CN105802983A - High-flux screening method of aliphatic hydrocarbon generation gene, obtained mutant and application - Google Patents

Abstract

The invention discloses a high-flux screening method of an aliphatic hydrocarbon generation gene, an obtained mutant and application, and belongs to the technical field of biological engineering.By means of the method, a carrier carrying a potential aliphatic hydrocarbon generation gene is introduced into a host cell carrying a detection element, the host cell is cultured, the expression situation of the target gene is authenticated by responding to a signal of the detection element, and the mutant with high aliphatic hydrocarbon yield is separated out.Meanwhile, the invention further provides the aldehyde deformylating oxygenase mutant obtained through the screening method.The aldehyde deformylating oxygenase mutant has higher aliphatic hydrocarbon synthesis efficiency compared with an enzyme wild type.By means of the method, the specific gene or mutant with higher aliphatic hydrocarbon yield can be easily and rapidly screened from candidate genes with potential aliphatic hydrocarbon generation capacity and a mutant library of the candidate genes.

Description

A high-throughput screening method for aliphatic hydrocarbon-producing genes and the obtained mutants and applications

technical field

The invention relates to a high-throughput screening method for aliphatic hydrocarbon-producing genes, the obtained mutant and its application, and belongs to the technical field of bioengineering.

Background technique

The development of biofuels is one of the effective ways to solve the problems of resources, energy and environment faced by human beings. Since aliphatic hydrocarbons are the main components of gasoline, diesel, aviation kerosene and other engine fuels, they have high energy density, low hygroscopicity and low volatility, and are compatible with existing transportation facilities and engine systems, bio-aliphatic hydrocarbons have been considered as a The most promising high-quality biofuel.

In recent years, it has been found in some organisms that some specific enzymes can convert intracellular aliphatic compounds (such as fatty acids and fatty aldehydes) into aliphatic hydrocarbons. For example, in cyanobacteria, there is mainly a two-step pathway for the synthesis of aliphatic hydrocarbons, that is, fatty acyl-ACP generates fatty aldehyde under the action of fatty-acyl-ACP reductase (AAR), and fatty aldehyde is then processed by fatty aldehyde deformyloxygenase ( ADO) catalyzed reformylation to aliphatic hydrocarbons and formic acid. In addition, there is a cytochrome P450OleT in Jeotgalicoccus bacteria, which can directly decarboxylate fatty acids to olefins with terminal unsaturated double bonds. At present, the above-mentioned pathways have been recombinantly expressed in Escherichia coli or cyanobacteria, and the synthesis of aliphatic hydrocarbons such as C15, C17, and C19 has been successfully detected, showing a good development prospect.

At present, some microorganisms with natural aliphatic hydrocarbon biosynthesis pathways have low efficiency in synthesizing aliphatic hydrocarbons and do not have the potential for industrial application. One key reason is the extremely low catalytic efficiency of catalysts. Therefore, improving the catalytic activity of key enzymes in the aliphatic hydrocarbon synthesis pathway through bioengineering technology is one of the effective ways to promote the biosynthesis of aliphatic hydrocarbons. For example, by randomly or semi-randomly introducing single or several amino acid insertions, deletions or substitutions into the primary structure of the enzyme, and through corresponding high-throughput screening methods, it is a method to change the catalytic properties of the enzyme and realize the improvement of the enzyme. effective means. Due to the large number of potential modification schemes, how to quickly isolate enzymes or mutants with strong hydrocarbon production ability through high-throughput screening methods is an urgent problem to be solved. However, limited by the physical and chemical properties of aliphatic hydrocarbons, how to quickly evaluate the ability of microorganisms to synthesize aliphatic hydrocarbons is a difficult point in research work. The traditional technical process is to first extract and concentrate the aliphatic hydrocarbons in the cells through organic solvents, and then analyze the content of aliphatic hydrocarbons in them by gas chromatography-mass spectrometry. The analysis process has low throughput and takes a long time. Higher cost (SchirmerA, et al, Science, 2010.329:559-562). Limited by the analysis method, there is no precedent for the implementation of a high-throughput screening method for aliphatic hydrocarbon-producing genes.

Contents of the invention

In order to solve the above problems, the present invention provides a high-throughput screening method for producing aliphatic hydrocarbon genes and the obtained mutants and applications. The technical scheme adopted is as follows:

The object of the present invention is to provide a high-throughput screening method for aliphatic hydrocarbon-producing genes. The method is to introduce a vector carrying a potential aliphatic hydrocarbon-producing gene into a host cell carrying a detection element, cultivate the host cell, and pass the detection element Signal Response Identify the expression profile of the gene of interest and isolate mutants that produce high aliphatic hydrocarbons.

The steps of the screening method are as follows:

1) amplify the aliphatic hydrocarbon gene, and introduce random mutation through error-prone PCR reaction to obtain the amplified product;

2) connecting the amplified product obtained in step 1) to a plasmid vector containing a resistance gene after digestion to obtain a recombinant plasmid;

3) constructing a host cell carrying the detection element, and transforming the recombinant plasmid obtained in step 2) into the host cell to obtain a transformant;

4) Cultivate the transformant obtained in step 3), detect and identify the expression of the target gene according to the signal response to the detection element and isolate the mutant with high aliphatic hydrocarbon production.

Preferably, the aliphatic hydrocarbon-producing gene in step 1) is the fatty aldehyde deformyloxygenase gene ADO.

Preferably, the detection element in step 3) includes an aliphatic hydrocarbon detection element and a reporting element.

More preferably, the aliphatic hydrocarbon detection element at least comprises a constitutive promoter, a transcriptional activator and a hydrocarbon-responsive promoter; the reporter element is green fluorescent protein, LacZ gene, luciferase or resistance one of the genes.

Preferably, the constitutive promoter is the promoter PalkS, whose nucleotide sequence is shown in SEQ ID NO.6; the transcription activator is a transcription activator AlkR, whose amino acid sequence is shown in SEQ ID NO.7; the hydrocarbon The responsive characteristic promoter is the promoter PalkM, and its nucleotide sequence is shown in SEQ ID NO.8.

Preferably, the host cell in step 3) is Escherichia coli, yeast or cyanobacteria.

Preferably, the signal of the detection element in step 4) refers to one or more of fluorescence intensity, light absorption value, chemiluminescence intensity and colony size.

Application of any of the screening methods in high-yield aliphatic hydrocarbon genetically engineered bacteria.

Another object of the present invention is to provide a mutant obtained by the screening method, the mutant has at least one of the following changes in the amino acid sequence of the fatty aldehyde deformyloxygenase shown in SEQ ID NO.1:

1) The 194th glutamic acid is mutated to leucine (E194K), and its amino acid sequence is shown in SEQ ID NO.2;

2) The 9th glutamic acid is mutated to glycine, and the 27th isoleucine is mutated to asparagine (I9G&I27N), the amino acid sequence of which is shown in SEQ ID NO.3;

3) The 204th glutamic acid is mutated to phenylalanine (I204F), the amino acid sequence of which is shown in SEQ ID NO.4.

The application of the mutants in the biosynthesis of aliphatic hydrocarbons is also within the protection scope of the present invention.

The beneficial effect that the present invention obtains is:

The fatty aldehyde deformyloxygenase mutant obtained by the screening method provided by the invention has higher aliphatic hydrocarbon synthesis efficiency than the enzyme wild type; the high-throughput screening method of high-yield aliphatic hydrocarbon gene provided by the present invention is helpful To quickly screen out specific genes or mutants with higher hydrocarbon production efficiency from candidate genes with potential hydrocarbon production capacity and their mutant library; specifically:

1) There is no public report on the mutation site of the fatty aldehyde deformyloxygenase mutant gene of the present invention before the present invention.

2) The high-throughput screening method of the present invention utilizes the aliphatic hydrocarbon detection element to directly detect the synthesis of aliphatic hydrocarbons in cells, which can be used for the first time according to the signal intensity of the reporter protein (such as fluorescence, chemiluminescence, enzyme activity, etc.) Specific genes and their mutants with hydrocarbon-producing ability and stronger hydrocarbon-producing ability can be quickly screened out of a large gene library. There has been no other similar high-throughput screening method for aliphatic hydrocarbon-producing genes.

Description of drawings

Fig. 1 is a schematic diagram of plasmid 1 carrying AAR-ADO hydrocarbon-producing gene.

Figure 2 GC-MS detection results of aliphatic hydrocarbons in Escherichia coli.

Among them, Figure A is the control sample, Figure B is the aliphatic hydrocarbon-producing Escherichia coli sample, the internal standard is n-eicosane, and the main aliphatic hydrocarbon products are n-pentadecane and heptadecene.

Figure 3 is a schematic diagram of plasmid 2 carrying an aliphatic hydrocarbon detection element.

Fig. 4 bacterium fluid fluorescence intensity changes with inducer concentration and culture time and the relation of aliphatic hydrocarbon content and fluorescence intensity;

Among them, Figure A is the change of the fluorescence intensity of the bacterial solution with the concentration of the inducer and the culture time; B is the relationship between the aliphatic hydrocarbon content and the fluorescence intensity.

Fig. 5 is a schematic diagram of the principle of the high-throughput screening method for high-yielding aliphatic hydrocarbon genes provided by the present invention.

Fig. 6 is a schematic diagram of flow sorting results provided by the embodiment of the present invention;

(In the figure, a is the result of flow cytometry analysis of uninduced cells; b is the result of flow cytometry of cells induced by 0.05mMIPTG).

The aliphatic hydrocarbon synthesis amount of the ADO dominant mutant obtained by directed evolution screening and the improvement level relative to the wild type in Fig. 7;

(wherein, wt: wild-type fatty aldehyde deformyloxygenase as a mutation template; 3-3: ADO mutant I (E194K); 5-3: ADO mutant II (E9G&I27N); 5-7: ADO mutation Body III (I204F); 3-3/5-3: ADO mutant IV (E194K & E9G & I27N).

detailed description

Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention, rather than limiting the scope of the invention. Various objects and advantages of this invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of a preferred method.

Embodiments of the present invention will be described in detail below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be considered as limiting the scope of the present invention.

Unless otherwise indicated, otherwise the molecular biology test method used in the present invention, basically with reference to people such as J.Sambroo, molecular cloning: laboratory handbook, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and F.M.Ausubel etc. People, Molecular Biology Laboratory Manual, 3rd Edition, John Wiley & Sons, Inc., 1995, performed according to the method described in or according to the product instructions. The reagents used were not indicated by the manufacturer, but were commercially available conventional products. Those skilled in the art understand that the examples describe the present invention by way of example and are not intended to limit the scope of the claimed invention.

Sequence listing information:

SEQ ID NO.1: Amino acid sequence of fatty aldehyde deformyloxygenase ADO.

SEQ ID NO.2: Amino acid sequence of ADO mutant II: E194K.

SEQ ID NO.3: Amino acid sequence of ADO mutant I: E9G & I27N.

SEQ ID NO.4: Amino acid sequence of ADO mutant III: I204F.

SEQ ID NO.5: Amino acid sequence of ADO mutant IV: E9G&I27N&E194K

SEQ ID NO.6: The deoxyribonucleic acid sequence of the constitutive promoter PalkS.

SEQ ID NO.7: Amino acid sequence encoding transcription regulator AlkR.

SEQ ID NO.8: The deoxyribonucleic acid sequence of the hydrocarbon-responsive promoter PalkM.

SEQ ID NO.9: Nucleotide sequence of primer ADO-For.

SEQ ID NO.10: Nucleotide sequence of primer ADO-Rev.

Example 1 Construction of Escherichia coli aliphatic hydrocarbon synthesis pathway and determination of aliphatic hydrocarbon content

Strains:

Escherichia coli BL21(DE3)ΔfadE

Carrier:

Plasmid 1 carrying fatty aldehyde deformyloxygenase ADO and fatty acyl-ACP reductase AAR: pACYCduet-ADO-AAR (Figure 1)

Medium:

LB medium (containing tryptone 10g/L, yeast extract powder 5g/L, NaCl10g/L, if it is a solid medium, add agar 15g/L per liter of medium)

Improved M9 medium (containing 6g/LNa ₂ HPO ₄ , 3g/LKH ₂ PO ₄ , 0.5g/LNaCl, 2g/LNH ₄ Cl, 0.25g/LMgSO ₄ 7H ₂ O, 11mg/LCaCl ₂ , 27mg/LFeCl ₃ 6H ₂ O, 2mg/LZnCl ₂ 4H ₂ O, 2mg/LNa ₂ MoO ₄ 2H ₂ O, 1.9mg/LCuSO ₄ 5H ₂ O, 0.5mg/LH ₃ BO ₃ , 1mg/L Vitamin B1, 200mMBis -Tris (pH7.25) and 0.1% (v/v) Triton-X100)

Implementation steps: transform plasmid 1 into Escherichia coli BL21(DE3)ΔfadE, culture the obtained transformant in resistant LB medium (containing 34 μg/mL chloramphenicol) overnight, and then transfer to the corresponding resistant modified M9 culture After culturing to the logarithmic phase (OD600≈0.6-0.8), add 1 mM IPTG to induce the expression of hydrocarbon-producing genes, continue culturing for about 20-40 hours, take 3-100 mL of bacterial liquid, and add n-eicosane standard substance to it A certain concentration (1 ~ 50mg/L), and add the same volume of chloroform:methanol mixture as the bacterial solution (chloroform and methanol are mixed at a volume ratio of 2:1), fully shake, and centrifuge (refer to the centrifugation condition 4°C, centrifugal force 8000×g , 15 min) and then remove the lower organic phase and blow dry, add 200-500 μL chromatographically pure n-hexane to dissolve the residual oil phase, centrifuge, transfer the supernatant to a chromatographic sampling bottle and use GC-MS to detect the aliphatic hydrocarbon content in the sample. Reference chromatographic detection conditions: Gas chromatography-mass spectrometry (GC-MS) detection uses Agilent7890A-5975C HP-INNOWax (30m×250μm×0.25μm) chromatographic column, using helium as the carrier gas, and the steady flow rate is 1mL/min . The inlet temperature was 250°C, the air flow ratio was 20:1, and the heating program was: keep the temperature at 100°C for 1 min, then raise the temperature to 150°C at 5°C/min, then raise the temperature to 250°C at 10°C/min, and keep the temperature for 15 min.

The results are shown in Figure 2, the n-eicosane peak eluting time as internal standard is about 28min, by comparing with negative control (E. The peaks of n-pentadecane and heptadecene erupt at about 17min and 22min respectively, and the content of synthetic aliphatic hydrocarbons can be calculated according to the respective peak areas and molecular weights of the two aliphatic hydrocarbons and the internal standard. However, because the detection process takes a long time and the extraction process of aliphatic hydrocarbons is complicated, only organic solvent extraction and GC-MS methods cannot achieve rapid analysis and high-throughput screening of aliphatic hydrocarbon-producing samples.

Example 2 Using aliphatic hydrocarbon components to detect the synthesis of aliphatic hydrocarbons in cells

Strains:

Escherichia coli BL21(DE3)ΔfadE; Escherichia coli DH5α

Carrier:

Plasmid 1 carrying fatty aldehyde deformyloxygenase ADO and fatty acyl-ACP reductase AAR: pACYCduet-ADO-AAR; plasmid 2 carrying aliphatic hydrocarbon detection element AlkR-PalkM::GFP (Figure 3): pCom8-AlkR- PalkM::GFP

Medium:

LB medium (containing tryptone 10g/L, yeast extract powder 5g/L, NaCl10g/L, if it is a solid medium, add agar 15g/L per liter of medium)

Improved M9 medium (containing 6g/LNa ₂ HPO ₄ , 3g/LKH ₂ PO ₄ , 0.5g/LNaCl, 2g/LNH ₄ Cl, 0.25g/LMgSO ₄ 7H ₂ O, 11mg/LCaCl ₂ , 27mg/LFeCl ₃ 6H ₂ O, 2mg/LZnCl ₂ 4H ₂ O, 2mg/LNa ₂ MoO ₄ 2H ₂ O, 1.9mg/LCuSO ₄ 5H ₂ O, 0.5mg/LH ₃ BO ₃ , 1mg/L vitamin B1, 200mMBis -Tris (pH7.25) and 0.1% (v/v) Triton-X100)

Implementation steps: Plasmid 1 and plasmid 2 are transformed into Escherichia coli BL21 (DE3) ΔfadE respectively or jointly, and the transformants obtained are grown in resistant LB medium (containing 34 μg/mL chloramphenicol and/or 50 μg/mL gentamicin , depending on the plasmid carried), after overnight culture, transfer to the corresponding resistant modified M9 medium, culture to the logarithmic phase (OD600≈0.6-0.8), add 0.05~1mMIPTG to induce the expression of hydrocarbon-producing genes, and continue to culture After about 10 hours to 20 hours, use a microplate reader (with a fluorescence module) (or fluorescence microscope, flow cytometer) to detect the fluorescence of the bacterial solution. And with reference to the method in embodiment 1, detect the aliphatic hydrocarbon content in the sample with GC-MS method

The results are shown in Figure 4, the fluorescence of transformants carrying both plasmid 1 and plasmid 2 was significantly enhanced after adding IPTG and cultured for about 10-20 hours, and the fluorescence intensity was positively correlated with the content of inducer and aliphatic hydrocarbons, indicating that the Cell fluorescence intensity to indirectly detect the relative content of aliphatic hydrocarbons.

Example 3 High-throughput screening of ADO mutant genes with high aliphatic hydrocarbon production ability

The ADO gene was amplified with primers ADO-For and ADO-Rev, and an error-prone PCR reaction system was used to introduce random base mutations. The primer sequences used are as follows (the underlined part is the restriction site):

ADO-For: TATA CCATGG CGCAGCTTGAAGCCAGCCT

ADO-Rev: CCTG GAATTC AAACGGCCGCAA

Specifically, in the PCR reaction, the plasmid carrying the ADO gene was used as a template, ADO-For and ADO-Rev were used as upstream and downstream primers respectively, and rTaqDNA polymerase from Takara Company was used. The reaction system is shown in the table below:

Table 1 PCR reaction system

The PCR reaction conditions are as follows: first 95°C for 2min; then 94°C for 1min, 55°C for 1min, 72°C for 1min, a total of 30 cycles; finally 72°C for 10min.

After the reaction, the PCR amplification product was detected by agarose gel electrophoresis (0.8% agarose concentration), and a band of about 700 bp was obtained. Double-cut the ADO gene fragment obtained by error-prone PCR with restriction endonucleases NcoI and EcoRI, and connect it into the same double-digested plasmid 2 (longer fragment) to replace the wild-type ADO fragment, and transform the ligated product into carrying plasmid 1 The host strain Escherichia coli BL21(DE3)ΔfadE competent cells; add about 1mL LB medium to 200μL transformation product, after incubation for about 1 hour, add gentamicin and chloramphenicol (concentrations are both 50μg/mL, the following same); after continuing to culture overnight, transfer to the modified M9 medium with resistance at a ratio of 1:20, culture to logarithmic phase (OD600≈0.6) at 37°C, add 0.05mMIPTG, and culture at 30°C for about 16 hours, Dilute the bacterial solution to about 10 ^-6 cells/mL, and use a flow sorter (such as BDFACSAriaIII) with a sorting device to sort (excitation light 485nm, emission light 535nm), and collect the 1 with the strongest fluorescence intensity. % cells (Figure 6). Spread the collected cells on the LB plate with corresponding resistance, incubate and culture, after the colony grows, pick a single clone and inoculate it into a centrifuge tube with resistant LB medium, after overnight culture, transfer to The resistant modified M9 medium was cultivated to the logarithmic phase, then added IPTG, continued to cultivate for 10-20 hours, and checked the fluorescence of the cells with a fluorescence microscope and a microplate reader (with a fluorescence module). By comparing the fluorescence intensities, three cells were obtained. The mutant strains are respectively designated as mutants I, II, and III, and the amino acid sequences of the ADO mutants are determined by sequencing as shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4.

Embodiment 4 utilizes ADO mutant to biosynthesize aliphatic hydrocarbon

Plasmid 2 carrying the three ADO mutant genes described in Example 3 (the ADO mutant gene replacing the wild-type ADO gene fragment in plasmid 2) was respectively cloned into the hydrocarbon-producing host Escherichia coli BL21(DE3)ΔfadE; in addition, for To further improve the potential hydrocarbon production capacity of the mutant, refer to mutant 3-3, replace glutamic acid at position 194 in the amino acid sequence of mutant 5-7 with leucine (E194K), and record it as mutant IV: E9G&I27N&E194K. Transform the above plasmid into the hydrocarbon-producing host Escherichia coli BL21(DE3)ΔfadE and obtain the transformants with reference to the culture conditions and aliphatic hydrocarbon detection method described in Example 1, measure the hydrocarbon production of the strains carrying the four mutants and compare with the wild-type ADO for comparison.

As shown in Figure 7, parallel experiments were carried out under the same culture conditions adopted, carrying the four fatty aldehyde deformyloxygenase mutants I (3-3, E194K, SEQ ID NO.2), II (5- 3, Escherichia coli of E9G&I27N, SEQIDNO.3), III (5-7, I204F, SEQIDNO.4), IV (3-3/5-3, E9G&I27N&E194K, SEQIDNO.5), the hydrocarbon production is wild-type ADO 2.0 to 2.9 times of that of ADO, indicating that the four mutants have higher hydrocarbon production efficiency than wild-type ADO. The mutation site has not been reported before.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore Therefore, the protection scope of the present invention should be defined by the claims.

Claims (10)

1. one kind produce aliphatic group because of high-throughput screening method, it is characterized in that, by carry potential product aliphatic group because of vector introduction in the host cell carrying detecting element, cultivate host cell, by the response of detecting element signal being differentiated the expression of screening genes of interest, and separate the mutant of high yield aliphatic hydrocarbon.

2. screening technique described in claim 1, it is characterised in that step is as follows:

1) amplification produce aliphatic group because of, and by fallibility PCR reaction introduce random mutation, it is thus achieved that amplified production；

2) by step 1) amplified production that obtains is connected in the plasmid vector containing resistant gene after enzyme action, it is thus achieved that recombiant plasmid；

3) building and carry the host cell of detecting element, and by step 2) recombinant plasmid transformed of gained is to host cell, it is thus achieved that transformant；

4) incubation step 3) gained transformant, the signal of detecting element is responded and carries out the qualification of destination gene expression and separate the mutant of high yield aliphatic hydrocarbon by detection basis.

3. screening technique described in claim 2, it is characterized in that, step 1) described product aliphatic group because of, include but not limited to one or more in following gene: fatty aldehyde piptonychia acyl monooxygenase gene ADO, decarboxylation of fatty acids enzyme OleT, terminal olefin synzyme Ols.

4. screening technique described in claim 2, it is characterised in that step 3) described detecting element, including aliphatic hydrocarbon detecting element and Reports component.

5. screening technique described in claim 4, it is characterised in that described aliphatic hydrocarbon detecting element, including at least the promoter of a constitutive promoter, a transcription regulatory factor and a hydrocarbon response characteristic；Described Reports component, is the one in green fluorescent protein, LacZ gene, luciferase or resistant gene.

6. screening technique described in claim 2, it is characterised in that step 3) described host cell is escherichia coli, yeast or cyanobacteria.

7. screening technique described in claim 2, it is characterised in that step 4) signal of described detecting element, refer to one or more in fluorescence intensity, absorbance value, chemiluminescence intensity and bacterium colony size.

8. arbitrary screening technique described in claim 1-7 is obtaining high yield aliphatic group because of the application in engineering bacteria.

9. one kind utilizes the mutant that either method described in claim 1-7 obtains, it is characterised in that following a kind of change at least occurs the aminoacid sequence of the fatty aldehyde piptonychia acyl oxygenase as shown in SEQIDNO.1:

1) the 9th glutamic acid mutation is glycine, and the 27th isoleucine mutation is agedoite, such as SEQIDNO.3；

2) the 194th glutamic acid mutation is lysine, such as SEQIDNO.2；

3) the 204th glutamic acid mutation is phenylalanine, such as SEQIDNO.4.

10. the application in aliphatic hydrocarbon biosynthesis of the mutant described in claim 9.