CN108949647A - A kind of engineering bacteria and its application in production l-tyrosine - Google Patents

Abstract

The invention discloses a kind of engineering bacteria and its applications in production l-tyrosine, belong to technical field of bioengineering.The production method for producing l-tyrosine the invention proposes the conversion lactic acid and phenol that do not generate hydrogen oxide constructs the engineering bacteria of three enzymes coexpression, realizes the efficient production of l-tyrosine on the basis of Escherichia coli transhipment and coenzyme synthetic system is transformed.The method of resting cell production l-tyrosine of the invention, process is simple, impurity is few, has important industrial application value.

Description

A kind of engineering bacterium and its application in producing L-tyrosine

technical field

The invention relates to an engineering bacterium and its application in producing L-tyrosine, belonging to the technical field of bioengineering.

Background technique

L-tyrosine (p-hydroxyphenylalanine) is an essential amino acid for the human body and has important applications in medicine and food. At present, L-tyrosine is extracted, enzymatically, and fermented by genetically engineered bacteria. The process of hydrolyzing and extracting L-tyrosine from wool method and other raw materials has a lot of pollution and low purity. Escherichia coli engineering bacteria use glucose as a raw material to synthesize L-tyrosine with a low yield, which is currently unable to compete with extraction and enzymatic methods.

Enzymatic production of L-tyrosine with tyrosine phenol lyase is the most widely used method. A variety of schemes have been designed (Synthesis of L-Tyrosine or 3,4-Dihydroxyphenyl-L-alanine from DL-Serine and Phenol or Pyrocathechol, Agric. Biol. Chem. 37 (1973) 493-499. Chinese Patent 201310168119.5), Among them, the conversion method using pyruvate and phenol as substrates has been implemented industrially. Pyruvate is a relatively expensive intermediate, so patent 201310289373.0 uses unpurified pyruvic acid feed solution to directly convert, or first oxidize lactic acid with lactic acid oxidase to generate pyruvic acid, and then further catalyze the production of L-tyrosine (multiple Enzyme-coupled biosynthesis of pyruvate and L-tyrosine, 2014, Master Thesis of Nanjing University), but pyruvate is easy to decompose, and the efficiency of this scheme is not high.

Contents of the invention

Based on the defects of various current methods, the present invention proposes a production method of converting lactic acid and phenol to produce L-tyrosine without generating hydrogen oxide, and constructs a co-expression of three enzymes on the basis of transforming E. coli transport and coenzyme synthesis system The engineered bacteria realized the high-efficiency production of L-tyrosine. The technical problem to be solved by the present invention is to provide a recombinant bacterium capable of producing L-tyrosine and reducing the generation of impurities, and at the same time, the present invention is to solve the technical problem of the construction and application of the strain.

The first object of the present invention is to provide recombinant Escherichia coli capable of producing pure doloxetamine at low cost; said recombinant Escherichia coli expresses exogenous L-lactate dehydrogenase, NADH oxidase and tyrosine phenol lyase simultaneously, And the L-tyrosine uptake gene was knocked out on the basis of the host Escherichia coli.

In one embodiment, the exogenous L-lactate dehydrogenase is L-lactate dehydrogenase derived from lactic acid bacteria. The exogenous NADH oxidase is NADH oxidase derived from lactic acid bacteria.

In one embodiment, the lactate dehydrogenase is from Lactococcus lactis ATCC 19257, Lactobacillus plantarum ATCC 14917.

In one embodiment, the amino acid sequence of the lactate dehydrogenase is the sequence whose accession NO is WP_003131075.1 and KRL33571.1 on NCBI.

In one embodiment, the nucleotide sequence of the lactate dehydrogenase is the sequence whose accession NO on NCBI is: NZ_JXJZ01000017REGION:18532..19509, AZEJ01000016REGION:16296..17249.

In one embodiment, the NADH oxidase is from Lactococcus lactis ATCC 19257, Lactobacillus sanfranciscensis DSM20451, Lactobacillus brevis ATCC 14869.

In one embodiment, the amino acid sequence of the NADH oxidase is the sequence of accession NO WP_032950924.1, WP_056958268.1 and ERK43827.1 on NCBI.

In one embodiment, the nucleotide sequence of the NADH oxidase is the accession NO on NCBI: NZ_JXJZ01000002REGION:complement(39571..40911), NZ_AYYM01000013REGION:complement(15875..17233), AWVK01000048REGION:complement(50022. .51416) sequence.

In one embodiment, the L-lactate dehydrogenase, NADH oxidase, and tyrosine phenol lyase are co-expressed by pETDuet-1.

In one embodiment, the tyrosine phenol lyase is derived from Erwinia herbicola, Citrobacter intermedius, Citrobacter freundii, Symbiobacterium thermophilum or Symbiobacterium toebii .

In one embodiment, the tyrosine phenol lyase is derived from Citrobacter freundii ATCC8090, and its amino acid sequence is the sequence whose accession NO is WP_003837154.1 on NCBI.

In one embodiment, the L-tyrosine uptake gene includes any one or a combination of L-tyrosine decomposing gene and phenol decomposing gene.

In one embodiment, the L-tyrosine decomposition gene is any one or a combination of hpaD and mhpB.

In one embodiment, the phenol decomposing gene is any one or a combination of hpaD and mhpB.

In one embodiment, the nucleotide sequences of the L-tyrosine decomposing gene and the phenol decomposing gene are on NCBI, and the accession NO on NCBI is: NC_012892REGION:complement (4505585..4506436) and NC_012892 REGION:339806. .340750.

In one embodiment, the recombinant Escherichia coli further expresses any one or more of lactate transporter genes, phenol transport-related genes, and ammonium ion transport genes that transport substrates into cells.

In one embodiment, the recombinant Escherichia coli further enhances the expression of any one or more of NAD synthesis genes and pyridoxal phosphate synthesis-related genes.

In one embodiment, the genes for enhanced expression are lldP (lactic acid transport gene), amtB (ammonium ion transport gene), hpaX (phenol transport gene), mhpT (phenol transport gene), icsA (NAD synthesis gene) Any one or more of , nadA (NAD synthesis gene), pdxJ (pyridoxal phosphate synthesis gene).

In one embodiment, the host bacteria is Escherichia coli BL21(DE3).

In one embodiment, the enhanced expression is achieved by adding a constitutive promoter in front of the gene to be expressed on the Escherichia coli BL21 (DE3) genome.

In one embodiment, the accession NO of the lldP on NCBI is: NC_012892REGION:3646638..3648293; amtB is NC_012892REGION:442006..443292; hpaX is; NC_012892REGION:complement(4502025..4503401); mtB is 92NC 344788..345999; icsA is NC_012892REGION:complement(2526116..2527330); nadA is NC_012892REGION:740487..741530; pdxJ is NC_012892REGION:complement(2567591..2568322).

The second object of the present invention is to provide a method for producing optically pure L-tyrosine by using the recombinant bacteria of the present invention.

In one embodiment, the production of L-tyrosine is carried out by transformation of whole cells.

In one embodiment, in the system produced by whole cell transformation, the wet cell weight is 1-200g/L, the L-lactic acid is 1-200g/L, the phenol concentration is 1-200g/L, the pH is 7.0-9.0, and the temperature is 15 -40°C, shaker speed 250 rpm; transformation time 1-24 hours.

The third object of the present invention is to provide the application of the recombinant bacterium of the present invention or the method of the present invention in the fields of chemical industry, food, medicine and the like.

Beneficial effects of the present invention:

The invention constructs a novel three-enzyme co-expression genetically engineered bacterium, which can be applied to the production of L-tyrosine. The selection scheme of the present invention does not produce hydrogen peroxide, the cells are not easy to decompose L-tyrosine, and the cells have relatively high NAD content. The production process is simple and the raw materials are easy to obtain, and has good industrial application prospect.

specific implementation plan

The functional core of the Escherichia coli engineering bacteria of the present invention can co-express three kinds of enzymes, which are respectively L-lactate dehydrogenase (L-Lactate dehydrogenase), NADH oxidase (NADH oxidase), tyrosinephenol lyase (tyrosinephenol- lyase). The principle is: in the whole cell of engineering bacteria, L-lactate dehydrogenase dehydrogenates L-lactate to pyruvate and NADH with NAD in the bacteria as a coenzyme; NADH oxidase oxidizes NADH to NAD, realizing the coenzyme NAD regeneration. Then, under the action of tyrosine phenol lyase, pyruvate, ammonium ion, and phenol are synthesized into L-tyrosine, and at the same time knockout or enhanced expression of related genes on the Escherichia coli genome promotes the transport of substrates and reduces products decomposition.

In order to solve the problems of the technologies described above, the technical scheme adopted in the present invention is as follows:

1. Bacterial strains and plasmids involved in the present invention

Lactobacillus plantarum ATCC 14917, Lactococcus lactis ATCC 19257, Lactobacillus brevis ATCC 14869, Citrobacterfreundii ATCC 8090 were purchased from American Culture Collection ATCC. coli BL21(DE3). Lactobacillus sanfranciscensis DSM20451 was purchased from DSMZ, the German Culture Collection of Microorganisms. pCasRed and pCRISPR-gDNA were purchased from Zhenjiang Aibimeng Biotechnology Co., Ltd.

2. Knockout and constitutive enhanced expression of related genes in Escherichia coli

(1) Knockout of Escherichia coli aroma compound degradation gene

Both phenol and L-tyrosine in the present invention are easily decomposed by enzymes in Escherichia coli, according to the literature (Biodegradation of Aromatic Compounds by Escherichia coli, Microbiol Mol BiolRev.2001,65(4):523-569.), Knock out related genes to avoid decomposition of products and substrates. The selected genes are hpaD and mhpB, and the accession NO on NCBI is: NC_012892REGION:complement(4505585..4506436) and NC_012892REGION:339806..340750.

(2) Constitutive enhanced expression of lactic acid, phenol, and ammonium ion transport genes in Escherichia coli

In the process of whole-cell transformation, the substrate needs to be transported into the cell. Enhancing the lactate transporter helps to maintain a high concentration of the intracellular substrate quickly and for a long time, which is conducive to the reaction. The gene related to lactate transport is lldP, and the accession NO on NCBI is: NC_012892REGION:3646638..3648293. The genes related to phenol transport are hpaX and mhpT, and the accession NO on NCBI is: NC_012892REGION:complement(4502025..4503401) and NC_012892REGION:344788..345999. The gene related to ammonium ion transport is amtB, and the accession NO on NCBI is: NC_012892REGION:442006..443292.

(3) Overexpression of genes related to NAD synthesis in Escherichia coli

In the process of lactate dehydrogenation, NAD is needed as a coenzyme, and the key enzymes of the NAD synthesis pathway in E. coli can be enhanced to increase the level of NAD in the bacteria, thereby facilitating the generation of pyruvate. The selected genes are icsA and nadA. The accession NO on NCBI is: NC_012892 REGION:complement(2526116..2527330), NC_012892REGION:740487..741530.

(4) Expression of genes related to Pyridoxal phosphate synthesis

Pyridoxal (amine) phosphate is a coenzyme of tyrosine lyase, overexpressing the core gene pdxJ in the coenzyme pathway is beneficial to the synthesis of L-tyrosine. The accession NO on NCBI is: NC_012892REGION:complement(2567591..2568322).

3. Selection of enzymes involved in conversion of lactic acid to pyruvate

(1) Selection of L-lactate dehydrogenase

L-lactic acid is the cheapest organic acid. At present, L-lactic acid oxidase is mainly used to oxidize L-lactic acid to produce pyruvate. During this process, hydrogen peroxide is produced, and hydrogen peroxide will oxidize pyruvate to produce acetic acid. L-lactate dehydrogenase widely exists in a variety of microorganisms. Usually, lactate dehydrogenase with NAD (NADP) as a coenzyme tends to use pyruvate as a substrate to synthesize lactic acid. However, when lactic acid is excessive or the carbon source is only lactic acid, some lactic acid Dehydrogenase will remove the hydrogen of lactic acid to generate pyruvate, and use L-lactic acid as a substrate to transfer the hydrogen generated on L-lactic acid to the coenzyme NAD or NADP, thereby generating NADH or NADPH.

The present invention obtains L-lactate dehydrogenase genes llldh (amino acid sequence is WP_003131075.1) and lpldh (amino acid sequence is KRL33571.1) respectively from Lactococcus lactis ATCC 19257 and Lactobacillus plantarum ATCC14917, and the expression products are used for the dehydrogenation of lactic acid.

(2) Selection of NADH oxidase

Lactate dehydrogenase dehydrogenates lactate to NADH pyruvate. NADH needs to be oxidized by NAD oxidase to regenerate NAD, so as to realize the continuous progress of the reaction. There are two types of NADH oxidase, the water-producing type and the hydrogen peroxide-producing type, and the water-producing NADH oxidase will not produce hydrogen peroxide toxicity. The present invention obtains water-producing NADH oxidase genes lsnox (amino acid sequence is WP_056958268.1), llnox (amino acid sequence is WP_003131075.1), lbnox (amino acid sequence is ERK43827) from Lactobacillus sanfranciscensis DSM20451, Lactococcus lactis ATCC 19257, and Lactobacillus brevis respectively , the expression product is used for the regeneration of NAD. .

(3) Selection of tyrosine phenol lyase

Tyrosine phenol lyases from Erwinia herbicola, Citrobacter intermedius, Citrobacter freundii, and thermophiles Symbiobacterium thermophilum and Symbiobacterium toebii are the most commonly studied. The present invention selects the enzyme from Citrobacter freundii with higher activity, and obtains the tyrosine phenol lyase gene cftpl (amino acid sequence is WP_003837154.1) from Citrobacter freundii ATCC 8090.

4. Construction of co-expression system and cell culture

At present, there are multiple methods for the co-expression of Escherichia coli multigenes (Escherichia coli multigene co-expression strategy, China Biotechnology Journal, 2012, 32 (4): 117-122). Production of shikimic acid and resveratrol, 2016, Shanghai Pharmaceutical Industry Research Institute, Ph. and RBS, so the expression intensity of genes is not greatly affected by the arrangement order. Each plasmid contains three genes, and the constructed plasmid is thermally transfected into E. coli competent cells, and coated on an antibiotic solid plate, and positive transformants are screened to obtain recombinant E. coli. Cell culture: According to the classical recombinant Escherichia coli culture and induced expression scheme, the recombinant Escherichia coli was transferred to LB fermentation medium (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L in volume ratio of 2%). In L), when the OD ₆₀₀ of the cells reached 0.6-0.8, IPTG with a final concentration of 0.4 mM was added, and expression was induced at 20°C for 8 hours. After induction of expression, the cells were collected by centrifugation at 20° C., 8000 rpm, and 20 minutes.

5. Whole Cell Transformation to Produce L-Tyrosine

The whole cell transformation system is: cell wet weight 1-200g/L, L-lactic acid 1-200g/L, phenol 1-200g/L, pH7.0-9.0, temperature 20-50℃, shaker speed 250 rpm minutes; conversion time 1-24 hours.

6. Detection and analysis of samples

The quantitative analysis of L-tyrosine was detected and analyzed by PerkinElmer Series 200 high-performance liquid chromatography equipped with a differential refractive index detector. The chromatographic conditions are as follows: mobile phase is methanol-0.1% formic acid water (40:60), Hanbang Megres C18 column (4.6×250mm, 5μm), flow rate 1ml/min, column temperature 30℃, injection volume 20μl.

In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer, the present invention will be described in detail below in conjunction with the embodiments. It should be noted that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

Example 1

According to the method described in the literature Large scale validation of an efficient CRISPR/Cas-based multigene editing protocol in Escherichia coli. Microbial Cell Factories, 2017, 16(1): 68, hpaD and mhpB on Escherichia coli BL21 (DE3) were singled out Or double knockout, wherein, the gene knockout plasmid used in the present invention is pCasRed and pCRISPR-gDNA (hpaD sgRNA) and homologous arm (hpaDdonor) are introduced together on Escherichia coli BL21 (DE3), Cas9/sgRNA induces host in hpaD gene A double-strand break occurred at the site, and the recombinase Red integrated the hpaD donor into the hpaD gene to achieve gene knockout, which was verified by sequencing. hpaD sgRNA, hpaD donor, mhpB sgRNA, and mhpB donor are respectively shown in the sequence listing SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. mhpB was knocked out in the same way.

Prepare a solution with a pH of 8, 1 g/L of phenol or L-tyrosine, 100 g/L of wet bacteria, and measure the concentration after standing at 35°C for 10 hours. Table 1 shows the remaining amount of phenol or L-tyrosine in the reaction system.

Table 1 Residual concentrations after substrate and product decomposition by different bacterial strains

strain Phenol g/L L-Tyrosineg/L Escherichia coli BL21(DE3) 0.6 0.4 Escherichia coli BL21 (ΔhpaDΔmhpB,DE3) 0.9 0.8 Escherichia coli BL21(ΔhpaD,DE3) 0.7 0.5 Escherichia coli BL21(ΔmhpB,DE3) 0.6 0.6

Obviously, Escherichia coli BL21 (ΔhpaDΔmhpB, DE3) had the best effect, so it was named Escherichia coli HM.

Example 2

Recombinant Escherichia coli construction: First, the genes encoding lactate dehydrogenase, NADH oxidase, and tyrosine phenolic acid lyase were respectively connected to the plasmid pETDuet-1. After obtaining various three-gene co-expression recombinant plasmids, the plasmids were transformed into Escherichia coli HM, and positive transformants were obtained by screening with ampicillin plate, that is, recombinant Escherichia coli was obtained.

Induced expression method: transfer recombinant Escherichia coli to LB fermentation medium (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L) at a volume ratio of 2%, when the cell OD ₆₀₀ reaches 0.6-0.8 Afterwards, IPTG with a final concentration of 0.4mM was added, and expression was induced at 20°C for 8h. After induction of expression, the cells were collected by centrifugation at 20° C., 8000 rpm, and 20 minutes.

The collected cells were analyzed for transformation, and the results are shown in Table 2. The whole cell transformation system in the transformation system is: cell wet weight 100g/L, L-lactic acid 50g/L, phenol 50g/L, pH 8.0, temperature 35°C, shaker speed 250 rpm; transformation time 10 hours.

Table 2 co-expression Escherichia coli produces L-tyrosine efficiency comparison

strain L-Tyrosineg/L Escherichia coli HM/pETDuet-1-lsnox-llldh-cftpl 43.5 Escherichia coli HM/pETDuet-1-lsnox-lpldh-cftpl 57.0 Escherichia coli HM/pETDuet-1-llnox-llldh-cftpl 66.2 Escherichia coli HM/pETDuet-1-llnox-lpldh-cftpl 57.7 Escherichia coli HM/pETDuet-1-lbnox-llldh-cftpl 38.8 Escherichia coli HM/pETDuet-1-lbnoxl-pldh-cftpl 52.2

It can be seen from the above table that Escherichia coli HM/pETDuet-1-llnox-llldh-cftpl has the best result.

Example 3

According to the strain construction method described in Example 2 (various types of plasmids were selected according to the instructions using different resistance plates to screen positive transformants) and induced expression method, various types of cells were collected for transformation analysis, and the results are shown in Table 3. The whole cell transformation system in the transformation system is: cell wet weight 50g/L, L-lactic acid 10g/L, phenol 10g/L, pH 7.0, temperature 30°C, shaker speed 250 rpm; transformation time 10 hours.

The various expression plasmids of table 3 are for the comparison of producing L-tyrosine

strain L-Tyrosineg/L Escherichia coli HM/pETDuet-1-llnox-llldh-cftpl 6.3 Escherichia coli HM/pACYCDuet-1-llnox-llldh-cftpl 4.3 Escherichia coli HM/pCOLADuet-1-llnox-llldh-cftpl 5.7 Escherichia coli HM/pRSFDuet-1-llnox-llldh-cftpl 5.3 Escherichia coli HM/pCDFDuet-1-llnox-llldh-cftpl 4.8

It can be seen from the above table that the effect is the best when pETDuet-1 is used for co-expression.

Example 4

Using the method described in the literature Large scale validation of an efficient CRISPR/Cas-based multigene editing protocol in Escherichia coli.Microbial Cell Factories, 2017,16(1):68, the corresponding gene on the Escherichia coli HM genome was increased before the Escherichia coli The moderate expression strength constitutive promoter (PG) in front of the 3-phosphate glyceraldehyde dehydrogenase gene (gpdA), the sequence is shown in SEQ ID NO:10.

When enhancing the expression of the gene lldP, the Escherichia coli HM genome was used as a template, and the primers lldP-FF/lldP-FR, lldP-gpdA-F/lldP-gpdA-R, lldP-RF/lldP-RR were used to amplify the upstream, promoter sub, downstream sequence, and lldP-FF and lldP-RR primers were fused into an expression cassette containing the gpdA promoter. Then, after the plasmid pCasRed and pCRISPR-gDNA (including lldP sgRNA) were transferred into Escherichia coli HM, Cas9/sgRNA induced the host to undergo a double-strand break at the lldP gene site, and the recombinase Red integrated the gpdA promoter into the front of the lldP gene, and Sequencing verification.

When enhancing the expression of the gene hpaX, a method similar to the method of enhancing the expression of the gene lldP was used to amplify the upstream, promoter, and downstream sequences first, and design primers to fuse to an expression cassette containing the gpdA promoter. Then, together with the plasmid pCasRed and pCRISPR-gDNA (including hpaX sgRNA) into Escherichia coli HM, Cas9/sgRNA induces a double-strand break in the host at the hpaX gene site, and the recombinase Red integrates the gpdA promoter into the front of the hpaX gene, and Sequencing verification

When enhancing the expression of the gene mhpT, a method similar to the method of enhancing the expression of the gene lldP is used to amplify the upstream, promoter, and downstream sequences first, and design primers to be fused to an expression cassette containing the gpdA promoter. Then, together with the plasmid pCasRed and pCRISPR-gDNA (including mhpT sgRNA) into Escherichia coli HM, Cas9/sgRNA induces a double-strand break in the host at the mhpT gene site, and the recombinase Red integrates the gpdA promoter into the front of mhpT, and then sequenced verify

When enhancing the expression of the gene amtB, a method similar to the method of enhancing the expression of the gene lldP is used to amplify the upstream, promoter, and downstream sequences first, and design primers to be fused to an expression cassette containing the gpdA promoter. Then, after the plasmid pCasRed and pCRISPR-gDNA (including amtB sgRNA) were transferred into Escherichia coli HM, Cas9/sgRNA induced a double-strand break at the amtB gene locus in the host, and the recombinase Red integrated the gpdA promoter into the front of the amtB gene, and Sequencing verification

The following table is the corresponding index of the primer name and sequence number.

Table 4 Comparison of primer names and sequence numbers

The expression was induced according to the method described in Example 2, and various types of cells were collected for transformation analysis. The results are shown in Table 5. The whole cell transformation system in the transformation system is: cell wet weight 10g/L, L-lactic acid 50g/L, phenol 10g/L, pH 8.0, temperature 40°C, shaker speed 250 rpm; transformation time 12 hours.

Table 5 Comparison of conversion results

strain L-Tyrosineg/L Escherichia coli HM(PG-lldP)/pETDuet-1-llnox-llldh-cftpl 8.3 Escherichia coli HM(PG-hpaX)/pETDuet-1-llnox-llldh-cftpl 7.5 Escherichia coli HM(PG-mhpT)/pETDuet-1-llnox-llldh-cftpl 7.8 Escherichia coli HM(PG-amtB)/pETDuet-1-llnox-llldh-cftpl 7.0 Escherichia coli HM (PG-hpaX, PG-mhpT)/pETDuet-1-llnox-llldh-cftpl 8.9 Escherichia coli HM (PG-lldP, PG-hpaX, PG-mhpT)/pETDuet-1-llnox-llldh-cftpl 9.7 Escherichia coli HM/pETDuet-1-llnox-llldh-cftpl 6.9

The best Escherichia coli HM (PG-lldP, PG-hpaX, PG-mhpT) was named Escherichia coli HMLHM.

Example 5

According to the method for example 4, the medium expression strength constitutive promoter (PG) before the 3-phosphate glyceraldehyde dehydrogenase gene (gpdA) of Escherichia coli before the icsA and/or nadA gene in Escherichia coli HMLHM is increased, and the sequence is as SEQID NO :22. The plasmid is then reintroduced.

When enhancing the expression of the gene icsA, a method similar to that of enhancing the expression of the gene lldP in Example 4 was used to amplify the upstream, promoter, and downstream sequences first, and design primers to be fused to an expression cassette containing the gpdA promoter. Then, after the plasmid pCasRed and pCRISPR-gDNA (including icsA-gRNA) were transferred into Escherichia coli HMLHM, Cas9/sgRNA induced a double-strand break in the host at the icsA gene locus, and the recombinase Red integrated the gpdA promoter into the front of the icsA gene, And sequence verification

When enhancing the expression of the gene nadA, a method similar to that of enhancing the expression of the gene lldP in Example 4 was used to first amplify the upstream, promoter, and downstream sequences, and design primers to be fused to an expression cassette containing the gpdA promoter. Then, after the plasmid pCasRed and pCRISPR-gDNA (including nadA-gRNA) were transferred into Escherichia coli HMLHM, Cas9/sgRNA induced a double-strand break in the host at the icsA gene locus, and the recombinase Red integrated the gpdA promoter into the front of the nadA gene, And sequence verification

When enhancing the expression of the gene pdxJ, use the method similar to the method of enhancing the expression of the gene lldP in Example 4 to first amplify the upstream, promoter, and downstream sequences, and design primers to be fused to an expression cassette containing the gpdA promoter. Then, together with the plasmid pCasRed and pCRISPR-gDNA (including pdxJ-gRNA) into Escherichia coli HMLHM, Cas9/sgRNA induces a double-strand break in the host at the icsA gene site, and the recombinase Red integrates the gpdA promoter before the pdxJ gene, And sequence verification

The following table is the corresponding index of the primer name and sequence number.

Table 6 Comparison of primer names and sequence numbers

name number in the sequence listing icsA sgRNA SEQ ID NO:2 nadA sgRNA SEQ ID NO:3 pdxJ sgRNA SEQ ID NO: 16

After the genetic modification is completed, the co-expression plasmid is introduced. The expression was induced according to the method described in Example 2, and various types of cells were collected for transformation analysis. The results are shown in Table 7. The whole cell transformation system in the transformation system is: cell wet weight 20g/L, L-lactic acid 200g/L, phenol 200g/L, pH 9.0, temperature 30°C, shaker speed 250 rpm; transformation time 24 hours.

Table 7 Conversion results comparison

strain L-Tyrosineg/L Escherichia coli HMLHM (PG-icsA, PG-nadA) pETDuet-1-llnox-llldh-cftpl 83.1 Escherichia coli HMLHM(PG-icsA)/pETDuet-1-llnox-llldh-cftpl 53.5 Escherichia coli HMLHM(PG-nadA)/pETDuet-1-llnox-llldh-cftpl 81.9 Escherichia coli HMLHM(PG-nadA,PG-pdxJ)/pETDuet-1-llnox-llldh-cftpl 94.0 Escherichia coli HMLHM/pETDuet-1-llnox-llldh-cftpl 54.9

Name the best Escherichia coli HMLHM (PG-nadA, PG-pdxJ) as Escherichia coliHP

Example 6

According to the induced expression method described in Example 2, after the induction and expression of Escherichia coli HP/pETDuet-1-llnox-llldh-cftpl was completed, the bacterial cells were collected, and in a 100ml reaction system, the cell wet weight was 1g/L, and the L-lactic acid was 1g/L. L, phenol 1g/L, pH 7.0, temperature 15°C, shaker speed 250 rpm; conversion time 1 hour. As a result of the measurement, the L-tyrosine concentration was 52 mg/L.

Example 7

According to the induced expression method described in Example 2, after the induced expression of Escherichia coli HP/pETDuet-1-llnox-llldh-cftpl was completed, the bacterial cells were collected, and in a 100ml reaction system, the cell wet weight was 200g/L, and the L-lactic acid was 200g/L. L, phenol 200g/L, pH 8.5, temperature 40°C, shaker speed 250 rpm; conversion time 24 hours. As a result of the measurement, the concentration of L-tyrosine was 402 g/L. L-tyrosine is poorly soluble in water and will precipitate out at high concentrations. This result is the result of measurement after dilution. Under the same conditions, the L-tyrosine concentration of Escherichia coli BL21(DE3)/pCOLADuet-1-llnox-llldh was 365g/L.

The above-mentioned transformation and construction of enzymes and their co-expression genetically engineered bacteria, the culture medium composition and culture method of the thalline and the whole cell biotransformation are only preferred embodiments of the present invention, and are not intended to limit the present invention. Generally speaking, other bacteria, filamentous fungi, actinomycetes, and animal cells can undergo genome modification and be used for whole-cell catalysis of multi-gene co-expression. Any modification and equivalent replacement made within the principle and spirit of the present invention.

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<213> Artificial sequence

<400> 9

taacacctga cccgcagtgt aaccg 25

<210> 10

<211> 1100

<212>DNA

<213>Escherichia coli BL21(DE3)

<400> 10

atgaatcatg ttgatgagtg gccgatcgct acgtgggaag aaaccacgaa actccattgc 60

gcaatacgct gcgataacca gtaaaaagac cagccagtga atgctgattt gtaaccttga 120

atatttattt tccataacat ttcctgcttt aacataattt tccgttaaca taacggggctt 180

ttctcaaaat ttcattaaat attgttcacc cgttttcagg taatgactcc aacttattga 240

tagtgtttta tgttcagata atgcccgatg actttgtcat gcagctccac cgattttgag 300

aacgacagcg acttccgtcc cagccgtgcc aggtgctgcc tcagattcag gttatgccgc 360

tcaattcgct gcgtatatcg cttgctgatt acgtgcagct ttcccttcag gcgggattca 420

tacagcggcc agccatccgt catccatatc accacgtcaa agggtgacag caggctcata 480

agacgcccca gcgtcgccat agtgcgttca ccgaatacgt gcgcaacaac cgtcttccgg 540

agcctgtcat acgcgtaaaa cagccagcgc tggcgcgatt tagccccgac atagccccac 600

tgttcgtcca tttccgcgca gacgatgacg tcactgcccg gctgtatgcg cgaggttacc 660

gactgcggcc tgagtttttt aagtgacgta aaatcgtgtt gaggccaacg cccataatgc 720

gggcagttgc ccggcatcca acgccattca tggccatatc aatgattttc tggtgcgtac 780

cgggttgaga agcggtgtaa gtgaactgca gttgccatgt tttacggcag tgagagcaga 840

gatagcgctg atgtccggcg gtgcttttgc cgttacgcac caccccgtca gtagctgaac 900

aggagggaca gctgatagaa acagaagcca ctggagcacc tcaaaaacac catcatacac 960

taaatcagta agttggcagc atcaccccgt tttcagtacg ttacgtttca ctgtgagaat 1020

ggagattgcc catcccgcca tcctggtcta agcctggaaa ggatcaattt tcatccgaac 1080

gttcctgaca ggagaaaacc 1100

<210> 11

<211> 20

<212>DNA

<213> Artificial sequence

<400> 11

tatgcccgtc gatcgcgccc 20

<210> 12

<211> 120

<212>DNA

<213> Artificial sequence

<400> 12

ccaagatcac gcacgtaccg tcgatgtatc tctctgaact gccagggaaa aaccacggtt 60

agatcagcaa gcgttgccgg gaaatgggcg tcgataccat tatcgttttc gacacccact 120

<210> 13

<211> 20

<212>DNA

<213> Artificial sequence

<400> 13

tcatcgagta cctcttgcgc 20

<210> 14

<211> 120

<212>DNA

<213> Artificial sequence

<400> 14

tagcctgata tgcacgctta tcttcactgt ctttcccact cgccgctggt gggatatgtc 60

aatggcgtga ttgccagcgc ccgcgagcgt attgcggctt tctcccctga actggtggtg 120

<210> 15

<211> 20

<212>DNA

<213> Artificial sequence

<400> 15

cgaacagaaa gacgatcagg 20

<210> 16

<211> 20

<212>DNA

<213> Artificial sequence

<400> 16

cgtcgcggtc agtaatgtga 20

<210> 17

<211> 20

<212>DNA

<213> Artificial sequence

<400> 17

ggtaatggct gcacctgcgg 20

<210> 18

<211> 20

<212>DNA

<213> Artificial sequence

<400> 18

gcgggatgaa gatgatgaag 20

Claims (10)

1. a kind of recombination bacillus coli, which is characterized in that the recombination bacillus coli express simultaneously external source l-lactate dehydrogenase, Nadh oxidase and tyrosine phenol lyase, and knocked out l-tyrosine on the basis of host e. coli and decomposed relevant base Because, phenol decompose in relevant gene any one or it is a variety of.

2. recombination bacillus coli according to claim 1, which is characterized in that the recombination bacillus coli expresses L- simultaneously Lactate dehydrogenase L lldh, nadh oxidase llnox and tyrosine phenol lyase cftpl, have knocked out aromatic compound degrading gene HpaD and mhpB, and overexpression Lactate Transport gene lldP, phenol transporter gene hpaX, phenol transporter gene mhpT, NAD synthesizes gene nadA, phosphoric acid Vitamin B6 synthesizes gene pdxJ.

3. a kind of method for producing optical voidness l-tyrosine, which is characterized in that the method is to utilize any institute of claim 1-2 The recombination bacillus coli stated.

4. according to the method described in claim 3, it is characterized in that, the external source Pfansteihl dehydrogenation of the expression of recombinant e. coli Enzyme is the l-lactate dehydrogenase of originating in lactic acid bacterium.

5. according to the method described in claim 3, it is characterized in that, the recombination bacillus coli knock out gene be hpaD, Any one in mhpB or two kinds of combinations.

6. according to the method described in claim 3, it is characterized in that, the recombination bacillus coli also overexpression Lactate Transport Gene, ammonia radical ion transporter gene, phenol transporter gene, NAD synthesis gene, phosphoric acid Vitamin B6 synthesis gene are a kind of or more Kind.

7. according to the method described in claim 6, it is characterized in that, the gene of the recombination bacillus coli overexpression is Any one or more in lldP, amtB, hpaX, mhpT, icsA, nadA, pdxJ.

8. method according to claim 6 or 7, which is characterized in that the overexpression is by by host e. coli Increase constitutive promoter before the gene of need to strengthen expression on genome.

9. according to the method described in claim 8, it is characterized in that, the l-lactate dehydrogenase of the expression of recombinant e. coli, Nadh oxidase, tyrosine phenol lyase are co-expressed by pETDuet-1.

10. according to any method of claim 3-9, which is characterized in that in the system of the resting cell production, carefully Born of the same parents weight in wet base 1-200g/L, Pfansteihl 1-200g/L, phenol concentration 1-200g/L, pH 7.0-9.0,15-40 DEG C of temperature, shaking table turns 250 revs/min of speed；Transformation time 1-24 hours.