CN108949657A - A kind of engineering bacteria and its application in danshensu and α-ketoglutaric acid coproduction - Google Patents
A kind of engineering bacteria and its application in danshensu and α-ketoglutaric acid coproduction Download PDFInfo
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Abstract
The invention discloses a kind of engineering bacteria and its applications in danshensu and α-ketoglutaric acid coproduction, belong to technical field of bioengineering.The present invention constructs a kind of three novel enzyme co-expression gene engineering bacterias, which can be applied to the production of optically pure 3- (3,4- dihydroxy phenyl) -2 hydroxy propanoic acid.(D/L)-alpha-hydroxy carboxylic acid compounds dehydrogenase that the present invention selects all has that Substratspezifitaet is poor, and the strong feature of optics specificity can produce optically pure D- danshensu and L- danshensu, while coproduction α-ketoglutaric acid.Further, by the decomposition of the transhipment that correlation gene is mutually promoted to substrate and reduction product in knockout or overexpression genome of E.coli, the production efficiency of recombinant bacterium is improved.Simple using the method for recombinant bacterium conversion production danshensu of the invention and α-ketoglutaric acid and raw material is easy to get, impurity is few, has good industrial applications prospect.
Description
Technical field
The present invention relates to a kind of engineering bacteria and its applications in danshensu and α-ketoglutaric acid coproduction, belong to bioengineering
Technical field.
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 Recombinant organism 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 present invention provides a kind of optically pure danshensus and α-ketoglutaric acid coproduction
Production method, and construct multienzyme coexpression engineering bacteria, realize the efficient production of danshensu.It is to be solved by this invention
Technical problem is to provide a kind of recombinant bacterium of energy low cost production danshensu.While it the invention solves the building of the bacterial strain and answering
The technical issues of using.
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 3 kinds of enzymes, respectively L-amino acid oxidase, Pidolidone dehydrogenase, alpha-hydroxy carboxylic acid compounds dehydrogenase, and in host's large intestine
Phenolic compound has been knocked out on the basis of bacillus decomposes relevant gene.
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 Pidolidone dehydrogenase from Escherichia coli BL21,
Rhodobacter sphaeroides ATCC BAA-808、Clostridium symbiosum ATCC 14940、
Bacillus subtilis 168。
In one embodiment, the amino acid sequence of Pidolidone dehydrogenase is that accession NO is WP_ on NCBI
000373021.1, WP_011338202.1, WP_003497202.1, WP_010886557.1 sequence.
In one embodiment, the nucleotide sequence of Pidolidone dehydrogenase is accession NO on NCBI are as follows: NC_
012892REGION:1786741..1788084、NC_007493REGION:complement(2129131..2130558)、
NZ_KE992901REGION:complement(17603..18955)、NC_000964REGION:complement
The sequence of (2402067..2403350).
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- oxygroup 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, be by encoded L-amino acids oxidizing ferment, alpha-hydroxy carboxylic acid compounds dehydrogenase and
The gene of the enzyme of Pidolidone dehydrogenation, is all connected on plasmid, and building obtains three gene co-expressing recombinant plasmids, then will recombination
Plasmid converts corresponding bacterial strain, obtains recombination engineering.
In one embodiment, it is that host constructs that the recombinant bacterium, which is with Escherichia coli BL21 (DE3),
It arrives.
In one embodiment, it is any one in hpaD, mhpB that the phenolic compound, which decomposes relevant gene,
Or two kinds of combinations.
In one embodiment, it is on NCBI that the phenolic compound, which decomposes the nucleotide sequence of relevant gene,
Accession NO are as follows: NC_012892REGION:complement (4505585..4506436) and NC_012892REGION:
339806..340750。
In one embodiment, the recombinant bacterium also overexpression glutamate transport gene, NAD synthesize gene, FAD
It is one or more kinds of to synthesize 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 gltS (glutamate transport gene), (NAD is closed nadA
At gene), any one or more in ribF (FAD synthesize gene).
In one embodiment, gltS accession NO on NCBI are as follows: NC_012892REGION:
complement(3694931..3696136)；NadA is NC_012892REGION:740487..741530；RibF is NC_
012892REGION:25479..26420。
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 resting cell production, including wet cell weight 1-200g/L, it is left-handed
DOPA 1-200g/L, Pidolidone 1-200g/L, pH 6.0-9.0；It is reacted in 15-40 DEG C, time 1-48 hour.
Beneficial effects of the present invention:
The present invention constructs a kind of three novel enzyme co-expression gene engineering bacterias, the bacterium can be applied to optically pure 3- (3,
4- dihydroxy phenyl) -2 hydroxy propanoic acid production.It is special that (D/L)-alpha-hydroxy carboxylic acid compounds dehydrogenase that the present invention selects all has substrate
One property is poor, the strong feature of optics specificity, can produce optically pure D- danshensu and L- danshensu, while coproducing alpha -one penta 2
Acid.Further, by knock out or overexpression genome of E.coli on mutually by correlation gene promote substrate transhipment and subtract
The decomposition of few product, improves the production efficiency of recombinant bacterium.Utilize recombinant bacterium conversion production danshensu of the invention and α -one penta
The method of diacid is simple and raw material is easy to get, and has good industrial applications prospect.
Specific embodiment
The leitungskern of engineering bacteria of the invention is that 3 kinds of enzymes, respectively L-amino acid oxidase, α-can be expressed simultaneously
Hydroxycarboxylic acid dehydrogenase and Pidolidone dehydrogenase.Its principle are as follows: entirely intracellular in engineering bacteria, Pidolidone dehydrogenase is with thallus
Pidolidone dehydrogenation is generated α-ketoglutaric acid and NADH for coenzyme by interior NAD；Levodopa is by L-amino acid oxidase deamination
Generate 3,4- dihydroxyphenyl pyruvic acid；Alpha-hydroxy carboxylic acid compounds dehydrogenase utilizes the NADH of glutamate dehydrogenase process generation by 3,4- dihydroxy
Base phenylpyruvic acid is reduced into danshensu while realizing the regeneration of coenzyme NAD.Knockout or overexpression bacillus coli gene simultaneously
The decomposition of the transhipment that correlation gene is mutually promoted to substrate and reduction product in group.
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、Bacillus
subtilis ATCC 13952、Escherichia coli BL21(DE3)、Proteus mirabilis ATCC 29906、
Cosenzaea myxofaciens ATCC19692,Morganella morganii ATCC 49993.Purchased from German microorganism
Bacillus coagulans DSM 1, the Weissella confusa strain DSM of Culture Collection Center DSMZ
20196,Providencia rettgeri DSM 1131,Ignatzschineria larvae DSM 13226.It is purchased from
PETDuet-1, pACYCDue-1, pCOLADuet-1, pRSFDuet-1 plasmid and Escherichia of Novagen company
coli BL21(DE3)。
2. the knockout of related gene and composing type overexpression in Escherichia coli
(1) Escherichia coli phenolic compound decomposes the knockout of relevant gene
Phenolic substances in the present invention is all easily decomposed by the enzyme in Escherichia coli, according to document (Biodegradation
OfAromatic 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 glutamate transport gene
, need to be substrate transport to just can be carried out into the cell during resting cell, enhancing glutamate transporter has
Help the high concentration for maintaining substrate intracellular quickly and for a long time, is conducive to the progress of reaction.Select the relevant base of glutamate transport
Because being gltS, the upper accession NO of NCBI are as follows: NC_012892REGION:complement (3694931..3696136).It is more
It is bar similar with aromatic amino acid, need to absorb amino acid etc. in cell cultivation process, therefore thallus itself can express largely
Amino acid transporter, without overexpression again.
(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 accessionNO of NCBI are as follows: NC_012892REGION:740487..741530.
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 accessionNO of NCBI are as follows: NC_012892REGION:25479..26420.
3. the selection of enzyme
(1) 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.Biotechnol.2013,97:9323-41).L-amino acid oxidase is by α amino and CαOn
It is most absolutely to utilize molecular oxygen direct oxidation reduced form FAD, regenerating oxidation type FAD in hydrogen migration to FAD, while generating peroxide
Change hydrogen.Such as Poljanac etc. generates 3,4- dihydroxy benzenes using east western diamond rattlesnake poison L-amino acid oxidase oxidation DOPA
Then pyruvic acid adds lactic dehydrogenase and hydrogenlyase to generate 3,4- dihydroxy benzenes lactic acid again, in addition necessary in the process
Catalase is added to eliminate toxicity (Modelling and Optimization of the (R)-(+)-of hydrogen peroxide
3,4-Dihydroxyphenyllactic Acid Production Catalyzed,Chem.Biochem.Eng.Q.2005,
19(4)351–358).In addition also a kind of L-amino acid oxidase is related to electron transport chain on cell membrane, and electronics is by breathing
Chain passes to cytochrome oxidase, makes reducing molecular oxygen water, to not generate hydrogen peroxide, this enzyme is primarily present in
Proteus (Proteus sp.), Providian Pseudomonas (Providencia sp.), Morganella (Morganella
(the Crystal structure of a membrane-bound l-amino acid deaminase in bacteriums such as sp.)
from Proteus vulgaris.J.Struct.Biol.2016,195:306-15).The present invention has selected 5 kinds not produce peroxide
The L- oxygroup acid oxidase for changing hydrogen, from Proteus mirabilis ATCC 29906, Cosenzaea myxofaciens
ATCC 19692、Providencia rettgeri DSM 1131、Morganella morganii ATCC 49993、
In Ignatzschineria 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,
The sequence of OAT30925.1, EFE55026.1, WP_036414800.1 or WP_026879504.1, these enzymes all have substrate wide
General and active strong feature.
(2) 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.
(3) selection of Pidolidone dehydrogenase
Pidolidone is a kind of amino acid the most cheap, after dehydrogenation at α-ketoglutaric acid added value with higher,
α-ketoglutaric acid is mainly produced with L-GLOD oxidation Pidolidone at present, is taken off on Pidolidone in the process
Hydrogen is wasted.L-Glu dehydrogenase is widely present in almost all creatures, will be on Pidolidone by substrate of Pidolidone
The hydrogen of generation passes to coenzyme NAD or NADP, to generate NADH or NADPH.NADH or NADPH can be used as hydroxyl above-mentioned
The hydrogen donor of base hydroxy acid dehydrogenase.The present invention is from Escherichia coli BL21, Rhodobacter sphaeroides
ATCC BAA-808, Clostridium symbiosum ATCC 14940, it respectively obtains in Bacillus subtilis 168
Pidolidone gene ecgdh (amino acid sequence is WP_000373021.1), (amino acid sequence is WP_ to rsgdh
011338202.1), csgdh (amino acid sequence is WP_003497202.1), (amino acid sequence is WP_ to bsgdh
010886557.1)。
4. the building of coexpression system and the culture of cell
There are many method (Escherichia coli polygenes coexpression strategy, Chinese biologicals for Escherichia coli polygenes coexpression at present
Engineering magazine, 2012,32 (4): 117-122), the present invention is using Liu Xianglei (synthetic biology technological transformation Escherichia coli production
Shikimic acid and resveratrol, 2016, Shanghai Institute of Pharmaceutical Industry, doctoral thesis) the method building is wrapped before each gene
Promoter containing T7 and RBS binding site, theoretically speaking because having T7 and RBS, the expression intensity of gene before each gene
It is influenced by arrangement order little.Include three genes on each plasmid, the plasmid heat built is transduceed into Escherichia coli sense
It by state cell, and is coated on antibiotic solid plate, screening obtains positive transformant to get to recombination bacillus coli.Carefully
The culture of born of the same parents: being 2% by recombination bacillus coli according to classical recombination bacillus coli culture and inducing expression scheme by volume
Amount be 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 optical voidness danshensu
The system of cell transformation production are as follows: wet cell weight 1-200g/L, levodopa concentration 1-200g/L, L- paddy ammonia
Acid concentration is 1-200g/L, pH 6.0-9.0, is reacted in 15-40 DEG C, time 1-48 hour.Liquid chromatogram measuring after conversion
Danshensu yield and configuration.Levodopa solubility is lower, is the suspension containing insoluble matter in the case of high concentration.
6. the detection and analysis of sample
The quantitative analysis of danshensu and α-ketoglutaric acid: conversion fluid uses 200 efficient liquid phase of PerkinElmer Series
Chromatograph tests and analyzes, and matches differential refraction detector.Chromatographic condition are as follows: mobile phase is -0.1% formic acid water of methanol (40:60), adopts
With 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.
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.
Embodiment 1
The screening of Pidolidone dehydrogenase, clones a variety of Pidolidone dehydrogenase genes from each bacterial strain respectively, and
It is expressed in Escherichia coli BL21 (DE3).Derivational expression method: being 2% by recombination bacillus coli by volume
Amount be 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.
According to document (clone, expression and the enzyme assay Shanghai traffic of bacillus natto glutamic acid dehydrogenase gene
College journal agricultural sciences version, 2010,1:82-86.) active, the described method measurement Pidolidone of broken born of the same parents' measurement crude enzyme liquid
Dehydrogenase is using NAD as the activity of coenzyme, and the results are shown in Table 1.Therefore selection is de- from the Pidolidone of bacillus subtilis
Hydrogen enzyme bsgdh is produced as most preferably for danshensu.
The expression activitiy of the different L-amino acid oxidases of table 1
| Recombinant bacterium | Active U/ml |
| Escherichia coli BL21(DE3)/pETDuet-1-ecgdh | 0.3 |
| Escherichia coli BL21(DE3)/pETDuet-1-rsgdh | 1.1 |
| Escherichia coli BL21(DE3)/pETDuet-1-csgdh | 1.6 |
| Escherichia coli BL21(DE3)/pETDuet-1-bsgdh | 3.5 |
Embodiment 2
According to document Large scale validation ofan 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:16, SEQ ID
NO:17, SEQ ID NO:18, shown in SEQ ID NO:19.MhpB is knocked out in the same way.
The solution that pH is 7, levodopa or D- danshensu 4g/L are configured, wet thallus amount 200g/L, 35 DEG C are placed 10 hours
After measure concentration, the surplus of levodopa and D- danshensu in reaction system is shown in table 2.
2 different strains of table are to the residual concentration after substrate and product decomposition
| Levodopa g/L | D- danshensu g/L | |
| Escherichia coli BL21(DE3) | 1.5 | 1.5 |
| Escherichia coli BL21(ΔhpaDΔmhpB,DE3) | 3.6 | 3.6 |
| Escherichia coli BL21(ΔhpaD,DE3) | 2.1 | 2.7 |
| Escherichia coli BL21(ΔmhpB,DE3) | 1.6 | 1.6 |
Escherichia coli BL21 (Δ hpaD Δ mhpB, DE3) effect is best, it is named as Escherichia
coli HM。
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 Pidolidone dehydrogenase, is connected on plasmid.Three gene co-expressing recombinant plasmids are obtained, plasmid are converted big
Enterobacteria Escherichia coli HM obtains positive transformant using antibiotic plate screening to get recombination large intestine bar is arrived
Bacterium.
Thallus will be collected after the completion of recombination bacillus coli inducing expression, in 100ml reaction volume, wet cell weight 40g/
L, levodopa concentration 40g/L, Pidolidone concentration are 30g/L, pH 8.0, are reacted in 35 DEG C, the time 12 hours.Conversion knot
Liquid chromatogram measuring danshensu yield and configuration after beam.
The comparison of the various recombinant bacteriums of table 3
Embodiment 4
Using document Large scale validation ofan 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:15 institute
Show.
When the gltS that enhances gene is expressed, using Escherichia coli HM genome as template, with primer gltS-FF/
GltS-FR, gltS-gpdA-F/gltS-gpdA-R, gltS-RF/gltS-RR amplify upstream, promoter, downstream sequence, and
The expression cassette containing gpdA promoter is fused to by primer of gltS-FF and gltS-RR.Then with plasmid pCasRed,
After pCRISPR-gDNA (sgRNA containing gltS) is transferred to Escherichia coli HM together, Cas9/sgRNA induces host and exists
Double-strand break occurs for gltS gene loci, before gpdA promoter is integrated into gltS 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
| Title | It is numbered in sequence table |
| gltS sgRNA | SEQ ID NO:1 |
| gltS-FF | SEQ ID NO:3 |
| gltS-FR | SEQ ID NO:4 |
| gltS-gpdA-F | SEQ ID NO:5 |
| gltS-gpdA-R | SEQ ID NO:6 |
| gltS-RF | SEQ ID NO:7 |
| gltS-RR | SEQ ID NO:8 |
According to method inducing expression described in embodiment 1, 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 5g/L, Pidolidone 50g/L, levodopa 20g/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-gltS) of effect is named as Escherichia coli HML.
Embodiment 5
Escherichia coli will be increased before nadA, ribF gene in Escherichia coli HML according to the method for example 4
Medium expression intensity constitutive promoter (PG) before glyceraldehyde 3-phosphate dehydro-genase gene (gpdA), sequence such as SEQ ID NO:
Shown in 15.Then plasmid is imported again.
When the nadA that enhances gene is expressed, using Escherichia coli HML genome as template, with primer nadA-FF/
NadA-FR, nadA-gpdA-F/nadA-gpdA-R, nadA-RF/nadA-RR amplify upstream, promoter, downstream sequence, and
The expression cassette containing gpdA promoter is fused to by primer of nadA-FF and nadA-RR.Then with plasmid pCasRed,
After pCRISPR-gDNA (sgRNA containing nadA) is transferred to Escherichia coli HML together, Cas9/sgRNA induces host and exists
Double-strand break occurs for nadA gene loci, before gpdA promoter is integrated into nadA gene by recombinase Red, and sequence verification.
When the ribF that enhances gene is expressed, using Escherichia coli HML genome as template, with primer ribF-FF/
RibF-FR, ribF-gpdA-F/ribF-gpdA-R, ribF-RF/ribF-RR amplify upstream, promoter, downstream sequence, and
The expression cassette containing gpdA promoter is fused to by primer of ribF-FF and ribF-RR.Then with plasmid pCasRed,
After pCRISPR-gDNA (sgRNA containing ribF) is transferred to Escherichia coli HML together, Cas9/sgRNA induces host and exists
Double-strand break occurs for 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:20 |
| nadA sgRNA | SEQ ID NO:2 |
| ribF-FF | SEQ ID NO:21 |
| ribF-FR | SEQ ID NO:22 |
| ribF-gpdA-F | SEQ ID NO:23 |
| ribF-gpdA-R | SEQ ID NO:24 |
| ribF-RF | SEQ ID NO:25 |
| ribF-RR | SEQ ID NO:26 |
| nadA-FF | SEQ ID NO:9 |
| nadA-FR | SEQ ID NO:10 |
| nadA-gpdA-F | SEQ ID NO:11 |
| nadA-gpdA-R | SEQ ID NO:12 |
| nadA-RF | SEQ ID NO:13 |
| nadA-RR | SEQ ID NO:14 |
After the completion of genetic modification, co-expression plasmid is imported.According to method inducing expression described in embodiment 1, 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,
Pidolidone 120g/L, levodopa 120g/L, pH 9.0, temperature are 30 DEG C, 250 revs/min of shaking speed；Transformation time 24
Hour.
7 conversion results of table compare
Best Escherichia coli HML (PG-nadA, PG-ribF) is named as Escherichia coli
HNR。
Embodiment 6
According to derivational expression method described in embodiment 1, by Escherichia coli HNR/pCOLADuet-1-
Thallus is collected after the completion of efmdhd-bsgdh-cmaao inducing expression, in 100ml reaction system, wet cell weight 1g/L, L- paddy
Propylhomoserin 1g/L, levodopa 1g/L, pH 6.0, temperature are 15 DEG C, 250 revs/min of shaking speed；Transformation time 1 hour.Measurement
As a result, R- danshensu concentration is 93mg/L, e.e% > 99.9.
Embodiment 7
According to derivational expression method described in embodiment 1, thallus will be collected after the completion of bacterial strain inducing expression in table 8, in 100ml
In reaction system, wet cell weight 200g/L, Pidolidone 200g/L, levodopa 200g/L, pH 8.5, temperature is 40 DEG C, is shaken
250 revs/min of revolving speed of bed；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 application in danshensu and α-ketoglutaric acid coproduction
<130> 2018.3.15
<160> 26
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<400> 1
ttatggcttc accaatgcga 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
ttaacggcgt cggcttcggg 20
<210> 3
<211> 25
<212> DNA
<213>artificial sequence
<400> 3
taagggttac gttgacggtt aagca 25
<210> 4
<211> 50
<212> DNA
<213>artificial sequence
<400> 4
tcggccactc atcaacatga ttcattgctt aaccgtcaac gtaaccctta 50
<210> 5
<211> 50
<212> DNA
<213>artificial sequence
<400> 5
taagggttac gttgacggtt aagcaatgaa tcatgttgat gagtggccga 50
<210> 6
<211> 50
<212> DNA
<213>artificial sequence
<400> 6
ttgctaaagt atcgagatga aacatggttt tctcctgtca ggaacgttcg 50
<210> 7
<211> 50
<212> DNA
<213>artificial sequence
<400> 7
cgaacgttcc tgacaggaga aaaccatgtt tcatctcgat actttagcaa 50
<210> 8
<211> 25
<212> DNA
<213>artificial sequence
<400> 8
agccagctcc cacagtttca gcccc 25
<210> 9
<211> 25
<212> DNA
<213>artificial sequence
<400> 9
tcgaatcctg cacgacccac cacta 25
<210> 10
<211> 50
<212> DNA
<213>artificial sequence
<400> 10
tcggccactc atcaacatga ttcatcgaca ttagcgtaat attcgctgtt 50
<210> 11
<211> 50
<212> DNA
<213>artificial sequence
<400> 11
aacagcgaat attacgctaa tgtcgatgaa tcatgttgat gagtggccga 50
<210> 12
<211> 50
<212> DNA
<213>artificial sequence
<400> 12
tgtctggatc aaacattacg ctcatggttt tctcctgtca ggaacgttcg 50
<210> 13
<211> 50
<212> DNA
<213>artificial sequence
<400> 13
cgaacgttcc tgacaggaga aaaccatgag cgtaatgttt gatccagaca 50
<210> 14
<211> 25
<212> DNA
<213>artificial sequence
<400> 14
catccacgga caatgcgcgc agctg 25
<210> 15
<211> 1100
<212> DNA
<213> Escherichia coli BL21(DE3)
<400> 15
atgaatcatg ttgatgagtg gccgatcgct acgtgggaag aaaccacgaa actccattgc 60
gcaatacgct gcgataacca gtaaaaagac cagccagtga atgctgattt gtaaccttga 120
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
<210> 16
<211> 20
<212> DNA
<213>artificial sequence
<400> 16
tatgcccgtc gatcgcgccc 20
<210> 17
<211> 120
<212> DNA
<213>artificial sequence
<400> 17
ccaagatcac gcacgtaccg tcgatgtatc tctctgaact gccagggaaa aaccacggtt 60
agatcagcaa gcgttgccgg gaaatgggcg tcgataccat tatcgttttc gacacccact 120
<210> 18
<211> 20
<212> DNA
<213>artificial sequence
<400> 18
tcatcgagta cctcttgcgc 20
<210> 19
<211> 120
<212> DNA
<213>artificial sequence
<400> 19
tagcctgata tgcacgctta tcttcactgt ctttcccact cgccgctggt gggatatgtc 60
aatggcgtga ttgccagcgc ccgcgagcgt attgcggctt tctcccctga actggtggtg 120
<210> 20
<211> 20
<212> DNA
<213>artificial sequence
<400> 20
cagcacacac ccttcttgcg 20
<210> 21
<211> 25
<212> DNA
<213>artificial sequence
<400> 21
aaggtctaat gaggagatat ttatg 25
<210> 22
<211> 50
<212> DNA
<213>artificial sequence
<400> 22
tcggccactc atcaacatga ttcatcataa atatctcctc attagacctt 50
<210> 23
<211> 50
<212> DNA
<213>artificial sequence
<400> 23
aaggtctaat gaggagatat ttatgatgaa tcatgttgat gagtggccga 50
<210> 24
<211> 50
<212> DNA
<213>artificial sequence
<400> 24
tatgtatgcc gcgtatcagc ttcatggttt tctcctgtca ggaacgttcg 50
<210> 25
<211> 50
<212> DNA
<213>artificial sequence
<400> 25
cgaacgttcc tgacaggaga aaaccatgaa gctgatacgc ggcatacata 50
<210> 26
<211> 25
<212> DNA
<213>artificial sequence
<400> 26
ttcatcacgc gcaatctgcg ctttc 25
Claims (10)
1. a kind of recombination bacillus coli, which is characterized in that the recombination bacillus coli expresses external source Pidolidone dehydrogenation simultaneously
Enzyme, alpha-hydroxy carboxylic acid compounds dehydrogenase and L-amino acid oxidase, and phenolic compound has been knocked out on the basis of host e. coli
Decompose relevant gene.
2. recombination bacillus coli according to claim 1, which is characterized in that it is hpaD, mhpB that the phenols, which decomposes gene,
In the combination of any one or two kinds.
3. recombination bacillus coli according to claim 1, which is characterized in that the recombination bacillus coli also overexpression
Glutamate transport gene, NAD synthesis gene, FAD synthesis gene are one or more kinds of.
4. recombination bacillus coli according to claim 1, which is characterized in that the gene of the overexpression be gltS,
Any one or more in nadA, ribF.
5. recombination bacillus coli according to claim 3 or 4, which is characterized in that the overexpression is by by host
Increase constitutive promoter before the gene of need to strengthen expression on genome of E.coli.
6. recombination bacillus coli according to claim 1, which is characterized in that the Pidolidone dehydrogenase, Alpha-hydroxy carboxylic
Acidohydrogenase, L-amino acid oxidase are co-expressed by pCOLADuet.
7. recombination bacillus coli according to claim 1, which is characterized in that the host e. coli is
Escherichia coli BL21(DE3)。
8. a kind of method for producing danshensu, which is characterized in that the method is to utilize the recombination as claimed in claim 1 to 7
Bacterium.
9. according to the method described in claim 8, it is characterized in that, the method is to carry out resting cell production.
10. according to the method described in claim 8, it is characterized in that, in the system of resting cell production, including cell
Weight in wet base 1-200g/L, levodopa 1-200g/L, Pidolidone 1-200g/L, pH 6.0-9.0；It is reacted in 15-40 DEG C, the time
1-48 hours.
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| CN201810352742.9A CN108949657B (en) | 2018-04-19 | 2018-04-19 | A kind of engineering bacteria and its application in danshensu and α-ketoglutaric acid coproduction |
| PCT/CN2018/111884 WO2019200873A1 (en) | 2018-04-19 | 2018-10-25 | Recombinant escherichia coli and method for producing tanshinol by using same |
| JP2020558016A JP7075505B2 (en) | 2018-04-19 | 2018-10-25 | Recombinant Escherichia coli and a method for producing salbianophosphate A using recombinant Escherichia coli |
| 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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| CN111088207A (en) * | 2020-01-07 | 2020-05-01 | 南京工业大学 | Biocatalysis method for improving yield of synthesized glutaric acid in escherichia coli |
| CN112695043A (en) * | 2021-01-18 | 2021-04-23 | 浙江中医药大学 | Application of SmbZIP2 gene in improving content of salvianolic acid in salvia miltiorrhiza |
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| 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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| 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 |
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| CN111088207A (en) * | 2020-01-07 | 2020-05-01 | 南京工业大学 | Biocatalysis method for improving yield of synthesized glutaric acid in escherichia coli |
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