CN105886450B - Engineering bacterium of tyrosine phenol lyase and construction method and application thereof - Google Patents
Engineering bacterium of tyrosine phenol lyase and construction method and application thereof Download PDFInfo
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- 241000894006 Bacteria Species 0.000 title claims abstract description 38
- 108091000100 Tyrosine Phenol-Lyase Proteins 0.000 title claims abstract description 23
- 238000010276 construction Methods 0.000 title abstract description 10
- WTDRDQBEARUVNC-LURJTMIESA-N L-DOPA Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-LURJTMIESA-N 0.000 claims abstract description 30
- WTDRDQBEARUVNC-UHFFFAOYSA-N L-Dopa Natural products OC(=O)C(N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000013613 expression plasmid Substances 0.000 claims abstract description 28
- 241000588724 Escherichia coli Species 0.000 claims abstract description 22
- 239000000758 substrate Substances 0.000 claims abstract description 22
- 238000006243 chemical reaction Methods 0.000 claims abstract description 21
- 238000000855 fermentation Methods 0.000 claims abstract description 16
- 230000004151 fermentation Effects 0.000 claims abstract description 16
- 108050001186 Chaperonin Cpn60 Proteins 0.000 claims abstract description 15
- 102000052603 Chaperonins Human genes 0.000 claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 7
- 238000004321 preservation Methods 0.000 claims abstract description 7
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 claims description 35
- 238000012258 culturing Methods 0.000 claims description 18
- 108090000623 proteins and genes Proteins 0.000 claims description 16
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 claims description 14
- 239000012634 fragment Substances 0.000 claims description 13
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 10
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 claims description 10
- 239000013612 plasmid Substances 0.000 claims description 9
- 239000013604 expression vector Substances 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- 229960005091 chloramphenicol Drugs 0.000 claims description 7
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 claims description 7
- 239000001963 growth medium Substances 0.000 claims description 7
- 229930027917 kanamycin Natural products 0.000 claims description 7
- 229960000318 kanamycin Drugs 0.000 claims description 7
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims description 7
- 229930182823 kanamycin A Natural products 0.000 claims description 7
- 229940054269 sodium pyruvate Drugs 0.000 claims description 7
- YCCILVSKPBXVIP-UHFFFAOYSA-N 2-(4-Hydroxyphenyl)ethanol Natural products OCCC1=CC=C(O)C=C1 YCCILVSKPBXVIP-UHFFFAOYSA-N 0.000 claims description 6
- DBLDQZASZZMNSL-QMMMGPOBSA-N L-tyrosinol Natural products OC[C@@H](N)CC1=CC=C(O)C=C1 DBLDQZASZZMNSL-QMMMGPOBSA-N 0.000 claims description 6
- 108090000856 Lyases Proteins 0.000 claims description 6
- 102000004317 Lyases Human genes 0.000 claims description 6
- 241001052560 Thallis Species 0.000 claims description 6
- 235000004330 tyrosol Nutrition 0.000 claims description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 5
- 235000019270 ammonium chloride Nutrition 0.000 claims description 5
- 230000001580 bacterial effect Effects 0.000 claims description 5
- 230000010355 oscillation Effects 0.000 claims description 5
- 235000010265 sodium sulphite Nutrition 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 241000588919 Citrobacter freundii Species 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 238000002407 reforming Methods 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 claims description 2
- 230000009466 transformation Effects 0.000 claims 1
- 230000014509 gene expression Effects 0.000 abstract description 12
- 230000000694 effects Effects 0.000 abstract description 9
- 229960004502 levodopa Drugs 0.000 abstract description 8
- 230000008569 process Effects 0.000 abstract description 4
- 230000002194 synthesizing effect Effects 0.000 abstract description 4
- 230000010473 stable expression Effects 0.000 abstract description 3
- 239000002699 waste material Substances 0.000 abstract description 3
- 238000009776 industrial production Methods 0.000 abstract description 2
- 239000005457 ice water Substances 0.000 description 15
- 210000004027 cell Anatomy 0.000 description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 12
- 238000002360 preparation method Methods 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 9
- 238000002156 mixing Methods 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 6
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 6
- 102000003425 Tyrosinase Human genes 0.000 description 6
- 108060008724 Tyrosinase Proteins 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 5
- 238000007664 blowing Methods 0.000 description 5
- 229940041514 candida albicans extract Drugs 0.000 description 5
- 239000011248 coating agent Substances 0.000 description 5
- 238000000576 coating method Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 230000035939 shock Effects 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 239000012137 tryptone Substances 0.000 description 5
- 239000012138 yeast extract Substances 0.000 description 5
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- -1 ammonia ions Chemical class 0.000 description 4
- 239000006227 byproduct Substances 0.000 description 4
- XQGPKZUNMMFTAL-UHFFFAOYSA-L dipotassium;hydrogen phosphate;trihydrate Chemical compound O.O.O.[K+].[K+].OP([O-])([O-])=O XQGPKZUNMMFTAL-UHFFFAOYSA-L 0.000 description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 description 4
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 description 4
- 229910021529 ammonia Inorganic materials 0.000 description 3
- 229960000723 ampicillin Drugs 0.000 description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 3
- 229940107700 pyruvic acid Drugs 0.000 description 3
- 229960004441 tyrosine Drugs 0.000 description 3
- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 description 2
- 241000588769 Proteus <enterobacteria> Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 235000007682 pyridoxal 5'-phosphate Nutrition 0.000 description 2
- 239000011589 pyridoxal 5'-phosphate Substances 0.000 description 2
- 229960001327 pyridoxal phosphate Drugs 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 241000588698 Erwinia Species 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 241000901842 Escherichia coli W Species 0.000 description 1
- 108090000301 Membrane transport proteins Proteins 0.000 description 1
- 102000003939 Membrane transport proteins Human genes 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007068 beta-elimination reaction Methods 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000009061 membrane transport Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 229910001414 potassium ion Inorganic materials 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
- C12P13/222—Phenylalanine
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- Bioinformatics & Cheminformatics (AREA)
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- General Health & Medical Sciences (AREA)
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Abstract
The invention relates to a tyrosine phenol lyase engineering bacterium and a construction method and application thereof, wherein the tyrosine phenol lyase gene is constructed into an expression plasmid, and is simultaneously introduced into a chaperonin expression plasmid to obtain the tyrosine phenol lyase gene which is identified as Escherichia coli, is named as Escherichia coli FPLF8 (Escherichia coli HPLF 8), is stored in China center for type culture collection with the preservation date of 2016 2 and 19 days and the preservation number of CCTCCNO: m2016065. The engineering bacteria can be used for synthesizing the levodopa through fermentation and conversion, has high expression efficiency, stable expression product and high activity, can be used for converting and synthesizing the levodopa under the condition of providing related substrates, and has the advantages of simple process, low cost, high yield, less three-waste discharge and industrial production application value.
Description
Technical Field
The invention relates to an engineering bacterium and a construction method thereof, in particular to a tyrosol lyase engineering bacterium and a construction method and application thereof, belonging to the field of genetic engineering.
Background
The chemical name of levodopa (3, 4-dihydroxyphenyl-L-ananine, L-DOPA for short) is 3, 4-dihydroxyphenylalanine, and the structural formula is as follows:
L-DOPA, an important bioactive substance, is an important intermediate in the biochemical metabolic pathway from L-tyrosine to catechol or melanin.
In the last 60 s, many foreign scholars began to focus on the study of the microbial enzymatic synthesis of L-DOPA. In order to increase the yield of L-DOPA and the substrate conversion rate, researchers have conducted a great deal of research on a process for synthesizing L-DOPA by a microbial enzyme method.
Tyrosine phenol lyase (TPL, e.c.4.1.99.2), also known as β -tyrosinase, uses pyridoxal phosphate (PLP) as a coenzyme, potassium ions and ammonia ions as cofactors, and TPL can catalyze L-Tyrosine to undergo a β -elimination reaction to produce phenol, pyruvic acid and ammonia. Since this reaction is reversible, L-DOPA can be produced from catechol, pyruvic acid and ammonia under the catalysis of TPL after the catechol is used instead of phenol. The precursor of levodopa has an inhibiting effect on enzyme activity at a higher concentration, wherein catechol and pyruvic acid have strong inhibiting effects and can also cause irreversible inactivation of enzyme, reaction conditions are difficult to control, byproducts are more, and the yield of L-DOPA is low.
There are also some uses of bacteria in nature, e.g.Escherichia、Proteus(variants)Genus Proteus),StizolobiumhassjooAndErwinia(Erwinia) and the like, and the TPL escherichia coli genetic engineering bacteria are transformed for 30 hours to generate 29.6 g/L-DOPA as reported in the review of levodopa enzymatic synthesis. As another example, Jang-Young Lee et al cloned p-hydroxyphenylacetate-3-hydroxylase (PHAH) derived from Escherichia coli W (ATCC11105) to convert L-tyrosine to L-DOPA and accumulated the product to 10g/L, which was filed for patent US 5837504. The researchers found that although the expression amount of TPL of the recombinant strains is higher than that of the wild strains, the final synthetic capacity of L-DOPA is not obviously improved or even lower than that of the wild strains. This is probably because the strain has relatively perfect substrate, product in and out cell membrane transport mechanism and substrate catechol inhibition enzyme activity tolerance besides TPL high activity to obtain higher L-DOPA synthesis ability. Generally speaking, the reaction conditions are difficult to control, the stability is poor, the number of byproducts is large, and the yield of L-DOPA is low.
Disclosure of Invention
One of the objects of the present invention is to provide an engineering bacterium; identified as Escherichia coli, named Escherichia coli (Escherichia coli) FPLF8, and preserved in China center for type culture Collection with the preservation address: the preservation date of Wuhan university Wuchang Lojia mountain is 2016, 2 and 19 months, and the preservation number is CCTCCNO: m2016065.
One of the purposes of the invention is to provide a construction method of the engineering bacteria, wherein tyrosine phenol lyase genes are constructed into expression plasmids, and meanwhile chaperonin expression plasmids are introduced to obtain Escherichia coli FPLF 8.
The third purpose of the invention is to provide the application of the engineering bacteria, and the levodopa can be synthesized by fermentation and conversion by using the engineering bacteria, so that the yield is high, the byproducts are few, the stability is good, the quality is good, and the three wastes are less in emission.
In order to achieve the purpose, the invention adopts the following technical scheme:
the engineering bacteria are obtained by connecting a tyrosine phenol lyase gene to an expression vector, constructing an expression plasmid pFPL, and introducing the expression plasmid pFPL into recipient bacteria escherichia coli.
Preferably, a chaperonin expression plasmid is further introduced into E.coli into which expression plasmid pFPL has been introduced, to obtain Escherichia coli FPLF 8. The chaperonin expression plasmid is introduced to promote the soluble expression of Escherichia coli FPLF8 enzyme, so as to raise the enzyme activity of unit thallus and the stability of fermentation enzyme activity unit.
Furthermore, the chaperonin expression plasmid is pG-KJE8, the effect is more obvious, and the expression of the target enzyme of Escherichia coli FPLF8 is better.
Preferably, the tyrosine phenol lyase gene is derived from Citrobacter freundii, is connected to an expression vector, has good fusion, stable subsequent expression, more efficient and more specific synthesis of levodopa by an expression product, few byproducts, great convenience for subsequent separation and purification, simplified separation steps and effectively reduced production cost.
Preferably, pET24a is adopted as the expression vector, so that the fusion property with the target gene fragment is better, and the subsequent expression is more stable.
Preferably, the recipient bacterium escherichia coli adopts BL21 (DE 3), and has high enzyme expression efficiency, stable expression, high yield and high genetic stability.
Preferably, the construction method specifically comprises: (1) integrating the cloned tyrosine phenol lyase whole gene fragment into an expression vector pET24a, and transforming the integrated reforming plasmid into BL21 (DE 3) to obtain FPL bacteria; (2) the chaperonin expression plasmid is introduced into FPL bacteria to obtain FPLF8 bacteria.
According to the scheme, the expression efficiency and the expression stability of the target gene fragment can be effectively improved by optimizing the expression vector and the receptor bacterium, and the activity and the stability of the tyrosine phenol lyase generated by the expression of the engineering bacterium are improved by means of the chaperonin expression plasmid.
The obtained engineering bacterium Escherichia coli FPLF8 is used for converting and generating L-dopa in the presence of a substrate solution.
The method comprises the following specific operation steps:
(1) selecting a single colony, inoculating the colony into a test tube containing an LB culture medium, adding kanamycin (100 mg/L) and chloramphenicol (25 mg/L), culturing at 35-37 ℃ and 220rpm for 12-16h to obtain a first-grade seed;
(2) inoculating the primary seed into a shake flask containing a fermentation culture medium, culturing for 2-5h at 35-37 ℃ and 250rpm under 200-; the fermentation medium comprises the following components: tryptone 12g/L, yeast extract 24g/L, glycerol 5g/L, potassium dihydrogen phosphate 2.31 g/L, dipotassium hydrogen phosphate trihydrate 16.43 g/L;
(3) centrifuging to collect bacteria to obtain bacteria;
(4) adding 60g of thalli into a substrate solution, stirring uniformly, and carrying out sealed oscillation reaction at 25 ℃; the substrate solution comprises 14-16g/L of sodium pyruvate, 10-12g/L of catechol, 40-45g/L of ammonium chloride, 2-5g/L of sodium sulfite and 1-3g/L of EDTA, and the pH value is adjusted to 7.5-8.5;
preferably, during the reaction in step (4), the substrates catechol and sodium pyruvate are added several times, and the catechol concentration is controlled not to exceed 10 g/L.
The invention has the following beneficial effects: the engineering bacteria obtained by the invention have high expression efficiency, stable expression product and high activity, can be used for converting and synthesizing levodopa under the condition of providing related substrates, and has the advantages of simple process, low cost, high yield, less three-waste discharge and industrial production application value.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1:
1. preparation of BL21 (DE 3) into competent cells
BL21 (DE 3) competence was obtained by using TAKARA competent cell preparation kit according to the instructions.
2. Total gene synthesis tyrosine phenol lyase gene fragment
According to Gene ID: the sequence provided by X66978.1, tyrosine phenol lyase whole gene fragment of synthetic origin.
3. Construction of tyrosinase expression plasmid pFPL
The tyrosinase whole gene fragment was subcloned into pET24a plasmid, restriction sites BamHI, XhoI.
4. Introduction of expression plasmid pFPL into competent cells
1) Taking out the competence from-70 ℃, and immediately inserting the mixture into an ice water bath for 3 min;
2) taking 1 microliter of plasmid pFPL from a super clean bench, adding competence, flicking, uniformly mixing, immediately inserting into an ice water bath for 25min, and standing;
3) gently transferring the competence to 42 ℃ water bath for heat shock for 1.5min, and immediately gently transferring to ice water bath for 5 min;
4) adding about 700 microliters of LB (Luria Bertani), and resuscitating at 150rpm and 37 ℃ for 60 min;
5)3500rpm 3min, discarding 600. ang. of supernatant and 700. mu.l, blowing and sucking the rest bacteria liquid, mixing uniformly, coating ampicillin (100 mg/L) LB plate, culturing for 16h at 37 ℃ to obtain Escherichia coli FPLF 8.
Example 2:
1. preparation of BL21 (DE 3) into competent cells
BL21 (DE 3) competence was obtained by using TAKARA competent cell preparation kit according to the instructions.
2. Total gene synthesis tyrosine phenol lyase gene fragment
According to Gene ID: the sequence provided by X66978.1, tyrosine phenol lyase whole gene fragment of synthetic origin.
3. Construction of tyrosinase expression plasmid pFPL
The tyrosinase whole gene fragment was subcloned into pET24a plasmid, restriction sites BamHI, XhoI.
4. Introduction of expression plasmid pFPL into competent cells
6) Taking out the competence from-70 ℃, and immediately inserting the mixture into an ice water bath for 3 min;
7) taking 1 microliter of plasmid pFPL from a super clean bench, adding competence, flicking, uniformly mixing, immediately inserting into an ice water bath for 25min, and standing;
8) gently transferring the competence to 42 ℃ water bath for heat shock for 1.5min, and immediately gently transferring to ice water bath for 5 min;
9) adding about 700 microliters of LB (Luria Bertani), and resuscitating at 150rpm and 37 ℃ for 60 min;
10) 3500rpm 3min, discarding 600 and 700 microliters of supernatant, uniformly blowing and sucking the residual bacterial liquid, coating an LB plate with ampicillin (100 mg/L), and culturing at 37 ℃ for 16h to obtain FPL bacteria;
preparation of FPL Strain competence
BL21 (DE 3) competence was obtained by using TAKARA competent cell preparation kit according to the instructions.
6. Introduction of chaperonin expression plasmid into FPL competent cell
1) Taking out the competence from-70 ℃, and immediately inserting the mixture into an ice water bath for 3 min;
2) taking 1 microliter of chaperonin expression plasmid pG-KJE8 from a super clean bench, adding competence, flicking, mixing uniformly, immediately inserting into ice water bath for 25min, and standing;
3) gently transferring the competence to 42 ℃ water bath for heat shock for 1.5min, and immediately gently transferring to ice water bath for 5 min;
4) adding about 700 microliters of LB (Luria Bertani), and resuscitating at 150rpm and 37 ℃ for 60 min;
5)3500rpm 3min, discarding 600. sup. st and 700. mu.l of supernatant, blowing and sucking the rest bacteria liquid, mixing well, coating with LB plate containing kanamycin (100 mg/L) and chloramphenicol (25 mg/L), culturing at 37 deg.C for 16h to obtain FPLF8 bacteria.
Example 3:
the difference from example 2 is that pKJE7 was used as the chaperonin expression plasmid.
Example 4:
1. preparation of BL21 (DE 3) into competent cells
BL21 (DE 3) competence was obtained by using TAKARA competent cell preparation kit according to the instructions.
2. Total gene synthesis tyrosine phenol lyase gene fragment
According to Gene ID: the sequence provided by X66978.1, tyrosine phenol lyase whole gene fragment of synthetic origin.
3. Construction of tyrosinase expression plasmid pFPL
The tyrosinase whole gene fragment was subcloned into pET24a plasmid, restriction sites BamHI, XhoI.
4. Introduction of expression plasmid pFPL into competent cells
11) Taking out the competence from-70 ℃, and immediately inserting the mixture into an ice water bath for 3 min;
12) taking 1 microliter of plasmid pFPL from a super clean bench, adding competence, flicking, uniformly mixing, immediately inserting into an ice water bath for 25min, and standing;
13) gently transferring the competence to 42 ℃ water bath for heat shock for 1.5min, and immediately gently transferring to ice water bath for 5 min;
14) adding about 700 microliters of LB (Luria Bertani), and resuscitating at 150rpm and 37 ℃ for 60 min;
15) 3500rpm 3min, discarding 600 and 700 microliters of supernatant, uniformly blowing and sucking the residual bacterial liquid, coating an LB plate with ampicillin (100 mg/L), and culturing at 37 ℃ for 16h to obtain FPL bacteria;
preparation of FPL Strain competence
BL21 (DE 3) competence was obtained by using TAKARA competent cell preparation kit according to the instructions.
6. Chaperonin plasmid introduced into FPL competent cell
6) Taking out the competence from-70 ℃, and immediately inserting the mixture into an ice water bath for 3 min;
7) taking 1 microliter of chaperonin expression plasmid pG-KJE8 from a super clean bench, adding competence, flicking, mixing uniformly, immediately inserting into ice water bath for 25min, and standing;
8) gently transferring the competence to 42 ℃ water bath for heat shock for 1.5min, and immediately gently transferring to ice water bath for 5 min;
9) adding about 700 microliters of LB (Luria Bertani), and resuscitating at 150rpm and 37 ℃ for 60 min;
10) 3500rpm 3min, discarding 600 and 700 microliters of supernatant, blowing and sucking the residual bacterial liquid, mixing uniformly, coating an LB plate added with kanamycin (100 mg/L) and chloramphenicol (25 mg/L), and culturing at 37 ℃ for 16h to obtain FPLF8 bacteria;
7. fermentation to produce tyrosine phenol lyase
LB culture medium: 10g/L of tryptone, 0.5g/L of yeast extract, 10g/L of sodium chloride and pure water.
Fermentation medium: tryptone 12g/L, yeast extract 24g/L, glycerol 5g/L, potassium dihydrogen phosphate 2.31 g/L, dipotassium hydrogen phosphate trihydrate 16.43 g/L and pure water.
1) Selecting a single colony, inoculating the single colony into a 4ml LB culture medium test tube, adding kanamycin (100 mg/L) and chloramphenicol (25 mg/L), culturing at 37 ℃ and 220rpm for 12h to obtain a first-grade seed;
2) inoculating the first seed into a shaking flask of 100ml fermentation medium, culturing at 37 deg.C and 220rpm for 4h, adding IPTG to final concentration of 1mM, at 25 deg.C and 220rpm, and culturing for 12 h;
3) and (3) centrifugally collecting the bacteria from the bacteria liquid in the step (2), and placing the bacteria in a refrigerator at the temperature of 20 ℃ below zero.
8. Conversion of tyrosol lyase to produce L-dopa
1)1L substrate solution: 14g/L of sodium pyruvate, 10g/L of catechol, 40g/L of ammonium chloride, 2g/L of sodium sulfite and 1g/L of EDTA, and the pH value is adjusted to 8.0;
2) adding 60g of thalli into 1L of substrate solution, stirring uniformly, and carrying out sealed oscillation reaction at 25 ℃;
3) when the catechol residual concentration is below 1g/L, the reaction is stopped, and the L-dopa concentration is accumulated to be above 85 g/L.
Example 5:
the difference from example 4 is that:
1. fermentation to produce tyrosine phenol lyase
4) Selecting a single colony, inoculating a 4ml LB test tube, adding kanamycin (100 mg/L) and chloramphenicol (25 mg/L), culturing at 35 ℃ and 200rpm for 14h to obtain first-grade seeds;
5) inoculating 100ml TB flask to the first test tube seed, culturing at 35 deg.C and 200rpm for 2h, adding IPTG to final concentration of 1.5mM, and culturing at 23 deg.C and 180rpm for 10 h; fermentation medium: tryptone 12g/L, yeast extract 24g/L, glycerol 5g/L, potassium dihydrogen phosphate 2.31 g/L, dipotassium hydrogen phosphate trihydrate 16.43 g/L.
6) Centrifuging to collect the bacteria, and placing in a refrigerator at-20 ℃.
2. Conversion of tyrosol lyase to produce L-dopa
4)1L substrate solution: 15g/L of sodium pyruvate, 11g/L of catechol, 43g/L of ammonium chloride, 3g/L of sodium sulfite and 2g/L of EDTA, and the pH value is adjusted to be 7.5;
5) adding 60g of thallus and PLP-100mg into 1L of substrate solution, stirring uniformly, and carrying out sealed oscillation reaction at 25 ℃;
6) adding substrates of catechol and sodium pyruvate in the conversion process, and keeping the concentration of the catechol to be 8 g/L;
7) accumulating the L-dopa to 65g/L, stopping increasing the substrate, stopping the reaction when the concentration of the catechol residue is below 1g/L, and accumulating the L-dopa concentration to more than 85 g/L.
Example 6:
the difference from example 4 is that:
the specific operation steps of fermentation and conversion to produce L-dopa in the presence of a substrate solution comprise:
(1) selecting a single colony, inoculating the single colony into a test tube containing an LB culture medium, adding kanamycin (100 mg/L) and chloramphenicol (25 mg/L), culturing at 36 ℃ and 250rpm for 16h to obtain a first-grade seed;
(2) inoculating the first-stage seeds into a shake flask containing a fermentation medium, culturing at 36 ℃ and 250rpm for 2-5h, adding IPTG to a final concentration of 1mM, at 27 ℃ and 220rpm, and culturing for 11 h; the fermentation medium comprises the following components: tryptone 12g/L, yeast extract 24g/L, glycerol 5g/L, potassium dihydrogen phosphate 2.31 g/L, dipotassium hydrogen phosphate trihydrate 16.43 g/L;
(3) centrifugally collecting thalli by the bacterial liquid in the step (2);
(4) adding 60g of thalli into a substrate solution, stirring uniformly, and carrying out sealed oscillation reaction at 25 ℃; the substrate solution comprises 16g/L of sodium pyruvate, 12g/L of catechol, 45g/L of ammonium chloride, 5g/L of sodium sulfite and 3g/L of EDTA, and the pH value is adjusted to 8.5;
(5) when the residual concentration of catechol is below 0.5g/L, the reaction is stopped.
Claims (4)
1. A tyrosol lyase engineering bacterium, which is called Escherichia coli (Escherichia coli) FPLF8, is preserved in China center for type culture collection with the preservation date of 2016 years, 2 months and 19 days, and the preservation number is CCTCC NO: m2016065; the engineering bacteria are obtained by connecting a tyrosine phenol lyase gene to an expression vector, constructing an expression plasmid pFPL, and introducing the expression plasmid pFPL into recipient bacteria escherichia coli; introducing a chaperonin expression plasmid into escherichia coli introduced with the expression plasmid pFPL to obtain escherichia coli FPLF 8; the chaperonin expression plasmid is pG-KJE 8; the tyrosine phenol lyase gene is derived from Citrobacter freundii; the expression vector adopts pET24 a; the recipient bacterium Escherichia coli is BL21 (DE 3).
2. The method of claim 1, comprising: (1) integrating the cloned tyrosine phenol lyase whole gene fragment into an expression vector pET24a, and transforming the integrated reforming plasmid into BL21 (DE 3) to obtain FPL bacteria; (2) the chaperonin expression plasmid is introduced into FPL bacteria to obtain FPLF8 bacteria.
3. The use of the tyrosol lyase engineering bacteria of claim 1 for the production of L-dopa by fermentation and conversion in the presence of a substrate solution.
4. The use of the tyrosol lyase engineering bacteria of claim 3, wherein the specific steps of fermentation and transformation to produce L-dopa in the presence of a substrate solution comprise:
(1) selecting a single colony to be inoculated into a test tube containing an LB culture medium, culturing 100mg/L of kanamycin and 25mg/L of chloramphenicol at 35-37 ℃ and 250rpm of 200-;
(2) inoculating the primary seed into a shake flask containing a fermentation culture medium, culturing for 2-5h at 35-37 ℃ and 250rpm under 200-;
(3) centrifugally collecting thalli by the bacterial liquid in the step (2);
(4) adding 60g of thalli into a substrate solution, stirring uniformly, and carrying out sealed oscillation reaction at 25 ℃; the substrate solution comprises 14-16g/L of sodium pyruvate, 10-12g/L of catechol, 40-45g/L of ammonium chloride, 2-5g/L of sodium sulfite and 1-3g/L of EDTA, and the pH value is adjusted to 7.5-8.5;
(5) when the residual concentration of catechol is below 1g/L, the reaction is stopped.
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| CN106591383B (en) * | 2016-12-16 | 2020-09-04 | 江南大学 | Method for efficiently synthesizing caffeic acid by using catechol as substrate |
| CN107325996A (en) * | 2017-05-03 | 2017-11-07 | 浙江绿创生物科技有限公司 | A kind of tyrosine phenol lyase engineering bacteria and its construction method and application |
| CN107964525B (en) * | 2017-05-03 | 2020-10-27 | 浙江绿创生物科技有限公司 | Engineering bacterium of tyrosine phenol lyase and construction method and application thereof |
| CN108642130B (en) * | 2018-03-29 | 2021-10-15 | 浙江工业大学 | High-throughput screening method for high-activity strain of tyrosine phenol lyase |
| CN108949652B (en) * | 2018-04-19 | 2022-08-09 | 江南大学 | Engineering bacterium and application thereof in producing caffeic acid |
| CN108949649B (en) * | 2018-04-19 | 2021-03-26 | 江南大学 | Engineering bacterium and application thereof in producing levodopa |
| CN108715827B (en) * | 2018-06-08 | 2021-07-20 | 鲁东大学 | Extracellular expression of tyrosine phenol lyase and application thereof |
| CN110055290A (en) * | 2018-08-10 | 2019-07-26 | 浙江工业大学 | A kind of method and application of immobilized enzyme catalysis production levodopa |
| CN110055292B (en) * | 2018-08-10 | 2020-06-19 | 浙江工业大学 | Pyruvic acid and levodopa co-production process and application |
| CN110055291A (en) * | 2018-08-10 | 2019-07-26 | 浙江工业大学 | A method of improving tyrosine phenol lyase catalytic production levodopa efficiency |
| CN109112122B (en) * | 2018-09-29 | 2021-10-22 | 山东鲁抗医药股份有限公司 | Induction expression method in fermentation process of tyrosine phenol lyase |
| CN110331153B (en) * | 2019-06-24 | 2021-04-30 | 浙江工业大学 | Kluyveromyces tyrosol lyase mutant and application thereof |
| CN111733152B (en) * | 2020-04-28 | 2022-02-01 | 江南大学 | Escherichia coli expressing inclusion body of activity of tyrosine phenol lyase and application of escherichia coli |
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