed flask culture mentioned above. The media are specified in the experiments below. Aliquots of 10–15 mL culture medium were collected for analysis.

2.2. Analysis of cells, PHA, and culture medium The optical density of culture medium was measured at 620 nm to monitor cell growth with a UV/Vis spectrophotometer (DU 530, Beckman, CA, USA). Aliquots of culture medium were centrifuged at 7000 rpm for 10 min to separate the supernatant (culture solution) from wet pellets (cells and PHA). The wet pellets were washed with 50 mL of mineral solution and then freeze-dried to determine the cell mass concentration and PHA content as mentioned below. The supernatant solution was analyzed with a HPLC system (VP series, Shimadzu, MD, USA). The organic acids were eluted with a water-sulfuric acid carrier (pH 2.0) at 0.8 mL/min through an organic acid column (Rezex ROA H+ (8%) 150 mm × 7.8 mm, Phenomenex, CA, USA) maintained at 60 ◦ C and measured with a UV detected at 210 nm. The glucose and fructose were eluted with a water–potassium phosphate carrier (15 mM K2 HPO4 ) at 0.5 mL/min through a carbohydrate column (Supelcogel K 300 mm × 7.8 mm, Sigma Aldrich, MO, USA) maintained at 80 ◦ C and measured with a RID detector. PHA content and composition in cells were determined after methanolysis of freeze-dried cell mass in methanol (3.2% v/v sulfuric acid) at 100 ◦ C for 8 h. The methyl hydroxyalkanoates were hydrolyzed to hydroxyalkanoic acid by adding 10 M NaOH solution and then measured with the HPLC above. The calibration was performed with PHA standards purchased from Sigma Aldrich.

295

2.3. Crude enzyme preparation The wet pellets harvested from 200 mL flask cultures on levulinic acid or acetic acid were washed and re-suspended in 5 mL phosphate buffer consisting of 60 mM K2 HPO4 and 2 mM MgSO4 . One milliliter of slurry was transferred into a 1.5 mL microcentrifuge tubes for cell disruption with an ultrasonic cell disrupter (Model 100, Fisher Scientific, TX, USA). The sonication was conducted at 40 W and 30 s each cycle for 3 cycles. Between the cycles, the suspension was left on ice for about 5 min to reduce the sonication heat. The membrane-bound enzymes and water-soluble enzymes from the disrupted cells were separated with centrifugation at 10,000 × g for 10 min. The clean supernatant solution containing water-soluble enzymes was kept at −20 ◦ C for later use. The wet pellets containing membrane-bound enzymes was washed in a phosphate buffer solution once, re-suspended in 0.9 mL phosphate buffer solution (pH 6), and kept at −20 ◦ C for later use. 2.4. In vitro bioconversion of levulinic acid All reactions were performed in 1.5 mL micro-centrifuge tubes at 30 ◦ C and 200 rpm in an orbital rotary incubator. A typical reaction solution consisted of 1 mM LA, 0.7 mM coenzyme A, 1.5 mM adenosine tri-phosphate (ATP), 30 mM K2 HPO4 , and 1 mM MgCl2 . The reaction was initiated by addition of crude enzymes at 25% vol/vol. Acetic acid was also used to replace LA for control. The organic acids were analyzed with the HPLC described above, but detected with a RID detector because of ATP interference with the organic acids in the UV detector. CoA, AcCoA, and PrpCoA were detected with a UV detector at 261 nm. Samples of 20 ␮L were eluted with a mobile solution consisting of 150 mM citrate buffer at pH 5.0 and 9% vol/vol acetonitrile through a C18 column (Premier C18 250 mm× 4.6 mm, Shimadzu, MD, USA). Under these conditions, the retention times of CoA, AcCoA and PrpCoA were 4.0 min, 5.7 min, and 10.7 min, respectively. The concentrations of CoAcontaining compounds were calculated from individual calibration curves of standard compounds obtained from Sigma Aldrich. 3. Results and discussion 3.1. LA utilization by C. necator Although levulinic acid is toxic to cells, the substrate can be effectively utilized by C. necator for growth and PHA production at low concentrations (below 2 g/L). Acetic acid and propionic acid are also utilized quickly after they are activated with CoA. Carbohydrates, however, must be catabolized through glycolysis into acetyl-CoA. Therefore, carbohydrate utilization may be slower than organic acid utilization. A kinetic analysis was performed for LA and compared with those of glucose and fructose, two carbohydrates abundant in renewable feedstocks and often used with organic acids in culture for production of PHA copolymers (Ramsay et al., 1990; Yu et al., 2009). In a kinetic analysis, substrate concentrations were prepared at 2 g/L or less and other nutrients were available in excess. A Monod model Eq. (1) is used to simulate the kinetics of cell growth and substrate utilization. max s = (1) Km + s Cell mass and substrate concentrations were measured to obtain the values of max and Km for each substrate. The Langmuir plot was used to calculate the max and Km . The Monod parameters of C. necator were determined with LA as limiting substrate in a bioreactor batch culture (Table 1). The specific consumption rate (V) for each substrate was determined from time courses of the residual

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Table 1 The Monod model parameters of C. necator on levulinic acid (LA), glucose, and fructose. Substrate

max (h−1 )

Km (g/L)

Vm (h−1 )

YX/S

LA Glucose Fructose

0.26 0.18 0.30

0.01 0.08 0.05

0.79 0.46 0.80

0.36 0.48 0.51

substrate concentrations. The overall yield (YX/S ) was determined from the slope of the cell density versus substrate consumption. LA was utilized at a high rate by C. necator at low concentrations. Of all substrates, C. necator had the highest affinity for LA with a Km of 0.01 g/L (Table 1). The organic acid also had a high max and Vm at 0.26 h−1 and 0.79 h−1 , respectively. Interestingly, LA had a YX/S of 0.36, which was much lower than glucose and fructose (Table 1). Some LA might be removed from culture by adsorbing to the cells as revealed above. Fructose had similar max and Vm values to LA, but the Km of fructose was significantly higher than the Km of LA (Table 1). Glucose was the least preferred by C. necator under these conditions. The carbon source had the lowest max , Km , and Vm at 0.18 h−1 , 0.08 g/L, and 0.46 h−1 , respectively. At low concentrations, LA is utilized at high rates than glucose and fructose, similar to other organic acids. The kinetics were further examined in a multiple substrate culture. A batch fermentation was prepared with glucose, fructose, and LA as the carbon source to examine the behavior of C. necator. The max of C. necator was 0.29 h−1 , which is similar to the max of LA as an individual substrate (Table 1). Interestingly, C. necator consumed LA almost exclusively in the presence of glucose and fructose (Fig. 2). Even under optimal conditions, glucose and fructose consumption was minimal. In 4.5 h, the utilization of LA, glucose and fructose were 93%, 9%, and 7%, respectively (Fig. 2). The maximum specific LA rate reached was 0.53 h−1 , which is 67% of the Vm of LA as the sole substrate (Table 1). The presence of LA regulates or inhibits the metabolic pathways of glucose and fructose. One possible mechanism is that the intermediates from LA metabolism may converge on the sugar pathways. Since LA is metabolized at a higher rate than glucose and fructose, the carbohydrate pathways will be halted because there is already a high concentration of metabolic intermediates in the cytoplasm. The results imply the possibility of an LA initial conversion to AcCoA and PrpCoA. In this case, LA would be incorporated into the main metabolism at a higher rate than glucose and fructose, which must first pass through glycolysis to form pyruvate and acetyl-CoA. The overall yield in the fermentation was 0.52, which is similar to the yields of C. necator grown on sugars (Table 1). The YX/S (0.52) is higher than the YX/S of LA alone (0.36), probably because of the contribution from the sugars.

Residual Substrate (g/L)

0.6 0.5 0.4 0.3 0.2

Glucose Fructose

0.1

LA

0.0 0

1

2 3 Time (hrs)

4

5

Fig. 2. The time course of residual substrate concentration (g/L).

Table 2 PHA formation after 48 h in C. necator grown on LA at different C/N ratios. C/N (w/w)

Cell density (g/L)

13 21 31 42

3.9 4.0 3.5 3.0

± ± ± ±

0.1 0.1 0.0 0.0

Residual cell mass (g/L)

PHA content (% w/w)

2.3 1.8 1.1 1.1

41 55 69 64

± ± ± ±

1 4 5 1

3HV/PHA (% w/w) 51 40 30 29

± ± ± ±

0 1 2 0

3.2. PHA formation on LA LA is converted into multiple monomers in the PHA metabolism of C. necator (Fig. 1). Analysis of the PHA composition from LA may provide insight about the intermediates in the metabolism of LA. Flask cultures with LA as the sole carbon source were prepared with different nitrogen concentrations to analyze the C/N ratio influence on PHA formation. Nitrogen limitation induces PHA accumulation in C. necator and may affect the PHA composition when grown on LA. Cultures contained 0.6, 0.8, 1.2, or 2 g/L (NH4 )2 SO4 , which provided C/N ratios of 42, 31, 21, or 13, respectively. Equal amounts of LA were added to culture at 0, 12, 24, and 36 h to a total concentration of 10 g/L. Cultures consisted of 95% v/v mineral solution, and 0.1% v/v trace solution. Cultures were harvested after 48 h and analyzed. Cell growth was limited at C/N ratios above 30 due to nitrogen limitation (Table 2). Cultures at 13 and 21 C/N showed no growth limitation and reached 4 g/L cell density after 48 h. The PHA content decreased as the C/N ratio decreased because carbon was directed towards cell growth rather than PHA synthesis. As mentioned above, 3HB can be synthesized from AcCoA and 3HV from condensation of AcCoA and PrpCoA. In all cultures, 3HB and 3HV were present at relatively high concentrations (Table 2). The high content of these monomers supports the possibility of LA split into AcCoA and PrpCoA. Interestingly, the 3HV content increased when the C/N ratio was decreased (Table 2). While the 3HV concentration increased slightly when the C/N ratio decreased, the overall PHA and 3HB concentration decreased significantly. In the 3HB pathway, two acetyl-CoA are condensed by ␤-ketothiolase A to eventually produce 3HB. When nitrogen is in excess, there may be increased utilization of acetyl-CoA for cell growth and/or ␤-ketothiolase A may be inhibited. Based on the 3HV concentration, the activity of the LA to 3HV pathway is largely unaffected by the C/N ratio change. The results suggest the formation of AcCoA and PrpCoA from LA and also reveal a phenomenon where the pathways of 3HB and 3HV from LA are affected differently when C. necator is subject to nitrogen limitation. 3.3. In vitro bio-conversion of LA An acyl-CoA synthetase is responsible for activation of the organic acid by binding CoA to LA. The activity of LA conversion was observed in the pellet slurry of LA-grown cells, while no activity was observed in the supernatant solution. Therefore, the LA acyl-CoA synthetase is a membrane-bound enzyme and the crude enzyme slurry includes cell debris and PHA granules. Also, the LA conversion activity was only present in the cells pre-grown on LA. When cells were grown on acetate, there was no LA conversion activity in the pellet slurry. Based on the results, LA acyl-CoA synthetase is therefore an inducible, not constitutive, membrane-bound enzyme. Conversion of LA by the crude enzymes was examined in an assay solution for 3.5 h. The in vitro reaction consisted of 25% v/v pellet slurry, 0.71 mM LA, CoA, and ATP in a phosphate buffer solution. The concentrations of LA and reaction products were monitored at 30 min intervals. Fig. 3 is the two HPLC chromatograms indicating formation of AcCoA and PrpCoA as the two major initial intermediates found in LA conversion in vitro. After 2 h, the initial

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297

Table 3 Conversion of acetyl-CoA (AcCoA) and propionyl-CoA (PrpCoA) catalyzed by wet pellet crude enzymes. Substrate

CoA (␮M) AcCoA (␮M) PrpCoA (␮M) CoA balance

LA

800

CoA

Ac-CoA

Prp-CoA

100 90 80 70 60 50 40 30 20 10 0

LA, CoA (µM)

700 600 500 400 300 200 100 0 0

1

2 Time (hrs)

3

Ac-CoA, Prp-CoA (µM)

Fig. 3. The chromatogram of CoA, AcCoA, and PrpCoA in the LA bio-conversion assay at 0 and 3.5 h.

4

Fig. 4. The formation of acetyl-CoA and propionyl-CoA from LA by the sonication pellet solution from LA cultured cells.

710 ␮M LA was depleted from the reaction (Fig. 3). In 3.5 h, the CoA concentration decreased by 212 ␮M, while AcCoA and PrpCoA increased by 93 ␮M and 50 ␮M, respectively (Fig. 4). The LA-CoA was not detected in the assay solution. Generally speaking, two reactions should occur for LA conversion to AcCoA and PrpCoA: binding of CoA to LA and LA-CoA split into AcCoA and PrpCoA. The observation implies that the two reactions may be catalyzed by the same enzyme, with LA-CoA not being released until another CoA is supplied and LA-CoA is split. Coincidentally, the almost immediate conversion of LA to AcCoA and PrpCoA indicates a split reaction over a beta-oxidation reaction. Beta-oxidation of LA would require multiple reactions with co-factors to form AcCoA and PrpCoA. In fact, no oxidation–reduction cofactors were provided in the in vitro assay solution. .Interestingly, the conversion yield of LA into AcCoA and PrpCoA was only 10% and the molar ratio of AcCoA to PrpCoA formation is 1.9, which should be 1 if LA is split into AcCoA and PrpCoA. The crude enzymes might also convert PrpCoA as well as AcCoA in the assay solution. 3.4. Other reactions catalyzed by the crude enzymes AcCoA and PrpCoA were used as the initial substrates in reactions by the crude enzymes. The solution consisted of 25% v/v pellet

LA 56% (400 µM)

CoA 44% (310 µM)

LA-CoA

AcCoA only

PrpCoA only

AcCoA + PrpCoA

0h

1h

0h

1h

0h

1h

0±0 100 ± 1 0±0 100%

7±2 87 ± 2 3±1 97%

0±0 0±0 117 ± 3 100%

39 ± 2 22 ± 2 49 ± 1 94%

0±0 193 ± 4 146 ± 2 100%

105 ± 2 144 ± 2 86 ± 1 99%

slurry, 188 ␮M AcCoA, 140 ␮M PrpCoA, and ATP in a phosphate buffer. In 1 h, the AcCoA and PrpCoA concentrations declined by 49 ␮M and 60 ␮M, respectively, at rates of 49 ␮M/h and 60 ␮M/h (Table 3). These rates are used to estimate the LA conversion to AcCoA and PrpCoA later. Interestingly, when AcCoA or PrpCoA were used individually as the substrate, very little AcCoA was converted, while PrpCoA was, to a large extent, converted to CoA and AcCoA (Table 3). After the reaction, the CoA balance for the AcCoA assay, PrpCoA assay, and AcCoA plus PrpCoA assay were 97%, 94%, and 99%, respectively (Table 3). PrpCoA is converted into AcCoA, which explains why PrpCoA is converted at a higher rate than AcCoA when both substrates are present in the assay (Table 3). This also explains why the AcCoA formation is greater than PrpCoA formation in the LA in vitro bio-conversion (Fig. 4). Compared with AcCoA only, more AcCoA is converted when PrpCoA is present, suggesting that the crude enzymes condense AcCoA and PrpCoA into a product at a higher rate than condensation of two AcCoAs. AcCoA and PrpCoA are known to condense to form 3-ketovaleryl-CoA (Doi et al., 1987; Slater et al., 1998). The low activity of membrane-bound crude enzymes in condensation of AcCoA also implies that the first reaction of 3HB synthesis primarily occurs in the cytoplasm solution. 3.5. LA adsorption and overall balance on wet pellets LA is more hydrophobic than short organic acid such as acetic acid and propionic acid, so a large portion of LA removed from the assay solution might be adsorbed or absorbed by the wet pellet, but not converted. To quantify LA adsorption, assay solutions were prepared without CoA. The reaction solution consisted of 917 ␮M LA, 25% v/v pellet slurry, and either 800 ␮M or no ATP in a phosphate buffer. The LA concentration was monitored over a 2 h period. The LA concentration declined to the same level in the assay solutions with or without ATP. About 36% of LA was removed from the solution at a rate of 200 ␮M/h. The rates of LA adsorption and continuous conversion PrpCoA was used to estimate the conversion of LA to AcCoA and PrpCoA (Fig. 5). As mentioned above, the LA was adsorbed and converted in 2 h and the LA membrane adsorption rate was 200 ␮M/h. Consequently, 400 ␮M LA was adsorbed by the wet pellets membrane, which accounts for 56% of the LA removed from the solution (Fig. 4). This leaves 44% of LA for conversion to AcCoA and PrpCoA. In the conversion of LA into PrpCoA and AcCoA, 1 mole LA is split into 1 mole AcCoA and 1 mole PrpCoA (Fig. 5). Therefore, the molar formation and continuous conversion of PrpCoA is equal to the molar conversion of LA because PrpCoA was only formed from LA. This is

CoA 44% (310 µM)

AcCoA + PrpCoA + Unaccounted 37% (260 µM)

7% (50 µM)

LA (adsorption) Fig. 5. LA molar mass balance in the in vitro bio-conversion. The formation and continuous conversion is accounted for in the molar mass % PrpCoA.

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not the case for AcCoA because AcCoA was formed from both LA and PrpCoA (Table 3). Within the 3.5 h, 210 ␮M PrpCoA was converted to other products. Taking the free PrpCoA (50 ␮M) in the assay solution, 260 ␮M PrpCoA is actually formed. It accounted for 84% of the 310 ␮M LA not adsorbed by the wet pellets (Fig. 5). The remaining 7% LA was unaccounted. This mass balance analysis on PrpCoA indicates that acetyl-CoA and propionyl-CoA are the major intermediates in the initial metabolism of LA by C. necator (Fig. 1). 4. Conclusion In this study, we investigated the in vitro bioconversion of levulinic acid with Cupriavidus necator crude enzymes and, for the first time, showed direct evidence of acetyl-CoA and propionyl-CoA formation from LA. The results reveal the initial metabolic intermediates through which C. necator synthesizes a PHA copolymer consisting primarily of 3HB and 3HV monomers. The acyl-CoA synthetase responsible for LA activation was found to be membrane-associated and inducible. Also, it was demonstrated that the PHA composition could be altered by controlling the carbon nitrogen ratio when LA is the sole carbon source. Under appropriate conditions, LA can be utilized at a much higher rate than glucose and fructose, which is characteristic of other short chain organic acids. The results provide insight into manipulation of the 3HV content and therefore the material properties of PHA. Acknowledgements The authors appreciate the financial support from EGI Technologies LLC, Hawaii Natural Energy Institute, and the Office of Naval Research (N00014-09-1-0709). References Assary, R.S., Redfern, P.C., Hammond, J.R., Greeley, J., Curtiss, L.A., 2010. Computational studies of the thermochemistry for conversion of glucose to levulinic acid. The Journal of Physical Chemistry B 114, 9002–9009. Bengtsson, S., Pisco, A.R., Reis, M.A.M., Lemos, P.C., 2010. Production of polyhydroxyalkanoates from fermented sugar cane molasses by a mixed culture enriched in glycogen accumulating organisms. Journal of Biotechnology 145, 253–263. Bozell, J.J., Moens, L., Elliott, D.C., Wang, Y., Neuenscwander, G.G., Fitzpatrick, S.W., Bilski, R.J., Jarnefeld, J.L., 2000. Production of levulinic acid and use as a platform chemical for derived products. Resources, Conservation and Recycling 28, 227–239. Bramer, C.O., Steinbuchel, A., 2001. The methylcitric acid pathway in Ralstonia eutropha: new genes identified involved in propionate metabolism. Microbiology 147, 2203–2214. ´ M., Rha, C., Sinskey, A., 2011. Budde, C., Riedel, S., Hübner, F., Risch, S., Popovic, Growth and polyhydroxybutyrate production by Ralstonia eutropha in emulsified plant oil medium. Applied Microbiology and Biotechnology 89, 1611–1619. Chung, S.H., Choi, G.G., Kim, H.W., Rhee, Y.H., 2001. Effect of levulinic acid on the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Ralstonia eutropha KHB-8862. The Journal of Microbiology 39, 79–82. Doi, Y., Kunioka, M., Nakamura, Y., Soga, K., 1987. Biosynthesis of copolyesters in Alcaligenes eutrophus H16 from carbon-13 labeled acetate and propionate. Macromolecules 20, 2988–2991. Du, G., Chen, J., Yu, J., Lun, S., 2001. Kinetic studies on poly-3-hydroxybutyrate formation by Ralstonia eutropha in a two-stage continuous culture system. Process Biochemistry 37, 219–227. Gross, R.A., Kalra, B., 2002. Biodegradable polymers for the environment. Science 297, 803–807.

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