CN104120141A - A method of synthesizing beta-caryophyllene by microbial catalysis and a reconstituted cell capable of synthesizing the beta-caryophyllene - Google Patents
A method of synthesizing beta-caryophyllene by microbial catalysis and a reconstituted cell capable of synthesizing the beta-caryophyllene Download PDFInfo
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Abstract
A method of synthesizing beta-caryophyllene by microbial catalysis and a reconstituted cell capable of synthesizing the beta-caryophyllene are disclosed. Acetyl coenzyme A used in the method is obtained from simple initial materials such as glucose. The method includes: A) a step of constructing the reconstituted cell capable of synthesizing the beta-caryophyllene from the acetyl coenzyme A, wherein the reconstituted cell comprises gene segments comprising acetyl-CoA acyltransferase, 3-hydroxy-3-methylglutaryl coenzyme A synthetase, hydroxymethylglutaryl-CoA reductase, mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase, isopentenyl diphosphate isomerase, geranyl pyrophosphate synthase, farnesyl pyrophosphate synthase, and beta-caryophyllene synthase; and B) a step of culturing the reconstituted cell in a culture medium containing glucose, inducing with a proper inducer, separating and purifying to obtain the beta-caryophyllene.
Description
Technical field
The present invention relates to a kind of method of utilizing Biological preparation terpene compound, particularly, relate to the method for the synthetic β-caryophyllene of a kind of microorganism catalysis and can synthesize the reconstitution cell of β-caryophyllene, wherein acetyl-CoA used is to obtain from the simple parent material such as glucose.
Background technology
Terpene compound---β-caryophyllene, is a kind of Important Platform compound of dicyclo sesquiterpene type, is the important source material of medicine, food, makeup and high-density propellant industry.β-caryophyllene, as spices, has been applied in makeup and foodstuff additive; In pharmaceutical industries, β-caryophyllene has the effects such as local anaesthesia, anti-inflammatory, mosquito-repelling, anxiety, antidepressant, its derivative β-caryophyllenol also can be applicable in cough-relieving and phlegm-eliminating medicine, and β-caryophyllene oxide has the effects such as analgesia and anti-inflammatory, antimycotic, anti-cell toxicity; The product of β-caryophyllene hydrogenation also can be used as the raw material of high-density propellant simultaneously.
At present, the preparation of β-caryophyllene mainly comprise from natural phant, adopt series of chemical extract or utilize related raw material to synthesize under suitable condition two kinds of methods, because the content of β-caryophyllene in natural phant is lower, cause its extraction cost very high; Because the structure of β-caryophyllene is more complicated, make the technique of chemosynthesis loaded down with trivial details, productive rate is low simultaneously, and energy consumption is large.Therefore the operational path of finding a kind of alternative sustainable preparation β-caryophyllene is significant.
Utilize the operational path of microorganism catalysis synthesis of chemicals and material, there is the advantages such as raw material is renewable, environmental protection, reaction conditions gentleness because of it, and paid attention to by scientist gradually.In organism, two kinds of natural pathways metabolisms of main existence are carried out the biosynthesizing of terpene compound, i.e. mevalonic acid (MVA) approach and methyl E4P (MEP) approach.MVA approach is mainly present in the enchylema of eukaryote, archeobacteria and higher plant, and MEP approach is present in plastid, bacterium and the algae of plant, the final product of this two classes pathways metabolism is all the precursor substance dimethylallylpyrophosphate (dimethylallyldiphosphate that forms isoprene, DMAPP) and isopentenyl pyrophosphate (IPP), pass through afterwards geraniol ester diphosphate synthase, farnesyl pyrophosphate synthase and terpenes synthetic enzyme catalysis DMAPP and IPP to terpene compound.Before this, also do not utilize microorganism fermentation to obtain the relevant report of β-caryophyllene.The present invention has introduced the novel method of utilizing microorganism fermentation preparation β-caryophyllene first.
Summary of the invention
The invention discloses the reconstitution cell that a kind of microorganism catalysis synthesizes the method for β-caryophyllene and can synthesize β-caryophyllene, utilize renewable resources glucose for raw material, prepare terpene compound by biological catalyst---β-caryophyllene, set up β-caryophyllene synthesis route of Sustainable development.
Mainly by genetic engineering means, to MVA approach, synthetic terpene compound---β-caryophyllene relative enzyme gene carries out heterogenous expression in intestinal bacteria etc. build by genetic engineering technique the reconstitution cell forming in the present invention, finally in reconstitution cell, has successfully set up a kind of terpene compound---the biosynthetic pathway of β-caryophyllene.
The present invention adopts following technical scheme:
Microorganism catalysis synthesizes a method for β-caryophyllene, and described method comprises the steps:
A) building can be from the reconstitution cell of the synthetic β-caryophyllene of acetyl-CoA, and described reconstitution cell comprises following gene fragment: acetyl-CoA acyltransferase, 3-Hydroxy-3-methylglutaryl CoA A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, Mevalonic kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-bisphosphate decarboxylase, isopentenylpyrophosphate isomerase, spiceleaf ester group pyrophosphate synthase, farnesyl pyrophosphate synthase and β-caryophyllene synthase;
B) utilize A) described in reconstitution cell in the substratum that comprises glucose, cultivate, induce by inductor, after separation and purification, can obtain β-caryophyllene.
Wherein said acetyl-CoA acyltransferase gene source in: 1) yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) is (GenBank:BK006949.2), or 2) derive from other bacterium, preferably enterococcus faecalis (Enterococcus faecalis) (GenBank:AAG02438.2), (GenBank:AIA26811.1) brown bacillus (Phaeobacter inhibens) is (GenBank:AFO90074.1) for aurococcus (Staphylococcus aureus), hookworm is coveted copper bacterium (Cupriavidus necator) (GenBank:AEI82514.1), intestinal bacteria (Escherichia coli) (GenBank:ADX49537.1), or streptococcus pyogenes (Streptococcus pyogenes) (GenBank:NP_607695.1), or 3) derive from other organism, and acetyl-CoA acyltransferase gene do not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.Described 3-Hydroxy-3-methylglutaryl CoA A synthase gene derives from: 1) yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) (GenBank:YM4987.09C); Or 2) derive from other bacterium, preferably enterococcus faecalis (Enterococcus faecalis) (GenBank:EEU26497.1), aurococcus (Staphylococcus aureus) (GenBank:YP_501316.1), faecium (Enterococcus faecium) (GenBank:AAG02443.1), (GenBank:YP_849629.1), or streptococcus pyogenes (Streptococcus pyogenes) (GenBank:AAL97584.1) for Wei Shi Li Site bacterium (Listeria welshimeri); Or 3) derive from other organism, and 3-Hydroxy-3-methylglutaryl CoA A synthase gene do not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
Described 3-hydroxy-3-methylglutaryl coenzyme A reductase gene source in: 1) yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) is (GenBank:YLR450W); Or 2) derive from other bacterium, preferably enterococcus faecalis (Enterococcus faecalis) (GenBank:ESU74184.1), aurococcus (Staphylococcus aureus) (GenBank:AAG02423.1), (GenBank:AGS75052.1), or streptococcus pyogenes (Streptococcus pyogenes) (GenBank:AAL97583.1) for faecium (Enterococcus faecium); Or 3) derive from other organism, and 3-hydroxy-3-methylglutaryl coenzyme A reductase gene do not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.Described Mevalonic kinase gene source in: 1) yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) is (GenBank:NP_013935.1); Or 2) derive from other bacterium, preferably enterococcus faecalis (Enterococcus faecalis) (GenBank:EPI38248.1), aurococcus (Staphylococcus aureus) (GenBank:ABR51486.1), (GenBank:EFS06266.1), or streptococcus pyogenes (Streptococcus pyogenes) (GenBank:AFV37808.1) for faecium (Enterococcus faecium); Or 3) derive from other organism, and Mevalonic kinase gene do not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
Described mevalonic acid-5-phosphokinase gene source in: 1) yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) is (GenBank:NP_013947.1); Or 2) derive from other bacterium, preferably enterococcus faecalis (Enterococcus faecalis) (GenBank:WP_016626966.1), aurococcus (Staphylococcus aureus) (GenBank:AIA27148.1), (GenBank:WP_016629841.1), or streptococcus pyogenes (Streptococcus pyogenes) (GenBank:WP_023613167.1) for faecium (Enterococcus faecium); Or 3) derive from other organism, and mevalonic acid-5-phosphokinase gene do not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.Described mevalonic acid-5-bisphosphate decarboxylase gene derives from: 1) yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) (GenBank:AY757921.1); Or 2) derive from other bacterium, preferably enterococcus faecalis (Enterococcus faecalis) (GenBank:YP_005707688.1), (GenBank:EPI24610.1), or streptococcus pyogenes (Streptococcus pyogenes) (GenBank:AAL97580.1) for faecium (Enterococcus faecium); Or 3) derive from other organism, and mevalonic acid-5-bisphosphate decarboxylase gene do not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
Described isopentenylpyrophosphate isomerase gene derives from: 1) yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) (GenBank:NP_015208.1); Or 2) derive from other bacterium, preferably enterococcus faecalis (Enterococcus faecalis) (GenBank:NP_814639.1), aurococcus (Staphylococcus aureus) (GenBank:YP_501084.1), (GenBank:ERK34722.1), or streptococcus pyogenes (Streptococcus pyogenes) (GenBank:YP_001128672.1) for faecium (Enterococcus faecium); Or 3) derive from other organism, and isopentenylpyrophosphate isomerase gene do not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
Described geraniol ester diphosphate synthase gene source in: 1) intestinal bacteria (Escherichia coli) (GenBank:AHY68945.1), or Acinetobacter baumannii (Acinetobacter baumannii) (GenBank:BAN86603.1); Or 2) derive from abies grandis (Abies grandis) (GenBank:AAN01134.1), mango (Mangifera indica) (GenBank:AFJ52722.1), (GenBank:AEZ55677.1), or grape (Vitis vinifera) (GenBank:AAR08151.1) for the red sage root (Salvia Miltiorrhiza); Or 3) derive from other organism, and geraniol ester diphosphate synthase gene do not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
Described farnesyl phosphate synthase gene derives from: 1) intestinal bacteria (Escherichia coli) (GenBank:AHY68945.1), or Neuraspora crassa (Neurospora crassa) (GenBank:CAA65645.1); Or 2) Chunlan (Cymbidium goeringii) (GenBank:AFP19446.1), mango (Mangifera indica) (GenBank:AFJ52720.1), (GenBank:AGH33733.1), or long capsule water cloud (Ectocarpus siliculosus) (GenBank:CBN75787.1) for asparagus (Asparagus officinalis); Or 3) derive from other organism, and farnesyl phosphate synthase gene do not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
Described β-caryophyllene synthase gene derives from: 1) the climing Herba Lycopi of little Hua (Mikania micrantha) (GenBank:FJ767894.1); Or 2) Xin Keliduoniya santal (Santalum austrocaledonicum) (GenBank:ADO87005.1); Or 3) Herba Artemisiae annuae (Artemisia annua) (GenBank:AF472361.1); Or 4) corn (Zea mays) (GenBank:ABY79213.1), (GenBank:BAN81914.1), or apple (Malus domestica) (GenBank:AGB14624.1) for absinth (Artemisia absinthium); Or 5) derive from other organism, and β-caryophyllene synthase gene do not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
The invention still further relates to a kind of reconstitution cell that can synthesize from acetyl-CoA β-caryophyllene, described reconstitution cell comprises following gene fragment: acetyl-CoA acyltransferase, 3-Hydroxy-3-methylglutaryl CoA A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, Mevalonic kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-bisphosphate decarboxylase, isopentenylpyrophosphate isomerase, geraniol ester diphosphate synthase, farnesyl pyrophosphate synthase and β-caryophyllene synthase.Wherein, reconstitution cell is by bacterial cell, as intestinal bacteria, and subtilis or fungal cell (as yeast saccharomyces cerevisiae), or micro-algae etc. is built and is formed by genetic engineering technique.About can from the synthetic β-caryophyllene of acetyl-CoA the structure of reconstitution cell can be referring to description above.
Those skilled in the art should understand that, for expressing better in reconstitution cell, the gene order of above-mentioned various enzymes or the nucleotide sequence by its derivative coding with the albumen of same or analogous function can carry out codon optimized according to the codon preference of host cell used.
The nucleotide sequence that those skilled in the art be also to be understood that the gene order of above-mentioned various enzymes or had the albumen of same or analogous function by its derivative coding can be cloned in host cell according to conventional molecule clone technology.In addition, these nucleotide fragments can also for example, be operably connected with suitable expression controlling elements (, promotor, enhanser etc.).These all, within those skilled in the art's limit of power, do not need to pay performing creative labour.These nucleotide fragments operability connect can also can be without joint by means of joint, and this can carry out appropriate selection according to actual needs by those skilled in the art.
Those skilled in the art should understand that, well can be from the reconstitution cell of the synthetic β-caryophyllene of acetyl-CoA at the constructing host cell that selection is suitable, those skilled in the art can test and (for example determine suitable culture condition according to technology general knowledge or through limited number of time, the parameters such as the temperature of fermentation culture, stirring velocity, pH value, dissolved oxygen rate, fermentation time), also can select suitable inductor, determine the opportunity that adds inductor, etc.This does not need to pay performing creative labour.
Beneficial effect of the present invention:
Compared with traditional preparation technology, the route that utilizes microorganism catalysis to synthesize β-caryophyllene has following advantage: (1) is owing to there being narrow spectrum β-caryophyllene synthetic enzyme, so the product generating has highly selective, industrially can greatly reduce separation costs; (2) use raw material for ligocellulose degradation obtain glucose be renewable resources; (3) whole process is to carry out at normal temperatures and pressures, and energy consumption is low.Therefore, utilize biocatalysis means to prepare β-caryophyllene and will become the inexorable trend of β-caryophyllene industrial development from now on.In addition, microorganism has fast growth, fermentation period is short, genetic background is clear, be easy to through engineering approaches operation, can utilize the features such as cheap renewable resources, and therefore microorganism has become the effective means of the chemical of production bio-based in recent years as biological catalyst.
Brief description of the drawings
In detailed description below in conjunction with accompanying drawing, above-mentioned feature and advantage of the present invention will be more obvious, wherein:
Fig. 1 utilizes acetyl-CoA biosynthesizing terpene compound---β-caryophyllene pathways metabolism schematic diagram;
Fig. 2 is pYJM50 plasmid map;
Fig. 3 is pYJM51 plasmid map;
Fig. 4 is pYJM14 plasmid map;
Fig. 5 is that the GC of tunning β-caryophyllene structure analyzes collection of illustrative plates;
Fig. 6 shows that engineering bacteria YJM51 produces the time curve of β-caryophyllene; Wherein β-caryophyllene output (▲) of engineering bacteria YJM51 and Growth of Cells (■), when engineering bacteria Growth of Cells 12h starts to carry out inducing culture;
Fig. 7 shows the impact of inducing temperature on engineering bacteria β-caryophyllene: as the cell OD of engineering bacteria YJM51
600while reaching 0.6-0.9, in substratum, add the IPTG that final concentration is 1mM, at different temperature, carry out respectively inducing culture 29h:25 DEG C (white), 30 DEG C (light gray), 34 DEG C (grey), 37 DEG C (Dark grey); Above-mentioned testing data is mean value in triplicate;
Fig. 8 shows that IPTG concentration produces the impact of β-caryophyllene on engineering bacteria: as the cell OD of engineering bacteria YJM51
600while reaching 0.6-0.9, be 30 DEG C at inducing temperature, under the condition of different final concentration IPTG, induce 66h:0.1mM (■), 0.25mM (◆), 0.5mM (▲), 1mM (●); Above-mentioned testing data is mean value in triplicate.
Embodiment
Further describe the present invention below with reference to specific embodiment, but it should be appreciated by those skilled in the art that the present invention is not limited to these specific embodiments.
Embodiment 1
Derive from acetyl-CoA acyltransferase gene/3-hydroxy-3-methylglutaryl coenzyme A reductase gene (mvaE) of enterococcus faecalis (Enterococcus faecalis) by co expression in intestinal bacteria, 3-Hydroxy-3-methylglutaryl CoA A synthase gene (mvaS); Derive from the Mevalonic kinase gene (ERG12) of yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), mevalonic acid-5-phosphokinase gene (ERG8), mevalonic acid-5-bisphosphate decarboxylase gene (ERG19), isopentenylpyrophosphate isomerase gene (IDI1); Derive from the geraniol ester diphosphate synthase gene (GPPS2) of abies grandis (Abies grandis); Derive from the farnesyl phosphate synthase gene (FPPS) of Neuraspora crassa (Neurospora crassa) and derive from β-caryophyllene synthase gene (QHS1) of Herba Artemisiae annuae (Artemisia annua), utilize glucose degradation intermediate product acetyl-CoA biosynthesizing isoprene derivatives---β-caryophyllene.
The clone of 1.1 foreign genes and the structure of expression vector
1.1.1 the clone of foreign gene
1.1.1.1 the clone of enterococcus faecalis MVA upstream pathways metabolism gene
The mvaS gene (GenBank:EEU26497.1) that comes from enterococcus faecalis (Enterococcusfaecalis), mvaE gene (GenBank:AAG02438.2) is obtained by chemical synthesis process by Shanghai JaRa company.Be connected with carrier pGH (purchased from Shanghai Jierui Biology Engineering Co., Ltd) respectively afterwards and obtain pGH/mvaS, pGH/mvaE.
1.1.1.2GPPS2, the clone of ispA and QHS1 gene
Respectively to coming from Abies grandis GPPS2 gene (GenBank:AAN01134.1) and Artemisia annua QHS1 gene (GenBank:AF543530.1) sequence is carried out rare codon analysis (http://www.genscript.com/cgi-bin/tools/rare_codon_analysis), and its rare codon is optimized for to the codon (http://www.jcat.de/) of E.coli preference.GPP synthase gene (GPPS2) after optimization and β-caryophyllene synthase gene (QHS1) are served extra large JaRa company and are carried out chemosynthesis, and are connected into and on pGH carrier, form respectively pGH-GPPS2 and pGH-QHS1 carrier.
Taking E.coli genome as template, utilize primer ispA-F (5 '-CATGGACGT CATGGACTTTCCGCAGCAACTC-3 ') and ispA-R (5 '-TTATTTATTACG CTGGATGATGTCTCGAGCGG-3 ') pcr amplification ispA gene (GenBank:AHY68945.1).
1.1.2 the structure of expression vector
1.1.2.1pYJM24 vector construction
PGH-GPPS2 carrier and pACYCDuet-1 carrier (Novagen) are carried out to double digestion with NdeI and BglII respectively, carrier and the external source fragment ratio of 1:5 in molar ratio, 4 DEG C of connections are spent the night or 16 DEG C of connection 4～6h, connect product Transformed E .coli DH5 α, then coating is added with 34 μ gmL
-1the LB solid plate of paraxin, PCR screening positive clone extracts after recombinant plasmid pYJM24 (pACY-GPPS2) from positive colony, then by restriction enzyme digestion and order-checking qualification.
1.1.2.2pYJM26 vector construction
PACY-mvaE-mvaS-ispSPa carrier (pYJM20) and pACY-GPPS2 (pYJM24) carrier are carried out to double digestion with NcoI and PstI respectively, carrier pACY-GPPS2 and the external source fragment mvaE-mvaS ratio of 1:5 in molar ratio, 4 DEG C of connections are spent the night or 16 DEG C of connection 4～6h, connect product Transformed E .coli DH5 α, then coating is added with 34 μ gmL
-1the LB solid plate of paraxin, PCR screening positive clone extracts after recombinant plasmid pYJM26 (pACY-mvaE-mvaS-GPPS2) from positive colony, then by restriction enzyme digestion and order-checking qualification.
1.1.2.3pYJM50 vector construction
PGH-QHS1 carrier and pYJM26 (pACY-mvaE-mvaS-GPPS2) carrier are carried out to double digestion with BglII and Fse I respectively, carrier pACY-mvaE-mvaS-GPPS2 and the external source fragment QHS1 ratio of 1:5 in molar ratio, 4 DEG C of connections are spent the night or 16 DEG C of connection 4～6h, connect product Transformed E .coli DH5 α, then coating is added with 34 μ gmL
-1the LB solid plate of paraxin, PCR screening positive clone extracts after recombinant plasmid pYJM50 (pACY-mvaE-mvaS-GPPS2-QHS1) from positive colony, then by restriction enzyme digestion and order-checking qualification.
1.1.2.4pYJM51 vector construction
IspA gene fragment and pYJM50 (pACY-mvaE-mvaS-GPPS2-QHS1) carrier are carried out to double digestion with Aat II and Xho I respectively, carrier pACY-mvaE-mvaS-GPPS2-QHS1 and the ispA gene fragment ratio of 1:5 in molar ratio, 4 DEG C of connections are spent the night or 16 DEG C of connection 4～6h, connect product Transformed E .coli DH5 α, then coating is added with 34 μ gmL
-1the LB solid plate of paraxin, PCR screening positive clone extracts after recombinant plasmid pYJM51 (pACY-mvaE-mvaS-GPPS2-QHS1-ispA) from positive colony, then by restriction enzyme digestion and order-checking qualification.
1.1.2.5pYJM14 (pTrc-low) vector construction
DNA assembling (Lego DNA assembling) method of setting up according to laboratory builds pTrc-low carrier: the method is that a kind of multiple DNA fragmentation unwinds by sex change in vitro, annealing is assembled into the fast method of recombinant plasmid, in connection procedure without any Restriction Enzyme.In the process building, by PCR (simple PCR, overlap extension PCR, long tailed primer PCR) method amplify a series of continuous fragments (successive substrate fragments, SFs), when these fragments of design amplification, need between these adjacent segment, there is one section of long lap, after these fragments are mixed by equimolar ratio example, sex change, annealing.Because the overlap between fragment and fragment is longer, account for the 1/3-2/3 of whole fragment length, the strand that makes the strand after sex change be easy to form with adjacent segment is hybridized, and finally forms ring-type, product after annealing is transformed after intestinal bacteria, finally can form recombinant plasmid.PTrc-low vector construction process is as follows:
Respectively taking pTrchis2B skeleton (from Invitrogen company) and yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) genome as template, amplify the Partial Fragment SFIBhB of plasmid pTrchis2B, SFB12hB, and the fragment ERG12 (SF128h12, SFB12h12) of 4 genes in downstream of yeast saccharomyces cerevisiae MVA approach; ERG8 (SF128h8, SF819h8); ERG19 (SF819h19, SF19IhI); IDI (SF19IhI, SFIBhI), then, taking these fragments or pTrchis2B skeleton as template, goes out to assemble 6 SFs fragment SF128 of plasmid, SF819, SF19I, SFIB, SFB12, SFB by regular-PCR or overlapping pcr amplification.4 gene ERG12, ERG8, ERG19,3 RBS sequences between IDI are in the time of pcr amplification, during by design primer, introduce, and make 4 genes by expressing under single trc (trp-lac promoter) promotor effect.
Table 1 is primer and the template of fragment amplification in the process of pTrc-low plasmid construction, and table 2 gathers for primer sequence used.
The primer of table 1 fragment amplification and template
Table1PCR?templates?and?primers?for?fragment?construction
Table 2pTrc-low builds primer sequence and gathers
Table2Primer?sequences?used?to?construct?pTrc-low
1.1.2.6pTrc-low the structure of plasmid and qualification
By 6 SFs fragment SF128 of amplification, SF819, SF19I, SFIB, SFB12, SFB mixes in 1.5mLEP pipe by equimolar ratio example, after sealing, (flooding) boiled and made its sex change in the beaker that distilled water is housed, and is then placed under room temperature, to allow its naturally cooling (annealing).Get appropriate volume and connect product Transformed E .coli DH5 α, then coating is added with the LB solid plate of ammonia benzyl mycin, PCR screening positive clone extracts after recombinant plasmid pYJM14 (pTrc-low) from positive colony, then by restriction enzyme digestion and order-checking qualification.
Embodiment 2
The plasmid building is transformed in competent cell, by shake flask fermentation and two kinds of methods of ferment tank, recombinant bacterium is carried out to fermentation culture, utilize chromatography of gases technology tunning to be carried out to the detection of quantitative and qualitative analysis.
The structure of 2.1E.coli recombinant bacterial strain
By pYJM51 (pACY-mvaE-mvaS-GPPS2-QHS1-ispA) and pYJM14 (pTrc-low) recombinant plasmid common thermal shock Transformed E .coli BL21 (DE3) competent cell, coat and be added with paraxin and the antibiotic LB solid plate of ammonia benzyl mycin, screen and obtain positive colony by PCR, obtain thus the engineering colon bacillus that contains pYJM51 and pYJM14.
The cultivation of 2.2 engineering colon bacillus
Engineering colon bacillus after activation is inoculated in the LB liquid medium that contains paraxin and ammonia benzyl mycin in the ratio of 1:100, and 37 DEG C, shaking culture under 180rpm condition, works as OD
600nmduring for 0.6-0.8, in bacterium liquid, add inductor IPTG to final concentration 0.5mmolL
-1, then proceed at 30 DEG C, under 180rpm condition, continue to cultivate.When after engineering strain induction 24h, get head space gas 1ml, utilize GC qualitative detection.
2.3 engineering bacterium fermentation tests
Picking mono-clonal is to 50ml M9 seed culture medium (1L M9salts:20g Glucose, 6g Na
2hPO
4, 3g KH
2pO
4, 1g NH
4cl, 0.5g NaCl, 0.24g MgSO
4, 121 DEG C of high pressure steam sterilization 15min.) in, 37 DEG C, 180rpm activates spend the night (18-24h).Seed is seeded to and contains 2L fermention medium (19.6g K by 10% inoculum size
2hPO
4.3H
2o; 4.2g citric acid.H
2o; 0.6g ferric ammonium citrate; The 0.8ml vitriol oil; 40g glucose, (NH
4)
6mo
7o
24.4H
2o0.123mg; ZnSO
4.7H
2o0.097mg; H
3bO
40.823mg; CuSO
4.5H
2o0.083mg; MnCl
24H
2o0.527mg, 4ml1M MgSO
4, 1900ml distilled water) 5L small-sized fermentation tank in, air flow 1.3VVM, rotating speed 400rpm, 37 DEG C are cultured to OD
600be about at 12 o'clock, 0.5mM IPTG, 37 DEG C of abduction deliverings, adjust pH with ammoniacal liquor, control pH 7.0, add IPTG one time every 8h.β-caryophyllene the product obtaining carries out qualitative and quantitative analysis by GC to it.In culturing process, remaining glucose in fermented liquid is detected, and add by variable flow the liquid glucose that concentration is 800g/L, maintain remaining sugar concentration below 0.5g/L.Every 4h gets fermented liquid 5ml, measures cell OD
600, glucose concn; Every 15min gets tail gas 1ml, utilizes gas chromatographic detection product isoprene concentration.Until OD no longer changes, till product no longer produces.
Testing conditions: GC system adopts the auspicious rainbow SP-6890 of Shandong Lunan type gas chromatograph, and chromatographic column is that (m), detector is fid detector to μ m × 0.2,25m × 250 μ to HP-INNOWAX column; 200 DEG C of vaporizer temperature, 230 DEG C of detector temperatures, flow rate of carrier gas: 1ml/min.
Post heating schedule is: 50 DEG C of insulation 0.5min,
4 DEG C/min rises to 100 DEG C,
25 DEG C/min rises to 250 DEG C, insulation 5min.
As seen from the figure: fermentation target product accumulative total content is up to 0.97g/L.
Embodiment 3
Different fermentation conditions, as inducing temperature, rotating speed, inductor concentration, nitrogenous source, concentration of substrate, Medium's PH Value and composition proportion etc., can affect the output of tunning firpene.This patent has detected different inducing temperatures, the impact on β-caryophyllene output of inductor concentration and nitrogenous source.
The structure of 3.1E.coli recombinant bacterial strain
By pYJM51 (pACY-mvaE-mvaS-GPPS2-QHS1-ispA) and pYJM14 (pTrc-low) recombinant plasmid common thermal shock Transformed E .coli BL21 (DE3) competent cell, coat and be added with paraxin and the antibiotic LB solid plate of ammonia benzyl mycin, screen and obtain positive colony by PCR, obtain thus the engineering colon bacillus that contains pYJM27 and pYJM14.
The impact of the different inducing temperatures of 3.2 research on β-caryophyllene output
Picking mono-clonal is cultivated activation and is spent the night in 5mlLB bottle, is inoculated in 100ml contains in the antibiotic liquid fermentation medium of Cm+Amp by 1%, and 37 DEG C, shaking culture under 180rpm condition, works as OD
600nmduring for 0.6-0.8, in bacterium liquid, add inductor IPTG to final concentration 0.5mmolL
-1, then proceed under differing temps (25 DEG C, 30 DEG C, 34 DEG C, 37 DEG C) and carry out inducing culture.Get 1ml head space gas at different time points and carry out GC mensuration.
3.3 impacts of research different IP TG concentration on β-caryophyllene output
Picking mono-clonal is cultivated activation and is spent the night in 5ml LB bottle, being inoculated in 100ml by 1% contains in the antibiotic liquid fermentation medium of Cm+Amp, 37 DEG C of shaking culture 4h, when OD600=0.6-1.0 left and right, add different concns IPTG (0.1mM, 0.25mM, 0.5mM, 1mM) under 30 DEG C of conditions, carry out inducing culture.Get 1ml head space gas at different time points and carry out GC mensuration.
Should be appreciated that, although with reference to its exemplary embodiment, the present invention is shown particularly and described, but will be understood by those skilled in the art that, under the condition not deviating from by the spirit and scope of the present invention as defined in the claims, the variation of various forms and details can be carried out therein, the arbitrary combination of various embodiments can be carried out.
Claims (10)
1. a method for the synthetic β-caryophyllene of microorganism catalysis, is characterized in that, described method comprises the steps:
A) building can be from the reconstitution cell of the synthetic β-caryophyllene of acetyl-CoA, and described reconstitution cell comprises following gene fragment: acetyl-CoA acyltransferase, 3-Hydroxy-3-methylglutaryl CoA A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, Mevalonic kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-bisphosphate decarboxylase, isopentenylpyrophosphate isomerase, spiceleaf ester group pyrophosphate synthase, farnesyl pyrophosphate synthase and β-caryophyllene synthase;
B) utilize A) described in reconstitution cell in the substratum that comprises glucose, cultivate, induce by inductor, after separation and purification, can obtain β-caryophyllene.
2. method according to claim 1, is characterized in that, wherein said acetyl-CoA acyltransferase gene source in: 1) yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) is (GenBank:BK006949.2); Or 2) derive from other bacterium, preferably enterococcus faecalis (Enterococcus faecalis) (GenBank:AAG02438.2), aurococcus (Staphylococcus aureus) (GenBank:AIA26811.1), brown bacillus (Phaeobacter inhibens) (GenBank:
aFO90074.1), hookworm covet copper bacterium (Cupriavidus necator) (GenBank:
aEI82514.1), intestinal bacteria (Escherichia coli) (GenBank:
aDX49537.1), or streptococcus pyogenes (Streptococcus pyogenes) (GenBank:
nP_607695.1); Or 3) derive from other organism, and acetyl-CoA acyltransferase gene do not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function; Described 3-Hydroxy-3-methylglutaryl CoA A synthase gene derives from: 1) yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) (GenBank:YM4987.09C); Or 2) derive from other bacterium, preferred enterococcus faecalis (Enterococcus faecalis) (GenBank:
eEU26497.1), aurococcus (Staphylococcus aureus) (GenBank:
yP_501316.1), faecium (
enterococcus faecium) (GenBank:
aAG02443.1), Wei Shi Li Site bacterium (Listeria welshimeri) (GenBank:
yP_849629.1), or streptococcus pyogenes (Streptococcus pyogenes) (GenBank:
aAL97584.1); Or 3) derive from other organism, and 3-Hydroxy-3-methylglutaryl CoA A synthase gene do not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
3. method according to claim 1, is characterized in that, wherein said 3-hydroxy-3-methylglutaryl coenzyme A reductase gene source in: 1) yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) is (GenBank:YLR450W); Or 2) derive from other bacterium, preferred enterococcus faecalis (Enterococcus faecalis) (GenBank:
eSU74184.1), aurococcus (Staphylococcus aureus) (GenBank:
aAG02423.1), faecium (Enterococcus faecium) (GenBank:
aGS75052.1), or streptococcus pyogenes (Streptococcus pyogenes) (GenBank:
aAL97583.1); Or 3) derive from other organism, and 3-hydroxy-3-methylglutaryl coenzyme A reductase gene do not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function; Described Mevalonic kinase gene source in: 1) yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) (GenBank:
nP_013935.1); Or 2) derive from other bacterium, preferred enterococcus faecalis (Enterococcus faecalis) (GenBank:
ePI38248.1), aurococcus (Staphylococcus aureus) (GenBank:
aBR51486.1), faecium (Enterococcus faecium) (GenBank:
eFS06266.1), or streptococcus pyogenes (Streptococcus pyogenes) (GenBank:
aFV37808.1); Or 3) derive from other organism, and Mevalonic kinase gene do not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
4. method according to claim 1, is characterized in that, wherein said mevalonic acid-5-phosphokinase gene source in: 1) yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) (GenBank:
nP_013947.1); Or 2) derive from other bacterium, preferably enterococcus faecalis (Enterococcus faecalis) (GenBank:WP_016626966.1), aurococcus (Staphylococcus aureus) (GenBank:AIA27148.1), (GenBank:WP_016629841.1), or streptococcus pyogenes (Streptococcus pyogenes) (GenBank:WP_023613167.1) for faecium (Enterococcus faecium); Or 3) derive from other organism, and mevalonic acid-5-phosphokinase gene do not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function; Described mevalonic acid-5-bisphosphate decarboxylase gene derives from: 1) yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) (GenBank:AY757921.1); Or 2) derive from other bacterium, preferred enterococcus faecalis (Enterococcus faecalis) (GenBank:
yP_005707688.1), faecium (Enterococcus faecium) (GenBank:
ePI24610.1), or streptococcus pyogenes (Streptococcus pyogenes) (GenBank:
aAL97580.1); Or 3) derive from other organism, and mevalonic acid-5-bisphosphate decarboxylase gene do not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
5. method according to claim 1, is characterized in that, wherein said isopentenylpyrophosphate isomerase gene derives from: 1) yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) (GenBank:
nP_015208.1); Or 2) derive from other bacterium, preferred enterococcus faecalis (Enterococcus faecalis) (GenBank:
nP_814639.1), aurococcus (Staphylococcus aureus) (GenBank:
yP_501084.1), faecium (Enterococcus faecium) (GenBank:
eRK34722.1), or streptococcus pyogenes (Streptococcus pyogenes) (GenBank:
yP_001128672.1); Or 3) derive from other organism, and isopentenylpyrophosphate isomerase gene do not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
6. method according to claim 1, is characterized in that, wherein said geraniol ester diphosphate synthase gene source in: 1) intestinal bacteria (Escherichia coli) (GenBank:
aHY68945.1), or Acinetobacter baumannii (Acinetobacter baumannii) (GenBank:
bAN86603.1); Or 2) derive from abies grandis (Abies grandis) (GenBank:
aAN01134.1) mango (Mangifera indica) (GenBank:
aFJ52722.1), the red sage root (Salvia Miltiorrhiza) (GenBank:
aEZ55677.1), or grape (Vitis vinifera) (GenBank:
aAR08151.1); Or 3) derive from other organism, and geraniol ester diphosphate synthase gene do not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
7. method according to claim 1, is characterized in that, wherein said farnesyl phosphate synthase gene derives from: 1) intestinal bacteria (Escherichia coli) (GenBank:
aHY68945.1), or Neuraspora crassa (Neurospora crassa) (GenBank:
cAA65645.1); Or 2) Chunlan (Cymbidium goeringii) (GenBank:
aFP19446.1), mango (Mangifera indica) (GenBank:
aFJ52720.1), asparagus (Asparagus officinalis) (GenBank:
aGH33733.1), or long capsule water cloud (Ectocarpus siliculosus) (GenBank:
cBN75787.1); Or 3) derive from other organism, and farnesyl phosphate synthase gene do not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
8. method according to claim 1, is characterized in that, wherein said β-caryophyllene synthase gene derives from: 1) the climing Herba Lycopi of little Hua (Mikania micrantha) (GenBank:FJ767894.1); Or 2) Xin Keliduoniya santal (Santalum austrocaledonicum) (GenBank:ADO87005.1); Or 3) Herba Artemisiae annuae (Artemisia annua) (GenBank:AF472361.1); Or 4) corn (Zea mays) (GenBank:ABY79213.1), absinth (Artemisia absinthium) (GenBank:
bAN81914.1), or apple (Malus domestica) (GenBank:
aGB14624.1); Or 5) derive from other organism, and β-caryophyllene synthase gene do not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
9. one kind can be synthesized from acetyl-CoA the reconstitution cell of β-caryophyllene; it is characterized in that, described reconstitution cell comprises following gene fragment: acetyl-CoA acyltransferase, 3-Hydroxy-3-methylglutaryl CoA A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, Mevalonic kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-bisphosphate decarboxylase, isopentenylpyrophosphate isomerase, geraniol ester diphosphate synthase, farnesyl pyrophosphate synthase and β-caryophyllene synthase.
10. reconstitution cell according to claim 9, is characterized in that, is by bacterial cell, as intestinal bacteria, and subtilis or fungal cell (as yeast saccharomyces cerevisiae), or micro-algae etc. is built and is formed by genetic engineering technique.
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| CN108893483A (en) * | 2018-06-22 | 2018-11-27 | 中国医学科学院药用植物研究所 | Radix Salviae Miltiorrhizae Terpene synthase gene SmTPS11, its cloning primer, expression vector, catalysate and application |
| CN108893483B (en) * | 2018-06-22 | 2021-09-17 | 中国医学科学院药用植物研究所 | Salvia miltiorrhiza terpene synthase gene SmTPS11, cloning primer, expression vector, catalytic product and application thereof |
| CN110964678A (en) * | 2018-09-29 | 2020-04-07 | 中国科学院青岛生物能源与过程研究所 | Genetically engineered bacterium for synthesizing farnesene and construction method and application thereof |
| CN110964678B (en) * | 2018-09-29 | 2022-11-11 | 中国科学院青岛生物能源与过程研究所 | Genetically engineered bacterium for synthesizing farnesene and construction method and application thereof |
| CN111004763A (en) * | 2019-12-26 | 2020-04-14 | 中国科学院青岛生物能源与过程研究所 | Engineering bacterium for producing β -caryophyllene and construction method and application thereof |
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| CN116286767A (en) * | 2023-03-21 | 2023-06-23 | 武汉软件工程职业学院(武汉开放大学) | Beta-caryophyllene synthetase, gene, strain and application |
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