CN108949647B - Engineering bacterium and application thereof in production of L-tyrosine - Google Patents
Engineering bacterium and application thereof in production of L-tyrosine Download PDFInfo
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Abstract
The invention discloses an engineering bacterium and application thereof in producing L-tyrosine, belonging to the technical field of biological engineering. The invention provides a production method for producing L-tyrosine by converting lactic acid and phenol without generating hydrogen oxide, constructs three-enzyme coexpression engineering bacteria on the basis of modifying an escherichia coli transfer and coenzyme synthesis system, and realizes the high-efficiency production of L-tyrosine. The method for producing L-tyrosine by whole cell transformation has simple process and less impurities, and has important industrial application value.
Description
Technical Field
The invention relates to an engineering bacterium and application thereof in producing L-tyrosine, belonging to the technical field of biological engineering.
Background
L-tyrosine (p-hydroxyphenylalanine) is an essential amino acid of human body and has important application in the aspects of medicine and food. The prior L-tyrosine extraction method, enzyme method, gene engineering bacteria fermentation method and the like. The L-tyrosine extracted by hydrolysis from raw materials such as wool method has large pollution and low purity. The yield of the Escherichia coli engineering bacteria for synthesizing the L-tyrosine by using glucose as a raw material is low, and the Escherichia coli engineering bacteria cannot compete with an extraction method and an enzyme method at present.
The enzymatic production of L-tyrosine by using a tyrosine phenol lyase is the most widely used method. Various schemes have been devised (Synthesis of L-Tyrosine or 3,4-Dihydroxyphenyl-L-alanine from DL-Serine and Phenol or pyrocatechol, Agric. biol. chem.37(1973) 493-499, Chinese patent 201310168119.5) in which a conversion process using pyruvic acid and Phenol as substrates has been industrially carried out. Pyruvate is an expensive intermediate, so that the patent 201310289373.0 adopts unpurified pyruvate liquid to directly convert, or firstly uses lactate oxidase to oxidize lactate to generate pyruvate, and then further catalytically produces L-tyrosine (research on multi-enzyme coupling biosynthesis of pyruvate and L-tyrosine, 2014, Master thesis of Nanjing university), but the efficiency of the scheme is not high.
Disclosure of Invention
Based on the defects of various methods at present, the invention provides a production method for producing L-tyrosine by converting lactic acid and phenol without generating hydrogen oxide, constructs three-enzyme co-expressed engineering bacteria on the basis of modifying an escherichia coli transfer and coenzyme synthesis system, and realizes the high-efficiency production of L-tyrosine. The technical problem to be solved by the invention is to provide a recombinant strain capable of producing L-tyrosine and reducing impurity generation, and simultaneously, the technical problems of construction and application of the strain are solved.
The first object of the present invention is to provide a recombinant E.coli strain capable of producing pure poly L-fenbacide at a low cost; the recombinant escherichia coli expresses exogenous L-lactate dehydrogenase, NADH oxidase and tyrosine phenol lyase at the same time, and an L-tyrosine absorption gene is knocked out on the basis of host escherichia coli.
In one embodiment, the exogenous L-lactate dehydrogenase is a lactic acid bacteria-derived L-lactate dehydrogenase. The exogenous NADH oxidase is NADH oxidase derived from lactobacillus.
In one embodiment, the lactate dehydrogenase is from Lactobacillus lactis ATCC 19257, Lactobacillus plantarum ATCC 14917.
In one embodiment, the amino acid sequence of the lactate dehydrogenase is the sequence of accession NO WP _003131075.1, KRL33571.1 at NCBI.
In one embodiment, the nucleotide sequence of the lactate dehydrogenase is that of an accession NO on NCBI: NZ _ JXJZ01000017REGION:18532..19509, AZEJ01000016REGION:16296.. 17249.
In one embodiment, the NADH oxidase is from Lactobacillus lactis ATCC 19257, Lactobacillus sanfranciscisciensis DSM20451, Lactobacillus brevicis ATCC 14869.
In one embodiment, the amino acid sequence of the NADH oxidase is the sequence WP _032950924.1, WP _056958268.1, ERK43827.1 on NCBI for access NO.
In one embodiment, the nucleotide sequence of said NADH oxidase is that of the accession NO on NCBI: NZ _ JXJZ01000002REGION: compensation (39571..40911), NZ _ AYYM01000013REGION: compensation (15875..17233), AWVK01000048REGION: compensation (50022.. 51416).
In one embodiment, the L-lactate dehydrogenase, NADH oxidase, and tyrosol lyase are co-expressed by pETDuet-1.
In one embodiment, the tyrosol lyase is derived from Erwinia herbicola (Erwinia herbicoloa), Citrobacter intermedius (Citrobacter intermedius), Citrobacter freundii (Citrobacterfreundii), thermobacter thermophilus (sambucobacter thermophilus) or symbiobacter toebi.
In one embodiment, the tyrosol lyase is derived from Citrobacter freundii ATCC 8090 and has the amino acid sequence WP _003837154.1 for access NO on NCBI.
In one embodiment, the L-tyrosine uptake gene comprises any one of an L-tyrosine decomposition gene, a phenol decomposition gene, or a combination of both.
In one embodiment, the L-tyrosine lyase is any one of hpaD, mhpB, or a combination of both.
In one embodiment, the phenol degradation gene is any one of hpaD, mhpB, or a combination of both.
In one embodiment, the nucleotide sequence of the L-tyrosine-degrading gene and the phenol-degrading gene is at NCBI, where access NO is: NC _012892REGION: completion (4505585..4506436) and NC _012892REGION:339806.. 340750.
In one embodiment, the recombinant escherichia coli further enhances expression of any one or more of a lactate transporter gene, a phenol transport-associated gene, and an ammonia ion transporter gene for transporting a substrate into a cell.
In one embodiment, the recombinant escherichia coli further enhances expression of any one or more of an NAD synthesis gene and a pyridoxal phosphate synthesis-related gene.
In one embodiment, the expression-enhanced gene is any one or more of lldP (lactate transporter gene), amtB (ammonium ion transporter gene), hpaX (phenol transporter gene), mhpT (phenol transporter gene), icsA (NAD synthase gene), nadA (NAD synthase gene), pdxJ (pyridoxal phosphate synthase gene).
In one embodiment, the host bacterium is Escherichia coli BL21(DE 3).
In one embodiment, the enhanced expression is achieved by adding a constitutive promoter in front of the gene to be enhanced on the genome of Escherichia coli BL21(DE 3).
In one embodiment, the lldP is access NO at NCBI: NC _012892REGION 3646638.. 3648293; amtB is NC-012892 REGION 442006.. 443292; hpaX is; NC _012892REGION: compensation (4502025.. 4503401); mhpT NC-012892 REGION 344788.. 345999; icsA is NC _012892REGION: completion (2526116.. 2527330); nadA is NC _012892REGION 740487.. 741530; pdxJ is NC _012892REGION: completion (2567591.. 2568322).
The second object of the present invention is to provide a method for producing optically pure L-tyrosine using the recombinant bacterium of the present invention.
In one embodiment, the production of L-tyrosine is by whole cell transformation.
In one embodiment, the whole cell transformation production system comprises 1-200g/L wet weight of cells, 1-200 g/L-lactic acid, 1-200g/L phenol concentration, 7.0-9.0 pH, 15-40 ℃ and 250 rpm of shaking table rotation speed; the conversion time is 1-24 hours.
The third purpose of the invention is to provide the application of the recombinant bacterium or the method of the invention in the fields of chemical industry, food, medicine and the like.
The invention has the beneficial effects that:
the invention constructs a novel three-enzyme co-expression gene engineering bacterium which can be applied to the production of L-tyrosine. The selection scheme of the invention does not produce hydrogen peroxide, the cells are not easy to decompose the L-tyrosine, and the cells have higher NAD content. The production process is simple, the raw materials are easy to obtain, and the method has a good industrial application prospect.
Detailed description of the preferred embodiments
The functional core of the escherichia coli engineering bacteria is that three enzymes can be co-expressed, namely L-Lactate dehydrogenase (L-Lactate dehydrogenase), NADH oxidase (NADH oxidase) and tyrosinol lyase (tyrosine phenol-lyase). The principle is as follows: in the whole cell of the engineering bacteria, L-lactate dehydrogenase takes NAD in the bacteria as coenzyme to dehydrogenate L-lactate to generate pyruvic acid and NADH; NADH oxidase oxidizes NADH to generate NAD, and the regeneration of coenzyme NAD is realized. Then under the action of tyrosine phenol lyase, pyruvic acid, ammonia radical ion and phenol are synthesized into L-tyrosine, and simultaneously, related genes on the genome of the escherichia coli are knocked out or enhanced to promote the transport of substrates and reduce the decomposition of products.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
1. the invention relates to a strain and a plasmid
Lactobacillus plantarum ATCC 14917, Lactobacillus lactis ATCC 19257, Lactobacillus brevis ATCC 14869, Citrobacter freundii ATCC 8090, which are available from American type culture Collection ATCC, were purchased from pETDuet-1, pACYCDue-1, pCOLADuet-1, pRSFDuet-1 plasmid and Escherichia coli BL21(DE3), which are available from Novagen. Lactobacillus sanfranciscensis DSM20451 was purchased from German Collection of microorganisms and cell cultures DSMZ. pCasRed and pCRISPR-gDNA were purchased from Zhenjiang Aibiemeng Biotech Ltd.
2. Knockout and constitutive enhanced expression of related genes in escherichia coli
(1) Knockout of Escherichia coli aromatic compound degradation gene
Both phenol and L-tyrosine in the present invention are very easily decomposed by enzymes in E.coli, and according to the literature (Biodegradation of Aromatic Compounds by Escherichia coli, Microbiol Mol Biol Rev.2001,65(4): 523-. The genes selected were hpaD and mhpB, with access NO at NCBI: NC _012892REGION: completion (4505585..4506436) and NC _012892REGION:339806.. 340750.
(2) Constitutive enhanced expression of escherichia coli lactic acid, phenol and ammonia radical ion transport gene
In the whole cell transformation process, the substrate is required to be transported into the cell, and the enhanced lactic acid transporter is beneficial to quickly and long-term maintaining the high concentration of the intracellular substrate and facilitating the reaction. The lactate transport-associated gene was selected to be lldP, with access NO at NCBI as: NC _012892REGION:3646638.. 3648293. Genes related to phenol transport are hpaX and mhpT, and access NO on NCBI is: NC _012892REGION: completion (4502025..4503401) and NC _012892REGION:344788.. 345999. The gene related to the transport of the ammonia radical ion is amtB, and the access NO on NCBI is as follows: NC _012892REGION:442006.. 443292.
(3) Overexpression of genes involved in NAD synthesis in Escherichia coli
In the process of lactate dehydrogenation, NAD is required to be used as coenzyme, the key enzyme of the synthesis pathway of the NAD of the escherichia coli is enhanced and expressed, the level of the NAD in the bacteria can be improved, and the generation of pyruvic acid is facilitated. The selected genes are icsA and nadA. Access NO on NCBI is: NC _012892REGION: compensation (2526116..2527330), NC _012892REGION:740487.. 741530.
(4) Expression of Pyridoxal phosphate synthesis-related genes
The pyridoxal phosphate (amine) is a coenzyme of tyrosine lyase, and the core gene pdxJ in the coenzyme pathway is overexpressed, thereby being beneficial to the synthesis of L-tyrosine. Access NO on NCBI is: NC _012892REGION: completion (2567591.. 2568322).
3. Selection of enzymes associated with conversion of lactate to pyruvate
(1) Selection of L-lactate dehydrogenase
L-lactic acid is the cheapest organic acid, and at present, L-lactic acid oxidase is mainly used for oxidizing L-lactic acid to produce pyruvic acid, hydrogen peroxide is produced in the process, and the hydrogen peroxide can oxidize the pyruvic acid to produce acetic acid. L-lactate dehydrogenase exists widely in various microorganisms, and lactate dehydrogenase, which generally uses NAD (NADP) as a coenzyme, tends to synthesize lactate using pyruvate as a substrate, but some lactate dehydrogenase removes hydrogen from lactate to produce pyruvate when lactate is excessive or the carbon source has only lactate, and transfers hydrogen produced on L-lactate to coenzyme NAD or NADP using L-lactate as a substrate to produce NADH or NADPH.
The L-lactate dehydrogenase gene llldh (with an amino acid sequence of WP _003131075.1) and lpldhh (with an amino acid sequence of KRL33571.1) are respectively obtained from Lactobacillus lactis ATCC 19257 and Lactobacillus plantarum ATCC 14917, and expression products are used for the dehydrogenation of lactic acid.
(2) Selection of NADH oxidase
Lactate dehydrogenase dehydrogenates lactate to pyruvate NADH. NADH needs to be oxidized by NAD oxidase to regenerate NAD, thereby realizing the continuous reaction. The NADH oxidase has two types of water-producing type and hydrogen peroxide-producing type, and the water-producing type NADH oxidase does not produce hydrogen peroxide toxicity. The water-producing NADH oxidase genes lsnox (with an amino acid sequence of WP _056958268.1), llnox (with an amino acid sequence of WP _003131075.1) and lbnox (with an amino acid sequence of ERK43827) are obtained from Lactobacillus sanfranciscensis DSM20451, Lactobacillus lactis ATCC 19257 and Lactobacillus brevis respectively, and the expression products are used for the regeneration of NAD. .
(3) Selection of tyrosine phenol lyase
Tyrosine phenol lyase derived from Erwinia herbicola (Erwinia herbicolo), Citrobacter intermedium (Citrobacter intermedium), Citrobacter freundii (Citrobacter freundii) and the thermophilic bacteria Symbiobacterium thermophilum and Symbiobacterium toebi and the like are the most commonly studied. The invention selects enzyme with higher activity from the citric acid bacteria Freund, and obtains tyrosine phenol lyase gene cftpl (the amino acid sequence is WP _003837154.1) from Citrobacter freundii ATCC 8090.
4. Construction of Co-expression System and culture of cells
At present, various methods for the polygene co-expression of escherichia coli (polygene co-expression strategy of escherichia coli, Chinese birth)The invention relates to a pharmaceutical engineering journal, 2012, 32(4):117-122), which is constructed by adopting the method of Liu Jie (the synthetic biology technology is used for transforming Escherichia coli to produce shikimic acid and resveratrol, 2016, Shanghai pharmaceutical industry research institute, doctor paper), wherein each gene comprises a T7 promoter and an RBS binding site in front, theoretically, because each gene is provided with a T7 and an RBS in front, the expression intensity of the genes is not greatly influenced by the arrangement order. Each plasmid contains three genes, the constructed plasmids are thermally transduced into escherichia coli competent cells, and are coated on an antibiotic solid plate, and positive transformants are obtained through screening, so that the recombinant escherichia coli is obtained. And (3) culturing the cells: according to the classical recombinant Escherichia coli culture and induction expression scheme, transferring the recombinant Escherichia coli into LB fermentation medium (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L) according to the volume ratio of 2%, when the cell OD600After reaching 0.6-0.8, IPTG was added to a final concentration of 0.4mM, and expression-induced culture was carried out at 20 ℃ for 8 hours. After the induction expression was completed, the cells were collected by centrifugation at 8000rpm for 20 minutes at 20 ℃.
5. Production of L-tyrosine by whole cell transformation
The whole cell transformation system is as follows: the wet weight of the cells is 1-200g/L, the L-lactic acid is 1-200g/L, the phenol is 1-200g/L, the pH is 7.0-9.0, the temperature is 20-50 ℃, and the rotating speed of a shaking table is 250 r/min; the conversion time is 1-24 hours.
6. Detection analysis of samples
Quantitative analysis of L-tyrosine adopts PerkinElmer Series 200 high performance liquid chromatograph for detection and analysis, and a differential refraction detector is arranged. The chromatographic conditions are as follows: the mobile phase was methanol-0.1% formic acid water (40:60) and a Hebang Megres C18 chromatographic column (4.6X 250mm, 5 μm) with a flow rate of 1ml/min, a column temperature of 30 ℃ and a sample volume of 20 μ l.
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more apparent, the present invention is described in detail below with reference to the embodiments. It should be noted that the specific embodiments described herein are only for explaining the present invention and are not used to limit the present invention.
Example 1
The hpaD and mhpB on Escherichia coli BL21(DE3) are subjected to single or double knockout according to the method described in the document Large scale validation of an effective CRISPR/Cas-based multi-gene editing protocol in Escherichia coli. Microbiological Cell Factories,2017,16(1):68, wherein the gene knockout plasmids used in the invention are pCasRed and pCRISPR-gDNA (hpaD sgRNA) which are introduced into Escherichia coli BL21(DE3) together with a homology arm (hpaD doror), the Cas9/sgRNA induces double strand breaks in the hpaD gene locus of the host, and Red integrates the hpaD doror into the hpaD gene, thereby realizing gene knockout and sequencing and verifying recombinase. The hpaD sgRNA, hpaD donor, mhpB sgRNA, and mhpB donor are shown in sequence tables SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, and SEQ ID NO 14, respectively. mhpB was knocked out in the same way.
A solution having a pH of 8, phenol or L-tyrosine 1g/L, and wet cell mass 100g/L was prepared, and the concentration was measured after standing at 35 ℃ for 10 hours. The remaining amount of phenol or L-tyrosine in the reaction system is shown in Table 1.
TABLE 1 residual concentrations of different strains after substrate and product decomposition
| Bacterial strains | Phenol g/L | L-tyrosine g/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 |
It is clear that Escherichia coli BL21(Δ hpaD Δ mhpB, DE3) works best and is named Escherichia coli HM.
Example 2
Constructing recombinant escherichia coli: first, genes encoding lactate dehydrogenase, NADH oxidase, and tyrosine phenol lyase were ligated to plasmid pETDuet-1, respectively. After various three-gene co-expression recombinant plasmids are obtained, the plasmids are transformed into Escherichia coli HM, and a positive transformant is obtained by screening an ampicillin plate, so that the recombinant Escherichia coli is obtained.
The induction expression method comprises the following steps: transferring the recombinant Escherichia coli into LB fermentation medium (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L) at a volume ratio of 2%, when cell OD600After reaching 0.6-0.8, IPTG was added to a final concentration of 0.4mM, and expression-induced culture was carried out at 20 ℃ for 8 hours. After the induction expression was completed, the cells were collected by centrifugation at 8000rpm for 20 minutes at 20 ℃.
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 as follows: the wet weight of the cells is 100g/L, the L-lactic acid is 50g/L, the phenol is 50g/L, the pH is 8.0, the temperature is 35 ℃, and the rotating speed of a shaking table is 250 r/min; the conversion time was 10 hours.
TABLE 2 comparison of the efficiency of L-tyrosine production by co-expression of E.coli
| Bacterial strains | L-tyrosine g/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 |
From the above table, it can be seen that the best results are obtained for Escherichia coli HM/pETDuet-1-llnox-llldh-cftpl.
Example 3
According to the strain construction method (each plasmid is used for screening positive transformants by using different resistance plates according to the specification) and the induced expression method described in example 2, each cell was collected for transformation analysis, and the results are shown in Table 3. The whole cell transformation system in the transformation system is as follows: the wet weight of the cells is 50g/L, the L-lactic acid is 10g/L, the phenol is 10g/L, the pH is 7.0, the temperature is 30 ℃, and the rotating speed of a shaking table is 250 r/min; the conversion time was 10 hours.
TABLE 3 comparison of various expression plasmids for L-tyrosine production
| Bacterial strains | L-tyrosine g/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 co-expression using pETDuet-1 works best.
Example 4
The medium expression strength constitutive Promoter (PG) in front of the corresponding gene of Escherichia coli 3-glyceraldehyde phosphate dehydrogenase gene (gpdA) is increased on Escherichia coli HM genome by adopting the method described in the document Large scale identification of an effective CRISPR/Cas-based multi-gene expression protocol in Escherichia coli, Microbiological Cell industries, 2017,16(1):68, and the sequence is shown as SEQ ID NO: 10.
When the expression of the gene lldP is enhanced, an Escherichia coli HM genome is used as a template, primers lldP-FF/lldP-FR and lldP-gpdA-F/lldP-gpdA-R, lldP-RF/lldP-RR are used to amplify an upstream sequence, a promoter and a downstream sequence, and the lldP-FF and the lldP-RR are used as primers to fuse into an expression frame containing a gpdA promoter. Then after being transformed into Escherichia coli HM together with plasmids pCasRed and pCRISPR-gDNA (containing lldP sgRNA), Cas9/sgRNA induces double strand break of host at lldP gene site, recombinase Red integrates gpdA promoter in front of lldP gene, and sequencing and verification are carried out.
When the expression of the enhanced gene hpaX is carried out, a method similar to the expression of the enhanced gene lldP is adopted, an upstream sequence, a promoter sequence and a downstream sequence are amplified firstly, and a primer is designed and fused into an expression frame containing a gpdA promoter. Then after being transformed into Escherichia coli HM together with plasmids pCasRed and pCRISPR-gDNA (containing hpaX sgRNA), Cas9/sgRNA induces double strand break of host at hpaX gene locus, recombinase Red integrates gpdA promoter in front of hpaX gene, and sequencing verifies
When enhancing the expression of the gene mhpT, a method similar to the method for enhancing the expression of the gene lldP is adopted, an upstream sequence, a promoter and a downstream sequence are amplified, and a primer is designed and fused into an expression frame containing a gpdA promoter. Then after being transformed into Escherichia coli HM together with plasmids pCasRed and pCRISPR-gDNA (containing mhpT sgRNA), Cas9/sgRNA induces double strand break of host at mhpT gene site, recombinase Red integrates gpdA promoter in front of mhpT, and sequencing verifies
When the gene amtB is enhanced, a method similar to the enhanced gene lldP expression is adopted, an upstream sequence, a promoter sequence and a downstream sequence are amplified firstly, and a primer is designed and fused into an expression frame containing a gpdA promoter. Then after being transformed into Escherichia coli HM together with plasmids pCasRed and pCRISPR-gDNA (containing amtB sgRNA), Cas9/sgRNA induces double strand break of host at amtB gene site, recombinase Red integrates gpdA promoter in front of amtB gene, and sequencing verifies
The following table is the corresponding index of the primer name and sequence number in the sequence listing.
TABLE 4 comparison of primer names with sequence Listing numbers
Expression was induced according to the method described in example 2, and various types of cells were collected for transformation analysis, and the results are shown in Table 5. The whole cell transformation system in the transformation system is as follows: the wet weight of the cells is 10g/L, the L-lactic acid is 50g/L, the phenol is 10g/L, the pH is 8.0, the temperature is 40 ℃, and the rotating speed of a shaking table is 250 r/min; the conversion time was 12 hours.
TABLE 5 comparison of transformation results
| Bacterial strains | L-tyrosine g/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 most effective Escherichia coli HM (PG-lldP, PG-hpaX, PG-mhpT) was named Escherichia coli HMLHM.
Example 5
The medium expression strength constitutive Promoter (PG) in Escherichia coli before the gene for icsA and/or nadA was increased according to the method of example 4 before the gene for glyceraldehyde-3-phosphate dehydrogenase (gpdA) of Escherichia coli, and the sequence is shown in SEQ ID NO: 22. The plasmid is then introduced.
When enhancing the expression of gene icsA, the method similar to that of enhancing the expression of gene lldP in example 4 is adopted, the upstream, promoter and downstream sequences are amplified, and the primers are designed and fused into an expression cassette containing the gpdA promoter. Then after being transferred into Escherichia coli HMLHM together with plasmids pCasRed and pCRISPR-gDNA (containing icsA-gRNA), Cas9/sgRNA induces double strand break of the host at the icsA gene site, recombinase Red integrates the gpdA promoter in front of the icsA gene, and sequencing verifies
When the expression of gene nadA is enhanced, the method similar to the method for enhancing the expression of gene lldP in example 4 is adopted, the upstream, promoter and downstream sequences are amplified firstly, and the primer fusion is designed to be an expression frame containing the gpdA promoter. Then after being transferred into Escherichia coli HMLHM together with plasmids pCasRed and pCRISPR-gDNA (containing nadA-gRNA), Cas9/sgRNA induces double strand break of host at icsA gene site, recombinase Red integrates gpdA promoter in front of nadA gene, and sequencing verifies
When the expression of the gene pdxJ is enhanced, the method similar to the method for enhancing the expression of the gene lldP in example 4 is adopted, the upstream, promoter and downstream sequences are amplified firstly, and a primer fusion is designed to be an expression frame containing a gpdA promoter. Then after being transferred into Escherichia coli HMLHM together with plasmids pCasRed and pCRISPR-gDNA (containing pdxJ-gRNA), Cas9/sgRNA induces double strand break of host at icsA gene site, recombinase Red integrates gpdA promoter in front of pdxJ gene, and sequencing verifies
The following table is the corresponding index of the primer name and sequence number in the sequence listing.
TABLE 6 comparison of primer names with sequence Listing numbers
| Name (R) | Number in sequence listing |
| icsA sgRNA | SEQ ID NO:2 |
| nadA sgRNA | SEQ ID NO:3 |
| pdxJ sgRNA | SEQ ID NO:16 |
After the gene transformation is completed, the co-expression plasmid is introduced. Expression was induced according to the method described in example 2, and various types of cells were collected for transformation analysis, and the results are shown in Table 7. The whole cell transformation system in the transformation system is as follows: the wet weight of the cells is 20g/L, the L-lactic acid is 200g/L, the phenol is 200g/L, the pH is 9.0, the temperature is 30 ℃, and the rotating speed of a shaking table is 250 r/min; the conversion time was 24 hours.
TABLE 7 comparison of transformation results
| Bacterial strains | L-tyrosine g/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 |
The best Escherichia coli HMLHM (PG-nadA, PG-pdxJ) was named Escherichia coli HP
Example 6
According to the inducible expression method of the embodiment 2, thalli are collected after the Escherichia coli HP/pETDuet-1-llnox-llldh-cftpl is subjected to inducible expression, and in a 100ml reaction system, the wet weight of cells is 1g/L, the wet weight of L-lactic acid is 1g/L, the wet weight of phenol is 1g/L, the pH value is 7.0, the temperature is 15 ℃, and the rotating speed of a shaking table is 250 r/min; the conversion time was 1 hour. As a result, the concentration of L-tyrosine was 52 mg/L.
Example 7
According to the inducible expression method of the embodiment 2, thalli are collected after the Escherichia coli HP/pETDuet-1-llnox-llldh-cftpl is subjected to inducible expression, and in a 100ml reaction system, the wet weight of cells is 200g/L, the wet weight of L-lactic acid is 200g/L, the wet weight of phenol is 200g/L, the pH value is 8.5, the temperature is 40 ℃, and the rotating speed of a shaking table is 250 r/min; the conversion time was 24 hours. As a result, the concentration of L-tyrosine was 402 g/L. L-tyrosine is hardly soluble in water and precipitates at a high concentration, and this result is measured after dilution. Under the same conditions, the L-tyrosine concentration of Escherichia coli BL21(DE3)/pCOLADuet-1-llnox-llldh is 365 g/L.
The modification and construction of the enzyme and its co-expressed genetically engineered bacteria, the culture medium composition and culture method of the bacteria, and the whole cell biotransformation described above are only preferred embodiments of the present invention, and are not intended to limit the present invention, and theoretically, other bacteria, filamentous fungi, actinomycetes, and animal cells can be used for genome modification and whole cell catalysis of multigene co-expression. Any modification, equivalent replacement, made within the principle and spirit of the present invention.
Sequence listing
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Claims (9)
1. A recombinant Escherichia coli is characterized in that the recombinant Escherichia coli expresses exogenous L-lactate dehydrogenase, NADH oxidase and tyrosine phenol lyase at the same time, and knocks out any one or more of genes related to L-tyrosine decomposition and genes related to phenol decomposition on the basis of host Escherichia coli, wherein the knocked-out genes of the recombinant Escherichia coli are any one or combination of hpaD and mhpB; the L-lactate dehydrogenase is L-lactate dehydrogenase Llldh with an amino acid sequence of WP _003131075.1, or L-lactate dehydrogenase Llpldh with an amino acid sequence of KRL 33571.1; the NADH oxidase is NADH oxidase lsnox with an amino acid sequence of WP _056958268.1, or NADH oxidase llnox with an amino acid sequence of WP _032950924.1, or NADH oxidase lbnox with an amino acid sequence of ERK 43827.1; the amino acid sequence of the tyrosine phenol lyase is WP _003837154.1 tyrosine phenol lyase cftpl; NCBI access NO of the knocked-out gene hpaD is NC-012892 REGION: compensation (4505585.. 4506436); the NCBI access NO of the knocked-out gene mhpB is NC-012892 REGION 339806.. 340750.
2. The recombinant Escherichia coli of claim 1, wherein the recombinant Escherichia coli expresses L-lactate dehydrogenase Llldh, NADH oxidase llnox and tyrosol lyase cftpl simultaneously, knockouts out genes hpaD and mhpB, and enhances expression of lactate transporter lldP, phenol transporter hpaX, phenol transporter mhpT, NAD synthesis gene nadA, and pyridoxal phosphate synthesis gene pdxJ.
3. A method for producing optically pure L-tyrosine using the recombinant Escherichia coli of any one of claims 1 to 2.
4. The method according to claim 3, wherein the exogenous L-lactate dehydrogenase expressed by the recombinant Escherichia coli is lactic acid bacteria-derived L-lactate dehydrogenase.
5. The method of claim 3, wherein the recombinant Escherichia coli further enhances expression of one or more of a lactate transporter gene, an ammonia root ion transporter gene, a phenol transporter gene, an NAD synthetic gene, and a pyridoxal phosphate synthetic gene.
6. The method of claim 5, wherein the recombinant E.coli enhanced expression gene is any one or more of lldP, amtB, hpaX, mhpT, icsA, nadA and pdxJ.
7. The method of claim 6, wherein the enhanced expression is achieved by adding a constitutive promoter in front of the gene to be enhanced on the genome of E.coli of the host.
8. The method of claim 7, wherein the recombinant Escherichia coli-expressed L-lactate dehydrogenase, NADH oxidase, and tyrosol lyase are co-expressed by pETDuet-1.
9. The method according to any one of claims 3 to 8, wherein the recombinant Escherichia coli is used for producing L-tyrosine by whole-cell transformation, and in a system for producing L-tyrosine by whole-cell transformation, the wet weight of cells 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, the temperature is 15-40 ℃, and the rotating speed of a shaking table is 250 revolutions per minute; the conversion time is 1-24 hours.
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| CN103224972A (en) * | 2013-05-09 | 2013-07-31 | 湖北新生源生物工程股份有限公司 | L-tyrosine preparation method through enzymatic conversion |
| CN103343149A (en) * | 2013-07-10 | 2013-10-09 | 南京大学 | Preparation method of L-tyrosine or L-tyrosine derivative employing enzyme conversion |
| CN104498555A (en) * | 2014-12-08 | 2015-04-08 | 宁波市镇海海德生化科技有限公司 | Supplementation and enzymatic conversion based ultrasonic-assisted preparation method of L-tyrosine |
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| CN103343149A (en) * | 2013-07-10 | 2013-10-09 | 南京大学 | Preparation method of L-tyrosine or L-tyrosine derivative employing enzyme conversion |
| CN104498555A (en) * | 2014-12-08 | 2015-04-08 | 宁波市镇海海德生化科技有限公司 | Supplementation and enzymatic conversion based ultrasonic-assisted preparation method of L-tyrosine |
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