KR101743603B1 - Method for Isobutanol production from an engineered Shewanella oneidensis - Google Patents
Method for Isobutanol production from an engineered Shewanella oneidensis Download PDFInfo
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Abstract
The present invention relates to a method for increasing the productivity of isobutanol through improved production and supply of isobutanol from Shewanella oneidensis , and more particularly, to a method for increasing the productivity of isobutanol through N-acetylglucosamine (NAG) , Pyruvate and lactate as a carbon source to produce isobutanol from a strain of Schwannella onesidense and a method for producing iso-butanol from an externally supplied electricity using an electric reactor, The present invention relates to a method for significantly increasing the productivity of isobutanol from onionidensis.
Description
The present invention relates to improved Shewanella < RTI ID = 0.0 > oneidensis ), and a method for increasing the productivity of isobutanol by supplying electricity to the improved Schwannellaonodesis using an electric reactor from the outside.
Isobutanol is one of the structural isomers of butanol that is widely used in conventional petroleum-based alcohols. It is usually produced naturally during the fermentation of carbohydrates in microorganisms. Chemical methods are also obtained by oxo - or propylene - based synthesis or by air oxidation of propene and butane. It can be obtained as a by-product in the synthesis of methanol, and is used not only as a starting material for organic synthesis but also as a solvent in chemical reactions. It is also used as a raw material for ester synthesis for fruit essence and as a solvent for paints or varnishes.
Because of this wide range of use, Hanwha Chemical and LG Chemical are currently mass-producing them in Korea through chemical synthesis methods. In addition, researches for the development and industrialization of isobutanol production technology based on bios have continued.
However, the production process by chemical synthesis of isobutanol is not only using petroleum as a raw material but also producing a large amount of organic solvent and toxic substance as a by-product, thereby causing environmental pollution problem.
Therefore, in order to replace such a petrochemical-derived isobutanol production method, research on the production of microorganism-derived iso-butanol has been carried out. Especially, Escherichia The production of isobutanol using microorganisms such as E. coli , Ralstonia eutropha and Bacillus subtilis has been extensively studied, and most of the production routes of isobutanol in organisms have been found.
Various efforts have been made to increase the productivity of isobutanol by introducing the isobutanol production pathway into various industrial strains or by removing genes related to the byproduct production pathway through a metabolic engineering approach in order to secure more advanced technology for producing isobutanol However, due to the limitations of existing genetic engineering and metabolic engineering approaches, various attempts have been made to optimize other substrates and to optimize them in the culture process, to extract after production, and to develop purification technology.
The problem of conventional isobutanol production technology has been mainly studied for the purpose of developing strains through genetic engineering and metabolic engineering approaches. However, the construction and optimization of the metabolic pathway through it is very difficult, It has to be totally dependent on its metabolic pathway and yield.
In the isobutanol production pathway, NAD (P) H is required as a cofactor. The cofactor is involved in the regulation of productivity of major metabolites. NAD (P) H / NAD + ratio, production yield of the target product is greatly affected. Therefore, in order to increase the concentration of NAD (P) H in the microorganism, studies were conducted mainly to remove the overexpression of related genes or the pathway consuming NAD (P) H.
Accordingly, the present inventors have made intensive efforts to develop a strain and a production method that produce more efficient iso-butanol than the existing technology, and as a result, have found that the technology capable of producing isobutanol from Shewanella oneidensis and the introduction of an electric reactor The present inventors have completed the present invention by developing a technique for increasing the productivity through cofactor conversion through the use of a catalyst.
It is an object of the present invention to provide an improved strain of Shewanella oneidensis for the production of isobutanol.
It is another object of the present invention to provide a method for efficiently producing isobutanol from the improved Schwannella annenidisc.
It is another object of the present invention to provide a method for increasing the production of isobutanol through cofactor conversion by introducing an electric reactor into the improved Schwannella donepensis.
In order to accomplish the above object, the present invention provides a method for producing ketoisovalerate decarboxylase ( kivD ) gene and alcohol dehydrogenase ( adh ) gene in Shewanella oneidensis And an isobutanol productivity is increased, and a recombinant Schwannella ondensis strain is provided.
The present invention also relates to a method for producing isobutanol from a recombinant Schwannella ondensis strain comprising culturing a recombinant Schwannella ondensis strain according to the present invention and recovering isobutanol from the cultured medium ≪ / RTI >
The present invention also relates to a method for producing a recombinant Schwannella enteric strain according to the present invention in a culture medium containing NAG (N-acetylglucosamine), pyruvate or lactate as a carbon source, or a combination of two or more thereof Culturing, and recovering isobutanol. The present invention also provides a method for increasing isobutanol production from a recombinant Schwannella ondensis strain.
The present invention also relates to a method for producing recombinant Schwannella ondensis strain, which comprises culturing a recombinant Schwannella ondensis strain according to the present invention while supplying electricity using an electric reactor, and then recovering isobutanol, Thereby providing a method for increasing butanol production.
In addition, the present invention relates to an optimal combination and optimum concentration of carbon sources (NAG is 1 to 4% (w / v), pyruvic acid is 0.5 To 1.5% (w / v) of lactic acid, and 1-2% (w / v) of lactic acid) and then recovering the isobutanol from the recombinant Schwannella ondensis strain Provides a method for increasing iso-butanol production.
The invention optimum conditions of carbon source using the improved S. oneidensis, by being cultivated in a culture medium containing (optimum composition and concentration), S. oneidensis improved to increase about 4-5 times is less productive of isobutanol from Also, the productivity of isobutanol can be increased about 2-3 times from S. oneidensis, which is improved through electric supply using an electric reactor.
In the case of E. coli or R. eutropha , a mediator, which is a substance that helps electrons to pass through the cell membrane, was not used. However, in the case of S. oneidensis , And it has an advantage of being able to accommodate external electron supply itself without needing additional protein expression by producing various proteins which act as a mediator.
In addition, S. oneidensis has a high resistance to various metal ions that inhibit the growth of microorganisms such as heavy metal ions, and has a special metabolic pathway to chitin, one of the representative biomass of the next generation. Thus, production of isobutanol And the like.
1 is a schematic diagram showing a metabolic pathway for producing isobutanol.
Figure 2 is a schematic diagram showing fufuryl alcohol conversion via cofactor engineering.
Figure 3 is a graphical representation of the Shewanella < RTI ID = 0.0 > from oneidensis) is a schematic view showing the path of producing isobutanol by introducing electric reactor.
Figure 4 is a graph showing the results of the production of isobutanol from the conditions of 0-2% NAG and pyruvate from the modified Schwannellaonidensis:
A: Variation of growth of strains according to NAG concentration;
B: Isobutanol production by NAG concentration;
C: Variation of strain growth with concentration of pyruvic acid; And
D: Production of isobutanol according to concentration of pyruvic acid.
FIG. 5 is a graph showing the results of the production of isobutanol from the conditions of 0-4% lactate and pyruvic acid from the improved Schwannellaonidensis:
A: Variation of growth of strains according to lactate concentration;
B: Production of isobutanol by lactic acid concentration;
C: Variation of growth of strains by using optimal NAG and lactic acid as a carbon source; And
D: Production of isobutanol by using optimal NAG and lactic acid as a carbon source.
Figure 6 is a graph showing the results of the production of isobutanol over time from the conditions of optimal NAG, pyruvic acid and lactic acid from the improved Schwannella onesensis:
A: Variation of strain growth by NAG concentration; And
B: Production of isobutanol according to NAG concentration.
7 is a photograph showing the production process of isobutanol through the operation of an electric reactor.
FIG. 8 is a graph showing the results of production of isobutanol from an improved Schwannella annenidus through external electrical supply using an electric reactor:
A: Variation of strain growth by electricity supply; And
B: Production of isobutanol according to electricity supply.
Hereinafter, the present invention will be described in detail.
The invention Schwarzer Nella ohneyi den sheath (Shewanella one is an expression vector containing one or more genes selected from the group consisting of ketoisovalerate decarboxylase ( kivD ) gene and alcohol dehydrogenase ( adh ) gene, and isobutanol productivity , ≪ / RTI > recombinant Schwannella onesidis strain.
The kivD gene may be composed of the nucleotide sequence of SEQ ID NO: 1, and the adh gene may be composed of the nucleotide sequence of SEQ ID NO: 2.
Preferably, the kivD gene is derived from Lactococcus lactis and the adh gene is derived from R. eutropha , and is not limited to the origin of the gene.
The kivD gene and the adh gene are respectively a nucleotide sequence in which one or more nucleotide sequences are substituted, added or deleted in a natural gene derived from Lactococcus lactis ( L. lactis ) and R. eutropha Lt; / RTI >
The expression vector may be a vector capable of expressing the kivD gene and the adh gene in Schwannellaonodescis , and the pBBR1MCS-2 vector may be used.
The Schwannella onesensis is preferably a strain of S. oneidensis MR-1, but is not limited thereto.
The recombinant Schwannella ondensis strain
Constructing a pJL23 vector by cloning a ketoisovalerate decarboxylase (kivD) enzyme gene and an alcohol dehydrogenase (adh) enzyme gene into an expression vector;
Next, E. coli strain was transformed with E. coli by transfecting the pJL23 vector constructing a pJL23 strain; And
Next, the host S. oneidensis Strain and E. coli culturing the pJL23 strain together through a conjugation method, and then selecting a strain of S. oneidensis pJL23 into which the pJL23 vector has been introduced.
The present invention also relates to a method for producing a recombinant Shwe Nelendonecec strain according to the present invention; And
And recovering isobutanol from the cultured culture. The present invention also provides a method for producing isobutanol from a recombinant Schwannella ondensis strain.
In the above method, the culture is preferably carried out in a culture medium containing NAG (N-acetylglucosamine) and pyruvate or lactate as a carbon source, and all of the three carbon sources It is more preferable to culture in an added medium.
In the above method, the culture is carried out by adding NAG at a concentration of 1 to 4% (w / v) as a carbon source, 0.5 to 1.5% (w / v) concentration of pyruvic acid or 1 to 2% (w / v) It is preferable to cultivate in one culture medium and cultivate in a medium supplemented with 2% (w / v) concentration of NAG, 1.5% (w / v) concentration of pyruvic acid or 2% (w / v) Is more preferable.
The culturing is preferably carried out at 25 to 35 DEG C for 20 to 100 hours, more preferably at 30 DEG C for 24 to 48 hours.
The present invention also relates to a method for producing iso-butanol, comprising culturing a strain of Schwannella onesidense or an improved strain of Schwannella ondensis in a medium having an optimal carbon source, and recovering isobutanol from the cultured medium A method for increasing the productivity of isobutanol from a strain of Wanella onidenesis or an improved strain of Shuwenella onesidense.
In this method, the optimal composition of the carbon source may include one or more of pyruvic acid or lactic acid in the NAG as a component or all three, and the concentration for this combination is 2% (w / v) for NAG, It is preferred that the amount of rubic acid is 1.5% (w / v) or that of lactic acid is 2% (w / v).
In addition, the present invention relates to a method for culturing a strain of Schwannella oniumdensis or an improved Schwannella ondensis strain through an electric power supply using an electric reactor in a medium having an optimal carbon source, Wherein the step of recovering isobutanol from a strain of Schwannella ondensis or an improved strain of Schwannella ondensis comprises increasing the yield of isobutanol.
The electric reactor is preferably a bioelectrical reactor (BER), but it is not limited thereto, and any type of electric reactor capable of supplying electrons can be used.
The electric reactor is an H-type reactor having a dual chamber. One chamber has a working electrode, the other chamber has a counter electrode, It is preferable to use a structure separated by a proton exchange membrane.
The electric reactor preferably maintains a constant voltage within a range of -50 mV to -150 mV, and more preferably maintains a constant voltage of -100 mV to supply electricity.
The culture was carried out in a medium supplemented with 1 to 4% (w / v) concentration of NAG as a carbon source, 0.5 to 1.5% (w / v) concentration of pyruvic acid or 1 to 2% (w / v) .
The culture is preferably carried out at 25 to 35 DEG C for 20 hours, and more preferably at 30 DEG C for 48 hours or more.
Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples. However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the present invention is not limited to the following Examples and Experimental Examples.
< Example 1> Shuwaniella Oneyidensis Isobutanol Introduction and culture of production route
pBBR1MCS-2 vector (Broad-host-range cloning vector (Kan r)): from (Kovach, ME, et al, Gene 166. 175-176, 1995 reference) 2 cake toy Nassau multiple rate (2-ketoisovalerate) in iso-butanoyl Cain via the aldehyde (isobutaldehyde) derived from L. lactis required to switch to isobutanol (isobutanol) Toy Nassau multiple rate decarboxylase (ketoisovalerate decarboxylase, kivD) gene (SEQ ID NO: 1) derived from alcohols and R.eutropha The pJL23 vector was constructed by cloning the alcohol dehydrogenase (adh) enzyme gene (SEQ ID NO: 2) (Lu JN, et al., Appl Microbiol Biot 96: 283-297, 2012).
Here, the primer base sequence used is as follows.
KF: GCTGCT aagctt ATGTATACAGTAGGAGATT (SEQ ID NO: 3)
KR: TTATGATTTATTTTGTTCAGCAAATAGTTT (SEQ ID NO: 4)
AF: TTTTGTTCAGCAAATAGTTTAggagagctactctgtcagtcATGACCGCAATGATGA (SEQ ID NO: 5)
AR: Gctgct ggatcc TCAGTGCGGCTTGATGG (SEQ ID NO: 6)
Then, E. coli strain S17-1 transduction (heat shock transformation) to the thermal shock (Tp R Sm R rec A, thi, pro, hsdR -M + RP4: 2-Tc:: Mu Km :: Tn7 λpir) method To construct the E. coli S17-1 / pJL23 strain by transfecting the pJL23 vector.
Then, a strain of S. oneidensis MR-1 (Lake Oneida isolate) (Reimann A, et al., Appl Microbiol Biot 50: 47-50, 1996) and E. coli S17-1 / pJL23 strain were added to 5 ml of LB browth (Aerobic condition, 30 ° C, 200 rpm, 24 hours culture).
Then, using the conjugation method, S. oneidensis 1 ml of each of MR-1 and E. coli S17-1 / pJL23 was harvested and cultured on LB agar medium (30 ° C., 24 hours) and streaked on an antibiotic plate to obtain pJL23 The S. oneidensis strain containing the vector was obtained.
< Example 2> Electric reactor install
The bioelectrical reactor (BER) (Pyrex) was an H-type reactor with a dual-chamber (450 mL each) with a slight modification (Choi O, et al., Biotechnol Bioeng 109: 2494-2502, 2012). The bio-electrical reactor configuration consisted of a graphite felt electrode (3.5 cm x 9.5 cm) as a working electrode and a Pt plate electrode (3.5 cm x 9.5 cm) as a counter electrode, , And reference cells consisting of Ag / AgCl, 3 M KCl (BASI, West Lafayette, Ind.) Are contained in a cathode compartment. The compartments are separated by Nafion 117 cation exchange membrane (Naracelltech, Seoul, South Korea). The reactor temperature was maintained at 30 ° C by heating tapes (Daihan, Wonju, South Korea) and the temperature sensor was inserted into the anode compartment via a stainless steel rod.
< Experimental Example 1> Improved Schwannella From Oneidensis Isobutanol production
<1-1> As the carbon source NAG and Pyruvic Identify optimal concentration
S. oneidensis MR-1 / pJL23 was isolated from a variety of carbon sources such as NAG, acetate, glucose, lactate and galactose to produce isobutanol, . This is because it does not provide enough energy for the production of isobutanol a single carbon atom is S. oneidensis MR-1 / pJL23 as well as growth.
To overcome this problem, NAG was used as a carbon source for growth and pyruvate was used as an additional ancillary substrate and precursor of isobutanol production.
To optimize the concentration of carbon source, NAG (N-acetylglucosamin) and pyruvic acid concentrations were used at various concentrations ranging from 0 to 2% (w / v). For the optimization of NAG concentration, NAG concentration was used variously for the fixed pyruvic acid concentration (1%). To optimize the pyruvic acid concentration, the pyruvic acid concentration Were used.
The optimum medium for production of isobutanol was determined by using M9 culture medium supplemented with 0.1% yeast extract, using NAG and pyruvic acid as carbon sources on the basis of 0 to 2%, respectively, as production media .
Growth of S. oneidensis MR-1 / pJL23 was determined by measuring OD at 595 nm after collecting 200 [mu] l of each culture sample from a 96 well microtiter plate (Tecan, Mannedorf, Switzerland).
The amount of isobutanol was confirmed by Gas Chromatography (GC) analysis (Chen X, et al., Biotechnol Biofuels 4:21, 2011). Specifically, 1 mL of the production medium supernatant was transferred to a separate organic solvent container, 1 mL of chloroform was added thereto, and the mixture was mixed. The solution was filtered with a PVDF filter, and a fused silica capillary column iso-butanol was analyzed using a silica capillary column (Supelco SPB-5, 30 m × 0.32 mm, id 0.25 μm film) and GC (Gas chromatography) equipped with helium as a carrier gas (Agilent, CA, USA). The analysis conditions are shown in Table 1 below.
As a result, cell growth and isobutanol production showed a positive correlation with the amount of NAG in the medium (FIGS. 4A and 4B). The production of isobutanol was the highest at the addition of 1.5% of pyruvic acid, but at 2% of pyruvic acid concentration, both cell growth and isobutanol production were significantly inhibited (FIGS. 4C and D).
<1-2> Additional As the carbon source Determination of optimum concentration of lactic acid
In order to optimize the lactate concentration as a carbon source, M9 culture medium supplemented with 0.1% yeast extract was used as a carbon source on the basis of 0 to 4% of lactic acid, respectively, and the optimal production medium for isobutanol The conditions were confirmed. It was confirmed that the production of isobutanol increased through the combination of NAG and lactic acid as a carbon source. Production of isobutanol was confirmed by GC analysis.
As a result, although S. oneidensis was a bacterium using lactic acid as an electron donor, isobutanol was hardly produced when lactic acid alone was used as a carbon source. On the other hand, the addition of 1 and 2% lactic acid to NAG increased the production of isobutanol to 1.7 mg / L and 3.5 mg / L, respectively (Figs. 5A and 5B), in particular the combination of 2% NAG and 2% , Strain growth was good and the production of isobutanol increased to 9.9 mg / L (Fig. 5, C and D).
<1-3> NAG, Pyruvic acid And synergistic effects by combination of lactic acid
Using M9 culture medium supplemented with 0.1% yeast extract, using optimal concentrations of NAG, pyruvic acid and lactic acid (2% NAG and 1.5% pyruvic acid added with 2% lactic acid) as the carbon source, (Monitored for 72 hours). Production of isobutanol was confirmed by GC analysis.
As a result, the strain growth pattern was observed to be similar regardless of whether lactic acid was present or not, but 10.3 mg / L of isobutanol was produced 48 hours after culture in a medium supplemented with 2% NAG, 1.5% pyruvic acid and 2% lactic acid (A and B in Fig. 6). In contrast, isobutanol was scarcely produced (1.0 mg / L) in the medium without 2% lactic acid.
This has a significant effect on the production of isobutanol from the improved S. oneidensis by the addition of lactic acid and lactic acid acts as a pyruvic acid precursor which increases the flow of pyruvic acid in the valine pathway resulting in an increase of isobutanol production You can do it.
< Experimental Example 2> Electric reactor Improved Schwannella by using electricity supply Oneyidensis Increased productivity of isobutanol from
250 ml of 50 mM phosphate buffer was filled in a chamber having a counter electrode in an H-type electric reactor and a chamber containing a working electrode was charged with isobutanol production medium (2% NAG , 1.5% pyruvic acid and 2% lactic acid). The middle portion was fixed with a Nafion 117 proton membrane. The voltage was kept constant at -100 mV using potentiostat / galvanostat (EG & G Princeton Applied Research, Model 273A, Princeton, NJ) and incubated at 30 ° C for 48 hours. After collecting the culture, the production of isobutanol was confirmed by the above-mentioned method.
As a result, S. oneidensis MR-1 / pJL23 markedly increased growth and production of isobutanol (19.3 mg / L), and increased with time (Figs. 8A and 8B).
As described above, the productivity of isobutanol can be increased by increasing the internal NAD (P) H through electron supply through an electric reactor using the modified Shewanella oneidensis according to the present invention Therefore, by using Schwannellaonodesense as a production strain, it is possible to mass-produce isobutanol efficiently without artificially expressing the protein complex necessary for electric supply by the electric reactor. .
<110> Konkuk University Industrial Cooperation Corp <120> Method for Isobutanol production from an engineered Shewanella oneidensis <130> NP15-1160 <160> 6 <170> KoPatentin 3.0 <210> 1 <211> 1647 <212> DNA <213> Lactococcus lactis <400> 1 atgtatacagag taggagatta cctattagac cgattacacg agttaggaat tgaagaaatt 60 tttggagtcc ctggagacta taacttacaa tttttagatc aaattatttc ccgcaaggat 120 atgaaatggg tcggaaatgc taatgaatta aatgcttctt atatggctga tggctatgct 180 cgtactaaaa aagctgccgc atttcttaca acctttggag taggtgaatt gagtgcagtt 240 aatggattag caggaagtta cgccgaaaat ttaccagtag tagaaatagt gggatcacct 300 acatcaaaag tccaaaatga aggaaaattt gttcatcata cgctggctga cggtgatttt 360 aaacacttta tgaaaatgca cgaacctgtt acagcagctc gaactttact gacagcagaa 420 aatgcaaccg ttgaaattga ccgagtactt tctgcactac taaaagaaag aaaacctgtc 480 tatatcaact taccagttga tgttgctgct gcaaaagcag agaaaccctc actccctttg 540 aaaaaagaaa atccaacttc aaatacaagt gaccaagaga ttttgaataa aattcaagaa 600 agcttgaaaa atgccaaaaa accaatcgtg attacaggac atgaaataat tagctttggc 660 ttagaaaata cagtcactca atttatttca aagacaaaac tccctattac gacattaaac 720 tttggaaaaa gttcagttga tgaaactctc ccttcatttt taggaatcta taatggtaaa 780 ctctcagagc ctaatcttaa agaattcgtg gaatcagccg acttcatcct gatgcttgga 840 gttaaactca cagactcttc aacaggagca tttacccatc atttaaatga aaataaaatg 900 atttcactga acatagacga aggaaaaata tttaacgaaa gcatccaaaa ttttgatttt 960 gaatccctca tctcctctct cttagaccta agcggaatag aatacaaagg aaaatatatc 1020 gataaaaagc aagaagactt tgttccatca aatgcgcttt tatcacaaga ccgcctatgg 1080 caagcagttg aaaacctaac tcaaagcaat gaaacaatcg ttgctgaaca agggacatca 1140 ttctttggcg cttcatcaat tttcttaaaa ccaaagagtc attttattgg tcaaccctta 1200 tggggatcaa ttggatatac attcccagca gcattaggaa gccaaattgc agataaagaa 1260 agcagacacc ttttatttat tggtgatggt tcacttcaac ttacagtgca agaattagga 1320 ttagcaatca gagaaaaaat taatccaatt tgctttatta tcaataatga tggttataca 1380 gtcgaaagag aaattcatgg accaaatcaa agctacaatg atattccaat gtggaattac 1440 tcaaaattac cagaatcatt tggagcaaca gaagaacgag tagtctcgaa aatcgttaga 1500 actgaaaatg aatttgtgtc tgtcatgaaa gaagctcaag cagatccaaa tagaatgtac 1560 tggattgagt tagttttggc aaaagaagat gcaccaaaag tactgaaaaa aatgggtaaa 1620 ctatttgctg aacaaaataa atcataa 1647 <210> 2 <211> 1101 <212> DNA <213> Ralstonia eutropha <400> 2 atgaccgcaa tgatgaaagc cgccgttttt gtcgagcctg gccggatcga actggcagac 60 aagccgatcc cggatatcgg ccccaacgat gccctggtgc gtatcaccac caccaccatc 120 tgcggcaccg acgtgcacat cctcaagggt gagtacccgg tggcgaaggg cctgaccgtg 180 ggccatgagc ccgtcggcat cattgaaaag ctcggcagcg cggtgacggg ataccgcgaa 240 ggccagcgcg tgatcgccgg cgcaatctgc cccaacttca actcctacgc ggcgcaggac 300 ggcgtggcct cgcaggatgg cagctacctg atggccagcg gccagtgcgg ctgccacggc 360 tacaaggcga ccgcgggctg gcgcttcggc aacatgatcg acggtaccca ggcggaatac 420 gtgctggtgc ccgacgccca ggccaacctg acgccaatcc ccgatggcct caccgacgag 480 caggtgctga tgtgccccga catcatgtcc accggcttca agggcgcgga aaacgccaat 540 atccgcatcg gcgacaccgt ggccgtgttc gcgcagggcc cgatcgggct atgcgcgacc 600 gccggcgcgc ggctgtgcgg cgccaccacc atcatcgcca tcgacggcaa cgaccaccgg 660 ctggagatcg cgcgcaagat gggcgcggac gtggtcctga acttccgcaa ctgcgacgtg 720 gtggacgagg tcatgaagct gaccggcggg cgcggcgtgg atgcctcgat cgaggcgctg 780 ggcacgcagg caaccttcga gcagtcgctg cgcgtgctca agcccggcgg cacgctgtcc 840 agcctggggg tctattcaag cgacctgacc attccgctgt cggctttcgc cgcggggctg 900 ggcgaccaca agatcaacac cgcgctgtgc cccggcggca aggaacgcat gcggcggctg 960 atcaatgtga tcgagtcggg gcgggtcgac ctgggagcgc tggtgacgca ccagtacagg 1020 ctggacgaca tcgtcgcggc ctacgacctg ttcgccaacc agcgcgacgg cgtgctgaag 1080 atcgccatca agccgcactg a 1101 <210> 3 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> KF primer <400> 3 gctgctaagc ttatgtatac agtaggagat t 31 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> KR primer <400> 4 ttatgattta ttttgttcag caaatagttt 30 <210> 5 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> AF primer <400> 5 ttttgttcag caaatagttt aggagagcta ctctgtcagt catgaccgca atgatga 57 <210> 6 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> AR primer <400> 6 gctgctggat cctcagtgcg gcttgatgg 29
Claims (15)
2) The recombinant Schwannella ondensis strain prepared in the above step 1) was used as a carbon source at a concentration of 1.5 to 2% (w / v) of NAG (N-acetylglucosamine) and 1 to 2% (w / v) Or a concentration of NAG (N-acetylglucosamine) of 1.5 to 2% (w / v), pyruvate of 1 to 1.5% (w / v) and a lactate of 1 to 2% w / v) in the medium; And
3) recovering isobutanol from the culture medium cultured in step 2);
≪ / RTI > wherein said recombinant strain is iso-butanol.
2) The recombinant Schwannella ondensis strain prepared in the above step 1) was used as a carbon source at a concentration of 1.5 to 2% (w / v) of NAG (N-acetylglucosamine) and 1 to 2% (w / v) Or a concentration of NAG (N-acetylglucosamine) of 1.5 to 2% (w / v), pyruvate of 1 to 1.5% (w / v) and a lactate of 1 to 2% w / v) in an electric furnace; And
3) recovering isobutanol from the culture medium cultured in step 2);
Wherein the isobutanol is produced from a recombinant Schwannella ondensis strain using an electric reactor.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110511882A (en) * | 2019-07-16 | 2019-11-29 | 天津科技大学 | A kind of salt tolerant and the bacterial strain with electricity production characteristic and its application in microbiological fuel cell |
| CN110511882B (en) * | 2019-07-16 | 2022-01-14 | 天津科技大学 | Salt-tolerant bacterial strain with electrogenesis characteristic and application thereof in microbial fuel cell |
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