CN103898037B - A kind of coproduction geraniol and the genetic engineering bacterium and its construction method of nerol and application - Google Patents
A kind of coproduction geraniol and the genetic engineering bacterium and its construction method of nerol and application Download PDFInfo
- Publication number
- CN103898037B CN103898037B CN201410086945.XA CN201410086945A CN103898037B CN 103898037 B CN103898037 B CN 103898037B CN 201410086945 A CN201410086945 A CN 201410086945A CN 103898037 B CN103898037 B CN 103898037B
- Authority
- CN
- China
- Prior art keywords
- geraniol
- gene
- nerol
- acid
- synthase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of coproduction geraniol and the genetic engineering bacterium and its construction method of nerol and application, belong to gene engineering technology field.The present invention expresses acetyl-CoA acyltransferase/3-hydroxy-3-methylglutaryl coenzyme A reductase, the methyl glutaryl coenzyme A synthase of 3 hydroxyl 3, mevalonate kinase, the phosphokinase of mevalonic acid 5, the diphosphonic acid decarboxylase of mevalonic acid 5, isopentenylpyrophosphate isomerase, geraniol ester diphosphate synthase, and geraniol synzyme or phosphatase in genetic engineering bacterium.The present invention has been successfully established the metabolic pathway of synthesis geraniol and nerol using genetic engineering means in Escherichia coli body, glucose biological directly can be converted into geraniol and nerol.
Description
Technical field
The present invention relates to a kind of coproduction geraniol and the genetic engineering bacterium and its construction method of nerol and application, belong to base
Because of field of engineering technology.
Background technology
Geraniol（Trans- 3,7- methyl -2,6- octadiene -1- alcohol, also known as Geraniol, Geraniol）And its isomerism
Body nerol（Cis- 3,7- dimethyl -2,6- octadienol, nerol）A kind of acyclic monoterpene alcohols compound, be attar of rose,
One of main component of essential oil such as Martin's sesame oil and citronella oil.Geraniol and nerol and its ester can be used for essence and edible perfume
Essence, it is the host of rose system essence, also can be widely used to medicine, tobacco, food ingredient.In addition, also act as natural low toxicity
Insect-proof agent, a kind of new cancer chemoprotective preparation.At present the global annual requirement of nerol is at 5000 tons or so, wherein China
Demand is also not less than 500 tons, and global production capacity only has 3000 tons or so.
Geraniol and nerol are naturally occurring in the various plants of fish pelargonium, cymbopogon distans, lemongrass, rose etc. 50.From day
Volatile oil is extracted in right plant, is still the main source of in the market.But because cultivation climate has a great influence, and
With the rising of human cost, the quantity and cost of natural geraniol are difficult to stabilization, and supply can not be guaranteed.
Industrial production geraniol and nerol are using laurene as raw material, and one-level chloride and the sodium acetate of laurene are total to
Heat, obtain the acetate mixture of geraniol and nerol.Then by this thick ester saponification, redistillation containing about 60% Geraniol and
The mixture of 40% nerol, carefully fractionation can obtain high-grade geraniol.Yellow space equality synthesizes perfume (or spice) using linalool as raw material
Leaf-alcohol and nerol.The existing many reports of synthetic method of nerol geraniol are prepared using citral as raw material, such as are catalyzed hydrogen
Change method, aluminium alcoholates method, sodium borohydride method.Yin Xianhong is improved research to sodium borohydride method, makees mixed solvent with water and benzene, substitutes
Pure organic solvent.Using phase transfer catalyzed methods, reaction rate is improved.But there is environmental pollution, condition harshness etc. in chemical method
Problem, and the product impurity synthesized is more, have impact on the quality and downstream application of geraniol.
The content of the invention
The problem to be solved in the present invention is to provide a kind of gene work that geraniol and nerol are synthesized using glucose as substrate
Journey bacterium.
The genetic engineering bacterium is that geraniol synzyme base has been imported in the microorganism that can express geranyl pyrophosphate
The genetic engineering bacterium that cause or phosphatase gene obtain.
The microorganism is the microorganism for being usually used in molecular biology transformation, preferably Escherichia coli.
The genetic engineering bacterium has co-expressed acetyl-CoA acyltransferase/3-hydroxy-3-methylglutaryl coenzyme A reductase, 3-
Hydroxyl -3- methyl glutaryl coenzyme As synthase, mevalonate kinase, mevalonic acid -5- phosphokinases, mevalonic acid -5- diphosphonic acid take off
Carboxylic acid, isopentenylpyrophosphate isomerase, geraniol ester diphosphate synthase, and geraniol synzyme or phosphatase.
Acetyl-CoA acyltransferase/the 3-hydroxy-3-methylglutaryl coenzyme A reductase derives from saccharomyces cerevisiae
(Saccharomyces cerevisiae), or bacterium, or other organisms, preferably enterococcus faecalis
（Enterococcusfaecalis）.
The 3-Hydroxy-3-methylglutaryl CoA A synthase derives from：Saccharomyces cerevisiae (Saccharomyces
Cerevisiae), or bacterium, or other organisms, preferred enterococcus faecalis（Enterococcusfaecalis）.
The mevalonate kinase derives from saccharomyces cerevisiae (Saccharomyces cerevisiae), or bacterium, or its
Its organism, preferably saccharomyces cerevisiae.
Mevalonic acid -5- the phosphokinases derive from saccharomyces cerevisiae (Saccharomyces cerevisiae), or carefully
Bacterium, or other organisms, preferably saccharomyces cerevisiae.
Mevalonic acid -5- diphosphonic acid the decarboxylase derives from saccharomyces cerevisiae (Saccharomyces cerevisiae),
Or bacterium, or other organisms, preferably saccharomyces cerevisiae.
The isopentenylpyrophosphate isomerase derives from saccharomyces cerevisiae (Saccharomyces cerevisiae), or carefully
Bacterium, or other organisms, preferably saccharomyces cerevisiae.
The geraniol ester diphosphate synthase derives from Escherichia coli (Escherichia coli), or abies grandis
(Abiesgrandis) other organisms, preferably abies grandis.
The geraniol synzyme derives from Herba Lysimachiae foenumgraeci (Sweet Basil), or Cinnamomum tenuipilum (Cinnamomum
), or other organisms tenuipilum.The geraniol synzyme of Herba Lysimachiae foenumgraeci is preferably derived from, its nucleotide sequence is such as
GenBank:Shown in AY362553.1.
The phosphatase derives from yeast, or bacterium, or other organisms, preferably saccharomyces cerevisiae (Saccharomyces
cerevisiae).The phosphatidic acid phosphatase DPP1 of preferably saccharomyces cerevisiae（NCBI Reference Sequence:NM_
001180592.1）, or the phosphatidic acid phosphatase LPP1 from saccharomyces cerevisiae（NCBI Reference Sequence:NM_
001180811.3）, or the ADP- ribose pyrophosphatase genes EcNudF from Escherichia coli（GenBank:U22009.1）,
Or the alkaline phosphatase from Escherichia coli（phoA）, or the phosphatase phoE from Bacillus subtillis（GenBank:
CP003783.1）, or from Klebsiella pneumoniae（Klebsiella pneumoniae）Alkaline phosphatase phoA
（GenBank:AF453253.1）.
The present invention also provides a kind of method for building the genetic engineering bacterium, is that can express the micro- of geranyl pyrophosphate
Geraniol synthase gene or phosphatase gene have been imported in biology.
The construction method mainly comprises the steps：
（1）By acetyl-CoA acyltransferase gene/3-hydroxy-3-methylglutaryl coenzyme A reductase gene, 3- hydroxyl -3- methyl
Glutaryl coenzyme A synthase gene and geraniol ester diphosphate synthase gene cloning recombinate matter to plasmid pACYCDuet-1, structure
Grain pACY-mvaE-mvaS-GPPS2.
Acetyl-CoA acyltransferase gene/3-hydroxy-3-methylglutaryl coenzyme A reductase the gene, 3- hydroxyl -3- methylpents
Two acyl coenzyme A synthase genes are preferably derived from enterococcus faecalis（Enterococcus faecalis）.The geraniol ester diphosphonic acid closes
Abies grandis is derived from into enzyme gene（Abiesgrandis）.
（2）By mevalonate kinase gene, mevalonic acid -5- phosphokinases gene, mevalonic acid -5- diphosphonic acid decarboxylases
Gene and isopentenylpyrophosphate isomerase gene are cloned into pTrcHis2B and obtain recombinant plasmid pTrc-low.
The mevalonate kinase gene, mevalonic acid -5- phosphokinases gene, mevalonic acid -5- diphosphonic acid decarboxylases
Gene, isopentenylpyrophosphate isomerase gene are preferably derived from saccharomyces cerevisiae（Saccharomyces cerevisiae）.
（3）Geraniol synzyme or the gene cloning of phosphatase or pyrophosphatase will be encoded to plasmid pACY-mvaE-
On mvaS-GPPS2.
First demonstration that phosphatase can be catalyzed GPP synthesis geraniols and nerol.
Present invention also offers a kind of method for producing geraniol and nerol, is that can express geranyl pyrophosphate
Microorganism in import geraniol synthase gene or phosphatase gene, utilize engineering bacteria fermentation production geraniol and flores aurantii
Alcohol.
Acetyl-CoA acyltransferase/3-hydroxy-3-methylglutaryl coenzyme A reductase, 3- hydroxyl -3- first are co-expressed in microorganism
It is base glutaryl coenzyme A synthase, mevalonate kinase, mevalonic acid -5- phosphokinases, mevalonic acid -5- diphosphonic acid decarboxylase, different
Amylene pyrophosphoric acid isomerase, geraniol ester diphosphate synthase, and geraniol synzyme or phosphatase；Fermented and given birth to using recombinant bacterium
Produce geraniol and nerol.
It is preferred that in microorganism co-express acetyl-CoA acyltransferase/3-hydroxy-3-methylglutaryl coenzyme A reductase, 3- hydroxyls-
3- methyl glutaryl coenzyme As synthase, mevalonate kinase, mevalonic acid -5- phosphokinases, mevalonic acid -5- diphosphonic acid decarboxylations
Enzyme, isopentenylpyrophosphate isomerase, geraniol ester diphosphate synthase and phosphatase；Utilize recombinant bacterium fermenting and producing geraniol and orange
Flower alcohol.
The phosphatase is the phosphatidic acid phosphatase DPP1 from saccharomyces cerevisiae, encodes the nucleotide sequence such as NCBI of the enzyme
Reference Sequence:Shown in NM_001180592.1；Or the phosphatidic acid phosphatase LPP1 from saccharomyces cerevisiae, coding
The nucleotide sequence of the enzyme such as NCBI Reference Sequence:Shown in NM_001180811.3；Or from large intestine bar
ADP- the ribose pyrophosphatase gene EcNudF, its nucleotide sequence such as GenBank of bacterium:Shown in U22009.1；Or derive from
The alkaline phosphatase gene phoA of Escherichia coli, its nucleotide sequence is as shown in SEQ ID NO.1；Or from Ko subtilis
The phosphatase phoE of bacillus, its nucleotide sequence such as GenBank:Shown in CP003783.1；Or from citric acid pneumonia
The alkaline phosphatase phoA of bacterium, its nucleotide sequence such as GenBank:Shown in AF453253.1.
The present invention proposes that using cheap, renewable resource glucose be raw material, and perfume (or spice) is directly prepared by biocatalyst
Leaf-alcohol and nerol.The route raw material is cheap, sustainable supply, has the potentiality of large-scale development.With traditional preparation technology
Compare, the route for synthesizing firpene using microorganism catalysis has following advantage：（1）The raw material used is ligocellulose degradation
The glucose of acquisition, it is renewable resource.（2）Whole process is to carry out at normal temperatures and pressures, and energy consumption is low.（3）With from plant
Extraction method is identical, and the geraniol and nerol of bioanalysis synthesis are natural prodcuts, and market demand degree is high.（4）Using glucose as original
Material, geraniol and nerol are obtained by engineering bacteria one-step fermentation, possess industrialization production potentiality.Therefore, living things catalysis is utilized
Means prepare geraniol and nerol by as the inexorable trend of industrial development from now on.In addition, Escherichia coli have the speed of growth
Hurry up, fermentation period is short, genetic background understands, be easy to engineering operation, using cheap renewable resource the features such as, therefore greatly
Enterobacteria has turned into the effective means for producing biological-based chemicals in recent years as biocatalyst.
Brief description of the drawings
Fig. 1 is plasmid pLWG2 structure schematic diagrames.
Fig. 2 is plasmid pLWG3 structure schematic diagrames.
Fig. 3 is plasmid pLWG4 structure schematic diagrames.
Fig. 4 is plasmid pLWG5 structure schematic diagrames.
Fig. 5 geraniols and nerol hybrid standard product GC-MS elution curves.
Fig. 6 nerol molecular fragment mass spectrograms.
Fig. 7 geraniol molecular fragment mass spectrograms.
The geraniol GC-MS figures that Fig. 8 engineering colon bacillus LWG2 synthesizes.
Fig. 9 engineering colon bacillus LWG3 GC-MS figures.
Figure 10 engineering colon bacillus LWG4 GC-MS figures.
The GC of Figure 11 geraniols and nerol hybrid standard product detection figures.
The nerol and the GC detection figures of geraniol that Figure 12 engineering colon bacillus LWG5 synthesizes.
The nerol and the GC detection figures of geraniol that Figure 13 engineering colon bacillus LWG6 synthesizes.
Embodiment
The present invention, using Escherichia coli as type strain, synthesis spiceleaf has been successfully established at it in vivo using genetic engineering means
The metabolic pathway of alcohol and nerol, glucose biological directly can be converted into geraniol and nerol.
The detection method of nerol and geraniol：Zymotic fluid 1ml and then 12000 × 1min centrifugations are taken, take the μ l of supernatant 500 simultaneously
Isometric ethyl acetate is added in mixing 5min on turbula shaker, 10min is then stood, after taking upper organic phase to be filtered
It is placed on to be measured in liquid phase bottle.Testing conditions：GC-MS INSTRUMENT MODELs：Angilent7890；Separate column type number：DB-5；Ion
Source：EI；Sample size：0.01ml；Detector：Level Four bar；Column temperature：50 DEG C of startings are warming up to 280 DEG C with 10 DEG C/min speed, protect
Warm 5min；Sample detection：Liquid in liquid phase bottle is taken to be detected.GC INSTRUMENT MODELs：SP-6890 separates cylindricality number：HP-
INNOWAX sample sizes：1ul detectors：Hydrogen flame detector（FID）Column temperature：50 DEG C of startings are warming up to 10 DEG C/min speed
250 DEG C, it is incubated 5min sample detections：Upper organic phase is taken to be detected.
Plasmid：
PYJM26, i.e. pACY-mvaE-mvaS-GPPS2, carried from enterococcus faecalis（Enterococcus
faecalis）Acetyl-CoA acyltransferase gene/3-hydroxy-3-methylglutaryl coenzyme A reductase gene (mvaE)（GenBank
No.AAG02438）, 3-Hydroxy-3-methylglutaryl CoA A synthase genes (mvaS)（GenBank No.AAG02439）；And come
Come from abies grandis（Abiesgrandis）Geraniol ester diphosphate synthase gene (GPPS2) (GenBank
No.AF513112.1 pACYCDuet-1).PYJM26 construction method is referring to Jianming Yang, et al.Metabolic
engineering of Escherichia coli for the biosynthesis of alpha-
pinene.Biotechnology for Biofuels,2013,6:60。
PYJM14, i.e. pTrc-low, carried from saccharomyces cerevisiae（Saccharomyces cerevisiae）'s
Mevalonate kinase gene (ERG12) NCBI Reference Sequence:NM_001182715.1, mevalonic acid -5- phosphoric acid
Kinase gene (ERG8) NCBI Reference Sequence:NM_001182727.1, mevalonic acid -5- diphosphonic acid decarboxylases
Gene (ERG19) GenBank:X97557.1, isopentenylpyrophosphate isomerase gene (IDI1) NCBI Reference
Sequence:NM_001183931.1 pTrcHis2B.The construction method of the plasmid is referring to Jianming Yang, et
al.Metabolic engineering of Escherichia coli for the biosynthesis of alpha-
pinene.Biotechnology for Biofuels,2013,6:60。
The primer sequence of table 1
Phosphatidic acid phosphatase DPP1 biosynthesis nerol and geraniol of the expression of embodiment 1 from saccharomyces cerevisiae
By in Escherichia coli co expression derive from enterococcus faecalis acetyl-CoA acyltransferase gene/hydroxyl first
Base glutaryl CoA reductase gene (mvaE), 3-Hydroxy-3-methylglutaryl CoA A synthase genes (mvaS)；From wine brewing
The mevalonate kinase gene (ERG12) of yeast, mevalonic acid -5- phosphokinases gene (ERG8), mevalonic acid -5- diphosphonic acid
Decarboxylase gene (ERG19), isopentenylpyrophosphate isomerase gene (IDI1)；Geraniol ester diphosphonic acid from abies grandis closes
Into enzyme gene (GPPS2) and the phosphatidic acid phosphatase DPP1 from saccharomyces cerevisiae（NCBI Reference Sequence:NM_
001180592.1）, geraniol is synthesized using glucose biological.
（1）The structure of DPP1 gene cloning and expression carriers
Using Saccharomyces Cerevisiae in S 288c genomic DNAs masterplate, DPP1-rbs-F2 and DPP1-XhoR are that primer enters performing PCR expansion
Increase, PCR amplification conditions：98 DEG C of pre-degeneration 30s；98 DEG C of denaturation 5s, 60 DEG C of 5s that anneal, 72 DEG C of extension 1min30s, above denaturation,
After annealing, three steps of extension repeat 35 circulations, 72 DEG C of extension 10min.Utilize glue reclaim kit（Purchased from Fermentas,
Cat.No.K0692）Reclaim target gene fragment.Gained genetic fragment and pYJM26 carriers are carried out with BglII and XhoI respectively
Double digestion, carrier and exogenous sequences in molar ratio 1:5 ratio, 4 DEG C of connections are overnight or 16 DEG C of 4~6h of connection, connection product turn
Change E.coli DH5 α, be then coated with added with 34 μ gmL-1The LB solid plates of chloramphenicol, PCR screening positive clones, from the positive
Recombinant plasmid pLWG2 (pACY-mvaE-mvaS-GPPS2- ' DPP1) is extracted in clone（Fig. 1）Afterwards, then by restricted digestion and
Sequencing identification.
（2）The structure of E.coli recombinant bacterial strains
Common thermal shock Transformed E .coli BL21 (DE3) competent cells of pLWG2 and pYJM4 are coated on containing chloramphenicol
With the LB solid plates of ammonia benzyl mycin antibiotic, screened by PCR and obtain positive colony, be derived from containing pLWG2 and pYJM4
Engineering colon bacillus LWG2.
（3）Fermentation production nerol and geraniol
Picking step（2）The LWG2 of acquisition white monoclonal（Grown in 12h）3ml LB (Cm+Amp, 1 ‰) in,
Activated in 37 DEG C of shaking table cultures.It is 1.0 or so to be forwarded to 30ml LB that bacterium is dense（Amp+Cm）In spread cultivation, as seed.
The bacterium solution after spreading cultivation is taken to access 100ml fermentation mediums by 1% inoculum concentration（K2HPO4·3H2O0.98g；One hydration lemon
Lemon acid 0.21g；Ferric citrate 0.03g；MD powdered beefs 0.9g；Glucose 0.2g；MgSO4(1M)200ul；100 μ of trace element
l（(NH4) 6Mo7O244H2O7.4g/L, ZnSO47H2O5.8g/L, H3BO449.4g/L, CuSO45H2O5g/L,
MnCl2·4H2O31.6g/L）；The μ g/mL of ampicillin 50；The μ g/mL of chloramphenicol 34）, 37 DEG C are shaken bacterium, the dense length of bacterium to 1.0 or so
Final concentration of 0.5mM IPTG is added, adds bottle stopper, carries out anaerobic fermentation.30 DEG C, 180rpm shakes bacterium.（4）Product detection
Ferment after 24-48h, take final zymotic fluid detection yield, GC-MS results show（Fig. 8）, obtain 451mg/L's
The geraniol of nerol and 400mg/L.
Phosphatidic acid phosphatase LPP1 biosynthesis nerol and geraniol of the expression of embodiment 2 from saccharomyces cerevisiae
By in Escherichia coli co expression derive from enterococcus faecalis acetyl-CoA acyltransferase gene/hydroxyl first
Base glutaryl CoA reductase gene (mvaE), 3-Hydroxy-3-methylglutaryl CoA A synthase genes (mvaS)；From wine brewing
The mevalonate kinase gene (ERG12) of yeast, mevalonic acid -5- phosphokinases gene (ERG8), mevalonic acid -5- diphosphonic acid
Decarboxylase gene (ERG19), isopentenylpyrophosphate isomerase gene (IDI1)；Geraniol ester diphosphonic acid from abies grandis closes
Into enzyme gene (GPPS2) and the phosphatidic acid phosphatase LPP1 from saccharomyces cerevisiae（NCBI Reference Sequence:NM_
001180811.3）, geraniol is synthesized using glucose biological.
（1）The structure of LPP1 gene cloning and expression carriers
Using Saccharomyces Cerevisiae in S 288c genomic DNAs masterplate, LPP1-rbs-F2 and LPP1-XhoR are that primer enters performing PCR expansion
Increase, PCR amplification conditions：98 DEG C of pre-degeneration 30s；98 DEG C of denaturation 5s, 62 DEG C of 5s that anneal, 72 DEG C of extension 1min30s, above denaturation,
After annealing, three steps of extension repeat 35 circulations, 72 DEG C of extension 10min.Utilize glue reclaim kit（Purchased from Fermentas,
Cat.No.K0692）Reclaim target gene fragment.Gained genetic fragment and pYJM26 carriers are carried out with BglII and XhoI respectively
Double digestion, carrier and exogenous sequences in molar ratio 1:5 ratio, 4 DEG C of connections are overnight or 16 DEG C of 4~6h of connection, connection product turn
Change E.coli DH5 α, be then coated with the LB solid plates added with 34 μ gmL-1 chloramphenicol, PCR screening positive clones, from the positive
After extracting recombinant plasmid pLWG3 (pACY-mvaE-mvaS-GPPS2- ' LPP1) in clone, then pass through restricted digestion and sequencing
Identification.
（2）The structure of E.coli recombinant bacterial strains
By pLWG3 (pACY-mvaE-mvaS-GPPS2- ' LPP1) and pYJM14 recombinant plasmids（Common thermal shock conversion
E.coli BL21 (DE3) competent cell, the LB solid plates added with chloramphenicol and ammonia benzyl mycin antibiotic are coated on, are passed through
PCR screenings obtain positive colony, are derived from the engineering colon bacillus LWG3 containing pLWG3 and pYJM4.
（3）Fermentation production nerol and geraniol
M9 seed culture mediums（/L）：20g glucose, 6g Na2HPO4, 3g KH2PO4, 1g NH4Cl, 0.5gNaCl, 0.24g
MgSO4, 121 DEG C of high pressure steam sterilization 15min.
Fermentation medium（/2L）：19.6g K2HPO4·3H2O, 4.2g citric acidH2O, 0.6g ironic citrate
Ammonium, the 0.8ml concentrated sulfuric acids, 40g glucose, 0.123mg (NH4)6Mo7O2·4H2O, 0.097mg ZnSO4·7H2O, 0.823mg
H3BO4, 0.083mg CuSO4·5H2O, 0.527mg MnCl2·4H2O, 4ml1M MgSO4, 1900ml distilled water.
Picking monoclonal into 50ml M9 seed culture mediums, 37 DEG C, 180rpm activation overnight（18-24h）.Seed is pressed
10% inoculum concentration is seeded in the 5L small-sized fermentation tanks containing 2L fermentation mediums, throughput 1.3VVM, rotating speed 400rpm, and 37
DEG C culture is to OD600When about 12, final concentration of 0.1-1mM IPTG is added, 25 DEG C of -37 DEG C of induced expressions, pH is adjusted with ammoniacal liquor,
PH is controlled 7.0.Obtained firpene product carries out qualitative and quantitative analysis by GC-MS to it.To zymotic fluid in incubation
Middle residual glucose is detected, and by non-uniform flow add concentration be 800g/L liquid glucose, maintain remaining sugar concentration 0.5g/L with
Under.Zymotic fluid 5ml, measure cell OD600, concentration of glucose are taken per 4h；Tail gas 1ml is taken per 15min, utilizes gas chromatographic detection
Product isoprene concentrations.Until OD no longer changes, untill product no longer produces.
GC-MS results show（Fig. 9）, obtain 1.35g/L nerol and 1.98g/L geraniol.
Embodiment 3 expression from Escherichia coli ADP- ribose pyrophosphatase gene EcNudF biosynthesis nerols and
Geraniol
By in Escherichia coli co expression derive from enterococcus faecalis acetyl-CoA acyltransferase gene/hydroxyl first
Base glutaryl CoA reductase gene (mvaE), 3-Hydroxy-3-methylglutaryl CoA A synthase genes (mvaS)；From wine brewing
The mevalonate kinase gene (ERG12) of yeast, mevalonic acid -5- phosphokinases gene (ERG8), mevalonic acid -5- diphosphonic acid
Decarboxylase gene (ERG19), isopentenylpyrophosphate isomerase gene (IDI1)；Geraniol ester diphosphonic acid from abies grandis closes
ADP- ribose pyrophosphatase genes EcNudF into enzyme gene (GPPS2) and from Escherichia coli（GenBank:
U22009.1）, geraniol is synthesized using glucose biological.
（1）The structure of EcNudF gene cloning and expression carriers
Using e. coli k12 genomic DNA as masterplate, eNudF-rbs-BglF and eNudF-XhoR are that primer enters performing PCR
Amplification, PCR amplification conditions：98 DEG C of pre-degeneration 30s；98 DEG C of denaturation 5s, 58 DEG C of annealing 5s, 72 DEG C extend 1min30s, and the above becomes
Property, annealing, extension three steps repeat 35 circulation after, 72 DEG C extension 10min.Utilize glue reclaim kit（It is purchased from
Fermentas, Cat.No.K0692）Reclaim target gene fragment.Gained genetic fragment and pYJM26 carriers are used into BglII respectively
Double digestion is carried out with XhoI, carrier and exogenous sequences in molar ratio 1:5 ratio, 4 DEG C of connections are overnight or 16 DEG C connect 4~6h,
Connection product Transformed E .coli DH5 α, are then coated with the LB solid plates added with 34 μ gmL-1 chloramphenicol, and PCR screenings are positive
Clone, after extracting recombinant plasmid pLWG4 (pACY-mvaE-mvaS-GPPS2-EcNudF) from positive colony, then pass through limitation
Property digestion and sequencing identification.
（2）The structure of E.coli recombinant bacterial strains
By common thermal shock Transformed E .coli BL21 (DE3) competent cells of pLWG4 and pYJM14, it is coated on mould added with chlorine
Element and ammonia benzyl mycin antibiotic LB solid plates, by PCR screen obtain positive colony, be derived from containing pLWG4 with
PYJM4 engineering colon bacillus LWG4.
（3）Fermentation production nerol and geraniol
Engineering colon bacillus LWG4 culture and fermentation process are the same as embodiment 1.GC-MS results show, obtain 135mg/L
Nerol and 198mg/L geraniol.
The expression of embodiment 4 derives from Herba Lysimachiae foenumgraeci geraniol synthase gene GES biosynthesis nerol and geraniol
By in Escherichia coli co expression derive from enterococcus faecalis acetyl-CoA acyltransferase gene/hydroxyl first
Base glutaryl CoA reductase gene (mvaE), 3-Hydroxy-3-methylglutaryl CoA A synthase genes (mvaS)；From wine brewing
The mevalonate kinase gene (ERG12) of yeast, mevalonic acid -5- phosphokinases gene (ERG8), mevalonic acid -5- diphosphonic acid
Decarboxylase gene (ERG19), isopentenylpyrophosphate isomerase gene (IDI1)；Geraniol ester diphosphonic acid from abies grandis closes
Into enzyme gene (GPPS2) and from Herba Lysimachiae foenumgraeci geraniol synthase gene GES（GenBank:AY362553.1）, utilize grape
Sugared biosynthesis geraniol.
（1）The structure of GES gene cloning and expression carriers
According to gene order GenBank:AY362553.1 synthesis geraniol synthase genes (GES), and it is connected into pGH carriers
Upper formation pGH-GES carriers.Using pGH-GES as masterplate, GES-rbs-Bgl II and GES-xhol I are primer, and PCR is expanded,
PCR amplification conditions：98 DEG C of pre-degeneration 30s；98 DEG C of denaturation 5s, 57 DEG C of annealing 5s, 72 DEG C of extension 1min30s, above denaturation, are moved back
After fire, three steps of extension repeat 35 circulations, 72 DEG C of extension 10min.Utilize glue reclaim kit（Purchased from Fermentas,
Cat.No.K0692）Reclaim target gene fragment.
The genetic fragment of recovery and pYJM26 carriers are subjected to double digestion, carrier and external source piece with BglII and XhoI respectively
Section in molar ratio 1:5 ratio, 4 DEG C of connections are overnight or 16 DEG C connect 4~6h, connection product Transformed E .coli DH5 α, then apply
Cloth PCR screening positive clones, extracts recombinant plasmid added with the LB solid plates of 34 μ gmL-1 chloramphenicol from positive colony
After pLWG1 (pACY-mvaE-mvaS-GPPS2-GES), then pass through restricted digestion and sequencing identification.
（2）Engineering colon bacillus LWG6 structure
By common thermal shock Transformed E .coli BL21 (DE3) competent cell of pLWG1 and pYJM14 recombinant plasmids, it is coated on
Added with chloramphenicol and the LB solid plates of ammonia benzyl mycin antibiotic, screened by PCR and obtain positive colony, be derived from containing
PLWG1 and pYJM4 engineering colon bacillus LWG5.
（3）Fermentation production nerol and geraniol
Fermentation process carries out quantitative detection with embodiment 2 using GC（Figure 12）, it is finally obtained 150mg/L nerol
With 1.98g/L geraniol.
Alkaline phosphatase of the expression of embodiment 5 from Escherichia coli（phoA）Biosynthesis nerol and geraniol
By in Escherichia coli co expression derive from enterococcus faecalis acetyl-CoA acyltransferase gene/hydroxyl first
Base glutaryl CoA reductase gene (mvaE), 3-Hydroxy-3-methylglutaryl CoA A synthase genes (mvaS)；From wine brewing
The mevalonate kinase gene (ERG12) of yeast, mevalonic acid -5- phosphokinases gene (ERG8), mevalonic acid -5- diphosphonic acid
Decarboxylase gene (ERG19), isopentenylpyrophosphate isomerase gene (IDI1)；Geraniol ester diphosphonic acid from abies grandis closes
Into enzyme gene (GPPS2) and the alkaline phosphatase from Escherichia coli（phoA）, as shown in SEQ ID NO.1, utilize grape
Sugared biosynthesis geraniol.
（1）The clone of phoA genes and plasmid construction
Using e. coli k12 genomic DNA as masterplate, phoA-rbs-F2 and phoA-R are that primer enters performing PCR amplification, PCR
Amplification condition：94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 2min, 35 circulate；72 DEG C are prolonged
Stretch 10min.Utilize glue reclaim kit（Purchased from Fermentas, Cat.No.K0692）Reclaim target gene fragment.
The genetic fragment of recovery and pYJM26 carriers are subjected to double digestion, carrier and external source piece with BglII and XhoI respectively
Section in molar ratio 1:5 ratio, 4 DEG C of connections are overnight or 16 DEG C connect 4~6h, connection product Transformed E .coli DH5 α, then apply
Cloth PCR screening positive clones, extracts recombinant plasmid added with the LB solid plates of 34 μ gmL-1 chloramphenicol from positive colony
After pLWG5 (pACY-mvaE-mvaS-GPPS2-phoA), then pass through restricted digestion and sequencing identification.
（2）E.coli recombinant bacterial strains LWG5 structure
By common thermal shock Transformed E .coli BL21 (DE3) competent cell of pLWG5 and pYJM14 recombinant plasmids, it is coated on
Added with chloramphenicol and the LB solid plates of ammonia benzyl mycin antibiotic, screened by PCR and obtain positive colony, be derived from containing
PLWG5 and pYJM4 engineering colon bacillus LWG5.
（3）Fermentation production nerol and geraniol
Method shows with embodiment 1, GC results（Figure 13）, obtain 20mg/L nerol and 637mg/L geraniol.
Phosphatase phoE biosynthesis nerol and geraniol of the expression of embodiment 6 from Bacillus subtillis
By in Escherichia coli co expression derive from enterococcus faecalis acetyl-CoA acyltransferase gene/hydroxyl first
Base glutaryl CoA reductase gene (mvaE), 3-Hydroxy-3-methylglutaryl CoA A synthase genes (mvaS)；From wine brewing
The mevalonate kinase gene (ERG12) of yeast, mevalonic acid -5- phosphokinases gene (ERG8), mevalonic acid -5- diphosphonic acid
Decarboxylase gene (ERG19), isopentenylpyrophosphate isomerase gene (IDI1)；Geraniol ester diphosphonic acid from abies grandis closes
Into enzyme gene (GPPS2) and from Bacillus subtillis phosphatase phoE as shown in SEQ ID NO.2（GenBank:
CP003783.1）.Plasmid method：Two sections of gene phoE for carrying BglII and XhoI restriction enzyme sites are synthesized by Shanghai life work, formed
Plasmid pGH/phoE, the plasmid and pYJM26 carriers are subjected to double digestion, connection, conversion and fermentation with BglII and XhoI respectively
Etc. process with embodiment 5.Glucose finally is utilized, 15mg/L nerol and 35mg/L spiceleaf are obtained after fermented and cultured
Alcohol.
The expression of embodiment 7 derives from Klebsiella pneumoniae（Klebsiella pneumoniae）Alkaline phosphatase
PhoA biosynthesis nerol and geraniol
By in Escherichia coli co expression derive from enterococcus faecalis acetyl-CoA acyltransferase gene/hydroxyl first
Base glutaryl CoA reductase gene (mvaE), 3-Hydroxy-3-methylglutaryl CoA A synthase genes (mvaS)；From wine brewing
The mevalonate kinase gene (ERG12) of yeast, mevalonic acid -5- phosphokinases gene (ERG8), mevalonic acid -5- diphosphonic acid
Decarboxylase gene (ERG19), isopentenylpyrophosphate isomerase gene (IDI1)；Geraniol ester diphosphonic acid from abies grandis closes
Into enzyme gene (GPPS2) and from Klebsiella pneumoniae（Klebsiella pneumoniae）Alkaline phosphatase phoA
（GenBank:AF453253.1）, using obtaining 60mg/L nerol and 348mg/L spiceleaf after glucose fermentation culture
Alcohol.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, it can all do various change and modification, therefore the protection model of the present invention
Enclose being defined of being defined by claims.
Claims (1)
- A kind of 1. method for producing geraniol and nerol, it is characterised in that fragrant using glucose production by genetic engineering bacterium Leaf-alcohol and nerol, the genetic engineering bacterium are that acetyl-CoA acyltransferase/methylol penta has been co-expressed in Escherichia coli Two acyl coenzyme A reductases, 3-Hydroxy-3-methylglutaryl CoA A synthase, mevalonate kinase, mevalonic acid -5- phosphokinases, Mevalonic acid -5- diphosphonic acid decarboxylase, isopentenylpyrophosphate isomerase, geraniol ester diphosphate synthase and phosphatase；It is described Phosphatase gene is the phosphatidic acid phosphatase LPP1 from saccharomyces cerevisiae, encodes the nucleotide sequence such as NCBI of the enzyme Reference Sequence:Shown in NM_001180811.3；Wherein acetyl-CoA acyltransferase gene/methylol penta 2 Acyl coenzyme A reductase genes, 3-Hydroxy-3-methylglutaryl CoA A synthase genes derive from enterococcus faecalis, the wherein phosphorus of geraniol ester two Acid synthase gene derives from abies grandis；Wherein mevalonate kinase gene, mevalonic acid -5- phosphokinases gene, first hydroxyl Valeric acid -5- diphosphonic acid decarboxylase gene, isopentenylpyrophosphate isomerase gene derive from saccharomyces cerevisiae.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410086945.XA CN103898037B (en) | 2014-03-11 | 2014-03-11 | A kind of coproduction geraniol and the genetic engineering bacterium and its construction method of nerol and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410086945.XA CN103898037B (en) | 2014-03-11 | 2014-03-11 | A kind of coproduction geraniol and the genetic engineering bacterium and its construction method of nerol and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103898037A CN103898037A (en) | 2014-07-02 |
| CN103898037B true CN103898037B (en) | 2017-11-17 |
Family
ID=50989615
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410086945.XA Active CN103898037B (en) | 2014-03-11 | 2014-03-11 | A kind of coproduction geraniol and the genetic engineering bacterium and its construction method of nerol and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103898037B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105420135B (en) * | 2016-01-06 | 2019-02-26 | 山东大学 | One plant height produces recombinant Saccharomyces cerevisiae bacterial strain and its application of monoterpene geraniol |
| CN110658292B (en) * | 2018-06-29 | 2021-03-26 | 华中科技大学 | Method for detecting geranyl ester pyrophosphate content based on prenylation reaction |
| CN109810999B (en) * | 2019-03-11 | 2023-03-24 | 湖北工业大学 | Method for producing nerol by microbial fermentation |
| CN110438145A (en) * | 2019-07-15 | 2019-11-12 | 天津大学 | The corynebacterium glutamicum of synthesis geraniol and construction method and application |
| CN112391371B (en) * | 2019-08-14 | 2022-05-20 | 中国科学院青岛生物能源与过程研究所 | Tobacco monoterpene synthetase TPS2b, and coding gene and application thereof |
| CN112391327B (en) * | 2019-08-14 | 2022-08-05 | 中国农业科学院烟草研究所 | Engineering bacterium for co-production of geraniol and nerol and construction method and application thereof |
| CN111286482A (en) * | 2020-05-13 | 2020-06-16 | 中国科学院烟台海岸带研究所 | Escherichia coli engineering bacterium capable of rapidly producing geraniol and construction method and application thereof |
| CN113215131B (en) * | 2021-06-04 | 2023-06-20 | 天津大学佐治亚理工深圳学院 | Phosphate hydrolase and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102851253A (en) * | 2012-08-03 | 2013-01-02 | 江南大学 | Escherichia coli engineering strain having high phenethyl alcohol yield and application thereof |
| WO2013170265A1 (en) * | 2012-05-11 | 2013-11-14 | Donald Danforth Plant Science Center | Methods for high yield production of terpenes |
-
2014
- 2014-03-11 CN CN201410086945.XA patent/CN103898037B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013170265A1 (en) * | 2012-05-11 | 2013-11-14 | Donald Danforth Plant Science Center | Methods for high yield production of terpenes |
| CN102851253A (en) * | 2012-08-03 | 2013-01-02 | 江南大学 | Escherichia coli engineering strain having high phenethyl alcohol yield and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| Inheritance of berry volatile compounds in two half-sib grape(Vitis vinifera) populations.;B.H.Wu;《Euphytica》;20130228;第189卷(第3期);第351-364页 * |
| Metabolic engineering of Escherichia coli for the biosynthesis of alpha-pinene;Jianming Yang et al.;《Biotechnology for biofuels》;20130430;第6卷(第1期);图1、图1图注 * |
| 植物萜类化合物的天然合成途径及其相关合酶;张长波等;《植物生理学通讯》;20070831;第43卷(第4期);第783页第2栏第2段 * |
| 香叶醇的研究进展;孙立宏等;《西北药学杂志》;20091031;第24卷(第5期);第428-430页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103898037A (en) | 2014-07-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103898037B (en) | A kind of coproduction geraniol and the genetic engineering bacterium and its construction method of nerol and application | |
| Henke et al. | Patchoulol production with metabolically engineered Corynebacterium glutamicum | |
| Jia et al. | Yarrowia lipolytica construction for heterologous synthesis of α-santalene and fermentation optimization | |
| CN104946575B (en) | A kind of E. coli expression strains and its application of high yield tyrosol and/or rhodioside and icariside D2 | |
| CN104372017B (en) | A kind of method and application for improving genetic engineering bacterium isoprene and its derivative yield | |
| CN104031872B (en) | A kind of product isoprene gene engineering bacteria and application thereof | |
| CN104846000B (en) | Using glucose production to the recombination bacillus coli and purposes of hydroxy-benzyl alcohol or Gastrodin | |
| CN106032538B (en) | One plant of metabolic engineering bacteria and its application in utilization a variety of substrates production vanillic aldehyde | |
| CN112695003B (en) | Genetically engineered bacterium for high yield of cembratriene-alcohol and construction method and application thereof | |
| CN104120141A (en) | A method of synthesizing beta-caryophyllene by microbial catalysis and a reconstituted cell capable of synthesizing the beta-caryophyllene | |
| CN104004701A (en) | Method for building high-yield 5-aminolevulinic acid escherichia coli engineering strains | |
| CN109402189A (en) | The method and its extracting method of fermenting and producing pentanediamine | |
| CN107354118B (en) | Genetically engineered bacterium with gamma-terpinene synthesis capacity and construction method and application thereof | |
| CN107119001B (en) | The production method of genetically engineered hydrogenlike silicon ion and preparation method thereof and farnesol | |
| CN111286482A (en) | Escherichia coli engineering bacterium capable of rapidly producing geraniol and construction method and application thereof | |
| Özgen et al. | The Synthesis of Chiral γ‐Lactones by Merging Decatungstate Photocatalysis with Biocatalysis | |
| CN109810999A (en) | A method of nerol is produced using microbial fermentation | |
| CN104789512B (en) | The production bacterium of isoprene and the method for producing isoprene | |
| CN107460220A (en) | A kind of preparation method of rhodioside and the like | |
| CN103540557B (en) | A kind of method of biological process synthesis isoprene alcohol | |
| CN108753852A (en) | A kind of method that bioanalysis prepares raspberry ketone | |
| CN112391327B (en) | Engineering bacterium for co-production of geraniol and nerol and construction method and application thereof | |
| CN103540558B (en) | A kind of method of biological process synthesis prenol | |
| CN105802895A (en) | Fermentation medium and fermentation method of Streptomyces sp. | |
| CN105755095B (en) | A kind of method of biological enzyme synthesis (R) -2- hydroxy acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CB03 | Change of inventor or designer information |
Inventor after: Liu Wei Inventor after: Xian Mo Inventor after: Tian Ning Inventor after: Jiang Yudong Inventor before: Xian Mo Inventor before: Liu Wei Inventor before: Tian Ning Inventor before: Jiang Yudong |
|
| CB03 | Change of inventor or designer information |