A genetically engineered bacterium for synthesizing natural benzaldehyde and its derivatives and its application

Abstract

The invention discloses a genetically engineered bacterium for synthesizing natural benzaldehyde and its derivatives and its application, relates to the field of biotechnology, the genetically engineered bacterium includes genes for expressing mandelate racemase, D-mandelate dehydrogenase and benzoylformate decarboxylase; wherein the amino acid sequence of mandelate racemase is as shown in SEQ ID NO:1, the amino acid sequence of D-mandelate dehydrogenase is as shown in SEQ ID NO:2, and the amino acid sequence of benzoylformate decarboxylase is as shown in SEQ ID NO:3. The application of genetically engineered bacteria in preparing benzyl alcohol, benzoic acid and benzylamine. The genetically engineered bacteria provided by the present invention enables it to carry out cofactor self-circulation, realizes stable efficient conversion of racemic mandelic acid (DL-mandelic acid) into benzaldehyde and its derivatives (benzyl alcohol, benzoic acid and benzylamine), and has important application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a genetic engineering bacterium for synthesizing natural benzaldehyde and its derivatives and application thereof. Background Art

[0002] Benzaldehyde is the simplest aromatic aldehyde with a special bitter almond smell. It is widely used in medicine, chemical industry and food. As one of the most widely used spices in the world, benzaldehyde has been used in the production of food such as roasted nuts, dried fruits, chocolate, candy and jelly. Due to consumers' preference for natural products as food additives, natural benzaldehyde has gradually become a high-value-added product in short supply. Various derivatives of benzaldehyde are also widely used in many fields such as spices and medicine. For example, benzyl alcohol is a commonly used fixative. It is an indispensable spice for the preparation of jasmine, moonflower, ylang-ylang and other flavors. It can also be used as a food fixative for fruit juice drinks; benzoic acid can be used in the production of food preservatives and drugs for the prevention and treatment of skin diseases; benzylamine can be used in the synthesis of drugs such as fluphenazine, nebivolol and moxifloxacin. The process of chemical synthesis of benzaldehyde and its derivatives is long, with high pollutant emissions and chlorine-containing products, which greatly limits their application and low product prices. Natural benzaldehyde and its derivatives are mainly extracted from plants such as apricot, cherry, laurel leaves and jasmine, but the content of these substances in plants is extremely low (usually less than 0.01%), and extraction and purification are difficult. Therefore, with the increasingly stringent environmental protection requirements and the increasing market demand for high-quality natural products, it is urgent to develop efficient and green methods for synthesizing benzaldehyde and its derivatives.

[0003] With the rapid development of synthetic biology technology and biocatalysis, microorganisms can be designed and modified and used in the production of raw materials such as drugs, fragrances and materials. At present, there are not many studies on the synthesis of natural benzaldehyde and its derivatives using microorganisms or biocatalysis. However, due to the activity of key enzymes such as mandelate synthase, these methods use substrates such as phenylalanine, phenylpyruvate or glucose to synthesize benzaldehyde and its derivatives with relatively low yields, and there are also problems such as low conversion rate and poor cofactor circulation efficiency.

[0004] Therefore, those skilled in the art are committed to developing an efficient microbial or biocatalytic technology to carry out green synthesis of natural benzaldehyde and its derivatives. Summary of the invention

[0005] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to develop an efficient microorganism or biocatalysis technology to carry out green synthesis of natural benzaldehyde and its derivatives.

[0006] To achieve the above-mentioned object, the present invention provides a genetically engineered bacterium for synthesizing natural benzaldehyde and its derivatives, comprising genes for expressing mandelate racemase, D-mandelate dehydrogenase and benzoylformate decarboxylase; wherein the amino acid sequence of mandelate racemase is shown in SEQ ID NO:1, the amino acid sequence of D-mandelate dehydrogenase is shown in SEQ ID NO:2, and the amino acid sequence of benzoylformate decarboxylase is shown in SEQ ID NO:3.

[0007] Furthermore, the nucleotide sequence of mandelate racemase is shown in SEQ ID NO:8; the nucleotide sequence of D-mandelate dehydrogenase is shown in SEQ ID NO:9; the nucleotide sequence of benzoylformate decarboxylase is shown in SEQ ID NO:10; and the expression host of the genetically engineered bacteria is the Escherichia coli BL21 (DE3) strain.

[0008] Furthermore, the mandelate racemase is selected from Burkholderia aromatica; the D-mandelate dehydrogenase is selected from Lactobacillus schrenkieri; and the benzoylformate decarboxylase is selected from Pseudomonas syringae.

[0009] Furthermore, the genetically engineered bacteria also include a gene for expressing phenylacetaldehyde reductase; wherein the amino acid sequence of phenylacetaldehyde reductase is shown in SEQ ID NO:4, the nucleotide sequence of phenylacetaldehyde reductase is shown in SEQ ID NO:11, and the phenylacetaldehyde reductase is selected from Salvia splendens.

[0010] Furthermore, the genetically engineered bacteria also include a gene for expressing benzaldehyde dehydrogenase; wherein the amino acid sequence of benzaldehyde dehydrogenase is shown in SEQ ID NO:5, the nucleotide sequence of benzaldehyde dehydrogenase is shown in SEQ ID NO:12, and the benzaldehyde dehydrogenase is selected from Pseudomonas montogenii.

[0011] Furthermore, the genetically engineered bacteria also include genes for expressing transaminase and alanine dehydrogenase; wherein the amino acid sequence of the transaminase is shown in SEQ ID NO:6; the amino acid sequence of the alanine dehydrogenase is shown in SEQ ID NO:7, the nucleotide sequence of the transaminase is shown in SEQ ID NO:13, the nucleotide sequence of the alanine dehydrogenase is shown in SEQ ID NO:14, the transaminase is selected from Rutgeria atlanticum; and the alanine dehydrogenase is selected from Bacillus carinii.

[0012] Furthermore, the benzaldehyde derivatives include benzyl alcohol, benzoic acid and / or benzylamine; genetically engineered bacteria are used as transformation bacteria, and racemic mandelic acid is used as a substrate; the final concentration of racemic mandelic acid is 5-100mM, pH 6-8, the transformation temperature is 25-45°C, and the transformation time is 6-12h.

[0013] The present invention also provides an application of a genetically engineered bacterium for synthesizing natural benzaldehyde and its derivatives in the preparation of benzyl alcohol.

[0014] The present invention also provides an application of a genetically engineered bacterium for synthesizing natural benzaldehyde and its derivatives in the preparation of benzoic acid.

[0015] The present invention also provides an application of a genetically engineered bacterium for synthesizing natural benzaldehyde and its derivatives in the preparation of benzylamine. Burkholderia aromatica, Lactobacillus schreiberis, Pseudomonas syringae, Pseudomonas monteri, Rutgeria atlanticum and Bacillus carnosus are all commercial strains and can be purchased on the market.

[0016] In preferred embodiment 1 of the present invention, the synthetic pathway for producing benzaldehyde and its derivatives (benzyl alcohol, benzoic acid or benzylamine) by genetically engineered bacteria is described in detail;

[0017] In another preferred embodiment 2 of the present invention, the process of constructing the genetically engineered bacteria Lumy-E212 is described in detail;

[0018] In another preferred embodiment 3 of the present invention, the process of constructing the genetically engineered bacteria Lumy-E213 is described in detail;

[0019] In another preferred embodiment 4 of the present invention, the process of constructing the genetically engineered bacteria Lumy-E214 is described in detail;

[0020] In another preferred embodiment 5 of the present invention, the process of constructing the genetically engineered bacteria Lumy-E215 is described in detail;

[0021] In another preferred embodiment 6 of the present invention, the process of converting racemic mandelic acid to prepare benzaldehyde and its derivatives by genetically engineered bacteria is described in detail.

[0022] The beneficial technical effects of the present invention are:

[0023] The present invention provides a genetically engineered bacterium, and is used for converting racemic mandelic acid to prepare benzaldehyde, benzyl alcohol, benzoic acid or benzylamine. Relative to the current biosynthesis method, the present invention uses racemic mandelic acid as a substrate, provides a new synthesis path, and avoids the problem of low yield and conversion rate of related products caused by low activity of key enzymes such as mandelic acid synthase in previous methods. The genetically engineered bacterium provided by the present invention can stably and efficiently convert racemic mandelic acid into benzaldehyde or its derivatives, and is a green biosynthesis technology with broad application prospects.

[0024] The genetically engineered bacteria provided by the present invention can carry out cofactor self-circulation, realize stable and efficient conversion of racemic mandelic acid (DL-mandelic acid) into benzaldehyde and its derivatives (benzyl alcohol, benzoic acid and benzylamine), and have important application prospects.

[0025] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram of a synthetic pathway for preparing benzaldehyde and its derivatives by transforming racemic mandelic acid with genetically engineered bacteria according to a preferred embodiment 1 of the present invention;

[0027] Figure 2 This is a graph showing the yield of benzaldehyde and its derivatives produced by genetically engineered bacteria according to a preferred embodiment 6 of the present invention. DETAILED DESCRIPTION

[0028] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0029] The strains and growth conditions used in the following examples are as follows:

[0030] The cloning host DH5α and the expression host BL21 (DE3) were purchased from Invitrogen. All E. coli were cultured in LB medium containing 100 mg / l ampicillin, kanamycin or chloramphenicol at 37°C.

[0031] The LB liquid culture medium formula is: peptone 10g / L, yeast extract 5g / L, NaCl 10g / L, pH 7.0; LB solid culture medium is added with 20g / L agar in LB liquid culture medium; 121°C high temperature and high pressure steam sterilization for 20min.

[0032] All plasmids were derived from pETDuet-1, pRSFDuet-1 or pACYCDuet-1 (purchased from Novagen) and were used to express target genes.

[0033] Example 1 Synthetic pathway for producing benzaldehyde and its derivatives (benzyl alcohol, benzoic acid or benzylamine) using genetically engineered bacteria

[0034] like Figure 1 As shown, genetically engineered bacteria are used as transformants, and racemic mandelic acid is used as a substrate, including D-mandelic acid and L-mandelic acid.

[0035] When the genetically engineered bacteria contain genes for expressing mandelate racemase (PaMR), D-mandelate dehydrogenase (SsDMDH) and benzoylformate decarboxylase (PsBFD), it is used to prepare benzaldehyde; when the genetically engineered bacteria contain genes for expressing mandelate racemase, D-mandelate dehydrogenase, benzoylformate decarboxylase and phenylacetaldehyde reductase, it is used to prepare benzyl alcohol; when the genetically engineered bacteria contain genes for expressing mandelate racemase, D-mandelate dehydrogenase, benzoylformate decarboxylase and benzaldehyde dehydrogenase, it is used to prepare benzoic acid; when the genetically engineered bacteria contain genes for expressing mandelate racemase, D-mandelate dehydrogenase, benzoylformate decarboxylase, transaminase and alanine dehydrogenase, it is used to prepare benzylamine.

[0036] Example 2 Construction of genetically engineered bacteria Lumy-E212

[0037] (1) Synthetic mandelate racemase (PaMR) encoding gene (mar) fragment, D-mandelate dehydrogenase (SsDMDH) encoding gene (dmdh) fragment, benzoylformate decarboxylase (PsBFD) encoding gene (bfd) fragment and NADH oxidase (LrNOX) encoding gene (nox) fragment:

[0038] Using the online software JCat, the mar gene in Paraburkholderia aromaticivorans, the dmdh gene in Schleiferilactobacillus shenzhenensis, the bfd gene in Pseudomonas syringae and the nox gene in Limosilactobacillus reuteri were codon optimized according to the codon preference of Escherichia coli. The nucleotide sequence of mar after optimization is shown in SEQ ID NO: 8, the nucleotide sequence of dmdh is shown in SEQ ID NO: 9, the nucleotide sequence of bfd is shown in SEQ ID NO: 10, and the nucleotide sequence of nox is shown in SEQ ID NO: 15. The above gene sequences were sent to GENEWIZ for gene synthesis, mar was synthesized between the BamHI and HindIII restriction sites of pETDuet-1 to obtain pETDuet-mar, dmdh was synthesized between the NdeI and XhoI restriction sites of pETDuet-mar to obtain the recombinant expression plasmid pETDuet-mar-dmdh; bfd was synthesized between the BamHI and HindIII restriction sites of pRSFDuet-1 to obtain pRSFDuet-bfd, and nox was synthesized between the NdeI and XhoI restriction sites of pRSFDuet-bfd to obtain the recombinant expression plasmid pRSFDuet-bfd-nox.

[0039] (2) Obtaining the genetically engineered bacteria Lumy-E212:

[0040] In order to avoid the conversion of benzaldehyde by endogenous alcohol dehydrogenase in Escherichia coli, the gene knockout of Escherichia coli BL21 (DE3) was carried out according to the method described in the Molecular Cloning Experiment Guide (Third Edition), and 7 genes (dkgB, yqhC, yqhD, yahK, yeaE, dkgA, yjgB) were knocked out to obtain Escherichia coli KARE. 5 μL of the recombinant plasmid pETDuet-mar-dmdh obtained in step (1) and 5 μL of the recombinant plasmid pRSFDuet-bfd-nox obtained in step (1) were transformed into Escherichia coli KARE competent cells by heat shock. The heat-shocked bacterial solution was spread on an LB solid culture medium plate containing 100 mg / L ampicillin and kanamycin, and cultured in a constant temperature incubator at 37°C for 12 hours. A single colony was picked from the plate and placed in 5 mL LB liquid culture medium (containing 100 mg / L ampicillin and kanamycin), and cultured in a shaker at 37°C with a shaker speed of 200 rpm; the cultured bacterial solution was subjected to PCR amplification verification to obtain the genetically engineered strain Lumy-E212, as shown in FIG. Figure 1 As shown, Lumy-E212 can be used to convert racemic mandelic acid to prepare benzaldehyde, and the introduction of NADH oxidase can regenerate the cofactor NAD+.

[0041] Example 3 Construction of genetically engineered bacteria Lumy-E213

[0042] (1) Synthetic phenylacetaldehyde reductase (SsPAR) encoding gene (par) fragment:

[0043] The par gene in Salvia splendens was codon optimized according to the codon preference of Escherichia coli using the online software JCat, and the nucleotide sequence of par after optimization is shown in SEQ ID NO: 11. The par gene sequence was sent to GENEWIZ for gene synthesis, and synthesized between the NdeI and XhoI restriction sites of pRSFDuet-bfd in step (1) of Example 1 to obtain the recombinant expression plasmid pRSFDuet-bfd-par.

[0044] (2) Obtaining the genetically engineered bacteria Lumy-E213:

[0045] 5 μL of the recombinant plasmid pETDuet-mar-dmdh obtained in step (1) of Example 1 and 5 μL of the recombinant plasmid pRSFDuet-bfd-par obtained in step (1) were transformed into competent Escherichia coli BL21 (DE3) cells by heat shock. The heat-shocked bacterial solution was spread on an LB solid culture medium plate containing 100 mg / L ampicillin and kanamycin, and cultured in a 37°C constant temperature incubator for 12 hours. A single colony was picked from the plate and added to 5 mL of LB liquid culture medium (containing 100 mg / L ampicillin and kanamycin), and cultured in a shaker at 37°C with a shaker speed of 200 rpm; the cultured bacterial solution was subjected to PCR amplification verification to obtain the genetically engineered strain Lumy-E213, as shown in Figure 1 As shown, Lumy-E213 can be used to convert racemic mandelic acid to prepare benzyl alcohol, and NAD+ and NADH can be recycled between D-mandelate dehydrogenase and phenylacetaldehyde reductase.

[0046] Example 4 Construction of genetically engineered bacteria Lumy-E214

[0047] (1) Synthesize the benzaldehyde dehydrogenase (PmBADH) encoding gene (badh) fragment:

[0048] The badh gene in Pseudomonas monteilii was codon optimized according to the codon preference of Escherichia coli using online software JCat, and the nucleotide sequence of badh after optimization is shown in SEQ ID NO: 12. The badh gene sequence was sent to GENEWIZ for gene synthesis and synthesized between the BamHI and HindIII restriction sites of pACYCDuet-1 to obtain pACYCDuet-badh.

[0049] (2) Obtaining the genetically engineered bacteria Lumy-E214:

[0050] 3 μL of the recombinant plasmid pETDuet-mar-dmdh obtained in step (1) of Example 1, 3 μL of pRSFDuet-bfd-nox, and 3 μL of the recombinant plasmid pACYCDuet-badh obtained in step (1) were transformed into Escherichia coli KARE competent cells by heat shock. The heat-shocked bacterial solution was spread on an LB solid culture medium plate containing 100 mg / L ampicillin and kanamycin and 20 mg / L chloramphenicol, and cultured in a 37°C constant temperature incubator for 12 hours. A single colony was picked from the plate and added to 5 mL of LB liquid culture medium (containing 100 mg / L ampicillin and kanamycin and 20 mg / L chloramphenicol), and cultured in a shaker at 37°C with a shaker speed of 200 rpm; the cultured bacterial solution was subjected to PCR amplification verification to obtain the genetically engineered strain Lumy-E214, as shown in Figure 1 As shown, Lumy-E214 can be used to convert racemic mandelic acid to produce benzoic acid, and the introduction of NADH oxidase can regenerate the cofactor NAD+.

[0051] Example 5 Construction of genetically engineered bacteria Lumy-E215

[0052] (1) Synthetic transaminase (RaATA) encoding gene (ata) fragment, alanine dehydrogenase (BcAlaDH) encoding gene (adh) fragment:

[0053] Using the online software JCat, the ata gene in Ruegeria atlantica and the adh gene in Bacillus cabrialesii were codon optimized according to the codon preference of Escherichia coli. The nucleotide sequence of ata after optimization is shown in SEQ ID NO: 13, and the nucleotide sequence of adh is shown in SEQ ID NO: 14. The ata and adh gene sequences were sent to GENEWIZ for gene synthesis. Ata was synthesized between the BamHI and HindIII restriction sites of pACYCDuet-1 to obtain pACYCDuet-ata, and adh was synthesized between the NdeI and XhoI restriction sites of pACYCDuet-ata to obtain the recombinant expression plasmid pACYCDuet-ata-adh.

[0054] (2) Obtaining the genetically engineered bacteria Lumy-E215:

[0055] 3 μL of the recombinant plasmid pETDuet-mar-dmdh obtained in step (1) of Example 1, 3 μL of pRSFDuet-bfd-nox, and 3 μL of the recombinant plasmid pACYCDuet-ata-adh obtained in step (1) were transformed into Escherichia coli KARE competent cells by heat shock. The heat-shocked bacterial solution was spread on an LB solid culture medium plate containing 100 mg / L ampicillin and kanamycin and 20 mg / L chloramphenicol, and cultured in a 37°C constant temperature incubator for 12 hours. A single colony was picked from the plate and added to 5 mL of LB liquid culture medium (containing 100 mg / L ampicillin and kanamycin and 20 mg / L chloramphenicol), and cultured in a shaker at 37°C with a shaker speed of 200 rpm; the cultured bacterial solution was subjected to PCR amplification verification to obtain the genetically engineered strain Lumy-E215, as shown in Figure 1 As shown, Lumy-E215 can be used to convert racemic mandelic acid to prepare benzylamine, and the introduction of NADH oxidase can regenerate the cofactor NAD+.

[0056] Example 6 Genetically engineered bacteria transform racemic mandelic acid to prepare benzaldehyde and its derivatives

[0057] (1) Preparation of whole-cell catalysts from genetically engineered bacteria:

[0058] The genetically engineered bacteria Lumy-E212 or Lumy-E213 obtained in Example 2 or Example 3 were inoculated into 50 mL LB liquid culture medium (containing 100 mg / L ampicillin and 100 mg / L kanamycin) with an inoculation loop, and the genetically engineered bacteria Lumy-E214 or Lumy-E215 obtained in Example 4 or Example 5 were inoculated into 50 mL LB liquid culture medium (containing 100 mg / L ampicillin and 100 mg / L kanamycin and 20 mg / L chloramphenicol) with an inoculation loop for strain activation, and cultured at 37° C. overnight; subsequently, the activated bacterial solution was inoculated into 5 L LB liquid culture medium at an inoculum volume of 1% (volume ratio), and cultured at 37° C., 200 rpm, for 3-5 hours until OD600 reached 0.6-0.8, and 0.2 mM IPTG was used for induction culture at 16°C and 200 rpm for 16 h. The culture solution was collected and centrifuged at 4°C and 3500 rpm for 15 min to collect the bacterial cells, which were then washed twice with phosphate buffer for later use.

[0059] (2) Lumy-E212 conversion of racemic mandelic acid to prepare benzaldehyde

[0060] The reaction system was constructed using the Lumy-E212 bacteria collected in step (1) as a catalyst, the amount of bacteria added was a final concentration of 20 g / L, 100 mM racemic mandelic acid was added, pH = 8, the reaction conditions were 30°C, the stirring speed was 200 rpm, and the reaction time was 12 h. The amount of benzaldehyde generated was detected by HPLC, using an Agilent 1260 liquid chromatograph, an Eclipse XDB-C18 column (4.6×150 mm), a column temperature of 30°C, a detection wavelength of 255 nm, mobile phase A was water (containing 1% trifluoroacetic acid), mobile phase B was acetonitrile (containing 1% trifluoroacetic acid), the flow rate was 1 mL / min, and the gradient elution program was: 0 minutes, 95% mobile phase A + 5% mobile phase B; 8 minutes, 20% mobile phase A + 80% mobile phase B; 10 minutes, 20% mobile phase A + 80% mobile phase B; 14 minutes, 95% mobile phase A + 5% mobile phase B, flow rate 1 mL / min. Figure 2 As shown, racemic mandelic acid was transformed by genetically recombinant bacteria to obtain 8.79 g / L of benzaldehyde.

[0061] (3) Lumy-E213 conversion of racemic mandelic acid to prepare benzyl alcohol

[0062] The reaction system was constructed using the Lumy-E213 cells collected in step (1) as a catalyst, the cell addition amount was 20 g / L at a final concentration, 100 mM racemic mandelic acid was added, pH = 8, the reaction conditions were 30 ° C, the stirring speed was 200 rpm, and the reaction time was 12 h. The amount of benzyl alcohol produced was detected by HPLC, using an Agilent 1260 liquid chromatograph, an Eclipse XDB-C18 column (4.6×150 mm), a column temperature of 30° C., a detection wavelength of 215 nm, mobile phase A was water (containing 1% trifluoroacetic acid), mobile phase B was acetonitrile (containing 1% trifluoroacetic acid), the flow rate was 1 mL / min, and the gradient elution program was: 0 minutes, 95% mobile phase A + 5% mobile phase B; 8 minutes, 20% mobile phase A + 80% mobile phase B; 10 minutes, 20% mobile phase A + 80% mobile phase B; 14 minutes, 95% mobile phase A + 5% mobile phase B, flow rate 1 mL / min. Figure 2 As shown, racemic mandelic acid was transformed by genetically recombinant bacteria to obtain 10.15 g / L of benzyl alcohol.

[0063] (4) Lumy-E214 conversion of racemic mandelic acid to benzoic acid

[0064] The reaction system was constructed using the Lumy-E214 cells collected in step (1) as a catalyst, the cell addition amount was 20 g / L at a final concentration, 100 mM racemic mandelic acid was added, pH = 8, the reaction conditions were 30 ° C, the stirring speed was 200 rpm, and the reaction time was 12 h. The amount of benzoic acid produced was detected by HPLC, using an Agilent 1260 liquid chromatograph, an Eclipse XDB-C18 column (4.6×150 mm), a column temperature of 30°C, a detection wavelength of 215 nm, mobile phase A was water (containing 1% trifluoroacetic acid), mobile phase B was acetonitrile (containing 1% trifluoroacetic acid), the flow rate was 1 mL / min, and the gradient elution program was: 0 minutes, 95% mobile phase A + 5% mobile phase B; 8 minutes, 20% mobile phase A + 80% mobile phase B; 10 minutes, 20% mobile phase A + 80% mobile phase B; 14 minutes, 95% mobile phase A + 5% mobile phase B, flow rate 1 mL / min. Figure 2 As shown, racemic mandelic acid was transformed by genetically recombinant bacteria to obtain 9.88 g / L of benzoic acid.

[0065] (5) Lumy-E215 conversion of racemic mandelic acid to prepare benzylamine

[0066] The reaction system was constructed using the Lumy-E215 cells collected in step (1) as a catalyst, the cell addition amount was 20 g / L at a final concentration, 100 mM racemic mandelic acid was added, pH = 8, the reaction conditions were 30 ° C, the stirring speed was 200 rpm, and the reaction time was 12 h. The amount of benzoic acid produced was detected by HPLC, using an Agilent 1260 liquid chromatograph, an Eclipse XDB-C18 column (4.6×150 mm), a column temperature of 30°C, a detection wavelength of 215 nm, mobile phase A was water (containing 1% trifluoroacetic acid), mobile phase B was acetonitrile (containing 1% trifluoroacetic acid), the flow rate was 1 mL / min, and the gradient elution program was: 0 minutes, 95% mobile phase A + 5% mobile phase B; 8 minutes, 20% mobile phase A + 80% mobile phase B; 10 minutes, 20% mobile phase A + 80% mobile phase B; 14 minutes, 95% mobile phase A + 5% mobile phase B, flow rate 1 mL / min. Figure 2 As shown, racemic mandelic acid was transformed by genetically recombinant bacteria to obtain 6.71 g / L of benzylamine.

[0067] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A genetically engineered bacterium for synthesizing benzaldehyde and its derivatives, characterized in that: The genetically engineered bacteria include genes for expressing mandelate racemase, D-mandelate dehydrogenase and benzoylformate decarboxylase; wherein the amino acid sequence of the mandelate racemase is shown in SEQ ID NO: 1, the amino acid sequence of the D-mandelate dehydrogenase is shown in SEQ ID NO: 2, and the amino acid sequence of the benzoylformate decarboxylase is shown in SEQ ID NO: 3; the nucleotide sequence of the mandelate racemase is shown in SEQ ID NO: 8; the nucleotide sequence of the D-mandelate dehydrogenase is shown in SEQ ID NO: 9; the nucleotide sequence of the benzoylformate decarboxylase is shown in SEQ ID NO: NO:10; the expression host of the genetically engineered bacteria is Escherichia coli BL21 (DE3) strain; the mandelate racemase is selected from Burkholderia aromatica; the D-mandelate dehydrogenase is selected from Lactobacillus schrenkieri of Shenzhen; the benzoylformate decarboxylase is selected from Pseudomonas syringae; the genetically engineered bacteria also includes a gene for expressing phenylacetaldehyde reductase; wherein the amino acid sequence of the phenylacetaldehyde reductase is shown in SEQ ID NO:4, the nucleotide sequence of the phenylacetaldehyde reductase is shown in SEQ ID NO:11, and the phenylacetaldehyde reductase is selected from Salvia sp.

2. Use of the genetically engineered bacteria as claimed in claim 1 in the preparation of benzyl alcohol.

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