Hexuronate C4-epimerase variant having improved D-tagatose conversion activity, and D-tagatose production method using same

Abstract

Provided are a hexuronate C4-epimerase variant with improved activity in converting D-fructose by D-tagatose of hexuronate C4-epimerase and a method for production of D-tagatose using them.

Claims

1. A hexuronate C4-epimerase variant comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1, in which one or more amino acid residue selected from the group consisting of leucine (L) as an amino acid residue at position corresponding to 77, alanine (A) residue at position corresponding to 158, and proline (P) residue at position corresponding to 351, from the N-terminus of a hexuronate C4-epimerase of the amino acid sequence set forth in SEQ ID NO: 1 are mutated.

2. The hexuronate C4-epimerase variant according to claim 1, wherein the leucine (L) residue at position corresponding to 77 is replaced by proline (P) or arginine (R).

3. The hexuronate C4-epimerase variant according to claim 1, wherein the alanine (A) residue at position corresponding to 158 is replaced by threonine (T).

4. The hexuronate C4-epimerase variant according to claim 1, wherein the proline (P) residue at position corresponding to 351 is replaced by serine (S).

5. The hexuronate C4-epimerase variant according to claim 1, wherein in case the leucine (L) residue at position corresponding to 77 is replaced by proline (P) or arginine (R), one or more amino acid residue selected from the group consisting of serine (S) residue at position corresponding to 125, the alanine (A) residue at position corresponding to 158, the proline (P) residue at position corresponding to 351, from the N-terminus of the hexuronate C4-epimerase of the amino acid sequence set forth in SEQ ID NO: 1 are further mutated.

6. The hexuronate C4-epimerase variant according to claim 1, wherein in case the alanine (A) residue at position corresponding to 158 is replaced by threonine (T), one or more amino acid residue selected from the group consisting of serine (S) residue at position corresponding to 125, the glutamine (Q) residue at position corresponding to 149, the valine (V) residue at position corresponding to 267, and the proline (P) residue at position corresponding to 351, from the N-terminus of the hexuronate C4-epimerase of SEQ ID NO: 1 are further mutated.

7. The hexuronate C4-epimerase variant according to claim 1, wherein in case the proline (P) residue at position corresponding to 351 is replaced by serine (S), one or more amino acid residue selected from the group consisting of serine (S) residue at position corresponding to 125, and the valine (V) residue at position corresponding to 267, from the N-terminus of the hexuronate C4-epimerase are further mutated.

8. The hexuronate C4-epimerase variant according to claim 1, wherein serine (S) residue at position corresponding to 125 from the N-terminus of a hexuronate C4-epimerase of the amino acid sequence set forth in SEQ ID NO: 1 is further mutated.

9. The hexuronate C4-epimerase variant according to claim 8, wherein the serine (S) residue at position corresponding to 125 is replaced by aspartic acid (D), glutamine (Q), glutamic acid (E), threonine (T), asparagine (N), cysteine (C), or tyrosine (Y).

10. The hexuronate C4-epimerase variant according to claim 8, in which serine (S) as an amino acid residue at position corresponding to 125, lysine (K) as an amino acid residue at position corresponding to 164, aspartic acid (D) as an amino acid residue at position corresponding to 168, and glutamic acid (E) as an amino acid residue at position corresponding to 175 from the N-terminus of a hexuronate C4-epimerase of the amino acid sequence set forth in SEQ ID NO: 1 are mutated.

11. The hexuronate C4-epimerase variant according to claim 8, in which serine (S) as an amino acid residue at position corresponding to 125, glutamine (Q) as an amino acid residue at position corresponding to 149, and valine (V) as an amino acid residue at position corresponding to 267 from the N-terminus of a hexuronate C4-epimerase of the amino acid sequence set forth in SEQ ID NO: 1 are mutated.

12. The hexuronate C4-epimerase variant according to claim 1, wherein one or more amino acid residue selected from the group consisting of glutamine (Q) residue at position corresponding to 149, lysine (K) residue at position corresponding to 164, aspartic acid (D) residue at position corresponding to 168, glutamic acid (E) residue at position corresponding to 175, and valine (V) residue at position corresponding to 267, from the N-terminus of the hexuronate C4-epimerase of the amino acid sequence set forth in SEQ ID NO: 1 are further mutated.

13. A nucleic acid encoding the hexuronate C4-epimerase variant according to claim 12.

14. A method for D-tagatose production comprising bringing the hexuronate C4-epimerase variant according to claim 12, a microorganism or a culture thereof expressing the variant into contact with D-fructose.

15. A nucleic acid encoding the hexuronate C4-epimerase variant according to claim 1.

16. A method for D-tagatose production comprising bringing the hexuronate C4-epimerase variant according to claim 1, a microorganism or a culture thereof expressing the variant into contact with D-fructose.

17. The hexuronate C4-epimerase variant according to claim 1, wherein the hexuronate C4-epimerase variant comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1.

18. The hexuronate C4-epimerase variant according to claim 1, wherein the hexuronate C4-epimerase variant comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1.

19. The hexuronate C4-epimerase variant according to claim 1, wherein the hexuronate C4-epimerase variant comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1.

20. The hexuronate C4-epimerase variant according to claim 1, wherein the hexuronate C4-epimerase variant comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1.

Description

BEST MODE

(1) Hereinafter, the present invention is described in more detail with reference to the following Examples. However, the following Examples are merely examples of the present invention, and the contents of the present invention should not be construed as being limited thereto.

EXAMPLE

Example 1. Improved Target Site Design and Analysis

(2) Amino acids predicted to be functionally important were firstly selected based on analysis of the tertiary structure model of the active site of the ortholog (a homologous gene predicted to have the same function in different microbial species) which has identity with an amino acid of a hexuronate C4-epimerase derived from Thermotoga neapolitana (hereinafter referred to as wild-type). Then, based on analysis results of the docking model between D-fructose and the refined active site structure after the alanine scanning mutagenesis, a modified target site was designed for improvement of the unit activity of the conversion reaction of D-fructose by C4-epimerization. The details thereof are described as follows.

(3) 1-1. Ortholog Analysis

(4) The homologous genes (ortholog) having identity with the wild-type amino acid sequence (SEQ ID NO: 1) [about 60 homologous genes with 80% sequence coverage and 50% or more homology] were screened using GenBank gene database. Through multiple sequence alignment analysis among amino acid sequences of the selected homologous genes, conserved amino acid residues predicted to be functionally important in the wild-type amino acid sequence were identified.

(5) 1-2. Analysis of Enzyme Tertiary Structure Model

(6) There was no protein structure that appears to have 30% or more amino acid sequence identity with the homologous genes of the wild-type in Protein Data Bank database, and thus it was expected that accuracy in the prediction of the tertiary structure model of the wild-type by a homology modeling method would be low. Accordingly, the active sites among the tertiary structure models obtained from various modeling servers (RaptorX, Robetta, ModWeb, M4T, HHpred, PHYRE2, ITASSER and SWISS-MODEL) were compared and analyzed to obtain information about the structure sites that were predicted as the same.

(7) 1-3. Alanine Scanning Mutagenesis and Docking Binding Analysis

(8) The amino acids that were selected based on the amino acid sequence analysis and the analysis of the tertiary structural model of the active site among the homologous genes as described above were substituted and mutated with alanine, and these recombinant mutation enzymes were produced in Escherichia coli. Then, characteristics of each mutation site were analyzed. Amino acids predicted to be functionally important were selected through the docking simulation between D-fructose and the refined active site structure after the alanine scanning mutagenesis was analyzed. Then, the modified target site was designed for the improvement of the unit activity of the conversion reaction of D-fructose by C4-epimerization. The amino acid sites of which activity is completely lost through the alanine scanning mutagenesis [assuming catalytic metal ion binding residues and deprotonation/protonation involved catalytic residues] were excluded from the target site for activity improvement.

Example 2. Production of Mutation Enzyme and Selection of Activity-Modified Mutation Enzyme

(9) Single-site saturation mutagenesis libraries of 54 target sites designed in Example 1 (amino acid residues at position Nos: 9, 21, 60, 62, 68, 77, 91, 97, 125, 126, 140, 141, 145, 149, 157, 158, 160, 163, 164, 166, 167, 168, 175, 176, 177, 185, 202, 218, 221, 231, 241, 242, 267, 268, 272, 276, 284, 295, 297, 302, 306, 316, 337, 351, 361, 366, 386, 388, 402, 403, 415, 429, 440, and 441 from the N-terminal of the wild-type hexuronate C4-epimerase) were constructed, and mutation sites of which the unit activity was improved and amino acids were screened. The multiple mutation enzyme was made by integrating the information of the screened modified sites to develop a mutation enzyme having improved unit activity of the conversion reaction of D-fructose by C4-epimerization.

(10) 2-1. Saturation Mutagenesis

(11) The recombinant expression vector constructed for expression of wild-type enzyme gene, wild-type Escherichia coli BL21 (DE3) (which expresses the recombinant enzyme in which the wild-type was introduced into the NdeI and XhoI restriction enzyme sites of pET21a and 6×His-tag is bound at the C-terminal of the wild-type) was used as a template for saturation mutagenesis for producing a variant library. Inverse PCR-based saturation mutagenesis was used in consideration of diversity of mutation distribution and yield of variants (2014. Anal. Biochem. 449: 90-98), NDT, VMA, ATG and TGG mixed primers in which termination codon was excluded and rare codons of E. coli were minimized in order to minimize the screening scale of the constructed variant library (i.e., to minimize the number of codons introduced during saturation mutagenesis) were designed and used (2012. Biotechniques 52:149-158). Specifically, a mixed primer including 15 bp for the front base, 3 bp (NDT, VMA, ATG and TGG, respectively) for substituting the displaced site, and 15 bp for the back base of the respective mutated sites, i.e., 33 bp in total length was constructed and used. The PCR was repeated 30 times under conditions of denaturation at 94° C. for 2 minutes, denaturation at 94° C. for 30 seconds, annealing at 60° C. for 30 seconds, extension at 72° C. for 10 minutes, and extension at 72° C. for 60 minutes. After constructing the saturation mutagenesis libraries for each mutation site, variants for each library were randomly selected (<mutation 11), and base sequences were analyzed to evaluate amino acid mutation distribution. Based on the analysis results, the screening scale of 90% or more of the sequence coverage for each library was determined (2003. Nucleic Acids Res. 15; 31:e30).

(12) 2-2. Screening of Activity-Modified Mutation Enzyme and Construction of Multiple Mutation Enzyme

(13) A chromogenic assay was used to specifically quantify D-fructose in order to rapidly screen large quantities of activity-modified mutation enzymes in the produced saturation mutagenesis libraries. Specifically, a 70% folin-ciocalteu reagent (SIGMA-ALDRICH) and a substrate reaction solution were mixed at a ratio of 15:1 and reacted at 80° C. for 5 minutes. The OD values measured at 900 nm were compared and analyzed.

(14) 54 variants in the mutation site with increased activity (D-tagatose production by conversion of D-fructose) as compared to the relative activity of the wild-type enzyme (SEQ ID NO: 1) were firstly selected. The base sequences of the corresponding genes were analyzed and the amino acid mutation information was analyzed (Tables 2 to 10).

(15) The firstly selected mutation enzymes were reacted with D-fructose using a purified enzyme solution (His-tag affinity chromatography), and the reaction products were used to finally select 236 variants with the increased activity in producing D-tagatose by conversion from D-fructose as compared to the wild-type enzyme by using HPLC (column Shodex SUGAR SP-G, column analysis temperature of 80° C., mobile phase H.sub.2O, flow rate of 0.6 ml/min, Refractive

(16) Index Detector).

Example 3. Comparative Evaluation of Activity-Modified Mutation Enzyme Characteristics

(17) In order to evaluate the relative activity of the D-fructose C4-epimerization on the mutation enzyme for a single site with improved unit activity and on the mutation enzyme for a multiple site in combination thereof, each enzyme was expressed in E. coli BL21 (DE3) by a conventional method (see Sambrook et al. 1989) and purified (by His-tag affinity chromatography). Then, in the presence of NiSO.sub.4, each enzyme at a concentration of 10 units/ml was added to 25% (w/v) D-fructose substrate and reacted at pH 8.0 [50 mM potassium phosphate buffer] and at 65° C. for 2 hours, and the relative activity of D-fructose C4-epimerization as compared to the wild-type recombinase (wild-type, SEQ ID NO: 1) derived from Thermotoga neapolitana was measured.

(18) TABLE-US-00002 TABLE 2 Number of Relative name 77 125 158 185 272 403 variants activity WT — 100 M1 C 1 193 M2 Y 1 116 M3 Q 1 165 M4 E 1 202 M5 T 1 211 M6 N 1 131 M7 D 1 303 M8 K 1 114 M9 R 1 114 M10 H 1 118 M11 Q 1 107 M12 A 1 102 M13 F 1 104 M14 E 1 120 M15 D 1 121 M16 Q 1 116 M17 S 1 133 M18 V 1 117 M19 R 1 105 M20 K 1 117 M21 S 1 114 M22 T 1 130 M23 Q 1 119 M24 F 1 110 M25 V 1 117 M26 I 1 128 M27 A 1 119 M28 P D 2 479 M29 P D 2 487 M30 R D 2 426 M31 D T 2 494 M32 D K 2 543 M33 D R 2 430 M34 D H 2 493 M35 D Q 2 584 M36 D A 2 447

(19) TABLE-US-00003 TABLE 3 Number of Relative name 77 125 149 158 185 267 268 272 351 403 variants Activity M37 D G 2 481 M38 D S 2 355 M39 D P 2 199 M40 D D 2 343 M41 D H 2 259 M42 D Q 2 347 M43 D N 2 222 M44 D K 2 351 M45 D H 2 343 M46 D Y 2 305 M47 D S 2 421 M48 D S 2 377 M49 D F 2 380 M50 D T 2 431 M51 D Q 2 371 M52 D V 2 372 M53 P D T 3 572 M54 P D T 3 452 M55 P D S 3 473 M56 D R T 3 557 M57 D R M 3 594 M58 D T M 3 608 M59 D T S 3 605 M60 D T S 3 480 M61 D Q C 3 422 M62 D Q C 3 422 M63 D K D 3 638 M64 D K V 3 402 M65 D K I 3 515 M66 D K L 3 506 M67 D K M 3 540 M68 D K Q 3 628 M69 D K T 3 790 M70 D Q T 3 746 M71 D M S 3 613 M72 D D F 3 281 M73 D D S 3 274 M74 D D V 3 178 M75 D D I 3 314

(20) TABLE-US-00004 TABLE 4 name 9 21 60 62 68 77 97 125 140 149 158 164 166 168 175 M76 R D T M77 D M78 D M79 R D T M80 D M81 D M82 D M83 D M84 D M85 Y D P D T M86 G D M87 L D M88 D P M E G M89 D R M90 D M E G M91 D R M92 D M93 D M94 D M95 F I D R M96 F I D R M97 F I D R M98 F I D R Number of Relative name 185 231 267 268 272 297 306 316 351 386 403 415 variants Activity M76 T 4 441 M77 H M 4 495 M78 Q D M 4 548 M79 Q V T 5 437 M80 C D M 5 526 M81 Q M T D 5 451 M82 Q M T D 5 510 M83 R M T D V 5 555 M84 M S V T 5 445 M85 E 6 427 M86 Q M M T 6 489 M87 M T D V 6 695 M88 V 6 564 M89 M T D V 6 496 M90 T K 6 498 M91 M T D V 6 592 M92 Q M T D T 6 691 M93 M T D S V 6 553 M94 Q M C D M 6 588 M95 M F S 7 540 M96 M F S 7 454 M97 M F S 7 498 M98 M F S 7 500

(21) TABLE-US-00005 TABLE 5 Number of Relative name 91 125 141 164 168 175 176 185 267 268 272 306 403 variants Activity M99 W D Q M T D T 7 478 M100 I D Q M T D T 7 560 M101 N D Q M T D T 7 486 M102 D F Q M T D T 7 496 M103 D M E G M C D 7 437 M104 D H Q M T D I 7 610 M105 D F Q M T D I 7 539 M106 D Y Q M T D I 7 662 M107 D Q M T D Q 7 822 M108 D Q M T D I 7 1011 M109 D Q M T D L 7 728 M110 D Q M T D A 7 749 M111 D Q M T D P 7 728 M112 D Q M T D V 7 1023 M113 D Q M T D W 7 682 M114 D Q M T D R 7 607 M115 D Q M T D H 7 948 M116 D Q M T D F 7 956 M117 D Q M T D K 7 536 M118 D Q M T D N 7 932 M119 D Q M T D E 7 400 M120 D Q M T D D 7 476 M121 D Q M T D C 7 457 M122 D Q M T D T 7 690

(22) TABLE-US-00006 TABLE 6 Number of Relative name 125 126 164 166 168 175 185 231 267 268 272 297 306 386 388 403 variants Activity M123 D Q M T D M V 7 326 M124 D Q M T D V T 7 693 M125 D Q M T M V T 7 822 M126 D Q M D M V T 7 558 M127 D Q T D M V T 7 655 M128 D M T D M V T 7 597 M129 Q M T D M V T 7 589 M130 D G Q M T D V T 8 521 M131 D M E G T K V T 8 445 M132 D R Q M T D M T 8 697 M133 D Q R M T D M T 8 640 M134 D Q M T G M V M 8 487 M135 D Q M T D M V M 8 786 M136 D Q M T D M V G 8 809 M137 D Q M T D H V T 8 440 M138 D Q M T D V V T 8 649 M139 D Q M T D F V T 8 740 M140 D Q M T D M V I 8 1006 M141 D Q M T V M V I 8 699 M142 D Q M T A M V I 8 540 M143 D Q M T D M V T 8 495 M144 D Q M T E M V I 8 931 M145 D Q M T D M V G 8 557 M146 D Q M T D M V D 8 625

(23) TABLE-US-00007 TABLE 7 Number of Relative name 60 97 125 126 145 163 164 166 185 202 221 231 241 242 267 268 272 276 284 306 386 403 415 variants Activity M147 D Q M T D M V N 8 408 M148 D M C D A M T E 8 418 M149 D D T F F M T P T 9 643 M150 A D Q M T D M V T 9 672 M151 L D Q M T D M V T 9 695 M152 D F Q M T D M V T 9 661 M153 D L Q M T D M V T 9 656 M154 D P Q M T D M V T 9 636 M155 D I Q M T D M V T 9 667 M156 D T Q M T D M V T 9 670 M157 D A Q M T D M V T 9 518 M158 D G Q M T D M V I 9 682 M159 D R Q M T D M V I 9 553 M160 D A Q M T D M V T 9 553 M161 D A Q M T D M V T 9 664 M162 D Q Q M T D M V T 9 597 M163 D M Q M T D M V T 9 634 M164 D R Q M T D M V T 9 752 M165 D Q R M T D M V T 9 733 M166 D Q N M T D M V I 9 699 M167 D Q T M T D M V I 9 697 M168 D Q S M T D M V I 9 736 M169 D Q M T D E M V I 9 601 M170 D Q M T D A M V I 9 586

(24) TABLE-US-00008 TABLE 8 Number of Relative name 97 125 157 160 163 164 166 167 185 231 267 268 272 306 337 366 386 402 403 429 440 variants Activity M171 D Q M T D M T V I 9 1093 M172 D Q M T D M Y V I 9 1093 M173 D Q M T D M N V I 9 1489 M174 D Q M T D M P V I 9 1408 M175 D Q M T D M S V I 9 1180 M176 D Q M T D M S V I 9 771 M177 D Q M T D M G V I 9 567 M178 D Q M T D M C V I 9 476 M179 D Q M T D M V F I 9 677 M180 D Q M T D M V C I 9 668 M181 D Q M T D M V Y I 9 644 M182 D Q M T D M V T P 9 585 M183 D Q M T D M V T A 9 764 M184 L D Q M T D M V T 10 550 M185 L D Q M T D M V T 10 706 M186 L D Q R M T D M V T 10 613 M187 D R Q M T D M W V I 10 1268 M188 D L Q M T D M W V I 10 1429 M189 D F Q M T D M W V I 10 982 M190 D R Q M T D M W V I 10 565 M191 D Y Q M T D M W V I 10 668 M192 D M Q R M T D M V T 10 617 M193 D A Q M T D M W V I 10 1803 M194 D W Q M T D M W V I 10 1854

(25) TABLE-US-00009 TABLE 9 Number of Relative name 125 167 177 185 218 267 268 272 295 306 337 386 403 variants Activity M195 D I Q M T D M W V I 10 1678 M196 D K Q M T D M W V I 10 1432 M197 D M Q M T D M W V I 10 1770 M198 D V Q M T D M W V I 10 1351 M199 D S Q M T D M W V I 10 1951 M200 D Y Q M T D M W V I 10 911 M201 D H Q M T D M W V I 10 733 M202 D L Q M T D M W V I 10 1489 M203 D Q I M T D M W V I 10 818 M204 D Q S M T D M W V I 10 1294 M205 D Q L M T D M W V I 10 1348 M206 D Q F M T D M W V I 10 1350 M207 D Q C M T D M W V I 10 1204 M208 D Q M T D C M W V I 10 1000 M209 D Q M T D R M W V I 10 485 M210 D Q M T D Y M W V I 10 1261

(26) TABLE-US-00010 TABLE 10 Number of Relative name 125 185 267 268 272 302 306 337 361 366 386 403 441 variants Activity M211 D Q M T D C M W V I 10 1222 M212 D Q M T D M W K V I 10 966 M213 D Q M T D M W E V I 10 630 M214 D Q M T D M W V V I 10 586 M215 D Q M T D M W W V I 10 783 M216 D Q M T D M W Y V I 10 781 M217 D Q M T D M W M V I 10 549 M218 D Q M T D M W R V I 10 760 M219 D Q M T D M W Q V I 10 731 M220 D Q M T D M W L V I 10 638 M221 D Q M T D M W R V I 10 879 M222 D Q M T D M W Y V I 10 1428 M223 D Q M T D M W C V I 10 856 M224 D Q M T D M W L V I 10 589 M225 D Q M T D M F S V I 10 1306 M226 D Q M T D M E S V I 10 1246 M227 D Q M T D M S S V I 10 1271 M228 D Q M T D M W S V I 10 1306 M229 D Q M T D M W V I E 10 1160 M230 D Q M T D M W V I W 10 1150 M231 D Q M T D M W V I H 10 1250 M232 D Q M T D M W V I K 10 1270 M233 D Q M T D M W V I A 10 1250 M234 D Q M T D M W V I R 10 1220 M235 D Q M T D M W V I S 10 1449 M236 D Q M T D M W V I F 10 1294

(27) From the above results, it could be confirmed that the C4-epimerase variants of the present invention had the increased L7-fructose C4-epimerization activity as compared to that of the wild-type enzyme, and in particular, the enzyme variant of M199 was analyzed as having increased the unit activity about 20 times, and thus, it could be confirmed that the activity of producing tagatose of the present invention was remarkably increased as compared to the wild-type enzyme.