A recombinant bacterium for producing L-tyrosine and its construction method and application

Abstract

This invention discloses a recombinant bacterium for producing L-tyrosine, its construction method, and its applications. The genetically engineered bacterium of this invention incorporates genes for threonine aldolase, threonine deaminase, leucine dehydrogenase, phenylalanine hydroxylase, pterin 4α-methanolamine dehydratase, and dihydropterin reductase, enabling its use in the biosynthesis of L-tyrosine. This invention also discloses a method for constructing and applying a recombinant *Escherichia coli* engineered bacterium. The production of L-tyrosine using the recombinant bacterium of this invention is highly efficient, environmentally friendly, and low-cost, showing promising prospects for industrial application.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant bacterium for producing L-tyrosine, a construction method and an application thereof. Background Art

[0002] L-Tyrosine is an important amino acid and a raw material for the preparation of p-hydroxycinnamic acid, levodopa and p-hydroxystyrene. It is widely used in the food, medicine and cosmetics industries.

[0003] The main methods for preparing L-tyrosine include extraction, chemical synthesis and biotransformation. The extraction method mainly uses natural protein resources as raw materials, and extracts L-tyrosine through steps such as hydrolysis, decolorization and crystallization. The high cost and low yield of this method limit its industrial application. The chemical synthesis method is synthesized by hydroxylation of L-phenylalanine or by condensation of p-hydroxybenzaldehyde and hydantoin, alkaline hydrolysis, transamination and other steps. This method has complex steps, high energy consumption and produces a large amount of pollutants, and has been gradually eliminated. The biotransformation method has the advantages of high specificity, green environmental protection, mild reaction conditions and no need for multi-step separation and purification, and has received widespread attention.

[0004] Biotransformation methods are divided into microbial fermentation and enzyme catalysis. The process control of microbial fermentation is complex. The process conditions must be suitable for the growth of microorganisms and conducive to the accumulation of metabolites. The production cycle is long and the efficiency is low, which is not suitable for industrial application. Therefore, an effective and mild enzyme catalysis method is the best choice for preparing L-tyrosine. At present, the literature has reported a variety of enzyme catalysis preparation routes for L-tyrosine. Sha Xu et al. screened out tyrosine phenol lyase (TPL) from the bacterium Erwinia herbicola, and used pyruvate, ammonia and phenol as substrates to convert and prepare L-tyrosine, and finally obtained 48.5g / L of L-tyrosine. However, the price of pyruvate is relatively expensive. Therefore, Chen Mingliang et al. used glycine and formaldehyde as substrates, and converted them into pyruvate by aldolase and D-serine dehydratase, and then added ammonia, phenol and TPL to synthesize L-tyrosine. After 11h of conversion, the L-tyrosine yield reached 117g / L. Chinese patent CN103224972 uses a mixed amino acid solution rich in L-serine as a substrate, adds an appropriate amount of phenol, and synthesizes L-tyrosine using a dual enzyme catalysis of serine deaminase and tyrosine phenol lyase.

[0005] The above-reported catalytic preparation routes of L-tyrosine all involve phenol or formaldehyde, which is highly toxic. This poses a great challenge to safety management during the scale-up production process. In addition, these two harmful substances may remain in the final L-tyrosine product, limiting its scope of application. Therefore, it is urgent to create a method for preparing L-tyrosine with high efficiency and low toxicity.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The object of the present invention is to provide a recombinant bacterium for producing L-tyrosine and a construction method and application thereof. The recombinant bacterium is used to produce L-tyrosine, which has high production efficiency, is environmentally friendly and low cost, and has good prospects for industrial application.

[0008] The present invention is achieved in that:

[0009] In a first aspect, the present invention provides a recombinant bacterium for producing L-tyrosine, wherein the recombinant bacterium contains exogenous genes; the exogenous genes include genes encoding threonine aldolase, threonine deaminase, leucine dehydrogenase, phenylalanine hydroxylase, pterin 4a-methanolamine dehydratase and dihydropterin reductase.

[0010] The present invention proposes for the first time that benzaldehyde and glycine are converted into β-phenylserine by threonine aldolase (LTA), β-phenylserine is converted into phenylpyruvate by threonine deaminase (TD), phenylpyruvate is converted into L-phenylalanine by leucine dehydrogenase (LeuDH) and NADH, L-tyrosine is generated under the action of phenylalanine hydroxylase (P4H), O2 and tetrahydropterin (MH4), pterin 4a-methanolamine dehydratase (PCD) and dihydropterin reductase (DHMR) are used for the recycling of MH4, and the NADH required in the reaction process is provided by glucose metabolism by the bacteria. The reaction principle is shown in the following figure:

[0011]

[0012] According to the above reaction principle, the inventors introduced genes encoding threonine aldolase, threonine deaminase, leucine dehydrogenase, phenylalanine hydroxylase, pterin 4a-methanolamine dehydratase and dihydropterin reductase into a host bacterium to obtain a recombinant bacterium. The recombinant bacterium is used as a biocatalyst to convert cheap and low-toxic substrates benzaldehyde and glycine into L-tyrosine.

[0013] In some embodiments, the threonine aldolase includes AhLTA and PsLTA, the amino acid sequence of AhLTA is shown as SEQ ID NO.1; the amino acid sequence of PsLTA is shown as SEQ ID NO.2.

[0014] In the present invention, AhLTA is derived from Aeromonas hydrophila, with Genbank number CAD7528144.1; PsLTA is derived from Pseudomonas aeruginosa, with Genbank number OPF27990.1. After obtaining the amino acid sequences of threonine aldolase AhLTA and PsLTA, the inventors optimized codons according to the preference of Escherichia coli and synthesized two optimized nucleotide sequences by total synthesis.

[0015] In some embodiments, the nucleotide sequence of AhLTA is shown as SEQ ID NO.3; the nucleotide sequence of PsLTA is shown as SEQ ID NO.4.

[0016] In some embodiments, the threonine deaminase includes VaTD and PvTD, the amino acid sequence of VaTD is shown as SEQ ID NO.5; the amino acid sequence of PvTD is shown as SEQ ID NO.6.

[0017] In the present invention, VaTD is derived from Vibrio aestuarianus, with a Genbank number of CAD7528144.1; PvTD is derived from Pseudomonas veronii, with a Genbank number of CAD0262619.1. After obtaining the amino acid sequences of threonine deaminase VaTD and PvTD, the inventors optimized codons according to the preference of Escherichia coli and synthesized two optimized nucleotide sequences by total synthesis.

[0018] In some embodiments, the nucleotide sequence of VaTD is shown as SEQ ID NO.7; the nucleotide sequence of PvTD is shown as SEQ ID NO.8.

[0019] In some embodiments, the leucine dehydrogenase includes FgLeuDH and AmLeuDH, the amino acid sequence of FgLeuDH is shown in SEQ ID NO.9; the amino acid sequence of AmLeuDH is shown in SEQ ID NO.10.

[0020] In the present invention, FgLeuDH is derived from Flavobacterium gilvum, with a Genbank number of KFC59129.1; AmLeuDH is derived from Alteromonas mediterranea, with a Genbank number of AGQ00979.1. After obtaining the amino acid sequences of leucine dehydrogenase FgLeuDH and AmLeuDH, the inventors optimized the codons according to the preference of Escherichia coli and synthesized the two optimized nucleotide sequences by a total synthesis method.

[0021] In some embodiments, the nucleotide sequence of FgLeuDH is shown as SEQ ID NO.11; the nucleotide sequence of AmLeuDH is shown as SEQ ID NO.12.

[0022] In some embodiments, the phenylalanine hydroxylase includes VcP4H and PaP4H, the amino acid sequence of VcP4H is shown in SEQ ID NO.13; the amino acid sequence of PaP4H is shown in SEQ ID NO.14.

[0023] In the present invention, VcP4H is derived from Vibrio cholerae, with a Genbank number of KFE12728.1; PaP4H is derived from Pseudoalteromonas atlantica, with a Genbank number of ABG41507.1. After obtaining the amino acid sequences of phenylalanine hydroxylase VcP4H and PaP4H, the inventors optimized codons according to the preference of Escherichia coli and synthesized two optimized nucleotide sequences by a total synthesis method.

[0024] In some embodiments, the nucleotide sequence of VcP4H is shown as SEQ ID NO.15; the nucleotide sequence of PaP4H is shown as SEQ ID NO.16.

[0025] In some embodiments, the pterin 4a-carbinolamine dehydratase comprises ApPCD and GaPCD, wherein the amino acid sequence of ApPCD is shown as SEQ ID NO.17; and the amino acid sequence of GaPCD is shown as SEQ ID NO.18.

[0026] In the present invention, ApPCD is derived from Actinoplanes philippinensis, with a Genbank number of SFF49499.1; GaPCD is derived from Geodermatophilus aquaeductus, with a Genbank number of SMO77949.1. After obtaining the amino acid sequences of pterin 4a-methanolamine dehydratase ApPCD and GaPCD, the inventors optimized the codons according to the preference of Escherichia coli, and synthesized the two optimized nucleotide sequences by a total synthesis method.

[0027] In some embodiments, the nucleotide sequence of ApPCD is shown as SEQ ID NO.19; and the nucleotide sequence of GaPCD is shown as SEQ ID NO.20.

[0028] In some embodiments, the dihydropterin reductase includes PvDHMR and KmDHMR, the amino acid sequence of PvDHMR is shown in SEQ ID NO.21; the amino acid sequence of KmDHMR is shown in SEQ ID NO.22.

[0029] In the present invention, PvDHMR is derived from Pectobacterium versatile, with a Genbank number of AVT58569.1; KmDHMR is derived from Klebsiella michiganensis, with a Genbank number of QSW16815.1. After obtaining the amino acid sequences of dihydropterin reductase PvDHMR and KmDHMR, the inventors optimized the codons according to the preference of Escherichia coli, and synthesized the two optimized nucleotide sequences by a total synthesis method.

[0030] In some embodiments, the nucleotide sequence of PvDHMR is shown as SEQ ID NO.23; the nucleotide sequence of KmDHMR is shown as SEQ ID NO.24.

[0031] In some embodiments, the host of the above-mentioned recombinant bacteria is Escherichia coli.

[0032] In some embodiments, the Escherichia coli is selected from any one of Escherichia coli BL21, Escherichia coli DH5α and Escherichia coli XL-Blue.

[0033] In some embodiments, the recombinant bacteria contain an expression vector, and the expression vector includes pETDuet-1 plasmid, pCDFDuet-1 plasmid and pACYCDuet-1 plasmid.

[0034] In a second aspect, the present invention also provides a method for constructing the above-mentioned recombinant bacteria, which comprises: connecting the genes of threonine aldolase, threonine deaminase, leucine dehydrogenase, phenylalanine hydroxylase, pterin 4a-methanolamine dehydratase and dihydropterin reductase to an expression vector, and then introducing the obtained recombinant expression vector into the host bacteria to obtain the recombinant bacteria.

[0035] In the present invention, one enzyme is selected from each of the above-mentioned threonine aldolase, threonine deaminase, leucine dehydrogenase, phenylalanine hydroxylase, pterin 4a-methanolamine dehydratase and dihydropterin reductase, and the six enzymes are co-expressed in combination. The method of introducing the plasmid carrying the coding genes of the above-mentioned enzymes can be that any two of the coding genes are present on the same plasmid, or that the six genes are present on different plasmids respectively, or other introduction methods can be used, and the present invention is not limited to this.

[0036] Preferably, pETDuet-1 plasmid, pCDFDuet-1 plasmid and pACYCDuet-1 plasmid are used to co-express the coding genes of the six enzymes.

[0037] The two-by-two combinations of the coding genes for the six enzymes can be any combination of coding genes for two enzymes. The preferred combination is a combination of genes encoding threonine aldolase and threonine deaminase, a combination encoding leucine dehydrogenase and phenylalanine hydroxylase, and a combination encoding pterin 4a-methanolamine dehydratase and dihydropterin reductase. A more preferred combination is that the pETDuet-1 plasmid is connected with genes encoding threonine aldolase and threonine deaminase, the pCDFDuet-1 plasmid is connected with genes encoding leucine dehydrogenase and phenylalanine hydroxylase, and the pACYCDuet-1 plasmid is connected with genes encoding pterin 4a-methanolamine dehydratase and dihydropterin reductase.

[0038] In some embodiments, restriction sites EcoRI and HindIII are added at both ends of the nucleotide sequence of threonine aldolase; restriction sites NdeI and XhoI are added at both ends of the nucleotide sequence of threonine deaminase; restriction sites EcoRI and HindIII are added at both ends of the nucleotide sequence of leucine dehydrogenase; restriction sites NdeI and XhoI are added at both ends of the nucleotide sequence of phenylalanine hydroxylase; restriction sites EcoRI and HindIII are added at both ends of the nucleotide sequence of pterin 4a-methanolamine dehydratase; restriction sites NdeI and XhoI are added at both ends of the nucleotide sequence of dihydropterin reductase.

[0039] In a third aspect, the present invention provides a method for producing L-tyrosine, which comprises inducing the culture of the above-mentioned recombinant bacteria and then adding them to a transformation system for whole-cell transformation to obtain L-tyrosine.

[0040] Whole cell transformation refers to the process of chemical transformation using a complete biological organism (i.e., whole cell, tissue, or even individual) as a catalyst. In the present invention, the organism of whole cell transformation is mainly a microorganism-recombinant bacteria, and its essence is to use the enzyme in the recombinant bacteria for catalysis.

[0041] In some embodiments, the conversion system comprises benzaldehyde at a concentration of 1-50 g / L, glycine at a concentration of 1-40 g / L, and glucose at a concentration of 20-80 g / L.

[0042] In some embodiments, the amount of fresh cells of the recombinant bacteria in the transformation system is 1-20 g / L.

[0043] In some embodiments, the pH of the conversion system is 6.0-9.0.

[0044] In some embodiments, the conversion temperature of the conversion system is 15-40°C.

[0045] In some embodiments, the transformation time of the transformation system is 6-24 h.

[0046] In some embodiments, the induction culture of the recombinant bacteria includes inoculating the recombinant bacteria into a selective culture medium to obtain a seed solution, then inoculating the seed solution into a basal culture medium and culturing until the OD600nm reaches 0.7, adding an inducer for induction, and cleaning the wet bacteria after completion.

[0047] In some embodiments, the recombinant bacteria are cultured in a selective medium at a temperature of 30-40° C. for 10-15 hours.

[0048] In some embodiments, the culturing temperature of the seed solution in the basal medium is 30-40°C.

[0049] In some embodiments, the induction temperature in the induction step is 25-30°C and the time is 12-18 hours.

[0050] The present invention has the following beneficial effects:

[0051] The present invention provides a novel method for producing L-tyrosine, which uses a recombinant Escherichia coli to produce L-tyrosine using benzaldehyde and glycine as substrates. Benzaldehyde and glycine are ideal substrates with wide sources, low toxicity, simple preparation process and low price. The enzyme selected by the present invention has the advantages of high activity and strong optical specificity. Therefore, the recombinant bacteria of the present invention are used to transform and produce L-tyrosine, which has high production efficiency, is green and environmentally friendly, and has low cost, and has good industrial application prospects. DETAILED DESCRIPTION

[0052] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0053] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0054] The pETDuet-1 plasmid, pCDFDuet-1 plasmid, pACYCDuet-1 plasmid, Escherichia coli BL21, Escherichia coli DH5α, and Escherichia coli XL-Blue used in the following examples were purchased from Novagen.

[0055] Example 1

[0056] This embodiment provides a recombinant Escherichia coli and a method for constructing the same. The specific steps are as follows:

[0057] The fully synthetic LTA recombinant plasmid and the pETDuet-1 vector were double-digested with restriction endonucleases EcoRI and HindIII, and the fully synthetic TD recombinant plasmid and the pETDuet-1 vector were double-digested with restriction endonucleases NdeI and XhoI, respectively. LTA and TD from different sources were ligated to the pETDuet-1 vector in pairs using T4 DNA ligase to obtain recombinant plasmid 1; the fully synthetic LeuDH recombinant plasmid and the pCDFDuet-1 vector were double-digested with restriction endonucleases EcoRI and HindIII, and the fully synthetic P4H recombinant plasmid and the pCDFDuet-1 vector were double-digested with restriction endonucleases NdeI and XhoI, and LeuDH and P4H from different sources were ligated to the pCDFDuet-1 vector in pairs using T4 DNA ligase to obtain recombinant plasmid 2; the fully synthetic LeuDH recombinant plasmid and the pCDFDuet-1 vector were double-digested with restriction endonucleases EcoRI and HindIII, and the fully synthetic P4H recombinant plasmid and the pCDFDuet-1 vector were double-digested with restriction endonucleases NdeI and XhoI, and LeuDH and P4H from different sources were ligated to the pCDFDuet-1 vector in pairs using T4 DNA ligase to obtain recombinant plasmid 2; The fully synthetic PCD recombinant plasmid and the pACYCDuet-1 vector were double-digested with restriction endonucleases NdeI and XhoI, respectively; the fully synthetic DHMR recombinant plasmid and the pACYCDuet-1 vector were double-digested with restriction endonucleases NdeI and XhoI, respectively; PCD and DHMR from different sources were ligated in pairs to the pACYCDuet-1 vector using T4 DNA ligase to obtain recombinant plasmid 3; different recombinant plasmids 1, 2, and 3 were combined and transformed into E. coli BL21 (DE3) competent cells, respectively, to obtain recombinant E. coli.

[0058] Example 2

[0059] This example is to induce the culture of the recombinant Escherichia coli in Example 1, and the specific steps are as follows:

[0060] The recombinant E. coli was inoculated into LB medium containing 50 mg / L ampicillin, 50 mg / L streptomycin and 50 mg / L chloramphenicol, and cultured at 37°C and 200 rpm for 12 hours to obtain seed liquid. The seed liquid was inoculated into fresh LB medium at a 2% inoculation rate, and cultured at 37°C and 200 rpm until the bacterial concentration OD600nm reached 0.7. 0.5 mM IPTG was added, and after induction at 28°C for 15 hours, the culture was centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded, and the wet bacteria were washed twice with 0.9% saline, centrifuged, and set aside.

[0061] Example 3

[0062] This example is a comparison of the transformation abilities of various recombinant E. coli

[0063] The collected recombinant E. coli was resuspended in a 50 mL system, with a final cell concentration of 20 g / L, a benzaldehyde concentration of 50 g / L, a glycine concentration of 40 g / L, and a glucose concentration of 80 g / L, pH 8.0, and reacted at 30°C, a shaking speed of 200 rpm, and a conversion time of 24 h. After the conversion, the yield of L-tyrosine was determined by HPLC.

[0064] Table 1 Comparison of L-tyrosine production of various recombinant bacteria

[0065]

[0066] Example 4

[0067] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21 (DE3) / pETDuet-PsLTA-PvTD + pCDFDuet-FgLeuDH-PaP4H + pACYCDuet-ApPCD-PvDHMR was completed, the cells were collected and placed in a 50mL system with a cell wet weight of 1g / L, benzaldehyde of 1g / L, glycine of 1g / L, glucose of 20g / L, pH 8.0, temperature of 30°C, shaker speed of 200rpm, and conversion time of 24h. The HPLC determination results showed that the L-tyrosine yield was 1.7g / L.

[0068] Example 5

[0069] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21 (DE3) / pETDuet-PsLTA-PvTD + pCDFDuet-FgLeuDH-PaP4H + pACYCDuet-ApPCD-PvDHMR was completed, the cells were collected and placed in a 50mL system with a cell wet weight of 3g / L, benzaldehyde of 5g / L, glycine of 4g / L, glucose of 20g / L, pH 8.0, temperature of 35°C, shaker speed of 200rpm, and conversion time of 24h. The HPLC determination results showed that the L-tyrosine yield was 8.4g / L.

[0070] Example 6

[0071] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21 (DE3) / pETDuet-PsLTA-PvTD + pCDFDuet-FgLeuDH-PaP4H + pACYCDuet-ApPCD-PvDHMR was completed, the cells were collected and placed in a 50mL system with a cell wet weight of 5 g / L, benzaldehyde of 8 g / L, glycine of 6.5 g / L, glucose of 20 g / L, pH of 8.0, temperature of 30°C, shaker speed of 200 rpm, and conversion time of 24 h. The HPLC determination results showed that the L-tyrosine yield was 13.4 g / L.

[0072] Example 7

[0073] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21 (DE3) / pETDuet-PsLTA-PvTD + pCDFDuet-FgLeuDH-PaP4H + pACYCDuet-ApPCD-PvDHMR was completed, the cells were collected and placed in a 50mL system with a cell wet weight of 8 g / L, benzaldehyde of 12 g / L, glycine of 10 g / L, glucose of 20 g / L, pH 8.0, temperature of 30°C, shaker speed of 200 rpm, and conversion time of 24 h. The HPLC determination results showed that the L-tyrosine yield was 20.2 g / L.

[0074] Example 8

[0075] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21 (DE3) / pETDuet-PsLTA-PvTD + pCDFDuet-FgLeuDH-PaP4H + pACYCDuet-ApPCD-PvDHMR was completed, the cells were collected and placed in a 50mL system with a cell wet weight of 10 g / L, benzaldehyde 20 g / L, glycine 18 g / L, glucose 40 g / L, pH 8.0, temperature 30°C, shaker speed 200 rpm, and conversion time 24 h. The HPLC determination results showed that the L-tyrosine yield was 34.1 g / L.

[0076] Example 9

[0077] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21 (DE3) / pETDuet-PsLTA-PvTD + pCDFDuet-FgLeuDH-PaP4H + pACYCDuet-ApPCD-PvDHMR was completed, the cells were collected and placed in a 50 mL system with a cell wet weight of 15 g / L, benzaldehyde 40 g / L, glycine 35 g / L, glucose 60 g / L, pH 8.0, temperature 30°C, shaker speed 200 rpm, and conversion time 24 h. The HPLC determination results showed that the L-tyrosine yield was 66.3 g / L.

[0078] Example 10

[0079] According to the induction expression method described in Example 2, the cells of E. coli BL21 (DE3) / pETDuet-PsLTA-PvTD + pCDFDuet-FgLeuDH-PaP4H + pACYCDuet-ApPCD-PvDHMR were collected after the induction expression was completed. In a 50mL system, the wet weight of the cells was 18 g / L, benzaldehyde was 45 g / L, glycine was 40 g / L, glucose was 70 g / L, pH was 8.0, the temperature was 30°C, the shaking speed was 200 rpm, and the conversion time was 24 hours. The HPLC determination results showed that the L-tyrosine yield was 75.2 g / L.

[0080] Embodiment 11

[0081] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21 (DE3) / pETDuet-PsLTA-PvTD + pCDFDuet-FgLeuDH-PaP4H + pACYCDuet-ApPCD-PvDHMR was completed, the cells were collected and placed in a 50mL system with a cell wet weight of 20 g / L, benzaldehyde of 30 g / L, glycine of 25 g / L, glucose of 50 g / L, pH 8.0, temperature of 30°C, shaker speed of 200 rpm, and conversion time of 12 h. The HPLC determination results showed that the L-tyrosine yield was 50.2 g / L.

[0082] Example 12

[0083] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21 (DE3) / pETDuet-PsLTA-PvTD + pCDFDuet-FgLeuDH-PaP4H + pACYCDuet-ApPCD-PvDHMR was completed, the cells were collected and placed in a 50mL system with a cell wet weight of 20 g / L, benzaldehyde of 15 g / L, glycine of 12 g / L, glucose of 30 g / L, pH 8.0, temperature of 30°C, shaker speed of 200 rpm, and conversion time of 6 hours. The HPLC determination results showed that the L-tyrosine yield was 25.4 g / L.

[0084] Embodiment 13

[0085] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21 (DE3) / pETDuet-PsLTA-PvTD + pCDFDuet-FgLeuDH-PaP4H + pACYCDuet-ApPCD-PvDHMR was completed, the cells were collected and placed in a 50mL system with a cell wet weight of 15 g / L, benzaldehyde 10 g / L, glycine 8 g / L, glucose 20 g / L, pH 8.0, temperature 30°C, shaker speed 200 rpm, and conversion time 8 hours. The HPLC determination results showed that the L-tyrosine yield was 16.7 g / L.

[0086] Embodiment 14

[0087] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21 (DE3) / pETDuet-PsLTA-PvTD + pCDFDuet-FgLeuDH-PaP4H + pACYCDuet-ApPCD-PvDHMR was completed, the cells were collected and placed in a 50mL system with a cell wet weight of 12 g / L, benzaldehyde 20 g / L, glycine 16 g / L, glucose 40 g / L, pH 8.0, temperature 30°C, shaker speed 200 rpm, and conversion time 15 h. The HPLC determination results showed that the L-tyrosine yield was 33.7 g / L.

[0088] Embodiment 15

[0089] According to the induction expression method described in Example 2, the cells of E. coli BL21 (DE3) / pETDuet-PsLTA-PvTD + pCDFDuet-FgLeuDH-PaP4H + pACYCDuet-ApPCD-PvDHMR were collected after the induction expression was completed. In a 50mL system, the wet weight of the cells was 10g / L, benzaldehyde was 15g / L, glycine was 12g / L, glucose was 30g / L, pH was 6.0, the temperature was 35°C, the shaking speed was 200rpm, and the conversion time was 12h. The HPLC determination results showed that the L-tyrosine yield was 25.2g / L.

[0090] Example 16

[0091] According to the induction expression method described in Example 2, the cells of E. coli BL21 (DE3) / pETDuet-PsLTA-PvTD + pCDFDuet-FgLeuDH-PaP4H + pACYCDuet-ApPCD-PvDHMR were collected after the induction expression was completed. In a 50mL system, the wet weight of the cells was 15g / L, benzaldehyde was 25g / L, glycine was 20g / L, glucose was 50g / L, pH was 7.0, the temperature was 35°C, the shaking speed was 200rpm, and the conversion time was 12h. The HPLC determination results showed that the L-tyrosine yield was 42.3g / L.

[0092] Embodiment 17

[0093] According to the induction expression method described in Example 2, the cells of E. coli BL21 (DE3) / pETDuet-PsLTA-PvTD + pCDFDuet-FgLeuDH-PaP4H + pACYCDuet-ApPCD-PvDHMR were collected after the induction expression was completed. In a 50mL system, the wet weight of the cells was 13g / L, benzaldehyde was 20g / L, glycine was 17g / L, glucose was 40g / L, pH was 7.5, the temperature was 35°C, the shaking speed was 200rpm, and the conversion time was 12h. The HPLC determination results showed that the L-tyrosine yield was 33.4g / L.

[0094] Embodiment 18

[0095] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21 (DE3) / pETDuet-PsLTA-PvTD + pCDFDuet-FgLeuDH-PaP4H + pACYCDuet-ApPCD-PvDHMR was completed, the cells were collected and placed in a 50mL system with a cell wet weight of 7 g / L, benzaldehyde 7 g / L, glycine 6 g / L, glucose 20 g / L, pH 8.5, temperature 35°C, shaker speed 200 rpm, and conversion time 12 h. The HPLC determination results showed that the L-tyrosine yield was 11.8 g / L.

[0096] Embodiment 19

[0097] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21 (DE3) / pETDuet-PsLTA-PvTD + pCDFDuet-FgLeuDH-PaP4H + pACYCDuet-ApPCD-PvDHMR was completed, the cells were collected and placed in a 50mL system with a cell wet weight of 4g / L, benzaldehyde 5g / L, glycine 4g / L, glucose 20g / L, pH 9.0, temperature 35°C, shaker speed 200rpm, and conversion time 12h. The HPLC determination results showed that the L-tyrosine yield was 8.2g / L.

[0098] Embodiment 20

[0099] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21 (DE3) / pETDuet-PsLTA-PvTD + pCDFDuet-FgLeuDH-PaP4H + pACYCDuet-ApPCD-PvDHMR was completed, the cells were collected and placed in a 50mL system with a cell wet weight of 16 g / L, benzaldehyde 40 g / L, glycine 32 g / L, glucose 60 g / L, pH 7.5, temperature 15°C, shaker speed 200 rpm, and conversion time 24 h. HPLC determination results showed that the L-tyrosine yield was 67.7 g / L.

[0100] Embodiment 21

[0101] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21 (DE3) / pETDuet-PsLTA-PvTD + pCDFDuet-FgLeuDH-PaP4H + pACYCDuet-ApPCD-PvDHMR was completed, the cells were collected and placed in a 50mL system with a cell wet weight of 10 g / L, benzaldehyde 25 g / L, glycine 20 g / L, glucose 50 g / L, pH 7.5, temperature 25°C, shaker speed 200 rpm, and conversion time 24 h. HPLC determination results showed that the L-tyrosine yield was 42.5 g / L.

[0102] Embodiment 22

[0103] According to the induction expression method described in Example 2, the cells of E. coli BL21 (DE3) / pETDuet-PsLTA-PvTD + pCDFDuet-FgLeuDH-PaP4H + pACYCDuet-ApPCD-PvDHMR were collected after the induction expression was completed. In a 50mL system, the wet weight of the cells was 11g / L, benzaldehyde was 30g / L, glycine was 24g / L, glucose was 50g / L, pH was 7.5, the temperature was 40°C, the shaking speed was 200rpm, and the conversion time was 24h. The HPLC determination results showed that the L-tyrosine yield was 51.1g / L.

[0104] Embodiment 23

[0105] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21 (DE3) / pETDuet-PsLTA-PvTD + pCDFDuet-FgLeuDH-PaP4H + pACYCDuet-ApPCD-PvDHMR was completed, the cells were collected and placed in a 50mL system with a cell wet weight of 30 g / L, benzaldehyde of 60 g / L, glycine of 50 g / L, glucose of 80 g / L, pH 7.5, temperature of 35°C, shaker speed of 200 rpm, and conversion time of 36 h. The HPLC determination results showed that the L-tyrosine yield was 11.2 g / L.

[0106] Embodiment 24

[0107] According to the induction expression method described in Example 2, the cells of E. coli BL21 (DE3) / pETDuet-PsLTA-PvTD + pCDFDuet-FgLeuDH-PaP4H + pACYCDuet-ApPCD-PvDHMR were collected after the induction expression was completed. In a 50mL system, the wet weight of the cells was 25g / L, benzaldehyde was 40g / L, glycine was 35g / L, glucose was 80g / L, pH was 5.5, the temperature was 35°C, the shaking speed was 200rpm, and the conversion time was 36h. The HPLC determination results showed that the L-tyrosine yield was 8.5g / L.

[0108] Embodiment 25

[0109] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21 (DE3) / pETDuet-PsLTA-PvTD + pCDFDuet-FgLeuDH-PaP4H + pACYCDuet-ApPCD-PvDHMR was completed, the cells were collected and placed in a 50mL system with a cell wet weight of 15 g / L, benzaldehyde 55 g / L, glycine 45 g / L, glucose 90 g / L, pH 9.5, temperature 35°C, shaker speed 200 rpm, and conversion time 36 h. HPLC determination results showed that the L-tyrosine yield was 6.7 g / L.

[0110] Embodiment 26

[0111] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21 (DE3) / pETDuet-PsLTA-PvTD + pCDFDuet-FgLeuDH-PaP4H + pACYCDuet-ApPCD-PvDHMR was completed, the cells were collected and placed in a 50mL system with a cell wet weight of 20 g / L, benzaldehyde 40 g / L, glycine 50 g / L, glucose 80 g / L, pH 7.5, temperature 10°C, shaker speed 200 rpm, and conversion time 36 h. HPLC determination results showed that the L-tyrosine yield was 10.2 g / L.

[0112] Embodiment 27

[0113] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21 (DE3) / pETDuet-PsLTA-PvTD + pCDFDuet-FgLeuDH-PaP4H + pACYCDuet-ApPCD-PvDHMR was completed, the cells were collected and placed in a 50mL system with a cell wet weight of 30 g / L, benzaldehyde 50 g / L, glycine 45 g / L, glucose 90 g / L, pH 8.0, temperature 45°C, shaker speed 200 rpm, and conversion time 36 h. HPLC determination results showed that the L-tyrosine yield was 5.9 g / L.

[0114] Embodiment 28

[0115] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21 (DE3) / pETDuet-PsLTA-PvTD + pCDFDuet-FgLeuDH-PaP4H + pACYCDuet-ApPCD-PvDHMR was completed, the cells were collected and placed in a 50mL system with a cell wet weight of 0.5 g / L, benzaldehyde of 0.5 g / L, glycine of 0.4 g / L, glucose of 5 g / L, pH 8.0, temperature of 30°C, shaker speed of 200 rpm, and conversion time of 36 h. The HPLC determination results showed that the L-tyrosine yield was 0.2 g / L.

[0116] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A recombinant bacterium for producing L-tyrosine, characterized in that: The recombinant bacteria contain exogenous genes; the exogenous genes include genes encoding threonine aldolase, threonine deaminase, leucine dehydrogenase, phenylalanine hydroxylase, pterin 4a-methanolamine dehydratase and dihydropterin reductase; The host of the recombinant bacteria is Escherichia coli.

2. The recombinant bacterium according to claim 1, characterized in that The threonine aldolase includes AhLTA and PsLTA. The amino acid sequence of AhLTA is shown in SEQ ID NO.1; the amino acid sequence of PsLTA is shown in SEQ ID NO.

2.

3. The recombinant bacterium according to claim 1, characterized in that The threonine deaminase includes VaTD and PvTD. The amino acid sequence of VaTD is shown as SEQ ID NO.5; the amino acid sequence of PvTD is shown as SEQ ID NO.

6.

4. The recombinant bacterium according to claim 1, characterized in that The leucine dehydrogenase includes FgLeuDH and AmLeuDH. The amino acid sequence of FgLeuDH is shown in SEQ ID NO.9; the amino acid sequence of AmLeuDH is shown in SEQ ID NO.

10.

5. The recombinant bacterium according to claim 1, characterized in that The phenylalanine hydroxylase includes VcP4H and PaP4H. The amino acid sequence of VcP4H is shown in SEQ ID NO.13; the amino acid sequence of PaP4H is shown in SEQ ID NO.

14.

6. The recombinant bacterium according to claim 1, characterized in that The pterin 4a-methanolamine dehydratase includes ApPCD and GaPCD, the amino acid sequence of ApPCD is shown as SEQ ID NO.17; the amino acid sequence of GaPCD is shown as SEQ ID NO.

18.

7. The recombinant bacterium according to claim 1, characterized in that The dihydropterin reductase includes PvDHMR and KmDHMR, the amino acid sequence of PvDHMR is shown as SEQ ID NO.21; the amino acid sequence of KmDHMR is shown as SEQ ID NO.

22.

8. The recombinant bacterium according to claim 1, characterized in that The nucleotide sequence of AhLTA in the threonine aldolase is shown as SEQ ID NO.3; the nucleotide sequence of PsLTA is shown as SEQ ID NO.

4.

9. The recombinant bacterium according to claim 1, characterized in that The nucleotide sequence of VaTD in the threonine deaminase is shown as SEQ ID NO.7; the nucleotide sequence of PvTD is shown as SEQ ID NO.

8.

10. The recombinant bacterium according to claim 1, characterized in that The nucleotide sequence of FgLeuDH in the leucine dehydrogenase is shown as SEQ ID NO.11; the nucleotide sequence of AmLeuDH is shown as SEQ ID NO.

12.

11. The recombinant bacterium according to claim 1, characterized in that The nucleotide sequence of VcP4H in the phenylalanine hydroxylase is shown as SEQ ID NO.15; the nucleotide sequence of PaP4H is shown as SEQ ID NO.

16.

12. The recombinant bacterium according to claim 1, characterized in that The nucleotide sequence of ApPCD in the pterin 4a-methanolamine dehydratase is shown as SEQ ID NO.19; the nucleotide sequence of GaPCD is shown as SEQ ID NO.

20.

13. The recombinant bacterium according to claim 1, characterized in that The nucleotide sequence of PvDHMR in the dihydropterin reductase is shown as SEQ ID NO.23; the nucleotide sequence of KmDHMR is shown as SEQ ID NO.

24.

14. The recombinant bacterium according to claim 1, characterized in that The Escherichia coli is selected from any one of Escherichia coli BL21, Escherichia coli DH5α and Escherichia coli XL-Blue.

15. The recombinant bacterium according to claim 1, characterized in that The recombinant bacteria contain expression vectors, which include pETDuet-1 plasmid, pCDFDuet-1 plasmid and pACYCDuet-1 plasmid.

16. The method for constructing a recombinant bacterium according to any one of claims 1 to 15, characterized in that: The genes of threonine aldolase, threonine deaminase, leucine dehydrogenase, phenylalanine hydroxylase, pterin 4a-methanolamine dehydratase and dihydropterin reductase are connected to an expression vector, and then the obtained recombinant expression vector is introduced into a host bacterium to obtain the recombinant bacterium.

17. The method for constructing a recombinant bacterium according to claim 16, characterized in that: The recombinant bacteria simultaneously contain pETDuet-1 plasmid, pCDFDuet-1 plasmid and pACYCDuet-1 plasmid.

18. The method for constructing a recombinant bacterium according to claim 16, wherein the pETDuet-1 plasmid is connected with genes encoding threonine aldolase and threonine deaminase.

19. The method for constructing a recombinant bacterium according to claim 16, wherein the pCDFDuet-1 plasmid is connected with genes encoding leucine dehydrogenase and phenylalanine hydroxylase.

20. The method for constructing a recombinant bacterium according to claim 16, wherein the pETDuet-1 plasmid is connected with genes encoding pterin 4a-carbinolamine dehydratase and dihydropterin reductase.

21. A method for producing L-tyrosine, characterized in that: The method comprises inducing and culturing the recombinant bacteria constructed by the construction method of the recombinant bacteria according to any one of claims 16 to 20, adding the recombinant bacteria to a transformation system for whole-cell transformation to obtain L-tyrosine; wherein the recombinant bacteria synthesizes L-tyrosine using benzaldehyde and glycine as substrates.

22. The method for producing L-tyrosine according to claim 21, characterized in that The conversion system comprises benzaldehyde with a concentration of 1-50 g / L, glycine with a concentration of 1-40 g / L, and glucose with a concentration of 20-80 g / L.

23. The method for producing L-tyrosine according to claim 21, characterized in that The amount of fresh bacteria of the recombinant bacteria in the transformation system is 1-20 g / L.

24. The method for producing L-tyrosine according to claim 21, characterized in that The pH of the conversion system is 6.0-9.

0.

25. The method for producing L-tyrosine according to claim 21, characterized in that The conversion temperature of the conversion system is 15-40°C.

26. The method for producing L-tyrosine according to claim 21, characterized in that The transformation time of the transformation system is 6-24 h.

27. The method for producing L-tyrosine according to claim 21, characterized in that The induction culture of the recombinant bacteria includes inoculating the recombinant bacteria into a selective culture medium to obtain a seed solution, then inoculating the seed solution into a basic culture medium and culturing until the OD600nm reaches 0.7, adding an inducer for induction, and washing the wet bacteria after completion.

28. The method for producing L-tyrosine according to claim 27, characterized in that The recombinant bacteria are cultured in a selective culture medium at a temperature of 30-40° C. for 10-15 hours.

29. The method for producing L-tyrosine according to claim 27, characterized in that The culture temperature of the seed solution in the basic culture medium is 30-40°C.

30. The method for producing L-tyrosine according to claim 27, characterized in that The induction temperature in the induction step is 25-30° C. and the time is 12-18 h.

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