CN117757708A - Construction method and application of engineering bacteria for producing methyl salicylate - Google Patents
Construction method and application of engineering bacteria for producing methyl salicylate Download PDFInfo
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- CN117757708A CN117757708A CN202311694161.0A CN202311694161A CN117757708A CN 117757708 A CN117757708 A CN 117757708A CN 202311694161 A CN202311694161 A CN 202311694161A CN 117757708 A CN117757708 A CN 117757708A
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- OSWPMRLSEDHDFF-UHFFFAOYSA-N methyl salicylate Chemical compound COC(=O)C1=CC=CC=C1O OSWPMRLSEDHDFF-UHFFFAOYSA-N 0.000 title claims abstract description 105
- 229960001047 methyl salicylate Drugs 0.000 title claims abstract description 52
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- 238000010276 construction Methods 0.000 title abstract description 9
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- 108090000623 proteins and genes Proteins 0.000 claims abstract description 14
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229960004889 salicylic acid Drugs 0.000 claims abstract description 10
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- 101710089841 Isochorismate pyruvate lyase Proteins 0.000 claims abstract description 8
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- 108060004795 Methyltransferase Proteins 0.000 claims description 6
- 102000016397 Methyltransferase Human genes 0.000 claims description 6
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- 239000000411 inducer Substances 0.000 claims description 3
- 241000228245 Aspergillus niger Species 0.000 claims description 2
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- 244000063299 Bacillus subtilis Species 0.000 claims 1
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- 230000015572 biosynthetic process Effects 0.000 abstract description 7
- 238000003786 synthesis reaction Methods 0.000 abstract description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 4
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- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 abstract description 3
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- 238000005886 esterification reaction Methods 0.000 abstract description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 abstract 1
- 238000001308 synthesis method Methods 0.000 abstract 1
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- 239000012634 fragment Substances 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
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- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a construction method and application of engineering bacteria for producing methyl salicylate. Engineering bacteria comprising co-expressing genes encoding isochorismate synthase, isochorismate pyruvate lyase, salicylic acid methylase in a host. By using the engineering bacteria for producing methyl salicylate, glucose is used as a carbon source to produce methyl salicylate, so that the synthesis of methyl salicylate from the head is realized. The invention does not have high-temperature esterification reaction, does not add exogenous salicylic acid, does not introduce impurity phenol, and has no report on a milligram-grade biological method methyl salicylate synthesis method at present.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a methyl salicylate production strain, a construction method and application thereof.
Background
Methyl salicylate has the English name Methyl Salicylate, meSA for short and chemical formula C 8 H 8 O 3 CAS number: 119-36-8, structural formula:
methyl salicylate is an organic ester naturally produced by green leaf plants, also called wintergreen oil, which is also a well-known, internationally recognized, important ester compound, widely used in pesticides, color fixatives, external analgesics, etc., and added as a flavoring agent and a spice ingredient in foods such as chewing gum, syrup, and beverage. The current market price of methyl salicylate is 19500 yuan/ton. The chemical synthesis of methyl salicylate is mainly carried out by esterifying, heating salicylic acid and methanol under catalysis of sulfuric acid, and esterifying. However, salicylic acid is extremely easy to generate side reactions in the reaction, so that oxidative discoloration or peculiar smell is generated, and the salicylic acid is not easy to recycle once the salicylic acid is deteriorated. And the chemical method is utilized to industrially synthesize the methyl salicylate and introduce the impurity phenol with corrosive action on the skin, while the biological method is not used for synthesizing the methyl salicylate, and no milligram-grade biological method methyl salicylate is reported at present because the esterification reaction at high temperature does not exist and no exogenous salicylic acid is added.
Disclosure of Invention
In view of the above, it is clearly necessary to provide an engineering bacterium for producing methyl salicylate, and a construction method and application thereof, so as to solve the above problems.
It is therefore a first object of the present invention to screen out enzymes which are still catalytically efficient in vitro from a large number of organisms or microorganisms capable of synthesizing methyl salicylate for the heterologous synthesis of methyl salicylate. For this purpose, the invention prefers genes related to enzymes derived from bacteria, fungi or protein engineering, by expression of which biosynthesis of methyl salicylate is achieved with a simple carbon source.
A second object of the present invention is to provide a host for high yield of methyl salicylate, which is biosynthesized by introducing a route for high efficiency production of methyl salicylate into original or engineered bacteria, fungi, yeast.
In order to achieve the above object, the present invention provides an engineering bacterium for producing methyl salicylate, comprising: genes encoding E.coli-derived isochorismate synthase (EntC), pseudomonas fluorescens-derived isochorismate pyruvate lyase (PchB) and fairy fan-derived methyltransferase (CbSAMT) were co-expressed in the host.
Wherein the host is a bacterium, yeast or fungus, wherein the bacterium or fungus is original or engineered. Preferably, the host is E.coli, B.subtilis, C.glutamicum, saccharomyces cerevisiae or A.niger.
The invention also provides a construction method of the engineering bacteria for producing methyl salicylate, which comprises the following steps:
the recombinant expression plasmid is obtained by connecting genes encoding isochorismate synthase (EntC), isochorismate pyruvate lyase (PchB) and methyltransferase (CbSAMT) to the expression plasmid;
and (3) transforming the recombinant plasmid vector into an escherichia coli host to obtain engineering bacteria for producing methyl salicylate. Wherein, the expression plasmid can be pZE12-luc or pCS27.
The invention also provides application of engineering bacteria for producing methyl salicylate, wherein the engineering bacteria are inoculated into a culture medium according to the inoculation amount of 1-5% by volume, and an inducer is added for fermentation treatment at 28-40 ℃ to prepare the methyl salicylate; the carbon source in the culture medium is glucose.
Based on the above, the medium comprises: 1g of·L -1 3-morpholinopyuvic acid 5-40 g.L -1 Glucose, 1-5 g.L -1 Yeast powder 5-8g.L -1 NaHPO 4 ，0.3～2g·L -1 NaCl，3g·L -1 KH 2 PO 4 ，1～5g·L -1 NH 4 Cl，240～250mg·L -1 MgSO 4 ，14～15.5mg·L- 1 CaCl 2 The balance being water.
Therefore, as shown in fig. 1, in the fermentation culture process of using glucose carbon source, glucose in the engineering bacteria is subjected to biological enzymolysis and is converted into salicylic acid under the action of isochorismate synthase (EntC) and isochorismate pyruvate lyase (PchB), and salicylic acid is converted into methyl salicylate under the action of methyltransferase (CbSAMT), so that methyl salicylate is synthesized from glucose by the engineering bacteria, and therefore, the engineering bacteria for producing methyl salicylate can realize the synthesis process of methyl salicylate, avoid the introduction of impurity phenol, realize the synthesis of methyl salicylate by using glucose as a source through a biological enzymolysis method, have low environmental pollution and are beneficial to industrial production of methyl salicylate.
Therefore, the engineering bacteria for producing the methyl salicylate provided by the invention are obtained by screening out the enzyme with activity in vitro; based on the engineering bacteria, a biosynthesis way of the methyl salicylate shown in figure 1 is designed, so that the methyl salicylate can be efficiently synthesized by adopting a biological method by taking a glucose simple carbon source as a raw material.
Drawings
FIG. 1 is a pathway diagram of biosynthesis of methyl salicylate provided by the present invention.
Fig. 2 is a diagram of fermentation results of producing methyl salicylate glycoside by engineering bacterium BW1 provided in example 3 of the present invention.
FIGS. 3A and B are graphs showing HPLC detection results of the strain fermentation product and methyl salicylate standard provided in example 3, respectively.
The specific embodiment is as follows:
the technical scheme of the invention is further described in detail through the following specific embodiments.
In the present invention, there is no particular requirement for the type of expression plasmid, and it is considered that the construction method for expressing the target gene in E.coli can be carried out by various methods commonly used in the art, such as the method of connecting the target gene to a vector after the cleavage treatment, and the details are not repeated.
In the following examples, E.coli strain BW25113, trans 5. Alpha. And BL21 (DE 3) were all commonly used E.coli strains, commercially available, with trans 5. Alpha. Being used for vector construction, BL21 (DE 3) for protein expression, and BW25113 as strains for fermentation. Among them, plasmids and strains used in each example are shown in the following Table 1.
Example 1
Recombinant plasmid pCs-entC-pchB, pZE-CbSAMT.
The recombinant plasmid pCs-entC-pchB provided in this example is obtained by connecting genes entC and pchB to an E.coli expression vector pCs, and the recombinant plasmid pZE-CbSAMT is obtained by connecting gene CbSAMT to an E.coli expression vector pZE 12-luc.
The construction method of the recombinant plasmid provided by the embodiment specifically comprises the following steps. Genes encoding isochorismate synthase (EntC), isochorismate pyruvate lyase (PchB) and methyltransferase (CbSAMT) derived from bacterial, fungal or protein engineering were selected. Obtaining target fragments by PCR amplification of target genes encoding isochorismate synthase (EntC) and isochorismate pyruvate lyase (PchB), then carrying out enzyme digestion on the target fragments and the vector by using proper enzymes, recovering the digested fragments, and then inserting the fragments into an expression plasmid pCS27 to obtain pCs-entC-pchB recombinant plasmids (see Table 1); the target gene of methyltransferase (CbSAMT) was amplified by PCR to obtain a target fragment, then the target fragment and the vector were digested with an appropriate enzyme, and the digested fragment was recovered and then inserted into the expression plasmid pZE12-luc to obtain a pZE-CbSAMT recombinant plasmid (see Table 1).
Example 2
Engineering bacteria for producing methyl salicylate: recombinant E.coli BW1
The engineering bacteria for producing methyl salicylate provided by the invention have no special requirements on the type of host strain used for constructing expression plasmids, and BW25113 strain is adopted as an initial host for constructing plasmids in the embodiment of the invention.
Firstly, picking fresh BW25113 colony, inoculating the colony into 4mL of LB culture medium, culturing for 8-12 h at 37 ℃, then inoculating 1mL into 100mL of LB culture medium, culturing at 37 ℃ until OD600 is up to 0.6, centrifuging at 6000rpm for 10min at 4 ℃ to collect thalli, washing with 10mL of 10% precooled glycerol, centrifuging at 6000rpm for 10min, repeating the glycerol washing step again, centrifuging, pouring the residual glycerol as much as possible, and finally adding a proper amount of 10% glycerol to resuspend cells to obtain competent cells. Taking 90 mu L of competent recombinant plasmid pCs-entC-pchB and 2 mu L of recombinant plasmid pZE-CbSAMT, placing on ice for two minutes, adding 600 mu L of LB culture medium after electric transformation, washing out cells after electric transformation, resuscitating for 1h at 37 ℃, coating the cells on an ampicillin resistance plate, culturing overnight in a constant temperature incubator at 37 ℃, and culturing for 8-10 h at 37 ℃ in 4mL of LB culture medium containing ampicillin resistance after bacterial colonies grow on the plate, thus obtaining engineering bacteria for producing methyl salicylate: the E.coli strain containing the recombinant plasmid pCs-entC-pchB and the recombinant plasmid pZE-CbSAMT is represented by recombinant E.coli BW 1.
The inducer IPTG with a final concentration of 0.5mM was initially added to the fermentation, and a part of the fermentation broth was taken out every 12 hours for measuring the growth of the cells and the yield of the target product methyl salicylate, and the results are shown in FIG. 2.
And detecting the target product methyl salicylate by adopting an HPLC analysis method, wherein the detection conditions are as follows: chromatographic column: separation column: diamond C18, ID 5 μm, 250X 4.6mm;
mobile phase: a is methanol, B is 1 per mill trifluoroacetic acid aqueous solution, the column temperature is 40 ℃, and the detection wavelength is 280nm at the flow rate of 1 mL/min. The gradient elution procedure is shown in the following table:
700 mu L of the fermentation broth is taken as a sample, a filtering membrane is adopted, HPLC analysis is carried out on the liquid after the membrane is taken, and the analysis result is shown in FIG. 3B in FIG. 3. The standard aqueous solution containing methyl salicylate was analyzed by HPLC using the method described above, and the analysis results are shown in FIG. 3A of FIG. 3, and FIG. 3A is a standard chart. As can be seen from fig. 3A: the retention time of the characteristic peak of the methyl salicylate is about 20.76min (20.757 min); from fig. 3B, it can be seen that there is a characteristic peak at about 20.76min (20.758 min), so that it can be determined that the characteristic peak with a retention time of 19.288min in fig. 3B is methyl salicylate, and thus methyl salicylate can be prepared by the method provided in this embodiment. The final yield of methyl salicylate can reach 765mg/L.
Table 1 list of plasmids and strains used in the examples of the invention
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same; while the invention has been described in detail with reference to the preferred embodiments, those skilled in the art will appreciate that: modifications may be made to the specific embodiments of the present invention or equivalents may be substituted for part of the technical features thereof; without departing from the spirit of the invention, it is intended to cover the scope of the invention as claimed.
Claims (4)
1. An engineering bacterium for producing methyl salicylate, which is characterized by comprising the following components: co-expressing genes encoding isochorismate synthase (EntC), isochorismate pyruvate lyase (PchB) and salicylic acid methylase (CbSAMT) in a host; the host is a bacterium, yeast or fungus, which is either original or engineered.
2. The engineering bacterium according to claim 1, comprising: the host is Escherichia coli, bacillus subtilis, corynebacterium glutamicum, saccharomyces cerevisiae or Aspergillus niger.
3. The method for constructing the engineering bacteria according to claim 1, which comprises the following steps:
the recombinant expression plasmid is obtained by connecting genes encoding isochorismate synthase (EntC), isochorismate pyruvate lyase (PchB) and methyltransferase (CbSAMT) to the expression plasmid; and (3) transforming the recombinant plasmid vector into an escherichia coli host to obtain engineering bacteria for producing methyl salicylate. Wherein the expression plasmid is pZE12-luc or pCS27.
4. The application of the engineering bacteria according to claim 1, wherein the engineering bacteria are inoculated into a culture medium according to the inoculum size of 1-5% by volume, and an inducer is added to perform fermentation treatment at 28-40 ℃ to prepare methyl salicylate;
the culture medium comprises: 1 g.L -1 3-morpholinopyuvic acid 5-40 g.L -1 Glucose, 1-5 g.L -1 Yeast powder 5-8g.L -1 NaHPO 4 ，0.3～2g·L -1 NaCl，3g·L -1 KH 2 PO 4 ，1～5g·L -1 NH 4 Cl，240～250mg·L -1 MgSO 4 ，14～15.5mg·L -1 CaCl 2 The balance being water.
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