CN119685184A - Construction and application of an engineered strain of Saccharomyces cerevisiae with high vanillin production - Google Patents

Abstract

The invention discloses the construction and application of an engineering strain of Saccharomyces cerevisiae for synthesizing vanillin using ferulic acid as a substrate, and belongs to the field of genetic engineering technology. The invention provides a recombinant Saccharomyces cerevisiae, wherein a series of genes for redox vanillin to generate vanillic acid and vanillyl alcohol are knocked out, and feruloyl-CoA synthetase FCS and para-hydroxycinnamoyl-CoA hydratase ECH obtained by screening from the extreme environment macrogene library of hot springs in western Sichuan are overexpressed. The engineering strain VAN002 with high vanillin production obtained by the invention has an accumulation of 16.8 g/L of vanillin, and a molar conversion rate of catalyzing ferulic acid to synthesize vanillin reaches 93%, which is the highest level reported for the synthesis of vanillin by Saccharomyces cerevisiae.

Description

Construction and application of saccharomyces cerevisiae engineering strain for high-yield vanillin

Technical Field

The invention relates to construction and application of a saccharomyces cerevisiae engineering strain for high-yield vanillin, and belongs to the technical field of genetic engineering.

Background

Vanillin, which is known by the chemical name 3-methoxy-4-hydroxybenzaldehyde (3-methoxy-4-hydrobenzaldehyde), has a molecular formula of C ₈H₈O₃ and a relative molecular mass of 152.12. Vanillin, one of the derivatives of ferulic acid, also known as vanillin, is a widely used spice known for its intense vanilla taste. It was originally extracted from the pods of orchids, particularly vanilla (Vanilla planifolia) in mexico. After the pods are ripe, the vanilla extract containing vanillin can be extracted by fermentation and drying. Due to the complex planting and extraction processes of vanilla, elaborate manual operations and suitable climatic conditions are required, which lead to high costs of the natural vanilla extract. Vanillin provides a sweet and warm vanilla flavor to a variety of foods and beverages, such as ice cream, cake, candy, and soft drinks. It is also used as a fragrance in cosmetics, perfumes, and as a bitter-masking flavoring in certain pharmaceuticals. In addition, vanillin is also used in plastics and rubber articles to impart specific odors. The wide use of vanillin and the unique aroma make it a key ingredient of the global fragrance market.

At present, vanillin is produced mainly by three means, extraction from natural plants, chemical synthesis and bioconversion. The vanillin extracted naturally is pure and safe, but is limited by low yield, long production period, limited resources and high cost, and cannot meet the market demands due to the limitation of climate and land resources. Chemical synthesis, while low cost and fast, can be environmentally friendly and, due to lack of substrate selectivity, reduces production efficiency and increases downstream processing costs. In contrast, bioconversion, particularly methods utilizing microbial cells to convert different substrates, has become a promising trend in recent years due to their mild reaction conditions, high specificity and efficient catalytic capabilities.

In the field of biosynthesis, vanillin generally has eugenol, isoeugenol, lignin or ferulic acid as precursor substances. Ferulic acid is considered to be the most potential substrate for vanillin production because of its wide and abundant sources and low toxicity to microorganisms. The existing genetic engineering bacteria using ferulic acid as a substrate comprise escherichia coli, pichia pastoris and the like, however, the genetic engineering bacteria have low enzyme activity and poor stability so as to influence the yield and the production efficiency of target products, in addition, the high concentration of ferulic acid can interfere the normal physiological functions of cells, and the accumulation of vanillin can feedback to inhibit the activity of related enzymes, so that the reaction is difficult to continuously and efficiently carry out, and the yield of vanillin is limited.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a method for synthesizing vanillin by taking ferulic acid as a substrate, which selects saccharomyces cerevisiae as an expression metabolism host, screens feruloyl-CoA synthase with catalytic activity remarkably higher than that of other species sources reported at present in the heterologous expression of the saccharomyces cerevisiae, coexpresses key genes in a vanillin synthesis pathway, realizes the efficient synthesis of ferulic acid to vanillin by whole-cell catalysis, and meets the market demand for vanillin.

The first technical scheme provided by the invention is a recombinant saccharomyces cerevisiae which is characterized in that feruloyl-coenzyme A synthetase FCS and p-hydroxycinnamoyl-coenzyme A hydratase ECH which are derived from a Sichuan hot spring metagenome library are simultaneously overexpressed in chassis strains.

In certain embodiments, the feruloyl-coa synthetase FCS has an amino acid sequence shown in SEQ ID No.1 and the hydroxycinnamoyl-coa hydratase ECH has an amino acid sequence shown in SEQ ID No. 2.

In certain embodiments, the nucleotide sequence encoding feruloyl-coa synthetase gene Fcs is shown in SEQ ID No.3 and the nucleotide sequence of hydroxycinnamoyl-coa hydratase gene Ech is shown in SEQ ID No. 4.

In certain embodiments, the chassis strain is saccharomyces cerevisiae IMX581 with the adh6(Gene ID:855368)、gre2(Gene ID:854014)、gcy1(Gene ID:854287),ypr1(Gene ID:851974),aad3(Gene ID:850471) gene of saccharomyces cerevisiae itself knocked out.

The second technical scheme provided by the invention is a construction method of recombinant saccharomyces cerevisiae, wherein the method is to knock out adh6, gre2, gcy1, ypr1 and aad3 genes of an original strain, and over-express feruloyl coenzyme A synthase FCS and p-hydroxycinnamoyl coenzyme A hydratase ECH which are derived from a Sichuan hot spring metagenome library on the original strain.

In certain embodiments, the feruloyl-coa synthetase FCS has an amino acid sequence shown in SEQ ID No.1 and the hydroxycinnamoyl-coa hydratase ECH has an amino acid sequence shown in SEQ ID No. 2.

In certain embodiments, the nucleotide sequence encoding feruloyl-coa synthetase gene Fcs is shown in SEQ ID No.3 and the nucleotide sequence of hydroxycinnamoyl-coa hydratase gene Ech is shown in SEQ ID No. 4.

In certain embodiments, the method comprises the steps of:

(1) Taking IMX581 strain as an original strain, knocking out adh6, gre2, gcy1, ypr1 and aad3 genes, and obtaining a chassis strain VAN001;

(2) And (3) over-expressing the feruloyl-CoA synthetase gene Fcs and the hydroxycinnamoyl-CoA hydratase gene Ech on the chassis strain VAN001 in the step (1) to obtain the recombinant saccharomyces cerevisiae.

In certain embodiments, the Fcs gene and the Ech gene are combined by gene overlap techniques to construct a donor gene expression cassette, which is integrated into the multiple copy site Ty1 transposon of the VAN001 chassis strain to obtain the VAN002 strain.

The third technical scheme provided by the invention is a method for producing vanillin, wherein the method is to convert the recombinant saccharomyces cerevisiae into ferulic acid to synthesize vanillin.

In certain embodiments, the recombinant Saccharomyces cerevisiae seed solution is added to a culture system using glucose as a carbon source, cultured at 30 ℃ and 200rpm for about 24 hours to OD ₆₀₀ about 6, then D-galactose with a final concentration of 2g/L is added as an inducer, 3-6 g/L ferulic acid is added to the reaction system, and the reaction is performed on a 30 ℃ and 200rpm constant temperature shaking table to prepare vanillin.

In certain embodiments, the recombinant s.cerevisiae is subjected to fed-batch fermentation on a 5L fermenter.

In certain embodiments, the fermentation medium is an inorganic salt medium comprising glucose, KH ₂PO₄,(NH₄)₂SO₄,MgSO₄·7H₂ O, trace elements, vitamins, and defoamers.

In some embodiments, the glucose is added in an amount of 20g/L.

In some embodiments, glucose is added at a flow rate of 1g/L/h beginning at the time of the second sudden increase in dissolved oxygen in the culture system.

In some embodiments, the addition amount of the recombinant saccharomyces cerevisiae is 5% -10%.

In some embodiments, the reaction conditions are 30 ℃, the rotation speed of the stirring paddle is 300-700 rpm, the pH of the fermentation liquor is controlled to be 5.0, and the ventilation rate is 1vvm.

The fourth technical scheme provided by the invention is the recombinant saccharomyces cerevisiae described in the first technical scheme, or the method described in the second technical scheme, or the application of the method described in the third technical scheme in the aspect of producing vanillin or vanillin-containing products.

The invention has the following technical effects:

(1) According to the invention, the saccharomyces cerevisiae which takes the genes encoding aldehyde ketone reductase and alcohol dehydrogenase adh6, gre2, gcy1, ypr1 and aad3 as chassis cells is knocked out, so that chassis strain VAN001 capable of stably accumulating vanillin is obtained;

(2) The feruloyl-CoA synthetase FCS and the p-hydroxycinnamayl-CoA hydratase ECH of the Sichuan hot spring metagenome library are heterologously expressed in the engineering strain VAN001, so that the synthesis of vanillin with higher yield is realized;

(3) The engineering strain VAN002 for high yield of vanillin obtained by the invention can reach 7.4g/L of yield of vanillin fermented in a shake flask, fed-batch fermentation is carried out in a 5L fermentation tank, the accumulation of vanillin reaches 16.8g/L after 120h of fermentation, and the method is the highest level of vanillin synthesized by the engineering strain of Saccharomyces cerevisiae reported at present.

Drawings

FIG. 1 is a metabolic scheme of ferulic acid synthesis vanillin.

FIG. 2 shows the chromatograms of vanillin and ferulic acid, wherein A is the liquid chromatogram of the standard product of vanillin and ferulic acid, and B is the liquid chromatogram of the fermentation sample.

FIG. 3 is an evaluation of vanillin stability yield in VAN001 strain.

FIG. 4 is an evaluation of the yield of vanillin catalyzed by the ferulic acid synthesis in shake flasks of the VAN002 strain.

FIG. 5 is an evaluation of vanillin yield from the catalytic ferulic acid synthesis on a VAN002 strain fermenter.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail below with reference to the accompanying drawings 1 to 5.

The pROS plasmid referred to in the examples below, saccharomyces cerevisiae IMX581, which is a laboratory-resident, wherein the pROS plasmid is described in MANS ET AL FEMS YEAST Res.2015Mar;15 (2) pii.fov004.doi 10.1093/femsyr/Fov004.Epub 20151r4, saccharomyces cerevisiae IMX581 (genotype: MATA Ura3-52 can1Δ:: cas9-natNT2 TRP1 LEU2 HIS 3) genome with integrated Cas9 protein expression cassettes in the CRISPR-Cas9 system, and with Ura auxotroph selection markers, is described in ：CRISPR/Cas9:a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae. in the examples below in the following:

Seed culture medium (YPD) (1L) comprises yeast extract 10g, tryptone 20g, and glucose 20g.

Shake flask fermentation medium (SC-U) (1L) YNB 6.7g,DO Supplement-Ura 1.29g, glucose 0.5g/L

The fermentation medium of the fermenter comprises (1L) 20g of glucose, (NH ₄)SO₄ 7.5g,KH₂PO₄14.4 g,MgSO₄7H₂ O0.5 g, trace element (ZnSO₄·7H₂O(4.5g·L^-1),MnCl₂·2H₂O(1.0g·L^-1),CuSO₄·6H₂O(0.3g·L^-1),CoCl₂·6H₂O(0.3g·L^-1),Na₂MoO₄·2H₂O(0.4g·L^-1),CaCl₂·2H₂O(4.5g·L^-1),FeSO₄·7H₂O(3.0g·L^-1),Na₂EDTA(19g·L^-1),H₃BO₃(1.0g·L^-1),KI(0.1g·L^-1)) is 500 times mother solution, the addition amount of each L is 2mL, vitamin (biotin (0.05 g.L ^-1), calcium pantothenate (1 g.L ^-1), nicotinic acid (1 g.L ^-1), inositol (25 g.L ^-1), thiamine hydrochloride (1 g.L ^-1), pyridoxal hydrochloride (1 g.L ^-1), p-aminobenzoic acid (0.2 g.L ^-1)) is 1000 times mother solution, the addition amount of each L is 1mL, vitamin solution is 12mL, and defoamer.

The strain information referred to in the following examples is shown in Table 1 below:

TABLE 1 major plasmids and Strain information related to the invention

The primer sequences involved in the following examples are shown in tables 2 to 3:

TABLE 2 primers used in example 1

TABLE 3 primers used in example 2

The detection methods of vanillin and ferulic acid involved in the following examples are as follows:

1. sample preparation fermentation broth samples were centrifuged at 12000rpm for 1min, and the supernatant was diluted with 20% aqueous methanol by an appropriate factor and filtered through a 0.22 μm filter.

2. The liquid phase conditions were a chromatographic column InfinityLab Poroshell HPH-C18, 4.6X105 mm,2.7-Micron, mobile phase A ddH ₂ O (0.1% acetic acid), mobile phase B methanol. The elution process comprises 0-6 min,20% of B, 6-10 min, 20-40% of B, 10-15 min,40% of B, 15-16 min, 40-20% of B, 16-20 min and 20% of B. The flow rate is 0.7mL/min, the column temperature is 30 ℃, and the sample injection volume is 10 mu L. The detector is a diode array detector, and the detection wavelength is 280nm (vanillin) and 323nm (ferulic acid). The target substance is determined by comparison with the retention time and the spectrogram of the standard substance, and the measured substance is quantitatively analyzed according to the peak area.

Example 1 construction of Chassis cells, namely Van001, a Van-stable strain of vanillin

The method comprises the following specific steps:

(1) The genome of the saccharomyces cerevisiae strain is used as a template, primers ADH6-UF and ADH6-UR and ADH6-DF and ADH6-DR are used for respectively amplifying to obtain DNA fragments of 500bp at the upstream and downstream of the ADH6 gene, and then the two fragments are connected by using overlap PCR to be used as Donor DNA for gene editing, and the construction method of the rest Donor DNA is the same as the construction method;

(2) The pROS original plasmid is used as a template, and the primer ADH6-gRNA is used for PCR amplification to obtain the pROS-ADH 6 plasmid. The resulting Donor DNA and pROS10-adh6 plasmid were co-transformed into a competent strain of Saccharomyces cerevisiae IMX581 expressing Cas9 protein by chemical transformation. The single colony obtained was identified by PCR using the primer ADH6-JD-F, ADH-JD-R, and analyzed by combining the PCR result with the sequencing comparison to obtain the strain from which the ADH6 gene was knocked out.

Referring to the above procedure, gre2, gcy, ypr, aad3 genes were deleted by the same gene editing method, thereby obtaining a chassis strain designated as VAN001.

EXAMPLE 2 Chassis VAN001 Van consumption evaluation

Van001 strain obtained in example 1 was inoculated into DelFT medium at an inoculum size of 1%, cultured overnight at 30℃and 200rpm, added with 10g/L of vanillin solution, shake-flask cultured for 48 hours, sampled at 4 hours, 8 hours, 24 hours and 48 hours, respectively, and the vanillin residue and the synthesis of vanillin and vanillyl alcohol were measured by HPLC detection.

The results showed that the control strain (IMX 581 wild strain) showed a gradual decrease in vanillin residue during the 48h cultivation with vanillin addition, an increase in vanillic acid and vanillyl alcohol accumulation, a 1.5g/L vanillyl alcohol cultivation with a 3.8g/L vanillic acid and a stable vanillin content with only small amounts of vanillic acid and vanillyl alcohol accumulation (FIG. 3).

The VAN001 engineering strain is taken as a chassis cell, and the reaction of vanillin oxidation reduction to form vanillyl alcohol and vanillic acid is weakened due to the knockout of various oxidation-reduction enzymes, so that vanillin can be stably accumulated, and the chassis strain has stronger advantages as a production strain.

EXAMPLE 3 construction of engineering Strain VAN002

The method comprises the following specific steps:

(1) The genome of Saccharomyces cerevisiae strain is used as template, the primers Ty1-37-up-F and Ty1-37-up-R and Ty1-37-dn-F and Ty1-37-dn-R are used for respectively amplifying to obtain DNA fragments at the upstream and downstream of Ty1 transposon, the primers FCS-F and FCS-R are used for respectively amplifying to obtain FCS and ECH sequence fragments (the nucleotide sequence for encoding the feruloyl-CoA synthase FCS is shown as SEQ ID NO.3, the nucleotide sequence for encoding the p-hydroxycinnamoyl-CoA hydratase ECH is shown as SEQ ID NO. 4), and then overlapping PCR is used for connecting a plurality of fragments to be used as Donor DNA for gene editing;

(2) The pROS original plasmid is used as a template, and primer Ty1-gRNA is used for PCR amplification to obtain pROS-Ty 1 plasmid. The resulting Donor DNA was co-transformed with pROS-Ty 1 plasmid into a VAN001 competent strain by chemical transformation. The single colony obtained was identified by PCR using the primers ty1-up-F and ty1-up-R, and analyzed by combining sequencing comparison according to the PCR result, the strain VAN002 over-expressing the multicopy fcs and ech genes was obtained.

The PCR reaction system and PCR amplification conditions are shown in tables 4 and 5:

TABLE 4 PCR reaction System

TABLE 5 PCR amplification conditions

Note that a, the annealing temperature is determined according to the Tm value of the primer, and b, the extension time is determined according to the length of the target product.

EXAMPLE 4 VaN002 Strain catalyzes the evaluation of conversion efficiency of ferulic acid into vanillin

The method comprises the following specific steps:

(1) The engineering strain VAN002 prepared in example 3 was transferred to YPD liquid medium and cultured at 30℃and 200rpm for 24 hours to prepare seed solution.

(2) The seed solution obtained by the preparation was transferred to a shake flask containing 100mL of SD-U liquid medium at an inoculum size of 1% (v/v), cultured at 30℃and 200rpm for about 24 hours to OD ₆₀₀ about 6, followed by adding D-galactose as an inducer at a final concentration of 2g/L and ferulic acid at a final concentration of 3g/L to the reaction system, and reacted on a 30℃and 200rpm constant temperature shaker, and when the ferulic acid was consumed, the addition of ferulic acid solution at a final concentration of 3g/L was continued for about 24 hours. Samples were taken every 4 hours, and vanillin production and ferulic acid residue were measured, and the results are shown in fig. 4.

The result shows that after 3g/L of ferulic acid is added to the reaction system for the first time, only 0.4g/L of ferulic acid residue can be detected after 24 hours, 2.15g/L of vanillin is produced, the molar conversion rate reaches 91.4%, the same content of ferulic acid is added again, after the reaction is continued for 24 hours, 0.6g/L of ferulic acid remains, and finally 3.92g/L of vanillin is produced, and the molar conversion rate reaches 92.6%.

Example 5 fed batch fermentation validation of Vanillin

In view of the better experimental results obtained in the shake flask experiments of example 4, using ferulic acid as a substrate to synthesize vanillin, the potential of vanillin to achieve amplified production was evaluated in this embodiment, and fed-batch fermentation was performed in a 5L fermentor using VAN002 strain, as follows:

(1) Inoculating the engineering bacteria VAN002 strain prepared in the example 3 into YPD culture medium, culturing for 24 hours at 30 ℃ and 200rpm to prepare primary seed liquid, inoculating the primary seed liquid into 250mL of YPD culture medium according to the inoculum size of 1% (v/v), and culturing for 48 hours at 30 ℃ and 200rpm to obtain secondary seed liquid;

(2) Inoculating the prepared secondary seed solution into a 5L fermentation tank containing an inorganic salt fermentation medium according to an inoculum size of 10% (v/v), controlling the fermentation initial temperature to be 30 ℃ before the fermentation starts, setting the fermentation initial rotating speed to be 300rpm, controlling the pH of the fermentation process to be 5.0 by using 10% ammonia water, setting dissolved oxygen to be 100%, increasing ventilation to 1vvm to 3vvm when the dissolved oxygen is reduced to 30%, and then controlling the dissolved oxygen to be more than 15% by stirring. In the culture process, two sudden increases of dissolved oxygen are observed, the first time is that glucose is consumed, the second time is that ethanol produced by fermentation is consumed, and at the moment, glucose is fed at a speed of 6 g/L/h. The feeding rate is changed once per hour until the dissolved oxygen can not be maintained by more than 15% after stirring is regulated to the maximum, and then the feeding rate is reduced to 1g/L/h. Sampling and measuring the thallus density of the fermentation liquid, when the OD ₆₀₀ reaches 100 (about 60 h), properly reducing the sugar supplementing speed and the stirring rotating speed due to the reduced activity of the strain, adding D-galactose with the final concentration of 2g/L into a fermentation tank, inducing the expression of protein, starting feeding ferulic acid when the fermentation is about 68h, maintaining the flow acceleration at 1-3 g/L/h, detecting the residue of the ferulic acid and the generation condition of vanillin by a liquid phase, and monitoring the synthesis condition of side products vanillic acid and vanillyl alcohol.

The result shows that along with the increase of the cell density, the flow acceleration of the ferulic acid at the beginning of the reaction is maintained at 3g/L/h, and the flow acceleration is adjusted to be 1-3 g/L/h according to the residual condition of the ferulic acid at the time of sampling during the reaction. Finally, after 120h fermentation, the yield of vanillin reaches 16.8g/L (figure 5), the total added amount of ferulic acid is 75g, and the molar conversion rate is 93%.

Comparative example 1

The specific embodiment is the same as in example 3 except that ECH is replaced with Actinobacteria-derived gene obtained by screening from the extreme link sample gene library, and fermentation is performed according to the method of example 4, and the result shows that the vanillin yield is 1.86g/L and the molar conversion is 63.9%.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

1. A recombinant saccharomyces cerevisiae is characterized in that feruloyl-coa synthetase FCS and p-hydroxycinnamayl-coa hydratase ECH which are derived from a Sichuan hot spring metagenome library are simultaneously overexpressed in a chassis strain, the amino acid sequence of the feruloyl-coa synthetase FCS is shown as SEQ ID No.1, and the amino acid sequence of the hydroxycinnamayl-coa hydratase ECH is shown as SEQ ID No. 2.

2. The recombinant s.cerevisiae according to claim 1, wherein the chassis strain is s.cerevisiae IMX581 in which the adh6, gre2, gcy1, ypr1, aad3 genes of s.cerevisiae itself are knocked out.

3. The recombinant s.cerevisiae according to claim 2, wherein the adh6, gre2, gcy1, ypr, the aad3 genes have Gene IDs 855368, 854014, 854287, 851974, 850471, respectively.

4. A construction method of recombinant saccharomyces cerevisiae is characterized in that adh6, gre2, gcy1, ypr1 and aad3 genes of an original strain are knocked out, feruloyl-coa synthetase FCS and p-hydroxycinnamoyl-coa hydratase ECH derived from a Sichuan hot spring metagenome library are overexpressed on the original strain, the amino acid sequence of the feruloyl-coa synthetase FCS is shown as SEQ ID NO.1, and the amino acid sequence of the hydroxycinnamoyl-coa hydratase ECH is shown as SEQ ID NO. 2.

5. The method according to claim 4, characterized in that it comprises the steps of:

(1) Taking IMX581 strain as an original strain, knocking out adh6, gre2, gcy1, ypr1 and aad3 genes, and obtaining a chassis strain VAN001;

(2) And (3) over-expressing the feruloyl-CoA synthetase gene Fcs and the hydroxycinnamoyl-CoA hydratase gene Ech on the chassis strain VAN001 in the step (1) to obtain the recombinant saccharomyces cerevisiae.

6. The method according to claim 5, wherein the Fcs gene and the Ech gene are combined to construct a donor gene expression cassette by gene overlapping technology, and the donor gene expression cassette is integrated into a multiple copy site Ty1 transposon of the VAN001 chassis strain to obtain the VAN002 strain.

7. A method for producing vanillin, characterized in that the method is to convert ferulic acid into vanillin by using the recombinant saccharomyces cerevisiae according to any one of claims 1-3.

8. The method according to claim 7, wherein the recombinant saccharomyces cerevisiae seed solution according to any one of claims 1 to 3 is added into a culture system using glucose as a carbon source for culture, D-galactose is used as an inducer, and 3-6g/L ferulic acid is added into the reaction system for conversion reaction, so as to prepare vanillin.

9. The method according to claim 7, wherein after adding the recombinant saccharomyces cerevisiae seed solution according to any one of claims 1 to 3 to a culture system using glucose as a carbon source, starting feeding glucose when the dissolved oxygen of the culture system suddenly increases for the second time, and feeding ferulic acid at a speed of 1 to 3g/L/h by using D-galactose as an inducer to prepare vanillin.

10. Use of the recombinant saccharomyces cerevisiae according to any of claims 1-3, or the method according to any of claims 4-6, or the method according to any of claims 7-9 for the production of vanillin or vanillin-containing products.