A method for constructing a self-regulating bidirectional transport system of vanillin in Escherichia coli to improve the yield of vanillin

Abstract

The present invention discloses a method for constructing a self-regulating bidirectional vanillin transport system in Escherichia coli to improve vanillin production. Specifically, the vanillin synthesis genes Ech and Fcs from Amycolatopsis sp., the coding gene of ferulic acid transporter Todx and the coding gene of vanillin transporter PP_0179 under the regulation of the vanillin self-inducing promoter are heterologously expressed in Escherichia coli, and vanillin is produced by batch fermentation. Using this method for shake flask fermentation, 1.52 mM of vanillin is produced within 24 h, which is 1.57 times higher than that of the control strain, and the tolerance of the strain is improved. The cell concentration in the fermentation broth is 1.39 times higher than that of the control strain.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to a method for constructing a self-regulating bidirectional transport system of vanillin in Escherichia coli to improve the yield of vanillin. Background Art

[0002] Vanillin has a unique creamy vanilla fragrance and is one of the largest broad-spectrum spice varieties in the world. It is widely used in the fields of food, medicine, daily chemical industry, and agriculture, and has very important economic value. Vanillin synthesized by the microbial method using renewable natural substrates has a quality close to that of natural products and a low production cost, becoming a promising green synthesis method.

[0003] Using Escherichia coli as a chassis and utilizing the key enzymes for vanillin synthesis, feruloyl-CoA synthetase (FCS) and feruloyl-CoA hydratase / aldolase (ECH), to construct a microbial cell factory for converting the renewable substrate ferulic acid into vanillin is one of the important development directions of vanillin biosynthesis. However, the slow entry of the substrate into the cell and the inhibitory effect of the product on the chassis cell during the conversion process greatly limit the production capacity of the cell factory. Therefore, there are still huge challenges in the efficient and sustainable production of vanillin using Escherichia coli cell factories. Research has shown that by regulating the expression and activity of aromatic compound transporters, the concentration of aromatic compounds in the cell can be dynamically adjusted, effectively improving the tolerance of microorganisms to aromatic compounds and promoting the secretion of products. The vanillin efflux transporter helps increase the tolerance of microorganisms to vanillin and effectively avoids further degradation of vanillin, while the ferulic acid influx transporter can efficiently uptake the substrate ferulic acid from the environment. Introducing the above transporters into the Escherichia coli cell factory and designing and constructing a self-regulating bidirectional transport system of vanillin using the vanillin self-inducible promoter can solve the bottleneck problem restricting the production of vanillin by the cell factory and effectively improve the synthesis efficiency. Currently, there is no relevant report on improving the yield of vanillin by constructing a self-regulating bidirectional transport system of vanillin with Escherichia coli as the chassis cell. Summary of the Invention

[0004] The purpose of the present invention is to provide an application of a method for constructing a self-regulating bidirectional transport system of vanillin in Escherichia coli to improve the yield of vanillin.

[0005] The technical solution adopted by the present invention is as follows:

[0006] A method for constructing a self-regulating bidirectional transport system of vanillin in Escherichia coli to improve the yield of vanillin, characterized in that the preparation method comprises the following steps:

[0007] (1) Obtain the target gene;

[0008] (2) Construct a recombinant vector;

[0009] (3) Transform the recombinant vector into Escherichia coli, and heterologously express the vanillin synthesis genes ech from Amycolatopsis sp. in Escherichia coli R37M , fcs, the encoding gene of ferulic acid transporter Todx and the encoding gene of vanillin transporter PP_0179 under the regulation of the vanillin self-inducible promoter in Escherichia coli to obtain Escherichia coli containing the vanillin self-regulatory bidirectional transport system;

[0010] (4) Produce vanillin by fermenting the Escherichia coli with the vanillin self-regulatory bidirectional transport system obtained in step (3), and that's it.

[0011] The method for constructing a vanillin self-regulatory bidirectional transport system in Escherichia coli to improve vanillin production, characterized in that the target genes in step (1) are the fcs gene and ech R37M gene; the preparation method is as follows: First, obtain the FCS and ECH protein sequence information respectively, replace the arginine at the 37th position of the ECH protein with methionine, optimize the gene sequence according to the usage principle of Escherichia coli preferred codons, and then perform PCR amplification with primers fcs-F / R and ech-F / R respectively and recover by agarose gel electrophoresis to obtain the fcs gene and ech R37M gene.

[0012] The method for constructing a vanillin self-regulatory bidirectional transport system in Escherichia coli to improve vanillin production, characterized in that the recombinant vector is pETuet-Ech-Fcs-adh7-Todx-PP_0179, and in step (2), ech R37M , fcs, todx, pp_0179 genes and the adh7 promoter are ligated to the pETuet-1 vector by Gibson to construct the recombinant vector pETuet-Ech-Fcs-adh7-Todx-PP_0179.

[0013] The method for constructing a vanillin self-regulatory bidirectional transport system in Escherichia coli to improve vanillin production, characterized in that the fermentation method in step (4) is: inoculate the overnight cultured Escherichia coli seed liquid into the fermentation medium at an inoculation amount of 1.2-1.4%, culture at 30-35 °C and 220-250 rpm, the final concentration of IPTG inducer is 1-1.2 mM, and the final concentration of the substrate ferulic acid is 10-11 mM.

[0014] The method for constructing a vanillin self-regulatory bidirectional transport system in Escherichia coli to improve vanillin production, characterized in that the fermentation medium is LB medium, which contains 10-12 g / L of tryptone, 5-6 g / L of yeast extract, 10-12 g / L of sodium chloride, and the rest is deionized water.

[0015] The method for constructing a self-regulating bidirectional vanillin transport system in Escherichia coli to improve vanillin production, characterized in that, in step (2), ech R37M and fcs genes are under the regulation of a strong promoter, and Todx and PP_0179 genes are under the regulation of a vanillin-inducible promoter.

[0016] The method for constructing a self-regulating bidirectional vanillin transport system in Escherichia coli to improve vanillin production, characterized in that the strong promoter is P T7 , and the vanillin-inducible promoter is P adh7 .

[0017] The nucleic acid sequence of SEQ ID NO.1 contains the sequences of three genes, todxpp_0179 and adh7. The nucleic acid sequence numbered 1-911 is the adh7 nucleic acid sequence; the nucleic acid sequence numbered 912-2270 is the todx nucleic acid sequence; the nucleic acid sequence numbered 2271-3689 is the pp_0179 nucleic acid sequence

[0018] The beneficial effects of the present invention are as follows:

[0019] (1) In the vanillin synthesis pathway with Escherichia coli as the chassis cell, the present invention constructs a self-regulating bidirectional vanillin transport system to improve vanillin production and the tolerance of the chassis cell to vanillin by expressing the ferulic acid transporter Todx and the vanillin transporter PP_0179 under the regulation of the vanillin-inducible promoter adh7.

[0020] (2) In the technical solution of the present application, overexpression of the ferulic acid transporter can increase the intracellular ferulic acid concentration and improve the utilization rate of the substrate in the cell; overexpression of the vanillin transporter can promote the tolerance of the chassis cell to vanillin, and at the same time reduce product inhibition and promote vanillin synthesis by timely efflux of the product; using the vanillin-inducible promoter adh7 to initiate each transporter gene can effectively control the expression level of the transporter and avoid a large metabolic burden on the strain caused by overexpression of the transporter, thereby affecting the normal growth of the strain. The method of the present invention can increase the vanillin production (1.57 times increase) and increase the concentration of the bacterial solution in the medium (OD 600 1.39 times increase). Description of the Drawings

[0021] Figure 1 : Vector map of pETuet-Ech-Fcs-adh7-Todx-PP_0179

[0022] Figure 2 : Graph of cell growth concentration change during shake flask batch fermentation

[0023] Figure 3:The yield comparison chart of vanillin fermentation in shake flask batches is displayed by concentration, where the vertical axis is the concentration of vanillin, the left column in the horizontal axis represents pETuet-Ech-Fcs, and the right column represents pETuet-Ech-Fcs-adh7-Todx-PP_0179 DETAILED DESCRIPTION

[0024] Example 1

[0025] The experimental materials involved in the following examples are as follows:

[0026] The protein expression strain Escherichia coli BL21 (DE3) was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; the plasmid pETduet-1 was purchased from Novagen; the restriction endonucleases EcoRI, HindⅢ, NotI, BglII, XhoI, and FseI were all purchased from Thermo Fisher Scientific; PrimeSTAR Max DNA Polymerase was purchased from Takara; 2×F8FastLong PCRMaster Mix was purchased from Beijing Aidelai Biotechnology Co., Ltd.; the Gibson ligation kit was purchased from Rambolide Biotechnology Co., Ltd.; and ferulic acid and vanillin standards were purchased from Aladdin.

[0027] The instruments and equipment used are conventional instruments and equipment in this field

[0028] Step (1)

[0029] Construction of the expression vector PETuet-ech-fcs for the synthesis pathway of vanillin

[0030] 1. Acquisition of target gene

[0031] First, the FCS and ECH protein sequence information was obtained from NCBI (www.ncbi.nlm.nih.gov / ), the arginine at position 37 of the ECH protein was replaced with methionine, the gene sequence was optimized according to the principle of using the dominant codons of Escherichia coli, and the gene was synthesized by Huada Qinglan Biotechnology (Wuxi) Co., Ltd. Then, PCR amplification was performed with primers fcs-F / R and ech-F / R, respectively, and agarose gel recovery was performed to obtain the fcs gene and ech R37M Gene.

[0032] The PCR system was: template 1 μL, primer-F 1 μL, primer-R 1 μL, PrimeSTAR Max 25 μL, and sterile water 22 μL.

[0033] PCR program: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 10 s, annealing at 60°C for 15 s, extension at 72°C at 1 kb / min, and 72°C for 10 min, for 30 cycles.

[0034] The primer sequences are as follows:

[0035]

[0036] 2. Construction of the recombinant vector PETuet-Ech-Fcs by Gibson assembly

[0037] The empty vector PETuet-1 was double digested with restriction endonucleases EcoRⅠ and HindⅢ, and the digested vector was recovered using an enzyme digestion product recovery kit. The vector and the target fragment were ligated using a Gibson assembly kit (Lamboid Biotechnology Co., Ltd.). The recombinant vector PETuet-Ech-Fcs was successfully obtained by colony PCR verification. The expression of Ech and Fcs was controlled by the strong promoter P T7 Control the expression of Ech and Fcs.

[0038] Gibson assembly system: 5 μL of DNA Assembly Mix, 2 μL of linearized vector, 3 μL of target fragment, and ligate at 50 °C for 1 h.

[0039] Colony PCR system for colonies: colonies, 1 μL of primer-F, 1 μL of primer-R, 5 μL of 2×F8FastLong PCR Master Mix, and 2 μL of sterile water.

[0040] Colony PCR program for colonies: pre-denaturation at 98 °C for 10 min, denaturation at 98 °C for 10 s, annealing at 56 °C for 15 s, extension at 72 °C at 1 kb / min, 72 °C for 10 min, for 30 cycles.

[0041] Step (2)

[0042] Construction of the vanillin self-regulating bidirectional transport system in Escherichia coli

[0043] 1. Obtaining the target genes

[0044] First, the sequence information of ferulic acid transporter Todx, vanillin transporter PP_0179, and vanillin self-inducible promoter adh7 was obtained from NCBI (www.ncbi.nlm.nih.gov / ). The coding gene sequences of Todx and PP_0179 were optimized according to the usage principle of Escherichia coli preferred codons, and the above genes and promoter were synthesized by BGI-Qinglan Biotechnology (Wuxi) Co., Ltd. Then, the coding genes of Todx and PP_0179 and the adh7 promoter sequence were obtained by PCR amplification using primers Todx-F / R, PP_0179-F / R, adh7-F / R respectively and agarose gel recovery.

[0045] The PCR system is as follows: 1 μL of template, 1 μL of primer - F, 1 μL of primer - R, 25 μL of PrimeSTARMax, and 22 μL of sterile water.

[0046] PCR program: Pre - denaturation at 95°C for 5 min, denaturation at 95°C for 10 s, annealing at 60°C for 15 s, extension at 72°C at 1 kb / min, 72°C for 10 min, for 30 cycles.

[0047] The primer sequences are as follows:

[0048]

[0049] 2. Construction of the recombinant vector pETuet - Ech - Fcs - adh7 - Todx - PP_0179 by Gibson assembly

[0050] The vector pETuet - Ech - Fcs was double - digested with restriction endonucleases NotI and BglⅡ, and the digested vector was recovered using an enzyme - digestion product recovery kit; the vector and the target fragment were ligated using a Gibson assembly kit (Lanbolide Biotechnology Co., Ltd.), and the recombinant vector pETuet - Ech - Fcs - adh7 was successfully obtained through colony PCR verification; pETuet - Ech - Fcs - adh7 was double - digested with restriction endonucleases BglⅡ and XhoI, and ligated with the Todx gene encoding ferulic acid transporter using the Gibson assembly method to obtain the recombinant vector pETuet - Ech - Fcs - adh7 - Todx; after double - digestion with restriction endonucleases BglⅡ and XhoI, it was ligated with the PP_0179 gene encoding vanillin transporter using the Gibson assembly method to obtain the recombinant vector pETuet - Ech - Fcs - adh7 - Todx - PP_0179. The plasmids constructed by this method are shown in the appendix Figure 1 。

[0051] Gibson assembly system: 5 μL of DNA Assembly Mix, 2 μL of linearized vector, 3 μL of target fragment, ligated at 50°C for 1 h.

[0052] Colony PCR system for colonies: colonies, 1 μL of primer - F, 1 μL of primer - R, 5 μL of 2×F8FastLong PCR Master Mix, 2 μL of sterile water.

[0053] Colony PCR system for colonies: Pre - denaturation at 98°C for 10 min, denaturation at 98°C for 10 s, annealing at 56°C for 15 s, extension at 72°C at 1 kb / min, 72°C for 10 min, for 30 cycles.

[0054] Step (3)

[0055] Effect of Vanillin Self-Regulating Bidirectional Transporter System on Vanillin Production and Tolerance in Escherichia coli

[0056] 1. Fermentation culture: (1) Transform the recombinant vectors pETuet-Ech-Fcs and pETuet-Ech-Fcs-adh7-Todx-PP_0179 into Escherichia coli BL21(DE3) respectively, coat them on the LB plate with ampicillin resistance, and culture overnight at 37 °C. (2) Pick a single colony into 10 mL of LB liquid medium containing ampicillin resistance, and culture overnight at 30 °C with 220 rpm / min. (3) Inoculate the overnight culture into 20 mL of fresh LB liquid medium containing ampicillin resistance, and inoculate to the final concentration of OD 600 = 0.1, and culture at 30 °C with 220 rpm until OD 600 = 0.6, add IPTG inducer with a final concentration of 1 mM, and continue to culture at 30 °C with 220 rpm until OD 600 = 0.8, add ferulic acid with a final concentration of 10 mM as the substrate, and continue to culture at 30 °C with 220 rpm.

[0057] 2. Sample treatment:

[0058] Take 1 mL of the fermentation broth, centrifuge at 12000 rpm for 2 min at 4 °C to separate the supernatant and precipitate. Transfer the supernatant to a new EP tube, add an equal volume of absolute ethanol, and filter through a 0.22 μm organic filter membrane into an HPLC sample vial. Add 1 mL of 1×PBS buffer to the precipitate sample, gently pipette and mix well, centrifuge at 12000 rpm for 2 min, discard the supernatant to remove the compounds remaining on the cell surface, repeat the washing twice, add 200 μL of 1×PBS to dissolve the cells, then add an equal volume of absolute ethanol and an appropriate amount of quartz sand, vortex, and filter through a 0.22 μm organic filter membrane into an HPLC sample vial with an inner insert tube.

[0059] 2. HPLC reaction conditions:

[0060] Chromatographic column: ZORBAX StableBond C18, 4.6×250 nm, 5 μm; Ultraviolet absorption wavelength: 308 nm; Flow rate: 0.5 ml / min; Injection volume: 10 μL / 20 μL. Mobile phase: Mobile phase A is an aqueous solution of 0.1% formic acid; Mobile phase B is a methanol solution of 0.1% formic acid. The mobile phase is gradient eluted, and the elution program is: 60% mobile phase A from 0 - 8.5 min; 15% mobile phase A from 9.5 - 18 min; 60% mobile phase A from 19 - 25 min.

[0061] The results showed that the construction of a self-regulating bidirectional vanillin transport system in Escherichia coli could effectively improve the tolerance of recombinant Escherichia coli to vanillin and increase vanillin production. The cell concentration in the fermentation broth increased by 1.39 times compared with the control strain (as Figure 2 shown), and the vanillin production after 24 h of fermentation reached 1.52 mM in terms of concentration, which was 1.57 times higher than that of the control strain (as Figure 3 shown).

Claims

1. A method for constructing a self-regulating bidirectional transport system of vanillin in Escherichia coli to improve the yield of vanillin, characterized in that, The preparation method comprises the following steps: (1) Obtain the target gene; (2) The vanillin synthesis genes ECH from Amycolatopsis orientalis R37M , FCS and the todx and pp_0179 genes under the regulation of the adh7 promoter were ligated to the pETuet-1 vector by Gibson to construct the recombinant vector pETuet-Ech-Fcs-adh7-Todx-PP_0179; (3) Transform the recombinant vector into Escherichia coli and heterologously express ECH in Escherichia coli R37M , FCS, and the genes encoding ferulic acid transporter Todx and vanillin transporter PP_0179 under the regulation of the vanillin self-inducible promoter adh7 promoter to obtain Escherichia coli containing a vanillin self-regulating bidirectional transport system; The nucleic acid sequence of the todx coding gene is positions 912 - 2270 of SEQ ID NO.1; the nucleic acid sequence of the pp_0179 coding gene is positions 2271 - 3689 of SEQ ID NO.1; (4) Produce vanillin by fermenting the Escherichia coli containing the vanillin self-regulating bidirectional transport system obtained in step (3), thus obtaining the product.

2. The method for constructing a self-regulating bidirectional transport system of vanillin in Escherichia coli to improve the yield of vanillin according to claim 1, characterized in that, The target genes described in step (1) are the FCS gene and the ECH R37M gene; the preparation method is as follows: First, obtain the protein sequence information of FCS and ECH respectively, replace the 37th arginine of the ECH protein with methionine, optimize the gene sequence according to the usage principle of the dominant codons of Escherichia coli, and then perform PCR amplification and agarose gel recovery respectively to obtain the FCS gene and the ECH R37M gene.

3. The method for constructing a self-regulating bidirectional transport system of vanillin in Escherichia coli to improve the yield of vanillin according to claim 1, characterized in that, The fermentation method in step (4) is as follows: inoculate the overnight cultured Escherichia coli seed liquid into the fermentation medium at an inoculation amount of 1.2 - 1.4%, culture at 30 - 35°C with 220 - 250 rpm, the final concentration of the IPTG inducer is 1 - 1.2 mM, and the final concentration of the substrate ferulic acid is 10 - 11 mM.

4. The method for constructing a self-regulating bidirectional transport system of vanillin in Escherichia coli to improve the yield of vanillin according to claim 3, characterized in that, The fermentation medium described is LB medium, which contains 10 - 12 g / L of tryptone, 5 - 6 g / L of yeast extract, 10 - 12 g / L of sodium chloride, and the remaining component is deionized water.

5. The method for constructing a self-regulating bidirectional transport system of vanillin in Escherichia coli to improve the yield of vanillin according to claim 4, characterized in that, The ECH described in step (2) R37M and the FCS gene are under the regulation of a strong promoter.

6. The method for constructing a self-regulating bidirectional transport system of vanillin in Escherichia coli to improve the yield of vanillin according to claim 5, characterized in that, The strong promoter described is PT7.

7. The method for constructing a self-regulating bidirectional transport system of vanillin in Escherichia coli to improve the yield of vanillin according to claim 6, characterized in that, The nucleic acid sequence of the adh7 promoter is positions 1 - 911 of SEQ ID NO.1.

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