Ralstonton recombinant strain for producing isobutanol by using fructose and carbon dioxide as well as construction method and application of Ralstonton recombinant strain

Abstract

The invention relates to construction of a Ralstonia strain capable of producing isobutanol by utilizing fructose and carbon dioxide and application of the Ralstonia strain. The construction method comprises the following steps: firstly, knocking out bkdA and bkdB genes in tricarboxylic acid cycle of the Ralstonia H16 to obtain a recombinant strain H1601; then, a PHB operator phaCAB in the H1601 is knocked out, and an alsS gene of bacillus subtilis and ilvC and ilvD genes of Ralstonia are introduced into the site, so that a recombinant strain H1602 is obtained. And finally, introducing yqhD and kivD gene expression cassettes of escherichia coli into the H1602, and constructing a recombinant strain H1603 capable of synthesizing isobutanol by using fructose and carbon dioxide. The strain can be used for synthesizing isobutanol through heterotrophic or autotrophic fermentation by taking carbon dioxide and fructose as carbon sources, the operation is simple and convenient, and higher yield can be obtained.

Description

Technical Field

[0001] The invention relates to the field of biosynthesis, in particular to the construction and application of a Ralstonia strain capable of producing isobutanol by utilizing fructose and carbon dioxide. Background Art

[0002] As an important microbial metabolite, isobutanol has been widely used in many fields, such as food, medicine and chemical industry. Isobutanol can be mixed with gasoline or diesel at any concentration and can be used directly without modifying the car engine. This makes it not only an ideal additive for existing fuels, but also an alternative fuel. In addition, isobutanol is also an efficient solvent and food flavoring, and is recognized as one of the food flavorings allowed for use by GB 2760-2014. It can also be used to produce chemical products such as petroleum additives, antioxidants, plasticizers, synthetic rubber and drugs. Another important use of isobutanol is as a precursor of isobutylene, which is widely used in rubber, specialty chemicals and refining industries. The global isobutanol market has exceeded US$1 billion and is growing at an annual rate of 6.4%.

[0003] Ralstonia Ralstonia eutropha ) is a facultative chemoautotrophic Gram-negative bacterium that can utilize a variety of carbon sources, such as fructose and fat for heterotrophic growth, and can also grow autotrophically in a mixture of hydrogen, carbon dioxide and oxygen. Existing high-yield isobutanol fermentation strains, such as Bacillus subtilis and Clostridium, are mostly Class II pathogens and are not suitable for large-scale industrial applications. The use of expensive glucose as a fermentation substrate also limits the economic feasibility of isobutanol production. Ralstonia has become an ideal choice for reducing the cost of isobutanol production due to its rich metabolic pathways and ability to utilize a variety of substrates (such as carbon dioxide, fructose, formic acid, etc.) Summary of the invention

[0004] The present invention aims to solve the deficiencies in the prior art and provide a recombinant strain of Ralstonia that can synthesize isobutanol from fructose and carbon dioxide. The present invention also provides a method for constructing the recombinant strain and its application in isobutanol production.

[0005] The first object of the present invention is to provide a method for constructing a recombinant Ralstonia strain that produces isobutanol using fructose and carbon dioxide.

[0006] The second object of the present invention is to construct a recombinant strain of Ralstonia that produces isobutanol using fructose and carbon dioxide.

[0007] The third object of the present invention is to construct a recombinant strain of Ralstonia that uses hydrogen generated by CoP-doped C3N4 under light-driven conditions as an electron donor and uses carbon dioxide to de novo synthesize isobutanol for use in the production of isobutanol.

[0008] The technical solution of the present invention is summarized as follows: 1. The TCA cycle in Ralstonia bkdA and bkdB The gene was knocked out to obtain the recombinant strain H16_01.

[0009] 2. Knockout of the PHB operon in the recombinant strain H16_01 phaCAB (Include phaA , phaB1 and phaC1 ), and introduced the Bacillus subtilis from alsS The recombinant strain H16_02 was constructed by expressing the ilvC and ilvD genes of Ralstonia spp.

[0010] 3. Introduction of E. coli into the recombinant strain H16_02 yqhD and kivD The gene expression cassette was finally obtained, and the recombinant Ralstonia strain H16_03 capable of producing isobutanol from fructose and carbon dioxide was obtained.

[0011] Said bkdA The nucleotide sequence of the gene is shown in SEQ ID NO.1; bkdB The nucleotide sequence of the gene is shown in SEQ ID NO.2; phaA The nucleotide sequence of the gene is shown in SEQ ID NO.3; phaB1 The nucleotide sequence of the gene is shown in SEQ ID NO.4; phaC1 The nucleotide sequence of the gene is shown in SEQ ID NO.5; α-acetolactate synthase alsS The nucleotide sequence is shown in SEQ ID NO.6; alpha-ketoacid reductoisomerase ilvC The nucleotide sequence is shown in SEQ ID NO.7; dihydroxyacid dehydratase ilvD The nucleotide sequence is shown in SEQ ID NO.8; Alcohol dehydrogenase yqhD The nucleotide sequence is shown in SEQ ID NO.9; α-Ketoisovalerate decarboxylase kivD The nucleotide sequence is shown in SEQ ID NO.10; The Ralstonia recombinant strain constructed by the above method can produce isobutanol by utilizing fructose and carbon dioxide.

[0012] Beneficial effects of the present invention: The Ralstonia recombinant strain of the present invention can utilize a variety of renewable resources for fermentation due to its metabolic versatility. Through the optimization of metabolic engineering, the utilization range of the strain for different substrates is expanded. The strain can quickly reach a higher cell density, and with the continuous development of genetic tools, it has become an ideal platform strain widely used in biosynthesis. Although the production cost of isobutanol is still high, the recombinant strain of the present invention can be used to ferment and synthesize isobutanol using relatively low-priced fructose and carbon dioxide, which can not only reduce costs but also expand the scope of substrate utilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The synthetic pathway of 2,3-butanediol in the recombinant Ralstonia strain; Figure 2 The isobutanol production-time graph of the recombinant strain H16_03 of Ralstonia spp. for producing isobutanol from fructose and carbon dioxide in fructose fermentation; Figure 3 A comparison of isobutanol production by the recombinant Ralstonia strain H16_03 in gas fermentation using fructose and carbon dioxide.

[0014] Figure 4 This is a comparison of the isopentanol production of the recombinant Ralstonia strain H16_03 that produces isobutanol using fructose and carbon dioxide under different inducer concentrations. DETAILED DESCRIPTION

[0015] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0016] Original wild type Ralstonia Ralstonia eutropha H16, purchased from the ATCC official website in October 2016.

[0017] Origin of genes: bkdA Gene: From Ralstonia Ralstonia eutropha H16) bkdB Gene: From Ralstonia Ralstonia eutropha H16) phaA Gene: From Ralstonia Ralstonia eutropha H16) phaB1 Gene: From Ralstonia Ralstonia eutropha H16) phaC1 : From Ralstonia Ralstonia eutropha H16) α-acetolactate synthase alsS : From Bacillus subtilis ( Bacillus subtilis 168) alpha-ketoacid reductoisomerase ilvC : From Ralstonia Ralstonia eutropha H16) dihydroxyacid dehydratase ilvD : From Ralstonia Ralstonia eutropha H16) Alcohol dehydrogenase yqhD :From Escherichia coli ( Escherichia coli ) α-Ketoisovalerate decarboxylase: from Ralstonia Escherichia coli ) Implementation Cases: 1. Module construction A method for constructing a recombinant strain of Ralstonia that produces isobutanol using fructose and carbon dioxide comprises the following steps: Knockout of 2-oxoisovalerate dehydrogenase gene in TCA cycle by site-specific homologous recombination bkdA、bkdB , and obtained the recombinant strain H16_01.

[0018] Knockout of PHB degradation-related operons using site-specific homologous recombination phaCAB (Include phaA , phaB1 ,and phaC1 ), and used site-specific homologous recombination to Bacillus subtilis 168 genes encoding acetoin synthesis alsS and α-ketoacid synthesis genes from wild-type strains of Ralstonia ilvC and ilvD The gene expression cassette was integrated into this site to obtain the recombinant strain H16_02.

[0019] Using the combined transfer method, the code from E.coli Alcohol dehydrogenase ( yqhD ) and α-ketoisovalerate decarboxylase ( kivD ) was introduced into the production strain H16_02 to obtain the recombinant strain H16_03.

[0020] bkdA The nucleotide sequence of the gene is shown in SEQ ID NO.1; bkdB The nucleotide sequence of the gene is shown in SEQ ID NO.2; phaA The nucleotide sequence of the gene is shown in SEQ ID NO.3; phaB1The nucleotide sequence of the gene is shown in SEQ ID NO.4; phaC1 The nucleotide sequence of the gene is shown in SEQ ID NO.5; α-acetolactate synthase alsS The nucleotide sequence is shown in SEQ ID NO.6; alpha-ketoacid reductoisomerase ilvC The nucleotide sequence is shown in SEQ ID NO.7; dihydroxyacid dehydratase ilvD The nucleotide sequence is shown in SEQ ID NO.8; Alcohol dehydrogenase yqhD The nucleotide sequence is shown in SEQ ID NO.9; α-Ketoisovalerate decarboxylase kivD The nucleotide sequence is shown in SEQ ID NO.10; The specific steps are: (1) A method for constructing a recombinant Ralstonia strain H16_01 that produces isobutanol using fructose and carbon dioxide, comprising the following steps: The screening resistance gene KanR comes from plasmid pK18mobSacB (purchased from Addgene, Inc. WWW.addgene.org).

[0021] Obtained from NCBI database Ralstonia eutropha H16-derived genes bkdA、bkdB The gene sequence of Ralstonia eutropha On the H16 genome, bkdA、bkdB Located in the same operon bkdAB Therefore, a knockout plasmid was constructed containing these two genes.

[0022] bkdAB The knockout plasmid of gene was wild type Ralstonia eutropha H16 genome was used as template. bkdAB -up and bkdAB -down as the homologous upper arm and homologous lower arm (SEQ ID NO.11 and SEQ ID NO.12), respectively. bkdAB up-F (SEQ ID NO. 13), bkdAB up-R (SEQ ID NO. 14) and bkdAB down-F (SEQ ID NO.15), bkdAB down-R (SEQ ID NO. 16) is a primer to amplify the homology arms bkdAB- up (SEQ ID NO. 11) and bkdABdown (SEQ ID NO. 12); The DNA PCR enzyme used in the present invention is Fast Pfu polymerase from Quanshijin Biotechnology Co., Ltd. The 50 μL PCR amplification system is as follows: DNA template, 1 μL; 2.5 μL each of the front primer (10 μM) and the rear primer (10 μM); 5 μL of dNTP (10 mM); 10 μL of 5×PfuBuffer; 1 μL of Fast Pfu polymerase; and finally, double distilled water is used to make up to 50 μL.

[0023] The amplification program was set on the PCR instrument. The amplification conditions were 95°C pre-denaturation for 2 min (1 cycle); 95°C denaturation for 20 sec, 55°C annealing for 20 sec, 72°C extension for 30 s (35 cycles); 72°C extension for 5 min (1 cycle).

[0024] Each pair of homologous upper and lower arm fragments obtained by PCR amplification was mixed with the pK18mobSacB linearized suicide plasmid vector cut with HindIII and EcoRI restriction endonucleases, and the three fragments were spliced ​​into a complete recombinant vector using the BM Seamless Connection Kit (Bomed Biotechnology Co., Ltd.). The molar ratio of each fragment was 1:1, and the total amount of fragments reached 1 μg. The connection volume was as follows: DNA fragment 5μL, 2× Seamless Cloning Mix 5μL. The connection program was set on the PCR instrument. The connection conditions were 50℃ 15min, 16℃ 10min.

[0025] The recombinant vector pK18mobSacB- bkdAB The cells were introduced into Trans1-T1 Phage Resistant Chemically Competent Cell produced by Quanshijin Company by heat shock method, and single colonies were grown on LB-Kan50 agar plates (10 g / L NaCl, 10 g / L tryptone and 50 g / L yeast extract, 50 g / L kanamycin was added at 1:1000). After incubation at 37℃ 220 rpm for 12 h, an appropriate number of single colonies were picked and cultured in LB-Kan50 liquid medium at 37℃ ℃ After incubation at 220 rpm for 12 h, the samples were sent to GeneWeichi for sequencing.

[0026] After the sequencing results were correct, the constructed knockout plasmid was extracted using the kit of Quanshijin Company, and the heat shock method was still used to introduce it into the competent state of the s17-1 strain prepared in advance by chemical methods. Single colonies grew on LB-Kan50 agar plates (10 g / L NaCl, 10 g / L tryptone and 50 g / L yeast extract, 50 g / L kanamycin was added at 1:1000). After incubation at 37℃ 220 rpm for 12 h. Pick an appropriate number of single colonies, grow them in LB-Kan50 liquid culture medium, incubate them at 37℃ 220 rpm for 12 h, and send them to Jinweizhi Company for sequencing.

[0027] After the sequencing results were confirmed, the s17-1 strain with the correct sequencing was cultured in LB-Kan50 liquid medium at 37°C and 220 rpm until OD 600 = 0.6-0.8, take 1 mL and add the same volume and concentration of wild type Ralstonia eutropha H16 was resuspended in 200 μL sterile water by centrifugation, and finally mixed at a ratio of 2:3, and incubated at 30°C and 230 rpm for 36-48 h in NB agarose medium (0.5 g / L (NH4)2SO4, 10 g / L tryptone, 10 g / L yeast extract, 0.6 g / L beef extract powder) to allow the constructed knockout plasmid to be introduced into the wild-type Ralstonia eutropha H16.

[0028] After the mixed strains grow to a suitable state, scrape an appropriate amount of the mixed strains and resuspend them in 50 μL of sterile water. After diluting them to an appropriate multiple, culture them on NB-Kan200+Gm10 agarose (NB medium with 200 g / L penicillin resistance and 10 g / L gentamicin resistance added at 1:1000) medium at 30°C 230 rpm for the initial screening of single exchange. The single colonies grown were used for the initial verification of the results of the single exchange using the colony PCR method.

[0029] The colony PCR enzyme used in the present invention is Fastaq polymerase from Quanshijin Biotechnology Co., Ltd. The 15 μL PCR amplification system is as follows: 0.3 μL each of the front primer (10 μM) and the back primer (10 μM); 1.2 μL of dNTP (10 mM); 1.5 μL of 10×FastaqBuffer; 0.15 μL of Fastaq polymerase; 10.15 μL of double distilled water, and finally pick an appropriate amount of colonies.

[0030] The amplification program was set on the PCR instrument. The amplification conditions were 94°C pre-denaturation for 5 min (1 cycle); 95°C denaturation for 30 sec, 58°C annealing for 20 sec, 72°C extension for 30 sec (30 cycles); 72°C extension for 5 min (1 cycle).

[0031] Select a single colony after successful verification, culture it in NB liquid culture medium at 30℃ 280 rpm for 6 h, then culture it on NB-15% sucrose solid culture medium (10 g / L tryptone, 5 g / L yeast extract, 150 g / L sucrose, 15 g / L agar powder) at 30℃ 230 rpm to an appropriate size, verify the result of the second exchange by colony PCR, and obtain the desired knockout bacteria.

[0032] Knockout once by the above method bkdA、bkdB Finally, the recombinant Ralstonia strain H16_01 that produces isobutanol using fructose and carbon dioxide was obtained.

[0033] (2) A method for constructing a recombinant Ralstonia strain H16_02 that produces isobutanol using fructose and carbon dioxide, comprising the following steps: The screening resistance gene KanR comes from plasmid pK18mobSacB (purchased from Addgene, Inc. WWW.addgene.org).

[0034] Obtained from NCBI database Ralstonia eutropha H16-derived genes phaCAB (Include phaA , phaB1 ,and phaC1 ) gene sequence and construct a plasmid to knock out the operon.

[0035] Obtained from NCBI database Bacillus subtilis 168 genes from α-acetolactate decarboxylase alsD , the nucleotide sequence (SEQ ID NO.6) was obtained by artificial synthesis; Obtained from NCBI database Ralstonia eutropha H16-derived gene α-ketoacid reductoisomerase ilvC , the nucleotide sequence (SEQ ID NO.7) was obtained by artificial synthesis; Obtained from NCBI database Ralstonia eutropha H16-derived dihydroxyacid dehydratase ilvD , the nucleotide sequence (SEQ ID NO.8) was obtained by artificial synthesis; Construct the expression cassettes for these two genes: α-Acetolactate decarboxylase alsD, α-ketoacid reductoisomerase ilvC , dihydroxyacid dehydratase ilvD The expression cassette uses P BAD Promoter and rrnB T1 terminator; by site-specific homologous recombination, to the recombinant strain H16_02 of Ralstonia that produces isobutanol using fructose and carbon dioxide phaCAB α-acetolactate decarboxylase alsD , α-ketoacid reductoisomerase ilvC , dihydroxyacid dehydratase ilvD ; phaCAB The knockout plasmid of the operon is based on the wild type Ralstonia eutropha H16 genome was used as template. phaC -up and phaB -down as the homologous upper arm and homologous lower arm (SEQ ID NO.17 and SEQ ID NO.18), respectively. phaC up-F (SEQ ID NO. 19), phaC up alsSD -R (SEQ ID NO. 20) and phaB down alsSD -F (SEQ ID NO. 21), phaB down-R (SEQ ID NO. 22) is a primer to amplify the homology arms phaC- up (SEQ ID NO. 11) and phaB- down (SEQ ID NO. 12); Synthetic α-acetolactate decarboxylase alsD Expression plasmid PHG12-pBAD- alsS As a template, phaCAB - alsS-F (SEQ ID NO. 23) and alsS-ilvCD-R (SEQ ID NO. 24) primer, amplify the BAD alpha acetolactate decarboxylase promoter and rrnB T1 terminator alsD Gene expression cassette P BAD - alsS .

[0036] Wild-type Ralstonia eutropha The H16 genome was used as a template. alsS- ilvCD-F (SEQ ID NO. 25) and phaCAB - ilvCD-R (SEQ ID NO.26) primer, amplify α-ketoacid reductoisomerase containing rrnB T1 terminator ilvC , dihydroxyacid dehydratase ilvD Gene expression cassette P BAD - ilvCD .

[0037] After the above PCR amplification, P BAD - alsS With P BAD - ilvCD The BM seamless ligation kit (Bomed Biotech Co., Ltd.) was used to splice into a complete gene expression cassette P BAD - alsS-ilvCD The molar ratio of each fragment is 1:1, the total amount of fragments reaches 1 μg, and the ligation volume is as follows: 5 μL DNA fragment, 5 μL 2× Seamless Cloning Mix. Set the ligation program on the PCR instrument. The ligation conditions are 50℃ 15min, 16℃ 10min.

[0038] The PCR amplified phaC up、 phaB down、pBAD- alsS - ilvCD The four fragments were mixed with the pK18mobSacB linearized suicide plasmid vector cut with HindIII and EcoRI restriction endonucleases and spliced ​​into a complete recombinant vector using the BM Seamless Ligation Kit (Bomed Biotechnology Co., Ltd.). The molar ratio of each fragment was 1:1, and the total amount of fragments reached 1 μg. The ligation volume was as follows: 5μL DNA fragment, 5μL 2× Seamless Cloning Mix. The ligation program was set on the PCR instrument. The ligation conditions were 50℃ 15min, 16℃ 10min.

[0039] The obtained recombinant vector was introduced into the recombinant strain H16_01 of Ralstonia spp. that produces isobutanol using fructose and carbon dioxide. The above-mentioned homologous recombination principle was used to clone the recombinant vector in the H16_01 genome. phaCBA α-acetolactate decarboxylase alsD , α-ketoacid reductoisomerase ilvC , dihydroxyacid dehydratase ilvD The gene sequence was obtained to obtain the recombinant Ralstonia strain H16_02 that produces isobutanol using fructose and carbon dioxide.

[0040] (3) A method for constructing a recombinant Ralstonia strain H16_03 that produces isobutanol using fructose and carbon dioxide, comprising the following steps: Obtained from NCBI database E.coli Alcohol dehydrogenase ( yqhD ) gene, and the nucleotide sequence (SEQ ID NO.9) was obtained by artificial synthesis; Obtained from NCBI database E.coli and α-ketoisovalerate decarboxylase ( kivD ) gene, and the nucleotide sequence (SEQ ID NO.10) was obtained by artificial synthesis; yqhD Gene expression cassette using P BAD promoter and rrnB T1 terminator; kivD Gene expression cassette using P BAD promoter and rrnB T1 terminator; The recombinant strain H16_02 of Ralstonia spp. that produces isobutanol from fructose and carbon dioxide was introduced by conjugative transfer. E.coli Alcohol dehydrogenase ( yqhD ) and α-ketoisovalerate decarboxylase ( kivD ) expression plasmid, and obtained the Ralstonia recombinant strain H16_03 that produces isobutanol using fructose and carbon dioxide (see Figure 1 ).

[0041] The synthetic alcohol dehydrogenase ( yqhD ) and α-ketoisovalerate decarboxylase ( kivD ) gene expression plasmid PHG-pBAD- yqhD-kivD The cells were introduced into Trans1-T1 Phage Resistant Chemically Competent Cell produced by Quanshijin Company through heat shock method, and single colonies were grown on LB-Kan50 agar plates (10 g / L NaCl, 10 g / L tryptone and 50 g / L yeast extract, 50 g / L kanamycin was added at 1:1000). After incubation at 37℃ 220 rpm for 12 h. An appropriate number of single colonies were picked, cultured in LB-Kan50 liquid medium, incubated at 37℃ 220 rpm for 12 h, and sent to Genwis for sequencing.

[0042] After the sequencing results were correct, the constructed expression plasmid was extracted using the kit of Quanshijin Company, and the heat shock method was still used to introduce it into the competent state of the s17-1 strain prepared in advance by chemical methods. The single colony grew on the LB-Kan50 agar plate (10 g / L NaCl, 10 g / L tryptone and 50 g / L yeast extract, 50 g / L kanamycin was added at 1:1000). After incubation at 37℃ 220 rpm for 12 h. Pick an appropriate number of single colonies, grow them in LB-Kan50 liquid culture medium, incubate them at 37℃ 220 rpm for 12 h, and send them to Jinweizhi Company for sequencing.

[0043] After the sequencing results were confirmed, the s17-1 strain with correct sequencing was cultured in LB-Kan50 liquid medium at 37°C and 220 rpm until OD 600 = 0.6-0.8, take 1mL and add the same volume and concentration of wild type Ralstonia eutropha H16 was resuspended in 200 μL sterile water by centrifugation, and finally mixed at a ratio of 2:3, and incubated at 30°C and 230 rpm for 36-48 h in NB agarose medium (0.5 g / L (NH4)2SO4, 10 g / L tryptone, 10 g / L yeast extract, 0.6 g / L beef extract powder), so that the constructed expression plasmid was introduced into the constructed Ralstonia recombinant strain H16_02 that produces isobutanol using fructose and carbon dioxide, thereby obtaining the Ralstonia recombinant strain H16_03 that produces isobutanol using fructose and carbon dioxide.

[0044] 2. Fermentation method Seed culture medium: 10 g / L yeast extract, 10 g / L tryptone, 6 g / L beef extract powder, 5 g / L ammonium sulfate, and the rest is water.

[0045] Fructose culture medium: fructose 10 g / L, Na2HPO4 3.57 g / L, KH2PO4 1.5 g / L, ammonium sulfate 0.167 g / L, sodium bicarbonate 0.02 g / L, and the rest is water.

[0046] CO2 gas fermentation medium: Na2HPO4 3.57 g / L, KH2PO4 1.5 g / L, ammonium sulfate 0.167 g / L, sodium bicarbonate 0.02 g / L, and the rest is water.

[0047] Inducer: Arabinose Control group: wild type Ralstonia eutropha H16 was inoculated with 4 mL of seed culture medium and cultured at 30°C 230 rpm for 24 h. Then 3 mL of bacterial culture was transferred to 60 mL of fresh fructose culture medium and cultured at 30°C 230 rpm until the stationary phase. The culture medium was taken for product analysis.

[0048] Experimental Group: (1) The recombinant strain H16_03 was inoculated into 4 mL of seed culture medium at 30°C and 230 rpm for 24 h. Then 3 mL of the bacterial culture was transferred to 60 mL of fresh fructose culture medium containing different arabinose concentrations at 30°C and 230 rpm. The culture was cultured until the plateau phase. 1 mL of the culture medium was taken at regular intervals to detect the OD value of the solution. 600 The remaining solution was centrifuged and the supernatant was taken for product analysis.

[0049] (2) The recombinant strain H16_03 was inoculated into 4 mL of seed culture medium at 30°C and 230 rpm for 24 h. Then 3 mL of the bacterial solution was transferred into 60 mL of fresh CO2 gas fermentation medium at 30°C and 230 rpm for cultivation until the plateau phase. 1 mL of the culture solution was taken at regular intervals to detect the OD value of the solution. 600 The remaining solution was centrifuged and the supernatant was taken for product analysis.

[0050] 3. Product Analysis Isobutanol is the target product of this experiment, and isopentanol is the by-product of this experiment, which is detected using a high performance liquid chromatography.

[0051] Use a disposable syringe with a capacity of 1 mL to transfer 200 μL of supernatant to the gas phase inlet bottle, cover the lid and put it into the FID gas detector to prepare for injection. The instrument parameters are as follows: the gas chromatography column uses a DB-WAX column (0.25 mm×30 m), the column oven temperature is 35°C; the injection port temperature is 240°C, the column temperature is 230°C; the injection volume is 10 μL; the carrier gas is N2, the purge flow rate is 35 mL / min, no splitting, and the tail flow rate is 350 mL / min.

[0052] Content calculation: Draw a standard curve based on the test results of isobutanol and isopentanol standards, and substitute the test results into the standard curve to calculate the isobutanol and isopentanol contents in the sample.

[0053] 4. Results In this study, we first used homologous recombination to knock out two genes related to pyruvate production. bkdA、bkdB In order to introduce the isobutanol biosynthesis pathway and further increase the biomass in this pathway, α-acetolactate decarboxylase was mutated by site-specific homologous recombination. alsD , α-ketoacid reductoisomerase ilvC , dihydroxyacid dehydratase ilvD The gene expression cassette was integrated into the genome of the isobutanol-producing strain H16_01 phaCAB The strain H16_02 was obtained, and then the gene encoding alcohol dehydrogenase ( yqhD ) and α-ketoisovalerate decarboxylase ( kivD ) was introduced into the production strain H16_02 to obtain the recombinant strain H16_03. After 36 hours of fermentation in fructose fermentation medium containing 0.025% arabinose inducer, the recombinant strain H16_03 finally obtained 39.8 mg of isobutanol, proving that the strain was successfully constructed. At the same time, under gas fermentation, 8.3 mg of isobutanol and isopentanol were produced in 120 hours, proving its ability to synthesize isobutanol using CO2 autotrophic fermentation.

Claims

1. A method for constructing a recombinant strain of Ralstonia for synthesizing isobutanol from fructose and carbon dioxide, characterized in that: The following steps are involved: (1) knocking out the bkdA and bkdB genes in the tricarboxylic acid cycle of Ralstonia to obtain the recombinant strain H16_01; (2) The PHB degradation operon phaCAB (containing phaA, phaB1, and phaC1) in the recombinant strain H16_01 was knocked out, and the alsS gene from Bacillus subtilis 168 and the ilvC and ilvD genes from Ralstonia were introduced into the site to construct the recombinant strain H16_02; (3) The yqhD and kivD gene expression cassettes in Escherichia coli were introduced into the recombinant strain H16_02 to construct the Ralstonia recombinant strain H16_03 that can synthesize isobutanol from fructose and carbon dioxide. The nucleotide sequence of the bkdA gene is shown in SEQ ID NO.1; The nucleotide sequence of the bkdB gene is shown in SEQ ID NO.2; The nucleotide sequence of the phaA gene is shown in SEQ ID NO. 3; The nucleotide sequence of the phaB1 gene is shown in SEQ ID NO.4; The nucleotide sequence of the phaC1 gene is shown in SEQ ID NO.5; The nucleotide sequence of α-acetolactate synthase alsS is shown in SEQ ID NO.6; The nucleotide sequence of α-ketoacid reductoisomerase ilvC is shown in SEQ ID NO.7; The nucleotide sequence of dihydroxyacid dehydratase ilvD is shown in SEQ ID NO.8; The nucleotide sequence of alcohol dehydrogenase yqhD is shown in SEQ ID NO.9; The nucleotide sequence of α-ketoisovalerate decarboxylase kivD is shown in SEQ ID NO.

10.

2. The construction method according to claim 1, wherein the constructed recombinant Ralstonia strain is used to produce isobutanol from fructose and carbon dioxide.

3. A method for producing isobutanol using the recombinant strain according to claim 2, characterized in that: The recombinant strain is fermented in a fermentation medium with fructose or carbon dioxide as a carbon source to synthesize isobutanol.

4. The production method according to claim 3, characterized in that: The formula of fructose fermentation medium is: fructose 10 g / L, Na2HPO4 3.57 g / L, KH2PO4 1.5 g / L, (NH4)SO4 0.167 g / L, NaHCO3 0.02 g / L, and the balance is water; the formula of carbon dioxide gas fermentation medium is: Na2HPO4 3.57 g / L, KH2PO4 1.5 g / L, (NH4)SO4 0.167 g / L, NaHCO3 0.02 g / L, and the balance is water.

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