Patent Application
20250230473
This application claims the priority benefit of Taiwan application serial no. 113101406, filed Jan. 12, 2024, the full disclosure of which is incorporated herein by reference.
This application includes a Sequence Listing filed electronically as an XML file named “Sequence Listing_PROFN-24004-USPT.xml”, created on Apr. 10, 2024, with a size of 15,808 bytes. The Sequence Listing is incorporated herein by reference.
The disclosure relates to a method for producing 3-hydroxy-3-methylbutyrate. More particularly, the disclosure relates to a method for producing 3-hydroxy-3-methylbutyrate using microorganisms.
Currently, there are two main methods for producing 3-hydroxy-3-methylbutyric acid (HMB): chemical synthesis and biosynthesis.
In the chemical synthesis method, a typical approach involves using diacetone alcohol and oxidizing agents as raw materials. After acidification and organic solvent extraction, the resulting product reacts with calcium hydroxide or calcium chloride to form salts of 3-hydroxy-3-methylbutyric acid. This method yields approximately 50 kg of product from the reaction of about 80 kg of diacetone alcohol and 1200 kg of 10% sodium hypochlorite, with the product containing 98.7 wt % 3-hydroxy-3-methylbutyric acid. Thus, the conversion rate of diacetone alcohol in this reaction is approximately 61%. Subsequently, 250 kg of ethanol is added, followed by treatment with calcium hydroxide to obtain 95 kg of solid calcium salt of HMB. However, this chemical synthesis method faces several challenges, including stringent reaction conditions requiring precise control of temperature with high heat and low-temperature environments, which demands sophisticated equipment and personnel. The oxidizing agents used are corrosive, and the resulting liquid contains high salt content, necessitating extensive organic solvent extraction. The conversion efficiency of the synthesis reaction to form salts with calcium is typically below 62%, and there is a high dependency on petrochemical raw materials.
To address the challenges of the chemical synthesis method, some research teams have begun investigating biosynthesis, which mainly includes enzymatic and microbial conversion pathways. One application of enzymatic biosynthesis involves the use of crude protein from cells of Galactomyces reessii, which contains hydratase enzymes. This method employs the precursor 3-methylbutyric acid for hydration, resulting in the production of 3-hydroxy-3-methylbutyric acid. However, this approach has drawbacks, including the high cost and large quantity requirement for purifying hydratase from mycelium, as well as difficulties in maintaining the enzymatic reaction's activity. Therefore, developing a more feasible method that utilizes renewable resources as carbon sources and employs microorganisms as metabolic factories for HMB production is desirable.
Currently, some research teams utilize Escherichia coli as a host to construct the production pathway of HMB and conduct fermentation using glucose as the carbon source. However, the yield of this method is not yet satisfactory. Additionally, there are efforts using Yarrowia lipolytica as a host to construct production pathways, also employing glucose as the microbial carbon source for fermentation. However, the main culture medium in this method typically relies on complex media, often requiring the addition of yeast extract or casein tryptone, further increasing the cultivation costs.
In one aspect, the present invention is directed to a transgenic strain for producing 3-hydroxy-3-methylbutyric acid. The transgenic strain comprises a host cell and a plurality of exogenous genes. The host cell has an anabolic pathway for converting a carbon source into acetyl-CoA, wherein the host cell is Escherichia coli, cyanobacteria, or yeast, and the carbon source comprises at least one of glucose, xylose, acetic acid or acetate, methanol, and carbon dioxide. The exogenous genes are located in the host cell to assemble an anabolic pathway from acetyl-CoA (Ac-COA) to 3-hydroxy-3-methylbutyrate (HMB). The exogenous genes comprise a first, second, third, fourth, and fifth exogenous genes. The first exogenous gene is a gene encoding acetyl-CoA acetyltransferase (AtoB) or acetyl-CoA thiolase (NphT7), or a gene with 80% or more sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2 and possessing AtoB activity or NphT7 activity. The second exogenous gene is a gene encoding hydroxymethylglutaryl-CoA synthase (MvaS), or a gene with 70% or more sequence identity to SEQ ID NO: 3 and possessing MvaS activity. The third exogenous gene is a gene encoding 3-hydroxy-3-methylglutaryl CoA dehydratase (LiuC), or gene with 50% or more sequence identity to SEQ ID NO: 4 and possessing LiuC activity. The fourth exogenous gene is a gene encoding 3-methylglutaryl-CoA decarboxylase (AibAB), or a gene with 70% or more sequence identity to SEQ ID NO: 5 and SEQ ID NO: 6 and possessing AibAB activity when expressed and combined. The fifth exogenous gene is a gene encoding a thioester hydrolase (YciA, TesB, MenI, or YqiA), or a gene with 60% or more sequence identity to SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, and possessing YciA, TesB, MenI, or YqiA activity when expressed.
According to an embodiment of this invention, the transgenic strain further comprises at least a recombinant plasmid carrying the exogenous genes.
According to another embodiment of this invention, the exogenous genes are integrated into a genome of the host cell.
According to another embodiment of this invention, the Escherichia coli comprises a plurality of substrains directly utilizing exogenous carbon sources to synthesize acetyl-CoA in vitro, and the Escherichia coli substrains comprise BW25113, XL1-Blue, DH5a, W3110, NEBExpress®, Rosetta (DE3), W strain, crooks, and the probiotic E. coli Nissle 1917.
According to another embodiment of this invention, the Escherichia coli has gene deletions of metabolic pathways competing with the anabolic pathway of 3-hydroxy-3-methylbutyratethe.
According to another embodiment of this invention, a gene of the Escherichia coli encoding pyruvate oxidase (PoxB) is knockout to reduce the production of acetate byproducts in the pathway synthesizing acetate from pyruvate.
According to another embodiment of this invention, a gene of the Escherichia coli encoding citrate synthase (GltA) in the TCA cycle is knockout to increase the yield of 3-hydroxy-3-methylbutyrate as the main product.
According to yet another embodiment of this invention, the cyanobacteria comprise Synechococcus elongatus.
According to yet another embodiment of this invention, the yeast comprises Pichia pastoris.
In another aspect, this invention is directed to a method for the production of 3-hydroxy-3-methylbutyrate. The method comprises culturing the transgenic strains as described above to allow the transgenic strain to produce 3-hydroxy-3-methylbutyrate. The culturing conditions comprise a first culturing temperature before inducing the expression of the exogenous genes in the transgenic strain is 37±2° C., a second culturing temperature after inducing the expression of the exogenous genes in the transgenic strain is 30±2° C., and a dissolved oxygen level in a bacterial solution of the transgenic strain is 30%.
According to an embodiment of this invention, a culture medium used is M9 minimal medium when the transgenic strain is the Escherichia coli.
According to another embodiment of this invention, a culture medium used is modified BG-11 medium containing 50±10 mM NaHCO3 when the transgenic strain is the cyanobacteria.
According to yet another embodiment of this invention, a culture medium used is YPB complex medium when the transgenic strain is the yeast.
From the above embodiments of the present invention, it can be seen that compared to the current chemical methods using petroleum derivatives as raw materials for producing HMB, the embodiments of the present invention enable microorganisms to directly utilize organic carbon sources (such as glucose, glycerol, xylose, etc.) for HMB production. This not only reduces the reliance on petrochemical raw materials in the HMB market but also reduces the generation of harmful byproducts. Furthermore, there is currently no literature or patents proving that the same HMB anabolic pathway can be expressed in Escherichia coli, Synechococcus elongatus, or Pichia pastoris as in the embodiments described above, and serve as metabolic factories for HMB production. Especially, Escherichia coli can achieve high yields of HMB with just the minimal medium M9 with pure salts, showing significant potential for industrial application and development.
The foregoing presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key/critical elements of the present invention or delineate the scope of the present invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later. Many of the attendant features will be more readily appreciated as the same becomes better understood by reference to the following detailed description considered in connection with the accompanying drawings.
In order to make the above and other aspects, features, advantages, and embodiments of the present invention more apparent and understandable, the description of the accompanying drawings is as follows.
Based on the above, a transgenic strain and a method for producing 3-hydroxy-3-methylbutyric acid (HMB) will be provided. In the following description, exemplary microorganisms and exemplary production methods for producing HMB will be introduced.
To provide a comprehensive description of the embodiments of the present invention, corresponding figures will be included below, along with explanatory descriptions of various aspects and specific embodiments of the invention. However, not all embodiments of the present invention require these technical details. That is, the implementation or application of the present invention is not limited to any particular form but may involve combining features and method steps from multiple embodiments.
First, it is necessary to design a synthetic metabolic pathway for 3-hydroxy-3-methylbutyric acid (HMB) within microorganisms, enabling the use of microorganisms as production tools for HMB.
In this embodiment, the microbes to be used include Escherichia coli, Synechococcus elongatus, and Pichia pastoris.
In
Synechococcus elongatus 120 is a photosynthetic microorganism capable of utilizing carbon dioxide directly. Through the Calvin cycle of photosynthesis, Synechococcus elongatus 120 first produces pyruvate, and then, via a series of synthetic metabolic steps, obtains acetyl-CoA. Synechococcus elongatus 120 serves as a model organism with abundant genetic tools. Hence in reaction step 101b, it is designed to synthesize acetoacetyl-CoA from acetyl-CoA, via the expression of acetyl-CoA thiolase encoded by the nphT7 gene from the Streptomyces species. In
Since Escherichia coli 110, Synechococcus elongatus 120, and Pichia pastoris 130 do not naturally possess all the enzymes required for the synthesis pathway of 3-hydroxy-3-methylbutyric acid (HMB) as depicted in
Table 1 outlines the enzymes required for the synthesis metabolic pathway of 3-hydroxy-3-methylbutyric acid (HMB) as depicted in
E. coli
Streptomyces sp.
Staphylococcus
aureus
Stigmatella
aurantiaca
E. coli
Experiment 1: Cultivation of Escherichia coli
Unless otherwise stated, all chemicals were purchased from TCI (Portland, OR). J. T. Baker (Phillipsburg. NJ. USA), or Sigma-Aldrich (St. Louis, MO. USA). Complex media such as Luria broth (LB), yeast extract, and LB agar were obtained from FocusBio (Guangzhou, China). All seed cultures were grown in LB medium at 37° C. with shaking at 200 rpm in a shaking incubator.
The culture medium used for HMB-producing strains was M9 minimal medium supplemented with glucose at a concentration below 40 g/L. The composition of M9 medium per liter included 12.8 g of Na2HPO4·7H2O, 3 g of KH2PO4, 0.5 g of NaCl, 0.5 g of NH4Cl, 1 mM of MgSO4, 1 mg/mL of vitamin B1, and 0.1 mM of CaCl2).
Additionally, when necessary, an appropriate concentration of antibiotics was added to the medium to facilitate strain selection. For example, 50 mg/mL kanamycin (kan), 100 μg/mL ampicillin (Amp), or 50 μg/mL chloramphenicol (Cm) could be added.
For strains with the glta gene knocked out, the medium used contained less than 40 g/L glucose and an additional nitrogen source compound at 5 g/L.
The cell growth of the strains was measured at a wavelength of 600 nm (OD600) using a Biotek Epoch 2 microplate spectrophotometer, with the optical path length adjusted to 1.
Experiment 1-2: Gene Electroporation in Escherichia coli
The entire process of electroporation was basically conducted on ice.
The 1% (V/V) bacterial suspension of E. coli cultured overnight was inoculated into a shaking flask containing 20 mL of LB medium. When the bacterial suspension in the flask reached an OD600 of 0.4 to 0.6, it was placed on ice and transferred to a 50 mL Falcon tube, then was centrifuged at 4° C. at a speed of 4,020 rpm for 10 minutes. The supernatant was discarded, and the cells were resuspended in 1 mL of 10 vol % glycerol and transferred to 2 mL microcentrifuge tubes. The tubes were centrifuged at 5,000×g for 2 minutes, and this washing step was repeated three times. Finally, the cells were resuspended and concentrated 30-fold in 10 vol % glycerol.
Next, 50 μL of concentrated bacterial culture and 50-100 ng of plasmid were mixed in a 1.5 mL centrifuge tube, and then the mixture was placed into a 1 mm electroporation cuvette (BioRad) and pulsed using the Ec1 program (V=1.8 kV) on a MicroPulser (Bio-Rad), with a time constant of 5.4 to 5.9 ms. After electroporation, the cells were mixed with 200 μL of LB medium and cultured at 37° C. and 200 rpm shaking for 1 hour. Subsequently, 100 μL and 1 μL of the bacterial solution were plated on LB agar plates containing the corresponding antibiotics.
The next day, three individual colonies were randomly picked from the LB agar plates to serve as three biological replicates, and each was inoculated into 1 mL of LB broth. The overnight cultures were then inoculated at 1% (V/V) into 2 mL of M9 medium containing glucose at a concentration below 40 g/L and the corresponding antibiotics.
Experiment 1-3: Fermentation of Escherichia coli in a Bioreactor
The fermentation medium for E. coli utilized M9 medium supplemented with trace metals and 40 g/L of glucose. The salt composition per liter of M9 medium included 6 g of Na2HPO4, 3 g of KH2PO4, 0.5 g of NaCl, and 1 g of NH4Cl. Additionally, the M9 medium contained 1 mM of MgSO4, 0.1 mg/mL of thiamine, 0.1 mM of CaCl2), 40 g/L of glucose, and 10 ml/L of trace metal solution.
The trace metal solution was prepared by dissolving 5 g of EDTA in 800 mL of water and adjusting the pH to 7.5 using 3M NaOH. Subsequently, the remaining trace metals were added, and water was added to achieve a final volume of 1 liter. The concentrations of the remaining trace metals were as follows: 498 mg FeCl3 and 84 mg ZnCl2 per liter of water, along with 0.1 M Cu Cl2·2H2O, 0·2 M Co Cl2·6 H2O, 0.1 M H3BO3, and 1 M MnCl2·4H2O per liter of trace metal solution.
Two days before fermentation, seed cultures were prepared following the steps below. Firstly, a single colony was randomly selected from an LB plate and inoculated into test tubes containing 4 mL of LB broth. These tubes were then shaken at 37° C. overnight. The following day, 2.5% (V/V) of the overnight culture was inoculated into shake flasks containing 50 mL of fermentation medium, and the flasks were shaken at 37° C. for 12-14 hours.
The fermentation was conducted in a 5 L bioreactor (Biostat® A, Sartorius) using a fed-batch strategy. The working volume of the bioreactor was 1 L. After centrifugation of the seed culture, the seed culture was concentrated using the fermentation medium. The fermentation started with an initial OD600 of 0·2. The pH was automatically controlled by a computer-controlled pump with 3 M NaOH to maintain a pH of 7.0, and the dissolved oxygen level was set to 30%. When the OD600 reached 2, 0.1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to induce the expression of RNA polymerase in E. coli for the expression of exogenous genes, followed by shaking the cultivation at 30° C. The feeding solution for the bioreactor consisted of M9 minimal medium containing a trace metal solution, supplemented with 50 wt % glucose, 0.1 mM IPTG, and 50 μg/mL kanamycin. Additionally, 0.02 M (NH4) 2SO4 was fed into the bioreactor every 12 hours as a nitrogen source for E. coli growth until the bacteria entered the stationary growth phase.
Experiment 1-4: Quantification and Analysis of Fermentation Products in Escherichia coli
1 mL of the bacterial culture was sampled into a 1.5 mL centrifuge tube and centrifuged at 18,000×g for 2 minutes. After centrifugation, the supernatant was collected for subsequent product analysis.
Non-volatile compound analysis in the fermentation products of Escherichia coli was primarily conducted using High-Performance Liquid Chromatography (HPLC). With the Shimadzu LC-2030C HPLC system, most compounds in the supernatant could be separated and purified, followed by analysis using Photodiode Array Detector (PDA) and Refractive Index Detector (RID-20A). The operating conditions included 5 mM H2SO4 as the mobile phase with a flow rate of 0.6 mL/min and an injection volume of 20 μL. Analysis was carried out using a Bio-Rad Aminex HPX-87H HPLC organic acid column and a Micro-Guard Cation H+ guard column at 35° C. Specifically, HMB, acetic acid, and glucose were detected using the RID detector, while other compounds were analyzed based on their absorbance at 210 nm using the PDA detector. Standard curves were established using 0.1-20 mM standard solutions to determine the concentrations of various products in the supernatant. The glucose consumption by E. coli was determined by the difference between the initial concentration and the final concentration of glucose in the culture medium of the experimental group.
For volatile chemical compounds such as ethanol in the fermentation products of Escherichia coli, quantification was performed using a Shimadzu GC-2010 Gas Chromatograph (GC) equipped with a Flame Ionization Detector (FID). The gas chromatography column used was an SH-Rtx-wax column (30 m×0.32 mm, df 0.5 μm). The column oven temperature was initially held at 40° C. for 2 minutes, then ramped at a gradient of 45° C. per minute to 85° C. and held for 2 minutes, followed by a continuous increase at a rate of 45° C. per minute until reaching 230° C., and held for 1 minute. The FID detector was maintained at 230° C., while the injector temperature was set at 225° C. Nitrogen was used as the carrier gas, with a split ratio of 1:15, for injecting 1 μL of the sample into the analytical column. 1-pentanol was used as the internal standard.
Experiment 1-5: Utilization of Escherichia coli Dual-Plasmid Expression System I with Homologous Genes carried by the Second Plasmid
In this experiment, in addition to using the pRM09 plasmid carrying genes atoB, mvaS, liuC, aibA, and aibB corresponding to reaction steps 101a-105 in
In this experiment, the first plasmid carried genes atoB, mvaS, liuC, aibA, and aibB for the HMB synthesis pathway under the control of the PLlacO1 promoter. The genes liuC, aibA, and aibB were sourced from Myxococcus xanthus. The second plasmid was utilized to carry genes encoding thioesterases from microorganisms other than E. coli to assess their catalytic efficiency in reaction step 106 of the pathway depicted in
Experiment 1-7: Escherichia coli Monoplasmid Expression System
In this experiment, the PLlacO1 promoter was used to carry all genes involved in the HMB synthesis pathway, and genes responsible for reaction steps 103-105 in
Myxococcales
Stigmatella aurantiaca DW4/3-1
Myxococcus xanthus
Archangium gephyra DSM 2261
Melittangium boletus DSM 14713
Cystobacter fuscus DSM 2262
Pyxidicoccus parkwaysis
Hyalangium minutum DSM 14724
Labilithrix luteola DSM 27648
Experiment 1-8: Single-plasmid Expression System III in Escherichia coli of Different Strains
In this experiment, a plasmid containing the PLlacol expression system regulated by the lacI gene, which can be expressed in most Escherichia coli strains, was constructed to carry the aforementioned genes atoB, mvaS, liuC, aibA, and aibB. The constructed PLlaco1 expression system plasmid was then transferred into different E. coli strains, and the HMB production of each strain was observed after 24 hours of fermentation.
Experiment 1-9: The Impact of Different Culture Media on HMB Production in Escherichia coli
In this experiment, different culture media were used to cultivate Escherichia coli to observe the impact of different culture media on HMB production. The results obtained are shown in
The results revealed that the culture medium used for HMB biosynthesis does not require the addition of any additional nitrogen sources, such as lysogeny broth (LB) or yeast extract. Instead, the highest HMB yield (approximately 3.53 g/L) and the least amount of acetic acid byproduct were achieved using the M9 minimal medium composed mainly of salts. Therefore, using only M9 minimal medium can reduce the cultivation cost of Escherichia coli and offers significant commercial advantages.
Experiment 1-10: The Influence of Different Carbon Sources on HMB Production in Escherichia coli
In this experiment, various compounds were employed as carbon sources for Escherichia coli to observe the impact of different carbon sources on HMB production.
Experiment 1-11: The Impact of Continuous Fermentation Duration on HMB Production in Escherichia coli
In this experiment, genetically modified Escherichia coli were cultivated on a large scale using a bioreactor, with a volume of 1 liter, while maintaining dissolved oxygen levels above 30%. Before adding the inducer, rapid cultivation was conducted at 37° C., followed by a temperature reduction to 30° C. after adding the inducer. Throughout the fermentation process, the pH was maintained at 7, and after a period of glucose consumption, feedings were added approximately every 50 hours to restore the glucose concentration to around 20 g/L. Under these cultivation conditions, the HMB-producing strain sustained production for approximately 11 days.
When Escherichia coli enters a state of glucose starvation, the protein expression of RpoS increases. Acting as a regulator, the RpoS protein can modulate the transcription of hundreds of genes in Escherichia coli upon entering a state of carbon starvation, allowing the organism to resist various stresses (Peterson et al., 2005). This signifies a shift in cellular metabolism. Such a shift could potentially affect the expression of exogenous genes or the yield of products. Therefore, conventional fermentation strategies for E. coli typically aim to maintain glucose concentrations above a certain threshold. Given that producing HMB using E. coli results in significant acetate byproduct formation, this experiment was designed to allow E. coli to utilize acetate as an alternative carbon source, thereby sustaining HMB production.
Experiment 1-12: Assessing the Impact of Knocking Out Genes of Competitive Pathway on HMB Production in Escherichia coli
In this experiment, it was attempted to enhance HMB production and reduce acetate formation in Escherichia coli by knocking out genes involved in competing pathways. Both wild-type and gene-knockout strains of Escherichia coli were cultured in M9 minimal medium supplemented with less than 40 g/L glucose and 5 g/L nitrogen source compounds. The wild-type strain used in the experiment was BL21 (DE3).
Therefore, further testing was conducted on the expression of the E. coli strain with both genes gltA and poxB knocked out (BL21 ΔgltA ΔpoxB). The results showed that after 24 hours of fermentation, a higher HMB production was obtained. Compared to the wild-type strain, the HMB production of the E. coli strain (BL21 ΔgltA ΔpoxB) was approximately 1.7 times higher, at 3.33 g/L compared to the wild-type HMB production of 1.96 g/L. Additionally, the HMB yield of the E. coli strain (BL21 ΔgltA ΔpoxB) was approximately 1.9 times higher, at 0.32 g/g glucose compared to the wild-type HMB yield of 0.17 g/g glucose. However, the acetate production of the E. coli strain (BL21 ΔgltA ΔpoxB) was only about 15% of the wild type.
Experiment 2-1: Cultivation Method for Synechococcus elongatus PCC 7942
All Synechococcus elongatus strains were cultured using modified BG-11 medium. The composition of BG-11 medium include 1.5 g/L NaNO3, 0.036 g/L CaCl2)·2H2O, 0.012 g/L ferric ammonium citrate, 0.0022 g/L Na2EDTA·2H2O, 0.040 g/L K2HPO4, 0.070 g/L MgSO4·7H2O, 0.020 g/L Na2CO3, 0.00882 g/L sodium citrate, and a 1000× dilution from a trace mineral solution (containing 1.43 g H3BO3, 0.905 g MnCl2·4H2O, 0.111 g ZnSO4·7H2O, 0.195 g Na2MoO4·2H2O, 0.0395 g CuSO4·5H2O, and 0.0245 g Co(NO3)2·6 H2O per 500 mL water).
Using a 1 μL inoculation loop, cells of S. elongatus cyanobacteria from a BG-11 petri dish were inoculated into fresh 40 mL modified BG-11 medium containing 50 mM NaHCO3 and cultured in 250 mL baffled shake flasks. The cultures were then transferred to a shaking incubator (Hi-point 600SR) and grown under the following conditions: light intensity ranging from 22 to 38 μE/s/m2, temperature at 30° C., and agitation at 110 rpm.
When the cell optical density at 730 nm (OD730 nm) reached between 0.4 and 0.6, the cyanobacteria were induced to express RNA polymerase by adding IPTG to a final concentration of 1 mM, facilitating the expression of exogenous genes in the cyanobacteria. After induction, 1 mL of culture was taken out for analysis of cell growth and HMB concentration. Following sampling, 1 mL of BG-11 medium containing 500 mM NaHCO3, appropriate antibiotics, and 1 mM IPTG was immediately added back to the culture to ensure adequate carbon supply. Cell growth was quantified by measuring absorbance at 730 nm wavelength using a Biotek Epoch 2 microplate spectrophotometer, with the path length adjusted to 1 cm.
In this experiment, we demonstrated for the first time the feasibility of using the photoautotrophic cyanobacterium, Synechococcus elongatus PCC 7942, as a host for expressing the HMB metabolic pathway and synthesizing HMB.
Experiment 3: Cultivation Method for Methylotrophic Yeast (Pichia pastoris X-33)
In this experiment, for the first time, we demonstrated the use of the eukaryotic organism, methylotrophic yeast (Pichia pastoris X-33), as a host for HMB production. Methylotrophic yeast has traditionally been widely employed as a host for heterologous protein expression systems, owing to its low production cost, capacity for post-translational modifications, and ability to regulate protein production using methanol.
Using YNB basic medium supplemented with 3 vol % methanol, the Pichia pastoris yeast was cultured in shake flasks at 30° C. and 220 rpm for 120 hours, with samples taken every 24 hours.
In this experiment, the Pichia pastoris X-33 yeast was cultured in YPB complex medium supplemented with 3 vol % methanol at a temperature of 30° C. and a shaking speed of 220 rpm for 120 hours in shake flasks, with samples taken every 24 hours.
The above results indicate that using YPB complex medium for culturing Pichia pastoris yeast resulted in approximately 67% higher yield compared to using YNB basal medium. Therefore, for the production of HMB using Pichia pastoris yeast, the complex medium would be the preferable choice.
From the above embodiments of the present invention, it is evident that compared to the current chemical methods using petroleum-derived materials for preparing HMB, the embodiments of the present invention allow for microbial production of HMB directly utilizing organic carbon sources such as glucose, glycerol, and xylose. This not only reduces the dependency of the HMB market on petrochemical raw materials but also minimizes the generation of harmful by-products. Moreover, there is currently no literature or patents demonstrating that the same HMB biosynthetic pathway can be expressed in Escherichia coli, Synechococcus elongatus, or Pichia pastoris yeast as metabolic factories for HMB production in the embodiments described above. Especially noteworthy is the fact that Escherichia coli can achieve high yields of HMB using only the minimal M9 basal medium with pure salts, showcasing significant potential for industrial application.
Currently, the price of glucose ranges from approximately $2.73 to $4 per kilogram, while the price of HMB ranges from about $20 to $30 per kilogram, with an estimated global market value of $90 million. The theoretical yield of HMB produced from glucose is 1.5 moles of glucose producing 1 mole of HMB, equivalent to 118.13 grams of HMB from 270 grams of glucose. Therefore, the theoretical yield is approximately 0.44 g/g of glucose. If the current cost of glucose is $2.73 per kilogram and the selling price of HMB is $20 per kilogram, the breakeven point is achieved when the yield of HMB exceeds or equals 0.1365 grams of HMB per gram of glucose. In the embodiments of the present invention, the highest HMB yield from Escherichia coli is up to 0.32 grams of HMB per gram of glucose, demonstrating significant commercial viability.
All the features disclosed in this specification (including any accompanying claims, abstract, and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, each feature disclosed is one example only of a generic series of equivalent or similar features.
| Number | Date | Country | Kind |
|---|---|---|---|
| 113101406 | Jan 2024 | TW | national |
