TW202020140A - Method of improving production yield of isoprene by cyanobacteria by gene transferring and encoding carbon fixation genes - Google Patents

Abstract

The present invention provides a method of improving production yield of isoprene by transgenic cyanobacteria, which improves the production yield of isoprene by cyanobacteria through gene transferring and encoding carbon fixation genes such as bicarbonate transporter and carbonic anhydrase. The present invention also provides a transgenic cyanobacterium, and an incubating method of the transgenic cyanobacteria for producing isoprene.

Description

Method for improving yield of isoprene produced by blue-green fungus

The invention relates to a method for increasing the yield of isoprene produced by transgenic blue-green bacteria. The invention also relates to a genetically transformed blue-green bacteria and a method for cultivating the genetically transformed blue-green bacteria to produce isoprene.

Isoprene is the basic raw material for synthetic rubber and an emerging alternative biofuel. In consideration of the current isoprene manufacturing, chemical synthesis methods are still used, for example: generated by pyrolysis of petroleum, in addition to the problems of high energy consumption, high pollution and high technical complexity, there are also low production efficiency and petroleum raw materials. The inherent dilemma of non-renewable resources. Therefore, the method of producing isoprene by biotechnology is gradually developing.

There are two pathways for biosynthesis of isoprene in nature: Methylerythritol phosphate pathway (hereinafter referred to as MEP pathway) and mevalonic acid pathway (hereinafter referred to as MVA pathway), the former Mostly exist in the chloroplasts of bacteria, blue-green bacteria, green algae and higher plants, the latter mainly in eukaryotes, archaea and higher plants.

In 2003, Martin and others published the use of GM Escherichia coli to produce isoprene. In 2011, Zhao and others published that the dxs and dxr genes from Bacillus subtilis were inserted into E. coli by gene transfer. A large number of exogenous gene expression, and found that compared with the endogenous group of E. coli dxs gene and dxr gene mass expression, the group with exogenous gene production capacity of isoprene is 2.3 times higher.

Turn in the development of blue-green bacteria colonization of 2010, Lin Boge (Lindberg) et al to turn blue-green bacteria colonization isoprene synthase gene from Pueraria montana (Pueraria montana) of (isoprene synthase gene, hereinafter referred to as IspS ), and for exogenous gene expression, the production of isoprene is 50μg/biomass/day. Finally, Bentley et al. carried out the genetic transformation of blue-green bacteria in 2014 by introducing 7 genes from the exogenous MVA pathway: AtoB , HmgS , HmgR , MK , PMK , PMD and Fni , and Cooperate with the transformation of IspS gene to increase the production capacity of isoprene, but the group of 7 genes embedded in MVA pathway and IspS gene through gene transformation, its production capacity is only more than the group of pure embedded IspS gene It is 2.5 times higher, and the output of isoprene is only about 125μg/biomass/day, so how to effectively improve the production efficiency of isoprene in the organism is still a problem to be solved.

In order to use organisms to efficiently produce isoprene, the present invention provides a method for increasing the yield of isoprene produced by transgenic blue-green bacteria, in particular by transgenic methods to enhance transgenic blue-green The yield of isoprene produced by bacteria.

Another aspect of the present invention is to provide a genetically transformed blue-green bacteria and a method for cultivating the genetically transformed blue-green bacteria to produce isoprene.

The invention provides a method for improving the yield of isoprene produced by genetically transformed cyanobacteria, characterized in that a expression vector is transformed into the genetically transformed cyanobacteria, and the expression vector has a coding bicarbonate The nucleotide sequence of the salt transporter (Bicarbonate transporter) gene has a sequence similarity of at least 90% to SEQ ID NO: 1, and a nucleotide sequence encoding a carbonic anhydrase gene, and has a SEQ ID NO: 2 at least 90% sequence similarity; and express or overexpress these genes in the transgenic blue-green bacteria.

Preferably, the nucleotide sequence encoding the bicarbonate operator gene possessed by the above expression vector has a sequence similarity of at least 95% or at least 99% with SEQ ID NO: 1; more preferably, the above expression vector The nucleotide sequence encoding the bicarbonate operator gene is shown in SEQ ID NO:1.

Preferably, the above-mentioned bicarbonate operator gene line as shown in SEQ ID NO: 1 is selected from Synechococcus elongatus PCC7942. The Synechococcus elongatus PCC7942 is purchased from the French blue-green algae provenance Center (The Pasteur Culture Collection of Cyanobacteria).

Preferably, the nucleotide sequence of the expression vector and the gene encoding carbonic anhydrase has a sequence similarity to SEQ ID NO: 2 of at least 95% or at least 99%; more preferably, The nucleotide sequence of the code carbonic anhydrase gene possessed by the above expression vector is shown in SEQ ID NO: 2.

Preferably, the above-mentioned gene encoding carbonic anhydrase as shown in SEQ ID NO: 2 is also selected from Synechococcus elongatus PCC7942.

The "sequence similarity" referred to in the present invention refers to the same degree of comparison of bases of different nucleotide sequences, which can be compared by a known software to obtain a similarity ratio. The known software is more common: BLAST at http://blast.ncbi.nlm.nih.gov/Blast.cgi. The higher the sequence similarity, it usually means that it will have a similar function or biological activity, or it will have a close kinship in evolution.

A nucleotide sequence having a sequence similarity of at least 90%, 95%, or 99% according to the present invention refers to the nucleotide sequence disclosed in the present invention is added, deleted, and/or replaced after being added, deleted, and/or substituted. The sequences disclosed in the invention have at least 90%, 95% or 99% of the same degree, and still maintain the same function or biological activity. Therefore, as long as a gene is at least 90%, 95%, or 99% similar to the nucleotide sequence disclosed in the present invention, and the function or biological activity of the sequence is not changed, it is included in the present invention.

In one embodiment of the present invention, the above-mentioned gene-transforming blue-green bacteria has a plurality of exogenous genes, which includes a nucleoside encoding an isopentenyl-diphosphate Delta-isomerase I gene Acid sequence, and at least 90% sequence similarity to SEQ ID NO: 3, and a nucleotide sequence encoding an isoprene synthase (isoprene synthase) gene, and at least 90% to SEQ ID NO: 4 Sequence similarity; and express or overexpress these genes in the transgenic blue-green bacteria.

The "exogenous gene" (exogenous gene) referred to in the present invention, also translated as "heterologous gene", refers to the endogenous genome not from the host cell or the target cell itself, but from other species Or a gene or a nucleotide fragment of a cell, and introduced into the host cell or target cell by genetic engineering technology. The "exogenous gene" can also be a synthetic gene or a nucleotide fragment.

The term "co-transplantation" as used in the present invention refers to the transformation of a plurality of expression vectors each carrying a different gene to be expressed into the same host cell or the same target cell in a sequential or sequential manner. The "co-transgenic strain" referred to in the present invention refers to a cell strain or strain in which different genes to be expressed are embedded in different segments of its own genome after the step of transformation, and each of the different genes is processed. Performance or excessive performance.

Preferably, the above nucleotide sequence encoding the isopentenyl diphosphate isomerase gene has a sequence similarity of at least 95% or at least 99% with SEQ ID NO: 3; more preferably, the encoding isopentenyl The nucleotide sequence of the alkenyl diphosphate isomerase gene is shown in SEQ ID NO:3.

Preferably, the nucleotide sequence encoding the isoprene synthase gene has a sequence similarity of at least 95% or at least 99% to SEQ ID NO: 4; more preferably, the isoprene encoding The nucleotide sequence of the enzyme gene is shown in SEQ ID NO:4.

In another embodiment of the present invention, the above-mentioned gene-transforming blue-green bacteria are further transformed to embed a large subunit and a small subunit encoding ribulose 1,5-diphosphate carboxylase/oxygenase (Rubisco) The nucleotide sequence of the unit gene, and has a sequence similarity of at least 90% with SEQ ID NO: 5; and express or overexpress the gene in the transgenic blue-green bacteria of the gene.

Preferably, the nucleotide sequence of the gene encoding ribulose 1,5-diphosphate carboxylase/oxygenase (Rubisco) large and small subunits has at least 95% of SEQ ID NO: 5 Or at least 99% sequence similarity; more preferably, the nucleotide sequence of the gene encoding ribulose 1,5-diphosphate carboxylase/oxygenase (Rubisco) large and small subunits is such as SEQ ID NO: 5 shows.

In one embodiment of the present invention, the expression vector used to transform any of the above genes into the blue-green bacteria transgenic gene is selected from pAM1573 transfection vector, pAM1303 transfection vector, pNS3 transfection vector or a combination thereof; Preferably, the expression vector used to transform the gene encoding the bicarbonate operator and the gene encoding the carbonic anhydrase into the transgenic blue-green bacteria is the pAM1573 transfection vector and/or the above exogenous gene line is transformed using the pAM1303 transgenic vector to The gene transfects blue-green bacteria.

In one embodiment of the present invention, the nucleotide sequence of the gene encoding the ribulose 1,5-diphosphate carboxylase/oxygenase (Rubisco) large subunit and small subunit is transformed into the gene transfer The expression vector used by Chlorocyanobacterium is a pNS3 transfer vector; preferably, the pNS3 transfer vector is a transfer vector with an rbcL promoter: pNS3-PrbcL transfer vector.

In one embodiment of the present invention, the above-mentioned expression vector includes restriction sites, which are selected from: Bma HI, Sma I, Pme I, Zra I, Xba I, Sal I, Eco RV, Sac I, Spe I, Eco RI, Hind III, Apa I, Kpn I, Xho I, Sac II, Xma I, Not I, Nde I, Nhe I, Pvu II, Sca I, Pst I, Hinc II or Dra I or A combination of these; preferably any one or a combination of BmaH I, Sma I, Pme I, Zra I, Xba I, Sal I or Eco RV.

In one embodiment of the present invention, the above expression vector includes a promoter, which is a ribulose 1,5-diphosphate carboxylase/oxygenase large subunit (PrbcL) promoter, and has a SEQ ID NO: 6 has a sequence similarity of at least 90%, 95% or 99%; more preferably, its nucleotide sequence is shown in SEQ ID NO: 6.

In one embodiment of the present invention, the gene-transforming blue-green fungus strain is selected from Synechococcus sp . , Synechocystis sp . , Thermosynechococcus sp . ), any one of a group of unicellular cyanobacteria genus (Cyanothece sp.), Anabaena (Anabaena sp.) and Nostoc (Nostoc sp.) composed of a combination thereof. Preferably, the genetically transformed blue-green bacteria is Synechococcus elongatus .

The present invention also provides a gene-transforming blue-green fungus whose genome is transformed with a gene encoding a bicarbonate operator embedded therein and has a sequence similar to SEQ ID NO: 1 of at least 90%, at least 95%, or at least 99% Degree, a gene encoding carbonic anhydrase, and having a sequence similarity to SEQ ID NO: 2 of at least 90%, at least 95%, or at least 99%, a gene encoding exogenous isopentenyl diphosphate isomerase, and Has a sequence similarity to SEQ ID NO: 3 of at least 90%, at least 95%, or at least 99%, and a gene encoding an exogenous isoprene synthase, and has a sequence similar to SEQ ID NO: 4 of at least 90%, at least 95% or at least 99% sequence similarity; and express or overexpress these genes in the transgenic blue-green bacteria.

Preferably, the gene encoding the bicarbonate operator embedded in the genome of the transgenic blue-green bacteria is shown in SEQ ID NO: 1, and the gene encoding carbonic anhydrase is shown in SEQ ID NO: 2, encoding exogenous The isoprenyl diphosphate isomerase gene is shown in SEQ ID NO: 3, and the gene encoding the exogenous isoprene synthase is shown in SEQ ID NO: 4.

In another embodiment of the present invention, the gene-transforming blue-green bacteria is transformed with at least one ribulose 1,5-diphosphate carboxylase/oxygenase large subunit (PrbcL) promoter embedded therein, and The gene encoding the bicarbonate operator and the gene encoding the carbonic anhydrase are each embedded in the downstream of a ribulose 1,5-diphosphate carboxylase/oxygenase large subunit (PrbcL) promoter, and/or The gene encoding exogenous isoprenyl diphosphate isomerase and the gene encoding exogenous isoprene synthase are both embedded in a 1,5-diphosphate ribulose carboxylase/oxygenase major Unit (PrbcL) downstream of the promoter.

In another embodiment of the present invention, the gene-transformed blue-green bacteria is further transformed to embed large subunits and small subunits encoding ribulose 1,5-diphosphate carboxylase/oxygenase (Rubisco) The nucleotide sequence of the unit gene has a sequence similarity to SEQ ID NO: 5 of at least 90%, at least 95%, or at least 99%; and expresses or overexpresses the gene in the transgenic blue-green bacteria. Preferably, the nucleotide sequence of the gene encoding ribulose 1,5-diphosphate carboxylase/oxygenase (Rubisco) large and small subunits is shown in SEQ ID NO:5.

In another embodiment of the present invention, the genes encoding ribulose 1,5-diphosphate carboxylase/oxygenase (Rubisco) large and small subunits are embedded in a ribonone 1,5-diphosphate Downstream of the sugar carboxylase/oxygenase large subunit (PrbcL) promoter.

The names and sources of the aforementioned enzymes and the genes encoding the enzymes are shown in Table 1.

Table 1

The invention also provides a method for producing isoprene, which is characterized by cultivating the above-mentioned gene-transgenic blue-green bacteria. Preferably, the step of cultivating the transgenic blue-green bacteria includes adding at least 30 millimoles (mM) of bicarbonate or carbonate to the culture solution. Preferably, the bicarbonate or carbonate concentration is 40 millimolar (mM) to 60 mM, and more preferably 50 mM bicarbonate or carbonate is added.

Preferably, the bicarbonate or the carbonate is calcium carbonate, magnesium carbonate, cesium carbonate, potassium carbonate, sodium carbonate, calcium bicarbonate, magnesium bicarbonate, potassium bicarbonate, sodium bicarbonate, ammonium bicarbonate or bicarbonate Sodium sesquicarbonate; more preferably, the bicarbonate or carbonate is sodium bicarbonate.

Finally, as shown in FIG. 11, compared with the prior art directly improving the pathway of biosynthesis of isoprene in nature, the present invention starts from the cycle of carbon fixation in cells and finds that the exogenous idi gene and The blue-green fungus group of exogenous ispS gene can further increase the yield of isoprene produced by the blue-green fungus after further colonization and over expression of carbon-fixing genes: ictB gene and ecaA gene. At least 60 times more, and the production of isoprene reaches 1209.8μg/biomass/day, which is significantly higher than the output of the blue-green fungus transgenic plants disclosed by Linberg: 50μg/biomass/day, and that revealed by Bentley Production of blue-green bacteria transgenic plants: 125μg/biomass/day.

In addition, the ribulose 1,5-diphosphate carboxylase/oxygenase (Rubisco) large subunit rbcL and the ribulose 1,5-diphosphate carboxylase/oxygenase (Rubisco) small subunit were transferred. In the blue-green fungus group of rbcS , the yield of isoprene is 0.91 μg/biomass/hr. Compared with the blue-green fungus group transfecting the ictB gene and the ecaA gene, the yield of isoprene is 50.41 μg. / biomass/hr. It can be seen that although the genes transferred by the two groups are the same as the catalytic enzyme genes encoding the carbon cycle pathway, the productivity of the blue-green bacteria group that transferred the ictB gene and the ecaA gene is significantly higher, and up to 55 times . Therefore, by transforming the two carbon-fixing genes of ictB and ecaA into blue-green bacteria with isoprene production capacity through expression vectors, the yield of isoprene production by blue-green bacteria can be effectively improved, and the excellent improvement effect Unreasonable expectations.

Example 1: Preparation of isoprene-producing S. sphaeroides transgenic strain (containing idi - ispS gene)

1. Acquisition of idi gene

The isopentenyl-diphosphate Delta-isomerase I gene ( idi gene) is derived from Populus euphratica and is based on Populus euphratica 's encoding isopentenyl diphosphate isomerase ) sequences (GeneID: after 105,112,811), and codon optimized in accordance with the S. elongatus PCC7942 DNA sequence, obtain nucleotide sequences optimized idi gene, obtained through chemical synthesis optimization idi gene, and placed in a pUC57 vector, becomes pUC57-Fragment 1 plastid, the optimization and chemical synthesis of the aforementioned genes were entrusted to Genewiz of the United States.

2. Obtaining the ispS gene

The isoprene synthase gene (i.e. ispS gene) is derived from Populus nigra ( Populus nigra ), deleted based on the Populus nigra encoding isoprene synthase (ispS) sequence (GenBank accession No. HQ684728.1) After the N-terminal signal peptide, codon optimization was performed according to the S. elongatus PCC7942 DNA sequence to obtain the optimized ispS gene nucleotide sequence, and then the optimized ispS gene was obtained by chemical synthesis and placed in the pUC57 vector to become pUC57-Fragment 2 Plastids, optimization and chemical synthesis of the aforementioned genes were commissioned by Genewiz of the United States.

Construction of PrbcL-idi-PrbcL-ispS gene transfer vector

The transfection vector pAM1303 (Andersson et al., 2000) was treated with the restriction enzyme BamH I and then treated with Alkaline Phosphatase (New England Biolabs, USA) to prevent the DNA from self-adhesion. After cutting the idi gene fragment in pUC57-Fragment 1 plastid with restriction enzyme BamH I (New England Biolabs, USA), the entire gene fragment was inserted into the transgenic vector pAM1303 of Synechococcus elongatus PCC7942 through ligation. In the BamH I cut position, the conjugated plastid DNA was transformed into E. coli strain DH5α by heat shock to obtain the idi gene transfer vector of Synechococcus elongatus PCC7942: pAM1303-idi.

After transfecting the aforementioned pAM1303-idi gene with the restriction enzyme Sma I, it was treated with Alkaline Phosphatase (New England Biolabs, USA) to prevent the DNA from adhering to itself. After cutting the ispS gene fragment in pUC57-Fragment 2 with restriction enzyme Sma I (New England Biolabs, USA), the entire gene fragment was inserted into the Sma I cleavage site of the pAM1303-idi gene transfer vector by conjugation , The conjugated plastid DNA was transformed into E. coli strain DH5α by heat shock to obtain the idi-ispS gene transfer vector of Synechococcus elongatus PCC7942: pAM1303-idi-ispS.

After transfecting the aforementioned pAM1303-idi-ispS gene vector with the restriction enzyme Pme I, it was treated with Alkaline Phosphatase (New England Biolabs, USA) to prevent DNA self-adhesion. Then use the restriction enzyme Sma I (New England Biolabs, USA) to synthesize the Synechococcus elongatus PCC7942 rbcL promoter gene fragment (as shown in SEQ ID NO: 6) in pYT&A-rbcL plastid (Te-Jin Chow, Fooyin University) After excision, the entire gene fragment was inserted into the Pme I cleavage site of the aforementioned pAM1303-idi-ispS gene transfer vector by conjugation, and the conjugated plastid DNA was transformed into E. coli strain DH5α by heat shock. The gene transfer vector of PrbcL-idi-ispS of Synechococcus elongatus PCC7942 was obtained: pAM1303-PrbcL-idi-ispS.

After transfecting the aforementioned pAM1303-PrbcL-idi-ispS gene vector with the restriction enzyme Zra I, it was treated with Alkaline Phosphatase (New England Biolabs, USA) to prevent DNA self-adhesion. Then, the restriction enzyme Sma I (New England Biolabs, USA) was used to cut the Synechococcus elongatus PCC7942 rbcL promoter gene fragment in pYT&A-rbcL plastid (Te-Jin Chow, Fooyin University) to remove the restriction enzyme, and the whole was ligated for conjugation. The gene fragment was inserted into the Zra I cleavage site of the aforementioned pAM1303-PrbcL-idi-ispS gene transfer vector, and the joined plastid DNA was transformed into E. coli strain DH5α by heat shock to obtain PrbcL-idi of Synechococcus elongatus PCC7942 -PrbcL-ispS gene transfer vector: pPrbcL-idi-PrbcL-ispS, part of its structure is shown in Figure 1.

4. Preparation of Synechococcus elongatus PCC7942 isoprene (idi-ispS) transgenic plants

The aforementioned pPrbcL-idi-PrbcL-ispS transfection vector was transformed into Synechococcus elongatus PCC7942 wild type cells by transformation, and the transgenic Synechococcus sp. was selected with Spectinomycin antibiotic. First, collect 10 mL of Synechococcus elongatus PCC7942 wild-type cells by centrifugation. After removing the culture solution, add 5 mL of 10 mM NaCl solution. After mixing, centrifuge at 3,980 rpm for 10 minutes. After removing the supernatant, add 1 mL of 10 mM. BG-11 liquid medium of EPPS was used to suspend algae cells, and 1.5 μg of pPrbcL-idi-PrbcL-ispS plastid DNA was added, and cultured at 28°C in the dark with shaking overnight. After 6 hours of incubation, the cells were centrifuged at 14,000 rpm. algal cells collected 2 minutes, then add 300 μL of 10 mM EPPS BG-11 liquid medium for suspending the algal cells, diluted 10-fold in a row, from each added 100 μL 10 mM EPPS applied and containing Spectinomycin (20 μg mL ^{- 1} ) The BG-11 solid medium is placed at 28°C and irradiated until the algal colonies grow. The algal colonies grown on the solid medium are selected with a sterile toothpick to the BG-11 solid medium supplemented with 10 M EPPS and Spectinomycin (20 μg mL ^-1 ). After irradiating the culture, the algae strains that grow well are cultured in the supplement Spectinomycin (20 μg mL ^-1 ) liquid medium.

The transformed algal strains are cultured in antibiotic-containing medium. Scrape about one loop or 1.5 mL of algal bacteria, and first use colony polymerase chain reaction (Polymerase chain reaction, PCR) to detect whether Synechococcus elongatus has the idi and ispS genes. Take algae colonies and suspend algae cells with TE-triton solution (TE, pH 8.0 + 1% Triton X-100), then treat them at 95℃ for 3.5 minutes, and then extract them twice with chloroform, then take the supernatant and separate Perform PCR reaction with the idi and ispS primer pairs (as shown in Table 2 below) to detect whether the transgenic S. sphaeroides has the idi and ispS genes. Those who are confirmed to have the idi and ispS genes by the test are the production of isoprene. The successful transgenic strain of Synechococcus elongatus.

The schematic diagram of the idi and ispS gene expression vectors embedded in the first neutral position (NSI) of Synechococcus elongatus genome is shown in Figure 1, and the test results are shown in Figure 2. The group No. 1 uses sterile water as a negative control group. The group No. 2 uses plastid DNA as a positive control group, and the group No. 3 is a transgenic plant.

Table 2. Idi and ispS primer pairs

5. Synechococcus elongatus PCC7942-idi-ispS transgenic plants produce isoprene

After transfecting PCC7942-idi-ispS with 200 μmol m ^-2 s ^-1 light intensity and 50 mM NaHCO _{3 for} five days, the isoprene was measured by gas chromatography, and the analysis results are shown in Figure 3. The signal that appeared at 1.660 minutes was consistent with that of isoprene, while the wild type of the control group did not appear at 1.660 minutes. Therefore, it was confirmed from the above results that a transgenic strain of Synechococcus elongatus PCC7942-idi-ispS capable of producing isoprene was obtained.

Example 2: Preparation of isoprene-producing S. sphaeroides transgenic strains (containing idi-ispS and ictB-ecaA genes)

1. Acquisition of ictB gene

The bicarbonate transporter gene (that is, the ictB gene) was selected from Synechococcus elongatus PCC7942, and a pair of ictB gene primers was designed (as shown in Table 3 below), using the chromosomal DNA of Synechococcus elongatus PCC7942 as a template, Perform polymerase chain reaction (PCR) with the ictB gene primer pair. The PCR reaction solution contains 1X PCR buffer solution, 0.4 mM dNTP, 2 mM MgCl ₂ , 1 unit Takara ex Taq DNA polymerase (polymerase), 0.5 μM primers (ictB-f, ictB-r), total volume 50 μL, reaction conditions at 95°C for 3 minutes; 32 cycles: 95°C for 1 minute, 55°C for 1 minute, 72°C for 2 minutes; finally extended by 72°C After maintaining at 4°C for 10 minutes, PCR was performed to amplify the ictB gene fragment, and the amplified ictB gene was bound to the yT&A (Yeastern Biotech Co., Ltd.) plastid with T4 DNA ligase (ligase) to obtain ictB. The pyT&A-ictB plasmid of the gene.

Table 3. ictB primer pair

2. Acquisition of ecaA gene

The carbonic anhydrase gene ( ecaA gene for short) was selected from Synechococcus elongatus PCC7942, and the ecaA gene primer pair was designed (shown in Table 4 below). Using the chromosome gene of Synechococcus elongatus PCC7942 as a template, the ecaA gene primer pair was used Polymerase chain reaction, PCR reaction solution contains 1X PCR buffer solution, 0.4 mM dNTP, 2 mM MgCl2, 1 unit Takara ex Taq DNA polymerase, 0.5 μM primers (ecaA-f, ecaA-r), total volume 50 μL, The reaction conditions were 95°C for 3 minutes; 32 cycles: 95°C for 1 minute, 55°C for 1 minute, 72°C for 1 minute; finally extended 72°C for 10 minutes, maintained at 4°C, PCR was performed to amplify the ecaA gene fragment, and the amplified The ecaA gene was bound to yT&A (Yeastern Biotech Co., Ltd.) plastid with T4 DNA ligase to obtain pyT&A-ecaA plastid with ecaA gene.

Table 4. ecaA primer pairs

3. Construction of PrbcL-ictB-ecaA gene transfer vector

After transfection vector pAM1573-PrbcL (Te-Jin Chow, Fooyin University) with rbcL promoter was treated with restriction enzyme Xba I, it was treated with DNA Polymerase I, Large (Klenow) Fragment, and both ends of DNA were modified into blunt ends , Treated with Alkaline Phosphatase (New England Biolabs, USA) to prevent the DNA from adhering to itself.

After cutting the ictB gene fragment in the pyT&A-ictB plastid with the restriction enzyme BamH I (New England Biolabs, USA), it was treated with DNA Polymerase I, Large (Klenow) Fragment, and the two ends of the DNA were modified to blunt ends. The whole gene fragment was inserted into the Xba I cleavage site of the transgenic vector pAM1573-PrbcL of Synechococcus elongatus PCC7942 by conjugation, and the conjugated plastid DNA was transformed into E. coli strain DH5α by heat shock to obtain Synechococcus elongatus PCC7942 The ictB gene transfer vector.

After transfecting the pAM1573-PrbcL-ictB vector with the restriction enzyme Sal I, it was treated with DNA Polymerase I, Large (Klenow) Fragment. After modifying both ends of the DNA to blunt ends, it was performed with Alkaline Phosphatase (New England Biolabs, USA) Treatment to prevent DNA from sticking to itself.

The pAM1573-PrbcL-ictB transfection vector was then cut with the restriction enzyme BamH I (New England Biolabs, USA) to cut the ecaA gene fragment in pyT&A-ecaA, and the entire gene fragment was inserted into pAM1573-PrbcL-ictB for conjugation. In the Sal I cut position of the transfer vector, the conjugated DNA was transformed into E. coli strain DH5α by heat shock to obtain the ictB-ecaA gene transfer vector of Synechococcus elongatus PCC7942: pPrbcL-ictB-ecaA, and its partial structure is as follows Figure 4 shows.

4. Preparation of Synechococcus elongatus PCC7942 isoprene (idi-ispS) strain co-transformed bicarbonate operator (ictB) and carbonic anhydrase (ecaA) gene transgenic strains

The aforementioned pPrbcL-ictB-ecaA transfection vector was transformed into Synechococcus elongatus PCC7942 isoprene (idi-ispS) strain by transformation, and the transgenic algae were screened with Spectinomycin/Chloramphenicol antibiotics. Collect 10 mL of Synechococcus elongatus PCC7942 isoprene (idi-ispS) bacterial cells by centrifugation. After removing the culture medium, add 5 mL of 10 mM NaCl solution, mix well, and centrifuge at 3,980 rpm for 10 minutes. Remove the supernatant and then 1 mL Add 10 mM EPPS in BG-11 liquid medium to suspend algae cells, and add 1.5 μg of pPrbcL-ictB-ecaA plastid DNA, incubate at 28°C in the dark and shake overnight. After 6 hours of incubation on the next day, centrifuge at 14,000 rpm After collecting algae cells for 2 minutes, and then suspending the algae cells in 300 μL of BG-11 liquid medium supplemented with 10 mM EPPS, make a serial 10-fold dilution, and take 100 μL each for the addition of 10 mM EPPS and ophthalmicin/chloramphenicol (20 μg mL ^-1 /15 μg mL ^-1 ) of BG-11 solid medium, placed at 28 ℃, light culture, until algae colonies grow. The algal colonies grown on the solid medium were selected with a sterile toothpick to the BG-11 solid medium supplemented with 10 M EPPS and containing spectinomycin/chloramphenicol (20 μg mL ^-1 /15 μg mL ^-1 ) and cultured under light. After that, the algae strain with good growth is cultivated to the liquid medium supplemented with guanycin/chloramphenicol (20 μg mL ^-1 /15 μg mL ^-1 ).

Cultivate these transformed strains on antibiotic-containing medium. Scrape about one fungus ring or 1.5 mL of Synechococcus elongatus, and first use colony PCR to detect whether Synechococcus elongatus has genes for ictB and ecaA. Take algae colonies and suspend the cells with TE-triton solution (TE, pH 8.0 + 1% Triton X-100), then treat them at 95°C for 3.5 minutes, and then extract them twice with chloroform. Take the supernatant and test-PrbcL- For, ecaA-rev primer pair (shown in Table 5 below) PCR reaction to detect whether the transgenic S. sclerotiorum has the genes of ictB and ecaA. Those who have confirmed that they have the genes of ictB and ecaA by the test are considered successful. Transformation strain.

Table 5. Test-PrbcL-for, ecaA -rev primer pairs

The schematic diagram of the ictB and ecaA gene expression vectors embedded in the second neutral position (NSII) of Synechococcus elongatus genome is shown in FIG. 4, and the detection results are shown in FIG. 5, wherein the group No. 1 uses sterile water as a negative control group. The group No. 2 uses plastid DNA as a positive control group, and the group No. 3 is a transgenic plant.

Example 3: Preparation of a transgenic strain of Synechococcus elongatus (containing idi-ispS and rbcL&S genes)

1. Acquisition of rbcL&S gene

The ribulose 1,5-diphosphate carboxylase/oxygenase (Rubisco) large and small subunit genes (ie, rbcL&S genes) were selected from Synechococcus elongatus PCC7942, and the rbcL&S gene primer pairs were designed (see Table 6 below) (Shown), using Chromosomal DNA of Synechococcus elongatus PCC7942 as template, and polymerase chain reaction (PCR) using rbcL&S gene primer pairs. The PCR reaction solution contains 1X PCR buffer solution, 0.4 mM dNTP, 2 mM MgCl2, 1 unit Takara ex Taq DNA polymerase, 0.5 μM primers (rbcL&S-f, rbcL&S-r), total volume 50 μL, reaction conditions 95°C 3 minutes; 32 cycles: 95°C 1 minute, 55°C 1 minute, 72°C 1 Minutes; lastly extend 72℃ for 10 minutes, maintain at 4℃, perform PCR to amplify rbcL&S gene fragments, and bind the amplified rbcL&S gene to yT&A (Yeastern Biotech Co., Ltd.) plastid with T4 DNA ligase, to A pyT&A-rbcL&S plasmid with rbcL&S gene was obtained.

Table 6. rbcL&S primer pair

2. Construction of PrbcL-rbcL&S gene transfer vector

After transfection vector pNS3-PrbcL (Te-Jin Chow, Fooyin University) with rbcL promoter was treated with restriction enzyme BamH I, it was treated with DNA Polymerase I, Large (Klenow) Fragment, and both ends of DNA were modified into blunt ends. , Treated with Alkaline Phosphatase (New England Biolabs, USA) to prevent DNA self-adhesion.

The rbcL&S gene fragment in the pyT&A-rbcL&S plastid was cut with the restriction enzyme Eco RV (New England Biolabs, USA), and the entire gene fragment was inserted into Synechococcus elongatus PCC7942 transgenic vector pNS3-PrbcL BamH by conjugation. In the I-cut position, the conjugated plastid DNA was transformed into E. coli strain DH5α by heat shock to obtain the rbcL&S gene transfer vector of Synechococcus elongatus PCC7942: pNS3-PrbcL-rbcL&S, and its partial structure is shown in FIG. 6.

3. Synechococcus elongatus PCC7942 isoprene (idi-ispS) strain co-transformed Rubisco large subunit and small subunit gene (rbcL&S) transgenic strains

The aforementioned pNS3-PrbcL-rbcL&S transfection vector was transformed into Synechococcus elongatus PCC7942 isoprene (idi-ispS) strain by transformation, and the transgenic algae were screened with Spectinomycin/Kanamycin antibiotics. Collect 10 mL of Synechococcus elongatus PCC7942 bacterial cells by centrifugation. After removing the culture solution, add 5 mL of 10 mM NaCl solution. After mixing, centrifuge at 3,980 rpm for 10 minutes. Remove the supernatant and add 1 mL of 10 mM EPPS BG- 11 Suspend the algae cells in liquid medium and add 1.5 μg of pNS3-PrbcL-rbcL&S plastid DNA, incubate at 28°C in the dark and shake overnight. After cultivating for 6 hours on the next day, centrifuge at 14,000 rpm for 2 minutes to collect algae cells. After suspending the algae cells in 300 μL of BG-11 liquid medium supplemented with 10 mM EPPS, make a serial 10-fold dilution. Take 100 μL each and apply to 10 mM EPPS supplemented with Kanamycin/Kanamycin (20 μg mL ^-1 /15 μg mL ^-1 ) of BG-11 solid medium, placed at 28 ℃, light culture, until algae colonies grow. The algal colonies grown on the solid medium were selected with a sterile toothpick to add 10 M EPPS and BG-11 solid medium containing komycin/kanamycin (20 μg mL ^-1 /15 μg mL ^-1 ), and cultured under light. Afterwards, the algae strains with good growth are cultured to liquid medium supplemented with komycin/kanamycin (20 μg mL ^-1 /15 μg mL ^-1 ).

Cultivate these transformed strains on antibiotic-containing medium. Scrape about one fungus ring or 1.5 mL of Synechococcus elongatus, and first check whether Synechococcus elongatus has the rbcL&S gene by colony PCR. Take algae colonies and suspend the cells with TE-triton solution (TE, pH 8.0 + 1% Triton X-100), then treat them at 95℃ for 3.5 minutes, and then extract twice with chloroform, take the supernatant and test-PrbcL respectively -For, rbcL&S-rev primer pairs (shown in Table 7 below) perform PCR reaction to detect whether the transformed Synechococcus elongatus has the rbcL&S gene, and the person confirmed to have the rbcL&S gene by the test is the successful transformed strain.

Table 7. rbcL&S primer pair

The schematic diagram of the rbcL&S gene expression vector embedded in the third neutral position (NSIII) of Synechococcus elongatus genome is shown in Figure 6, and the test results are shown in Figure 7, where the group No. 1 uses sterile water as a negative control group, No. 2 The plastid DNA was used as a positive control group, and the group No. 3 was a transgenic plant.

Example 4: Preparation of a transgenic strain of Synechococcus elongatus (containing idi-ispS, ictB-ecaA and rbcL&S genes)

The aforementioned pNS3-PrbcL-rbcL&S transgenic vector was transformed into Synechococcus elongatus PCC7942 isoprene (idi-ispS) to co-transform the Bicarbonate transporter and the carbonic anhydrase gene (ictB-ecaA) strain, using Spectinomycin/ Chlorine Chloramphenicol/Kanamycin antibiotics are used to screen transgenic algae. Centrifuge 10 mL of Synechococcus elongatus PCC7942 isoprene (idi-ispS) to co-transform ictB-ecaA cells. After removing the culture medium, add 5 mL of 10 mM NaCl solution, mix well, and centrifuge at 3,980 rpm for 10 minutes to remove the supernatant. Suspend the algae cells in 1 mL of BG-11 liquid medium supplemented with 10 mM EPPS, and add 1.5 μg of pNS3-PrbcL-rbcL&S plastid DNA, incubate at 28°C in the dark and shake overnight, then incubate for 6 hours on the next day. , Collect algae cells by centrifuging at 14,000 rpm for 2 minutes, and then suspend algae cells in 300 μL of BG-11 liquid medium supplemented with 10 mM EPPS, then make a serial 10-fold dilution, and take 100 μL each for 10 mM EPPS and guanycin-containing / Chloramphenicol/Kanamycin (20 μg mL ^-1 /15 μg mL ^-1 /15 μg mL ^-1 ) BG-11 solid medium, placed at 28 ℃, light culture, until algae colonies grow. The algae colonies grown on the solid medium were selected with a sterile toothpick to add 10 M EPPS and containing guanycin/chloramphenicol/kanamycin (20 μg mL ^-1 /15 μg mL ^-1 /15 μg mL ^-1 ) On the BG-11 solid medium, after illuminating the culture, the algae with good growth is cultivated to add guanycin/chloramphenicol/kanamycin (20 μg mL ^-1 /15 μg mL ^-1 /15 μg mL ^-1 ) of liquid culture medium.

Cultivate these transformed strains on antibiotic-containing medium. Scrape about one fungus ring or 1.5 mL of Synechococcus elongatus, and first check whether Synechococcus elongatus has the rbcL&S gene by colony PCR. Take algae colonies and suspend the cells with TE-triton solution (TE, pH 8.0 + 1% Triton X-100), then treat them at 95℃ for 3.5 minutes, and then extract twice with chloroform, take the supernatant and test-PrbcL respectively -For and rbcL&S-rev primer pairs perform PCR reaction to detect whether the transgenic S. sphaeroides has the rbcL&S gene. Those who have confirmed the rbcL&S gene after testing are successful transformed strains. The test results are shown in Figure 8. The group No. 1 uses sterile water as a negative control group, the group No. 2 uses plastid DNA as a positive control group, and the group No. 3 is a transgenic plant.

Example 5: Comparative experiment on growth and photosynthesis of transgenic plants

The algae fluid of the transformed strains, which had been cultured for about 3 weeks, was inoculated into 100 ml of BG11+EPPS broth containing spectinomycin/chloramphenicol (20 μg mL ^-1 /15 μg mL ^-1 ). The culture was carried out with a gas containing 5% carbon dioxide (CO ₂ )/air (Air), the aeration rate was 0.1 vvm, the culture temperature was 28° C., the light intensity was 200 μmol m ^-2 s ^-1 , and the culture was carried out in a 24-hour light cycle. The fresh BG-11 culture medium was used as a blank control group, and the experimental group was id i-ispS transgenic plant, idi-ispS-ictB-ecaA co-transformed plant, idi-ispS-rbcL&S co-transformed plant and idi- ispS-ictB-ecaA-rbcL&S co-transformed strain. The OD value of the culture solution at a wavelength of 750 nm was measured daily with an ultraviolet-visible spectrophotometer (CT-5600, ChromTech, USA). According to the OD750 absorbance value, the growth curve of Synechococcus elongatus PCC7942 grown under the culture condition of 5% carbon dioxide/air is plotted, as shown in FIG. 9.

As can be seen from Fig. 9, the three co-transgenic plants of idi-ispS-ictB-ecaA, co-transgenic plants of idi-ispS-rbcL&S and co-transgenic plants of idi-ispS-ictB-ecaA-rbcL&S co-transformed due to excessive carbon sequestration genes Performance (over expression), so it has better growth compared to the idi-ispS transgenic plant, in which the OD750 of the idi-ispS-ictB-ecaA co-transformed plant and the idi-ispS-ictB-ecaA-rbcL&S co-transformed plant It can reach 4.96 and 5.36, compared with the OD750 of the idi-ispS transgenic plant is only 3.39, which is an increase of 1.46 times and 1.58 times.

Photosynthesis and respiration are measured by the oxygen electrode of Hansatech Inc (DW1, Hansatech, Norfolk, England). Take 10ml of algae fluid in an oxygen electrode chamber (O ₂ electrode chamber), the light intensity is 100 E m ^-2 s ^-1 , and then add sodium bicarbonate during the measurement to make the final concentration 3mM, light (Halogen Deko-Lampen, SYLVANIA, Tokyo, Japan) After 1 to 2 minutes, measure the amount of oxygen released in the unit, which is the net photosynthetic rate; then, replace the algae with a new one after the oxygen consumption rate stabilizes (about 5 To 10 minutes), calculate the amount of oxygen consumed per unit time without light, this is the respiration rate (respiration rate), the sum of the two is the total photosynthesis rate (gross photosynthetic rate), the result is shown in Figure 10 Shown.

As can be seen from Figure 10, the total photosynthesis rate of the three co-transgenic plants idi-ispS-ictB-ecaA, co-transgenic plants idi-ispS-rbcL&S and co-transgenic plants idi-ispS-ictB-ecaA-rbcL&S is also obvious The total photosynthesis rate of the co-transgenic plants of idi-ispS-ictB-ecaA, idi-ispS-rbcL&S and idi-ispS-ictB-ecaA-rbcL&S can reach 387.6, 355.1 and 435.4, compared with The total photosynthesis rate of idi-ispS transgenic plants was only 293.7, which was increased by 1.32 times, 1.21 and 1.48 times, respectively.

Example 6: Comparative experiment on the yield of isoprene in transgenic plants

The algae fluid of the transformed strains cultured for about 3 weeks was inoculated into 100 ml of BG11+EPPS containing 50 mM NaHCO ₃ culture fluid containing spectinomycin/chloramphenicol (20 μg mL-1/15 μg mL-1 ). The shaking rate was 100 rpm, the incubation temperature was 28°C, the light intensity was 200 μmol m ^-2 s ^-1 , and the cultivation period was 24 hours. After three days of incubation, the isoprene concentration was analyzed with a gas chromatograph/flame ion detector (Agilent 6890N GC/FID).

From the isoprene production results shown in Figure 11, it can be found that the idi-ispS transplants, the idi-ispS-ictB-ecaA co-transplants, the idi-ispS-rbcL&S co-transplants and the idi-ispS-ictB-ecaA- The 3-day isoprene yield of rbcL&S co-transformed plants was 0.79, 50.41, 0.91 and 53.29 µg/g biomass/hr, respectively. Therefore, after co-transformation of ictB and ecaA genes or co-transformation of ictB, ecaA and rbcL&S genes, the production of isoprene increased by 63-fold and 67-fold, respectively, and their daily production was 1209.8 and 1278.96 µg/g, respectively. biomass/day.

To sum up, it can be seen that by transposing the two carbon-fixing genes of ictB and ecaA in the present invention, the effect of increasing the yield of isoprene is significantly better than that of the growth and photosynthesis, so the two carbon-fixing genes of ictB and ecaA are also transgenic It is unexpectedly highly specific to increase the synthesis of isoprene, rather than increasing the production of genetically transformed blue-green bacteria. In addition, the transgenic blue-green bacteria were not harmful to the growth of the transgenic blue-green bacteria due to the competition of carbon source during the synthesis of isoprene. Therefore, the two carbon-fixing genes of ictB and ecaA were transferred to the transgenic blue. Chlorella has two advantages: "Highly specific promotion of the synthesis of isoprene by transgenic blue-green bacteria" and "Promotion of the growth of transgenic blue-green bacteria."

(no)

FIG. 1 is a partial schematic diagram of an expression vector containing an exogenous idi gene and an exogenous ispS gene and a partial schematic diagram of the expression vector embedded in the Synechococcus elongatus genome.

Fig. 2 is a PCR detection confirmation diagram of the transfer of exogenous idi gene and exogenous ispS gene.

FIG. 3 is a gas chromatograph analysis result of a product of a wild type cell of Synechococcus elongatus and a transgenic strain of Synechococcus elongatus idi + ispS in a control group.

FIG. 4 is a partial schematic diagram of an expression vector containing the ictB gene and ecaA gene and a partial schematic diagram of the expression vector embedded in the Synechococcus elongatus genome.

Fig. 5 is a PCR detection confirmation diagram of the transfer results of the idi , ispS , ictB and ecaA genes.

6 is a partial schematic diagram of an expression vector containing the rbcL&S gene and a partial schematic diagram of the expression vector embedded in the Synechococcus elongatus genome.

Fig. 7 is a PCR detection confirmation diagram of the transfer results of the idi , ispS and rbcL&S genes.

Figure 8 is a PCR detection confirmation diagram of the results of the gene transfer of idi , ispS , ictB , ecaA and rbcL&S .

Fig. 9 is a graph showing the growth curves of four species of Synechococcus elongatus.

Figure 10 is a comparison chart of photosynthesis of four species of Synechococcus elongatus.

Fig. 11 is a graph comparing the yield of isoprene in four strains of Synechococcus elongatus.

(no)

＜110＞ Fuying University of Science and Technology＜120＞ Method for improving the yield of isoprene produced by blue-green fungi＜160＞ 17 ＜170＞ PatentIn version 3.5 ＜210＞ 1 ＜211＞ 1404 ＜212＞ DNA ＜213＞ Slender poly alga (Synechococcus elongatus PCC7942) <400> 1 atgactgtct ggcaaactct gacttttgcc cattaccaac cccaacagtg gggccacagc 60 agtttcttgc atcggctgtt tggcagcctg cgagcttggc gggcctccag ccagctgttg 120 gtttggtctg aggcactggg tggcttcttg cttgctgtcg tctacggttc ggctccgttt 180 gtgcccagtt ccgccctagg gttggggcta gccgcgatcg cggcctattg ggccctgctc 240 tcgctgacag atatcgatct gcggcaagca acccccattc actggctggt gctgctctac 300 tggggcgtcg atgccctagc aacgggactc tcacccgtac gcgctgcagc tttagttggg 360 ctagccaaac tgacgctcta cctgttggtt tttgccctag cggctcgggt tctccgcaat 420 ccccgtctgc gatcgctgct gttctcggtc gtcgtgatca catcgctttt tgtcagtgtc 480 tacggcctca accaatggat ctacggcgtt gaagagctgg cgacttgggt ggatcgcaac 540 tcggttgccg acttcacctc acgggtttac agctatctgg gcaaccccaa cctgctggct 600 gcttatctgg tgccgacgac tgccttttct gcagcagcga tcggggtgtg gcgcggctgg 660 ctccccaagc tgctggcgat cgctgcg aca ggtgcgagca gcttatgtct gatcctcacc 720 tacagtcgcg gtggctggct gggttttgtc gccatgattt ttgtctgggc gttattaggg 780 ctctactggt ttcaaccccg tctacccgca ccctggcgac gctggctatt cccagtcgta 840 ttgggtggac tagtcgcggt gctcttggtg gcggtgcttg gacttgagcc gttgcgcgtg 900 cgcgtgttga gcatctttgt ggggcgtgaa gacagcagca acaacttccg gatcaatgtc 960 tggctggcgg tgctgcagat gattcaagat cggccttggc tgggcatcgg ccccggcaat 1020 accgccttta acctggttta tcccctctat caacaggcgc gctttacggc gttgagcgcc 1080 tactccgtcc cgctggaagt cgcggttgag ggcggactac tgggcttgac ggccttcgct 1140 tggctgctgc tggtcacggc ggtgacggcg gtgcggcagg tgagccgact gcggcgcgat 1200 cgcaatcccc aagccttttg gttgatggct agcttggccg gtttggcagg aatgctgggt 1260 cacggtctgt ttgataccgt gctctatcga ccggaagcca gtacgctctg gtggctctgt 1320 attggagcga tcgcgagttt ctggcagccc caaccttcca agcaactccc tccagaagcc 1380 gagcattcag acgaaaaaat gtag 1404 <210> 2 <211> 714 <212> DNA <213 ＞ Synechococcus elongatus ( Sy nechococcus elongatus PCC7942) <400> 2 atgcgccggc gatcgctcct agcagctctt ggcggcagct gcatcggttg gttgggcagc 60 agtcaacccg tctgggccag tgcagactgg gactatagtc gccgccgagg tcctcgccag 120 tgggccaagc ttgatccggc ctatgccatt tgtcagcagg gtcgtcaaca atccccaatt 180 aacctgacgg gtcagcctga cagaacacca ctggattacc gcgatcgccc gtttaagggc 240 attctgcagc aggctcccca ttccctccgc attgactgtc cagctgggaa tggtttctgg 300 gaagctggca ccttctatga gctgctgcaa tttcacttcc acacgcccag tgagcaccag 360 caccaaggcc aacgtttccc agcagaaatt catttcgtcc atcgcagcga tcgcgatcag 420 ctcgctgtgg ttggtgtttt tctggcagcg ggcgatcgcc cccttccgat tctggacacc 480 ctcttagccg tgccaccgtc aactgacaat caactgcttt caacagccat tcaaccgaca 540 gacctcatgc cccgcgatcg caccgtctgg cgctactctg ggtctctaac aaccccaccc 600 tgctctgaac ccgttctctg gcgggtctgc gatcgcccac tgtttgtagc tcggcaacaa 660 ctccggcagc tccggcaacg gttgggtatg aatgctcgac cactgcaagc ctaa 714 <210> 3 <211> 945 ＜212＞ DNA ＜213＞ Populus euphratica ＜400＞ 3 atg agcctga cctcgcgcat cctgtttacc cccccgacca ccattaaact gtcgccgagc 60 ctgccccccc ccagcgcctt ttgtccgctg aaaacgccca gcctgtcgtt tatcagcccc 120 caccccagca ttctgcgctt cacccacatc accaagagcc ccagcagcct ctttgcccgc 180 gtgtttagca gctttaaccc caccgccacc accaccccgc ccaccatggg tgatgccccc 240 gatgcgggta tggatgcggt ccaacgccgc ctcatgtttg gcgacgaatg catcctggtc 300 gatgagaacg atcgcgtggt gggccacgac agcaagtata actgccacct gtgggaaaac 360 atcctgaagg gcaacgccct gcaccgcgcc tttagcgtct ttctcttcaa cagcaagcac 420 gagctgctcc tgcagcaacg cagcgcgacc aaagtcacct tccccctggt ctggaccaat 480 acctgctgca gccacccgct gtaccgcgag agcgaactga ttgacgaaga tgccctgggt 540 gtccgcaatg ccgcccagcg caaactgttt gatgaactgg gcatcccggc cgaagatgtg 600 cccgtggatc agtttagccc cctgggccgt attttataca aggcccccag cgatggcaaa 660 tggggcgagc acgagctcga ttacctgctc ttcattgtcc gcgatgtcag cgtgaacccg 720 aaccccgatg aggtggccga cattaagtac gtcaaccagg atgagctgaa ggagctgctg 780 cgcaaagcgg atgcgggcga agaaggcctg aaactgag cc cctggttccg cctggtcgtg 840 gataacttcc tgttcaagtg gtgggatcac gtggagaagg gcacgctgga agaagcggcc 900 gatatgaagg cgatccataa gctgcatcac caccatcacc actag 945 <210> 4 <211> 1647 <212> DNA <213> black poplar (Populus nigra) <400> 4 atgcgccgca gcgcgaacta cgagccgaac agctgggact atgactacct gctgtcgtcg 60 gacaccgacg agtcgatcga ggtgtacaag gacaaagcga agaagctgga agcggaagtg 120 cggcgcgaga tcaacaacga aaaggccgaa ttcctgaccc tgcccgaact catcgataac 180 gtccaacgcc tgggcctggg ctatcgcttc gagagcgata tccggcgggc cctggatcgc 240 ttcgtctcga gcggtggctt tgatgccgtc accaagacca gcctgcacgc caccgccctc 300 agcttccgcc tgctgcgcca acacggcttc gaagtcagcc aggaggcctt cagcggcttc 360 aaggaccaga acggcaactt cctgaagaac ctgaaggagg acattaaggc catcctgagc 420 ctctacgagg cctcgtttct ggccctcgag ggcgagaaca ttctggacga ggcgaaagtc 480 tttgcgatca gccacctgaa ggaactgagc gaggaaaaga tcggcaagga tctggcggaa 540 caggtgaacc acgccctgga actcccgctg catcgccgca cccaacgcct cgaggccgtc 600 t ggtcgatcg aggcctatcg caagaaggaa gatgccgacc aggtcctgct ggagctcgcc 660 atcctcgact acaacatgat ccaatcggtc taccagcgcg atctccgcga aacgagccgc 720 tggtggcgcc gggtcggtct cgccaccaaa ctgcatttcg cccgcgaccg cctgattgaa 780 agcttttact gggccgtggg cgtggccttc gaaccccaat acagcgactg ccgcaatagc 840 gtcgccaaga tgttctcgtt cgtgaccatc atcgatgaca tctacgacgt gtacggcacc 900 ctggatgaac tggaactctt cacggatgcc gtggagcgct gggatgtcaa tgccatcgat 960 gatctgcccg actacatgaa gctgtgcttc ctggccctct acaacaccat caacgagatc 1020 gcgtacgata atctgaagga caaaggcgaa aacattctgc cctacctcac gaaggcctgg 1080 gcggacctgt gcaatgcgtt cctgcaggag gcgaagtggc tgtacaacaa aagcaccccc 1140 acgttcgacg agtacttcgg caacgcgtgg aaaagcagca gcggccccct gcaactcgtc 1200 tttgcgtact tcgcggtggt gcagaacatc aaaaaggagg agatcgataa cctccagaag 1260 taccacgaca ttatcagccg gcccagccac atctttcgcc tgtgtaacga cctggccagc 1320 gcgagcgccg aaatcgcgcg cggtgagacc gccaactcgg tgagctgcta catgcgcacg 1380 aagggcatca gcgaggagct cgcgacggag tcggtg atga acctgatcga tgagacctgg 1440 aagaagatga acaaagagaa gctgggcggc agcctgttcg cgaagccctt tgtggaaacc 1500 gccatcaatc tggcgcgcca gagccattgc acctaccata acggcgatgc ccataccagc 1560 ccggatgagc tcacccgcaa acgcgtgctg agcgtcatca ccgaacccat cctcccgttc 1620 gaacgccacc atcatcacca ccactag 1647 <210> 5 <211> 1845 <212> DNA <213> Synechococcus elongatus (of Synechococcus elongatus PCC7942) <400> 5 ttagtagcgg ccgggacgat gaacgatgaa gctcacggtt tggcactgct tgatgttgtc 60 gaagccagcg acacggatgt agcaatcacc gtattcgctg cggcactcac gcacttcatc 120 gaggacttgc tgagggctct tgcagtcaaa cagggggagc ttccacatcg tccagtagaa 180 ctcttccgga ttcgagtgct cgttgaactc gatcaagggg tggaagcctt gctcgatcat 240 gtactcgatt tgtgcagcga tttggcgatc gctgagggga ggcaggtacg agaaagtctc 300 gaaacgacgc tctttgggca gagttttcat gctcatgatt cagaaatcct gaaaggttaa 360 gtcgaaaggc tggggagaaa gctttacgca gcaacgctca ctcccccagc gatagtcaga 420 ggctccttag agcttgtcca tcgtttcgaa ttcgaacttg atctctttcc agaggtcgag 480 ggcagcagcc agttcaggcg accacttgcc agcttcacga aggatgtcgc cgccttcacg 540 gtagaggtcg cgaccttcgt tccgagcttg gacgcaagct tccaaggcaa cacggttcgc 600 ggttgcacca ggagcattac cccaggggtg acccaaggtg ccgccaccga actggagaac 660 ggagtcatca ccgaagattt ccaccagtgc gggcatgtgc cacacgtgga taccaccgga 720 agcaaccggc agcacgcccg gcatcgacgc ccaatcttgg gtgaagaaga ccccacggct 780 gcggtcagct tcgatgtggt cttcgcgcat caagtcaaca aagcccaagg tcgaagcttt 840 gtcgccttcc agtttgccga cgacggtgcc ggagtggagg tggtcaccac cggacagacg 900 caaacacttg gccaagacac ggaagtgaat cccgtggtta cgctgacggt cgatcaccgc 960 gtgcattgca cggtggatgt gcagcaggac gccgttgtcg cggcaccatt ttgccaaggt 1020 ggtgttggcg gtgaaaccag ccgtcaagaa gtcatgcatg atgatcggca tgccgagttc 1080 tttagcgaac tcagcccgtt tcatcatttc ttcgcaggtc ggcgcggtca cgttcaggta 1140 gtgacctttg atttcaccgg tttctgcttg cgatttgtgg attgcatcag ccacaaacag 1200 gaagcgatcg cgccagcgtt ggaacggctg cgagttgatg ttttcgtcgt ctttggtgaa 1260 gtccag accg ccgcgcagac attcgtagac ggcacgaccg tagtttttcg ccgacagacc 1320 gagttttggt ttgatcgtgc aacccagcat cggacggccg tacttgttca gcaggtcgcg 1380 ctcgacttgg ataccgtggg gaggaccttg gaaggttttg accaaggcga cggggaagcg 1440 gatgtcttcc agacgcagcg aacggatagc tttgaagcca aacacgttac cgacgatcga 1500 ggtcaggatg ttggtgaccg acccttcttc aaacaggtcg agcgggtaag cgatgaacgc 1560 aaagtaggag ttctcttcgc cttgcaccgg ctcgatgtgg tagcacttgc ctttgtaccg 1620 atccatgtcg gtcagcaagt cggtccacac ggtggtccag gtaccggtcg aagattcagc 1680 cgcgatcgcc gcaccagctt cgtcagcagg gacacccggc tgagggctga agcggaaagc 1740 cgccagcagg tcagtgtctt tgggggtgta atcgggggtg taataggtga gtttgtagtc 1800 cttcaccccg gccttatagc ctgcggcaga ttgcgtcttg ggcat 1845 <210> 6 <211> 361 <212> DNA <213> Synechococcus elongatus (Synechococcus elongatus PCC7942) <400> 6 gcggctgaaa gtttcggact cagtagacct aagtacagag tgatgtcaac gccttcaagc 60 tagacgggag gcggcttttg ccatggttca gcgatcgctc ctcatcttca ataagcaggg 120 catgagccag cgttaagcaa atcaaatcaa a tctcgcttc tgggcttcaa taaatggttc 180 cgattgatga taggttgatt catgaggaat ctaaggctta attctccaca aaagaattaa 240 gcgtccgtcg caacggaatg ctccgctgga cttgcgctgt gggactgcag ctttacaggc 300 tccccctgcc agaaatcctg aatcgtcgag catatctgac atatctctag ggagagacga 360 c 361 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <400> 7 atgagcctga cctcgcgcat cctgt 25 <210> 8 <211> 25 <212> DNA <213> artificial sequence <400> 8 cagcttatgg atcgccttca tatcg 25 <210> 9 <211> 25 <212> DNA <213> artificial sequence <400> 9 atgcgccgca gcgcgaacta cgagc 25 <210> 10 <211> 25 <212> DNA <213> artificial sequence <400> 10 gcgttcgaac gggaggatgg gttcg 25 <210> 11 <211> 60 <212> DNA <213> artificial sequence <400> 11 aattgatatc tttaagaagg agatatacat atgactgtct ggcaaactct gacttttgcc 60 ＜210＞ 12 ＜211＞ 40 ＜212＞ DNA ＜213＞ Artificial sequence＜400＞ 12 aattga tatc ctacattttt tcgtctgaat gctcggcttc 40 <210> 13 <211> 60 <212> DNA <213> artificial sequence <400> 13 aattggatcc tttaagaagg agatatacat atgcgccggc gatcgctcct agcagctctt 60 <210> 14 <211> 40 <212><211> 40 <212><400> 14 aattggatcc ttaggcttgc agtggtcgag cattcatacc 40 <210> 15 <211> 30 <212> DNA <213> artificial sequence <400> 15 gactgcagct ttacaggctc cccctgccag 30 <210> 16 <211> 52 <212> DNA <213> artificial Sequence <400> 16 aattgatatc tttaagaagg agatatacat atgcccaaga cgcaatctgc cg 52 <210> 17 <211> 32 <212> DNA <213> Artificial sequence <400> 17 aattgatatc ttagtagcgg ccgggacgat ga 32

(no)

Claims (10)

A method for improving the yield of isoprene produced by transgenic blue-green bacteria, which is characterized by transforming an expression vector into genetically transformed blue-green bacteria, the expression vector having a nucleoside encoding a bicarbonate operator gene Acid sequence, and at least 90% sequence similarity to SEQ ID NO:1, and a nucleotide sequence encoding a carbonic anhydrase gene, and at least 90% sequence similarity to SEQ ID NO:2; and These genes are expressed in the transgenic blue-green bacteria. The method according to claim 1, wherein the transgenic blue-green bacteria has a plurality of foreign genes, which includes a nucleotide sequence encoding a prenyl diphosphate isomerase gene, and has the same sequence as SEQ ID NO: 3 At least 90% sequence similarity, and a nucleotide sequence encoding the isoprene synthase gene, and having at least 90% sequence similarity to SEQ ID NO: 4; In the performance of these genes. The method according to claim 1, wherein the transgenic blue-green bacteria is further transformed to embed a gene encoding ribulose 1,5-diphosphate carboxylase/oxygenase large and small subunits Nucleotide sequence, and has at least 90% sequence similarity with SEQ ID NO: 5; and express the gene in the transgenic blue-green bacteria. The method according to any one of claims 1 to 3, wherein the expression vector used to transform any of the genes into the blue-green bacteria transgenic gene is selected from pAM1573 transfection vector, pAM1303 transfection vector, pNS3 transfection vector Or a combination thereof. The method of claim 1, wherein the transgenic blue-green bacteria is selected from Synechococcus, Synechocystis, Thermophilic Blue-green Algae, Unicellular Cyanobacteria, Anabaena and Candida Any one or combination of groups. A genetically transformed cyanobacterium, whose genome has been transformed and embedded with a gene encoding a bicarbonate operator and having a sequence similarity of at least 90% to SEQ ID NO: 1, a gene encoding carbonic anhydrase and having SEQ ID NO: 2 at least 90% sequence similarity, a gene encoding exogenous isopentenyl diphosphate isomerase, and having at least 90% sequence similarity to SEQ ID NO: 3, and a coding exogenous Sex isoprene synthase gene, and it has at least 90% sequence similarity with SEQ ID NO: 4; and express these genes in the transgenic blue-green bacteria. The transgenic blue-green bacteria as described in claim 6, which is further transformed with a nucleoside encoding a gene encoding ribulose 1,5-diphosphate carboxylase/oxygenase large and small subunits The acid sequence has a sequence similarity of at least 90% with SEQ ID NO: 5; and the gene is expressed in the transgenic blue-green bacteria. The transgenic blue-green bacteria according to claim 2 or 6, which has been transformed and embedded with at least one ribulose 1,5-diphosphate carboxylase/oxygenase large subunit (PrbcL) promoter, and The gene encoding the bicarbonate operator and the gene encoding the carbonic anhydrase are each embedded in the downstream of a ribulose 1,5-diphosphate carboxylase/oxygenase large subunit (PrbcL) promoter, and/or The gene encoding exogenous isoprenyl diphosphate isomerase and the gene encoding exogenous isoprene synthase are both embedded in a 1,5-diphosphate ribulose carboxylase/oxygenase major Unit (PrbcL) downstream of the promoter. A method for producing isoprene, characterized by cultivating the transgenic blue-green bacteria as described in claim 6. The method according to claim 9, wherein the culturing step includes adding at least 30 mM bicarbonate or carbonate to the culture solution.

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