CN108949647A - A kind of engineering bacteria and its application in production l-tyrosine - Google Patents
A kind of engineering bacteria and its application in production l-tyrosine Download PDFInfo
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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
Technical field
The present invention relates to a kind of engineering bacteria and its applications in production l-tyrosine, belong to technical field of bioengineering.
Background technique
L-tyrosine (p-hydroxyphenylalanine) is the necessary amino acid of human body, is had in terms of medicine and food
There is important application.L-tyrosine comes extraction method, enzyme process, genetic engineering fungus fermentation method etc. at present.It is hydrolyzed from raw materials such as hair methods
Extraction l-tyrosine process contamination is big and purity is lower.Colibacillus engineering is that raw material accent synthesizes L- junket ammonia using glucose
The yield of acid is lower, can not compete at present with extraction method and enzyme process.
It is most widely used method with tyrosine phenol lyase Production by Enzymes l-tyrosine.Existing kinds of schemes is set
Count (Synthesis of L-Tyrosine or 3,4-Dihydroxyphenyl-L-alanine from DL-Serine
(1973) 493-499. Chinese patent of and Phenol or Pyrocathechol, Agric.Biol.Chem.37
201310168119.5), wherein industrially having implemented using pyruvic acid and phenol as the method for transformation of substrate.Pyruvic acid is more
A kind of expensive intermediate, therefore patent 201310289373.0 is directly converted using unpurified pyruvic acid feed liquid, or
Person first uses lactate oxidase Oxidation of Lactic to generate pyruvic acid, and then further (multienzyme coupling is biological for catalytic production l-tyrosine
It synthesizes pyruvic acid and L-- tyrosine is studied, 2014, Nanjing University's Master's thesis), but pyruvic acid easily decomposes, this scheme efficiency
It is not high.
Summary of the invention
Based on the defect of current various methods, the invention proposes the conversion lactic acid for not generating hydrogen oxide and phenol to produce L- junket
The production method of propylhomoserin constructs the work of three enzymes coexpression on the basis of Escherichia coli transhipment and coenzyme synthetic system is transformed
Journey bacterium realizes the efficient production of l-tyrosine.L- junket ammonia can be produced technical problem to be solved by the invention is to provide a kind of
The technical issues of acid simultaneously reduces the recombinant bacterium that impurity generates, while the invention solves the building of the bacterial strain and applications.
The first purpose of the invention is to provide can pure how left-handed bar of the recombination bacillus coli of low cost production；The recombination
Escherichia coli express external source l-lactate dehydrogenase, nadh oxidase and tyrosine phenol lyase simultaneously, and in host's large intestine bar
L-tyrosine has been knocked out on the basis of bacterium absorbs gene.
In one embodiment, the external source l-lactate dehydrogenase is the l-lactate dehydrogenase of originating in lactic acid bacterium.External source
Nadh oxidase be originating in lactic acid bacterium nadh oxidase.
In one embodiment, the lactic dehydrogenase from Lactococcus lactis ATCC 19257,
Lactobacillus plantarum ATCC 14917。
In one embodiment, the amino acid sequence of the lactic dehydrogenase is that accession NO is WP_ on NCBI
003131075.1, the sequence of KRL33571.1.
In one embodiment, the nucleotide sequence of the lactic dehydrogenase is accession NO on NCBI are as follows: NZ_
The sequence of JXJZ01000017REGION:18532..19509, AZEJ01000016REGION:16296..17249.
In one embodiment, the nadh oxidase from Lactococcus lactis ATCC 19257,
Lactobacillus sanfranciscensis DSM20451、Lactobacillus brevis ATCC 14869。
In one embodiment, the amino acid sequence of the nadh oxidase is that accession NO is WP_ on NCBI
032950924.1, WP_056958268.1, ERK43827.1 sequence.
In one embodiment, the nucleotide sequence of the nadh oxidase is accession NO on NCBI are as follows: NZ_
JXJZ01000002REGION:complement(39571..40911)、NZ_AYYM01000013REGION:complement
The sequence of (15875..17233), AWVK01000048REGION:complement (50022..51416).
In one embodiment, the l-lactate dehydrogenase, nadh oxidase, tyrosine phenol lyase are to pass through
PETDuet-1 coexpression.
In one embodiment, the tyrosine phenol lyase is from the raw Erwinia (Erwinia of grass
Herbicola), intermediate citric acid fungus (Citrobacter intermedius), C. freundii
(Citrobacterfreundii), Thermophilic Bacteria Symbiobacterium thermophilum or Symbiobacterium
toebii。
In one embodiment, the tyrosine phenol lyase derives from Citrobacter freundii ATCC
8090, amino acid sequence is the sequence that accession NO is WP_003837154.1 on NCBI.
In one embodiment, it includes that l-tyrosine decomposes gene, phenol decomposes base that the l-tyrosine, which absorbs gene,
Any one because in or two kinds of combinations.
In one embodiment, the l-tyrosine decomposition gene is any one in hpaD, mhpB or two kinds
Combination.
In one embodiment, the phenol decomposition gene is any one in hpaD, mhpB or two kinds of combinations.
In one embodiment, it is NCBI that the l-tyrosine, which decomposes gene and the nucleotide sequence of phenol decomposition gene,
On, the upper accession NO of NCBI are as follows: NC_012892REGION:complement (4505585..4506436) and NC_
012892 REGION:339806..340750。
In one embodiment, the recombination bacillus coli also overexpression is by substrate transport to intracellular lactic acid
Transporter gene, phenol transhipment related gene, in ammonia radical ion transporter gene any one or it is a variety of.
In one embodiment, the recombination bacillus coli also further overexpression NAD synthesizes gene, phosphoric acid pyrrole
In more aldehyde synthesis related genes any one or it is a variety of.
In one embodiment, the gene of the overexpression is lldP (Lactate Transport gene), amtB (ammonia radical ion
Transporter gene), hpaX (phenol transporter gene), mhpT (phenol transporter gene), icsA (NAD synthesize gene), (NAD is closed nadA
At gene), any one or more in pdxJ (phosphoric acid Vitamin B6 synthesize gene).
In one embodiment, the host strain is Escherichia coli BL21 (DE3).
In one embodiment, the overexpression is by by Escherichia coli BL21 (DE3) genome
Increase constitutive promoter before the gene of upper need to strengthen expression.
In one embodiment, lldP accession NO on NCBI are as follows: NC_012892REGION:
3646638..3648293；AmtB is NC_012892REGION:442006..443292；HpaX is；NC_012892REGION:
complement(4502025..4503401)；MhpT is NC_012892REGION:344788..345999；IcsA is NC_
012892REGION:complement(2526116..2527330)；NadA is NC_012892REGION:
740487..741530；PdxJ is NC_012892REGION:complement (2567591..2568322).
A second object of the present invention is to provide a kind of method for producing optical voidness l-tyrosine, the method is to utilize this
The recombinant bacterium of invention.
In one embodiment, the production l-tyrosine is to carry out resting cell production.
In one embodiment, in the system of the resting cell production, wet cell weight 1-200g/L, Pfansteihl 1-
200g/L, phenol concentration 1-200g/L, pH 7.0-9.0,15-40 DEG C of temperature, 250 revs/min of shaking speed；Transformation time 1-
24 hours.
Third object of the present invention is to provide recombinant bacteriums of the present invention or the method for the present invention in chemical industry, food, medicine etc.
The application in field.
Beneficial effects of the present invention:
The present invention constructs a kind of three novel enzyme co-expression gene engineering bacterias, which can be applied to the life of l-tyrosine
It produces.Selection scheme of the present invention does not produce hydrogen peroxide, and cell is not easily decomposed l-tyrosine, and has higher NAD content into the cell.
The production process is simple and raw material is easy to get, and has good industrial applications prospect.
Specific embodiment
The leitungskern of colibacillus engineering of the invention is can to co-express three kinds of enzymes, respectively l-lactate dehydrogenase
(L-Lactate dehydrogenase), nadh oxidase (NADH oxidase), tyrosine phenol lyase (tyrosine
phenol-lyase).Its principle are as follows: entirely intracellular in engineering bacteria, l-lactate dehydrogenase is newborn by L- by coenzyme of endobacillary NAD
Sour dehydrogenation generates pyruvic acid and NADH；NADH is aoxidized and is generated NAD by nadh oxidase, realizes the regeneration of coenzyme NAD.Then exist
Pyruvic acid, ammonia radical ion, phenol synthesis are l-tyrosine under the action of tyrosine phenol lyase, while knockout or overexpression are big
The decomposition of the transhipment that correlation gene is mutually promoted to substrate and reduction product on enterobacteria genome.
In order to solve the above technical problems, The technical solution adopted by the invention is as follows:
1. bacterial strain according to the present invention and plasmid
Lactobacillus plantarum ATCC 14917 purchased from American Type Culture Collecti ATCC,
Lactococcus lactis ATCC 19257、Lactobacillus brevis ATCC 14869、Citrobacter
Freundii ATCC 8090 is purchased from pETDuet-1, pACYCDue-1, pCOLADuet-1, pRSFDuet- of Novagen company
1 plasmid and Escherichia coli BL21 (DE3).Lactobacillus sanfranciscensis DSM20451 is purchased from
Germany Microbiological Culture Collection Center DSMZ.PCasRed, pCRISPR-gDNA must the limited public affairs of dream biotechnology purchased from Zhenjiang love
Department.
2. the knockout of related gene and composing type overexpression in Escherichia coli
(1) knockout of Escherichia coli aromatic compound degrading gene
Phenol and l-tyrosine in the present invention are all easily decomposed by the enzyme in Escherichia coli, according to document
(Biodegradation of Aromatic Compounds by Escherichia coli, Microbiol Mol Biol
Rev.2001,65 (4): 523-569.), related gene being knocked out, and avoids the decomposition of product and substrate.The gene of selection is hpaD
And the upper accession NO of mhpB, NCBI are as follows: NC_012892REGION:complement (4505585..4506436) and NC_
012892REGION:339806..340750。
(2) the composing type overexpression of Escherichia coli lactic acid, phenol, ammonia radical ion transporter gene
, need to be substrate transport to just can be carried out into the cell during resting cell, enhancing Lactate Transport albumen helps
In the high concentration for quickly and for a long time maintaining substrate intracellular, be conducive to the progress of reaction.Selecting the relevant gene of Lactate Transport is
The upper accession NO of lldP, NCBI are as follows: NC_012892REGION:3646638..3648293.Phenol transports relevant gene
It is hpaX and mhpT, the upper accession NO of NCBI are as follows: NC_012892REGION:complement
(4502025..4503401) and NC_012892REGION:344788..345999.Ammonia radical ion transports relevant gene
The upper accession NO of amtB, NCBI are as follows: NC_012892REGION:442006..443292.
(3) overexpression of Escherichia coli NAD synthesis related gene
It is needed during lactic dehydrogenase using NAD as coenzyme, the key enzyme of overexpression Escherichia coli NAD route of synthesis,
It is horizontal that endobacillary NAD can be improved, to be conducive to the generation of pyruvic acid.The gene of selection has icsA, nadA.On NCBI
Accession NO are as follows: NC_012892 REGION:complement (2526116..2527330), NC_012892
REGION:740487..741530。
(4) expression of phosphoric acid Vitamin B6 (Pyridoxal phosphate) synthesis related gene
Phosphoric acid Vitamin B6 (amine) is the coenzyme of tyrosine lyases, the core gene pdxJ being overexpressed in the coenzyme approach,
Be conducive to the synthesis of l-tyrosine.The upper accession NO of NCBI are as follows: NC_012892REGION:complement
(2567591..2568322)。
3. the selection that lactic acid is converted into pyruvic acid relevant enzyme
(1) selection of l-lactate dehydrogenase
Pfansteihl is organic acid the most cheap, mainly produces pyruvic acid at present with Pfansteihl oxydasis Pfansteihl,
Hydrogen peroxide is generated in the process, and hydrogen peroxide can oxide acetylacetonate acid generation acetic acid.L-lactate dehydrogenase is widely present a variety of
In microorganism, usually tended to the lactic dehydrogenase that NAD (NADP) is coenzyme using pyruvic acid as substrate synthesizing lactic acid, but work as
Lactic acid excess or carbon source only have the hydrogen that some lactic dehydrogenases can take off lactic acid when lactic acid to generate pyruvic acid, using Pfansteihl as substrate
The hydrogen generated on Pfansteihl is passed into coenzyme NAD or NADP, to generate NADH or NADPH.
The present invention is from Lactococcus lactis ATCC 19257 and Lactobacillus plantarum ATCC
L-lactate dehydrogenase gene llldh (amino acid sequence is WP_003131075.1) and lpldh (amino are respectively obtained in 14917
Acid sequence is KRL33571.1), expression product is used for the dehydrogenation of lactic acid.
(2) selection of nadh oxidase
Lactic dehydrogenase dehydrogenation from lactic acid generates pyruvic acid NADH.NADH needs are regenerated by NAD oxydasis
NAD, to realize the lasting progress of reaction.Nadh oxidase, which has, produces two kinds of peroxidating Hydrogen of water type and production, produces the NADH of water type
Oxidizing ferment will not generate hydrogen peroxide toxicity.The present invention is respectively from Lactobacillus sanfranciscensis
DSM20451, Lactococcus lactis ATCC 19257, it obtains in Lactobacillus brevis producing water type NADH oxygen
Change enzyme gene lsnox (amino acid sequence is WP_056958268.1), llnox (amino acid sequence is WP_003131075.1),
Lbnox (amino acid sequence is ERK43827), expression product are used for the regeneration of NAD..
(3) selection of tyrosine phenol lyase
From the raw Erwinia (Erwinia herbicola) of grass, intermediate citric acid fungus (Citrobacter
Intermedius), C. freundii (Citrobacterfreundii) and Thermophilic Bacteria Symbiobacterium
The tyrosine phenol lyase of thermophilum and Symbiobacterium toebii etc. is most often to study.The present invention therefrom selects
The higher enzyme from Citrobacter freundii of activity is selected, obtains junket from Citrobacter freundii ATCC 8090
Propylhomoserin phenols cracking enzyme gene cftpl (amino acid sequence is WP_003837154.1).
4. the building of coexpression system and the culture of cell
At present Escherichia coli polygenes coexpression there are many method, (Escherichia coli polygenes coexpression strategy, China are raw
Object engineering magazine, 2012,32 (4): 117-122), (synthetic biology technological transformation Escherichia coli are raw using Liu Xianglei by the present invention
Produce shikimic acid and resveratrol, 2016, Shanghai Institute of Pharmaceutical Industry, doctoral thesis) the method building, before each gene
Comprising T7 promoter and RBS binding site, theoretically speaking the expression of gene is strong because having T7 and RBS before each gene
Degree is influenced little by arrangement order.Include three genes on each plasmid, the plasmid heat built is transduceed into Escherichia coli
It in competent cell, and is coated on antibiotic solid plate, screening obtains positive transformant to get recombination bacillus coli is arrived.
The culture of cell: according to classical recombination bacillus coli culture and inducing expression scheme, it is by volume by recombination bacillus coli
2% amount is transferred in LB fermentation medium (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L), as cell OD600Reach
After 0.6-0.8, the IPTG of final concentration of 0.4mM is added, in 20 DEG C of inducing expression culture 8h.After inducing expression, 20 DEG C,
8000rpm, cell is collected by centrifugation within 20 minutes.
5. resting cell produces l-tyrosine
Resting cell system are as follows: wet cell weight 1-200g/L, Pfansteihl 1-200g/L, phenol 1-200g/L, pH
7.0-9.0,20-50 DEG C of temperature, 250 revs/min of shaking speed；Transformation time 1-24 hours.
6. the detection and analysis of sample
The quantitative analysis of l-tyrosine is tested and analyzed using 200 high performance liquid chromatograph of PerkinElmer Series, is matched
Differential refraction detector.Chromatographic condition are as follows: mobile phase is -0.1% formic acid water of methanol (40:60), using Chinese nation Megres C18
Chromatographic column (4.6 × 250mm, 5 μm), flow velocity 1ml/min, 30 DEG C of column temperature, 20 μ l of sample volume.
In order to which technical problems, technical solutions and advantages to be solved are more clearly understood, tie below
Embodiment is closed, the present invention will be described in detail.It should be noted that specific embodiment described herein is only to explain
The present invention is not intended to limit the present invention.
Embodiment 1
According to document Large scale validation of an efficient CRISPR/Cas-based multi
gene editing protocol in Escherichia coli.Microbial Cell Factories,2017,16
(1): method described in 68 by Escherichia coli BL21 (DE3) hpaD and mhpB carry out single or double knockout,
In, the plasmid of gene knockout used in the present invention is pCasRed and pCRISPR-gDNA (hpaD sgRNA) and homology arm (hpaD
Donor it) imports on Escherichia coli BL21 (DE3) together, Cas9/sgRNA induces host and sends out in hpaD gene loci
HpaD donor is integrated on hpaD gene by raw double-strand break, recombinase Red, realizes the knockout of gene, and sequence verification.
HpaD sgRNA, hpaD donor, mhpB sgRNA, mhpB donor are respectively such as sequence table SEQ ID NO:11, SEQ ID
NO:12, SEQ ID NO:13, shown in SEQ ID NO:14.MhpB is knocked out in the same way.
Configure the solution that pH is 8, phenol or l-tyrosine 1g/L, wet thallus amount 100g/L, 35 DEG C place 10 hours after survey
Determine concentration.It is shown in reaction system in table 1, the surplus of phenol or l-tyrosine.
1 different strains of table are to the residual concentration after substrate and product decomposition
| Bacterial strain | 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 |
Obviously Escherichia coli BL21 (Δ hpaD Δ mhpB, DE3) effect is best, it is named as
Escherichia coli HM。
Embodiment 2
Recombination bacillus coli building: first by encoding lactate dehydrogenase, nadh oxidase, tyrosine phenolic acid lyases base
Cause is connected respectively on plasmid pETDuet-1.After obtaining various three gene co-expressings recombinant plasmids, plasmid is converted into large intestine bar
Bacterium Escherichia coli HM screens to obtain positive transformant to get recombination large intestine bar is arrived using ampicillin plate
Bacterium.
Derivational expression method: being that 2% amount is transferred to LB fermentation medium (peptone by recombination bacillus coli by volume
10g/L, yeast powder 5g/L, NaCl 10g/L) in, as cell OD600After reaching 0.6-0.8, it is added final concentration of 0.4mM's
IPTG, in 20 DEG C of inducing expression culture 8h.After inducing expression, 20 DEG C, 8000rpm, cell is collected by centrifugation within 20 minutes.
The cell being collected into carries out transformation assay, and the results are shown in Table 2.Resting cell system in transformation system are as follows: thin
Born of the same parents weight in wet base 100g/L, Pfansteihl 50g/L, phenol 50g/L, pH 8.0, temperature are 35 DEG C, 250 revs/min of shaking speed；Conversion
Time 10 hours.
The efficiency that table 2 co-expresses Escherichia coli generation l-tyrosine compares
| Bacterial strain | 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 |
Escherichia coli HM/pETDuet-1-llnox-llldh-cftpl result is most as can be seen from the above table
It is good.
Embodiment 3
According to strain construction method described in example 2, (all kinds of plasmids are sieved according to the bright book of explanation using different resistant panels
Select positive transformant) and derivational expression method, it collects various types of cells and carries out transformation assay, the results are shown in Table 3.In transformation system
Resting cell system are as follows: wet cell weight 50g/L, Pfansteihl 10g/L, phenol 10g/L, pH 7.0, temperature are 30 DEG C, and shaking table turns
250 revs/min of speed；Transformation time 10 hours.
Comparison of the various expression plasmids of table 3 for production l-tyrosine
| Bacterial strain | 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 is best using effect when pETDuet-1 coexpression as can be seen from the above table.
Embodiment 4
Using document Large scale validation of an efficient CRISPR/Cas-based multi
gene editing protocol in Escherichia coli.Microbial Cell Factories,2017,16
(1): method described in 68 will correspond to the 3- phosphoric acid for increasing Escherichia coli before gene on Escherichia coli HM genome
Medium expression intensity constitutive promoter (PG) before glyceraldehyde dehydrogenase gene (gpdA), sequence such as SEQ ID NO:10 institute
Show.
When the lldP that enhances gene is expressed, using Escherichia coli HM genome as template, with primer lldP-FF/
LldP-FR, lldP-gpdA-F/lldP-gpdA-R, lldP-RF/lldP-RR amplify upstream, promoter, downstream sequence, and
The expression cassette containing gpdA promoter is fused to by primer of lldP-FF and lldP-RR.Then with plasmid pCasRed,
After pCRISPR-gDNA (sgRNA containing lldP) is transferred to Escherichia coli HM together, Cas9/sgRNA induces host and exists
Double-strand break occurs for lldP gene loci, before gpdA promoter is integrated into lldP gene by recombinase Red, and sequence verification.
When the hpaX that enhances gene is expressed, using the method for similar lldP expression of enhancing gene, upstream, starting are first amplified
Son, downstream sequence, and design primer is fused to the expression cassette containing gpdA promoter.Then with plasmid pCasRed, pCRISPR-
After gDNA (sgRNA containing hpaX) is transferred to Escherichia coli HM together, Cas9/sgRNA induces host in hpaX gene
Double-strand break occurs for site, before gpdA promoter is integrated into hpaX gene by recombinase Red, and sequence verification
When the mhpT that enhances gene is expressed, using the method for similar lldP expression of enhancing gene, upstream, starting are first amplified
Son, downstream sequence, and design primer is fused to the expression cassette containing gpdA promoter.Then with plasmid pCasRed, pCRISPR-
After gDNA (sgRNA containing mhpT) is transferred to Escherichia coli HM together, Cas9/sgRNA induces host in mhpT gene
Double-strand break occurs for site, before gpdA promoter is integrated into mhpT by recombinase Red, and sequence verification
When the amtB that enhances gene is expressed, using the method for similar lldP expression of enhancing gene, upstream, starting are first amplified
Son, downstream sequence, and design primer is fused to the expression cassette containing gpdA promoter.Then with plasmid pCasRed, pCRISPR-
After gDNA (sgRNA containing amtB) is transferred to Escherichia coli HM together, Cas9/sgRNA induces host in amtB gene
Double-strand break occurs for site, before gpdA promoter is integrated into amtB gene by recombinase Red, and sequence verification
Following table is the manipulative indexing of Primer and sequence table serial number.
4 Primer of table is compareed with sequence table serial number
According to method inducing expression as described in example 2, collects various types of cells and carry out transformation assay, the results are shown in Table 5.
Resting cell system in transformation system are as follows: wet cell weight 10g/L, Pfansteihl 50g/L, phenol 10g/L, pH 8.0, temperature are
40 DEG C, 250 revs/min of shaking speed；Transformation time 12 hours.
5 conversion results of table compare
| Bacterial strain | 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 best Escherichia coli HM (PG-lldP, PG-hpaX, PG-mhpT) of effect is named as
Escherichia coli HMLHM。
Embodiment 5
Large intestine will be increased before icsA and/or nadA gene in Escherichia coli HMLHM according to the method for example 4
Medium expression intensity constitutive promoter (PG) before the glyceraldehyde 3-phosphate dehydro-genase gene (gpdA) of bacillus, sequence such as SEQ
Shown in ID NO:22.Then plasmid is imported again.
When the icsA that enhances gene is expressed, using the method for lldP expression of enhancing gene similar in embodiment 4, first amplify
Trip, promoter, downstream sequence, and design primer are fused to the expression cassette containing gpdA promoter.Then with plasmid pCasRed,
After pCRISPR-gDNA (containing icsA-gRNA) is transferred to Escherichia coli HMLHM together, Cas9/sgRNA induces host
Double-strand break occurs in icsA gene loci, before gpdA promoter is integrated into icsA gene by recombinase Red, and sequence verification
When the nadA that enhances gene is expressed, using the method for lldP expression of enhancing gene similar in embodiment 4, first amplify
Trip, promoter, downstream sequence, and design primer are fused to the expression cassette containing gpdA promoter.Then with plasmid pCasRed,
After pCRISPR-gDNA (containing nadA-gRNA) is transferred to Escherichia coli HMLHM together, Cas9/sgRNA induces host
Double-strand break occurs in icsA gene loci, before gpdA promoter is integrated into nadA gene by recombinase Red, and sequence verification
When the pdxJ that enhances gene is expressed, using the method for lldP expression of enhancing gene similar in embodiment 4, first amplify
Trip, promoter, downstream sequence, and design primer are fused to the expression cassette containing gpdA promoter.Then with plasmid pCasRed,
After pCRISPR-gDNA (containing pdxJ-gRNA) is transferred to Escherichia coli HMLHM together, Cas9/sgRNA induces host
Double-strand break occurs in icsA gene loci, before gpdA promoter is integrated into pdxJ gene by recombinase Red, and sequence verification
Following table is the manipulative indexing of Primer and sequence table serial number.
6 Primer of table is compareed with sequence table serial number
| Title | It is numbered in sequence table |
| icsA sgRNA | SEQ ID NO:2 |
| nadA sgRNA | SEQ ID NO:3 |
| pdxJ sgRNA | SEQ ID NO:16 |
After the completion of genetic modification, co-expression plasmid is imported.According to method inducing expression as described in example 2, collect each
Class cell carries out transformation assay, and the results are shown in Table 7.Resting cell system in transformation system are as follows: wet cell weight 20g/L,
Pfansteihl 200g/L, phenol 200g/L, pH 9.0, temperature are 30 DEG C, 250 revs/min of shaking speed；Transformation time 24 hours.
7 conversion results of table compare
| Bacterial strain | 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 |
Best Escherichia coli HMLHM (PG-nadA, PG-pdxJ) is named as Escherichia coli
HP
Embodiment 6
According to derivational expression method described in embodiment 2, by Escherichia coli HP/pETDuet-1-llnox-
Thallus is collected after the completion of llldh-cftpl inducing expression, in 100ml reaction system, wet cell weight 1g/L, Pfansteihl 1g/L,
Phenol 1g/L, pH 7.0, temperature are 15 DEG C, 250 revs/min of shaking speed；Transformation time 1 hour.Measurement result, l-tyrosine
Concentration is 52mg/L.
Embodiment 7
According to derivational expression method described in embodiment 2, by Escherichia coli HP/pETDuet-1-llnox-
Thallus is collected after the completion of llldh-cftpl inducing expression, in 100ml reaction system, wet cell weight 200g/L, Pfansteihl
200g/L, phenol 200g/L, pH 8.5, temperature are 40 DEG C, 250 revs/min of shaking speed；Transformation time 24 hours.Measurement knot
Fruit, l-tyrosine concentration are 402g/L.L-tyrosine is insoluble in water, can be precipitated under high concentration, measures after the dilution of this result
As a result.Escherichia coli BL21 (DE3)/pCOLADuet-1-llnox-llldh l-tyrosine is dense under similarity condition
Degree is 365g/L.
The transformation and building of above-described enzyme and its co-expression gene engineering bacteria, the culture medium composition of thallus and culture side
Method and Whole Cell Bioconversion are only presently preferred embodiments of the present invention, are not intended to restrict the invention, theoretically speaking its
Its bacterium, filamentous fungi, actinomyces, zooblast can carry out the transformation of genome, and for the complete of polygenes coexpression
Cell catalysis.All made any modifications, equivalent replacement within principle and spirit of the invention.
Sequence table
<110>Southern Yangtze University
<120>a kind of engineering bacteria and its application in production l-tyrosine
<130> 2018.3.15
<160> 18
<170> PatentIn version 3.3
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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.
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