KR101960501B1 - New ketol-acid reductoisomerase with a cofactor preference to NADH developed - Google Patents

Abstract

The present invention relates to an enzymatic modification of ketol-acid reductoisomerase essential for the production of isobutanol. More specifically, the enzyme was modified through the Rossman loop remodeling to make Escherichia coli-derived ketol-acid reductoisomerase having affinity for NADPH have affinity for NADH.

Description

NADH-friendly new ketol-acid reductoisomerase with a cofactor preference to NADH developed.

The present invention relates to a novel NADH-friendly ketolanhydride isomerase.

As the development and use of excessive fossil fuel resources cause environmental problems, it is urgent to search for environmentally friendly renewable energy sources to replace existing fossil fuels. In particular, Korea is highly dependent on fossil fuels (85%), and most of them are imported from abroad, so the search for renewable energy sources is very important.

Generally, eco-friendly bioenergy refers to liquid fuels produced by the fermentation of microorganisms from renewable resources such as lignocellulose in herbal leagues. Although bioethanol produced mainly by yeast has been spotted as bioenergy to date, it has been searching for improved bioenergy having high energy density and hygroscopicity as compared with ethanol. An example is isobutanol. Isobutanol is mainly produced by Escherichia coli transformed with a gene of a heterologous microorganism E. coli , yeast ( Saccharomyces cerevisiae ), Bacillus subtilus , and Corynebacterium glutaminicum . However, the biggest obstacle to the production of isobutanol is that first, high concentration of isobutanol inhibits the growth and fermentation of microorganisms. It has been reported in many literature that this problem can be overcome by high cell density fermentation. Second, in the process of isobutanol biosynthesis, five enzymes (AlsS, IlvC, ilvD, Kdc, and Adh) are essential as shown in FIG. 1 of the accompanying drawings. Two molecules of NADH produced in the process and one molecule Of NADPH is a metabolic stream that is repeatedly produced and consumed, so that the cofactor redox balance of the coenzyme is destroyed.

In general, the cofactor balance means that NADH / NAD ⁺ and NADPH / NADP ⁺ are paired to maintain the carbon flux and life cycle while maintaining the redox balance it means. However, if the balance of the coenzyme is destroyed, it interferes with the growth and metabolism of the cell, and eventually decreases the productivity of isobutanol. To date, many studies have been conducted to maintain the equilibrium of coenzyme in balance. Representative methods include 1) promoter engineering at the cellular level, genome scale engineering, Approaches such as synthetic biotechnology and cofactor regeneration through the introduction of foreign genes and 2) approach to protein engineering at the protein level . In particular, the enzyme engineering method utilizes information obtained through whole enzyme random mutagenesis and structural analysis of enzymes, and uses saturated random mutagenesis of a specific amino acid of the enzyme, Screening of enzymes is typical.

As shown in Fig. 1, the isobutanol biosynthesis process using microbial fermentation is a metabolic flow in which the two molecules of NADH produced in the process and the one molecule of NADPH consumed in the biosynthesis process continue to be repeatedly produced and consumed, The cofactor redox balance is destroyed. As a result, the functions of many enzymes involved in the synthesis and degradation of substances related to the redox reaction of cells are weakened, the overall physiological activity is reduced, and the productivity of isobutanol is remarkably reduced.

Domestic patent registration No. 1573775

Therefore, the present inventors tried to solve the co-enzyme imbalance of NADPH / NADP ⁺ and NADH / NAD ⁺ on the isobutanol biosynthesis pathway by dissolving NADH in place of NADPH to convert 2-acetolactate into 2,3-dihydroxyisovalerate (2,3- (Amino acid sequence 67-78) which is involved in the binding of phosphate of NADPH in the enzyme. As a result, it was found that the NADPH- The mutant library was constructed through targeted random mutagenesis (concept of random mutation), and the enzyme having affinity to NADH was screened. Among the initial 400 mutant enzymes, 42 enzymes were screened. Finally, two kinds of enzymes having high affinity for NADH were discovered to complete the present invention.

Accordingly, it is an object of the present invention to provide two kinds of NADH-friendly ketolanilide isomerase mutants.

The present invention provides NADH-friendly ketolanhydride isomerase variants.

The present invention also provides a method for producing isobutanol using the ketolanidduct isomerase enzyme.

Hereinafter, the problem solving means of the present invention will be described in more detail.

Ketolanideductoisomerase (ilvC) is a very important enzyme that makes an intermediate product essential for the production of branched-chain amino acid. Molecular chain amino acid has recently been used in animal feed, dietary supplements, It is said that 1,500 tons are produced. IlvC is also an important enzyme in the production of various bioenergies including isobutanol and 1-butanol.

According to one embodiment of the present invention, the NADH-friendly ketolan reductoisomerase variant is Escherichia coli origin Ketol acid reductoisomerase represented by the amino acid sequence of SEQ ID NO: 1. The wild-type (IlvC_W) enzyme has amino acid Arg at positions 68, 76 and 78 directly involved in binding with phosphate of NADPH in the rosemann loop May include substituted variants. Specifically, the NADH-compatible ketolanidyl isomerase mutant is a mutant in which Arg at position 68 is substituted with Ser, Arg at position 76 is substituted with Phe, Arg at position 78 is substituted with Asp (IlvC_CH11) or a mutant in which the Arg at position 68 of the wild type enzyme is substituted with Phe, the Arg at position 76 is substituted with Phe, the Arg at position 78 is substituted with Ala (hereinafter referred to as IlvC_CH13) Lt; / RTI >

A gene coding for the ketolan reductoisomera mutant IlvC_CH11, which can be represented by the following SEQ ID NO: 4.

The gene encoding the ketolan reductoisomera mutant IlvC_CH13, which is represented by the nucleotide sequence of SEQ ID NO: 6 below.

SEQ ID NO: 1

IlvC_W amino acid sequence

M A N Y F N T L N L R Q Q L A Q L G K C R F M G R D E F A D G A S Y L Q G K K V V I V G C G A Q G L N Q G L N M R D S G L D I S Y A L R K E A I A E K R A S W R K A T E N G F K V G T Y E E L I P Q A D L V I N L T P D K Q H S D V V R T V Q P L M K D G A A L G Y S H G F N I V E V G E Q I R K D I T V V M V A P K C P G T E V R E E Y K R G F G V P T L I A V H P E N D P K G E G M A I A K A W A A A T G G H R A G V L E S S F V A E V K S D L M G E Q T I L C G M L Q A G S L L C F D K L V E E G T D P A Y A E K L I Q F G W E T I T E A L K Q G G I T L M M D R L S N P A K L R A Y A L S E Q L K E I M A P L F Q K H M D D I I S G E F S S G M M A D W A N D D K K L L T W R E E T G K T A F E T A P Q Y E G K I G E Q E Y F D K G V L M I A M V K A G V E L A F E T M V D S G I I E E S A Y Y E S L H E L P L I A N T I A R K R L Y E M N V V I S D T A E Y G N Y L F S Y A C V P L L K P F M A E L Q P G D L G K A I P E G A V D N G Q L R D V N E A I R S H A I E Q V G K K L R G Y M T D M K R I A V A G

SEQ ID NO: 2

IlvC_W nucleotide sequence

GCCGCAGTATGAAGGCAAAATCGGCGAGCAGGAGTACTTCGATAAAGGCGTACTGATGATTGCGATGGTGAAAGCGGGCGTTGAACTGGCGTTCGAAACCATGGTCGATTCCGGCATCATTGAAGAGTCTGCATATTATGAATCACTGCACGAGCTGCCGCTGATTGCCAACACCATCGCCCGTAAGCGTCTGTACGAAATGAACGTGGTTATCTCTGATACCGCTGAGTACGGTAACTATCTGTTCTCTTACGCTTGTGTGCCGTTGCTGAAACCGTTTATGGCAGAGCTGCAACCGGGCGACCTGGGTAAAGCTATTCCGGAAGGCGCGGTAGATAACGGGCAACTGCGTGATGTGAACGAAGCGATTCGCAGCCATGCGATTGAGCAGGTAGGTAAGAAACTGCGCGGCTATATGACAGATATGAAACGTATTGCTGTTGCGGGT

SEQ ID NO: 3

IlvC_CH11 amino acid sequence

M A N Y F N T L N L R Q Q L A Q L G K C R F M G R D E F A D G A S Y L Q G K K V V I V G C G A Q G L N Q G L N M R D S G L D I S Y A E S A L V V W L S F D D W R K A T E N G F K V G T Y E E L I P Q A D L V I N L T P D K Q H S D V V R T V Q P L M K D G A A L G Y S H G F N I V E V G E Q I R K D I T V V M V A P K C P G T E V R E E Y K R G F G V P T L I A V H P E N D P K G E G M A I A K A W A A A T G G H R A G V L E S S F V A E V K S D L M G E Q T I L C G M L Q A G S L L C F D K L V E E G T D P A Y A E K L I Q F G W E T I T E A L K Q G G I T L M M D R L S N P A K L R A Y A L S E Q L K E I M A P L F Q K H M D D I I S G E F S S G M M A D W A N D D K K L L T W R E E T G K T A F E T A P Q Y E G K I G E Q E Y F D K G V L M I A M V K A G V E L A F E T M V D S G I I E E S A Y Y E S L H E L P L I A N T I A R K R L Y E M N V V I S D T A E Y G N Y L F S Y A C V P L L K P F M A E L Q P G D L G K A I P E G A V D N G Q L R D V N E A I R S H A I E Q V G K K L R G Y M T D M K R I A V A G

SEQ ID NO: 4

IlvC_CH11 base sequence

GCCGCAGTATGAAGGCAAAATCGGCGAGCAGGAGTACTTCGATAAAGGCGTACTGATGATTGCGATGGTGAAAGCGGGCGTTGAACTGGCGTTCGAAACCATGGTCGATTCCGGCATCATTGAAGAGTCTGCATATTATGAATCACTGCACGAGCTGCCGCTGATTGCCAACACCATCGCCCGTAAGCGTCTGTACGAAATGAACGTGGTTATCTCTGATACCGCTGAGTACGGTAACTATCTGTTCTCTTACGCTTGTGTGCCGTTGCTGAAACCGTTTATGGCAGAGCTGCAACCGGGCGACCTGGGTAAAGCTATTCCGGAAGGCGCGGTAGATAACGGGCAACTGCGTGATGTGAACGAAGCGATTCGCAGCCATGCGATTGAGCAGGTAGGTAAGAAACTGCGCGGCTATATGACAGATATGAAACGTATTGCTGTTGCGGGT

SEQ ID NO: 5

IlvC_CH13 amino acid sequence

M A N Y F N T L N L R Q Q L A Q L G K C R F M G R D E F A D G A S Y L Q G K K V V I V G C G A Q G L N Q G L N M R D S G L D I S Y A D F G E F S L R L F G A W R K A T E N G F K V G T Y E E L I P Q A D L V I N L T P D K Q H S D V V R T V Q P L M K D G A A L G Y S H G F N I V E V G E Q I R K D I T V V M V A P K C P G T E V R E E Y K R G F G V P T L I A V H P E N D P K G E G M A I A K A W A A A T G G H R A G V L E S S F V A E V K S D L M G E Q T I L C G M L Q A G S L L C F D K L V E E G T D P A Y A E K L I Q F G W E T I T E A L K Q G G I T L M M D R L S N P A K L R A Y A L S E Q L K E I M A P L F Q K H M D D I I S G E F S S G M M A D W A N D D K K L L T W R E E T G K T A F E T A P Q Y E G K I G E Q E Y F D K G V L M I A M V K A G V E L A F E T M V D S G I I E E S A Y Y E S L H E L P L I A N T I A R K R L Y E M N V V I S D T A E Y G N Y L F S Y A C V P L L K P F M A E L Q P G D L G K A I P E G A V D N G Q L R D V N E A I R S H A I E Q V G K K L R G Y M T D M K R I A V A G

SEQ ID NO: 6

IlvC_CH13 base sequence

GCCGCAGTATGAAGGCAAAATCGGCGAGCAGGAGTACTTCGATAAAGGCGTACTGATGATTGCGATGGTGAAAGCGGGCGTTGAACTGGCGTTCGAAACCATGGTCGATTCCGGCATCATTGAAGAGTCTGCATATTATGAATCACTGCACGAGCTGCCGCTGATTGCCAACACCATCGCCCGTAAGCGTCTGTACGAAATGAACGTGGTTATCTCTGATACCGCTGAGTACGGTAACTATCTGTTCTCTTACGCTTGTGTGCCGTTGCTGAAACCGTTTATGGCAGAGCTGCAACCGGGCGACCTGGGTAAAGCTATTCCGGAAGGCGCGGTAGATAACGGGCAACTGCGTGATGTGAACGAAGCGATTCGCAGCCATGCGATTGAGCAGGTAGGTAAGAAACTGCGCGGCTATATGACAGATATGAAACGTATTGCTGTTGCGGGT

In the present invention, ketol-acid reductoisomerase (IlvC) enzyme, an enzyme that converts 2-acetolactate, which is involved in isobutanol biosynthesis, into 2,3-dihydroxyisovalerate using NADPH Of NADPH to NADH. For this purpose, a mutant library was constructed through targeted random mutagenesis as a concept of remodeling the Rossmann loop region (amino acid sequence No. 67-78) involved in the binding of phosphate of NADPH in the enzyme , And an enzyme having affinity for NADH was screened. Of the initial 400 mutagenic enzymes, 42 enzymes were screened. Finally, two enzymes (IlvC_CH11, IlvC_CH13) with high affinity for NADH were screened. The mutant enzymes were 2.5-3 times higher in catabolic efficiency than NADH enzymes in terms of the rate of enzyme reaction after production and purification of two enzymes transformed into E. coli. And 4.3-4.5 times lower for NADPH. Finally, the two mutant enzymes have been shown to alter the amino acids Arg68, Arg76, and Ser78, which bind to NADPH with phosphate in the Rossman loop, to Ser68, Phe76, Asp78 and Phe68, Phe76, and Ala78, It is believed that there is a decisive reason for the affinity of NADH to be increased because the affinity is reduced.

When the mutant enzyme of ketol-acid reductoisomerase (IlvC) having high NADH affinity developed in the present invention is applied to the conventional method of producing isobutanol, since NADH produced in the process is consumed by IlvC, The problem of imbalance is solved and the productivity of isobutanol can be improved (Fig. 2).

In particular, the technology for directly increasing the production of isobutanol and the like by directly applying the ketol acid reductoisomerase, which has been developed in the present invention, to the bioenergy biosynthetic microorganism strain is a method for producing environmentally- It is very meaningful in.

When the mutant enzyme of ketol-acid reductoisomerase (IlvC) having high NADH affinity according to the present invention is applied to the production of isobutanol, the problem of cofactor redox balance is solved and isobutanol Productivity is expected to increase.

Figure 1 schematically illustrates the biosynthetic pathway of isobutanol.
Figure 2 is a schematic representation of the biosynthetic pathway of isobutanol using the IlvC mutant of the present invention.
FIG. 3 shows the procedure for constructing a library for the development of a coenzyme NADH-favored IlvC enzyme [(a) amino acid sequence and position of the Rossman loop, which is a target of a random mutation of IlvC enzyme, and (b) assembly PCR strategy for producing a random mutant library. , (c) Obtaining random library through Assmebly PCR: M, marker; lane 1, fragment 1, 198 bp; lane 2, fragment 1 + 2, 271 bp; lane 3, fragment 3, 1242 bp; lane 4, mutant ilvC of the Rossman loop, 1476 bp].
FIG. 4 shows the chemical synthesis of 2-acetolactate (hereinafter referred to as 2-AL) for IlvC enzyme screening [(a) schematic diagram for 2-AL chemical synthesis, (b) total amount obtained through GC / TOF MS Ion mass spectrometry (c) synthesized 2-AL mass spectrum].
Figure 5 shows NADH consumption by wild type and screened mutant IlvC [C, empty vector; W, wild type IlvC; 1-42, mutant IlvC].
Figure 6 shows the results of purification of the finally selected IlvC_CH11 and IlvC_CH13 mutants and the wild type IlvC enzyme [M, protein size marker (kDa); lane 1, wild type IlvC enzyme; lane 2, IlvC_CH11; lane 3, IlvC_CH13].
Figure 7 is a Lineweaver-Burk plot for determining the selectivity of the IlvC mutation and the wild-type enzyme kinetic parameters [(a) enzymatic reaction with various concentrations of NADH as a coenzyme, (b) varying concentrations of NADPH as a coenzyme To the enzyme reaction].
Fig. 8 shows the enzymatic reaction of IlvC_CH11 and IlvC_CH13. [Total ion chromatogram (total ion chromatogram) obtained by GC / TOF MS analysis of (a) enzyme reaction sample using NADH as enzyme coenzyme purified, (b ) Mass spectrum of the sample after enzyme reaction. (c) mass spectrum of the reference material 2,3-DHIV.
9 shows the results of (a) DNA sequence analysis and (b) protein sequence analysis of IlvC_CH11 and IlvC_CH13.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited by the following examples.

[ Example ]

Example  One: Ketoxylic acid Reductoisomerase ( ketol - acid 재ductoisomerase , IlvC ) Rosmann Loop for enzyme development ( Rossman loop ) Random mutant library

In order to convert the coenzyme affinity of NADPH (nicotinamide adenine dinucleotide phosphate), which was possessed by IlvC enzyme, to NADH (nicotinamide adenine dinucleotide), attention was paid to the difference between two coenzymes, "phosphate" A Rossman loop was randomly mutated to screen mutant enzymes with high affinity to NADH (Fig. 3 (a)). For this, a random mutation was constructed by using assembly PCR as shown in the schematic diagram of FIG. 3 (b) using 'Sd_' primers of the ilvC gene shown in Table 1 below. As a result, the step-by-step as shown in (c) of Figure 3 ilvC DNA fragments were obtained. Finally, all the fragments were assembled to construct a desired ilvC mutant library (Fig. 3 (c)).

In order to facilitate screening, a protein surface expression vector was prepared (pATLIC_ ilvC ^{mut in} Table 1) and transformed into E. coli Top10. As a result, 3.2 × 10 ⁴ colonies (CFU) were obtained and the transformation efficiency was 3.2 × 10 ³ CFU / μg DNA. In the meantime, a random selection of 20 colonies revealed that 36 bp of the rosemann loop had a mutation probability of 60%, while the remaining 40% had a mutation but most of the single base pair deletion And a frame shift mutation due to the mutation.

▶ Experimental method 1-1) Construction of ilvC gene mutant library :

Genomic DNA was isolated from E. coli strain K12, and the ilvC_pET_Fw and ilvC_pET_Rv primers shown in Table 1 were used to carry out ilvC Gene. Subsequently, assembly PCR was carried out using 'Sd_' primers shown in Table 1 with the obtained gene as a template (see Table 2). The target of the mutation, the Rhuman Loop, used the primer SD_R2 as its own template, and the primers used for mutagenesis had overlapping regions (15 bp overlapping region) of 5 'and 3' -62 < [deg.] ≫ C.

[Table 1]

The bacteria, plasmids and primers used in this experiment

[Table 2]

(NEB; New England Biolabs, Ipswich, MA, USA) using the Q5 polymerase for the ilvC mutagenesis.

Example  2: Coenzyme On NADH Affinity  Mutant enzyme screening

Prior to mutagenic screening, 2-AL (2-acetolactate) used as a substrate was first synthesized from ethyl 2-acetoxy-2-methylacetoacetate (EAMAA) through saponification using 0.1 M NaOH a)). The synthesized 2-AL was confirmed by gas chromatography / mass spectrometry. FIG. 4 (b) is a total ion chromatogram of synthesized 2-AL, and the retention time can be confirmed. FIG. 3 (c) shows the result of the mass spectrum of the synthesized 2-AL. As a result of the mass spectrum of the derivatized form through a unique peak on the ionized and separated mass fragmentation pattern, -AL could be confirmed. Afterwards, 400 mutagenic colonies were selected and overexpressed, followed by reaction with substrate (2-AL) and NADH, resulting in screening of 42 mutant enzymes that consume the most NADH ( 5). Among them, 13 enzymes were selected again, and 13 enzymes were overexpressed in E. coli BL21 (DE3), followed by in vitro reaction. Finally, two enzymes were selected and identified as IlvC_CH11 (SEQ ID NO: 3) And IlvC_CH13 (SEQ ID NO: 5).

▶ Experimental method 2-1) 2- chemical synthesis and identification of acetolactate (AL-2) (1):

For the screening of IlvC enzyme, the substrate 2-AL was synthesized from ethyl 2-acetoxy-2-methylacetoacetate (EAMAA). Then, the synthesized 2-AL sample was completely dried using a vacuum concentrator and pyridine was added to 40 mg / mL of methoxyamine hydrochloride in pyridine (Sigma- Aldrich, St. Louis, Mo., USA) at 30 ° C and 200 rpm for 90 minutes. Then, the reaction was performed with 45 μL of N-methyl-N- (trimethylsilyl) trifluoroacetamide (Fluka, Buchs, Switzerland) at 37 ° C. and 200 rpm for 30 minutes.

▶ Experimental method 2-2) 2- acetolactate (chemical synthesis and identification of 2-AL) (2):

The sample prepared in Experimental Method 2-1) was transferred to a vial and analyzed by gas chromatography / mass spectrometry (Agilent Technologies, 7890B CC, Wilmington, DE, USA). The GC column temperature was maintained at 50 ° C for 5 minutes, then the temperature was increased to 330 ° C, and the temperature was maintained for 1 minute. The temperature of the column was measured by RTX-5Sil MS capillary column (30 m length, 25 mm inner diameter, 0.25 mm film thickness) . The samples were injected with 1 μL spitless and the transfer line temperature and ion source temperature were maintained at 280 ° C and 250 ° C, respectively. The final GC / TOF MS results were matched with the BinBase library and confirmed to be 2-AL.

▶ Experimental method 2-3) Mutant IlvC enzyme screening :

A single colony of E. coli transformed with pATLIC_ ilvC ^mut , a vector for surface expression of the mutant protein, was cultured in LB (Luria-Bertani) liquid medium at 37 ° C for 16 hours. Thereafter, the cell culture medium was transferred to a new LB liquid medium so that the cell density became OD ₆₀₀ = 0.02. Then, when the cell culture medium reached OD ₆₀₀ = 0.5, 0.2% (w / v) And arabinose was added thereto. After 12 hours, 2 mL of the cell culture was taken, and then cells were obtained after centrifugation at 5000 xg for 10 minutes. 2.5 mM 2-AL and 200 μM NADH were incubated in phosphate buffer (100 mM sodium phosphate, 1 mM DTT, 10 mM MgCl _2, pH 7.0) for 30 min at 37 ° C for enzyme reaction. Finally, the amount of NADH consumed by the IlvC mutation expressed on the cell surface was confirmed by absorbance at 340 nm using a microplate reader. As a negative control, NADH consumption by wild type IlvC enzyme was also compared.

Example  3: IlvC _CH11 and IlvC Kinetic Coefficient of _CH13 Enzyme ( kinetic parameter ) Determination

As shown in FIG. 6, purified wild-type IlvC enzymes and selected mutant IlvC enzymes were mixed with 2.5 mM 2-AL and various concentrations of NADPH and NADH (0.05-0.5 mM) to a concentration of 0.1 mg / phosphate buffer at 37 ° C for 30 minutes. The consumed amounts of NADPH and NADH were then monitored for 1 hour every 5 minutes at 340 nm using a microplate reader. As a result, substrate, NADH, and after obtaining the initial enzyme reaction velocity (initial velocity) per concentration of NADPH Lineweaver-Burk plot based on the (7) was able to calculate the K m value and V max values, by using this K cat value And catabolic efficiency ( K cat / K m) (Table 3). As a result, it was confirmed that the catabolic efficiency of IlvC_CH11 and IlvC_CH13 on NADH was increased 2.9 and 2.6 times, respectively, compared to wild type IlvC. On the other hand, NADPH decreased 4.3 and 4.6 times compared to wild-type IlvC (Table 3). As a result, it was concluded that the selected IlvC_CH11 and CH13 mutant enzymes had higher affinity for NADH than the wild type.

▶ Experimental method 3-1) After obtaining the ilvC _CH11 _CH13 and ilvC gene by PCR, and the ilvC_Fw ilvC_Rv in Table 1 to the selected mutant enzymes and overexpression of the wild type from the use IlvC pATILC_ilvC_CH11 and pATILC_ilvC_CH13, pET21a (+) vector to construct pET21_ilvC, pET21_ilvC_CH11 and pET21_ilvC_CH11 plasmids. Plasmids were transformed into E. coli BL21 (DE3). For expression of the cells, a single colony of E. coli BL21 (DE3) was cultured in 10 mL of LB medium containing 100 μg / mL of ampicillin at 37 ° C for 16 hours. The cell density was then adjusted to an absorbance (600 nm) of 0.02 in 1 L fresh LB medium. After the incubation, the absorbance was 0.5, 0.5 mM IPTG (isopropyl-β-D- galactopyranoside) was added and cultured at 16 ° C for 16 hours. Cells were then resuspended in 30 mL of phosphate buffer (100 mM sodium phosphate, 20 mM sodium chloride, pH 7.0) and sonicated. After centrifugation, the supernatant was taken and reacted with 1 mL of Ni ^{2 +} -NTAsepharose (Qiagen, Valencia, CA, USA) for 1 hour at 4 ° C. To 1000 mM). Finally, the purified protein was quantified using the BCA method.

[Table 3]

Characteristics of the two mutants and wild-type enzymes developed in the present invention

Example  4: Selected IlvC  Mutant enzyme NADH  Identify active activity

Whether the selected IlvC mutant enzymes using NADH as a coenzyme from a 2-acetolactate synthesis of 2,3-dihydroxylisovalerate (2,3-DHIV) in vitro experiments. The reaction conditions were as follows: 0.1 mg / mL of enzyme was reacted with 2.5 mM 2-acetolactate and 0.5 mM NADH in a final volume of 200 μL of phosphate buffer at 37 ° C for 10 minutes. 2-2), GC / TOF MS analysis was performed after derivatization of the sample 8). As a result, it was confirmed that the substrate 2-AL was converted to 2,3-DHIV by using the NADH as a coenzyme by the IlvC_ mutant enzyme (FIG. 8 (a)). On the other hand, in the absence of the control enzyme, 2-AL was not changed and there was no reaction product (FIG. 8 (a)).

Finally, GC / TOF MS analysis showed that the mass spectra of the enzyme reaction products were compared with the standard mass of 2,3-dihydroxylisovalerate (Sigma-Aldrich, St. Louis, Mo., USA) (Fig. 8 (b), (c)).

Example 5: DNA and protein sequence analysis of selected IlvC mutant enzymes

Sequence analysis of the selected DNA and protein of IlvC mutant enzymes revealed that a total of 36 bases and a total of 12 amino acids between amino acids 67-78aa between 198-233bp of the gene corresponding to the Rossman loop corresponded to the wild type ilvC (or IlvC (Fig. 9 (a), (b)), and it was confirmed that the remodeling was completely performed

Particularly, it was confirmed that the amino acids 68, 76 and 78 directly involved in binding with the phosphate of NADPH in the Rhodamine loop were substituted with Arg68Ser, Arg76Phe and Arg78Asp in IlvC_CH11 and Arg68Phe, Arg76Phe and Arg78Ala in IlvC_CH13 (Fig. 9 (b)).

<110> Korea University Industrial & Academic Collaboration Foundation <120> New ketol-acid reductoisomerase with a cofactor preference to          NADH developed <130> P17U13C0754 <160> 6 <170> KoPatentin 3.0 <210> 1 <211> 491 <212> PRT <213> Escherichia coli <400> 1 Met Ala Asn Tyr Phe Asn Thr Leu Asn Leu Arg Gln Gln Leu Ala Gln   1 5 10 15 Leu Gly Lys Cys Arg Phe Met Gly Arg Asp Glu Phe Ala Asp Gly Ala              20 25 30 Ser Tyr Leu Gln Gly Lys Lys Val Val Ile Val Gly Cys Gly Ala Gln          35 40 45 Gly Leu Asn Gln Gly Leu Asn Met Arg Asp Ser Gly Leu Asp Ile Ser      50 55 60 Tyr Ala Leu Arg Lys Glu Ala Ile Ala Glu Lys Arg Ala Ser Trp Arg  65 70 75 80 Lys Ala Thr Glu Asn Gly Phe Lys Val Gly Thr Tyr Glu Glu Leu Ile                  85 90 95 Pro Gln Ala Asp Leu Val Ile Asn Leu Thr Pro Asp Lys Gln His Ser             100 105 110 Asp Val Val Arg Thr Val Gln Pro Leu Met Lys Asp Gly Ala Ala Leu         115 120 125 Gly Tyr Ser His Gly Phe Asn Ile Val Glu Val Gly Glu Gln Ile Arg     130 135 140 Lys Asp Ile Thr Val Val Met Ala Pro Lys Cys Pro Gly Thr Glu 145 150 155 160 Val Arg Glu Glu Tyr Lys Arg Gly Phe Gly Val Pro Thr Leu Ile Ala                 165 170 175 Val His Pro Glu Asn Asp Pro Lys Gly Glu Gly Met Ala Ile Ala Lys             180 185 190 Ala Trp Ala Ala Ala Thr Gly Gly His Arg Ala Gly Val Leu Glu Ser         195 200 205 Ser Phe Val Ala Glu Val Lys Ser Asp Leu Met Gly Glu Gln Thr Ile     210 215 220 Leu Cys Gly Met Leu Gln Ala Gly Ser Leu Leu Cys Phe Asp Lys Leu 225 230 235 240 Val Glu Glu Gly Thr Asp Pro Ala Tyr Ala Glu Lys Leu Ile Gln Phe                 245 250 255 Gly Trp Glu Thr Ile Thr Glu Ale Leu Lys Gln Gly Gly Ile Thr Leu             260 265 270 Met Met Asp Arg Leu Ser Asn Pro Ala Lys Leu Arg Ala Tyr Ala Leu         275 280 285 Ser Glu Gln Leu Lys Glu Ile Met Ala Pro Leu Phe Gln Lys His Met     290 295 300 Asp Asp Ile Ile Ser Gly Glu Phe Ser Ser Gly Met Met Ala Asp Trp 305 310 315 320 Ala Asn Asp Asp Lys Lys Leu Leu Thr Trp Arg Glu Glu Thr Gly Lys                 325 330 335 Thr Ala Phe Glu Thr Ala Pro Gln Tyr Glu Gly Lys Ile Gly Glu Gln             340 345 350 Glu Tyr Phe Asp Lys Gly Val Leu Met Ile Ala Met Val Lys Ala Gly         355 360 365 Val Glu Leu Ala Phe Glu Thr Met Val Asp Ser Gly Ile Ile Glu Glu     370 375 380 Ser Ala Tyr Tyr Glu Ser Leu His Glu Leu Pro Leu Ile Ala Asn Thr 385 390 395 400 Ile Ala Arg Lys Arg Leu Tyr Glu Met Asn Val Val Ile Ser Asp Thr                 405 410 415 Ala Glu Tyr Gly Asn Tyr Leu Phe Ser Tyr Ala Cys Val Pro Leu Leu             420 425 430 Lys Pro Phe Met Ala Glu Leu Gln Pro Gly Asp Leu Gly Lys Ala Ile         435 440 445 Pro Glu Gly Ala Val Asp Asn Gly Gln Leu Arg Asp Val Asn Glu Ala     450 455 460 Ile Arg Ser His Ala Ile Glu Gln Val Gly Lys Lys Leu Arg Gly Tyr 465 470 475 480 Met Thr Asp Met Lys Arg Ile Ala Val Ala Gly                 485 490 <210> 2 <211> 1473 <212> DNA <213> Escherichia coli <400> 2 atggctaact acttcaatac actgaatctg cgccagcagc tggcacagct gggcaaatgt 60 cgctttatgg gccgcgatga attcgccgat ggcgcgagct accttcaggg taaaaaagta 120 gtcatcgtcg gctgtggcgc acagggtctg aaccagggcc tgaacatgcg tgattctggt 180 ctcgatatct cctacgctct gcgtaaagaa gcgattgccg agaagcgcgc gtcctggcgt 240 aaagcgaccg aaaatggttt taaagtgggt acttacgaag aactgatccc acaggcggat 300 ctggtgatta acctgacgcc ggacaagcag cactctgatg tagtgcgcac cgtacagcca 360 ctgatgaaag acggcgcggc gctgggctac tcgcacggtt tcaacatcgt cgaagtgggc 420 gagcagatcc gtaaagatat caccgtagtg atggttgcgc cgaaatgccc aggcaccgaa 480 gtgcgtgaag agtacaaacg tgggttcggc gtaccgacgc tgattgccgt tcacccggaa 540 aacgatccga aaggcgaagg catggcgatt gccaaagcct gggcggctgc aaccggtggt 600 caccgtgcgg gtgtgctgga atcgtccttc gttgcggaag tgaaatctga cctgatgggc 660 gagcaaacca tcctgtgcgg tatgttgcag gctggctctc tgctgtgctt cgacaagctg 720 gtggaagaag gtaccgatcc agcatacgca gaaaaactga ttcagttcgg ttgggaaacc 780 atcaccgaag cactgaaaca gggcggcatc accctgatga tggaccgtct ctctaacccg 840 gcgaaactgc gtgcttatgc gctttctgaa cagctgaaag agatcatggc acccctgttc 900 cagaaacata tggacgacat catctccggc gaattctctt ccggtatgat ggcggactgg 960 gccaacgatg ataagaaact gctgacctgg cgtgaagaga ccggcaaaac cgcgtttgaa 1020 accgcgccgc agtatgaagg caaaatcggc gagcaggagt acttcgataa aggcgtactg 1080 atgattgcga tggtgaaagc gggcgttgaa ctggcgttcg aaaccatggt cgattccggc 1140 atcattgaag agtctgcata ttatgaatca ctgcacgagc tgccgctgat tgccaacacc 1200 atcgcccgta agcgtctgta cgaaatgaac gtggttatct ctgataccgc tgagtacggt 1260 aactatctgt tctcttacgc ttgtgtgccg ttgctgaaac cgtttatggc agagctgcaa 1320 ccgggcgacc tgggtaaagc tattccggaa ggcgcggtag ataacgggca actgcgtgat 1380 gtgaacgaag cgattcgcag ccatgcgatt gagcaggtag gtaagaaact gcgcggctat 1440 atgacagata tgaaacgtat tgctgttgcg ggt 1473 <210> 3 <211> 491 <212> PRT <213> Artificial Sequence <220> <223> IlvC_CH11 <400> 3 Met Ala Asn Tyr Phe Asn Thr Leu Asn Leu Arg Gln Gln Leu Ala Gln   1 5 10 15 Leu Gly Lys Cys Arg Phe Met Gly Arg Asp Glu Phe Ala Asp Gly Ala              20 25 30 Ser Tyr Leu Gln Gly Lys Lys Val Val Ile Val Gly Cys Gly Ala Gln          35 40 45 Gly Leu Asn Gln Gly Leu Asn Met Arg Asp Ser Gly Leu Asp Ile Ser      50 55 60 Tyr Ala Glu Ser Ala Leu Val Val Trp Leu Ser Phe Asp Asp Trp Arg  65 70 75 80 Lys Ala Thr Glu Asn Gly Phe Lys Val Gly Thr Tyr Glu Glu Leu Ile                  85 90 95 Pro Gln Ala Asp Leu Val Ile Asn Leu Thr Pro Asp Lys Gln His Ser             100 105 110 Asp Val Val Arg Thr Val Gln Pro Leu Met Lys Asp Gly Ala Ala Leu         115 120 125 Gly Tyr Ser His Gly Phe Asn Ile Val Glu Val Gly Glu Gln Ile Arg     130 135 140 Lys Asp Ile Thr Val Val Met Ala Pro Lys Cys Pro Gly Thr Glu 145 150 155 160 Val Arg Glu Glu Tyr Lys Arg Gly Phe Gly Val Pro Thr Leu Ile Ala                 165 170 175 Val His Pro Glu Asn Asp Pro Lys Gly Glu Gly Met Ala Ile Ala Lys             180 185 190 Ala Trp Ala Ala Ala Thr Gly Gly His Arg Ala Gly Val Leu Glu Ser         195 200 205 Ser Phe Val Ala Glu Val Lys Ser Asp Leu Met Gly Glu Gln Thr Ile     210 215 220 Leu Cys Gly Met Leu Gln Ala Gly Ser Leu Leu Cys Phe Asp Lys Leu 225 230 235 240 Val Glu Glu Gly Thr Asp Pro Ala Tyr Ala Glu Lys Leu Ile Gln Phe                 245 250 255 Gly Trp Glu Thr Ile Thr Glu Ale Leu Lys Gln Gly Gly Ile Thr Leu             260 265 270 Met Met Asp Arg Leu Ser Asn Pro Ala Lys Leu Arg Ala Tyr Ala Leu         275 280 285 Ser Glu Gln Leu Lys Glu Ile Met Ala Pro Leu Phe Gln Lys His Met     290 295 300 Asp Asp Ile Ile Ser Gly Glu Phe Ser Ser Gly Met Met Ala Asp Trp 305 310 315 320 Ala Asn Asp Asp Lys Lys Leu Leu Thr Trp Arg Glu Glu Thr Gly Lys                 325 330 335 Thr Ala Phe Glu Thr Ala Pro Gln Tyr Glu Gly Lys Ile Gly Glu Gln             340 345 350 Glu Tyr Phe Asp Lys Gly Val Leu Met Ile Ala Met Val Lys Ala Gly         355 360 365 Val Glu Leu Ala Phe Glu Thr Met Val Asp Ser Gly Ile Ile Glu Glu     370 375 380 Ser Ala Tyr Tyr Glu Ser Leu His Glu Leu Pro Leu Ile Ala Asn Thr 385 390 395 400 Ile Ala Arg Lys Arg Leu Tyr Glu Met Asn Val Val Ile Ser Asp Thr                 405 410 415 Ala Glu Tyr Gly Asn Tyr Leu Phe Ser Tyr Ala Cys Val Pro Leu Leu             420 425 430 Lys Pro Phe Met Ala Glu Leu Gln Pro Gly Asp Leu Gly Lys Ala Ile         435 440 445 Pro Glu Gly Ala Val Asp Asn Gly Gln Leu Arg Asp Val Asn Glu Ala     450 455 460 Ile Arg Ser His Ala Ile Glu Gln Val Gly Lys Lys Leu Arg Gly Tyr 465 470 475 480 Met Thr Asp Met Lys Arg Ile Ala Val Ala Gly                 485 490 <210> 4 <211> 1473 <212> DNA <213> Artificial Sequence <220> <223> IlvC_CH11 <400> 4 atggctaact acttcaatac actgaatctg cgccagcagc tggcacagct gggcaaatgt 60 cgctttatgg gccgcgatga attcgccgat ggcgcgagct accttcaggg taaaaaagta 120 gtcatcgtcg gctgtggcgc acagggtctg aaccagggcc tgaacatgcg tgattctggt 180 ctcgatatct cctacgctga atccgcattg gtcgtctggt tgagctttga cgactggcgt 240 aaagcgaccg aaaatggttt taaagtgggt acttacgaag aactgatccc acaggcggat 300 ctggtgatta acctgacgcc ggacaagcag cactctgatg tagtgcgcac cgtacagcca 360 ctgatgaaag acggcgcggc gctgggctac tcgcacggtt tcaacatcgt cgaagtgggc 420 gagcagatcc gtaaagatat caccgtagtg atggttgcgc cgaaatgccc aggcaccgaa 480 gtgcgtgaag agtacaaacg tgggttcggc gtaccgacgc tgattgccgt tcacccggaa 540 aacgatccga aaggcgaagg catggcgatt gccaaagcct gggcggctgc aaccggtggt 600 caccgtgcgg gtgtgctgga atcgtccttc gttgcggaag tgaaatctga cctgatgggc 660 gagcaaacca tcctgtgcgg tatgttgcag gctggctctc tgctgtgctt cgacaagctg 720 gtggaagaag gtaccgatcc agcatacgca gaaaaactga ttcagttcgg ttgggaaacc 780 atcaccgaag cactgaaaca gggcggcatc accctgatga tggaccgtct ctctaacccg 840 gcgaaactgc gtgcttatgc gctttctgaa cagctgaaag agatcatggc acccctgttc 900 cagaaacata tggacgacat catctccggc gaattctctt ccggtatgat ggcggactgg 960 gccaacgatg ataagaaact gctgacctgg cgtgaagaga ccggcaaaac cgcgtttgaa 1020 accgcgccgc agtatgaagg caaaatcggc gagcaggagt acttcgataa aggcgtactg 1080 atgattgcga tggtgaaagc gggcgttgaa ctggcgttcg aaaccatggt cgattccggc 1140 atcattgaag agtctgcata ttatgaatca ctgcacgagc tgccgctgat tgccaacacc 1200 atcgcccgta agcgtctgta cgaaatgaac gtggttatct ctgataccgc tgagtacggt 1260 aactatctgt tctcttacgc ttgtgtgccg ttgctgaaac cgtttatggc agagctgcaa 1320 ccgggcgacc tgggtaaagc tattccggaa ggcgcggtag ataacgggca actgcgtgat 1380 gtgaacgaag cgattcgcag ccatgcgatt gagcaggtag gtaagaaact gcgcggctat 1440 atgacagata tgaaacgtat tgctgttgcg ggt 1473 <210> 5 <211> 491 <212> PRT <213> Artificial Sequence <220> <223> IlvC_CH13 <400> 5 Met Ala Asn Tyr Phe Asn Thr Leu Asn Leu Arg Gln Gln Leu Ala Gln   1 5 10 15 Leu Gly Lys Cys Arg Phe Met Gly Arg Asp Glu Phe Ala Asp Gly Ala              20 25 30 Ser Tyr Leu Gln Gly Lys Lys Val Val Ile Val Gly Cys Gly Ala Gln          35 40 45 Gly Leu Asn Gln Gly Leu Asn Met Arg Asp Ser Gly Leu Asp Ile Ser      50 55 60 Tyr Ala Asp Phe Gly Glu Phe Ser Leu Arg Leu Phe Gly Ala Trp Arg  65 70 75 80 Lys Ala Thr Glu Asn Gly Phe Lys Val Gly Thr Tyr Glu Glu Leu Ile                  85 90 95 Pro Gln Ala Asp Leu Val Ile Asn Leu Thr Pro Asp Lys Gln His Ser             100 105 110 Asp Val Val Arg Thr Val Gln Pro Leu Met Lys Asp Gly Ala Ala Leu         115 120 125 Gly Tyr Ser His Gly Phe Asn Ile Val Glu Val Gly Glu Gln Ile Arg     130 135 140 Lys Asp Ile Thr Val Val Met Ala Pro Lys Cys Pro Gly Thr Glu 145 150 155 160 Val Arg Glu Glu Tyr Lys Arg Gly Phe Gly Val Pro Thr Leu Ile Ala                 165 170 175 Val His Pro Glu Asn Asp Pro Lys Gly Glu Gly Met Ala Ile Ala Lys             180 185 190 Ala Trp Ala Ala Ala Thr Gly Gly His Arg Ala Gly Val Leu Glu Ser         195 200 205 Ser Phe Val Ala Glu Val Lys Ser Asp Leu Met Gly Glu Gln Thr Ile     210 215 220 Leu Cys Gly Met Leu Gln Ala Gly Ser Leu Leu Cys Phe Asp Lys Leu 225 230 235 240 Val Glu Glu Gly Thr Asp Pro Ala Tyr Ala Glu Lys Leu Ile Gln Phe                 245 250 255 Gly Trp Glu Thr Ile Thr Glu Ale Leu Lys Gln Gly Gly Ile Thr Leu             260 265 270 Met Met Asp Arg Leu Ser Asn Pro Ala Lys Leu Arg Ala Tyr Ala Leu         275 280 285 Ser Glu Gln Leu Lys Glu Ile Met Ala Pro Leu Phe Gln Lys His Met     290 295 300 Asp Asp Ile Ile Ser Gly Glu Phe Ser Ser Gly Met Met Ala Asp Trp 305 310 315 320 Ala Asn Asp Asp Lys Lys Leu Leu Thr Trp Arg Glu Glu Thr Gly Lys                 325 330 335 Thr Ala Phe Glu Thr Ala Pro Gln Tyr Glu Gly Lys Ile Gly Glu Gln             340 345 350 Glu Tyr Phe Asp Lys Gly Val Leu Met Ile Ala Met Val Lys Ala Gly         355 360 365 Val Glu Leu Ala Phe Glu Thr Met Val Asp Ser Gly Ile Ile Glu Glu     370 375 380 Ser Ala Tyr Tyr Glu Ser Leu His Glu Leu Pro Leu Ile Ala Asn Thr 385 390 395 400 Ile Ala Arg Lys Arg Leu Tyr Glu Met Asn Val Val Ile Ser Asp Thr                 405 410 415 Ala Glu Tyr Gly Asn Tyr Leu Phe Ser Tyr Ala Cys Val Pro Leu Leu             420 425 430 Lys Pro Phe Met Ala Glu Leu Gln Pro Gly Asp Leu Gly Lys Ala Ile         435 440 445 Pro Glu Gly Ala Val Asp Asn Gly Gln Leu Arg Asp Val Asn Glu Ala     450 455 460 Ile Arg Ser His Ala Ile Glu Gln Val Gly Lys Lys Leu Arg Gly Tyr 465 470 475 480 Met Thr Asp Met Lys Arg Ile Ala Val Ala Gly                 485 490 <210> 6 <211> 1473 <212> DNA <213> Artificial Sequence <220> <223> IlvC_CH13 <400> 6 atggctaact acttcaatac actgaatctg cgccagcagc tggcacagct gggcaaatgt 60 cgctttatgg gccgcgatga attcgccgat ggcgcgagct accttcaggg taaaaaagta 120 gtcatcgtcg gctgtggcgc acagggtctg aaccagggcc tgaacatgcg tgattctggt 180 ctcgatatct cctacgctga ttttggtgaa ttttcgttac gtctgttcgg tgcctggcgt 240 aaagcgaccg aaaatggttt taaagtgggt acttacgaag aactgatccc acaggcggat 300 ctggtgatta acctgacgcc ggacaagcag cactctgatg tagtgcgcac cgtacagcca 360 ctgatgaaag acggcgcggc gctgggctac tcgcacggtt tcaacatcgt cgaagtgggc 420 gagcagatcc gtaaagatat caccgtagtg atggttgcgc cgaaatgccc aggcaccgaa 480 gtgcgtgaag agtacaaacg tgggttcggc gtaccgacgc tgattgccgt tcacccggaa 540 aacgatccga aaggcgaagg catggcgatt gccaaagcct gggcggctgc aaccggtggt 600 caccgtgcgg gtgtgctgga atcgtccttc gttgcggaag tgaaatctga cctgatgggc 660 gagcaaacca tcctgtgcgg tatgttgcag gctggctctc tgctgtgctt cgacaagctg 720 gtggaagaag gtaccgatcc agcatacgca gaaaaactga ttcagttcgg ttgggaaacc 780 atcaccgaag cactgaaaca gggcggcatc accctgatga tggaccgtct ctctaacccg 840 gcgaaactgc gtgcttatgc gctttctgaa cagctgaaag agatcatggc acccctgttc 900 cagaaacata tggacgacat catctccggc gaattctctt ccggtatgat ggcggactgg 960 gccaacgatg ataagaaact gctgacctgg cgtgaagaga ccggcaaaac cgcgtttgaa 1020 accgcgccgc agtatgaagg caaaatcggc gagcaggagt acttcgataa aggcgtactg 1080 atgattgcga tggtgaaagc gggcgttgaa ctggcgttcg aaaccatggt cgattccggc 1140 atcattgaag agtctgcata ttatgaatca ctgcacgagc tgccgctgat tgccaacacc 1200 atcgcccgta agcgtctgta cgaaatgaac gtggttatct ctgataccgc tgagtacggt 1260 aactatctgt tctcttacgc ttgtgtgccg ttgctgaaac cgtttatggc agagctgcaa 1320 ccgggcgacc tgggtaaagc tattccggaa ggcgcggtag ataacgggca actgcgtgat 1380 gtgaacgaag cgattcgcag ccatgcgatt gagcaggtag gtaagaaact gcgcggctat 1440 atgacagata tgaaacgtat tgctgttgcg ggt 1473

Claims (3)

An NADH-friendly ketolan lidto isomerase variant consisting of the amino acid sequence of SEQ ID NO: 3
5. An NADH-friendly ketolan reductoisomerase variant comprising the amino acid sequence of SEQ ID NO: 5.
A method for producing isobutanol using the ketolanilductoisomerase mutant of claim 1 or 2.

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