Patent Application
20230235370
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The present invention relates to a method for biosynthesis polyhydroxybutyrate, and more particularly to a method for biosynthesis polyhydroxybutyrate by a yeast transformant.
Unsustainability degradation of petroleum plastic threatens the stability of ecosystem on land and marine. Increasing plastic consumption in the world encourages the transformation of raw materials plastic to become biodegradable plastic. The most common type of biodegradable polymers of plastics is polyhydroxyalkanoates (PHAs). Polyhydroxybutyrate (PHB) is one of the most studied PHAs. Both of them were the best examples of biopolymer energy storage and having similar biosynthesis properties to synthetic biopolymers.
Since, PHB was discovered by Lemoigne in 1926 in Bacillus megaterium as cytoplasmic inclusion. It has attracted much industrial attention as a biodegradable and biocompatible thermoplastic. Moreover, it can be generated using renewable and non-renewable resources to obtain biodegradable polymers. PHB is the most important example of a biocompatible and bio-degradable hydrophobic polymer at high melting temperatures and crystallinity.
Some tremendous potential applications of PHB are used in biomedical, pharmacological, waste management, veterinary, agricultural, and novel biofuel. Some microorganisms successfully produce PHB by natural products from wild-type or recombinant organisms, such as bacterial, cyanobacteria and green algae. In controlled aerobic and anaerobic cultivation, about 300 species of bacteria and archaea have been discovered to produce PHA and PHB.
Some technologies have been improved PHB content from microbial cell factories from both wildtype and recombinant, such as Cupriavidus necator, Bacillus sp., Synechocystis sp., Recombinant E. coli and Halomonas sp. Nevertheless, the cost production in bacterial technologies was very expensive and involving many complicated methods.
The information disclosed in this “BACKGROUND OF THE INVENTION” section is only for enhancement understanding of the background of the invention and therefore it may contain information that does not form the prior art that is already known to a person of ordinary skill in the art. Furthermore, the information disclosed in this “BACKGROUND OF THE INVENTION” section does not mean that one or more problems to be solved by one or more embodiments of the invention were acknowledged by a person of ordinary skill in the art.
The present invention provides a method for biosynthesis polyhydroxybutyrate by a yeast transformant, which provides a way of cheaper, faster and flexibility in biotechnology metabolism to improve PHB production.
Other advantages and objects of the invention may be further illustrated by the technical features broadly embodied and described as follows.
In order to achieve one or a portion of or all of the objects or other objects, a method for biosynthesis polyhydroxybutyrate by a yeast transformant provided in an embodiment of the invention includes the following steps: (1) transforming a polyhydroxybutyrate biosynthesis related gene into an oleaginous yeast to obtain an yeast transformant. (2) screening the yeast transformant. (3) cultivating the yeast transformant to obtain the polyhydroxybutyrate.
In one embodiment of the invention, the oleaginous yeast is Rhodotorula glutinis.
In one embodiment of the invention, a Rhodotorula glutinis strain is BCRC 22360.
In one embodiment of the invention, the polyhydroxybutyrate biosynthesis related gene comprises at least one of PhaA gene, PhaB gene or PhaC gene.
In one embodiment of the invention, the polyhydroxybutyrate biosynthesis related gene is PhaA gene, PhaB gene and PhaC gene.
In one embodiment of the invention, the polyhydroxybutyrate biosynthesis related gene comprises at least one of a first gene having at least 90% sequence identity with a sequence of PhaA gene, a second gene having at least 90% sequence identity with a sequence of PhaB gene or a third gene having at least 90% sequence identity with a sequence of PhaC gene.
In one embodiment of the invention, the method of transforming a polyhydroxybutyrate biosynthesis related gene into an oleaginous yeast to obtain the yeast transformant comprises inserting the polyhydroxybutyrate biosynthesis related gene into a linearized plasmid, and transforming the linearized plasmid into the oleaginous yeast.
In one embodiment of the invention, a DNA of the yeast transformant has the polyhydroxybutyrate biosynthesis related gene.
In one embodiment of the invention, the method of screening the yeast transformant comprises screening with a primer pair, one of the primer of the primer pair has a sequence as SEQ ID NO: 4, and the other primer has a sequence as SEQ ID NO: 5.
In one embodiment of the invention, the method of screening the yeast transformant comprises screening with a primer pair, one of the primer of the primer pair has a sequence as SEQ ID NO: 6, and the other primer has a sequence as SEQ ID NO: 7.
In one embodiment of the invention, the method of screening the yeast transformant comprises screening with a primer pair, one of the primer of the primer pair has a sequence as SEQ ID NO: 8, and the other primer has a sequence as SEQ ID NO: 9.
In one embodiment of the invention, the method of cultivating the yeast transformant to obtain the polyhydroxybutyrate comprises cultivating under aerobic condition.
In one embodiment of the invention, the method of cultivating the yeast transformant to obtain the polyhydroxybutyrate comprises cultivating the yeast transformant with oil.
In one embodiment of the invention, a yield of polyhydroxybutyrate increase when cultivating the yeast transformant with glucose.
In one embodiment of the invention, a production capacity of polyhydroxybutyrate per cell increase when cultivating the yeast transformant with glycerol or oil.
In one embodiment of the invention, the method of cultivating the yeast transformant to obtain the polyhydroxybutyrate comprises cultivating the yeast transformant in seawater.
Since yeast has flexibility in physiology, is novel in biotechnology metabolism and larger than bacteria, it provides a cheaper and faster way to improve PHB production. In the method of the embodiment of the invention, an oleaginous yeast is used in PHB production, which belongs to a type of yeast that has ability to provide lipids as a substrate for PHB synthesis in metabolic plasticity. It is demonstrated that an increasing of PHB production in oleaginous yeast by using optimation some carbon sources. Therefore, comparing with the method of using bacteria in the conventional technology, the oleaginous yeast of the embodiment of the invention provides a way of cheaper, faster and flexibility in biotechnology metabolism, and may further increase the PHB production.
Other objectives, features and advantages of The invention will be further understood from the further technological features disclosed by the embodiments of The invention wherein there are shown and described preferred embodiments of this invention, simply by way of illustration of modes best suited to carry out the invention.
The present invention will become more readily apparent to those ordinarily skilled in the art after reviewing the following detailed description and accompanying drawings, in which:
The present invention will now be described more specifically with reference to the following embodiments. It is to be noted that the following descriptions of preferred embodiments of this invention are presented herein for purpose of illustration and description only. It is not intended to be exhaustive or to be limited to the precise form disclosed.
Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those with ordinary knowledge in the technical field to which the present invention belongs.
The article “a”, “an” and “the” used here refers to one or more (ie, at least one) grammatical acceptors of the article.
In the embodiment, the basic growth media for plasmid transformant contained 25 g/L LB broth with antibiotics (for example, Ampicillin). Rhodotorula glutinis are cultured in YP2D at 24° C. at 250 rpm several times, as follows: 1% yeast extract, 1% peptone and 20% glucose. The medium for transformant Rhodotorula glutinis is 1% yeast extract, 2% peptone, 20% galactose, and antibiotics (G418). The medium for seed screening and seed culture of transformants Rhodotorula glutinis are provided on a defined medium (21 g/L YM broth) in a-250 flasks using 50 ml as working volume. The fermentation medium (per liter) (in shaking flask cultivation and aerobic batch bioreactor) is comprised of 2 g of yeast extract, 2 g of (NH4)2SO4, 1 g of KH2PO4, 0.5 g of MgSO4·7H2O, 0.1 g of CaCl2 and 0.1 g of NaCl, which is expressed as the standard medium in the embodiment. Carbon sources mix in fermentation media when in shaking flask cultivation comprised of glucose (30 g/L; 45 g/L and 60 g/L), galactose (30 g/L), crude glycerol (30 g/L) from biodiesel industry and WCO (30 g/L). Carbon sources mix in aerobic batch fermentation media using glucose and crude glycerol in similar concentrations (30 g/L). In the crude glycerol experiment, carbon sources mix in aerobic batch fermentation media using glucose (30 g/L) and crude glycerol (30 g/L; 60 g/L and 90 g/L). In the NaCl experiment, the fermentation medium (per liter) in the aerobic batch bioreactor was comprised of 2 g of yeast extract, 2 g of (NH4)2SO4, 1 g of KH2PO4, 0.5 g of MgSO4·7H2O, 0.1 g of CaCl2 and (0.1 g as control; 1 g; 2 g; 3 g; 4 g and 5 g) of NaCl. Carbon sources mixed in aerobic batch fermentation media using glucose and crude glycerol in similar concentrations (30 g/L).
The biosynthesis pathway of PHB involves three enzymes and there in order essential reactions including acetyl-CoA C-acetyltransferase, NADPH-dependent acetoacetyl-CoA reductase, and PHA synthase as shown in
In the embodiment, the polyhydroxybutyrate biosynthesis related gene comprises at least one of PhaA gene (GenBank: KP681582, SEQ ID NO: 1), PhaB gene (GenBank: KP681583, SEQ ID NO: 2) or PhaC gene (GenBank: KP681584, SEQ ID NO: 3). That is, the PHB biosynthesis related gene of the invention may be PhaA gene and genes other than PhaB gene and PhaC gene, PhaB gene and genes other than PhaA gene and PhaC gene, PhaC gene and genes other than PhaA gene and PhaB gene, PhaA gene and PhaB gene and genes other than PhaC gene, PhaB gene and PhaC gene and genes other than PhaA gene, PhaA gene and PhaC gene and genes other than PhaB gene, and PhaA gene, PhaB gene and PhaC gene. In another preferred embodiment, the PHB biosynthesis related gene is PhaA gene, PhaB gene and PhaC gene.
In the biosynthesis pathway of PHB, the acetyl-CoA C-acetyltransferase is encoded by PhaA gene or a first gene having at least 90% sequence identity with a sequence of PhaA gene. The NADPH-dependent acetoacetyl-CoA reductase is encoded by PhaB gene of a second gene having at least 90% sequence identity with a sequence of PhaB gene. The PHA synthase is encoded by PhaC gene or a third gene having at least 90% sequence identity with a sequence of PhaC gene. Therefore, the PHB biosynthesis related gene may also be regarded as comprising at least one of the first gene, the second gene or the third gene.
In the embodiment, the PHB biosynthesis related gene is PhaA gene, PhaB gene and PhaC gene is taken as an example, but not limited thereto. The PhaA gene, the PhaB gene, and the PhaC gene from Cupriavidus necator were codon-optimized towards S. cerevisiae and synthesized (TWIST BIOSCIENCE, USA). Sequence design & optimization for three PHB pathway constructions (pKLAC2-na-PhaA, pKLAC2-na-PhaB, pKLAC2-na-PhaC) are inserted in the plasmid (pKLAC2-na-).
To transform the PHB biosynthesis related gene into the oleaginous yeast Rhodotorula glutinis, the PHB biosynthesis related gene is first inserted into a linearized plasmid pKLAC2-na- (SacII digestion). Then, Rhodotorula glutinis strain BCRC 22360 are transformed with the linearized plasmid pKLAC2-na- according to Pi H W, Anandharaj M, Kao Y Y, et al. (2018) Engineering the oleaginous red yeast Rhodotorula glutinis for simultaneous β-carotene and cellulase production. Sci Rep 8:2-11. After the transformation, the linearized plasmid is pasted to a DNA of the oleaginous yeast Rhodotorula glutinis without being existed independently in the body of the oleaginous yeast, that is, a DNA of the yeast transformant has the PHB biosynthesis related gene. The advantage of pasting the linearized plasmid to the DNA of the oleaginous yeast Rhodotorula glutinis is when cultivating the yeast transformant, the PHB biosynthesis related gene may follow the DNA replication of the oleaginous yeast more stably and reduce the occurrence of errors.
The Rhodotorula glutinis are cultivated in 5 ml YP2D from the single colony at 30° C. at 250 rpm and then 0.2 OD cells are sub-cultured into 50 ml YP2D until reaching 0.6˜1.4 OD. Cells are harvested at 3000 rpm for 3 min (4° C.) and washed with 5 ml ice-cold distilled H2O two times. Then, cells are resuspended in cooled TMLSD buffer (10 mM Tris-HCl buffer (pH 8.0) containing 2 mM MgCl2, 100 mM lithium acetate, 270 mM sucrose, 10 mM dithiothreitol) and incubated at 24° C. for 1 h with shaking for 250 rpm. After the incubation, cells are harvested as described above and first washed using with 5 ml ice-cold distilled H2O and second with 1 ml TMS buffer (10 mM Tris-HCl buffer (pH 8.0) containing 2 mM MgCl2, 270 mM sucrose). Finally, competent cells are resuspended in 250 μL TMS buffer and prepared for electroporation analysis.
Electroporation procedure referred to Pi H W, Anandharaj M, Kao Y Y, et al. (2018) Engineering the oleaginous red yeast Rhodotorula glutinis for simultaneous β-carotene and cellulase production. Sci Rep 8:2-11, by mixing the 10-15 μl DNA with 40 μl competent cells and kept on ice for 15 min. Then cells are transferred to the ice-cold aluminum cuvette (0.2 cm gap Gene Pulser/MicroPulser Electroporation Cuvettes, Bio-Rad, USA) and electroporation is performed (1000 V, 400 Ω, and 25 μF capacitance), using a MicroPulser (Bio-Rad Laboratories, USA). After electroporation, cells are resuspended in 1 mL ice-cold YP2D and transferred into new tubes on ice for 15 min, and then incubated at 30° C. for 12 h. The cell suspension is spread onto YP2D plates containing selection markers (G418) and incubated at 30° C. for 4-5 days.
Next, Step S102: screening the yeast transformant. The successfully engineered colonies are validated by PCR amplification of the integrated gene, using extracted genomic DNA as a template. More than 380 single colonies are confirmed by PCR using gene-specific primer pairs. For example, screening the PhaA gene with a primer pair, one of the forward primer of the primer pair has a sequence as SEQ ID NO: 4, and the other reverse primer has a sequence as SEQ ID NO: 5, but not limited thereto. For example, screening the PhaB gene with a primer pair, one of the forward primer of the primer pair has a sequence as SEQ ID NO: 6, and the other reverse primer has a sequence as SEQ ID NO: 7, but not limited thereto. For example, screening the PhaC gene with a primer pair, one of the forward primer of the primer pair has a sequence as SEQ ID NO: 8, and the other reverse primer has a sequence as SEQ ID NO: 9, but not limited thereto.
Western Blot Analysis
Yeast protein extraction was determined by standard method procedure from a company (Bio Basic). Briefly, ten candidate transformant Rhodotorula glutinis are cultivated in YM broth at 24° C. until OD600 of yeast cell density reaches ˜1.0 or so. The culture cell is centrifugated at 8000 rpm for one minute, then discard supernatant and washed with sterilized water and kept yeast paste. Subsequently, yeast paster is added 500 μl isosmotic buffer, 5 μl Snailase buffer and 1 μl mercaptoethanol per 50 mg wet yeast paste. Then mixs gently with up and down solution and yeast paste to fully re-suspend yeast cells. Incubate at 37° C. for 2 hours and occasionally invert the tube for several times. Subsequently, centrifugate at 5000 rpm for one minute and discard supernatant and kept precipitates. Precipitates are washed with 500 μl isosmotic buffer, then centrifugate at 5000 rpm for one minute, and kept protoplasmic precipitates. This step could repeat once more. In the last step, 500 μl hypoosmotic buffer and 5 μl PMSF solution are added in protoplasmic precipitates and then vortex to fully re-suspend protoplasmic. The lysed solutions are kept at −20° C. for 30 minutes, and then thaw at room temperature. This step could repeat once more and store at −20° C. for further western blot.
Western blot analysis is performed using Lee M H, Hsu T L, Lin J J, et al. (2020) Constructing a human complex type N-linked glycosylation pathway in Kluyveromyces marxianus. PLoS One 15:1-16. Firstly, for measuring total protein concentration, the lysate of protein is mixed with bicinchoninic acid from Pierce™ BCA Protein Assay Kit. Then, the concentration of protein is measured using Sunrise, Tecan apparatus completely with Elisa reader. Subsequently, 10-50 μg of lysate is loaded to Tris-glycine SDS-Polyacrylamide Gel Electrophoresis using a combination of the Resolving gels (bottom gel) and 5% of the Stacking gels (upper gel). The Resolving gels are comprised of 30% Acrylamide mix, 1.5M Tris (pH 8.8), 10% SDS, 10% Ammonium Persulfate, TEMED and ddH2O. The Stacking gels are comprised of 30% Acrylamide mix, 1.0M Tris (pH 6.8), 10% SDS, 10% Ammonium persulfate, TEMED and ddH2O. The electrophoresis runs in 1X running buffer in 70V at room temperature for 30 min and continued in 120V at the room temperature for 35 min. After electrophoresis, the SDS-PAGE is transferred to the Polyscreen® PVDF transfer membrane (NEF1002001PK) at 120V and 4° C. for 100 min in 1X transfer buffer. The membrane is blocked in 5% skim milk with shaking at 70 rpm for 1 hour at room temperature. Then, the membrane is washed with PBST (Phosphate Buffered Saline with 0.1% Tween-20) for three-time with shaking 100 rpm for 5 min at room temperature. HRP-conjugated Mouse anti His-Tag mAB as the primary and secondary antibody is diluted by fresh 0.1% PBST and added 1000-fold to the membrane for 1 hour with shaking 70 rpm at room temperature. Then, the membrane is washed with 0.1% PBST for one time with shaking 100 rpm for 15 min and continued to second and third washed in same condition for each 5 min at room temperature. The last, membrane is imaged for detecting protein on a Multige121 imaging system.
Next, Step S103: cultivating the yeast transformant to obtain the PHB and performing analysis.
Screening PHB production from cultivation selected engineered Rhodotorula glutinis
Screening of PHB production is performed in shaking flasks. 1 ml of nine selected candidate strains of transformant Rhodotorula glutinis and wild-type (WT) are inoculated into a 250-ml flask containing the seed medium of a 50-mL working volume, pH 5.5. It is shaken at 150 rpm at 24° C. for 48h under aerobic conditions. Subsequently, 5 ml of seed medium (10%) are transferred into 250-ml flasks containing designated amounts of fermentation medium of a 50-ml working volume, pH 5.5, as described previously. In this part, glucose 30 g/L and galactose 30 g/L are used as carbon sources. It is cultured at 24° C. and shaken at 150 rpm under aerobic conditions. All shaking culture conditions are provided in triplicate to express the values as the mean±standard deviation.
Shaking flask cultivation of specific Rhodotorula glutinis strain #100-29
1 ml seed of specific Rhodotorula glutinis strain #100-29 is inoculated in a similar condition above. It is shaken at 150 rpm at 24° C. for 48h under aerobic conditions. Subsequently, it is cultured in a medium appropriate and condition for similar above. In this part, glucose 30 g/L, 45 g/L and 60 g/L and galactose, crude glycerol, oil each 30 g/L are used as carbon sources. The oil may be general oil such as animal oil, vegetable oil etc., or may be waste oil, the invention does not particularly limit the type of oil. All shaking culture conditions are provided in triplicate to express the values as the mean±standard deviation. In the NaCl experiment, 5 ml of seed culture was transferred into a 250-L flask shaking of a 50-L working volume of fermentation medium, pH 5.5. It was shaken at 150 rpm at 24° C. for 48h under aerobic conditions. In this part, carbon sources mixed in aerobic batch fermentation media using glucose (30 g/L) and crude glycerol in similar concentrations (30 g/L).
Aerobic batch bioreactor operation of specific Rhodotorula glutinis strain #100-29
First, 300 ml of seed culture is transferred into a 5-L batch bioreactor (model BTF-A, Biotop Ltd., Taiwan) of a 3-L working volume of fermentation medium (as described previously). The samples are carried out for interval times: 12h, 24 h, 36h, 48, 72h, 96h, 120h and 144h. The pH level is automatically maintained at 5.5 by automatically feeding 0.8M NaOH solution and 0.8M HCl into the medium. The fermentor is operated at 24° C. with dissolved oxygen controlled at a 30±10% saturation level. The agitation during the process is limited to a range of 200-400 rpm (300 rpm) with a 1.5 vvm aeration rate.
Biomass Measurement and Glucose Analysis
Biomass concentration is measured using an infrared balance (Denver Instrument, IR 35) and the procedure is prepared according to Yen H W, Hu C Y, Liang W S (2019) A cost efficient way to obtain lipid accumulation in the oleaginous yeast Rhodotorula glutinis using supplemental waste cooking oils (WCO). J Taiwan Inst Chem Eng 97:80-87. The concentration of carbon sources residues is quantified in the culture supernatant using an Ultimate 3000 HPLC refraction index (RI) detector (Agilent series 1100, Agilent Technologies, Santa Clara, Calif.) equipped with a Coregel 87H3 Column, ICE-99-9861, Serial No. 12528142. It was operated at 60° C. (glucose) and 55° C. (glycerol) and a flow rate of 0.6 mL/min of 0.01 N H2SO4 (glucose) and 0.008 N H2SO4 (glycerol).
Dried Cell Mass Determination
10 ml volume of culture samples are centrifuged at 7.000 rpm for 10 min and the pellets are washed once with distilled water and centrifuged in a similar condition. The pellet is kept in the freezer overnight. To lyophilize the biomass, the recovered cell pellet is immediately frozen in a freeze-dryer (CT-series, Panchum coin.) for 24h. The dry cell weight is determined and the pellet was kept at 4° C. for further analysis.
Measurement of PHB
PHB was analyzed as described in Karr D B, Waters J K, Emerich D W (1983) Analysis of poly-β-hydroxybutyrate in Rhizobium japonicum bacteroids by ion-exclusion high-pressure liquid chromatography and UV detection. Appl Environ Microbiol 46:1339-1344. Tyo K E, Zhou H, Stephanopoulos G N (2006) High-throughput screen for poly-3-hydroxybutyrate in Escherichia coli and synechocystis sp. strain PCC6803. Appl Environ Microbiol 72:3412-3417. Kocharin K, Chen Y, Siewers V, Nielsen J (2012) Engineering of acetyl-CoA metabolism for the improved production of polyhydroxybutyrate in Saccharomyces cerevisiae. AMB Express 2:1-11. Kocharin K, Siewers V, Nielsen J (2013) Improved polyhydroxybutyrate production by Saccharomyces cerevisiae through the use of the phosphoketolase pathway. Biotechnol Bioeng 110:2216-2224. Briefly, 40-50 mg of dried cells are weighed and boiled in 2 mL (95° C.) of concentrated sulfuric acid for 60 min. Samples are centrifuged (10 min, 7.000 rpm) to remove cell debris. Subsequently, the supernatant is diluted 20X of 0.014 N H2SO4, and filtered in membrane nylon B13NY045, Basefil Syringe Filter, 0.45 μm, area 1.09 cm2, diameter 13 mm. The extract is analyzed in an HPLC-UV detector using a Coregel 87H3 Column, ICE-99-9861, Serial No. 13722909. Commercially standard PHB (Sigma-Aldrich, St. Louis, Mo.), processed in parallel with the samples, is used as a standard. The HPLC is operated at 60° C. and a flow rate of 0.6 ml/min of 0.014 N H2SO4.
Total Lipid Analysis
Extraction of lipids from wet biomass is based on a modification of the procedure used by Bligh, E. G. and Dyer W J (1959) Canadian Journal of Biochemistry and Physiology. Can J Biochem Physiol 37. Around 50-100 mg dry biomass from a crude powder is blended with 5 ml chloroform/methanol (2:1) and the mixture is agitated for 3 min at room temperature in an orbital shaker. The solvent phase is recovered by centrifugation at 7000 rpm for 10 min. The whole solvent is evaporated and dried under vacuum conditions.
Results
1. Expression of PhaA gene, PhaB gene, and PhaC gene in engineered Rhodotorula glutinis strains and accumulation of recombinant PHB
The transformation of three gene cassettes expression using strong promotor (pKLAC), PhaA gene, PhaB gene, and PhaC gene, are successfully integrated into the Rhodotorula glutinis genome, using modified lithium acetate competent cells in the electroporation method. The yeast transformants are screened using YP2D supplemented with G418 (5 μg/ml, 10 μg/ml, 25 μg/ml, 50 μg/ml, 100 μg/ml and 200 μg/ml) and the wild type without expression cassette is used as a control.
In the embodiment, candidates from three combination concentrations of antibiotics G418 in YPAD galactose (25 μg/ml, 50 μg/ml and 100 μg/ml) are screened.
2. Characterization of overexpression PhaA gene, PhaB gene, and PhaC gene by Western Blot analysis
10 candidates recombinant Rhodotorula glutinis transformed with PhaA gene, PhaB gene and PhaC gene are tested for expression of the PHA synthase by Western blot analysis using an anti His-Tag, as shown in
The result shows a novel for Rhodotorula glutinis as oleaginous yeast successfully capable used to microbial cell factory to PHB production. Simultaneously, it could be the best and specific host to express three keys enzymes for the PHB production: acetyl-CoA C-acetyltransferase (encoded by PhaA), NADPH-dependent acetoacetyl-CoA reductase (encoded by PhaB), and PHA synthase (encoded by PhaC) in bacteria and some yeast. It is an important thing that the invention proves not only transformation in Saccharomyces cerevisiae capable to product the PHB, but Rhodotorula glutinis no doubtful more competent host because it also has many benefits for industrial manufacturing.
3. The screening of heterologous expression of PhaA gene, PhaB gene, and PhaC gene in nine candidates of selected transformants Rhodotorula glutinis
The investigation of heterologous expression of PHB has been successfully carried out on candidate transformant Rhodotorula glutinis using the western blot and HPLC method.
In the embodiment, a method for PHB extraction and recovery from yeast cells using chemical disruption by purifying sulphuric acid (as described in the method) are demonstrated. The presence of PHB in intracellular of transformant Rhodotorula glutinis is confirmed by HPLC analysis, using commercial PHB standard control, as shown in
The result of PHB production was shown in
4. Evaluation of growth and PHB production of selected engineered Rhodotorula glutinis strain #100-29 using shaking flask cultivation
The selected engineered Rhodotorula glutinis strain #100-29 is evaluated of PHB production capacity in 250 ml shaken flasks of a 50-ml working volume for 48h. Firstly, Rhodotorula glutinis strain #100-29 is grown on media with different carbon sources in same concentration, such as glucose, galactose, crude glycerol and oil, each for 30 g/L (pH 5.5) to provide completely carbon and energy sources. Metabolite products are analyzed for biomass, PHB production, lipid and residue of carbon sources. The performance of engineered Rhodotorula glutinis strain #100-29 is displayed in
The average residue of carbon sources from
For evaluating the concentration of glucose, the selected engineered Rhodotorula glutinis strains #100-29 were cultured in 30 g/L, 45 g/L, and 60 g/L of glucose as shown in
Different ranges of glucose (30 g/L, 45 g/L and 60 g/L) are utilized to optimize the best glucose range necessary for the maximum PHB production. Glucose-rich medium enhances the PHB production capability of yeast under nitrogen and phosphate limitation sources. Thus, glucose source as supplement carbon is added in the microorganism medium.
The ability to use a lower concentration of glucose as a carbon source is an important advantage, a cheap and abundant substrate. The maximum yield of PHB concerning the carbon source is very high in the organism. Nutrient limitation is necessary to trigger PHB accumulation, and generally, ammonia is used as the critical control factor for uncoupling the growth of cells and PHB production. A recombinant E. coli strain gave the maximum PHB content (about 60% PHB of DCW) at a specific combination of yeast extract and peptone. Similar results are obtained from the cultivation of Anaerobiospirillum succimproducens and Phaffia rhodozyma in the presence of yeast extract, and a combination of yeast extract and peptone. Furthermore, Table 3 shows that engineered Rhodotorula glutinis strain #100-29 had higher productivity PHB (PHB concentration, PHB yield and PHB content) when cultured in glucose and crude glycerol each 30 g/L rather than in transformant Saccharomyces cerevisiae in the previous study (references [1], [2], [4]-[6] and [8]). The maximum amount of PHB accumulated in engineered Saccharomyces cerevisiae strain C13ABYS86 6.7%, however in the embodiment of the invention are obtained up to 28.87% and 62% in glucose and crude glycerol, respectively.
S. cerevisiae
Arxulaadenin
ivorans
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
R. glutinis
5. Performance of selected engineered Rhodotorula glutinis strain #100-29 using aerobic batch bioreactor for PHB production
To further explore PHB production from engineered Rhodotorula glutinis strain #100-29, two independent fermentation aerobic batch bioreactors are carried out. The fermentation is performed in glucose 30 g/L and crude glycerol 30 g/L as carbon sources and pH-controlled (5.5) of 5 L bioreactor. Under the conditions, full glucose and glycerol consumption are observed within 12-144h, as shown in
6. Performance of engineered Rhodotorula glutinis strain #100-29 using varieties concentrations of crude glycerol in the aerobic batch bioreactor for increasing PHB production
For evaluating the concentration of crude glycerol, the selected engineered R. glutinis strains #100-29 are cultured in 30 g/L, 60 g/L, and 90 g/L of crude glycerol. The production biomass during fermentation time is increased parallel in both conditions (crude glycerol 30 g/L and 60 g/L), as shown in
The PHB production in a 5 L aerobic batch bioreactor may improve the PHB production up to more than 3 times (as shown in Table 3) from shaking flask cultivation. Thus, significantly higher biomass could be obtained in a larger scale bioreactor. In the embodiment, PHB accumulation in crude glycerol when cultured in a batch bioreactor is higher than glucose in the same concentration (as shown in Table 3). Continuous culture offers many advantages for industrial production, provided that contamination is avoided and the stability of the strain is guaranteed. The advantages include simplicity of culture control, homogeneity of the production, and constancy of culture conditions.
The result in the invention represents that production PHB yield from engineered Rhodotorula glutinis strain #100-29 is higher than Rhodotorula glutinis var. glutinis 60 in the previous study, when it is cultured in a batch bioreactor using crude glycerol 30 g/L for the maximum time. Table 3 indicates that engineered Rhodotorula glutinis strain #100-29 has the highest PHB yield up to 43.35 mg/g in glucose and 95.53 mg/g in crude glycerol, rather than Saccharomyces cerevisiae strain RKS in glucose and galactose conditions. In addition, in the batch bioreactor, the higher concentration PHB and PHB yield are obtained from Rhodotorula glutinis strain #100-29 rather than Saccharomyces cerevisiae strain SCKK006 when it cultured with glucose. Moreover, Table 3 illustrates that in the batch bioreactor, the higher PHB content is discovered in glucose and crude glycerol (up to 28.87% and 62%, respectively) rather than Saccharomyces cerevisiae strain TMB 4444 when it cultured with xylose.
The utilization of crude glycerol for the production of PHA is an interesting alternative to support biodiesel production from fats and oils. Crude glycerol as a by-product of biodiesel production from rape is such a promising carbon source. This is an alternative and renewable energy source that will result in a surplus of glycerol. About 3.2 million tons of biodiesel per year were produced in Europe during 2005. From the invention, the utilization of crude glycerol as an ideal carbon source for industrial fermentation to produce PHB is successfully demonstrated.
As a co-product stream from biodiesel production, generally, composition minerals from crude glycerol are glycerol, free fatty acids (FFA), fatty acid methyl esters (FAME), and some traces of salts. And it will depend on the feedstock material, the transesterification process (catalytic way) and the recovery technology employed. Fatty acids and fatty acid methyl ester are the important components played role in acetyl-CoA. Acetyl-CoA is a central metabolite in carbon and energy metabolism which connects glycolysis, tricarboxylic acid cycle, β-oxidation, and de novo biosynthesis of fatty acids. Starting from acetyl-CoA, the biosynthesis of PHB, the simplest and most well-studied member of PHA family, is more suitable and faster catalyzed by three enzymes: β-ketothiolase, NADPH-dependent acetoacetyl-CoA reductase, and PHA synthase.
Conversion of the crude glycerol into PHAs is a promising route to offset the production cost of biodiesel and valorize the crude glycerol. The PHB content of engineered Rhodotorula glutinis strain #100-29 achieved a maximum 62% when cultured in crude glycerol (as shown in Table 3).
7. PHB production from engineered Rhodotorula glutinis strain #100-29 using varieties concentrations of NaCl in shaking flask
For evaluating the PHB production, the selected engineered R. glutinis strains #100-29 are cultured in control (0.1 g/L), 1, 2, 3, 4 and 5 g/L of NaCl (
Also referring to Table 3, the data shows that in culture fermentation condition crude glycerol 30 g/L using 4 g/L NaCl is the optimum concentration of NaCl that can provide the adequate stress for the optimal PHB accumulation (PHB concentration, PHB yield and PHB content) rather than in another concentration. One plausible correlation is between osmotic stress and salt concentration. Therefore, NaCl addition may cause the yeast to eliminate water and cell content, to make more space for PHB granules and it become more packed under osmotic stress. As a result, the experiments show that the PHB production from engineered Rhodotorula glutinis may be carry out in brine or seawater. Compared to using pure water or distilled water in the experiment, seawater is cheap and easy to obtain, when the PHB production from engineered Rhodotorula glutinis is used in mass production, the advantage of seawater will be greater than pure water or distilled water.
In the embodiment of the invention, the enhancement of PHB production is improved by appropriated for genetic modifications in Rhodotorula glutinis. The engineered Rhodotorula glutinis from strain #100-29 as an eukaryotic organism has the capability to enhance PHB production (PHB concentration, PHB yield and PHB content). The improvement of PHB accumulation is obtained in flask shake culture with glucose, galactose, crude glycerol and oil as carbon sources, in which the lipid content in oil is higher than glucose. However, fermentation in the bioreactor is known more advantageous with crude glycerol as carbon sources. Crude glycerol is one of significant effort has been invested in the invention by using inexpensive carbon substrates and developing more efficient fermentation processes to reduce PHB production cost. Further, the implementation of biofuels along the biodegradable plastics could be a green outlet for petrochemical plastics and fuels, using common methods in the polymer industry. Rhodotorula glutinis strain #100-29 is a new PHB-producing strain adapted to convert crude glycerol. These microbial polyesters have high polymer accumulation rates which lead to future prospects concerning the optimization of cultivation parameters and the increase of cellular productivity. Furthermore, optimizations of the fermentation conditions are required to improve productivity. This result is applied to the next-generation industrial biotechnology (NGIB) offers extensive opportunities for competitive bioproduction.
In summary, since yeast has flexibility in physiology, is novel in biotechnology metabolism and larger than bacteria, it provides a cheaper and faster way to improve PHB production. In the method of the embodiment of the invention, an oleaginous yeast Rhodotorula glutinis is used in PHB production, which belongs to a type of yeast that has ability to provide lipids as a substrate for PHB synthesis in metabolic plasticity. It is demonstrated that an increasing of PHB production in oleaginous yeast by using optimation some carbon sources. Therefore, comparing with the method of using bacteria in the conventional technology, the oleaginous yeast of the embodiment of the invention provides a way of cheaper, faster and flexibility in biotechnology metabolism, and may further increase the PHB production.
While the invention has been described in terms of what is presently considered to be the most practical and preferred embodiments, it is to be understood that the invention needs not be limited to the disclosed embodiment. On the contrary, it is intended to cover various modifications and similar arrangements included within the spirit and scope of the appended claims which are to be accorded with the broadest interpretation so as to encompass all such modifications and similar structures.
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