Method for efficiently producing vanillin by utilizing vibrio natriureus

Abstract

The invention discloses a method for efficiently producing vanillin by utilizing vibrio natriticus, and relates to the field of biochemistry, chassis cells are vibrio natriticus, and plasmids are introduced into electrotransfection competent cells of the vibrio natriticus by an electrotransfection method to obtain recombinant bacteria; the plasmids are feruloyl coenzyme A synthase of syntrophic alkane degrading bacteria and enoyl coenzyme A hydratase from saccharomonospora vibrio, and recombinant plasmids are obtained by codon optimization of vibrio, gene synthesis and combined expression. The method for producing vanillin comprises the following steps: introducing the carrier into vibrio natrieti, carrying out whole-cell catalysis, and carrying out biotransformation by taking ferulic acid as an initial substrate to generate vanillin. The vibrio natriticus with higher protein synthesis efficiency and higher growth speed is introduced as a chassis cell, so that the cell culture time and the protein expression time can be further shortened, and the reaction efficiency is improved. Meanwhile, the reaction is carried out under a high-temperature condition, so that the vanillin synthesis rate is higher.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biochemistry, and particularly relates to a method for efficiently producing vanillin by using Vibrio natriegens. BACKGROUND

[0002] Vanillin, the chemical name of which is 4-hydroxy-3-methoxybenzaldehyde, is also known as vanilla aldehyde and vanilla aldehyde. It is a phenolic aldehyde with aldehyde, hydroxyl and ether as functional groups. It is known as the "Queen of Spices" due to its unique aroma and can be used to enhance flavor and taste in food. It can also be used as a food preservative additive. In addition to the food industry, vanillin is widely used in the pharmaceutical, cosmetic and agricultural industries. In the pharmaceutical industry, vanillin has health care and medical functions and is also an important raw material for the synthesis of various drugs. In the daily necessities industry, vanillin can be used as a flavoring agent in cosmetics and perfumes. In agricultural production, vanillin can also be used as a ripening agent and yield enhancer for crops. In addition, vanillin can also be used as a conductive agent, oxidation aid, defoaming agent, etc. and is widely used in industry.

[0003] The yield of vanillin extracted from plants is low, the maintenance cost is high, and the climate requirements for the growth of vanilla plants are strict. The annual yield of plant-based vanillin is about 7500 tons, while the global market demand is as high as several million tons, and plant-based vanillin is far from enough. In addition, the production of plant-based vanillin has high labor costs, and its ex-factory price is 50-200 times higher than that of synthetic vanillin.

[0004] In order to meet the growing global market demand, chemically synthesized vanillin has replaced natural vanillin. In 2025, the global vanillin market share is expected to reach 724.5 million US dollars. The price of natural vanillin extracted from plants is between 1200 and 1400 US dollars per kilogram, while the price of chemically synthesized vanillin is less than 15 US dollars per kilogram. Although chemically synthesized vanillin has high yield and low price, most food safety regulatory agencies only allow natural vanillin to be used in food and flavoring applications. Therefore, the production of natural vanillin using genetically engineered microorganisms and plants is expected to reduce the cost of chemical synthesis of vanillin and improve the environmental problems caused by the synthesis process.

[0005] Microbial transformation of vanillin breaks through the limitations of traditional plant extraction method, has the advantages of short production cycle, not limited by natural environment, avoids the problems of high energy consumption and high pollution of chemical synthesis method, and its product is recognized as natural vanillin by the food safety regulations of the United States and the European Union, which meets the needs of consumers for healthy food. At present, many strains have been found that can transform ferulic acid into vanillin. Studies have found that many microorganisms, such as Pseudomonas putida, Aspergillus niger, Pseudomonas fluorescens, amyloid-like mold, Streptomyces, etc., can transform ferulic acid into vanillin. Microorganisms metabolize FA through five different pathways: (1) aldehyde-alcoholization reverse reaction independent of coenzyme A; (2) aldehyde-alcoholization reverse reaction dependent on coenzyme A; (3) beta-oxidation reaction; (4) non-oxidative decarboxylation reaction; (5) reduction reaction. The metabolic pathways of various microorganisms from FA to vanillin are similar, mainly involving feruloyl coenzyme A synthetase (FCS) and enoyl coenzyme A hydratase (ECH). In the presence of coenzyme A and ATP, FA is converted to feruloyl coenzyme A by FCS, and then to vanillin by ECH. In recent years, a variety of strains that can produce vanillin using ferulic acid as a substrate have been reported. Because vanillin can inhibit microbial growth, this leads to low yield of microbial transformation of vanillin. Halomonas sp. B15 can produce a maximum of 255 mg / L using 0.5 g / L ferulic acid within 48 h. In contrast, Streptomyces setonii (13.9 g / L) and Streptomyces V-1 (19.2 g / L) have higher vanillin yields, but the mycelium is too dense during fermentation, which poses higher requirements for product separation and purification processes, further increasing downstream processing costs and limiting the feasibility of its industrial application.

[0006] Through fermentation optimization and genetic engineering of these strains, the yield of vanillin can be significantly improved. For example, the fermentation yield of Streptomyces V1 can reach 19.2 g / L. In addition, synthetic biology methods have also been used to modify microorganisms such as Escherichia coli and yeast to produce vanillin using precursors, showing good prospects for industrial application.

[0007] During fermentation, the content of glucose and vanillin affects the accumulation of vanillin. Amycolatopsis sp. uses a multi-pulse feeding strategy to produce higher vanillin yield (0.46 g·L-1h-1), which is equivalent to producing 0.69 g of vanillin per 1 g of ferulic acid, which is one of the highest values reported for wild-type bacteria. Although the conversion rate is only 69%, this technology can recover biomass and remove vanillin from the culture medium, minimizing its inhibitory effect on cell growth.

[0008] In recent years, people have begun to study the extraction of ferulic acid from agroforestry biomass to reduce production costs. Recent studies have shown that under optimal fermentation conditions, Enterobacter hormaechei can bioconvert ferulic acid extracted from pomegranate peel in 8h and produce 4.2g / L of vanillin. Wheat bran, rice bran, coconut shell, and sugarcane bagasse can also be used to convert vanillin. Microbial conversion of crop waste to produce natural vanillin provides an environmentally friendly, economical, and sustainable approach. However, the main drawback is that the content of ferulic acid is low, and a large amount of biomass is needed to obtain enough reaction substrate. In addition, a large amount of space and labor is required to process the extracted biomass, which greatly increases the economic burden.

[0009] Therefore, the skilled person in the art is committed to developing a method for efficiently producing vanillin with recombinant bacteria, using a chassis cell with higher protein synthesis efficiency and faster growth rate, shortening the cell culture time and protein expression time, thereby improving the synthesis efficiency of vanillin. SUMMARY

[0010] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present application is to develop a method for efficiently producing vanillin with recombinant bacteria, using a chassis cell with higher protein synthesis efficiency and faster growth rate, shortening the cell culture time and protein expression time, thereby improving the synthesis efficiency of vanillin.

[0011] To achieve the above-mentioned purpose, the present application provides a recombinant bacterium of Vibrio natriegens, the chassis cell is Vibrio natriegens, which is obtained by introducing a plasmid into Vibrio natriegens electrocompetent cells by electroporation method; the plasmid is a feruloyl-CoA synthase of Syntrophaceae bacterium and an enoyl-CoA hydratase from Saccharomonospora viridis, which is codon optimized for Vibrio and synthesized by gene synthesis, and the obtained recombinant plasmid is expressed in combination.

[0012] The present application also provides a method for efficiently producing vanillin using a recombinant bacterium of Vibrio natriegens, comprising the following steps:

[0013] Step 1: Using FCS and ECH as control proteins, different sources of high-temperature enzymes with specific functions are mined; the high-temperature enzymes include ECH-1 from Saccharomonospora viridis DSM 43017, named ECH-1, and spFCS from Syntrophaceae bacterium; they are codon optimized for Vibrio and synthesized by gene synthesis, and expressed in combination to obtain vector pETDuet-spfcs-ech1;

[0014] Step 2: introducing the vector pETDuet-spfcs-ech1 obtained in step 1 into Vibrio natriegens V.natriegens Vmax strain to obtain a recombinant strain Vmax;

[0015] Step 3, whole-cell catalysis of the recombinant strain Vmax obtained in step 2 to obtain a recombinant bacterial liquid;

[0016] Step 4, bioconversion of the recombinant bacterial liquid obtained in step 3 using ferulic acid as the initial substrate.

[0017] Further, the high-temperature enzyme in step 1 also includes apFCS from Aeribacillus pallidus and scFCS from Syntrophorhabdaceae bacterium.

[0018] Further, step 2 also includes the preparation step of the sodium-dependent Vibrio electrotransformation competent cells:

[0019] (1) Take out the preserved strain Vmax, transfer to a shake tube of LB3 medium, and incubate at 37°C overnight to activate the culture;

[0020] (2) The next day, transfer the Vmax liquid obtained in step (1) to a fresh shake tube of LB3 medium for culture; when the strain grows to the logarithmic phase, take out the shake tube and place it on ice to obtain a first bacterial liquid;

[0021] (3) Transfer the first bacterial liquid obtained in step (2) to a centrifuge tube that has been sterilized and pre-cooled, centrifuge the bacteria at 4°C for the first time; then discard the supernatant, resuspend the bacteria with pre-sterilized and pre-cooled electrotransformation buffer, and centrifuge the bacteria for the second time, repeat three times to obtain a second bacterial liquid;

[0022] (4) Resuspend the second bacterial liquid obtained in step (2) with the electrotransformation buffer.

[0023] Further, the components of the electrotransformation buffer are 680 mM sucrose and 7 mM potassium phosphate.

[0024] Further, step 2 also includes the sodium-dependent Vibrio electrotransformation step;

[0025] (5) Mix the vector pETDuet-spfcs-ech1 with the competent cells of the sodium-dependent Vibrio, then use the electrotransformation instrument to perform the electroporation experiment, and obtain the electrotransformed bacteria;

[0026] (6) Incubate the electrotransformed bacteria obtained in step (5) in LB3 culture solution at 37°C in a constant temperature incubator, then uniformly spread on LB3 solid medium containing antibiotics, and incubate at 37°C in a constant temperature incubator.

[0027] Further, the electrotransformation parameters of the electroporation experiment in step (5) are as follows: voltage 1400V, resistance 200Ω, capacitance 25kF, and electrotransformation cup size 2mm.

[0028] Further, the whole-cell catalysis in step 3 further comprises:

[0029] Step 3.1, the glycerol tube of the recombinant strain Vmax was taken out from the refrigerator, and was transferred to fresh LB3 culture medium at an inoculation amount of 1%, and was cultured at 37°C, 200 rpm overnight to obtain activated bacterial liquid;

[0030] Step 3.2, the activated bacterial liquid of step 3.1 was transferred to new LB3 liquid culture medium at an inoculation amount of 1%, and was cultured at 37°C, 200 rpm; when the strain grew to the logarithmic phase, IPTG was added to a final concentration of 0.2 mM; then the protein expression was induced by continuing to culture at 37°C for 4 hours, and the third bacterial liquid was obtained;

[0031] Step 3.3, the third bacterial liquid obtained in step 3.2 was placed in a centrifugal cup and centrifuged at 4000 rpm for 20 minutes to collect the bacteria; the supernatant was discarded, and the bacteria were resuspended with physiological saline; centrifugation was continued at 4000 rpm for 20 minutes, and this process was repeated twice.

[0032] Step 3.4, the bacteria were resuspended with 100 mM PBS buffer, pH 7.2, to OD600=50; the reaction system was 10 mL; the reaction was carried out at different temperatures in a shaking bed at 200 rpm.

[0033] Further, the logarithmic phase in step 3.2 is the optical density OD600 at 600 nm to 0.6-0.8.

[0034] Further, the biotransformation temperature in step 4 is 50°C or 60°C.

[0035] Further, on the basis of the whole-cell factory of Escherichia coli, a sodium-dependent Vibrio V. natriegens with higher protein synthesis efficiency and faster growth rate was further constructed as a chassis cell, so as to shorten the cell culture time and protein expression time, thereby improving the synthesis efficiency of vanillin.

[0036] Further, two high-temperature-resistant key enzymes in the vanillin synthesis pathway were screened through bioinformatics means, i.e., feruloyl-CoA synthase (spFCS) from mutualistic alkane-degrading bacteria and enoyl-CoA hydratase (ECH1) from Micromonospora viridifaciens.

[0037] Further, in the presence of coenzyme A and ATP, ferulic acid is converted to feruloyl-CoA by the key enzyme feruloyl-CoA synthase (FCS), and then is converted to vanillin by another key enzyme enoyl-CoA hydratase (ECH). At the same time, vanillin is synthesized under high temperature conditions, which is easy to inactivate endogenous proteins and reduce the production of by-products.

[0038] In the preferred embodiment 1 of the present application, the high-temperature enzyme mining process based on bioinformatics is described in detail;

[0039] In another preferred embodiment 2 of the present application, the function process of verifying thermophilic enzyme by whole cell catalysis is described in detail.

[0040] In another preferred embodiment 3 of the present application, the preparation of Vibrio natriophilus electrotransformation competent cells and the electrotransformation method are described in detail.

[0041] In another preferred embodiment 4 of the present application, the recombinant Vibrio high-temperature whole cell catalysis process is described in detail.

[0042] In another preferred embodiment 5 of the present application, the construction process of recombinant strains is described in detail.

[0043] In another preferred embodiment 6 of the present application, the optimization process of E. coli catalytic production of vanillin is described in detail.

[0044] In another preferred embodiment 7 of the present application, the construction process of Vibrio natriophilus catalytic vanillin production system is described in detail.

[0045] The present application has the following beneficial technical effects:

[0046] The introduction of Vibrio natriophilus with higher protein synthesis efficiency and faster growth rate as the chassis cell can further shorten the cell culture time and protein expression time, thereby improving the reaction efficiency. At the same time, the reaction under high temperature conditions, the synthesis rate of vanillin is higher. Under the condition of 60℃, using 1.5g / L ferulic acid as the substrate for catalysis, the vanillin yield reached 1.12g / L within 20 minutes, and the yield reached 5.46g / L·h-1, which was 2.73 times of the yield of E. coli (2.16g / L·h-1), which also showed that Vibrio natriophilus was a chassis cell with great potential for vanillin synthesis.

[0047] The E. coli recombinant strain of ECH-1 was induced overnight at 16℃, 200rpm under the condition of IPTG final concentration of 0.2mM, and the vanillin yield was 3.79mM.

[0048] Using 1g / L ferulic acid as the substrate for catalysis at 50℃ for 0.5h, the ferulic acid yield of the E. coli recombinant strain spFCS-ECH1 can reach 3.88mM.

[0049] The concept, specific structure and technical effects of the present application will be further described in combination with the drawings, so as to fully understand the purpose, features and effects of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1is a schematic diagram of construction of spFCS and ECH1 expression vector of a preferred embodiment 2 of the present application;

[0051] Figure 2 is a graph of test results of catalytic ability of vanillin production candidate enzyme of a preferred embodiment 2 of the present application;

[0052] Figure 3 is a graph of test results of catalytic ability of FCS candidate enzyme of a preferred embodiment 2 of the present application;

[0053] Figure 4 is a schematic diagram of construction of recombinant plasmid of a preferred embodiment 5 of the present application;

[0054] Figure 5 is a graph of test results of catalytic ability of vanillin production candidate enzyme of a preferred embodiment 2 of the present application;

[0055] Figure 6 is a graph of test results of catalytic ability of vanillin production candidate enzyme of a preferred embodiment 2 of the present application;

[0056] Figure 7 is a catalytic curve of strain BL21 (pETDuet-spfcs-echl) of a preferred embodiment 6 of the present application;

[0057] Figure 8 is a graph of test results of optimal substrate concentration of strain BL21 (pETDuet-spfcs-echl) of a preferred embodiment 6 of the present application;

[0058] Figure 9 is a catalytic curve of strain BL21 (pETDuet-spfcs-echl) of a preferred embodiment 6 of the present application; DETAILED DESCRIPTION

[0059] The technical contents of the present application are more clearly and conveniently understood by the following reference to the drawings of the specification, which introduce a plurality of preferred embodiments of the present application. The present application can be embodied in many different forms, and the scope of protection of the present application is not limited to the embodiments mentioned herein.

[0060] Example 1 High-temperature enzyme mining based on bioinformatics

[0061] With FCS and ECH as control proteins, input CEM (http: / / cem.sjtu.edu.cn / ) website to mine high-temperature enzymes with specific functions from different sources. Set parameters and enzyme source range, screen enzymes from microorganisms with growth temperature greater than 50℃. According to the first comparison data between candidate enzymes, i.e. the sequence similarity score of candidate enzymes and control proteins. The higher the Score, the more likely the candidate enzyme is a high-temperature enzyme with the same function as the initial protein. In order to avoid repeated operation, select a part of candidate enzymes ranked in the front, and screen out candidate enzymes from the same genus and with high similarity of amino acid sequence. Further calculate the melting point index (Tm Index, TI) of the candidate enzyme on the website http: / / tm.life.nthu.edu.tw / index.htm, and the fatty coefficient on the website https: / / web.expasy.org / protparam / , and select the candidate enzymes with TI and fatty coefficient higher than the control proteins for testing.

[0062] FCS and ECH are key enzymes for the biosynthesis of vanillin from ferulic acid, and related enzymes need to be mined for the establishment of a high-temperature whole-cell catalytic production pathway of vanillin in cells. According to literature reports, FCS and ECH in Acidothermus sp. Y2 1 1 are vanillin production enzymes under high temperature conditions, and can effectively eliminate the side reaction of vanillyl alcohol production. Therefore, the protein sequences thereof are used as reference templates for mining on the CEM website. The optimal growth temperature of microorganisms is greater than 50℃ as a condition for screening. At the same time, the TI value and fatty coefficient of the candidate enzyme are predicted on the bioinformatics website.

[0063] The Tm (melting temperature) of protein is similar to the Tm of DNA, which refers to the temperature at which 50% of the protein is unfolded during thermal denaturation, and is an important indicator for measuring the thermal stability of protein. The higher the Tm value, the better the theoretical thermal stability. The website http: / / tm.life.nthu.edu.tw / index.htm assigns a TI index to the predicted Tm value of the protein, TI>1 represents that the predicted Tm is greater than 65℃, TI<0 represents that the predicted Tm of the protein is less than 55℃, and 0<TI<1 represents that the predicted Tm is between 55℃ and 65℃.

[0064] The fatty coefficient is another indicator for measuring the thermal stability of protein, which refers to the content of aliphatic side chain amino acids in protein, including alanine, valine, leucine, isoleucine, etc. These aliphatic amino acids stabilize the three-dimensional structure of protein through hydrophobic interaction in protein structure. According to theoretical simulation, the higher the fatty coefficient, the better the thermal stability of protein. The mining results of candidate enzymes are shown in Table 1.

[0065] Table 1 Mining results of candidate enzymes

[0066]

[0067] The ECH sequence in the literature reported in the heat-tolerant Amycolatopsis sp. was used as input content to mine heat-tolerant ECH. The output Score was arranged from high to low, the candidate enzymes with TI value higher than 1.0 of the control protein and fatty coefficient higher than 76.9 of the control protein were screened, and the candidate enzymes with high similarity to ECH were removed, to obtain ECH-1 from Saccharomonospora viridis DSM 43017. It was codon-optimized for E. coli and Vibrio, and then gene synthesis and subsequent function verification were performed.

[0068] The synthesized spFCS gene and the plasmid pETDuet-fcs-ech from strain VA1 (BL21) were simultaneously subjected to double enzyme digestion with BamH I and Not I, and after recovering the gene fragment, T4 ligation was performed to construct a recombinant plasmid, the gene sequence of spFCS replaced the gene fragment of FCS on the original plasmid vector, and the recombinant plasmid was used to verify the catalytic function of the spFCS heat-tolerant enzyme.

[0069] The synthesized ECH1 gene and the plasmid pETDuet-fcs-ech from strain VA1 (BL21) were simultaneously subjected to double enzyme digestion with Bgl II and Xho I, and after recovering the gene fragment, T4 ligation was performed to construct a recombinant plasmid, the gene sequence of ECH1 replaced the gene fragment of FCS on the original plasmid vector, and the recombinant plasmid was used to verify the catalytic function of the FCS candidate heat-tolerant enzyme.

[0070] Example 2 Whole-cell catalytic verification of the function of thermophilic enzyme

[0071] The steps are as follows:

[0072] (1) The obtained recombinant plasmid was introduced into strain BL21-1 (DE3) by chemical transformation method, and overnight culture was performed on a plate.

[0073] (2) The next day, a single colony was picked from the plate and inoculated into 5 mL of fresh LB medium, and 37°C, 200 rpm shaking culture was performed for 10-12 hours.

[0074] (3) The bacterial solution was transferred to 30 mL of liquid LB medium at an inoculation amount of 1%, and 37°C, 200 rpm shaking culture was performed to prepare seed liquid.

[0075] (4) After 3-4 hours, the seed liquid was transferred to new LB liquid medium at an inoculation amount of 1%, and 37°C, 200 rpm culture was continued.

[0076] (5) When the strain grows to the logarithmic phase and the optical density (OD600) at 600 nm reaches about 0.6-0.8, add IPTG to a final concentration of 0.2 mM and induce overnight at 16°C and 200 rpm.

[0077] (6) The next day, collect the bacterial suspension in a centrifuge cup at 4000 rpm for 20 minutes. Resuspend the cells in 30 mL of PBS buffer and centrifuge at 6000 rpm for 10 minutes. Repeat twice.

[0078] (7) Resuspend the cells in PBS buffer (pH = 7.2-7.4) to an OD600 of 50. Add 1 g / L ferulic acid as a reaction substrate to the reaction system. Take 500 μL of the sample at the corresponding time point and terminate the reaction by adding 1 / 100 (v / v) 6 M concentrated hydrochloric acid. Store the sample in a -20°C refrigerator for subsequent analysis.

[0079] (8) Sample treatment: Add an equal volume of methanol to the sample, shake thoroughly for 1 minute, and then centrifuge at 12,000 rpm for 10 minutes. Take the supernatant and filter it through a 0.22 μm filter and add it to a liquid phase vial.

[0080] (9) Liquid phase testing using Waters

[0081] The mobile phase consisted of methanol:water:glacial acetic acid (volume ratio: 35:65:0.05); the flow rate was 0.80 mL / min; the detection wavelength was 320 nm; the chromatographic column was an Agilent Eclipse XDB-C18 reversed-phase column (5 μM, 4.6 × 150 nm); the column temperature was 25°C; the injection volume was 20 μL; and the elution was isocratic. Qualitative determination was based on retention time. Quantitative determination was based on peak area.

[0082] (10) The two synthesized ECH genes and the plasmid pETDuet-fcs-ech from strain VA1 (BL21) were simultaneously digested with BglⅡ and XhoⅠ. After the gene fragments were recovered, T4 ligation was performed to construct a recombinant plasmid. The gene sequence of ECH-(1-2) replaced the gene fragment of FCS on the original plasmid vector. The recombinant plasmid was used to verify the catalytic function of the FCS candidate heat-resistant enzyme. Schematic diagram of the construction of the spFCS and ECH1 expression vector Figure 1 shown.

[0083] The recombinant plasmid was transformed into BL21-1 (DE3), and a whole-cell catalytic experiment was carried out at 50°C using 1 g / L ferulic acid as a substrate. Then, 500 μL of cells were taken after 0.5 hours of catalysis and processed as a sample. The concentrations of ferulic acid and vanillin in the catalytic system were detected by HPLC. The catalytic ability test results of the candidate enzyme for vanillin production are shown in the figure. Figure 2The results showed that no vanillin was produced and no ferulic acid was consumed after 0.5 hours of catalysis for all FCS candidate enzymes, so the results are not shown. Both ECH candidate enzymes produced vanillin and ferulic acid was consumed. According to the literature, strain VA1 had a maximum yield at 50°C, with a vanillin yield of 3.55 mM in 0.5 hours. VA1 was used as a control group for the experiment, and the recombinant strain expressing ECH-1 produced 3.79 mM of vanillin under the same conditions, and the recombinant strain expressing ECH-2 produced 3.35 mM of vanillin. Therefore, ECH-1 has a higher catalytic effect, and ECH-1 is selected for subsequent testing and catalysis.

[0084] The synthesized nine FCS genes were subjected to synchronous BamH I and Not I double enzyme digestion with the plasmid pETDuet-fcs-ech from strain VA1 (BL21), and the gene fragments were recovered and subjected to T4 ligation to construct recombinant plasmids. The gene sequence of FCS replaced the gene fragment of FCS on the original plasmid vector, and the recombinant plasmid was used to verify the catalytic function of the FCS candidate heat-resistant enzyme.

[0085] The nine candidate enzymes screened were subjected to codon optimization for E. coli and Vibrio, and the synthetic genes were obtained by a BamH I and Not I double enzyme digestion system, and the gene fragments were ligated to construct recombinant plasmids for transformation. Whole-cell catalysis experiments were performed in BL21-1 (DE3). After 0.5 hours of catalysis at 50°C with 1 g / L ferulic acid as the substrate, 500 μL of cells were also used as samples for processing, and HPLC was used to detect the concentrations of ferulic acid and vanillin in the catalytic system.

[0086] The FCS candidate enzyme catalytic ability test is as follows Figure 3VA1 is the control strain VA1 (BL21); bbFCS is the strain pETDuet-bbfcs-ech (BL21); tfFCS is the strain pETDuet-tffcs-ech (BL21); spFCS is the strain pETDuet-spfcs-ech (BL21); dbFCS is the strain pETDuet-dbfcs-ech (BL21); tsFCS is the strain pETDuet-tsfcs-ech (BL21); apFCS is the strain pETDuet-apfcs-ech (BL21); slFCS is the strain pETDuet-slfcs-ech (BL21); scFCS is the strain pETDuet-scfcs-ech (BL21); btFCS is the strain pETDuet-btfcs-ech (BL21), and the results show that, taking strain VA1 as the control group, among the 9 FCS candidate enzymes, the catalytic ability of spFCS, apFCS and scFCS is stronger than that of FCS-CG at high temperature. After high-temperature catalysis for 0.5 hours, the vanillin yield is 3.88 mM, 3.55 mM and 3.76 mM, respectively, and the conversion rate is as high as 81.8%. After whole-cell catalysis of the expression strains of bbFCS, tfFCS, dbFCS, tsFCS, slFCS and btFCS at 50°C for 0.5 hours, the vanillin yield is 2.74 mM, 1.98 mM, 1.75 mM, 2.22 mM, 2.18 mM and 1.98 mM, respectively. This shows that the candidate enzymes all have catalytic activity at 50°C. However, considering the yield and yield, spFCS, apFCS and scFCS are selected for the next test and expression.

[0087] Example 3 Preparation of Vibrio natriegens electrocompetent cells and electrotransformation method

[0088] Based on the method of reported in the literature for the electrotransformation of Vibrio natriegens ATCC 14048, the specific method for the strain Vmax electroporation experiment is as follows:

[0089] (1) Take the preserved strain Vmax glycerol tube from the refrigerator, transfer 1% to 5 mL of LB3 medium in a shaking tube, and incubate at 37°C in a shaking incubator at 200 rpm overnight.

[0090] (2) The next day, transfer the activated Vmax bacterial solution to 5 mL of fresh LB3 medium in a shaking tube and incubate. When the strain grows to the logarithmic phase and the optical density value at 600 nm OD600 is about 0.6-0.8, remove the shaking tube and place it on ice for 15-20 minutes.

[0091] (3) Transfer the bacterial solution to a sterilized and pre-chilled 15 mL centrifuge tube, centrifuge at 4500 rpm, 4°C for 10 minutes to collect the bacteria. Then discard the supernatant, resuspend the bacteria in 5 mL of pre-sterilized and pre-chilled electrotransformation buffer (680 mM sucrose and 7 mM potassium phosphate dibasic, pH 7), centrifuge at 4000 rpm, 4°C for 10 minutes to collect the bacteria, repeat three times.

[0092] (4) Resuspend the bacterial solution in 200 L of electrotransformation buffer (to ensure transformation efficiency, the whole process of preparing competent cells should be carried out under low temperature conditions).

[0093] (5) Take 1 μg of plasmid and mix it with the competent cells, then use the BioRad electroporator to perform the electroporation experiment. The electroporation parameters are as follows: voltage 1400 V, resistance 200 Ω, capacitance 25 kF, and 2 mm electrotransformation cup.

[0094] (6) After electroporation, quickly transfer the bacteria to 1 mL of sterilized LB3 culture solution, incubate in a 37°C constant temperature incubator for 2 hours, then evenly spread on LB3 solid medium containing appropriate antibiotic concentration, and incubate in a 37°C constant temperature incubator.

[0095] Example 4 Recombinant Vibrio high-temperature whole-cell catalysis

[0096] The steps are as follows:

[0097] 4.1 Take the recombinant Vibrio glycerol tube out of the refrigerator, transfer it to fresh LB3 culture medium at a 1% inoculation amount, and incubate at 37°C, 200 rpm overnight.

[0098] 4.2 The next day, take the activated bacterial solution and transfer it to new LB3 liquid medium at a 1% inoculation amount, continue to incubate at 37°C, 200 rpm. When the strain grows to the logarithmic phase, the optical density at 600 nm OD600 is about 0.6-0.8, add IPTG to a final concentration of 0.2 mM. Then continue to incubate at 37°C for 4 hours to induce protein expression.

[0099] 4.3 Take the bacterial solution and place it in a centrifuge cup, centrifuge at 4000 rpm for 20 minutes to collect the bacteria. Discard the supernatant, resuspend the bacteria with 30 mL of physiological saline. Continue to centrifuge at 4000 rpm for 20 minutes, repeat this process twice.

[0100] 4.4 Resuspend the bacteria in 100 mM PBS buffer (pH 7.2) to OD600 = 50. The reaction system is 10 mL. The reaction is carried out in a shaking incubator at different temperatures at 200 rpm. During the reaction, take 500 μL samples periodically, use 1 / 100 (V / V) concentrated hydrochloric acid to terminate the reaction, and store the samples in a -20°C refrigerator for subsequent concentration determination.

[0101] Construction of recombinant strains

[0102] The FCS with catalytic effect at high temperature was combined with ECH for expression. The gene fragment and linearized vector pETDuet-echl were obtained by double enzyme digestion system of BamHl and Notl, and then the recombinant plasmids pETDuet-apfcs-echl, pETDuet-spfcs-echl, pETDuet-scfcs-echl were obtained under the action of T4 ligase. The schematic diagram of recombinant plasmid construction is shown in Figure 4 The recombinant plasmid was transformed into BL21-1(DE3) competent to obtain the recombinant strain, and the whole cell catalysis was carried out according to the above method.

[0103] Example 6 Optimization of catalytic production of vanillin by E. coli

[0104] The steps are as follows:

[0105] 6.1 Optimization of temperature conditions

[0106] The obtained recombinant strains BL21(pETDuet-apfcs-echl), BL21(pETDuet-spfcs-echl), BL21(pETDuet-scfcs-echl) were subjected to whole cell catalysis under different temperature conditions, and their abilities to catalyze synthesis of vanillin were compared. The results of comparison of the abilities of E. coli BL21(DE3) recombinant strains expressing different FCS and ECH-1 to produce vanillin are shown in Figure 5 The whole cell catalytic reaction was carried out for 0.5 hours under different temperature conditions, and the initial concentration of ferulic acid was 1 g / L.

[0107] The recombinant strains BL21 (pETDuet-apfcs-ech1), BL21 (pETDuet-spfcs-ech1), BL21 (pETDuet-scfcs-ech1) all showed certain catalytic activity in the temperature range of 30-65°C. Among them, the optimal catalytic temperature of strain BL21 (pETDuet-apfcs-ech1) was 55°C, and the vanillin concentrations produced at 30°C, 37°C, 42°C, 50°C, 55°C, 60°C, 65°C were 3.08 mM, 3.26 mM, 3.51 mM, 3.68 mM, 3.85 mM, 3.49 mM, 2.99 mM, respectively. Compared with strain BL21 (pETDuet-apfcs-ech), the vanillin concentration obtained by the catalytic system after replacing ECH with ECH-1 at 50°C increased by 0.3 mM. The optimal catalytic temperature of strain BL21 (pETDuet-spfcs-ech1) was 60°C, and the vanillin concentrations produced at 30°C, 37°C, 42°C, 50°C, 55°C, 60°C, 65°C were 2.75 mM, 2.93 mM, 3.57 mM, 3.94 mM, 4.19 mM, 4.28 mM, 3.67 mM, respectively. Compared with strain BL21 (pETDuet-spfcs-ech), the vanillin concentration obtained by the catalytic system after replacing ECH with ECH-1 at 50°C increased by 0.06 mM. The optimal catalytic temperature of strain BL21 (pETDuet-spfcs-ech1) was 55°C, and the vanillin concentrations produced at 30°C, 37°C, 42°C, 50°C, 55°C, 60°C, 65°C were 2.97 mM, 3.24 mM, 3.55 mM, 3.87 mM, 4.11 mM, 3.69 mM, 3.16 mM, respectively. Compared with strain BL21 (pETDuet-spfcs-ech), the vanillin concentration obtained by the catalytic system after replacing ECH with ECH-1 at 50°C increased by 0.11 mM.

[0108] Among the recombinant strains BL21 (pETDuet-apfcs-ech1), BL21 (pETDuet-spfcs-ech1), BL21 (pETDuet-scfcs-ech1), the strain BL21 (pETDuet-spfcs-ech1) had higher catalytic effect under high temperature conditions, which indicated that the spFCS-ECH1 double enzyme system had higher heat resistance in intracellular expression, higher than the FCS-ECH1 double enzyme system.

[0109] 6.2 Optimization of pH conditions

[0110] The reaction system was then catalyzed under different pH conditions. Since the optimum pH of FCS and ECH in Chapter 3 is between 7.0 and 7.5, the pH gradient is set to 6.0, 6.5, 7.0, 7.5, and 8.0. The results of comparing the ability of E. coli BL21 (DE3) recombinant strains expressing different FCS and ECH-1 to produce vanillin are shown in Figure 6 The whole cell catalytic reaction was carried out under different pH conditions for 0.5 hours, and the initial concentration of ferulic acid was 1 g / L.

[0111] It can be seen from Figure 6 that the optimum catalytic pH of recombinant strains BL21 (pETDuet-apfcs-ech1), BL21 (pETDuet-spfcs-ech1), and BL21 (pETDuet-scfcs-ech1) is 7.0. At pH 6.0, 6.5, 7.0, 7.5, and 8.0, the vanillin production of strain BL21 (pETDuet-apfcs-ech1) is 2.52 mM, 3.12 mM, 3.62 mM, 3.17 mM, and 2.60 mM, respectively. At pH 6.0, 6.5, 7.0, 7.5, and 8.0, the vanillin production of strain BL21 (pETDuet-spfcs-ech1) is 2.76 mM, 3.33 mM, 3.94 mM, 3.54 mM, and 2.79 mM, respectively. At pH 6.0, 6.5, 7.0, 7.5, and 8.0, the vanillin production of strain BL21 (pETDuet-scfcs-ech1) is 2.61 mM, 3.14 mM, 3.83 mM, 3.45 mM, and 2.77 mM, respectively.

[0112] 6.3 Optimization of substrate concentration

[0113] Based on the determination of the optimum temperature and pH of the catalytic system of strains BL21 (pETDuet-apfcs-ech1), BL21 (pETDuet-spfcs-ech1), and BL21 (pETDuet-scfcs-ech1), we believe that recombinant strain BL21 (pETDuet-spfcs-ech1) has stronger heat resistance and stability, so we choose recombinant strain BL21 (pETDuet-spfcs-ech1) for further testing and optimization. During the catalytic process, samples were taken at 0h, 0.5h, 1.0h, 1.5h, 2.0h, 4.0h, 6.0h, 8.0h, 10.0h, and 12.0h to measure the consumption trend of ferulic acid and the production trend of vanillin.

[0114] The catalytic curve of strain BL21 (pETDuet-spfcs-ech1) is shown in Figure 7As shown, after 0.5 hours of catalysis at 50°C, ferulic acid has been almost completely consumed, while vanillin still has a small increase between 0.5 hours and 1 hour, which might indicate that the intracellular catalytic system can catalyze more than 1 g / L of ferulic acid. Therefore, we further increased the concentration of ferulic acid substrate to explore the maximum catalytic capacity of spFCS. The catalysis conditions were 50°C, and 1.0 g / L, 1.5 g / L, and 2.0 g / L of ferulic acid were added as the initial substrate, respectively, and the ferulic acid consumption and vanillin production were tested after 0.5 hours and 1 hour, respectively.

[0115] The results of the optimal substrate concentration test of strain BL21 (pETDuet-spfcs-echl) are shown in Table 2. Figure 8 As shown, when the added ferulic acid concentration was 1.0 g / L and 1.5 g / L, it was almost completely consumed after 0.5 hours, while when 2.0 g / L of ferulic acid was added as the initial substrate, there was still 0.38 g / L (1.96 mM) of ferulic acid remaining after 0.5 hours of catalysis. Under the conditions of 1.0 g / L, 1.5 g / L, and 2.0 g / L of substrate concentration, the vanillin content obtained after 0.5 hours was 4.18 mM (0.64 g / L), 6.54 mM (1.00 g / L), and 7.07 mM (1.08 g / L), respectively, and the molar conversion rate was 84.44%, 85.83%, and 72.89%, respectively. Therefore, strain BL21 (pETDuet-spfcs-echl) has the highest molar conversion rate when 1.5 g / L of ferulic acid is added as the initial catalytic substrate, and the yield reaches 2.16 g L-1h-1. Subsequently, this initial substrate concentration was used for whole-cell catalysis to improve the efficiency of ferulic acid bioconversion to vanillin.

[0116] Construction of a V. natriegens catalysis system for vanillin production

[0117] V. natriegens as an industrial chassis cell has the advantage of fast growth and shows potential for application in the field of biotechnology. With the improvement of genetic manipulation tools in V. natriegens, the establishment and optimization of protein expression tools, V. natriegens shows more and more advantages in biological production. For example, previous studies have shown that V. natriegens as a chassis cell for whole-cell catalysis to produce L-dopa and N-acetylneuraminic acid also shows a fast protein expression and production rate.

[0118] Based on this, in order to further improve the ability of spFCS-ECH1 combination to produce vanillin, the vector pETDuet-spfcs-ech1 was introduced into the V. natriegens Vmax strain to obtain a recombinant strain Vmax(pETDuet-spfcs-ech1). Then, the whole-cell catalysis was carried out according to the method of 4.2.4.4. The biotransformation was carried out at 60°C with 1.5 g / L ferulic acid as the initial substrate, and 0.5 hours later, 1.07 g / L vanillin was obtained. After 1 hour, the concentration of vanillin in the system decreased to 0.69 g / L. It was speculated that the reaction was reversed after reaching the maximum reaction limit within 0.5 hours, and the yield decreased. Therefore, the sampling interval was shortened, and the cell liquid was taken every 10 minutes for concentration determination, and the catalytic curve of the strain Vmax(pETDuet-spfcs-ech1) is shown in Figure 9 As can be seen, similar to the catalysis of E. coli, when 1.5 g / L ferulic acid is used as the initial substrate, the reaction proceeds rapidly, and reaches the maximum yield of 1.12 g / L (6.88 mM) at 20 minutes. Within 10 minutes of the reaction, the maximum yield is 5.46 g / L-h, which is 2.73 times that of E. coli under the same conditions, indicating that the strain V. natriegens Vmax has a significant production advantage compared with E. coli BL21.

[0119] The above describes in detail the preferred embodiments of the present application. It should be understood that those skilled in the art can make many modifications and changes to the present application without creative labor based on the concept of the present application. Therefore, any technical solution obtained by logical analysis, reasoning or limited experiments based on the prior art according to the concept of the present application should be within the protection scope defined by the claims.

Claims

1. A recombinant bacterium of Vibrio natriuresis, characterized in that The chassis cells of the recombinant bacteria are Vibrio natriuresis, and the plasmid is introduced into the electrotransformation competent cells of the Vibrio natriuresis by electrotransformation; the plasmids are feruloyl-CoA synthase from the syntrophic alkane-degrading bacteria and enoyl-CoA hydratase from Saccharomonas viridans, which are codon-optimized for Vibrio, and gene synthesis is performed to combine and express the recombinant plasmid.

2. A method for efficiently producing vanillin using the recombinant Vibrio natriuresis bacteria as described in claim 1, characterized in that: The method comprises the following steps: Step 1: Using FCS and ECH as control proteins, identify thermophilic enzymes from different sources with specific functions; the thermophilic enzymes include ECH-1 from Saccharomonospora viridis DSM 43017 and spFCS from Syntrophaceae bacteria; these enzymes are codon-optimized for Vibrio, synthesized, and expressed in combination to obtain the vector pETDuet-spfcs-ech1; Step 2, introducing the vector pETDuet-spfcs-ech1 obtained in step 1 into the V. natriegens Vmax strain to obtain the recombinant strain Vmax; Step 3, subjecting the recombinant strain Vmax obtained in step 2 to whole-cell catalysis to obtain a recombinant bacterial solution; Step 4: biotransform the recombinant bacterial solution obtained in step 3 using ferulic acid as an initial substrate to produce the vanillin.

3. The method according to claim 2, wherein The high-temperature enzyme in step 1 further comprises apFCS from Aeribacillus pallidus and scFCS from Syntrophorhabdaceae bacterium.

4. The method according to claim 2, wherein The step 2 also includes the step of preparing electroporation competent cells of Vibrio natrium: (1) Remove the preserved strain Vmax, transfer it to a shake tube containing LB3 medium, and activate the culture in a shaker at 37°C overnight; (2) On the second day, the activated Vmax bacterial solution from step (1) was transferred to a shake tube containing fresh LB3 medium for culture; when the strain grew to the logarithmic phase, the shake tube was removed and placed on ice to obtain the first bacterial solution; (3) Transfer the first bacterial solution obtained in step (2) to a sterilized and pre-cooled centrifuge tube, and centrifuge for the first time at 4°C to collect the bacteria; then discard the supernatant, resuspend the bacteria in a sterilized and pre-cooled electroporation buffer, and centrifuge for the second time to collect the bacteria, and repeat three times to obtain the second bacterial solution; (4) Resuspend the second bacterial solution obtained in step (2) using electroporation buffer.

5. The method according to claim 4, wherein The electrotransfer buffer comprises 680 mM sucrose and 7 mM potassium dihydrogen phosphate.

6. The method according to claim 4, wherein Said step 2 also includes a step of electrotransforming Vibrio natrium; (5) taking the vector pETDuet-spfcs-ech1 and mixing it with the competent cells of Vibrio natriuresis, and then performing an electroporation experiment using an electroporator to obtain electroporated bacteria; (6) The electroporated bacteria obtained in step (5) were rapidly incubated in LB3 culture medium in a 37°C constant temperature incubator, and then evenly spread onto LB3 solid culture medium containing antibiotics and cultured statically in a 37°C constant temperature incubator.

7. The method according to claim 6, wherein The electroporation parameters of the electroporation experiment in step (5) are as follows: voltage 1400 V, resistance 200 Ω, capacitance 25 kF, and electroporation cup size 2 mm.

8. The method according to claim 2, wherein The whole-cell catalysis in step 3 further comprises: Step 3.

1. Take the glycerol tube of the recombinant strain Vmax out of the refrigerator and transfer it to fresh LB3 medium at a 1% inoculum size for overnight culture at 37°C and 200 rpm to obtain an activated bacterial solution; Step 3.2: Transfer the activated bacterial solution from step 3.1 to a fresh LB3 liquid medium at a 1% inoculum size and continue culturing at 37°C and 200 rpm. When the strain reaches the logarithmic phase, add IPTG to a final concentration of 0.2 mM. Continue culturing at 37°C in a shaker for 4 hours to induce protein expression, thereby obtaining the third bacterial solution. Step 3.3: Place the third bacterial suspension obtained in step 3.2 in a centrifuge cup and centrifuge at 4000 rpm for 20 minutes to collect the bacteria; discard the supernatant and resuspend the bacteria in physiological saline; continue centrifugation at 4000 rpm for 20 minutes, and repeat this process twice; Step 3.4: Resuspend the cells to OD600 = 50 using 100 mM PBS buffer, pH 7.2; use a 10 mL reaction system; and perform the reaction in a shaker at 200 rpm at different temperatures.

9. The method according to claim 8, wherein The logarithmic phase in step 3.2 is when the optical density OD600 at 600 nm reaches 0.6 to 0.

8.

10. The method according to claim 2, wherein The bioconversion temperature in step 4 is 50°C or 60°C.

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