Gallic acid-producing strain and application thereof

Abstract

This invention provides a gallic acid-producing strain and its applications. The gallic acid-producing strain has a genome derived from the originating strain *Escherichia coli*. mbha site and ilvG Knock-in the gene encoding 3-dehydroshikimate dehydratase at each site. quiC In the genome yjip Knock-in of the gene encoding the p-hydroxybenzoic acid hydroxylase mutant pobA*** Further application of CRISPR-Cas9 technology to optimize gene expression in the starting strain by changing promoter strength and increasing copy number, thereby increasing the yield of gallic acid produced by fermentation using the aforementioned gallic acid-producing strain, achieving a carbon yield of 60% of the natural theoretical carbon yield, with low production cost and no need for external antibiotics or inducers. This invention also proposes an application of the aforementioned gallic acid-producing strain in the production of gallic acid derivatives, using the strain as the starting strain to construct an engineered strain for gallic acid derivative production, thereby enabling the synthesis of other high-value compounds using gallic acid as the starting compound.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and more specifically, this invention relates to a gallic acid producing strain and its application. Background Technology

[0002] Gallic acid (GA) is a polyphenolic organic compound with the molecular formula C7H6O5 and a molecular weight of 170.12. Its structural formula is: Gallic acid is soluble in hot water, ether, ethanol, acetone, and glycerol; sparingly soluble in cold water; and insoluble in benzene and chloroform. As a naturally occurring polyphenol, it is found in plants such as pomegranate, eucalyptus, and tea. Gallic acid and its derivatives possess various biological activities, including antibacterial, anti-inflammatory, antioxidant, antitumor, cardioprotective, and neuroprotective effects. Therefore, it is used in the food and cosmetic industries. Furthermore, gallic acid can react with Fe... 3+ Gallic acid forms a blue-black precipitate, which can be used as a raw material for making blue-black ink. Therefore, gallic acid has important applications in the fields of pharmaceutical and chemical synthesis.

[0003] Currently, the main methods for producing gallic acid both domestically and internationally include acid hydrolysis, alkaline hydrolysis, fermentation, and enzymatic methods. All of these methods suffer from limited raw material supply, demanding reaction conditions, environmental pollution, and low efficiency. With the development of metabolic engineering technology, *E. coli* biosynthesis, as a relatively mild and sustainable production method, can replace chemical synthesis. Chinese patent application CN 116769846A discloses a method for whole-cell catalytic synthesis of gallic acid using engineered strains, comprising: constructing a recombinant *E. coli* genetically engineered bacterium expressing fusobacterial tanninase; and using the recombinant *E. coli* genetically engineered bacterium to catalyze the synthesis of gallic acid from substrates. This method uses a newly derived tanninase to construct a recombinant *E. coli* genetically engineered bacterium as a whole-cell catalyst for gallic acid synthesis. It can catalyze the synthesis of gallic acid from tannin substrates under high-temperature conditions, and when using tara powder as a substrate, it exhibits good catalytic synthesis effects at 37°C. The overall reaction can reach equilibrium within 12 hours, and high-concentration substrates can be used to obtain high yields of gallic acid. However, the patent application does not disclose that the engineered strain can achieve de novo catalytic synthesis of gallic acid using a carbon source in its whole cells; moreover, the engineered strain is not stable enough, and antibiotics and inducers need to be added during the fermentation process, which increases the possibility of antibiotic pollution to the environment and the potential influx of resistance genes into the environment.

[0004] β-Glucogallin (BGG) is a plant secondary metabolite, mainly found in plants such as Phyllanthus emblica, pomegranate, and strawberry. Due to its free radical scavenging properties, BGG can prevent various diseases such as diabetes and related complications, including retinopathy, glaucoma, inflammation, liver damage, and UV-induced skin damage. Studies have also found that BGG can inhibit the proliferation and migration of cholangiocarcinoma cells. Currently, the synthetic mechanism of BGG remains unclear, therefore, there are no reports of de novo biosynthesis. Summary of the Invention

[0005] One objective of this invention is to provide a gallic acid producing strain that has good passage stability and can achieve gallic acid synthesis by fermentation using a carbon source.

[0006] Another objective of this invention is to provide an engineered bacterium for the production of gallic acid derivatives. This engineered bacterium, using the aforementioned gallic acid producing strain as the starting strain, can efficiently biosynthesize gallic acid derivatives using gallic acid as a substrate.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] A gallic acid-producing strain had the gene quiC, which encodes 3-dehydroshikimate dehydratase, knocked into the mbha and ilvG sites of the Escherichia coli genome, and the gene pobA***, which encodes a mutant of p-hydroxybenzoic acid hydroxylase, knocked into the yjip site of the genome.

[0009] Preferably, the 3-dehydroshikimate dehydratase quiC is derived from *Pseudomonas putida*; the hydroxybenzoic acid hydroxylase mutant is derived from a triple mutant of *Pseudomonas aeruginosa*, and more preferably, the nucleotide sequence of the gene encoding the hydroxybenzoic acid hydroxylase mutant is shown in SEQ ID NO. 1. Preferably, the 3-dehydroshikimate dehydratase quiC is derived from P… trc The promoter is overexpressed in multiple copies on the chromosomal DNA of *E. coli*; the p-hydroxybenzoic acid hydroxylase mutant pobA*** is expressed via P lpp The promoter is expressed on the E. coli genome.

[0010] The Escherichia coli strain used as the starting strain is preferably Escherichia coli BW25113, MG1655 or BL21.

[0011] Furthermore, the glycolysis pathway genes pykA and pykF were knocked out in the genome of the *E. coli*, the gene ptsG encoding a protein related to the phosphoenolpyruvate glycotransferase system was knocked out, and the gene glf encoding a glucose transporter and the gene glk encoding glucokinase were integrated at the yncI site in the genome. Additionally, the gene aroG encoding 2-dehydro-3-deoxyphosphoheptanate aldolase was integrated at the yghX site in the genome. fbr Preferably, glf, glk, and aroG fbr Through P trc The promoter was overexpressed or enhanced on the genome of the E. coli.

[0012] Furthermore, the transketolase-encoding gene tktA and the transaldolase-encoding gene talB are integrated into the yjiT site of the *E. coli* genome. This integration of the pentose phosphate pathway enhances the supply of erythrose-4-phosphate precursors in gallic acid-producing strains. Preferably, tktA and talB are connected via the constitutive promoter P. lpp It is overexpressed or enhanced on the genome.

[0013] Furthermore, the gene encoding 2-dehydro-3-deoxyphosphate-heptanoate aldolase, aroG, was integrated into the yeep site of the E. coli genome. fbr The genes encoding 3-dehydroquinoline synthase (aroB) and 3-dehydroquinoline dehydratase (aroD) are integrated into the shikimic acid pathway to increase the supply of 3-dehydroshikimic acid precursors. Preferably, aroG... fbr aroB and aroD are promoted through the constitutive promoter P. lpp It is overexpressed or enhanced on the genome.

[0014] Application of the above-mentioned gallic acid-producing strain in the fermentation production of gallic acid. Preferably, the culture medium used in the fermentation process comprises 10-20 g / L simple carbon source, 4-12 g / L yeast extract, 4-8 g / L Na₂HPO₄, 0.3-2 g / L NaCl, 2-4 g / L KH₂PO₄, 0.6-1.5 g / L NH₄Cl, 200-300 mg / L MgSO₄, and 10-25 mg / L CaCl₂. The simple carbon source is preferably glucose, glycerol, or a combination of both.

[0015] Further, the gallic acid-producing strain is inoculated into shake flask culture medium at an inoculation rate of 1% to 10% by volume, and the culture is carried out at 30℃ to 35℃ to produce gallic acid. Preferably, the shake flask culture medium comprises 10 to 20 g / L simple carbon source, 4 to 7 g / L yeast extract, 4 to 7 g / L Na₂HPO₄, 0.3 to 2 g / L NaCl, 2 to 4 g / L KH₂PO₄, 0.6 to 1.2 g / L NH₄Cl, 0.8 to 1.5 g / L MOPS (3-morpholinopropanesulfonic acid buffer), 200 to 300 mg / L MgSO₄, and 10 to 25 mg / L CaCl₂. Preferably, the stirring speed during shake flask fermentation is 200 to 220 rpm. This allows for the shake flask culture of the gallic acid-producing strain, achieving a gallic acid yield of 7.16 g / L.

[0016] Further, the gallic acid-producing strain is first inoculated into LB medium for shake-flask amplification culture to obtain a shake-flask seed culture; then, the shake-flask seed culture is inoculated into the fermenter medium at a volume fraction of 5%–10%, and fermentation is carried out at 30℃–35℃ and dissolved oxygen maintained at 10%–30% to produce gallic acid. Preferably, the fermenter medium includes 10–20 g / L simple carbon source, 8–12 g / L yeast extract, 4–7 g / L Na₂HPO₄, 0.3–2 g / L NaCl, 2–4 g / L KH₂PO₄, 0.6–1.2 g / L NH₄Cl, 200–300 g / L MgSO₄, 10–25 g / L CaCl₂, and 35–45 mg / L VB₁. The preferred process parameters for fermenter culture are: ventilation rate 3–5 m³ / h. 3 The stirring speed is 300-1000 rpm per hour. This allows for the effective scale-up production of gallic acid in fermenters, achieving a yield of 51.57 g / L, with a carbon recovery rate of 60% of the theoretical natural carbon recovery rate.

[0017] The application of the above-mentioned gallic acid-producing strain in the production of gallic acid derivatives. Preferably, the application involves constructing an engineered strain for gallic acid derivative production using the gallic acid-producing strain as the starting material. The synthesis of the gallic acid derivative can use gallic acid as a starting compound or intermediate compound. Preferably, the gallic acid derivative can be β-glucosidin, ellagic acid, or propyl gallate, etc.

[0018] Furthermore, the engineered gallic acid derivative production strain includes a gallic acid-producing strain expressing the glycosidase (RiGT2). Preferably, the engineered gallic acid derivative production strain includes a glycosidase encoding gene expressed on a plasmid of the gallic acid-producing strain. The expression vector is preferably pET, pZE, or pCS, etc. The glycosidase can preferably be derived from raspberry (Rubus idaeus L.), rice (Oryza sativa L.), strawberry (Fragaria × annassa), wild strawberry (Fragaria vesca L.), etc.

[0019] Furthermore, the engineered bacteria for gallic acid derivative production are inoculated into the culture medium at an inoculation rate of 1% to 10% by volume, and fermented at 30°C to 35°C to produce gallic acid derivatives. Preferably, the engineered bacteria for gallic acid derivative production are inoculated into a 30-50 mL shake flask containing 250 mL of liquid at an inoculation rate of 1% to 5% by volume, and fermented at 30°C to 35°C and 200-220 rpm. In this way, the de novo synthesis of β-glucosamine is achieved, with a yield of up to 92.42 mg / L.

[0020] The gallic acid-producing strains and gallic acid derivative-producing engineered strains described in this invention are constructed using existing technologies and methods. This invention optimizes the expression levels of genes related to enzymes in the gallic acid metabolic pathway, achieving stable and high yields. Compared to existing technologies, the gallic acid-producing strains provided by this invention exhibit higher passage stability, more stable fermentation results, and higher carbon conversion rates and yields. Furthermore, the gallic acid-producing strains provided by this invention do not require antibiotics throughout the fermentation process, avoiding environmental pollution from antibiotics and the potential influx of resistance genes into the environment. The gallic acid-producing strains provided by this invention not only efficiently produce gallic acid but can also be used as starting strains for gallic acid derivative engineered strains, enabling the biosynthesis of other high-value compounds based on gallic acid as a starting or intermediate compound, such as the de novo biosynthesis of β-glucosidin. Attached Figure Description

[0021] Figure 1 It is the biosynthetic pathway of gallic acid and its derivatives.

[0022] Figure 2 This is an HPLC chromatogram of gallic acid standard.

[0023] Figure 3 This is an HPLC chromatogram of the fermentation product.

[0024] Figure 4 This is a diagram showing the results of shake-flask fermentation of a gallic acid-producing strain.

[0025] Figure 5 This is a diagram showing the fermentation results of the gallic acid producing strain in a fermenter.

[0026] Figure 6 This is an HPLC chromatogram of the β-glucosamine standard.

[0027] Figure 7 This is an HPLC chromatogram of the fermentation product of β-glucosamine gallin.

[0028] Figure 8 This is a diagram showing the results of shake-flask fermentation of a β-glucosamine-producing strain. Detailed Implementation

[0029] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0030] In this invention, it is assumed that the construction and transformation of plasmid vectors, as well as the methods for E. coli gene knockout, knock-in, and gene mutation, can employ various methods commonly used in the field, which will not be elaborated upon further.

[0031] In the following embodiments, *Escherichia coli* strains BW25113 and trans5α are commonly used *E. coli* strains and are commercially available. All raw materials and reagents used are common items and are commercially available; for example, LB medium includes 1% peptone, 1% NaCl, and 0.5% yeast extract. The plasmids and strains used in the embodiments of this invention are shown in Tables 1 and 2, the promoter DNA sequences are shown in Table 3, and the primer DNA sequences used for amplification during genome integration are shown in Table 4.

[0032] Table 1 List of plasmids involved in the embodiments of the present invention

[0033] pTartget-1 gRNA plasmids specifically recognize the mbha site. pTartget-2 gRNA plasmids specifically recognize the ilvG site. pTartget-3 gRNA plasmids specifically recognize the yjiP site. pTartget-4 gRNA plasmids specifically recognize the yjiT site. pTartget-5 gRNA plasmids specifically recognize the yeep site. pET-RiGT2 <![CDATA[P L lacO1,colE ori,luc,Amp R B contains the RiGT2 gene.

[0034] Table 2 List of strains involved in the embodiments of the present invention

[0035]

[0036] The construction method of strain GJ 0 was as follows: using Escherichia coli BW25113 as the starting strain, pykA and pykF were first knocked out to reduce the conversion of PEP to pyruvate; subsequently, to further increase the supply of precursor PEP, the key gene ptsG of the PTS system was knocked out, and P was injected into the yncI site of the genome via P 10trc The promoter integrates the glucose-assisted diffusion system genes glucose-promoting protein glf and glucokinase glk; then P is used 10trc The promoter integrates aroG at the yghX site in the genome. fbrThe initial strain GJ 0 was constructed. For details of the construction process, please refer to the paper: Ning A, Chong X, Shubin Z, et al. Establishing a growth-coupled mechanism for high-yield production of β-arbutin from glycerol in Escherichia coli[J]. Bioresource Technology, 2023, 369128491-128491.

[0037] Table 3. Promoter DNA Sequence List

[0038]

[0039] The culture media used in each example are as follows:

[0040] 1) Shake-flask fermentation medium for each strain: 6.78 g / L NaHPO4, 3 g / L KH2PO4, 1 g / L NH4Cl, 1 g / L MOPS, 1.5 g / L NaCl, 20 g / L glucose, 5 g / L yeast extract, 250 mg / L MgSO4, 15 mg / L CaCl2, with water as the solvent.

[0041] 2) Fermentation medium for the strain: 1L fermenter medium contains 6.78g NaHPO4, 3g KH2PO4, 1g NH4Cl, 1.5g NaCl, 10g yeast extract, 250mg MgSO4, 15mg CaCl2, and 30mg VB1. After sterilization, 15g glucose and trace elements (0.03mg H3BO3, 0.38mg CuCl2, 0.4mg Na2EDTA, 0.5mg CoCl2, 0.94mg ZnCl2, 1.6mg MnCl2, and 3.6mg FeSO4) are added, with water as the solvent.

[0042] 3) LB medium: yeast extract 5g / L, peptone 10g / L, NaCl 10g / L.

[0043] Biosynthetic pathways of gallic acid and its derivatives, such as Figure 1 As shown, a simple carbon source is converted into protocatechuic acid via glycolysis and shikimic acid dehydration under the action of 3-dehydroshikimic acid dehydratase. Protocatechuic acid is then converted into gallic acid under the catalysis of a p-hydroxybenzoic acid hydroxylase mutant. Gallic acid is then converted into β-glucosidase galloquinone under the catalysis of glycosidase. The technical solution to be protected by this invention is further illustrated below with specific embodiments.

[0044] Example 1

[0045] This embodiment provides a protocatechuic acid-producing strain, which is mainly a genetically engineered bacterium constructed by integrating and expressing quiC, a 3-dehydroshikimate dehydratase derived from *Pseudomonas putida*, into the chromosomal DNA of *Escherichia coli* strain GJ0. The specific construction method includes the following steps:

[0046] The primer DNA sequences used in the quiC integration process are shown in Table 4:

[0047] Table 4. Primer DNA sequence list used for quiC integration.

[0048]

[0049] Step (1) Construction of the three-in-one fragment and pTarget plasmid: Using the genome of *Pseudomonas putida* as a template, the quiC gene of Gene ID: 88363944 was amplified. Using the chromosomal DNA of strain BW25113 as a template, and using mbha-UF, mbha-UR, mbha-DF, and mbha-DR as primers, the upstream and downstream homologous arms of mbha were amplified by PCR. Then, using mbha-UF and mbha-DR as primers, the above DNA fragment was coupled with pTarget plasmid. trc10 P trc30 P trc50 The promoter was used for overlap PCR, and the PCR product was detected by agarose gel electrophoresis and purified by gel excision to obtain the upstream homologous arm -P of the donor fragment mbha. trc -quiC-mbha downstream homologous arm.

[0050] The plasmid pTartget-1, which specifically recognizes the pseudogene mbha, was constructed using the pTartget vector as a template. The N20 primers containing the mbha-specific recognition primer were subjected to PCR annealing with the following program: pre-denaturation at 95℃ for 5 min; annealing at 30-50℃ for 1 min. The annealed fragment was ligated into the pTartget vector using the ClonExpress II One Step Cloning Kit (Nanjing Novizan Medical Technology Co., Ltd.) and transformed into DH5α. The plasmid pTartget-1 was obtained by screening and sequencing on LB solid medium containing 100 μg / mL ampicillin.

[0051] By using the upstream homologous arm -P of the aforementioned donor fragment mbha trcThe downstream homologous arm of the -mbha-ygay plasmid and the pTartget-1 plasmid were electroporated into strain GJ 0 carrying the pCas9 plasmid. The bacterial culture was then plated onto LB agar containing 100 μg / mL ampicillin and 100 μg / mL spectinomycin, and positive strains were obtained by colony PCR verification. These positive strains were then transferred to LB tubes containing 100 μg / mL spectinomycin and 0.2% arabinose and incubated at 30°C for 12 h to eliminate the pTartget-1 plasmid. The strains with the pTartget-1 plasmid eliminated were then transferred to new LB tubes and incubated overnight at 42°C to eliminate the pCas9 plasmid, yielding strains GJ 1, GJ 2, and GJ 3.

[0052] The recombinant strains GJ1, GJ2, and GJ3 were streaked onto LB agar and cultured for 12 hours to obtain single colonies. Three uniformly sized single colonies were picked and inoculated into 5 mL of liquid LB agar, and cultured in a shaker at 37°C for 12 hours. Then, 1 mL of the bacterial culture was inoculated into 50 mL shake flasks at a volume ratio of 2% for fermentation. The fermentation conditions were 33°C, 220 rpm, and 48 hours. The concentration of the precursor protocatechuic acid was determined by high-performance liquid chromatography (HPLC). The strains GJ1, GJ2, and GJ3 produced 5.09 g / L, 4.41 g / L, and 3.49 g / L of protocatechuic acid, respectively.

[0053] HPLC detection conditions for protocatechuic acid and gallic acid: Diamonsil C18, ID 5μm, 250×4.6mm, gradient elution, organic phase was 100% methanol, aqueous phase was 2‰ trifluoroacetic acid, specific conditions are shown in Table 5.

[0054] Table 5 HPLC detection conditions

[0055] 0 20 16 100 7 50 17 20 15 100 22 20

[0056] Step (2): As can be seen from the above, the optimal promoter strength is P. trc10 Continue to increase the copy number of this expression cassette. Construct the three-in-one fragment and pTarget plasmid: Using the *Pseudomonas putida* genome as a template, amplify the quiC gene cluster. Using chromosomal DNA from strain BW25113 as a template, and with ilvG-UF, ilvG-UR, ilvG-DF, and ilvG-DR as primers, amplify the upstream and downstream homologous arms of ilvG by PCR. Then, using ilvG-UF and ilvG-DR as primers, combine the above DNA fragment with pTarget plasmid. 10trc The promoter was used for overlap PCR. The PCR product was detected by agarose gel electrophoresis and purified by gel excision to obtain the upstream homologous arm -P of the donor fragment ilvG. trc-quiC-ilvG downstream homologous arm.

[0057] Using ilvG-pTarget-F and pTarget-R as primers and the original linear pTarget plasmid as a template, a linear pTarget plasmid at the ilvG site was amplified by PCR. The resulting linear pTarget plasmid was then circularized using Basic-mix homologous recombination enzyme to obtain the pTarget plasmid at the ilvG site. This pTarget plasmid expresses sgRNA. The plasmid pTartget-2 was obtained by screening and sequencing on LB solid medium containing 100 μg / mL ampicillin.

[0058] By using the upstream homologous arm -P of the aforementioned donor fragment ilvG trc The downstream homologous arm of the -ilvG-ygay plasmid and the pTartget-1 plasmid were electroporated into strain GJ 3 carrying the pCas9 plasmid. The bacterial culture was then plated onto LB agar containing 100 μg / mL ampicillin and 100 μg / mL spectinomycin, and positive strains were obtained by colony PCR verification. These positive strains were then transferred to LB tubes containing 100 μg / mL spectinomycin and 0.2% arabinose and incubated at 30°C for 12 h to eliminate the pTartget-1 plasmid. The strain with the pTartget-1 plasmid eliminated was then transferred to a new LB tube and incubated overnight at 42°C to eliminate the pCas9 plasmid, yielding strain GJ 4.

[0059] The above-mentioned strain GJ 4 was streaked on LB medium and cultured for 12 h to obtain single colonies. Three single colonies of uniform size were picked and inoculated into 5 mL of liquid LB medium, and cultured in a shaker at 37℃ for 12 h. Then, 1 mL of the bacterial culture was inoculated into 50 mL of fermentation medium at a volume ratio of 2%, wherein the fermentation medium consisted of 5 g / L NaHPO4, 3 g / L KH2PO4, 1 g / L NH4Cl, 1 g / L MOPS, 1 g / L NaCl, 20 g / L glucose, 5 g / L yeast extract, 250 mg / L MgSO4, 15 mg / L CaCl2, and water as solvent. Fermentation was carried out at 33℃ and 220 rpm for 48 h, and the concentration of the precursor protocatechuic acid was determined by high performance liquid chromatography. The protocatechuic acid yield of strain GJ 4 was 6.08 g / L.

[0060] Example 2

[0061] This embodiment provides a gallic acid producing strain, which is mainly constructed by a genetically engineered strain that expresses a single copy of pobA*** on chromosomal DNA.

[0062] This embodiment provides a method for constructing the above-mentioned gallic acid producing strain, which is basically similar to the method for constructing the protocatechuic acid producing strain provided in Example 1. The main difference is that the high-yielding strain GJ 4 provided in Example 1 is used as the operating object, the integration site is the yjip site, and the integrated gene is pobA*** shown in SEQ ID NO.1. Specifically, the primers used in step (1) of Example 1 are replaced by yjip-UF, yjip-UR, mbha-DF, mbha-DR, quiC-F, quiC-R, mbha-pTarget-F, and pTarget-R, respectively, with yjip-UF, yjip-UR, yjip-pobA***-F, yjip-pobA***-R, yjip-DF, yjip-DR, and yjip-pTarget-F, and the promoter is replaced with Plpp1.9, Plpp4.0, and Plpp4.2 to obtain recombinant strains GJ 5, GJ 6, and GJ 7. The DNA sequences of the primers used for the integrated gene pobA*** are shown in Table 6.

[0063] Table 6. Primer DNA sequence list used for integrating pobA***

[0064]

[0065] The recombinant bacterial strains GJ 5, GJ 6, and GJ 7 were inoculated at a rate of 2% (v / v) into 50 mL shake flask culture medium. Fermentation was carried out at 33°C and 220 rpm for 48 h to obtain the corresponding fermentation products. The concentration of gallic acid in the fermentation products was determined by high-performance liquid chromatography (HPLC). The HPLC chromatogram of the gallic acid standard is shown below. Figure 2 As shown, the HPLC detection chromatogram of the fermentation products of strain GJ 5 is as follows. Figure 3 As shown in the figure, the fermentation results are as follows: Figure 4 The gallic acid yields of strains GJ5, GJ6, and GJ7 were measured to be 4.30 g / L, 4.67 g / L, and 5.2 g / L, respectively.

[0066] Example 3

[0067] This embodiment provides a gallic acid producing strain, which is mainly constructed by increasing the supply of erythrose-4-phosphate precursor. The strain integrates expression cassettes of the pentose phosphate pathway transketolase tktA and transaldolase talB, and the expression cassettes are driven by the constitutive promoter Plpp.

[0068] This embodiment provides a method for constructing the above-mentioned gallic acid-producing strain, which is basically similar to the construction method provided in Example 1. The main difference is that the high-yielding strain GJ 7 provided in Example 2 is used as the target, the integration site is the yjiT site, and the integrated gene is tktA-talB. Specifically, the primers are replaced with yjip-UF, yjiT-UR, yjiT-tktA-talB-F, yjiT-tktA-talB-R, yjiT-DF, yjiT-DR, and yjiT-pTarget-F, and the promoter is replaced with Plpp1.9, Plpp4.0, and Plpp4.2 to obtain recombinant strains GJ 8, GJ 9, and GJ 10. The DNA sequences of the primers used to integrate the gene of the pentose phosphate pathway are shown in Table 7.

[0069] Table 7. Primer DNA sequence list used for genes integrating the pentose phosphate pathway.

[0070]

[0071] The recombinant strains GJ 8, GJ 9, and GJ 10 were inoculated at a rate of 2% (v / v) into 50 mL shake flask culture medium. Fermentation was carried out at 33°C and 220 rpm for 48 h to obtain the corresponding fermentation products. The concentration of gallic acid in the fermentation products was determined by high-performance liquid chromatography (HPLC). The gallic acid yields of strains GJ 8, GJ 9, and GJ 10 were found to be 5.80 g / L, 6.19 g / L, and 6.23 g / L, respectively.

[0072] Example 4

[0073] This embodiment provides a gallic acid-producing strain, which is mainly constructed by increasing the supply of 3-dehydroshikimic acid precursor. The constructed strain integrates the 2-dehydro-3-deoxyphosphoheptanate aldolase aroG from the shikimic acid pathway. fbr Expression cassettes for 3-dehydroquinoline synthase aroB and 3-dehydroquinoline dehydratase aroD, said expression cassettes being driven by the constitutive promoter Plpp.

[0074] This embodiment provides a method for constructing the gallic acid-producing strain described above, which is basically similar to the construction method provided in Example 1. The main difference is that this embodiment uses the high-yielding strain GJ 10 provided in Example 3 as the operating object, the integration site is the yeep site, and the integrated gene is aroG. fbr -aroB-aroD; specifically, replace the primers with yeep-UF, yeep-UR, or yeep-aroG. fbr -aroB-aroD-F, yeep-aroG fbrBy using the primers -aroB-aroD-R, yeep-DF, yeep-DR, and yeep-pTarget-F, and replacing the promoters with Plpp1.9, Plpp4.0, and Plpp4.2, recombinant strains GJ 11, GJ 12, and GJ 13 were obtained. The DNA sequences of the primers used to integrate the shikimic acid pathway are shown in Table 8.

[0075] Table 8. Primer DNA sequence list used for genes integrating the shikimic acid pathway.

[0076]

[0077] The recombinant strains GJ 11, GJ 12, and GJ 13 were inoculated at a volume ratio of 2%, with 1 mL of bacterial culture added to 50 mL of shake flask culture medium. Fermentation was carried out at 33℃ and 220 rpm for 48 h to obtain the corresponding fermentation products. The concentration of gallic acid in the fermentation products was determined by high-performance liquid chromatography (HPLC). The gallic acid yields of strains GJ 11, GJ 12, and GJ 13 were found to be 6.54 g / L, 6.75 g / L, and 7.16 g / L, respectively.

[0078] Example 5

[0079] This embodiment provides a method for scale-up production of gallic acid using a gallic acid-producing strain in a fermenter. This embodiment uses the gallic acid-producing strain GJ 13 prepared in Example 4 for fermentation scale-up production, and the specific production method is as follows:

[0080] First, the gallic acid-producing strain GJ 13 was inoculated into LB medium for shake-flask amplification culture to obtain shake-flask seed culture. Then, the shake-flask seed culture was inoculated at an 8% (v / v) rate into a 3L fermenter containing 1L of fermentation medium for fermentation culture at 33°C to de novo synthesize gallic acid. The process parameters for fermentation in the fermenter were: ventilation rate 3-5 m³ / h. 3 The stirring speed was 800 rpm, dissolved oxygen was maintained at approximately 20%, and a glucose solution with a concentration of 650 g / L was added. 25% (w / v) ammonia was added to adjust the pH of the fermentation broth to 6.5. Then, fermentation products were collected at 12 h, 24 h, 36 h, and 48 h, and gallic acid concentration was determined by high-performance liquid chromatography. The fermentation results are as follows: Figure 5 As shown.

[0081] from Figure 5 It can be seen that the gallic acid producing strain can effectively achieve the scale-up production of gallic acid in fermenters, with a yield of 51.57 g / L and a carbon yield of 60% of the natural theoretical carbon yield.

[0082] It should be noted that, during fermentation in the fermentation tank, those skilled in the art can adjust the above conditions to a certain extent according to the actual situation, without affecting the achievement of the purpose of the present invention.

[0083] Example 6

[0084] This embodiment provides an engineered bacterium that produces β-glucosidin de novo. It is mainly constructed by using strain GJ 13 obtained in Example 4 as the substrate bacteria and transforming it with the raspberry-derived glycosidase RiGT2 shown in SEQ ID NO.37.

[0085] The specific construction method includes: streaking the above-mentioned gallic acid producing strain GJ 13 on an antibiotic-free plate, culturing it overnight at 37°C, picking positive single-clone transformants to obtain a genetically engineered strain of gallic acid, electrochemically transferring it into the β-glucosamine production plasmid pET-RiGT2 to obtain the β-glucosamine engineered strain GJ 14.

[0086] This embodiment also provides a method for biosynthesizing β-glucosamine using the engineered strain GJ 14, which produces β-glucosamine. The method includes: inoculating the engineered strain GJ 14 into a 250 mL 40 mL shake flask at a volume fraction of 5%, and fermenting it at 33°C and 200–220 rpm to synthesize β-glucosamine. The concentration of β-glucosamine in the fermentation product of strain GJ 14 is determined by high-performance liquid chromatography (HPLC). The HPLC chromatogram of the β-glucosamine standard is shown below. Figure 6 As shown, the HPLC detection chromatogram of the fermentation products of strain GJ 14 is as follows. Figure 7 As shown in the figure, the fermentation results are as follows: Figure 8 As shown.

[0087] HPLC detection employed gradient elution. Mobile phase A consisted of 100% methanol, and mobile phase B consisted of 2‰ trifluoroacetic acid. The flow rate was 1 ml / min. At a detection temperature of 40°C, the HPLC program was set as follows: 5%-50% methanol for 20 minutes, 100% methanol for 2 minutes, 100%-5% methanol for 2 minutes, and 5% methanol for an additional 5 minutes.

[0088] from Figure 8 As can be seen, the engineered bacteria of β-glucosamine can achieve de novo synthesis of β-glucosamine, and finally completed the construction of the artificial synthesis pathway of β-glucosamine for the first time, with a yield of 92.42 mg / L.

[0089] Therefore, the gallic acid-producing strain provided in this embodiment of the invention, combined with a metabolic engineering strategy, utilizes CRISPR Cas9 technology to optimize gene expression intensity in the host by changing promoter strength and increasing copy number. First, it completes pathway construction by integrating genes related to the gallic acid synthesis pathway; second, it increases the supply of precursors phosphoenolpyruvate and erythrose-4-phosphate, knocking out or integrating genes related to their metabolism; finally, it strengthens the shikimic acid pathway by integrating genes related to it. This achieves the biosynthesis of gallic acid from simple carbon sources such as glucose. The β-glucosidin engineered strain provided in this embodiment of the invention is mainly constructed using the gallic acid-producing strain as the substrate bacteria, realizing the construction of recombinant engineered strains of gallic acid derivatives using the gallic acid-producing strain as the starting strain.

[0090] In summary, this invention utilizes metabolic engineering techniques to modify *E. coli*, obtaining a recombinant strain that produces gallic acid. Compared to engineered strains based on plasmid vectors, this strain offers advantages such as high synthesis efficiency, genetic stability, and the elimination of the need for additional inducing agents. This invention not only provides a genetically engineered bacterium that produces high levels of gallic acid but can also be used to synthesize other high-value compounds starting from gallic acid, such as the first-ever de novo biosynthesis of β-glucosinolate.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in the present invention.

Claims

1. A gallic acid-producing strain, characterized by: In the genome of E. coli mbha site and ilvG Knock-in the gene encoding 3-dehydroshikimate dehydratase at each site. quiC In the genome yjip Knock-in of the gene encoding the p-hydroxybenzoic acid hydroxylase mutant pobA ; The 3-dehydroshikimate dehydratase quiC Through P trc The promoter is overexpressed in multiple copies on the genome; The p-hydroxybenzoate hydroxylase mutant pobA Through P lpp The promoter is expressed on the genome; The Escherichia coli was Escherichia coli BW25113; The 3-dehydroshikimate dehydratase quiC is derived from *Pseudomonas putida*. Pseudomonas putida The hydroxybenzoic acid hydroxylase mutant pobA The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1; Knockout of glycolysis pathway genes in the genome pykA and pykF Knock out genes encoding proteins related to the phosphoenolpyruvate glucotransferase system. ptsG and in the genome yncI Site-integrated glucose transporter encoding gene glf and the gene encoding glucokinase glk And in the genome yghX The gene encoding 2-dehydro-3-deoxyphosphate heptanoate aldolase is integrated at this site. aroG fbr ; The gene encoding the glucose transporter glf The gene encoding glucokinase glk The gene encoding 2-dehydro-3-deoxyphosphate heptanoate aldolase aroG fbr Through P trc The promoter is overexpressed or enhanced on the genome; In the genome yjiT Site-integrated transketolase encoding gene tktA and the gene encoding transaldolase talB ; The gene encoding the transketolase tktA and the gene encoding transaldolase talB Through the constitutive promoter P lpp Overexpressed or enhanced on the genome; In the genome yeep The site integrates the encoding gene of the 2-dehydro-3-deoxyphosphate heptanoate aldolase. aroG fbr The gene encoding 3-dehydroquinoline synthase aroB and the gene encoding 3-dehydroquinoline dehydratase aroD ; The gene encoding the 2-dehydro-3-deoxyphosphoheptanate aldolase aroG fbr The gene encoding 3-dehydroquinoline synthase aroB and the gene encoding 3-dehydroquinoline dehydratase aroD Through the constitutive promoter P lpp In the genome yeep The site is overexpressed or enhanced.

2. The application of the gallic acid producing strain according to claim 1 in the fermentation production of gallic acid.

3. The application according to claim 2, characterized in that, The culture medium used in the fermentation process includes 10-20 g / L simple carbon source, 4-12 g / L yeast extract, 4-8 g / L Na2HPO4, 0.3-2 g / L NaCl, 2-4 g / L KH2PO4, 0.6-1.5 g / L NH4Cl, 200-300 mg / L MgSO4, and 10-25 mg / L CaCl2.

4. The application according to claim 3, characterized in that, The simple carbon source is glucose, glycerol, or a combination of both.

5. The application according to claim 2, characterized in that: The gallic acid producing strain was inoculated into shake flask culture medium at an inoculation rate of 1% to 10% by volume, and the culture was carried out at 30℃ to 35℃ to produce gallic acid.

6. The application according to claim 5, characterized in that, The shake flask culture medium comprises 10-20 g / L simple carbon source, 4-7 g / L yeast extract, 4-7 g / L Na2HPO4, 0.3-2 g / L NaCl, 2-4 g / L KH2PO4, 0.6-1.2 g / L NH4Cl, 0.8-1.5 g / L MOPS, 200-300 mg / L MgSO4, and 10-25 mg / L CaCl2.

7. The application according to claim 5, characterized in that, The stirring speed for shake flask fermentation is 200~220 rpm.

8. The application according to claim 2, characterized in that: First, the gallic acid producing strain is inoculated into LB medium for shake-flask amplification culture to obtain shake-flask seed liquid; then, the shake-flask seed liquid is inoculated into fermenter medium at a volume fraction of 5%~10%, and fermenter culture is carried out at 30℃~35℃ and dissolved oxygen is maintained at 10%~30% to produce gallic acid.

9. The application according to claim 8, characterized in that, The process parameters for producing gallic acid in the fermenter are: ventilation volume 3~5 m³ / h. 3 / h, stirring speed 300~1000 rpm.

10. The application of the gallic acid-producing strain according to claim 1 in the production of gallic acid derivatives, characterized in that, The application uses the gallic acid-producing strain as the starting strain to construct an engineered strain for producing gallic acid derivatives; the gallic acid derivative is β-glucosamine. The engineered bacteria for producing gallic acid derivatives further expressed a gene encoding a glycosidase on the gallic acid producing strain, and the nucleotide sequence of the gene encoding the glycosidase is shown in SEQ ID NO.

37.

11. The application according to claim 10, characterized in that: The engineered bacteria for producing gallic acid derivatives were inoculated into the culture medium at a volume fraction of 1% to 10%, and fermented at 30℃ to 35℃ to produce gallic acid derivatives.

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