CN103898037A - Genetically engineered bacterium co-generating geraniol and nerol and construction method and application thereof - Google Patents
Genetically engineered bacterium co-generating geraniol and nerol and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a genetically engineered bacterium co-generating geraniol and nerol and a construction method and an application thereof, and belongs to the technical field of generic field. According to the genetically engineered bacterium disclosed by the invention, acetyl coenzyme A acyltransferase/hydroxymethyl glutaryl coenzyme A reductase, 3-hydroxyl-3-methyl glutaryl coenzyme A synthase, mevalonic acid kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-diphosphonic acid decarboxylase, isopentenylpyrophosphate isomerase, geranyl diphosphonic acid synthetase and geraniol synthetase or phosphatase are expressed. The metabolic pathways of geraniol and nerol synthesized in escherichia coli are successfully constructed by genetic engineering means, and glucose is biologically converted into geraniol and nerol.
Description
Technical field
The present invention relates to genetic engineering bacterium and construction process and the application of a kind of coproduction Geraniol and vernol, belong to gene engineering technology field.
Background technology
Geraniol (trans-3,7-methyl-2,6-octadiene-1-alcohol, Geraniol, claim again Geraniol) and isomers vernol (cis-3,7-dimethyl-2,6-octadienol, nerol) being a kind of acyclic monoterpene alcohol compound, is one of main component of rose oil, Martin's sesame oil and lemongrass wet goods volatile oil.Geraniol and vernol and ester thereof can be used for essence and food flavour, are that rose is the host of essence, also can be widely used in medicine, tobacco, food ingredients.In addition, also can be used as natural low toxicity insect-proof agent, the cancer chemoprotective preparation that a class is novel.The global annual requirement of vernol is 5000 tons of left and right at present, and wherein the demand of China is also not less than 500 tons, and global throughput only has 3000 tons of left and right.
Geraniol and vernol are natural to be present in 50 various plants such as Flos Pelargonii, Remote Lemongrass Herb, lemongrass, Rose.From natural phant, extract volatile oil, be still the main source on market.But because cultivation climate impact is larger, and along with the rising of human cost, the quantity of natural Geraniol and cost are difficult to stablize, supply can not be guaranteed.
Industrial production Geraniol and vernol are take myrcene as raw material, and the one-level muriate of myrcene and sodium acetate be warm altogether, obtain the acetate mixture of Geraniol and vernol.Then by this thick ester saponification, redistillation must be approximately containing the mixture of 60% Geraniol and 40% vernol, and carefully fractionation can obtain high-grade Geraniol.Yellow space equality, take phantol as raw material, synthesizes Geraniol and vernol.Prepare the existing many reports of synthetic method of vernol Geraniol take citral as raw material, for example catalytic hydrogenation, aluminium alcoholates method, sodium borohydride method.Yin Xianhong improves research to sodium borohydride method, makes mixed solvent with water and benzene, instead of pure organic solvent.Adopt phase-transfer catalysis method, improve speed of reaction.But there is the problem such as environmental pollution, condition harshness in chemical method, and synthetic product impurity is many, has affected quality and the downstream application of Geraniol.
Summary of the invention
The problem to be solved in the present invention is to provide a kind of genetic engineering bacterium that synthesizes Geraniol and vernol take glucose as substrate.
Described genetic engineering bacterium is in the microorganism that can express geranyl tetra-sodium, to have imported the genetic engineering bacterium that Geraniol synthase gene or phosphatase gene obtain.
Described microorganism is the microorganism that is usually used in molecular biology transformation, preferably intestinal bacteria.
Described genetic engineering bacterium coexpression acetyl-CoA acyltransferase/3-hydroxy-3-methylglutaryl coenzyme A reductase, 3-Hydroxy-3-methylglutaryl CoA A synthase, Mevalonic kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-bisphosphate decarboxylase, isopentenylpyrophosphate isomerase, geraniol ester diphosphate synthase, and Geraniol synthetic enzyme or Phosphoric acid esterase.
Described acetyl-CoA acyltransferase/3-hydroxy-3-methylglutaryl coenzyme A reductase derives from yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), or bacterium, or other organism, preferably enterococcus faecalis (Enterococcusfaecalis).
Described 3-Hydroxy-3-methylglutaryl CoA A synthase derives from: yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), or bacterium, or other organism, preferably enterococcus faecalis (Enterococcusfaecalis).
Described Mevalonic kinase derives from yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), or bacterium, or other organism, preferably saccharomyces cerevisiae.
Described mevalonic acid-5-phosphokinase derives from yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), or bacterium, or other organism, preferably saccharomyces cerevisiae.
Described mevalonic acid-5-bisphosphate decarboxylase derives from yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), or bacterium, or other organism, preferably saccharomyces cerevisiae.
Described isopentenylpyrophosphate isomerase derives from yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), or bacterium, or other organism, preferably saccharomyces cerevisiae.
Described geraniol ester diphosphate synthase derives from intestinal bacteria (Escherichia coli), or other organism of abies grandis (Abiesgrandis), preferably abies grandis.
Described Geraniol synthetic enzyme derives from Herba Lysimachiae foenumgraeci (Sweet Basil), or Cinnamomum tenuipilum (Cinnamomum tenuipilum), or other organism.The Geraniol synthetic enzyme that preferably derives from Herba Lysimachiae foenumgraeci, its nucleotide sequence is as shown in GenBank:AY362553.1.
Described Phosphoric acid esterase derives from yeast, or bacterium, or other organism, preferably saccharomyces cerevisiae (Saccharomyces cerevisiae).The phosphatidic acid phosphatase DPP1(NCBI Reference Sequence:NM_001180592.1 of preferably saccharomyces cerevisiae), or derive from the phosphatidic acid phosphatase LPP1(NCBI Reference Sequence:NM_001180811.3 of yeast saccharomyces cerevisiae), or derive from colibacillary ADP-ribose pyrophosphatase gene EcNudF(GenBank:U22009.1), or derive from colibacillary alkaline phosphatase (phoA), or derive from the Phosphoric acid esterase phoE(GenBank:CP003783.1 of Bacillus subtillis), or derive from the alkaline phosphatase phoA(GenBank:AF453253.1 of Cray Bai Shi pulmonitis strain (Klebsiella pneumoniae)).
The present invention also provides a kind of method that builds described genetic engineering bacterium, is in the microorganism that can express geranyl tetra-sodium, to have imported Geraniol synthase gene or phosphatase gene.
Described construction process mainly comprises the steps:
(1) acetyl-CoA acyltransferase gene/3-hydroxy-3-methylglutaryl coenzyme A reductase gene, 3-Hydroxy-3-methylglutaryl CoA A synthase gene and the gene clone of geraniol ester diphosphate synthase are arrived to plasmid pACYCDuet-1, construction recombination plasmid pACY-mvaE-mvaS-GPPS2.
Described acetyl-CoA acyltransferase gene/3-hydroxy-3-methylglutaryl coenzyme A reductase gene, 3-Hydroxy-3-methylglutaryl CoA A synthase gene preferably derive from enterococcus faecalis (Enterococcus faecalis).Described geraniol ester diphosphate synthase gene source is in abies grandis (Abiesgrandis).
(2) Mevalonic kinase gene, mevalonic acid-5-phosphokinase gene, mevalonic acid-5-bisphosphate decarboxylase gene and isopentenylpyrophosphate isomerase gene are cloned into pTrcHis2B and obtain recombinant plasmid pTrc-low.
Described Mevalonic kinase gene, mevalonic acid-5-phosphokinase gene, mevalonic acid-5-bisphosphate decarboxylase gene, isopentenylpyrophosphate isomerase gene preferably derive from yeast saccharomyces cerevisiae (Saccharomyces cerevisiae).
(3) by the gene clone of coding Geraniol synthetic enzyme or Phosphoric acid esterase or Pyrophosphate phosphohydrolase to plasmid pACY-mvaE-mvaS-GPPS2.
The present invention proves that Phosphoric acid esterase can the synthetic Geraniol of catalysis GPP and vernol first.
The present invention also provide a kind of produce Geraniol and vernol method, be in the microorganism that can express geranyl tetra-sodium, to import Geraniol synthase gene or phosphatase gene, utilize genetic engineering bacterium fermentative production Geraniol and vernol.
Coexpression acetyl-CoA acyltransferase/3-hydroxy-3-methylglutaryl coenzyme A reductase, 3-Hydroxy-3-methylglutaryl CoA A synthase, Mevalonic kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-bisphosphate decarboxylase, isopentenylpyrophosphate isomerase, geraniol ester diphosphate synthase in microorganism, and Geraniol synthetic enzyme or Phosphoric acid esterase; Utilize recombinant bacterium fermentative production Geraniol and vernol.
Preferably coexpression acetyl-CoA acyltransferase/3-hydroxy-3-methylglutaryl coenzyme A reductase, 3-Hydroxy-3-methylglutaryl CoA A synthase, Mevalonic kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-bisphosphate decarboxylase, isopentenylpyrophosphate isomerase, geraniol ester diphosphate synthase and Phosphoric acid esterase in microorganism; Utilize recombinant bacterium fermentative production Geraniol and vernol.
Described Phosphoric acid esterase is the phosphatidic acid phosphatase DPP1 from yeast saccharomyces cerevisiae, and the nucleotide sequence of this enzyme of encoding is as shown in NCBI Reference Sequence:NM_001180592.1; Or from the phosphatidic acid phosphatase LPP1 of yeast saccharomyces cerevisiae, the nucleotide sequence of this enzyme of encoding is as shown in NCBI Reference Sequence:NM_001180811.3; Or deriving from colibacillary ADP-ribose pyrophosphatase gene EcNudF, its nucleotide sequence is as shown in GenBank:U22009.1; Or deriving from colibacillary alkaline phosphatase gene phoA, its nucleotide sequence is as shown in SEQ ID NO.1; Or deriving from the Phosphoric acid esterase phoE of Bacillus subtillis, its nucleotide sequence is as shown in GenBank:CP003783.1; Or deriving from the alkaline phosphatase phoA of Cray Bai Shi pulmonitis strain, its nucleotide sequence is as shown in GenBank:AF453253.1.
That the present invention proposes to utilize is cheap, renewable resources glucose is raw material, directly prepares Geraniol and vernol by biological catalyst.This route raw material cheapness, sustainable supply, have the potentiality of large-scale development.Compared with traditional preparation technology, utilize the route of the synthetic firpene of microorganism catalysis to there is following advantage: the raw material that (1) is used is the glucose that ligocellulose degradation obtains, and is renewable resources.(2) whole process is to carry out at normal temperatures and pressures, and energy consumption is low.(3) identical with extraction method from plant, the Geraniol that biological process is synthetic and vernol are natural product, and market requirement degree is high.(4) take glucose as raw material, obtain Geraniol and vernol by engineering bacteria one-step fermentation, possess industrialization production potential.Therefore, utilize biocatalysis means to prepare Geraniol and vernol will become the inexorable trend of industrial development from now on.In addition, intestinal bacteria have 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 intestinal bacteria have become the effective means of the chemical of production bio-based in recent years as biological catalyst.
Accompanying drawing explanation
Fig. 1 is that plasmid pLWG2 builds schematic diagram.
Fig. 2 is that plasmid pLWG3 builds schematic diagram.
Fig. 3 is that plasmid pLWG4 builds schematic diagram.
Fig. 4 is that plasmid pLWG5 builds schematic diagram.
Fig. 5 Geraniol and vernol hybrid standard product GC-MS elution curve.
Fig. 6 vernol molecular fragment mass spectrum.
Fig. 7 Geraniol molecular fragment mass spectrum.
The Geraniol GC-MS figure that Fig. 8 engineering colon bacillus LWG2 synthesizes.
The GC-MS figure of Fig. 9 engineering colon bacillus LWG3.
The GC-MS figure of Figure 10 engineering colon bacillus LWG4.
The GC of Figure 11 Geraniol and vernol hybrid standard product detects figure.
The GC detection figure of the vernol that Figure 12 engineering colon bacillus LWG5 synthesizes and Geraniol.
The GC detection figure of the vernol that Figure 13 engineering colon bacillus LWG6 synthesizes and Geraniol.
Embodiment
The present invention utilizes genetic engineering means take intestinal bacteria as type strain, has successfully set up the pathways metabolism of synthetic Geraniol and vernol in its body, can directly glucose biological be converted into Geraniol and vernol.
The detection method of vernol and Geraniol: then 12000 × 1min is centrifugal to get fermented liquid 1ml, get supernatant 500 μ l and add equal-volume ethyl acetate to mix 5min on vortex vibrator, then leave standstill 10min, get after upper organic phase is filtered, be placed in liquid phase bottle to be measured.Testing conditions: GC-MS instrument model: Angilent7890; Separator column model: DB-5; Ion source: EI; Sample size: 0.01ml; Detector: level Four bar; Column temperature: 50 ℃ of startings are warming up to 280 ℃ with the speed of 10 ℃/min, insulation 5min; Sample detection: get liquid in liquid phase bottle and detect.GC instrument model: SP-6890 separates cylindricality number: HP-INNOWAX sample size: 1ul detector: hydrogen flame detector (FID) column temperature: 50 ℃ of startings are warming up to 250 ℃ with the speed of 10 ℃/min, insulation 5min sample detection: get upper organic phase and detect.
Plasmid:
PYJM26, be pACY-mvaE-mvaS-GPPS2, to carry the acetyl-CoA acyltransferase gene/3-hydroxy-3-methylglutaryl coenzyme A reductase gene (mvaE) (GenBank No.AAG02438) that derives from enterococcus faecalis (Enterococcus faecalis), 3-Hydroxy-3-methylglutaryl CoA A synthase gene (mvaS) (GenBank No.AAG02439); And derive from the pACYCDuet-1 of the geraniol ester diphosphate synthase gene (GPPS2) (GenBank No.AF513112.1) of abies grandis (Abiesgrandis).The construction process of pYJM26 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, be pTrc-low, to carry Mevalonic kinase gene (ERG12) the NCBI Reference Sequence:NM_001182715.1 that derives from yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), mevalonic acid-5-phosphokinase gene (ERG8) NCBI Reference Sequence:NM_001182727.1, mevalonic acid-5-bisphosphate decarboxylase gene (ERG19) GenBank:X97557.1, the pTrcHis2B of isopentenylpyrophosphate isomerase gene (IDI1) NCBI Reference Sequence:NM_001183931.1.The construction process of this 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.
Table 1 primer sequence
Derive from acetyl-CoA acyltransferase gene/3-hydroxy-3-methylglutaryl coenzyme A reductase gene (mvaE) of 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, 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 and derive from the phosphatidic acid phosphatase DPP1(NCBI Reference Sequence:NM_001180592.1 of yeast saccharomyces cerevisiae), utilize glucose biological to synthesize Geraniol.
(1) clone of DPP1 gene and the structure of expression vector
Take Saccharomyces Cerevisiae in S 288c genomic dna as masterplate, DPP1-rbs-F2 and DPP1-XhoR are that primer carries out pcr amplification, pcr amplification condition: 98 ℃ of denaturation 30s; 98 ℃ of sex change 5s, 60 ℃ of annealing 5s, 72 ℃ are extended 1min30s, and above sex change, annealing, extension three steps repeat after 35 circulations, and 72 ℃ are extended 10min.Utilize glue to reclaim test kit (purchased from Fermentas, Cat.No.K0692) and reclaim goal gene fragment.Gained gene fragment and pYJM26 carrier are carried out to double digestion with BglII and XhoI respectively, carrier and the external source fragment ratio of 1:5 in molar ratio, 4 ℃ of connections are spent the night or 16 ℃ of connection 4～6h, connect product Transformed E .coli DH5 α, and then coating is added with 34 μ gmL
-1the LB solid plate of paraxin, PCR screening positive clone, from positive colony, extract recombinant plasmid pLWG2 (pACY-mvaE-mvaS-GPPS2-' DPP1) (Fig. 1) after, then identify by restriction enzyme digestion and order-checking.
(2) structure of E.coli recombinant bacterial strain
By pLWG2 and the common thermal shock Transformed E of pYJM4 .coli BL21 (DE3) competent cell, coat and contain paraxin and the antibiotic LB solid plate of ammonia benzyl mycin, screen and obtain positive colony by PCR, obtain thus the engineering colon bacillus LWG2 that contains pLWG2 and pYJM4.
(3) vernol and Geraniol are produced in fermentation
The white mono-clonal (growing in 12h) of the LWG2 that picking step (2) obtains, in 3ml LB (Cm+Amp, 1 ‰), is cultivated activation in 37 ℃ of shaking tables.Bacterium is dense is that 1.0 left/right rotations are connected to 30ml LB(Amp+Cm) in spread cultivation, as seed.
The bacterium liquid of getting after spreading cultivation accesses 100ml fermention medium (K by 1% inoculum size
2hPO
43H2O0.98g; Monohydrate potassium 0.21g; Ferric ammonium citrate 0.03g; MD beef powder 0.9g; Glucose 0.2g; MgSO
4(1M) 200ul; Trace element 100 μ l((NH4) 6Mo7O244H2O7.4g/L, ZnSO47H2O5.8g/L, H3BO449.4g/L, CuSO45H2O5g/L, MnCl24H2O31.6g/L); Penbritin 50 μ g/mL; Paraxin 34 μ g/mL), 37 ℃ are shaken bacterium, and bacterium is dense to be grown to 1.0 left and right to add final concentration is the IPTG of 0.5mM, adds bottle stopper, carries out anaerobically fermenting.30 ℃, 180rpm shakes bacterium.(4) product detects
After fermentation 24-48h, get final fermented liquid and detect output, GC-MS result shows (Fig. 8), has obtained the vernol of 451mg/L and the Geraniol of 400mg/L.
Derive from acetyl-CoA acyltransferase gene/3-hydroxy-3-methylglutaryl coenzyme A reductase gene (mvaE) of 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, 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 and derive from the phosphatidic acid phosphatase LPP1(NCBI Reference Sequence:NM_001180811.3 of yeast saccharomyces cerevisiae), utilize glucose biological to synthesize Geraniol.
(1) clone of LPP1 gene and the structure of expression vector
Take Saccharomyces Cerevisiae in S 288c genomic dna as masterplate, LPP1-rbs-F2 and LPP1-XhoR are that primer carries out pcr amplification, pcr amplification condition: 98 ℃ of denaturation 30s; 98 ℃ of sex change 5s, 62 ℃ of annealing 5s, 72 ℃ are extended 1min30s, and above sex change, annealing, extension three steps repeat after 35 circulations, and 72 ℃ are extended 10min.Utilize glue to reclaim test kit (purchased from Fermentas, Cat.No.K0692) and reclaim goal gene fragment.Gained gene fragment and pYJM26 carrier are carried out to double digestion with BglII and XhoI respectively, carrier and the external source fragment ratio of 1:5 in molar ratio, 4 ℃ of connections are spent the night or 16 ℃ of connection 4～6h, connect product Transformed E .coli DH5 α, then coating is added with the LB solid plate of 34 μ gmL-1 paraxin, PCR screening positive clone extracts after recombinant plasmid pLWG3 (pACY-mvaE-mvaS-GPPS2-' LPP1) from positive colony, then identifies by restriction enzyme digestion and order-checking.
(2) structure of E.coli recombinant bacterial strain
By pLWG3 (pACY-mvaE-mvaS-GPPS2-' LPP1) and pYJM14 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 LWG3 that contains pLWG3 and pYJM4.
(3) vernol and Geraniol are produced in fermentation
M9 seed culture medium (/L): 20g glucose, 6g Na
2hPO
4, 3g KH
2pO
4, 1g NH
4cl, 0.5gNaCl, 0.24g MgSO
4, 121 ℃ of high pressure steam sterilization 15min.
Fermention medium (/ 2L): 19.6g K
2hPO
43H
2o, 4.2g citric acidH
2o, 0.6g ferric ammonium citrate, the 0.8ml vitriol oil, 40g glucose, 0.123mg (NH
4)
6mo
7o
24H
2o, 0.097mg ZnSO
47H
2o, 0.823mg H
3bO
4, 0.083mg CuSO
45H
2o, 0.527mg MnCl
24H
2o, 4ml1M MgSO
4, 1900ml distilled water.
Picking mono-clonal in 50ml M9 seed culture medium, 37 ℃, 180rpm activation spend the night (18-24h).Seed is seeded to by 10% inoculum size in the 5L small-sized fermentation tank that contains 2L fermention medium, air flow 1.3VVM, rotating speed 400rpm, 37 ℃ are cultured to OD
600be about at 12 o'clock, the IPTG that interpolation final concentration is 0.1-1mM, 25 ℃ of-37 ℃ of abduction deliverings, adjust pH with ammoniacal liquor, control pH 7.0.The firpene product obtaining carries out qualitative and quantitative analysis by GC-MS 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 OD600, 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.
GC-MS result shows (Fig. 9), has obtained the vernol of 1.35g/L and the Geraniol of 1.98g/L.
Derive from acetyl-CoA acyltransferase gene/3-hydroxy-3-methylglutaryl coenzyme A reductase gene (mvaE) of 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, 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 and derive from colibacillary ADP-ribose pyrophosphatase gene EcNudF(GenBank:U22009.1), utilize glucose biological to synthesize Geraniol.
(1) clone of EcNudF gene and the structure of expression vector
Take e. coli k12 genomic dna as masterplate, eNudF-rbs-BglF and eNudF-XhoR are that primer carries out pcr amplification, pcr amplification condition: 98 ℃ of denaturation 30s; 98 ℃ of sex change 5s, 58 ℃ of annealing 5s, 72 ℃ are extended 1min30s, and above sex change, annealing, extension three steps repeat after 35 circulations, and 72 ℃ are extended 10min.Utilize glue to reclaim test kit (purchased from Fermentas, Cat.No.K0692) and reclaim goal gene fragment.Gained gene fragment and pYJM26 carrier are carried out to double digestion with BglII and XhoI respectively, carrier and the external source fragment ratio of 1:5 in molar ratio, 4 ℃ of connections are spent the night or 16 ℃ of connection 4～6h, connect product Transformed E .coli DH5 α, then coating is added with the LB solid plate of 34 μ gmL-1 paraxin, PCR screening positive clone extracts after recombinant plasmid pLWG4 (pACY-mvaE-mvaS-GPPS2-EcNudF) from positive colony, then identifies by restriction enzyme digestion and order-checking.
(2) structure of E.coli recombinant bacterial strain
By pLWG4 and the common thermal shock Transformed E of pYJM14 .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 LWG4 that contains pLWG4 and pYJM4.
(3) vernol and Geraniol are produced in fermentation
The cultivation of engineering colon bacillus LWG4 and fermentation process are with embodiment 1.GC-MS result shows, has obtained the vernol of 135mg/L and the Geraniol of 198mg/L.
Derive from acetyl-CoA acyltransferase gene/3-hydroxy-3-methylglutaryl coenzyme A reductase gene (mvaE) of 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, 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 and derive from Herba Lysimachiae foenumgraeci Geraniol synthase gene GES(GenBank:AY362553.1), utilize glucose biological to synthesize Geraniol.
(1) clone of GES gene and the structure of expression vector
According to the synthetic Geraniol synthase gene (GES) of gene order GenBank:AY362553.1, and be connected into and on pGH carrier, form pGH-GES carrier.Take pGH-GES as masterplate, GES-rbs-Bgl II and GES-xhol I are primer, pcr amplification, pcr amplification condition: 98 ℃ of denaturation 30s; 98 ℃ of sex change 5s, 57 ℃ of annealing 5s, 72 ℃ are extended 1min30s, and above sex change, annealing, extension three steps repeat after 35 circulations, and 72 ℃ are extended 10min.Utilize glue to reclaim test kit (purchased from Fermentas, Cat.No.K0692) and reclaim goal gene fragment.
The gene fragment of recovery and pYJM26 carrier are carried out to double digestion with BglII and XhoI respectively, carrier and the external source fragment ratio of 1:5 in molar ratio, 4 ℃ of connections are spent the night or 16 ℃ of connection 4～6h, connect product Transformed E .coli DH5 α, then coating is added with the LB solid plate of 34 μ gmL-1 paraxin, PCR screening positive clone extracts after recombinant plasmid pLWG1 (pACY-mvaE-mvaS-GPPS2-GES) from positive colony, then identifies by restriction enzyme digestion and order-checking.
(2) structure of engineering colon bacillus LWG6
By pLWG1 and the common thermal shock Transformed E of pYJM14 recombinant plasmid .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 LWG5 that contains pLWG1 and pYJM4.
(3) vernol and Geraniol are produced in fermentation
Fermentation process, with embodiment 2, utilizes GC to carry out detection by quantitative (Figure 12), has finally obtained the vernol of 150mg/L and the Geraniol of 1.98g/L.
Derive from acetyl-CoA acyltransferase gene/3-hydroxy-3-methylglutaryl coenzyme A reductase gene (mvaE) of 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, 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 and derive from colibacillary alkaline phosphatase (phoA), as shown in SEQ ID NO.1, utilizing glucose biological to synthesize Geraniol.
(1) clone of phoA gene and plasmid construction
Take e. coli k12 genomic dna as masterplate, phoA-rbs-F2 and phoA-R are that primer carries out pcr amplification, pcr amplification condition: 94 ℃ of denaturation 3min; 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 2min, 35 circulations; 72 ℃ are extended 10min.Utilize glue to reclaim test kit (purchased from Fermentas, Cat.No.K0692) and reclaim goal gene fragment.
The gene fragment of recovery and pYJM26 carrier are carried out to double digestion with BglII and XhoI respectively, carrier and the external source fragment ratio of 1:5 in molar ratio, 4 ℃ of connections are spent the night or 16 ℃ of connection 4～6h, connect product Transformed E .coli DH5 α, then coating is added with the LB solid plate of 34 μ gmL-1 paraxin, PCR screening positive clone extracts after recombinant plasmid pLWG5 (pACY-mvaE-mvaS-GPPS2-phoA) from positive colony, then identifies by restriction enzyme digestion and order-checking.
(2) structure of E.coli recombinant bacterial strain LWG5
By pLWG5 and the common thermal shock Transformed E of pYJM14 recombinant plasmid .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 LWG5 that contains pLWG5 and pYJM4.
(3) vernol and Geraniol are produced in fermentation
Method is with embodiment 1, and GC result shows (Figure 13), has obtained the vernol of 20mg/L and the Geraniol of 637mg/L.
Derive from acetyl-CoA acyltransferase gene/3-hydroxy-3-methylglutaryl coenzyme A reductase gene (mvaE) of 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, mevalonic acid-5-phosphokinase gene (ERG8), mevalonic acid-5-bisphosphate decarboxylase gene (ERG19), isopentenylpyrophosphate isomerase gene (IDI1); The Phosphoric acid esterase phoE that derives from the geraniol ester diphosphate synthase gene (GPPS2) of abies grandis and derive from Bacillus subtillis is as shown in SEQ ID NO.2 (GenBank:CP003783.1).Plasmid method: by the synthetic two sections of gene phoE with BglII and XhoI restriction enzyme site of the raw work in Shanghai, form plasmid pGH/phoE, this plasmid and pYJM26 carrier are carried out to double digestion with BglII and XhoI respectively, and the processes such as connection, conversion and fermentation are with embodiment 5.Finally utilize glucose, after fermentation culture, obtained the vernol of 15mg/L and the Geraniol of 35mg/L.
Derive from acetyl-CoA acyltransferase gene/3-hydroxy-3-methylglutaryl coenzyme A reductase gene (mvaE) of 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, 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 and derive from the alkaline phosphatase phoA(GenBank:AF453253.1 of Cray Bai Shi pulmonitis strain (Klebsiella pneumoniae)), obtain the vernol of 60mg/L and the Geraniol of 348mg/L after utilizing glucose fermentation to cultivate.
Although the present invention with preferred embodiment openly as above; but it is not in order to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, therefore protection scope of the present invention should be with being as the criterion that claims were defined.
Claims (10)
1. a genetic engineering bacterium for coproduction Geraniol and vernol, is characterized in that, is in the microorganism that can express geranyl tetra-sodium, to have imported the genetic engineering bacterium that Geraniol synthase gene or phosphatase gene obtain.
2. genetic engineering bacterium according to claim 1; it is characterized in that; described genetic engineering bacterium coexpression acetyl-CoA acyltransferase/3-hydroxy-3-methylglutaryl coenzyme A reductase, 3-Hydroxy-3-methylglutaryl CoA A synthase, Mevalonic kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-bisphosphate decarboxylase, isopentenylpyrophosphate isomerase, geraniol ester diphosphate synthase, and Geraniol synthetic enzyme or Phosphoric acid esterase.
3. genetic engineering bacterium according to claim 2, is characterized in that, described Geraniol synthetic enzyme derives from Herba Lysimachiae foenumgraeci, and the nucleotide sequence of this enzyme of encoding is as shown in GenBank:AY362553.1.
4. genetic engineering bacterium according to claim 2, is characterized in that, described Phosphoric acid esterase is the phosphatidic acid phosphatase DPP1 from yeast saccharomyces cerevisiae, and the nucleotide sequence of this enzyme of encoding is as shown in NCBI Reference Sequence:NM_001180592.1; Or from the phosphatidic acid phosphatase LPP1 of yeast saccharomyces cerevisiae, the nucleotide sequence of this enzyme of encoding is as shown in NCBI Reference Sequence:NM_001180811.3; Or deriving from colibacillary ADP-ribose pyrophosphatase gene EcNudF, its nucleotide sequence is as shown in GenBank:U22009.1; Or deriving from colibacillary alkaline phosphatase gene phoA, its nucleotide sequence is as shown in SEQ ID NO.1; Or deriving from the Phosphoric acid esterase phoE of Bacillus subtillis, its nucleotide sequence is as shown in GenBank:CP003783.1; Or deriving from the alkaline phosphatase phoA of Cray Bai Shi pulmonitis strain, its nucleotide sequence is as shown in GenBank:AF453253.1.
5. a method that builds genetic engineering bacterium described in claim 1 is in the microorganism that can express geranyl tetra-sodium, to have imported Geraniol synthase gene or phosphatase gene.
6. method according to claim 5, is characterized in that, concrete steps are as follows:
(1) acetyl-CoA acyltransferase gene/3-hydroxy-3-methylglutaryl coenzyme A reductase gene, 3-Hydroxy-3-methylglutaryl CoA A synthase gene and the gene clone of geraniol ester diphosphate synthase are arrived to plasmid pACYCDuet-1, construction recombination plasmid pACY-mvaE-mvaS-GPPS2;
Described acetyl-CoA acyltransferase gene/3-hydroxy-3-methylglutaryl coenzyme A reductase gene, 3-Hydroxy-3-methylglutaryl CoA A synthase gene derive from enterococcus faecalis (Enterococcus faecalis), and described geraniol ester diphosphate synthase gene source is in abies grandis (Abiesgrandis);
(2) Mevalonic kinase gene, mevalonic acid-5-phosphokinase gene, mevalonic acid-5-bisphosphate decarboxylase gene and isopentenylpyrophosphate isomerase gene are cloned into pTrcHis2B and obtain recombinant plasmid pTrc-low;
Described Mevalonic kinase gene, mevalonic acid-5-phosphokinase gene, mevalonic acid-5-bisphosphate decarboxylase gene, isopentenylpyrophosphate isomerase gene preferably derive from yeast saccharomyces cerevisiae (Saccharomyces cerevisiae);
(3) by the gene clone of coding Geraniol synthetic enzyme or Phosphoric acid esterase or Pyrophosphate phosphohydrolase to plasmid pACY-mvaE-mvaS-GPPS2.
7. produce Geraniol and vernol a method, it is characterized in that, in the microorganism that can express geranyl tetra-sodium, import Geraniol synthase gene or phosphatase gene, utilize genetic engineering bacterium fermentative production Geraniol and vernol.
8. method according to claim 7, it is characterized in that, coexpression acetyl-CoA acyltransferase/3-hydroxy-3-methylglutaryl coenzyme A reductase, 3-Hydroxy-3-methylglutaryl CoA A synthase, Mevalonic kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-bisphosphate decarboxylase, isopentenylpyrophosphate isomerase, geraniol ester diphosphate synthase in microorganism, and Geraniol synthetic enzyme or Phosphoric acid esterase; Utilize recombinant bacterium fermentative production Geraniol and vernol.
9. method according to claim 8, it is characterized in that coexpression acetyl-CoA acyltransferase/3-hydroxy-3-methylglutaryl coenzyme A reductase, 3-Hydroxy-3-methylglutaryl CoA A synthase, Mevalonic kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-bisphosphate decarboxylase, isopentenylpyrophosphate isomerase, geraniol ester diphosphate synthase and Phosphoric acid esterase in microorganism; Utilize recombinant bacterium fermentative production Geraniol and vernol.
10. method according to claim 9, is characterized in that, described Phosphoric acid esterase is the phosphatidic acid phosphatase DPP1 from yeast saccharomyces cerevisiae, and the nucleotide sequence of this enzyme of encoding is as shown in NCBI Reference Sequence:NM_001180592.1; Or from the phosphatidic acid phosphatase LPP1 of yeast saccharomyces cerevisiae, the nucleotide sequence of this enzyme of encoding is as shown in NCBI Reference Sequence:NM_001180811.3; Or deriving from colibacillary ADP-ribose pyrophosphatase gene EcNudF, its nucleotide sequence is as shown in GenBank:U22009.1; Or deriving from colibacillary alkaline phosphatase gene phoA, its nucleotide sequence is as shown in SEQ ID NO.1; Or deriving from the Phosphoric acid esterase phoE of Bacillus subtillis, its nucleotide sequence is as shown in GenBank:CP003783.1; Or deriving from the alkaline phosphatase phoA of Cray Bai Shi pulmonitis strain, its nucleotide sequence is as shown in GenBank:AF453253.1.
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