Synergistic antibacterial effect of the combination of chlorhexidine and chlorhexidine digluconate

Abstract

This invention provides an engineered microbial strain for the biosynthesis of syringin and its application. The engineered microbial strain includes a host microbial strain and HpaBC, COMT, and UGT72E2 co-expressed in the host microbial strain. This engineered microbial strain, in conjunction with p-coumarol fermentation, can biosynthesize syringin. This invention also provides an engineered microbial strain for the biosynthesis of syringin and its application. This engineered microbial strain includes a p-coumarol biosynthetic engineered microbial strain and HpaBC, COMT, and UGT72E2 co-expressed in the p-coumarol biosynthetic engineered microbial strain. The p-coumarol biosynthetic engineered microbial strain includes a host microbial strain and TAL, 4CL1, CCR, and ADH6 co-expressed in the host microbial strain. AroG is also co-expressed in the host microbial strain. fbr and TyrA fbr This engineered bacterium for the biosynthesis of syringin can achieve high-efficiency biosynthesis of syringin using a simple carbon source.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biosynthesis, in particular to a Syringin biosynthesis engineering bacteria and application. BACKGROUND

[0002] Syringin, also known as Eleutheroside B, is a white powder at room temperature, soluble in hot water and ethanol, slightly soluble in cold water, and almost insoluble in ether. It is a major bioactive phenylpropanol glycoside compound in Acanthopanax senticosus, and is the main active ingredient of traditional Chinese medicines such as Acanthopanax, Ruxiang, Taxillus and Violaceum. It has the effects of lowering blood lipids and blood sugar, relieving myocardial hypertrophy, protecting liver, and preventing and treating osteoporosis. Animal experiments show that Syringin has protective effects on mitochondrial damage caused by tissue ischemia and hypoxia followed by reoxygenation during liver surgery, can inhibit hepatocyte apoptosis, activate the production of tumor necrosis factor in liver tissue, and has the effects of clearing free radicals, protecting nerve cell damage, and preventing and treating coronary heart disease, angina pectoris, stroke, and other effects. In addition, it is also used in combination therapy for treating tumors and emergency rapid hemostatic patches. Many studies have shown that Syringin is used to make various oral preparations and injection preparations, which are safe, non-toxic, have strong pharmacological effects, good efficacy, and good prospects for medicinal use.

[0003] Currently, Syringin is mainly extracted from plants by high-temperature organic solvent extraction, macroporous resin extraction, ultrasonic extraction, or a combination of existing extraction methods. For example, CN101307079A discloses a method for extracting and separating Syringin from Ilex rotunda, and benzene and acetone are used as solvents for purification and separation; CN106188173A discloses a method for separating and preparing Syringin from Ruxiang medicinal materials using macroporous resin; and CN113234110A discloses a method for preparing Syringin, Eleutheroside B, and Atractylodes lactone III from Codonopsis pilosula based on PRE-HPLC.

[0004] Existing methods for extracting Syringin from plants all use a large amount of organic reagents for extraction and purification, and some processes are accompanied by high temperature and the generation of wastewater, waste gas, and waste liquid, which are not environmentally friendly, increase the difficulty of subsequent separation and purification, and are not conducive to large-scale preparation. In recent years, with more and more microbial cell factories being used to produce various fine chemicals, constructing a microbial cell factory for producing Syringin from renewable resources has become a feasible solution to the above problems. Although Syringin is a natural substance synthesized in Acanthopanax, its natural synthesis pathway has not been completely elucidated. Therefore, it is of great significance to develop a method for biosynthesis of Syringin. SUMMARY

[0005] Therefore, one object of the present application is to provide a biosynthesis engineering strain of syringin and application, which mainly expresses key enzymes required for biosynthesis of syringin in a host strain, the key enzymes are screened from a large number of enzymes capable of synthesizing syringin in vivo of organisms or microorganisms, and the heterologous efficient synthesis of syringin is realized by enzymes with catalytic efficiency in vitro. The biosynthesis engineering strain of syringin can efficiently synthesize syringin in combination with the fermentation culture of p-coumaric acid.

[0006] Another object of the present application is to provide a biosynthesis engineering strain of syringin and application capable of realizing efficient biosynthesis of syringin with simple carbon sources such as glucose and glycerol as sources, and the yield of syringin can reach 645.26 mg / L by using the engineering strain.

[0007] Therefore, in order to achieve the above object, the present application provides the following technical solutions:

[0008] A biosynthesis engineering strain of syringin comprises a host strain and key enzymes required for biosynthesis of syringin co-expressed in the host strain, the key enzymes comprise 4-hydroxyphenylacetate 3-hydroxylase (HpaBC), caffeic acid O-methyltransferase (COMT) and glycosyltransferase (UDP-Glycosyltransferase 72E2, UGT72E2).

[0009] In the present application, the host strain is an original or modified bacterium or fungus, for example, an original or modified Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae or Pichia pastoris, etc.

[0010] In the present application, the caffeic acid methoxyltransferase is abbreviated as COMT, the 4-hydroxyphenylacetate 3-hydroxylase is abbreviated as HpaBC, and the glycosyltransferase is abbreviated as UGT72E2.

[0011] In the present application, the key enzymes are enzymes derived from bacteria, fungi or protein engineering.

[0012] The HpaBC is preferably derived from Klebsiella pneumoniae, Saccharothrix espanaensis, Streptomyces lunaelactis, Nocardia farcinica, Rhodococcus ruber.

[0013] The COMT is preferably derived from A. thaliana, Triticum aestivum, Hordeum vulgare, Festuca arundinacea, Lolium perenne.

[0014] The UGT72E2 is preferably derived from A. thaliana, Zea mays, Nicotiana tabacum, Dendrobium catenatum, Medicago sativa L.

[0015] A biosynthesis method of syringin, comprising: fermenting the syringin biosynthesis engineering bacteria in a fermentation medium containing p-coumaric acid to obtain syringin. The addition amount of p-coumaric acid in the fermentation medium is 0.5-1.5 g / L. The carbon source in the fermentation medium is monosaccharide, disaccharide or any combination thereof. Preferably, the carbon source in the fermentation medium is one or any combination of glycerol, glucose, sucrose, fructose, xylose. Preferably, the fermentation medium is M9 medium.

[0016] A syringin biosynthesis engineering bacteria, comprising a p-coumaric acid biosynthesis engineering bacteria and key enzymes required for syringin biosynthesis co-expressed in the p-coumaric acid biosynthesis engineering bacteria, the key enzymes comprising HpaBC, COMT and UGT72E2.

[0017] The p-coumaric acid biosynthesis engineering bacteria comprises a host bacteria and tyrosine ammonia lyase (TAL), 4-coumaryl CoA ligase (4CL1), cinnamoyl-CoA reductase (CCR), alcohol dehydrogenase (ADH6) co-expressed in the host bacteria.

[0018] The host bacteria also co-express 3-deoxy-D-arabino-heptolosonate-7-phosphate synthetase (AroG fbr ) and prephenate dehydrogenase (TyrA fbr ), so as to mainly improve the yield of tyrosine synthesized by the shikimic acid pathway, thereby achieving the purpose of improving the yield of p-coumaric acid synthesized by the p-coumaric acid biosynthesis engineering bacteria, and further achieving the purpose of improving the yield of biosynthesis of syringin by the syringin biosynthesis engineering bacteria.

[0019] The genes encoding pyruvate kinase I PykF, pyruvate kinase II PykA, prephenate dehydratase pheA and tyrosine transcriptional repressor TyrR are knocked out in the host bacteria, so as to mainly achieve the purpose of improving the yield of tyrosine by inhibiting the competitive pathway, thereby improving the yield of p-coumaric acid synthesized by the p-coumaric acid biosynthesis engineering bacteria, and further achieving the purpose of improving the yield of biosynthesis of syringin by the syringin biosynthesis engineering bacteria.

[0020] In the present application, the English abbreviation of tyrosine aminolyase is TAL, the English abbreviation of 4-coumarate coenzyme A ligase is 4CL1, the English abbreviation of cinnamoyl coenzyme A reductase is CCR, the English abbreviation of alcohol dehydrogenase is ADH6, the English abbreviation of 3-deoxy-D-arabino-heptolosonate-7-phosphate synthetase is AroG fbr , the English abbreviation of prephenate dehydrogenase is TyrA fbr .

[0021] The AroG fbr , TyrA fbr , TAL, 4CL1, CCR and ADH6 are respectively derived from bacteria, fungi or protein engineering enzymes.

[0022] The TAL is preferably derived from Rhodobacter sphaeroides, R. glutinis, Streptomyces albus, Rhodobacter capsulatus and Micromonospora echinofusca.

[0023] The 4CL1 is preferably derived from A. thaliana, Oryza sativa, Nicotiana tabacum, Populus trichocarpa and Petunia hybrida.

[0024] The CCR is preferably derived from A. thaliana, Leucaena leucocephala, Picea abies, Sorghum bicolor, Populus trichocarpa.

[0025] The ADH6 is preferably derived from Saccharomyces cerevisiae, Rhodococcus ruber, Pseudomonas aeruginosa, Zymomonas mobilis, Acetobacter aceti.

[0026] A biosynthesis method of syringin, comprising the following steps: fermenting the syringin biosynthesis engineering bacteria in a fermentation medium to obtain syringin. The carbon source in the fermentation medium is monosaccharide, disaccharide or any combination thereof. Preferably, the carbon source in the fermentation medium is one or any combination of glycerol, glucose, sucrose, fructose and xylose. Preferably, the fermentation medium is M9 medium.

[0027] Please refer to Figure 1 The main reason why the above-mentioned syringin biosynthesis engineering bacteria provided by the application can synthesize syringin is that simple carbon sources such as glucose and glycerol are converted into tyrosine under the action of enzymes AroG fbr , TyrA fbr under the action of the shikimic acid pathway of the engineering bacteria, tyrosine is converted into p-coumaric acid under the catalysis of enzyme TAL, p-coumaric acid is converted into p-coumarol under the catalysis of enzymes 4CL1, CCR and ADH6 in turn, p-coumarol is converted into sinapyl alcohol under the catalysis of enzymes HpaBC and COMT, and sinapyl alcohol is converted into syringin under the action of enzyme UGT72E2. As can be seen, the syringin biosynthesis engineering bacteria provided by the application can realize efficient biosynthesis of syringin from simple carbon sources as a source in the process of fermentation culture; and syringin can also be efficiently synthesized by adding p-coumarol.

[0028] Therefore, the above-mentioned syringin biosynthesis engineering bacteria provided by the application are obtained by screening key enzymes with activity in vitro; and the key enzymes for synthesizing syringin are used as the basis to design the above-mentioned syringin biosynthesis engineering bacteria. Figure 1The invention illustrates the biosynthetic pathway of syringin and utilizes molecular biology and metabolic regulation to optimize the synthesis route, thereby achieving efficient biosynthesis of syringin. The production of syringin using the engineered bacteria for syringin biosynthesis provided by this invention has advantages such as simplicity, low cost, and high conversion rate, which is conducive to industrial-scale production, reduces production costs, and provides important evidence for the industrial production of syringin. Attached Figure Description

[0029] Figure 1 The biosynthetic pathway diagram of syringin provided by this invention.

[0030] Figure 2 The image shows the fermentation results of the syringin biosynthesis engineered strain BW1 provided in Example 1 of this invention, when p-coumarol was added to synthesize syringin.

[0031] Figure 3 This is the HPLC detection chromatogram of the syringin standard used in Example 1 of this invention.

[0032] Figure 4 This is an HPLC chromatogram of the fermentation product synthesized from engineered bacteria BW1 with p-coumarol, as provided in Example 1 of this invention.

[0033] Figure 5 This is a diagram showing the fermentation results of the de novo synthesis of syringin by the engineered strain BW2, which is used in Example 2 of this invention.

[0034] Figure 6 This is an HPLC chromatogram of the fermentation product synthesized by the engineered bacteria BW2 in Example 2 of the present invention using a simple carbon source. Detailed Implementation

[0035] The technical solution of the present invention will be further described in detail below through specific embodiments. In the present invention, there are no special requirements for the type of expression plasmid. It can be considered that the construction method for expressing the target gene in Escherichia coli can adopt various methods commonly used in the art, such as ligating the target gene into a vector after enzyme digestion treatment, which will not be described in detail hereafter.

[0036] 1) Enzymes used in the examples

[0037] The enzymes used in the following examples are all existing enzymes. In addition to the key enzymes shown in Table 1, other enzymes used in the examples, such as the various enzymes used in the pathway of synthesizing shikimic acid from carbon sources, are also existing commonly used enzymes.

[0038] Table 1 Enzymes used in each embodiment

[0039]

[0040] 2) Medium used in the examples

[0041] The medium used in the following examples is as follows:

[0042] LB medium: yeast powder 5 g / L, peptone 10 g / L, sodium chloride 10 g / L.

[0043] M9 medium: glycerol or glucose 10 g / L, yeast powder 2.5 g / L, disodium hydrogen phosphate 6.78 g / L, potassium dihydrogen phosphate 3 g / L, ammonium chloride 1 g / L, sodium chloride 0.5 g / L, MOPS 2 g / L, magnesium sulfate 0.241 g / L, calcium chloride 0.011 g / L.

[0044] 3) Plasmids and strains used in the examples

[0045] In the following examples, both Escherichia coli strain BW25113 and trans5α are commonly used Escherichia coli strains, and are commercially available, wherein trans5α is used for vector construction, and BW25113 is used as a fermentation strain. The plasmids and strains used in each example are shown in Table 2.

[0046] Table 2 Plasmids used in each example

[0047]

[0048] 4) Detection conditions for HPLC analysis used in the examples

[0049] The conditions for detecting syringin standard and fermentation products by HPLC analysis method in the examples are as follows:

[0050] Column: separation column: Diamonsil C18, ID 5 μm, 250 x 4.6 mm;

[0051] Mobile phase: A is methanol, B is 1 ‰ formic acid aqueous solution, column temperature is 35 ℃, flow rate is 0.8 mL / min, detection wavelength is 260 nm. The gradient elution program is shown in Table 3:

[0052] Table 3 Gradient elution program

[0053] 0 5 95 30 100 0 35 5 95 38 5 95

[0054] Example 1

[0055] Construction of syringin engineering strain for biosynthesis of syringin by in vitro addition of p-coumaric acid

[0056] As Figure 1As shown, the p-coumaric acid is converted into the sinapyl alcohol under the catalysis of the enzymes HpaBC and COMT, and the sinapyl alcohol is converted into the syringin under the catalysis of the enzyme UGT72E2. The present embodiment provides a syringin biosynthesis engineering bacterium, which comprises an Escherichia coli and HpaBC, COMT and UGT72E2 co-expressed in the Escherichia coli.

[0057] The present embodiment also provides a method for constructing the syringin biosynthesis engineering bacterium, which comprises the following steps:

[0058] The genes of the enzymes HpaBC, COMT and UGT72E2 are respectively obtained by PCR amplification, and the fragments and the vector are subjected to enzyme digestion with appropriate restriction enzymes. The fragments after enzyme digestion are recovered by gel recovery or column recovery, and then the target genes are inserted into the plasmid pZE12-luc (high copy) to obtain the recombinant plasmid pZE-HpaBC-COMT-UGT72E2 (see Table 2).

[0059] The competent cells of BW25113 are prepared by the electroporation method, and 90 μL of the competent cells are divided into 1.5 mL EP tubes for transformation. 1.8 μL of the constructed recombinant plasmid pZE-HpaBC-COMT-UGT72E2 is added into the centrifuge tube containing 90 μL of the competent cells, and mixed uniformly. Then the plasmid is electroporated into the competent cells by using an electroporation instrument. After the electroporation is completed, LB medium is added, and the mixture is transferred into a 1.5 mL centrifuge tube for recovery for 60 min. Then the bacterial solution is inoculated on a plate containing ampicillin, and incubated at 37°C overnight to prepare the syringin biosynthesis engineering strain BW1: BW25113 (pZE-HpaBC-COMT-UGT72E2).

[0060] Fermentation of the syringin biosynthesis engineering strain BW1

[0061] Shaking flask fermentation for synthesizing syringin: three single colonies of the strain BW1 for producing syringin are respectively inoculated into 4 mL of liquid LB medium with ampicillin resistance, and incubated at 37°C for 12 h. Then the bacterial solution is respectively transferred into 50 mL of M9 fermentation medium, and 0.5 mM of IPTG is added for induction. 1 g / L of the substrate p-coumaric acid is added, and the corresponding antibiotic is added according to the actual situation. In the present embodiment, the carbon source in the M9 fermentation medium is glucose. Part of the fermentation solution is taken out at 12 h, 24 h, 36 h and 48 h of fermentation, and the bacterial growth and fermentation are determined by HPLC. The results are shown in Figure 2 As shown in Figure 2 It can be seen from the above that the engineering strain BW1 of the present embodiment can synthesize 267.75 mg / L of syringin by using p-coumaric acid in a shaking flask.

[0062] Syringin standard solutions were prepared at concentrations of 0 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, and 500 mg / L, and analyzed by HPLC. Figure 3 The HPLC chromatogram for a 500 mg / L syringin standard shows a peak elution time of 16.378 min. Figure 4 The HPLC chromatogram of the fermentation product shows that the peak elution time at approximately 16.374 min is consistent with that of the standard peak, which proves that the recombinant Escherichia coli BW1 provided in this example can synthesize syringin through in vitro fermentation with p-coumarol.

[0063] Example 2

[0064] Construction of a metabolic pathway for the de novo heterologous synthesis of syringin from simple carbon sources in microorganisms

[0065] After achieving the biotransformation of coumarin to syringin, the key enzyme AroG in the shikimic acid pathway was overexpressed. fbr TyrA fbr The de novo synthesis of syringin was achieved by knocking out genes encoding pyruvate kinase I (PykF), pyruvate kinase II (PykA), and prebenzoic acid dehydratase (pheA) in the shunt pathway, and by knocking out the gene of TyrR, a tyrosine transcriptional repressor with a repressive effect, to enhance the supply of shikimic acid in the upstream pathway and increase tyrosine production. The de novo synthesis of syringin was achieved by overexpressing TAL, 4CL1, CCR, and ADH6 to increase the conversion rate of tyrosine to p-coumarol. This embodiment provides a syringin biosynthetic engineered bacterium, comprising a p-coumarol biosynthetic engineered bacterium and HpaBC, COMT, and UGT72E2 co-expressed in the p-coumarol biosynthetic engineered bacterium. The p-coumarol biosynthetic engineered bacterium includes the knockout of genes encoding PykF, PykA, pheA, and TyrR in *E. coli*, and the introduction of AroG co-expressed in *E. coli*. fbr TyrA fbr TAL, 4CL1, CCR and ADH6.

[0066] This embodiment also provides a method for constructing the above-mentioned engineered bacteria for the biosynthesis of syringin, including:

[0067] The recombinant plasmids pZE-HpaBC-COMT-UGT72E2 and pCS-TAL-4CL1-CCR-ADH6-AroG were obtained respectively using the method provided in Example 1. fbr -TyrA fbr ;

[0068] The method of Red recombination was used to knock out the coding genes PykF, PykA, pheA and TyrR in the host bacteria BW25113 to obtain the knockout host bacteria BWΔPykFΔPykAΔpheAΔTyrR; the recombinant plasmid pCS-TAL-4CL1-CCR-ADH6-AroG was introduced into the knockout host bacteria BWΔPykFΔPykAΔpheAΔTyrR by the method provided in Reference Example 1 to obtain the recombinant bacteria BW2: BW25113ΔPykFΔPykAΔpheAΔTyrR (pCS-TAL-4CL1-CCR-ADH6-AroG). fbr -TyrA fbr The knockout host bacteria BWΔPykFΔPykAΔpheAΔTyrR was co-introduced to prepare the BW25113 high coumaric alcohol-producing strain;

[0069] The recombinant plasmid pZE-HpaBC-COMT-UGT72E2 was introduced into the BW25113 high coumaric alcohol-producing strain by the method provided in Reference Example 1 to prepare the syringin-producing strain BW2: BW25113 high coumaric alcohol-producing strain (pZE-HpaBC-COMT-UGT72E2).

[0070] Fermentation of the syringin biosynthesis engineering bacteria BW2

[0071] Shaking flask fermentation of the above engineering bacteria BW2: three single colonies were picked from the plate of the syringin biosynthesis engineering bacteria BW2, respectively, and inoculated into 4 mL liquid LB medium with ampicillin resistance and kanamycin resistance, and cultured at 37°C for 12 h, and then the bacterial liquid was transferred into 50 mL M9 fermentation medium, 0.25-1 mM IPTG was added for induction, and the corresponding antibiotics were added according to the actual situation, wherein the carbon source in the M9 fermentation medium used in this example was glycerol. Part of the fermentation liquid was taken out at 12 h, 24 h, 36 h and 48 h of fermentation, and the fermentation condition was detected by HPLC, and the results are shown in Figure 5 From the results in Figure 5 , it can be seen that the engineering strain BW2 of this example can synthesize 645.26 mg / L of syringin using simple carbon sources such as glycerol in a shaking flask.

[0072] The fermentation liquid was detected by the method provided in Reference Example 1, and HPLC detection of syringin production was detected under the same liquid phase conditions, and the peak time was 16.370 min, as shown in Figure 6 ; which is consistent with the standard peak 16.378 min peak time, which can prove that the recombinant Escherichia coli BW2 provided in this example can realize the biosynthesis of syringin with simple carbon sources as the source.

[0073] It should be pointed out finally that the above examples are only used to illustrate the technical solutions of the present application but not to limit it; although the present application has been explained in detail with reference to the preferred embodiments, those skilled in the art should understand that the specific embodiments of the present application can be modified or some technical features can be replaced by the equivalent ones without departing from the spirit of the technical solutions of the present application, and all of them should be covered in the technical solution range claimed by the present application.

Claims

1. A biosynthesis engineering bacteria of Syringa, characterized in that: The bacteria are obtained by co-expressing the genes encoding key enzymes required for biosynthesis of syringin in an engineering bacteria for biosynthesis of p-coumaric acid, including 4-hydroxyphenylacetic acid-3-hydroxylase HpaBC, caffeic acid methyltransferase COMT and glycosyltransferase UGT72E2, wherein the engineering bacteria for biosynthesis of p-coumaric acid is obtained by co-expressing the genes encoding tyrosine amino-cleavage enzyme TAL, 4-coumarate coenzyme A ligase 4CL1, cinnamoyl coenzyme A reductase CCR, ethanol dehydrogenase ADH6 and 3-deoxy-D-arabino-heptulosonate-7-phosphate synthetase AroG in Escherichia coli BW25113, wherein the genes encoding pyruvate kinase I PykF, pyruvate kinase II PykA, prephenate dehydratase pheA and tyrosine transcriptional repressor TyrR are knocked out in the Escherichia coli BW25113 fbr and prephenate dehydrogenase TyrA fbr , wherein the accession number of the 4-hydroxyphenylacetic acid-3-hydroxylase HpaBC is WP_000801472.1, the accession number of the caffeic acid methyltransferase COMT is NP_200227.1, the accession number of the glycosyltransferase UGT72E2 is NP_201470.1, the accession number of the tyrosine amino-cleavage enzyme TAL is AAA33883.1, the accession number of the 4-coumarate coenzyme A ligase 4CL1 is NP_175579.1, the accession number of the cinnamoyl coenzyme A reductase CCR is ABL01801.3, the accession number of the ethanol dehydrogenase ADH6 is NP_014051.3, the accession number of the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthetase AroG fbr is WP_001109196.1, and the accession number of the prephenate dehydrogenase TyrA fbr is WP_000225229.

1.

2. A method of biosynthesis of syringin, comprising: The step of obtaining syringin by fermenting the syringin biosynthesis engineering bacteria of claim 1 in a fermentation medium, wherein the carbon source of the fermentation medium is monosaccharide, disaccharide or any combination thereof.

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