Method for synthesizing vanillin and vanillic acid by escherichia coli engineering bacteria based on temperature regulation

Abstract

The application provides a method for synthesizing vanillin and vanillic acid by E. coli engineering bacteria based on temperature regulation, which comprises three stages of bacterial growth, induction expression and product synthesis, and the temperature of the induction expression and product synthesis stages is differentially regulated, so as to regulate the key reductase activity and intracellular carbon flow distribution, vanillin can be synthesized, and the precursor vanillic acid can be directionally enriched. The application realizes efficient synthesis of different products in the same strain and the same fermentation system, greatly improves the process production flexibility while ensuring high fermentation yield, meets the flexible production needs of two high-value aromatic products in industrialization, solves the technical problems of poor metabolic matching, uncontrollable products and low yield in constant temperature fermentation in the traditional process, and has a good popularization and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of industrial microbial technology, specifically relating to a method for synthesizing vanillin and vanillic acid using engineered Escherichia coli based on temperature control. Background Technology

[0002] Vanillin and vanillic acid are both high-value-added aromatic natural products, widely used in food flavorings, daily chemical fragrances, biopharmaceuticals, and fine chemicals, with stable market demand and high added value. Vanillin is the most widely used natural flavor monomer in industry, with a pure aroma and a wide range of applications. Vanillic acid is more stable, with excellent antioxidant and antibacterial properties, and can be used as a key precursor and intermediate in vanillin biosynthesis, as well as as an independent raw material in cosmetics, pharmaceutical preservatives, and biosynthesis. Both have extremely high industrial application value. Compared to traditional high-pollution chemical synthesis processes, de novo fermentation synthesis of natural aromatic products by microorganisms is greener, safer, and produces products with higher purity, meeting natural product certification standards, and is currently the mainstream development direction in the industry.

[0003] Escherichia coli, with its clear genetic background, rapid proliferation rate, ease of genetic modification, and high-density fermentation, is a superior substrate strain for vanillin biosynthesis. Existing high-yield engineered bacteria generally employ a de novo synthesis pathway using glucose as the sole carbon source. Through intracellular stepwise metabolic catalysis, vanillic acid is ultimately used as the core intermediate, further reducing it to the final product, vanillin. This process utilizes inexpensive raw materials, is simple, and environmentally friendly, possessing excellent prospects for industrialization.

[0004] However, current fermentation processes still suffer from several key technical defects, severely limiting the product selectivity and fermentation yield of de novo glucose synthesis systems. Firstly, existing technologies all employ isothermal fermentation, making it impossible to achieve controllable selective synthesis of vanillin and vanillic acid. Traditional processes maintain fixed induction and synthesis temperatures throughout the entire process, resulting in a fixed intracellular carbon flux distribution pattern, unidirectional metabolic pathway progression, and a single fermentation product—vanillin or vanillic acid. Since these two products have independent market applications and high added value, traditional single-product fermentation models suffer from poor production flexibility and low equipment utilization, requiring two separate process systems. This significantly increases production and setup costs, failing to meet the flexible production needs of producing two products in a single fermentation.

[0005] Secondly, the constant-temperature process cannot match the temperature differences required for cell growth, protein induction, and product synthesis, resulting in limited yield and severe metabolic blockage. The de novo synthesis of vanillin from glucose is a multi-stage cascade metabolic process, with significant differences in the optimal temperature windows for cell proliferation, key enzyme induction and expression, and vanillic acid catalytic conversion. Traditional constant-temperature processes struggle to adapt to the physiological characteristics of each stage: excessively high temperatures easily lead to denaturation of key synthetic enzymes, increased inclusion bodies, cell stress damage, and activation of byproduct metabolic pathways; excessively low temperatures result in insufficient protein induction, low catalytic efficiency, incomplete carbon source metabolism, and the retention of large amounts of vanillic acid intermediates that cannot be converted, ultimately leading to low product conversion rates and a significant bottleneck in overall fermentation potency.

[0006] Furthermore, the lack of differentiated control between induction and synthesis temperatures is the core reason for the uncontrollable product and low conversion rate. The specific reduction process of vanillic acid to vanillin is highly dependent on temperature-sensitive key functional enzymes, whose protein soluble expression, catalytic activity, and substrate selectivity are all highly sensitive to temperature. Current technologies do not distinguish the temperature logic between the induction and synthesis stages, using uniform parameters throughout, which makes it impossible to achieve precise pathway control: insufficient low temperatures lead to low expression levels of related enzymes, while unsuitable high temperatures hinder catalytic conversion. It is impossible to achieve a controllable switch between "remaining in vanillic acid" or "completely converting to vanillin," resulting in poor product selectivity and extremely low freedom of process control.

[0007] Finally, current industry research generally focuses on optimizing the yield of a single product, neglecting the role of temperature difference control in the switching of stepwise metabolic pathways, and has not yet formed a segmented temperature control system that can flexibly switch between two products. Conventional process optimization is limited to fine-tuning conventional parameters such as pH, dissolved oxygen, and inducer concentration, which cannot solve the fundamental defects such as stepwise inhibition of glucose metabolism, unreasonable carbon flow distribution, and single product solidification. As a result, the yield and selectivity of vanillin and vanillic acid synthesized de novo by existing engineered bacteria cannot meet the requirements of flexible and efficient industrial production.

[0008] In summary, to address the technical problems of existing isothermal fermentation processes, such as single product, mismatched metabolic stages, poor product selectivity, disordered intermediate accumulation, and limited fermentation yield, it is crucial to develop a three-stage temperature-controlled fermentation process that can precisely match cell growth, protein induction, and product synthesis. This process, which achieves controllable and high-yield synthesis of vanillin and vanillic acid through temperature difference regulation during induction and synthesis, is a pressing technical challenge in this field. Summary of the Invention

[0009] The technical problem to be solved: Existing fermentation processes for the de novo synthesis of vanillin from glucose using *E. coli* generally employ isothermal culture, which cannot flexibly switch between the synthesis of vanillin and vanillic acid. Furthermore, the temperature requirements for each stage of cell growth, protein induction expression, and product synthesis are mismatched, easily leading to metabolic pathway imbalances, intermediate accumulation, low product conversion rates, and insufficient production flexibility. This invention provides a method for synthesizing vanillin and vanillic acid using engineered *E. coli* strains based on temperature control. This method is a three-stage temperature-controlled fermentation, dividing the fermentation process into three independent temperature-controlled stages: cell growth, induction expression, and product synthesis. By differentially controlling the temperature during the induction expression and product synthesis stages, the activity of key reductases and intracellular carbon flux distribution are adjusted, achieving efficient synthesis of both vanillin and vanillic acid products within the same fermentation system.

[0010] Technical solution: This invention provides a method for synthesizing vanillin and vanillic acid using temperature-controlled engineered Escherichia coli. The method employs BL21-AC6 recombinant engineered bacteria and uses a three-stage gradient temperature control strategy to precisely match the metabolic requirements of cell growth, functional enzyme induction expression, and product catalytic synthesis, thereby achieving flexible and efficient synthesis of vanillin and vanillic acid in the same fermentation system.

[0011] The present invention also provides specific steps of the above method:

[0012] S1. The BL21-AC6 recombinant engineered bacteria were inoculated into LB liquid medium and cultured with shaking to obtain the primary seed culture; the primary seed culture was transferred to LB liquid medium and cultured with shaking to obtain the secondary seed culture;

[0013] S2. Add trace element stock solution to the basal fermentation medium, adjust the pH of the system to 6.8-6.9 with ammonia, stir under aeration, inoculate the secondary seed culture into the system, and incubate at 30-37℃ and dissolved oxygen 20-30% for 4-6 h until the system OD reaches its limit. 600 It is 20-25;

[0014] S3. Add IPTG to induce expression, and add feed medium. Maintain the pH of the system at 7.0 with ammonia water, and induce culture at 16-20℃ for 12-18 h.

[0015] S4. After induction, raise the temperature and continue fermentation for 14-30 h to produce vanillic acid or vanillin; the temperature for producing vanillic acid is 29-31℃; the temperature for producing vanillin is 24-26℃.

[0016] In step S1 above, the BL21-AC6 recombinant engineered bacteria used *Escherichia coli* BL21(DE3) as the starting strain and overexpressed the following genes: dehydroshikimate dehydratase (DSD), 3-O-methyltransferase (OMT), carboxylic acid reductase (CAR), vanillyl alcohol oxidase (VaoA), 5'-methyladenosine nucleosidase (Mtn), S-ribose homocysteine ​​lyase (LuxS), and citrate synthase mutant gene (gltA). R119L .

[0017] Preferably, the above-mentioned dehydroshikimate dehydratase gene DSD is derived from Propionibacterium, and its nucleotide sequence is shown in SEQ ID NO.1.

[0018] Preferably, the 3-O-methyltransferase gene OMT is derived from Leptospira, and its nucleotide sequence is shown in SEQ ID NO.2.

[0019] Preferably, the carboxylic acid reductase gene CAR is derived from Nocardia iowanensis, and its nucleotide sequence is shown in SEQ ID NO. 3.

[0020] Preferably, the vanillyl oxidase gene VaoA is derived from Penicillium spp., and its nucleotide sequence is shown in SEQ ID NO.4.

[0021] Preferably, the 5'-methionine adenosine nucleosidase gene Mtn is derived from Escherichia coli, and its nucleotide sequence is shown in SEQ ID NO.5.

[0022] Preferably, the S-ribosomal homocysteine ​​lyase gene LuxS is derived from Escherichia coli, and its nucleotide sequence is shown in SEQ ID NO.6.

[0023] Preferably, the above-mentioned citrate synthase mutant is obtained by mutating arginine at position 119 of the amino acid sequence of wild-type citrate synthase to leucine; the citrate synthase mutant gene gltA R119L The nucleotide sequence is shown in SEQ ID NO. 7.

[0024] Preferably, the inoculation amount of the primary seed liquid in step S1 is 1-5%.

[0025] Preferably, the basic fermentation medium in step S2 above comprises the following components: glucose 10-20 g / L, magnesium sulfate 0.125-0.585 g / L, ammonium sulfate 1-4 g / L, citric acid 1-1.7 g / L, tryptone 10-30 g / L, potassium dihydrogen phosphate 3.5-10 g / L, yeast extract 5-15 g / L, dipotassium hydrogen phosphate 3.35-5.35 g / L, and 0.1-0.5% polyether defoamer, with water as the solvent; optionally, the basic fermentation medium also contains 8-15 g of glycerol.

[0026] Preferably, the amount of trace element mother liquor added in step S2 is 0.1-0.5%; the trace element mother liquor includes the following components: copper sulfate 3-7 mg / mL, manganese sulfate 0.05-0.1 mg / mL, calcium chloride 0.2 mg / mL, ferrous sulfate 1 mg / mL, zinc sulfate 0.010-0.225 mg / mL, and water as the solvent.

[0027] Preferably, the inoculation amount of the secondary seed liquid in step S2 above is 5-10%.

[0028] Preferably, the final concentration of IPTG in step S3 is 0.5-1 mM.

[0029] Preferably, the feeding medium in step S3 comprises the following components: glucose 150-225 g / L, magnesium sulfate 0.125-0.585 g / L, tryptone 10-30 g / L, yeast extract 5-15 g / L, L-methionine 10-15 g / L, and water as the solvent; the flow rate of the feeding medium is 6-24 mL / h.

[0030] Preferably, the temperature for producing vanillic acid in step S4 is 30°C; the temperature for producing vanillin is 25°C. This invention also provides the application of the above method in the production of vanillic acid or vanillin.

[0031] Beneficial effects:

[0032] 1. This invention precisely controls the intracellular reductive metabolic pathway by regulating the temperature during the growth, induced expression, and synthesis stages of the bacterial strain. At low temperatures, the vanillic acid conversion pathway can be blocked, achieving targeted enrichment of vanillic acid. Increasing the temperature activates the catalytic conversion pathway, stabilizing the synthesis of vanillin. This enables the production of vanillic acid or vanillin within the same fermentation system, with maximum yields of 3095.5 mg and 640.9 mg / L for vanillic acid and vanillin, respectively. This overcomes the limitation of traditional processes that can only produce a single product, achieving flexible production of two products using the same strain and process.

[0033] 2. Staged temperature control is highly adapted to the physiological metabolism of the cells, effectively reducing the accumulation of by-products, improving the utilization rate of glucose carbon source, reducing impurities in the fermentation system, and significantly reducing the difficulty and production cost of subsequent product separation and purification.

[0034] 3. This invention is simple to operate, highly controllable, and has good process repeatability. It does not require additional modification of the strain sequence and can switch product types simply by fine-tuning the temperature, achieving flexible production of high-value-added products and significantly improving the applicability and industrial value of the fermentation process.

[0035] 4. This invention can be stably implemented with conventional E. coli fermentation culture medium and conventional fermentation equipment, significantly improving equipment utilization and production flexibility, and adapting to the needs of industrialized mass production. Attached Figure Description

[0036] Figure 1 The liquid chromatogram of vanillic acid in Example 1 (5-fold dilution);

[0037] Figure 2 The liquid chromatogram of vanillin in Example 5 (5-fold dilution). Detailed Implementation

[0038] The present invention will be further described below with reference to embodiments. These embodiments are illustrative of the present invention, but the present invention is not limited to these embodiments:

[0039] The BL21-AC6 recombinant engineered bacteria involved in this embodiment and comparative example were preserved in the Zhongyuan Food Laboratory. The BL21-AC6 recombinant engineered bacteria used *Escherichia coli* BL21(DE3) as the starting strain and overexpressed the following genes: *Propionibacterium* dehydroshikimate dehydratase gene PodDSD, *Leptospira* 3-O-methyltransferase gene LiOMT, *Nocardia iowandrae* carboxylic acid reductase gene NiCAR, *Penicillium jaundiceum* vanillyl alcohol oxidase gene VaoA, *Escherichia coli* 5'-methyladenosine nucleosidase gene Mtn, *Escherichia coli* S-ribosomalcysteine ​​lyase gene LuxS, and citrate synthase mutant gene gltA. R119L .

[0040] Example 1

[0041] This embodiment describes a method for synthesizing vanillic acid using the BL21-AC6 recombinant engineered bacteria through temperature control, including the following steps:

[0042] S1. Culture medium preparation and sterilization:

[0043] (1) Basic fermentation medium: Weigh 10 g of glucose, 0.585 g of magnesium sulfate heptahydrate, 4 g of ammonium sulfate, 1.7 g of citric acid, and bring the volume to 200 mL with deionized water. Sterilize at 115℃ for 30 min. Separately weigh 20 g of tryptone, 3.5 g of potassium dihydrogen phosphate, 5 g of yeast extract, 5.35 g of dipotassium hydrogen phosphate, 1 mL of polyether defoamer CS4809, and bring the volume to 800 mL with deionized water. Sterilize at 121℃ for 30 min. After the two groups of mediums have cooled to room temperature, mix them under aseptic conditions to obtain a basic fermentation medium with a total volume of 1 L, which is suitable for a 3 L fermenter system.

[0044] (2) Feed culture medium: Weigh 150 g of glucose, 0.585 g of magnesium sulfate heptahydrate, 20 g of tryptone, 10 g of yeast extract, 10 g of L-methionine, and make up to 1 L with deionized water. Sterilize at 115℃ for 30 min and cool to room temperature for later use.

[0045] (3) Trace element mother liquor: Weigh out 70 mg of copper sulfate pentahydrate, 0.5 mg of manganese sulfate, 2 mg of calcium chloride, 10 mg of ferrous sulfate heptahydrate, and 2.25 mg of zinc sulfate heptahydrate, respectively, and make up to 10 mL with deionized water. Filter the solution through a 0.22 μm sterile filter head for sterilization, dispense and store. Add 1 mL of this trace element mother liquor per liter of fermentation system.

[0046] (4) 1 M IPTG induction solution: Weigh 2.83 g of IPTG, dissolve it in 10 mL of sterile deionized water, filter it through a 0.45 μm filter membrane for sterilization, dispense it into aliquots and store it at -20℃ for later use.

[0047] S2. Preparation of secondary seed culture: BL21-AC6 recombinant engineered bacteria were inoculated into 10 mL LB liquid medium and cultured with shaking at 37℃ and 220 rpm for 8 h to obtain primary seed culture; 400 μL of primary seed culture was transferred into 40 mL LB liquid medium and cultured with shaking at 37℃ and 220 rpm for 12 h to obtain stable secondary seed culture for fermenter inoculation;

[0048] S3. Rapid cell growth stage (37℃): 1 L of basic fermentation medium was added to a 3 L fermenter, and pH, dissolved oxygen, and temperature electrodes were connected in sequence; under aseptic conditions, 1 mL of trace element stock solution was added, and the pH of the fermentation system was adjusted to 6.8-6.9 using 25% ammonia water; the mixture was pre-stirred at 100 rpm for 1 h under aeration, and then the stirring speed was adjusted to 1200 rpm, and the dissolved oxygen was calibrated to 100%.

[0049] Add 50 mL of secondary seed culture to the fermenter, set the fermentation temperature to 37℃, and maintain the dissolved oxygen in the fermentation system at a stable 30% by controlling the stirring speed in series. Incubate at this temperature for 6 h until the bacterial OD of the culture reaches zero. 600 By 20, high-density bacterial biomass accumulation was completed;

[0050] S4. Low-temperature functional enzyme induction stage (16℃): OD of the bacterial culture 600 When the concentration reached 20, IPTG with a final concentration of 1 mM was added to induce expression; at the same time, constant feed was started with a feed flow rate of 10 mL / h, 25% ammonia was used to maintain the pH of the system at 7.0, the fermentation temperature was adjusted to 16℃, and constant temperature induction culture was carried out for 18 h.

[0051] S5. Product synthesis stage: After the induction at 16℃, the temperature of the fermentation system is adjusted to 30℃ and cultured for 18h;

[0052] S6. Sampling and Product Detection: After fermentation, samples were taken to detect vanillic acid content. The bacterial culture OD was measured after the samples were diluted 100 times. 600 Take 200 mL of fermentation broth, add four times the volume of methanol, sonicate for 1 h, filter, and then use high performance liquid chromatography (HPLC) to detect the product content.

[0053] Example 2

[0054] The difference between this embodiment and Embodiment 1 is that 10 g of glycerol is added to the basic fermentation medium in this embodiment, while the other steps are the same as in Embodiment 1.

[0055] Example 3

[0056] The difference between this embodiment and Embodiment 1 is that the amount of glucose added to the feed culture medium in this embodiment is 225g.

[0057] Example 4

[0058] The difference between this embodiment and embodiment 1 is that the induction time in S4 of this embodiment is 16 hours, while the other steps are the same as in embodiment 1.

[0059] To further illustrate the technical effects of the present invention, a comparative example is also provided, as follows:

[0060] Comparative Example 1

[0061] The difference between this comparative example and Example 1 is that the fermentation temperature in this comparative example S5 is 20°C, while the other steps are the same as in Example 1.

[0062] Comparative Example 2

[0063] The difference between this comparative example and Example 1 is that the fermentation temperature in this comparative example S5 is 25°C, while the other steps are the same as in Example 1.

[0064] Example 5

[0065] This embodiment describes a method for synthesizing vanillin using BL21-AC6 recombinant engineered bacteria through temperature control. The specific steps are the same as in Example 1, except that the fermentation temperature in S5 is 25°C.

[0066] Example 6

[0067] The difference between this embodiment and embodiment 5 is that the inoculation amount of the secondary seed liquid in S2 of this embodiment is 8%, while the remaining steps are the same as in embodiment 5.

[0068] Example 7

[0069] The difference between this embodiment and embodiment 5 is that the final concentration of IPTG in S4 of this embodiment is 0.5 mM, while the other steps are the same as in embodiment 5.

[0070] Comparative Example 3

[0071] The difference between this comparative example and Example 5 is that the fermentation temperature in this comparative example S5 is 20°C, while the other steps are the same as in Example 5.

[0072] Comparative Example 4

[0073] The difference between this comparative example and Example 5 is that the induction temperature in S4 of this comparative example is 25°C, while the other steps are the same as in Example 5.

[0074] Table 1. Liquid chromatography detection results of Examples 1-4 and Comparative Examples 1-2

[0075]

[0076] Table 2. Liquid chromatography detection results of Examples 4- and Comparative Examples 3-4

[0077]

[0078] This invention achieves the controllable synthesis of vanillic acid and vanillin by regulating the cell growth temperature and protein induction temperature. As shown in Table 1, the three-stage temperature-controlled fermentation process of this invention can stably achieve a vanillic acid fermentation yield of 2721.3~3095.5 mg / L and a vanillin fermentation yield of 594.6~640.9 mg / L. The process has good repeatability, is simple to operate, and has high production flexibility, showing good prospects for industrial application.

[0079] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A method for synthesizing vanillin and vanillic acid using temperature-controlled engineered Escherichia coli, characterized in that, Includes the following steps: S1. The BL21-AC6 recombinant engineered bacteria were inoculated into LB liquid medium and cultured with shaking to obtain the primary seed culture; The primary seed culture was transferred to LB liquid medium and cultured with shaking to obtain the secondary seed culture. S2. Add trace element stock solution to the basal fermentation medium, adjust the pH of the system to 6.8-6.9 with ammonia, stir under aeration, inoculate the secondary seed culture into the system, and incubate at 30-37℃ and dissolved oxygen 20-30% for 4-6 h until the system OD reaches its limit. 600 It is 20-25; S3. Add IPTG to induce expression, and add feed medium. Maintain the pH of the system at 7.0 with ammonia water, and induce culture at 16-20℃ for 12-18 h. S4. After induction, raise the temperature and continue fermentation for 14-30 h to produce vanillin or vanillic acid; the temperature for producing vanillin is 24-26℃; the temperature for producing vanillic acid is 29-31℃.

2. The method according to claim 1, characterized in that: In step S1, the BL21-AC6 recombinant engineered bacteria uses *Escherichia coli* BL21(DE3) as the starting strain and overexpresses the following genes: dehydroshikimate dehydratase (DSD), 3-O-methyltransferase (OMT), carboxylic acid reductase (CAR), vanillyl alcohol oxidase (VaoA), 5'-methyladenosine nucleosidase (Mtn), S-ribose homocysteine ​​lyase (LuxS), and citrate synthase mutant gene (gltA). R119L .

3. The method according to claim 2, characterized in that: The dehydroshikimate dehydratase gene DSD, 3-O-methyltransferase gene OMT, carboxylic acid reductase gene CAR, vanillyl alcohol oxidase gene VaoA, 5'-methylthioadenosine nucleosidase gene Mtn, S-ribosomalcysteine ​​lyase gene LuxS, and citrate synthase mutant gene gltA are mentioned. R119L The nucleotide sequences are shown in SEQ ID NO.1~7 respectively.

4. The method according to claim 1, characterized in that: The inoculation amount of the primary seed solution in step S1 is 1-5%.

5. The method according to claim 1, characterized in that, The basic fermentation medium in step S2 comprises the following components: glucose 10-20 g / L, magnesium sulfate 0.125-0.585 g / L, ammonium sulfate 1-4 g / L, citric acid 1-1.7 g / L, tryptone 10-30 g / L, potassium dihydrogen phosphate 3.5-10 g / L, yeast extract 5-15 g / L, dipotassium hydrogen phosphate 3.35-5.35 g / L, and 0.1-0.5% polyether defoamer, with water as the solvent; optionally, the basic fermentation medium also contains 8-15 g of glycerol.

6. The method according to claim 1, characterized in that: The amount of trace element mother liquor added in step S2 is 0.1-0.5%; the trace element mother liquor includes the following components: copper sulfate 3-7 mg / mL, manganese sulfate 0.05-0.1 mg / mL, calcium chloride 0.2 mg / mL, ferrous sulfate 1 mg / mL, zinc sulfate 0.010-0.225 mg / mL, and water as the solvent.

7. The method according to claim 1, characterized in that: The inoculation amount of the secondary seed liquid in step S2 is 5-10%.

8. The method according to claim 1, characterized in that: The final concentration of IPTG in step S3 is 0.5-1 mM.

9. The method according to claim 1, characterized in that, The feeding medium in step S3 comprises the following components: glucose 150-225 g / L, magnesium sulfate 0.125-0.585 g / L, tryptone 10-30 g / L, yeast extract 5-15 g / L, L-methionine 10-15 g / L, and water as the solvent; the flow rate of the feeding medium is 6-24 mL / h.

10. The use of the method according to any one of claims 1 to 9 in the production of vanillin or vanillic acid.

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