CN102851253A - Escherichia coli engineering strain having high phenethyl alcohol yield and application thereof - Google Patents
Escherichia coli engineering strain having high phenethyl alcohol yield and application thereof Download PDFInfo
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- CN102851253A CN102851253A CN2012102764913A CN201210276491A CN102851253A CN 102851253 A CN102851253 A CN 102851253A CN 2012102764913 A CN2012102764913 A CN 2012102764913A CN 201210276491 A CN201210276491 A CN 201210276491A CN 102851253 A CN102851253 A CN 102851253A
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Abstract
The invention discloses an Escherichia coli engineering strain having high phenethyl alcohol yield and application thereof. An Escherichia coli gene engineering bacterium capable of substantially accumulating phenylalanine (L-Phe) is used as an original strain (the fermentation level of the shake flask fermented phenylalanine can be up to 6.0g/L); phenylpyruvic acid decarboxylase (KDC) and alcohol dehydrogenase (ADH1) are subjected to independent expression and appropriate gene combination to co-express two key enzyme genes so as to construct a recombinant strain; the recombinant strain capable of co-expressing phenylpyruvic acid decarboxylase and alcohol dehydrogenase can produce 130mg/L of beta-phenethyl alcohol, and the phenylalanine yield is 5.0g/L; and the accumulation of phenethyl alcohol can not be detected in the whole fermentation process of the Escherichia coli having high L-Phe yield of the original strain used as a control strain. The invention provides a method for enabling Escherichia coli to directly produce phenethyl alcohol through the fermentation of glucose used as a unique carbon source by means of a metabolic engineering means by using enzyme genes which can realize over-expression and are derived from anabolic pathways of different hosts.
Description
Technical field
The present invention relates to a kind of colibacillus engineering strain and application thereof of high yield phenylethyl alcohol, particularly a kind of colibacillus engineering strain of recombination bacillus coli overexpression phenylpyruvate decarboxylase (KDC) and the ethanol dehydrogenase (ADH1) at the high yield phenylalanine belongs to the metabolic engineering field.
Background technology
(β-Phenylethanol), also claim 2 phenylethyl alcohol, beta-phenyl ethanol, phenylethyl alcohol, chemical name are beta-phenyl ethanol (2-pheny ethyl alcohol is called for short PEA) to bata-phenethyl alcohol, and molecular formula is C8H10O, and molecular weight is 122.16.
Bata-phenethyl alcohol is a kind of aromatic alcohol with simple and elegant fine and smooth rose scent, in the natural essential oil that is present in a lot of flowers and plant, such as rose, jacinthe, jasmine, narcissus, lily etc., other product that contains natural bata-phenethyl alcohol is the food that relates to fermentation during some are produced, as tealeaves, cola, coffee, bread, liquor, hard cider, cheese and soy sauce etc.
The rose aromatic odour is quite popular to people, so that bata-phenethyl alcohol has a wide range of applications in food, medicine, makeup, tobacco and cosmetics of everyday use, it is not only the basal component of all rose scent fragrance, and has synergism and synergism, is the required component of multiple odor type prescription.
Bata-phenethyl alcohol has the aromatic odour of Rose, and its graceful soft fragrance is very popular.This is so that bata-phenethyl alcohol has a wide range of applications in food, makeup, tobacco and cosmetics of everyday use, and it is not only the basal component of all rose scent fragrance, and has synergism and synergism, is the required component of multiple odor type prescription.As spice additive, its usage quantity is only second to vanillin food grade,1000.000000ine mesh, be second largest fragrance component, can be used for the preparation that rose, caramel, honey and other fruit flavour type food flavor(ing) and various wine are used essence and flavouring essence for tobacco, it is indispensable material in rose and other plant local flavor; To stablizing so that it can ad hoc be used for soap scent of alkali; A small amount of bata-phenethyl alcohol is as the flavour ingredient of food, such as soft drink, candy, biscuit etc.Derivative take bata-phenethyl alcohol as raw material production such as Phenylethyl ethanoate, also is to have the spices that very high spices is worth.Bata-phenethyl alcohol also is pharmaceutically traditional fungistat.
Nearly ten thousand tons of the annual production of whole world bata-phenethyl alcohol, except a part seldom be from natural Rose or Flos Rosae Rugosae quintessence oil, extract, all be to adopt the industrial chemicals benzene of cheapness or vinylbenzene by chemical synthesis process production basically.The bata-phenethyl alcohol market value of chemosynthesis is about 3.50 dollars/kg, and natural bata-phenethyl alcohol price up to 1,000 dollar/more than the kg.The production of natural bata-phenethyl alcohol, someone estimates that annual production is several tons now, from rose or other essential oil, extract expensive, needs that therefore far away can not satisfying the market.
Since last century Mo, the raising that quality of the life is required along with people and to the concern of health, the human consumer more and more pays attention to the security of food, daily necessities etc., more advocates " green " and " natural ", and the productions such as food, makeup also more and more tend to use natural additive.In recent years, along with detecting of the generation of adverse events in the food service industry and the toxicant such as carcinogenic, various countries are to all more value and strengthen management of food safety, and are also day by day clear and definite to some restriction provision of processed food in various countries' trade mark act.At US and European, seasonings and the perfume compound that can be marked as " natural " must be to adopt physical method extraction and enzyme catalysis or Production by Microorganism Fermentation from natural materials.In the market or the supermarket, often can see at the extrusion position of food product pack the printed words of " natural ", " containing natural perfume ", " without adding synthetic ", " without adding chemical seasoning " and so on.Its purpose obviously is in order to cater to the human consumer to the needs of " natural " food.Because people have certain conflict and even the feared state of mind to the foodstuff additive of chemosynthesis, thereby would rather pay the price that is higher than several times of syntheticss and even tens times for safety, health and go to choose natural product, some high-grade food of the U.S. would rather adopt expensive natural extract bata-phenethyl alcohol and need not cheap synthetic bata-phenethyl alcohol.
Natural bata-phenethyl alcohol mainly is to produce with microbial transformation L-Phe method in the world, mainly concentrates on the developed countries such as France, Germany, Japan, and its current turnout far can not satisfy consumers demand.
The research that China produces aspect the natural bata-phenethyl alcohol at microbial method still is in the starting stage, there is no at present the producer that produces natural bata-phenethyl alcohol, therefore needs to strengthen the research work of this aspect.
The natural bata-phenethyl alcohol of Biological preparation
(1) can synthesize the microorganism of bata-phenethyl alcohol
Multiple yeast has the ability of de novo synthesis and bioconversion on L-phenylalanine to generation bata-phenethyl alcohol, such as kluyveromyces marxianus (Kluyveromyces marxianus) fermentation 5d, can produce the bata-phenethyl alcohol that concentration is 400mg/L, fermentation pichia spp (Pichia fermentans) fermentation 16h can form the bata-phenethyl alcohol that concentration is 505.5mg/L.In addition, also have Saccharomyces aceti (Saccharomyces vini), produce Ruan's torulopsis (Torulopsisu tilis), thamnidium (Cladosporiumc ladosporioides), Kluyveromyces lactis (Kluyveromyces lactis), yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), Hansenula anomala (Hansenula anomala) etc., can both be in process of growth a certain amount of bata-phenethyl alcohol of de novo synthesis.
Many fungies also have de novo synthesis or transform L-Phe is the ability of bata-phenethyl alcohol, such as phelliuns igniarius (Phellinus igniarius), smooth layer pore fungi (Phellinus laevigatus), poplar shelf fungus (Phellinus tremulae), st-yrax thin skin pore fungi (Ischnoderma benzoinum), geotrichum penicillatum (Geotrichum penicillatum), Hericium erinaceus (Bull. Ex Fr.) Pers. (Hericiumerinaceus), hard black pore fungi (Nigroporus durus) and aspergillus niger (Aspergillus niger) etc., but synthetic or conversion capability is relatively low.
(2) pathways metabolism of synthetic bata-phenethyl alcohol
1. de novo synthesis---phenyl-pyruvic acid approach
In some yeast cell, bata-phenethyl alcohol can be by the shikimic acid pathway de novo synthesis of synthetic aromatic amino acid.After namely forming branched acid through shikimic acid pathway, branched acid is transformed into prephenic acid under the mutase effect, through forming phenyl-pyruvic acid after dehydration, the decarboxylation; The phenyl-pyruvic acid decarboxylation produces phenylacetic aldehyde; The phenylacetic aldehyde dehydrogenation just generates bata-phenethyl alcohol; Pathways metabolism is seen Fig. 1.
2. phenylalanine path for transformation---Emhorn approach
In mammalian cell, L-Phe changes tyrosine into, and tyrosine is degraded to fumaric acid and etheric acid through homogentisic acid again.And in microorganism cells, may there be several approach in the katabolism of L-Phe.One of them is the styracin approach, and the first step of degradation process is that the phenylalanine deaminize produces trans-cinnamic acid, as in shadow yeast (Sporobolomyces roseus) and glutinous rhodotorula (Rhodotorula glutinis) cell.For the microorganism that amino acid is only utilized as nitrogenous source, styracin has not had further necessity of degraded; And for the microorganism that can utilize amino acid as carbon source, styracin then further is degraded to 3-ketone group oxalic acid through Protocatechuic Acid, and 3-ketone group oxalic acid finally enters into the TCA circulation.
In microorganism cells, another approach that L-Phe decomposes is to generate phenyl-pyruvic acid by amino acid whose transamination, or forms phenylethylamine under decarboxylation, and decarboxylation or amine oxidation form phenylacetic aldehyde again, and then generates bata-phenethyl alcohol.This approach at first is to be found in 1907 by Ehrlich.
Studies show that, in cell, bata-phenethyl alcohol synthesizes to walk the kind which bar approach depends on nitrogenous source in the substratum, only has when L-Phe exists as only nitrogen source, and the Emhorn approach just can be preponderated.If there is other nitrogenous source that more easily utilizes to exist in the environment, even under higher L-Phe concentration conditions, still some passes through other approach by metabolism to L-Phe, enter the TCA circulation as be degraded to 3-ketone group oxalic acid by the styracin approach, and this degradation pathway can not be fully suppressed, so under any condition, L-Phe can not be converted into bata-phenethyl alcohol fully.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of colibacillus engineering strain of high yield phenylethyl alcohol, and its single expression derives from pichia spp (Pichia pastoris GS115) phenylpyruvate decarboxylase and yeast saccharomyces cerevisiae (Saccharomyces cerevisiae S288c) ethanol dehydrogenase or tandem expression.
The present invention also provides a kind of method that makes up the colibacillus engineering strain of high yield phenylethyl alcohol, take the intestinal bacteria of high yield phenylalanine as starting strain, utilize expression vector pCL1920 to express two enzyme KDC, ADH1 that derive from phenylethyl alcohol route of synthesis in pichia spp and the yeast saccharomyces cerevisiae, obtain the colibacillus engineering strain of high yield phenylethyl alcohol.
Another technical problem that the present invention will solve provides a kind of method of fermentative production phenylethyl alcohol, and concrete grammar is as follows:
1 bacterial strain: E.coli pAP-B03/pCL1920-adh1-kdc
2 substratum:
Slant medium (g/L): peptone 10, sodium-chlor 10, yeast powder 5, glucose 5; Slant medium adds agar 20, pH7.0;
Ferment-seeded substratum (g/L): LB substratum (peptone 10, sodium-chlor 10, yeast powder 5), pH7.0, liquid amount 20mL/250mL adds Kan50mg/L, Spec80mg/L in case of necessity in substratum.
Fermention medium (g/L): glucose 35, (NH
4)
2SO
45, KH
2PO
43, MgSO
47H
2O3, NaCl 1, Na-Citrate1.5, CaCl
22H
2O 0.015, FeSO
47H
2O 0.1125, and Thiamine-HCl 0.075, and L-Tyr 0.4, peptone 4, yeast powder 2, Kan0.04, microelement nutritious liquid (TES) 1.5mL/L, pH6.8 ± 0.1; 12g/L CaCO
3Be used for regulating pH.Liquid amount is 50mL/250mL
TES(g/L)：Al
2(SO
4)
3·18H
2O 2.0，CoSO
4·7H
2O 0.75，CuSO
4·5H
2O 2.5，H
3BO
3 0.5，MnSO
4·7H
2O24，Na
2MoO
4·2H
2O 3.0，NiSO
4·6H
2O 2.5，ZnSO
4·7H
2O15。
3 culture condition:
(1) seed culture: picking list colony inoculation is cultivated to triangular flask from the LB flat board, and liquid amount is 50mL/250mL, 37 ° of C, and 200r/min cultivates 10-14h.
(2) shake flask fermentation is cultivated: with the inoculum size of cultured seed culture fluid by 10% (v/v), be seeded in the 500mL triangular flask that the 50mL fermention medium is housed and cultivate, 33 ° of C of leavening temperature, shaking speed 200r/min, when thalli growth to OD
610During for 1.5-2.0, go to rapidly 38 ° of C shaking tables, continue to be cultured to 48h.
The mensuration of 4 phenylethyl alcohols: high performance liquid chromatography (HPLC).
(1) chromatographic condition:
Chromatography column: Synergi Hrdro-RP C
18(4.6mm * 250mm, 4 μ m);
Guard column: Analytical KJO-4282(4.0mm * 2.0mm);
Moving phase: methanol-water=50: 50(v/v);
Detect wavelength: 210nm; Flow velocity: 1mL/min; Sample size: 20 μ L; Column temperature: 30 ° of C.
(2) preparation of standardized solution:
The bata-phenethyl alcohol working fluid: get 10mL bata-phenethyl alcohol standard reserving solution in the volumetric flask of 100mL, the aqueous ethanolic solution constant volume with 70%, its concentration is 100mg/L.Be diluted to respectively 50,20,10,5,1 and 0.5mg/L with moving phase again.
(3) sample preparation:
After the fermentation ends, 10000r/min is centrifugal to be obtained fermented supernatant fluid 3mL and adds 7mL aqueous ethanolic solution constant volume 10mL, shakes up, and centrifugally goes out foreigh protein removing etc., can the sample introduction analysis through 0.45 μ m membrane filtration.
Colibacillus engineering strain provided by the invention can produce bata-phenethyl alcohol 130mg/L, effectively utilizes the flux of the synthetic path of L-Phe to promote the generation of output phenylethyl alcohol, thereby realizes that microbe fermentation method direct fermentation glucose produces phenylethyl alcohol.
Description of drawings
Fig. 1: goal gene amplification.Figure (a) swimming lane 1:DL 2000Marker; Swimming lane 2:adh1; Figure (b) swimming lane 1:DL 2000Marker; Swimming lane 2:kdc
Fig. 2: key gene adh1/kdc connects the cloning vector checking
(a) 1:DL5000Marker; The 2:pMD19-adh1 double digestion obtains adh1; 3:adh1;
(b) 1:DL10000 Marker; 2:pMD 19-kdc; 3:pMD19-kdc double digestion kdc.
Fig. 3: key gene expression vector establishment and enzyme are cut checking
1:pCL1920-adh1; The 2:pCL1920-adh1 double digestion obtains adh1; 3:DL10000; 5:pCL1920-adh1-kdc; The 5:pCL1920-adh1-kdc double digestion obtains kdc
Fig. 4 recombinant bacterium fermentation checking
Embodiment
Structure and the evaluation of embodiment 1 recombinant plasmid
Pichia spp (Pichia pastoris GS115) and yeast saccharomyces cerevisiae (Saccharomyces cerevisiae S288c) genome are that template is obtained kdc and adh1 respectively,
Primer following (circle partly is RBS, and underscore partly is the primer restriction enzyme site)
kdc F:TCC
CCCGGG ATGGCCCCAGTTCCAGATATAGCA
R:C
GAGCTCTTAACCTACGATTTTGGCTTTGTTCTTG
adh1 F：AAAA
CTGCAGGTAA 
ATGTCTATCCCAGAAACTCAAAAAGGTGT
R:：CGC
GGATCCGAATTTTCGTTTTAAAACCTAAGAGTCACTTTA
Reaction conditions is: 94 ° of C5min; 94 ° of C30s again, 62 ° of C (adh1)/63 ° C (kdc) 30s, 72 ° of C70s (adh1)/108s (kdc) (30 circulations); 72 ° of C10min carry out the PCR reaction, and with the checking of 0.8% agarose gel electrophoresis and recovery pcr amplification product, the result obtains the adh1/kdc gene fragment (Fig. 1 (a)-swimming lane 2, Fig. 1 (b)-swimming lane 2) that conforms to the bibliographical information size for amplification.With adh1/kdc gene and the cloning vector pMD19 behind restriction enzymes double zyme cutting digestion and the purifying, with the T4DNA ligase enzyme in 16 ℃ of connections of spending the night, connect product and transform Host Strains JM109, transformed bacteria liquid is coated on LB (Ampr) flat board, 37 ° of C incubated overnight are selected positive colony and are extracted plasmid (adh1 Fig. 2 (a)-swimming lane 4; Kdc Fig. 2 (b)-swimming lane 2), cut (adh1 Fig. 2 (a)-swimming lane 2 with enzyme; Kdc Fig. 2 (b)-swimming lane 3) and PCR(adh1 Fig. 2 (a)-swimming lane 3) method validation, and serve the sea and give birth to worker's sequence verification, select the correct transformant of order-checking, and make up recombinant expression vector, such as Fig. 3.The L-Phe that recombinant plasmid pCL1920-adh1, pCL1920-kdc, pCL1920-adh1-kdc is transformed this laboratory early development is produced bacterium intestinal bacteria WSH-Z06(pAP-B03) (CCTCC M 2010009), realized suitability for industrialized production, but in process of production, easily be subject to the pollution of phage, cause a large amount of tank switchings, had a strong impact on the economic benefit of manufacturing enterprise.
The impact that embodiment 2 coexpression ADH1, KDC enzyme ferment on the recombination bacillus coli phenylethyl alcohol
Different recombinant bacterium intestinal bacteria carry out the fermenting experiment contrast with the bacterium that sets out.Recombinant bacterium E.coli pAP-B03/pCL-adh1-kdc, carry out fermenting experiment, measure phenylalanine output and bata-phenethyl alcohol output as shown in Figure 4: can both produce bata-phenethyl alcohol in the situation by independent and co expression phenylpyruvate decarboxylase (KDC) and ethanol dehydrogenase (ADH1), and the output of expressing simultaneously bata-phenethyl alcohol in the situation of two enzyme genes is the highest, high energy reaches 130mg/L, and the bacterium E.coli pAP-B03 that sets out can't detect phenylethyl alcohol output (Fig. 4).
Claims (5)
1. the colibacillus engineering strain of a high yield phenylethyl alcohol is characterized in that expressing pichia spp (Pichia pastorisGS115) phenylpyruvate decarboxylase and yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) ethanol dehydrogenase.
2. engineering strain claimed in claim 1 is characterized in that described single expression or the tandem expression of being expressed as.
3. the construction process of the described engineering strain of claim 1, it is characterized in that intestinal bacteria take the high yield phenylalanine are as starting strain, utilize expression vector pCL1920 to express two enzyme KDC, ADH1 that derive from phenylethyl alcohol route of synthesis in pichia spp and the yeast saccharomyces cerevisiae, obtain the colibacillus engineering strain of high yield phenylethyl alcohol.
4. the method for the described engineering strain fermentative Production of claim 1 phenylethyl alcohol, it is characterized in that the engineering strain activation after, in the inoculum size access fermention medium by 10% (v/v), 33 ° of C of leavening temperature, shaking speed 200r/min, when thalli growth to OD
610During for 1.5-2.0, go to rapidly 38 ° of C shaking tables, continue to be cultured to 48h.
5. method claimed in claim 4 is characterized in that fermentative medium formula is: (g/L): glucose 35, (NH
4)
2SO
45, KH
2PO
43, MgSO
47H
2O3, NaCl1, Na-Citrate1.5, CaCl
22H
2O 0.015, FeSO
47H
2O 0.1125, Thiamine-HCl0.075, L-Tyr0.4, peptone 4, yeast powder 2, Kan0.04, microelement nutritious liquid (TES) 1.5mL/L, pH6.8 ± 0.1; 12g/L CaCO
3Be used for regulating pH; Liquid amount is 50mL/250mL;
TES(g/L)：Al
2(SO
4)
3·18H
2O2.0，CoSO
4·7H
2O0.75，CuSO
4·5H
2O2.5，H
3BO
30.5，MnSO
4·7H
2O24，Na
2MoO
4·2H
2O3.0，NiSO
4·6H
2O2.5，ZnSO
4·7H
2O15。
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| CN103898037A (en) * | 2014-03-11 | 2014-07-02 | 中国科学院青岛生物能源与过程研究所 | Genetically engineered bacterium co-generating geraniol and nerol and construction method and application thereof |
| CN106011184A (en) * | 2016-06-21 | 2016-10-12 | 华南农业大学 | Noncellular synthetic biology based preparation method of 2-phenylethanol and application |
| CN106957878A (en) * | 2017-04-19 | 2017-07-18 | 波顿（上海）生物技术有限公司 | A kind of method that living things catalysis produces 2 benzyl carbinols |
| CN107586752A (en) * | 2017-08-04 | 2018-01-16 | 江南大学 | A kind of engineering bacteria and its application |
| CN108624626A (en) * | 2018-05-15 | 2018-10-09 | 上海应用技术大学 | A kind of bacterial strain and method producing bata-phenethyl alcohol |
| CN110438055A (en) * | 2019-08-01 | 2019-11-12 | 湖北大学 | A kind of whole-cell catalyst containing phenylpyruvate decarboxylase mutant and the application in production benzyl carbinol |
| CN112760243A (en) * | 2021-03-03 | 2021-05-07 | 华东理工大学 | Fermentation medium for saccharomyces cerevisiae and method for producing phenethyl alcohol by saccharomyces cerevisiae fermentation |
| CN115261297A (en) * | 2022-09-15 | 2022-11-01 | 杭州唯铂莱生物科技有限公司 | Escherichia coli recombinant strain and method for producing 3, 4-dihydroxy phenylethanol by using same |
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| CN103898037B (en) * | 2014-03-11 | 2017-11-17 | 中国科学院青岛生物能源与过程研究所 | A kind of coproduction geraniol and the genetic engineering bacterium and its construction method of nerol and application |
| CN103898037A (en) * | 2014-03-11 | 2014-07-02 | 中国科学院青岛生物能源与过程研究所 | Genetically engineered bacterium co-generating geraniol and nerol and construction method and application thereof |
| CN106011184B (en) * | 2016-06-21 | 2019-09-20 | 华南农业大学 | The acellular synthetic biology preparation method and application of 2 phenylethyl alcohol |
| CN106011184A (en) * | 2016-06-21 | 2016-10-12 | 华南农业大学 | Noncellular synthetic biology based preparation method of 2-phenylethanol and application |
| CN106957878A (en) * | 2017-04-19 | 2017-07-18 | 波顿（上海）生物技术有限公司 | A kind of method that living things catalysis produces 2 benzyl carbinols |
| CN106957878B (en) * | 2017-04-19 | 2020-10-30 | 波顿（上海）生物技术有限公司 | Method for producing 2-phenethyl alcohol by biological catalysis |
| CN107586752A (en) * | 2017-08-04 | 2018-01-16 | 江南大学 | A kind of engineering bacteria and its application |
| CN107586752B (en) * | 2017-08-04 | 2019-12-24 | 江南大学 | Engineering bacterium and application thereof |
| CN108624626A (en) * | 2018-05-15 | 2018-10-09 | 上海应用技术大学 | A kind of bacterial strain and method producing bata-phenethyl alcohol |
| CN110438055A (en) * | 2019-08-01 | 2019-11-12 | 湖北大学 | A kind of whole-cell catalyst containing phenylpyruvate decarboxylase mutant and the application in production benzyl carbinol |
| CN110438055B (en) * | 2019-08-01 | 2022-05-27 | 湖北大学 | Whole-cell catalyst containing phenylpyruvate decarboxylase mutant and application of whole-cell catalyst in production of phenethyl alcohol |
| CN112760243A (en) * | 2021-03-03 | 2021-05-07 | 华东理工大学 | Fermentation medium for saccharomyces cerevisiae and method for producing phenethyl alcohol by saccharomyces cerevisiae fermentation |
| CN112760243B (en) * | 2021-03-03 | 2023-04-07 | 华东理工大学 | Fermentation medium for saccharomyces cerevisiae and method for producing phenethyl alcohol by saccharomyces cerevisiae fermentation |
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