Biosynthesis of polyhydroxyalkanotes in wildtype yeasts

Biosynthesis of the biodegradable polymers polyhydroxyalkanotes (PHAs) are studied extensively in wild type and genetically modified prokaryotic cells, however the content and structure of PHA in wild type yeasts are not well documented. The purpose of this study was to screen forty yeast isolates collected from different Egyptian ecosystems for their ability to accumulate PHAs. Identification of the isolates and characterization of PHAs produced by the positive strains in the Nile-red staining assay was envisaged. One positive isolates which was identified using the API 20C yeast identification system as Rhodotorula minuta strain RY4 produced 2% of PHA in biomass, in glucose, oleic acid and tween 60 containing medium, over a growth period of 96 h. The nature of the PHA thus produced was analyzed by infrared spectroscopy and nuclear magnetic resonance (H and C NMR) and found to contain polyhydroxybutyrate and polyhydroxyvalerate. This study shows for the first time monomer detection by H and/or C NMR of PHA polymers synthesized in wild type yeasts. @ JASEM Plastic materials have become an integral part of contemporary life because they possess many desirable properties, including durability and resistance to degradation. Over the past 10-20 years, their widespread uses have been increasingly regarded as a source of environmental and waste management problems (Anderson and Dawes 1990). These problems have created much interest in the development and production of biodegradable plastics. Estimates of the current global market for these biodegradable plastics range up to 1.3 billion kg per year (Kalia et al. 2000, Song et al. 1999). Among the various biodegradable plastics available, there is a growing interest in the group of polyhydroxyalkanotes (PHAs). These are natural polymers produced by a variety of bacteria and they are 100% biodegradable (Doi and Abe 1990, Holmes 1988). PHAs have been shown to occur in over 90 genera of Gram-positive and Gram-negative bacteria species (Steinbuchel 1991). Bacteria synthesize and accumulate PHAs as carbon and energy storage materials or as a sink for redundant reducing power under the condition of limiting nutrients in the presence of excess carbon sources (Steinbuchel and Valentin. 1995). The major commercial drawback of the so-produced bacterial PHAs is their high production cost, making them substantially more expensive than synthetic plastics (Poirier et al. 1995). Therefore, looking for eukaryotic cell systems like yeast able to accumulate PHAs seems to be a beneficial alternative to the production of PHAs in bacteria. PHA-producing yeast may have potential commercial applications. First large scale fermentation and separations technology are well developed for this organism. Another consideration is that yeast may perform better than bacteria on cheap carbon sources such as molasses. Finally niche PHA-producing yeast might be found as part of a co-generation scheme in which cells are cultivated for ethanol as another primary product. Therefore, the purpose of this study was to screen and characterize wildtype yeast isolates for their ability to accumulate PHAs. MATERIALS AND METHODS Yeast isolates and screening for PHAs accumulation: Forty yeast strains were isolated from the following samples; molasses, foodstuffs, concentrated juices and soils. Samples were seeded by dilution in YEPD medium (containing 2% (wt/v) yeast extract, peptone and dextrose) and the strains were incubated at 30°C for 24h according to Sanni and lonner (Sanni and Lonner 1993). PHA accumulation was screened in yeast isolates on YEPD agar plates containing 25 μg Nile-Red (Sigma) per liter as described elsewhere (Spiekermann et al. 1999). Three lighted isolates were recorded positives; subsequently isolates were subjected for identification using the standard biochemical test API 20C Aux system (BioMerieux Vitek, Inc., Hazelwood, Mo.). PHA production and extraction: For PHA production, a stationary-phase culture grown in YEPD medium was harvested by centrifugation and cells were washed once in water and resuspended in mineral salts medium (MSM) (Schlegel et al. 1961) supplanted with 0.1% glucose, 0.5% of the detergent Tween 60 (Sigma, St. Louis, Mo.), and 0.1% fatty acid (oleic acid). Cells were grown at 30°C for an additional 1 to 6 days before harvest of the cells for PHB analysis. The pH of the growth media was 6.0. Biosynthesis of polyhydroxyalkanotes DESOUKY A.M. ABD-EL-HALEEM; SAHAR A. ZAKI; ASHRAF T. ABUELHAMD; AMRO AMARA; GADALLAH M.S. ABOELREESH 6 PHB extraction procedure was performed according to Findlay and White (1983). Lyophilized yeast cell sediments were placed in a Soxhlet extractor lined with glass wool and wrapped with a resistance strip heater. Enough chloroform to amply cover the sample was added, and the sample was sonicated for 10 min. The sample was extracted for overnight in a total of 125 ml of chloroform. The extraction thimble of the Soxhlet extractor was heated so that the chloroform present boiled, maintaining solubility of the polymers. The chloroform was recovered and removed in a rotary evaporator in vacuum. Subsequently, the polymer was redissolved in hot chloroform and PHA was recovered from the chloroform by nonsolvent precipitation and filtration. Methanol was used as the nonsolvent (4–6 volumes). To determine PHB yield, the correlation between production of PHB and dry cell weight was determined by Spearman's test (Conver 1971). In vitro PhaC enzyme assays: PhaC synthase activity was determined spectrophotometrically by monitoring the release of CoA at 412 nm (Valentin and Steinbüchel. 1994). The standard assay contained 1 mM DTNB dissolved in Tris/HCl buffer 50 mM Ph 7.5), 20 mM MgCL2 and 0.75, 1.25, 2.5, 3.7 and 5 μM of DL-β-Hydroxybutyryl CoA respectively in Tris/HCl buffer, (150 mM, pH 7.5) at 37C. The kinetic parameters for the release of CoA were determined using Lineweaver-Burk plots. In each assay, blank tubes (without enzyme) were measured to correct for spontaneous hydrolysis of substrate. One U of enzyme is defined as the amount which produced 1 μmol product per minute under assay conditions. Spectroscopic analysis of PHAs: The Infra Red (IR) spectroscopic analysis was taken with IR (Bruker). The NMR spectra were recorded on a JOEL ECA 500 spectrometer. The 500 MHz H-NMR spectra were recorded from a CDCl3 solution of the PHA (30 mg/ml) at 20°C, 1.30809856 s acquisition times and 12.5250501 KHz spectral width. The C-NMR spectra were recorded from a CDCl3 solution of the samples using H-decoupling, 0.69206016 s acquisition time and 47.348485 KHz spectral width. H-NMR and C-NMR chemical shifts were referred to CHCl3 and CDCl3 (c= 7.24 ppm and c = 77.0 ppm, respectively). RESULTS AND DISCUSSION Yeast isolates and screening for PHA accumulation: It is established that a considerable number of both Gram-negative and Gram-positive bacteria are able to accumulate PHA and can grow on different carbon sources. According to the number of carbon atoms in the monomeric units of the PHA, PHA-producing bacteria can be generally classified into two groups (Steinbuchel. and Valentin 1995). The first class of bacteria, including Ralstonia eutropha, produces short chain length PHA with monomer units ranged from C3 to C5, while the other class, including Pseudomonas oleovorans, produces medium chain length PHB with monomer units from C6 to C14 (Anderson and Dawes 1990). In contrast, PHA in wild type yeast has received brief mention in the literature. Nuti and Lepidi (1974) and Safak et al. (2002) reported the presence of PHA in wild type yeasts, this is not in agreement with our experience, and it appears that the methods employed by theses authors could not distinguish between PHB and monomeric 3HB moieties. Oligomeric PHB of 120-200 subunits has been reported in yeast at about 0.0002% of dry weight (Seebach et al. 1994), and is believed to function not as a storage material but as a membrane transport channel in complex with calcium ion and polyphosphate in all living cells. In the present work, screening among yeast isolates for presence/absence of PHA accumulation was performed using the Nile-red staining assay. Under the UV transilluminor, three isolates (Y1, Y4 and Y8) exhibited a strong fluorescence in comparing to other yeast isolates. This finding is considered an evidence for the specificity of the Nile-red staining approach to screen new microbial isolates for PHAs production. It is known that the Nile-red stain emitted strongly positive red fluorescence signals only with hydrophobic compounds like PHAs and lipids. Nilered intended to show any lipid particles inside the cells and it did not react with any tissue constituent except by solution and could be detected by fluorescence spectroscopy or flow cytometery (Gorenflo et al. 1999, Spiekermann et al. 1999). Subsequently, the three positive isolates were subjected for biochemical identification using API 20C Aux system. The results showed correct identifications of the isolates without the need to supplement extra tests. The isolates identified as Kebeckera spp. (K-Y1), Rhodotorula minuta (R-Y4) and Candida norvegensis (C-Y8), respectively. In vivo PhaC enzyme assays and PHA production: Afterward, biochemical identified isolates K-Y1, RY4 and C-Y8 were subjected for In vivo PhaC enzyme assay using crude extracts of each strain. The apparent Km and Vmax for the strains is presented in Table 1. Biosynthesis of polyhydroxyalkanotes DESOUKY A.M. ABD-EL-HALEEM; SAHAR A. ZAKI; ASHRAF T. ABUELHAMD; AMRO AMARA; GADALLAH M.S. ABOELREESH 7 Table 1: PhaC synthase activity in strains KY1, RY8 and CY4 Strain Km Vmax R-Y4 63.99 329.8

ABSTRACT: Biosynthesis of the biodegradable polymers polyhydroxyalkanotes (PHAs) are studied extensively in wild type and genetically modified prokaryotic cells, however the content and structure of PHA in wild type yeasts are not well documented.The purpose of this study was to screen forty yeast isolates collected from different Egyptian ecosystems for their ability to accumulate PHAs.Identification of the isolates and characterization of PHAs produced by the positive strains in the Nile-red staining assay was envisaged.One positive isolates which was identified using the API 20C yeast identification system as Rhodotorula minuta strain RY4 produced 2% of PHA in biomass, in glucose, oleic acid and tween 60 containing medium, over a growth period of 96 h.The nature of the PHA thus produced was analyzed by infrared spectroscopy and nuclear magnetic resonance (1 H and 13 C NMR) and found to contain polyhydroxybutyrate and polyhydroxyvalerate.This study shows for the first time monomer detection by 1 H and/or 13 C NMR of PHA polymers synthesized in wild type yeasts.@ JASEM Plastic materials have become an integral part of contemporary life because they possess many desirable properties, including durability and resistance to degradation.Over the past 10-20 years, their widespread uses have been increasingly regarded as a source of environmental and waste management problems (Anderson and Dawes 1990).These problems have created much interest in the development and production of biodegradable plastics.Estimates of the current global market for these biodegradable plastics range up to 1.3 billion kg per year (Kalia et al. 2000, Song et al. 1999).Among the various biodegradable plastics available, there is a growing interest in the group of polyhydroxyalkanotes (PHAs).These are natural polymers produced by a variety of bacteria and they are 100% biodegradable (Doi andAbe 1990, Holmes 1988).PHAs have been shown to occur in over 90 genera of Gram-positive and Gram-negative bacteria species (Steinbuchel 1991).Bacteria synthesize and accumulate PHAs as carbon and energy storage materials or as a sink for redundant reducing power under the condition of limiting nutrients in the presence of excess carbon sources (Steinbuchel and Valentin. 1995).
The major commercial drawback of the so-produced bacterial PHAs is their high production cost, making them substantially more expensive than synthetic plastics (Poirier et al. 1995).Therefore, looking for eukaryotic cell systems like yeast able to accumulate PHAs seems to be a beneficial alternative to the production of PHAs in bacteria.PHA-producing yeast may have potential commercial applications.First large scale fermentation and separations technology are well developed for this organism.Another consideration is that yeast may perform better than bacteria on cheap carbon sources such as molasses.Finally niche PHA-producing yeast might be found as part of a co-generation scheme in which cells are cultivated for ethanol as another primary product.Therefore, the purpose of this study was to screen and characterize wildtype yeast isolates for their ability to accumulate PHAs.

Yeast isolates and screening for PHAs accumulation:
Forty yeast strains were isolated from the following samples; molasses, foodstuffs, concentrated juices and soils.Samples were seeded by dilution in YEPD medium (containing 2% (wt/v) yeast extract, peptone and dextrose) and the strains were incubated at 30°C for 24h according to Sanni and lonner (Sanni and Lonner 1993).PHA accumulation was screened in yeast isolates on YEPD agar plates containing 25 µg Nile-Red (Sigma) per liter as described elsewhere (Spiekermann et al. 1999).Three lighted isolates were recorded positives; subsequently isolates were subjected for identification using the standard biochemical test API 20C Aux system (BioMerieux Vitek, Inc., Hazelwood, Mo.).

PHA production and extraction:
For PHA production, a stationary-phase culture grown in YEPD medium was harvested by centrifugation and cells were washed once in water and resuspended in mineral salts medium (MSM) (Schlegel et al. 1961) supplanted with 0.1% glucose, 0.5% of the detergent Tween 60 (Sigma, St. Louis, Mo.), and 0.1% fatty acid (oleic acid).Cells were grown at 30°C for an additional 1 to 6 days before harvest of the cells for PHB analysis.The pH of the growth media was 6.0.

6
PHB extraction procedure was performed according to Findlay and White (1983).Lyophilized yeast cell sediments were placed in a Soxhlet extractor lined with glass wool and wrapped with a resistance strip heater.Enough chloroform to amply cover the sample was added, and the sample was sonicated for 10 min.The sample was extracted for overnight in a total of 125 ml of chloroform.The extraction thimble of the Soxhlet extractor was heated so that the chloroform present boiled, maintaining solubility of the polymers.The chloroform was recovered and removed in a rotary evaporator in vacuum.Subsequently, the polymer was redissolved in hot chloroform and PHA was recovered from the chloroform by nonsolvent precipitation and filtration.Methanol was used as the nonsolvent (4-6 volumes).To determine PHB yield, the correlation between production of PHB and dry cell weight was determined by Spearman's test (Conver 1971).
In vitro PhaC enzyme assays: PhaC synthase activity was determined spectrophotometrically by monitoring the release of CoA at 412 nm (Valentin and Steinbüchel. 1994).The standard assay contained 1 mM DTNB dissolved in Tris/HCl buffer 50 mM Ph 7.5), 20 mM MgCL 2 and 0.75, 1.25, 2.5, 3.7 and 5 µM of DL-β-Hydroxybutyryl CoA respectively in Tris/HCl buffer, (150 mM, pH 7.5) at 37 o C. The kinetic parameters for the release of CoA were determined using Lineweaver-Burk plots.In each assay, blank tubes (without enzyme) were measured to correct for spontaneous hydrolysis of substrate.One U of enzyme is defined as the amount which produced 1 µmol product per minute under assay conditions.

Spectroscopic analysis of PHAs:
The Infra Red (IR) spectroscopic analysis was taken with IR (Bruker).The NMR spectra were recorded on a JOEL ECA 500 spectrometer.The 500 MHz 1 H-NMR spectra were recorded from a CDCl 3 solution of the PHA (30 mg/ml) at 20°C, 1.30809856 s acquisition times and 12.5250501 KHz spectral width.The 13 C-NMR spectra were recorded from a CDCl 3 solution of the samples using 1 H-decoupling, 0.69206016 s acquisition time and 47.348485 KHz spectral width. 1 H-NMR and 13 C-NMR chemical shifts were referred to CHCl 3 and CDCl 3 (c= 7.24 ppm and c = 77.0ppm, respectively).

RESULTS AND DISCUSSION
Yeast isolates and screening for PHA accumulation: It is established that a considerable number of both Gram-negative and Gram-positive bacteria are able to accumulate PHA and can grow on different carbon sources.According to the number of carbon atoms in the monomeric units of the PHA, PHA-producing bacteria can be generally classified into two groups (Steinbuchel. and Valentin 1995).The first class of bacteria, including Ralstonia eutropha, produces short chain length PHA with monomer units ranged from C3 to C5, while the other class, including Pseudomonas oleovorans, produces medium chain length PHB with monomer units from C6 to C14 (Anderson and Dawes 1990).
In contrast, PHA in wild type yeast has received brief mention in the literature.Nuti and Lepidi (1974) and Safak et al. (2002) reported the presence of PHA in wild type yeasts, this is not in agreement with our experience, and it appears that the methods employed by theses authors could not distinguish between PHB and monomeric 3HB moieties.Oligomeric PHB of 120-200 subunits has been reported in yeast at about 0.0002% of dry weight (Seebach et al. 1994), and is believed to function not as a storage material but as a membrane transport channel in complex with calcium ion and polyphosphate in all living cells.
In the present work, screening among yeast isolates for presence/absence of PHA accumulation was performed using the Nile-red staining assay.Under the UV transilluminor, three isolates (Y1, Y4 and Y8) exhibited a strong fluorescence in comparing to other yeast isolates.This finding is considered an evidence for the specificity of the Nile-red staining approach to screen new microbial isolates for PHAs production.It is known that the Nile-red stain emitted strongly positive red fluorescence signals only with hydrophobic compounds like PHAs and lipids.Nilered intended to show any lipid particles inside the cells and it did not react with any tissue constituent except by solution and could be detected by fluorescence spectroscopy or flow cytometery (Gorenflo et al. 1999, Spiekermann et al. 1999).
Subsequently, the three positive isolates were subjected for biochemical identification using API 20C Aux system.The results showed correct identifications of the isolates without the need to supplement extra tests.The isolates identified as Kebeckera spp.(K-Y1), Rhodotorula minuta (R-Y4) and Candida norvegensis (C-Y8), respectively.In vivo PhaC enzyme assays and PHA production: Afterward, biochemical identified isolates K-Y1, R-Y4 and C-Y8 were subjected for In vivo PhaC enzyme assay using crude extracts of each strain.The apparent K m and V max for the strains is presented in Table 1.The results revealed that PhaC synthase is highly expressed in strain R-Y4 in compare to the other two isolates.Kinetic parameters for release of CoA at different substrate condition were determined as in Figure 1.It is known that PHA synthase requires a phosphopantheine prosthetic group for activity (Gerngross et al. 1994).Thus, the results obtained indicated that RY4 cells could perform this process.

Biosynthesis of polyhydroxyalkanotes DESOUKY A.M. ABD-EL-HALEEM
To confirm these results, cells of each strain were grown in 250 ml Erlenmeyer flasks containing 50 ml MSM medium at 30°C on a rotary shaker at 200rev/min.All grown flasks were exposed to the same conditions, and three separate flasks were inoculated from the same culture in each case.Data from shack flasks indicated that strains K-Y1 and C-Y8 did not contain measurable PHA.In contrast, strain R-Y4 that grown under the same conditions contained 2% PHA of the cell dry weight; therefore, PHA produced by Rhodotorula minuta strain R-Y4 was subjected for further spectroscopic analysis.

Spectroscopic analysis of PHAs:
The determination of the monomer composition of Poly(3HB) and Poly(3HB-co-3HV) is relatively simple, and can easily be acquired by means of GC, GC-MS, and HPLC.However, with these methods it is not possible to detect the presence of two or more distinctive polymers, because the polymers are hydrolyzed before analysis (Anderson andDawes 1990, Doi 1995).It is, however, possible to determine this by means of NMR: the complexity of the carbonyl signals in the 13 C-NMR spectrum can be used to determine whether PHA consists of homopolymers or a copolymer.In addition, NMR has also been used in the determination of the chain dynamics, the crystallinity and the pathways involved in PHA synthesis (Doi et al. 1985, Anderson and Dawes 1990, Doi 1995, Williams. and Fleming 1996).Therefore, the extracted PHA-like polymers from strain RY4 were subjected for IR and NMR ( 1 H and 13 C) spectroscopic analysis.IR spectroscopy showed intense absorptions typical to PHA at 1728-1740 cm -1 and at 1280 cm -1 corresponding to C=O and C-O stretching groups, respectively (Fig. 2).IR analysis indicated also that the polymer is mostly polyhydroxybutyrate (PHB) with polyhydroxyvalerate (PHV).
As shown in Figure 3, both 1 H and 13 C Nuclear magnetic resonance spectra confirmed poly(3HB-co-3HV-co-5HV) copolymer production in strain R-Y4.The spectra demonstrated the presence of signals indicating 3-PHB, 3-HV and 5-HV side chains.Out of the above results, it appears that yeast isolate R-Y4 produce a mixed polymer and that their yields and types may possibly be varied using various other substrates and cultural conditions.Thus, these results attempted for the first time monomer detection by spectroscopic analysis of PHA polymers synthesized in wild type yeasts.PHAs in wild type yeast were originally reported by Reusch (1989) followed by Seebach et al. (1992) and Leaf et al. (1996).Theses authors have not attempted monomer detection by 1 H and/or 13 C NMR, though such species may be relatively insoluble in chloroform.However, Reusch (1989) suggests that native low molecular mass PHB may be complexes with various lipids or proteins, making its solubility behavior unpredictable.

Fig 3 :
Fig 3: 1 H NMR (A) and 13 C NMR (B) spectra analysis of the polymer produced by the wildtype yeast strain RY4