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

Abstract

The invention belongs to the field of biochemical industry, in particular to a production method of medium-carbon paraffins. Alkanes are mainly derived from oil and natural gas, and can also be produced by the metabolism of some species in nature, but the amount of alkanes produced by these microorganisms is very low. With the rapid development of modern molecular biology, especially synthetic biology, regulating the expression of related enzymes in the process of alkane synthesis, so that microbial cells can produce alkane through simple metabolism, is a cutting-edge technology for the production of renewable energy. This method provides an excellent solution to the problems of insufficient supply of traditional fossil fuels and the inability of new biofuels to completely replace fossil fuels. Compared with other strains, Escherichia coli has the advantages of clear genetic background, easy transformation, fast growth rate, and suitable for high-density fermentation. It is an ideal strain for microbial synthesis of chemicals and fuels. Using Escherichia coli to produce bioalkane is a new way of indisputable, unreliable and sustainable production.

Description

A kind of production method of medium-chain alkanes

technical field

The invention belongs to the field of biochemical industry, in particular to a production method of medium-carbon paraffins. the

Background technique

Alkanes are the main components of fossil energy, such as gasoline, diesel and aviation fuel, which are the separation products of petroleum, and are composed of alkanes with different carbon chain lengths. In recent years, with the depletion of fossil energy and the sharp increase in crude oil consumption, the shortage of fossil energy supply and how to achieve sustainable development have become issues that human beings must pay attention to. Therefore, research on how to use renewable energy to replace fossil fuels has gradually become a new hot topic. the

Currently the most widely used fossil energy substitutes mainly include fuel ethanol, fuel butanol and biodiesel. However, there are many problems in the application of these alternatives, such as alcohol gasoline is easy to form stratification and corrode the engine rubber, the cost of biodiesel raw materials and the energy consumption of the production process are high, making it impossible for them to completely replace gasoline , diesel and other hydrocarbons. Therefore, it is imminent to seek a better renewable alternative to fossil energy, that is, a low-cost, high-efficiency method for alkane production. the

Alkanes are mainly derived from oil and natural gas, and can also be produced through the metabolism of some species in nature, such as cuticle wax of plants, pheromones of insects, etc. Very few. With the rapid development of modern molecular biology, especially synthetic biology, genetic engineering is used to regulate the expression of related enzymes in the process of alkanes synthesis, so that microbial cells can produce alkanes only through simple metabolism, which is the key to the production of renewable energy. cutting-edge technology. This method provides an excellent solution to the problems of insufficient supply of traditional fossil fuels and the inability of new biofuels to completely replace fossil fuels. Compared with other strains, Escherichia coli has the advantages of clear genetic background, easy transformation, fast growth, suitable for high-density fermentation, etc. It is an ideal strain for microbial synthesis of chemicals and fuels. Using Escherichia coli to produce bioalkane is a new way of indisputable, unreliable and sustainable production. the

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

(1) The direct use of alkanes metabolized by microorganisms using renewable biomass as raw materials can greatly alleviate the problem of insufficient supply of fossil energy. the

(2) The alkane obtained by the present invention is no different from the alkane in petroleum, and is closer to the properties of fossil fuel itself, overcomes a series of problems existing in fuel ethanol, fuel butanol, biodiesel, etc., and has a very high utilization rate. the

(3) Microorganisms can produce alkanes only through simple metabolism, the production cycle is short, it is not affected by factors such as site and season, and it does not occupy arable land resources, so it is easy to realize industrial production. the

Contents of the invention

The object of the present invention is to provide a renewable energy process route for synthesizing alkanes in recombinant Escherichia coli (Escherichia coli) with a large amount of renewable fatty acids in nature as raw materials, so that microorganisms can be used as cell factories to ferment and produce alkanes. the

The present invention firstly overexpresses the acyl-CoA synthetase gene from Escherichia coli K12 strain to construct a genetically engineered bacterium, that is, the gene fadD is cloned into the prokaryotic expression vector pACYCDuet-1 to obtain the recombinant plasmid pACYCDuet-fadD, plasmid 1. The expressed acyl-CoA synthetase can catalyze fatty acid to produce metabolite ester acyl-CoA by adding medium-chain fatty acid such as C14:0/C16:0 exogenously, and make this precursor substance obtain in host cells accumulation. the

Then heterologous expression of acyl-CoA reductase from Acinetobacter baylyi ADP1 (Acinetobacter baylyi). Specifically, the acyl-CoA reductase gene acr in Acinetobacter was cloned into the prokaryotic expression vector pETDuet-1 to obtain the recombinant plasmid pETDuet-acr, plasmid 2. The expressed acyl-CoA reductase can catalyze the reduction of acyl-CoA accumulated in cells to C14/C16 fatty aldehydes. the

Finally, heterologous expression of aldehyde decarbonylase from Nostoc punctiforme. That is, the aldehyde decarbonylase gene dc was cloned into the prokaryotic expression vector plasmid 2 to obtain recombinant plasmids pETDuet-acr-dc and plasmid 3 . The expressed aldehyde decarbonylase can catalyze the decarbonylation of aliphatic aldehydes to generate alkanes. the

The recombinant plasmid 1/plasmid 3 is co-transformed into E. coli BL21(DE3), and IPTG induces the co-expression of the recombinant plasmids, and the fatty acids can be gradually metabolized by adding C14:0/C16:0 fatty acids exogenously. For alkanes with different carbon chain lengths such as C13/C15, the alkane yield obtained by adding C14:0 fatty acid exogenously is 2.17mg/L, and the alkane yield obtained by adding C16:0 fatty acid exogenously is 1.81mg/L , so as to obtain the renewable resource process route of directly synthesizing alkanes by recombinant Escherichia coli. the

The method provided by the present invention expresses in Escherichia coli the key enzyme gene that specifically regulates the synthesis of acyl-CoA, the reduction of acyl-CoA and the decarbonylation reaction of aldehydes, and can use the alkanes obtained from the metabolism of natural fatty acids by microorganisms and carry out high-density fermentation to obtain The synthesis of renewable alkanes opens a new route. the

Description of drawings

Figure 1: DNA Agarose Gel Electrophoresis. The right side is the purified Escherichia coli acyl-CoA synthetase gene PCR product, 1686bp. On the left is the DNA size standard. the

Figure 2: Map of the constructed recombinant vector pACYCDuet-fadD. The fadD gene is cloned into the pACYCDuet-1 vector with NCOⅠ and EcoRI sites at both ends of the gene. It was numbered as plasmid 1. the

Figure 3: DNA agarose gel electrophoresis. The right side is the purified Acinetobacter acyl-CoA reductase gene PCR product, 888bp. On the left is the DNA size standard. the

Figure 4: Map of the constructed recombinant vector pETDuet-acr. The acr gene is cloned in the pETDuet-1 vector, with NCOⅠ and BamHI sites at both ends of the gene. It was numbered plasmid 2. the

Figure 5: DNA agarose gel electrophoresis. The right side is the purified aldehyde decarbonylase gene PCR product in Nostoc algae, 699bp. On the left is the DNA size standard. the

Figure 6: Map of the constructed recombinant vector pETDuet-acr-dc. The dc gene is cloned into the pETDuet-acr vector, with BglⅡ and HindⅢ sites at both ends of the gene. It was numbered plasmid 3. the

Figure 7: The production of alkanes in the culture medium of engineered Escherichia coli containing plasmid 1/plasmid 3 induced by exogenously fed C14:0 fatty acid; C13 means tridecane; C15 means pentadecane. the

Figure 8: The production of alkane in the culture medium of engineered Escherichia coli containing plasmid 1/plasmid 3 induced by exogenously fed C16:0 fatty acid; C13 means tridecane; C15 means pentadecane. the

Detailed ways

The invention provides a renewable energy process route for directly synthesizing alkanes by using recombinant Escherichia coli, so that natural fatty acids can be effectively utilized. The present invention solves this technical problem through the following scheme: first, using Escherichia coli genomic DNA as a template, designing corresponding primers and adding corresponding cloning enzyme cutting sites to amplify the fadD gene, the nucleic acid sequence of which is shown in sequence 1, each encoded The amino acid sequence of is shown in sequence 2. After PCR amplification, the DNA fragment of the target gene was recovered and ligated to the corresponding restriction site of the prokaryotic expression vector pACYCDuet-1 after enzyme digestion, and the ligation product was transformed into E. coli competent cells, and the positive recombinant plasmid pACYCDuet-fadD was obtained by screening (plasmid 1), its plasmid map is shown in Figure 2. The plasmid was purified, detected by electrophoresis, and sequenced to identify the target gene. the

Then use the whole gene synthesis method to synthesize the acr gene respectively, the nucleic acid sequence of which is shown in sequence 3, and the amino acid sequence encoded by each is shown in sequence 4, and the corresponding cloning enzyme cutting site is added, and the restriction enzyme is connected to the prokaryotic expression vector On the corresponding restriction site of pETDuet-1, the ligation product was transformed into E. coli competent cells, and the positive recombinant plasmid pETDuet-acr (plasmid 2) was obtained by screening, its plasmid map is shown in Figure 4, and the same method was used to Identify the gene of interest. the

Finally, the dc genes were synthesized using the method of whole gene synthesis, the nucleic acid sequence of which is shown in sequence 5, and the amino acid sequences encoded by each are shown in sequence 6, and the corresponding cloning restriction sites were added, and then ligated into prokaryotic expression On the corresponding restriction site of vector pETDuet-acr, the ligation product was transformed into Escherichia coli competent cells, and the positive recombinant plasmid pETDuet-acr-dc (plasmid 3) was obtained by screening, its plasmid map is shown in Figure 4, and The same method was used to identify the target gene. the

After co-inducing expression of recombinant plasmid 1/3 in Escherichia coli BL21(DE3) and adding exogenous fatty acid, the culture continued, and alkanes with different carbon chain lengths such as C13/C15 could be obtained in the recombinant E. coli cells. the

Experimental steps

1) The strains used

Escherichia coli BL21(DE3)

2) The medium used

LB medium (peptone 10g/L, yeast extract 5g/L, NaCl 5g/L, 121°C damp heat sterilization for 20min), if plate medium is prepared, 1.5% agar powder should be added; selective LB medium: 50ml Add about 50 μL of 0.1% ampicillin solution and chloramphenicol solution to LB medium. the

3) Experimental method:

Respectively, the genome of Escherichia coli (Escherichia coli) K12 strain extracted by using the Bomide kit method, and an acyl-CoA reductase [acyl- CoA reductase] gene DNA and an aldehyde decarbonylase [aldehyde decarboxylase] gene DNA from Nostoc punctiforme were used as templates, and acyl-CoA synthetase [acyl- CoA synthetase] gene, acyl-CoA reductase gene and aldehyde decarbonylase gene. the

Use the gel recovery kit to recover the target fragment, digest the acyl-CoA synthetase gene fadD fragment and the prokaryotic expression vector pACYCDuet-1 with the corresponding restriction enzymes, and connect them overnight at 16°C, which is the constructed prokaryotic expression Carrier plasmid 1; then use the same method to connect the acr fragment of the acyl-CoA reductase gene to the prokaryotic expression vector pETDuet-1, which is the constructed prokaryotic expression vector plasmid 2; finally use the same method to connect the aldehyde decarbonylase gene dc The fragment is connected with plasmid 2, which is the constructed prokaryotic expression vector plasmid 3. the

The constructed recombinant plasmid 1/plasmid 3 is co-transformed into E. coli BL21(DE3) by electroporation to obtain engineering E. coli producing acyl-CoA synthetase, acyl-CoA reductase and aldehyde decarbonylase . Inoculate engineering Escherichia coli into 50mL LB liquid medium at an inoculum amount of 1%, rapidly shake culture at 37°C, add inducer IPTG and exogenous feeding fatty acid to the culture medium and continue to culture at 30°C for 48h to induce expression Target protein; extract the alkanes in the fermentation broth, measure the yield of alkanes such as C13/C15, that is, the prepared alkanes with different carbon chain lengths. the

Concrete method of the present invention is described further below in conjunction with embodiment:

Example 1

1 Cloning of acyl-CoA synthetase in Escherichia coli and construction of recombinant vector

1.1 Cloning of acyl-CoA synthetase in Escherichia coli

The genome of Escherichia coli (Escherichia coli) K12 strain was extracted by using the Bomide kit method, using the Escherichia coli genomic DNA as a template, primers were designed according to the GenBank sequence, and corresponding enzyme cutting sites (NCOI and EcoRI) and protective bases were added, The acyl-CoA synthetase [acyl-CoA synthetase] gene encoded by fadD was amplified by PCR, and the acyl-CoA synthetase induced by this gene can metabolize fatty acids to generate ester acyl-CoA. the

The primers used for PCR amplification of acyl-CoA synthetase gene fadD are as follows:

Upstream primer: 5'-CATG CCATGG GCAAGAAGGTTTGGCTTAACC-3'

Downstream primer: 5'-CG GAATTC CGGCTCAGGCTTTATTGTCCACT-3'

The PCR reaction system contained 0.2 μL of genomic DNA, 0.25 μL of upstream and downstream primers, 1.6 μL of dNTPs mixture, 2 μL of 10×Buffer, 0.4 μL of pyrobest polymerase, and added double distilled water to make up to 20 μL. The reaction conditions were pre-denaturation at 94°C for 4min, denaturation at 94°C for 1min, annealing at 55°C for 1min, extension at 72°C for 1min and 50s, 30 cycles, 72°C for 10min, and storage at 4°C. the

In this way, the target acyl-CoA synthetase gene fadD can be PCR amplified and cloned:

The length of the gene sequence is 1686bp. After the PCR amplification, the target gene is recovered using a PCR recovery kit and detected by 1% agarose gel electrophoresis. The resulting target DNA band is 1686bp (as shown in Figure 1). the

1.2 Construction of recombinant vector plasmid 1

The cloned fadD gene and the pACYCDuet-1 vector (purchased from Novagen) were double digested with the same enzymes (NCOI and EcoRI), digested at 37°C for 4 hours, and the molar ratio of the vector to the foreign gene fragment was 1:3- Use NEB T4 DNA ligase at a ratio of 1:10 to connect overnight at 16°C, use the ligation product to transform Escherichia coli Top10 competent cells, spread chloramphenicol-resistant LB agar plates, culture overnight at 37°C, and pick An appropriate amount of single colony was cultured overnight in LB medium, and colony PCR was performed. Use the kit to extract the plasmid, carry out plasmid digestion verification, and the positive recombinant vector will be obtained if the sequencing result is correct. Its sequence is determined as shown in sequence 1, and the encoding amino acid sequence is shown in sequence 2. Since the start codon of this gene is a rare start codon TTG, it was changed to a common start codon ATG when PCR amplifying the gene, hereby explain. the

Map of the constructed recombinant vector plasmid 1 (see Figure 2). the

Example 2

1 Cloning of acyl-CoA reductase in Acinetobacter and construction of recombinant vector

1.1 Cloning of acyl-CoA reductase in Acinetobacter

The acyl-CoA reductase [acyl-CoA reductase] gene DNA in Acinetobacter baylyi was synthesized by the method of total gene synthesis. The gene-induced expression of acyl-CoA reductase can catalyze the reduction reaction of ester acyl-CoA accumulated in cells to generate C14/C16 fatty aldehydes. the

Using the acyl-CoA reductase gene DNA synthesized above as a template, primers were designed according to the GenBank sequence, and corresponding restriction sites (NCOⅠ and BamHI) and protective bases were added. the

The primers used for PCR amplification of acyl-CoA reductase gene acr are as follows:

Upstream primer: 5'-GCAGCCCATGGCTAGCATGAACGCTAAAC-3'

Downstream primer: 5'-CCGCTGGATCCTTACCAGTGTTCACCTGG-3'

The PCR reaction system contained 0.2 μL of genomic DNA, 0.25 μL of upstream and downstream primers, 1.6 μL of dNTPs mixture, 2 μL of 10×Buffer, 0.4 μL of pyrobest polymerase, and added double distilled water to make up to 20 μL. The reaction conditions were pre-denaturation at 94°C for 4min, denaturation at 94°C for 1min, annealing at 55°C for 1min, extension at 72°C for 1min, 30 cycles, 72°C for 10min, and storage at 4°C. the

In this way, PCR amplification can be cloned into the target acyl-CoA reductase gene acr:

The length of the gene sequence is 888bp. After the PCR amplification, the target gene is recovered with a PCR recovery kit and detected by 1% agarose gel electrophoresis. The obtained target DNA band is 888bp (as shown in Figure 3). the

1.2 Construction of recombinant vector plasmid 2

The cloned acr gene and the pETDuet-1 vector (purchased from Novagen) were double digested with the same enzymes (NCO Ⅰ and BamH Ⅰ), digested at 37°C for 4 hours, and the molar ratio of the vector and the foreign gene fragment was 1: 3-1:10 ratio, use NEB T4 DNA ligase to connect overnight at 16°C, use the ligation product to transform Escherichia coli Top10 competent cells, coat LB agar plates with ampicillin resistance, culture overnight at 37°C, pick Take an appropriate amount of single colony, culture overnight in LB medium, and perform colony PCR. Use the kit to extract the plasmid, carry out plasmid digestion verification, and the positive recombinant vector will be obtained if the sequencing result is correct. Its sequence is determined as shown in sequence 3, and the coding amino acid sequence is shown in sequence 4. Since the start codon of this gene is a rare start codon GTG, it was changed to a common start codon ATG when synthesizing the gene, hereby explain. the

Map of the constructed recombinant vector plasmid 2 (see Figure 4). the

Example 3

1 Cloning of aldehyde decarbonylase from Nostoc algae and construction of recombinant vector

1.1 Cloning of aldehyde decarbonylase from Nostoc algae

The aldehyde decarbonylase [aldehyde decarboxylase] gene DNA in Nostoc punctiforme was synthesized by the method of total gene synthesis. The aldehyde decarbonylase induced by this gene can catalyze the decarbonylation of aliphatic aldehydes to generate alkanes. the

The gene is handed over to the company for synthesis, and after synthesis, it becomes the pUCE-dc plasmid and the strain containing the plasmid. Inoculate the glycerol tube of the strain into LB liquid medium for cultivation, and extract the plasmid with an omega kit, so that the plasmid containing the aldehyde decarbonylase gene dc can be cloned:

The length of the gene sequence is 699bp, detected by 1% agarose gel electrophoresis, and the obtained target DNA band is 699bp (as shown in FIG. 5 ). the

1.2 Construction of recombinant vector plasmid 3

The cloned dc gene and the recombinant vector plasmid 2 were double digested with the same enzymes (BglⅡ and HindⅢ), digested at 37°C for 4 hours, and the molar ratio of the vector and the foreign gene fragment was 1:3-1:10, Use NEB’s T4 DNA ligase to connect overnight at 16°C, transform Escherichia coli Top10 competent cells with the ligation product, spread the LB agar plate with ampicillin resistance, culture overnight at 37°C, pick an appropriate amount of single colony, and culture in LB culture overnight and perform colony PCR. Use the kit to extract the plasmid, carry out plasmid digestion verification, and the positive recombinant vector will be obtained if the sequencing result is correct. Its sequence is determined as shown in sequence 5, and the encoding amino acid sequence is shown in sequence 6. the

Map of the constructed recombinant vector plasmid 3 (see Figure 6). the

Example 4

1 Construction of Escherichia coli containing acr/dc genetic engineering

The constructed recombinant vector plasmid 3 containing acyl-CoA reductase acr/aldehyde decarbonylase dc was transformed into Escherichia coli BL21 (DE3) competent cells by chemical transformation (ie heat shock at 42°C), Clothed with ampicillin-resistant LB agar plate, cultured overnight at 37°C, picked positive colonies, and obtained the engineering Escherichia coli plasmid 3 containing the acyl-CoA reductase/aldehyde dehydrogenase expressed by acr/dc,- Store the strain at 80°C. the

Example 5

1 Construction of Escherichia coli containing fadD/acr/dc genetic engineering

1.1 Production of Escherichia coli Competent Cells Containing acr/dc

The Escherichia coli BL21(DE3) strain containing plasmid 3 was inoculated into the LB liquid test tube culture medium according to the inoculum amount of 1‰, and shaken overnight at 37°C and 160rpm to prepare the primary seed solution. Inoculate this primary seed liquid into 50 mL of LB liquid medium (containing 50 μL of ampicillin) according to the inoculum amount of 1%, culture at 37°C with shaking at 160 rpm for about 2.5 hours, and when the OD ₆₀₀ is about 0.5, put the bacterial liquid into Cool on ice for 30 min. Centrifuge at 4000rpm for 5min at 4°C to collect the bacteria, and resuspend the cells with pre-cooled 10% glycerol, repeat this process 4-5 times, and finally resuspend the cells with 1 mL of pre-cooled 10% glycerol Aliquot on top to prepare Escherichia coli competent cells containing plasmid 3, and store the cells at -80°C.

1.2 Construction of genetically engineered Escherichia coli containing fadD/acr/dc

The constructed recombinant vector plasmid 1 containing the acyl-CoA synthetase gene fadD was transformed into Escherichia coli BL21 (DE3) competent cells containing plasmid 3 by electroporation, and coated with ampicillin and chloramphenicol After culturing overnight at 37°C on a double-resistant LB agar plate, the positive colonies were picked, and the project containing the acyl-CoA synthetase/acyl-CoA reductase/aldehyde dehydrogenase expressed by fadD/acr/dc was obtained Escherichia coli plasmid 1/plasmid 3, store the strain at -80°C. the

Example 6

1 Gas chromatography-mass spectrometry detection method for alkanes

The alkanes extracted by methyl tert-butyl ether are detected by gas chromatography-mass spectrometry, and the gas chromatography-mass spectrometry conditions are as follows: Shimadzu GCMS-QP2010, HP-5ms chromatographic column, carrier gas: high-purity helium (99.999%), Injection port temperature: 280°C, carrier gas column head pressure: 100.2kPa, split ratio 10:1; temperature program: initial 60°C, maintain 4min; 5°C/min to 160°C; 80°C/min to 300°C , hold for 3min; mass spectrometry running time: 2.5-25min, scan fragment: 80-800amu. the

Example 7

Production of Alkanes by Adding C14:0 Fatty Acids Exogenously

1 Synthesis of medium paraffins

Inoculate engineering Escherichia coli plasmid 1/plasmid 3 into 50 mL of LB liquid culture medium (containing 50 μL of ampicillin and 50 μL of chloramphenicol) at a 1% inoculum size, culture at 37°C with shaking at 160 rpm for about 3 hours, when OD ₆₀₀ At about 0.6, add 0.4mM inducer IPTG to the bacterial solution, and at the same time add C14:0 fatty acid exogenously, until the final concentration is 1‰ of the culture solution, and continue shaking culture at 30°C for 48h. Glucose was added exogenously every 12 hours to a final concentration of 1% of the culture solution. The culture solution was centrifuged at 8000rpm for 10min, and the cells were broken under the conditions of working for 3s, resting for 3s, and working 70 times. Take the cell disruption solution containing alkanes and save it for subsequent detection.

2 Extraction of medium paraffins

Take 1.5mL of cell disruption solution, add 400μL of methyl tert-butyl ether, shake on the shaker for 1min, and shake up and down to fully extract, centrifuge at 8000rpm for 2min, separate and collect the organic layer. In this way, a certain concentration of medium-chain alkanes can be obtained. the

3 Gas detection of medium paraffins

The alkanes extracted with methyl tert-butyl ether were detected by gas chromatography-mass spectrometry. The gas chromatography conditions are as shown in Example 6, and the results are shown in FIG. 7 . From the analysis of the final gas chromatogram, it can be concluded that the alkane yield of C13/C15 produced by plasmid 1/plasmid 3 recombinant Escherichia coli is 2.17mg/L. the

Example 8

Production of Alkanes by Adding C16:0 Fatty Acids Exogenously

1 Synthesis of medium paraffins

Inoculate engineering Escherichia coli plasmid 1/plasmid 3 into 50 mL of LB liquid culture medium (containing 50 μL of ampicillin and 50 μL of chloramphenicol) at a 1% inoculum size, culture at 37°C for about 3 hours with shaking at 160 rpm, and when the OD600 is about When the concentration is 0.6, add 0.4mM inducer IPTG to the bacterial solution, and at the same time add C16:0 fatty acid exogenously, to a final concentration of 1‰ of the culture solution, and continue shaking culture at 30°C for 48h. Glucose was added exogenously every 12 hours to a final concentration of 1% of the culture solution. The culture solution was centrifuged at 8000rpm for 10min, and the cells were broken under the conditions of working for 3s, resting for 3s, and working 70 times. Take the cell disruption solution containing alkanes and save it for subsequent detection. the

2 Extraction of medium paraffins

Take 1.5mL of cell disruption solution, add 400μL of methyl tert-butyl ether, shake on the shaker for 1min, and shake up and down to fully extract, centrifuge at 8000rpm for 2min, separate and collect the organic layer. In this way, a certain concentration of medium-chain alkanes can be obtained. the

3 Gas detection of medium paraffins

The alkanes extracted with methyl tert-butyl ether were detected by gas chromatography-mass spectrometry. The gas chromatography conditions are as shown in Example 6, and the results are shown in FIG. 8 . From the analysis of the final gas chromatogram, it can be concluded that the alkane yield of C13/C15 produced by plasmid 1/plasmid 3 recombinant Escherichia coli is 1.81 mg/L. the

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.