Engineering bacterium for synthesizing phloretin as well as construction method and application of engineering bacterium

Abstract

The invention provides an engineering bacterium for synthesizing phloretin as well as a construction method and application of the engineering bacterium. The engineering bacteria comprise escherichia coli and genes which are co-expressed in the escherichia coli and are used for coding tyrosine deaminase, p-coumaroyl-coenzyme A ligase, chalcone synthase and reductase, the tyrosine deaminase is derived from rhodotorula or hypericum monogynum, the p-coumaroyl-coenzyme A ligase is derived from arabidopsis A.thaliana, petunia or apple, and the chalcone synthase is derived from chalcone synthase. The chalcone synthase gene is derived from petunia hybrida, and the reductase gene is derived from clostridium or acetyl clostridium. Efficient synthesis of phloretin can be realized by utilizing the engineering bacteria, and the yield of the phloretin synthesized from the beginning can reach 244.55 mg / L. The invention also provides an application of the engineering bacterium in biosynthesis of phloretin derivatives such as 3-hydroxy phloretin, hesperetin dihydrochalcone, hesperetin dihydrochalcone glucoside, neohesperidin dihydrochalcone and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering technology. More specifically, this invention relates to an engineered bacterium for synthesizing phloretin, its construction method, and its application. Background Technology

[0002] Phloretin (Pht) is a dihydrochalcone natural product widely found in plants of the genus Malus in the family Rosaceae. Its molecular formula is C1. 15 H 14 O5.

[0003] With a relative molecular mass of 274.27, pure crystals are light red, readily soluble in methanol, ethanol, and acetone, almost insoluble in water, and soluble in alkaline solutions. Numerous studies have shown that Phlorizin possesses physiological functions such as antioxidant, antibacterial, tyrosinase inhibition, α-glucosidase inhibition, hypoglycemic, cardiovascular protection, anti-tumor, and cosmetic benefits. Currently, phlorizin is primarily produced chemically, and its production in the market is mainly through chemical synthesis. Phlorizin in natural products mainly exists in the form of glycosides. Phlorizin is prepared by acid hydrolysis of phlorizin glycoside derivatives such as phlorizin. Alternatively, naringin can be obtained by catalytic hydrogenation to naringin dihydrochalcone, followed by hydrolysis under acidic conditions to produce phlorizin. Phlorizin can also be synthesized from phloroglucinol and p-hydroxyphenylpropionic acid under BF3Et2O catalysis.

[0004] However, current chemical methods for synthesizing phlorizin suffer from problems such as seasonal limitations of raw materials, high costs, low efficiency, chemical waste discharge, and severe environmental pollution. Therefore, based on the advantages of biosynthesis technology, the biosynthetic pathway of phlorizin has become a research hotspot. To this end, Chinese invention patent application CN 108138151 A discloses a method using yeast as the substrate bacteria, starting with tyrosine, catalyzed by deaminase TAL to p-coumaric acid, p-coumaric acid to p-coumaroyl-CoA via p-coumaric acid-coenzyme A ligase 4CL, p-coumaric acid-CoA to dihydrocoumaric acid-CoA via endogenous reductase ENR / DBR, and dihydrocoumaric acid-CoA to phlorizin via chalcone synthase CHS. Although this invention patent application provides an engineered strain for biosynthesizing phlorizin, the substrate bacteria of this engineered strain are mainly yeast, which is not suitable for Escherichia coli, and the yield of phlorizin is low, which is not conducive to industrial application. Summary of the Invention

[0005] In view of this, the main objective of this invention is to provide an engineered bacterium that uses Escherichia coli as the substrate bacteria and can efficiently synthesize and produce phloretin, as well as its construction method and application.

[0006] This invention primarily aims to construct a novel biosynthetic pathway for phlorin from *E. coli*, based on existing analyses of heterologous pathways for phlorin synthesis, and to screen suitable exogenous enzymes to achieve efficient phlorin synthesis. Experimental results show that the *E. coli* engineered strain can achieve a phlorin yield of 369.23 mg / L when supplemented with tyrosine in vitro, and a final yield of 244.55 mg / L when using glycerol and glucose as simple carbon sources.

[0007] Specifically, the technical solution provided by this invention is as follows: An engineered bacterium for synthesizing phlorizin comprises *Escherichia coli* and genes co-expressed in the *E. coli* encoding tyrosine deaminase (TAL), p-coumaroyl-CoA ligase (4CL), chalcone synthase (CHS), and reductase, wherein the tyrosine deaminase (TAL) is derived from *Rhodotorula rubra* or *Hypericum perforatum*, and the p-coumaroyl-CoA ligase (4CL) is derived from *Arabidopsis thaliana*. A. thaliana Petunias, apples, etc., the chalcone synthase gene (CHS) is derived from petunias, and the reductase gene is derived from Clostridium ( Eubacterium ramulus (hereinafter referred to as "ERED") or Clostridium acetobutylicum (hereinafter referred to as "ER").

[0008] The Escherichia coli can be the original Escherichia coli, BL21, BW25113, JCL16, etc., or it can be a recombinant Escherichia coli that has been genetically engineered, such as tyrosine-producing Escherichia coli.

[0009] To further increase phloretin production, the *E. coli* is a tyrosine-producing *E. coli*; preferably, the tyrosine-producing *E. coli* includes *E. coli* BW25113 and a key enzyme encoding tyrosine synthesis co-expressed in BW25113; preferably, the key enzyme encoding tyrosine synthesis includes a tryptophan synthase gene. aroG and prephenyl acid dehydrogenase gene tyrA .

[0010] To further enhance the yield of naringenin dihydrochalcone, a precursor for phlorizin synthesis, a molecular chaperone (ibpAB) was introduced into the engineered strain. The NCBI database accession number for ibpAB is CP016018.1. Thus, the engineered strain can achieve a phlorizin yield of 80.13 mg / L by adding 0.5 g / L of tyrosine in vitro, and a final phlorizin yield of 244.55 mg / L using glycerol and glucose as simple carbon sources.

[0011] The present invention also provides a method for constructing the above-mentioned engineered bacteria for synthesizing phloretin, comprising: The recombinant expression plasmid was constructed by ligating the encoding genes of tyrosine deaminase (TAL), p-coumaroyl-CoA ligase (4CL), chalcone synthase (CHS), and reductase (ERED) into the expression plasmid. The engineered bacteria were constructed, and the recombinant plasmid was transformed into Escherichia coli to obtain an engineered bacteria that synthesizes phloretin.

[0012] The expression plasmid can be pCS27 (medium copy), pSA74, pZE12-luc (high copy), pET-lac, etc. Preferably, after module optimization, the encoding genes of chalcone synthase (CHS) and reductase (ERED) are ligated to pET-lac to form the recombinant plasmid pET-lac-CHS-ERED; the encoding genes of tyrosine ammonia-lyase (TAL) and coumaroyl-CoA ligase (4CL) are ligated to pCS27 to form the recombinant plasmid pCS27-lac-TAL-4CL.

[0013] A method for biosynthesizing phlorizin includes: fermenting the engineered bacteria for synthesizing phlorizin in a fermentation medium at an inoculum concentration of 1% to 10% by volume to obtain phlorizin. Preferably, the inoculum concentration is 1% to 5%.

[0014] Furthermore, tyrosine is added to the fermentation medium to achieve in vitro synthesis of phlorizin from tyrosine. Preferably, the amount of tyrosine added is 0.1-2 g / L.

[0015] The carbon source in the fermentation medium is glycerol, monosaccharides, disaccharides, or any combination thereof. Preferably, the carbon source in the fermentation medium is one or any combination of glycerol, glucose, sucrose, fructose, and xylose.

[0016] During the fermentation process, 0.25–1 mM of the inducing agent IPTG is added; the fermentation temperature is preferably 30℃–40℃.

[0017] The fermentation medium comprises: 1–5 g / L MOPS, 5–20 g / L simple carbon source, 1–5 g / L yeast extract, 5–8 g / L Na2HPO4, 0.3–2 g / L NaCl, 2.3–4.0 g / L KH2PO4, and 1–5 g / L NH4Cl; wherein the simple carbon source is one or any combination of glycerol, glucose, sucrose, fructose, and xylose.

[0018] Therefore, the engineered bacteria for synthesizing phlorizin provided by the present invention can not only synthesize phlorizin efficiently with a yield of 369.23 mg / L, but also achieve a carbon yield of 12.23%.

[0019] The application of an engineered bacterium that synthesizes phlorizin in the biosynthesis of phlorizin derivatives. The phlorizin derivatives may be 3-hydroxyphlorizin, hesperidin dihydrochalcone, hesperidin dihydrochalcone glucoside, or neohesperidin dihydrochalcone. Attached Figure Description

[0020] Figure 1 This is a diagram illustrating the biosynthetic pathway of phloretin and its derivatives provided by the present invention.

[0021] Figure 2 This is a diagram showing the fermentation results of the engineered strain BW1, which synthesizes phloretin by adding tyrosine in vitro, as provided in an embodiment of the present invention.

[0022] Figure 3 This is a diagram showing the fermentation results of the engineered strain for de novo phloretin synthesis and BW2, provided in an embodiment of the present invention.

[0023] Figure 4 This is a diagram showing the fermentation results of engineered strains BW3 and BW4, which synthesize phloretin by adding tyrosine in vitro, as provided in embodiments of the present invention.

[0024] Figure 5 This is a diagram showing the fermentation results of the engineered strain BW5, which synthesizes phlorizin de novo, as provided in an embodiment of the present invention.

[0025] Figure 6 The above diagram shows the fermentation results of engineered strains BW6, BW7, BW8, and BW9, which synthesize phlorizin derivatives de novo, according to embodiments of the present invention. Detailed Implementation

[0026] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0027] In this invention, there are no special requirements for the type of expression plasmid. It can be assumed that the construction method for expressing the target gene in Escherichia coli can adopt various methods commonly used in the field, such as ligating the target gene into a vector after enzyme digestion. Further details will not be elaborated hereafter.

[0028] 1) Enzymes used in the examples and comparative examples All enzymes involved in this invention are derived from commonly used substances, and the sources of these enzymes are not limited to those listed in this invention. Any enzyme with a similarity of less than 80% to the enzymes listed in this invention is within the scope of protection of this invention. In the following examples and comparative examples, in addition to the key enzymes shown in Table 1, other enzymes, such as the various enzymes used in the pathway for synthesizing shikimic acid from a carbon source, are also existing commonly used enzymes.

[0029] Table 1. Enzyme or Protein Source Table

[0030] 2) Culture media used in the examples and comparative examples LB medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L; Fermentation medium: 2 g / L MOPS, 10 g / L glucose, 2.5 g / L yeast extract, 6.78 g / L Na2HPO4, 0.5 g / L NaCl, 3 g / L KH2PO4 and 1 g / L NH4Cl.

[0031] 3) Plasmids and strains used in the examples and comparative examples In the following examples and comparative examples, the *E. coli* strains BW25113 and trans5α are commonly used *E. coli* strains and are commercially available. Trans5α was used for vector construction, while BW25113 (rrnBT14 ΔlacZWJ16 hsdR514 ΔaraBADAH33 ΔrhaBADLD78) was used as the fermentation strain. The plasmid pET: P L lacO1, colE ori, luc,Amp R plasmid pCS: P L lacO1, p15A ori, Kan R .

[0032] The tyrosine-producing strain BW0 is designated as BW25113 ::50 aroG ::30 tyrA. The construction method of this strain can be found in Ping Jurong, et al. Synergetic engineering of Escherichia coli for efficient production of l-tyrosine, Synthetic and Systems Biotechnology, Published: 2023-12, Issue: 4, Volume: 8, Page: 724-731.

[0033] 4) Detection conditions for HPLC analysis used in the examples The conditions for detecting phloretin and its derivatives standards and fermentation products using HPLC analysis in the examples are as follows: Chromatographic column: Separation column: Diamonsil C18, ID 5μm, 250 × 4.6 mm; Mobile phase: A is methanol, B is 1‰ formic acid aqueous solution; column temperature: 40℃; flow rate: 1 mL / min; detection wavelength: 280 nm. Constant gradient elution was used. The gradient elution program is shown in Table 2 below: Table 2 Gradient elution program

[0034] Standard solutions of phlorizin, 3-hydroxyphlorizin, hesperidin dihydrochalcone, hesperidin dihydrochalcone glucoside, and neohesperidin dihydrochalcone were prepared with concentrations of 0 mg / L, 100 mg / L, 200 mg / L, and 500 mg / L, respectively. Cells were lysed with 1:1 methanol for derivatization and HPLC detection.

[0035] The technical solution protected by this invention will be described in detail below with specific embodiments.

[0036] I. Engineered bacteria for in vitro tyrosine synthesis of phloretin Example 1 like Figure 1 As shown, the pathway for synthesizing phlorizin can be as follows: During metabolism, L-tyrosine is converted to p-coumaric acid by enzyme TAL, p-coumaric acid is converted to p-hydroxyphenylpropionic acid by enzyme 4CL, p-hydroxyphenylpropionic acid is converted to p-dihydrocoumaryl-CoA by enzyme 4CL, and p-dihydrocoumaryl-CoA is converted to phlorizin by enzyme CHS. Based on this, this embodiment provides an engineered bacterium BW1 for synthesizing phlorizin, including Escherichia coli BW25113 and the encoding genes for TAL, 4CL, CHS and ER co-expressed in Escherichia coli.

[0037] Construction of engineered bacteria This invention also provides a method for constructing the above-mentioned engineered bacteria that synthesize phloretin. First, the overlap technique is used to ligate TAL and 4CL, and CHS and ER together. Then, homologous recombination is used to construct recombinant plasmids pCS-TAL-4CL and pET-CHS-ER. Specifically, the steps include: first, selecting tyrosine deaminase TAL, p-coumaroyl-CoA ligase 4CL, chalcone synthase CHS, and reductase ER, and obtaining the corresponding gene fragments using PCR. Next, the corresponding gene fragments and the vector are double-digested with endonucleases. The digested fragments are then recovered by gel extraction or column extraction. Finally, the target gene is inserted into plasmids pET-lac (high copy number) and pCS27 (medium copy number), respectively, to obtain the pET-CHS-ER and pCS-TAL-4CL recombinant plasmids (Table 2). Among them, the recombinant plasmid pET-CHS-ER is mainly obtained by ligating the genes encoding CHS and ER into the same vector plasmid pET-lac; the recombinant plasmid pCS-TAL-4CL is mainly obtained by ligating the genes encoding TAL and 4CL into the same vector plasmid pCS27.

[0038] To prepare competent BW25113 cells by electroporation, fresh BW25113 colonies were first inoculated into 4 mL of LB medium and cultured at 37°C for 8–12 h. Then, 1 mL of the culture was inoculated into 100 mL of LB medium and cultured at 37°C until the OD reached the target cell count. 600 When the cells reached 0.6, they were collected by centrifugation at 6000 rpm for 10 min at 4℃. They were washed with 10 mL of 10% pre-cooled glycerol and centrifuged at 6000 rpm for 10 min. The glycerol washing step was repeated. After centrifugation, the remaining glycerol was drained as much as possible. Finally, an appropriate amount of 10% glycerol was added to resuspend the cells to obtain competent cells. Take 90 μL of competent cells, add 2 μL of recombinant plasmids pCS-TAL-4CL and pET-CHS-ERED, and place on ice for 2 min. After electroporation, add 600 μL of LB medium, wash out the electroporated cells, and revive at 37 ℃ for 1 h. Spread on kanamycin and ampicillin resistant plates and incubate overnight at 37 ℃. After the bacteria grow on the plates, pick the bacteria and incubate them at 37 ℃ for 8-10 h in 4 mL of LB medium containing kanamycin resistant bacteria to obtain the engineered bacterium BW1: BW25113 (pCS-TAL-4CL, pET-CHS-ER) that synthesizes phlorizin. This engineered bacterium BW1 is an Escherichia coli strain containing recombinant plasmids pCS-TAL-4CL and pET-CHS-ER.

[0039] application Fresh, engineered single colonies of recombinant *E. coli* BW1 were picked from plates and inoculated into corresponding 4 mL LB tubes containing the appropriate antibiotics. After incubation at 37°C for 8 h, the colonies were transferred to shake flasks containing 50 mL M9 medium and 0.5 g / L tyrosine for fermentation. The inoculum size was 1%–10%, the fermentation temperature was 30°C–40°C, and the rotation speed was 200 rpm. The M9 medium consisted of 2 g / L MOPS, 10 g / L glucose, 2 g / L yeast extract, 6.78 g / L Na₂HPO₄, 0.5 g / L NaCl, 3.0 g / L KH₂PO₄, and 1.0 g / L NH₄Cl, with appropriate antibiotics added as needed. IPTG inducer at a final concentration of 0.5 mM was added at the beginning of fermentation. Fermentation broth was collected at 12 h, 24 h, 36 h, and 48 h to determine the growth status of the corresponding engineered strains and the yield of the target product. The results are as follows: Figure 2 As shown.

[0040] from Figure 2 It can be seen that this pathway can generate phlorin from tyrosine. When 0.5 g / L of tyrosine was added for 24 h, the phlorin production of engineered strain BW1 reached 5.11 mg / L. The carbon yield of engineered strain BW1 was calculated to be 1%.

[0041] Validation of the phloretin synthesis pathway This validation experiment provides an engineered bacterium, the main difference between which is the engineered bacterium BW1 provided in Example 1: it only co-expresses the coding genes for TAL and ERED in Escherichia coli BW25113, while other genes remain unchanged; that is, this engineered bacterium is BW25113 (pCS-TAL, pET-ERED). The engineered bacterium BW25113 (pCS-TAL, pET-ERED) was fermented using the same method as in Example 1, and the fermentation products were detected by HPLC.

[0042] Tests showed no formation of p-hydroxyphenylpropionic acid (phloroglucinic acid). This indicates that the reductase ERED cannot convert p-coumaric acid to p-hydroxyphenylpropionic acid.

[0043] Example 2 To further increase the yield of phloretin, this embodiment provides an engineered bacterium BW2 that synthesizes phloretin. The main difference between this engineered bacterium and the engineered bacterium BW1 provided in Example 1 is that the *Escherichia coli* BW25113 also co-expresses the coding gene for the molecular chaperone ibpAB of CHS.

[0044] This embodiment also provides a method for constructing engineered strain BW2, which is basically the same as the method for constructing BW1. First, the genes encoding CHS, ER, and ibpAB are ligated into the same vector plasmid pET-lac to obtain the recombinant plasmid pET-CHS-ER-ibpAB. Then, the genes encoding TAL and 4CL are ligated into the same vector plasmid pCS27 to obtain the recombinant plasmid pCS-TAL-4CL. Next, using homologous recombination technology, the recombinant plasmids pET-CHS-ER-ibpAB, pET-CHS-ER-ibpAB, and pCS-TAL-4CL are electroporated into *Escherichia coli* BW25113 to obtain the above-mentioned engineered strain BW2:BW25113 (pCS-TAL-4CL, pET-CHS-ER-ibpAB). The engineered strain BW2 is fermented using the same method as in Example 1, and the fermentation products are detected by HPLC. The detection results are as follows: Figure 3 As shown. From Figure 3 The results show that adding the molecular chaperone ibpAB to the phlorizin synthesis pathway using engineered strain BW2 can increase phlorizin yield. Adding 0.5 g / L tyrosine resulted in 80.13 mg / L phlorizin production after 24 h, representing a yield increase of approximately 15-fold, with a carbon yield of 5.14%. This is primarily because the introduction of the molecular chaperone can promote protein folding more quickly and effectively, reducing the production of ineffective proteins.

[0045] Example 3 To further increase the synthesis yield of phloretin, this embodiment provides another pathway for synthesizing phloretin. During metabolism, L-tyrosine is converted to p-coumaric acid by enzyme TAL, p-coumaric acid is converted to p-coumaryl-CoA by enzyme 4CL, p-coumaryl-CoA is converted to naringenin chalcone by enzyme CHS, and naringenin chalcone is reduced to phloretin by enzyme ERED.

[0046] Based on this, this embodiment provides an engineered bacterium BW3 that synthesizes phloretin. The main difference between this engineered bacterium and the engineered bacterium BW1 provided in Example 1 is that the reductase ERED derived from Clostridium is used instead of the reductase ER in Example 1, while the rest remains unchanged; that is, the engineered bacterium BW3 that synthesizes phloretin is: BW25113 (pCS-TAL-4CL, pET-CHS-ERED).

[0047] The engineered strain BW3 was fermented using the same method as in Example 1, and the fermentation products were detected by HPLC. The results are as follows: Figure 4 As shown. From Figure 4 It can be seen that when 0.5 g / L tyrosine was added and fermented for 24 h, the phloretin production of engineered strain BW3 reached 127.12 mg / L; the carbon yield of engineered strain BW3 was calculated to be 7.55%.

[0048] Example 4 To further increase the yield of phloretin, this embodiment provides a new engineered bacterium, BW4, for synthesizing phloretin. The main difference between this engineered bacterium and the engineered bacterium BW3 provided in Example 2 is that the *Escherichia coli* BW25113 also co-expresses the coding gene for the molecular chaperone ibpAB of CHS.

[0049] This embodiment also provides a method for constructing engineered strain BW4, which is basically the same as the method for constructing BW2. First, the genes encoding CHS, ERED, and ibpAB are ligated into the same vector plasmid pET-lac to obtain the recombinant plasmid pET-CHS-ERED-ibpAB. Then, the genes encoding TAL and 4CL are ligated into the same vector plasmid pCS27 to obtain the recombinant plasmid pCS-TAL-4CL. Then, homologous recombination technology is used to electroporate the recombinant plasmids pET-CHS-ERED-ibpAB, pET-CHS-ERED-ibpAB, and pCS-TAL-4CL into Escherichia coli BW25113 to obtain the above-mentioned engineered strain BW4:BW25113 (pCS-TAL-4CL, pET-CHS-ERED-ibpAB).

[0050] The engineered strain BW4 was fermented using the same method as in Example 1, and the fermentation products were analyzed by HPLC to determine the growth status of the corresponding engineered strain and the yield of the target product. The results are as follows: Figure 4 As shown. From Figure 4 The results show that when engineered strain BW4 was supplemented with 0.5 g / L tyrosine, it produced 244.55 mg / L phloretin after 24 h of fermentation, with a carbon yield of 12.23%. The yield and carbon yield were approximately twice that of BW3.

[0051] II. Example 5: Engineered bacteria for de novo synthesis of phlorizin and its application This invention provides an engineered bacterium, BW5, for de novo synthesis of phloretin. The main difference between this engineered bacterium and the engineered strain BW4 is that the engineered strain BW5 uses the tyrosine-producing engineered bacterium BW0 as the substrate bacteria, replacing the substrate bacteria BW25113 of strain BW3. In other words, the engineered strain BW4 is BW0 (pCS-TAL-4CL, pET-CHS-ERED-ibpAB).

[0052] The construction method of engineered strain BW5 is basically the same as that of engineered strain BW4.

[0053] Engineered strains BW4 and BW5 were fermented, and the fermentation products were analyzed by HPLC to determine the growth status of the corresponding engineered strains and the yield of the target product. The results are as follows: Figure 5 As shown. In this embodiment, the fermentation culture method of engineered bacteria BW4 and BW5 is basically the same as that of engineered bacteria BW1 in Example 1. The main difference is that in this embodiment, engineered bacteria BW4 and BW5 do not need to add tyrosine during the fermentation synthesis of phloretin in M9 medium.

[0054] from Figure 5 As can be seen, both engineered bacteria BW4 and BW5 can synthesize phlorizin de novo. Engineered bacteria BW5 produced 369.23 mg / L of phlorizin, approximately 11 times the 33.91 mg / L produced by engineered bacteria BW4. This is mainly because engineered bacteria BW5 uses the high-tyrosine-producing engineered bacteria BW0 as its substrate. BW0 can synthesize large amounts of tyrosine de novo in M9 medium and promote carbon source conversion, thus achieving efficient phlorizin synthesis with a carbon recovery rate of 33.12%.

[0055] III. Engineered bacteria for de novo synthesis of 3-hydroxyphlorin, its construction methods, and applications. The biosynthetic pathway of 3-hydroxyphlorin: Based on the biosynthetic pathway of phlorin, under the action of metabolism, phlorin is converted into 3-hydroxyphlorin by flavonoid hydroxylase.

[0056] Example 6: Engineered strain BW6 synthesizing 3-hydroxyphlorin This embodiment provides an engineered bacterium, BW6, for synthesizing 3-hydroxyphlorin, comprising an engineered bacterium for phlorin synthesis and a gene encoding the flavonoid hydroxylase HpaBC expressed in the engineered bacterium. The flavonoid hydroxylase HpaBC can be derived from Escherichia coli, Klebsiella pneumoniae, Rhodococcus erythropoietin, etc. Pseudomonas aeruginosa or Rhodococcus opacus In this embodiment, the engineered bacterium that synthesizes phloretin is the aforementioned engineered bacterium BW5, and HpaBC is the RoHpaBC mutant Y215A. This mutant is mainly obtained by using existing gene mutation methods to mutate the 215th site of the original hydroxylase RoHpaBC, specifically by mutating the site from "Y" to "A".

[0057] This embodiment also provides a method for constructing an engineered bacterium that synthesizes 3-hydroxyphloretin. The method is basically the same as that used for BW1. First, the gene encoding the hydroxylase RoHpaBC mutant Y215A is ligated into plasmid pCS27 to obtain the recombinant plasmid pCS-RoHpaBC / Y215A. Then, the recombinant plasmid pCS-HpaBCY215A is electroporated into the engineered bacterium BW5 using homologous recombination technology to obtain the above-mentioned engineered strain BW6:BW5 (pCS-RoHpaBC / Y215A).

[0058] Application of engineered bacteria BW6 The engineered strain BW6 was fermented, and the fermentation products were analyzed by HPLC to determine the growth status of the corresponding engineered strain and the yield of the target product. The results are as follows: Figure 6 As shown. In this embodiment, the fermentation culture method of engineered bacteria BW6 is basically the same as that of engineered bacteria BW5.

[0059] from Figure 6 It can be seen that the engineered bacterium BW6 can achieve de novo synthesis of 3-hydroxyphloretin using a simple carbon source, and the yield reaches 204.11 mg / L.

[0060] IV. Engineered bacteria for synthesizing hesperidin dihydrochalcone, their construction methods, and applications The biosynthetic pathway of hesperidin dihydrochalcone is based on the biosynthetic pathway of 3-hydroxyphlorin. Under the action of metabolism, 3-hydroxyphlorin is converted into hesperidin dihydrochalcone by methyltransferase.

[0061] Example 7: Engineered strain BW7 for synthesizing hesperidin dihydrochalcone This embodiment provides an engineered bacterium, BW7, for synthesizing hesperidin dihydrochalcone, comprising an engineered bacterium that synthesizes phloretin and encoding genes for flavonoid hydroxylase HpaBC and methyltransferase OMT co-expressed in the phloretin-synthesizing engineered bacterium. The methyltransferase OMT is derived from cyanobacteria, *Mycobacterium chrysogenum*, *Mycobacterium tuberculosis*, *Sclerotium sessiliflorum*, and other cyanobacteria, such as... Rauwolfia serpentina , Myxococcus xanthus In this embodiment, the engineered bacterium that synthesizes phloretin is the aforementioned engineered bacterium BW5, HpaBC is the RoHpaBC mutant Y215A, and OMT is MxOMT.

[0062] This embodiment also provides a method for constructing an engineered bacterium that synthesizes hesperidin dihydrochalcone. The method is basically the same as that used for BW1. First, the gene encoding the RoHpaBC mutant hydroxylase is ligated into the vector plasmid pCS27 to obtain the recombinant plasmid pCS-RoHpaBC / Y215A. Then, the gene encoding MxOMT is ligated into the vector plasmid pET-lac to obtain the recombinant plasmid pET-MxOMT. Then, using homologous recombination technology, the recombinant plasmids pCS-RoHpaBC / Y215A and pET-MxOMT are co-electropoized into Escherichia coli BW5 to obtain the above-mentioned engineered strain BW7:BW5 (pCS-RoHpaBC / Y215A, pET-MxOMT). This engineered bacterium BW7 is also equivalent to BW6 (pET-MxOMT).

[0063] Application of engineered bacteria BW7 The engineered strain BW7 was fermented, and the fermentation products were analyzed by HPLC to determine the growth status of the corresponding engineered strain and the yield of the target product. The results are as follows: Figure 6 As shown. In this embodiment, the fermentation culture method of engineered bacteria BW7 is basically the same as that of engineered bacteria BW5.

[0064] from Figure 6 It can be seen that the engineered strain BW11 can achieve de novo synthesis of hesperidin dihydrochalcone, with a yield of 127.29 mg / L.

[0065] V. Engineered bacteria for synthesizing hesperidin dihydrochalcone glucoside, their construction methods, and applications The biosynthetic pathway of hesperidin dihydrochalcone glucoside is based on the biosynthetic pathway of hesperidin dihydrochalcone. Under the action of metabolism, hesperidin dihydrochalcone is converted into hesperidin dihydrochalcone glucoside by glycosyltransferase.

[0066] Example 8: Engineered strain BW8 synthesizing hesperidin dihydrochalcone glucoside This embodiment provides an engineered bacterium, BW8, for synthesizing hesperidin dihydrochalcone glucoside. It includes an engineered bacterium that synthesizes phloretin and genes encoding flavonoid hydroxylase HpaBC, methyltransferase OMT, and glycosyltransferase OGT, co-expressed within the phloretin-synthesizing engineered bacterium. The glycosyltransferase OGT is derived from plants such as kudzu root, japonica rice, Arabidopsis thaliana, tobacco, and Bacillus cereus. Pueraria lobata , Arabidopsis thaliana In this embodiment, the engineered bacterium that synthesizes phloretin is the aforementioned engineered bacterium BW5, HpaBC is the RoHpaBC mutant Y215A, OMT is MxOMT, and OGT is OGT72E2.

[0067] This embodiment also provides a method for constructing an engineered bacterium that synthesizes hesperidin dihydrochalcone glucoside. The method is essentially the same as that used for BW1. First, the gene encoding the RoHpaBC mutant hydroxylase is ligated into the vector plasmid pCS27 to obtain the recombinant plasmid pCS-RoHpaBC / Y215A. Then, the genes encoding MxOMT and OGT72E2 are ligated into the vector plasmid pET-lac to obtain the recombinant plasmid pET-MxOMT-OGT72E2. Next, using homologous recombination technology, the recombinant plasmids pCS-RoHpaBC / Y215A and pET-MxOMT-OGT72E2 are co-electropoized into *Escherichia coli* BW5 to obtain the engineered strain BW8:BW5 (pCS-RoHpaBC / Y215A, pET-MxOMT-OGT72E2). This engineered bacterium BW8 is also equivalent to BW7 (pET-OGT72E2).

[0068] Application of engineered bacteria BW8 The engineered strain BW8 was fermented, and the fermentation products were analyzed by HPLC to determine the growth status of the corresponding engineered strain and the yield of the target product. The results are as follows: Figure 6 As shown. In this embodiment, the fermentation culture method of engineered bacteria BW14 is basically the same as that of engineered bacteria BW5.

[0069] from Figure 6 It can be seen that the engineered bacterium BW14 can achieve de novo synthesis of hesperidin dihydrochalcone glucoside using a simple carbon source, and the yield reaches 183.2 mg / L.

[0070] VI. Engineered bacteria for synthesizing neohesperidin dihydrochalcone, its construction method, and its application The biosynthetic pathway of neohesperidin dihydrochalcone is based on the biosynthetic pathway of hesperidin dihydrochalcone glucoside. Under the action of metabolism, hesperidin dihydrochalcone glucoside is converted into neohesperidin dihydrochalcone by rhamnosyltransferase.

[0071] Example 9: Engineered strain BW9 synthesizing novel hesperidin dihydrochalcone This embodiment provides an engineered bacterium, BW9, for synthesizing the neohesperidin dihydrochalcone. It includes an engineered bacterium that synthesizes phloretin and genes encoding flavonoid hydroxylase HpaBC, methyltransferase OMT, glycosyltransferase OGT, and rhamnosyltransferase 1,2RhaT, co-expressed within the phloretin-synthesizing engineered bacterium. The rhamnosyltransferase Cm1,2RhaT can be derived from citrus, tea, etc., for example... Citrus maxima In this embodiment, the engineered bacterium that synthesizes phloretin is the aforementioned engineered bacterium BW5, HpaBC is the RoHpaBC mutant Y215A, OMT is MxOMT, OGT is OGT72E2, and 1,2RhaT is Cm1.2RhaT.

[0072] This embodiment also provides a method for constructing an engineered bacterium that synthesizes the new hesperidin dihydrochalcone. The method is essentially the same as that used for BW1. First, the gene encoding the hydroxylase RoHpaBC mutant is ligated into the vector plasmid pCS27 to obtain the recombinant plasmid pCS-RoHpaBC / Y215A. Then, the genes encoding MxOMT, OGT72E2, and Cm1.2RhaT are ligated into the same vector plasmid pET-lac to obtain the recombinant plasmid pET-MxOMT-P1OGT2. -Cm1.2RhaT; then, using homologous recombination technology, the recombinant plasmids pCS-RoHpaBC / Y215A and pET-MxOMT-P1OGT2-Cm1.2RhaT are co-electrotransferred into Escherichia coli BW5 to obtain the above-mentioned engineered strain BW9:BW5 (pCS-RoHpaBC / Y215A, pET-RsOMT-P1OGT2-Cm1.2RhaT); this engineered strain BW9 is also equivalent to BW8 (pET-Cm1.2RhaT).

[0073] Application of engineered bacteria BW9 The engineered strain BW9 was fermented, and the fermentation products were analyzed by HPLC to determine the growth status of the corresponding engineered strain and the yield of the target product. The results are as follows: Figure 6 As shown. In this embodiment, the fermentation culture method of engineered bacteria BW9 is basically the same as that of engineered bacteria BW5.

[0074] from Figure 6 It can be seen that the engineered bacterium BW9 can achieve de novo synthesis of the new hesperidin dihydrochalcone using a simple carbon source, and the yield reaches 98.36 mg / L.

[0075] 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. An engineered bacterium that synthesizes phlorizin, characterized in that: The invention includes *Escherichia coli* and genes co-expressed in *E. coli* encoding tyrosine deaminase, p-coumaroyl-CoA ligase, chalcone synthase, and reductase, wherein the tyrosine deaminase is derived from *Rhizopus rubrum* or *Hypericum perforatum*, the p-coumaroyl-CoA ligase is derived from *Arabidopsis thaliana*, *Petunia spp.*, or *Apple*, the chalcone synthase gene is derived from *Petunia spp.*, and the reductase gene is derived from *Clostridium* or *Clostridium acetylatedum*.

2. The engineered bacteria according to claim 1, characterized in that: the Escherichia coli is the original Escherichia coli or a recombinant Escherichia coli that has been genetically engineered.

3. The engineered bacteria according to claim 1, characterized in that: the Escherichia coli is a tyrosine-producing Escherichia coli; Preferably, the tyrosine-producing Escherichia coli includes Escherichia coli BW25113 and a key enzyme encoding tyrosine synthesis co-expressed in BW25113.

4. The engineered bacteria according to claim 3, characterized in that: the key enzymes for synthesizing tyrosine include the tryptophan synthase gene aroG and the prephenylacetic acid dehydrogenase gene tyrA.

5. The engineered bacteria according to any one of claims 1-4, characterized in that: it further incorporates the molecular chaperone ibpAB; Preferably, the NCBI database registration number of the molecular chaperone ibpAB is CP016018.

1.

6. A method for constructing the engineered bacteria according to any one of claims 1-5, comprising: The recombinant expression plasmid was constructed by ligating the encoding genes for tyrosine deaminase, p-coumaroyl-CoA ligase, chalcone synthase, and reductase into the expression plasmid. The engineered bacteria were constructed, and the recombinant plasmid was transformed into Escherichia coli to obtain an engineered bacteria that synthesizes phloretin.

7. The construction method according to claim 6, characterized in that: the step of constructing the recombinant expression plasmid includes ligating the encoding genes of the chalcone synthase and reductase to the same expression plasmid pET-lac to form the recombinant plasmid pET-lac-CHS-ER; and ligating the encoding genes of the tyrosine ammonia-lyase and p-coumaroyl-CoA ligase to the same expression plasmid pCS27 to form the recombinant plasmid pCS27-lac-TAL-4CL.

8. A method for the biosynthesis of phlorizin, comprising: The engineered bacteria described in any one of claims 1-5 are fermented in a fermentation medium at an inoculation rate of 1% to 10% by volume. Preferably, the inoculum amount is 1-5%, and the fermentation temperature is 30℃-40℃; Preferably, the carbon source in the fermentation medium is glycerol, monosaccharide, disaccharide, or any combination thereof; Preferably, the fermentation medium comprises: 1–5 g / L MOPS, 5–20 g / L simple carbon source, 1–5 g / L yeast extract, 5–8 g / L Na2HPO4, 0.3–2 g / L NaCl, 2.3–4.0 g / L KH2PO4, and 1–5 g / L NH4Cl; wherein the simple carbon source is one or any combination of glycerol, glucose, sucrose, fructose, and xylose.

9. The biosynthesis method according to claim 8, characterized in that: Add tyrosine to the fermentation medium; Preferably, the amount of tyrosine added is 0.1-2 g / L.

10. The use of the engineered bacteria according to any one of claims 1-5 in the biosynthesis of phloretin derivatives; Preferably, the phlorizin derivative is 3-hydroxyphlorizin, hesperidin dihydrochalcone, hesperidin dihydrochalcone glucoside, or neohesperidin dihydrochalcone.

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