CN104004790A - Method for producing medium-chain alkanes - Google Patents

Abstract

The invention belongs to the field of biochemical engineering, and particularly relates to a method for producing medium-chain alkanes. The alkanes are mainly derived from petroleum and natural gas and also can be produced from metabolism of species in the natural world, however the quantity of the alkanes of microorganisms is low. With the rapid development of modern molecular biology, particularly the development of synthetic biology, the expression of related enzymes in the synthetic process of the alkanes is regulated, so that microorganism cells can generate the alkanes only through simple metabolism, and an advanced technology for producing renewable energy is achieved. The method for producing medium-chain alkanes provides an excellent solution for the problems that traditional fossil fuel supply is insufficient, and novel biofuel can not completely replace the fossil fuel. Compared with other bacterial strains, escherichia coli has the advantages of being clear in genetic background, easy to improve, high in growing speed, suitable for high-density fermentation and the like, and is the ideal bacterial strain for synthetizing chemicals and fuel in a microbiological method. The method for producing the biological alkanes by the escherichia coli is a new way which does not occupy the space, does not depend on the weather, and can achieve sustainable production.

Description

A kind of production method of middle paraffinic hydrocarbons

Technical field

The invention belongs to biological chemical field, particularly a kind of production method of middle carbochain alkane.

Background technology

Alkane, is the main ingredient of fossil energy, such as separated product gasoline, diesel oil and the Aviation Fuel etc. of oil, all the alkane of different carbon chain lengths, consists of.And in recent years, increasingly exhausted along with fossil energy, and the surge of crude oil consumption amount, fossil energy feed rate is not enough and how to realize Sustainable development, has become the problem that mankind institute must concern.Therefore,, about how utilizing the research of renewable energy source substitute fossil fuels, become gradually newborn hot subject.

The fossil energy substitute being most widely used at present, mainly comprises alcohol fuel, fuel butanols and biofuel etc.But these several surrogates are in application process, all there are many problems, such as alcohol gasoline very easily forms layering and corrosion engine rubber, biodiesel raw material cost and energy consumption in production process are all more high, cause their hydrocarbons such as replacing gasoline, diesel oil completely.So, for fossil energy, seek a kind of better renewable surrogate, a kind of low cost, high efficiency alkane production method, be extremely urgent.

Alkane is mainly derived from oil and natural gas, also can produce by the metabolism of some species of occurring in nature, such as the epicuticular wax of plant, the pheromone of insect etc., but academia is few for utilizing microorganism this " cell factory " to produce the research of alkane.Develop rapidly along with modern molecular biology especially synthetic biology, utilize engineered means, in regulation and control alkane building-up process, the expression of relevant enzyme, makes microorganism cells only need just can produce alkane by simple metabolism, is the cutting edge technology of production renewable energy source.The method for traditional fossil fuel undersupply, novel biological fuel can not substitute the problems such as fossil fuel completely that an excellent solution is provided.Compare with other bacterial strains, intestinal bacteria have genetic background clear, be easy to transformation, fast growth, be applicable to the advantages such as high density fermentation, be the desirable strain of microbial method synthesis of chemicals and fuel.Utilize intestinal bacteria produce biological alkane be a kind of ground out of question, by day, the new way of Sustainable Production.

Compare with known technology, the present invention has the following advantages:

(1) directly utilize microorganism to take the alkane that renewable biomass obtains as raw material metabolism, can greatly alleviate the problem of fossil energy undersupply.

(2) alkane obtaining by the present invention, is as good as with the alkane in oil, more approaches the character of fossil oil itself, has overcome the series of problems that alcohol fuel, fuel butanols, biofuel etc. exist, and utilization ratio is high.

(3) microorganism only need just can produce alkane by simple metabolism, with short production cycle, not affected by the factors such as place, season, and the resource that do not occupy cultivated land, easily realizes suitability for industrialized production.

Summary of the invention

The object of the present invention is to provide a kind of at recombination bacillus coli (Escherichia coli), the occurring in nature of take exists in a large number and reproducible lipid acid is raw material, the renewable energy source operational path of synthesizing alkanes, makes microorganism can be used as cell factory, fermentative production alkane.

First the present invention carried out expression to the acyl-CoA synthetase gene from e. coli k12 strain, built genetic engineering bacterium, was about to gene fadD and was cloned in prokaryotic expression carrier pACYCDuet-1, obtained recombinant plasmid pACYCDuet-fadD, plasmid 1.The acyl-CoA synthetase of expressing can add by exogenous stream the mode of the medium chain fatty acids such as C14:0/C16:0, and catalysis lipid acid generates meta-bolites ester acyl coenzyme A, and this precursor substance is accumulated in host cell.

Heterogenous expression is from the acyl-CoA reductase enzyme in acinetobacter calcoaceticus ADP1 (Acinetobacter baylyi) again.Be specially acyl-CoA reductase gene acr in acinetobacter calcoaceticus is cloned in prokaryotic expression carrier pETDuet-1, obtain recombinant plasmid pETDuet-acr, plasmid 2.The ester acyl coenzyme A that the acyl-CoA reductase enzyme of expressing accumulates in can catalysis cell is reduced to the alkanoic of C14/C16.

Finally, heterogenous expression is from the aldehyde decarbonylation base enzyme in nostoc (Nostoc punctiforme).Be about to aldehyde decarbonylation base enzyme gene dc and be cloned in prokaryotic expression carrier plasmid 2, obtain recombinant plasmid pETDuet-acr-dc, plasmid 3.The aldehyde decarbonylation base enzyme of expressing can be sloughed carbonyl by catalysis alkanoic, generates alkane.

Recombinant plasmid 1/ plasmid 3 is proceeded in e. coli bl21 (DE3) jointly, IPTG induction recombinant plasmid carries out coexpression, can add by exogenous stream the mode of C14:0/C16:0 lipid acid, make lipid acid progressively metabolism generate the alkane that the carbon chain lengths such as C13/C15 do not wait, wherein to add the alkane output that C14:0 lipid acid obtains be 2.17mg/L to exogenous stream, it is 1.81mg/L that exogenous stream adds the alkane output that C16:0 lipid acid obtains, thereby obtains utilizing the renewable resources operational path of the direct synthesizing alkanes of recombination bacillus coli.

Method provided by the invention; the key gene of, acyl-CoA reduction synthetic at expression in escherichia coli specificity regulation and control acyl-CoA and aldehyde de-carbonyl reaction; the alkane that can utilize microbial metabolism natural acid to obtain; and carry out high density fermentation, for renewable alkane synthetic opened up a new approach.

Accompanying drawing explanation

Fig. 1: DNA agarose gel electrophoresis.Right side is acyl-CoA synthetase gene PCR product in the intestinal bacteria after purifying, 1686bp.Left side is DNA size standards.

Fig. 2: the collection of illustrative plates of the recombinant vectors pACYCDuet-fadD of structure, fadD gene clone is in pACYCDuet-1 carrier, and gene two ends are with NCO I and EcoR I site.Be numbered plasmid 1.

Fig. 3: DNA agarose gel electrophoresis.Right side is acyl-CoA reductase gene PCR product in the acinetobacter calcoaceticus after purifying, 888bp.Left side is DNA size standards.

Fig. 4: the collection of illustrative plates of the recombinant vectors pETDuet-acr of structure, acr gene clone is in pETDuet-1 carrier, and gene two ends are with NCO I and BamH I site.Be numbered plasmid 2.

Fig. 5: DNA agarose gel electrophoresis.Right side is aldehyde decarbonylation base enzyme gene PCR product in the nostoc after purifying, 699bp.Left side is DNA size standards.

Fig. 6: the collection of illustrative plates of the recombinant vectors pETDuet-acr-dc of structure, dc gene clone is in pETDuet-acr carrier, and gene two ends are with Bgl II and Hind III site.Be numbered plasmid 3.

Fig. 7: the engineering colon bacillus that contains plasmid 1/ plasmid 3 is after induction, and exogenous stream adds C14:0 lipid acid, the condition of production of alkane in its nutrient solution; C13 represents tridecane; C15 represents pentadecane.

Fig. 8: the engineering colon bacillus that contains plasmid 1/ plasmid 3 is after induction, and exogenous stream adds C16:0 lipid acid, the condition of production of alkane in its nutrient solution; C13 represents tridecane; C15 represents pentadecane.

Embodiment

A kind of renewable energy source operational path that utilizes the direct synthesizing alkanes of recombination bacillus coli provided by the invention, is utilized effectively natural acid.The present invention solves this technical problem by following scheme: first take genome of E.coli DNA as template, design corresponding primer and add corresponding clone's restriction enzyme site, amplification fadD gene, its nucleotide sequence is as shown in sequence 1, and coded aminoacid sequence is as shown in sequence 2 separately.After pcr amplification, reclaim and obtain goal gene DNA fragmentation, enzyme is connected to after cutting on the corresponding restriction enzyme site of prokaryotic expression carrier pACYCDuet-1, connection product is transformed in competent escherichia coli cell, screening obtains positive recombinant plasmid pACYCDuet-fadD (plasmid 1), and its plasmid map as shown in Figure 2.Plasmid is purified, electrophoresis detection, order-checking are identified goal gene.

Recycle the synthetic method of full gene synthetic acr gene respectively, its nucleotide sequence is as shown in sequence 3, coded aminoacid sequence is as shown in sequence 4 separately, and add corresponding clone's restriction enzyme site, enzyme is connected to after cutting on the corresponding restriction enzyme site of prokaryotic expression carrier pETDuet-1, and connection product is transformed in competent escherichia coli cell, and screening obtains positive recombinant plasmid pETDuet-acr (plasmid 2), its plasmid map as shown in Figure 4, and is identified goal gene with same method.

Finally, utilize the synthetic method of full gene synthetic dc gene respectively, its nucleotide sequence is as shown in sequence 5, coded aminoacid sequence is as shown in sequence 6 separately, and add corresponding clone's restriction enzyme site, enzyme is connected to after cutting on the corresponding restriction enzyme site of prokaryotic expression carrier pETDuet-acr, connection product is transformed in competent escherichia coli cell, screening obtains positive recombinant plasmid pETDuet-acr-dc (plasmid 3), its plasmid map as shown in Figure 4, and is identified goal gene with same method.

Recombinant plasmid 1/3 common abduction delivering in e. coli bl21 (DE3) is added after lipid acid with exogenous stream, continue to cultivate, in recombinant Bacillus coli cells, can obtain the not alkane of grade of the carbon chain lengths such as C13/C15.

Experimental procedure

1) bacterial classification adopting

E. coli bl21 (DE3)

2) substratum adopting

LB substratum (peptone 10g/L, yeast extract 5g/L, NaCl5g/L, 121 ℃ of moist heat sterilization 20min), if preparation plate culture medium needs to add 1.5% agar powder; The penbritin solution and the chloromycetin solution that in the LB substratum of selectivity LB substratum: 50ml, add 50 μ L left and right 0.1%.

3) experimental technique:

Respectively with, intestinal bacteria (Escherichia coli) the K12 strain gene group of utilizing Bo Maide test kit method to extract, utilizing full gene synthesis technology synthetic is template from acyl-CoA reductase enzyme [acyl-CoA reductase] gene DNA in acinetobacter calcoaceticus ADP1 (Acinetobacter baylyi) and one from aldehyde decarbonylation base enzyme [aldehyde decarboxylase] gene DNA in nostoc (Nostoc punctiforme), pcr amplification obtains acyl-CoA synthetase [acyl-CoA synthetase] gene with corresponding clone's restriction enzyme site, acyl-CoA reductase gene and aldehyde decarbonylation base enzyme gene.

With glue, reclaim test kit and reclaim object fragment, with corresponding restriction enzyme respectively enzyme cut acyl-CoA synthetase gene fadD fragment and prokaryotic expression carrier pACYCDuet-1, and in 16 ℃ of connections of spending the night, be the prokaryotic expression carrier plasmid 1 building; By identical method, acyl-CoA reductase gene acr fragment is connected with prokaryotic expression carrier pETDuet-1 again, is the prokaryotic expression carrier plasmid 2 building; Finally by identical method, aldehyde decarbonylation base enzyme gene dc fragment is connected with plasmid 2, is the prokaryotic expression carrier plasmid 3 building.

The recombinant plasmid building 1/ plasmid 3 is utilized to electric method for transformation, be jointly transformed in e. coli bl21 (DE3), can obtain the engineering colon bacillus that produces acyl-CoA synthetase, acyl-CoA reductase enzyme and aldehyde decarbonylation base enzyme.Engineering colon bacillus is inoculated in 50mL LB liquid nutrient medium by 1% inoculum size, and 37 ℃ of quick oscillation are cultivated, and continue at 30 ℃ cultivation 48hs, abduction delivering target protein after adding inductor IPTG and exogenous stream to add lipid acid in nutrient solution; Extract the alkane in fermented liquid, measure the output of the alkane such as C13/C15, be the alkane of the different carbon chain lengths preparing.

Below in conjunction with embodiment, concrete grammar of the present invention is described further:

Embodiment 1

The clone of acyl-CoA synthetase and the structure of recombinant vectors in 1 intestinal bacteria

The clone of acyl-CoA synthetase in 1.1 intestinal bacteria

Utilize Bo Maide test kit method to extract intestinal bacteria (Escherichia coli) K12 strain gene group; take genome of E.coli DNA as template; according to GenBank primers; and add corresponding restriction enzyme site (NCO I and EcoR I) and protect base; acyl-CoA synthetase [acyl-CoA synthetase] gene of pcr amplification fadD coding, the acyl-CoA synthetase of this gene induced expression can make lipid acid generate ester acyl coenzyme A by metabolism.

Pcr amplification acyl-CoA synthetase gene fadD the primer is as follows:

Upstream primer: 5 '-CATG cCATGGgCAAGAAGGTTTGGCTTAACC-3 '

Downstream primer: 5 '-CG gAATTCcGGCTCAGGCTTTATTGTCCACT-3 '

PCR reaction system contains 0.2 μ L genomic dna, each 0.25 μ L of upstream and downstream primer, and dNTPs mixed solution 1.6 μ L, 10 * Buffer2 μ L, pyrobest polysaccharase 0.4 μ L, adds distilled water and mends to 20 μ L.Reaction conditions is 94 ℃ of 4min of denaturation, 94 ℃ of 1min of sex change, and the 55 ℃ of 1min that anneal, extend 72 ℃ of 1min50s, circulate 30 times, 72 ℃ of 10min, 4 ℃ of preservations.

Can be cloned into object acyl-CoA synthetase gene fadD by pcr amplification like this:

This gene order length is 1686bp, utilizes PCR to reclaim test kit and reclaims goal gene, and utilize 1% agarose gel electrophoresis detection after pcr amplification finishes, and resulting target DNA band is 1686bp (as shown in Figure 1).

The structure of 1.2 transfer vector plasmids 1

Clone's fadD gene is carried out to double digestion with identical enzyme (NCO I and EcoR I) for pACYCDuet-1 carrier (being purchased from Novagen company), 37 ℃ of enzymes are cut 4 hours, carrier and exogenous genetic fragment be the ratio of 1:3-1:10 in molar ratio, utilize 16 ℃ of connections of T4DNA ligase enzyme of NEB to spend the night, with this, connect product and transform intestinal bacteria Top10 competent cell, coating is with chlorampenicol resistant LB agar plate, after 37 ℃ of incubated overnight, the appropriate single bacterium colony of picking, incubated overnight in LB substratum, and carry out bacterium colony PCR.With test kit, extract plasmid, carry out plasmid enzyme restriction checking, sequencing result correctly obtains positive recombinant vectors.Measure its sequence as shown in sequence 1, the aminoacid sequence of coding as shown in sequence 2.Because the initiator codon of this gene is rare initiator codon TTG, therefore changed into common initiator codon ATG, hereby explanation when pcr amplification gene.

The collection of illustrative plates (seeing Fig. 2) of constructed transfer vector plasmid 1.

Embodiment 2

The clone of acyl-CoA reductase enzyme and the structure of recombinant vectors in 1 acinetobacter calcoaceticus

The clone of acyl-CoA reductase enzyme in 1.1 acinetobacter calcoaceticus

Utilize the synthetic method of full gene to synthesize acyl-CoA reductase enzyme [acyl-CoA reductase] gene DNA in acinetobacter calcoaceticus (Acinetobacter baylyi).The ester acyl coenzyme A generation reduction reaction that the acyl-CoA reductase enzyme of this gene induced expression accumulates in can catalysis cell, generates the alkanoic of C14/C16.

The acyl-CoA reductase gene DNA in above-mentioned synthetic acinetobacter calcoaceticus of take is template, according to GenBank primers, and adds corresponding restriction enzyme site (NCO I and BamH I) and protection base.

Pcr amplification acyl-CoA reductase gene acr the primer is as follows:

Upstream primer: 5 '-GCAGCCCATGGCTAGCATGAACGCTAAAC-3 '

Downstream primer: 5 '-CCGCTGGATCCTTACCAGTGTTCACCTGG-3 '

PCR reaction system contains 0.2 μ L genomic dna, each 0.25 μ L of upstream and downstream primer, and dNTPs mixed solution 1.6 μ L, 10 * Buffer2 μ L, pyrobest polysaccharase 0.4 μ L, adds distilled water and mends to 20 μ L.Reaction conditions is 94 ℃ of 4min of denaturation, 94 ℃ of 1min of sex change, and the 55 ℃ of 1min that anneal, extend 72 ℃ of 1min, circulate 30 times, 72 ℃ of 10min, 4 ℃ of preservations.

Can be cloned into object acyl-CoA reductase gene acr by pcr amplification like this:

This gene order length is 888bp, utilizes PCR to reclaim test kit and reclaims goal gene, and utilize 1% agarose gel electrophoresis detection after pcr amplification finishes, and resulting target DNA band is 888bp (as shown in Figure 3).

The structure of 1.2 transfer vector plasmids 2

Clone's acr gene is carried out to double digestion with identical enzyme (NCO I and BamH I) for pETDuet-1 carrier (being purchased from Novagen company), 37 ℃ of enzymes are cut 4 hours, carrier and exogenous genetic fragment be the ratio of 1:3-1:10 in molar ratio, utilize 16 ℃ of connections of T4DNA ligase enzyme of NEB to spend the night, with this, connect product and transform intestinal bacteria Top10 competent cell, coating is with amicillin resistance LB agar plate, after 37 ℃ of incubated overnight, the appropriate single bacterium colony of picking, incubated overnight in LB substratum, and carry out bacterium colony PCR.With test kit, extract plasmid, carry out plasmid enzyme restriction checking, sequencing result correctly obtains positive recombinant vectors.Measure its sequence as shown in sequence 3, the aminoacid sequence of coding as shown in sequence 4.Because the initiator codon of this gene is rare initiator codon GTG, therefore changed into common initiator codon ATG, hereby explanation when synthetic this gene.

The collection of illustrative plates (seeing Fig. 4) of constructed transfer vector plasmid 2.

Embodiment 3

The clone of aldehyde decarbonylation base enzyme and the structure of recombinant vectors in 1 nostoc

The clone of aldehyde decarbonylation base enzyme in 1.1 nostocs

Utilize the synthetic method of full gene to synthesize aldehyde decarbonylation base enzyme [aldehyde decarboxylase] gene DNA in nostoc (Nostoc punctiforme).The aldehyde decarbonylation base enzyme of this gene induced expression can be sloughed carbonyl by catalysis alkanoic, generates alkane.

This gene gives company synthetic, is pUCE-dc plasmid and the bacterial strain that contains this plasmid after synthesizing.Bacterial strain glycerine pipe is inoculated in LB liquid nutrient medium and is cultivated, and extract plasmid with omega test kit, can be cloned into like this plasmid that contains aldehyde decarbonylation base enzyme gene dc:

This gene order length is 699bp, utilizes 1% agarose gel electrophoresis detection, and resulting target DNA band is 699bp (as shown in Figure 5).

The structure of 1.2 transfer vector plasmids 3

Clone's the dc gene enzyme (Bgl II and Hind III) identical with transfer vector plasmid 2 use carried out to double digestion, 37 ℃ of enzymes are cut 4 hours, carrier and exogenous genetic fragment be the ratio of 1:3-1:10 in molar ratio, utilize 16 ℃ of connections of T4DNA ligase enzyme of NEB to spend the night, connect product transform intestinal bacteria Top10 competent cell with this, coating is with amicillin resistance LB agar plate, after 37 ℃ of incubated overnight, the appropriate single bacterium colony of picking, incubated overnight in LB substratum, and carry out bacterium colony PCR.With test kit, extract plasmid, carry out plasmid enzyme restriction checking, sequencing result correctly obtains positive recombinant vectors.Measure its sequence as shown in sequence 5, the aminoacid sequence of coding as shown in sequence 6.

The collection of illustrative plates (seeing Fig. 6) of constructed transfer vector plasmid 3.

Embodiment 4

1 structure that contains acr/dc gene engineering colibacillus

By the transfer vector plasmid 3 that contains acyl-CoA reductase enzyme acr/ aldehyde decarbonylation base enzyme dc building; by the method for chemical conversion (i.e. 42 ℃ of heat shocks); be transformed in e. coli bl21 (DE3) competent cell; coating is with amicillin resistance LB agar plate; after 37 ℃ of incubated overnight; the positive bacterium colony of picking, has obtained 3 ,-80 ℃ of these bacterial strains of preservation of engineering colon bacillus plasmid that contain the expressed acyl-CoA reductase enzyme/aldehyde dehydrogenase of acr/dc.

Embodiment 5

1 structure that contains fadD/acr/dc gene engineering colibacillus

1.1 making that contain acr/dc competent escherichia coli cell

The e. coli bl21 that contains plasmid 3 (DE3) bacterial strain is inoculated in LB liquid tube substratum according to 1 ‰ inoculum size, and 37 ℃, 160rpm shaken overnight, makes primary seed solution.This primary seed solution is inoculated in 50mL LB liquid nutrient medium and (includes 50 μ L penbritins) according to 1% inoculum size, and 37 ℃, the about 2.5h of 160rpm shaking culture, works as OD ₆₀₀be about at 0.5 o'clock, bacterium liquid is placed in to cooled on ice 30min.In 4 ℃, with 4000rpm, centrifugal 5min, collects thalline, and with 10% glycerine re-suspended cell of precooling, repeat this process 4-5 time, finally use the glycerine re-suspended cell of the 1mL10% of precooling, and in packing on ice, just make the competent escherichia coli cell that contains plasmid 3, preserve this cell for-80 ℃.

1.2 structures that contain fadD/acr/dc gene engineering colibacillus

By the transfer vector plasmid 1 that contains acyl-CoA synthetase gene fadD building; the method transforming by electricity; be transformed in e. coli bl21 (DE3) competent cell that contains plasmid 3; coating is with the dual resistance LB agar plate of penbritin and paraxin; after 37 ℃ of incubated overnight; the positive bacterium colony of picking; 3 ,-80 ℃ of these bacterial strains of preservation of engineering colon bacillus plasmid 1/ plasmid that contain the expressed acyl-CoA synthetase/acyl-CoA reductase enzyme/aldehyde dehydrogenase of fadD/acr/dc have been obtained.

Embodiment 6

The gas chromatography-mass spectrography detection method of 1 alkane

The alkane that methyl tert-butyl ether is extracted carries out gas chromatography-mass spectrography detection, gas chromatography-mass spectrum condition is as follows: Shimadzu GCMS-QP2010, HP-5ms chromatographic column, carrier gas: high-purity helium (99.999%), injector temperature: 280 ℃, carrier gas column cap is pressed: 100.2kPa, splitting ratio 10:1; Temperature programming: initial 60 ℃, maintain 4min; 5 ℃/min is warming up to 160 ℃; 80 ℃/min is warming up to 300 ℃, keeps 3min; Mass spectrum working time: 2.5-25min, scanning fragment: 80-800amu.

Embodiment 7

Exogenous stream adds C14:0 lipid acid and produces alkane

In 1, paraffinic hydrocarbons is synthetic

Engineering colon bacillus plasmid 1/ plasmid 3 is inoculated in 50mL LB liquid medium and (includes the penbritin of 50 μ L and the paraxin of 50 μ L) by 1% inoculum size, and 37 ℃, the about 3h of 160rpm shaking culture, works as OD ₆₀₀be about at 0.6 o'clock, in bacterium liquid, add 0.4mM inductor IPTG, simultaneously exogenous stream adds C14:0 lipid acid, to final concentration be nutrient solution 1 ‰, continuation is shaking culture 48h under 30 ℃ of conditions, and in culturing process, every the exogenous stream of 12h, to add glucose to final concentration be nutrient solution 1%.Nutrient solution under 8000rpm condition, centrifugal 10min, and at work 3s, intermittently carry out cytoclasis under the condition of 3s, work 70 times.Get the cytoclasis liquid that contains alkane and preserve, for subsequent detection work is prepared.

The extraction of paraffinic hydrocarbons in 2

Get 1.5mL cytoclasis liquid, add 400 μ L methyl tert-butyl ethers, the 1min that vibrates on shaker, and turn upside down and shake up, fully extract the centrifugal 2min of 8000rpm, separated and collected organic layer.Can obtain so certain density middle paraffinic hydrocarbons.

In 3, the makings of paraffinic hydrocarbons detects

Alkane to methyl tert-butyl ether extraction carries out gas chromatography-mass spectrography detection, and GC conditions as described in Example 6, the results are shown in Figure 7.From final gas phase collection of illustrative plates, can analyze and draw: the alkane output of the C13/C15 that plasmid 1/ plasmid 3 recombination bacillus colis produce is 2.17mg/L.

Embodiment 8

Exogenous stream adds C16:0 lipid acid and produces alkane

In 1, paraffinic hydrocarbons is synthetic

Engineering colon bacillus plasmid 1/ plasmid 3 is inoculated in 50mL LB liquid medium and (includes the penbritin of 50 μ L and the paraxin of 50 μ L) by 1% inoculum size, 37 ℃, the about 3h of 160rpm shaking culture, when OD600 is about 0.6, in bacterium liquid, add 0.4mM inductor IPTG, exogenous stream adds C16:0 lipid acid simultaneously, to final concentration be nutrient solution 1 ‰, continuation is shaking culture 48h under 30 ℃ of conditions, and in culturing process, every the exogenous stream of 12h, to add glucose to final concentration be nutrient solution 1%.Nutrient solution under 8000rpm condition, centrifugal 10min, and at work 3s, intermittently carry out cytoclasis under the condition of 3s, work 70 times.Get the cytoclasis liquid that contains alkane and preserve, for subsequent detection work is prepared.

The extraction of paraffinic hydrocarbons in 2

Get 1.5mL cytoclasis liquid, add 400 μ L methyl tert-butyl ethers, the 1min that vibrates on shaker, and turn upside down and shake up, fully extract the centrifugal 2min of 8000rpm, separated and collected organic layer.Can obtain so certain density middle paraffinic hydrocarbons.

In 3, the makings of paraffinic hydrocarbons detects

Alkane to methyl tert-butyl ether extraction carries out gas chromatography-mass spectrography detection, and GC conditions as described in Example 6, the results are shown in Figure 8.From final gas phase collection of illustrative plates, can analyze and draw: the alkane output of the C13/C15 that plasmid 1/ plasmid 3 recombination bacillus colis produce is 1.81mg/L.

Claims (8)

1. in, a production method for paraffinic hydrocarbons, is characterized in that, comprising:

Step 1, first the acyl-CoA synthetase gene fadD from e. coli k12 strain is cloned into prokaryotic expression carrier; obtain recombinant plasmid 1; through IPTG, induce; can add by exogenous stream the mode of the medium chain fatty acids such as C14:0/C16:0, catalysis lipid acid generates meta-bolites ester acyl coenzyme A.

Step 2, again the acyl-CoA reductase gene acr in acinetobacter calcoaceticus is cloned into prokaryotic expression carrier, obtains recombinant plasmid 2, through IPTG induction, the ester acyl coenzyme A accumulating in can catalysis cell is reduced to the alkanoic of C14/C16.

Step 3, finally the aldehyde decarbonylation base enzyme gene dc in nostoc is cloned into prokaryotic expression carrier, obtains recombinant plasmid 3, through IPTG induction, can slough carbonyl by catalysis alkanoic, generate alkane.

Step 4, the recombinant plasmid obtaining 1/3 is gone in BL21 (DE3) jointly, after exogenous stream adds the medium chain fatty acids such as C14:0/C16:0 and IPTG abduction delivering, paraffinic hydrocarbons in can producing.

2. method according to claim 1, is characterized in that, the described nucleotide sequence from acyl-CoA synthetase gene fadD in intestinal bacteria is as shown in sequence 1, and coded aminoacid sequence is as shown in sequence 2 separately.

3. method according to claim 1, is characterized in that, fadD gene clone, to double promoter expression vector pACYCDuet-1, is obtained to recombinant plasmid pACYCDuet-fadD, plasmid 1, and wherein acyl-CoA synthetase gene is placed in T ₇promoter1 downstream, by T ₇promoters driven is expressed.

4. method according to claim 1, is characterized in that, the described nucleotide sequence from acyl-CoA reductase gene acr in acinetobacter calcoaceticus is as shown in sequence 3, and coded aminoacid sequence is as shown in sequence 4 separately.

5. method according to claim 1, is characterized in that, described acr gene clone, to double promoter expression vector pETDuet-1, obtains recombinant plasmid pETDuet-acr, plasmid 2, and wherein acyl-CoA reductase gene is placed in T ₇promoter1 downstream, by T ₇promoters driven is expressed.

6. method according to claim 1, is characterized in that, the described nucleotide sequence from aldehyde decarbonylation base enzyme gene dc in nostoc is as shown in sequence 5, and coded aminoacid sequence is as shown in sequence 6 separately.

7. method according to claim 1, is characterized in that, described dc gene clone, to double promoter expression vector pETDuet-acr, obtains recombinant plasmid pETDuet-acr-dc, plasmid 3, and wherein aldehyde decarbonylation base enzyme gene is placed in T ₇promoter1 downstream, by T ₇promoters driven is expressed.

8. method according to claim 1; it is characterized in that; proceeded to the recombination bacillus coli of recombinant plasmid 1/3; after adding 0.4mM IPTG induction acyl-CoA synthetase, acyl-CoA reductase enzyme, aldehyde dehydrogenase expression; can add through exogenous stream the mode of the medium chain fatty acids such as C14:0/C16:0, catalysis fatty acid metabolism generates the middle paraffinic hydrocarbons that the carbon chain lengths such as C13/C15 do not wait.