Escherichia coli engineering strain for producing beta, gamma type dihydric alcohol and construction method and application thereof

Abstract

The present application relates to a kind of engineering strains of escherichia coli for producing β, γ-type diol and its construction method and application, which method is by constructing the recombinant plasmid pET-ARO10 of gene ARO10 expressing phenylpyruvic acid decarboxylase;Construction of the recombinant plasmid pRSF-ScIlv2c of gene ScIlv2 expressing acetyl-hydroxy acid synthase;The recombinant plasmid pET-ARO10 and the recombinant plasmid pRSF-ScIlv2c are introduced into escherichia coli, to obtain the engineering strains of escherichia coli for producing β, γ-type diol.The engineering strains of escherichia coli can produce β, γ-type diol 4-methyl pentane-2, 3-diol, 5-methyl hexane-2, 3-diol, 4-methyl hexane-2, 3-diol, realize the efficient synthesis of β, γ-type diol in escherichia coli, can be applied to medicine, biofuel, novel polymer material and the like industry.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly relates to an Escherichia coli engineering strain for producing β,γ-diol, a construction method thereof, and an application thereof. Background Art

[0002] Diols are a class of important chemical raw materials, which are widely used in daily necessities and fine chemical industries such as fuels, solvents, polymer monomers, and pharmaceutical precursors. The traditional diol synthesis process relies on the processing of petroleum raw materials, which has an impact on the environment and resources. In recent years, many progress have been made in the biosynthesis of diols. With the establishment of cell factories and the proposal of various biosynthetic pathways, structurally diverse diols can be obtained by fermenting various microbial chassis such as Escherichia coli, Corynebacterium glutamicum, Klebsiella pneumoniae, Bacillus licheniformis, or Bacillus amyloliquefaciens using renewable raw materials such as glycerol, glucose, xylose, sucrose, and hydrolysates of cellulose. Currently, the biosynthesis of some short-chain diols has made important developments and achieved commercialization. For example, DuPont in the United States successfully used an engineered bacterium to convert glucose hydrolyzed from corn into 1,3-propanediol (α,γ-diol).

[0003] β,γ-diol, compared with diols with the same molecular structure, has a higher melting point, a lower boiling point, and a higher flash point. The advantages in its physical properties make it possible to be used as a rocket propellant fuel, a component of synthetic rubber, and polymers. However, at present, except for 2,3-butanediol, there are still bottlenecks in the biosynthesis technology for synthesizing β,γ-diol. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems in the related technologies. For this reason, an object of the present invention is to provide an Escherichia coli engineering strain for producing β,γ-diol, a construction method thereof, and an application thereof; realizing the conversion of various renewable raw materials glycerol or glucose into β,γ-diol with a C6-C7 structure in an Escherichia coli chassis.

[0005] For this reason, in the first aspect of the present invention, the present invention proposes a construction method for an Escherichia coli engineering strain for producing β,γ-diol, which includes the following steps:

[0006] Construct a recombinant plasmid pET-ARO10 expressing the gene ARO10 of phenylpyruvate decarboxylase;

[0007] Construct a recombinant plasmid expressing the gene Sc Ilv2 of acetohydroxyacid synthase Sc pRSF-

[0008] Combine the recombinant plasmid pET-ARO10 and the recombinant plasmid pRSF- ScIlv2c was introduced into Escherichia coli to obtain an engineered Escherichia coli strain that produces β,γ-type diols.

[0009] According to the present invention, a method for constructing an engineered Escherichia coli strain for producing β,γ-diols is provided, wherein the method involves introducing the phenylpyruvate decarboxylase gene ARO10 and the acetylhydroxy acid synthase gene into Escherichia coli. Sc Ilv2 enables the constructed Escherichia coli engineered strain to produce β,γ-type diols such as 4-methylpentane-2,3-diol, 5-methylhexane-2,3-diol, and 4-methylhexane-2,3-diol, achieving efficient synthesis of β,γ-type diols in Escherichia coli. This technology can be applied in industries such as pharmaceuticals, biofuels, and novel polymer materials.

[0010] Optionally, the nucleotide sequence of the gene ARO10 encoding the phenylpyruvate decarboxylase is shown in SEQ ID NO: 1; the gene encoding the acetylhydroxy acid synthase... Sc The nucleotide sequence of Ilv2 is shown in SEQ ID NO: 2.

[0011] Optionally, the *Escherichia coli* is *Escherichia coli*. E. coli K-12 MG1655 RARE .

[0012] Optionally, the recombinant plasmid pET-ARO10 is constructed using Saccharomyces cerevisiae. Saccharomyces cerevisiae S288C Using the genome as a template, and the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 as primers, the ARO10 gene fragment was amplified by PCR and ligated into the vector pETDuet-1 to obtain the recombinant plasmid pET-ARO10 with ampicillin resistance.

[0013] Optionally, the recombinant plasmid pRSF- Sc Ilv2c is constructed using brewer's yeast. Saccharomyces cerevisiae S288C Using the genome as a template and the sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 as primers, PCR amplification was performed to obtain... Sc The Ilv2 gene fragment was ligated into pRSFDuet-1 to obtain the recombinant plasmid pRSF- which possesses kanamycin resistance. Sc Ilv2c.

[0014] In a second aspect, the present invention provides an engineered strain of *Escherichia coli* that produces β,γ-type diols, obtained by the above-described construction method.

[0015] In a third aspect, the present invention provides a method for producing β,γ-type diols by fermentation using the aforementioned engineered strain of Escherichia coli.

[0016] Alternatively, the engineered strain of *Escherichia coli* can be fermented in a modified M9 medium using glycerol or glucose as a carbon source to produce β,γ-type diols.

[0017] Optionally, the β,γ type diol includes 4-methylpentane-2,3-diol, 5-methylhexane-2,3-diol, and 4-methylhexane-2,3-diol.

[0018] Optionally, the concentration of the glycerol or glucose is 40 g / L.

[0019] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0020] Figure 1 This is a gel electrophoresis image of the PCR amplification of the target gene according to an embodiment of the present invention;

[0021] Figure 2 The image shows the recombinant plasmid pET-ARO10 according to an embodiment of the present invention.

[0022] Figure 3 The recombinant plasmid pRSF- according to an embodiment of the present invention Sc The graph of Ilv2c;

[0023] Figure 4 This is a gas chromatographic detection diagram of diol production by recombinant strains according to an embodiment of the present invention;

[0024] Figure 5 This is a gas chromatography-mass spectrometry (GC-MS) result of diols produced by the recombinant strain according to an embodiment of the present invention.

[0025] Figure 6 A graph showing the yield of diols produced by the recombinant strain of the present invention using different carbon sources during fermentation.

[0026] Figure 7 To compare the biomass (OD600) of recombinant strains using different carbon sources according to embodiments of the present invention;

[0027] Figure 8 This is a schematic diagram of the synthesis pathway of diols according to an embodiment of the present invention. Detailed Implementation

[0028] The technical solution of the present invention is illustrated below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or defining the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0029] To better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0030] The test materials used in this invention are all commercially available products and can be purchased on the market; unless otherwise specified, the experiments involved are all conventional experimental methods.

[0031] Escherichia coli K-12 MG1655 RARE is available commercially (Addgene: E. coli K-12 MG1655 RARE).

[0032] Table 1: Primers used for PCR amplification

[0033]

[0034] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0035] Example 1: Target Gene Amplification and Recombinant Plasmid Construction

[0036] (1) According to NCBI data Saccharomyces cerevisiae S288C The ARO10 gene (Gene ID: 851987) encoding phenylpyruvate decarboxylase was used to design the ARO10-F and ARO10-R primer sequences (Table 1). Based on data from... Saccharomyces cerevisiae S288C The gene encoding acetylhydroxyl synthase Sc Ilv2 (Gene ID: 855135), designed to obtain Sc Ilv2-F and Sc Ilv2-R primer sequences (Table 1).

[0037] (2) Using the sequences shown in SEQ ID NO:3 and SEQ ID NO:4 as upstream and downstream primers (Table 1), using Saccharomyces cerevisiae. Saccharomyces cerevisiae S288C The genome of *Saccharomyces cerevisiae* (Gene ID: 851987) was used as a template for PCR amplification of the ARO10 gene fragment. The nucleotide sequence of the ARO10 gene fragment is shown in SEQ ID NO: 1. Using the sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 as upstream and downstream primers (Table 1), *Saccharomyces cerevisiae* was used for PCR amplification. Saccharomyces cerevisiae S288C The genome (Gene ID: 855135) was used as a template for PCR amplification. Sc Ilv2 gene fragment, Sc The nucleotide sequence of the Ilv2 gene fragment is shown in SEQ ID NO: 2. The PCR enzyme used for amplification was Phusion® High-Fidelity DNA Polymerases from New England Biolabs. The reaction system is shown in Table 2; the PCR reaction conditions are shown in Table 3.

[0038] Table 2 PCR reaction system

[0039]

[0040] Table 3 PCR amplification program

[0041]

[0042] The PCR amplification products were verified by agarose gel electrophoresis. The PCR results for the ARO10 gene are as follows: Figure 1 As shown in Figure A, the size of the amplified fragment is between 1kb and 2kb, which is basically consistent with the size of the ARO10 gene. Sc The PCR results for Ilv2 are as follows: Figure 1 As shown in B, the amplified fragment size is around 3kb, which is basically consistent with... Sc Ilv2c gene size. The target band was cut, and the target fragment ARO10 was purified and recovered using a BioFlux DNA purification kit. Sc Ilv2.

[0043] (3) Using restriction endonucleases from New England Biolabs BamHI- HF® and XhoI For the target fragment ARO10, Sc Ilv2 and the vectors pRSFDuet-1 and pETDuet-1 were digested with enzymes, and the reaction system is shown in Table 4.

[0044] Table 4 Enzyme digestion system

[0045]

[0046] Fragments and vectors cut by restriction endonucleases were purified using a DNA purification kit from BioFlux.

[0047] (4) The fragments digested by restriction endonucleases and the vector were ligated using T4 ligase from New England Biolabs. The gene for phenylpyruvate decarboxylase ARO10 was inserted into the BamHI and XhoI sites of pETDuet-1; acetylhydroxy acid synthase... Sc The Ilv2c gene was inserted into the BamHI and XhoI sites of pRSFDuet-1. The ligation system is shown in Table 5.

[0048] Table 5 Connection System

[0049]

[0050] After ligation at room temperature for 3-4 hours, the ligation product was transformed into *E. coli* DH5α competent cells. Positive clones were screened using ampicillin and kanamycin, respectively. Colony PCR was used to verify positive single clones, and sequencing was performed. Once confirmed, the plasmid was extracted to obtain the recombinant plasmid pET-ARO10. Figure 2 As shown; and the recombinant plasmid pRSF- Sc Ilv2c, such as Figure 3 As shown.

[0051] Example 2 Construction of recombinant strains

[0052] Original strain E. coli K-12MG1655 RARE After culturing overnight in 2 mL of antibiotic-free LB medium, electrocompetent cells were prepared. The preparation method for electrocompetent cells was as follows: 1) Inoculate the bacterial culture into 100 mL of LB medium at a ratio of 1:100. Shake for approximately 2-3 hours until the OD600 reaches between 0.6 and 0.8. 2) Incubate the bacterial culture on ice for 10 minutes, then centrifuge at 7000 rpm for 10 minutes at 4°C, and remove the supernatant. Resuspend the bacterial pellet in double-distilled water. 3) Centrifuge at 7000 rpm for 10 minutes at 4°C, remove the supernatant, and resuspend the bacterial pellet in 10% glycerol. 4) Repeat step 3) once. 5) Add 5 mL of 10% glycerol to resuspend the bacterial pellet, aliquot into EP, freeze in liquid nitrogen, and store at -80°C.

[0053] The recombinant plasmid pET-ARO10 and recombinant plasmid pRSF- were converted by electroporation. Sc Ilv2c transfer E. coli K-12 MG1655 RARE Competent cells were revived by adding 1 mL of LB medium for one hour.

[0054] Screening was performed using an LB platform supplemented with ampicillin and kanamycin. Positive single clones were obtained after overnight culture and named MR- Sc Ilv2-ARO10. Positive bacterial culture was stored at -80°C after adding 20% ​​glycerol.

[0055] Example 3: Application of recombinant strains in the production of β,γ-type diols

[0056] (1) Culture of engineered strains: The recombinant strain MR- obtained in Example 2 was cultured. Sc Ilv2-ARO10 was removed and streaked onto a solid LB agar plate containing kanamycin and ampicillin, and incubated overnight at 37°C. The recombinant strain MR- was picked the next day. Sc Ilv2-ARO10 monoclonal isolates were inoculated into liquid LB medium supplemented with kanamycin and ampicillin and incubated at 37°C with shaking at 250 rpm for 8–10 hours as the fermentation seed culture. The seed culture was then inoculated at a 1% inoculation rate into 10 mL of M9 medium supplemented with kanamycin and ampicillin; incubated at 37°C with shaking at 250 rpm for 4 hours until the OD600 reached a range of 0.8–10. IPTG was added to a final concentration of 100 μM. The culture was then further incubated at 30°C with shaking at 250 rpm for 108 hours, with samples taken every 24 hours for analysis.

[0057] The other components of the M9 culture medium include: 11.1 µg / L CaCl2, 240.7 mg / L MgSO4, 17.08 g / L Na2HPO4·12H2O, 3 g / L KH2PO4, 1 g / L NH4Cl, 0.5 g / L NaCl; trace elements: 50 mg / L EDTA, 8.3 mg / L FeCl3·6H2O, 0.84 mg / L ZnCl2, 0.13 mg / L CuCl2·2H2O, 0.1 mg / L CoCl2·6H2O, 0.1 mg / L H3BO3, 16 mg / L MnCl2·6H2O, 0.3 mg / L Na2MoO4·4H2O, 5 mg / L thiamine hydrochloride, 10 mg / L nicotinic acid, 0.1 mg / L biotin (pH 7.2), 7 g / L yeast extract, and 4% (w / v) [unspecified ingredient]. Glucose or 4% (w / v) glycerol.

[0058] (2) Diol extraction

[0059] Take 300 μL of the well-mixed fermentation broth and transfer it to a 1 mL EP tube. Mix with 300 μL of ethyl acetate. Vortex for 2 minutes to extract the diol from the fermentation broth layer to the ethyl acetate layer. Centrifuge at 14000 rpm for 5 minutes, and take 200 μL of ethyl acetate for analysis.

[0060] (3) Diol detection

[0061] Diols were determined by gas chromatography using a Nexis GC-2030 instrument equipped with a flame ionization detector (FID) on an SH-Rtx-5 capillary column (25 m × 0.32 mm × 0.5 μm). A split syringe was used, with 1 μL of sample injected per column. Nitrogen was used as the carrier gas at a flow rate of 1.0 mL / min. The column oven temperature program was: initial temperature 40 °C (hold for 2 min), then ramped to 250 °C at a rate of 15 °C / min (hold for 2 min). The FID detector was operated at 350 °C. Quantification of diols was achieved using external standard calibration.

[0062] The results are as follows Figure 4 As shown, this is the recombinant strain MR- Sc The gas chromatogram of diol production by Ilv2-ARO10 shows that the recombinant strain MR- Sc Ilv2-ARO10 can produce β,γ-type diols: 4-methylpentane-2,3-diol, 5-methylhexane-2,3-diol, and 4-methylhexane-2,3-diol, achieving efficient synthesis of β,γ-type diols in Escherichia coli.

[0063] (4) Identification of diol structure

[0064] GC / MS analysis was performed on an Agilent 7890B gas chromatograph coupled with a mass spectrometer to confirm the formation of the target diol. An Agilent DB-5ms UI capillary column (30 m × 250 μm × 0.25 μm) was used. A split syringe operating at 250 °C was used, with 1 μL of sample injected per column. Helium was used as the carrier gas, and the column flow rate was 1.0 mL / min. The column oven temperature program was: initial temperature 40 °C (hold for 2 min), then ramped to 250 °C at a rate of 15 °C / min (hold for 2 min). MS was run in scan mode of 2–500 amu with a solvent delay of 4.5 min. The MS ion source and MS Quad temperatures were set to 230 °C and 150 °C, respectively.

[0065] The results are as follows Figure 5 As shown, by comparing the mass spectra with those in the NIST database to confirm the same MS fragment peaks, the formation of β,γ-type diols: 4-methylpentane-2,3-diol, 5-methylhexane-2,3-diol, and 4-methylhexane-2,3-diol was confirmed.

[0066] Example 4: Fermentation optimization of recombinant strains

[0067] The fermentation method was the same as in Example 3 above; glycerol and glucose were selected as the two basic carbon sources for comparison on the culture medium. 40 g / L glycerol or 40 g / L glucose was added to the basic M9 medium to prepare a modified M9 medium. Fermentation was carried out at 30°C and 250 rpm for 108 hours. Samples were taken every 24 hours to test the OD600 and metabolite parameters, comparing the effects of different carbon sources on biomass and diol production.

[0068] The results are as follows Figure 6 and Figure 7 As shown, comparisons of different carbon sources revealed that using glycerol as a carbon source resulted in higher diol yields and biomass than using glucose. The yields of 4-methylpentane-2,3-diol reached 4.4 ± 0.3 mM, 5-methylhexane-2,3-diol reached 4.4 ± 0.5 mM, and 4-methylhexane-2,3-diol reached 0.2 mM.

[0069] In summary, according to embodiments of the present invention, such as Figure 8 As shown, the phenylpyruvate decarboxylase ARO10 gene and acetylhydroxy acid synthase from Saccharomyces cerevisiae were used. Sc Ilv2 gene introduced into E. coli E. coli K-12 MG1655 RARE In the process, recombinant strains were obtained. The construction method of recombinant strains is simple, and fermentation experiments have shown that recombinant strains can produce β,γ-type diols such as 4-methylpentane-2,3-diol, 5-methylhexane-2,3-diol, and 4-methylhexane-2,3-diol. This realizes the conversion of various renewable raw materials, glycerol, into C6-C7 structured β,γ-type diols on the E. coli chassis. Furthermore, the recombinant strains are fermented using renewable carbon sources such as glycerol or glucose, which is a green and sustainable method for diol synthesis.

[0070] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. The illustrative expressions of the above terms in this specification should not be construed as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0071] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for constructing an engineered strain of *Escherichia coli* that produces β,γ-type diols, characterized in that, Includes the following steps: A recombinant plasmid pET-ARO10 expressing the gene ARO10 of phenylpyruvate decarboxylase was constructed; the nucleotide sequence encoding the gene ARO10 of phenylpyruvate decarboxylase is shown in SEQ ID NO: 1; Construct a gene expressing acetylhydroxyl synthase Sc Ilv2 recombinant plasmid pRSF- Sc Ilv2c; the gene encoding the acetylhydroxy acid synthase. Sc The nucleotide sequence of Ilv2 is shown in SEQ ID NO: 2; The recombinant plasmid pET-ARO10 and the recombinant plasmid pRSF- Sc Ilv2c was introduced into Escherichia coli to obtain an engineered Escherichia coli strain that produces β,γ-type diols.

2. The construction method according to claim 1, characterized in that, The *E. coli* is *Escherichia coli*. E. coli K- 12 MG1655 RARE .

3. The construction method according to claim 1, characterized in that, The recombinant plasmid pET-ARO10 was constructed using Saccharomyces cerevisiae. Saccharomyces cerevisiae S288C Using the genome as a template, and the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 as primers, the ARO10 gene fragment was amplified by PCR and ligated into the vector pETDuet-1 to obtain the recombinant plasmid pET-ARO10 with ampicillin resistance.

4. The construction method according to claim 1, characterized in that, The recombinant plasmid pRSF- Sc Ilv2c is constructed using brewer's yeast. Saccharomyces cerevisiae S288C Using the genome as a template, and the sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 as primers, PCR amplification was performed to obtain... Sc The Ilv2 gene fragment was ligated into pRSFDuet-1 to obtain the recombinant plasmid pRSF- which possesses kanamycin resistance. Sc Ilv2c.

5. An engineered strain of *Escherichia coli* that produces β,γ-type diols, characterized in that, It is constructed by the construction method of any one of claims 1-4.

6. A method for producing β,γ-type diols, characterized in that, The engineered strain of Escherichia coli described in claim 5 is used to produce β,γ-type diols through fermentation. The engineered strain of *Escherichia coli* was fermented in a modified M9 medium with glycerol or glucose as the carbon source to produce β,γ-type diols. The β,γ type diols include 4-methylpentane-2,3-diol, 5-methylhexane-2,3-diol, and 4-methylhexane-2,3-diol.

7. The method as described in claim 6, characterized in that, The concentration of the glycerol or glucose is 40 g / L.

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