Engineering bacterium for producing dihydrochalcone compound as well as construction method and application of engineering bacterium

Abstract

The invention provides an engineering bacterium for producing dihydrochalcone compounds as well as a construction method and application of the engineering bacterium. The engineering bacterium comprises a chassis bacterium and a coding gene of flavone hydroxylase HpaBC transferred into the chassis bacterium for expression. A coding gene of methyltransferase OMT which is co-expressed in the chassis bacteria, a coding gene of co-expressed glycosyl transferase OGT and a coding gene of co-expressed rhamnosyl transferase 1, 2RhaT are also transferred into the engineering bacteria. By utilizing the engineering bacteria, not only can biosynthesis of dihydrochalcone compounds such as 3-hydroxy phloretin, hesperetin dihydrochalcone, hesperetin dihydrochalcone glucoside, neohesperidin dihydrochalcone and the like by heterologous in-vitro phloretin addition be realized, but also efficient biosynthesis of the dihydrochalcone compounds by taking a simple carbon source as a source can be realized, and the method has the advantages of high efficiency, high yield and high yield. And the yield of the 3-hydroxy phloretin, the yield of the hesperetin dihydrochalcone, the yield of the hesperetin dihydrochalcone glucoside and the yield of the neohesperidin dihydrochalcone can reach 204.11 mg / L, 127.29 mg / L, 183.2 mg / L and 98.36 mg / L respectively.

Description

Technical Field

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

[0002] Neohesperidin dihydrochalcone (NHDC) is a flavonoid derivative extracted from natural citrus and orange plants through hydrogenation. Its molecular formula is C2. 28 H 36 O 15 .

[0003] NHDC is a non-toxic, high-sweetness, low-calorie sweetener with functions of sweetening, enhancing aroma, masking bitterness, and modifying flavor. It also possesses pharmacological activities such as antioxidant, anti-inflammatory, lipid-lowering, liver and kidney protection, and gastrointestinal improvement, showing potential for development in the food and pharmaceutical fields. Currently, phloretin is mainly produced through chemical synthesis. Neohesperidin undergoes ring-opening under alkaline conditions to obtain neohesperidin chalcone, which is then hydrogenated in a hydrogen environment using a nickel or palladium carbon skeletal catalyst to yield the target product, neohesperidin dihydrochalcone.

[0004] However, current chemical synthesis methods for neohesperidin dihydrochalcone and other similar compounds suffer from problems such as seasonal limitations of raw materials, high costs, low efficiency, chemical waste discharge, and severe environmental pollution. Therefore, the biosynthesis of neohesperidin dihydrochalcone and similar compounds has become a research hotspot. The biosynthetic pathways of dihydrochalcone compounds, such as neohesperidin dihydrochalcone, in microorganisms are long and complex, posing numerous challenges to the design, construction, and optimization of engineered bacteria using fermentation sugars as raw materials. Among these biosynthetic pathways, phlorizin can serve as a platform for the synthesis of dihydrochalcone compounds, making the biosynthetic pathway of dihydrochalcone compounds a research focus. For example, Chinese invention patent application CN 108138151 A discloses a method using yeast as the substrate bacteria, starting with tyrosine, and catalyzing the deaminase TAL to convert it into p-coumaric acid. The p-coumaric acid is then converted into p-coumaryl-CoA by p-coumaric acid-coenzyme A ligase 4CL, and p-coumaryl-CoA is converted into dihydrocoumaryl-CoA by endogenous reductase ENR / DBR. Dihydrocoumaryl-CoA is then converted into phlorizin by chalcone synthase CHS. Although this invention patent application provides an engineered bacteria for biosynthesizing phlorizin, the substrate bacteria of this engineered bacteria are mainly yeast, which is not suitable for Escherichia coli, and the yield of phlorizin is low, which is not conducive to industrial application.

[0005] Therefore, achieving efficient biosynthesis of dihydrochalcone compounds such as 3-hydroxyphloretin, hesperidin dihydrochalcone, hesperidin dihydrochalcone glucoside, and neohesperidin dihydrochalcone still faces many challenges. Summary of the Invention

[0006] In view of this, the main objective of the present invention is to provide an engineered bacterium that uses Escherichia coli as the substrate bacteria and can efficiently synthesize and produce dihydrochalcone compounds, and its application.

[0007] This invention mainly studies the biosynthetic pathway of dihydrochalcone compounds and screens suitable exogenous enzymes. Based on this, an engineered bacterium that heterologously produces dihydrochalcone compounds is constructed to achieve the biosynthesis of dihydrochalcone compounds.

[0008] Specifically, the technical solution provided by this invention is as follows: An engineered bacterium that produces dihydrochalcone compounds includes a sclerotium and a gene encoding the flavonoid hydroxylase HpaBC expressed in the sclerotium.

[0009] The substrate bacteria can be primitive microorganisms such as Escherichia coli, yeast, and Rhodococcus, or recombinant microorganisms that have undergone genetic engineering. The Escherichia coli can be primitive Escherichia coli, BL21, BW25113, JCL16, etc. The recombinant microorganisms can be recombinant Escherichia coli, such as Escherichia coli that synthesizes 3-hydroxyphloretin, or tyrosine-producing Escherichia coli.

[0010] The flavonoid hydroxylase HpaBC can be derived from Escherichia coli, Klebsiella pneumoniae, Rhodococcus erythropoietin, etc. Pseudomonas aeruginosa or Rhodococcus opacus wait.

[0011] Based on the above, the engineered bacteria that produce dihydrochalcone compounds include engineered bacteria that produce dihydrochalcone compounds.

[0012] Based on the above, the engineered bacteria producing dihydrochalcone compounds include engineered bacteria that synthesize hesperidin dihydrochalcone, and also include the encoding gene for the methyltransferase OMT co-expressed in the substrate bacteria. The methyltransferase OMT is derived from cyanobacteria, *Mycobacterium chrysogenum*, *Mycobacterium tuberculosis*, *Sclerotium sclerotium*, and other cyanobacteria, such as... Rauwolfia serpentina , Myxococcus xanthus wait.

[0013] Based on the above, the engineered bacteria producing dihydrochalcone compounds include engineered bacteria that synthesize hesperidin dihydrochalcone glucoside, and also include the encoding gene for the glycosyltransferase OGT co-expressed in the substrate bacteria. The glycosyltransferase OGT is derived from kudzu root, japonica rice, Arabidopsis thaliana, tobacco, Bacillus cereus, etc. Pueraria lobata , Arabidopsis thaliana wait.

[0014] Based on the above, the engineered bacteria producing dihydrochalcone compounds include engineered bacteria that synthesize neohesperidin dihydrochalcone, and also include the gene encoding rhamnosyltransferase 1,2RhaT co-expressed in the substrate bacteria. The rhamnosyltransferase 1,2RhaT can be derived from citrus, tea, etc., for example... Citrus maxima .

[0015] Based on the above, the substrate bacteria are engineered bacteria that synthesize phloretin, including *Escherichia coli* and genes co-expressed in the *Escherichia coli* encoding tyrosine deaminase (TAL), p-coumaroyl-CoA ligase (4CL), chalcone synthase (CHS), and reductase. The tyrosine deaminase (TAL) is derived from *Rhododendron simsii* 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 The term "Escherichia coli" (hereinafter referred to as "ERED") or Clostridium acetobutylicum (hereinafter referred to as "ER") can refer to either primitive Escherichia coli, BL21, BW25113, JCL16, or recombinant Escherichia coli that has undergone genetic engineering, such as tyrosine-producing Escherichia coli.

[0016] 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 .

[0017] To further enhance the yield of naringenin dihydrochalcone, a precursor of phloretin synthesis, a molecular chaperone (ibpAB) was introduced into the engineered bacteria. The NCBI database accession number of the molecular chaperone (ibpAB) is CP016018.1.

[0018] A method for constructing an engineered bacterium that produces dihydrochalcone compounds includes: Recombinant expression plasmids were constructed by ligating the gene encoding flavonoid hydroxylase HpaBC into the expression plasmid. The engineered bacteria were constructed, and the recombinant plasmid was transformed into substrate bacteria to obtain engineered bacteria that produce dihydrochalcone compounds.

[0019] The expression plasmid can be pCS27 (medium copy), pSA74, pZE12-luc (high copy), pET-lac, etc.

[0020] A method for the biosynthesis of dihydrochalcone compounds includes: fermenting engineered bacteria producing dihydrochalcone compounds in a fermentation medium at an inoculum concentration of 1% to 10% by volume to obtain dihydrochalcone compounds. Preferably, the inoculum concentration is 1% to 5%. Preferably, during the fermentation process, 0.25 to 1 mM of the inducer IPTG is added; the fermentation temperature is preferably 30°C to 40°C.

[0021] Furthermore, phlorizin is added to the fermentation medium to enable the in vitro synthesis of dihydrochalcone compounds such as 3-hydroxyphlorizin, hesperidin dihydrochalcone, hesperidin dihydrochalcone glucoside, and neohesperidin dihydrochalcone. Preferably, the amount of phlorizin added is 0.1-1 g / L.

[0022] 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.

[0023] 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.

[0024] Please see Figure 1 During metabolism, the basal bacteria convert simple carbon sources into L-tyrosine. L-tyrosine is converted into phlorizin by enzymes TAL, 4CL, CHS, and ER or ERED. Phlorizin is converted into 3-hydroxyphlorizin by flavonoid hydroxylase HpaBC. 3-hydroxyphlorizin is converted into hesperidin dihydrochalcone by methyltransferase OMT. Hesperidin dihydrochalcone is converted into hesperidin dihydrochalcone glucoside by glycosyltransferase OGT. Hesperidin dihydrochalcone glucoside is converted into neohesperidin dihydrochalcone by rhamnosyltransferase 1,2RhaT.

[0025] Therefore, the engineered bacteria for producing dihydrochalcone compounds provided by this invention can not only achieve heterologous in vitro biosynthesis of dihydrochalcone compounds such as 3-hydroxyphlorin, hesperidin dihydrochalcone, hesperidin dihydrochalcone glucoside, and neohesperidin dihydrochalcone by adding phlorin, but also achieve efficient biosynthesis of dihydrochalcone compounds using simple carbon sources. The yields of 3-hydroxyphlorin, hesperidin dihydrochalcone, hesperidin dihydrochalcone glucoside, and neohesperidin dihydrochalcone can reach 204.11 mg / L, 127.29 mg / L, 183.2 mg / L, and 98.36 mg / L, respectively. Attached Figure Description

[0026] Figure 1 This is a diagram illustrating the biosynthetic pathway of phloretin and its derivatives provided by the present invention.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] Figure 6 This is a fermentation result diagram of the engineered strain BW6, which synthesizes 3-hydroxyphlorin in vitro by adding phlorin, as provided in an embodiment of the present invention.

[0032] Figure 7 This is a fermentation result diagram of the engineered strain BW7, which synthesizes 3-hydroxyphlorin through in vitro addition of phlorizin, provided in an embodiment of the present invention. Figure 8 This is a fermentation result diagram of the engineered strain BW9, which synthesizes hesperidin dihydrochalcone by adding phloretin in vitro, as provided in an embodiment of the present invention.

[0033] Figure 9 This is a fermentation result diagram of the engineered strain BW10, which synthesizes hesperidin dihydrochalcone by adding phloretin in vitro, as provided in an embodiment of the present invention.

[0034] Figure 10 This is a fermentation result diagram of the engineered strain BW12, which synthesizes hesperidin dihydrochalcone glucoside in vitro with the addition of phloretin, provided in an embodiment of the present invention.

[0035] Figure 11 This is a fermentation result diagram of the engineered strain BW13, which synthesizes hesperidin dihydrochalcone glucoside in vitro with the addition of phloretin, provided in an embodiment of the present invention.

[0036] Figure 12 This is a fermentation result diagram of the engineered strain BW15, which synthesizes neohesperidin dihydrochalcone by adding phloretin in vitro, according to an embodiment of the present invention.

[0037] Figure 13 The above diagram shows the fermentation results of engineered strains BW8, BW11, BW14, and BW16, which synthesize phlorizin derivatives de novo, according to embodiments of the present invention. Detailed Implementation

[0038] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0039] 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.

[0040] 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.

[0041] Table 1. Enzyme or Protein Source Table 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.

[0042] 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 .

[0043] 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.

[0044] 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 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.

[0045] The technical solution protected by this invention will be described in detail below with specific embodiments.

[0046] 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.

[0047] 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.

[0048] 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. 600When 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.

[0049] 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.

[0050] 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%.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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).

[0057] 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%.

[0058] 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.

[0059] 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).

[0060] 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.

[0061] 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).

[0062] The construction method of engineered strain BW5 is basically the same as that of engineered strain BW4.

[0063] 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.

[0064] 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%.

[0065] III. Engineered bacteria for synthesizing 3-hydroxyphlorin, their 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.

[0066] Example 6: Engineered bacteria BW6, BW7, and BW8 for the synthesis of 3-hydroxyphlorin This embodiment provides an engineered bacterium for synthesizing 3-hydroxyphloretin, including Escherichia coli and the gene encoding the flavonoid hydroxylase HpaBC expressed in the Escherichia coli.

[0067] This embodiment also provides a method for constructing engineered bacteria that synthesize 3-hydroxyphlorin, which is basically the same as the construction method of BW1. First, the genes encoding hydroxylases PaHpaBC and RoHpaBC are ligated into the vector plasmid pCS27 to obtain recombinant plasmids pCS-PaHpaBC and pCS-RoHpaBC, respectively. Then, the genes encoding TAL, 4CL, ibpAB, and the hydroxylase RoHpaBC mutant Y215A are ligated into the same vector plasmid pCS27 to obtain the recombinant plasmid pCS-TAL-4CL-ibpAB-Y215A. Finally, the genes encoding CHS and ERED are ligated into the same vector plasmid pET-lac to obtain the recombinant plasmid pET. -CHS-ERED; then, using homologous recombination technology, the recombinant plasmids pCS-PaHpaBC and pCS-RoHpaBC were electroporated into Escherichia coli BW25113 to obtain the above-mentioned engineered strains BW6:BW25113 (pCS-PaHpaBC) and BW7:BW25113 (pCS-RoHpaBC). pET-CHS-ERED and pCS-TAL-4CL-ibpAB-Y215A were electroporated into the tyrosine-producing engineered strain BW0 to obtain the engineered strain BW8:BW0 (pCS-TAL-4CL-ibpAB-Y215A, pET-CHS-ERED).

[0068] In this embodiment, the RoHpaBC mutant Y215A of hydroxylase is mainly obtained by mutating the 215th site of the original RoHpaBC using existing gene mutation methods, specifically by mutating the site from "Y" to "A".

[0069] (1) Synthesis of 3-hydroxyphlorin by in vitro addition of phlorizin Engineered strains BW6 and BW7 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 6 and Figure 7 As shown. In this embodiment, the fermentation culture method of engineered bacteria BW6 and BW7 is basically the same as that of engineered bacteria BW1 in Example 1. The main difference is that in this embodiment, 0.5 g / L of phloretin is added instead of tyrosine added in Example 1 during the fermentation of engineered bacteria BW6 and BW7 in M9 medium.

[0070] from Figure 6 and Figure 7 It can be seen that both engineered bacteria BW6 and BW7 can synthesize 3-hydroxyphlorin in vitro with the addition of phlorizin. After 12 h of fermentation, engineered bacteria BW7 produced 401.73 mg / L of 3-hydroxyphlorin, which is much higher than the 56.23 mg / L produced by engineered bacteria BW6 after 12 h of fermentation.

[0071] (2) De novo synthesis of 3-hydroxyphloretin The engineered strain BW8 is actually equivalent to the coding gene of the RoHpaBC mutant of flavonoid hydroxylase expressed in the engineered strain BW5 that synthesizes phloretin. That is, the engineered strain BW8 can also be represented as BW5 (pCS-Y215A).

[0072] 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 13 As shown. In this embodiment, the fermentation culture method of engineered bacteria BW8 is basically the same as that of engineered bacteria BW6 and BW7. The main difference is that in this embodiment, engineered bacteria BW8 does not require the addition of phloretin during fermentation in M9 medium.

[0073] from Figure 13 It can be seen that the engineered bacterium BW8 can achieve de novo synthesis of 3-hydroxyphloretin, with a yield of 204.11 mg / L.

[0074] 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.

[0075] Example 7: Engineered bacteria BW9, BW10, and BW11 for synthesizing hesperidin dihydrochalcone This embodiment provides an engineered bacterium for synthesizing hesperidin dihydrochalcone, including Escherichia coli and the encoding genes for flavonoid hydroxylase HpaBC and methyltransferase OMT expressed in the Escherichia coli.

[0076] This embodiment also provides a method for constructing an engineered bacterium that synthesizes hesperidin dihydrochalcone. The method is essentially the same as that used for BW1. First, the gene encoding the hydroxylase RoHpaBC is ligated into the vector plasmid pCS27 to obtain the recombinant plasmid pCS-RoHpaBC. Then, the genes encoding TAL, 4CL, ibpAB, and the RoHpaBC mutant Y215A are ligated into the same vector plasmid pCS27 to obtain the recombinant plasmid pCS-TAL-4CL-ibpAB-Y215A. Next, the genes encoding RsOMT and MxOMT are ligated into the vector plasmid pET-lac to obtain the recombinant plasmids pET-RsOMT and pET-MxOMT, respectively. Finally, the genes encoding CHS, ERED, and MxOMT are ligated into the same vector plasmid pET-lac to obtain the recombinant plasmid pET-CH. S-ERED-MxOMT; then, using homologous recombination technology, the recombinant plasmid pCS-RoHpaBC was co-electropoverted into Escherichia coli BW25113 with pET-RsOMT and pET-MxOMT, respectively, to obtain the above-mentioned engineered strains BW9:BW25113 (pCS-RoHpaBC, pET-RsOMT) and BW10:BW25113 (pCS-RoHpaBC, pET-MxOMT). pET-CHS-ERED-MxOMT and pCS-TAL-4CL-ibpAB-Y215A were electroporated into the tyrosine-producing engineered bacterium BW0 to obtain the engineered strain BW11:BW0 (pCS-TAL-4CL-ibpAB-Y215A, pET-CHS-ERED-MxOMT).

[0077] (1) Synthesis of hesperidin dihydrochalcone by adding phloretin in vitro Engineered strains BW9 and BW10 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 8 and Figure 9 As shown. In this embodiment, the fermentation culture method for engineered bacteria BW9 and BW10 is the same as that for engineered bacteria BW6 and BW7.

[0078] from Figure 8 and Figure 9 The results show that both engineered bacteria BW9 and BW10 can synthesize hesperidin dihydrochalcone in vitro with the addition of phloretin. After 24 hours of fermentation, engineered bacteria BW10 produced 233.28 mg / L of hesperidin dihydrochalcone, significantly higher than the 45.66 mg / L produced by engineered bacteria BW9 after the same period.

[0079] (2) De novo synthesis of hesperidin dihydrochalcone The engineered strain BW11 is actually equivalent to the gene encoding the flavonoid hydroxylase RoHpaBC mutant and the methyltransferase MxOMT co-expressed in the engineered strain BW5 that synthesizes phloretin. That is, the engineered strain BW11 can also be represented as BW5 (pCS-Y215A, pET-MxOMT) or BW8 (pET-MxOMT).

[0080] The engineered strain BW11 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 13 As shown. In this embodiment, the fermentation culture method of engineered bacteria BW11 is basically the same as that of engineered bacteria BW9 and BW10. The main difference is that in this embodiment, engineered bacteria BW11 does not require the addition of phloretin during fermentation in M9 medium.

[0081] from Figure 13 As can be seen, the engineered strain BW11 can achieve de novo synthesis of hesperidin dihydrochalcone, with a yield of 127.29 mg / L.

[0082] 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.

[0083] Example 8: Engineered bacteria BW12, BW13, and BW14 for synthesizing hesperidin dihydrochalcone glucoside This embodiment provides an engineered bacterium for synthesizing hesperidin dihydrochalcone glucoside, including Escherichia coli and the encoding genes for flavonoid hydroxylase HpaBC, methyltransferase OMT, and glycosyltransferase OGT expressed in the Escherichia coli.

[0084] 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 hydroxylase RoHpaBC is ligated into the vector plasmid pCS27 to obtain the recombinant plasmid pCS-RoHpaBC. Then, the genes encoding TAL, 4CL, ibpAB, and the RoHpaBC mutant Y215A are ligated into the same vector plasmid pCS27 to obtain the recombinant plasmid pCS-TAL-4CL-ibpAB-Y215A. Next, the genes encoding MxOMT and OGT from different sources are ligated into the same vector plasmid pET-lac to obtain the recombinant plasmids pET-MxOMT-P1OGT2 and pET-MxOMT-OGT72E2, respectively. Finally, the genes encoding CHS, ERED, MxOMT, and OGT72E2 are ligated into the same vector plasmid pET-lac to obtain the recombinant plasmid pET-CHS-ERED-MxOMT. -OGT72E2; then, using homologous recombination technology, the recombinant plasmid pCS-RoHpaBC was co-electrotransformed with pET-MxOMT-P1OGT2 and pET-MxOMT-OGT72E2 into Escherichia coli BW25113 to obtain the above-mentioned engineered strains BW12:BW25113 (pCS-RoHpaBC, pET-MxOMT-P1OGT2) and BW13:BW25113 (pCS-R oHpaBC, pET-MxOMT-OGT72E2), pET-CHS-ERED-MxOMT-OGT72E2 and pCS-TAL-4CL-ibpAB-Y215A are electroporated into the tyrosine-producing engineered bacterium BW0 to obtain the engineered strain BW14:BW0 (pCS-TAL-4CL-ibpAB-Y215A, pET-CHS-ERED-MxOMT-OGT72E2).

[0085] (1) Synthesis of hesperidin dihydrochalcone glucoside by adding phloretin in vitro Engineered strains BW12 and BW13 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 10 and Figure 11 As shown. In this embodiment, the fermentation culture method for engineered bacteria BW9 and BW10 is the same as that for engineered bacteria BW6 and BW7.

[0086] from Figure 10 and Figure 11The results show that both engineered bacteria BW12 and BW13 can synthesize hesperidin dihydrochalcone glucoside in vitro with the addition of phloretin. After 24 hours of fermentation, engineered bacteria BW13 produced 287.46 mg / L of hesperidin dihydrochalcone glucoside, significantly higher than the 23.72 mg / L produced by engineered bacteria BW13 after the same fermentation period.

[0087] (2) De novo synthesis of hesperidin dihydrochalcone The engineered strain BW14 is actually equivalent to the encoding genes of the flavonoid hydroxylase RoHpaBC mutant, methyltransferase MxOMT, and glycosyltransferase OGT72E2 co-expressed in the engineered strain BW5 that synthesizes phloretin. That is, the engineered strain BW14 can also be represented as BW5 (pCS-Y215A, pET-MxOMT-OGT72E2) or BW11 (pET-OGT72E2).

[0088] The engineered strain BW14 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 13 As shown. In this embodiment, the fermentation culture method of engineered bacteria BW14 is basically the same as that of engineered bacteria BW12 and BW13. The main difference is that in this embodiment, engineered bacteria BW14 does not require the addition of phloretin during fermentation in M9 medium.

[0089] from Figure 13 It can be seen that the engineered strain BW14 can achieve de novo synthesis of hesperidin dihydrochalcone glucoside, with a yield of 183.2 mg / L.

[0090] 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.

[0091] Example 9: Engineered bacteria BW15 and BW16 for the synthesis of neohesperidin dihydrochalcone This embodiment provides an engineered bacterium for synthesizing a new hesperidin dihydrochalcone, including Escherichia coli and the encoding genes for flavonoid hydroxylase HpaBC, methyltransferase OMT, glycosyltransferase OGT and rhamnosyltransferase 1,2RhaT expressed in Escherichia coli.

[0092] 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 is ligated into the vector plasmid pCS27 to obtain the recombinant plasmid pCS-RoHpaBC. Then, the genes encoding TAL, 4CL, ibpAB, and the RoHpaBC mutant Y215A are ligated into the same vector plasmid pCS27 to obtain the recombinant plasmid pCS-TAL-4CL-ibpAB-Y215A. Next, 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. Finally, the genes encoding CHS, ERED, MxOMT, OGT72E2, and Cm1.2RhaT are ligated into the same vector plasmid pET-lac to obtain the recombinant plasmid pET-CHS- ERED-MxOMT-OGT72E2-Cm1.2RhaT; then, using homologous recombination technology, the recombinant plasmids pCS-RoHpaBC and pET-MxOMT-P1OGT2-Cm1.2RhaT were co-electropoverted into E. coli BW25113 to obtain the above-mentioned engineered strain BW15:BW25113 (pCS-RoHpaBC, pET-MxOMT-P1OGT2-Cm1.2RhaT). By electroporating pET-CHS-ERED-MxOMT-OGT72E2-Cm1.2RhaT and pCS-TAL-4CL-ibpAB-Y215A into the tyrosine-producing engineered bacterium BW0, the engineered strain BW16:BW0 (pCS-TAL-4CL-ibpAB-Y215A, pET-CHS-ERED-MxOMT-OGT72E2-Cm1.2RhaT) can be obtained.

[0093] (1) Synthesis of neohesperidin dihydrochalcone by adding phloretin in vitro The engineered strain BW15 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 12 As shown. In this embodiment, the fermentation culture method of engineered bacteria BW15 is the same as that of engineered bacteria BW6 and BW7.

[0094] from Figure 12 It can be seen that the engineered strain BW15 can synthesize neohesperidin dihydrochalcone in vitro by adding phloretin, and the yield of neohesperidin dihydrochalcone after 24 h of fermentation reaches 104.64 mg / L.

[0095] (2) De novo synthesis of neohesperidin dihydrochalcone The engineered strain BW16 is actually equivalent to the encoding genes of the flavonoid hydroxylase RoHpaBC mutant, methyltransferase MxOMT, glycosyltransferase OGT72E2, and rhamnosyltransferase Cm1,2RhaT co-expressed in the engineered strain BW5 that synthesizes phloretin. That is, the engineered strain BW16 can also be represented as BW5 (pCS-Y215A, pET-RsOMT-P1OGT2-Cm1.2RhaT) or BW14 (pET-Cm1.2RhaT).

[0096] The engineered strain BW16 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 13 As shown. In this embodiment, the fermentation culture method of engineered bacteria BW16 is basically the same as that of engineered bacteria BW15. The main difference is that in this embodiment, engineered bacteria BW16 does not require the addition of phloretin during fermentation in M9 medium.

[0097] from Figure 13 As can be seen, the engineered strain BW16 can achieve de novo synthesis of the new hesperidin dihydrochalcone, with a yield of 98.36 mg / L.

[0098] 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 producing dihydrochalcone compounds, characterized in that: This includes the basal bacteria and the gene encoding the flavonoid hydroxylase HpaBC expressed in the basal bacteria; Preferably, the flavonoid hydroxylase HpaBC is derived from Escherichia coli, Klebsiella pneumoniae, or Rhodococcus erythropoietin.

2. The engineered bacteria producing dihydrochalcone compounds according to claim 1, characterized in that: it includes engineered bacteria producing dihydrochalcone compounds.

3. The engineered bacteria that produce dihydrochalcone compounds according to claim 2, characterized in that: it includes engineered bacteria that synthesize hesperidin dihydrochalcone, and further includes the encoding gene for methyltransferase OMT co-expressed by the chassis bacteria. Preferably, the methyltransferase OMT is derived from cyanobacteria, *Mycobacterium chrysogenum*, *Mycobacterium tuberculosis*, *Sophora japonica*, or cyanobacteria.

4. The engineered bacteria that produce dihydrochalcone compounds according to claim 3, characterized in that: it includes engineered bacteria that synthesize hesperidin dihydrochalcone glucoside, and further includes the encoding gene of glycosyltransferase OGT co-expressed in the chassis bacteria. Preferably, the glycosyltransferase OGT is derived from kudzu root, japonica rice, Arabidopsis thaliana, tobacco, or Bacillus cereus.

5. The engineered bacteria that produce dihydrochalcone compounds according to claim 4, characterized in that: it includes engineered bacteria that synthesize neohesperidin dihydrochalcone, and further includes the gene encoding rhamnosyltransferase 1,2RhaT co-expressed by the chassis bacteria. Preferably, the rhamnosyltransferase 1,2RhaT is derived from citrus or tea trees.

6. The engineered bacteria producing dihydrochalcone compounds according to any one of claims 1-5, characterized in that: the chassis bacteria are primitive microorganisms or recombinant microorganisms modified by genetic engineering; Preferably, the microorganism is Escherichia coli, yeast, or Rhodococcus; Preferably, the substrate bacteria are primitive Escherichia coli, BL21, BW25113, JCL16, Escherichia coli that synthesizes 3-hydroxyphloretin, or tyrosine-producing Escherichia coli.

7. The engineered bacteria producing dihydrochalcone compounds according to claim 6, characterized in that: the basal bacteria are engineered bacteria synthesizing phloretin, comprising *Escherichia coli* and genes co-expressed in the *Escherichia coli* encoding tyrosine deaminase, p-coumaroyl-CoA ligase, chalcone synthase, and reductase, wherein, The tyrosine deaminase is derived from red yeast or Hypericum, the p-coumaroyl-CoA ligase is derived from Arabidopsis thaliana, petunia or apple, the chalcone synthase gene is derived from petunia, and the reductase gene is derived from Clostridium or Clostridium acetylatede. Preferably, 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; Preferably, the key enzymes for tyrosine synthesis include the tryptophan synthase gene aroG and the prephenylacetic acid dehydrogenase gene tyrA.

8. A method for constructing an engineered bacterium that produces dihydrochalcone compounds, comprising: Recombinant expression plasmids were constructed by ligating the gene encoding flavonoid hydroxylase HpaBC into the expression plasmid. The engineered bacteria were constructed, and the recombinant plasmid was transformed into substrate bacteria to obtain engineered bacteria that produce dihydrochalcone compounds.

9. A method for biosynthesizing dihydrochalcone compounds, comprising the steps of: fermenting the engineered bacteria that produce dihydrochalcone compounds according to any one of claims 1-7 in a fermentation medium at an inoculum amount of 1% to 10% by volume to obtain dihydrochalcone compounds; 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; Preferably, the dihydrochalcone compound is 3-hydroxyphloretin, hesperidin dihydrochalcone, hesperidin dihydrochalcone glucoside, or neohesperidin dihydrochalcone.

10. The biosynthesis method according to claim 9, characterized in that: Add phloretin to the fermentation medium; Preferably, the amount of phloretin added is 0.1-1 g / L.

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