JP2023019310A - Acetolactate synthase and method for producing isobutanol using the same - Google Patents

Abstract

To provide a mutant having a change in the reaction of converting pyruvate into acetolactate, which is an important reaction for isobutanol biosynthesis, and further provide a production technique that increases the production of isobutanol by culturing the Escherichia coli having the mutant enzyme introduced thereto.SOLUTION: The present invention provides a mutant of acetolactate synthase and a microorganism having the mutant introduced thereto.SELECTED DRAWING: None

Description

The present invention relates to acetolactate synthase and a method for producing isobutanol using the same.

Butanol is an important industrial chemical, useful as a fuel additive, as a raw material chemical in the plastics industry, and as a food grade extractant in the food and fragrance industry. With 10-12 billion pounds of butanol produced annually by petrochemical means, the demand for this commodity chemical is expected to grow.

Chemical synthesis methods for isobutanol are known, such as the oxo synthesis method, the catalytic hydrogenation of carbon monoxide (1), and the Guerbet condensation of methanol with n-propanol (2). These methods use starting materials derived from petrochemicals and are generally expensive and environmentally damaging. Isobutanol production from plant-derived materials minimizes greenhouse gas emissions and represents an advance in the art.

Isobutanol is produced biologically as a by-product of yeast fermentation. It is a constituent of "fusel oil" and is formed by this fungal group as a result of incomplete metabolism of amino acids. Isobutanol is specifically produced from the catabolism of L-valine. The α-keto acid obtained after collecting the amino group of L-valine as a nitrogen source is decarboxylated and reduced to isobutanol by enzymes of the so-called Ehrlich pathway (Non-Patent Document 3). The yields of fusel oil and/or its constituents achieved during beverage fermentation are typically low. For example, the concentration of isobutanol produced during beer fermentation is reported to be less than 16 parts per million (Non-Patent Document 4). Addition of exogenous L-valine to fermentation increases the isobutanol yield as described in (Non-Patent Document 3), providing a concentration of 20 g/L of L-valine during fermentation reduces the yield to 3 g/L. An isobutanol yield of L is reportedly obtained. However, the use of valine as a raw material is prohibitively expensive for isobutanol production on an industrial scale. Biosynthesis of isobutanol directly from sugars is economically viable and represents an advance in the art.

In general, attempts have been made to increase the production efficiency of useful substances by introducing multiple genes into microorganisms to construct new reaction pathways in the cells, and by imparting or enhancing the ability to produce specific substances. ing. For example, in Patent Document 1, recombinant organisms endowed with isobutanol-producing ability have been successfully produced by introducing an isobutanol biosynthetic gene group into a plurality of microorganisms that cannot produce isobutanol.

At this time, five types of genes were introduced to impart isobutanol-producing ability: acetolactate synthase, which converts intracellular pyruvate to acetolactate, and acetolactate reductase, which converts acetolactate to isovaleric acid. , isovaleric acid converting enzyme that converts isovaleric acid to 2-keto acid, 2-keto acid decarboxylase that converts 2-keto acid to isobutyraldehyde, and alcohol dehydrogenase that converts isobutyraldehyde to isobutanol. . Among them, the reaction of acetolactate synthase, which catalyzes the initial reaction, is considered important for the production of the final product, isobutanol. Non-Patent Document 1 focused on acetolactate synthase in the isobutanol biosynthetic gene group, and succeeded in improving the catalytic efficiency of the enzyme by introducing mutations into the gene. However, the production of isobutanol was unexpectedly decreased, and the reason was unknown.

That is, in the isobutanol biosynthetic pathway, the initial reaction, acetolactate synthesis, is important for isobutanol production, but the improvement in isobutanol production cannot be predicted based solely on the improvement in enzymatic activity. The development of enzymes that can improve isobutanol production involves making full use of information such as improved enzyme stability to create mutant enzymes with altered reactivity and actually verifying their effects on isobutanol production. It is considered possible.

Japanese Patent No. 5276986

"Ullmann's Encyclopedia of Industrial Chemistry", 6th Edition, Volume 5, pages 716-719, Wiley-VCH Verlag GmbH and Co. , Weinheim, Germany, 2003 Carlini et al., J. Am. Mol. Catal. A: Chem. 220:215-220, 2004 Dickinson et al., J. Am. Biol. Chem. 273(40):25752-25756, 1998. Garcia et al., Process Biochemistry 29:303-309, 1994

In efforts to build a bio-economy society and address environmental issues such as the SDGs, the conversion of chemical raw materials through bioprocesses is once again attracting attention. For example, isobutanol is widely used as a raw material for lacquers and paints, and as a plasticizer for plastics and rubber. In general, the key to bioprocesses is the ability of microorganisms to produce chemicals, and many studies have been conducted. If we can improve the productivity of isobutanol by increasing the activity of the enzyme that converts pyruvic acid, the primary metabolite of microorganisms, to acetolactate and introducing it into microorganisms, we can contribute to the conversion of raw materials for chemical products and bio-products. It can be expected to expand into the chemical industry, which is undergoing a shift.
The subject of the present invention is to create a mutant of acetolactate synthase, which is important for isobutanol biosynthesis, to change the reaction that converts pyruvate to acetolactate, and to prepare Escherichia coli into which the mutant is introduced. The object is to improve the production of isobutanol by culturing.

The inventor focused on acetolactate synthase, an enzyme that catalyzes the reaction that produces acetolactate from pyruvate, and by introducing computational design technology, succeeded in producing mutant enzymes with altered enzymatic activity. bottom. Furthermore, by introducing this mutant enzyme into microorganisms, the inventors succeeded in increasing the amount of isobutanol produced, leading to the creation of the present invention.

That is, the present invention includes the following aspects:
One aspect of the present invention is
[1] Regarding acetolactate synthase, which is any protein shown in any of the following (1) to (3):
(1) A protein consisting of an amino acid sequence represented by SEQ ID NO: 1, 9, or 13 (2) In the protein described in (1), substitution of lysine at position 40 with tryptophan and substitution of aspartic acid at position 42 with tryptophan substitution, histidine at position 123 to leucine, phenylalanine at position 267 to valine, and glutamine at position 497 to leucine (3) SEQ ID NOs: 1 and 9 , or a protein consisting of an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence represented by 13, and having the activity of converting pyruvate to acetolactate

Another aspect of the present invention relates to [2] a polynucleotide encoding any of the acetolactate synthase described in [1] above.

Another aspect of the present invention relates to [3] a vector incorporating the polynucleotide of [2] above.
Another aspect of the present invention relates to [4] a transformant characterized by being transformed with the recombinant vector of [3] above.
Here, in one embodiment, the transformant of the present invention is the transformant according to [5] above [4], further comprising isovaleric acid converting enzyme, 2-keto acid decarboxylase, and alcohol dehydrogenase. characterized in that it is transformed to express

Another aspect of the present invention relates to [6] a method for producing isobutanol, which comprises culturing the transformant of [4] or [5] above.

The acetolactate synthase of the present invention has altered acetolactate synthesis activity compared to before the introduction of the mutation. The E. coli transformant introduced with this enzyme produced 1.02-1.64 times more isobutanol at a culture temperature of 37°C compared to the E. coli transformant introduced with the enzyme before the mutation was introduced. bottom. That is, by using this mutant, it is possible to improve the isobutanol production efficiency of the microorganism.

FIG. 1 shows a plasmid map of the plasmid prepared in Example 1 below. FIG. 1A shows the plasmid map of pCDFDuet-1/alsS-ilvCD and FIG. 1B shows the plasmid map of pCOLADuet-1/kivd-ADH2. FIG. 2A shows an image obtained by subjecting a crude extract derived from Escherichia coli having an enzyme expression plasmid containing a wild-type alsS gene to a nickel column and further to SDS-PAGE, which was performed in Example 3 below. FIG. 2B is an image obtained by subjecting a crude extract derived from Escherichia coli having an enzyme-expressing plasmid having an alsS gene of mutant 1-5 to a nickel column and further subjecting it to SDS-PAGE, which was performed in Example 3 below. indicates FIG. 3 shows the amount of isobutanol produced (g/ L) or is a graph showing isobutanol production amount/cell mass. FIG. 4 shows the isobutanol production amount (g/L ) or isobutanol production/cell mass.

One aspect of the present invention provides an acetolactate synthase that is any protein shown in any of the following (1) to (3):
(1) A protein consisting of an amino acid sequence represented by SEQ ID NO: 1, 9, or 13 (2) In the protein described in (1), substitution of lysine at position 40 with tryptophan and substitution of aspartic acid at position 42 with tryptophan substitution, histidine at position 123 to leucine, phenylalanine at position 267 to valine, and glutamine at position 497 to leucine (3) SEQ ID NOs: 1 and 9 , or a protein consisting of an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence represented by 13, and having the activity of converting pyruvate to acetolactate

By "acetolactate synthase" is meant an enzyme that catalyzes the conversion of pyruvate to acetolactate and _CO2 . One embodiment of the acetolactate synthase according to the present invention includes (1) a mutant (SEQ ID NO: 1) in which lysine at position 40 in the wild-type amino acid sequence (SEQ ID NO: 21) is substituted with tryptophan, and histidine at position 123. is substituted with leucine (SEQ ID NO: 9), and a mutant with phenylalanine at position 267 substituted with valine (SEQ ID NO: 13).

Further, one embodiment of the acetolactate synthase according to the present invention is (2) substitution of tryptophan for lysine at position 40 in the protein consisting of the amino acid sequence represented by SEQ ID NO: 1, 9, or 13 (K40W) , 42nd aspartic acid to tryptophan (D42W), 123rd histidine to leucine (H123L), 267th phenylalanine to valine (F267V), 497th glutamine to leucine (Q497L) from has at least one mutation selected from the group consisting of
The amino acid substitution mutations listed above can be introduced as a combination of one, two, three, four, or all five. Further, mutations in acetolactate synthase of the present invention include all combinations of mutations listed above.

Further, one embodiment of the acetolactate synthase according to the present invention is (3) a protein consisting of an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 1, 9, or 13. and a protein that has the activity of converting pyruvate to acetolactate. In a preferred embodiment, the sequence identity with the amino acid sequence represented by SEQ ID NO: 1, 9, or 13 is 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% 97% or more, 98% or more, or 99% or more.
As one embodiment, the acetolactate synthase according to the present invention is derived from an amino acid sequence represented by SEQ ID NO: 1, 9, or 13 in which one or more amino acids are deleted, substituted, or added. and having the activity of converting pyruvate to acetolactate. Here, "an amino acid sequence in which one or more amino acids are deleted, substituted, or added" means 1 to 57, 1 to 50, 1 to 40, 1 to 30, 1 to 20 , 1 to 10 amino acids are deleted, substituted, or added.
The acetolactate synthase of the present invention has the activity of converting pyruvate to acetolactate in addition to the above-listed amino acid substitution mutations (K40W, D42W, H123L, F267V, Q497L) or apart from the substitution mutations. It may have another mutation as long as it has.

The activity of acetolactate synthase to convert pyruvate into acetolactate can be evaluated according to the method of Example 4 below, for example. When it has the activity of converting pyruvate to acetolactate, for example, when evaluated according to Example 4 below, it means that it exhibits an activity of 0.01 U / mg or more at 30 ° C. or 37 ° C. say.

In a preferred embodiment of the acetolactate synthase according to the present invention, a gene encoding the acetolactate synthase is introduced into recombinant Escherichia coli having a reaction pathway that produces isobutanol from pyruvate, and pyruvate is produced under anaerobic conditions. When used in a reaction pathway that produces isobutanol from acid, it shows improved isobutanol production per dry cell weight compared to the wild-type enzyme.
That is, in one embodiment, the acetolactate synthase of the present invention comprises an acetolactate synthase comprising the following proteins:
(3) 'A protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 1, 9, or 13, and from pyruvic acid A protein that shows improved isobutanol production per dry cell weight compared to the wild-type enzyme when a gene encoding the protein is introduced into a recombinant Escherichia coli having a reaction pathway that produces isobutanol.

In order to construct a reaction pathway for producing isobutanol from pyruvate in a host cell, in addition to the acetolactate synthase gene, an acetolactate reductase gene, an isovaleric acid convertase gene, a 2-ketoacid decarboxylase gene, An alcohol dehydrogenase gene may be introduced into a host cell. Such a method for producing a transformant for isobutanol production from pyruvic acid is known, and can be carried out with reference to the disclosure of the present specification, Japanese Patent No. 5276986, and the like.
Evaluation of the amount of isobutanol produced per dry cell weight when acetolactate synthase is used in the reaction pathway for isobutanol production is, for example, the method described in Example 7 (isobutanol production under anaerobic conditions). can be done according to

In a preferred embodiment of the acetolactate synthase according to the present invention, a gene encoding the acetolactate synthase is introduced into recombinant Escherichia coli having a reaction pathway that produces isobutanol from pyruvate, and When used in a reaction pathway that produces isobutanol from pyruvate, it shows an improved isobutanol production amount per dry cell weight compared to the wild-type enzyme.

In another aspect, the invention provides a polynucleotide encoding any of the acetolactate synthases described above. One embodiment of a polynucleotide according to the present invention comprises a polynucleotide consisting of a DNA sequence represented by SEQ ID NO:2, 6, 10, 14 or 18. In preferred embodiments, the polynucleotide according to the present invention is a polynucleotide consisting of the DNA sequence represented by SEQ ID NO:2, 10 or 14. A polynucleotide consisting of a DNA sequence represented by SEQ ID NO: 2, 6, 10, 14, or 18 encodes a protein consisting of an amino acid sequence represented by SEQ ID NO: 1, 5, 9, 13, or 17, respectively. do. Polynucleotides according to the present invention are not limited to the above examples, and can be provided as polynucleotides having optimal codons that are appropriately preferred by host cells into which they are introduced.

In another aspect, the present invention provides a vector incorporating a polynucleotide encoding the acetolactate synthase of the present invention. The vector contains the polynucleotide so as to express the acetolactate synthase according to the present invention. As for the vector and its construction, a well-known vector suitable for expressing the acetolactate synthase (for example, the type of host cell) can be employed.
A vector incorporating a polynucleotide encoding the acetolactate synthase of the present invention further includes an acetolactate reductase gene, an isovaleric acid convertase gene, a 2-ketoacid decarboxylase gene, and an alcohol dehydrogenase gene. It may contain a polynucleotide encoding one or more genes selected from the group consisting of

In another aspect, the present invention provides a transformant characterized by being transformed with the above vector.
Host cells for isobutanol production are not limited as long as they are cells capable of expressing acetolactate synthase. Cyanobacteria, Clostridium thermocellum, Clostridium cellulotycum, Ralstonia eutropha, Geobacillus thermoglucosidasius, Hydrogenophilus ther and the like can be mentioned.
Methods for obtaining transformants by introducing the vector of the present invention into host cells are known, and suitable methods such as electroporation can be employed as appropriate.

In one embodiment of the transformant of the present invention, in addition to expressing acetolactate synthase, acetolactate reductase, isovaleric acid converting enzyme, 2-ketoacid decarboxylase, and alcohol dehydrogenase are further expressed. Transformed to express Genes encoding the above five enzymes can be introduced into host cells using one or more vectors.

In another aspect, the present invention provides a method for producing isobutanol, which comprises culturing the transformant of the present invention.
Usual media containing a carbon source such as glucose can be used as the culture medium.
The culture temperature can be 8-47° C., and the culture time can be 12-120 hours. Shaking culture is preferable for culture, and the shaking condition can be 100 to 300 rpm. Also, the culture can be performed under anaerobic conditions or under aerobic conditions.
In a preferred embodiment, the method for producing isobutanol according to the present invention includes a step of culturing a transformant capable of expressing acetolactate synthase under aerobic conditions.
The aerobic conditions herein are achieved by using a feathered flask, shake flask, jar fermenter, or the like to entrain a large amount of air into the culture solution during stirring, thereby significantly improving the aeration efficiency. . At this time, the shaking condition is preferably 100 to 300 rpm. Also, as the flask with feathers, for example, 016310-500A manufactured by SIBATA, 4551FK500R manufactured by IWAKI, etc. can be used.

EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited to the following examples.

(Example 1)
In order to construct a reaction pathway that converts pyruvate to isobutanol in E. coli, five enzymes (acetolactate synthase (derived from Bacillus subtilis), acetolactate reductase (derived from Escherichia coli), isovaleric acid converting enzyme ( (derived from Escherichia coli), 2-keto acid decarboxylase (derived from Lactococcus lactis), and alcohol dehydrogenase (derived from Saccharomyces cerevisiae)) were obtained (SEQ ID NOS: 23, 25, 27, or 29; and the corresponding amino acid sequences are shown as SEQ ID NOs: 24, 26, 28 and 30). The obtained sequence information was designed to have a codon sequence suitable for Escherichia coli on the HP of Thermo Fisher, and genes with codon-optimized base sequences were synthesized respectively (acetolactate synthase gene-containing plasmid: pMA-T /alsS, plasmid containing acetolactate reductase gene and isovaleric acid converting enzyme gene: pMK-RQ/ilvCD, plasmid containing 2-keto acid decarboxylase gene: pMA-T/kivd, plasmid containing alcohol dehydrogenase gene: pMA -T/ADH2). The acetolactate synthase gene alsS and the 2-ketoacid decarboxylase gene kivd were excised with the restriction enzymes NcoI and BamHI, and the acetolactate reductase gene and isovaleric acid converting enzyme gene ilvCD and the alcohol dehydrogenase gene ADH2 were cut with the restriction enzymes NcoI and BamHI. Excised with NdeI and XhoI. The resulting acetolactate synthase gene, acetolactate reductase gene and isovaleric acid converting enzyme gene were inserted into the E. coli expression vector pCDFDuet-1, and the 2-keto acid decarboxylase gene and alcohol dehydrogenase gene were It was inserted into the E. coli expression vector pCOLADuet-1 to prepare the plasmids shown in FIG. 1 (pCDFDuet-1/alsS-ilvCD, pCOLADuet-1/kivd-ADH2).

(Example 2)
Among the genes described in Example 1, for the acetolactate synthase gene, a mutant enzyme was designed using Rosetta (software). Specifically, first, using Spartan'18 (software), the movable range of the substrate molecule was calculated based on the molecular mechanics method for the chemical structure of the substrate. Next, using the crystal structure of the enzyme (PDB ID: 4RJK) registered in the Protein Data Bank (PDB) and the calculated substrate molecule, the most stable state of the enzyme-substrate complex is docked by Rosetta. Calculated by simulation. In this case, the term "stable" refers to a state in which the energy of the entire structure indicating the binding energy between the enzyme and the substrate and the stability of the entire complex is low, based on Rosetta's unique energy function. 2000 docking models were calculated. The resulting 2000 model stabilities, i.e., enzyme-substrate binding energies and overall structure energies indicative of overall complex stability, were ranked using Jupyter Notebook (software) and the top 10 enzyme-substrate A complex model was extracted. Furthermore, using molecular modeling display software, we narrowed down to one best model that forms an ideal catalytic structure from the bond distance and bond angle between the enzyme and the substrate.

Using Rosetta, in one ideal best model, the total stability when all types of amino acids (20 types) are substituted for catalytic amino acid residues whose distance from the residue is within 9 Å Calculated per hit. At that time, 1000 docking models were calculated. Here, we added the constraint energy of the catalyst structure as an indicator of stability, ranked them using Jupyter Notebook, and extracted the top 10 enzyme-substrate complex models that are candidates for mutation sites. A solution has emerged that it is best to mutate five at the same time.

Mutant 1 is a mutant (SEQ ID NO: 1) in which lysine at position 40 is substituted with tryptophan, using pMA-T/alsS as a template and two types of oligonucleotides (SEQ ID NOS: 3 and 4) as primers. It was amplified by PCR and introduced a sequence substitution (SEQ ID NO: 2). Mutant 2 is a mutant (SEQ ID NO: 5) in which aspartic acid at position 42 is substituted with tryptophan, using pMA-T/alsS as a template and two types of oligonucleotides (SEQ ID NOS: 7 and 8) as primers. It was amplified by PCR and introduced a sequence substitution (SEQ ID NO: 6). Mutant 3 is a mutant (SEQ ID NO: 9) in which histidine at position 123 is substituted with leucine, using pMA-T/alsS as a template and two oligonucleotides (SEQ ID NOS: 11 and 12) as primers. It was amplified by PCR and introduced a sequence substitution (SEQ ID NO: 10). Mutant 4 is a mutant (SEQ ID NO: 13) in which phenylalanine at position 267 is substituted with valine, using pMA-T/alsS as a template and two oligonucleotides (SEQ ID NOS: 15 and 16) as primers. It was amplified by PCR and introduced a sequence substitution (SEQ ID NO: 14). Mutant 5 is a mutant (SEQ ID NO: 17) in which glutamine at position 497 is substituted with leucine, using pMA-T/alsS as a template and two types of oligonucleotides (SEQ ID NOS: 19 and 20) as primers. It was amplified by PCR and introduced a sequence substitution (SEQ ID NO: 18).

(Example 3)
Recombinant E. coli carrying an enzyme expression plasmid containing the wild-type (SEQ ID NO: 22) or mutant 1-5 alsS gene (SEQ ID NO: 2, 6, 10, 14, or 18) was incubated with 20 g/L lactose. After culturing in LB medium, the resulting cells were disrupted by sonication to prepare a crude extract. The resulting crude extract was applied to a nickel column to prepare a purified sample of each mutant enzyme (Fig. 2).

(Example 4)
The activity of each acetolactate synthase was measured as follows. 50 μg of the purified enzyme preparation was added to a reaction solution containing 10 mM MOPS (pH 7.0), 1 mM magnesium chloride, 0.1 mM thiamine pyrophosphate, and 10 mM pyruvic acid, and reacted at 37° C. for 10 minutes. % (v/v) sulfuric acid, reacted at 37° C. for 25 minutes, centrifuged at 20,000×g at 4° C. for 5 minutes, added 0.5 mL of 2 M sodium hydroxide, 1 mL of 5 g/L to the supernatant. Creatine and 1 mL of 50 g/L α-naphthol were added, reacted at 37° C. for 10 minutes, and then absorbance at 530 nm was measured with a spectrophotometer. 1 U is defined as the amount of enzyme that can convert 1 μmol of pyruvate to acetolactate per minute. Protein amounts were measured with the Qubit Protein Assay Kit. As a result of activity measurement under 37 ° C conditions using purified preparations of each enzyme, the activity of the wild-type enzyme before the introduction of mutation was 2.40 U / mg, the activity of mutant 1 was 0.06 U / mg, the mutation The activity of form 2 was 0.35 U/mg, the activity of variant 3 was 0.05 U/mg, the activity of variant 4 was 1.07 U/mg, and the activity of variant 5 was 0.83 U/mg. In addition, as a result of activity measurement under 30 ° C. conditions, the activity of the wild-type enzyme is 1.75 U / mg, the activity of mutant 1 is 0.05 U / mg, the activity of mutant 2 is 0.32 U / mg, Mutant 3 had an activity of 0.04 U/mg, Mutant 4 had an activity of 1.04 U/mg, and Mutant 5 had an activity of 0.80 U/mg.

(Example 5)
The plasmid described in FIG. 1, which has a wild-type (SEQ ID NO: 22) or mutant 1-5 alsS gene (SEQ ID NOS: 2, 6, 10, 14, or 18) as an acetolactate synthase gene. built. Each plasmid was introduced into DE3-treated E. coli by electroporation to prepare genetically modified E. coli. The recombinant E. coli was cultured in M9 minimal medium containing 20 g/L of yeast extract and 50 g/L of glucose using a feather flask at 30° C. for 48 hours with shaking at 180 rpm. By using a flask with a feather, air is easily entrained in the culture medium during stirring, resulting in more aerobic conditions than in a normal flask. The amount of cells is shown by converting the OD600 value into the dry cell weight. Glucose, acetic acid and isobutanol were quantified by the HPLC method. As a result, mutants 3 and 4 showed higher isobutanol production during 30 and 48 hours of culture compared to the wild-type enzyme (Fig. 3). Mutant 3 also produced more isobutanol than the wild type even after 24 hours of culture. Furthermore, when comparing the isobutanol production per dry cell weight, it was found that mutants 3 and 4 exceeded the wild-type isobutanol production in all of the 24, 30, and 48 hour cultures ( Figure 3). That is, it was found that the isobutanol production per cell was improved by introducing the mutants 3 and 4 into E. coli.

(Example 6)
Each recombinant E. coli prepared in Example 5 was cultured in M9 minimal medium containing 20 g/L of yeast extract and 50 g/L of glucose using a screw cap flask at 30° C. for 48 hours with shaking at 100 rpm. By using a screw cap flask, oxygen supply from the outside is cut off, and the conditions are more anaerobic than in a normal flask. The amount of cells is shown by converting the OD600 value into the dry cell weight. Glucose, acetic acid and isobutanol were quantified by the HPLC method. As a result, mutants 1, 3, and 4 showed higher isobutanol production during 21, 24, 30, and 48 hours of culture compared to the wild-type enzyme (Fig. 4). Mutants 3 and 4 also exceeded wild-type isobutanol production at 48 hours. Furthermore, when comparing the isobutanol production per dry cell weight, it was found that mutants 1, 3, and 4 exceeded the wild-type isobutanol production in any of 21, 24, and 30 hours of culture. (Fig. 4). Mutants 3 and 4 also produced more isobutanol than the wild type in 48-hour culture. That is, it was found that introduction of mutants 1, 3, and 4 into E. coli improved the isobutanol production per cell.

Since the mutant enzyme of the present invention changes the reaction that irreversibly converts pyruvate to acetolactate, the normal flow of using pyruvate for cell synthesis and production of various substances in organisms such as E. coli also changes. As a result, the pathway toward the synthesis of acetolactate is also changed in the cells cultured with glucose or the like, and isobutanol production can be improved.

Claims (6)

Acetolactate synthase, which is any protein shown in any of the following (1) to (3):
(1) A protein consisting of an amino acid sequence represented by SEQ ID NO: 1, 9, or 13 (2) In the protein described in (1), substitution of lysine at position 40 with tryptophan and substitution of aspartic acid at position 42 with tryptophan substitution, histidine at position 123 to leucine, phenylalanine at position 267 to valine, and glutamine at position 497 to leucine (3) SEQ ID NOs: 1 and 9 , or a protein consisting of an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence represented by 13, and having the activity of converting pyruvate to acetolactate A polynucleotide encoding any of the acetolactate synthase according to claim 1 . A vector incorporating the polynucleotide of claim 2 . A transformant characterized by being transformed with the vector according to claim 3 . 5. The transformant according to claim 4, which is transformed to express isovaleric acid converting enzyme, 2-ketoacid decarboxylase and alcohol dehydrogenase. A method for producing isobutanol, comprising a step of culturing the transformant according to claim 4 or 5.

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