CN108949648B - A kind of engineering bacteria and its with the application of cheap substrates production danshensu - Google Patents
A kind of engineering bacteria and its with the application of cheap substrates production danshensu Download PDFInfo
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Abstract
The invention discloses a kind of engineering bacteria and its with the application of cheap substrates production danshensu, belong to technical field of bioengineering.The present invention provides can the inexpensive recombinant bacterium for producing optical voidness danshensu；The recombinant bacterium expresses 4 kinds of enzymes, respectively tyrosine phenol lyase, L-amino acid oxidase, l-lactate dehydrogenase, alpha-hydroxy carboxylic acid compounds dehydrogenase simultaneously；Further, recombinant bacterium of the invention has also knocked out phenolic substances and has decomposed gene, overexpression Lactate Transport gene, catechol transporter gene, any one or more in coenzyme synthesis related gene.The present invention realizes the efficient production of danshensu, and method 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 with the application of cheap substrates production danshensu, belong to biotechnology neck
Domain.
Background technique
Extract from the danshensu of Radix Salviae Miltiorrhizae, scientific name R- (+) -3- (3,4- dihydroxy phenyl) -2 hydroxy propanoic acid, D- (+)-β -
(3,4- dihydroxy phenyl) lactic acid, English name are as follows: Danshensu, D-DSS, R-DSS, (R)-(+) -3- (3,4-
Dihydroxyphenyl)-lactic acid, (R)-(+) -3- (3,4-Dihydroxyphenyl) -2-
Hydroxypropanoic acid is a kind of dextrorotation phenolic acid compound.There is currently no natural left-handed danshensus.
Danshensu is the important effective ingredient in Salvia miltiorrhiza Bge water extract, and the country obtained and reflected from Salvia miltiorrhiza Bge water extract in 1980
Structure (research of Determination of water-soluble active constituents of radix, the structure of II .D (+) β (3,4- dihydroxy phenyl) lactic acid, Shanghai first are determined
Medical college's journal, 1980,05 (7), 384-385), it is various research shows that danshensu has important pharmacological effect effect, in the heart
Treatment of cranial vascular disease etc. has unique treatment effect.
Current danshensu, which is mainly extracted from Radix Salviae Miltiorrhizae, obtains (patent CN200810038853.9).Danshensu is in Radix Salviae Miltiorrhizae
Content is lower, and danshensu planting cost height and limits throughput, therefore not only price is high but also much can not for current danshensu
Meets the needs of market.Patent CN201310559498.0 proposes a kind of building bacillus coli gene engineering drawing and utilizes glucose
The method that fermentation produces danshensu, since metabolic pathway of synthesizing relates to the use of hydroxylase, which is easy to make metabolic process product
The yield of danshensu is aoxidized and influenced, simultaneously because Escherichia coli fermentation is high oxygen process, can also aoxidize danshensu, therefore work as
Preceding this method yield is lower, and cost will be higher than plant extract process.Patent CN201210190171.6 proposes the red phenol of hydrolysis
The method of sour B production danshensu, tanshin polyphenolic acid B need to be extracted from Radix Salviae Miltiorrhizae, and chemical hydrolysis process has a large amount of side reactions, same uncomfortable
For large-scale production.The catalyst of chirality synthesis danshensu (patent CN201210420488.4) is prohibitively expensive, currently also only
Rest on laboratory level.
It is proposed early in Roth in 1988 et al. and corresponding 3,4- dihydroxy benzenes is first obtained with chemical Treatment levodopa
Pyruvic acid, then the method for enzymatic clarification S- (+) -3- (3,4- dihydroxy phenyl) -2 hydroxy propanoic acid (S-DSS, L-DSS)
(Enzymatic Synthesis of (S)-(-) -3- (3,4-Dihydroxyphenyl) lactic Acid, Arch.Pharm.
(Weinheim) (1988) 321,179-180).Z.Findrik, et al. levodopa is converted using snake venom amino acid oxidase
At 3,4- dihydroxyphenyl pyruvic acid, is then restored again with D-lactic acid dehydrogenase and generate D- (3,4- dihydroxy phenyl) lactic acid
(Modelling and Optimization of the(R)-(+)-3,4-dihydroxyphenyllactic Acid
Production Catalyzed with D-lactateDehydrogenase from Lactobacillus
Leishmannii Using Genetic Algorithm, Chem.Biochem.Eng.Q.19 (4) 351-358 (2005)).This
Two methods prepare the higher cost of 3,4- dihydroxyphenyl pyruvic acid intermediate, and complicated for operation.
Summary of the invention
Based on the defect of current various methods, the invention proposes a kind of production methods of novel danshensu, and construct
The engineering bacteria of multienzyme coexpression, realizes the efficient production of danshensu.Technical problem to be solved by the invention is to provide one
Kind can efficiently produce the recombinant bacterium of danshensu with cheap substrates, while the invention solves the technologies of the building of the bacterial strain and application
Problem.
The first purpose of the invention is to provide can the inexpensive recombinant bacterium for producing optical voidness danshensu；The recombinant bacterium is same
When express 4 kinds of enzymes, respectively tyrosine phenol lyase, L-amino acid oxidase, l-lactate dehydrogenase, alpha-hydroxy carboxylic acid compounds dehydrogenation
Enzyme.
In one embodiment, the tyrosine phenol lyase is from Erwinia herbicola ATCC
214344。
In one embodiment, the amino acid sequence of the tyrosine phenol lyase is that accession NO is on NCBI
P31011.2。
In one embodiment, the alpha-hydroxy carboxylic acid compounds dehydrogenase is D type alpha-hydroxy carboxylic acid compounds dehydrogenase, is come from
Lactobacillus plantarum ATCC 14917, Enterococcus faecalis ATCC 35038 or
Lactobacillus fermentum ATCC 14931。
In one embodiment, the alpha-hydroxy carboxylic acid compounds dehydrogenase is L-type alpha-hydroxy carboxylic acid compounds dehydrogenase, is come from
Bacillus coagulans DSM 1, Weissella confusa strain DSM 20196 or Lactobacillus
fermentum ATCC 14931。
In one embodiment, the alpha-hydroxy carboxylic acid compounds dehydrogenase is D- alpha-hydroxy carboxylic acid compounds dehydrogenase, amino acid sequence
Column are the sequences that accession NO. is WP_003643296.1, WP_002335374.1 or EEI22188.1 on NCBI；α-
Hydroxycarboxylic acid dehydrogenase is L- alpha-hydroxy carboxylic acid compounds dehydrogenase, and amino acid sequence is that accession NO is WP_ on NCBI
013858488.1, the sequence of WP_003607654.1 or WP_035430779.1.
In one embodiment, the nucleotide sequence of D- alpha-hydroxy carboxylic acid compounds dehydrogenase is accession NO. on NCBI
For NZ_GL379761REGION:COMPLEMENT (533562..534560), NZ_KB944641REGION:
161892..162830, the sequence of ACGI01000078REGION:20793..21791；The nucleosides of L- alpha-hydroxy carboxylic acid compounds dehydrogenase
Acid sequence is that accession NO. is NZ_ATUM01000014REGION:39316..40254, NZ_ on NCBI
The sequence of JQAY01000006REGION:69708..70640, NZ_GG669901REGION:45517..46470.
In one embodiment, the l-lactate dehydrogenase comes from Lactococcus lactis ATCC 19257.
In one embodiment, the amino acid sequence of the l-lactate dehydrogenase is that accession NO is on NCBI
WP_003131075.1 sequence.
In one embodiment, the nucleotide sequence of the l-lactate dehydrogenase is accession NO on NCBI are as follows:
The sequence of NZ_JXJZ01000017REGION:18532..19509.
In one embodiment, what the L-amino acid oxidase was comes from Proteus mirabilis ATCC
29906、Cosenzaea myxofaciens ATCC 19692、Morganella morganii ATCC 49993、
Peroxidating is not produced in Providencia rettgeri DSM 1131 or Ignatzschineria larvae DSM 13226
The L-amino acid oxidase of hydrogen.
In one embodiment, the amino acid sequence of L-amino acid oxidase is that accession NO is WP_ on NCBI
004244224.1, the sequence of OAT30925.1, EFE55026.1, WP_036414800.1 or WP_026879504.1.
In one embodiment, in the nucleotide sequence of L-amino acid oxidase such as sequence table: NZ_
GG668576REGION:1350390..1351805、LXEN01000066REGION:20563..21963、
ACCI02000030REGION:21025..22443、NZ_LAGC01000006REGION:309569..310993、NZ_
KI783332REGION:35799..37217。
In one embodiment, the recombinant bacterium, including by encoding tyrosine phenols cracking enzyme, L-amino acid oxidase,
The gene of alpha-hydroxy carboxylic acid compounds dehydrogenase and the enzyme of Pfansteihl dehydrogenation is connected on 2 plasmids, then by recombinant plasmid transformed host
Escherichia coli obtain recombination engineering.
In one embodiment, the alpha-hydroxy carboxylic acid compounds dehydrogenase gene and l-lactate dehydrogenase gene are attached to matter
After expression, L-amino acid oxidase and tyrosine phenol lyase gene are attached to plasmid pACYCDue-1 after grain pETDuet-1
Expression.
In one embodiment, the host strain is Escherichia coli BL21 (DE3).
In one embodiment, the recombinant bacterium has also knocked out phenolic substances and has decomposed gene.
In one embodiment, the knockout phenolic substances decompose gene be hpaD, mhpB in any one or
Two kinds of person combinations.
In one embodiment, the nucleotide sequence that the phenolic substances decomposes gene is accession NO on NCBI
Are as follows: NC_012892REGION:complement (4505585..4506436) and NC_012892REGION:
339806..340750。
In one embodiment, the recombinant bacterium also overexpression Lactate Transport gene, catechol transporter gene,
Any one or more in coenzyme synthesis related gene.
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, the gene of the overexpression is lldP (Lactate Transport gene), hpaX (catechol
Transporter gene), mhpT (catechol transporter gene), nadA (NAD synthesize gene), pdxJ (phosphoric acid Vitamin B6 synthesizes gene),
Any one or more in ribF (FAD synthesize gene).
In one embodiment, lldP accession NO on NCBI are as follows: NC_012892REGION:
3646638..3648293；HpaX is；NC_012892REGION:complement(4502025..4503401)；MhpT is
NC_012892REGION:344788..345999；NadA is NC_012892REGION:740487..741530；PdxJ is NC_
012892REGION:complement(2567591..2568322)；RibF is NC_012892REGION:25479..26420.
In one embodiment, the recombinant bacterium is on the basis for the escherichia coli host for having knocked out hpaD and mhpB
On, overexpression lldP, hpaX, mhpT, nadA, pdxJ and ribF, and at the same time express tyrosine phenol lyase, L- ammonia
Base acid oxidase, l-lactate dehydrogenase and alpha-hydroxy carboxylic acid compounds dehydrogenase.
A second object of the present invention is to provide a kind of method for producing danshensu, the method is to utilize weight of the invention
Group bacterium.
In one embodiment, the production danshensu is to carry out resting cell production.
In one embodiment, in the system of the resting cell production, wet cell weight 1-200g/L, adjacent benzene two
Phenol concentration is 1-200g/L, and Pfansteihl concentration is 1-200g/L, pH 6.0-9.0, ammonia radical ion concentration 1-30g/L；In 15-40
DEG C reaction, time 1-48 hour.Liquid chromatogram measuring danshensu yield and configuration after conversion.
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 four novel enzyme co-expression gene engineering bacterias, which can be applied to optical voidness danshensu
Production.(D/L)-alpha-hydroxy carboxylic acid compounds dehydrogenase that the present invention selects all has that Substratspezifitaet is poor, the strong feature of optics specificity,
Optically pure D- danshensu and L- danshensu can be produced.The production process is simple and raw material is easy to get, and there is good industrialization to answer
Use prospect.
Specific embodiment
The leitungskern of engineering bacteria of the invention is that 4 kinds of enzymes, respectively tyrosine phenol lyase, L- can be expressed simultaneously
Amino acid oxidase, alpha-hydroxy carboxylic acid compounds dehydrogenase and l-lactate dehydrogenase.Its principle are as follows:, Pfansteihl entirely intracellular in engineering bacteria
Pfansteihl dehydrogenation is generated pyruvic acid and NADH using endobacillary NAD as coenzyme by dehydrogenase；Tyrosine phenol lyase is catalyzed acetone
Acid, ammonia radical ion, catechol generate levodopa；Levodopa then generates 3,4- dihydroxy by L-amino acid oxidase deamination
Phenylpyruvic acid；3,4- dihydroxyphenyl pyruvic acid is reduced by the NADH that alpha-hydroxy carboxylic acid compounds dehydrogenase is generated using lactic dehydrogenase process
Danshensu, the regeneration for realizing coenzyme NAD simultaneously.Simultaneously knock out or overexpression genome of E.coli on mutually by correlation gene
Promote the transhipment of substrate and reduces the decomposition of product.
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,
Enterococcus faecalis ATCC 35038、Lactobacillus fermentum ATCC 14931、
Escherichia coli BL21(DE3)、Proteus mirabilis ATCC 29906、Cosenzaea myxofaciens
ATCC 19692、Morganella morganii ATCC 49993、Lactococcus lactis ATCC 19257、
Erwinia herbicola ATCC 214344,Aeromonas phenologenes ATCC 7966.Purchased from German microorganism
Weissella confusa strain DSM 20196, the Providencia rettgeri DSM of Culture Collection Center DSMZ
1131,Ignatzschineria larvae DSM 13226.PETDuet-1, pACYCDue-1 purchased from Novagen company,
PCOLADuet-1, pRSFDuet-1 plasmid and Escherichia coli BL21 (DE3).PCasRed, pCRISPR-gDNA purchase
From Zhenjiang Ai Bi dream Biotechnology Co., Ltd.
2. the knockout of related gene and composing type overexpression in Escherichia coli
(1) Escherichia coli phenolic substances decomposes the knockout of gene
Phenolic substances in the present invention is 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 is knocked out, avoids the decomposition of product and substrate.The gene of selection is hpaD and mhpB, on NCBI
Accession NO are as follows: NC_012892REGION:complement (4505585..4506436) and NC_012892REGION:
339806..340750。
(2) the composing type overexpression of Escherichia coli lactic acid, catechol 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.Catechol transhipment is relevant
Gene is hpaX and mhpT, the upper accession NO of NCBI are as follows: NC_012892REGION:complement
(4502025..4503401) and NC_012892REGION:344788..345999.
(3) Escherichia coli coenzyme synthesizes the composing type overexpression of related important gene
It is needed in alpha-hydroxy carboxylic acid compounds dehydrogenase reduction process using NADH as coenzyme, overexpression Escherichia coli NAD synthesis
Endobacillary NAD level can be improved, to be conducive to the generation of danshensu in the key enzyme of approach.The gene of selection has nadA.
The upper accession NO of NCBI are as follows: NC_012892REGION:740487..741530.
Phosphoric acid Vitamin B6 (amine) is the coenzyme of tyrosine phenol lyase, the core gene being overexpressed in the coenzyme approach
PdxJ is conducive to the synthesis of levodopa.The upper accession NO of NCBI are as follows: NC_012892REGION:complement
(2567591..2568322)。
FAD is the coenzyme of L-amino acid oxidase, and the important gene ribF being overexpressed in the coenzyme approach is conducive to strengthen
L-amino acid oxidase activity.The upper accession NO of NCBI are as follows: NC_012892REGION:25479..26420.
3. the selection of enzyme in four enzyme coupled catalytic reactions
(1) selection of l-lactate dehydrogenase
Pfansteihl is organic acid the most cheap, after dehydrogenation at pyruvic acid added value with higher.At present mainly with
Pfansteihl oxydasis Pfansteihl produces pyruvic acid, and the hydrogen taken off on Pfansteihl in the process is wasted.Also have with ferment
The method of female fermenting and producing ketone acid.L-lactate dehydrogenase is widely present in multiple-microorganism, is substrate by Pfansteihl using Pfansteihl
The hydrogen of upper generation passes to coenzyme NAD or NADP, to generate NADH or NADPH.NADH or NADPH can be used as above-mentioned
The hydrogen donor of 'alpha '-hydroxy acids dehydrogenase.Generally tended to the lactic dehydrogenase that NAD (NADP) is coenzyme with pyruvic acid
For substrate synthesizing lactic acid, but the hydrogen that lactic dehydrogenase can take off lactic acid when lactic acid excess generates pyruvic acid.
The present invention obtains l-lactate dehydrogenase gene llldh (amino from Lactococcus lactis ATCC 19257
Acid sequence is WP_003131075.1).
(2) selection of tyrosine phenol lyase
Tyrosine phenol lyase (Tyrosine phenol lyase, TPL, E.C.4.1.99.2) also known as β-tyrosine
Enzyme, tyrosine phenol lyase can be catalyzed l-tyrosine and β-elimination reaction generation phenol, pyruvic acid and ammonia occur, can also be catalyzed more
Ba Fasheng β-elimination reaction generates catechol, pyruvic acid and ammonia.The reaction is reversible, and catechol, pyruvic acid and ammonia can
L-3,4 dihydroxyphenylalanine is generated under tyrosine phenol lyase catalysis.The present invention distinguishes from Erwinia herbicola ATCC 214344
Clone obtains tyrosine phenol lyase gene ehtpl, and amino acid sequence is P31011.2.
(3) selection of L-amino acid oxidase
L-amino acid oxidase is widely present in bacterium, fungi, mammalian cell, snake venom, insect toxins and algae
(L-amino acid oxidase as biocatalyst:a dream too far.Appl.Microbiol.Biotechn
ol.2013,97:9323-41).L-amino acid oxidase is by α amino and CαOn hydrogen migration to FAD on, it is most absolutely using point
Sub- oxygen direct oxidation reduced form FAD, regenerating oxidation type FAD, while generating hydrogen peroxide.Such as Poljanac etc. uses east water chestnut
It carries on the back crotalin L-amino acid oxidase oxidation DOPA and generates 3,4- dihydroxyphenyl pyruvic acid, then add lactic dehydrogenase and first again
Acidohydrogenase is generated into 3,4- dihydroxy benzenes lactic acid, must add catalase in addition in the process to eliminate hydrogen peroxide
Toxicity (Modelling and Optimization of the (R)-(+) -3,4-Dihydroxyphenyllactic
Acid Production Catalyzed,Chem.Biochem.Eng.Q.2005,19(4)351–358).In addition there are also a kind of
L-amino acid oxidase is related to electron transport chain on cell membrane, and electronics passes to cytochrome oxidase by respiratory chain, makes
Reducing molecular oxygen is water, to not generate hydrogen peroxide, this enzyme be primarily present in Proteus (Proteus sp.),
(Crystal in the bacteriums such as Providian Pseudomonas (Providencia sp.), Morganella (Morganella sp.)
structure of a membrane-bound l-amino acid deaminase from Proteus
vulgaris.J.Struct.Biol.2016,195:306-15).The present invention has selected 5 kinds of l-amino acids for not producing hydrogen peroxide
Oxidizing ferment, from Proteus mirabilis ATCC 29906, Cosenzaea myxofaciens ATCC 19692,
Providencia rettgeri DSM 1131、Morganella morganii ATCC 49993、Ignatzschineria
In larvae DSM 13226 respectively clone obtain L-amino acid oxidase gene pmaao, cmaao, praao, mmaao,
Ilaao, amino acid sequence be on NCBI accession NO. be WP_004244224.1, OAT30925.1,
The sequence of EFE55026.1, WP_036414800.1 or WP_026879504.1, these enzymes all have substrate extensively and activity is strong
The characteristics of.
(4) selection of alpha-hydroxy carboxylic acid compounds dehydrogenase
The case where according to most suitable substrate, alpha-hydroxy carboxylic acid compounds dehydrogenase include lactic dehydrogenase, alpha-hydroxy acid isocaproic acid dehydrogenation
Enzyme, mandelate dehydrogenase, glyoxylate reductase etc., these enzyme energy wide applications generate alpha-hydroxy carboxylic acid compounds in a variety of substrates, usually
It is named according to the substrate of its most suitable effect.The present invention therefrom select it is optical strong and to 3,4- dihydroxyphenyl pyruvic acid have compared with
Strongly active enzyme, the production for D or L danshensu.From Lactobacillus plantarum ATCC 14917,
It is cloned respectively in Enterococcus faecalis ATCC 35038, Lactobacillus fermentum ATCC 14931
D type alpha-hydroxy carboxylic acid compounds dehydrogenase gene lpldhd, efmdhd, lfldhd are obtained, amino acid sequence is accession on NCBI
It NO. is the sequence of WP_003643296.1, WP_002335374.1, EEI22188.1.From Bacillus coagulans DSM
1, in Weissella confusa strain DSM 20196, Lactobacillus fermentum ATCC 14931 respectively
Clone obtains L-type alpha-hydroxy carboxylic acid compounds dehydrogenase gene bcldhl, wcldhl, lfldhl, and amino acid sequence is on NCBI
Accession NO is the sequence of WP_013858488.1, WP_003607654.1, WP_035430779.1.
4. the building of coexpression system and the culture of cell
It will be every in L-amino acid oxidase selected above, (D/L)-alpha-hydroxy carboxylic acid compounds dehydrogenase, tyrosine phenol lyase
Optional one carries out four enzymes with l-lactate dehydrogenase and combines coexpression.
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.It include two bases on each plasmid using two kinds of plasmids of pACYCDue-1 and pETDuet-1
Cause, by the plasmid built, heat is transduceed in competent escherichia coli cell simultaneously, and is coated on the solid of dual anti-(Kan and Cm)
On plate, screening obtains positive transformant to get recombination bacillus coli is arrived.The culture of cell: according to classical recombination large intestine bar
Bacterium culture and inducing expression scheme, are 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.
4. resting cell produces optical voidness danshensu
The system of cell transformation production are as follows: wet cell weight 1-200g/L, catechol concentration are 1-200g/L, Pfansteihl
Concentration is 1-200g/L, pH 6.0-9.0, ammonia radical ion concentration 1-30g/L；It is reacted in 15-40 DEG C, time 1-48 hour.Conversion
After liquid chromatogram measuring danshensu yield and configuration.
5. the detection and analysis of sample
The quantitative analysis of danshensu: conversion fluid is using the detection point of 200 high performance liquid chromatograph of PerkinElmer Series
UV detector is matched in analysis.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, Detection wavelength 280nm.
Chiral analysis: 200 high performance liquid chromatograph of PerkinElmer Series tests and analyzes, with showing UV detector,
Chiralcel OD-H chiral column (4.6 × 250mm), mobile phase volume ratio are n-hexane: isopropanol: trifluoroacetic acid=80:20:
0.1, flow velocity 0.5mL/min, 25 DEG C of column temperature, sample volume 20 μ L, Detection wavelength 280nm.
Danshensu solubility is lower, and conversion process measures after then diluting if any crystallization is precipitated.
The optical purity of danshensu is evaluated by enantiomeric excess value (%e.e).
When producing R- danshensu,
Enantiomeric excess value %e.e=[(SR-SS)/(SR+SS) × 100%]
When producing S- danshensu,
Enantiomeric excess value %e.e=[(SS-SR)/(SR+SS) × 100%]
S in formulaSFor the peak area of S- danshensu in conversion fluid, SRFor the liquid chromatogram peak area of R- danshensu in conversion fluid.
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.
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.Its
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:10, SEQ ID
NO:11, SEQ ID NO:12, shown in SEQ ID NO:13.MhpB is knocked out in the same way.
The solution that pH is 8, catechol or D- danshensu 2g/L are configured, wet thallus amount 100g/L, 35 DEG C are placed 10 hours
After measure concentration.It is shown in reaction system in table 1, the surplus of catechol and D- danshensu.
1 different strains of table are to the residual concentration after substrate and product decomposition
| Catechol g/L | D- danshensu g/L | |
| Escherichia coli BL21(DE3) | 0.7 | 0.7 |
| Escherichia coli BL21(ΔhpaDΔmhpB,DE3) | 1.8 | 1.9 |
| Escherichia coli BL21(ΔhpaD,DE3) | 1.3 | 1.6 |
| Escherichia coli BL21(ΔmhpB,DE3) | 1.4 | 1.3 |
Obviously Escherichia coli BL21 (Δ hpaD Δ mhpB, DE3) effect is best, it is named as
Escherichia coli HM。
Embodiment 2
The comparison of alpha-hydroxy carboxylic acid compounds dehydrogenase zymologic property.Usual this enzyme may also have reduction pyruvic acid to generate cream
Acid ability, therefore cannot restore or it is atomic it is weak reduction pyruvic acid enzyme be relatively good.Using pyruvic acid as substrate, compare not
It is (clonal expression of serratia marcescens H3010 fermented type D-lactic acid dehydrogenase gene, pure according to document with the reducing power of enzyme
Change and zymologic property research industrial microorganism, 2012,42 (04): 30-37.) described in method measurement using NAD be coenzyme reduction
The activity of pyruvic acid, experimental result are as shown in table 1.
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 various alpha-hydroxy carboxylic acid compounds dehydrogenases of table 2 restore pyruvic acid expression activitiy
| Recombinant bacterium | Active U/ml |
| Escherichia coli HM/pETDuet-1-lpldhd | 6.6 |
| Escherichia coli HM/pETDuet-1-efmdhd | 0 |
| Escherichia coli HM/pETDuet-1-lfldhd | 0.7 |
| Escherichia coli HM/pETDuet-1-bcldhl | 5.2 |
| Escherichia coli HM/pETDuet-1-wcldhl | 0.2 |
| Escherichia coli HM/pETDuet-1-lfldhl | 6.3 |
Embodiment 3
Recombination bacillus coli building: encoding tyrosine phenols cracking enzyme, L-amino acid oxidase, alpha-hydroxy carboxylic acid compounds are taken off first
The gene of hydrogen enzyme and l-lactate dehydrogenase is connected respectively on pETDuet-1 or pACYCDuet-1 plasmid.Obtain two kinds it is biradical
Because co-expressing recombinant plasmid, two kinds of plasmids are converted into Escherichia coli Escherichia coli HM, it is green using chloramphenicol and ammonia benzyl
Mycin plate screening obtains positive transformant to get recombination bacillus coli is arrived.
Thallus will be collected after the completion of recombination bacillus coli inducing expression, in 100ml reaction volume, wet cell weight 20g/
L, catechol concentration are 10g/L, and Pfansteihl concentration is 10g/L, pH 8.0, ammonia radical ion concentration 30g/L；It is reacted in 35 DEG C,
Time 12 hours.Liquid chromatogram measuring danshensu yield and configuration after conversion.
The comparison of the various recombinant bacteriums of table 3
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 is as shown in SEQ ID NO:9.
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
Following table is the manipulative indexing of Primer and sequence table serial number.
4 Primer of table is compareed with sequence table serial number
| Title | It is numbered in sequence table |
| lldP sgRNA | SEQ ID NO:1 |
| hpaX sgRNA | SEQ ID NO:14 |
| mhpT sgRNA | SEQ ID NO:15 |
| lldP-FF | SEQ ID NO:3 |
| lldP-FR | SEQ ID NO:4 |
| lldP-gpdA-F | SEQ ID NO:5 |
| lldP-gpdA-R | SEQ ID NO:6 |
| lldP-RF | SEQ ID NO:7 |
| lldP-RR | SEQ ID NO:8 |
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 200g/L, catechol 10g/L, pH 8.0, temperature
Degree is 40 DEG C, 250 revs/min of shaking speed；Transformation time 12 hours.
5 conversion results of table compare
The best Escherichia coli HM (PG-lldP, PG-hpaX, PG-mhpT) of effect is named as
Escherichia coli HMLHM。
Embodiment 5
It will be increased before nadA, pdxJ, ribF gene in Escherichia coli HMLHM according to the method for embodiment 4 big
Medium expression intensity constitutive promoter (PG) before the glyceraldehyde 3-phosphate dehydro-genase gene (gpdA) of enterobacteria, sequence is such as
Shown in SEQ ID NO:9.Then plasmid is imported again.
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 nadA 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 pdxJ gene loci, before gpdA promoter is integrated into pdxJ gene by recombinase Red, and sequence verification
When the ribF 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 ribF gene loci, before gpdA promoter is integrated into ribF 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 |
| ribF sgRNA | SEQ ID NO:17 |
| nadA sgRNA | SEQ ID NO:2 |
| 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, Catechol 2 00g/L, pH 9.0, temperature are 30 DEG C, 250 revs/min of shaking speed；Transformation time 24 is small
When.
7 conversion results of table compare
Best Escherichia coli HMLHM (PG-nadA, PG-ribF, PG-pdxJ) is named as
Escherichia coli NPR。
Embodiment 6
According to derivational expression method described in embodiment 2, by Escherichia coli NPR/pETDuet-1-wcldhl-
Thallus is collected after the completion of llldh+pACYCDuet-1-cmaao-ehtpl inducing expression, in 100ml reaction system, cell is wet
Weight 1g/L, Pfansteihl 1g/L, catechol 1g/L, pH 6.0, temperature are 15 DEG C, 250 revs/min of shaking speed；Transformation time 1
Hour.Measurement result, S- danshensu concentration are 78mg/L.
Embodiment 7
According to derivational expression method described in embodiment 2, thallus will be collected after the completion of bacterial strain inducing expression in table 7, in 100ml
In reaction system, wet cell weight 200g/L, Pfansteihl 200g/L, Catechol 2 00g/L, pH 8.5, temperature is 40 DEG C, shaking table
250 revs/min of revolving speed；Transformation time 48 hours.Precipitating is all diluted into measurement result after dissolution.
8 conversion results of table compare
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 with cheap substrates production danshensu application
<130> 2018.3.15
<160> 17
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<400> 1
gattgccacc gtccacgagg 20
<210> 2
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<400> 2
ttaacggcgt cggcttcggg 20
<210> 3
<211> 25
<212> DNA
<213>artificial sequence
<400> 3
aaatacaatc tctgtaggtt cttct 25
<210> 4
<211> 50
<212> DNA
<213>artificial sequence
<400> 4
tcggccactc atcaacatga ttcatgagtc tgttgctcat ctccttgtca 50
<210> 5
<211> 50
<212> DNA
<213>artificial sequence
<400> 5
tgacaaggag atgagcaaca gactcatgaa tcatgttgat gagtggccga 50
<210> 6
<211> 50
<212> DNA
<213>artificial sequence
<400> 6
cgtagttttg ttgccagaga ttcatggttt tctcctgtca ggaacgttcg 50
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<400> 7
cgaacgttcc tgacaggaga aaaccatgaa tctctggcaa caaaactacg 50
<210> 8
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<213>artificial sequence
<400> 8
taacacctga cccgcagtgt aaccg 25
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<213> Escherichia coli BL21(DE3)
<400> 9
atgaatcatg ttgatgagtg gccgatcgct acgtgggaag aaaccacgaa actccattgc 60
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atatttattt tccataacat ttcctgcttt aacataattt tccgttaaca taacgggctt 180
ttctcaaaat ttcattaaat attgttcacc cgttttcagg taatgactcc aacttattga 240
tagtgtttta tgttcagata atgcccgatg actttgtcat gcagctccac cgattttgag 300
aacgacagcg acttccgtcc cagccgtgcc aggtgctgcc tcagattcag gttatgccgc 360
tcaattcgct gcgtatatcg cttgctgatt acgtgcagct ttcccttcag gcgggattca 420
tacagcggcc agccatccgt catccatatc accacgtcaa agggtgacag caggctcata 480
agacgcccca gcgtcgccat agtgcgttca ccgaatacgt gcgcaacaac cgtcttccgg 540
agcctgtcat acgcgtaaaa cagccagcgc tggcgcgatt tagccccgac atagccccac 600
tgttcgtcca tttccgcgca gacgatgacg tcactgcccg gctgtatgcg cgaggttacc 660
gactgcggcc tgagtttttt aagtgacgta aaatcgtgtt gaggccaacg cccataatgc 720
gggcagttgc ccggcatcca acgccattca tggccatatc aatgattttc tggtgcgtac 780
cgggttgaga agcggtgtaa gtgaactgca gttgccatgt tttacggcag tgagagcaga 840
gatagcgctg atgtccggcg gtgcttttgc cgttacgcac caccccgtca gtagctgaac 900
aggagggaca gctgatagaa acagaagcca ctggagcacc tcaaaaacac catcatacac 960
taaatcagta agttggcagc atcaccccgt tttcagtacg ttacgtttca ctgtgagaat 1020
ggagattgcc catcccgcca tcctggtcta agcctggaaa ggatcaattt tcatccgaac 1080
gttcctgaca ggagaaaacc 1100
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<210> 12
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tagcctgata tgcacgctta tcttcactgt ctttcccact cgccgctggt gggatatgtc 60
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<210> 14
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cgaacagaaa gacgatcagg 20
<210> 15
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<400> 15
gcgggatgaa gatgatgaag 20
<210> 16
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Claims (16)
1. a kind of recombinant bacterium, which is characterized in that the recombinant bacterium expresses 4 kinds of enzymes, respectively tyrosine phenol lyase, L- ammonia simultaneously
Base acid oxidase, l-lactate dehydrogenase, alpha-hydroxy carboxylic acid compounds dehydrogenase；
Accession number of the tyrosine phenol lyase on NCBI is P31011.2；
The alpha-hydroxy carboxylic acid compounds dehydrogenase be on NCBI accession number be WP_003643296.1, WP_002335374.1 or
The D- alpha-hydroxy carboxylic acid compounds dehydrogenase or NCBI accession number of EEI22188.1 be WP_013858488.1, WP_003607654.1 or
The L- alpha-hydroxy carboxylic acid compounds dehydrogenase of WP_035430779.1；
Accession number of the L-amino acid oxidase on NCBI be WP_004244224.1, OAT30925.1, EFE55026.1,
WP_036414800.1 or WP_026879504.1.
Accession number of the l-lactate dehydrogenase on NCBI is WP_003131075.1.
2. recombinant bacterium according to claim 1, which is characterized in that the recombinant bacterium has also knocked out phenolic substances and decomposed base
Cause；It is protocatechuic acid 2,3- dioxygenase gene hpaD, 2,3- dihydroxyphenyl propionate 1,2 pairs that the phenolic substances, which decomposes gene,
Any one in monooxygenase gene mhpB or two kinds of combinations.
3. recombinant bacterium according to claim 1 or 2, which is characterized in that the recombinant bacterium also overexpression Lactate Transport
Gene, catechol transporter gene, any one or more in coenzyme synthesis related gene.
4. recombinant bacterium according to claim 3, which is characterized in that the overexpression is by by e. coli bl21
(DE3) increase constitutive promoter before the gene of need to strengthen expression on genome.
5. recombinant bacterium according to claim 3, which is characterized in that the gene of the overexpression is Lactate Transport gene
LldP, catechol transporter gene hpaX, catechol transporter gene mhpT, NAD synthesis gene nadA, the synthesis of phosphoric acid Vitamin B6
Gene pdxJ, FAD synthesize any one or more in gene ribF.
6. recombinant bacterium according to claim 1 or 2, which is characterized in that the recombinant bacterium is to knock out protocatechuic acid 2,
The basis of the escherichia coli host of 1,2 dioxygenase gene mhpB of 3- dioxygenase gene hpaD and 2,3- dihydroxyphenyl propionate
On, overexpression Lactate Transport gene lldP, catechol transporter gene hpaX, catechol transporter gene mhpT, NAD are closed
Gene ribF is synthesized at gene nadA, the synthesis of phosphoric acid Vitamin B6 gene pdxJ and FAD, and at the same time expressing tyrosine phenols cracking
Enzyme, L-amino acid oxidase, l-lactate dehydrogenase and alpha-hydroxy carboxylic acid compounds dehydrogenase.
7. a kind of method for producing danshensu, which is characterized in that the method is any described using claim 1~2,4~5
Recombinant bacterium.
8. a kind of method for producing danshensu, which is characterized in that the method is to utilize recombinant bacterium as claimed in claim 3.
9. a kind of method for producing danshensu, which is characterized in that the method is to utilize recombinant bacterium as claimed in claim 6.
10. the method according to the description of claim 7 is characterized in that the production danshensu is to carry out resting cell production.
11. method according to claim 8 or claim 9, which is characterized in that the production danshensu is to carry out resting cell life
It produces.
12. claim 1~2,4~5 any recombinant bacteriums are in chemical industry, food, the application for preparing drug field.
13. recombinant bacterium as claimed in claim 3 is in chemical industry, food, the application for preparing drug field.
14. method of claim 7 is in chemical industry, food, the application for preparing drug field.
15. method described in claim 8~10 is in chemical industry, food, the application for preparing drug field.
16. method described in claim 11 is in chemical industry, food, the application for preparing drug field.
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| JP2020558016A JP7075505B2 (en) | 2018-04-19 | 2018-10-25 | Recombinant Escherichia coli and a method for producing salbianophosphate A using recombinant Escherichia coli |
| PCT/CN2018/111884 WO2019200873A1 (en) | 2018-04-19 | 2018-10-25 | Recombinant escherichia coli and method for producing tanshinol by using same |
| DE112018007299.1T DE112018007299T5 (en) | 2018-04-19 | 2018-10-25 | Method of making Danshensu |
| US16/536,406 US10829790B2 (en) | 2018-04-19 | 2019-08-09 | Recombinant E. coli and method of producing Danshensu by using same |
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| CN101565694A (en) * | 2008-04-25 | 2009-10-28 | 天津天士力制药股份有限公司 | Salvianolic acid enzyme and mixed enzyme of same and ginsenoside and method for converting same into medicinal materials |
| CN103667371A (en) * | 2013-11-11 | 2014-03-26 | 天津大学 | Biological production method of tanshinol |
| CN107299072A (en) * | 2017-08-02 | 2017-10-27 | 江南大学 | A kind of engineering bacteria and its application |
| CN107916245A (en) * | 2017-10-31 | 2018-04-17 | 天津大学前沿技术研究院有限公司 | A kind of application for the method and the recombination engineering for producing L tyrosine recombination engineerings |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101565694A (en) * | 2008-04-25 | 2009-10-28 | 天津天士力制药股份有限公司 | Salvianolic acid enzyme and mixed enzyme of same and ginsenoside and method for converting same into medicinal materials |
| CN103667371A (en) * | 2013-11-11 | 2014-03-26 | 天津大学 | Biological production method of tanshinol |
| CN107299072A (en) * | 2017-08-02 | 2017-10-27 | 江南大学 | A kind of engineering bacteria and its application |
| CN107916245A (en) * | 2017-10-31 | 2018-04-17 | 天津大学前沿技术研究院有限公司 | A kind of application for the method and the recombination engineering for producing L tyrosine recombination engineerings |
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