CN109136158A - It is a kind of using biomass hydrolysate as the genetic engineering bacterium of Material synthesis styrene and its construction method and application - Google Patents

It is a kind of using biomass hydrolysate as the genetic engineering bacterium of Material synthesis styrene and its construction method and application Download PDF

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CN109136158A
CN109136158A CN201710498760.3A CN201710498760A CN109136158A CN 109136158 A CN109136158 A CN 109136158A CN 201710498760 A CN201710498760 A CN 201710498760A CN 109136158 A CN109136158 A CN 109136158A
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gene
plasmid
genetic engineering
styrene
engineering bacterium
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咸漠
张海波
刘长青
刘会洲
刘炜
徐鑫
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Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
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    • C12Y504/99005—Chorismate mutase (5.4.99.5)

Abstract

The present invention provides a kind of using biomass hydrolysate as the genetic engineering bacterium of Material synthesis styrene and its construction method and application, is related to gene engineering technology field and fermentation engineering field.The genetic engineering bacterium is overexpressed phenylalanine ammonia-lyase cDNA, cortex cinnamomi pyruvate decarboxylase gene, 3- deoxidation-D- Arab's ketoheptose -7- phosphate synthase gene and bifunctional enzyme chorismate mutase-prephenate dehydratase gene.Its construction step is as follows: clone's phenylalanine ammonia-lyase cDNA and cortex cinnamomi pyruvate decarboxylase gene are connected to plasmid；3- deoxidation-D- Arab ketoheptose -7- phosphate synthase gene and bifunctional enzyme gene are cloned, plasmid is connected to；Plasmid is imported into E. coli competent, obtains genetic engineering bacterium.It is Material synthesis styrene that said gene engineering bacteria, which can be used for biomass hydrolysate,.With green and sustainability, and it is higher by the yield of the styrene produced using glucose as carbon source significantly, realizes the breakthrough that cannot achieve all the time.

Description

It is a kind of using biomass hydrolysate as the genetic engineering bacterium of Material synthesis styrene and its structure Construction method and application
Technical field
The present invention relates to a kind of using biomass hydrolysate as the genetic engineering bacterium of Material synthesis styrene and its construction method With application, belong to gene engineering technology field and fermentation engineering field.
Background technique
Styrene is the monomer of synthetic rubber and plastics, for producing butadiene-styrene rubber, polystyrene, foamed polystyrene； It is also used for being copolymerized the engineering plastics for manufacturing a variety of different purposes from other monomers.Tree such as is made with acrylonitrile, butadiene copolymer Rouge is widely used in various household electrical appliance and industrial；The resin of impact resistance, bright in color is obtained with acrylonitrile compolymer；With A kind of thermoplastic elastomer obtained by butadiene copolymer is widely used as polyvinyl chloride, polyacrylic modifying agent etc..Styrene master It is used to produce styrene series resin and butadiene-styrene rubber, and production one of ion exchange resin and the raw material of pharmaceuticals, this Outside, styrene can also be used in the industries such as pharmacy, dyestuff, pesticide and ore dressing.
The source of styrene is mainly chemical synthesis at present, mainly includes ethylbenzene catalytic dehydrogenation method and ethylbenzene conjugated oxidation. Wherein 550~600 DEG C of dehydrogenations produce styrene to ethylbenzene catalytic dehydrogenation method under the effect of the catalyst, so that technique is by equipment It is required that the high and high limitation of energy consumption.And there is the disadvantages of reflection is complicated, by-product is more, long technical process in ethylbenzene conjugated oxidation.Together When above two method all influenced by the unsustainable equal bottlenecks of raw material.It is more and more with the development of synthetic biology Chemicals it is mild by reaction condition, the synthesis of environment amenable biological catalysis.Also, biosynthesis process uses can The hydrolysate glucose of regenerated biomass material is primary raw material, is had the advantages that sustainable.Azeem,Muhammad1 Et al. discovery can produce styrene with direct fermentation forest waste using Penicillium expansum, but overall hair The lower up to 52 μ g/h of ferment efficiency.John J.Beck2Et al. continuously fermented 60 days using Fusarium oxysporum, The styrene of separable amount is obtained, production efficiency is equally its restrictive factor (US 9,057,080B1).Currently, David R.Nielsen3Et al. to have been realized in using glucose be that raw material by engineering colon bacillus or Engineering Yeast produces benzene second Alkene needs additionally to add phenylalanine to engineered strain although relatively high before production efficiency is opposite, and phenylalanine It is expensive.In addition to this, the price of glucose sugar is higher also becomes a key factor for limiting its application.Biomass by hydrolyzation Technology can effectively reduce the cost of fermentation raw material, but will form a variety of fermentation inhibitors in product, such as furfural, hydroxyl first Base furfural, acetic acid, phenolic compound etc..To inhibit fermentation, so that biomass hydrolysate is that fermenting raw materials produce styrene Yield be restricted.And it is 251 ± 3mg L that glucose, which is the amount of styrene of carbon source production,-1, at present with biomass by hydrolyzation Liquid is that fermenting raw materials synthesizing styrene is difficult to be more than this yield.Fermentation inhibitor can generate toxic effect to bacterial strain simultaneously, Further limit styrene yield.
Summary of the invention
To make full use of biomass resource and solving a variety of fermentation inhibitors pair formed in above-mentioned biomass hydrolysate It is big that strain growth generates inhibiting effect, toxicity, and then the problems such as limit output, in order to mitigate fermentation inhibitor to the poison of bacterial strain Property effect, the present invention provides it is a kind of utilize biomass hydrolysate tolerance host e. coli, with biomass hydrolysate be original The genetic engineering bacterium and its construction method for expecting synthesizing styrene, using one plant of tolerance ligno-cellulose hydrolysate host's large intestine Bacillus strain (the bacterial strain granted patent number: CN201310473790.0, bacterial strain deposit number are CGMCC No.8028) is carried out The synthetic work of biology base styrene is taken so that realizing using biomass hydrolysate is that raw material efficiently synthesizes styrene Technical solution is as follows:
One of the objects of the present invention is to provide a kind of using biomass hydrolysate as the genetic engineering of Material synthesis styrene Bacterium, the genetic engineering bacterium are overexpressed phenylalanine ammonia-lyase cDNA, cortex cinnamomi pyruvate decarboxylase gene, 3- deoxidation-D- Arab heptanone Sugar -7- phosphate synthase gene and bifunctional enzyme chorismate mutase-prephenate dehydratase gene.
Preferably, said gene engineering bacteria starting strain is the Escherichia coli bacterium of one plant of tolerance ligno-cellulose hydrolysate Strain, the bacterial strain have been named as bacterial strain qibebt-3, and it is common which is preserved in China Committee for Culture Collection of Microorganisms Microorganism center, deposit number are CGMCC No.8028.
Preferably, the phenylalanine ammonia lyase is respectively derived from the benzene of arabidopsis (Arabidopsis thaliana) Alanine ammonolysis enzyme gene AtPAL or the phenylalanine ammonia-lyase cDNA of parsley (Petroselinum crispum) FtPAL；The cortex cinnamomi pyruvate decarboxylase gene, for derived from the cinnamic acid of saccharomyces cerevisiae (Saccharomyces cerevisiae) Decarboxylase gene FDC1；3- deoxidation-D- Arab ketoheptose -7- the phosphate synthase gene, for derived from the 3- of Escherichia coli Deoxidation-D- Arab ketoheptose -7- phosphate synthase gene aroF；The bifunctional enzyme chorismate mutase-prephenic acid dehydration Enzyme gene is derived from bifunctional enzyme chorismate mutase-prephenate dehydratase gene pheA of Escherichia coli.
Preferably, the phenylalanine ammonia-lyase cDNA AtPAL from arabidopsis (Arabidopsis thaliana) GeneBank accession number are as follows: 822493；The phenylalanine ammonia from parsley (Petroselinum crispum) Solve the UniProtKB/Swiss-Prot:P24481.1 of enzyme gene FtPAL；The cortex cinnamomi pyruvate decarboxylase gene FDC1's GeneBank accession number are as follows: 330443520；3- deoxidation-D- Arab ketoheptose -7- phosphate synthase gene the aroF's GeneBank accession number are as follows: 947084；The bifunctional enzyme chorismate mutase-prephenate dehydratase gene pheA GeneBank accession number are as follows: 947080.
The present invention also provides the construction methods of said gene engineering bacteria, and steps are as follows:
(1) clone is derived from the phenylalanine ammonia-lyase cDNA AtPAL of arabidopsis, derived from the cortex cinnamomi acid decarboxylase of saccharomyces cerevisiae Gene FDC1, and plasmid pTrcHis2B is connected to as restriction enzyme gene by obtained by, obtain the first recombinant plasmid；Gram The grand phenylalanine ammonia-lyase cDNA FtPAL derived from parsley, derived from the cortex cinnamomi pyruvate decarboxylase gene FDC1 of saccharomyces cerevisiae, And plasmid pTrcHis2B is connected to as restriction enzyme gene by obtained by, obtain second recombinant plasmid.
(2) 3- deoxidation-D- Arab ketoheptose -7- phosphate synthase gene aroF and double function of the clone derived from Escherichia coli Energy enzyme chorismate mutase-prephenate dehydratase gene pheA, and plasmid is connected to as restriction enzyme gene by obtained by On pET-duet-1, third recombinant plasmid is obtained；
(3) resulting first recombinant plasmid of step 1) and the resulting third recombinant plasmid of step 2) imported into biomass by hydrolyzation In the competent cell of liquid resistant strain qibebt-3, genetic engineering bacterium 1 is obtained；Resulting second recombinant plasmid of step 1) The competent cell of biomass hydrolysate resistant strain qibebt-3 is imported into the resulting third recombinant plasmid of step 2) In, obtain genetic engineering bacterium 2.
In addition, the present invention also provides said gene engineering bacterias using biomass hydrolysate as answering in Material synthesis styrene With.
The biomass is one of stalk, timber, duckweed, seaweed or corn or two kinds or more.
The biomass hydrolysate is by dilute acid hydrolysis method, diluted alkaline Hydrolyze method, lime Hydrolyze method, the hydrolysis of hydroxyl oxygen radical What method or enzymatic isolation method obtained.
The preparation process of the biomass hydrolysate is minced using purpose biomass powder, and dilute acid hydrolysis method or dilute is utilized Alkali hydrolysis method or lime Hydrolyze method or hydroxyl oxygen radical Hydrolyze method or/and enzymatic isolation method mince to biomass powder and handle, filtering Residue is removed, after centrifugation removes residue, after being 7.0 ± 0.1 using NaOH adjusting pH value, adds 1g/L laccase or horseradish Peroxidase or beans shell cross peroxidase, and 37 DEG C of warm bath 1h use 1g/L laccase or horseradish peroxidase again after centrifugation Enzyme or beans shell cross 37 DEG C of warm bath 1h of peroxidase, and the carbon source of fermentation is made in centrifugation of gained hydrolyzate；If in order into one Step improves the content of glucose in hydrolyzate, by above-mentioned resulting hydrolyzate addition cellulase 40FPU/g substrate and β-grape Glycosidase 20CBU/g substrate vibrates 50h in 45 DEG C of oscillators, and filtering gained hydrolyzate does the carbon source of fermentation.
Biomass hydrolysate the preparation method comprises the following steps:
Dilute acid hydrolysis method: taking 20g biomass powder to mince, and 3~5h is hydrolyzed at 75~95 DEG C using 1% sulfuric acid, filtering Residue is removed, after centrifugation removes residue, after being 7.0 ± 0.1 using NaOH adjusting pH value, adds 1g/L laccase or horseradish Peroxidase or beans shell cross peroxidase, and 37 DEG C of warm bath 1h use 1g/L laccase or horseradish peroxidase again after centrifugation Enzyme or beans shell cross 37 DEG C of warm bath 1h of peroxidase, and centrifugation gained hydrolyzate does the carbon source of fermentation.
Diluted alkaline Hydrolyze method: taking 20g biomass powder to mince, and 3~5h is hydrolyzed at 75~95 DEG C using 1% NaOH, filtering Residue is removed, after centrifugation removes residue, after being 7.0 ± 0.1 using HCl adjusting pH value, adds 1g/L laccase or horseradish mistake Oxide enzyme or beans shell cross peroxidase, and 37 DEG C of warm bath 1h use 1g/L laccase or horseradish peroxidase again after centrifugation Or beans shell crosses 37 DEG C of warm bath 1h of peroxidase, centrifugation gained hydrolyzate does the carbon source of fermentation.
Lime treatment: taking 20g biomass powder to mince, and is diluted to 100g/L using sterile water, and the Ca (OH) of 20g/l is added2 In 60 DEG C of warm bath 1h, it is filtered to remove residue, after centrifugation removes residue, after being 7.0 ± 0.1 using HCl adjusting pH value, addition 1g/L laccase or horseradish peroxidase or beans shell cross peroxidase, and 37 DEG C of warm bath 1h use 1g/L laccase again after centrifugation Either horseradish peroxidase or beans shell cross 37 DEG C of warm bath 1h of peroxidase, and centrifugation gained hydrolyzate does the carbon of fermentation Source.
The processing of hydroxyl oxygen radical: taking 20g biomass powder to mince, and is diluted to 100g/L using sterile water, sodium hypochlorite is added With hydrogen peroxide (volume ratio of the two is 10:1) in 60 DEG C of warm bath 1h, it is filtered to remove residue, after centrifugation removes residue, is utilized After acid or alkali adjusting pH value are 7.0 ± 0.1, add 1g/L laccase or horseradish peroxidase or beans shell crosses peroxidating Object enzyme, 37 DEG C of warm bath 1h cross 37 DEG C of peroxidase with 1g/L laccase or horseradish peroxidase or beans shell again after centrifugation Warm bath 1h, centrifugation gained hydrolyzate do the carbon source of fermentation.
Enzymatic isolation method: taking and weigh 20g biomass powder and mince, and the citric acid solution of 0.1M pH 4.5 is added, keeps substrate dense Degree reaches 10g/l, adds cellulase 40FPU/g substrate and beta-glucosidase 20CBU/g substrate, shakes in 45 DEG C of oscillators 50h is swung, filtering gained hydrolyzate does the carbon source of fermentation.
In a preferred embodiment in accordance with this invention, a kind of utilization renewable biomass hydrolyzate synthesis benzene is provided The method of ethylene, this method include that the recombinant escherichia coli strain of the method building in the present invention is being suitable for what it grew Under the conditions of comprising biomass hydrolysate as being cultivated in the culture medium of carbon source and inducer, during the cultivation process constantly It is passed through air, from being able to detect that styrene in tail gas in tunning.
Preferably, the biomass hydrolysate main component is carbohydrate, oils (for example, vegetable oil such as cottonseed oil, palm Oil or soybean oil), it is fatty acid (such as unsaturated fatty acid, saturated fatty acid or polyunsaturated fatty acid), lipid, phosphatide, sweet Grease (for example, monoglyceride, diglyceride, triglycerides etc.), polypeptide (for example, microorganism or vegetable protein or peptide), yeast Extract, the ingredient from yeast extract, polymer, acid, alcohol, aldehyde, ketone, amino acid, succinate, acetic acid esters etc..At this In invention, carbon source is preferably renewable carbohydrate, such as hydrolyzing biomass, including bagasse, corn stover, wood pulp, inverted sugar, light Cooperate product (including, but are not limited to glucose) and other monosaccharide (for example, fructose, mannose, galactolipin, xylose or Arabinose etc.), disaccharides, polysaccharide etc..It is minced using the biomass powder of 20~40g mesh, is hydrolyzed using dilute acid hydrolysis method or diluted alkaline Method or lime Hydrolyze method or hydroxyl oxygen radical Hydrolyze method or/and enzymatic isolation method processing, are filtered to remove residue, after centrifugation removes residue, After being 7.0 ± 0.1 using NaOH adjusting pH value, adds 1g/L laccase or horseradish peroxidase or beans shell crosses peroxide Compound enzyme, 37 DEG C of warm bath 1h, is crossed at peroxidase with laccase or horseradish peroxidase or beans shell again after centrifugation The carbon source of fermentation is made in reason, centrifugation of gained hydrolyzate, and ingredient is mainly glucose, xylose, furfural, additionally containing few Measure hydroxymethylfurfural, acetic acid, phenolic compound.
What the present invention obtained has the beneficial effect that:
In the method for biosynthesis styrene of the invention, medium to the big rule for being suitable for engineering colon bacillus can be used Any fluid nutrient medium of mould culture, such as M9 fluid nutrient medium can add and the engineering in the fluid nutrient medium The corresponding antibiotic of the antibiotic resistance of Escherichia coli or combination are to improve growth selectivity, for example, if in engineering large intestine Two kinds of antibiotic of chloramphenicol and ammonia benzyl mycin have been used in the screening process of bacillus respectively, then (such as have been shaken in biosynthetic process Bottle culture or fermentation tank culture) in, above two antibiotic can be added in the medium.In addition, being closed in biology of the invention At conventional inducer such as IPTG in the process, can be added in the medium to carry out Fiber differentiation.
The engineering colon bacillus bacterial strain of bacterial strain preparation method preparation according to the present invention can obtain the benzene second of one-component Alkene.
Styrene biological synthesis method according to the present invention, the shaking flask culture yield of styrene are more than 320mg/L, close 13.3 Mg/L/h is higher by tradition significantly with 52 μ g/h of biomass ferment production styrene, realize cannot achieve all the time it is prominent It is broken.
Method of the invention has green and sustainable using the hydrolysis sugar from biomass as Material synthesis styrene Property.
The engineering colon bacillus bacterial strain of bacterial strain preparation method preparation according to the present invention can obtain the benzene second of one-component Alkene.
The present invention provides a kind of using biomass hydrolysate as the genetic engineering bacterium of Material synthesis styrene and its building side Method, and using high-cellulose hydrolyzate tolerance Escherichia coli is that host is generated with solving fermentation inhibitor to strain growth The problems such as inhibiting effect.Economic, green of the invention, fundamentally provides one and utilizes sustainable method synthesizing styrene The problem of for alleviating styrene synthesis industry insufficient raw material.
Definition involved in the present invention and abbreviation:
Following abbreviation or abbreviation used herein:
High-cellulose hydrolyzate tolerance Escherichia coli (Escherichia coli): E.coli qibebt-3CGMCC No.8028
Saccharomyces cerevisiae (Saccharomyces cerevisiae): S.cerevisiae
Arabidopsis (Arabidopsis thaliana): A.Thaliana
Parsley (Petroselinum crispum): P.Crispum
" genetic fragment " is understood as according to specific use occasion opposite with internal specific gene sequence herein The isolated nucleic acid molecules or nucleic acid sequence answered, rather than the genetic fragment being present in the intracorporal genome of organism.
" overexpression " or " overexpression " refers to that expression of the specific gene in organism is more than normal level, for example, logical Crossing enhances endogenous expression or introducing foreign gene to realize.
Detailed description of the invention
The flow chart of Fig. 1 biomass hydrolysate synthesizing styrene.
Fig. 2 styrene biosynthetic metabolism approach.
Fig. 3 phenylalanine ammonia lyase, cortex cinnamomi acid decarboxylase coexpression vector schematic diagram, wherein the AtPAL gene in Fig. 3 A FtPAL gene in arabidopsis, Fig. 3 B is derived from parsley.
Fig. 4 3- deoxidation-D- Arab's ketoheptose -7- phosphate synthase and can be catalyzed chorismate be prephenic acid and Catalysis prephenic acid is converted into the bifunctional enzyme coexpression vector schematic diagram of phenylpyruvic acid.
The gas phase of Fig. 5 engineering bacteria synthesizing styrene-Mass Spectrometer Method figure；
(wherein, A: gas phase map, B: mass-spectrogram).
Specific embodiment
The present invention will be further described combined with specific embodiments below, but the present invention should not be limited by the examples.
Following embodiment agents useful for same, material, method and instrument are this field conventional reagent, material without specified otherwise Material, method and instrument, those skilled in the art can be obtained by commercial channel.
Embodiment 1
(1) phenylalanine ammonia lyase related gene, cortex cinnamomi acid decarboxylase related gene vector plasmid (pTrcHis plasmid) It constructs (Fig. 3)
The benzene of arabidopsis (Arabidopsis thaliana) is derived from by co-expressing in Escherichia coli (E.coli) Alanine ammonolysis enzyme gene AtPAL (AtPAL, ID:824493), cortex cinnamomi pyruvate decarboxylase gene (FDC1, GI:330443520) obtain Obtain first group of plasmid.
By co-expressing in Escherichia coli (E.coli) from parsley (Petroselinum crispum) Phenylalanine ammonia-lyase cDNA (FtPAL, (EC:4.3.1.24)), cortex cinnamomi pyruvate decarboxylase gene (FDC1, GI:330443520) Obtain second group of plasmid.
The above gene belongs to known, the method that preparation method carries out PCR as template using genome, Huo Zhezhi The synthesis of chemical gene method is connect, above method belongs to mature molecular biology method, and particularity is not present.Required for this experiment Gene carry out being sent into Hua Da synthetic gene sequence after codon optimization, obtain pGH-AtPAL, pGH-FtPAL, pGH- FDC1。
After obtaining target gene, phenylalanine ammonia-lyase cDNA (AtPAL, ID:824493) utilizes restriction enzyme Nco I and Xho I is connected to first expression sites of pTrcHis2B plasmid, cortex cinnamomi pyruvate decarboxylase gene in a manner of contacting (FDC1, GI:330443520) is connected to pTrcHis2B restriction enzyme Bgl II and Kpn I in a manner of contacting Second expression sites of plasmid.(as shown in Figure 3A) digestion system is as shown in table 1.
1 double digestion system of table
Reaction condition: 37 DEG C, digestion 1h.
After cutting target fragment, glue recycling is carried out using the plastic recovery kit of Omega Bio-Tek company.
Enzyme linked system is as shown in table 2:
2 enzyme linked system of table
16 DEG C of connections overnight.
The above plasmid construction method is related to round pcr, nucleic acid synthesis techniques, Plasmid restriction enzymatic cleavage methods, digestion piece Section recovery method, endonuclease bamhi connection method etc..Above method belongs to mature molecular biology method, and particularity is not present.
(2) 3- deoxidation-D- Arab ketoheptose -7- phosphate synthase gene is prephenic acid with that can be catalyzed chorismate And catalysis prephenic acid is converted into bifunctional enzyme chorismate mutase-prephenate dehydratase genophore plasmid of phenylpyruvic acid The building (Fig. 4) of (pACYC plasmid)
3- deoxidation-the D- of Escherichia coli (E.coli) is derived from by overexpression common in Escherichia coli (E.coli) Arabic ketoheptose -7- phosphate synthase gene (aroF, GI:947084), catalysis chorismate are that prephenic acid and catalysis are pre- Benzoic acid is converted into bifunctional enzyme chorismate mutase-prephenate dehydratase (pheA, ID:947080) of phenylpyruvic acid.
Specific experiment operation is as follows:
PCR amplification target fragment aroF and pheA, using E.coli BL21 (DE3) genome as template, respectively with aroF- BamH I/aroF-Not I and pheA-Bgl II/pheA-Xho I is primer, expands aroF and pheA gene respectively.
PCR reaction system is as shown in table 3:
Table 3PCR reaction system
PCR amplification program:
After obtaining target gene, 3- deoxidation-D- Arab's ketoheptose -7- phosphate synthase gene (aroF, GI:947084) First expression position of pACYCDuet-1 plasmid is connected in a manner of contacting restriction enzyme BamH I and Not I Point.Catalysis chorismate is that prephenic acid and catalysis prephenic acid are converted into the bifunctional enzyme chorismate mutase-of phenylpyruvic acid in advance Benzoic acid dehydrase gene (pheA, ID:947080) is connected in the way of contacting by restriction enzyme Bgl II with Xho I To second expression sites (as shown in Figure 4) of pACYCDuet-1 plasmid.
The above plasmid construction method is related to round pcr, nucleic acid synthesis techniques, Plasmid restriction enzymatic cleavage methods, digestion piece Section recovery method, endonuclease bamhi connection method etc..Above method belongs to mature molecular biology method, and particularity is not present.
(3) prepared by competence bacterial strain:
The single colonie that one diameter of picking is about 2 to 3 millimeters out of 37 DEG C overnight (16-20h) culture dish.As Single colonie is inoculated in 30 milliliters of sterilizing test tubes equipped with 5 milliliters of LB meat soup, and shaken cultivation is stayed overnight at 37 DEG C.Transfer 0.2 milliliter of overnight culture shaken cultivation at 37 DEG C in 50 milliliters of sterilizing triangular flasks equipped with 15 or 20 milliliters of LB 2 to 2.5 hours (bacterium is in logarithmic growth phase at this time).At room temperature, 4000rpm is centrifuged 5 minutes collection logarithmic phase cells.It abandons Culture medium retains cell precipitation.10 milliliters of ice-cold MgCl2-CaCl2 solution are added, and slightly beat.At 4 DEG C, 4000rpm It is centrifuged 10 minutes collection cells.MgCl2-CaCl2 solution is abandoned, cell precipitation is retained.0.8 milliliter of (every 25 milliliters initial training is added Support object and be added 1 milliliter) the CaCl2 solution of ice-cold 0.1M, and slightly beat.Ice bath placement several hours are best.At this time Competent cell can directly carry out conversion operation, can also dispense, and be added after glycerol in -70 DEG C of frost preservations.
(4) plasmid converts
By above-mentioned steps (1) prepare plasmid be transformed into the competence of Escherichia coli qibebt-3, that is, construct to Biomass hydrolysate is the genetic engineering bacterium of Material synthesis styrene, specifically: plasmid conversion: take 3~5uL of plasmid to be added big In the competence of enterobacteria BL21 (DE3), mix；Ice bath 30min, 42 DEG C of heat shock 55s；The LB culture medium of 450 μ L is added, 1h is activated on 37 DEG C of shaking tables；4 000rpm are centrifuged 2min, abandon most of supernatant, mix, and apply the Double plate of Cm and Amp, 37 DEG C of culture 12h, picking monoclonal, it is related that building obtains single-turn phenylalanine ammonia lyase related gene, cortex cinnamomi acid decarboxylase The bacterial strain of gene plasmid (pTrcHis2B plasmid).
Plasmid prepared by above-mentioned steps (1), (2) is transformed into the competence of Escherichia coli qibebt-3, that is, is constructed To using biomass hydrolysate as the genetic engineering bacterium of Material synthesis styrene, specifically: plasmid conversion: 3~5uL of plasmid is taken to add In the competence for entering e. coli bl21 (DE3), mix；Ice bath 30min, 42 DEG C of heat shock 55s；The LB culture of 450 μ L is added Base activates 1h on 37 DEG C of shaking tables；4 000rpm are centrifuged 2min, abandon most of supernatant, mix, it is Double to apply Cm and Amp Plate, 37 DEG C of culture 12h, picking monoclonal, building are converted above-mentioned pTrcHis2B plasmid simultaneously and contain 3- deoxidation-D- Arabic ketoheptose -7- phosphate synthase and can be catalyzed chorismate be prephenic acid and catalysis prephenic acid be converted into propiophenone The bacterial strain of the bifunctional enzyme carrier pACYCDuet-1 plasmid of acid.
Embodiment 2 is using biomass hydrolysate as fermenting raw materials synthesizing styrene
(1) preparation (Fig. 1) of biomass hydrolysate
Dilute acid hydrolysis method: take 20g biomass powder mince (can be one of stalk, timber, duckweed, seaweed or corn, It is also possible to the two or more mixtures of these substances, mass ratio 1；1) it, is hydrolyzed at 75~95 DEG C using 1% sulfuric acid 5h, is filtered to remove residue, and centrifugation is adjusted after pH value is 7.0 ± 0.1 using NaOH except after residue, add 1g/L laccase or Person's horseradish peroxidase or beans shell cross peroxidase, and 37 DEG C of warm bath 1h use 1g/L laccase or horseradish mistake again after centrifugation Oxide enzyme or beans shell cross 37 DEG C of warm bath 1h of peroxidase, and centrifugation gained hydrolyzate does the carbon source of fermentation.
Diluted alkaline Hydrolyze method: taking 20g biomass powder to mince, and hydrolyzes 5h at 75~95 DEG C using 1% NaOH, is filtered to remove Residue after centrifugation removes residue, after being 7.0 ± 0.1 using HCl adjusting pH value, adds 1g/L laccase or horseradish peroxidating Object enzyme or beans shell cross peroxidase, 37 DEG C of warm bath 1h, after centrifugation again with 1g/L laccase or horseradish peroxidase or Beans shell crosses 37 DEG C of warm bath 1h of peroxidase, and centrifugation gained hydrolyzate does the carbon source of fermentation.
Lime treatment: taking 20g biomass powder to mince, and is diluted to 100g/l using sterile water, and the Ca (OH) of 20g/l is added2 In 60 DEG C of warm bath 1h, it is filtered to remove residue, after centrifugation removes residue, after being 7.0 ± 0.1 using HCl adjusting pH value, addition 1g/L laccase or horseradish peroxidase or beans shell cross peroxidase, and 37 DEG C of warm bath 1h use 1g/L laccase again after centrifugation Either horseradish peroxidase or beans shell cross 37 DEG C of warm bath 1h of peroxidase, and centrifugation gained hydrolyzate does the carbon of fermentation Source.
The processing of hydroxyl oxygen radical: taking 20~40g biomass powder to mince, and is diluted to 100g/l using sterile water, time chlorine is added Sour sodium and hydrogen peroxide (volume ratio of the two is 10:1) are filtered to remove residue in 60 DEG C of warm bath 1h, after centrifugation removes residue, After being 7.0 ± 0.1 using acid or alkali adjusting pH value, adds 1g/L laccase or horseradish peroxidase or beans shell was crossed Oxide enzyme, 37 DEG C of warm bath 1h, crosses peroxidase with 1g/L laccase or horseradish peroxidase or beans shell again after centrifugation 37 DEG C of warm bath 1h, centrifugation gained hydrolyzate do the carbon source of fermentation.
Enzymatic isolation method: taking and weigh 20~40g biomass powder and mince, and the citric acid solution of 0.1M pH 4.5 is added, makes bottom Object concentration reaches 10g/l, adds cellulase 40FPU/g substrate and beta-glucosidase 20CBU/g substrate, vibrates in 45 DEG C Device vibrates 50h, and filtering gained hydrolyzate does the carbon source of fermentation.
(2) the fermentation synthesis of styrene
It is cultivated respectively in the culture medium that two kinds of monoclonals obtained by 1 step of embodiment (3) are inoculated in respectively in culture bottle.
Above-mentioned culture medium prescription are as follows: tryptone 5g/L, yeast extract 10g/L, biomass hydrolysate (above five kinds of water Solve liquid) 10ml, 170mmol/l potassium dihydrogen phosphate, 720mmol/l dipotassium hydrogen phosphate, phenylalanine 1g/L.Cultivate bacterium solution When OD600 to 0.8 or so, the IPTG of final concentration of 0.4mM is added.Add bottle stopper simultaneously, forms anaerobic environment.Shaking flask is transferred to 30 DEG C, cultivate for 24 hours in 180rpm shaking flask, head space gas phase-mass spectroscopy styrene.The method of the quantitative use of styrene is, The ethyl acetate of 20ml is added into the shaking flask after induction for 24 hours, 6000rpm is centrifuged 10min, takes the organic phase solution mistake of 1ml 0.22 μm of organic phase film, obtained liquid carry out gas phase and quantify styrene.
(3) styrene product measurement (Fig. 5)
Styrene obtained in above-mentioned steps (2) is passed through into gas-chromatography (GC) and gas chromatograph-mass spectrometer (GC-MS) (GC- MS measurement) is analyzed it.GC uses CP-FFAP CB capillary chromatographic column (25m × 0.25mm；0.2 μm), method 50 DEG C maintain 1 minute, then 20 DEG C/min of temperature programmings are warming up to 240 DEG C, 240 DEG C of maintenance 5min for 30 DEG C/min to 120 DEG C, 260 DEG C of detector temperature.GC-MS uses Agilent HP-INNOWax capillary chromatographic column (30m × 0.32mm, 0.25 μ M.), method is 50 DEG C of maintenances 2 minutes, and then 10 DEG C/min of temperature programmings to 240 DEG C, 240 DEG C maintain 3 minutes.Experimental result The mass spectrogram of display, the retention time of sample peak and styrene as fig. 5 a and fig. 5b, according to vapor detection as a result, detection The yield of styrene.
Wherein, single-turn phenylalanine ammonia lyase related gene, cortex cinnamomi acid decarboxylase related gene plasmid (pTrcHis2B Plasmid) culture of the bacterial strain in step (2) styrene yield be 255mg/L；It converts simultaneously above-mentioned PTrcHis2B plasmid and containing 3- deoxidation-D- Arab's ketoheptose -7- phosphate synthase and chorismate can be catalyzed it is Prephenic acid and catalysis prephenic acid are converted into the bacterial strain of the bifunctional enzyme carrier pACYCDuet-1 plasmid of phenylpyruvic acid, in step (2) in the case of the culture in, yield can effectively be improved to 320mg/L.
Although the present invention has been disclosed in the preferred embodiment as above, it is not intended to limit the invention, any to be familiar with this The people of technology can be various changes and modification, therefore guarantor of the invention without departing from the spirit and scope of the present invention Protecting range, this is subjected to the definition of the claims.

Claims (8)

1. a kind of using biomass hydrolysate as the genetic engineering bacterium of Material synthesis styrene, which is characterized in that be overexpressed phenylpropyl alcohol ammonia Sour ammonolysis enzyme gene, cortex cinnamomi pyruvate decarboxylase gene, 3- deoxidation-D- Arab's ketoheptose -7- phosphate synthase gene and double function It can enzyme chorismate mutase-prephenate dehydratase gene.
2. genetic engineering bacterium according to claim 1, which is characterized in that the phenylalanine ammonia lyase is respectively derived from The phenylalanine ammonia-lyase cDNA AtPAL or parsley (Petroselinum of arabidopsis (Arabidopsis thaliana) Crispum phenylalanine ammonia-lyase cDNA FtPAL)；The cortex cinnamomi pyruvate decarboxylase gene, for derived from saccharomyces cerevisiae The cortex cinnamomi pyruvate decarboxylase gene FDC1 of (Saccharomyces cerevisiae)；3- deoxidation-D- Arab ketoheptose-the 7- Phosphate synthase gene, for derived from the 3- deoxidation-D- Arab ketoheptose -7- phosphate synthase gene aroF of Escherichia coli；Institute Bifunctional enzyme chorismate mutase-prephenate dehydratase gene is stated, the bifunctional enzyme chorismate mutase-of Escherichia coli is derived from Prephenate dehydratase gene pheA.
3. genetic engineering bacterium according to claim 2, which is characterized in that described to come from arabidopsis (Arabidopsis Thaliana the GeneBank accession number of phenylalanine ammonia-lyase cDNA AtPAL) are as follows: 822493；It is described to come from parsley The UniProtKB/Swiss-Prot of the phenylalanine ammonia-lyase cDNA FtPAL of (Petroselinum crispum): P24481.1；The GeneBank accession number of the cortex cinnamomi pyruvate decarboxylase gene FDC1 are as follows: 330443520；3- deoxidation-D- the Ah Draw the GeneBank accession number of primary ketoheptose -7- phosphate synthase gene aroF are as follows: 947084；The bifunctional enzyme chorismic acid becomes Position enzyme-prephenate dehydratase gene pheA GeneBank accession number are as follows: 947080.
4. genetic engineering bacterium according to claim 1, which is characterized in that starting strain is one plant of tolerance lignocellulosic The coli strain of hydrolyzate, the bacterial strain have been named as qibebt-3, which is preserved in Chinese microorganism strain preservation Administration committee's common micro-organisms center, deposit number are CGMCC No.8028.
5. a kind of construction method of any genetic engineering bacterium of claim 1-4, which is characterized in that steps are as follows:
1) clone is derived from the phenylalanine ammonia-lyase cDNA AtPAL of arabidopsis, derived from the cortex cinnamomi pyruvate decarboxylase gene of saccharomyces cerevisiae FDC1, and plasmid pTrcHis2B is connected to as restriction enzyme gene by obtained by, obtain the first recombinant plasmid；Clone source In the phenylalanine ammonia-lyase cDNA FtPAL of parsley, derived from the cortex cinnamomi pyruvate decarboxylase gene FDC1 of saccharomyces cerevisiae, and pass through Gained gene is connected to plasmid pTrcHis2B by restriction enzyme, obtains second recombinant plasmid.
2) clone is derived from the 3- deoxidation-D- Arab ketoheptose -7- phosphate synthase gene aroF and bifunctional enzyme of Escherichia coli Chorismate mutase-prephenate dehydratase gene pheA, and plasmid pET- is connected to as restriction enzyme gene by obtained by On duet-1, third recombinant plasmid is obtained；
3) resulting first recombinant plasmid of step 1) and resulting second recombinant plasmid of step 2) imported into e. coli bl21 (DE3) in competent cell, genetic engineering bacterium is obtained.
6. any genetic engineering bacterium of claim 1-4 is using biomass hydrolysate as answering in Material synthesis styrene With.
7. application according to claim 6, which is characterized in that the biomass is stalk, timber, duckweed, seaweed or jade Rice one of or two kinds or more.
8. application according to claim 6, which is characterized in that the biomass hydrolysate is by dilute acid hydrolysis method, dilute What alkali hydrolysis method, lime Hydrolyze method, hydroxyl oxygen radical Hydrolyze method or enzymatic isolation method obtained.
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