Method for constructing stably inherited genetic engineering strain for efficient biosynthesis of p-hydroxyphenylacetic acid and application thereof
By integrating pathway genes and Saccharomyces cerevisiae genes in host bacteria, a genetically engineered strain with high yield of hydroxyphenylacetic acid was solved, and the problem of plasmid instability affected production was achieved, efficient and stable hydroxyphenylacetic acid biosynthesis was achieved, and production costs were reduced. .
Patent Information
- Application Number
- CN202410763495.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-13
- Publication Date
- 2025-01-17
AI Technical Summary
In the existing biosynthesis methods, the instability of the plasmid affects the activity and yield of engineered E. coli, resulting in high production cost and low catalytic efficiency of hydroxyphenylacetic acid.
By integrating pathway genes into the genome of the host bacteria, overexpressing the shikimate pathway gene, and integrating the phenylphenate decarboxylase and phenylacetaldehyde dehydrogenase genes from Saccharomyces cerevisiae, genetically engineered strains with high yield of hydroxyphenylacetic acid were constructed and PTS was modified. Systematic, knock out competition pathway genes to avoid instability in plasmid expression.
The yield of hydroxyphenylacetic acid is increased to 3.6±0.03g/L, which improves production stability, reduces production costs, avoids the problems of plasmid loss and structural instability, and achieves efficient biosynthesis.
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Figure CN119320734A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of bioengineering, and in particular to the construction of a high-yield p-hydroxyphenylacetic acid strain and a method and application thereof. Background Art
[0002] p-Hydroxyphenylacetic acid is an important synthetic intermediate of phenolic acids. The application of p-Hydroxyphenylacetic acid covers many fields such as medicine, pesticides, and cosmetics. In the pharmaceutical industry, p-Hydroxyphenylacetic acid can be aminated to obtain p-Hydroxyphenylacetamide, etherified to obtain methoxyphenylacetic acid, esterified to obtain p-Hydroxyphenylacetic acid methyl ester, and reduced to obtain p-Hydroxyphenylethanol. These derivatives are all good pharmaceutical intermediates. The drugs synthesized from it mainly include cardiovascular drug atenolol, anti-inflammatory and analgesic drug benoxaprofen, antibiotic drug latamoxef sodium, etc. In the pesticide industry, p-Hydroxyphenylacetic acid can be used as a pesticide raw material to synthesize the pyrethroid insecticide cypermethrin. At the same time, p-Hydroxyphenylacetic acid is also widely used in various cosmetics and skin care products, such as exfoliants, whitening products, and anti-acne products. p-Hydroxyphenylacetic acid is also the active ingredient of salidroside and the Chinese herbal medicine Aster. There are two main methods for obtaining p-hydroxyphenylacetic acid: chemical synthesis and biosynthesis. Due to the high cost of raw materials and low catalytic efficiency, chemical synthesis of p-hydroxyphenylacetic acid is not the first choice. The biosynthesis method can use glucose as a carbon source to construct a plasmid to transform the pathway gene into the host strain to synthesize p-hydroxyphenylacetic acid from scratch. However, the unstable genetics of the plasmid affect the activity of the production strain and reduce the maintenance coefficient of the engineered Escherichia coli.
[0003] The present invention integrates some pathway genes into the genome of the host bacteria, and p-hydroxyphenylacetic acid can be produced without an expression plasmid, thereby improving the stability of the production function of the engineered strain and saving production costs. At the same time, the shikimic acid pathway genes are overexpressed to increase the carbon flux of the p-hydroxyphenylacetic acid production pathway, thereby increasing the output of p-hydroxyphenylacetic acid by 9 times, and the output of p-hydroxyphenylacetic acid is 3.6±0.03 g / L. Summary of the invention
[0004] 1. An object of the present invention is to provide a genetically engineered bacterium that produces high-yield p-hydroxyphenylacetic acid.
[0005] 2. In the above-mentioned genetically engineered bacteria, the target bacteria is Escherichia coli.
[0006] 3. The genetically engineered bacteria constructed by the present invention overexpress the shikimic acid pathway, transform the PTS system and knock out the genes of the competitive pathway to construct a genetically engineered strain with high p-hydroxyphenylpyruvic acid production. Then, genes encoding phenylpyruvic acid decarboxylase (ARO10) and phenylacetaldehyde dehydrogenase (feaB) from Saccharomyces cerevisiae are integrated into the engineered bacteria to construct a strain with high p-hydroxyphenylacetic acid production. The experimental results show that the engineered strain can produce p-hydroxyphenylacetic acid with a final yield of 3.6±0.03 g / L using glucose as a simple carbon source.
[0007] 4. The above-mentioned genetically engineered bacteria with high yield of p-hydroxyphenylacetic acid: the genes encoding phosphate carrier protein (ptsH), pyruvate kinase (pykA, pykF), tyrosine aminotransferase (tyrB), aspartate aminotransferase (aspC), and branched-chain amino acid aminotransferase (ilvE) are knocked out in the host, and then the genes encoding phenylpyruvate decarboxylase (ARO10) and phenylacetaldehyde dehydrogenase (feaB) from Saccharomyces cerevisiae are integrated into the host, and finally the genes encoding 3-deoxy-7-phosphoheptanoate synthase (AroG), shikimate kinase (AroL), chorismate synthase (AroC), 3-dehydroquinoline synthase (AroB), and 3-dehydroquinoline dehydratase (AroD) are integrated into the host.
[0008] 5. The present invention also provides a method for constructing the above-mentioned high-yield p-hydroxyphenylacetic acid engineering bacteria, comprising the steps of:
[0009] Construction of genetically engineered strains: The crispr cas9 technology was used to knock out the genes encoding phosphate carrier protein (ptsH), pyruvate kinase (pykA, pykF), tyrosine aminotransferase (tyrB), aspartate aminotransferase (aspC), and branched-chain amino acid aminotransferase (ilvE) in the host to construct a strain capable of producing p-hydroxyphenylpyruvate. Then, the crispr cas9 technology was used to integrate the genes encoding phenylpyruvate decarboxylase (ARO10) and phenylacetaldehyde dehydrogenase (feaB) from Saccharomyces cerevisiae into the host to construct an engineered strain capable of producing p-hydroxyphenylacetic acid. Finally, the genes encoding 3-deoxy-7-phosphoheptanoate synthase (AroG), shikimate kinase (AroL), chorismate synthase (AroC), 3-dehydroquinoline synthase (AroB), and 3-dehydroquinoline dehydratase (AroD) were integrated into the host to construct an engineered strain capable of stably and highly producing p-hydroxyphenylacetic acid.
[0010] 6. In the integration site of the genetically engineered bacteria, genes ARO10 and feaB replace the pseudo-sense gene mbhA on the genome, and genes aroG, aroL, aroC, aroB, and aroD replace the pseudo-sense gene ilvG on the genome.
[0011] 7. The integration sites described in the genetically engineered bacteria are selected because the genes mbhA and ilvG are meaningless sites in E. coli, so replacing them with other genes will not affect the normal growth of E. coli.
[0012] 8. The present invention also provides an application of a genetically engineered bacterium with high p-hydroxyphenylacetic acid production, wherein the engineered bacterium is streaked onto a plate without antibiotics, cultured overnight at 37°C, and positive monoclonal transformants are picked to obtain a genetically engineered strain with high p-hydroxyphenylacetic acid production, which is inoculated into a fermentation medium and fermented at 33°C to produce p-hydroxyphenylacetic acid from scratch.
[0013] 9. Based on the above, the fermentation medium includes 20g / L glucose, 5g / L yeast powder, 2g / L MOPS, 5g / LNa2HPO4, 0.5g / L NaCl, 3g / L KH2PO4, 1g / L NH4Cl, 250mg / L MgSO4, 15mg / L CaCl2, and the solvent is water.
[0014] 10. The genetically engineered bacteria provided by the present invention are constructed by transforming the PTS system, integrating the pathway gene into the genome of the host bacteria, and overexpressing the shikimic acid pathway gene. First, a genetically engineered strain capable of high-yield p-hydroxyphenylpyruvate is constructed, and then genes encoding phenylpyruvate decarboxylase (ARO10) and phenylacetaldehyde dehydrogenase (feaB) derived from Saccharomyces cerevisiae are co-expressed in the above-mentioned engineered bacteria to synthesize p-hydroxyphenylacetic acid from scratch, and the final p-hydroxyphenylacetic acid yield can reach 3.6±0.03 g / L.
[0015] 11. Compared with the original recombinant engineered bacteria that need to introduce plasmids to produce p-hydroxyphenylacetic acid, the genetically engineered bacteria provided by the present invention have the following advantages: (1) the production of the genetically engineered bacteria is more stable, because the unstable genetics of the plasmids affect the activity of the production strain, which reduces the maintenance coefficient of the engineered Escherichia coli. This is due to 1) the loss of plasmids, that is, the uneven distribution of plasmids to daughter cells leads to plasmid-free cells; 2) the structural instability of the plasmids, in which the DNA sequences of some plasmids are prone to mutation, resulting in incorrect expression of the desired protein; the above two reasons cause a large number of inactive bacteria in the fermentation system, resulting in a waste of carbon sources and a reduction in the yield of the target product. (2) The production of the genetically engineered bacteria is more cost-effective, because a strain that naturally produces p-hydroxyphenylacetic acid is constructed by genetically modifying wild-type Escherichia coli, that is, no antibiotics and inducers are required, and as long as there is sufficient carbon source, the efficient biosynthesis of p-hydroxyphenylacetic acid can be achieved while ensuring the stable genetics of the strain.
[0016] 12. The genetically engineered bacteria provided by the present invention achieve high production of p-hydroxyphenylacetic acid, avoiding the problems of high raw material cost and low catalytic efficiency; and the constructed integrated strain can achieve high production of p-hydroxyphenylacetic acid without the need for an expression plasmid, thereby alleviating the effect of multiple plasmid expressions on the unstable production of the engineered strain, and does not require the addition of antibiotics to maintain strain growth, nor does it require the addition of inducers to induce gene expression, thereby reducing the production cost of p-hydroxyphenylacetic acid and being more conducive to the industrial production of p-hydroxyphenyl ethyl segment. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 The present invention provides a biosynthetic pathway for p-hydroxyphenylacetic acid.
[0018] Figure 2 This is a diagram showing the fermentation results of BW producing p-hydroxyphenylacetic acid provided in Example 1 of the present invention.
[0019] Figure 3A This is the HPLC analysis result of the standard product p-hydroxyphenylacetic acid; Figure 3B This is a HPLC detection chart of the fermentation product of the strain provided in Example 1.
[0020] Figure 4 This is a diagram showing the fermentation results of the engineering bacteria BW2 provided in Example 3 of the present invention producing p-hydroxyphenylacetic acid.
[0021] Figure 5 This is a diagram showing the fermentation results of the engineering bacteria BW3 provided in Example 5 of the present invention producing p-hydroxyphenylacetic acid. DETAILED DESCRIPTION
[0022] The present invention will be further described below with reference to the accompanying drawings and implementation examples.
[0023] 1. Construction of the control strain pZE-pCS / BW for producing p-hydroxyphenylacetic acid using plasmid
[0024] (1) Construction of recombinant plasmid: control strain
[0025] i. The recombinant plasmid pZE-ARO10-feaB is mainly obtained by replacing the DNA fragment between the kpnI and XbaI restriction sites of the pZE vector with the gene-ARO10-feaB, and finally constructing pZE-ARO10-feaB.
[0026] ii. The recombinant plasmid pCS-tyrA-aroG is mainly obtained by replacing the DNA fragment between the kpnI and BamHI restriction sites of the pCS vector with the gene -tyrA-aroG, and finally constructing pCS-tyrA-aroG.
[0027] (2) The recombinant vectors pZE-ARO10-feaB and pCS-tyrA-aroG were introduced into the genetically engineered bacteria by electroporation. The positive clones were selected on ampicillin and kanamycin plates and named pZE-pCS / BW. The transformants were cultured at 37°C overnight to prepare a recombinant strain that produces p-hydroxyphenylacetic acid.
[0028] (3) Pick a single colony from the plate of the above recombinant strain, inoculate it into 4 ml of liquid LB medium containing ampicillin and canadensis penicillin, and culture it at 37°C for 12 hours. Transfer the bacterial liquid into 50 ml of fermentation medium and add IPTG for induction. Then, take samples at 12, 24, 36, 48, 60, and 72 hours and determine the concentration of the target product p-hydroxyphenylacetic acid by high performance liquid chromatography. The final yield is shown in the figure below. Figure 2 described.
[0029] (4) The generated product p-hydroxyphenylacetic acid was detected by high performance liquid chromatography (HPLC) analysis method, and the detection conditions were as follows:
[0030] Chromatographic column: Separation column: Diamonsil C18, ID 5 μm, 250 × 4.6 mm;
[0031] Mobile phase: The organic phase is pure methanol, the mobile phase is 1 / 1000 trifluoroacetic acid aqueous solution, the column temperature is 40°C, the flow rate is 1mL / min, and the detection wavelength is 255nm. The gradient elution program is shown in the following table:
[0032] Time (min) Organic phase A% Mobile phase B% 0 5 95 17 15 85 18 10 90 20 5 95
[0033] Take a sample of 500 μl of the fermentation broth and dissolve it in 500 μl of dimethyl sulfoxide. Filter the membrane. Take the liquid after passing through the membrane and perform high performance liquid chromatography analysis using the above method. The analysis results are shown in Figure 3. Figure 3B The above method was used to dissolve the standard product containing p-hydroxyphenylacetic acid in dimethyl sulfoxide in a ratio of 1:1 for high performance liquid chromatography analysis. The analysis results are shown in Figure 3. Figure 3A As shown, Figure 3A This is a standard product picture. Figure 3A It can be seen that the characteristic peak retention time of p-hydroxyphenylacetic acid is 9.326min; Figure 3B It can be seen that there is also a characteristic peak at 9.317min, from which we can judge Figure 3B The characteristic peak with a retention time of 9.317 min is p-hydroxyphenylacetic acid. Therefore, the method provided in this embodiment can prepare p-hydroxyphenylacetic acid.
[0034] 2. Example 2 Construction of genetically engineered Escherichia coli BW1
[0035] The genes ptsH, pykA, pykF, tyrB, aspC, and ilvE were knocked out using crispr cas9 technology. The specific implementation method is as follows:
[0036] (1) The vector pCas9 was introduced into Escherichia coli BW by electroporation, and the cells were cultured on a spectinomycin plate at 30°C for 20 h to screen for positive clones and transformants named BW-pCas9. A single BW-pCas9 clone grown on the plate was picked and inoculated into LB liquid medium containing 1.5 ul / mL spectinomycin and cultured at 30°C.
[0037] (2) Construction of ptsH gene site sgRNA plasmid
[0038] i. The targeting sequences used by sgRNA in this study are shown in Table 1
[0039] Table 1. Targeting sequences used by sgRNA in the ptsH gene
[0040]
[0041] ii. The primer sequences used in this study are shown in Table 2
[0042] Table 2 Primer sequence list
[0043]
[0044] iii. Construction of ptsH gene site sgRNA plasmid
[0045] P1 / P2 were used as primers and pTarget plasmid was used as template. The nucleotide sequence containing sgRNA obtained by PCR was 2200 kbp in length. After agarose gel electrophoresis, the PCR product was purified and recovered using a gel recovery kit. The PCR purification solution was chemically transformed into Escherichia coli DH5α competent cells, in which it was recombined and self-ligated to form the pTarget plasmid with ampicillin.
[0046] (3) Construction of knockout fragments
[0047] i. The primer sequences used in this study are shown in Table 3
[0048] Table 3 Primer sequence list
[0049]
[0050] ii. Construction of knockout fragments
[0051] iii. Using Escherichia coli as a template and P3 / P4 and P5 / P6 as primers, PCR amplification was performed to obtain two homology arms of the gene ptsH. Using the amplified homology arms at both ends as templates and P3 / P6 as primers, an integrated fragment was obtained by PCR overlap extension method. Agarose gel electrophoresis was performed, and then the PCR product was purified and recovered using a gel recovery kit.
[0052] (4) The sgRNA plasmid and the knockout fragment were introduced into the engineered bacteria carrying the BW Cas9 plasmid in (1) by electroporation. In the bacteria, the sgRNA guided the cas9 protein to recognize the knockout site sequence and cut it. The repair function of the bacteria itself allowed the knockout fragment to homologously recombine and replace the original site, thereby obtaining a genetically engineered strain carrying spectinomycin and ampicillin, which was cultured at 30°C on a plate with corresponding resistance for 24 hours.
[0053] (5) Eliminate the sgRNA plasmid and pCas plasmid to obtain genetically engineered bacteria that do not require antibiotics
[0054] ] Eliminate the sgRNA plasmid, culture the above genetically engineered strain in liquid LB containing spectinomycin, add 10mmol / L arabinose at the same time, and culture at 30℃ for 24h to induce cas9 protein expression and degrade the sgRNA plasmid to obtain genetically engineered bacteria with pCas plasmid for the next step of knocking out the gene pykA.
[0055] The method for knocking out the pykA gene is the same as that for knocking out the ptsH gene. The required target gene list and primer list are shown in Tables 4-6. Genetically engineered bacteria with pCas plasmids that knock out ptsH and pykA are obtained for the next step of knocking out the pykF gene.
[0056] Table 4. Targeting sequences used by sgRNA in the pykA gene
[0057]
[0058] Table 5. Primer sequences of sgRNA for gene pykA
[0059]
[0060] Table 6. Primer sequences for the integration fragment of the gene pykA
[0061]
[0062] The method for knocking out the pykF gene is the same as that for knocking out the ptsH gene. The required target gene list and primer list are shown in Tables 7-9. Genetically engineered bacteria with pCas plasmids that knock out ptsH, pykA, and pykF are obtained for the next step of knocking out the tyrB gene.
[0063] Table 7. Targeting sequences used by sgRNA in the gene pykF site
[0064]
[0065] Table 8. Primer sequences of sgRNA for gene pykF
[0066]
[0067] Table 9. Primer sequences for the integration fragment of the gene pykF
[0068]
[0069] The method for knocking out the tyrB gene is the same as that for knocking out the ptsH gene. The required target gene list and primer list are shown in Tables 10-12. Genetically engineered bacteria with pCas plasmids that knock out ptsH, pykA, pykF, and tyrB are obtained for the next step of knocking out the aspC gene.
[0070] Table 10. Targeting sequences used by sgRNA in gene tyrB site
[0071]
[0072] Table 11. Primer sequences of sgRNA for gene tyrB
[0073]
[0074] Table 12. Primer sequences for gene tyrB integration fragment
[0075]
[0076] The method for knocking out the aspC gene is the same as that for knocking out the ptsH gene. The required target gene list and primer list are shown in Tables 13-15. Genetically engineered bacteria with pCas plasmids that knock out ptsH, pykA, pykF, tyrB, and aspC are obtained for knocking out the ilvE gene in the next step.
[0077] Table 13. Targeting sequences used by sgRNA in the aspC gene
[0078]
[0079]
[0080] Table 14. Primer sequences of sgRNA for gene aspC
[0081]
[0082] Table 15. Primer sequences for the integration fragment of the gene aspC
[0083]
[0084] The gene ilvE was knocked out in the same manner as the above-mentioned gene ptsH. The required target gene list and primer list are shown in Tables 16-18. Genetically engineered bacteria carrying pCas plasmids with ptsH, pykA, pykF, tyrB, aspC, and ilvE knocked out were obtained.
[0085] Table 16. Targeting sequences used by sgRNA for the ilvE gene site
[0086]
[0087] Table 17. Primer sequences of sgRNA for gene ilvE
[0088]
[0089] Table 18. Primer sequences for integration fragments of the gene ilvE
[0090]
[0091] The genetically engineered bacteria carrying the pCas plasmid with ptsH, pykA, pykF, tyrB, aspC, and ilvE knocked out were placed in a 42°C shaker for 12 hours.
[0092] A genetically engineered bacterium BW1 was obtained that can stably produce p-hydroxyphenylpyruvate without the need for antibiotics.
[0093] 3. Example 3 Construction of recombinant Escherichia coli BW2
[0094] Using crispr cas9 technology, genes ARO10 and feaB replaced the pseudo-sense gene mbhA on the genome. Specific implementation method
[0095] (1) The vector pCas9 was introduced into Escherichia coli BW1 by electroporation, and the cells were cultured on spectinomycin plates at 30°C for 20 h to screen positive clones and transformants named BW-pCas9. The BW1-pCas9 single clone grown on the plate was picked and inoculated into LB liquid medium containing 1.5 ul / mL spectinomycin and cultured at 30°C.
[0096] (2) Construction of sgRNA plasmid for gene mbhA site
[0097] i. The targeting sequences used by sgRNA in this study are shown in Table 19
[0098] Table 19. Targeting sequences used by sgRNA in the mbhA gene site
[0099]
[0100] ii. The primer sequences used in this study are shown in Table 20
[0101] Table 20 Primer sequence list
[0102]
[0103] iii. Construction of sgRNA plasmid for gene mbhA site
[0104] P27 / P2 was used as primers and pTarget plasmid was used as template. The nucleotide sequence containing sgRNA obtained by PCR was 2200 kbp in length. After agarose gel electrophoresis, the PCR product was purified and recovered using a gel recovery kit. The PCR purification solution was chemically transformed into Escherichia coli DH5α competent cells, in which it was recombined and self-ligated to form the pTarget plasmid with ampicillin.
[0105] (3) Construction of integrated fragments
[0106] i. The primer sequences used in this study are shown in Table 21
[0107] Table 21 Primer sequence list
[0108]
[0109] ii. Construction of integration fragment
[0110] iii. Using Escherichia coli as a template and P28 / P29 and P32 / P33 as primers, PCR amplification was performed to obtain two homologous arms of the gene mbhA. Using gene ARO10-feaB as a template and P30 / P31 as primers, PCR amplification was performed to obtain the gene ARO10-feaB fragment. Then, using these three fragments as templates and P28 / P33 as primers, the PCR overlap extension method was used to obtain an integrated fragment, which was subjected to agarose gel electrophoresis, and then the PCR product was purified and recovered using a gel recovery kit.
[0111] (4) The sgRNA plasmid and integration fragment were introduced into the BW1 Cas9 strain in (1) by electroporation. In the bacteria, the sgRNA guided the cas9 protein to recognize the integration site sequence and cut it. The repair function of the bacteria itself enabled the integration fragment to homologously recombine and replace the pseudosense site, thereby obtaining a genetically engineered strain with spectinomycin and ampicillin, which was cultured at 30°C for 24 h on a plate with corresponding resistance.
[0112] (5) Eliminate the sgRNA plasmid and pCas plasmid to obtain a genetically engineered bacterium that does not require antibiotics
[0113] i. Eliminate the sgRNA plasmid, culture the above genetically engineered strain in liquid LB containing spectinomycin, add 10 mmol / L arabinose, and culture at 30°C for 24 hours to induce cas9 protein expression and degrade the sgRNA plasmid.
[0114] ii. Eliminate the pCas plasmid. Cultivate the genetically engineered bacteria from which the sgRNA plasmid has been eliminated in antibiotic-free liquid LB at 42°C for 24 hours to degrade the temperature-sensitive pCas plasmid.
[0115] (6) A genetically engineered strain BW2 was obtained that can achieve stable genetic production of p-hydroxyphenylacetic acid without the need for antibiotics.
[0116] 4. Example 4 Application of genetically engineered strain BW2: Fermentation culture to synthesize p-hydroxyphenylacetic acid from scratch
[0117] (6) The genetically engineered strain BW2 was spread on a plate without antibiotics and cultured overnight at 37°C. The positive single clone transformant was picked into a 4 ml LB test tube, cultured at 37°C for 10 h, and transferred into 50 ml of fermentation medium without antibiotics. The inoculation amount was 2% by volume, the fermentation temperature was 33°C, and the rotation speed was 220 rpm. The medium included 20 g / L glucose, 5 g / L yeast powder, 2 g / L MOPS, 5 g / L Na2HPO4, 0.5 g / L NaCl, 3 g / L KH2PO4, 1 g / L NH4Cl, 250 mg / L MgSO4, 15 mg / LCaCl2, and the solvent was water.
[0118] Part of the fermentation liquid was taken out every 12 hours to determine the growth of the bacteria and the yield of the target product p-hydroxyphenylacetic acid. The HPLC fermentation results were as follows: Figure 4 shown.
[0119] 5. Example 5: Engineered bacteria with high production of p-hydroxyphenylacetic acid: recombinant Escherichia coli BW3.
[0120] The crispr cas9 technology was used to integrate the genes aroG, aroL, aroC, aroB, and aroD into the pseudogene site ilvG. The specific implementation method.
[0121] (1) The vector pCas9 was introduced into E. coli BW2 by electroporation. The cells were cultured on spectinomycin plates at 30°C for 20 h and the positive clones were selected and named BW2-pCas9. The BW2-pCas9 single clone grown on the plate was selected and inoculated into
[0122] The cells were cultured in LB liquid medium containing 1.5ul / mL spectinomycin at 30°C.
[0123] (2) Construction of ilvG site sgRNA plasmid
[0124] i. The targeting sequences used by sgRNA in this study are shown in Table 22
[0125]
[0126] ii. The primer sequences used in this study are shown in Table 23
[0127] Table 23. Primer sequences
[0128]
[0129] Construction of sgRNA plasmid targeting ilvG gene
[0130] P34 / P2 was used as primers and pTarget plasmid was used as template. The nucleotide sequence containing sgRNA obtained by PCR was 2200 kbp in length. After agarose gel electrophoresis, the PCR product was purified and recovered using a gel recovery kit. The PCR purification solution was chemically transformed into Escherichia coli DH5α competent cells, in which it was recombined and self-ligated to form the pTarget plasmid with ampicillin.
[0131] (3) Construction of integrated fragments
[0132] i. The primer sequences used in this study are shown in Table 24
[0133] Table 24 Primer sequence table
[0134]
[0135] ii. Construction of integration fragment
[0136] Using Escherichia coli as a template and P35 / P36 and P39 / P40 as primers, PCR amplification was performed to obtain two homologous arms of the gene ilvG. Using the gene aroG / L / C / B / D as a template and P37 / P38 as primers, PCR amplification was performed to obtain the gene aroG / L / C / B / D fragment. Then, using these three fragments as templates and P35 / P40 as primers, the PCR overlap extension method was used to obtain an integrated fragment, which was subjected to agarose gel electrophoresis, and then the PCR product was purified and recovered using a gel recovery kit.
[0137] (4) The sgRNA plasmid and integration fragment were introduced into BW2 with Cas 9 plasmid in Example 3 by electroporation. In the bacteria, sgRNA guided cas9 protein to recognize the integration site sequence and cut it. The repair function of the bacteria itself enabled the integration fragment to homologously recombine and replace the pseudosense site, thus obtaining a genetically engineered strain with spectinomycin and ampicillin, which was cultured at 30°C on a plate with corresponding resistance for 24 h.
[0138] The sgRNA plasmid and the pCas plasmid are eliminated to obtain a genetically engineered bacterium that does not require antibiotics, and the method is the same as described in (5) of Example 3.
[0139] (5) A genetically engineered strain BW3 was obtained that can achieve stable genetic production of p-hydroxyphenylacetic acid without the need for antibiotics.
[0140] 6. Example 5 Application of genetically engineered strain BW3
[0141] Referring to Example 4, the genetically engineered strain BW3 was placed in the culture medium, and 1 mL of the sample was taken every 12 hours to measure the bacterial growth and target product yield. The results are as follows: Figure 5 shown.
Claims
1. A method for constructing a genetically engineered strain for efficient biosynthesis of p-hydroxyphenylacetic acid with stable inheritance, characterized in that: The following steps are involved: Firstly, genes encoding phosphate carrier protein (ptsH), pyruvate kinase (pykA, pykF), tyrosine aminotransferase (tyrB), aspartate aminotransferase (aspC), and branched-chain amino acid aminotransferase (ilvE) were knocked out in the host to construct a high-yield strain of p-hydroxyphenylpyruvate, a key intermediate in the production process of p-hydroxyphenylacetic acid; then, genes encoding phenylpyruvate decarboxylase (ARO10) and phenylacetaldehyde dehydrogenase (feaB) from Saccharomyces cerevisiae were integrated into the host to construct a genetically engineered strain capable of stably genetically producing p-hydroxyphenylacetic acid; finally, genes encoding 3-deoxy-7-phosphoheptanoate synthase (AroG), shikimate kinase (AroL), chorismate synthase (AroC), 3-dehydroquinoline synthase (AroB), and 3-dehydroquinoline dehydratase (AroD) were integrated into the host to obtain a genetically engineered strain with stable genetically efficient biosynthesis of p-hydroxyphenylacetic acid.
2. The method according to claim 1, characterized in that: The genes ptsH (Genebank sequence number: 946886), pykA (Genebank sequence number: 946527), pykF (Genebank sequence number: 946179), tyrB (Genebank sequence number: 948563), aspC (Genebank sequence number: 945553), and ilvE (Genebank sequence number: 948278) were deleted using the crispr cas9 system, and the gene ARO10 (from Saccharomyces cerevisiae S288C, Genebank sequence number: 851987), feaB (Genebank sequence number: 945933) replace the pseudo-significant gene mbhA on the genome, and the genes aroG (Genebank sequence number: 945605), aroL (Genebank sequence number: 945031), aroC (Genebank sequence number: 946814), aroB (Genebank sequence number: 947927), and aroD (Genebank sequence number: 946210) replace the pseudo-significant gene ilvG on the genome.
3. Application of the genetically engineered bacteria prepared by the method of claim 1, characterized in that: The constructed genetically engineered strain for efficient biosynthesis of p-hydroxyphenylacetic acid was spread on an antibiotic-free plate and cultured overnight at 37°C. A single clone was picked and cultured in liquid LB for 12 hours, inoculated into a fermentation medium, and fermented at 33°C to produce p-hydroxyphenylacetic acid from scratch.
4. The use according to claim 3, characterized in that: The fermentation medium includes 20 g / L glucose, 5 g / L yeast powder, 2 g / L MOPS, 5 g / L Na2HPO4, 0.5 g / L NaCl, 3 g / L KH2PO4, 1 g / L NH4Cl, 250 mg / L MgSO4, 15 mg / L CaCl2, and the solvent is water.