Recombinant microorganism for glycerol biosynthesis from carbon source and method for producing glycerol using same

Abstract

The present invention relates to a recombinant microorganism for glycerol biosynthesis from a carbon source and, more specifically, to a recombinant microorganism for glycerol biosynthesis from a carbon source by using a novel gene derived from a Bacillus licheniformis strain, and a method for producing glycerol using same. According to the present invention, the recombinant microorganism of the present invention provides a glycerol 1-phosphate pathway for glycerol biosynthesis, and thus can provide a novel synthesis pathway for glycerol biosynthesis as an alternative to the conventional glycerol 3-phosphate pathway.

Description

Recombinant microorganisms that biosynthesize glycerol from carbon sources and methods for producing glycerol using the same

[0001] The present invention relates to a recombinant microorganism that biosynthesizes glycerol from a carbon source, and more specifically, to a recombinant microorganism that biosynthesizes glycerol from a carbon source using a novel gene derived from a Bacillus licheniformis strain, and a method for producing glycerol using the same.

[0002] Glycerol is widely used as a raw material in pharmaceuticals, cosmetics, personal care products, and foodstuffs, and has numerous applications in various industries, including paints, printing inks, analytical reagents, soaps, polishes, and antifreeze. Glycerol esters are also important in the fat and oil industries. Furthermore, glycerol is a highly functionalized molecule compared to petrochemically produced hydrocarbons, allowing it to undergo a variety of chemical reactions to produce numerous high-value-added chemical products.

[0003] Glycerol can be produced through various methods, including hydrolysis from animal fats and similar raw materials, saponification during soap manufacturing, and esterification during biodiesel production. However, large-scale production primarily relies on esterification. However, the glycerol produced in this process contains impurities such as catalysts, water, soap, salts, and esters, resulting in low purity and requiring a separate purification process.

[0004] Glycerol can be produced through microbial fermentation. Previously, the natural biosynthesis of glycerol from glucose was developed from Saccharomyces cerevisiae. More specifically, Saccharomyces cerevisiae synthesizes dihydroxyacetone phosphate (DHAP) from glucose, then synthesizes glycerol 3-phosphate (G3P) from DHAP using glycerol 3-phosphate dehydrogenase (G3PDH) (DAR1, GPD1), and then synthesizes glycerol from G3P using glycerol 3-phosphatase (glycerol 3-phosphate phosphohydrolase; G3PP). That is, Saccharomyces cerevisiae biosynthesizes glycerol from glucose via the glycerol 3-phosphate pathway.

[0005] However, since there was a demand for the development of a new microorganism that provides an alternative pathway for industrial biosynthesis of glycerol, the present invention has been completed by providing a recombinant microorganism that biosynthesizes glycerol from a carbon source through the glycerol 1-phosphate alternative pathway using a novel gene derived from the Bacillus licheniformis (KCTC14485BP) strain.

[0006] The purpose of the present invention is to provide a novel recombinant microorganism that biosynthesizes glycerol, which provides a glycerol 1-phosphate alternative pathway for glycerol biosynthesis from a carbon source.

[0007] Another object of the present invention is to provide glycerol produced from the recombinant microorganism and a method for producing the same.

[0008] Another object of the present invention is to provide a use for producing glycerol from the recombinant microorganism.

[0009] The purposes of the present invention are not limited to those mentioned above. Other purposes and advantages of the present invention not mentioned above can be understood through the following description and will be more clearly understood through the embodiments of the present invention. Furthermore, it will be readily apparent that the purposes and advantages of the present invention can be realized by the means and combinations thereof set forth in the claims.

[0010] In order to achieve the above purpose, according to the present invention, a novel recombinant microorganism that biosynthesizes glycerol can be provided, which provides a glycerol 1-phosphate alternative pathway for glycerol biosynthesis.

[0011] In addition, according to the present invention, glycerol biosynthesized from the recombinant microorganism can be provided.

[0012] In addition, according to the present invention, a method for producing glycerol from the novel strain can be provided.

[0013] In addition, according to the present invention, a use of the novel strain for producing glycerol can be provided.

[0014] The recombinant microorganism of the present invention provides a glycerol 1-phosphate pathway for biosynthesizing glycerol from a carbon source, thereby providing a new glycerol synthesis pathway for biosynthesizing glycerol by a method other than the conventional glycerol 3-phosphate pathway.

[0015] The recombinant microorganism of the present invention provides an alternative pathway for more efficient biosynthesis of glycerol.

[0016] The recombinant microorganism of the present invention provides a useful alternative route for producing glycerol in larger quantities.

[0017] In addition to the effects described above, specific effects of the present invention are described below while explaining specific details for carrying out the invention.

[0018] Figure 1 shows a glycerol biosynthetic pathway according to one embodiment of the present invention.

[0019] Figure 2 is a graph showing a comparison of the glycerol production capacity of E. coli as an example of a recombinant microorganism into which a glycerol biosynthetic pathway has been introduced.

[0020] Figure 3 is a graph showing a comparison of the glycerol production capacity of C. glutamicum as an example of a recombinant microorganism into which a glycerol biosynthetic pathway has been introduced.

[0021] The aforementioned purposes, features, and advantages are described in detail below, so that those skilled in the art can easily practice the technical concepts of the present invention. In describing the present invention, detailed descriptions of known technologies related to the present invention will be omitted if they are deemed to unnecessarily obscure the gist of the present invention. The terms described below are terms that were described in consideration of their functions and actions in the present invention, and the meaning of each term should be interpreted based on the contents throughout this specification.

[0022]

[0023] Hereinafter, a recombinant microorganism that biosynthesizes glycerol according to the present invention, which provides a glycerol biosynthesis method according to the glycerol 1-phosphate pathway, glycerol biosynthesized using the same, and a method for producing the same are described in detail.

[0024] The present invention provides a recombinant microorganism for producing glycerol from a carbon source via the glycerol 1-phosphate pathway.

[0025] The abbreviations used in the present invention are as follows.

[0026] GLC, glucose;

[0027] DHAP, dihydroxyacetone phosphate;

[0028] GLY, glycerol;

[0029] G3P, glycerol-3-phosphate;

[0030] G3PDH, G3PD, GPD1, glycerol-3-phosphate dehydrogenase;

[0031] G3PP, GPP2, glycerol-3-phosphate phosphatase;

[0032] G1P, glycerol-1-phosphate;

[0033] G1PDH (G1PD or AraM) glycerol-1-phosphate dehydrogenase;

[0034] G1PP (DGP), glycerol-1-phosphate phosphatase;

[0035]

[0036] Figure 1 illustrates a pathway by which a recombinant microorganism produces glycerol from a carbon source, more specifically, a reaction pathway by which a carbon source is converted into DHAP and then glycerol is produced from DHAP. According to the glycerol production pathway provided by this application invention, DHAP is converted into glycerol 1-phosphate by glycerol 1-phosphate dehydrogenase, which is then converted into glycerol by glycerol 1-phosphatase.

[0037] According to one aspect disclosed herein, a novel recombinant microorganism is provided.

[0038] The recombinant microorganism may be a recombinant bacterium. For example, the recombinant microorganism may be Escherichia coli. For example, the recombinant microorganism may be Corynebacterium. For example, the recombinant microorganism may be Corynebacterium glutamicum. However, the recombinant microorganism strain is not limited thereto.

[0039] The recombinant microorganism may comprise glycerol 1-phosphate dehydrogenase (G1PDH). The recombinant microorganism may comprise glycerol 1-phosphatase (glycerol 1-phosphate phosphohydrolase; G1PP).

[0040] The recombinant microorganism may comprise glycerol 1-phosphate dehydrogenase (G1PDH) for synthesizing glycerol 1-phosphate (G1P) from DHAP. The recombinant microorganism may comprise glycerol 1-phosphatase (glycerol 1-phosphate phosphohydrolase; G1PP) for synthesizing glycerol from G1P.

[0041] Here, the G1PDH may be derived from the g1pD gene and the araM gene encoding 1-phosphate dehydrogenase (Glycerol 1-phosphate dehydrogenase; G1PDH) derived from Bacillus lichenfiormis (KCTC14485BP). The g1pD gene may include the sequence of SEQ ID NO: 1. The araM gene may include the sequence of SEQ ID NO: 2.

[0042] Here, the G1PP may be derived from the dgp gene encoding glycerol 1-phosphatase of Bacillus lichenfiormis (KCTC14485BP). The dgp gene may include the sequence of SEQ ID NO: 3.

[0043] The above Bacillus licheniformis strain was deposited at the Microbial Resource Center of the Korea Research Institute of Bioscience and Biotechnology on March 5, 2021 (KCTC14485BP).

[0044] In one embodiment, the recombinant microorganism provides an alternative pathway for synthesizing glycerol from a carbon source. Specifically, the recombinant microorganism synthesizes DHAP from a carbon source, then synthesizes glycerol 1-phosphate (G1P) from DHAP using glycerol 1-phosphate dehydrogenase (G1PDH), and then biosynthesizes glycerol from the G1P using glycerol 1-phosphatase (glycerol 1-phosphate phosphohydrolase; G1PP). The carbon source may be sucrose. The carbon source may be glucose. The carbon source may be fructose. The carbon source may be DHAP. The carbon source may be metabolized to provide DHAP. The carbon source may be a monosaccharide, an oligosaccharide, a polysaccharide or a single carbon substrate.

[0045] In another embodiment, the strain provides an alternative pathway for synthesizing glycerol from DHAP. Specifically, the strain synthesizes glycerol 1-phosphate (G1P) from DHAP using glycerol 1-phosphate dehydrogenase (G1PDH), and biosynthesizes glycerol from G1P using glycerol 1-phosphate phosphohydrolase (G1PP).

[0046] According to another aspect disclosed by the present specification, glycerol produced from a novel recombinant microorganism is provided.

[0047] According to another aspect disclosed by the present specification, a method for producing glycerol from a novel recombinant microorganism is provided.

[0048] According to another aspect disclosed herein, a use of the novel recombinant microorganism is provided for producing glycerol.

[0049] According to one embodiment, glycerol may be produced by culturing the recombinant microorganism in a medium containing a carbon source and recovering glycerol secreted into the culture medium.

[0050] According to another embodiment, glycerol can be obtained by providing a carbon source to the recombinant microorganism. The carbon source can be glucose. The glycerol can be secreted into the fermentation broth of the strain. The glycerol can be identified and quantified by high performance liquid chromatography (HPLC) and gas chromatography / mass spectrometry (GC / MS) analysis on cell-free extracts. Preferably, the fermentation medium is analyzed on an analytical ion exchange column using a mobile phase of 0.01 N sulfuric acid in a uniform manner. Methods for recovering glycerol from the fermentation medium are known in the art. For example, glycerol can be obtained from the cell medium by performing the steps of filtration of the reaction mixture, removal of water, extraction with an organic solvent, and fractional distillation (U.S. Patent No. 2,986,495).

[0051] Hereinafter, the present invention will be described in more detail through examples and experimental examples. However, the following examples and experimental examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples and experimental examples.

[0052] <Example 1> Production of a recombinant microorganism containing a gene derived from Bacillus licheniformis GSC4071

[0053] Bacillus licheniformis GSC4071 (KCTC 14485BP), a wild type (WT) strain isolated from a soil sample near Daejeon, was used. The heterologous strain, Escherichia coli W3110, was used as a model microorganism, and the heterologous strain, Corynebacterium glutamicum ATCC13032, was used as a model microorganism.

[0054] <1-1> Construction of a plasmid expressing glycerol biosynthesis genes in E. coli

[0055] For gene expression in E. coli, the universal plasmid pTrc99A was utilized. First, to introduce the g1pD or araM gene, the genomic DNA of Bacillus licheniformis GSC4071 strain was used as a template, and primers 4 / 5 and 6 / 7 were used to amplify the PCR to obtain sequence numbers 1 and 2, respectively. The obtained fragments were then treated with restriction enzymes EcoR1 / BamH1, respectively, and introduced into the pTrc99A plasmid. This resulted in the pTrc99A-g1pD or pTrc99A-araM plasmid. Next, to introduce the dgp gene into each plasmid, the genomic DNA of the Bacillus licheniformis GSC4071 strain was used as a template, and PCR amplification was performed using primers 8 / 9 to obtain sequence number 3. The obtained fragment was digested with BamH1 / Xba1 and introduced into pTrc99A-g1pD or pTrc99A-araM. As a result, pTrc99A-g1pD-dgp or pTrc99A-araM-dgp plasmid was obtained.

[0056] <1-2> Construction of a plasmid expressing glycerol biosynthesis genes in Cornebacterium

[0057] For gene expression in Cornebacterium, the universal plasmid pCES208-H36 was utilized. Using pTrc99A-g1pD-dgp and pTrc99A-araM-dgp, previously constructed for E. coli expression, as templates, g1pD-dgp and araM-dgp were amplified using primers 10 / 12 or 11 / 12, respectively. After digesting pCES208-H36 with BamH1 / Xba1, pCES208-H36-g1pD-dgp and pCES208-H36-araM-dgp were constructed, respectively, through Gibson assembly.

[0058] <1-3> Production of recombinant microorganisms

[0059] To construct an Escherichia coli strain for glycerol production, the model microorganism W3110 strain was used. For plasmid introduction, it was transferred to the W3110 strain via electroporation under the conditions of 25 uF, 200 Ω, and 2.5 kV / cm. To select strains containing the plasmid, screening was performed on LB agar medium containing 50 μg / mL Ampicillin, and gene expression was induced by adding 0.1 mM IPTG.

[0060] To construct a Corynebacterium strain for glycerol production, ATCC13032 strain was used as a model microorganism. Plasmids were introduced into the ATCC13032 strain via electroporation under conditions of 25 uF, 200 Ω, and 2.5 kV / cm. To select strains containing the plasmid, screening was performed on BHI agar medium containing 200 μg / mL spectinomycin. Gene expression was performed using a constitutive expression system, and no separate inducer was added.

[0061] The E. coli W3110 strain was used as Comparative Example 1, and the Corynebacterium ATCC13032 strain was used as Comparative Example 2. The strain in which the pTrc99A-g1pD-dgp plasmid was introduced into the E. coli W3110 strain was used as Production Example 1, and the strain in which the pTrc99A-araM-dgp plasmid was introduced into the E. coli W3110 strain was used as Production Example 2. The strain in which the pTrc99A-g1pD-dgp plasmid was introduced into the Corynebacterium ATCC13032 strain was used as Production Example 3, and the strain in which the pTrc99A-araM-dgp plasmid was introduced into the Corynebacterium ATCC13032 strain was used as Production Example 4.

[0062] [Table 1]

[0063]

[0064]

[0065] <Example 2> Verification of glycerol production by recombinant E. coli

[0066] The glycerol production ability was evaluated for the recombinant E. coli strains W3110 of Comparative Example 1 and Manufacturing Examples 1 and 2.

[0067] First, flask culture was performed for the above microorganisms as follows. Modified MR medium was used for culture, and 20 g / L glucose was added as a carbon source. A 250 mL Erlenmeyer flask was used with a working volume of 25 mL. Culture was performed for 24 hours at an initial pH of 7.0, a temperature of 37°C, and a shaking incubator at 200 rpm. The detailed composition of the modified MR medium is as follows. After the completion of culture for the wild-type and recombinant E. coli, samples were collected, and the collected samples were centrifuged at 13,000 rpm for 10 minutes, and the glycerol concentration of the supernatant was analyzed by high-performance liquid chromatography (HPLC).

[0068] [Table 2]

[0069]

[0070] As a result, it was confirmed that Comparative Example 1 did not produce glycerol at all. It was confirmed that Manufacturing Example 1 produced 1.78 g / L of glycerol. It was confirmed that Manufacturing Example 2 produced 0.13 g / L of glycerol. That is, while wild-type E. coli cannot produce glycerol at all, it was confirmed that recombinant E. coli transformed with a Bacillus licheniformis-derived gene is capable of glycerol biosynthesis, and it was confirmed that the case transformed with the g1pD gene secured a higher glycerol production ability than the case transformed with the araM gene (see Fig. 2).

[0071] <Example 3> Verification of glycerol production by recombinant Corynebacterium

[0072] The glycerol production ability was evaluated for ATCC13032 of Comparative Example 2 and the recombinant Corynebacterium of Manufacturing Examples 3 and 4.

[0073] First, flask culture was performed for the above microorganisms as follows.

[0074] Modified CGXII medium was used for cultivation, and 100 g / L glucose was added as a carbon source. A 250 mL baffled flask was used, and the working volume was 25 mL. Cultivation was performed for 24 hours at an initial pH of 7.0, a temperature of 30°C, and a 200 rpm shaking incubator. The detailed modified CGXII medium composition is as follows. Samples were collected after the completion of cultivation for the wild type and recombinant Corynebacterium, and the collected samples were centrifuged at 13,000 rpm for 10 minutes, and the glycerol concentration of the supernatant was analyzed by high-performance liquid chromatography (HPLC).

[0075] [Table 3]

[0076]

[0077] As a result, it was confirmed that Comparative Example 2 did not produce glycerol at all. It was confirmed that Manufacturing Example 3 produced 1.10 g / L of glycerol. It was confirmed that Manufacturing Example 4 produced 0.16 g / L of glycerol. That is, while wild-type Corynebacterium cannot produce glycerol at all, it was confirmed that recombinant Corynebacterium transformed with a Bacillus licheniformis-derived gene is capable of glycerol biosynthesis, and it was confirmed that the case transformed with the g1pD gene secured a higher glycerol production ability than the case transformed with the araM gene (see Fig. 3).

[0078] Although the embodiments of this specification have been described in more detail above, this specification is not necessarily limited to these embodiments, and various modifications may be implemented without departing from the technical spirit of this specification. Therefore, the embodiments disclosed in this specification are not intended to limit the technical spirit of the present invention, but to explain it, and the scope of the technical spirit of the present invention is not limited by these embodiments. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The protection scope of this specification and the present invention should be interpreted by the claims, and all technical ideas within a scope equivalent thereto should be interpreted as being included in the scope of rights of this specification and the present invention.

[0079] Name of depositor: Korea Research Institute of Bioscience and Biotechnology

[0080] Accession number: KCTC14485BP

[0081] Date of acceptance: 20210305

[0082]

Claims

1. A recombinant microorganism having a glycerol biosynthetic pathway, Pathway that converts DHAP to glycerol 1-phosphate (G1P); and Pathway from the above G1P to glycerol; A recombinant microorganism comprising:

2. In paragraph 1, The recombinant microorganism is a recombinant microorganism containing glycerol 1-phosphate dehydrogenase (G1PDH).

3. In paragraph 2, The above G1PDH is a recombinant microorganism encoded by the base sequence of sequence number 1.

4. In paragraph 2, The above G1PDH is a recombinant microorganism encoded by the base sequence of sequence number 2.

5. In paragraph 1, The above recombinant microorganism is a recombinant microorganism containing glycerol 1-phosphatase (glycerol 1-phosphate phosphohydrolase; G1PP).

6. In paragraph 5, The above G1PP is a recombinant microorganism encoded by the base sequence of sequence number 3.

7. In paragraph 1, The above recombinant microorganism is a bacterial recombinant microorganism.

8. In paragraph 1, The above recombinant microorganism is Escherichia coli, a recombinant microorganism.

9. In paragraph 1, The above recombinant microorganism is a recombinant microorganism, Corynebacterium glutamicum.

10. A step of producing glycerol by culturing the microorganism of paragraph 1 in a medium containing a carbon source; and Step of recovering glycerol; A method for producing glycerol, comprising:

11. In paragraph 10, A method for producing glycerol, wherein the carbon source comprises at least one selected from the group consisting of sucrose, glucose and fructose.

12. In paragraph 10, A method for producing glycerol, wherein the carbon source is glucose.

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