Genetically engineered bacterium for producing 3-methoxy-4-hydroxybenzaldehyde as well as construction method and application of genetically engineered bacterium

Abstract

The invention discloses a genetically engineered bacterium for producing 3-methoxy-4-hydroxybenzaldehyde as well as a construction method and application of the genetically engineered bacterium, and belongs to the technical field of genetic engineering. According to the genetically engineered bacterium, a vanillin dehydrogenase vdh gene is knocked out from amycolatopsis, and exogenous isocitrate lyase aceA and malic acid synthetase aceB genes are expressed. Through the genetically engineered bacterium, an efficient 3-methoxy-4-hydroxybenzaldehyde synthesis route is constructed, and the 3-methoxy-4-hydroxybenzaldehyde is produced by using the strain, so that not only can the yield of the 3-methoxy-4-hydroxybenzaldehyde be increased, but also the fermentation period can be shortened. Therefore, the genetically engineered bacterium and the production method of the 3-methoxy-4-hydroxybenzaldehyde, provided by the invention, have good industrial application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular, to a genetically engineered bacterium for producing 3-methoxy-4-hydroxybenzaldehyde, a construction method thereof, and an application thereof. Background Art

[0002] 3-Methoxy-4-hydroxybenzaldehyde, also known as vanillin, as one of the most important aromatic compounds in food and personal care products, is widely used in the fields of food, medicine, agriculture, cosmetics, etc. It is estimated that the annual consumption exceeds 12,000 tons, while natural 3-methoxy-4-hydroxybenzaldehyde accounts for less than 1% of the total demand.

[0003] At present, the production methods of 3-methoxy-4-hydroxybenzaldehyde on the market mainly include three categories: plant extraction, chemical synthesis, and microbial fermentation. According to current US and European legislation, chemically synthesized 3-methoxy-4-hydroxybenzaldehyde cannot be called "natural" 3-methoxy-4-hydroxybenzaldehyde, while 3-methoxy-4-hydroxybenzaldehyde produced by biotechnology can be considered natural. Therefore, the production of 3-methoxy-4-hydroxybenzaldehyde by microbial conversion of natural substrates has attracted much attention.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The object of the present invention is to provide a genetically engineered bacterium for producing 3-methoxy-4-hydroxybenzaldehyde, a construction method thereof, and an application thereof. Applying the genetically engineered bacterium of the present invention to the production of 3-methoxy-4-hydroxybenzaldehyde can increase the yield of 3-methoxy-4-hydroxybenzaldehyde and shorten the fermentation cycle.

[0006] Since coenzyme A can be generated through the TCA cycle and the glyoxylate shunt, and the influx of acetyl coenzyme A into the glyoxylate shunt is twice that of the TCA cycle, acetyl coenzyme A can be effectively consumed and coenzyme A can be generated. By overexpressing isocitrate lyase (aceA) (cleaving isocitrate) and malate synthase (aceB) (generating coenzyme A) in the glyoxylate shunt, the influx of isocitrate into the glyoxylate bypass enables more effective conversion of acetyl coenzyme A into coenzyme A. Based on the above content, the present invention proposes a method for strengthening the 3-methoxy-4-hydroxybenzaldehyde synthesis pathway by increasing the amount of coenzyme A.

[0007] To implement this scheme, the present invention first provides a genetically engineered bacterium for producing 3-methoxy-4-hydroxybenzaldehyde. This genetically engineered bacterium uses Amycolatopsis as the starting strain, knocks out the vanillin dehydrogenase (vdh) gene of this strain, and introduces exogenous isocitrate lyase (aceA) and malate synthase (aceB) genes.

[0008] Among them, the strain number of Amycolatopsis is ATCC 39116.

[0009] In some embodiments, aceA is derived from Kineosporia babensis and is denoted as KbaceA; aceB is derived from Micromonospora polyrhachis and is denoted as MpaceB. According to the host preference, the present invention performs codon optimization on the nucleotide sequences of KbaceA and MpaceB, and the optimized nucleotide sequences of KbaceA and MpaceB are shown in SEQ ID NO: 1-2.

[0010] For the construction of the above-mentioned genetically engineered bacteria, the present invention provides the following method:

[0011] (1) Construction of a knockout suicide vector containing the upstream and downstream homology arms of the vdh gene and apramycin resistance

[0012] An apramycin resistance gene (Apra) was synthesized, and its gene sequence was shown in SEQ ID NO: 3. Apra-f / Apra-r were used as primers to amplify the Apra sequence by PCR; vdh-up-f / vdh-up-d were used as primers and the genome of Amycolatopsis was used as a template to amplify the vdh-up gene sequence by PCR; vdh-down-f / vdh-down-d were used as primers and the genome of Amycolatopsis was used as a template to amplify the vdh-down gene sequence by PCR;

[0013] The above three fragments were used as templates, and vdh-up-f / vdh-down-r primers were used to amplify the vdh knockout sequence by overlap PCR, which was then cloned into the pUC vector to obtain the knockout suicide vector pUC-vdh-Apra (abbreviated as pUC-V) with upstream and downstream homology arms of the vdh gene and apramycin resistance.

[0014] (2) Construction of a vdh knockout suicide vector containing a strong promoter permE and expressing isocitrate lyase KbaceA and malate synthase MpaceB

[0015] Synthesize the strong promoter permE, whose gene sequence is shown in SEQ ID NO: 4. Using the permE-f1 / permE-r1 primers, PCR amplify the permE-1 sequence; using the permE-f2 / permE-r2 primers, PCR amplify the permE-2 sequence; using KbaceA-f / KbaceA-d as primers and the optimized isocitrate lyase KbaceA from Kineosporia babensis as a template, PCR amplify the KbaceA gene sequence; using MpaceB-f / MpaceB-d as primers and the optimized malate synthase MpaceB from Micromonospora polyrhachis as a template, PCR amplify the MpaceB gene sequence;

[0016] Using the above 4 fragments as templates and the permE-f1 / MpaceB-d primers, perform overlap PCR amplification to obtain the permE-KbaceA-permE-MpaceB expression sequence;

[0017] Using the apra-r1 / vdh-down-f1 primers and the suicide vector pUC-V constructed in (1) as a template, PCR amplify to obtain a linearized pUC-V vector fragment, and perform seamless cloning with the permE-KbaceA-permE-MpaceB expression sequence to obtain the vdh knockout suicide vector pUC-vdh-Apra-permE-KbaceA-permE-MpaceB (abbreviated as pUC-VAB) containing the strong promoter permE, expressing isocitrate lyase KbaceA, and malate synthase MpaceB;

[0018] (3) Construct recombinant Amycolatopsis

[0019] Using the method of electrotransformation, transform the suicide vector obtained in step (2) into Amycolatopsis. Screen positive strains according to apramycin resistance. The obtained strains are verified correctly by PCR to obtain Amycolatopsis strains that knockout vdh and express KbaceA and MpaceB at the same time.

[0020] In some embodiments, the apramycin resistance screening is to coat the obtained transformed Amycolatopsis on Bennet solid medium containing 50 μg / mL apramycin and culture it at 30 °C for 5 days to grow single colonies. The strains verified correctly by PCR are the positive strains of correct homologous recombination.

[0021] The primer information involved in the above construction method is shown in Table 1:

[0022] Table 1 Primer sequence information

[0023]

[0024]

[0025] Furthermore, on this basis, the present invention also provides a whole-cell catalyst containing the above-mentioned genetically engineered bacterium.

[0026] Knocking out vdh in the above-mentioned genetically engineered bacterium reduces the metabolism of 3-methoxy-4-hydroxybenzaldehyde. KbaceA derived from Kineosporia babensis and MpaceB derived from Micromonospora polyrhachis can convert acetyl-CoA into coenzyme A more effectively, strengthening the 3-methoxy-4-hydroxybenzaldehyde synthesis pathway by increasing the amount of coenzyme A. Using the above-mentioned genetically engineered bacterium as a whole-cell catalyst can obtain 3-methoxy-4-hydroxybenzaldehyde with a higher yield.

[0027] The present invention also provides the use of the above-mentioned genetically engineered bacterium or whole-cell catalyst in the production of 3-methoxy-4-hydroxybenzaldehyde. Based on this, the present invention can use the genetically engineered bacterium or whole-cell catalyst to produce 3-methoxy-4-hydroxybenzaldehyde, and the method is as follows:

[0028] Inoculate the above-mentioned genetically engineered bacterium into a culture medium, obtain a seed solution and then add it to a fermenter for fermentation, and add the substrate ferulic acid during the fermentation process.

[0029] Specifically, the production method of 3-methoxy-4-hydroxybenzaldehyde provided by the present invention is as follows:

[0030] (1) Inoculate the above-mentioned genetically engineered bacterium in Bennet liquid medium, culture at 30-45 °C for 24-72 h, add the seeds to the fermenter at 5%, and the fermentation conditions are: temperature is 30-45 °C, rotation speed is 200-800 rpm for fermentation, and the aeration ratio is 1-2 vvm;

[0031] (2) Add 10-20 g / L of ferulic acid substrate after 8-14 h of fermentation culture, and add it again when the remaining ferulic acid substrate is 0-5 g / L, and ferment for 48 h.

[0032] The present invention has the following beneficial effects:

[0033] The present invention provides a genetically engineered bacterium that knocks out the vanillin dehydrogenase gene and simultaneously expresses the heterologous citrate lyase gene and malate synthase gene. Applying this strain to the production of 3-methoxy-4-hydroxybenzaldehyde can not only increase the yield of 3-methoxy-4-hydroxybenzaldehyde, but also shorten the fermentation cycle; an efficient 3-methoxy-4-hydroxybenzaldehyde synthesis pathway has been constructed in Amycolatopsis, and it has good industrial application prospects. Detailed implementation mode

[0034] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0035] The features and performance of the present invention will be further described in detail below in conjunction with embodiments.

[0036] The host bacterium used in the embodiments of the present invention is Amycolatopsis sp. (ATCC 39116).

[0037] The culture medium formula involved in the present invention is as follows:

[0038] LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, and agar powder 20 g / L is added to make solid medium;

[0039] Bennet liquid medium: glucose 10 g / L, peptone 2 g / L, yeast extract 1 g / L, beef extract 1 g / L, glycerol 10 g / L, and agar powder 20 g / L is added to make solid medium;

[0040] CRM liquid medium: glucose 10 g / L, TSB 500 mL / L, yeast extract 0.5 g / L, beef extract 0.1 g / L, calcium chloride 5 mM, magnesium sulfate heptahydrate 5 mM;

[0041] Fermentation tank fermentation medium: yeast extract 10 - 50 g / L, glucose 10 - 50 g / L, sodium chloride 0.4 - 1.6 g / L, potassium dihydrogen phosphate 2 - 8 g / L, magnesium sulfate heptahydrate 0.1 - 0.5 g / L, calcium chloride 0.01 - 0.3 g / L.

[0042] The electrotransformation experiment used in the following embodiments includes the following steps:

[0043] (1) Streak the spores of the initial strain of Amycolatopsis sp. ATCC39116 onto Bennet medium and culture at 30 °C for 3 days. Scrape the spores and inoculate them into 25 mL of CRM liquid medium, and culture at 30 °C for 48 h. Inoculate 5% into the CRM medium containing glycine and KNO 3 and culture at 30 °C for 20 h. Collect the bacterial cells, add sterile water, resuspend and mix evenly, and then resuspend with electrotransformation buffer (0.5 M sucrose, 15% glycerol) to prepare electrotransformation competent cells;

[0044] (2) Transform the constructed suicide vector plasmid into the ET12567(pUZ8002) strain and extract the plasmid. Take 2 - 5 μg of the plasmid and place it in a pre-cooled EP tube. Add 60 μL of competent cells, put them into an electroporation cuvette, and perform electroporation at 15 kV / cm. Resuspend the electroporated bacterial solution in liquid medium and culture it at 30 °C for 8 h. Pipette 100 μL of the bacterial solution and spread it on a Bennet plate containing 50 μg / mL apramycin resistance. Gently pipette and mix well before spreading on the plate. Incubate the plate upside down at 30 °C for 5 days to grow transformants. Pick the transformants for verification by colony PCR.

[0045] Example 1

[0046] This example is about the construction of a genetically engineered bacterium, and the specific steps are as follows:

[0047] To construct Amycolatopsis sp. that knocks out the vdh gene and simultaneously expresses KbaceA and MpaceB, first construct a suicide vector pUC-VAB for knocking out the vdh gene and simultaneously expressing KbaceA and MpaceB.

[0048] The sequence of the strong promoter permE is as shown in SEQ ID NO: 4, and it is synthesized by total gene synthesis. Using the permE-f1 / permE-r1 primers and the synthesized strong promoter permE as a template, PCR amplify the permE-1 sequence; using the KbaceA-f / KbaceA-d primers and the optimized isocitrate lyase KbaceA from Kineosporia babensis as a template, PCR amplify the KbaceA gene sequence; using the permE-f2 / permE-r2 primers, PCR amplify the permE-2 sequence; using the MpaceB-f / MpaceB-d primers and the optimized malate synthase MpaceB from Micromonospora polyrhachis as a template, PCR amplify the MpaceB gene sequence;

[0049] Using the above 4 fragments as templates and the permE-f1 / MpaceB-d primers, perform overlap PCR amplification to obtain the permE-KbaceA-permE-MpaceB expression sequence;

[0050] Using the apra-r1 / vdh-down-f1 primers and the pUC-V plasmid as a template, PCR amplify to obtain a linearized pUC-V vector fragment, and perform seamless cloning with the permE-KbaceA-permE-MpaceB expression sequence using the ExnII kit for exonuclease to obtain the plasmid pUC-VAB;

[0051] Using the method of electrotransformation, the pUC-VAB suicide vector was transformed into Amycolatopsis pseudobranchiata, and mutant strains were screened according to apramycin resistance. The engineered strain of Amycolatopsis pseudobranchiata with vdh knocked out / Δvdh::permE-KbaceA::permE-MpaceB::Apra was obtained by PCR verification.

[0052] Comparative Example 1

[0053] This comparative example is Amycolatopsis pseudobranchiata with the vdh gene knocked out, and its construction is as follows:

[0054] The sequence of the apramycin resistance gene (Apra) is shown in SEQ ID NO: 3 and was synthesized in full length. To construct the suicide vector for knocking out vdh, first, using vdh-up-f / vdh-up-d as primers and the Amycolatopsis pseudobranchiata genome as a template, the vdh-up gene sequence was amplified by PCR; using vdh-down-f / vdh-down-d as primers and the Amycolatopsis pseudobranchiata genome as a template, the vdh-down gene sequence was amplified by PCR; using Apra-f / Apra-r as primers and the synthesized apramycin resistance gene (Apra) as a template, the Apra sequence was amplified by PCR;

[0055] Using the above 3 fragments as templates and vdh-up-f / vdh-down-r primers, the vdh knockout sequence was amplified by overlap PCR, and the vdh knockout fragment was cloned into the pUC vector through a TA cloning kit to obtain plasmid pUC-V;

[0056] Using the method of electrotransformation, the pUC-V suicide vector was transformed into Amycolatopsis pseudobranchiata, and mutant strains were screened according to apramycin resistance. The engineered strain of Amycolatopsis pseudobranchiata with vdh knocked out / Δvdh::Apra was obtained by PCR verification.

[0057] Comparative Example 2

[0058] This comparative example is Amycolatopsis pseudobranchiata that expresses the optimized KbaceA from Kineosporia babensis while knocking out the vdh gene, and its construction is as follows:

[0059] Using a method similar to that in Example 1, the permE-1 sequence and the optimized malate synthase KbaceA gene sequence from Kineosporia babensis were amplified by PCR respectively;

[0060] Using the above 2 fragments as templates, the permE-KbaceA expression sequence was amplified by overlap PCR;

[0061] The linearized pUC-V vector fragment was seamlessly cloned with the permE-KbaceA expression sequence using the ExnII exonuclease kit to obtain the plasmid pUC-permE-KbaceA;

[0062] Using the method of electrotransformation, the pUC-permE-KbaceA suicide vector was transformed into Amycolatopsis sp., and mutant strains were screened according to apramycin resistance. The engineered strain of Amycolatopsis sp. with vdh knocked out, / Δvdh::permE-KbaceA::Apra, was obtained by PCR verification.

[0063] Comparative Example 3

[0064] This comparative example is an Amycolatopsis sp. that expresses the optimized MtaceA from Marinactinospora thermotolerans while knocking out the vdh gene. Its construction is as follows:

[0065] Using a method similar to that in Example 1. The permE-1 sequence and the optimized isocitrate lyase MtaceA from Marinactinospora thermotolerans were amplified by PCR respectively. Its gene sequence is shown in SEQ ID NO: 21;

[0066] Using the above two fragments as templates, overlap PCR was used to amplify the permE-MtaceA expression sequence;

[0067] The linearized pUC-V vector fragment was seamlessly cloned with the permE-MtaceA expression sequence using the ExnII exonuclease kit to obtain the plasmid pUC-permE-MtaceA;

[0068] Using the method of electrotransformation, the pUC-permE-MtaceA suicide vector was transformed into Amycolatopsis sp., and mutant strains were screened according to apramycin resistance. The engineered strain of Amycolatopsis sp. with vdh knocked out, / Δvdh::permE-MtaceA::Apra, was obtained by PCR verification.

[0069] Comparative Example 4

[0070] This comparative example is an Amycolatopsis sp. that expresses the optimized SlaceA from Streptomyces lunaelactis while knocking out the vdh gene. Its construction is as follows:

[0071] Using a method similar to that in Example 1. The permE-1 sequence and the optimized isocitrate lyase SlaceA from Streptomyces lunaelactis were amplified by PCR respectively. Its gene sequence is shown in SEQ ID NO: 22;

[0072] Using the above two fragments as templates, the permE-SlaceA expression sequence was obtained by overlap PCR amplification;

[0073] The linearized pUC-V vector fragment and the permE-SlaceA expression sequence were seamlessly cloned using the ExnII exonuclease kit to obtain the plasmid pUC-permE-SlaceA;

[0074] Using the method of electroporation, the pUC-permE-SlaceA suicide vector was transformed into Amycolatopsis sp., and mutant strains were screened according to apramycin resistance. The engineered Amycolatopsis sp. strain with vdh knocked out / Δvdh::permE-SlaceA::Apra was obtained by PCR verification.

[0075] Comparative Example 5

[0076] This comparative example is an Amycolatopsis sp. that expresses the optimized MpaceB from Micromonospora polyrhachis while knocking out the vdh gene. Its construction is as follows:

[0077] Using a method similar to that in Example 1, the permE-1 sequence and the optimized malate synthase MpaceB gene sequence from Micromonospora polyrhachis were amplified by PCR respectively;

[0078] Using the above two fragments as templates, the permE-MpaceB expression sequence was obtained by overlap PCR amplification;

[0079] The linearized pUC-V vector fragment and the permE-MpaceB expression sequence were seamlessly cloned using the ExnII exonuclease kit to obtain the plasmid pUC-permE-MpaceB;

[0080] Using the method of electroporation, the pUC-permE-MpaceB suicide vector was transformed into Amycolatopsis sp., and mutant strains were screened according to apramycin resistance. The engineered Amycolatopsis sp. strain with vdh knocked out / Δvdh::permE-MpaceB::Apra was obtained by PCR verification.

[0081] Comparative Example 6

[0082] This comparative example is an Amycolatopsis sp. that expresses the optimized NaaceB from Nonomuraea aridisoli while knocking out the vdh gene. Its construction is as follows:

[0083] Use a method similar to Example 1. PCR amplify the permE-1 sequence and the optimized malate synthase NaaceB derived from Nonomuraea aridisoli respectively. Its gene sequence is shown in SEQ ID NO: 23;

[0084] Using the above two fragments as templates, perform overlap PCR amplification to obtain the permE-NaaceB expression sequence;

[0085] The linearized pUC-V vector fragment and the permE-NaaceB expression sequence are seamlessly cloned using the ExnII exonuclease kit to obtain the plasmid pUC-permE-NaaceB;

[0086] Using the method of electrotransformation, transform the pUC-permE-NaaceB suicide vector into Amycolatopsis sp., and screen for mutant strains according to apramycin resistance. Verify by PCR to obtain the engineered Amycolatopsis sp. strain with vdh knocked out / Δvdh::permE-NaaceB::Apra.

[0087] Comparative Example 7

[0088] This comparative example is an Amycolatopsis sp. that expresses the optimized AaaceB derived from Amycolatopsis arida while knocking out the vdh gene. Its construction is as follows:

[0089] Use a method similar to Example 1. PCR amplify the permE-1 sequence and the optimized malate synthase AaaceB derived from Amycolatopsis arida respectively. Its gene sequence is shown in SEQ ID NO: 24;

[0090] Using the above two fragments as templates, perform overlap PCR amplification to obtain the permE-AaaceB expression sequence;

[0091] The linearized pUC-V vector fragment and the permE-AaaceB expression sequence are seamlessly cloned using the ExnII exonuclease kit to obtain the plasmid pUC-permE-AaaceB;

[0092] Using the method of electrotransformation, transform the pUC-permE-AaaceB suicide vector into Amycolatopsis sp., and screen for mutant strains according to apramycin resistance. Verify by PCR to obtain the engineered Amycolatopsis sp. strain with vdh knocked out / Δvdh::permE-AaaceB::Apra.

[0093] Experimental Example

[0094] Fermentation comparison between the original strain (ATCC 39116) and the recombinant strain

[0095] Fermentation experiments for the production of 3 - methoxy - 4 - hydroxybenzaldehyde by ferulic acid conversion were carried out using Amycolatopsis pseudobranchialis ATCC 39116, the engineered strain Δvdh::Apra of Comparative Example 1, the engineered strain Δvdh::Apra::permE - KbaceA::Apra of Comparative Example 2, the engineered strain Δvdh::Apra::permE - MtaceA::Apra of Comparative Example 3, the engineered strain Δvdh::Apra::permE - SlaceA::Apra of Comparative Example 4, the engineered strain Δvdh::Apra::permE - MpaceB::Apra of Comparative Example 5, the engineered strain Δvdh::Apra::permE - NaaceB::Apra of Comparative Example 6, the engineered strain Δvdh::Apra::permE - AaaceB::Apra of Comparative Example 7, and the Δvdh::permE - KbaceA::permE - MpaceB::Apra strain constructed in Example 1. The experimental method is as follows:

[0096] (1) The above - mentioned strains were respectively inoculated into Bennet liquid medium and cultured at 30 °C for 48 h. The seeds were added to the fermenter at 5%, and fermented at 45 °C and 600 rpm with an aeration ratio of 2 vvm;

[0097] (2) Ferulic acid substrate at 20 g / L was added after 10 h of fermentation culture. When the remaining ferulic acid substrate was 1 g / L, it was added again. Fermentation was carried out for 48 h. The fermentation broth was diluted 10 times with water, and the supernatant was taken by centrifugation for HPLC detection of the concentration of 3 - methoxy - 4 - hydroxybenzaldehyde.

[0098] The fermentation results are shown in Table 2:

[0099] Table 2 Fermentation results of wild - type Amycolatopsis pseudobranchialis, Amycolatopsis pseudobranchialis in Comparative Examples 1 - 7 and Example 1

[0100] Strain Concentration of ferulic acid substrate (g / L) Concentration of vanillin (g / L) Concentration of remaining ferulic acid (g / L) Original strain 21.8 14.8 0.3 Comparative example 1 27 19.2 0.2 Comparative example 2 30.4 21.3 0.3 Comparative example 3 28.7 20.5 0.2 Comparative example 4 27.9 19.8 0.1 Comparative example 5 31.2 22.6 0.4 Comparative example 6 30.1 21.1 0.3 Comparative example 7 29.6 20.8 0.1 Example 1 33 24.8 0.2

[0101] The above results show that when the vdh gene is knocked out and the optimized isocitrate lyase KbaceA derived from Kineosporia babensis is overexpressed, the vanillin concentration is increased the most compared with aceA from the other two sources; when the vdh gene is knocked out and the optimized malate synthase MpaceB derived from Micromonospora polyrhachis is overexpressed, the vanillin concentration is increased the most compared with aceB from the other two sources;

[0102] Furthermore, by simultaneously knocking out the vdh gene and expressing the optimized isocitrate lyase KbaceA derived from Kineosporia babensis and the optimized malate synthase MpaceB derived from Micromonospora polyrhachis in the Amycolatopsis engineering bacteria, the conversion of 33 g / L ferulic acid into vanillin was achieved, and the concentration of vanillin in the fermentation broth reached 24.8 g / L. Compared with the initial strain Amycolatopsis ATCC39116 and the control strain Δvdh::Apra, Example 1 could significantly increase the yield of vanillin.

[0103] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A genetically engineered bacterium for producing 3-methoxy-4-hydroxybenzaldehyde, characterized in that: The genetically engineered bacteria is prepared by knocking out the vanillin dehydrogenase vdh gene in Amycolatopsis spp. and expressing exogenous isocitrate lyase aceA and malate synthase aceB genes.

2. The genetically engineered bacterium according to claim 1, characterized in that: The isocitrate lyase aceA includes KbaceA derived from Kineosporia babensis, and the malate synthase aceB includes MpaceB derived from Micromonospora polyrhachis.

3. The genetically engineered bacterium according to claim 2, characterized in that: The nucleotide sequence of the isocitrate lyase KbaceA is shown in SEQ ID NO: 1, and the nucleotide sequence of the malate synthase MpaceB is shown in SEQ ID NO:

2.

4. The genetically engineered bacterium according to claim 1, characterized in that: The strain number of the Amycolatopsis spp. is ATCC39116.

5. The method for constructing a genetically engineered bacterium according to any one of claims 1 to 4, characterized in that: include: The suicide vector containing a strong promoter, expressing isocitrate lyase aceA, malate synthase aceB and vanillin dehydrogenase vdh gene knockout is transformed into the Amycolatopsis spp., and then the genetically engineered bacteria are obtained through screening and verification.

6. The construction method according to claim 5, characterized in that: The method for constructing the suicide vector comprises: Using vdh-up-f / vdh-up-d as primers and the genome of Amycolatopsis simulans as a template, the vdh-up gene sequence was amplified; using vdh-down-f / vdh-down-r as primers and the genome of Amycolatopsis simulans as a template, the vdh-down gene sequence was amplified; using Apra-f / Apra-r as primers and the synthetic apramycin resistance gene Apra as a template, the apramycin resistance gene Apra sequence was amplified; The obtained vdh-up gene sequence, vdh-down gene sequence and apramycin resistance gene Apra sequence were used as templates, and vdh-up-f / vdh-down-r primers were used to amplify the vdh knockout sequence, and the vdh knockout fragment was cloned into the pUC vector by TA cloning to obtain the knockout suicide vector pUC-vdh-Apra with upstream and downstream homology arms of the vdh gene and apramycin resistance; The permE-1 sequence is amplified using permE-f1 / permE-r1 primers; the permE-2 sequence is amplified using permE-f2 / permE-r2 primers; the KbaceA-f / KbaceA-d primers are used, the isocitrate lyase KbaceA gene is used as a template, and the KbaceA gene sequence is amplified; the MpaceB-f / MpaceB-d primers are used, the malate synthase MpaceB is used as a template, and the MpaceB gene sequence is amplified; The permE-1 sequence, permE-2 sequence, KbaceA gene sequence and MpaceB gene sequence obtained were used as templates, and permE-f1 / aceB-d primers were used to amplify the permE-KbaceA-permE-MpaceB expression sequence; Using primer apra-r1 / vdh-down-f1, suicide vector pUC-vdh-Apra was used as template to amplify the linearized pUC-vdh-Apra vector fragment, which was seamlessly cloned with the permE-KbaceA-permE-MpaceB expression sequence to obtain the vanillin dehydrogenase vdh knockout suicide vector pUC-vdh-Apra-permE-KbaceA-permE-MpaceB containing a strong promoter permE and expressing isocitrate lyase KbAceA and malate synthase MpAceB; Preferably, the nucleotide sequences of the apramycin resistance gene and the strong promoter permE are as shown in SEQ ID NO: 3-4; The sequences of the vdh-up-f / vdh-up-d primers are shown in SEQ ID NOs: 5-6; The sequences of the Apra-f / Apra-r primers are shown in SEQ ID NOs: 7-8; The sequences of the vdh-down-f / vdh-down-r primers are shown in SEQ ID NOs: 9-10; The sequence of the apra-r1 / vdh-down-f1 primer is shown in SEQ ID NO: 11-12; The sequence of the permE-f1 / permE-r1 primer is shown in SEQ ID NO: 13-14; The sequences of the KbaceA-f / KbaceA-d primers are shown in SEQ ID NOs: 15-16; The sequences of the permE-f2 / permE-r2 primers are shown in SEQ ID NOs: 17-18; The sequences of the MpaceB-f / MpaceB-d primers are shown in SEQ ID NOs: 19-20.

7. A whole cell catalyst, characterized in that Contains the genetically engineered bacteria according to any one of claims 1 to 4.

8. Use of the genetically engineered bacteria according to any one of claims 1 to 4 or the whole-cell catalyst according to claim 7 in the production of 3-methoxy-4-hydroxybenzaldehyde.

9. A method for producing 3-methoxy-4-hydroxybenzaldehyde, characterized in that: include: The genetically engineered bacteria are inoculated into a culture medium, and the obtained seed liquid is then added into a fermentation tank for fermentation, and the substrate ferulic acid is added during the fermentation process.

10. The method according to claim 9, characterized in that The fermentation conditions in the fermenter are: under the conditions of temperature 30-45° C., rotation speed 200-800 rpm, ventilation ratio 1-2 vvm, adding 10-20 g / L of ferulic acid after fermentation for 8-14 hours, adding again when the remaining ferulic acid substrate is 0-5 g / L, and fermenting for 48 hours; Preferably, the culture conditions of the seed solution are: 30-45° C. for 24-72 hours.

Images