Biosynthetic production of 2-fucosyllactose

Abstract

The present invention provides a novel biosynthetic production process that converts L-galactose into 2'-fucosyllactose through four enzyme-catalyzed reaction steps.The process of the present invention is designed so that the cofactors required by the process are regenerated within the four reaction steps, thus making the process cost-effective and efficient.This process can be carried out in vitro in a cell-free system.The present invention also provides mutant enzymes that can be used to increase the production level of 2'-fucosyllactose, whether using the new pathway described herein or the mannose-dependent pathway known in the art.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 067,858 (entitled "Biosynthetic Production of 2-Fucosyllactose," filed August 19, 2020) and U.S. Provisional Patent Application No. 63 / 199,978 (entitled "Biosynthetic Production of 2-Fucosyllactose," filed February 5, 2021), the entire contents of each of which are incorporated herein by reference.

[0002] The field of the present invention relates to the production of 2'-fucosyllactose. More specifically, the present disclosure provides a novel biosynthetic pathway that converts L-galactose to 2'-fucosyllactose through four enzyme-catalyzed reaction steps, including the regeneration of various cofactors used in the pathway.

[0003] Reference to sequence listings submitted as text files via EFS-WEB This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy created on August 19, 2021 is named C149770041WO00-SEQ-ZJG and is 243,945 bytes in size. [Background technology]

[0004] Human milk oligosaccharides (HMOs) are the third most abundant solid component of human milk, after lactose and lipids. However, human milk oligosaccharides are not found in comparable abundance in other natural sources, including cow's milk, sheep's milk, or goat's milk. Breast-fed infants appear to thrive better, with lower incidences of diarrhea, respiratory illness, and otitis media compared with formula-fed infants. Clinical data indicate that many of the benefits of breast milk can be attributed to HMOs.

[0005] The trisaccharide 2'-fucosyllactose (2'-FL, whose chemical structure is shown in Figure 1) is one of the most abundant and clinically proven HMOs, and 2'-FL has the potential to be a nutritional supplement and therapeutic agent. In particular, there has been great interest in incorporating 2'-FL as a functional additive in infant formula. However, the availability of breast milk is limited, and the chemical synthesis of 2'-FL is complex, limiting supply and cost-effectiveness. In recent years, industry and academia have explored producing 2'-FL via biosynthesis by utilizing microbial strains (primarily Escherichia coli (E. coli) strains) engineered for fermentative production.

[0006] In the conventional biosynthesis process, microbial strains were engineered to overexpress α-1,2-fucosyltransferase (FutC), which catalyzes the production of 2'-FL from lactose and GDP-L-fucose. Two major approaches have been adopted to engineer GDP-L-fucose synthesis pathways for 2'-FL production. One approach (the "salvage pathway" shown in Figure 2) requires only the bifunctional enzyme FKP to directly convert L-fucose to GDP-L-fucose. The other approach (the "de novo synthesis" shown in Figure 2) uses glucose to synthesize GDP-L-fucose through a seven-step process.

[0007] Nevertheless, there are concerns among regulators and consumers that fermentation-produced foods, especially those intended for use in baby foods and infant formula, are susceptible to endotoxin and phage contamination. Furthermore, there is a need in the art for novel methods for producing 2'-FL that require fewer steps and are more cost-effective. Summary of the Invention

[0008] In one aspect, the present disclosure provides a novel biosynthetic production process that converts L-galactose to 2'-fucosyllactose through four enzyme-catalyzed reaction steps (Figure 3). The process of the present invention is designed so that the cofactors required by the process are regenerated within the four reaction steps, making the process cost-effective and efficient. This process can be carried out in vitro in a cell-free system.

[0009] In one embodiment, the disclosure provides a method for producing 2'-fucosyllactose, comprising: (a) administering NADPH and / or NADP for a period of time sufficient to convert GDP-L-galactose to GDP-L-fucose; + and (b) incubating GDP-L-galactose with a dehydratase and a reductase in the presence of 2'-fucosyllactose and GDP-L-fucose with lactose and an α-1,2-fucosyltransferase for a time sufficient to convert the GDP-L-fucose and lactose to 2'-fucosyllactose and GDP.

[0010] In some embodiments, the dehydratase can be GDP-mannose-4,6-dehydratase. Suitable dehydratase enzymes include enzymes comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:13. In certain embodiments, the dehydratase used in the method comprises the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:13.

[0011] In some embodiments, the reductase used in the method can be a GDP-4-keto-6-deoxy-mannose reductase. Suitable reductases include enzymes comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:7, SEQ ID NO:15, or SEQ ID NO:17. In certain embodiments, the reductase used in the method comprises the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:15, or SEQ ID NO:17.

[0012] Alpha-1,2-fucosyltransferases suitable for use in the present methods include enzymes comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and 61. In certain embodiments, the alpha-1,2-fucosyltransferase used in the present methods comprises the amino acid sequence set forth in any one of SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and 61. In certain embodiments, the α-1,2-fucosyltransferase used in the method comprises the amino acid sequence set forth in SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:39, SEQ ID NO:45, SEQ ID NO:55, or SEQ ID NO:61.

[0013] In some embodiments, the method further comprises incubating GDP-L-galactose in the presence of a first regenerating enzyme and a first substrate for said first regenerating enzyme, wherein said first regenerating enzyme is NADP +as a cofactor, thereby regenerating NADPH. For example, the first regenerating enzyme and the first substrate can be selected from the group consisting of: (a) malate dehydrogenase and malate, (b) formate dehydrogenase and formate, (c) phosphite dehydrogenase and phosphite, and (d) glucose dehydrogenase and glucose.

[0014] In some embodiments, the GDP-L-galactose used in the method is generated in situ. In certain embodiments, the GDP-L-galactose used in the method can be generated from GDP-mannose. In such embodiments, the method can further include incubating the GDP-mannose with GDP-mannose-3,5-epimerase for a time sufficient to convert the GDP-mannose to GDP-L-galactose. For example, the GDP-mannose-3,5-epimerase can be an enzyme comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 19. In certain embodiments, the GDP-mannose-3,5-epimerase can comprise the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 19.

[0015] Alternatively, the GDP-L-galactose used in the method can be produced from L-galactose. In such embodiments, the method can further include incubating L-galactose with fucokinase / guanylyltransferase in the presence of ATP and GTP for a time sufficient to convert the L-galactose to GDP-L-galactose. For example, the fucokinase / guanylyltransferase can be an enzyme comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In certain embodiments, the fucokinase / guanylyltransferase can comprise the amino acid sequence set forth in SEQ ID NO: 1.

[0016] In a preferred embodiment, the method further comprises incubating L-galactose in the presence of a second regenerating enzyme and a second substrate for the second regenerating enzyme, wherein the second regenerating enzyme catalyzes a reaction involving a second substrate that uses ADP as a cofactor, thereby regenerating ATP.In a more preferred embodiment, the method further comprises incubating L-galactose in the presence of a third regenerating enzyme and a third substrate for the third regenerating enzyme, wherein the third regenerating enzyme catalyzes a reaction involving a third substrate that uses GDP as a cofactor, thereby regenerating GTP.As mentioned above, GDP is produced as a by-product in the bioconversion of 2'-fucosyllactose from GDP-L-fucose.

[0017] The second regenerating enzyme and second substrate, and the third regenerating enzyme and third substrate may each independently be selected from the group consisting of: (a) pyruvate kinase and phospho(enol)pyruvate (PEP), (b) creatine kinase and creatine phosphate, (c) acetate kinase and acetyl phosphate, (d) polyphosphate kinase and polyphosphate, and (e) polyphosphate:AMP phosphotransferase, adenylate kinase and adenosine monophosphate.

[0018] In another aspect, the present disclosure relates to identifying novel enzymes that can be used to convert GDP-L-fucose to 2'-fucosyllactose. Such enzymes can be enzymes that contain an amino acid sequence that has at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and 61. In certain embodiments, the enzyme can contain an amino acid sequence set forth in any one of SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and 61. In certain embodiments, the enzyme may comprise the amino acid sequence set forth in SEQ ID NO:29, SEQ ID NO:61, SEQ ID NO:39, SEQ ID NO:45, SEQ ID NO:55, or SEQ ID NO:23.

[0019] Accordingly, the present disclosure also relates to a method for producing 2'-fucosyllactose, comprising incubating GDP-L-fucose with lactose and an alpha-1,2-fucosyltransferase for a time sufficient to convert the GDP-L-fucose and lactose to 2'-fucosyllactose, wherein the alpha-1,2-fucosyltransferase is an enzyme comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and 61. In certain embodiments, the α-1,2-fucosyltransferase can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and 61. In certain embodiments, the α-1,2-fucosyltransferase used in the methods can comprise the amino acid sequence set forth in SEQ ID NO: 29, SEQ ID NO: 61, SEQ ID NO: 39, SEQ ID NO: 45, SEQ ID NO: 55, or SEQ ID NO: 23.

[0020] In another aspect, the present disclosure relates to a method for producing 2'-fucosyllactose from L-galactose, the method comprising: (a) (i) fucokinase / guanylyltransferase; (ii) dehydratase; (iii) reductase; (iv) α-1,2-fucosyltransferase; (v) ATP; (vi) GTP; (vii) NADP; + and (viii) providing a reaction mixture containing NADPH, and (b) adding L-galactose to the reaction mixture; and (c) incubating the reaction mixture for a time sufficient to produce 2'-fucosyllactose. The reaction mixture comprises (ix) a first regenerating enzyme reacting with NADP +(x) a first regenerating enzyme and a first substrate for the first regenerating enzyme, wherein the first regenerating enzyme catalyzes a first reaction involving a first substrate using ADP as a cofactor, thereby regenerating NADPH; (x) a second regenerating enzyme and a second substrate for the second regenerating enzyme, wherein the second regenerating enzyme catalyzes a second reaction involving a second substrate using ADP as a cofactor, thereby regenerating ATP; and (xi) a third regenerating enzyme and a third substrate for the third regenerating enzyme, wherein the third regenerating enzyme catalyzes a third reaction involving a third substrate using GDP as a cofactor, thereby regenerating GTP.

[0021] In yet another aspect, the present disclosure relates to mutant enzymes that can be used to improve the production levels of 2'-fucosyllactose. These mutant enzymes include mutant dehydratase enzymes and mutant α-1,2-fucosyltransferase enzymes.

[0022] In the de novo pathway depicted in Figure 2 (left), the conversion of GDP-D-mannose to GDP-4-keto-6-deoxymannose is catalyzed by GDP-mannose-4,6-dehydratase (GMD). The resulting GDP-4-keto-6-deoxymannose is converted to GDP-L-fucose by a bifunctional 3,5-epimerase-4-reductase (e.g., WcaG from Escherichia coli) enzyme. However, it is well established that GDP-L-fucose acts as a negative feedback loop on the activity of the GMD enzyme (Figure 13). Inhibition is characterized as allosteric inhibition by human and Arabidopsis thaliana GMD. Therefore, it is beneficial to create mutant enzymes that target the allosteric binding pocket of GDP-L-fucose in Arabidopsis GMD (GMD, SEQ ID NO: 5) and human GMD (Hs GMD, SEQ ID NO: 9). Thus, in one embodiment, the disclosure relates to a mutated At GMD comprising the amino acid sequence of SEQ ID NO: 79, SEQ ID NO: 77, or SEQ ID NO: 75. In another embodiment, the disclosure relates to a mutated Hs GMD comprising the amino acid sequence of SEQ ID NO: 85, SEQ ID NO: 83, or SEQ ID NO: 81.

[0023] In another embodiment, the disclosure relates to a mutant enzyme having improved α-1,2-fucosyltransferase activity. Accordingly, in one embodiment, such a mutant α-1,2-fucosyltransferase can be a polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:109, SEQ ID NO:107, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, or SEQ ID NO:105.

[0024] In one aspect, the disclosure relates to a method for producing 2'-fucosyllactose, the method comprising providing the following enzymes in a culture medium containing L-galactose, wherein the enzymes are (i) a fucokinase comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1; (ii) a dehydratase comprising amino acids having at least 90% sequence identity to SEQ ID NO: 5, SEQ ID NO: 79, SEQ ID NO: 77, or SEQ ID NO: 75; (iii) a reductase comprising amino acids having at least 90% sequence identity to SEQ ID NO: 7; or (iv) an α-1,2-fucosyltransferase comprising an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 109; and incubating L-galactose with the enzymes for a sufficient time to produce 2'-fucosyllactose.

[0025] In another aspect, the disclosure relates to a method for producing 2'-fucosyllactose, the method comprising providing the following enzymes in a culture medium containing GDP-mannose, wherein the enzymes are (i) an epimerase comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3; (ii) a dehydratase comprising amino acids having at least 90% sequence identity to SEQ ID NO:5, SEQ ID NO:79, SEQ ID NO:77 or SEQ ID NO:75; (iii) a reductase comprising amino acids having at least 90% sequence identity to SEQ ID NO:7; and (iv) an α-1,2-fucosyltransferase comprising an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:109; and incubating GDP-mannose with the enzymes for a sufficient time to produce 2'-fucosyllactose.

[0026] The present disclosure also encompasses nucleic acid constructs containing nucleic acid sequences encoding at least the mutant anhydrases and / or mutant α-1,2-fucosyltransferases described herein, as well as microorganisms containing such nucleic acid constructs. The microorganisms or host cells can be induced to express the mutant anhydrases and / or mutant α-1,2-fucosyltransferases. To facilitate protein purification after expression, the nucleic acid sequences encoding the mutant anhydrases and / or mutant α-1,2-fucosyltransferases can contain a polyhistidine tag. The most common polyhistidine tag is formed from six histidine residues (6xHis tag) added to the N-terminus preceded by a methionine or to the C-terminus preceding the stop codon in the coding sequence of the protein of interest.

[0027] The present disclosure also relates to a recombinant microorganism for enhancing the production of 2'-fucosyllactose, wherein the microorganism comprises at least the following heterologous genes for producing 2'-fucosyllactose: (i) a first heterologous gene encoding a mutant anhydrase for producing GDP-L-fucose, wherein the mutant anhydrase is a polypeptide comprising an amino acid sequence selected from SEQ ID NO:79, SEQ ID NO:77, SEQ ID NO:75, SEQ ID NO:85, SEQ ID NO:83 and SEQ ID NO:81; and (ii) a second heterologous gene encoding a mutant α-1,2-fucosyltransferase for converting GDP-L-fucose to 2'-fucosyllactose, wherein the mutant α-1,2-fucosyltransferase is a polypeptide comprising an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:109. The microorganism may further comprise a heterologous gene for transporting 2'-fucosyllactose out of the cell.

[0028] While many aspects of the present disclosure relate to the enzymatic production of 2'-fucosyllactose, the present teachings also relate to the production of 2'-fucosyllactose by fermentation, particularly by culturing a microorganism engineered to contain (i) a first heterologous gene encoding a mutant anhydrase for producing GDP-L-fucose, wherein the mutant anhydrase is a polypeptide comprising an amino acid sequence selected from SEQ ID NO:79, SEQ ID NO:77, SEQ ID NO:75, SEQ ID NO:85, SEQ ID NO:83 and SEQ ID NO:81; and (ii) a second heterologous gene encoding a mutant α-1,2-fucosyltransferase for converting GDP-L-fucose to 2'-fucosyllactose, wherein the mutant α-1,2-fucosyltransferase is a polypeptide comprising an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:109. The microorganism can be cultured in a medium comprising at least one carbon source. The method can include separating the culture medium from the microorganism, and then isolating 2'-fucosyllactose from the culture medium.

[0029] Some embodiments of the present disclosure provide a method for producing 2'-fucosyllactose, comprising incubating GDP-L-fucose with an α-1,2-fucosyltransferase in a culture medium containing lactose for a period of time sufficient to convert the GDP-L-fucose and lactose to 2'-fucosyllactose and GDP, wherein the α-1,2-fucosyltransferase is selected from the group consisting of polypeptides comprising an amino acid sequence having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity to SEQ ID NO:109, SEQ ID NO:29, SEQ ID NO:107, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, or SEQ ID NO:105. In some embodiments, the α-1,2-fucosyltransferase is a polypeptide comprising the amino acid sequence of any one of SEQ ID NO:109, SEQ ID NO:29, SEQ ID NO:107, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, or SEQ ID NO:105.

[0030] In some embodiments, GDP-L-fucose is produced in situ in the culture medium from GDP-mannose or GDP-L-galactose in a reaction catalyzed by a dehydratase.

[0031] In some embodiments, the anhydrase is a polypeptide comprising an amino acid sequence having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity to SEQ ID NO: 5 or SEQ ID NO: 9. In some embodiments, the anhydrase is a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 9.

[0032] In some embodiments, the anhydrase is a polypeptide comprising an amino acid sequence having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity to SEQ ID NO: 79, SEQ ID NO: 77, or SEQ ID NO: 75. In some embodiments, the anhydrase is a polypeptide comprising the amino acid sequence of SEQ ID NO: 79, SEQ ID NO: 77, or SEQ ID NO: 75.

[0033] In some embodiments, the anhydrase is a polypeptide comprising an amino acid sequence having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity to SEQ ID NO: 85, SEQ ID NO: 83, or SEQ ID NO: 81. In some embodiments, the anhydrase is a polypeptide comprising the amino acid sequence of SEQ ID NO: 85, SEQ ID NO: 83, or SEQ ID NO: 81.

[0034] Further provided herein is a method for producing 2'-fucosyllactose, comprising the steps of incubating GDP-mannose and / or GDP-L-galactose with a dehydratase and a reductase in a culture medium in the presence of NADPH and / or NADP+ for a sufficient time to convert the GDP-mannose and / or GDP-L-galactose to GDP-L-fucose; and incubating the GDP-L-fucose with an α-1,2-fucosyltransferase for a sufficient time to convert the GDP-L-fucose and lactose to 2'-fucosyllactose and GDP, wherein the dehydratase is selected from the group consisting of SEQ ID NO: 79 , SEQ ID NO:77, SEQ ID NO:75, or SEQ ID NO:5; or a polypeptide comprising an amino acid sequence having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity to SEQ ID NO:85, SEQ ID NO:83, SEQ ID NO:81, or SEQ ID NO:9.

[0035] In some embodiments, the anhydrase is a polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 79, SEQ ID NO: 77, or SEQ ID NO: 75. In some embodiments, the anhydrase is a polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 85, SEQ ID NO: 83, SEQ ID NO: 81, or SEQ ID NO: 9.

[0036] In some embodiments, the alpha-1,2-fucosyltransferase is a polypeptide comprising an amino acid sequence having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity to SEQ ID NO: 109, SEQ ID NO: 29, SEQ ID NO: 107, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, or SEQ ID NO: 105. In some embodiments, the alpha-1,2-fucosyltransferase is a polypeptide comprising the amino acid sequence of SEQ ID NO: 109, SEQ ID NO: 29, SEQ ID NO: 107, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, or SEQ ID NO: 105.

[0037] In some embodiments, the reductase is a polypeptide that comprises amino acids having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity to SEQ ID NO:7, SEQ ID NO:15, or SEQ ID NO:17. In some embodiments, the reductase is a polypeptide that comprises the amino acids of SEQ ID NO:7, SEQ ID NO:15, or SEQ ID NO:17.

[0038] A recombinant microorganism for enhancing the production of 2'-fucosyllactose, the microorganism comprising at least the following heterologous genes for producing 2'-fucosyllactose: a first heterologous gene encoding a mutant anhydrase for producing GDP-L-fucose, wherein the mutant anhydrase is at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) of SEQ ID NO:79, SEQ ID NO:77, SEQ ID NO:75, SEQ ID NO:85, SEQ ID NO:83, and SEQ ID NO:81. ) sequence identity to SEQ ID NO: 109; and a second heterologous gene encoding a mutant α-1,2-fucosyltransferase for converting GDP-L-fucose to 2'-fucosyllactose, wherein the mutant α-1,2-fucosyltransferase is a polypeptide consisting of an amino acid sequence having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity to SEQ ID NO: 109.

[0039] In some embodiments, the mutant anhydrase is a polypeptide comprising the amino acid sequence of SEQ ID NO: 79, SEQ ID NO: 77, SEQ ID NO: 75, SEQ ID NO: 85, SEQ ID NO: 83, or SEQ ID NO: 81. In some embodiments, the mutant alpha-1,2-fucosyltransferase comprises the amino acid sequence of SEQ ID NO: 109.

[0040] In some embodiments, the microorganism further comprises a heterologous gene for transporting 2'-fucosyllactose out of the cell.

[0041] Another aspect of the present disclosure provides a method for producing 2'-fucosyllactose, comprising culturing the microorganism described herein in a culture medium comprising at least one carbon source.In some embodiments, the method further comprises separating the culture medium from the microorganism.In some embodiments, the method further comprises isolating 2'-fucosyllactose from the culture medium.

[0042] Also provided herein is a mutant anhydrase for producing GDP-L-fucose, which is a polypeptide comprising an amino acid sequence having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity to SEQ ID NO: 79, SEQ ID NO: 77, SEQ ID NO: 75, SEQ ID NO: 85, SEQ ID NO: 83, or SEQ ID NO: 81. In some embodiments, the mutant anhydrase comprises the amino acid sequence of SEQ ID NO: 79, SEQ ID NO: 77, SEQ ID NO: 75, SEQ ID NO: 85, SEQ ID NO: 83, or SEQ ID NO: 81.

[0043] Also provided herein is a mutant α-1,2-fucosyltransferase for producing 2'-fucosyllactose, which is a polypeptide comprising an amino acid sequence having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity to SEQ ID NO: 109, SEQ ID NO: 29, SEQ ID NO: 107, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, or SEQ ID NO: 105. In some embodiments, the mutant α-1,2-fucosyltransferase comprises the amino acid sequence of SEQ ID NO: 109, SEQ ID NO: 29, SEQ ID NO: 107, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, or SEQ ID NO: 105.

[0044] Nucleic acid constructs comprising a nucleic acid sequence encoding at least one of the mutant enzymes described herein, and microorganisms comprising such nucleic acid constructs, are also provided.

[0045] Further provided herein is a method for producing 2'-fucosyllactose, comprising incubating GDP-L-galactose with a dehydratase and a reductase in the presence of NADPH and / or NADP+ for a time sufficient to convert GDP-L-galactose to GDP-L-fucose; and incubating the GDP-L-fucose with lactose and α-1,2-fucosyltransferase for a time sufficient to convert the GDP-L-fucose and lactose to 2'-fucosyllactose and GDP.

[0046] In some embodiments, GDP-L-galactose is further incubated in the presence of a first regenerating enzyme and a first substrate for the first regenerating enzyme, where the first regenerating enzyme catalyzes a reaction involving the first substrate using NADP as a cofactor, thereby regenerating NADPH.

[0047] In some embodiments, the dehydratase is GDP-mannose-4,6-dehydratase.

[0048] In some embodiments, the dehydratase is an enzyme comprising an amino acid sequence having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity to SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:13. In some embodiments, the dehydratase is an enzyme comprising the amino acid sequence of SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:13.

[0049] In some embodiments, the reductase is a GDP-4-keto-6-deoxy-mannose reductase. In some embodiments, the dehydratase is an enzyme comprising an amino acid sequence having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity to SEQ ID NO:7, SEQ ID NO:15, or SEQ ID NO:17. In some embodiments, the dehydratase is an enzyme comprising the amino acid sequence of SEQ ID NO:7, SEQ ID NO:15, or SEQ ID NO:17.

[0050] In some embodiments, the method further comprises incubating GDP-mannose with GDP-mannose-3,5-epimerase for a time sufficient to convert the GDP-mannose to GDP-L-galactose. In some embodiments, the GDP-mannose-3,5-epimerase is an enzyme comprising an amino acid sequence having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity to SEQ ID NO:3 or SEQ ID NO:19. In some embodiments, the GDP-mannose-3,5-epimerase is an enzyme comprising the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:19.

[0051] In some embodiments, the method further comprises incubating L-galactose with fucokinase / guanylyltransferase in the presence of ATP and GTP for a time sufficient to convert said L-galactose to GDP-L-galactose.

[0052] In some embodiments, the fucokinase / guanylyltransferase is an enzyme comprising an amino acid sequence having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity to SEQ ID NO: 1. In some embodiments, the fucokinase / guanylyltransferase is an enzyme comprising an amino acid sequence having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity to SEQ ID NO: 1.

[0053] In some embodiments, the L-galactose is further incubated in the presence of a second regenerating enzyme and a second substrate for the second regenerating enzyme, where the second regenerating enzyme catalyzes a reaction involving the second substrate that uses ADP as a cofactor, thereby regenerating ATP.

[0054] In some embodiments, L-galactose is further incubated in the presence of a third regenerating enzyme and a third substrate for the third regenerating enzyme, and the third regenerating enzyme catalyzes a reaction involving the third substrate that uses GDP as a cofactor, thereby regenerating GTP.

[0055] In some embodiments, the first regenerating enzyme and first substrate are selected from the group consisting of: (a) malate dehydrogenase and malate, (b) formate dehydrogenase and formate, (c) phosphite dehydrogenase and phosphite, and (d) glucose dehydrogenase and glucose.

[0056] In some embodiments, the second regenerating enzyme and second substrate are selected from the group consisting of: (a) pyruvate kinase and phospho(enol)pyruvate (PEP), (b) creatine kinase and creatine phosphate, (c) acetate kinase and acetyl phosphate, (d) polyphosphate kinase and polyphosphate, and (e) polyphosphate:AMP phosphotransferase, adenylate kinase and adenosine monophosphate.

[0057] In some embodiments, the third regenerating enzyme and third substrate are selected from the group consisting of: (a) pyruvate kinase and phospho(enol)pyruvate (PEP), (b) creatine kinase and creatine phosphate, (c) acetate kinase and acetyl phosphate, (d) polyphosphate kinase and polyphosphate, and (e) polyphosphate:AMP phosphotransferase, adenylate kinase and adenosine monophosphate.

[0058] In some embodiments, the α-1,2-fucosyltransferase is an enzyme comprising an amino acid sequence having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity to any one of SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and 61. In some embodiments, the α-1,2-fucosyltransferase is an enzyme comprising the amino acid sequence of any one of SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and 61.

[0059] Also provided herein is a method for producing 2'-fucosyllactose, comprising incubating GDP-L-fucose with lactose and α-1,2-fucosyltransferase for a period of time sufficient to convert the GDP-L-fucose and lactose to 2'-fucosyllactose, wherein the α-1,2-fucosyltransferase is an enzyme comprising an amino acid sequence having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity to any one of SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and 61. In some embodiments, the α-1,2-fucosyltransferase is an enzyme comprising the amino acid sequence of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and 61.

[0060] Another aspect of the present disclosure is a method for producing 2'-fucosyllactose from L-galactose, comprising: (a) providing a reaction mixture comprising (i) fucokinase / guanylyltransferase, (ii) a dehydratase, (iii) a reductase, (iv) an α-1,2-fucosyltransferase, (v) ATP, (vi) GTP, (vii) NADP+, and (viii) NADPH; (b) adding L-galactose to the reaction mixture; and (c) incubating the reaction mixture for a time sufficient to produce 2'-fucosyllactose; wherein the reaction mixture undergoes (ix) a first reaction involving a first substrate in which a first regenerating enzyme uses NADP+ as a cofactor. (x) a first regenerating enzyme and a first substrate for the first regenerating enzyme, wherein the first regenerating enzyme can catalyze a second reaction involving a second substrate using ADP as a cofactor, thereby regenerating NADPH; (x) a second regenerating enzyme and a second substrate for the second regenerating enzyme, wherein the second regenerating enzyme can catalyze a second reaction involving a second substrate using ADP as a cofactor, thereby regenerating ATP; and (xi) a third regenerating enzyme and a third substrate for the third regenerating enzyme, wherein the third regenerating enzyme can catalyze a third reaction involving a third substrate using GDP as a cofactor, thereby regenerating GTP.

[0061] In some embodiments, the fucokinase / guanylyltransferase is an enzyme comprising an amino acid sequence having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity to SEQ ID NO: 1. In some embodiments, the fucokinase / guanylyltransferase is an enzyme comprising the amino acid sequence of SEQ ID NO: 1.

[0062] In some embodiments, the dehydratase is a GDP-mannose-4,6-dehydratase. In some embodiments, the dehydratase is an enzyme comprising an amino acid sequence having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity to SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:13. In some embodiments, the dehydratase is a GDP-mannose-4,6-dehydratase. In some embodiments, the dehydratase is an enzyme comprising the amino acids of SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:13.

[0063] In some embodiments, the reductase is a GDP-4-keto-6-deoxy-mannose reductase. In some embodiments, the reductase is an enzyme comprising an amino acid sequence having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity to SEQ ID NO:7, SEQ ID NO:15, or SEQ ID NO:17. In some embodiments, the reductase is a GDP-4-keto-6-deoxy-mannose reductase. In some embodiments, the reductase is an enzyme comprising the amino acids of SEQ ID NO:7, SEQ ID NO:15, or SEQ ID NO:17.

[0064] In some embodiments, the alpha-1,2-fucosyltransferase is an enzyme comprising an amino acid sequence having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity to any one of SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and 61. In some embodiments, the alpha-1,2-fucosyltransferase is an enzyme comprising the amino acid sequence of any one of SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and 61.

[0065] In some embodiments, the first regenerating enzyme and first substrate are selected from the group consisting of: (a) malate dehydrogenase and malate, (b) formate dehydrogenase and formate, (c) phosphite dehydrogenase and phosphite, and (d) glucose dehydrogenase and glucose.

[0066] In some embodiments, the second regenerating enzyme and second substrate are selected from the group consisting of: (a) pyruvate kinase and phospho(enol)pyruvate (PEP), (b) creatine kinase and creatine phosphate, (c) acetate kinase and acetyl phosphate, (d) polyphosphate kinase and polyphosphate, and (e) polyphosphate:AMP phosphotransferase, adenylate kinase and adenosine monophosphate.

[0067] In some embodiments, the third regenerating enzyme and third substrate are selected from the group consisting of: (a) pyruvate kinase and phospho(enol)pyruvate (PEP), (b) creatine kinase and creatine phosphate, (c) acetate kinase and acetyl phosphate, (d) polyphosphate kinase and polyphosphate, and (e) polyphosphate:AMP phosphotransferase, adenylate kinase and adenosine monophosphate.

[0068] While the present disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and are herein described in detail. It should be understood, however, that the drawings and detailed description presented herein are not intended to limit the disclosure to the particular embodiments disclosed; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0069] Other features and advantages of the present invention will become apparent in the following detailed description of preferred embodiments of the invention, which proceeds with reference to the accompanying drawings.

[0070] The following drawings form part of this specification and are included to further demonstrate certain aspects of the disclosure, which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0071] [Figure 1] FIG. 1 shows the chemical structure of 2′-fucosyllactose (2′-FL). [Figure 2] Figure 2 shows two prior art biosynthetic pathways for producing 2'-fucosyllactose. The key intermediate, GDP-L-fucose, is synthesized from L-fucose via the fucose-dependent salvage pathway or from D-glucose via a seven-step GDP-mannose-dependent de novo pathway. Glk: glucokinase; Pgi: phosphosparcoisomerase; ManA: mannose 6-phosphate isomerase; ManB: phosphomannomutase; ManC: α-D-mannose 1-phosphate guanylyltransferase; Gmd: GDP-mannose 6-dehydrogenase; WcaG: GDP-L-fucose synthase; Fkp: phosphofructokinase; FutC: α-1,2-fucosyltransferase. [Figure 3]Figure 3 shows a novel biosynthetic pathway for the production of 2'-FL from L-galactose according to the present disclosure. L-galactose can be converted to GDP-L-galactose by the Fkp enzyme. The resulting GDP-L-galactose can be converted to GDP-L-fucose by two enzymes, a dehydratase and a reductase. The GDP-L-fucose and lactose can then be converted to 2'-fucosyllactose (2'-FL) by α-1,2-fucosyltransferase (FutC). Alternatively, GDP-L-galactose can also be produced from GDP-D-mannose by GDP-mannose 3',5'-epimerase (GME). There are three cofactor regeneration systems that can be coupled with the bioconversion process: (1) ATP regeneration; (2) a GTP recycling system; and (3) an NADPH regeneration system. [Figure 4] Figure 4 shows LC-MS spectra confirming the conversion of L-galactose to GDP-L-galactose by FKP: (A) HPLC-UV chromatogram (254 nm) obtained from a sample without FKP ("No FKP"); (B) extracted total ion current (TIC) chromatogram of the GDP-L-galactose ion (604.05) from the same sample without FKP ("No FKP"); (C) HPLC-UV chromatogram of the FKP-added sample ("FKP Added"); (D) TIC chromatogram of the GDP-L-galactose ion (604.05) from the FKP-added sample ("FKP Added"); (E) mass spectrum in the 5.7-5.9 min region obtained from the "FKP Added" sample. [Figure 5]Figure 5 shows HPLC-UV chromatograms confirming the conversion of L-galactose to GDP-L-galactose via FKP in combination with an ATP-regenerating system. (A) Full HPLC-UV chromatogram (254 nm) obtained from a sample without FKP ("without FKP"); (B) Enlarged view of the region from 6.5 to 9.5 min of a partial UV chromatogram (254 nm) obtained from the same sample without FKP ("without FKP"); (C) Full UV chromatogram (254 nm) obtained from a sample with FKP ("with FKP"); (D) Enlarged view of the region from 6.5 to 9.5 min of a partial UV chromatogram obtained from the same sample with FKP ("with FKP"). [Figure 6] Figure 6 shows HPLC-UV chromatograms confirming the conversion of GDP-D-mannose to GDP-L-galactose by AtGME. (A) Full UV chromatogram (254 nm) obtained from a sample without AtGME ("without GME"). (B) Enlarged view of the 6-8 min region of a partial UV chromatogram obtained from the same sample without GME ("without GME"). (C) Full UV chromatogram (254 nm) obtained from a sample with GME ("with GME"). (D) Enlarged view of the 6-8 min region of a partial UV chromatogram obtained from the same sample with GME ("with GME"). (E) UV chromatogram (254 nm) of the product of the FKP reaction described in Figure 5 within the 6-8 min region. [Figure 7] Figure 7 shows HPLC-UV chromatograms confirming the conversion of GDP-L-galactose to GDP-L-fucose. Total UV (254 nm) chromatograms were obtained from (A) a control sample without enzyme ("No Enzyme"), (B) a sample under test reaction ("Test"), and (C) a 1 mM GDP-L-fucose standard. (D) A magnified overlay of the HPLC-UV chromatogram of the GDP-L-fucose standard over the UV chromatograms of the "No Enzyme" control and "Test" reactions within the 8.2-9.5 min region. [Figure 8]Figure 8 shows LC-MS spectra confirming the production of GDP-L-fucose from GDP-L-galactose. Total UV (254 nm) chromatograms were obtained from (A) a control sample without enzyme ("No Enzyme"), (B) a sample from the test reaction ("Test"), (C) a control sample without dehydratase ("No Dehydratase"), (D) a 1 mM GDP-L-fucose standard, and (E) a control sample without reductase ("No Reductase"). (F) A magnified overlay of the HPLC-UV chromatograms of the GDP-L-fucose standard onto the UV chromatograms of the "No Enzyme" control, the "No Dehydratase" control, the "No Reductase" control, and the "Test" reaction within the 10-10.8 min region. (G) Mass spectrum showing the 10.4 min peak obtained from a sample from the "Test" reaction. [Figure 9] Figure 9 shows LC-MS spectra confirming the bioconversion of GDP-L-galactose to 2'-FL. Extracted TIC chromatograms of the [MH]-ion of 2'-FL were obtained from (A) the 2'-FL standard; (B) a control without Gmd ("No Gmd"); (C) a "test" reaction sample; (D) a negative control without substrate ("No GDP-L-Gal"); (E) a negative control without WcaG enzyme ("No WcaG"); and (F) a negative control without FutC enzyme ("No FutC"). (G) Mass spectrum of the 18.9 min peak from the test reaction sample. [Figure 10] Figure 10 shows HPLC chromatograms confirming 2'-FL production by various FutC candidate enzymes. Refractive index units (μRIU) traces are shown for (A) 2'-FL standard, (B) FutC2, (C) FutC5, (D) FutC10, (E) FutC13, (F) FutC18, and (G) FutC21, respectively. Arrows indicate the 2'-FL peak. [Figure 11] Figure 11 shows various NTP regeneration systems according to the present teachings: (A) pyruvate kinase (PK) system; (B) creatine kinase system (CPK); (C) acetate kinase system (AckA); (D) polyphosphate kinase system (PPK); (E) polyphosphate:AMP phosphotransferase / adenylate kinase system (PAP / ADK). [Figure 12] 1 shows various NADPH regeneration systems according to the present disclosure: (A) the NADP-dependent malic enzyme (MaeB) system; (B) the formate dehydrogenase (FDH) system; (C) the phosphite dehydrogenase (PTDH) system; and (D) the glucose dehydrogenase (GDH) system. [Figure 13] FIG. 13 shows that GDP-L-fucose is a negative feedback loop for the wild-type GMD enzyme, which is known to catalyze the conversion of GDP-mannose to GDP-4-keto-6-deoxy-D-mannose in the de novo pathway. [Figure 14] FIG. 14 shows how GDP-L-fucose acts as a negative feedback on wild-type GMD, which can also be used to catalyze the conversion of GDP-L-galactose to GDP-4-keto-6-deoxy-L-galactose in a novel pathway according to the present teachings. [Figure 15] Figure 15 illustrates how GDP-L-fucose similarly acts as negative feedback to wild-type GMD, which can be used to catalyze the conversion of GDP-L-galactose to GDP-4-keto-6-deoxy-L-galactose, which can be derived from GDP-mannose using GDP-mannose 3',5'-epimerase (GME). [Figure 16] Figure 16 shows GDP-L-fucose inhibition data for At GMD and Hs GMD. (A) Relative activity of At GMD mutants (At M2, At M3, At M4), At GMD WT (At WT), and Ec GMD WT (Ec WT) at 0, 70, and 350 μM. (B) Relative activity of H GMD mutants (H M2, H M3, and H M4) compared to H GMD WT (H WT) and Ec GMD WT (Ec WT). [Figure 17] FIG. 17 shows FutC activity: (A) Activity screen of the ASR library; (B) Activity of various FutC enzymes compared to H. pylori FutC and the parent enzyme (FutC5) for the ASR12 construct. [Figure 18]FIG. 18 shows the novel biosynthetic pathway of the present invention for producing 2′-FL from L-galactose. [Figure 19] 19 shows the production of 2′-FL over time using a novel in vitro pathway that converts L-galactose to 2′-FL. ASR12 data are represented by circles, HpFutC data are represented by squares, ASR11 data are represented by triangles, and FutC5 data are represented by diamonds. DETAILED DESCRIPTION OF THE INVENTION

[0072] The present disclosure provides a novel multienzyme pathway for 2'-FL biosynthesis with the following advantages: (1) it uses L-galactose instead of L-fucose as the starting substrate; (2) it is a four-step process compared to the eight-step process required by the de novo mannose-dependent pathway (seven steps to synthesize GDP-fucose and eight steps to convert GDP-fucose to 2'-FL); and (3) the four-step pathway requires a GTP regeneration step, an ATP regeneration step, and an NAD(P) regeneration step. + The pathway includes a NAD(P)H recycling mechanism, thus significantly reducing the need for cofactors and associated costs. Furthermore, the pathway can be performed in vitro in a cell-free manner, which offers the following advantages over fermentation production: (1) it is a non-chemical and non-GMO process that uses all-natural biomolecules, such as enzymes, sugars, and cofactors, to synthesize 2'-FL; (2) as a cell-free process, it eliminates the possibility of endotoxin production and phage contamination, two major concerns of E. coli and other bacterial fermentations; (3) the enzymes can be expressed in the desired organism to ensure that all enzymes are in their most active form, and the process can be performed under favorable conditions without interference from other processes; and (4) the cell-free process results in simpler product purification steps.

[0073] Referring to FIG. 3, the present disclosure provides a method for the synthesis of GDP-L-galactose in four steps: (1) first, converting L-galactose to GDP-L-galactose via a reaction catalyzed by fucokinase / guanylyltransferase in the presence of ATP and GTP; (2) second, converting GDP-L-galactose to NAD(P)H in the presence of NAD(P)H as a cofactor; + (3) NADP + (3) a final reaction step utilizing the enzyme α-1,2-fucosyltransferase (futC) to convert GDP-fucose to 2'-FL in the presence of lactose, producing GDP as a by-product. With continued reference to Figure 3, the reaction system may include ATP regeneration, GTP regeneration, and NADPH regeneration systems, such that only small amounts of these cofactors are required to initiate the process, making the process significantly more cost-effective than existing methods.

[0074] Alternatively, this method can be a modification of the de novo synthesis pathway (Figure 2). Starting with D-glucose, the first five reaction steps can be carried out to obtain GDP-mannose. GDP-mannose can be converted to GDP-L-galactose in a reaction catalyzed by GDP-mannose-3,5-epimerase. Steps 2-4 of the four-step method described above can then be carried out to obtain 2'-FL.

[0075] Each step of this process is described in more detail below.

[0076] Synthesis of GDP-L-galactose FKP naturally catalyzes the conversion of L-fucose to GDP-L-fucose. It has been reported that FKP can generate GDP-L-galactose from L-galactose (Ohashi et al., 2017). Therefore, the first step of the present method can involve incubating L-galactose with fucokinase / guanylyltransferase in the presence of ATP and GTP for a time sufficient to convert L-galactose to GDP-L-galactose. For example, the fucokinase / guanylyltransferase can be an enzyme comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In certain embodiments, the fucokinase / guanylyltransferase can comprise the amino acid sequence set forth in SEQ ID NO: 1.

[0077] Alternatively, the GDP-L-galactose used in the present method can be produced from GDP-mannose. Thus, the present method can include a first step comprising incubating GDP-mannose with GDP-mannose-3,5-epimerase for a period of time sufficient to convert the GDP-mannose to GDP-L-galactose. For example, the GDP-mannose-3,5-epimerase can be an enzyme comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3 or SEQ ID NO:19. In certain embodiments, the GDP-mannose-3,5-epimerase can comprise the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:19.

[0078] Synthesis of GDP-L-fucose Without wishing to be bound by any particular theory, the present inventors believe that the enzyme capable of converting GDP-mannose to GDP-4-keto-6-deoxymannose can also convert GDP-L-galactose to GDP-4-keto-6-deoxy-L-galactose.

[0079] Although GDP-mannose 4,6-dehydratase (GMD) typically uses GDP-mannose as a substrate to produce GDP-4-keto-6-deoxymannose, the present inventors demonstrated in Example 3 below that GDP-mannose 4,6-dehydratase can also use GDP-L-galactose as a substrate to produce GDP-4-keto-6-deoxy-L-galactose. Therefore, suitable enzymes for catalyzing the conversion of GDP-L-galactose to GDP-4-keto-6-deoxy-L-galactose include GDP-mannose 4,6-dehydratases having the amino acid sequences set forth in SEQ ID NOs: 5, 9, 11, and 13. In some embodiments, suitable dehydratase enzymes include functional fragments or homologs of polypeptides having the amino acid sequences set forth in SEQ ID NOs: 5, 9, 11, or 13.

[0080] In a preferred embodiment, the GMD is a mutant that disrupts or inhibits allosteric binding of GDP-L-fucose by GMD. The GMD mutant can be a mutated At GMD comprising the amino acid sequence of SEQ ID NO: 79, SEQ ID NO: 77, or SEQ ID NO: 75. Alternatively, the GMD mutant can be an Hs GMD mutant comprising the amino acid sequence of SEQ ID NO: 85, SEQ ID NO: 83, or SEQ ID NO: 81.

[0081] GDP-4-keto-6-deoxy-L-galactose is then converted to GDP-L-fucose using a reductase. Suitable enzymes for catalyzing the conversion of GDP-4-keto-6-deoxy-L-galactose to GDP-L-fucose include reductases known to have GDP-4-keto-6-deoxy-mannose reductase activity. For example, a reductase having the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:15, or SEQ ID NO:17 may be used. In some embodiments, a suitable dehydratase may include a functional fragment or homolog of a polypeptide having the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:15, or SEQ ID NO:17.

[0082] Synthesis of 2'-fucosyllactose The final step of the method involves the conversion of GDP-L-fucose to 2'-fucosyllactose. This reaction is catalyzed by α-1,2-fucosyltransferase (futC). Exemplary enzymes that can function as futC include those listed in Table 3. Additional exemplary enzymes that can function as futC include those listed in Table 5 (ASR1-ASR12). In a preferred embodiment, the α-1,2-fucosyltransferase is selected from the group consisting of polypeptides comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:109, SEQ ID NO:29, SEQ ID NO:107, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, or SEQ ID NO:105.

[0083] Cofactor regeneration in the biotransformation of 2'-fucosyllactose ATP and GTP are essential for FKP activity and GDP-L-galactose production. The present method includes an NTP regeneration system, which helps provide a sustainable, cost-effective reaction system. NTP regeneration systems require a high-energy phosphate donor to add phosphate to NDP. Suitable systems include phospho(enol)pyruvate (PEP) and pyruvate kinase (A), creatine phosphate and creatine kinase (B), acetylphosphate and acetate kinase (C), polyphosphate and polyphosphate kinase (D), and polyphosphate:AMP phosphotransferase, adenylate kinase, and adenosine monophosphate (E) (Figure 11).

[0084] Furthermore, NADPH is an important cofactor for the reductase activity required for GDP-L-fucose production. + NADP is oxidized to +NADPH can be regenerated by incorporating a dependent oxidation reaction as part of the GDP-L-fucose synthesis disclosed herein. + Dependent oxidation reactions include the oxidation of malate to pyruvate, formate to CO, phosphite to phosphate, and glucose to gluconolactone (Figure 12). By including a donor substrate (malate, formate, phosphite, or glucose) and the corresponding dehydrogenase (malate dehydrogenase (MaeB, SEQ ID NO: 67), formate dehydrogenase (FDH, SEQ ID NO: 69), phosphite dehydrogenase (PTDH, SEQ ID NO: 71), and glucose dehydrogenase (GDH, SEQ ID NO: 73), respectively), NADPH can be continuously regenerated, further improving the production of GDP-L-fucose and 2'-FL.

[0085] Unless otherwise specified, the percent identity of two polypeptide or polynucleotide sequences refers to the percentage of identical amino acid residues or nucleotides over the entire length of the shorter of the two sequences.

[0086] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, the preferred materials and methods are described below.

[0087] The present disclosure will be more fully understood by consideration of the following non-limiting examples. It should be understood that these examples, while indicating preferred embodiments of the subject technology, are given by way of illustration only. From the above description and these examples, one skilled in the art will be able to ascertain the essential features of the subject technology and will be able to make various changes and modifications to the subject technology to adapt it to various applications and conditions without departing from the spirit and scope thereof.

[0088] Example Example 1: Screening of candidate enzymes Gene candidates were selected based on bioinformatic analysis. The following enzymes were screened for the desired activity: fucokinase / guanylyltransferase (FKP) from Bacteroides fragilis (SEQ ID NO: 1), GDP-mannose-3,5-epimerase from Arabidopsis thaliana (At GME) (SEQ ID NO: 3), and rice (Oryza sativa) (Os GME) (SEQ ID NO: 19), GDP-mannose-4,6-dehydratase from Escherichia coli (Ec GMD) (SEQ ID NO: 11), human (Hs GMD) (SEQ ID NO: 9), Arabidopsis thaliana (At GMD) (SEQ ID NO: 5), and Yersinia pseudotuberculosis (Yp DmhA) (SEQ ID NO: 13), GDP-mannose-4,6-dehydratase from Escherichia coli (WcaG) (SEQ ID NO: 7), and Campylobacter jejuni (Campylobacter jejuni). GDP-L-fucose synthase (GFS) or GDP-4-keto-6-deoxy-mannose reductase from M. jejuni (MlghC) (SEQ ID NO: 17) and Yersinia pseudotuberculosis (DmhB) (SEQ ID NO: 15), and 21 α-1,2-fucosyltransferases (FutC1-21) (odd-numbered SEQ ID NOs: 21-61 for each source organism listed in Table 2).

[0089] Full-length DNA fragments of all candidate genes were commercially synthesized. Almost all codons in the cDNA were changed to codons preferred by E. coli (Twist Bioscience, CA). The synthesized DNA was cloned into bacterial expression vectors (pET21 or pET28) to generate expression constructs.

[0090] Each expression construct was transformed into E. coli T7Express or BL21(DE3) cells, which were then grown at 37°C in LB medium containing 50 μg / mL ampicillin or 50 μg / mL kanamycin until the OD600 reached 0.4-0.8. Protein expression was induced by the addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and the cultures were further grown at 16°C for 16 hours. Cells were harvested by centrifugation (3,000 × g; 10 min; 4°C). The cell pellet was collected and used immediately or stored at -80°C.

[0091] Cells were resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 20 mM imidazole). After sonication, the lysate was clarified by centrifugation at 16,000 × g for 15 minutes. The clarified lysate was loaded onto an equilibrated (equilibration buffer: 50 mM Tris-HCl, pH 8.0, 20 mM imidazole, 150 mM NaCl, 20% glycerol) Talon metal affinity column (Takara Bio). After loading the protein sample, the column was washed with equilibration buffer to remove unbound contaminating proteins. The His-tagged recombinant polypeptide was eluted with equilibration buffer containing 250 mM imidazole. The protein was used for activity assays or aliquoted and stored at -80°C until needed.

[0092] All samples were analyzed by appropriate HPLC and LC-MS methods.

[0093] For LC-MS detection of GDP-L-fucose and GDP-L-galactose, the reaction was stopped by heating the sample to 99°C for 10 min, and the protein was removed by centrifugation. The column was a Phenomenex Luna C18(2) HST, 2.0 mm x 100 mm, with a particle size of 2.5 μm and a pore size of 100 Å. Mobile phase A was 10 mM triethylammonium acetate pH 7.0, and mobile phase B was 10 mM triethylamine acetate pH 7.0 (90%) and 10% acetonitrile. The column compartment was set to 25°C, and the flow rate was 0.3 mL / min. The pumping regime was 1% B for 0 min, 1% B for 1 min, 5% B for 8 min, 20% B for 8.1 min, 20% B for 10 min, 1% B for 10.1 min, and held for 15 min. The UV detector was set to 254 nm. The spray voltage was set to 2.7 kV, the capillary temperature to 300°C, the sheath gas to 40, the auxiliary gas to 8, the auxiliary gas to 2, the maximum spray current to 100, the probe heater temperature to 320°C, and the S-lens RF level to 60.

[0094] Samples were prepared as described above for HPLC detection of GDP-L-fucose, GDP-mannose, GDP-L-glucose, and GDP-L-galactose. The column was a Phenomenex Luna 5 μm C18(2) 100 Å, 4.6 × 250 mm. Mobile phase A was 10 mM triethylammonium acetate, pH 7.0, and mobile phase B was 10 mM triethylamine acetate, pH 7.0 (90%), and 10% acetonitrile. The column compartment was set to 25 °C, and the flow rate was 1.3 mL / min. The pumping regime was 0 min 1% B, 1 min 1% B, 8 min 5% B, 8.1 min 20% B, 10 min 20% B, 10.1 min 1% B, and held for 15 min. The UV detector was set to 254 nm.

[0095] The following method was used for LC-MS detection of 2'-FL. Samples were prepared as described above. Analytes were separated using a Thermo Fisher Hypercarb column, 2.1 x 100 mm, 3 μm particle size. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. The column section was set at 25 °C, and the flow rate was set at 0.2 mL / min. The total run time was 30 min. The pumping regime was 0% B at 0 min, 12% B at 21 min, 0% B at 22 min, and 0% B at 30 min. The spray voltage was 3.5 kV, the capillary temperature was 300 °C, the sheath gas was 50, the auxiliary gas was 10, the auxiliary gas was 2, the maximum spray current was 100, the probe heater temperature was 370 °C, and the S-lens RF level was 45.

[0096] For HPLC detection of 2'-FL, samples were prepared as described above. The HPLC instrument method was optimized by isocratic elution of the analytes with distilled water using an Aminex HPX-87H, 300 x 7.8 mm (BioRad) column, a flow rate of 0.6 mL / min, and a total run time of 12 min. The column compartment was set to 50°C. 2'-FL was monitored using a Refractomax 520.

[0097] Example 2: Identification of enzymes for GDP-L-galactose synthesis The first step in the novel pathway according to the present disclosure is the production of GDP-L-galactose. Figure 3 outlines two methods for producing GDP-L-galactose according to the present disclosure.

[0098] FKP naturally catalyzes the conversion of L-fucose to GDP-L-fucose. It has been reported that FKP can generate GDP-L-galactose from L-galactose (Ohashi et al., 2017). FKP was cloned into pET21 and expressed and purified as described in Example 1. The activity of FKP on L-galactose was assayed under the following conditions: 2 mM L-galactose, 2 mM ATP, 2 mM GTP, 4 mM MgCl2, 50 mM Tris-HCl, pH 7.5 (with or without 0.25 g / L FKP). Samples were incubated overnight at room temperature, the reaction was stopped by heating at 99°C for 10 minutes, and analyzed using LC-MS. The LC-MS results are shown in Figure 4.

[0099] Referring to Figure 4, several new peaks were observed in the FKP-added reaction (C) compared to the FKP-free reaction (A). The m / z of GDP-L-galactose was extracted to identify the GDP-L-galactose peak in the UV spectrum. The extracted chromatogram (D) shows the GDP-L-galactose elution at approximately 5.8 minutes, which was absent in the control without FKP. The mass spectrum of the 5.8-minute peak in the HPLC chromatogram is shown in Figure 4(E). The most abundant ion was 604.07, which corresponds to the [M-H] of GDP-L-galactose. - This demonstrates that FKP catalyzes the conversion of L-galactose to GDP-L-galactose.

[0100] Further studies were conducted to optimize the FKP reaction using L-galactose as a substrate by varying the reaction temperature, substrate concentration, and adding an ATP / GTP regeneration system. The reaction conditions were 50 mM Tris-HCl pH 7.5, 5 mM MgCl, 5 mM L-galactose, 2 mM ATP, 2 mM GTP, 10 mM PEP, and 1 U pyruvate kinase (with or without 0.8 mg / mL FKP). The reactions were incubated at 37 °C for 44 h. The reactions were stopped by heating the reactions at 99 °C for 10 min. Samples were analyzed using HPLC (Figure 5).

[0101] As shown in Figure 5, the reaction with FKP (D) produced GDP-L-galactose (retention time: 8.4 min) compared to the reaction without FKP (B). The final titer was 2.5 g / L. This data demonstrates that GDP-L-galactose was produced from L-galactose using the FKP production method.

[0102] In addition to producing GDP-L-galactose from L-galactose, GDP-L-galactose can also be produced from GDP-D-mannose according to the present teachings.

[0103] After enzymatic screening of various potential GDP-mannose-3,5-epimerase candidate enzymes, At GME (SEQ ID NO: 3) was found to exhibit higher activity than Os GME (SEQ ID NO: 19). The reaction conditions were 2 mM GDP-D-mannose, 1 mM NAD + , 50 mM Tris-HCl, pH 8.0, and with or without 0.33 mg / mL AtGME. The reactions were incubated at room temperature for 16 h, stopped by heating at 99 °C for 10 min, and analyzed using HPLC.

[0104] Figure 6 shows HPLC data confirming the conversion of GDP-D-mannose to GDP-L-galactose. In the reaction with AtGME, a decrease in GDP-mannose was observed, and two new peaks were formed (D) compared to the negative control (B). The two new peaks were identified as GDP-L-galactose and GDP-L-glucose. Specifically, the GDP-L-galactose peak was identified based on the product of the FKP reaction (E).

[0105] In summary, these data show that two methods for GDP-L-galactose production were used: the FKP approach led to higher titers;

[0106] Example 3: Identification of enzymes for GDP-L-fucose synthesis Having demonstrated the production of GDP-L-galactose, the next step was to generate GDP-L-fucose, which requires a dehydratase and a reductase. The list of GDP-mannose-4,6-dehydratases that dehydrate GDP-D-mannose is extensive. However, no GDP-L-galactose-4,6-dehydratases have been reported.

[0107] Four enzymes were screened: At Gmd (SEQ ID NO: 5), Hs Gmd (SEQ ID NO: 9), Ec Gmd (SEQ ID NO: 11), and Yp DmhA ​​(SEQ ID NO: 13), while Ec WcaG (SEQ ID NO: 7) is known to be a reductase. Among the potential dehydratases, At Gmd showed the highest initial activity. The reaction conditions are shown in Table 1. The reactions were incubated at 37°C for 16 hours and then stopped by heating at 99°C for 10 minutes. Samples were analyzed by both HPLC and LC-MS. [Table 1]

[0108] Figure 7 shows the HPLC data. In the test reaction, there is a small peak that coelutes with the GDP-L-fucose standard (B). To confirm that this peak is GDP-L-fucose, the sample was analyzed using LC-MS. The LC-MS data are shown in Figure 8. Figure 8 shows the full UV 254 nm traces for the (A) no enzyme, (B) test, (C) no dehydratase, (D) 1 mM GDP-L-fucose, and (E) no reductase samples, respectively. Comparing the "test" reaction sample (B) with the standard (D) reveals the presence of a peak eluting at a retention time similar to that of GDP-L-fucose (approximately 10.4 min) in the test conditions. Overlaying the various chromatograms (F) reveals the presence of a peak coeluting with GDP-L-fucose in the test reaction sample that is not present in the negative control. The mass spectrum of the 10.4 min peak from the test reaction sample is shown in Figure 8 (E). The most abundant ion is the [M-H] ion of GDP-L-fucose. -The data above demonstrate that At Gmd has dehydratase activity on GDP-L-galactose, which allows for the production of GDP-L-fucose.

[0109] Example 4: Bioconversion of GDP-L-galactose / L-galactose to 2'-FL To complete the novel pathway from GDP-L-galactose to 2'-FL, a series of reactions was set up to produce 2'-FL using GDP-L-galactose as a substrate. The reaction conditions are shown in Table 2. The reactions were incubated at 37°C for 16 hours and then terminated by heating at 99°C for 10 minutes. Samples were analyzed using LC-MS. [Table 2]

[0110] Four negative controls were set up: no Gmd, no WcaG, no FutC, and no GDP-L-galactose, and one test reaction sample containing substrate and all enzymes present. The LC-MS data are shown in Figure 9.

[0111] The chromatograms obtained from the test reaction sample and four negative controls respectively showed the [MH] of 2'-FL. - The ions were extracted. For the negative controls, no significant 2'-FL signal was observed ((B), (D), (E), and (F)).

[0112] A peak was observed at 18.9 minutes (C), which corresponds to the retention time of 2'-FL (A). The mass spectrum of the 18.9-minute peak from the test reaction is shown in Figure 9 (G), and the [M-H] of 2'-FL was observed. - ions and [M+FA-H] - The formate adducts are observed at m / z 487.17 and 533.17, respectively. Together, these data confirm that GDP-L-galactose is indeed converted to 2'-FL, thereby completing the de novo pathway.

[0113] Example 5: Identification of a novel FutC enzyme for 2'-FL production The final step in the novel pathway to 2'-FL requires an α-1,2-fucosyltransferase. Various FutC candidates were screened for soluble expression and fucosyltransferase activity in E. coli. A complete list of FutC candidates is shown in Table 3. The candidate enzymes exhibit various solubility and enzymatic activity for 2'-FL synthesis. Candidates with high soluble expression were tested for their activity in vitro using a de novo synthesis pathway. Reaction conditions were 50 mM Tris-HCl pH 8.0, 2 mM NADPH, 0.1 mM NADP. + The reaction mixture was 1 mM GDP-mannose, 80 mM lactose, 0.9 mg / mL Ec Gmd (SEQ ID NO: 11), 0.2 mg / mL Ec WcaG (SEQ ID NO: 7), and 0.2 mg / mL of fucosyltransferase candidate. The reaction was incubated at room temperature for 16 hours. The reaction was stopped by heating at 99°C for 10 minutes and then analyzed by HPLC.

[0114] Figure 10 shows focused μRIU traces from 6 to 8 minutes for five novel FutC candidates, a 2'-FL standard, and FutC from Helicobacter pylori (FutC21, SEQ ID NO: 61). The five novel FutC candidates were FutC2 (from Pisciglobus halotolerans, SEQ ID NO: 23), FutC5 (from Lachnospiraceae bacterium XBB2008, SEQ ID NO: 29), FutC10 (from Thermosynechococcus elongatus, SEQ ID NO: 39), FutC13 (from Candidatus Brocadia sapporoensis, SEQ ID NO: 45), and FutC18 (from Rhizobiales bacterium, SEQ ID NO: 55). All tested FutC candidates show peaks eluting at the same retention times (B), (C), (D), (E), and (F) as the 2'-FL standard (A). These data conclude that the five novel FutC candidates tested (SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 45, and SEQ ID NO: 55) have α-1,2-fucosyltransferase activity for 2'-FL production. [Table 3] TIFF0007810443000004.tif39162

[0115] Example 6: Cofactor regeneration in the biotransformation of 2'-FL ATP and GTP are essential for FKP activity and GDP-L-galactose production; however, they are expensive cofactors. To build a sustainable, cost-effective system, the present disclosure provides a method for the biological production of 2'-FL that includes an ATP and GTP regeneration system. The NTP regeneration system requires a high-energy phosphate donor to add phosphate to NDP.

[0116] Figure 11 shows several systems that can achieve this goal, including phospho(enol)pyruvate (PEP) and pyruvate kinase (A), creatine phosphate and creatine kinase (B), acetyl phosphate and acetate kinase (C), polyphosphate and polyphosphate kinase (D), and polyphosphate:AMP phosphotransferase, adenylate kinase, and adenosine monophosphate (E).

[0117] Furthermore, NADPH is a key cofactor for the reductase activity required for GDP-L-fucose production. During the reductase (WcaG)-catalyzed reaction, NADPH is converted to NADP + NADP is oxidized to + NADPH can be regenerated by incorporating a dependent oxidation reaction as part of the GDP-L-fucose synthesis disclosed herein. + Dependent oxidation reactions include the oxidation of malate to pyruvate, formate to CO, phosphite to phosphate, and glucose to gluconolactone (Figure 12). By including a donor substrate (malate, formate, phosphite, or glucose) and the corresponding dehydrogenase (malate dehydrogenase (MaeB, SEQ ID NO: 67), formate dehydrogenase (FDH, SEQ ID NO: 69), phosphite dehydrogenase (PTDH, SEQ ID NO: 71), and glucose dehydrogenase (GDH, SEQ ID NO: 73), respectively), NADPH can be continuously regenerated, further improving the production of GDP-L-fucose and 2'-FL.

[0118] Example 7: Screening of candidate mutant enzymes Full-length DNA fragments of all candidate genes were commercially synthesized. Almost all codons in the cDNA were changed to codons preferred by E. coli (Twist Bioscience, CA). The synthesized DNA was cloned into bacterial expression vectors (pET21 or pET28) to generate expression constructs.

[0119] Each expression construct was transformed into E. coli T7 Express or BL21(DE3) cells, which were then cultured at OD in LB medium containing 50 μg / mL ampicillin or 50 μg / mL kanamycin. 600 The cells were grown at 37°C until a RI of 0.4-0.8 was reached. Protein expression was induced by the addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and the cultures were further grown at 16°C for 16 h. Cells were harvested by centrifugation (3,000 × g; 10 min; 4°C). The cell pellet was collected and either used immediately or stored at -80°C.

[0120] Cells were resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 20 mM imidazole). After sonication, the lysate was clarified by centrifugation at 16,000 × g for 15 minutes. The clarified lysate was loaded onto an equilibrated (equilibration buffer: 50 mM Tris-HCl, pH 8.0, 20 mM imidazole, 150 mM NaCl, 20% glycerol) Talon metal affinity column (Takara Bio). After loading the protein sample, the column was washed with equilibration buffer to remove unbound contaminating proteins. The His-tagged recombinant polypeptide was eluted with equilibration buffer containing 250 mM imidazole. The protein was used for activity assays or aliquoted and stored at -80°C until needed.

[0121] All samples were analyzed by using appropriate HPLC and LC MS methods.

[0122] For LC-MS detection of GDP-L-fucose and GDP-L-galactose, the reaction was stopped by heating the sample to 99°C for 10 min, and the protein was removed by centrifugation. The column was a Phenomenex Luna C18(2) HST, 2.0 mm x 100 mm, with a particle size of 2.5 μm and a pore size of 100 Å. Mobile phase A was 10 mM triethylammonium acetate pH 7.0, and mobile phase B was 10 mM triethylamine acetate pH 7.0 (90%) and 10% acetonitrile. The column compartment was set to 25°C, and the flow rate was 0.3 mL / min. The pumping regime was 1% B for 0 min, 1% B for 1 min, 5% B for 8 min, 20% B for 8.1 min, 20% B for 10 min, 1% B for 10.1 min, and held for 15 min. The UV detector was set to 254 nm. The spray voltage was set to 2.7 kV, the capillary temperature to 300°C, the sheath gas to 40, the auxiliary gas to 8, the auxiliary gas to 2, the maximum spray current to 100, the probe heater temperature to 320°C, and the S-lens RF level to 60.

[0123] Samples were prepared as described above for HPLC detection of GDP-L-fucose, GDP-mannose, GDP-L-glucose, and GDP-L-galactose. The column was a Phenomenex Luna 5 μm C18(2) 100 Å, 4.6 × 250 mm. Mobile phase A was 10 mM triethylammonium acetate, pH 7.0, and mobile phase B was 10 mM triethylamine acetate, pH 7.0 (90%), and 10% acetonitrile. The column compartment was set to 25 °C, and the flow rate was 1.3 mL / min. The pumping regime was 0 min 1% B, 1 min 1% B, 8 min 5% B, 8.1 min 20% B, 10 min 20% B, 10.1 min 1% B, and held for 15 min. The UV detector was set to 254 nm.

[0124] The following method was used for LC-MS detection of 2'-FL. Samples were prepared as described above. Analytes were separated using a Thermo Fisher Hypercarb column, 2.1 x 100 mm, 3 μm particle size. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. The column section was set at 25 °C, and the flow rate was set at 0.2 mL / min. The total run time was 30 min. The pumping regime was 0% B at 0 min, 12% B at 21 min, 0% B at 22 min, and 0% B at 30 min. The spray voltage was 3.5 kV, the capillary temperature was 300 °C, the sheath gas was 50, the auxiliary gas was 10, the auxiliary gas was 2, the maximum spray current was 100, the probe heater temperature was 370 °C, and the S-lens RF level was 45.

[0125] For HPLC detection of 2'-FL, samples were prepared as described above. The HPLC instrument method was optimized by isocratic elution of the analytes with distilled water using an Aminex HPX-87 H, 300 x 7.8 mm (BioRad) column, a flow rate of 0.6 mL / min, and a total run time of 12 min. The column compartment was set to 50°C. 2'-FL was monitored using a Refractomax 520.

[0126] Example 8: Identification of a novel GDP-mannose-4,6-dehydratase enzyme for the production of GDP-L-fucose and 2'-FL In the de novo pathway, the conversion of GDP-D-mannose to GDP-4-keto-6-deoxymannose is catalyzed by GDP-mannose-4,6-dehydratase (GMD), which then converts the resulting GDP-4-keto-6-deoxymannose to GDP-L-fucose by the bifunctional 3,5-epimerase-4-reductase enzyme (e.g., WcaG from E. coli).

[0127] It is well established that GDP-L-fucose acts as a negative feedback loop on the activity of the GMD enzyme (Figure 13). Inhibition has been characterized as competitive inhibition in E. coli and allosteric inhibition by human and Arabidopsis GMD. See Somoza, JR et al., “Structural and Kinetic Analysis of Escherichia coli GDP-Mannose 4,6 Dehydratase Provides Insights into the Enzyme's Catalytic Mechanism and Regulation by GDP-L-fucose,” Structure, 8(2):123-125 (2000); and Pfeiffer, M. et al., “A Parsimonious Mechanism of Sugar Dehydration by Human GDP-Mannose-4,6-Dehydratase,” ACS Catalysis, 9(4):2962-2968 (2019).

[0128] To drive the production of 2'-FL, it would be beneficial to generate a large pool of GDP-L-fucose. The challenge posed by GDP-L-fucose negative feedback loop exists regardless of whether the novel pathway described above or the novel pathway described herein is used. Referring to Figure 14, it can be seen that after L-galactose is converted to GDP-L-galactose, GMD is used to convert the GDP-L-galactose to GDP-4-keto-6-deoxy-L-galactose, which is then converted to GDP-L-fucose by GDP-L-fucose synthase. Similarly, referring to Figure 15, in the modified de novo pathway described herein, GDP-mannose is converted to GDP-L-galactose by the GDP-mannose 3',5'-epimerase (GME) enzyme, GMD is used to convert the GDP-L-galactose to GDP-4-keto-6-deoxy-L-galactose, which is then converted to GDP-L-fucose by GDP-L-fucose synthase.

[0129] To alleviate the GDP-L-fucose inhibition present in all three pathways, we generated a series of mutations (Table 4) targeting the allosteric binding pocket of GDP-L-fucose in Arabidopsis GMD (At GMD, SEQ ID NO: 5) and human GMD (Hs GMD, SEQ ID NO: 9). [Table 4]

[0130] To test for inhibition, the mutants were expressed, purified, and assayed over a range of GDP-L-fucose concentrations as described in Example 7. Assay conditions were as follows: 50 mM Tris pH 7.5, 1 mM GDP-mannose, 0.5 mM NADP. + , 0.5 mg / mL dehydratase (GMD) enzyme, and 0 μM, 70 μM, or 350 μM GDP-L-fucose. The reactions were quenched at 99 °C for 10 min, and the activity of each enzyme was analyzed using a nucleotide sugar LC-MS method with GDP-4-keto-6-deoxymannose detection.

[0131] The relative activity of the GMD mutants was plotted as a function of GDP-L-fucose concentration (Figure 16). Referring to panel A of Figure 16, both the At GMD wild-type (At WT) and Ec GMD wild-type (Ec WT) were inhibited by GDP-L-fucose, with activity reduced by 95% (At WT) and 80% (Ec WT) at 350 μM GDP-L-fucose.

[0132] In comparison, the At GMD mutants show a marked improvement in their activity at 350 μM GDP-L-fucose, with At GMD M4 (At M4, SEQ ID NO: 79) retaining a remarkable 100% activity. The other two mutants, At GMD M3 (At M3, SEQ ID NO: 77) and At GMD M2 (At M2, SEQ ID NO: 75), retained 50% and 40% activity, respectively.

[0133] Similarly, referring to panel B of Figure 16, the Hs GMD wild-type enzyme (HWT) was significantly inhibited at 350 µM GDP-L-fucose, retaining less than 5% of its activity. Here, it can be seen that the Hs GMD mutants, particularly Hs GMD M4 (HM4, SEQ ID NO: 85) and Hs GMD M3 (HM3, SEQ ID NO: 83), surprisingly retained 100% activity at 350 µM GDP-L-fucose, demonstrating significant improvement in their activity. Furthermore, a third mutant, Hs GMD M2 (HM2, SEQ ID NO: 81), was also able to retain 80% activity.

[0134] The above data indicate that the present GMD mutant can be used to improve the yield of 2'-FL production by increasing the GDP-L-fucose pool.

[0135] Example 9: Identification of novel alpha-1,2-fucosyltransferases from ancestral sequence reconstruction One of the major limitations of 2'-FL production is FutC activity. After screening various FutC candidates as described in Example 5, we attempted to use bioinformatics to identify mutant FutC candidates with even higher activity and improved solubility. Based on ancestral sequence rearrangement (ASR) analysis, we designed a series of ASR mutants (Table 5) and screened them for solubility and activity. [Table 5]

[0136] The enzymes listed in Table 5 were expressed in E. coli and the clarified lysates were analyzed at OD 600 The standard curve was used for normalization and activity screening. The clarified lysates were supplemented with 50 mM Tris pH 7.5, 1 mM GDP-L-fucose, and 80 mM lactose, and the reaction was stopped by heating at 99°C for 10 minutes. The lysates were then analyzed using the 2'-FL HPLC method.

[0137] Referring to panel A of Figure 17, ASR12 showed both improved solubility and activity compared to the rest of the library. Assays were performed to compare the activity of the ASR11 and ASR12 enzymes with that of the parent construct, FutC#5 (SEQ ID NO: 29), and Helicobacter pylori FutC (HpFutC, SEQ ID NO: 61), an enzyme commonly used in 2'-FL production. Specifically, the assay conditions were 2 mM GDP-L-fucose, 40 mM lactose, 50 mM Tris pH 7.5, and 0.5 mg / mL FutC.

[0138] Referring to Figure 17, panel B, it can be seen that ASR12 (SEQ ID NO: 109), like the parent construct, outperforms Hp FutC and can be used to improve the overall potency of 2'-FL using the biosynthetic pathway described herein.

[0139] Example 10: Conversion of L-galactose to 2'-FL using an in vitro enzyme cascade As described herein, the inventors have developed a single-pot bioconversion process that can be used to produce 2'-FL from L-galactose. Referring to Figure 18, this enzymatic process uses four key enzymes that can be complemented by ATP recycling systems, GTP regeneration systems, and / or NADPH regeneration systems.

[0140] Figure 18 shows a four-enzyme in vitro pathway according to the present teachings. In the first step, L-galactose is converted to GDP-L-galactose using a phosphofructokinase (FKP) enzyme (e.g., SEQ ID NO: 1). In the second step, GDP-L-galactose is converted to GDP-4-keto-6-deoxy-L-galactose using a dehydratase (e.g., GMD, preferably the At GMD mutant M4 (SEQ ID NO: 79 and SEQ ID NO: 5)). In the third step, GDP-4-keto-6-deoxy-L-galactose is converted to GDP-L-fucose using a GDP-L-fucose synthase (e.g., a reductase such as WcaG, SEQ ID NO: 7). In the final step, 2'-FL is produced from GDP-L-fucose using FutC (e.g., FutC5, and preferably ASR12, SEQ ID NO: 29 and SEQ ID NO: 109). Acetate kinase for ATP recycling is also included.

[0141] To demonstrate this enzymatic process, all required enzymes were expressed and purified as previously described. The reaction conditions were 50 mM Tris pH 7.5, 10 mM L-galactose, 2 mM ATP, 2 mM GTP, 25 mM acetyl phosphate, 5 mM magnesium chloride, 5 mM potassium chloride, 0.15 mM NADP+, 0.5 mM NADPH, 40 mM lactose, 0.23 g / L phosphofructokinase (FKP, SEQ ID NO: 1), 0.06 g / L acetate kinase (GsAck, SEQ ID NO: 65) for the ATP recycling system, 0.3 g / L AtGMD (SEQ ID NO: 79), 0.75 g / L WcaG (SEQ ID NO: 7), and 0.2 g / L ASR12 (SEQ ID NO: 109). The reaction was stopped by heating at 99°C for 3 and 22 hours and analyzed using the 2'-FL LC-MS method. The LC-MS results are shown in Figure 19.

[0142] As expected, 2'-FL production was significantly higher using ASR12 FutC compared to the parent enzyme (FutC#5) and the commonly used Hp FutC. We have now demonstrated a novel method for producing 2'-FL in vitro with high product yields.

[0143] Destination array: FKP:AA (SEQ ID NO: 1) MQKLLSLPPNLVQSFHELERVNRTDWFCTSDPVGKKLGSGGGTSWLLEECYNEYSDGATFGEWLEKRILLHAGGQSRRLPGYAPSGKILTPVPVFRWERGQHLGQNLLSLQLPLYEKIMSLAPDKLHTLIASGDVYIRSEKPLQSIPEADVVCYGLWVDPSLATHHGVFASDRKHPEQLDFMLQKPSLAELESLSKTHLFLMDIGIWLLSDRAVEILMKRSHKESSEELKYYDLY SDFGLALGTHPRIEDEEVNTLSVAILPLPGGEFYHYGTSKELISSTLSVQNKVYDQRRIMHRKVKPNPAMFVQNAVVRIPLCAENADLWIENSHIGPKWKIASRHIITGVPENDWSLAVPAGVCVDVVPMGDKGFVARPYGLDDVFKGDLRDSKTTLTGIPFGEWMSKRGLSYTDLKGRTDDLQAASVFPMVNSVEELGLVLRWMLSEPELEEGKNIWLRSERFSADEISAGANLKR LYAQREEFRKGNWQALAVNHEKSVFYQLDLADAAEDFVRLGLDMPELLPEDALQMSRIHNRMLRARILKLDGKDYRPEEQAAFDLLRDGLLDGISNRKSTPKLDVYSDQIVWGRSPVRIDMAGGWTDTPPYSLYSGGNVVNLAIELNGQPPLQVYVKPCKDFHIVLRSIDMGAMEIVSTFDELQDYKKIGSPFSIPKAALSLAGFAPAFSAVSYASLEEQLKDFGAGIEVTLLAAIP AGSGLGTSILASTVLGAINDFCGLAWDKNEICQRTLVLEQLLTTGGGWQDQYGGVLQGVKLLQTEAGFAQSPLVRWLPDHLFTHPEYKDCHLLYYTGITRTAKGILAEIVSSMFNLNSSLHLNLLSEMKAHALDMNEAIQRGSFVEFGRLVGKTWEQNKALDSGTNPPAVEAIIDLIKDYTLGYKLPGAGGGGYLYMVAKDPQAAVRIRKILTENAPNPRAFVEMTLSDKGFQVSRS FKP:DNA(SEQ ID NO:2) At GME AA (SEQ ID NO: 3) MGTTNGTDYGAYTYKELEREQYWPSENLKISITGAGGFIASHIARRLKHEGHYVIASDWKKNEHMTEDMFCDEFHLVDLRVMENCLKVTEGVDHVFNLAADMGGMGFIQSNHSVIMYNNTMISFNMIEAARINGIKRFFYASSACIYPEFKQLETTNVSLKESDAWPAEPQDAYGLEKLATEELCKHY NKDFGIECRIGRFHNIYGPFGTWKGGREKAPAAFCRKAQTSTDRFEMWGDGLQTRSFTFIDECVEGVLRLTKSDFREPVNIGSDEMVSMMNEMAEMVLSFEEKKLPIHHIPGPEGVRGRNSDNNLIKEKLGWAPNMRLKEGLRITYFWIKEQIEKEKAKGSDVSLYGSSKVVGTQAPVQLGSLRAADGKE At GME DNA (SEQ ID NO: 4) At Gmd AA (Accession No. 5) MASENNGSRSDSESITAPKADSTVVEPRKIALITGITGQDGSYLTEFLLGKGYEVHGLIRRSSNFNTQRINHIYIDPHNVNKALMKLHYADLTDASSLRRWIDVIKPDEVYNLAAQSHVAVSFEIPDYTADVVATGALRLLEAVRSHTIDSGRTVKYYQAGSSEMFGSTPPPQSETTPFHPRSPYAASKCAAHWYTVNYREAYGLFACNGILFNHESPRRGENFVTRKITRALGRIKVGLQTKLFLGNLQASRDWGFAGDYVEAMWLMLQQEKPDDYVVATEEGHTVEEFLDVSFGYLGLNWKDYVEIDQRYFRPAEVDNLQGDASKAKEVLGWKPQVGFEKLVKMMVDEDLELAKREKVLVDAGYMDAKQQP At Gmd DNA (Accession No. 6) E. coli WcaG:AA (SEQ ID NO: 7) MSKQRVFIAGHRGMVGSAIRRQLEQRGDVELVLRTRDELNLLDSRAVHDFFASERIDQVYLAAAKVGGIVANNTYPADFIYQNMMIESNIIHAAHQNDVNKLLFLGSSCIYPKLAKQPMAESELLQGTLEPTNEPYAIAKIAGIKLCESYNRQYGRDYRS VMPTNLYGPHDNFHPSNSHVIPALLRRFHEATAQNAPDVVVWGSGTPMREFLHVDDMAAASIHVMELAHEVWLENTQPMLSHINVGTGVDCTIRELAQTIAKVVGYKGRVVFDASKPDGTPRKLLDVTRLHQLGWYHEISLEAGLASTYQWFLENQDRFRG E. coli WcaG: DNA (SEQ ID NO: 8) ATGAGCAAACAGCGCGTGTTATTGCCGGCCATCGTGGTATGGTTGGTAGCGCCATTCGTCGCCAGCTGGAACAGCGTGGTGATGGAGCTGGTGCTGCGTACCCGCGACGAACTGAATTTATTAGATAGCCGCGCCGTTCACGACTTTTTCGCCAGCGAACGCATCGACCAAGTTTATCTGGCCGCCGCAAAGTGGGCGGTATCGTTGCCAAACACCTATCCGGCCGACTTTATCT ATCAGAATATGATGATTGAAAGCAACATCCATGCCGCCCACCAGAACGACGTGAACAAACTGCTGTTTTTAGGTAGCAGCTGCATCTACCCGAAGCTGGCCAAACAGCCGATGGCGAAAGCGAACTGCTGCAAGGTACACTGGAACCGACCAACGAACCTTACGCAATTGCCAAGATCGCCGGCATTAAGCTGTGTGAGAGCTACAACCGCCAGTACGGTCGCGGATTATCGCAGCGT ATGCCGACCAATTTATATGGCCCGCATGATAACTTTCACCCGAGTAACAGCCACGTTATTCCGGCTTTATTACGCCGTTTCCACGAAGCAACGCCCAGAACGCCCGGATGTTGTTGTTTGGGGCAGCGGTACCCCTATGCGGCAGTTTTTACACGTTGATGATATGGCAGCAGCCAGCATCCATGTTATGGAACTGGCCCATGAAGTGTGGCTGGAGAACACACAGCCGATGCTGAGCC ATATCAATGTGGGCACTGGTGTGGATTGCACCATTCGTGAACTGGCCCAGACCATCGCAAAAGTGGTGGGCTACAAAGGTCGCGTGGTGTTTGATGCCAGCAAACCGGATGGCACACCGCGCAAACTGCTGGACGTGACCCGTTTACATCAGCTGGGCTGGTACCACGAAATCAGTTTAGAGGCTGGTTTAGCCAGCACCTACCAGTGGTTTTTAGAAAAATCAAGATCGCTTTGCGGTTGA Hs Gmd: AA MRNVALITGITGQDGSYLAEFLLEKGYEVHGIVRRSSSFNTGRIEHLYKNPQAHIEGNMKLHYGDLTDSTCLVKIINEVKPTEIYNLGAQSHVKISFDLAEYTADVDGVGTLRLLDAVKTCGLINSVKFYQASTSELYGKVQEIPQKETTPFYPRSPYGAAKLYAYWIVVNFREA YNLFAVNGILFNHESPRRGANFVTRKISRSVAKIYLGQLECFSLGNLDAKRDWGHAKDYVEAMWLMLQNDEPEDFVIATGEVHSVREFVEKSFLHIGKTIVWEGKNENEVGRCKETGKVHVTVDLKYYRPTEVDFLQGDCTKAKQKLNWKPRVAFDELVREMVHADVELMRTNPNA Hs Gmd:DNA(SEQ ID NO:10) Ec Gmd: AA (SEQ ID NO: 11) MSKVALITGVTGQDGSYLAEFLLEKGYEVHGIKRRASSFNTERVDHIYQDPHTCNPKFHLHYGDLSDTSNLTRILREVQPDEVYNLGAMSHVAVSFESPEYTADVDAMGTLRLLEAIRFLGLEKKTRFYQASTSELYGLVQEIPQKETTPFYPRSPYAVAKLYAYWITVNYRESYGMYACNGILFNHESPRRGETFVTRKITRAIANIAQGLESCLYLGNMDSLRDWGHAKDYVKMQWMMLQQEQPEDFVIATGVQYSVRQFVEMAAAQLGIKLRFEGTGVEEKGIVVSVTGHDAPGVKPGDVIIAVDPRYFRPAEVETLLGDPTKAHEKLGWKPEITLREMVSEMVANDLEAAKKHSLLKSHGYDVAIALES Ec Gmd: DNA (SEQ ID NO: 12) Yp DmhA: AA (SEQ ID NO: 13) MNNVLITGFTGQVGSQLADYILENTDDHVIGMMRWQESMDNIYHLTDRINKKDRISIQYADLNDLMSLYNLIDTVRPKFIFHLAAQSFPRTSFDIPIETLQTNIIGTANLLECIRKLKQQDGYDPVVHVCSSSEVYGRAKVGEALNEDTQFHGASPYSISKIGTDYLGQFYGEAYGIRTFVTRMGTHTGPRRSDVFFESTVAKQIALIEAGHQEPKLKVGNLASVRTFQDARDAVRAYYLLALESGKGNIPNGEVFNIAGDEAFKLPEVIELLLSFSTRNDIEVVTDTDRLRPIDADYQMFDSTKIKSYINWKPEIKAADMFRDLLQHWRNEIASGRIPLNR Yp DmhA: DNA (SEQ ID NO: 14) Yp DmhB:AA (SEQ ID NO: 15) MTKVFILGSNGYIGNNLMESLCDNIEVITVGRSNADIYINLESDDFQSLLNKVEFKDTVIFLSAISSPDECNNNYDYSYKINVKNTISLISLLLAKNVRVMFSSSDAVFGATQNLCDENSEKKPFGKYGEMKSEVEDY FTLEDDFFVVRFSYVLGRNDKFSMMIKEFYEQGKILDVFDGFERNVISINDVTAGIKNIICDWDSIKTRIVNFSGNELVSRQDIVNALVKEKYLNLKYKFTAAPESFWVGRPKKIHTKSNYLESILNRKLESYLEVIKE Yp DmhB: DNA (SEQ ID NO: 16) ATGACGAAAGTTTTCATTCTGGGCTCAAATGGTTACATAGGTAATAACCTGATGGAGTCGCTGTGTGATAATATTGAGGTGATCACGGTCGGTCGTTCAAACGCTGATATATACATTAACCTTGAATCCGACGATTTCCAGTCTCTGCTGAACAAAGTAGAGTTTAAAGATACAGTGATCTTCCTGAGCGCGATCAGTAGCCCGGACGAATGCAATAATAACTATGATTATAGCTATAAAATTAATGTGAAAAATACCATAAGCCTGATTAGCCTCTTACTAGCTAAAAACGTTCGCGTGATGTTCTCAAGCAGCGACGCGGTATTTGGCGCTACGCAAAATCTGTGCGATGAAAATTCCGAAAAAAAACCCTTTGGAAAGTATGGCGAAATGAAAAGCGAAGTTGAAGATTATTTCACCCTTGAGGATGATTTCTTTGTGGTCCGCTTCAGCTATGTGCTGGGGCGAAACGATAAATTTAGCATGATGATCAAAGAGTTTTACGAACAGGGTAAAATACTGGATGTGTTTGATGGCTTTGAACGTAACGTGATTAGCATAAATGACGTGACAGCGGGGATCAAAAACATCATTTGTGACTGGGATTCTATCAAAACTCGTATCGTCAATTTTTCCGGCAACGAATTAGTTTCTCGCCAGGACATCGTTAATGCGCTGGTGAAGGAAAAATACCTGAACCTCAAATACAAATTTACCGCCGCCCCCGAGTCGTTCTGGGTTGGCCGTCCCAAAAAGATTCACACCAAAAGCAATTACCTGGAATCGATTTTAAACCGTAAACTGGAAAGTTACCTGGAGGTCATCAAAGAGTAA Cp MlghC: AA (SEQ ID NO: 17) MSKKVLITGGAGYIGSVLTPILLEKGYEVCVIDNLMFDQISLLSCFHNKNFTFINGDAMDENLIRQEVAKADIIIPLAALVGAPLCKRNPKLAKMINYEAVKMISDFASPSQIFIYPNTNSGYGIGEKDAMCTEESPLRPISEYGIDKVHAEQYLLDKGNCVTFRLATVFGISPRMRLDLLVNDFTYRAYRDKFIVLFEEHFRRNYIHVRDVVKGFIHGIENYDKMKGQAYNMGLSSANLTKRQLAETIKKYIPDFYIHSANIGEDPDKRDYLVSNTKLEATGWKPDNTLEDGIKELLRAFKMMKVNRFANFN Cp MlghC:DNA (SEQ ID NO: 18) ATGTCCAAAAAGGTGCTGATCACCGGCGGCGCGGGCTATATCGGAAGTGTCCTGACCCCGATCCTGTTAGAAAAAGGCTATGAAGTCTGTGTTATCGACAATCTGATGTTTGACCAGATTTCTCTGCTTTCCTGTTTTCATAATAAGAATTTCACGTTCATAAACGGGGATGCGATGGATGAAAATCTGATTCGCCAGGAAGTAGCCAAAGCCGATATTATCATTCCGCTGGCGGCACTGGTCGGGGCGCCTCTGTGTAAACGCAACCCGAAACTGGCTAAAATGATCAACTACGAGGCAGTTAAGATGATTAGCGATTTTGCCTCCCCATCGCAGATCTTTATTTACCCAAACACCAATAGCGGTTACGGGATCGGCGAGAAAGATGCGATGTGCACCGAAGAATCGCCGCTGCGTCCGATTTCCGAGTATGGGATCGATAAAGTGCATGCTGAACAGTACCTGCTGGATAAAGGTAACTGCGTGACCTTTCGTTTAGCAACAGTCTTTGGAATTTCACCGCGTATGCGCCTTGATCTGCTCGTGAATGATTTTACATACCGCGCTTATCGTGACAAATTTATCGTTTTATTCGAAGAGCACTTTCGCCGCAACTATATTCACGTTCGTGATGTCGTGAAAGGCTTCATCCATGGGATAGAGAACTATGATAAAATGAAAGGCCAAGCGTACAACATGGGTCTGAGCTCGGCCAACCTAACCAAGCGCCAACTGGCCGAAACCATTAAGAAATATATTCCAGACTTCTACATCCATTCAGCGAACATTGGAGAAGATCCGGATAAACGCGACTATCTGGTTTCGAATACGAAGTTGGAAGCCACCGGTTGGAAACCTGATAATACTCTTGAGGATGGCATCAAAGAACTGTTACGTGCTTTTAAAATGATGAAGGTTAACCGCTTTGCGAATTTTAATTAA Os GME: AA (SEQ ID NO:  19) MGSSEKNGTAYGEYTYAELEREQYWPSEKLRISITGAGGFIGSHIARRLKSEGHYIIASDWKKNEHMTEDMFCHEFHLVDLRVMDNCLKVTNGVDHVFNLAADMGGMGFIQSNHSVIMYNNTMISFNMLEAARINGVKRFFYASSACIYPEFKQLETNVSLKESDAWPAEPQDAYGLEKLATEELCKHYTKDFGIECRVGRFHNIYGPFGTWKGGREKAPAAFCRKAKTSTDRFEMWGDGLQTRSFTFIDECVEGVLRLTKSDFREPVNIGSDEMVSMNEMAEIILSFEDRELPIHHIPGPEGVRGRNSDNTLIKEKLGWAPTMKLKDGLRFTYFWIKEQIEKEKTQGVDIAGYGSSKVVSTQAPVQLGSLRAADGKE Os GME:DNA FutC 1:AA (SEQ ID NO: 21) MVSIILRGGLGNQLFQYATGRAHSLRTNSTLFVNLSKLDSNLGPDVAKRSLHLEAFDLPVEYVDNETSHSFGRTIRRRIPQVVASINQLLATHLFKLYVEDQSLTFDPNVPNLPGNVTLDGYWQSERYFTEFTETLRREITVRNPVSGE NQRWYDLISDTGSVHVVRRGDYVDLGWALPPSYYRNALNQIQDETDVTDLFFFSDNIDWIRTNQKDLVPDHSDTNVHYVECNDGETAHEDLRLMRACDHHIVANSSFSWWGAWLDNSETKIVIAPDYWVHDPVNHLDIIPDRWDTVSW FutC 1: DNA (SEQ ID NO: 22) ATGGTCTCGATAATCCTACGCGGTGGACTCGGCAACCAACTATTCCAGTACGCGACGGGACGCGCACACTCACTCCGAACTAATTCTACTCTTTTTGTAAACCTCTCTAAACTTGACTCGAACCTTGGCCCCGACGTAGCGAAACGATCGCTACATCTTGAGGCGTTCGATCTTCCAGTTGAATATGTAGATAATGAGACAAGCCACAGTTTTGGCAGGACGATACGCAGACGGATCCCGCAGGTCGTCGCAAGTATAAACCAGTTACTAGCGACACATCTCTTCAAATTGTACGTCGAAGATCAGTCACTGACGTTCGATCCGAATGTCCCTAATCTACCTGGAAACGTCACACTCGACGGTTACTGGCAATCCGAACGCTATTTTACAGAGTTTACCGAGACGCTTCGGCGTGAAATTACGGTTCGTAATCCTGTGTCTGGTGAAAACCAACGGTGGTACGACCTCATCTCCGATACTGGCTCAGTAAGTGTACACGTCCGTCGTGGAGACTACGTTGATCTCGGCTGGGCACTTCCACCGTCCTACTACAGAAATGCCCTCAATCAGATTCAGGATGAAACTGACGTGACAGATCTGTTTTTTTTCTCCGACAACATTGACTGGATTCGTACCAACCAGAAAGACCTTGTGCCGGATCACAGCGATACCAACGTACACTACGTCGAGTGTAACGATGGAGAAACGGCCCACGAGGATCTCCGTCTGATGCGAGCCTGTGATCACCATATCGTCGCCAACAGCAGCTTCAGTTGGTGGGGTGCGTGGCTGGATAATTCTGAGACGAAAATTGTCATCGCTCCCGACTATTGGGTTCATGACCCGGTCAATCATCTCGATATTATTCCCGATCGATGGGATACCGTCAGTTGGTAG FutC 2: AA (SEQ ID NO: 23) MIYTRITSGLGNQMFQYAIAYSYSRKYDMPLILDLTNFKISKKRTYQLDKFKLNDYKKITFKNAPLEIKIFWLVEVLNMISIKLRKKEMKRKNNYNLKSTQFICEKYKEKYNINFDLTNKSLYLSGFWQSPLYFENYRDELIQQF SPNYVLSNKLKEYETKIINCRSVSVHIRRGDFLQHGLFKDVDYQKKAITYLEKKLDNPIFFFFSDDIEWTKEKFKNQKNCFFVSSDSKNSGIEEMYLMSKCENNIIANSTFSWWGAWLNQNQNKIVIAPSTGFGNKDILPKSWYTI FutC2: DNA (SEQ ID NO: 24) ATGATATATACACGAATAACGAGCGGTTTAGGGAACCAAATGTTTCAGTATGCTATTGCGTATTCGTATTCTAGGAAATATGATATGCCACTTATTCTTGATCTTACAAATTTTAAAATTTCAAAAAAGAGAACCTATCAATTAGATAAATTCAAACTTAATGATTATAAAAAGATAACATTTAAAAATGCTCCATTAGAAATAAAAATA TTTTGGTTGGTAGAGGTTTTAAACATGATTTCTATTAAACTAAGGAAAAAAGAAATGAAAAGAAAAAATAATTATAACTTGAAATCAACTCAATTTATATGCGAGAAATATAAAGAAAAATATAACATAAACTTTGATCTCACAAATAAATCACTTTATTTATCTGGATTTTGGCAAAGTCCTTTATATTTTGAAAACTATAGAGATGAA TTAATACAACAGTTTTCTCCTAATTATGTTTTATCAAATAAGTTAAAAGAATATGAAACTAAGATAATAAACTGTAGAAGCGTTTCTGTTCATATTAGAAGAGGAGATTTTTTACAACATGGTTTATTTAAAGATGTAGATTACCAAAAGAAAGCTATAACTTATTTAGAAAAGAAATTAGATAACCCTATTTTTTTCTTTTTCAGAC GATATTGAATGGACAAAAGAAAAATTTAAAAATCAAAAAAATTGTTTTTTTGTATCTTCAGATTCAAAAAATTCTGGTATAGAAGAAATGTATCTTATGTCTAAGTGTGAGAACAATATCATTGCAAATAGTACTTTTAGTTGGTGGGGAGCATGGTTAAATCAAAACCAAAATAAAATTGTAATAGCACCAAGCACTGGTTTTGGTAAT AAAGATATATTACCAAATCTTGGTATACAATTTAG FutC 3:AA (SEQ ID NO: 25) MLVVSMGCGLGNQMFEYAFYKHLCKKYTSEIIKLDIRHAFPFAHNGIELFDIFDLSGEVASKQEVLFLTSGYGLHGVGFEYKTIFHRIGEKVRKLFSLTPQTMKIQDDYTEYYNEFFNVMPGKSVYYLGVFANYHYFKEIQYDIKNIY KFPTIDDLKNKRYAEKMENCNSVSIHVRRGDYVSEGVKLTPLSFYRKAILKIEEKVKNAHFFVFADDVEYARSLFPDNDHYTFVEGNNGKNSFRDMQLMSLCKHNITANSTFSFWGAFLNSNPSKIVIAPNLPYTGAKYPFVCDDWVLI FutC3: DNA (SEQ ID NO: 26) ATGCTTGTTGTTAGTATGGGGTGTGGTTTGGGGAATCAGATGTTTGAATATGCATTTTATAAGCATTTATGTAAAAAATATACAAGCGAGATAATTAAACTTGATATAAGACACGCATTTCCGTTTGCTCATAATGGAATTGAGCTATTTGATATTTTTGATTTATCTGGAGAAGTTGCGAGTAAGCAAGAAGTTCTGTTTTTGACGTCAGGGTATGGCCTACATGGTGTTGGGTTTGAATATAAAACTATTTTTCACAGAATAGGAGAAAAAGTAAGAAAACTTTTCTCGTTGACACCACAAACTATGAAAATTCAAGATGATTATACAGAGTATTATAATGAATTTTTTAATGTGATGCCCGGTAAATCGGTGTACTATCTAGGTGTTTTCGCAAATTACCATTATTTTAAGGAGATACAATATGATATAAAAAATATATACAAATTTCCTACTATAGATGATCTGAAAAACAAAAGATATGCAGAAAAAATGGAAAATTGTAATTCAGTATCTATTCACGTTAGAAGAGGAGATTATGTAAGCGAAGGAGTAAAGCTTACGCCCTTATCATTTTATAGAAAAGCTATTTTAAAGATTGAAGAAAAGGTAAAAAATGCTCATTTTTTTGTCTTCGCAGATGATGTAGAGTATGCTCGTTCGCTTTTTCCTGATAATGATCATTATACGTTTGTAGAAGGAAATAATGGCAAGAATAGTTTTCGCGATATGCAACTTATGAGTTTATGTAAGCATAATATCACAGCAAACAGTACGTTTAGCTTTTGGGGAGCATTTTTAAATTCAAATCCTAGTAAAATAGTTATAGCGCCCAACTTGCCATATACAGGTGCAAAATATCCATTTGTATGTGATGATTGGGTGTTGATATAG FutC 4: AA (SEQ ID NO: 27) MIITRLIGGLGNQIFQYAVGRAVAARTNTPLLLDASGFPGYELRRYELDGFNVRAELVSAAQLARVGVTASAPHSLLERIKLRFFSQSTQKLPLREPILREASFTYDTRIEYVQAPIYLDGYWQSERYFSAIRMQLLQELTLKNEWGVG NEDMFAQIQAAGLGAVSLHVRRGDYVTNSHTATYHGVCSLDYYRAAVAYIAERVAAPHFFIFSDDHDWVSTNLQTGFPTTFVSVNSADHGIYDMMLMKTCRHHVIANSSFSWWGAWLNPYQDKIVVAPQRWFSGASHDISDLIPASWIRI FutC 4: DNA (SEQ ID NO: 28) ATGATCATTACTCGTCTAATTGGTGGTCTCGGCAATCAAATATTCCAATATGCAGTGGGTCGCGCCGTCGCCGCGCGCACGAACACGCCTCTGCTGCTGGACGCTTCCGGTTTTCCGGGTTATGAATTGCGGCGTTACGAGCTCGATGGTTTCAACGTCCGCGCCGAACTGGTCTCGGCTGCGCAACTGGCCCGCGTTGGGGTAACCGCCAGCGCTCCCCACTCTTTGCTGGAGCGAATCAAGCTCCGTTTTTTCTCTCAATCCACGCAGAAGCTACCTCTGCGGGAGCCGATCCTGCGCGAAGCCAGCTTTACCTACGATACCCGCATTGAATACGTACAGGCACCGATCTATCTGGATGGATATTGGCAGAGCGAGCGTTATTTCTCGGCTATCCGCATGCAGCTGCTGCAGGAGCTAACTCTCAAAAACGAGTGGGGAGTAGGAAACGAAGATATGTTTGCTCAGATCCAGGCTGCCGGACTCGGCGCCGTGTCGCTGCATGTCCGCCGGGGCGATTATGTGACAAATTCCCACACGGCTACTTATCACGGAGTATGCTCGCTGGATTACTACCGTGCGGCAGTGGCTTACATCGCCGAACGCGTGGCAGCGCCGCATTTTTTCATCTTTTCCGATGACCACGACTGGGTCAGCACCAATCTGCAGACCGGATTCCCGACCACTTTTGTCTCCGTTAATTCCGCTGACCATGGCATCTACGACATGATGCTGATGAAGACCTGCCGTCATCACGTAATCGCCAATAGCTCCTTCAGCTGGTGGGGCGCCTGGTTGAATCCTTATCAAGACAAGATCGTGGTTGCGCCGCAACGCTGGTTTAGCGGCGCATCGCACGACATAAGTGACCTCATTCCGGCTTCTTGGATCCGAATATGA FutC 5: AA (SEQ ID NO: 29) MIILQMSGGLGNQMFQYALYLKLKKLGREVKFDDETSYELDNARPVQLAVFDITYPRATRQEVTDMRDSSPAWKDRIRRKLKGRNLKQYTEANYSYDEHVFELDDTYLRGYFQTEKYFSDIRDEIYKTYTMRKDLITEQTTQYEEDILSHENS VSIHIRRGDYMTIEGGEIYAGICTDEFYDSAIKYVLERHPDAVFYLFTNDSSWAEYFCNIHSDVNIHVVEGNTEYFGYLDMYLMSRCKHHIVANSSFSWWGAWLGRDADGMVIAPDPWFNCSNCADIHTDRMILIDPKGELLTDDKGVRNESEE FutC5: DNA (SEQ ID NO: 30) ATGATCATATTACAGATGAGCGGCGGACTCGGGAATCAGATGTTCCAGTACGCTTTATATCTGAAACTTAAGAAGCTCGGCAGAGAAGTCAAATTCGATGATGAGACGAGCTATGAACTTGATAATGCGAGACCGGTACAGCTTGCCGTTTTTGACATAACCTATCCTCGTGCGACGAGACAGGAAGTCACCGACATGCGCGATTCTTCCCCCGCATGGAAGGACAGGATCAGACGTAAGTTAAAAGGCCGGAACCTGAAGCAGTACACCGAAGCAAACTACAGTTACGATGAACATGTATTCGAGCTGGACGATACGTATCTTCGGGGATATTTTCAGACCGAGAAGTATTTTTCCGATATCAGGGATGAGATCTACAAGACATACACGATGCGTAAGGATCTGATCACCGAACAGACTACGCAGTATGAGGAAGACATATTAAGTCATGAAAACAGTGTGAGCATCCATATACGCCGCGGCGATTACATGACCATAGAGGGCGGAGAGATATATGCCGGCATCTGTACGGACGAATTTTATGACTCAGCCATAAAGTATGTTCTTGAGAGACATCCGGATGCTGTATTTTATCTTTTTACCAATGACAGTTCATGGGCGGAGTATTTCTGTAACATACATTCCGATGTGAACATTCATGTCGTCGAAGGCAATACCGAATATTTCGGATACCTGGACATGTACCTGATGAGCAGGTGTAAGCATCATATCGTGGCAAACAGTTCTTTTTCATGGTGGGGAGCATGGCTCGGCAGGGATGCGGACGGTATGGTCATAGCACCGGATCCGTGGTTTAACTGCAGCAACTGTGCGGACATCCACACCGACAGGATGATCCTGATCGATCCCAAGGGTGAGCTGTTGACAGATGATAAGGGCGTAAGAAATG AGTCAGAAGAATAA FutC 6: AA (SEQ ID NO: 31) MIIARLFGGLGNQMFQYAAGKSLAERLGAELALDFRIIDERGTRRLTDVFDLDIVPATNLPATKHENLLRYGLWRAFGQSPKFRRETGLGYNAAFAEWSDDTYLHGYWQSEQYFSAISDHLRRVFQAVPAPSKENGAIAD DIRDCSAISLHVRRGDYLALGAHGVCDEAYYNAALSHIAPQLNQDPRVFVFSDDPQWAKDNLPLPFEKIVVDLNGPTTDYEDLRLMSLCDHNIIANSSFSWWGAWLNANPDKIVTAPANWFADAKLDNPDILPEGWQRITP FutC6: DNA (SEQ ID NO: 32) ATGATCATTGCAAGACTGTTCGGGGGTCTGGGAAACCAGATGTTCCAATATGCCGCAGGAAAGTCACTCGCTGAACGATTGGGTGCTGAGCTTGCACTCGATTTTAGAATAATTGATGAACGTGGCACCCGCCGCCTGACAGACGTGTTTGACCTCGACATTGTGCCGGCAACAAACCTTCCCGCCACCAAACATGAAAATCTTCTGAGATATGGGCTATGGCGTGCATTCGGTCAGTCCCCAAAATTTCGACGCGAGACAGGTCTTGGATACAATGCCGCCTTCGCGGAATGGAGCGACGACACTTATCTGCATGGCTATTGGCAGTCAGAGCAGTATTTTTCGGCAATCTCCGACCATTTACGCCGCGTGTTTCAAGCGGTGCCTGCACCGTCGAAAGAGAATGGTGCAATTGCAGATGACATTCGCGACTGCAGCGCGATCTCGCTGCATGTGCGCCGCGGGGACTACCTTGCCCTTGGGGCGCATGGCGTCTGTGATGAAGCCTATTACAATGCGGCGTTGTCTCATATCGCACCCCAATTGAACCAAGATCCACGTGTCTTTGTGTTTTCCGACGATCCGCAATGGGCCAAAGACAACCTTCCCCTGCCGTTTGAAAAGATTGTCGTCGATCTGAACGGCCCGACAACCGACTATGAAGACCTGCGATTGATGAGCCTGTGCGACCACAACATCATCGCAAACAGTTCATTTTCCTGGTGGGGCGCATGGTTAAACGCAAACCCTGACAAGATTGTCACCGCGCCAGCAAACTGGTTCGCGGATGCAAAGTTGGACAACCCCGACATTCTCCCTGAAGGCTGGCAAAGGATCACC CCCTGA FutC 7: AA (SEQ ID NO: 33) MYFQKKMIIVKLSGGLGNQLFQYALGRQLSIVNHTDLKMDTTNFSQPSGGTTRTFALGSFNIHAAQANKDEIKLLAGEPNRIFQRVRRKIGLMPIHYFKEPHFHFYQPVLSLQDGVYLDGYWQSEKYFAEIADRIREDLKPVGSFS NQYETFKQSIKQSVSVSVHIRRGDYTTTSKANRYLKPCEALYYQTAVEYLTKRISNLVFFVFSDDIEWAKAHIHFGFPMQYVEGNSAQEDLLLIASCQHHIIANSTFSWWGAWLNPHPDKIVIAPQKWFSTERFDTKDLLPESWILL FutC7: DNA (SEQ ID NO: 34) ATGGCTTACTTCCAGAAAAAGATGATCATCGTTAAACTGAGTGGCGGTCTGGGCAATCAGCTGTTTCAGTATGCCCTGGGTCGTCAGCTGAGCATTGTTAATCATACCGATCTGAAAATGGATACCACCAATTTTAGCCAGCCGAGCGGTGGCACCACCCGTACCTTTGCACTGGGCAGCTTTAATATTCATGCCGCACAGGCCAATAAGGATGAAATTAAGCTGCTGGCAGGCGAACCGAATCGCATTTTTCAGCGTGTTCGTCGCAAAATTGGCCTGATGCCGATTCATTATTTTAAAGAACCGCATTTTCACTTCTACCAGCCGGTGCTGAGTCTGCAGGATGGCGTGTATCTGGATGGCTATTGGCAGAGTGAAAAATATTTTGCCGAAATTGCCGATCGCATTCGCGAAGATCTGAAACCGGTGGGTAGTTTTAGCAATCAGTATGAAACCTTTAAGCAGAGCATTAAGCAGAGCGTTAGCGTTAGCGTGCATATTCGTCGTGGTGACTATACCACCACCAGTAAAGCCAATCGCTATCTGAAACCGTGTGAAGCACTGTATTATCAGACCGCAGTTGAATATCTGACCAAACGTATTAGCAATCTGGTTTTCTTTGTGTTTAGTGATGATATTGAGTGGGCCAAAGCCCATATTCATTTTGGTTTTCCGATGCAGTATGTTGAAGGCAATAGTGCCCAGGAAGATCTGCTGCTGATTGCAAGCTGCCAGCATCATATTATTGCCAATAGTACCTTTAGCTGGTGGGGTGCATGGCTGAATCCGCATCCGGATAAAATTGTGATTGCCCCGCAGAAATGGTTTAGTACCGAACGTTTTGATACCAAAGATCTGCTGCCGGAAAGCTGGATTCTGCTGTAA FutC 8: AA (SEQ ID NO: 35) MIISNIIGGLGNQMFQYAMARSLSLELKSDLLLDISSYDSYPLHQGYELDRVFKVRSSLAKVEDVKSVLGWQQNLFIHRVLRRPQFSWLRKKSLAIEPFFQYWEGVNFLPKNCYLFGYWQSEKYFNKFSEVIRQDFSFDSNMSE ENSFYSERIRKSNSVSVHIRRGDYLNNSVYASCSLEYYRSAIAHVSARSGNPVFFVFSDDIEWVKDNLEFEAESYFVAHNKAGESYNDMRLMSYCKHHVIANSSFSWWGAWLNPSPEKIVIAPKQWFTDGTNTKDLIPSEWMVL FutC 8: DNA (SEQ ID NO: 36) ATGGCTATCATCAGTAACATCATCGGCGGTCTGGGTAATCAGATGTTTCAGTATGCAATGGCTCGTAGTCTGAGTCTGGAACTGAAAAGCGATCTGCTGCTGGATATTAGCAGTTATGATAGCTATCCGCTGCATCAGGGCTATGAACTGGATCGTGTTTTTAAAGTTCGTAGTAGCCTGGCCAAAGTGGAAGATGTGAAAAGTGTGCTGGGCTGGCAGCAGAATCTGTTTATTCATCGCGTGCTGCGTCGTCCGCAGTTTAGCTGGCTGCGTAAAAAATCTCTGGCCATTGAACCGTTTTTCCAGTATTGGGAAGGCGTTAATTTTCTGCCGAAAAATTGTTATCTGTTCGGTTATTGGCAGAGCGAAAAATATTTTAATAAGTTCAGCGAGGTTATTCGTCAGGATTTTAGTTTTGATAGTAACATGAGTGAGGAAAATAGTTTTTACAGTGAACGTATTCGCAAAAGCAATAGCGTGAGTGTTCATATTCGTCGTGGTGACTATCTGAATAATAGCGTTTATGCCAGTTGTAGTCTGGAATATTATCGTAGTGCCATTGCACATGTGAGCGCCCGCAGCGGTAATCCGGTGTTTTTCGTTTTTAGTGATGATATTGAGTGGGTGAAAGATAATCTGGAATTTGAAGCAGAAAGTTATTTCGTTGCCCATAATAAGGCAGGCGAAAGTTATAATGATATGCGTCTGATGAGTTATTGTAAACATCATGTGATTGCCAATAGTAGCTTTAGCTGGTGGGGTGCCTGGCTGAATCCGAGCCCGGAAAAAATTGTTATTGCACCGAAACAGTGGTTTACCGATGGCACCAATACCAAAGATCTGATTCCGAGCGAATGGATGGTTCTGTAA FutC 9: AA (SEQ ID NO: 37) MVIIKMMGGLGNQMFQYALYKAFEQKHIDVYADLAWYKNKSVKFELYNFGIKINVASEKDINRLSDCQADFVSRIRRKIFGKKKSFVSEKNDSCYENDILRMDNVYLSGYWQTEKYFSNTREKLLEDYSFALVNSQVSE WEDSIRNKNSVSIHIRRGDYLQGELYGGICTSLYYAEAIEYIKMRVPNAKFFVFSDDVEWVKQQEDFKGFVIVDRNEYSSALSDMYLMSLCKHNIIANSSFSWWAAWLNRNEEKIVIAPRRWLNGKCTPDIWCKKWIRI FutC 9: DNA (SEQ ID NO: 38) ATGGTTATTATCAAGATGATGGGTGGTCTGGGCAATCAGATGTTTCAGTATGCCCTGTATAAAGCATTTGAACAGAAACATATCGACGTGTATGCCGATCTGGCATGGTATAAAAAATAAGAGCGTGAAATTTGAGCTGTATAATTTTGGCATTAAGATCAATGTGGCCAGTGAAAAAAGATTATAATCGTCTGAGCGATTGCCAGGCAGA TTTTGTTAGTCGCATTCGCCGCAAAATTTTTGCAAAAAGAAAAGTTTCGTGAGTGAAAAGAAGAATGATAGTTGTTATGAAAACGACATCCTGCGTATGGATAATGTGTATCTGAGCGGTTATTGGCAGACCGAAAAATTTTAGCAATACCCGTGAAAAGCTGCTGGAAGATTATAGCTTTGACTGGTTAATAGTCAGGTGAGCGAAT GGGAAGATAGCATTCGTAATAAGAATAGTGTTAGCATTCACATTCGCCGTGGTGACTATCTGCAGGGGCGAACTGTATGGCGGCATTTGTACCAGTCTGTATTATGCCGAAGCCATTGAATATATTAAGATGCGCGTTCCGAATGCCAAATTTTTCGTTTTTAGTGATGACGTGGAATGGGTGAAACAGCAGGAAGATTTTAAAGGTTT GTGATTGTTGACCGTAATGAATATAGCAGTGCACTGAGTGATATGTATCTGATGAGCCTGTGCAAACATAATATTATTGCCAATAGTAGCTTCAGCTGGTGGGCAGCATGGCTGAATCGTAATGAAAAAAATTGTTATCGCGCCCGCGCCGTTGGCTGAATGGCAAATGTACCCCGGATATTTGTGCAAAAATGGATTCGCATTTAA FutC 10:AA(SEQ ID NO:39) MIIVRLCGGLGNQMFQYAAGLAAAHRIGSEVKFDTHWFDATCLHQGLELRRVFGLELPEPSSKDLRKVLGACVHPAVRRLLSRRLLRALRPKSLVIQPHFHYWTGFEHLTDNVYLEGYWQSERYFSNIADIIRQQFRFVEPLDPHN AALMDEMQSGVSVSLHIRRGDYFNNPQMRRVHGVDLSEYYPAAVATMIEKTNAERFYVFSDDPQWVLEHLKLPVSYTVVDHNRGAASYRDMQLMSACRHHIIANSTFSWWGAWLNPRPDKVVIAPRHWFNVDVFDTRDLYCPEWIVL FutC 10: DNA (SEQ ID NO: 40) ATGGCTATCATCGTGCGTCTGTGCGGTGGTCTGGGTAATCAGATGTTTCAGTATGCCGCAGGTCTGGCAGCCGCACATCGCATTGGTAGTGAAGTGAAATTTGATACCCATTGGTTTGATGCAACCTGTCTGCATCAGGGCCTGGAACTGCGTCGTGTGTTTGGTCTGGAACTGCCGGAACCGAGCAGCAAAGATCTGCGCAAAGTTCTGGGTGCATGCGTGCATCCGGCAGTTCGCCGTCTGCTGAGTCGCCGTCTGTTACGTGCACTGCGTCCGAAAAGTCTGGTTATTCAGCCGCATTTTCATTATTGGACCGGTTTTGAACATCTGACCGATAATGTTTATCTGGAAGGTTATTGGCAGAGCGAACGCTATTTTAGCAATATTGCAGATATTATCCGCCAGCAGTTTCGCTTTGTGGAACCGCTGGACCCTCATAATGCCGCCCTGATGGATGAAATGCAGAGTGGCGTTAGTGTGAGCCTGCATATTCGCCGTGGTGACTATTTTAATAATCCGCAGATGCGCCGTGTTCATGGCGTTGATCTGAGTGAATATTATCCGGCCGCAGTGGCCACCATGATTGAAAAAACCAATGCCGAACGTTTTTATGTGTTTAGTGATGATCCGCAGTGGGTTCTGGAACATCTGAAACTGCCGGTTAGCTATACCGTGGTTGATCATAATCGTGGTGCCGCAAGTTATCGCGATATGCAGCTGATGAGTGCATGTCGCCATCATATTATTGCAAATAGCACCTTTAGTTGGTGGGGTGCATGGCTGAATCCGCGCCCGGATAAAGTGGTTATTGCCCCGCGTCATTGGTTTAATGTGGATGTTTTTGATACCCGTGATCTGTATTGCCCGGAATGGATTGTGCTGTAA FutC 11: AA (SEQ ID NO: 41) MIISKLKGGLGNQLFQYAIGRKMALEQGVELKLELSFFERQNNKTQARDFGLSCFNIDASIASSEDIRMILGPHFLRPLKRRLSKMGIPLFRWNYVRENSWAYHPEILKKKAPLILDGYWQSAAYFESIRDVLLSDFELKAECVSDK LRLLQKQITTESSVALHVRRGDYVTNPIVAKEFGICSYYEEAVSYMKALEGEPVFFVFSDDIDWCKKHFGEKAGTFVFVSGNQDYEDLMLMSACKHQIIANSSFSWWSAWLNKNPEKKVIAPKIWFADTQMYKTEHIVPQEWIRI FutC 11: DNA (SEQ ID NO: 42) ATGGCTATCATCAGCAAACTGAAAGGTGGCCTGGGGCAATCAGCTGTTTCAGTATGCCATTGGCCGCAAAATGGCCCTGGAACAGGGTGTTGAACTGAAACTGGAACTGAGTTTCTTTGAACGTCAGAATAAAGACCCAGGCCCGTGATTTTGGTCTGAGTTGTTTTAATATTGACGCCAGCATTGCAAGTAGCGAAGATATTCGTATGATTCTGGCCCG CATTTTCTGCGTCCGCTGAAACGTCGCCTGAGCAAAATGGGCATTCCGCTGTTTCGTTGGAATTATGTTCGCGAAAAATAGTTGGGCCTATCATCCGGAAATTCTGAAAAAAGAAAGCACCGCTGATTCTGGATGGTTATTGGCAGAGTGCAGCCTTATTTTGAAAGCATTCGTGATGTTCTGCTGAGCGATTTTGAACTGAAAGCAGAATGCGTTAGTGATAAA CTGCGTCTGCTGCAGAAACAGATTACCACCGAAAGTAGCGTGGCCCTGCATGTGCGCCGCGGTGACTATGTTACCAATCCGATTGTTGCAAAAGAATTGGTATTTGCAGTGAAAGTTACTATGAAAGAGCAGTTATTATATGAAGGCACTGGAAGGTGAACCGGTTTCTTTTGTGTTTAGCGATGATATTGATTGGTGTAAAAAGCATTTCGGCGAAAAA GCAGGTACCTTTGTGTTTGTGAGCGGTAATCAGGATTATGAAGATCTGATGCTGATGAGCGCATGTAAACATCAGATTATTGCAAATAGTAGCTTCAGCTGGTGGAGCCGCTGGCTGAATAAGAATCGGAAAGAAAGTTATTGCACCGAAAATTTGTTTGCAGATACCCAGATGTATAAAACCGAACATATTGTTCCGCAGGAATGGATTCGCATTTAA FutC 12:AA(SEQ ID NO:43) MITVSLIGGLGNQMFQYAAGKALAERHGVPLVLDLSGFRDYAVRSYLLDRLHVPEAGGALGQAESFQKFAARFARAKWKGRIDRLLGQVGLPKIVASSQEYREPHFHYDPAFEALGPSAVLFGYFQSERYFGSISESLSDWFSAREPFGDTA ADMLARIETSPLAISVHVRRGDYLNPGTAEFHGILGESYYRQALGRLERLCGQDSELFVFSDDPPAAEKVLDFASRSRLVHVRGDPERPWEDMALMARCHHHIIANSSFSWWGAWLNRSPHKHVVAPRAWFAPAELEKTNTADLYPAEWILV FutC 12: DNA (SEQ ID NO: 44) ATGGCTATCACCGTTAGTCTGATTGGCGGCCTGGGCAATCAGATGTTTCAGTATGCAGCCGGCAAAGCCCTGGCAGAACGTCATGGTGTGCCGCTGGTTCTGGATCTGAGTGGCTTTCGTGATTATGCAGTGCGCAGCTATCTGCTGGATCGCCTGCATGTTCCGGAAGCCGGCGGCGCTCTGGGCCAAGCAGAAAGCTTTCAGAAATTTGCCGCACGTTTTGCCCGCGCAAAATGGAAAGGTCGCATTGATCGTCTGCTGGGCCAGGTGGGTCTGCCGAAAATTGTTGCAAGCAGCCAGGAATATCGCGAACCGCATTTTCATTATGATCCGGCATTTGAAGCACTGGGTCCGAGCGCCGTGCTGTTTGGCTATTTTCAGAGCGAACGTTATTTTGGTAGCATTAGTGAAAGCCTGAGTGATTGGTTTAGCGCCCGTGAACCGTTTGGTGACACCGCCGCCGATATGCTGGCCCGTATTGAAACCAGCCCGCTGGCCATTAGCGTGCATGTTCGCCGCGGTGACTATCTGAATCCGGGCACCGCCGAATTTCATGGTATTCTGGGTGAAAGCTATTATCGCCAGGCACTGGGTCGCCTGGAACGCCTGTGCGGTCAGGATAGTGAACTGTTTGTGTTTAGTGATGATCCGCCGGCCGCAGAAAAAGTGCTGGATTTTGCCAGTCGCAGCCGTCTGGTTCATGTTCGCGGCGATCCGGAACGCCCGTGGGAAGATATGGCACTGATGGCCCGCTGCCATCATCATATTATTGCAAATAGTAGTTTCAGCTGGTGGGGCGCCTGGCTGAATCGTAGTCCGCATAAACATGTTGTGGCACCGCGTGCATGGTTTGCCCCGGCAGAACTGGAAAAAACCAATACCGCAGATCTGTATCCGGCCGAATGGATTCTGGTTTAA FutC 13: AA (SEQ ID NO: 45) MQLKRWPQLKPTDAAVFGSGKQTIMIIVKLMGGLGNQMFQYAAGRRLAEKLGVKLKLDIEMFKDNTLRKYELGAFNIQECFAAVEEIERLTVVKRGIVEKALDRVFKRPIRRPGGYVAEKYFVFDPSILQLPDQVYLDGYWQSEKYFAEIETIIREE FTIKYPQTDKNKVLSDSIKSGNSVTVHVRRGDYVNNPETNSLHGVCGIDYYQRCIDFIITKIANPHFFFFSDDPEWVKNNLKIKYESTVVEHNGAEKCYEDLRLLSQGKYHIIANSTFSWWGAWLNKNPEKMVVAPEKWFKKEDVNTKGFIPEDWIRL FutC 13: DNA (SEQ ID NO: 46) ATGGCTCAGCTGAAACGTTGGCCGCAGCTGAAACCGACCGATGCAGCCGTGTTTGGCAGTGGCAAACAGACCATTATGATTATTGTTAAACTGATGGGTGGTCTGGGCAATCAGATGTTTCAGTATGCCGCCGGCCGCCGTCTGGCCGAAAAACTGGGTGTTAAACTGAAACTGGATATTGAAATGTTCAAGGATAACACCCTGCGCAAATATGAACTGGGCGCATTCAATATTCAGGAATGTTTTGCCGCAGTTGAAGAAATTGAACGTCTGACCGTTGTTAAACGCGGTATTGTTGAAAAAGCCCTGGATCGCGTTTTTAAACGCCCGATTCGCCGTCCGGGTGGTTATGTTGCCGAAAAATATTTTGTTTTCGACCCGAGTATTCTGCAGCTGCCGGATCAGGTTTATCTGGATGGCTATTGGCAGAGTGAAAAATATTTCGCAGAAATTGAAACCATCATCCGCGAAGAATTCACTATTAAGTATCCGCAGACCGATAAAAATAAGGTTCTGAGCGATAGTATTAAGAGCGGCAATAGCGTGACCGTGCATGTGCGTCGTGGTGACTATGTTAATAATCCGGAAACCAATAGCCTGCATGGCGTGTGCGGTATTGATTATTATCAGCGCTGTATTGATTTCATTATCACCAAAATTGCGAACCCGCATTTCTTTTTCTTTAGTGATGATCCGGAATGGGTTAAAAATAATCTGAAAATTAAGTACGAGAGCACCGTGGTGGAACATAATGGCGCAGAAAAATGCTATGAAGATCTGCGTCTGCTGAGTCAGGGTAAATATCATATTATTGCCAATAGCACCTTCAGTTGGTGGGGTGCATGGCTGAATAAGAATCCGGAAAAAATGGTTGTTGCCCCGGAAAAATGGTTTAAAAAAGAAGATGTGAACACCAAAGGCTTTATTCCGGAAGATTGGATTCGTCTGTAA FutC 14: AA (SEQ ID NO: 47) MIVIKLIGGGLGNQMFQYATAKAIALHKNTTLKLDVSAFENYDLHDYSLDHFNITAKKYQQPPKWLKKIQNKLKPKTYYNEESFRYNSFLFDSNAKTILLNGYFQSEQYFLKYREEIIKDFSITSPLKPETKALLQK VHKTNAVSIHIRRGDLKHDVHNTFKEEYYKKAMKTIESKIDNPTYYLFSDDMPWVKLNFKSNFKTVYVDFNDAQTAFEDLVLMSNCKHNIIANSSFSWWAAWLNTNPSKIVIAPEQWFNGNKYDYTDVVPETWVKI FutC 14: DNA (SEQ ID NO: 48) ATGGCTATCGTGATTAAGCTGATTGGTGGTCTGGGTAATCAGATGTTTCAGTATGCCACCGCCAAAGCAATTGCCCTGCATAAAAATACCACCCTGAAACTGGATGTTAGTGCCTTTGAAAATTATGATCTGCATGATTATAGCCTGGATCATTTTAATATCACCGCAAAAAAGTACCAGCAGCCGCCGAAATGGCTGAAAAAGATTCAGAATAAGCTGAAACCGAAAACCTATTATAACGAAGAAAGTTTTCGCTATAACAGTTTTCTGTTTGATAGCAATGCCAAAACCATTCTGCTGAATGGTTATTTTCAGAGCGAACAGTATTTTCTGAAATATCGTGAAGAAATCATCAAGGATTTCAGTATTACCAGCCCGCTGAAACCGGAAACCAAAGCACTGCTGCAGAAAGTGCATAAAACCAATGCCGTTAGCATTCATATTCGCCGTGGCGATTTTCTGAAACATGATGTTCATAATACCTTCAAAGAGGAATATTACAAGAAGGCCATGAAAACCATTGAAAGCAAAATTGATAACCCGACCTATTATCTGTTTAGTGATGATATGCCGTGGGTTAAACTGAATTTTAAAAGCAATTTCAAGACCGTGTACGTGGATTTTAATGATGCCCAGACCGCATTTGAAGATCTGGTGCTGATGAGCAATTGTAAACATAATATTATCGCCAACAGCAGTTTTAGCTGGTGGGCCGCCTGGCTGAATACCAATCCGAGCAAAATTGTTATTGCACCGGAACAGTGGTTTAATGGTAATAAGTATGATTACACCGACGTTGTGCCGGAAACCTGGGTTAAAATTTAA FutC 15: AA (SEQ ID NO: 49) MIIIKFCGALGNQLFQYALYEKMRILGKDVKADISAFGDGNEKRFFYLDELGIEFNIASADEIAEYLNRKTIRFVPGFLQHRHYYFEKKPYVYNKKILSYDDCYLEGYWQNYRYFDDIKDELLKHMKFPCLPLEQKKL AEKMENENSVAVHVRMGDYLNLQDLYGGICDADYYDRAFSYIEGNISNPVYYGFSDDVDKASALLAKHKINWIDYNSEKGAIYDLILMSKCKNNIIANSSFSWWGAYLEYNNGKVVVSPNRWMNCFENSNIAYWGWISL FutC 15: DNA (SEQ ID NO: 50) ATGGCTATCATCATCAAGTTCTGGTGCCCTGGGTAATCAGCTGTTTCAGTATGCCCTGTATGAAAAAATGCGTATTCTGGGCAAAGATGTGAAAGCAGATATTAGCCCTTTGGCGATGGTAATGAAAAACGTTTCTTTATCTGGATGAGCTGGGTATTGAATTCAATATTGCCAGCGCAGATGAAATTGCAGAATATCTGAATCG TAAAACCATTCGTTTTGTTCCGGGTTTCTGCAGCATCGCCATTATTTTGAAAGAAAACCGTATGTGTACAACAAAAGATTCTGAGTTACGATGATTGCTATCTGGAAGGCTATTGGCAGAATTATCGTTATTTTGATGACATTAAGGACGAACTGCTGAAACATATGAAATTTCCGTGCCTGCCGCTGGAACAGAAAAAACTGG CCGAAAAAATGGAAAATGAAAATACGGTGGCAGTTCATGTTCGTATGGGCGATTATCTGAATCTGCAGGATCTGTATGGTGGTATTTGCGATGCAGATTATTATGATCGTGCATTTTCATATATCGAGGGTAATATTAGCAACCCGGTTTATTATGGTTTTAGCGATGATGTGATAAAGCAAGCGCACTGCTGGAAAACATAAAATT AATTGGATTGACTACAACAGCGAAAAAGGTGCAATCTATGATCTGATTCTGATGAGTAAATGTAAGAATAACATCATCGCCAATAGCAGCTTTAGCTGGTGGGGTGCATATCTGGAATATAATAATGGTAAAGTGGTGGTGAGTCCGAATCGCTGGATGAATTGCTTTGAAAATAGCAATATCGCCTATTGGGGCTGGATTAGCCTGTAA FutC 16:AA(SEQ ID NO:51) MSKKKPVIIEILGGIGNQMFQFALAKILAEKNDSELFIDTNFYKETSQNLKNFPRYFSVGIFDLQFKLATEKEKIFFKHPSLKNRLNRKLGLNYPKVFKEKSFNFDPELLTMKAPIFLKGYFQSYKYFAGTESKIRQLYEFPDEKLDSRN EEIKNRIITKTSVSVHIRRGDYVENRKTQDFHGNCSVEYYKKAVEYLSATIKDFNLVFFSDDIAWVQNQFKDLPYEKKFVTGNLYENSWKDMYLMSLCDHNIIANSSFSWWAAWLNKNPEKKVVAPKKWFADMDQEQKSLDLLPPDWVRI FutC 16: DNA (SEQ ID NO: 52) ATGGCTAGCAAAAGAAGCCGGTTATTATTGAAATTCTGGGTGGCATTGGCAATCAGATTGTTCAGTTTGCCCTGGCCAAAATTCTGGCAGAAAAGAATGATAGTGAACTGTTTATTGACACCAATTTTTACAAGGAAACCAGCCAGAATCTGAAAATTTTCCGCGTTATTTTAGCGTGGGTATTTTTGATCTGCAGTTTAAACTGGCAACGAAAAAGAAAAA TCTTTTTCAAGCACCCGAGCCTGAAAAATCGTCTGAATCGTAAACTGGGCCTGAATTACCGAAAGTGTTTAAAGAAAAGCTTTAATTTCGACCCGGAACTGCTGACCATGAAAGCCCCGATTTTTCTGAAAGGCTATTTTCAGAGCTATAAATATTTCGCAGGTACCGAAAGTAAAATTCGTCAGCTGTATGAATTTCGGATGAAAAAACTGATAGCCGCAAT GAAGAAATTAAGAATCGCATATTACCAAGACCAGTGTTAGCGTTCATATTCGTCGTGGCGATTATGTTGAAAATCGCAAAACCCAGGATTTTCATGGTAATTGCAGTGTGGAATATTATAAAAAGGCAGTTGAATACCTGAGCGCAACCATTAAGGATTTTAATCTGTTTTCTTTAGCGATGATATCGCATGGGTTCAGAATCAGTTTAAAGATCTGCCGTATG AAAAGAAATTCGTGACCGGTAATCTGTATGAAATAGTTGGAAAGATATGTACCTGATGAGTCTGTGCGATCATAATATTATTGCAAATAGTAGCTTCAGCTGGTGGGCAGCATGGCTGAATAAGAATCCGGAAAGAAGTTGTTGCCCCGAAAAAATGGTTTGCAGATATGGATCAGGAACAGAAAGCCTGGATCTGCTGCCGCCGGATTGGGTTCGTATTTAA FutC 17:AA(SEQ ID NO:53) MIVVRIIGGLGNQMFQYAFAKSLQQKGYQVKIDITKFKTYKLHGGYQLDKFKIDLETATTLENIISRLGFRRSTKERSLLFNKKFLEVPKREYIKGYFQTEKYFEDIKAILLKQFVVKNEISSSTLKYLKEIT IQQNACSLHIRRGDYVSDKKANSVHGTCDLAYYKEAIKVMKNKFNDTHFFIFSDDIAWVKQNLKVKNTTYIDHEVIPHEDIHLMSLCKHNITANSSFSWWGAWLNQHSNKVVIAPKQWYLNKENEIASKDWIKI FutC 17: DNA (SEQ ID NO: 54) ATGGCTATCGTGGTGCGCATTATTGGCGGCCTGGGTAATCAGATGTTTCAGTATGCCTTTGCCAAAAGTCTGCAGCAGAAAGGTTATCAGGTTAAAATTGATATCACCAAATTCAAGACCTACAAACTGCATGGTGGTTATCAGCTGGATAAATTCAAAATTGATCTGGAAACCGCCACCACCCTGGAAAATATTATTAGTCGCCTGGGTTTTCGCCGTAGTACCAAAGAACGCAGTCTGCTGTTTAATAAGAAATTTCTGGAAGTGCCGAAACGTGAATATATTAAGGGTTATTTTCAGACCGAAAAGTATTTTGAAGATATTAAGGCCATCCTGCTGAAACAGTTTGTGGTGAAAAATGAAATTAGCAGCAGCACCCTGAAATATCTGAAAGAAATTACCATTCAGCAGAATGCCTGTAGTCTGCATATTCGTCGCGGTGACTATGTGAGCGATAAAAAAGCCAATAGTGTGCATGGCACCTGTGATCTGGCATATTATAAAGAAGCAATTAAGGTTATGAAGAACAAGTTTAACGACACCCATTTCTTTATTTTCAGTGATGATATCGCCTGGGTGAAACAGAATCTGAAAGTGAAAAATACCACCTATATCGATCATGAAGTTATTCCGCATGAAGATATTCATCTGATGAGCCTGTGCAAACATAATATTACCGCCAATAGCAGTTTTAGTTGGTGGGGTGCATGGCTGAATCAGCATAGCAATAAGGTGGTTATTGCCCCGAAACAGTGGTATCTGAATAAGGAAAATGAAATTGCAAGCAAAGACTGGATTAAGATTTAA FutC 18: AA (SEQ ID NO: 55) MIVTRIVGGLGNQMFQYAVGRALSAKTGQEFKLDLSEMDRYKVHALQLDQFNIKGVRAGRHEIPFRPRKSFFGKILTALKNRNRIPQVFETTPSFDPSVLQRKGSCYLSGYWQSEKYFSDCSELIRADFSLKGPMSDERQAVL SQIRDAEAPVSVHVRRGDYVNTNTANSIHGTCEPEWYRQAMRKISDRTGDPTFFVFSDDPMWARSNLPTYEKMVFVEPRADGKDAEDMHLMSSCQSHIIANSTFSWWGAWLNPRQDKRVIAPARWFRAEDRDSTDLVPAQWERL FutC 18: DNA (SEQ ID NO: 56) ATGGCTATCGTTACCCGTATTGTGGGTGGCCTGGGTAATCAGATGTTTCAGTATGCAGTTGGCCGTGCCCTGAGTGCAAAAACCGGTCAGGAATTCAAACTGGATCTGAGCGAAATGGATCGCTATAAAGTTCATGCACTGCAGCTGGATCAGTTTAATATTAAGGGTGTTCGCGCCGGCCGTCATGAAATTCCGTTTCGTCCGCGCAAAAGTTTCTTTGGCAAAATTCTGACCGCACTGAAAAATCGCAATCGTATTCCGCAGGTTTTTGAAACCACCCCGAGCTTTGATCCGAGCGTGCTGCAGCGTAAAGGTAGCTGTTATCTGAGTGGTTATTGGCAGAGCGAAAAATATTTTAGCGATTGTAGCGAACTGATTCGTGCAGATTTTAGCCTGAAAGGTCCGATGAGCGATGAACGTCAGGCAGTGCTGAGTCAGATTCGTGATGCAGAAGCACCGGTGAGCGTTCATGTTCGCCGCGGCGATTATGTTACCAATACCACCGCCAATAGCATTCATGGCACCTGTGAACCGGAATGGTATCGTCAGGCCATGCGCAAAATTAGTGATCGTACCGGTGACCCGACCTTTTTCGTTTTTAGCGATGATCCGATGTGGGCACGCAGCAATCTGCCGACCTATGAAAAAATGGTTTTTGTGGAACCGCGTGCCGATGGTAAAGATGCCGAAGATATGCATCTGATGAGCAGCTGCCAGAGTCATATTATTGCAAATAGCACCTTTAGTTGGTGGGGTGCATGGCTGAATCCGCGCCAGGATAAACGCGTGATTGCACCGGCACGCTGGTTTCGCGCAGAAGATCGCGATAGCACCGATCTGGTTCCGGCCCAGTGGGAACGTCTGTAA FutC 19: AA (SEQ ID NO: 57) MIITHINGGLGNQMFQYAAGRALALRHGEELRLDTTREFDGKVQFGFGLDHFAIAARPGAPAELPPERRRDRLRYLAWRGFRLSPRLVRENGLGYNPGFAEIGDGAYLKGYWQSERYFRDVEATIRRDFTIITPPDPVNRAILD DLAASPAVSLHIRRGDYVVDPRTNATHGTCSMDYYARAVDLIAERMAETPVVYAFSDDPAWVRDNLELPCEIRVMDHNDSARNYEDLRLMSACRHHVIANSSFSWWGAWLNPSADKIVVSPARWFADPKLVNEDIWPTSWIRLS FutC 19: DNA (SEQ ID NO: 58) ATGGCTATCATCACCCATATTAACGGCGGTCTGGGCAATCAGATGTTTCAGTATGCCGCCGGTCGTGCACTGGCCCTGCGTCATGGTGAAGAACTGCGTCTGGATACCCGCGAATTTGATGGCAAAGTGCAGTTTGGTTTTGGTCTGGATCATTTTGCCATTGCCGCACGCCCGGGTGCCCCGGCAGAATTACCGCCTGAACGTCGTCGCGATCGCCTGCGCTATCTGGCCTGGCGTGGCTTTCGCCTGAGTCCGCGTCTGGTGCGTGAAAATGGTCTGGGCTATAATCCGGGTTTTGCCGAAATTGGTGACGGCGCATATCTGAAAGGTTATTGGCAGAGTGAACGCTATTTTCGCGATGTTGAAGCAACCATTCGTCGTGATTTTACCATTATTACCCCGCCGGACCCTGTGAATCGCGCCATTCTGGATGATCTGGCCGCCAGTCCGGCAGTGAGCCTGCATATTCGTCGTGGCGATTATGTTGTGGACCCTCGTACCAATGCCACCCACGGTACCTGTAGCATGGATTATTATGCCCGCGCAGTTGATCTGATTGCAGAACGTATGGCAGAAACCCCGGTGGTGTATGCATTTTCAGATGATCCGGCCTGGGTGCGCGATAATCTGGAACTGCCGTGCGAAATTCGCGTTATGGATCATAATGATAGCGCACGCAATTATGAAGATCTGCGCCTGATGAGTGCCTGCCGTCATCATGTTATTGCCAATAGTAGCTTTAGCTGGTGGGGCGCATGGCTGAATCCGAGCGCCGATAAAATTGTGGTTAGTCCGGCCCGTTGGTTTGCCGATCCGAAACTGGTTAATGAAGATATTTGGCCGACCAGTTGGATTCGTCTGAGTTAA FutC 20: AA (SEQ ID NO: 59) MVIVRVQGGLGNQMFQYGFAKYQELSNEEVYLDITDYQTHIHHYGFELEKVFSNLTYKTIDGERLNKVRANPNMLLNRMLNKVLNIQIVRGSEFREQPAVSVSKRYTYNKDIYFNGFWANNEYVDAVKDTLKKDFTFKYILEGR NRELMDFLQGKISVGVHVRRGDYLQEKELRDVCDPDYYRKAFEIFMKRDVKTVFIIFSDDIPWVRKNFHFSKNMVFVDWNSGGEKSHVDMQMMSLCNHNIIANSTFSWWGAWLNANKDKCVVAPRYWRNNSKNESLIYPKNWMLL FutC 20: DNA (SEQ ID NO: 60) ATGGTTATCGTTCGTGTGCAGGGCGGTCTGGGTAATCAGATGTTTCAGTATGGTTTTGCAAATATCAGGAACTGAGTAATGAAGAAGTTTATCTGGATATTACCGATTATCAGACCCATATTCATCATTATGGTTTTGAACTGGAAAAGGTGTTTAGTAATCTGACCTATAAAACCATTGACGGTGAACGTCTGAATAAGGTTCGCGCAAATCCGA ATATGCTGCTGAATCGCATGCTGAATAAGGTGCTGAATATTCAGATTGTGCGTGGTAGTGAATTTCGCGAACAGCCGGCAGTGAGCGTTAGCAAACGCTATACCTATAATAAGGATATCTATTTCAACGGCTTCTGGGCCAATAATGAATATGTGGATGCATGAAAGATACCCTGAAAAAAGATTTTACCTTCAAATACATCCTGGAAGGCCGCAAT CGTGAACTGATGGATTTTCTGCAGGGCAAAATTAGTGTGGGTGTGCATGTGCGTCGCGGCGATTATCTGCAGGAAAAAGAACTGCGCGATGTTTGTGATCCGGATTATTATCGCAAAGCATTTGAAATTTTCATGAAGCGCGATGTTAAAACCGTTTTATTATTTCAGCGACGATATTCCGTGGGTGCGCAAAAATTTCATTTTAGCAAAACA TGGTGTTCGTTGATTGGAATAGCGGCGGCGAAAAAAAGCCATGTTGATATGCAGATGATGAGCCTGTGTAATCATAATATTATCGCAAATAGCACCTTCAGCTGGTGGGGTGCATGGCTGAATGCCAATAAGGATAAATGTGTGGTTGCACCGCGTTATTGGCGTAATAATAGAAAAATGAAAGCCTGATCTATCCGAAAAAATTGGATGCTGCTGTAA FutC 21:AA(SEQ ID NO:61) MAFKVVQICGGLGNQMFQYAFAKSLQKHLNTPVLLDITSFDWSNRKMQLELFPIDLPYASAKEIAIAKMQHLPKLVRDTLKCMGFDRVSQEIVFEYEPGLLKPSRLTYFYGYFQDPRYFDAISPLIKQTFTLPPPENGNNKKKEEEYHR KLALILAAKNSVFVHVRRGDYVGIGCQLGIDYQKKALEYIAKRVPNMELFVFCEDLKFTQNLDLGYPFMDMTTRDKEEEAYWDMLLMQSCKHGIIANSTYSWWAAYLINNPEKIIIGPKHWLFGHENILCKEWVKIESHFEVKSKKYNA FutC 21: DNA (SEQ ID NO: 62) ATGGCATTCAAGGTGGTGCAGATTTGTGGCGGTCTGGGCAACCAGATGTTCCAGTATGCCTTCGCCAAGAGTCTGCAGAAGCATCTGAACACCCCGGTGCTGCTGGATATTACCAGTTTTGATTGGAGCAATCGCAAGATGCAGCTGGAGCTGTTCCCTATTGATCTGCCGTATGCCAGCGCCAAAGAGATCGCCATCGCCAAAATGCAGCATCTGCCGAAACTGGTGCGCGATACTTTAAAATGCATGGGCTTTGATCGTGTGAGCCAAGAAATCGTGTTTGAGTATGAGCCGGGTCTGCTGAAACCGAGCCGTTTAACCTATTTCTACGGCTATTTCCAAGATCCGCGCTACTTCGATGCCATCAGCCCGCTGATTAAGCAGACCTTCACTTTACCGCCGCCGGAGAATGGCAACAATAAGAAAAAAGAGGAAGAATATCATCGCAAGCTGGCTTTAATTCTGGCAGCCAAAAACAGCGTGTTCGTGCATGTTCGCCGCGGTGACTATGTGGGTATCGGCTGCCAGCTGGGCATCGACTATCAGAAGAAGGCTTTAGAATATATCGCAAAACGCGTGCCGAACATGGAGCTGTTTGTGTTTTGCGAGGATTTAAAATTTACCCAGAATTTAGATTTAGGCTACCCGTTTATGGACATGACCACCCGTGATAAAGAAGAAGAAGCCTATTGGGACATGCTGCTGATGCAGAGCTGCAAGCACGGCATCATTGCCAACAGCACCTATAGCTGGTGGGCCGCATATTTAATTAACAACCCGGAAAAGATCATCATCGGCCCGAAGCATTGGCTGTTCGGCCATGAGAACATTTTATGCAAGGAATGGGTTAAAATTGAGAGCCATTTCGAAGTGAAAAGCAAAAAGTATAACGCC Oc Pyruvate Kinase: AA (SEQ ID NO: 63) MSKSHSEAGSAFIQTQQLHAAMADTFLEHMCRLDIDSAPITARNTGIICTIGPASRSVET LKEMIKSGMNVARMNFSHGTHEYHAETIKNVRTATESFASDPILYRPVAVALDTKGPEIRTGLIKGSGTAEVELKKGATLKITLDNAYMEKCDENILWLDYKNICKVVDVGSKVYVD DGLISLQVKQKGPDFLVTEVENGGFLGSKKGVNLPGAAVDLPAVSEKDIQDLKFGVEQDVDMVFASFIRKAADVHEVRKILGEKGKNIKIISKIENHEGVRRFDEILEASDGIMVARG DLGIEIPAEKVFLAQKMIIGRCNRAGKPVICATQMLESMIKKPRPTRAEGSDVANAVLDGADCIMLSGETAKGDYPLEAVRMQHLIAREAEAAMFHRKLFEELARSSSHSTDLMEAMA MGSVEASYKCLAAALIVLTESGRSAHQVARYRPRAPIIAVTRNHQTARQAHLYRGIFPVVCKDPVQEAWAEDVDLRVNLAMNVGKARGFFKKGDVVIVLTGWRPGSGFTNTMRVVPVP Oc creatine kinase: AA (SEQ ID NO: 64) MPFGNTHNKYKLNYKSEEEYPDLSKHNNHMAKVLTPDLYKKLRDKETPSGFTLDDVIQTGVDNPGHPFIMTVGCVAGDEESYTVFKDLFDPIIQDRHGGFKPTDKHKTDLNHENLKGGDDDLDPHYVLSSRVRTGRSIKGYTLPPHCSRGERRAVEKLSVEALNSLTGEFKGKYYPLKSMTEQEQQQLIDD HFLFDKPVSPLLLASGMARDWPDARGIWHNDNKSFLVWVNEEDHLRVISMEKGGNMKEVFRRFCVGLQKIEEIFKKAGHPFMWNEHLGYVLTCPSNLGTGLRGGVHVKLAHLSKHPKFEEILTRLRLQKRGTGGVDTAAVGSVFDISNADRLGSSEVEQVQLVVDGVKLMVEMEKKLEKGQSIDDMIPAQK Gs AckA:AA (SEQ ID NO: 65) MAKVLAVNAGSSSLKFQLFDMPAETVLTKGIVERIGFDDAIFTIVVNGEKQREVTSIPNHAVAVKLLLDKLIRYGIIRSFDEIDGIGHRVVHGGEKFSDSVLITDEVIKQIEEVSELAPLHNPANLVGIRAFQEVLPNVPAVVFDTAFHQTMPEQSFLYSLPYEYYTKFGIRKYGFHGTSHKYVTQRAAELLGRPIE QLRLISCHLGNGASIAAVEGGKSIDTSMGFTPLAGVAMGTRSGNIDPALIPYIMEKTGMTVNEVIEVLNKKSGMLGISGISSDLRDLEKAAAEGNERAELALEVFANRIHKYIGSYAARMCGVDAIIFTAGIGENSEVVRAKVLRGLEFMGVYWDPILNKVRGKEAFISYPHSPVKVLVIPTNEEVMIARDVMRLANL Gs AckA: DNA (SEQ ID NO: 66) MaeB:AA (SEQ ID NO: 67) MDDQLKQSALDFHEFPVPGKIQVSPTKPLATQRDLALAYSPGVAAPCLEIEKDPLKAYKYTARGNLVAVISNGTAVLGLGNIGALAGKPVMEGKGVLFKKFAGIDVFDIEVDELDPDKFIEVVAALEPTFGGINLEDIKAPECFYIEQKLRERMNIPVFHDDQHGTAIISTAAILNGLRVVEKNISDVR MVVSGAGAAAIACMNLLVALGLQKHNIVVCDSKGVIYQGREPNMAETKAAYAVVDDGKRTLDDVIEGADIFLGCSGPKVLTQEMVKKMARAPMILALANPEPEILPPLAKEVRPDAIICTGRSDYPNQVNNVLCFPFIFRGALDVGATAINEEMKLAAVRAIAELAHAEQSEVVASAYGDQDLSFGPEYI IPKPFDPRLIVKIAPAVAKAAMESGVATRPIADDFDVYIDKLTEFVYKTNLFMKPIFSQARKAPKRVVLPEGEEARVLHATQELVTLGLAKPILIGRPNVIEMRIQKLGLQIKAGVDFEIVNNESDPRFKEYWTEYFQIMKRRGVTQEQAQRALISNPTVIGAIMVQRGEADAMICGTVGDYHEHFSVVKN VFGYRDGVHTAGAMNALLLPSGNTFIADTYVNDEPDAEELAEITLMAAETVRRFGIEPRVALLSHSNFGSSDCPSSSKMRQALELVRERAPELMIDGEMHGDAALVEAIRNDRMPDSSLKGSANILVMPNMEAARISYNLLRVSSSEGVTVGPVLMGVAKPVHVLTPIASVRRIVNMVALAVVEAQTQPL MaeB: DNA (SEQ ID NO: 68) FDH: AA (SEQ ID NO: 69) MKIVLVLYDAGKHAADEEKLYGCTENKLGIANWLKDQGHELITTSDKEGGNSVLDQHIPDADIIITTPFHPAYITKERIDKAKKLKLVVVAGVGSDHIDLDYINQTGKKISVLEVTGSNVVSVAEHVVMTMLVLVRNFVPAHEQIINHDWEVAAIAKDAYDIEGKTIATIGAGRIGYRVLERLVPFNPKELLYYQHQALPKDAEEKVGARRVENIEELVAQADIVTVNAPLHAGTKGLINKELLSKFKKGAWLVNTARGAICVAEDVAAALESGQLRGYGGDVWFPQPAPKDHPWRDMRNKYGAGNAMTPHYSGTTLDAQTRYAQGTKNILESFFTGKFDYRPQDIILLNGEYVTKAYGKHDKK FDH: DNA (SEQ ID NO: 70) PTDH:AA (SEQ ID NO: 71) MLPKLVITHRVHEEILQLLAPHCELITNQTDSTLTREEILRRCRDAQAMMAFMPDRVDADFLQACPELRVIGCALKGFDNFDVDACTARGVWLTFVPDLLTVPTAELAIGLAVGLGRHLRAADAFVRSGKFRGWQPRFYGTGLDNATVGFLGMGAIGLAMADRLQGWG ATLQYHARKALDTQTEQRLGLRQVACSELFASSDFILLALPLNADTLHLVNAELLALVRPGALLVNPCRGSVVDEAAVLAALERGQLGGYAADVFEMEDWARADRPQQIDPALLAHPNTLFTPHIGSAVRAVRLEIERCAAQNILQALAGERPINAVNRLPKANPAAD PTDH:DNA (SEQ ID NO:72) GDH:AA (SEQ ID NO: 73) MYPDLKGKVVAITGAASGLGKAMAIRFGKEQAKVVINYYSNKQDPNEVKEEVIKAGGEAVVVQGDVTKEEDVKNIVQTAIKEFGTLDIMINNAGLENPVPSHEMPLKDWDKVIGTNLTGAFLGSREAIKY FVENDIKGNVINMSSVHEVIPWPLFVHYAASKGGIKLMTETLALEYAPKGIRVNNIGPGAINTPINAEKFADPKQKADVESMIPMGYIGEPEEIAVAAWLASKEASYVTGITLFADGGMTQYPSFQAGRG GDH:DNA (SEQ ID NO:74) ATGTATCCTGATCTCAAGGGAAAAGTTGTAGCCATTACAGGTGCAGCCAGTGGACTTGGAAAAGCTATGGCGATTAGATTCGGGAAAGAACAAGCAAAGGTCGTCATCAACTATTATTCTAATAAGCAGGACCCCAACGAAGTAAAAGAAGAAGTAATCAAAGCAGGAGGTGAAGCCGTTGTGGTTCAGGGAGATGTTACCAAAGAAGAGGATGTCAAGAATATAGTTCAGACCGCGATTAAGGAATTTGGAACGTTAGATATTATGATTAATAATGCAGGTTTGGAAAACCCCGTACCTTCTCACGAAATGCCATTGAAGGATTGGGATAAGGTAATAGGAACGAATCTAACCGGAGCGTTCTTAGGCAGCAGAGAAGCCATCAAGTATTTTGTCGAGAACGATATAAAAGGAAATGTTATTAACATGTCATCCGTCCATGAGGTTATTCCATGGCCACTTTTCGTTCATTACGCTGCTAGTAAAGGTGGTATCAAATTAATGACAGAAACTTTGGCTCTGGAATATGCACCAAAAGGTATTAGAGTTAACAACATTGGACCAGGCGCTATTAATACTCCCATAAATGCTGAGAAATTTGCCGACCCAAAACAAAAAGCTGATGTTGAATCAATGATACCCATGGGATATATTGGAGAGCCTGAGGAAATAGCCGCTGTTGCTGCATGGCTTGCTTCCAAGGAAGCTTCTTATGTGACTGGGATCACTCTTTTCGCAGACGGAGGAATGACGCAATATCCATCCTTTCAGGCCGGGCGGGGCTAA Arabidopsis thaliana, At GMD M2: AA (SEQ ID NO: 75) MASENNGSRSDSESITAPKADSTVVEPRKIALITGITGQDGSYLTEFLLGKGYEVHGLIRRSSNFNTQRINHIYIDPANVNKALMKLHYADLTDASSLRRWIDVIKPDEVYNLAAQSHVAVSFEIPDYTADVVATGALRLLEAVRSHTIDSGRTVKYYQAGSSEMFGSTPPPQSETTPFHPRSPYAASKCAAHWYTVNYREAYGLFACNGILFNHESPRRGENFVTRKITRALGRIKVGLQTKLFLGNLQASRDWGFAGDYVEAMWLMLQQEKPDDYVVATEEGHTVEEFLDVSFGYLGLNWKDYVEIDQRYFRPAEVDNLQGDASKAKEVLGWKPQVGFEKLVKMMVDEDLELAKREKVLVDAGYMDAKQQP Arabidopsis thaliana, At GMD M2:DNA (SEQ ID NO: 76) Arabidopsis thaliana, At GMD M3:AA (SEQ ID NO: 77) MASENNGSRSDSESITAPKADSTVVEPRKIALITGITGQDGSYLTEFLLGKGYEVHGLIRRSSNFNTQRINHIYIDPHNVNKALMKLHYADLTDASSLRRWIDVIKPDEVYNLAAQSHVAVSFEIPDYTADVVATGALRLLEAVRSHTIDSGRTVKYYQAGSSEMFGSTPPPQSETTPFHPRSPYA ASKCAAHWYTVNYREAYGLFACNGILFNHESPRRGENFVTRAITRALGRIKVGLQTKLFLGNLQASRDWGFAGDYVEAMWLMLQQEKPDDYVVATEEGHTVEEFLDVSFGYLGLNWKDYVEIDQRYFRPAEVDNLQGDASKAKEVLGWKPQVGFEKLVKMMVDEDLELAKREKVLVDAGYMDAKQQP Arabidopsis thaliana, At GMD M3: DNA (SEQ ID NO: 78) Arabidopsis thaliana, At GMD M4:AA (SEQ ID NO: 79) MASENNGSRSDSESITAPKADSTVVEPRKIALITGITGQDGSYLTEFLLGKGYEVHGLIRRSSNFNTQRINHIYIDPHNVNKALMKLHYADLTDASSLRRWIDVIKPDEVYNLAAQSHVAVSFEIPDYTADVVATGALRLLEAVRSHTIDSGRTVKYYQAGSSEMFGSTPPPQSETTPFHPRSPYA ASKCAAHWYTVNYREAYGLFACNGILFNHESPRRGENFVTRKITAALGRIKVGLQTKLFLGNLQASRDWGFAGDYVEAMWLMLQQEKPDDYVVATEEGHTVEEFLDVSFGYLGLNWKDYVEIDQRYFRPAEVDNLQGDASKAKEVLGWKPQVGFEKLVKMMVDEDLELAKREKVLVDAGYMDAKQQP Arabidopsis thaliana, At GMD M4: DNA (SEQ ID NO: 80) Human, Hs GMD M2:AA (SEQ ID NO: 81) MRNVALITGITGQDGSYLAEFLLEKGYEVHGIVRRSSSFNTGRIEHLYKNPQAAIEGNMKLHYGDLTDSTCLVKIINEVKPTEIYNLGAQSHVKISFDLAEYTADVDGVGTLRLLDAVKTCGLINSVKFYQASTSELYGKVQEIPQKETTPFYPRSPYGAAKLYAYWIVVNFREAYNLFAVNGILFNHESPRRGANFVTRKISRSVAKIYLGQLECFSLGNLDAKRDWGHAKDYVEAMWLMLQNDEPEDFVIATGEVHSVREFVEKSFLHIGKTIVWEGKNENEVGRCKETGKVHVTVDLKYYRPTEVDFLQGDCTKAKQKLNWKPRVAFDELVREMVHADVELMRTNPNA Human, Hs GMD M2:DNA (SEQ ID NO: 82) Human, Hs GMD M3:AA (SEQ ID NO: 83) MRNVALITGITGQDGSYLAEFLLEKGYEVHGIVRRSSSFNTGRIEHLYKNPQAHIEGNMKLHYGDLTDSTCLVKIINEVKPTEIYNLGAQSHVKISFDLAEYTADVDGVGTLRLLDAVKTCGLINSVKFYQASTSELYGKVQEIPQKETTPFYPRSPYGAAKLYAYWIVVNFREA YNLFAVNGILFNHESPRRGANFVTRAISRSVAKIYLGQLECFSLGNLDAKRDWGHAKDYVEAMWLMLQNDEPEDFVIATGEVHSVREFVEKSFLHIGKTIVWEGKNENEVGRCKETGKVHVTVDLKYYRPTEVDFLQGDCTKAKQKLNWKPRVAFDELVREMVHADVELMRTNPNA Human, Hs GMD M3: DNA (SEQ ID NO: 84) Hs GMD M4:AA MRNVALITGITGQDGSYLAEFLLEKGYEVHGIVRRSSSFNTGRIEHLYKNPQAHIEGNMKLHYGDLTDSTCLVKIINEVKPTEIYNLGAQSHVKISFDLAEYTADVDGVGTLRLLDAVKTCGLINSVKFYQASTSELYGKVQEIPQKETTPFYPRSPYGAAKLYAYWIVVNFREAYNLFAVNGILFNHESPRRGANFVTRKISASVAKIYLGQLECFSLGNLDAKRDWGHAKDYVEAMWLMLQNDEPEDFVIATGEVHSVREFVEKSFLHIGKTIVWEGKNENEVGRCKETGKVHVTVDLKYYRPTEVDFLQGDCTKAKQKLNWKPRVAFDELVREMVHADVELMRTNPNA Hs GMD M4:DNA ASR 1:AA (SEQ ID NO: 87) MAFKVVQICGGLGNQMFQYAFAKSLQKHLNIPVLLDVTSFDWSNRKLQLELFPIDLPYASAKEIAMAKMQHLPKLVRDALKRMGFDRVSQEIVFEYEPKLLKPNRLTYFHGYFQDPRYFDGISPLIKQTFTLPPPPPENGNNKKKEEEYQ RKLSLILAAKNSVFVHIRRGDYVGIGCQLGIDYQKKAVEYMAKRVPNMELFVFCEDLEFTQNLDLGYPFMDMTTRDKEEEAYWDMMLMQSCKHGIIANSTYSWWAAYLINNPEKIIIGPKHWLFGHENILCKDWVKIESHFEVKSEKYNA ASR 1: DNA (SEQ ID NO: 88) ATGGCGTTTAAAGTCGTCCAGATTTGTGGAGGCTTAGGTAATCAAATGTTTCAGTATGCTTTTGCTAAGTCACTGCAAAAACACCTTAACATTCCTGTGCTTCTGGACGTTACCTCGTTTGATTGGTCGAATCGCAAATTACAGCTGGAGTTGTTTCCAATTGACTTGCCGTATGCCTCAGCCAAAGAAATCGCAATGGCGAAAATGCAGCATCTTCCGAAACTGGTGCGCGATGCGCTGAAACGCATGGGATTCGATCGCGTGTCCCAGGAAATCGTCTTTGAATATGAACCAAAGCTCCTGAAACCAAACCGCTTGACCTACTTTCATGGCTACTTTCAGGACCCCCGCTATTTCGACGGCATCTCTCCCTTAATTAAACAGACCTTCACACTCCCTCCTCCGCCGCCTGAAAACGGGAATAATAAAAAGAAAGAGGAGGAATATCAACGCAAACTGAGTCTGATTCTGGCGGCGAAAAACTCTGTTTTCGTCCACATCCGTCGCGGCGATTACGTCGGTATTGGTTGCCAGTTGGGCATTGATTACCAGAAAAAAGCGGTGGAATATATGGCGAAACGAGTGCCGAATATGGAACTATTTGTGTTTTGTGAGGATCTGGAGTTCACGCAGAACCTAGACTTGGGGTATCCATTTATGGATATGACCACGCGGGACAAGGAAGAGGAAGCCTACTGGGATATGATGCTGATGCAGTCATGCAAGCACGGTATTATCGCCAATAGCACCTACTCGTGGTGGGCCGCCTACTTAATTAACAATCCTGAGAAGATTATTATTGGTCCGAAACACTGGTTATTTGGCCACGAAAACATCCTCTGCAAGGATTGGGTTAAAATTGAATCGCACTTTGAAGTCAAATCTGAAAAATACAACGCA ASR 2: AA (SEQ ID NO: 89) MIIIRMSGGLGNQMFQYALYLKLKAMGKEVKIDDITEYEGDNARPIMLDVFGIDYDRATKEEVTELTDGSMDFLSRIRRKLFGRKSKEYREKSCNFDPQVLEMDPAYLEGYFQSEKYFQDVREQVRKAFRFRGIESGSIPLSEKTR ELQKQIEDSESVSIHIRRGDYLENGHGEVYGGICTDAYYKKAIEYMKEKFPDAKFYIFSNDTEWAKQHFKGENFVVVEGSTENTGYLDMFLMSKCRHHIIANSSFSWWGAWLNENPEKIVIAPSKWLNNRECKDIYTERMIRINPEV ASR 2: DNA (SEQ ID NO: 90) ATGATTATCATTCGCATGAGCGGGGGTCTGGGCAATCAGATGTTCCAGTATGCCCTCTATCTGAAGCTGAAAGCGATGGGCAAGGAAGTAAAAATCGATGATATAACCGAATACGAGGGCGATAATGCTCGCCCGATAATGCTGGACGTGTTTGGAATCGATTATGATCGTGCGACCAAAGAAGAAGTTACCGAACTCACCGACGGTTCTATGGACTTTCTGTCGCGCATCCGCCGTAAACTTTTCGGCCGCAAATCGAAAGAATACCGTGAAAAAAGCTGCAATTTTGACCCGCAAGTTTTGGAGATGGACCCGGCGTACCTGGAGGGCTATTTCCAGAGCGAAAAATATTTTCAAGATGTGCGCGAACAGGTTCGAAAAGCGTTCCGATTTCGTGGTATTGAATCAGGGTCCATTCCGCTGTCAGAAAAAACCCGCGAATTGCAGAAACAGATCGAAGATAGCGAGTCCGTTAGCATTCATATCCGTCGTGGTGACTATCTGGAGAACGGCCACGGCGAAGTGTACGGCGGAATCTGCACCGATGCCTATTACAAAAAAGCCATCGAATACATGAAGGAGAAATTCCCTGATGCCAAATTTTACATTTTTAGCAATGATACGGAGTGGGCAAAACAACATTTCAAGGGAGAGAACTTTGTGGTGGTTGAGGGCTCCACTGAAAATACTGGTTATCTTGATATGTTCCTGATGAGCAAATGTCGCCACCACATCATTGCGAATAGTTCGTTTAGCTGGTGGGGGGCGTGGTTGAACGAAAACCCGGAAAAAATCGTGATTGCCCCGAGCAAATGGCTGAATAACCGTGAATGTAAAGACATCTATACCGAACGCATGATCCGTATCAACCCCGAGGTG ASR 3: AA (SEQ ID No. 91) MIIIRIMGGLGNQMFQYALYRKLKSMGKEVKLDISWYDDHNQTHRSFELDVFGIDYDVASKEEISKFSNRSANFLSRIRRKLFGRKNKIYKEEDFNYDPEILELDDVYLEGYWQSEKYFEDIREQLRKEFTFPEELNEKNRELLE QMENENSVSIHIRRGDYLNNENADVYGGICTDDYYKKAIEYIRERIPDPKFYIFSDDIEWAKQQFKGDDFTIVDWNNGKDSYYDMYLMSKCKHNIIANSTFSWWGAWLNQNPEKIVISPKKWLNNHETSDIVCESWIRIDGQGEIR ASR 3: DNA (SEQ ID NO: 92) ATGATCATCATTCGCATTATGGGCGGCCTGGGTAATCAGATGTTTCAATACGCGCTGTATCGCAAACTGAAATCGATGGGAAAAGAAGTGAAACTGGACATCAGTTGGTACGATGATCATAATCAAACTCACCGCAGCTTTGAACTCGACGTCTTTGGTATTGATTATGATGTGGCATCCAAAGAGGAAATTAGCAAGTTTTCCAACCGCTCCGCGAATTTCCTGAGTAGAATTAGGCGAAAACTGTTTGGCCGAAAAAACAAAATTTATAAAGAGGAGGACTTTAACTACGATCCAGAAATCCTTGAATTAGATGATGTTTATCTGGAGGGCTATTGGCAAAGTGAGAAGTATTTCGAAGATATTCGCGAACAACTGCGTAAAGAGTTTACCTTTCCCGAAGAGCTGAACGAAAAGAATCGTGAGCTGCTGGAACAAATGGAAAACGAAAACTCGGTATCGATTCACATTCGTCGCGGAGATTATCTGAACAACGAGAACGCAGATGTATATGGTGGCATCTGCACAGATGATTACTATAAAAAAGCTATCGAATATATTCGTGAGCGCATTCCCGATCCAAAGTTTTATATATTCTCAGATGACATCGAATGGGCAAAACAACAGTTTAAAGGTGATGACTTCACCATCGTAGATTGGAACAATGGCAAAGACAGCTATTATGATATGTATCTGATGTCAAAGTGTAAACACAACATCATTGCTAATTCCACCTTTTCCTGGTGGGGCGCCTGGCTGAATCAAAATCCCGAGAAAATCGTGATTTCCCCTAAGAAATGGCTTAACAACCATGAAACCTCAGACATAGTATGCGAAAGTTGGATTAGGATTGACGGTCAAGGTGAAATTCGC ASR 4: AA (SEQ ID NO: ٩٣) MIIVRLTGGLGNQMFQYAMGRRLAEKHNTELKLDISGFENYKLRKYSLNHFNIQENFATPEEISRLTSVKQGRIEKLLRRILLRKRPKKPNTYIREKHFHFDPEILNLPDNVYLDGYWQSEKYFKDIEDIIRREFTIKNPQTGKNK EIAEQIQSCNSVSLHVRRGDYVTNPTTNQVHGVCGLDYYQRCVDYIAKKVENPHFFVFSDDPEWVKENLKIDYPTTFVDHNGADKDYEDLRLMSQCKHHIIANSTFSWWGAWLNSNPDKIVIAPKKWFNTSDMDTKDLIPENWIKL ASR 4: DNA (SEQ ID NO: 94) ATGATTATTGTCCGGCTTACGGGCGGCTTAGGCAACCAAATGTTTCAGTACGCAATGGGGCGCCGCTTAGCTGAAAAACATAATACCGAGCTGAAATTAGACATCAGCGGGTTTGAAAACTATAAACTGCGTAAATACAGCTTGAATCACTTTAATATTCAGGAAAATTTTGCCACACCGGAAGAGATTTCGCGGCTGACATCAGTTAAACAGGGCCGTATTGAAAAGTTGTTGCGCAGGATTCTGAGGAAGCGCCCAAAAAAACCGAATACGTATATCCGCGAGAAACACTTCCACTTTGATCCTGAAATTCTGAACCTCCCGGACAACGTTTACTTGGACGGTTACTGGCAGAGTGAGAAATACTTTAAGGACATTGAGGACATCATTCGCCGTGAGTTTACCATAAAAAATCCGCAGACCGGCAAAAACAAAGAGATCGCGGAACAGATCCAGAGTTGCAATAGTGTCTCACTGCATGTTCGTCGCGGTGATTACGTTACGAACCCCACTACCAACCAAGTCCACGGCGTCTGTGGGCTAGATTACTATCAACGTTGCGTGGATTATATCGCAAAAAAGGTTGAAAACCCACACTTCTTTGTTTTTAGCGATGATCCCGAGTGGGTGAAAGAAAACCTTAAAATCGATTATCCTACTACCTTCGTGGACCACAACGGTGCGGATAAAGACTATGAAGATTTACGTCTGATGTCACAATGCAAACATCATATCATTGCAAACTCTACCTTTAGTTGGTGGGGTGCCTGGCTCAATTCTAACCCTGACAAAATTGTGATTGCGCCGAAGAAGTGGTTCAACACTAGCGATATGGATACCAAAGATTTGATTCCAGAGAATTGGATCAAACTA ASR 5: AA (SEQ ID NO: 95) MIVVKLIGGLGNQMFQYAAAKALALEKNQKLRLDVSAFESYKLHNYGLNHFNITAKIYKKENKWLRKIKSFFKKNTYYKEQDFGYNPDLFDLKADNIFLEGYFQSEKYFLKYEKEIRKDFEIISPLKKQTKEMIEQ IQSVNSVSIHIRRGDYLTNPIHNTSKEEYYKKAMEFIESKIENPVFFVFSDDMDWVKENFKTNHETVFVDFNDASTNFEDLKLMSSCKHNIIANSSFSWWGAWLNKNPNKIVIAPKQWFNDDSINTSDIIPESWIKI ASR 5: DNA (SEQ ID NO: 96) ATGATCGTTGTAAAACTGATTGGTGGTCTTGGCAACCAGATGTTCCAGTACGCGGCGGCGAAAGCTCTGGCGCTCGAAAAAAACCAGAAGCTGCGTCTTGATGTCAGTGCTTTCGAATCATACAAACTGCACAATTATGGACTGAATCATTTCAACATAACCGCCAAAATCTATAAAAAAGAAAATAAGTGGTTACGCAAAATCAAAAGTTTCTTCAAAAAGAATACCTACTACAAAGAACAAGACTTTGGCTATAACCCGGATCTGTTTGATTTGAAAGCGGACAATATTTTTCTGGAGGGTTATTTCCAAAGCGAGAAATATTTTCTAAAGTACGAAAAAGAAATACGTAAAGATTTCGAGATCATCTCACCATTAAAAAAACAGACCAAAGAAATGATTGAACAAATTCAGTCTGTGAATAGTGTCTCGATACATATAAGGCGCGGTGATTATCTGACCAATCCGATTCATAATACGTCAAAAGAAGAATACTATAAGAAGGCAATGGAGTTTATTGAATCCAAAATTGAAAACCCGGTATTCTTCGTGTTTAGTGATGACATGGACTGGGTCAAAGAAAACTTTAAAACGAACCATGAGACTGTGTTCGTAGATTTCAATGATGCCAGCACCAACTTTGAGGACCTAAAGCTGATGTCCTCATGTAAACACAATATTATTGCGAACAGCTCTTTTAGCTGGTGGGGTGCTTGGCTGAATAAAAATCCGAACAAAATTGTTATCGCGCCAAAACAGTGGTTTAACGACGATAGCATTAATACTTCAGACATCATCCCGGAGTCCTGGATTAAAATA ASR 6: AA (Sequence number 97) MAFKVVQICGGLGNQMFQYAFAKSLQKHLNIPVLLDVTSFDSSNRKLQLELFPIDLPYASAKEIAMAKMQHLPKLVRDALKRMGFDRVSQEIVFEYEPKLLKPNRLTYFHGYFQDPRYFDGISPLIKQTFTLPPPPPENGNNKKKEEEYQ RKLSLILAAKNSVFVHIRRGDYVGIGCQLGIDYQKKAVEYMAKRVPNMELFVFCEDLEFTQNLDLGYPFMDMTTRDKEEEAYWDMMLMQSCKHGIIANSTYSWWAAYLINNPEKIIIGPKHWLFGHENILCKDWVKIESHFEVKSEKYNA ASR 6: DNA (SEQ ID NO: 98) ATGGCGTTTAAAGTGGTTCAGATTTGCGGCGGCTTAGGTAATCAGATGTTCCAGTATGCTTTTGCGAAAAGCCTGCAAAAACATCTGAATATTCCTGTCCTTTTAGACGTCACGAGCTTTGACTCCTCTAATAGAAAACTCCAATTAGAACTGTTCCCAATTGATCTGCCGTATGCAAGTGCAAAAGAGATTGCGATGGCAAAAATGCAGCACCTCCCAAAACTGGTTCGAGATGCCTTAAAGCGAATGGGATTCGACCGCGTCAGCCAGGAGATTGTTTTTGAATACGAACCTAAACTTCTTAAGCCAAACCGCCTGACGTACTTCCACGGTTACTTTCAAGATCCGCGCTATTTCGACGGAATCAGTCCGCTGATCAAGCAGACGTTCACCTTGCCGCCGCCGCCCCCTGAAAACGGTAATAATAAGAAAAAAGAAGAGGAATATCAGCGGAAGCTGAGCTTGATCCTGGCAGCCAAAAACAGTGTCTTTGTGCACATTCGTCGCGGCGACTATGTGGGCATTGGTTGTCAATTGGGGATTGATTACCAGAAAAAAGCGGTCGAGTACATGGCGAAACGAGTGCCCAATATGGAGCTGTTTGTTTTCTGCGAGGACTTAGAATTTACCCAGAATTTGGATCTGGGCTATCCGTTTATGGACATGACGACACGCGATAAAGAAGAGGAAGCCTACTGGGATATGATGCTGATGCAGAGCTGCAAGCACGGTATTATCGCTAACTCAACATATTCCTGGTGGGCCGCATATCTGATTAATAACCCCGAAAAGATTATCATCGGACCAAAACACTGGCTCTTCGGTCACGAAAATATCCTGTGCAAAGATTGGGTAAAGATTGAAAGCCACTTTGAAGTGAAAAGCGAAAAATATAACGCC ASR 7: AA (SEQ ID NO: 99) MIIIRMSGGLGNQMFQYALYLKLKSMGKEVKIDDITAYEGDNARPIMLDVFGIDYDRATKEEITEMTDSSMDFLSRIRRKLFGRKSKEYREKDFNFDPQVLEMDPAYLEGYFQSEKYFQDVREQVRKAFRFRKGSVPKELSEQTKELQKQIENS NSVSIHIRRGDYLENSHGEIYGGICTDAYYKKAIEYMKEKFPDAKFYIFSNDTEWAKQHFKGENFVIVEGSTENTGYLDMYLMSKCKHHIIANSSFSWWGAWLNDNPEKIVIAPSKWLNNRECKDIYTDRMIRIDAKGEVRSDDYGVRTNSTVK ASR 7: DNA (SEQ ID NO: 100) ATGATTATCATCCGCATGAGCGGCGGACTGGGCAACCAAATGTTCCAGTATGCCTTGTATCTGAAACTGAAAAGTATGGGTAAAGAAGTAAAATCGATGATATAACAGCCTATGAAGGGGATAACGCCCGCCCGATCATGCTGGACGTTTTCGGCATCGATTATGACCGTGCTACGAAAGAGGAGATTACCGAAATGACCGATTCCTCGATGGATTTCTGTCACGCATT CGTCGCAAACTGTTTGGACGTAAAAGTAAAGAATATCGCGAAAAGATTTCAATTTCGATCCGCAGGTCCTGGAGATGGACCCGGCGTACTTGGAAGGCTACTTCCAGTCCGAGAAATACTTTCAGGATTGCGCGGAACAGGTCCCGCAAGGCGTTCCGGTTCCCGCAAGGGAAGCGTACCGAAAGAATTGTCCGAACAGACCAAGGAACTGCAAAAACAGATTGAAACTCG AACTCAGTGTCAATTCATATCCGTGCGGCGACTATCTGGAAACTCACACGGTGAGATTTATGGGGGATTTGCACCGATGCTTACTATAAAAAAGCGATTGAATACATGAAAGAAAATTCCCGGATGCCAAATTCTATATTTCAGCAACGACACTGAATGGGCCAAGCAGCATTTTAAAGGCGAAAACTTTGTCATCGTTGAGGGCTCAACTGAAAATACCGGGTAC TTAGACATGTATCTGATGTCCAAATGTAAACACCACATTATTGCAAACTCTAGCTTTAGCTGGTGGGGTGCCTGGCTGAACGATAACCCGGAAAAATTGTAATCGCCCCGTCAAAATGGTTAAACAATCGCGAGTGCAAGGACATTTATACTGACCGCATGATTCGTATAGATGCAAAAGGCGAAGTCCGTAGCGATGATTATGGGTTCGTACGAACAGCACGGTGAAA ASR 8: AA MIIIRIMGGLGNQMFQYALYRKLKSMGKEVKLDISWYDDHNTHRSFELDVFGIEYDVASKKEISKFSNRSSNFLSRIRRKLFGKKNKIYQEEDFNYDPEILEMDDVYLEGYWQSEKYFEDIREQLRKEFTFPKEMNKQNKELLEQMENENS VSIHIRRGDYLNKENASIYGGICTDDYYKKAIEYIREKVSNPKFYIFSDDIEWAKQHFKGDDMTIVDWNNGKDSYYDMYLMSSCKHNIIANSTFSWWGAWLNQNPEKIVIAPKKWLNNHETSDIVCDNWIRIDGNGEIRSEEYGVRTGSTVK ASR 8: DNA (SEQ ID NO: 102) ATGATTATTATCCGCATTATGGGGGGCTTGGGCAACCAGATGTTCCAATATGCTCTGTATCGCAAACTAAAGTCCAATGGGTAAAGAGGTTAAATTGGATATTTCGTGGTATGACGATCATAATACCCATCGCTCATTTGAATTAGATGTTTTTGGCATTGAATATGACGTCGCATCCAAAAAAGAAATCTCGAAATTCTCTAACCGCTCAAGCAACTTTTTGTCTCG AATCCGCCGGAAGTTGTTCGGAAAAAAGAATAAAATCTATCAGGAGGAGGACTTCAACTATGACCCGGAGATCCTGGAAATGGATGATGTGTACCTGGAAGGGTACTGGCAGTCGAAAAATATTTTGAGGATATTCTGAACAGTTACGTAAAGAATTTACCTTCCCGAAAGAGATGAACAAACAGAACAAGGAACTGCTGGAACAGATGGAAACGAAAATTCCG TGTCCATCCATATTCGTCGTGGAGATTATTTAAACAAAGAAAACGCAAGCATTTATGGAGGAATCTGCACCGATGATTATTATAAAAAGGCAATTGAGTATATTCGCGAGAAAGTTAGTAACCCGAAGTTCTATATTTTTCGGATGATATAGAGTGGGCAAAACAGCATTTCAAAGGGGACGATATGACCATCGTGGACTGGAATAACGGCAAAGATTCCTATTAC GATATGTACCTGATGCAGTTGTTAAACACAACATATTGCCAACTCCACGTTTTCATGGTGGGGCCTGGCTGAACCAAAACCCGGAAAAGATTGTGATCGCTCCGAAAAATGGCTTAACAATCATGAAACTAGCGATATTGTTTGCGATAACTGGATTCGTATCGATGGTAATGGAGAAATTCGGTCGGAGGAATATGGGTCCGCACCGGAAGCACCGTGAAA ASR 9: AA MIIVRLTGGLGNQMFQYAMGRRLAEKHNTELKLDISAFENYKLRKYSLHHFNIQENFATPEEISRLTSVKQNKIEKLLHKILRKKPKKSNTYIKEKHFHFDPNILNLPDNVYLDGYWQSEKYFKDIEDIIRKEFTIKYPQTGKNK EIAEKIQSCNSVSIHIRRGDYVTNPTTNQVHGVCGLDYYQRCIDYIAKKVENPHFFVFSDDPEWVKENLKIQYPTTYVDHNNTDKDYEDLRLMSQCKHHIIANSTFSWWGAWLNSNPDKIVIAPKKWFNTSDYNTKDLIPENWIKL ASR 9: DNA (SEQ ID NO: 104) ATGATTATTGTCCGACTCACCGGCGGTCTGGGCAATCAAATGTTCCAATATGCAATGGGTCGCCGTTTAGCGGAAAAACACAATACAGAACTCAAACTGGACATTAGCGCGTTCGAGAATTATAAACTGCGAAAGTATAGTCTGCACCATTTTAATATCCAAGAAAATTTTGCAACCCCAGAAGAGATTAGTCGTTTAACGAGCGTAAAACAAAACAAGATCGAAAAACTGTTGCACAAAATCCTTCGCAAGAAACCGAAAAAATCAAACACCTACATTAAGGAGAAACATTTTCATTTTGATCCGAATATACTGAATCTGCCGGATAATGTATACTTAGATGGATACTGGCAAAGCGAAAAATACTTCAAGGATATTGAAGATATTATTCGTAAAGAATTTACAATCAAATATCCACAGACGGGTAAAAACAAGGAAATTGCGGAGAAAATTCAGTCTTGCAACTCTGTAAGTATACACATTCGTCGCGGTGATTATGTAACCAACCCGACCACTAACCAGGTTCATGGTGTTTGTGGCCTGGATTATTATCAGAGGTGCATCGACTATATTGCGAAAAAGGTGGAGAACCCGCACTTTTTTGTTTTCTCTGATGATCCTGAATGGGTAAAAGAAAATCTTAAAATCCAGTATCCAACCACGTATGTGGACCATAATAACACAGATAAAGATTACGAAGATTTGCGTCTGATGTCGCAGTGTAAACACCACATCATCGCGAACTCTACCTTTAGCTGGTGGGGTGCCTGGCTGAATAGTAATCCAGATAAAATAGTGATTGCTCCGAAAAAATGGTTTAATACGAGCGACTACAATACCAAAGACTTAATACCTGAAAATTGGATCAAACTG ASR 10: AA (Sequence No. 105) MIVVKLIGGLGNQMFQYAAAKALALEKNQKLRLDVSAFETYKLHNYGLNHFNITAKIYKKENKWLRKIKSFFKKNTYYKEQDFGYNPDLFNLKADNIFLEGYFQSEKYFLKYEKEIRKDFEIISPLKKQTKEMIEK IQSVNSVSIHIRRGDYLTNPIHNTSKEEYYKKAMKFIESKIENPVFFVFSDDMDWVKENFKTNHETVFVDFNDASTNFEDIKLMSSCKHNIIANSSFSWWGAWLNQNPNKIVIAPKQWFNDDSINTSDIIPESWIKI ASR 10: DNA (SEQ ID NO: 106) ATGATCGTCGTTAAACTTATCGGTGGTCTGGGGAACCAAATGTTTCAGTATGCCGCGGCGAAGGCTCTGGCGCTCGAAAAAAACCAAAAACTGCGCTTGGACGTTAGTGCATTTGAAACTTATAAATTACACAACTATGGCCTCAATCATTTCAATATCACGGCGAAAATTTACAAAAAGGAAAACAAGTGGTTACGCAAAATAAAATCATTCTTTAAAAAAAACACCTATTATAAAGAGCAGGACTTCGGATACAATCCTGACCTGTTTAACTTGAAAGCTGATAACATCTTTCTTGAAGGGTATTTCCAATCGGAAAAATATTTCCTCAAATATGAAAAAGAGATTCGAAAAGACTTCGAAATTATTAGTCCTCTGAAAAAACAAACGAAAGAAATGATCGAAAAAATCCAATCCGTGAACTCTGTCTCTATCCATATCCGTCGCGGCGACTACCTCACGAATCCCATACATAACACCTCCAAGGAGGAATACTATAAAAAAGCAATGAAATTTATTGAGTCGAAAATCGAAAACCCCGTGTTCTTTGTATTTTCGGATGATATGGACTGGGTGAAAGAAAACTTTAAAACGAACCATGAGACTGTATTCGTGGATTTCAATGATGCGAGCACAAATTTCGAAGATATTAAGCTGATGTCATCGTGTAAACACAATATCATTGCGAACAGTTCCTTCTCTTGGTGGGGGGCCTGGCTGAATCAGAATCCAAATAAAATTGTGATCGCTCCGAAGCAATGGTTTAATGATGATTCGATTAATACCTCGGATATTATTCCTGAGAGTTGGATCAAAATC ASR 11: AA (SEQ ID NO: 107) MIIIRMSGGLGNQMFQYALYRKLKAMGKEVKIDDVTGYEDDNQRPIMLDVFGIDYDRATKEEVTELTDSSMDFLSRIRRKLFGRKSKEYREEDCNFDPQVLEMDDAYLEGYFQSEKYFQDVREQLRKEFRFRSGSVPLSEKTREL QKQIENSNSVSIHIRRGDYLENGHAEVYGGICTDDYYKKAIEYMKEKFPDAKFYIFSNDVEWAKQHFKGENFVVVEGSEENTGYLDMFLMSKCRHHIIANSSFSWWGAWLNENPEKIVIAPSKWLNNRECKDIYTERMIRISAEV ASR 11: DNA (SEQ ID NO: 108) ATGATCATTATTCGCATGTCAGGCGGGCTGGGCAACCAGATGTTTCAGTATGCCCTCTATCGCAAGTTGAAAGCTATGGGCAAAGAGGTTAAAATTGACGACGTAACGGGATATGAAGATGACAATCAACGTCCGATCATGCTGGACGTGTTTGGTATCGATTACGACCGTGCGACCAAAGAAGAAGTGACCGAACTCACCGACTCCTCAATGACT TTCTGTCCCGTATCCGCCGTAAGCTGTTTGGCCGCAAATCTAAAAGAATATCGTGAAGAAGATTGTAATTTTGATCCGCAGGTGCTTGAAATGGATCCATACCTGGAGGGTTATTTCCAGAGCGAAAAAACTTTCAGGATGTTAGGGAACAGCTGCGCAAAGAGTTTCGATTTCGTTCAGGTTCAGTGCCGCTGTCGGAAAGACGCGGGAATTA CAGAAAACAGATTGAGAACAGCAACTCTGTGAGTATCCATATCAGACGTGGTGACTACCTGGAAAATGGTCATGCAGAAGTTTATGGTGGCATCTGTACGGACGACTACTATAAAAAAGCCATCGAATACATGAAAGAGAAATTCCCGGATGCGAAGTTCTACATTTTTTCTAATGATGTCGAATGGGCTAAGCAGCATTTTAAAGGCGAAAATTTTG TGGTTGTGGAAGGTTCGGAAGAAAATACCGGCTATTTAGATATGTTTCTTATGAGCAAGTGTCGCCATCATATAATTGCCAACTCTAGTTTTAGCTGGTGGGGCCATGGCTCAAATGAAAACCCAGAAAAAGATTGTAATCGCGCGTCTAAATGGCTGAACAACCGTGAATGCAAAGATATTTATACCGAACGTATGATTCGTATTTCCGCAGAAGTA ASR 12: AA MIIIRMSGGLGNQMFQYALYRKLKSMGKEVKIDDITGYEDDNQRSIMLDVFGIDYDKATKEEITKLTDSSMDFLSRIRRKLFGRKSKEYQEEDFNFDPQVLEMDDAYLEGYFQSEKYFQDVREQLRKEFTFRKNSVPELSEQTKELRKQIENS NSVSIHIRRGDYLENSHAEIYGGICTDDYYKKAIEYMKEKFPDAKFYIFSNDIEWAKQHFKGENFVIVDASEENTGYADMYLMSKCKHHIIANSSFSWWGAWLNDNPEKIVIAPSKWLNNKECKDIYTDRMIKIDAKGEVRSEDYGVRTSTVK ASR 12: DNA (SEQ ID NO: 110) ATGATTATTATACGTATGAGTGGCGGCCTGGGTAATCAAATGTTTCAGTATGCCCTGTACCGCAAATTGAAATCGATGGGGAAAGAGGTGAAAATAGACGACATCACCGGGTATGAGGACGATAACCAGCGTTCTATCATGCTCGATGTGTTTGGGATTGATTACGACAAAGCAACCAAAGAAGAGATAACCAAGCTGACCGACAGTAGCATGGACTTTCTGTCTCGCATTCGTCGCAAACTGTTTGGCCGCAAATCGAAGGAGTACCAGGAAGAAGATTTTAATTTTGACCCACAAGTCCTGGAAATGGATGATGCCTACCTCGAAGGGTACTTCCAAAGTGAAAAGTATTTCCAGGATGTGCGGGAGCAGCTGCGAAAAGAATTTACCTTTCGAAAAAACAGCGTGCCGGAACTGTCGGAACAGACGAAAGAACTGCGCAAACAAATTGAAAATAGCAACAGCGTGTCGATTCACATTCGCCGTGGTGACTATTTGGAAAACTCCCACGCCGAGATTTATGGCGGTATTTGTACTGACGATTACTACAAGAAAGCGATTGAGTACATGAAAGAGAAATTCCCGGATGCAAAGTTTTACATTTTCTCGAATGATATTGAATGGGCGAAACAGCACTTTAAAGGGGAGAATTTTGTAATTGTTGACGCATCAGAAGAGAACACTGGCTATGCGGATATGTACCTGATGAGCAAATGCAAACACCACATTATTGCCAATTCCTCCTTCTCGTGGTGGGGTGCCTGGCTGAACGATAACCCGGAAAAAATCGTGATTGCTCCGAGTAAATGGCTCAATAATAAAGAGTGCAAAGATATTTACACCGACCGCATGATTAAAATTGACGCCAAAGGTGAGGTCCGTTCAGAGGATTACGGCGTACGTACCAACTCTACCGTGAAA

Claims

1. A method for producing 2'-fucosyllactose, comprising: incubating GDP-L-fucose with α-1,2-fucosyltransferase in a culture medium containing lactose for a time sufficient to convert the GDP-L-fucose and lactose to 2'-fucosyllactose and GDP; The α-1,2-fucosyltransferase is a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

109. method.

2. 2. The method of claim 1, wherein the α-1,2-fucosyltransferase is a polypeptide comprising the amino acid sequence of SEQ ID NO:

109.

3. 3. The method of claim 1, wherein the GDP-L-fucose is produced in situ in the culture medium from GDP-mannose or GDP-L-galactose in a reaction catalyzed by a dehydratase.

4. The method of claim 3, wherein the dehydratase is a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO:

9.

5. 4. The method of claim 3, wherein the dehydratase is a polypeptide comprising the amino acid sequence of SEQ ID NO:79, SEQ ID NO:77 or SEQ ID NO:

75.

6. The method of claim 3, wherein the dehydratase is a polypeptide comprising the amino acid sequence of SEQ ID NO: 85, SEQ ID NO: 83 or SEQ ID NO:

81.

7. A method for producing 2'-fucosyllactose, comprising: (i) incubating GDP-mannose and / or GDP-L-galactose with a dehydratase and a reductase in the presence of NADPH and / or NADP+ in a culture medium for a time sufficient to convert the GDP-mannose and / or GDP-L-galactose to GDP-L-fucose; and (ii) incubating the GDP-L-fucose with α-1,2-fucosyltransferase and lactose for a time sufficient to convert the GDP-L-fucose and lactose to 2'-fucosyllactose and GDP; The dehydratase (a) a polypeptide comprising the amino acid sequence of SEQ ID NO:79, SEQ ID NO:77, SEQ ID NO:75, or SEQ ID NO:5; and (b) a polypeptide comprising the amino acid sequence of SEQ ID NO:85, SEQ ID NO:83, SEQ ID NO:81, or SEQ ID NO:9; is selected from the group consisting of The α-1,2-fucosyltransferase is a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

109. method.

8. 8. The method of claim 7, wherein the dehydratase is a polypeptide comprising the amino acid sequence of SEQ ID NO:79, SEQ ID NO:77 or SEQ ID NO:

75.

9. The method of claim 7 or claim 8, wherein the α-1,2-fucosyltransferase is a polypeptide comprising the amino acid sequence of SEQ ID NO:

109.

10. The method of any one of claims 6 to 9, wherein the reductase is a polypeptide comprising amino acids having at least 90% sequence identity to SEQ ID NO:7, SEQ ID NO:15 or SEQ ID NO:

17.

11. A recombinant microorganism for enhancing the production of 2'-fucosyllactose, wherein the microorganism comprises at least the following heterologous genes for producing 2'-fucosyllactose: (i) a first heterologous gene encoding a mutant anhydrase for producing GDP-L-fucose, wherein the mutant anhydrase is a polypeptide comprising an amino acid sequence selected from SEQ ID NO:79, SEQ ID NO:77, SEQ ID NO:75, SEQ ID NO:85, SEQ ID NO:83, and SEQ ID NO:81; and (ii) a second heterologous gene encoding a mutant α-1,2-fucosyltransferase for converting GDP-L-fucose to 2'-fucosyllactose, wherein the mutant α-1,2-fucosyltransferase is a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

109.

12. The microorganism of claim 11, further comprising a heterologous gene for transporting 2'-fucosyllactose out of the cell.

13. A method for producing 2'-fucosyllactose, comprising culturing the microorganism according to claim 11 or 12 in a culture medium containing at least one carbon source.

14. 14. The method of claim 13, further comprising separating the culture medium from the microorganism.

15. 15. The method of claim 14, further comprising isolating 2'-fucosyllactose from the culture medium.

16. A mutant α-1,2-fucosyltransferase for producing 2'-fucosyllactose, the mutant α-1,2-fucosyltransferase being a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

109.

17. A nucleic acid construct comprising a nucleic acid sequence encoding the mutant α-1,2-fucosyltransferase of claim 16.

18. A microorganism comprising the nucleic acid construct of claim 17.

19. A method for producing 2'-fucosyllactose, comprising: (a) NADPH and / or NADP for a period of time sufficient to convert GDP-L-galactose to GDP-L-fucose ＋ incubating the GDP-L-galactose with a dehydratase and a reductase in the presence of (b) incubating the GDP-L-fucose with lactose and α-1,2-fucosyltransferase for a time sufficient to convert the GDP-L-fucose and lactose to 2'-fucosyllactose and GDP; The α-1,2-fucosyltransferase is a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

109. method.

20. 20. The method of claim 19, further comprising incubating the GDP-L-galactose in the presence of a first regenerating enzyme and a first substrate for the first regenerating enzyme, wherein the first regenerating enzyme catalyzes a reaction involving the first substrate that uses NADP+ as a cofactor, thereby regenerating NADPH.

21. The method according to claim 19 or 20, wherein the dehydratase is GDP-mannose-4,6-dehydratase.

22. The method according to any one of claims 19 to 21, wherein the dehydratase is an enzyme comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

5.

23. The method according to any one of claims 19 to 21, wherein the dehydratase is an enzyme comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

9.

24. The method according to any one of claims 19 to 21, wherein the dehydratase is an enzyme comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

11.

25. The method according to any one of claims 19 to 21, wherein the dehydratase is an enzyme comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

13.

26. The method according to any one of claims 19 to 25, wherein the reductase is GDP-4-keto-6-deoxy-mannose reductase.

27. The method according to any one of claims 19 to 26, wherein the reductase is an enzyme comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

7.

28. The method according to any one of claims 19 to 26, wherein the reductase is an enzyme comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

15.

29. The method according to any one of claims 19 to 26, wherein the reductase is an enzyme comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

17.

30. 30. The method of any one of claims 19 to 29, further comprising incubating the GDP-mannose with GDP-mannose-3,5-epimerase for a time sufficient to convert the GDP-mannose to GDP-L-galactose.

31. The method of claim 30, wherein the GDP-mannose-3,5-epimerase is an enzyme comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

3.

32. The method of claim 30, wherein the GDP-mannose-3,5-epimerase is an enzyme comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

19.

33. 33. The method of any one of claims 19 to 32, further comprising the step of incubating L-galactose with fucokinase / guanylyltransferase in the presence of ATP and GTP for a period of time sufficient to convert the L-galactose to GDP-L-galactose.

34. 34. The method of claim 33, wherein the fucokinase / guanylyltransferase is an enzyme comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

1.

35. The method of claim 33 or 34, wherein the L-galactose is further incubated in the presence of a second regenerating enzyme and a second substrate for the second regenerating enzyme, and the second regenerating enzyme catalyzes a reaction involving the second substrate that uses ADP as a cofactor, thereby regenerating ATP.

36. The method of any one of claims 33 to 35, wherein the L-galactose is further incubated in the presence of a third regenerating enzyme and a third substrate for the third regenerating enzyme, and the third regenerating enzyme catalyzes a reaction involving the third substrate that uses GDP as a cofactor, thereby regenerating GTP.

37. 37. The method of any one of claims 20 to 36, wherein the first regenerating enzyme and the first substrate are selected from the group consisting of: (a) malate dehydrogenase and malate, (b) formate dehydrogenase and formate, (c) phosphite dehydrogenase and phosphite, and (d) glucose dehydrogenase and glucose.

38. 36. The method of claim 35, wherein the second regenerating enzyme and the second substrate are selected from the group consisting of: (a) pyruvate kinase and phospho(enol)pyruvate (PEP), (b) creatine kinase and creatine phosphate, (c) acetate kinase and acetyl phosphate, (d) polyphosphate kinase and polyphosphate, and (e) polyphosphate:AMP phosphotransferase, adenylate kinase and adenosine monophosphate.

39. 37. The method of claim 36, wherein the third regenerating enzyme and the third substrate are selected from the group consisting of: (a) pyruvate kinase and phospho(enol)pyruvate (PEP), (b) creatine kinase and creatine phosphate, (c) acetate kinase and acetyl phosphate, (d) polyphosphate kinase and polyphosphate, and (e) polyphosphate:AMP phosphotransferase, adenylate kinase and adenosine monophosphate.

40. A method for producing 2'-fucosyllactose from L-galactose, comprising: (a) (i) fucokinase / guanylyltransferase, (ii) dehydratase, (iii) reductase, (iv) α-1,2-fucosyltransferase, (v) ATP, (vi) GTP, (vii) NADP ＋ and (viii) providing a reaction mixture comprising NADPH; (b) adding L-galactose and lactose to the reaction mixture; and (c) incubating the reaction mixture for a period of time sufficient to produce 2'-fucosyllactose; the reaction mixture, (ix) the first regenerating enzyme is NADP ＋ (x) a first regenerating enzyme and a first substrate for the first regenerating enzyme, wherein the first regenerating enzyme catalyzes a first reaction involving a first substrate that uses ADP as a cofactor, thereby regenerating NADPH; (x) a second regenerating enzyme and a second substrate for the second regenerating enzyme, wherein the second regenerating enzyme catalyzes a second reaction involving a second substrate that uses ADP as a cofactor, thereby regenerating ATP; and (xi) a third regenerating enzyme and a third substrate for the third regenerating enzyme, wherein the third regenerating enzyme catalyzes a third reaction involving a third substrate that uses GDP as a cofactor, thereby regenerating GTP, The α-1,2-fucosyltransferase is a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

109. method.

41. 41. The method of claim 40, wherein the fucokinase / guanylyltransferase is an enzyme comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

1.

42. The method according to claim 40 or 41, wherein the dehydratase is GDP-mannose-4,6-dehydratase.

43. The method according to any one of claims 40 to 42, wherein the dehydratase is an enzyme comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

5.

44. The method according to any one of claims 40 to 42, wherein the dehydratase is an enzyme comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

9.

45. The method according to any one of claims 40 to 42, wherein the dehydratase is an enzyme comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

11.

46. The method according to any one of claims 40 to 42, wherein the dehydratase is an enzyme comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

13.

47. The method according to any one of claims 40 to 42, wherein the reductase is GDP-4-keto-6-deoxy-mannose reductase.

48. The method according to any one of claims 40 to 42, wherein the reductase is an enzyme comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

7.

49. The method according to any one of claims 40 to 42, wherein the reductase is an enzyme comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

15.

50. The method according to any one of claims 40 to 42, wherein the reductase is an enzyme comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

17.

51. 51. The method of any one of claims 40 to 50, wherein the first regenerating enzyme and the first substrate are selected from the group consisting of: (a) malate dehydrogenase and malate, (b) formate dehydrogenase and formate, (c) phosphite dehydrogenase and phosphite, and (d) glucose dehydrogenase and glucose.

52. 52. The method of any one of claims 40 to 51, wherein the second regenerating enzyme and the second substrate are selected from the group consisting of: (a) pyruvate kinase and phospho(enol)pyruvate (PEP), (b) creatine kinase and creatine phosphate, (c) acetate kinase and acetyl phosphate, (d) polyphosphate kinase and polyphosphate, and (e) polyphosphate:AMP phosphotransferase, adenylate kinase and adenosine monophosphate.

53. 53. The method of any one of claims 40 to 52, wherein the third regenerating enzyme and the third substrate are selected from the group consisting of: (a) pyruvate kinase and phospho(enol)pyruvate (PEP), (b) creatine kinase and creatine phosphate, (c) acetate kinase and acetyl phosphate, (d) polyphosphate kinase and polyphosphate, and (e) polyphosphate:AMP phosphotransferase, adenylate kinase and adenosine monophosphate.

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