3-Hydroxybutyrate Dehydrogenase with Enhanced Activity

Abstract

The present invention relates to a 3-hydroxybutyrate dehydrogenase (3HBDH) variant that converts levulinic acid (LA) to 4-hydroxyvaleric acid (4HV). The variant of the present invention exhibits significantly higher catalytic activity compared to conventional variants in the conversion reaction of levulinic acid to 4HV, and in particular, even under conditions of high concentration of levulinic acid, substrate inhibition does not occur or is significantly reduced, thereby stably maintaining high activity. Therefore, the present invention can effectively mitigate the substrate inhibition problem, which is a major cause of reduced productivity in industrial processes using high-concentration substrates, and as a result, has the effect of significantly improving the efficiency and process applicability of the levulinic acid-based 4HV production process.

Description

Technology Field

[0001] The present invention relates to a 3-hydroxybutyrate dehydrogenase variant and a method for producing the same.

[0002] This patented technology is the result of research conducted with funding from the government (Ministry of Science and ICT) and supported by the National Research Foundation of Korea (RS-2024-00352951).

[0003] In addition, this patented technology is the result of research funded by the Ministry of Trade, Industry and Energy and the Korea Institute of Industrial Technology Planning and Evaluation (KEIT) in 2026 (RS-2024-00488503). Background Technology

[0004] Levulinic acid (LA) is a bio-based precursor that can be derived from cellulose-based biomass, including lignocellulose, and is known to be produced through a relatively simple acid treatment process. As such, levulinic acid is attracting attention as an important platform chemical in the biomass-based chemical industry, as it can be utilized as a raw material for various high-value compounds such as pharmaceuticals, bioplastics, and biofuels.

[0005] In particular, among the compounds that can be derived from levulinic acid, 4-hydroxyvaleric acid (4HV) can be utilized as a monomer for biodegradable polyester or as a precursor for biopolyester, and furthermore, there is potential for conversion into various high-value-added chemicals. Therefore, the technology of converting levulinic acid into 4-hydroxyvaleric acid and applying it to the bioplastics and biofuel industries provides a significant foundation for the production of biomass-based high-value-added chemicals.

[0006] The above conversion can be carried out via an enzyme-catalyzed bioconversion method; for example, it is possible to convert levulinic acid to 4-hydroxyvaleric acid through a reduction reaction using 3-hydroxybutyrate dehydrogenase (3HBDH). 3HBDH is NAD + As a NAD-dependent oxidoreductase, + It is known as an enzyme that catalyzes the reversible redox reaction between D-3-hydroxybutyrate bound to NADH and acetoacetate bound to NADH.

[0007] Conversion reactions utilizing enzyme catalysts can generally be carried out under mild conditions, offering advantages over chemical conversion processes that may require high temperatures, high pressures, prolonged reactions, or expensive metal catalysts. Furthermore, specific enzymes provide stereoselectivity, which can be industrially advantageous as it enables the production of optically pure compounds without the need for separation processes for racemic mixtures.

[0008] Meanwhile, in the reduction reaction of levulinic acid using 3HBDH, enzyme activity is a key factor determining process efficiency. Generally, enzyme catalytic activity can be described by kinetic parameters such as the catalytic rate constant (kcat), substrate binding affinity (Km), and catalytic efficiency (kcat / Km). In particular, improving kcat can be an important technical challenge to ensure high productivity under high substrate concentration conditions.

[0009] Furthermore, under industrial conditions where substrates such as levulinic acid are applied at high concentrations, substrate inhibition may occur, which can lead to a decrease in reaction rate and reduced productivity. Therefore, reducing the effect of substrate inhibition to maintain activity even under high substrate concentration conditions in enzyme-based bioconversion processes can be recognized as an important technical challenge.

[0010] Approaches to enhance the catalytic activity or substrate specificity of enzymes include (i) a rational design that targets and mutates specific amino acid residues based on enzyme structural information and active site interactions, and (ii) a directed evolution method that secures superior variants through screening and selection after constructing a variant library containing random mutations.

[0011] Previously, studies aimed at improving the substrate specificity of 3HBDH for levulinic acid have been reported, and these studies can be understood as having been conducted with a focus on rational design that redesigns enzyme-substrate interactions primarily around the enzyme's active site. For example, Alcaligenes faecalis A variant with improved levulinic acid conversion characteristics has been proposed by introducing an active site mutation to the derived 3HBDH.

[0012] However, despite the improvement in substrate specificity as described above, there may still be limitations in enhancing catalytic activity (especially kcat) to the level required for actual industrial application and maintaining activity under high-concentration substrate conditions. In particular, certain variants exhibit substrate inhibition at levulinic acid concentrations of 50 mM or higher, which can lead to a decrease in efficiency under high-concentration substrate-based process conditions. This can act as a limiting factor in improving the productivity of the 4HV production process using levulinic acid.

[0013] Therefore, in the bioconversion process of converting levulinic acid to 4-hydroxyvaleric acid, there is a need to develop technology that provides a 3HBDH variant with (i) improved catalytic activity (e.g., kcat), (ii) reduced or suppressed substrate inhibition even under high-concentration substrate conditions, and (iii) excellent process applicability. The problem to be solved

[0014] In order to solve the above-mentioned problems, the inventors have made diligent research efforts to develop a 3-hydroxybutyrate dehydrogenase variant that can overcome the disadvantages of conventional 3-hydroxybutyrate dehydrogenase variants, which still exhibit low catalytic activity and substrate inhibition under high-concentration substrate conditions, and particularly improve kcat (catalytic rate constant) and reduce substrate inhibition effects under high-concentration levulinic acid conditions.

[0015] As a result, the present invention was completed by developing a dehydrogenase that has the effect of significantly improving the production efficiency of 4-hydroxyvaleric acid through the enzymatic reduction reaction of levulinic acid, by performing error-prone PCR-based directional evolution and screening on the entire enzyme structure, not limited to the active site, to enhance the catalytic activity of 3HBDH, thereby continuously maintaining high activity without substrate inhibition even under high concentration levulinic acid conditions.

[0016] Accordingly, the object of the present invention is to provide a 3-hydroxybutyrate dehydrogenase variant comprising one or more amino acid substitutions of V193F and G233R based on the amino acid sequence number of SEQ ID NO. 19.

[0017] Another objective of the present invention is to provide a polynucleotide encoding a 3-hydroxybutyrate dehydrogenase variant.

[0018] Another objective of the present invention is to provide a recombinant vector comprising a polynucleotide encoding a 3-hydroxybutyrate dehydrogenase variant.

[0019] Another objective of the present invention is to provide a transgenic microorganism into which a recombinant vector comprising a polynucleotide encoding a 3-hydroxybutyrate dehydrogenase variant has been introduced.

[0020] Another objective of the present invention is alkaline phaecalis ( Alcaligenes faecalis The present invention provides a method for producing 4-hydroxyvaleric acid, comprising an enzymatic reaction step of synthesizing 4-hydroxyvaleric acid by adding a substrate to a 3-hydroxybutyrate dehydrogenase variant derived from a strain.

[0021] Another objective of the present invention is to provide a method for preparing a 3-hydroxybutyrate dehydrogenase variant. means of solving the problem

[0022] The present invention will be described in more detail below.

[0023] One aspect of the present invention is a 3-hydroxybutyrate dehydrogenase variant comprising one or more amino acid substitutions of V193F and G233R based on the amino acid sequence number of SEQ ID NO. 19.

[0024] In the present invention, the 3-hydroxybutyrate dehydrogenase variant comprising the V193F amino acid substitution may be SEQ ID NO. 21, but is not limited thereto.

[0025] In the present invention, the 3-hydroxybutyrate dehydrogenase variant comprising a G233R amino acid substitution may be SEQ ID NO. 23, but is not limited thereto.

[0026] In the present invention, a 3-hydroxybutyrate dehydrogenase variant comprising V193F and G233R amino acid substitutions may be SEQ ID NO. 25, but is not limited thereto.

[0027] Another aspect of the present invention is a polynucleotide encoding a 3-hydroxybutyrate dehydrogenase variant comprising one or more amino acid substitutions of V193F and G233R based on the amino acid sequence number of SEQ ID NO. 19.

[0028] In the present invention, the polynucleotide may include SEQ ID NO. 22, SEQ ID NO. 24, or SEQ ID NO. 26, and, for example, may be composed of SEQ ID NO. 22, SEQ ID NO. 24, or SEQ ID NO. 26, but is not limited thereto.

[0029] Another aspect of the present invention is a recombinant vector comprising a polynucleotide encoding a 3-hydroxybutyrate dehydrogenase variant comprising one or more amino acid substitutions of V193F and G233R based on the amino acid sequence number of SEQ ID NO. 19.

[0030] In the present invention, the term “vector” refers to a means for expressing a target gene in a host cell. For example, it includes viral vectors such as plasmid vectors, cosmid vectors, bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors. Vectors that can be used as recombinant vectors may be produced by manipulating plasmids commonly used in the industry (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series and pUC19, etc.), phages (e.g., λgt4λ B, λ-Charon, λΔz1 and M13, etc.) or viruses (e.g., SV40, etc.), preferably pET22b(+) or pCDFDuet-1, but are not limited thereto.

[0031] In the present invention, the vector can typically be constructed as a vector for cloning or a vector for expression. The vector for expression may be any conventional one used in the art to express foreign proteins in plants, animals, or microorganisms, but is not limited thereto. The recombinant vector may be constructed through various methods known in the art.

[0032] In the present invention, the recombinant vector may be constructed using a prokaryotic cell or a eukaryotic cell as a host. For example, when the vector used is an expression vector and the host is a prokaryotic cell, it may include a potent promoter capable of proceeding transcription (e.g., pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription / translation termination sequence, but is not limited thereto.

[0033] In the present invention, the recombinant vector may be constructed using a prokaryotic cell or a eukaryotic cell as a host. For example, when the vector used is an expression vector and the host is a prokaryotic cell, it may include a potent promoter capable of proceeding transcription (e.g., pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription / translation termination sequence, but is not limited thereto.

[0034] Another aspect of the present invention is a transgenic microorganism into which a recombinant vector is introduced, comprising a polynucleotide encoding a 3-hydroxybutyrate dehydrogenase variant comprising one or more amino acid substitutions of V193F and G233R based on the amino acid sequence number of SEQ ID NO. 19.

[0035] In the present invention, the transformed microorganism may be obtained by introducing the recombinant vector into a suitable microorganism, but is not limited thereto.

[0036] In the present invention, the microorganism is a cell capable of stably and continuously cloning or expressing the recombinant vector, and any host cell known in the art may be used.

[0037] In the present invention, microorganisms may include Escherichia coli, yeast, animal cells, plant cells, or insect cells, etc. For example, prokaryotic cells include strains of the genus Bacillus such as E. coli JM109, E. coli DH5a, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, and Bacillus churingensis, as well as intestinal bacteria and strains such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species. When transforming into eukaryotic cells, host cells may include yeast (Saccharomyce cerevisiae), insect cells, plant cells, and animal cells, for example. E. coli , Saccharomyces , Pichia , Corynebacterium The following may be used, but are not limited thereto.

[0038] In the present invention, the transport (introduction) of the recombinant vector into a host cell may be performed using a transport method widely known in the art.

[0039] In the present invention, the transport method may, for example, use the CaCl2 method or the electroporation method when the microorganism is a prokaryotic cell, and when the microorganism is a eukaryotic cell, use the microinjection method, calcium phosphate precipitation method, electroporation method, liposome-mediated transfection method and gene bombardment method, but is not limited thereto.

[0040] In the present invention, the method for selecting transformed microorganisms can be easily carried out according to methods widely known in the art by utilizing the phenotype expressed by a selection marker. For example, if the selection marker is a specific antibiotic resistance gene, the transformed organism can be easily selected by culturing the transformed organism in a medium containing the antibiotic.

[0041] Another aspect of the present invention is a method for producing 4-hydroxyvaleric acid comprising the following steps:

[0042] Alkaline phaecalis ( Alcaligenes faecalis Enzyme reaction step for synthesizing 4-hydroxyvaleric acid by adding a substrate to a strain-derived 3-hydroxybutyrate dehydrogenase variant.

[0043] In this specification, Alkaline phaecalis is known as a Gram-negative aerobic rod-shaped bacterium that is widely distributed in soil and water environments and has biochemical characteristics of being oxidase-positive and catalase-positive.

[0044] In the present invention, the substrate may include levulinic acid, but is not limited thereto.

[0045] Levulinic acid in this specification is a C-5 chemical characterized by an acidic carboxyl group and a ketone carbonyl group, and can be represented by the following chemical formula 1.

[0046] [Chemical Formula 1]

[0047]

[0048] In the present invention, the 3-hydroxybutyrate dehydrogenase variant may be one in which the 3-hydroxybutyrate dehydrogenase comprises one or more amino acid substitutions among V193F and G233R based on the amino acid sequence number of SEQ ID NO. 19, but is not limited thereto.

[0049] Another aspect of the present invention is a method for preparing a 3-hydroxybutyrate dehydrogenase variant comprising the following steps, wherein the 3-hydroxybutyrate dehydrogenase comprises one or more amino acid substitutions of V193F and G233R based on the amino acid sequence number of SEQ ID NO. 19:

[0050] A step of providing a recombinant vector comprising a polynucleotide encoding the above variant; and

[0051] A step of culturing host cells transformed with the above recombinant vector to express the above variant.

[0052] In the present invention, the manufacturing method may include a step of recovering an expressed variant, but is not limited thereto.

[0053] In the present invention, the host cell is alkaline phaecalis ( Alcaligenes faecalis It may be a strain, but is not limited to this. Effects of the invention

[0054] The present invention relates to a 3-hydroxybutyrate dehydrogenase (3HBDH) variant that converts levulinic acid (LA) to 4-hydroxyvaleric acid (4HV). The variant of the present invention exhibits significantly higher catalytic activity compared to conventional variants in the conversion reaction of levulinic acid to 4HV, and in particular, even under conditions of high concentration of levulinic acid, substrate inhibition does not occur or is significantly reduced, thereby stably maintaining high activity.

[0055] Therefore, the present invention can effectively mitigate the substrate inhibition problem, which is a major cause of reduced productivity in industrial processes using high-concentration substrates, and as a result, has the effect of significantly improving the efficiency and process applicability of the levulinic acid-based 4HV production process. Brief explanation of the drawing

[0056] Figure 1 is a gene carrier (vector) map showing a pET-22b(+) vector with the Af-3HBDH gene inserted. Figure 2 is a gene carrier (vector) map showing the pCDFDuet-1 vector with FDH inserted. Figure 3 shows the results of confirming the Af-3HBDH gene product amplified by pET-22b(+) vector and error-prone PCR (ep-PCR) by agarose gel electrophoresis. Figure 4 is a figure showing the results of confirming the expression and purification of Af-3HBDH (wild type and variant) and FDH by SDS-PAGE. Figure 5 is a figure showing the results of performing a first round screening on the Af-3HBDH variant library. Figure 6 shows the results of a second round screening based on a triple mutant. Figure 7 is a graph showing the results of evaluating the effect of changes in levulinic acid (LA) concentration on the conversion reaction to 4-hydroxy acid (4HA) for Af-3HBDH wild type, double mutant, triple mutant, and quadruple mutant. Figure 8 is under conditions of a levulinic acid concentration of 100 mM. In vitro This is a graph showing the results of comparing the initial reaction rates between the wild type and variants by performing the reaction. Figure 9 is under conditions of a levulinic acid concentration of 50 mM. In vitro This is a graph showing the results of evaluating whether a 100% conversion rate was achieved by performing a response. Specific details for implementing the invention

[0057] The advantages and features of the present invention and the methods for achieving them will become clear by referring to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0059] 제조예 1. 3HBDH 유전자 및 재료 준비

[0060] The 3HBDH gene was commercially synthesized at Bioneer (Daejeon, South Korea). The primers were synthesized at Cosmogentech (Seoul, South Korea). Competent E. coli BL21 (DE3) and DH5α cells were purchased from Invitrogen (Carlsbad, CA, USA) and Novagen (Madison, WI, USA), respectively. The previously constructed Af-3HBDH (H144L / W187F) gene was prepared in the pET-22b(+) vector. DNA purification and extraction kits and Ni-NTA agarose for His-tag protein purification were purchased from Qiagen (Valencia, CA, USA). Levulinic acid, NADH, and ammonium formate were purchased from Sigma (St. Louis, MO, USA). A sodium levulinate solution was prepared as a substrate for enzymatic analysis. An Error-Prone PCR kit was purchased from Canvax. A Gel Purification Kit was purchased from Bioneer (Daejeon, South Korea). The In-Fusion®-HD Cloning Kit (Takara, Kusatsu, Japan) was used for cloning. All other chemicals were purchased from Sigma-Aldrich (St. Louis, Missouri, USA).

[0062] 제조예 2. 3HBDH 변이 라이브러리 구축

[0063] Ampicillin resistance was conferred by inserting the Af-3HBDH (H144L / W187F) variant gene into the pET-22b(+) vector (Fig. 1). In addition, C.boidinii and Pseudomonas To perform co-transformation with a formate dehydrogenase (FDH) gene derived from sp., the FDH gene was inserted into the pCDFDuet-1 vector (Fig. 2). The pCDFDuet-1 vector additionally provides streptomycin resistance as well as ampicillin resistance for double selection.

[0064] The primers designed for Gibson assembly-based cloning of the pCDFDuet-1 vector were configured to include an NdeI restriction enzyme recognition sequence at the 5′ end and an XhoI restriction enzyme recognition sequence at the 3′ end, and were additionally designed to include a His-tag sequence.

[0065] Meanwhile, after obtaining the Af-3HBDH(H144L / W187F) gene from the pET-22b(+) vector, various mutations were induced using an error-prone PCR kit. Specifically, PCR amplification was performed according to the conditions in Tables 2 and 3 using the primers listed in Table 1, and the amplification products were purified using a gel purification kit. Then, a random mutation library was constructed by cloning the purified PCR products using a cloning kit.

[0066] 서열번호 명칭 서열 5'→ 3' 1 pET-22b(+)_For GAGCTCCGTCGACAAGCTTG 2 pET-22b(+)_Rev TATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGG 3 Af-3HBDH_For AACTTAAGAAGGAGATACATATGCACCATCACCATC 4 Af-3HBDH_Rev CAAGCTTGTCGACGGAGCTCGAATTCTCATCTTGCGGTC 5 H144L_For AGCGCAC T CGGCCTGGTAGCATCTGTCAAT 6 H144L_Rev CAGGCCG A GTGCGCTCGCAATATTAATTAT 7 W187F_For CCGGGAT TC GTGCGTACGCCGCTGGTTGAG 8 W187F_Rev ACGCAC GA ATCCCGGGCAAATTGCATTACA 9 V193F_For CCGCTG T TTGAGAAACAAATC 10 V193F_Rev TTTCTCAA A CAGCGGCGTACG 11 G233R_For CAGTTAGGA A GAGCAGCAGTGTTTCTAAGT 12 G233R_Rev TGCTGCTC T TCCTAACTGTTCGGGCGT 13 pCDFDuet-1_For CTCGAGTCTGGTAAAGAAAC 14 pCDFDuet-1_Rev CATATGTATATCTCCTTCTTATACTTAAC 15 Ps-FDH_For CTTAGTATATTAGTTAAGTATAGAAGGAGATACATATGATGCACCACCACCACCAC 16 Ps-FDH_Rev GTTTCTTTACCAGACTCGAGTCAAACTGCTTTTTTGAATTTCGCGGCTT

[0067] * The underlined parts in Table 1 indicate sequences that are mutated through the primers.

[0068] Cloning PCR 조건:Pre-heating Template denaturation Primary annealing DNA elongation - Storage 95℃ 95℃ Primer Tm - 5℃ 72℃ 72℃ 4℃ 4min 30sec 30sec 1kb per 1min 5min - 1 cycle 15~25 cycles 1 cycle -

[0069] Mutant PCR 조건:Pre-heating Template denaturation Primary annealing DNA elongation - Storage 95℃ 95℃ Primer Tm - 5℃ 72℃ 72℃ 4℃ 4min 1min 30sec 1kb per 1min 1hr - 1 cycle 16~18 cycle 1 cycle -

[0071] The PCR amplification products and cloning products were verified by agarose gel electrophoresis, and the results are shown in Table 4 and Figure 3 below.

[0072] Sequence number designation Sequence 5'→ 3' 17 3-hydroxybutyrate dehydrogenase MLKGKKAVVT GSTSGIGLAM ATELAKAGAD VVINGFGQPE DIERERSTLE SKFGVKAYYL NADLSDAQAT RDFIAKAAEA LGGLDILVNN AGIQHTAPIE EFPVDKWNAI IALNLSAVFH GTAAALPIMQ KQGWGRIINI ASA H GLVASV NKSAYVAAKH GVVGLTKVTA LENAGKGITC NAICPG W GARDEN PL V EKQIEAI SQQKGIDIEA AARELLAEKQ PSLQFVTPEQ LG G AAVFLSS AAADQMTGTT LSLDGGWTAR 18 3-Hydroxybutyrate dehydrogenase nucleic acid sequence ATGCTTAAAGGTAAAAAAGCAGTCGTAACTGGTTCCACAAGCGGGATAGGACTGGCAATGGCAACTGAGCTTGCAAAAGCAGGCGCAGATGTAGTAATTAACGGTTTCGGTCAGCCAGAGGATATCGAACGCGAACGCTCAACCCTCGAATCTAAATTTGGCGTGAAAGCGTATTATCTGAACGCAGATCTCTCAGATGCACAGGCAACCCGGGATTTCATTGCAAAAGCAGCAGAAGCATTGGGTGGTCTGGACATCTTAGTCAATAACGCTGGTATCCAACATACCGCACCGATCGAAGAGTTTCCAGTGGACAAATGGAACGCAATTATTGCATTAAATTTAAGTGCTGTTTTTCATGGTACGGCAGCAGCACTTCCTATTATGCAGAAGCAGGGTTGGGGTCGTATAATTAATATTGCGAGCGCA CAC GGCCTGGTAGCATCTGTCAATAAATCGGCATACGTTGCAGCTAAGCATGGCGTTGTTGGTTTGACTAAAGTGACAGCTCTGGAAAATGCAGGTAAAGGTATAACGTGTAATGCAATTTGCCCGGGA TGG GTGCGTACGCCGCTG GTT GAGAAACAAATCGAAGCAATTAGTCAGCAAAAGGGAATAGACATCGAAGCTGCTGCACGTGAGTTGCTGGCAGAAAAACAGCCTAGCTTACAATTTGTTACGCCCGAACAGTTAGGA GGA GCAGCAGTGTTTCTAAGTTCCGCTGCAGCAGATCAGATGACAGGTACAACCCTGTCGCTGGATGGTGGTTGGACCGCAAGA 19 3-Hydroxybutyrate dehydrogenase dual variant H144L / W187F MLKGKKAVVT GSTSGIGLAM ATELAKAGAD VVINGFGQPE DIERERSTLE SKFGVKAYYL NADLSDAQAT RDFIAKAAEA LGGLDILVNN AGIQHTAPIE EFPVDKWNAI IALNLSAVFH GTAAALPIMQ KQGWGRIINI ASA L GLVASV NKSAYVAAKH GVVGLTKVTA LENAGKGITC NAICPG F VRT PLVEKQIEAI SQQKGIDIEA AARELLAEKQ PSLQFVTPEQ LGGAAVFLSS AAADQMTGTT LSLDGGWTAR 20 3-Hydroxybutyrate dehydrogenase dual variant H144L / W187F nucleic acid sequence ATGCTTAAAGGTAAAAAAGCAGTCGTAACTGGTTCCACAAGCGGGATAGGACTGGCAATGGCAACTGAGCTTGCAAAAGCAGGCGCAGATGTAGTAATTAACGGTTTCGGTCAGCCAGAGGATATCGAACGCGAACGCTCAACCCTCGAATCTAAATTTGGCGTGAAAGCGTATTATCTGAACGCAGATCTCTCAGATGCACAGGCAACCCGGGATTTCATTGCAAAAGCAGCAGAAGCATTGGGTGGTCTGGACATCTTAGTCAATAACGCTGGTATCCAACATACCGCACCGATCGAAGAGTTTCCAGTGGACAAATGGAACGCAATTATTGCATTAAATTTAAGTGCTGTTTTTCATGGTACGGCAGCAGCACTTCCTATTATGCAGAAGCAGGGTTGGGGTCGTATAATTAATATTGCGAGCGCA CTC GGCCTGGTAGCATCTGTCAATAAATCGGCATACGTTGCAGCTAAGCATGGCGTTGTTGGTTTGACTAAAGTGACAGCTCTGGAAAATGCAGGTAAAGGTATAACGTGTAATGCAATTTGCCCGGGA TTC GTGCGTACGCCGCTGGTTGAGAAACAAATCGAAGCAATTAGTCAGCAAAAGGGAATAGACATCGAAGCTGCTGCACGTGAGTTGCTGGCAGAAAAACAGCCTAGCTTACAATTTGTTACGCCCGAACAGTTAGGAGGAGCAGCAGTGTTTCTAAGTTCCGCTGCAGCAGATCAGATGACAGGTACAACCCTGTCGCTGGATGGTGGTTGGACCGCAAGA 21 3-Hydroxybutyrate dehydrogenase triple variant H144L / W187F / V193F MLKGKKAVVT GSTSGIGLAM ATELAKAGAD VVINGFGQPE DIERERSTLE SKFGVKAYYL NADLSDAQAT RDFIAKAAEA LGGLDILVNN AGIQHTAPIE EFPVDKWNAI IALNLSAVFH GTAAALPIMQ KQGWGRIINI ASA L GLVASV NKSAYVAAKH GVVGLTKVTA LENAGKGITC NAICPG F GARDEN PL F EKQIEAI SQQKGIDIEA AARELLAEKQ PSLQFVTPEQ LGGAAVFLSS AAADQMTGTT LSLDGGWTAR 22 3-Hydroxybutyrate dehydrogenase triple variant H144L / W187F / V193F nucleic acid sequence ATGCTTAAAGGTAAAAAAGCAGTCGTAACTGGTTCCACAAGCGGGATAGGACTGGCAATGGCAACTGAGCTTGCAAAAGCAGGCGCAGATGTAGTAATTAACGGTTTCGGTCAGCCAGAGGATATCGAACGCGAACGCTCAACCCTCGAATCTAAATTTGGCGTGAAAGCGTATTATCTGAACGCAGATCTCTCAGATGCACAGGCAACCCGGGATTTCATTGCAAAAGCAGCAGAAGCATTGGGTGGTCTGGACATCTTAGTCAATAACGCTGGTATCCAACATACCGCACCGATCGAAGAGTTTCCAGTGGACAAATGGAACGCAATTATTGCATTAAATTTAAGTGCTGTTTTTCATGGTACGGCAGCAGCACTTCCTATTATGCAGAAGCAGGGTTGGGGTCGTATAATTAATATTGCGAGCGCA CTC GGCCTGGTAGCATCTGTCAATAAATCGGCATACGTTGCAGCTAAGCATGGCGTTGTTGGTTTGACTAAAGTGACAGCTCTGGAAAATGCAGGTAAAGGTATAACGTGTAATGCAATTTGCCCGGGA TTC GTGCGTACGCCGCTG TTT GAGAAACAAATCGAAGCAATTAGTCAGCAAAAGGGAATAGACATCGAAGCTGCTGCACGTGAGTTGCTGGCAGAAAAACAGCCTAGCTTACAATTTGTTACGCCCGAACAGTTAGGAGGAGCAGCAGTGTTTCTAAGTTCCGCTGCAGCAGATCAGATGACAGGTACAACCCTGTCGCTGGATGGTGGTTGGACCGCAAGA 23 3-Hydroxybutyrate dehydrogenase triple variant H144L / W187F / G233R MLKGKKAVVT GSTSGIGLAM ATELAKAGAD VVINGFGQPE DIERERSTLE SKFGVKAYYL NADLSDAQAT RDFIAKAAEA LGGLDILVNN AGIQHTAPIE EFPVDKWNAI IALNLSAVFH GTAAALPIMQ KQGWGRIINI ASA L GLVASV NKSAYVAAKH GVVGLTKVTA LENAGKGITC NAICPG F VRT PLVEKQIEAI SQQKGIDIEA AARELLAEKQ PSLQFVTPEQ LG R AAVFLSS AAADQMTGTT LSLDGGWTAR 24 3-Hydroxybutyrate dehydrogenase triple variant H144L / W187F / G233R nucleic acid sequence ATGCTTAAAGGTAAAAAAGCAGTCGTAACTGGTTCCACAAGCGGGATAGGACTGGCAATGGCAACTGAGCTTGCAAAAGCAGGCGCAGATGTAGTAATTAACGGTTTCGGTCAGCCAGAGGATATCGAACGCGAACGCTCAACCCTCGAATCTAAATTTGGCGTGAAAGCGTATTATCTGAACGCAGATCTCTCAGATGCACAGGCAACCCGGGATTTCATTGCAAAAGCAGCAGAAGCATTGGGTGGTCTGGACATCTTAGTCAATAACGCTGGTATCCAACATACCGCACCGATCGAAGAGTTTCCAGTGGACAAATGGAACGCAATTATTGCATTAAATTTAAGTGCTGTTTTTCATGGTACGGCAGCAGCACTTCCTATTATGCAGAAGCAGGGTTGGGGTCGTATAATTAATATTGCGAGCGCA CTC GGCCTGGTAGCATCTGTCAATAAATCGGCATACGTTGCAGCTAAGCATGGCGTTGTTGGTTTGACTAAAGTGACAGCTCTGGAAAATGCAGGTAAAGGTATAACGTGTAATGCAATTTGCCCGGGA TTC GTGCGTACGCCGCTGGTTGAGAAACAAATCGAAGCAATTAGTCAGCAAAAGGGAATAGACATCGAAGCTGCTGCACGTGAGTTGCTGGCAGAAAAACAGCCTAGCTTACAATTTGTTACGCCCGAACAGTTAGGA AGA GCAGCAGTGTTTCTAAGTTCCGCTGCAGCAGATCAGATGACAGGTACAACCCTGTCGCTGGATGGTGGTTGGACCGCAAGA 25 3-Hydroxybutyrate dehydrogenase quadruple variant H144L / W187F / V193F / G233R MLKGKKAVVT GSTSGIGLAM ATELAKAGAD VVINGFGQPE DIERERSTLE SKFGVKAYYL NADLSDAQAT RDFIAKAAEA LGGLDILVNN AGIQHTAPIE EFPVDKWNAI IALNLSAVFH GTAAALPIMQ KQGWGRIINI ASA L GLVASV NKSAYVAAKH GVVGLTKVTA LENAGKGITC NAICPG F GARDEN PL F EKQIEAI SQQKGIDIEA AARELLAEKQ PSLQFVTPEQ LG R AAVFLSS AAADQMTGTT LSLDGGWTAR 26 3-Hydroxybutyrate dehydrogenase quadruple variant H144L / W187F / V193F / G233R nucleotide sequence ATGCTTAAAGGTAAAAAAGCAGTCGTAACTGGTTCCACAAGCGGGATAGGACTGGCAATGGCAACTGAGCTTGCAAAAGCAGGCGCAGATGTAGTAATTAACGGTTTCGGTCAGCCAGAGGATATCGAACGCGAACGCTCAACCCTCGAATCTAAATTTGGCGTGAAAGCGTATTATCTGAACGCAGATCTCTCAGATGCACAGGCAACCCGGGATTTCATTGCAAAAGCAGCAGAAGCATTGGGTGGTCTGGACATCTTAGTCAATAACGCTGGTATCCAACATACCGCACCGATCGAAGAGTTTCCAGTGGACAAATGGAACGCAATTATTGCATTAAATTTAAGTGCTGTTTTTCATGGTACGGCAGCAGCACTTCCTATTATGCAGAAGCAGGGTTGGGGTCGTATAATTAATATTGCGAGCGCA CTC GGCCTGGTAGCATCTGTCAATAAATCGGCATACGTTGCAGCTAAGCATGGCGTTGTTGGTTTGACTAAAGTGACAGCTCTGGAAAATGCAGGTAAAGGTATAACGTGTAATGCAATTTGCCCGGGA TTC GTGCGTACGCCGCTG TTT GAGAAACAAATCGAAGCAATTAGTCAGCAAAAGGGAATAGACATCGAAGCTGCTGCACGTGAGTTGCTGGCAGAAAAACAGCCTAGCTTACAATTTGTTACGCCCGAACAGTTAGGA AGA GCAGCAGTGTTTCTAAGTTCCGCTGCAGCAGATCAGATGACAGGTACAACCCTGTCGCTGGATGGTGGTTGGACCGCAAGA

[0074] Example 1. Variant expression and primary activity screening

[0075] For protein expression E. coli BL21(DE3) was used. For screening protein expression, pre-culture was performed by inoculating 600 μL of LB medium and incubating at 37°C and 200 rpm for 16 hours. Then, the culture medium was dispensed into a 96-deep-well plate, and isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM. Subsequently, protein expression was induced by incubating at 37°C and 300 rpm for 2 hours.

[0076] After inducing expression, the cells were recovered by centrifuging at 4,000 rpm (3,220 × g) for 20 minutes at 4 ℃. The recovered cells were lysed using BugBuster® (Merck Millipore, USA). Then, the lysate was centrifuged at 8,000 rpm (11,300 × g) for 20 minutes at 4 ℃ to remove cell debris, and the supernatant was separated to obtain crude protein (enzyme-containing supernatant).

[0077] The enzymatic activity of the Af-3HBDH variant was measured using the obtained crude protein. The reaction solution was prepared to contain 60 μL of Af-3HBDH crude protein, NADH (final concentration 0.25 mM), levulinic acid (LA; final concentration 5 mM), and 50 mM bis-Tris HCl buffer (pH 6.5), and the final reaction volume was adjusted to 200 μL.

[0078] The change in absorbance in the above reaction solution was measured at a wavelength of 340 nm for 10 minutes, and enzyme activity was calculated based on the rate of decrease in NADH absorbance. Based on the measured enzyme activity values, variants exhibiting relatively high activity were selected as primary candidates.

[0080] Example 2. Expression, purification, and kinetic analysis of selected variants

[0081] 2-1. Expression and Purification

[0082] Proteins were expressed and purified from Af-3HBDH (ep-PCR) variants selected for exhibiting high activity compared to Af-3HBDH (wild type), and kinetic analysis for levulinic acid was performed.

[0083] 100 μg mL of Af-3HBDH (ep-PCR) selected for showing higher activity than Af-3HBDH (wild type) - ¹ After inoculating into 5 mL LB medium containing ampicillin, the mixture was incubated overnight under aerobic conditions of 37 ℃ and 200 rpm.

[0084] For protein expression, 3 mL of the above pre-culture solution was inoculated into 200 mL of medium, and the absorbance (OD) at 600 nm 600 ) was cultured until it reached 0.5 to 0.6. Then, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.8 mM, and protein expression was induced at 20°C for 20 hours.

[0085] After inducing expression, cells were recovered by centrifugation at 4,000 rpm (3,220 × g) for 20 minutes at 4 ℃. The recovered cells were lysed using BugBuster® (Merck Millipore, USA), and then centrifuged at 8,000 rpm (11,300 × g) for 20 minutes at 4 ℃ to remove cell debris. Subsequently, proteins were purified by performing Ni-affinity chromatography (Hilden, Qiagen, Germany) using elution buffer (50 mM sodium phosphate, 300 mM NaCl, 250 mM imidazole).

[0086] The concentration of the purified protein was measured using the Bradford method with fetal bovine serum albumin as a standard. The expression / purification of the Af-3HBDH variant and FDH purified by the above Ni-affinity chromatography was confirmed by 10% SDS-PAGE.

[0087] As can be seen in Figure 4, a protein ladder was identified in Lane 1. In Lanes 2 through 5, protein bands corresponding to the wild type (27.1 kDa), bivariate (27.03 kDa), trivariate (27.04 kDa), and quadruplicate (27.05 kDa) of Af-3HBDH were identified, confirming that each Af-3HBDH protein was expressed. Additionally, in Lane 6, a protein band corresponding to FDH (44.14 kDa) was identified, confirming the expression of FDH.

[0088] Meanwhile, the samples from Lane 2 to Lane 6 were proteins eluted by nickel affinity chromatography, and through the verification of the bands, it was confirmed that Af-3HBDH wild type and variant and FDH were obtained in a purified state.

[0089] 2-2. Dynamic Analysis

[0090] The 96-well plate reaction mixture for kinetic analysis was composed of a total volume of 200 μL and contained 4.3787 μM Af-3HBDH (ep-PCR), 0.25 mM NADH, 10–0.3125 mM levulinic acid, and 50 mM bis-Tris HCl buffer (pH 6.5). Kinetic measurements were performed by measuring the NADH consumption rate at 340 nm absorbance.

[0091] As a result of the experiment, Triple mutant_H144L / W187F / V193F (M01-119) showed the highest activity in the first round (Fig. 5). Subsequently, in the second round performed based on the Triple mutant, Quadruple mutant_H144L / W187F / V193F / G233R (M02-344) showed the best activity (Fig. 6).

[0092] kcat, Km, and kcat / Km were calculated for the selected variants using levulinic acid as a substrate, and the results are shown in Table 5.

[0093] Kcat (sec -1 ) Km (mM) Kcat / Km (sec -1 mM -1 ) Double mutant H144L / W187F 0.09 0.5 0.18 Triple mutant H144L / W187F / V193F 0.19 3.69 0.05 Triple mutant H144L / W187F / G233R 0.07 0.97 0.07 Quadruple mutant H144L / W187F / V193F / G233R 0.56 12.73 0.04

[0095] As shown in Table 5, it was confirmed that the catalytic properties changed as kcat and Km for levulinic acid varied depending on the combination of mutations. In particular, the quadruple variant (H144L / W187F / V193F / G233R) showed an increase in kcat, which improved catalyst turnover, and this can be advantageous for increasing the reaction rate under high-concentration levulinic acid conditions. Therefore, when this variant is applied to a levulinic acid reduction-based 4-hydroxyvaleric acid production process, an improvement in process efficiency can be expected.

[0097] Example 3. 4HV in vitro enzymatic conversion reaction of levulinic acid (LA).

[0098] To convert levulinic acid to 4-hydroxyvaleric acid (4HV) in vitroEnzymatic reactions were performed. The reaction mixture was prepared by adding 3-hydroxybutyrate dehydrogenase (3HBDH) and formate dehydrogenase (FDH), quantified by the Bradford method, at concentrations of 0.4 mg / mL and 0.8 mg / mL, respectively, and containing sodium levulinate (Na-LA), sodium formate (Na-FA), NADH, and 50 mM bis-Tris buffer (pH 6.0). Specific conversion composition conditions are shown in Table 6 below.

[0099] composition Concentration 3HBDH 0.4 mg / mL FDH 0.8 mg / mL Na-LA 20~1,000 mM Na-FA 60~3,000 mM NADH 5 mM Bis-Tris buffer (pH 6.0) 50 mM Total 3~5 mL

[0101] The reaction mixture was configured to have a final volume of 3 to 5 mL, and the reaction mixture was incubated at 30 °C and 200 rpm. After collection, the reaction sample was heat-treated at 99 °C for 5 minutes, then centrifuged at 10,000 rpm for 10 minutes, and filtered through a 0.22 μm syringe filter for subsequent analysis.

[0102] As a result of the experiment, the wild type of Af-3HBDH showed no response. The Af-3HBDH double mutant exhibited excellent activity under LA conditions of 20 mM and 50 mM, but under high concentration conditions exceeding 100 mM, substrate inhibition occurred, leading to a decrease in activity. On the other hand, the Af-3HBDH triple mutant and quadruple mutant showed no substrate inhibition or significantly low inhibition even when the LA concentration exceeded 100 mM, confirming that they exhibited superior activity compared to the double mutant (Fig. 7). Furthermore, when the initial rate was measured while the reaction was sustained under LA concentrations of 50 mM and 100 mM, the initial rates of the triple and quadruple mutants were found to be 2 to 3 times faster than those of the double mutant (Figs. 8, 9).

[0104] Example 4. Analysis of 4HV and LA using HPLC

[0105] in vitro HPLC analysis was performed to confirm the consumption of levulinic acid and the generation of 4-hydroxyvaleric acid (4HV) in the reaction, and to quantify the conversion rate. 4HV and LA were analyzed using a Finnigan Surveyor Plus HPLC system (Thermo Scientific) equipped with a refractive index detector, and an Agilent ZORBAX SB-Aq reversed-phase column (4.6 mm × 150 mm, 3.5 μm) was used. A 25 mM ammonium formate aqueous solution (pH 2.0) was used as the mobile phase, the flow rate was set to 0.6 mL / min, and the column temperature was maintained at 60 ℃.

[0106] As a result of the experiment, the retention time under the above conditions was confirmed to be 3.1 minutes for 4HV and 3.5 minutes for LA.

Claims

Claim 1 A 3-hydroxybutyrate dehydrogenase variant comprising one or more amino acid substitutions of V193F and G233R based on the amino acid sequence number of SEQ ID NO.

19. Claim 2 A 3-hydroxybutyrate dehydrogenase variant according to claim 1, wherein the 3-hydroxybutyrate dehydrogenase variant comprising the V193F amino acid substitution is SEQ ID NO.

21. Claim 3 A 3-hydroxybutyrate dehydrogenase variant according to claim 1, wherein the 3-hydroxybutyrate dehydrogenase variant comprising the G233R amino acid substitution is SEQ ID NO.

23. Claim 4 A 3-hydroxybutyrate dehydrogenase variant according to claim 1, wherein the 3-hydroxybutyrate dehydrogenase variant comprising the V193F and G233R amino acid substitutions is SEQ ID NO.

25. Claim 5 A polynucleotide encoding the 3-hydroxybutyrate dehydrogenase variant of claim 1. Claim 6 In claim 5, the polynucleotide comprises SEQ ID NO. 22, SEQ ID NO. 24, or SEQ ID NO.

26. Claim 7 A recombinant vector comprising the polynucleotide of claim 5. Claim 8 Transformed microorganism into which the recombinant vector of claim 7 has been introduced. Claim 9 Method for preparing 4-hydroxyvaleric acid comprising the following steps: Alkaline phaecalis ( Alcaligenes faecalis Enzyme reaction step for synthesizing 4-hydroxyvaleric acid by adding a substrate to a strain-derived 3-hydroxybutyrate dehydrogenase variant. Claim 10 A method for producing 4-hydroxyvaleric acid according to claim 9, wherein the substrate comprises levulinic acid. Claim 11 A method for producing 4-hydroxyvaleric acid, wherein the 3-hydroxybutyrate dehydrogenase variant comprises 3-hydroxybutyrate dehydrogenase comprising one or more amino acid substitutions among V193F and G233R based on the amino acid sequence number of SEQ ID NO. 19.

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