CN112063678A - Biosynthesis method of Siamenoside I - Google Patents

Abstract

The invention relates to the technical field of natural product biosynthesis and food chemical industry, in particular to a biosynthesis method of Siamenoside I; the invention relates to a preparation method of Siamenoside I, which comprises the steps of selectively hydrolyzing mogroside V by using glycoside hydrolase to prepare mogroside IIIE, modifying the mogroside IIIE by using UDP-glucose dependent glycosyltransferase and mutants thereof through site-directed glycosylation, and synthesizing the Siamenoside I. The invention realizes green biosynthesis of the Siamenoside I and lays a material foundation for the development of the Siamenoside I as a sweetening agent or a flavoring agent.

Description

Biosynthesis method of Siamenoside I

Technical Field

The invention belongs to the technical field of natural product biosynthesis and food chemical industry, and particularly relates to a method for preparing mogroside IIIE by using glycoside hydrolase mogroside V, modifying the mogroside IIIE by UDP-glucose dependent glycosyltransferase fixed-point glucosylation, and biosynthesizing a natural non-nutritional sweet compound Siamenoside I.

Background

Sweeteners are important materials indispensable in food and beverage products. In recent years, the consumption of human beings has exponentially increased with the improvement of living standard of human beings. Sweeteners are classified by their source and can be classified into synthetic sweeteners and natural sweeteners. The natural sweetener is sweet component extracted from natural product, and mainly comprises glucose, fructose, sucrose, stevioside, rubusoside, mogroside, glycyrrhizin, etc. The artificial non-nutritive sweetener is one kind of artificial or semi-synthetic cane sugar substitute, and includes mainly aspartame, acesulfame potassium, sodium cyclamate, saccharin sodium, sucralose and other matters (Mooradian AD et al. clinical Nutrition ESPEN,2017,18: 1-8). The artificial synthetic sweetener has low cost, low calorie, and little metabolism, and can be widely used in food, beverage, and medicine. However, the safety of synthetic sweeteners has been a significant contributor to their market acceptance. Therefore, the development of natural sweeteners harmless to human health is sought after in the market.

Momordica grosvenori (Siraitia grosvenorii) is a cucurbitaceae plant which is specific in China, mainly grows in tropical and subtropical areas such as Guangxi, Hunan, Guangdong, Guizhou and the like, is sweet and cool, and has the effects of moistening lung and promoting fluid production (Liu C et al. The analysis of the components of the momordica grosvenori shows that the momordica grosvenori contains various vitamins, essential amino acids, flavones and a plurality of triterpene glycoside compounds, wherein the cucurbitane tetracyclic triterpene saponin (collectively called momordica grosvenori glycoside) is a main sweet source of the momordica grosvenori, the sweetness is extremely high, the sweetness of the mixed glycoside is about 250-fold and 300-fold of that of sucrose, and the heat quantity is basically zero. Mogrosides (mixtures) have been used globally as sweeteners and odorants by virtue of their safety, high sweetness and low calorie properties. China has approved mogroside as a food additive in 1996. In addition, modern pharmacological experiments prove that the mogroside has various biological activities (Qi XY et al Nutr. Res.,2008,28: 278-.

More than 30 compounds of the same type have been isolated and identified since the isolation of sweet compounds from Lo Han Guo by Takemoto et al in 1983 (Takemoto T et al Yakugaku Zasshi,1983,103: 1167-. The number of the 3, 11 and 24 bits of the mogrol mother nucleus connected with glucose is a main factor for determining the sweetness and the mouthfeel of the mogroside. Currently, the Siamenoside I with 3 glucosyl groups connected to the 24-position and 1 glucosyl group connected to the 3-position has the highest sweetness (563 times of the sweetness of 5% sucrose) and the mouthfeel is closest to that of sucrose in the discovered mogrosides. However, Siamenoside I is very low in Siamenoside I, the yield of extraction and purification is less than 0.045% (Matsumoto K et al. chem. pharm. Bull.,1990,38: 2030. 2032), the growth period of Siamenoside I is long, and the plantable range is small, so that Siamenoside I cannot be developed into a non-nutritive sweetener until now.

In addition, the existing chemical method is difficult to synthesize the Siamenoside I regioselectively and stereoselectively due to the existence of a plurality of glucose groups in the structure, and the biological catalysis technology taking enzyme as a catalyst presents the advantage which is difficult to match in the aspect of selective synthesis.

Disclosure of Invention

The invention discloses a brand new preparation method of Siamenoside I, which is characterized in that glycoside hydrolase is adopted to selectively hydrolyze mogroside V to prepare mogroside IIIE, UDP-glucose dependent glycosyltransferase and a brand new mutant thereof are used to modify the mogroside IIIE in a site-specific glycosylation manner, and Siamenoside I is biosynthesized.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

1) the glycoside hydrolase is EXG1 (amino acid sequence is shown as SEQ ID NO:1) from Saccharomyces cerevisiae and Asn (amino acid sequence is shown as SEQ ID NO: 2) from Aspergillus niger.

2) The UDP-glucose-dependent glycosyltransferase is derived from glycosyltransferase UGT94-289-2 of Siraitia grosvenorii and mutants thereof, and the amino acid sequence is shown as SEQ ID NO. 3.

3) The UGT94-289-2 mutant is a single-point or multi-point mutant of W16, K53, L93, M94, L97, I124, L125, T146, M150, T154, T181, T182, I194, E195, E275 and S308 in the amino acid sequence, and the mutation result is shown in the examples in detail.

4) The glycoside hydrolase and UDP-glucose dependent glycosyltransferase were used to construct plasmids separately.

5) The glycoside hydrolase and the UDP-glucose-dependent glycosyltransferase can be independently or co-expressed in Escherichia coli or Escherichia coli modified by UDP-glucose metabolic pathway to prepare the biocatalyst for biosynthesis of Siamenoside I, the heterologous expression host is only an example and is not limited to the scope of the invention, and the glycoside hydrolase and the UDP-glucose-dependent glycosyltransferase can also be expressed in lactobacillus, streptomyces, saccharomycetes, Bacillus subtilis and the like.

5) The Escherichia coli modified by the UDP-glucose metabolic pathway is a strain for constructing a sucrose synthase-UDP-glucose circulating system or a UDP-glucose metabolic pathway reconstructed strain (CN110699373A) constructed by the inventor in the early stage.

6.1) cell extract reaction system: breaking escherichia coli independently expressing or co-expressing glycoside hydrolase and UDP-glucose dependent glycosyltransferase, centrifuging to obtain a bacterial liquid, sequentially adding mogroside V and UDP-glucose to construct a biosynthesis system, and directionally synthesizing the Simenoside I.

6.2) resting cell reaction system: after the escherichia coli buffer solution for independently expressing or co-expressing the glycoside hydrolase and UDP-glucose dependent glycosyltransferase is resuspended, UDP-glucose is added or not added, a biosynthesis system is constructed, and the Siamenoside I is directionally synthesized.

6.3) in-situ reaction system: after the induction of escherichia coli co-expressing glycoside hydrolase and UDP-glucose dependent glycosyltransferase is finished, mogroside V is directly added into fermentation liquor to construct a biosynthesis system, and Siamenoside I is directionally synthesized.

Through reaction condition optimization, the biosynthesis process of the Siamenoside I disclosed by the invention shows a series of advantages: i) the concentration of the substrate is high and can reach 15 g/L; 2) the reaction efficiency is high, and the conversion rate can reach more than 95%; 3) the product is single and easy to purify; 4) the use of exogenous UDP-glucose is avoided, and the cost is greatly reduced. Compared with the reported process, the green synthesis process disclosed by the invention lays a foundation for the commercial development of Siamenoside I, and expands the imagination space for developing the single-component momordica glycoside sweetener.

The invention has the following advantages:

the UGT94-289-2 mutant disclosed by the invention is UDP-glucose dependent glycosyltransferase reported for the first time. At present, only the mogroside V (containing 5 glucose groups) with high natural content can be industrially prepared from reported mogrosides. By combining the factors, the invention adopts glycoside hydrolase to selectively hydrolyze the mogroside V and directionally synthesize the mogroside IIIE (containing 3 glucose groups); the single product Siamenoside I is synthesized in a green way by modifying the mogroside IIIE by utilizing a UDP-glucose dependent glycosyltransferase region and stereoselective glucosylation. Compared with the reported method for preparing the Siamenoside I by carrying out enzymolysis on the mogroside V (Wang R et al. food chem.,2019,276:43-49), the double-enzyme coupling synthesis process established by the invention has the advantages of high substrate concentration, single product, high conversion rate, easy purification of the product and the like, and lays a material foundation for the industrial development of the Siamenoside I sweetener.

In conclusion, the biosynthesis method of the Siamenoside I disclosed by the invention is green and environment-friendly, and is not reported in documents. Compared with the process for preparing the Siamenoside I by enzymolysis of the mogroside V, the method has the remarkable advantages of high efficiency and single product, and is expected to be used for large-scale production of the Siamenoside I. Therefore, the novel preparation process of the Siamenoside I disclosed by the invention has great potential application value.

Drawings

FIG. 1 is an SDS-PAGE analysis of Exg1 and Asn, where A is Exg1 and B is Asn.

FIG. 2 is an SDS-PAGE analysis of UGT94-289-2 and mutants thereof, wherein A-D is UGT94-289-2 and mutants thereof.

FIG. 3 is an HPLC analysis of mogroside V, IIIE and Siamenoside I.

FIG. 4 is a schematic diagram of a co-expression plasmid, wherein A is pETDute-L97W1-AtSUS1, and B is pETDute-AtSUS1-L97W 1.

Detailed Description

The following examples illustrate specific steps of the present invention, but the scope of the present invention is not limited by these examples.

Example 1 construction of glycoside hydrolase-expressing Strain

Exg 1A glycosyl transferase (amino acid sequence is shown in SEQ ID NO:1 and nucleotide sequence is shown in SEQ ID NO: 4) from Saccharomyces cerevisiae, its coding gene is entrusted to CRO company for synthesis, and pET28a (+) is used to construct expression plasmid pET28a-exg1, which is transformed to E.coli BL21 to obtain engineering strain E.coli-pExg.

Asn is a glycosyltransferase (amino acid sequence is shown as SEQ ID NO:2, nucleotide sequence is shown as SEQ ID NO: 5) from Aspergillus niger, the coding gene of Asn is consigned to CRO company for synthesis, and pET28a (+) is used for constructing expression plasmid pET28a-Asn, and the expression plasmid is transformed into E.coli BL21 to obtain the engineering strain E.coli-pAsn.

Example 2 construction of glycosyltransferase and mutants thereof

TABLE 1 mutant amino acids and mutant List

Note: the mutant nucleotide sequences of double-point mutation and three-point mutation are shown in SEQ ID NO. 7-24.

UGT94-289-2 is UDP-glucose dependent glycosyltransferase (amino acid sequence is shown as SEQ ID NO:3, nucleotide sequence is shown as SEQ ID NO: 6) from Siraitia grosvenorii, coding genes of the UDP-glucose dependent glycosyltransferase are entrusted to CRO company for synthesis, pET22b (+) is utilized to construct expression plasmid pET22b-UGT-1, and the expression plasmid is transformed to E.coli BL21 to obtain the engineering strain E.coli-pUgt-1.

Results of molecular docking showed that W16, K53, L93, M94, L97, I124, L125, T146, M150, T154, T181, T182, I194, E195, E275, S308 are key amino acid residues for interaction with the sugar donor UDP-glucose and the substrate mogroside IIIE. The invention constructs a series of mutants aiming at the amino acid residues, and the specific amino acid residue mutation conditions are shown in the table (note: the mutation of the nucleotide sequence follows the codon preference of escherichia coli and the enzyme cutting site avoidance principle). The mutants are all used for constructing expression vectors by using pET22b (+), and are transformed into E.coli BL21 to obtain a plurality of engineering strains.

Example 3 Induction of expression of glycoside hydrolases

The E.coli engineering bacteria containing the glycoside hydrolase gene are respectively inoculated into an LB culture medium (containing 50 mu g/mL kanamycin), and are cultured overnight at 37 ℃ and 220rpm to obtain corresponding seed liquid. The seed solution was transferred to a new LB medium (containing 50. mu.g/mL kanamycin) at an inoculum size of 1%, and cultured at 37 ℃ to OD₆₀₀When the concentration was 0.8. + -. 0.1, IPTG was added as an inducer to give a final concentration of 0.5mM, and induction culture was carried out at 15 ℃ for 12 hours. SDS-PAGE analysis showed that both glycoside hydrolase Exg1 and Asn were soluble and contained 12.8% and 14% of soluble protein (FIG. 1).

Example 4 inducible expression of glycosyltransferases and mutants thereof

The E.coli engineering bacteria containing the glycosyltransferase UGT94-289-2 and the mutant genes thereof are respectively inoculated into an LB culture medium (containing 100 mu g/mL ampicillin) and cultured overnight at 37 ℃ and 220rpm to obtain corresponding seed liquid. The seed solution was inoculated at 1% inoculum size to a new LB medium (containing 100. mu.g/mL ampicillin), and cultured at 37 ℃ to OD₆₀₀When the concentration is 0.8 + -0.1, IPTG as an inducer is added to give a final concentration of 0.6mM, induction culture at 20 ℃ for 16 hours. SDS-PAGE analysis results show that although the glycosyltransferase UGT94-289-2 and the mutant thereof can be expressed in a soluble manner, the content of the glycosyltransferase UGT94-289-2 and the mutant thereof is low, and the expression level of all the mutants only accounts for about 3-5% of the soluble protein (figure 2).

Example 5 HPLC analytical method

A chromatographic column: YMC-Pack ODS-A (150 mm. times.4.6 mm, 5 μm); mobile phase A: ultrapure water (containing 0.1% formic acid); mobile phase B: acetonitrile (0.1% formic acid); sample introduction amount: 5 mu L of the solution; column temperature: 30 ℃; detection wavelength: 205 nm; flow rate: 1.0 mL/min; gradient elution: 10% B-90% B, 25 min. Under the above HPLC analysis conditions, the retention times of mogroside V, IIIE and Siamenoside I were 9.1min, 10.2min and 9.3min, as shown in FIG. 3.

Example 6 enzymatic Synthesis of mogroside IIIE

A50 mL reaction system comprises a substrate mogroside V (1-15 g/L), 25mL of crude enzyme solution of glycoside hydrolase Exg1 (protein concentration is 6mg/mL), and 0.05M NaAC-HAC buffer solution (pH is 6.0). After the reaction is carried out for 5-24 hours at 40 ℃, the reaction solution is boiled for 5min, diluted to 1mg/mL, centrifuged, filtered and analyzed by HPLC. The results show that mogroside V can be completely converted to the single product mogroside IIIE by 20 hours of reaction when the substrate concentration is less than or equal to 5 g/L.

A50 mL reaction system comprises a substrate mogroside V (1-15 g/L), 25mL of crude enzyme solution of glycoside hydrolase Asn (protein concentration is 6.5mg/mL), and 0.05M NaAC-HAC buffer solution (pH is 4.5). After the reaction is carried out for 5-24 hours at 37 ℃, the reaction solution is boiled for 5min, diluted to 1mg/mL, centrifuged, filtered and analyzed by HPLC. The results show that under all conditions of substrate concentration and reaction time, mogroside V can be completely converted into a single product mogroside IIIE.

Example 7 Whole cell biosynthesis of mogroside IIIE

After the induction expression is finished, collecting E.coli-pExg thalli, diluting and resuspending by adopting 0.05M NaAC-HAC buffer solution (pH 6.0), and adding mogroside V (1-5 g/L) to construct a whole-cell biosynthesis system. After 20 hours at 40 ℃ HPLC analysis was performed. The results show that mogroside V can be completely converted to the single product mogroside IIIE when the substrate concentration is less than or equal to 2 g/L.

After the induction expression is finished, E.coli-pAsn thalli are collected, diluted and resuspended by adopting 0.05M NaAC-HAC buffer solution (pH 6.0), and mogroside V (1-15 g/L) is added to construct a whole-cell biosynthesis system. After 12 hours at 37 ℃ HPLC analysis was performed. The results show that mogroside V can be completely converted to the single product mogroside IIIE when the substrate concentration is less than or equal to 5 g/L.

Example 8 Synthesis of Siamenoside I from UGT94-289-2 and mutants thereof

The 5mL reaction system comprises a substrate of mogroside IIIE (10mg/mL), UDP-glucose (29.4mg/mL), glycosyltransferase UGT94-289-2 or a crude mutant enzyme solution 4mL (protein concentration is 6.5mg/mL), and 0.05M Tris-HCl buffer solution (pH 9.0). After 24 hours of reaction at 25 ℃ with shaking, the reaction solution was boiled for 5min and diluted to 1mg/mL, centrifuged, filtered and analyzed by HPLC. The results are shown in the table below, and both mutants I194G and L97W/T181D/I194G were able to completely convert mogroside IIIE to the single product Siamenoside I.

TABLE 2 UGT94-289-2 mutant catalytic Activity

Example 9 comparison of catalytic efficiency of mutants I194G and L97W/T181D/I194G

The 5mL reaction system comprises substrates of mogroside IIIE (12.5 mg/mL-20 mg/mL), UDP-glucose (29.4mg/mL), UGT94-289-2 mutant I194G, L97W/T181D/I194G crude enzyme solution 4mL (protein concentration is 6.5mg/mL), 0.05M Tris-HCl buffer solution (pH 9.0) and the like. After 24 hours of reaction at 25 ℃ with shaking, the reaction solution was boiled for 5min and diluted to 1mg/mL, centrifuged, filtered and analyzed by HPLC. The results are shown in the table below, and the mutant L97W/T181D/I194G is obviously superior to the mutant I194G in catalytic activity and can catalyze the substantial and complete conversion of 15mg/mL mogroside IIIE into Siamenoside I.

TABLE 3 catalytic efficiency of mutants I194G and L97W/T181D/I194G

Example 10 resting cell response of engineered Strain expressing mutant L97W/T181D/I194G

After the induction culture is completed, the thalli are collected by centrifugation and diluted and resuspended by using 0.05M Tris-HCl buffer solution (pH 9.0), and then mogroside IIIE (15mg/mL) and UDP-glucose (0 or 29.4mg/mL) are added to construct a whole-cell catalytic system. After 24 hours of shaking reaction at 25 ℃, the reaction solution was boiled for 5min, centrifuged to remove the bacterial cells, diluted to a substrate concentration of 1mg/mL, filtered and analyzed by HPLC. The results show that the yields of Siamenoside I are 47.5% and 82.8% respectively when the UDP-glucose concentration is 0 and 29.4 mg/mL.

Example 11 in situ catalysis of the engineered Strain expressing mutant L97W/T181D/I194G

After the induction culture is completed, glucose is added to the fermentation broth to 5mg/mL, and the pH value of the fermentation broth is adjusted to 9.0. Adding mogroside IIIE (15mg/mL) and UDP-glucose (0 or 29.4mg/mL) into the fermentation liquor to construct an in-situ catalytic system. After 24 hours of shaking reaction at 25 ℃, the reaction solution was boiled for 5min, centrifuged to remove the bacterial cells, diluted to a substrate concentration of 1mg/mL, filtered and analyzed by HPLC. The results show that the yields of Siamenoside I are 34.8% and 69.5% when the UDP-glucose concentration is 0 and 29.4mg/mL, respectively.

Example 12 sucrose synthase-UDP-glucose cycling System and biosynthesis of Siamenoside I

A co-expression plasmid (FIG. 4) of sucrose synthase (Arabidopsis thaliana sucrose synthase abbreviated as AtSUS1 and Vigna radiata sucrose synthase abbreviated as VrSUS1) and mutant L97W/T181D/I194G (abbreviated as L97W1) was constructed by using a co-expression vector pETDuet-1 and using restriction sites such as Nco I/Hind III and Nde I/Xho I, and E.coli BL21 was introduced to obtain co-expression strains E.coli-W01, E.coli-W02, E.coli-W03 and E.coli-W04. The induced expression condition of the engineering strain IPTG is 0.5mM IPTG, 15 ℃ and 16 hours. After induction expression is finished, collecting thalli, diluting and resuspending in 0.05M Tris-HCl buffer solution (pH 9.0), and constructing a whole-cell biocatalysis system: the substrate, mogroside IIIE, is 5mg/mL, sucrose is 500mg/mL, and the reaction is carried out for 12 hours at 25 ℃ with shaking. After the reaction is finished, the HPLC analysis result is shown in the following table, and the synthesis efficiency of Siamenoside I catalyzed by E.coli-W01 is the highest, which shows that: 1) the plasmid pETDuet-AtSUS1-L97W1 obtained by inserting the Arabidopsis thaliana sucrose synthase gene into the multiple cloning site 1 and inserting the mutant L97W/T181D/I194G gene into the multiple cloning site 2 is beneficial to synthesis of Simenoside I; 2) the activity matching degree of the Arabidopsis thaliana sucrose synthase and the mutant is higher, and the synthesis of the Simenoside I is facilitated.

TABLE 4 Synthesis efficiency of Siamenoside I

Example 13 modification of the UDP-glucose biosynthetic pathway and biosynthesis of Siamenoside I

The co-expression plasmid pETDuet-AtSUS1-L97W1 was introduced into the engineering strain E.coli CPM. DELTA.U &2(CN110699373A) which was a precursor strain for engineering UDP-glucose biosynthesis pathway and used in the construction of the engineering strain E.coli-W05. The induction expression conditions were the same as in example 12, and after the expression, the cells were collected, diluted and resuspended in 0.05M Tris-HCl buffer (pH 9.0), and a whole-cell biocatalysis system was constructed: the substrate, mogroside IIIE, is 5mg/mL, sucrose is 500mg/mL, and the reaction is carried out for 12 hours at 25 ℃ with shaking. After the reaction is finished, HPLC analysis and detection results show that the mogroside IIIE is completely converted into Siamenoside I.

Example 14 biosynthesis of substrate concentration with Simenoside I

Collecting E.coli-W05 thallus after induction expression, diluting and resuspending in 0.05M Tris-HCl buffer solution (pH 9.0), and constructing a whole-cell biocatalysis system: 5-15 mg/mL of mogroside IIIE serving as a substrate, 500mg/mL of cane sugar and shaking reaction at 25 ℃ for 12 hours. After the reaction is finished, HPLC analysis and detection results show that the yield of Siamenoside I is more than 97% when the concentration of the mogroside IIIE is less than or equal to 7.5 mg/mL; siamenoside I yields were 92.5%, 88.1%, and 82% at 10mg/mL, 12.5mg/mL, and 15mg/mL mogroside IIIE, respectively.

Example 15 biosynthesis of Simenoside I by glycoside hydrolase tandem E.coli-W05

A hydrolysis system (50mL) with Asn catalyzing 10mg/mL mogroside V was constructed as in example 6, after the reaction was completed, Asn was boiled and inactivated to obtain a reaction solution containing mogroside IIIE at about 5mg/mL, and pH was adjusted to 9.0 with NaAC to obtain suspension I. Dilute and resuspend e.coli-W05 with suspension I (induction completed) and add sucrose to 500mg/mL to construct a whole cell catalytic system. After 12 hours of reaction at 25 ℃ with shaking, HPLC analysis showed: asn catalyzes the mogroside IIIE prepared by hydrolyzing the mogroside V to be completely converted into Siamenoside I, and no mogroside V and IIIE residue exists.

Example 15 Synthesis of Simenoside I by Dual-bacterial catalysis of E.coli-pAsn and E.coli-W05

A whole-cell reaction system (50mL) was constructed according to example 7, e.coli-pAsn (induction completed) catalyzes hydrolysis of 5mg/mL mogroside V, and after the reaction was completed, Asn was boiled to inactivate to obtain a reaction solution containing about 2.5mg/mL mogroside IIIE, and pH was adjusted to 9.0 with NaAC to obtain suspension II. Dilute suspension II and resuspend e.coli-W05 (complete induction), add sucrose to 500mg/mL, construct whole cell catalytic system. After 12 hours of reaction at 25 ℃ with shaking, HPLC analysis showed: siamenoside I is a single product, and has no mogroside V and IIIE residues.

Example 16 E. construction of coli-W06 and Synthesis of Siamenoside I

pET28a-asn was introduced into E.coli-W05 to obtain an engineered strain E.coli-W06 co-expressing glycoside hydrolase and glycosyltransferase. After induction expression (0.5mM IPTG, 15 ℃, 16 hours), collecting thalli, and adopting buffer solutions with different pH values (pH4.5-9.0) to respectively construct a whole-cell biocatalysis system. In each reaction system, the mogroside V is 5mg/mL, the sucrose is 500mg/mL, and the reaction is carried out for 12 hours at 25 ℃ with shaking. After the reaction is finished, the HPLC result is shown in the following table, E.coli-W06 can catalyze the hydrolysis of mogroside V into IIIE, and the site-directed glycosylation generates Siamenoside I, but the efficiency needs to be further improved.

TABLE 5 E. coli-W06 catalytic Synthesis of Siamenoside I

Reaction system	Mogroside V	Mogroside III	Siamenoside I
				pH 4.5	0	92.5％	7.5％
pH 5.0	1.1％	90.0％	8.9％
				pH 5.5	7.4％	85.7％	6.9％
pH 6.0	23.7％	62.6％	13.7％
				pH 6.5	38.9％	42.6％	18.5％
pH 7.0	42.1％	10.3％	47.6％
				pH 7.5	51.5％	9.4％	39.1％
pH 8.0	88.4％	1.5％	10.1％
				pH 8.5	95.8％	0	4.2％
pH 9.0	98.6％	0	1.4％

Sequence listing

<110> university of Chinese pharmacy

<120> biosynthesis method of Siamenoside I

<160> 24

<170> SIPOSequenceListing 1.0

<210> 1

<211> 448

<212> PRT

<213> Exg1 amino acid sequence (2 Ambystoma laterale x Ambystoma jeffersonoanum)

<400> 1

Met Leu Ser Leu Lys Thr Leu Leu Cys Thr Leu Leu Thr Val Ser Ser

1 5 10 15

Val Leu Ala Thr Pro Val Pro Ala Arg Asp Pro Ser Ser Ile Gln Phe

20 25 30

Val His Glu Glu Asn Lys Lys Arg Tyr Tyr Asp Tyr Asp His Gly Ser

35 40 45

Leu Gly Glu Pro Ile Arg Gly Val Asn Ile Gly Gly Trp Leu Leu Leu

50 55 60

Glu Pro Tyr Ile Thr Pro Ser Leu Phe Glu Ala Phe Arg Thr Asn Asp

65 70 75 80

Asp Asn Asp Glu Gly Ile Pro Val Asp Glu Tyr His Phe Cys Gln Tyr

85 90 95

Leu Gly Lys Asp Leu Ala Lys Ser Arg Leu Gln Ser His Trp Ser Thr

100 105 110

Phe Tyr Gln Glu Gln Asp Phe Ala Asn Ile Ala Ser Gln Gly Phe Asn

115 120 125

Leu Val Arg Ile Pro Ile Gly Tyr Trp Ala Phe Gln Thr Leu Asp Asp

130 135 140

Asp Pro Tyr Val Ser Gly Leu Gln Glu Ser Tyr Leu Asp Gln Ala Ile

145 150 155 160

Gly Trp Ala Arg Asn Asn Ser Leu Lys Val Trp Val Asp Leu His Gly

165 170 175

Ala Ala Gly Ser Gln Asn Gly Phe Asp Asn Ser Gly Leu Arg Asp Ser

180 185 190

Tyr Lys Phe Leu Glu Asp Ser Asn Leu Ala Val Thr Thr Asn Val Leu

195 200 205

Asn Tyr Ile Leu Lys Lys Tyr Ser Ala Glu Glu Tyr Leu Asp Thr Val

210 215 220

Ile Gly Ile Glu Leu Ile Asn Glu Pro Leu Gly Pro Val Leu Asp Met

225 230 235 240

Asp Lys Met Lys Asn Asp Tyr Leu Ala Pro Ala Tyr Glu Tyr Leu Arg

245 250 255

Asn Asn Ile Lys Ser Asp Gln Val Ile Ile Ile His Asp Ala Phe Gln

260 265 270

Pro Tyr Asn Tyr Trp Asp Asp Phe Met Thr Glu Asn Asp Gly Tyr Trp

275 280 285

Gly Val Thr Ile Asp His His His Tyr Gln Val Phe Ala Ser Asp Gln

290 295 300

Leu Glu Arg Ser Ile Asp Glu His Ile Lys Val Ala Cys Glu Trp Gly

305 310 315 320

Thr Gly Val Leu Asn Glu Ser His Trp Thr Val Cys Gly Glu Phe Ala

325 330 335

Ala Ala Leu Thr Asp Cys Thr Lys Trp Leu Asn Ser Val Gly Phe Gly

340 345 350

Ala Arg Tyr Asp Gly Ser Trp Val Asn Gly Asp Gln Thr Ser Ser Tyr

355 360 365

Ile Gly Ser Cys Ala Asn Asn Asp Asp Ile Ala Tyr Trp Ser Asp Glu

370 375 380

Arg Lys Glu Asn Thr Arg Arg Tyr Val Glu Ala Gln Leu Asp Ala Phe

385 390 395 400

Glu Met Arg Gly Gly Trp Ile Ile Trp Cys Tyr Lys Thr Glu Ser Ser

405 410 415

Leu Glu Trp Asp Ala Gln Arg Leu Met Phe Asn Gly Leu Phe Pro Gln

420 425 430

Pro Leu Thr Asp Arg Lys Tyr Pro Asn Gln Cys Gly Thr Ile Ser Asn

435 440 445

<210> 2

<211> 860

<212> PRT

<213> Asn amino acid sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 2

Met Arg Phe Thr Leu Ile Glu Ala Val Ala Leu Thr Ala Val Ser Leu

1 5 10 15

Ala Ser Ala Asp Glu Leu Ala Tyr Ser Pro Pro Tyr Tyr Pro Ser Pro

20 25 30

Trp Ala Asn Gly Gln Gly Asp Trp Ala Glu Ala Tyr Gln Arg Ala Val

35 40 45

Asp Ile Val Ser Gln Met Thr Leu Ala Glu Lys Val Asn Leu Thr Thr

50 55 60

Gly Thr Gly Trp Glu Leu Glu Leu Cys Val Gly Gln Thr Gly Gly Val

65 70 75 80

Pro Arg Leu Gly Ile Pro Gly Met Cys Ala Gln Asp Ser Pro Leu Gly

85 90 95

Val Arg Asp Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn Val

100 105 110

Ala Ala Thr Trp Asp Lys Asn Leu Ala Tyr Leu Arg Gly Gln Ala Met

115 120 125

Gly Gln Glu Phe Ser Asp Lys Gly Ala Asp Ile Gln Leu Gly Pro Ala

130 135 140

Ala Gly Pro Leu Gly Arg Ser Pro Asp Gly Gly Arg Asn Trp Glu Gly

145 150 155 160

Phe Ser Pro Asp Pro Ala Leu Ser Gly Val Leu Phe Ala Glu Thr Ile

165 170 175

Lys Gly Ile Gln Asp Ala Gly Val Val Ala Thr Ala Lys His Tyr Ile

180 185 190

Ala Tyr Glu Gln Glu His Phe Arg Gln Ala Pro Glu Ala Gln Gly Tyr

195 200 205

Gly Phe Asn Ile Thr Glu Ser Arg Ser Ala Asn Leu Asp Asp Lys Thr

210 215 220

Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Ile Arg Ala Gly

225 230 235 240

Ala Gly Ala Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr Gly

245 250 255

Cys Gln Asn Ser Tyr Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu Gly

260 265 270

Phe Gln Gly Phe Val Met Ser Asp Trp Ala Ala His His Ala Gly Val

275 280 285

Ser Gly Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Val Asp

290 295 300

Tyr Asp Ser Gly Thr Ser Tyr Trp Gly Thr Asn Leu Thr Ile Ser Val

305 310 315 320

Leu Asn Gly Thr Ala Pro Gln Trp Arg Val Asp Asp Met Ala Val Arg

325 330 335

Ile Met Ala Ala Tyr Tyr Lys Val Gly Arg Asp Arg Leu Trp Thr Pro

340 345 350

Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly Phe Lys Tyr Tyr

355 360 365

Tyr Val Ser Glu Gly Pro Tyr Glu Lys Val Asn Gln Phe Val Asn Val

370 375 380

Gln Arg Asn His Ser Glu Leu Ile Arg Arg Ile Gly Ala Asp Ser Thr

385 390 395 400

Val Leu Leu Lys Asn Asp Gly Ala Leu Pro Leu Thr Gly Lys Glu Arg

405 410 415

Leu Val Ala Leu Ile Gly Glu Asp Ala Gly Ser Asn Pro Tyr Gly Ala

420 425 430

Asn Gly Cys Ser Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met Gly

435 440 445

Trp Gly Ser Gly Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu Gln

450 455 460

Ala Ile Ser Asn Glu Val Leu Lys Asn Lys Asn Gly Val Phe Thr Ala

465 470 475 480

Thr Asp Asn Trp Ala Ile Asp Gln Ile Glu Ala Leu Ala Lys Thr Ala

485 490 495

Ser Val Ser Leu Val Phe Val Asn Ala Asp Ser Gly Glu Gly Tyr Ile

500 505 510

Asn Val Asp Gly Asn Leu Gly Asp Arg Arg Asn Leu Thr Leu Trp Arg

515 520 525

Asn Gly Asp Asn Val Ile Lys Ala Ala Ala Ser Asn Cys Asn Asn Thr

530 535 540

Ile Val Ile Ile His Ser Val Gly Pro Val Leu Val Asn Glu Trp Tyr

545 550 555 560

Asp Asn Pro Asn Val Thr Ala Ile Leu Trp Gly Gly Leu Pro Gly Gln

565 570 575

Glu Ser Gly Asn Ser Leu Ala Asp Val Leu Tyr Gly Arg Val Asn Pro

580 585 590

Gly Ala Lys Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ala Tyr Gln

595 600 605

Asp Tyr Leu Tyr Thr Glu Pro Asn Asn Gly Asn Gly Ala Pro Gln Glu

610 615 620

Asp Phe Val Glu Gly Val Phe Ile Asp Tyr Arg Gly Phe Asp Lys Arg

625 630 635 640

Asn Glu Thr Pro Ile Tyr Glu Phe Gly Tyr Gly Leu Ser Tyr Thr Thr

645 650 655

Phe Asn Tyr Ser Asn Leu Gln Val Glu Val Leu Ser Ala Pro Ala Tyr

660 665 670

Glu Pro Ala Ser Gly Glu Thr Glu Ala Ala Pro Thr Phe Gly Glu Val

675 680 685

Gly Asn Ala Ser Asp Tyr Leu Tyr Pro Asp Gly Leu Gln Arg Ile Thr

690 695 700

Lys Phe Ile Tyr Pro Trp Leu Asn Ser Thr Asp Leu Glu Ala Ser Ser

705 710 715 720

Gly Asp Ala Ser Tyr Gly Gln Asp Ala Ser Asp Tyr Leu Pro Glu Gly

725 730 735

Ala Thr Asp Gly Ser Ala Gln Pro Ile Leu Pro Ala Gly Gly Gly Ala

740 745 750

Gly Gly Asn Pro Arg Leu Tyr Asp Glu Leu Ile Arg Val Ser Val Thr

755 760 765

Ile Lys Asn Thr Gly Lys Val Ala Gly Asp Glu Val Pro Gln Leu Tyr

770 775 780

Val Ser Leu Gly Gly Pro Asn Glu Pro Lys Ile Val Leu Arg Gln Phe

785 790 795 800

Glu Arg Ile Thr Leu Gln Pro Ser Lys Glu Thr Gln Trp Ser Thr Thr

805 810 815

Leu Thr Arg Arg Asp Leu Ala Asn Trp Asn Val Glu Thr Gln Asp Trp

820 825 830

Glu Ile Thr Ser Tyr Pro Lys Met Val Phe Ala Gly Ser Ser Ser Arg

835 840 845

Lys Leu Pro Leu Arg Ala Ser Leu Pro Thr Val His

850 855 860

<210> 3

<211> 459

<212> PRT

<213> UGT94-289-2 amino acid sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 3

Met Asp Ala Gln Gln Gly His Thr Thr Thr Ile Leu Met Leu Pro Trp

1 5 10 15

Val Gly Tyr Gly His Leu Leu Pro Phe Leu Glu Leu Ala Lys Ser Leu

20 25 30

Ser Arg Arg Lys Leu Phe His Ile Tyr Phe Cys Ser Thr Ser Val Ser

35 40 45

Leu Asp Ala Ile Lys Pro Lys Leu Pro Pro Ser Ile Ser Ser Asp Asp

50 55 60

Ser Ile Gln Leu Val Glu Leu Arg Leu Pro Ser Ser Pro Glu Leu Pro

65 70 75 80

Pro His Leu His Thr Thr Asn Gly Leu Pro Ser His Leu Met Pro Ala

85 90 95

Leu His Gln Ala Phe Val Met Ala Ala Gln His Phe Gln Val Ile Leu

100 105 110

Gln Thr Leu Ala Pro His Leu Leu Ile Tyr Asp Ile Leu Gln Pro Trp

115 120 125

Ala Pro Gln Val Ala Ser Ser Leu Asn Ile Pro Ala Ile Asn Phe Ser

130 135 140

Thr Thr Gly Ala Ser Met Leu Ser Arg Thr Leu His Pro Thr His Tyr

145 150 155 160

Pro Ser Ser Lys Phe Pro Ile Ser Glu Phe Val Leu His Asn His Trp

165 170 175

Arg Ala Met Tyr Thr Thr Ala Asp Gly Ala Leu Thr Glu Glu Gly His

180 185 190

Lys Ile Glu Glu Thr Leu Ala Asn Cys Leu His Thr Ser Cys Gly Val

195 200 205

Val Leu Val Asn Ser Phe Arg Glu Leu Glu Thr Lys Tyr Ile Asp Tyr

210 215 220

Leu Ser Val Leu Leu Asn Lys Lys Val Val Pro Val Gly Pro Leu Val

225 230 235 240

Tyr Glu Pro Asn Gln Glu Gly Glu Asp Glu Gly Tyr Ser Ser Ile Lys

245 250 255

Asn Trp Leu Asp Lys Lys Glu Pro Ser Ser Thr Val Phe Val Ser Phe

260 265 270

Gly Thr Glu Tyr Phe Pro Ser Lys Glu Glu Met Glu Glu Ile Ala Tyr

275 280 285

Gly Leu Glu Leu Ser Glu Val Asn Phe Ile Trp Val Leu Arg Phe Pro

290 295 300

Gln Gly Asp Ser Thr Ser Thr Ile Glu Asp Ala Leu Pro Lys Gly Phe

305 310 315 320

Leu Glu Arg Ala Gly Glu Arg Ala Met Val Val Lys Gly Trp Ala Pro

325 330 335

Gln Ala Lys Ile Leu Lys His Trp Ser Thr Gly Gly Leu Val Ser His

340 345 350

Cys Gly Trp Asn Ser Met Met Glu Gly Met Met Phe Gly Val Pro Ile

355 360 365

Ile Ala Val Pro Met His Leu Asp Gln Pro Phe Asn Ala Gly Leu Val

370 375 380

Glu Glu Ala Gly Val Gly Val Glu Ala Lys Arg Asp Ser Asp Gly Lys

385 390 395 400

Ile Gln Arg Glu Glu Val Ala Lys Ser Ile Lys Glu Val Val Ile Glu

405 410 415

Lys Thr Arg Glu Asp Val Arg Lys Lys Ala Arg Glu Met Gly Glu Ile

420 425 430

Leu Arg Ser Lys Gly Asp Glu Lys Ile Asp Glu Leu Val Ala Glu Ile

435 440 445

Ser Leu Leu Arg Lys Lys Ala Pro Cys Ser Ile

450 455

<210> 4

<211> 1347

<212> DNA

<213> Exg1 nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 4

atgctttcgc ttaaaacgtt actgtgtacg ttgttgactg tgtcatcagt actcgctacc 60

ccagtccctg caagagaccc ttcttccatt caatttgttc atgaggagaa caagaaaaga 120

tactacgatt atgaccacgg ttccctcgga gaaccaatcc gtggtgtcaa cattggtggt 180

tggttacttc ttgaaccata cattactcca tctttgttcg aggctttccg tacaaatgat 240

gacaacgacg aaggaattcc tgtcgacgaa tatcacttct gtcaatattt aggtaaggat 300

ttggctaaaa gccgtttaca gagccattgg tctactttct accaagaaca agatttcgct 360

aatattgctt cccaaggttt caaccttgtc agaattccta tcggttactg ggctttccaa 420

actttggacg atgatcctta tgttagcggc ctacaggaat cttacctaga ccaagccatc 480

ggttgggcta gaaacaacag cttgaaagtt tgggttgatt tgcatggtgc cgctggttcg 540

cagaacgggt ttgataactc tggtttgaga gattcataca agtttttgga agacagcaat 600

ttggccgtta ctacaaatgt cttgaactac atattgaaaa aatactctgc ggaggaatac 660

ttggacactg ttattggtat cgaattgatt aatgagccat tgggtcctgt tctagacatg 720

gataaaatga agaatgacta cttggcacct gcttacgaat acttgagaaa caacatcaag 780

agtgaccaag ttatcatcat ccatgacgct ttccaaccat acaattattg ggatgacttc 840

atgactgaaa acgatggcta ctggggtgtc actatcgacc atcatcacta ccaagtcttt 900

gcttctgatc aattggaaag atccattgat gaacatatta aagtagcttg tgaatggggt 960

accggagttt tgaatgaatc ccactggact gtttgtggtg agtttgctgc cgctttgact 1020

gattgtacaa aatggttgaa tagtgttggc ttcggcgcta gatacgacgg ttcttgggtc 1080

aatggtgacc aaacatcttc ttacattggc tcttgtgcta acaacgatga tatagcttac 1140

tggtctgacg aaagaaagga aaacacaaga cgttatgtgg aggcacaact agatgccttt 1200

gaaatgagag ggggttggat tatctggtgt tacaagacag aatctagttt ggaatgggat 1260

gctcaaagat tgatgttcaa tggtttattc cctcaaccat tgactgacag aaagtatcca 1320

aaccaatgtg gcacaatttc taactaa 1347

<210> 5

<211> 2583

<212> DNA

<213> Asn nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 5

atgaggttca ctttgatcga ggcggtggct ctgactgccg tctcgctggc cagcgctgat 60

gaattggcct actccccgcc gtattacccc tccccttggg ccaatggcca gggtgactgg 120

gcggaagcat accagcgcgc tgttgatatc gtctcgcaga tgacattggc tgagaaggtc 180

aatttgacta cgggaactgg atgggaattg gaattatgtg ttggtcagac tggaggtgtt 240

ccccgattgg gaattccggg aatgtgtgca caggatagcc ctctgggtgt tcgtgactcc 300

gactacaact ctgcgttccc tgccggtgtc aacgtggccg caacctggga caagaatctg 360

gcttaccttc gtggccaggc tatgggtcag gagtttagtg acaagggtgc tgatatccaa 420

ttgggtccag ctgccggccc tctcggtaga agtcccgacg gcggtcgtaa ctgggagggc 480

ttctcccccg acccggccct cagtggtgtg ctctttgcag agacaatcaa gggtattcag 540

gatgctggtg tggttgcaac ggctaagcac tacatcgcct acgagcagga gcatttccgt 600

caggcgcctg aagctcaagg ctacggattc aatattaccg agagtagaag cgcgaacctc 660

gacgataaga ctatgcatga gctgtacctc tggcccttcg cggatgccat ccgtgcaggt 720

gccggtgctg tgatgtgctc gtacaaccag atcaacaaca gctatggctg ccaaaacagc 780

tacactctga acaagctgct caaggctgag ctgggtttcc agggctttgt catgagtgat 840

tgggcggctc accatgccgg tgtgagtggt gctttggcgg gattggacat gtctatgccg 900

ggagacgtcg attacgacag tggcacgtct tactggggta ccaacttgac catcagtgtg 960

ctcaacggga cggcgcccca atggcgtgtt gatgacatgg ctgtccgcat catggccgcc 1020

tactacaagg tcggccgtga ccgtctgtgg actcctccca acttcagctc atggaccaga 1080

gatgaatacg gcttcaagta ctactatgtc tcggagggac cgtatgagaa ggtcaaccag 1140

ttcgtgaacg tgcaacgcaa ccatagcgag ttgatccgcc gtattggagc agacagcacg 1200

gtgctcctca agaacgatgg cgctcttccc ttgactggaa aggagcgctt ggtcgccctt 1260

atcggagaag atgcgggttc caatccttat ggtgccaacg gctgcagtga ccgtgggtgc 1320

gacaatggaa cattggcgat gggctgggga agtggcactg ccaactttcc ctacttggtg 1380

acccccgagc aggccatctc gaacgaggtg ctcaagaaca agaatggcgt attcactgcg 1440

accgataact gggctattga tcagattgag gcgcttgcta agaccgccag tgtctctctt 1500

gtctttgtca acgccgactc tggtgagggt tatatcaatg tcgacggaaa cctgggtgac 1560

cgcaggaacc tgaccctgtg gaggaacggc gacaatgtga tcaaggctgc tgctagcaac 1620

tgcaacaaca cgatcgttat tattcactct gtcggcccag tcttggttaa cgagtggtac 1680

gacaacccca atgttaccgc tattctctgg ggtggtcttc ccggtcagga gtctggcaac 1740

tccctcgccg acgtgctcta cggccgtgtc aaccccggtg ccaagtcgcc cttcacctgg 1800

ggcaagactc gtgaggccta ccaagattac ttgtacaccg agcccaacaa cggcaacgga 1860

gcgccccagg aagacttcgt cgagggcgtc ttcattgact accgcggatt tgacaagcgc 1920

aacgagactc ctatctatga gttcggctat ggtctgagct acaccacctt caactactcg 1980

aaccttcagg tggaggttct gagcgcccct gcgtacgagc ctgcttcggg cgagactgag 2040

gcagcgccga ctttcggaga ggtcggaaat gcgtcggatt acctctaccc cgatggactg 2100

cagagaatca ccaagttcat ctacccctgg ctcaacagta ccgatcttga ggcgtcttct 2160

ggggatgcta gctatgggca ggatgcctca gactatcttc ccgagggagc caccgatggc 2220

tctgcgcaac cgatcctgcc tgccggtggt ggtgctggcg gcaaccctcg cctgtacgac 2280

gagctcatcc gcgtgtcggt gactatcaag aacaccggca aggttgcggg tgatgaagtt 2340

cctcaactgt atgtttctct tggcggccct aacgaaccca agatcgtgct gcgtcaattc 2400

gagcgtatca cgctgcagcc gtcgaaagag acgcagtgga gcacaactct gacgcgccgt 2460

gaccttgcga actggaatgt tgagacgcag gactgggaga ttacgtcgta tcccaagatg 2520

gtgtttgccg gaagctcctc gcggaagctg ccgctccggg cgtctctgcc tactgttcac 2580

taa 2583

<210> 6

<211> 1380

<212> DNA

<213> UGT94-289-2 nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 6

atggatgccc aacaaggtca taccaccacc attttaatgc tgccgtgggt tggctatggc 60

catttactgc cgtttctgga actggccaaa tctttaagtc gccgtaaact gttccacatc 120

tatttctgca gcaccagcgt ttctttagac gccatcaaac ctaaactgcc gccgagtatc 180

agtagtgacg acagtatcca gctggttgag ctgcgtctgc ctagtagtcc ggaactgccg 240

ccgcatctgc acaccaccaa tggtctgcct agccatctga tgccggcttt acaccaagct 300

tttgtgatgg ccgcacagca ctttcaagtt attctgcaga ctttagcccc gcatttactg 360

atttacgaca ttttacaacc gtgggccccg caagttgcaa gctctttaaa cattccggcc 420

atcaacttta gcacaaccgg tgccagcatg ctgagccgta ctttacatcc gacacattac 480

ccgagcagca aattccctat cagcgaattc gtgctgcaca atcactggcg cgcaatgtat 540

accaccgccg acggcgcact gaccgaagaa ggtcacaaga tcgaggagac tttagccaat 600

tgtctgcaca ccagttgcgg cgtggttctg gtgaacagtt ttcgcgaact ggagaccaag 660

tatattgatt atctgagcgt tctgctgaat aagaaagtgg tgccggtggg tccgctggtg 720

tatgaaccga atcaagaagg cgaagacgaa ggctatagca gcattaaaaa ttggctggat 780

aagaaagagc cgagcagcac cgttttcgtg agcttcggca ccgagtactt tccgagtaaa 840

gaagaaatgg aggagattgc ctacggtctg gaactgagcg aggtgaactt tatctgggtt 900

ctgcgctttc cgcaaggtga cagtaccagc accatcgagg acgcactgcc taaaggcttt 960

ctggagcgtg ctggtgaacg tgccatggtg gtgaaaggct gggcaccgca agctaaaatt 1020

ttaaaacact ggagtaccgg cggtttagtt agccactgcg gttggaacag catgatggag 1080

ggcatgatgt ttggcgtgcc gatcatcgcc gttccgatgc atttagatca gccgtttaac 1140

gccggcttag tggaagaagc cggcgtgggt gttgaggcaa agcgtgacag cgatggcaaa 1200

attcagcgcg aagaggtggc caagagtatc aaagaagttg ttatcgagaa aacccgtgaa 1260

gacgtgcgta aaaaagcccg cgagatgggc gaaattttac gcagcaaagg tgatgagaaa 1320

atcgacgagt tagtggccga gatcagtctg ctgcgcaaaa aggccccgtg cagcatttaa 1380

<210> 7

<211> 1380

<212> DNA

<213> M150T/I194G nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 7

atggatgccc aacaaggtca taccaccacc attttaatgc tgccgtgggt tggctatggc 60

catttactgc cgtttctgga actggccaaa tctttaagtc gccgtaaact gttccacatc 120

tatttctgca gcaccagcgt ttctttagac gccatcaaac ctaaactgcc gccgagtatc 180

agtagtgacg acagtatcca gctggttgag ctgcgtctgc ctagtagtcc ggaactgccg 240

ccgcatctgc acaccaccaa tggtctgcct agccatctga tgccggcttt acaccaagct 300

tttgtgatgg ccgcacagca ctttcaagtt attctgcaga ctttagcccc gcatttactg 360

atttacgaca ttttacaacc gtgggccccg caagttgcaa gctctttaaa cattccggcc 420

atcaacttta gcacaaccgg tgccagcacc ctgagccgta ctttacatcc gacacattac 480

ccgagcagca aattccctat cagcgaattc gtgctgcaca atcactggcg cgcaatgtat 540

accaccgccg acggcgcact gaccgaagaa ggtcacaagg gcgaggagac tttagccaat 600

tgtctgcaca ccagttgcgg cgtggttctg gtgaacagtt ttcgcgaact ggagaccaag 660

tatattgatt atctgagcgt tctgctgaat aagaaagtgg tgccggtggg tccgctggtg 720

tatgaaccga atcaagaagg cgaagacgaa ggctatagca gcattaaaaa ttggctggat 780

aagaaagagc cgagcagcac cgttttcgtg agcttcggca ccgagtactt tccgagtaaa 840

gaagaaatgg aggagattgc ctacggtctg gaactgagcg aggtgaactt tatctgggtt 900

ctgcgctttc cgcaaggtga cagtaccagc accatcgagg acgcactgcc taaaggcttt 960

ctggagcgtg ctggtgaacg tgccatggtg gtgaaaggct gggcaccgca agctaaaatt 1020

ttaaaacact ggagtaccgg cggtttagtt agccactgcg gttggaacag catgatggag 1080

ggcatgatgt ttggcgtgcc gatcatcgcc gttccgatgc atttagatca gccgtttaac 1140

gccggcttag tggaagaagc cggcgtgggt gttgaggcaa agcgtgacag cgatggcaaa 1200

attcagcgcg aagaggtggc caagagtatc aaagaagttg ttatcgagaa aacccgtgaa 1260

gacgtgcgta aaaaagcccg cgagatgggc gaaattttac gcagcaaagg tgatgagaaa 1320

atcgacgagt tagtggccga gatcagtctg ctgcgcaaaa aggccccgtg cagcatttaa 1380

<210> 8

<211> 1380

<212> DNA

<213> T154F/I194G nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 8

atggatgccc aacaaggtca taccaccacc attttaatgc tgccgtgggt tggctatggc 60

catttactgc cgtttctgga actggccaaa tctttaagtc gccgtaaact gttccacatc 120

tatttctgca gcaccagcgt ttctttagac gccatcaaac ctaaactgcc gccgagtatc 180

agtagtgacg acagtatcca gctggttgag ctgcgtctgc ctagtagtcc ggaactgccg 240

ccgcatctgc acaccaccaa tggtctgcct agccatctga tgccggcttt acaccaagct 300

tttgtgatgg ccgcacagca ctttcaagtt attctgcaga ctttagcccc gcatttactg 360

atttacgaca ttttacaacc gtgggccccg caagttgcaa gctctttaaa cattccggcc 420

atcaacttta gcacaaccgg tgccagcatg ctgagccgtt tcttacatcc gacacattac 480

ccgagcagca aattccctat cagcgaattc gtgctgcaca atcactggcg cgcaatgtat 540

accaccgccg acggcgcact gaccgaagaa ggtcacaagg gcgaggagac tttagccaat 600

tgtctgcaca ccagttgcgg cgtggttctg gtgaacagtt ttcgcgaact ggagaccaag 660

tatattgatt atctgagcgt tctgctgaat aagaaagtgg tgccggtggg tccgctggtg 720

tatgaaccga atcaagaagg cgaagacgaa ggctatagca gcattaaaaa ttggctggat 780

aagaaagagc cgagcagcac cgttttcgtg agcttcggca ccgagtactt tccgagtaaa 840

gaagaaatgg aggagattgc ctacggtctg gaactgagcg aggtgaactt tatctgggtt 900

ctgcgctttc cgcaaggtga cagtaccagc accatcgagg acgcactgcc taaaggcttt 960

ctggagcgtg ctggtgaacg tgccatggtg gtgaaaggct gggcaccgca agctaaaatt 1020

ttaaaacact ggagtaccgg cggtttagtt agccactgcg gttggaacag catgatggag 1080

ggcatgatgt ttggcgtgcc gatcatcgcc gttccgatgc atttagatca gccgtttaac 1140

gccggcttag tggaagaagc cggcgtgggt gttgaggcaa agcgtgacag cgatggcaaa 1200

attcagcgcg aagaggtggc caagagtatc aaagaagttg ttatcgagaa aacccgtgaa 1260

gacgtgcgta aaaaagcccg cgagatgggc gaaattttac gcagcaaagg tgatgagaaa 1320

atcgacgagt tagtggccga gatcagtctg ctgcgcaaaa aggccccgtg cagcatttaa 1380

<210> 9

<211> 1380

<212> DNA/RNA

<213> L97W/I194G nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 9

atggatgccc aacaaggtca taccaccacc attttaatgc tgccgtgggt tggctatggc 60

catttactgc cgtttctgga actggccaaa tctttaagtc gccgtaaact gttccacatc 120

tatttctgca gcaccagcgt ttctttagac gccatcaaac ctaaactgcc gccgagtatc 180

agtagtgacg acagtatcca gctggttgag ctgcgtctgc ctagtagtcc ggaactgccg 240

ccgcatctgc acaccaccaa tggtctgcct agccatctga tgccggctug gcaccaagct 300

tttgtgatgg ccgcacagca ctttcaagtt attctgcaga ctttagcccc gcatttactg 360

atttacgaca ttttacaacc gtgggccccg caagttgcaa gctctttaaa cattccggcc 420

atcaacttta gcacaaccgg tgccagcatg ctgagccgta ctttacatcc gacacattac 480

ccgagcagca aattccctat cagcgaattc gtgctgcaca atcactggcg cgcaatgtat 540

accaccgccg acggcgcact gaccgaagaa ggtcacaagg gcgaggagac tttagccaat 600

tgtctgcaca ccagttgcgg cgtggttctg gtgaacagtt ttcgcgaact ggagaccaag 660

tatattgatt atctgagcgt tctgctgaat aagaaagtgg tgccggtggg tccgctggtg 720

tatgaaccga atcaagaagg cgaagacgaa ggctatagca gcattaaaaa ttggctggat 780

aagaaagagc cgagcagcac cgttttcgtg agcttcggca ccgagtactt tccgagtaaa 840

gaagaaatgg aggagattgc ctacggtctg gaactgagcg aggtgaactt tatctgggtt 900

ctgcgctttc cgcaaggtga cagtaccagc accatcgagg acgcactgcc taaaggcttt 960

ctggagcgtg ctggtgaacg tgccatggtg gtgaaaggct gggcaccgca agctaaaatt 1020

ttaaaacact ggagtaccgg cggtttagtt agccactgcg gttggaacag catgatggag 1080

ggcatgatgt ttggcgtgcc gatcatcgcc gttccgatgc atttagatca gccgtttaac 1140

gccggcttag tggaagaagc cggcgtgggt gttgaggcaa agcgtgacag cgatggcaaa 1200

attcagcgcg aagaggtggc caagagtatc aaagaagttg ttatcgagaa aacccgtgaa 1260

gacgtgcgta aaaaagcccg cgagatgggc gaaattttac gcagcaaagg tgatgagaaa 1320

atcgacgagt tagtggccga gatcagtctg ctgcgcaaaa aggccccgtg cagcatttaa 1380

<210> 10

<211> 1380

<212> DNA/RNA

<213> T154F/T182P nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 10

atggatgccc aacaaggtca taccaccacc attttaatgc tgccgtgggt tggctatggc 60

catttactgc cgtttctgga actggccaaa tctttaagtc gccgtaaact gttccacatc 120

tatttctgca gcaccagcgt ttctttagac gccatcaaac ctaaactgcc gccgagtatc 180

agtagtgacg acagtatcca gctggttgag ctgcgtctgc ctagtagtcc ggaactgccg 240

ccgcatctgc acaccaccaa tggtctgcct agccatctga tgccggcttt acaccaagct 300

tttgtgatgg ccgcacagca ctttcaagtt attctgcaga ctttagcccc gcatttactg 360

atttacgaca ttttacaacc gtgggccccg caagttgcaa gctctttaaa cattccggcc 420

atcaacttta gcacaaccgg tgccagcatg ctgagccgtt tcttacatcc gacacattac 480

ccgagcagca aattccctat cagcgaattc gtgctgcaca atcactggcg cgcaatgtat 540

accccggccg acggcgcact gaccgaagaa ggtcacaaga tcgaggagac tttagccaat 600

tgtctgcaca ccagttgcgg cgtggttctg gtgaacagtt ttcgcgaact ggagaccaag 660

tatattgatt atctgagcgt tctgctgaat aagaaagtgg tgccggtggg tccgctggtg 720

tatgaaccga atcaagaagg cgaagacgaa ggctatagca gcattaaaaa ttggctggat 780

aagaaagagc cgagcagcac cgttttcgtg agcttcggca ccgagtactt tccgagtaaa 840

gaagaaatgg aggagattgc ctacggtctg gaactgagcg aggtgaactt tatctgggtt 900

ctgcgctttc cgcaaggtga cagtaccagc accatcgagg acgcactgcc taaaggcttt 960

ctggagcgtg ctggtgaacg tgccatggtg gtgaaaggct gggcaccgca agctaaaatt 1020

ttaaaacact ggagtaccgg cggtttagtt agccactgcg gttggaacag catgatggag 1080

ggcatgatgt ttggcgtgcc gatcatcgcc gttccgatgc atttagatca gccgtttaac 1140

gccggcttag tggaagaagc cggcgtgggt gttgaggcaa agcgtgacag cgatggcaaa 1200

attcagcgcg aagaggtggc caagagtatc aaagaagttg ttatcgagaa aacccgtgaa 1260

gacgtgcgta aaaaagcccg cgagatgggc gaaattttac gcagcaaagg tgatgagaaa 1320

atcgacgagt tagtggccga gatcagtctg ctgcgcaaaa aggccccgtg cagcatttaa 1380

<210> 11

<211> 1380

<212> DNA/RNA

<213> T154F/T181D nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 11

atggatgccc aacaaggtca taccaccacc attttaatgc tgccgtgggt tggctatggc 60

catttactgc cgtttctgga actggccaaa tctttaagtc gccgtaaact gttccacatc 120

tatttctgca gcaccagcgt ttctttagac gccatcaaac ctaaactgcc gccgagtatc 180

agtagtgacg acagtatcca gctggttgag ctgcgtctgc ctagtagtcc ggaactgccg 240

ccgcatctgc acaccaccaa tggtctgcct agccatctga tgccggcttt acaccaagct 300

tttgtgatgg ccgcacagca ctttcaagtt attctgcaga ctttagcccc gcatttactg 360

atttacgaca ttttacaacc gtgggccccg caagttgcaa gctctttaaa cattccggcc 420

atcaacttta gcacaaccgg tgccagcatg ctgagccgtt tcttacatcc gacacattac 480

ccgagcagca aattccctat cagcgaattc gtgctgcaca atcactggcg cgcaatgtat 540

gacaccgccg acggcgcact gaccgaagaa ggtcacaaga tcgaggagac tttagccaat 600

tgtctgcaca ccagttgcgg cgtggttctg gtgaacagtt ttcgcgaact ggagaccaag 660

tatattgatt atctgagcgt tctgctgaat aagaaagtgg tgccggtggg tccgctggtg 720

tatgaaccga atcaagaagg cgaagacgaa ggctatagca gcattaaaaa ttggctggat 780

aagaaagagc cgagcagcac cgttttcgtg agcttcggca ccgagtactt tccgagtaaa 840

gaagaaatgg aggagattgc ctacggtctg gaactgagcg aggtgaactt tatctgggtt 900

ctgcgctttc cgcaaggtga cagtaccagc accatcgagg acgcactgcc taaaggcttt 960

ctggagcgtg ctggtgaacg tgccatggtg gtgaaaggct gggcaccgca agctaaaatt 1020

ttaaaacact ggagtaccgg cggtttagtt agccactgcg gttggaacag catgatggag 1080

ggcatgatgt ttggcgtgcc gatcatcgcc gttccgatgc atttagatca gccgtttaac 1140

gccggcttag tggaagaagc cggcgtgggt gttgaggcaa agcgtgacag cgatggcaaa 1200

attcagcgcg aagaggtggc caagagtatc aaagaagttg ttatcgagaa aacccgtgaa 1260

gacgtgcgta aaaaagcccg cgagatgggc gaaattttac gcagcaaagg tgatgagaaa 1320

atcgacgagt tagtggccga gatcagtctg ctgcgcaaaa aggccccgtg cagcatttaa 1380

<210> 12

<211> 1380

<212> DNA/RNA

<213> M150T/T181D nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 12

atggatgccc aacaaggtca taccaccacc attttaatgc tgccgtgggt tggctatggc 60

catttactgc cgtttctgga actggccaaa tctttaagtc gccgtaaact gttccacatc 120

tatttctgca gcaccagcgt ttctttagac gccatcaaac ctaaactgcc gccgagtatc 180

agtagtgacg acagtatcca gctggttgag ctgcgtctgc ctagtagtcc ggaactgccg 240

ccgcatctgc acaccaccaa tggtctgcct agccatctga tgccggcttt acaccaagct 300

tttgtgatgg ccgcacagca ctttcaagtt attctgcaga ctttagcccc gcatttactg 360

atttacgaca ttttacaacc gtgggccccg caagttgcaa gctctttaaa cattccggcc 420

atcaacttta gcacaaccgg tgccagcacc ctgagccgta ctttacatcc gacacattac 480

ccgagcagca aattccctat cagcgaattc gtgctgcaca atcactggcg cgcaatgtat 540

gacaccgccg acggcgcact gaccgaagaa ggtcacaaga tcgaggagac tttagccaat 600

tgtctgcaca ccagttgcgg cgtggttctg gtgaacagtt ttcgcgaact ggagaccaag 660

tatattgatt atctgagcgt tctgctgaat aagaaagtgg tgccggtggg tccgctggtg 720

tatgaaccga atcaagaagg cgaagacgaa ggctatagca gcattaaaaa ttggctggat 780

aagaaagagc cgagcagcac cgttttcgtg agcttcggca ccgagtactt tccgagtaaa 840

gaagaaatgg aggagattgc ctacggtctg gaactgagcg aggtgaactt tatctgggtt 900

ctgcgctttc cgcaaggtga cagtaccagc accatcgagg acgcactgcc taaaggcttt 960

ctggagcgtg ctggtgaacg tgccatggtg gtgaaaggct gggcaccgca agctaaaatt 1020

ttaaaacact ggagtaccgg cggtttagtt agccactgcg gttggaacag catgatggag 1080

ggcatgatgt ttggcgtgcc gatcatcgcc gttccgatgc atttagatca gccgtttaac 1140

gccggcttag tggaagaagc cggcgtgggt gttgaggcaa agcgtgacag cgatggcaaa 1200

attcagcgcg aagaggtggc caagagtatc aaagaagttg ttatcgagaa aacccgtgaa 1260

gacgtgcgta aaaaagcccg cgagatgggc gaaattttac gcagcaaagg tgatgagaaa 1320

atcgacgagt tagtggccga gatcagtctg ctgcgcaaaa aggccccgtg cagcatttaa 1380

<210> 13

<211> 1380

<212> DNA/RNA

<213> T181D/I194G nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 13

atggatgccc aacaaggtca taccaccacc attttaatgc tgccgtgggt tggctatggc 60

catttactgc cgtttctgga actggccaaa tctttaagtc gccgtaaact gttccacatc 120

tatttctgca gcaccagcgt ttctttagac gccatcaaac ctaaactgcc gccgagtatc 180

agtagtgacg acagtatcca gctggttgag ctgcgtctgc ctagtagtcc ggaactgccg 240

ccgcatctgc acaccaccaa tggtctgcct agccatctga tgccggcttt acaccaagct 300

tttgtgatgg ccgcacagca ctttcaagtt attctgcaga ctttagcccc gcatttactg 360

atttacgaca ttttacaacc gtgggccccg caagttgcaa gctctttaaa cattccggcc 420

atcaacttta gcacaaccgg tgccagcatg ctgagccgta ctttacatcc gacacattac 480

ccgagcagca aattccctat cagcgaattc gtgctgcaca atcactggcg cgcaatgtat 540

gacaccgccg acggcgcact gaccgaagaa ggtcacaagg gcgaggagac tttagccaat 600

tgtctgcaca ccagttgcgg cgtggttctg gtgaacagtt ttcgcgaact ggagaccaag 660

tatattgatt atctgagcgt tctgctgaat aagaaagtgg tgccggtggg tccgctggtg 720

tatgaaccga atcaagaagg cgaagacgaa ggctatagca gcattaaaaa ttggctggat 780

aagaaagagc cgagcagcac cgttttcgtg agcttcggca ccgagtactt tccgagtaaa 840

gaagaaatgg aggagattgc ctacggtctg gaactgagcg aggtgaactt tatctgggtt 900

ctgcgctttc cgcaaggtga cagtaccagc accatcgagg acgcactgcc taaaggcttt 960

ctggagcgtg ctggtgaacg tgccatggtg gtgaaaggct gggcaccgca agctaaaatt 1020

ttaaaacact ggagtaccgg cggtttagtt agccactgcg gttggaacag catgatggag 1080

ggcatgatgt ttggcgtgcc gatcatcgcc gttccgatgc atttagatca gccgtttaac 1140

gccggcttag tggaagaagc cggcgtgggt gttgaggcaa agcgtgacag cgatggcaaa 1200

attcagcgcg aagaggtggc caagagtatc aaagaagttg ttatcgagaa aacccgtgaa 1260

gacgtgcgta aaaaagcccg cgagatgggc gaaattttac gcagcaaagg tgatgagaaa 1320

atcgacgagt tagtggccga gatcagtctg ctgcgcaaaa aggccccgtg cagcatttaa 1380

<210> 14

<211> 1380

<212> DNA/RNA

<213> T181D/T182P nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 14

atggatgccc aacaaggtca taccaccacc attttaatgc tgccgtgggt tggctatggc 60

catttactgc cgtttctgga actggccaaa tctttaagtc gccgtaaact gttccacatc 120

tatttctgca gcaccagcgt ttctttagac gccatcaaac ctaaactgcc gccgagtatc 180

agtagtgacg acagtatcca gctggttgag ctgcgtctgc ctagtagtcc ggaactgccg 240

ccgcatctgc acaccaccaa tggtctgcct agccatctga tgccggcttt acaccaagct 300

tttgtgatgg ccgcacagca ctttcaagtt attctgcaga ctttagcccc gcatttactg 360

atttacgaca ttttacaacc gtgggccccg caagttgcaa gctctttaaa cattccggcc 420

atcaacttta gcacaaccgg tgccagcatg ctgagccgta ctttacatcc gacacattac 480

ccgagcagca aattccctat cagcgaattc gtgctgcaca atcactggcg cgcaatgtat 540

gacccggccg acggcgcact gaccgaagaa ggtcacaaga tcgaggagac tttagccaat 600

tgtctgcaca ccagttgcgg cgtggttctg gtgaacagtt ttcgcgaact ggagaccaag 660

tatattgatt atctgagcgt tctgctgaat aagaaagtgg tgccggtggg tccgctggtg 720

tatgaaccga atcaagaagg cgaagacgaa ggctatagca gcattaaaaa ttggctggat 780

aagaaagagc cgagcagcac cgttttcgtg agcttcggca ccgagtactt tccgagtaaa 840

gaagaaatgg aggagattgc ctacggtctg gaactgagcg aggtgaactt tatctgggtt 900

ctgcgctttc cgcaaggtga cagtaccagc accatcgagg acgcactgcc taaaggcttt 960

ctggagcgtg ctggtgaacg tgccatggtg gtgaaaggct gggcaccgca agctaaaatt 1020

ttaaaacact ggagtaccgg cggtttagtt agccactgcg gttggaacag catgatggag 1080

ggcatgatgt ttggcgtgcc gatcatcgcc gttccgatgc atttagatca gccgtttaac 1140

gccggcttag tggaagaagc cggcgtgggt gttgaggcaa agcgtgacag cgatggcaaa 1200

attcagcgcg aagaggtggc caagagtatc aaagaagttg ttatcgagaa aacccgtgaa 1260

gacgtgcgta aaaaagcccg cgagatgggc gaaattttac gcagcaaagg tgatgagaaa 1320

atcgacgagt tagtggccga gatcagtctg ctgcgcaaaa aggccccgtg cagcatttaa 1380

<210> 15

<211> 1380

<212> DNA/RNA

<213> T182P/I194G nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 15

atggatgccc aacaaggtca taccaccacc attttaatgc tgccgtgggt tggctatggc 60

catttactgc cgtttctgga actggccaaa tctttaagtc gccgtaaact gttccacatc 120

tatttctgca gcaccagcgt ttctttagac gccatcaaac ctaaactgcc gccgagtatc 180

agtagtgacg acagtatcca gctggttgag ctgcgtctgc ctagtagtcc ggaactgccg 240

ccgcatctgc acaccaccaa tggtctgcct agccatctga tgccggcttt acaccaagct 300

tttgtgatgg ccgcacagca ctttcaagtt attctgcaga ctttagcccc gcatttactg 360

atttacgaca ttttacaacc gtgggccccg caagttgcaa gctctttaaa cattccggcc 420

atcaacttta gcacaaccgg tgccagcatg ctgagccgta ctttacatcc gacacattac 480

ccgagcagca aattccctat cagcgaattc gtgctgcaca atcactggcg cgcaatgtat 540

accccggccg acggcgcact gaccgaagaa ggtcacaagg gcgaggagac tttagccaat 600

tgtctgcaca ccagttgcgg cgtggttctg gtgaacagtt ttcgcgaact ggagaccaag 660

tatattgatt atctgagcgt tctgctgaat aagaaagtgg tgccggtggg tccgctggtg 720

tatgaaccga atcaagaagg cgaagacgaa ggctatagca gcattaaaaa ttggctggat 780

aagaaagagc cgagcagcac cgttttcgtg agcttcggca ccgagtactt tccgagtaaa 840

gaagaaatgg aggagattgc ctacggtctg gaactgagcg aggtgaactt tatctgggtt 900

ctgcgctttc cgcaaggtga cagtaccagc accatcgagg acgcactgcc taaaggcttt 960

ctggagcgtg ctggtgaacg tgccatggtg gtgaaaggct gggcaccgca agctaaaatt 1020

ttaaaacact ggagtaccgg cggtttagtt agccactgcg gttggaacag catgatggag 1080

ggcatgatgt ttggcgtgcc gatcatcgcc gttccgatgc atttagatca gccgtttaac 1140

gccggcttag tggaagaagc cggcgtgggt gttgaggcaa agcgtgacag cgatggcaaa 1200

attcagcgcg aagaggtggc caagagtatc aaagaagttg ttatcgagaa aacccgtgaa 1260

gacgtgcgta aaaaagcccg cgagatgggc gaaattttac gcagcaaagg tgatgagaaa 1320

atcgacgagt tagtggccga gatcagtctg ctgcgcaaaa aggccccgtg cagcatttaa 1380

<210> 16

<211> 1380

<212> DNA/RNA

<213> T181D/T182P/I194G nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 16

atggatgccc aacaaggtca taccaccacc attttaatgc tgccgtgggt tggctatggc 60

catttactgc cgtttctgga actggccaaa tctttaagtc gccgtaaact gttccacatc 120

tatttctgca gcaccagcgt ttctttagac gccatcaaac ctaaactgcc gccgagtatc 180

agtagtgacg acagtatcca gctggttgag ctgcgtctgc ctagtagtcc ggaactgccg 240

ccgcatctgc acaccaccaa tggtctgcct agccatctga tgccggcttt acaccaagct 300

tttgtgatgg ccgcacagca ctttcaagtt attctgcaga ctttagcccc gcatttactg 360

atttacgaca ttttacaacc gtgggccccg caagttgcaa gctctttaaa cattccggcc 420

atcaacttta gcacaaccgg tgccagcatg ctgagccgta ctttacatcc gacacattac 480

ccgagcagca aattccctat cagcgaattc gtgctgcaca atcactggcg cgcaatgtat 540

gacccggccg acggcgcact gaccgaagaa ggtcacaagg gcgaggagac tttagccaat 600

tgtctgcaca ccagttgcgg cgtggttctg gtgaacagtt ttcgcgaact ggagaccaag 660

tatattgatt atctgagcgt tctgctgaat aagaaagtgg tgccggtggg tccgctggtg 720

tatgaaccga atcaagaagg cgaagacgaa ggctatagca gcattaaaaa ttggctggat 780

aagaaagagc cgagcagcac cgttttcgtg agcttcggca ccgagtactt tccgagtaaa 840

gaagaaatgg aggagattgc ctacggtctg gaactgagcg aggtgaactt tatctgggtt 900

ctgcgctttc cgcaaggtga cagtaccagc accatcgagg acgcactgcc taaaggcttt 960

ctggagcgtg ctggtgaacg tgccatggtg gtgaaaggct gggcaccgca agctaaaatt 1020

ttaaaacact ggagtaccgg cggtttagtt agccactgcg gttggaacag catgatggag 1080

ggcatgatgt ttggcgtgcc gatcatcgcc gttccgatgc atttagatca gccgtttaac 1140

gccggcttag tggaagaagc cggcgtgggt gttgaggcaa agcgtgacag cgatggcaaa 1200

attcagcgcg aagaggtggc caagagtatc aaagaagttg ttatcgagaa aacccgtgaa 1260

gacgtgcgta aaaaagcccg cgagatgggc gaaattttac gcagcaaagg tgatgagaaa 1320

atcgacgagt tagtggccga gatcagtctg ctgcgcaaaa aggccccgtg cagcatttaa 1380

<210> 17

<211> 1380

<212> DNA/RNA

<213> L97W/T181D/T182P nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 17

atggatgccc aacaaggtca taccaccacc attttaatgc tgccgtgggt tggctatggc 60

catttactgc cgtttctgga actggccaaa tctttaagtc gccgtaaact gttccacatc 120

tatttctgca gcaccagcgt ttctttagac gccatcaaac ctaaactgcc gccgagtatc 180

agtagtgacg acagtatcca gctggttgag ctgcgtctgc ctagtagtcc ggaactgccg 240

ccgcatctgc acaccaccaa tggtctgcct agccatctga tgccggctug gcaccaagct 300

tttgtgatgg ccgcacagca ctttcaagtt attctgcaga ctttagcccc gcatttactg 360

atttacgaca ttttacaacc gtgggccccg caagttgcaa gctctttaaa cattccggcc 420

atcaacttta gcacaaccgg tgccagcatg ctgagccgta ctttacatcc gacacattac 480

ccgagcagca aattccctat cagcgaattc gtgctgcaca atcactggcg cgcaatgtat 540

gacccggccg acggcgcact gaccgaagaa ggtcacaaga tcgaggagac tttagccaat 600

tgtctgcaca ccagttgcgg cgtggttctg gtgaacagtt ttcgcgaact ggagaccaag 660

tatattgatt atctgagcgt tctgctgaat aagaaagtgg tgccggtggg tccgctggtg 720

tatgaaccga atcaagaagg cgaagacgaa ggctatagca gcattaaaaa ttggctggat 780

aagaaagagc cgagcagcac cgttttcgtg agcttcggca ccgagtactt tccgagtaaa 840

gaagaaatgg aggagattgc ctacggtctg gaactgagcg aggtgaactt tatctgggtt 900

ctgcgctttc cgcaaggtga cagtaccagc accatcgagg acgcactgcc taaaggcttt 960

ctggagcgtg ctggtgaacg tgccatggtg gtgaaaggct gggcaccgca agctaaaatt 1020

ttaaaacact ggagtaccgg cggtttagtt agccactgcg gttggaacag catgatggag 1080

ggcatgatgt ttggcgtgcc gatcatcgcc gttccgatgc atttagatca gccgtttaac 1140

gccggcttag tggaagaagc cggcgtgggt gttgaggcaa agcgtgacag cgatggcaaa 1200

attcagcgcg aagaggtggc caagagtatc aaagaagttg ttatcgagaa aacccgtgaa 1260

gacgtgcgta aaaaagcccg cgagatgggc gaaattttac gcagcaaagg tgatgagaaa 1320

atcgacgagt tagtggccga gatcagtctg ctgcgcaaaa aggccccgtg cagcatttaa 1380

<210> 18

<211> 1380

<212> DNA/RNA

<213> L97W/T181D/I194G nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 18

atggatgccc aacaaggtca taccaccacc attttaatgc tgccgtgggt tggctatggc 60

catttactgc cgtttctgga actggccaaa tctttaagtc gccgtaaact gttccacatc 120

tatttctgca gcaccagcgt ttctttagac gccatcaaac ctaaactgcc gccgagtatc 180

agtagtgacg acagtatcca gctggttgag ctgcgtctgc ctagtagtcc ggaactgccg 240

ccgcatctgc acaccaccaa tggtctgcct agccatctga tgccggctug gcaccaagct 300

tttgtgatgg ccgcacagca ctttcaagtt attctgcaga ctttagcccc gcatttactg 360

atttacgaca ttttacaacc gtgggccccg caagttgcaa gctctttaaa cattccggcc 420

atcaacttta gcacaaccgg tgccagcatg ctgagccgta ctttacatcc gacacattac 480

ccgagcagca aattccctat cagcgaattc gtgctgcaca atcactggcg cgcaatgtat 540

gacaccgccg acggcgcact gaccgaagaa ggtcacaagg gcgaggagac tttagccaat 600

tgtctgcaca ccagttgcgg cgtggttctg gtgaacagtt ttcgcgaact ggagaccaag 660

tatattgatt atctgagcgt tctgctgaat aagaaagtgg tgccggtggg tccgctggtg 720

tatgaaccga atcaagaagg cgaagacgaa ggctatagca gcattaaaaa ttggctggat 780

aagaaagagc cgagcagcac cgttttcgtg agcttcggca ccgagtactt tccgagtaaa 840

gaagaaatgg aggagattgc ctacggtctg gaactgagcg aggtgaactt tatctgggtt 900

ctgcgctttc cgcaaggtga cagtaccagc accatcgagg acgcactgcc taaaggcttt 960

ctggagcgtg ctggtgaacg tgccatggtg gtgaaaggct gggcaccgca agctaaaatt 1020

ttaaaacact ggagtaccgg cggtttagtt agccactgcg gttggaacag catgatggag 1080

ggcatgatgt ttggcgtgcc gatcatcgcc gttccgatgc atttagatca gccgtttaac 1140

gccggcttag tggaagaagc cggcgtgggt gttgaggcaa agcgtgacag cgatggcaaa 1200

attcagcgcg aagaggtggc caagagtatc aaagaagttg ttatcgagaa aacccgtgaa 1260

gacgtgcgta aaaaagcccg cgagatgggc gaaattttac gcagcaaagg tgatgagaaa 1320

atcgacgagt tagtggccga gatcagtctg ctgcgcaaaa aggccccgtg cagcatttaa 1380

<210> 19

<211> 1380

<212> DNA/RNA

<213> T146A/T181D/I194G nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 19

atggatgccc aacaaggtca taccaccacc attttaatgc tgccgtgggt tggctatggc 60

catttactgc cgtttctgga actggccaaa tctttaagtc gccgtaaact gttccacatc 120

tatttctgca gcaccagcgt ttctttagac gccatcaaac ctaaactgcc gccgagtatc 180

agtagtgacg acagtatcca gctggttgag ctgcgtctgc ctagtagtcc ggaactgccg 240

ccgcatctgc acaccaccaa tggtctgcct agccatctga tgccggcttt acaccaagct 300

tttgtgatgg ccgcacagca ctttcaagtt attctgcaga ctttagcccc gcatttactg 360

atttacgaca ttttacaacc gtgggccccg caagttgcaa gctctttaaa cattccggcc 420

atcaacttta gcacagccgg tgccagcatg ctgagccgta ctttacatcc gacacattac 480

ccgagcagca aattccctat cagcgaattc gtgctgcaca atcactggcg cgcaatgtat 540

gacaccgccg acggcgcact gaccgaagaa ggtcacaagg gcgaggagac tttagccaat 600

tgtctgcaca ccagttgcgg cgtggttctg gtgaacagtt ttcgcgaact ggagaccaag 660

tatattgatt atctgagcgt tctgctgaat aagaaagtgg tgccggtggg tccgctggtg 720

tatgaaccga atcaagaagg cgaagacgaa ggctatagca gcattaaaaa ttggctggat 780

aagaaagagc cgagcagcac cgttttcgtg agcttcggca ccgagtactt tccgagtaaa 840

gaagaaatgg aggagattgc ctacggtctg gaactgagcg aggtgaactt tatctgggtt 900

ctgcgctttc cgcaaggtga cagtaccagc accatcgagg acgcactgcc taaaggcttt 960

ctggagcgtg ctggtgaacg tgccatggtg gtgaaaggct gggcaccgca agctaaaatt 1020

ttaaaacact ggagtaccgg cggtttagtt agccactgcg gttggaacag catgatggag 1080

ggcatgatgt ttggcgtgcc gatcatcgcc gttccgatgc atttagatca gccgtttaac 1140

gccggcttag tggaagaagc cggcgtgggt gttgaggcaa agcgtgacag cgatggcaaa 1200

attcagcgcg aagaggtggc caagagtatc aaagaagttg ttatcgagaa aacccgtgaa 1260

gacgtgcgta aaaaagcccg cgagatgggc gaaattttac gcagcaaagg tgatgagaaa 1320

atcgacgagt tagtggccga gatcagtctg ctgcgcaaaa aggccccgtg cagcatttaa 1380

<210> 20

<211> 1380

<212> DNA/RNA

<213> T146A/T181D nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 20

atggatgccc aacaaggtca taccaccacc attttaatgc tgccgtgggt tggctatggc 60

catttactgc cgtttctgga actggccaaa tctttaagtc gccgtaaact gttccacatc 120

tatttctgca gcaccagcgt ttctttagac gccatcaaac ctaaactgcc gccgagtatc 180

agtagtgacg acagtatcca gctggttgag ctgcgtctgc ctagtagtcc ggaactgccg 240

ccgcatctgc acaccaccaa tggtctgcct agccatctga tgccggcttt acaccaagct 300

tttgtgatgg ccgcacagca ctttcaagtt attctgcaga ctttagcccc gcatttactg 360

atttacgaca ttttacaacc gtgggccccg caagttgcaa gctctttaaa cattccggcc 420

atcaacttta gcacagccgg tgccagcatg ctgagccgta ctttacatcc gacacattac 480

ccgagcagca aattccctat cagcgaattc gtgctgcaca atcactggcg cgcaatgtat 540

gacaccgccg acggcgcact gaccgaagaa ggtcacaaga tcgaggagac tttagccaat 600

tgtctgcaca ccagttgcgg cgtggttctg gtgaacagtt ttcgcgaact ggagaccaag 660

tatattgatt atctgagcgt tctgctgaat aagaaagtgg tgccggtggg tccgctggtg 720

tatgaaccga atcaagaagg cgaagacgaa ggctatagca gcattaaaaa ttggctggat 780

aagaaagagc cgagcagcac cgttttcgtg agcttcggca ccgagtactt tccgagtaaa 840

gaagaaatgg aggagattgc ctacggtctg gaactgagcg aggtgaactt tatctgggtt 900

ctgcgctttc cgcaaggtga cagtaccagc accatcgagg acgcactgcc taaaggcttt 960

ctggagcgtg ctggtgaacg tgccatggtg gtgaaaggct gggcaccgca agctaaaatt 1020

ttaaaacact ggagtaccgg cggtttagtt agccactgcg gttggaacag catgatggag 1080

ggcatgatgt ttggcgtgcc gatcatcgcc gttccgatgc atttagatca gccgtttaac 1140

gccggcttag tggaagaagc cggcgtgggt gttgaggcaa agcgtgacag cgatggcaaa 1200

attcagcgcg aagaggtggc caagagtatc aaagaagttg ttatcgagaa aacccgtgaa 1260

gacgtgcgta aaaaagcccg cgagatgggc gaaattttac gcagcaaagg tgatgagaaa 1320

atcgacgagt tagtggccga gatcagtctg ctgcgcaaaa aggccccgtg cagcatttaa 1380

<210> 21

<211> 1380

<212> DNA/RNA

<213> L125A/T181D/I194G nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 21

atggatgccc aacaaggtca taccaccacc attttaatgc tgccgtgggt tggctatggc 60

catttactgc cgtttctgga actggccaaa tctttaagtc gccgtaaact gttccacatc 120

tatttctgca gcaccagcgt ttctttagac gccatcaaac ctaaactgcc gccgagtatc 180

agtagtgacg acagtatcca gctggttgag ctgcgtctgc ctagtagtcc ggaactgccg 240

ccgcatctgc acaccaccaa tggtctgcct agccatctga tgccggcttt acaccaagct 300

tttgtgatgg ccgcacagca ctttcaagtt attctgcaga ctttagcccc gcatttactg 360

atttacgaca ttgcccaacc gtgggccccg caagttgcaa gctctttaaa cattccggcc 420

atcaacttta gcacaaccgg tgccagcatg ctgagccgta ctttacatcc gacacattac 480

ccgagcagca aattccctat cagcgaattc gtgctgcaca atcactggcg cgcaatgtat 540

gacaccgccg acggcgcact gaccgaagaa ggtcacaagg gcgaggagac tttagccaat 600

tgtctgcaca ccagttgcgg cgtggttctg gtgaacagtt ttcgcgaact ggagaccaag 660

tatattgatt atctgagcgt tctgctgaat aagaaagtgg tgccggtggg tccgctggtg 720

tatgaaccga atcaagaagg cgaagacgaa ggctatagca gcattaaaaa ttggctggat 780

aagaaagagc cgagcagcac cgttttcgtg agcttcggca ccgagtactt tccgagtaaa 840

gaagaaatgg aggagattgc ctacggtctg gaactgagcg aggtgaactt tatctgggtt 900

ctgcgctttc cgcaaggtga cagtaccagc accatcgagg acgcactgcc taaaggcttt 960

ctggagcgtg ctggtgaacg tgccatggtg gtgaaaggct gggcaccgca agctaaaatt 1020

ttaaaacact ggagtaccgg cggtttagtt agccactgcg gttggaacag catgatggag 1080

ggcatgatgt ttggcgtgcc gatcatcgcc gttccgatgc atttagatca gccgtttaac 1140

gccggcttag tggaagaagc cggcgtgggt gttgaggcaa agcgtgacag cgatggcaaa 1200

attcagcgcg aagaggtggc caagagtatc aaagaagttg ttatcgagaa aacccgtgaa 1260

gacgtgcgta aaaaagcccg cgagatgggc gaaattttac gcagcaaagg tgatgagaaa 1320

atcgacgagt tagtggccga gatcagtctg ctgcgcaaaa aggccccgtg cagcatttaa 1380

<210> 22

<211> 1380

<212> DNA/RNA

<213> L125A/T181D nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 22

atggatgccc aacaaggtca taccaccacc attttaatgc tgccgtgggt tggctatggc 60

catttactgc cgtttctgga actggccaaa tctttaagtc gccgtaaact gttccacatc 120

tatttctgca gcaccagcgt ttctttagac gccatcaaac ctaaactgcc gccgagtatc 180

agtagtgacg acagtatcca gctggttgag ctgcgtctgc ctagtagtcc ggaactgccg 240

ccgcatctgc acaccaccaa tggtctgcct agccatctga tgccggcttt acaccaagct 300

tttgtgatgg ccgcacagca ctttcaagtt attctgcaga ctttagcccc gcatttactg 360

atttacgaca ttgcccaacc gtgggccccg caagttgcaa gctctttaaa cattccggcc 420

atcaacttta gcacaaccgg tgccagcatg ctgagccgta ctttacatcc gacacattac 480

ccgagcagca aattccctat cagcgaattc gtgctgcaca atcactggcg cgcaatgtat 540

gacaccgccg acggcgcact gaccgaagaa ggtcacaaga tcgaggagac tttagccaat 600

tgtctgcaca ccagttgcgg cgtggttctg gtgaacagtt ttcgcgaact ggagaccaag 660

tatattgatt atctgagcgt tctgctgaat aagaaagtgg tgccggtggg tccgctggtg 720

tatgaaccga atcaagaagg cgaagacgaa ggctatagca gcattaaaaa ttggctggat 780

aagaaagagc cgagcagcac cgttttcgtg agcttcggca ccgagtactt tccgagtaaa 840

gaagaaatgg aggagattgc ctacggtctg gaactgagcg aggtgaactt tatctgggtt 900

ctgcgctttc cgcaaggtga cagtaccagc accatcgagg acgcactgcc taaaggcttt 960

ctggagcgtg ctggtgaacg tgccatggtg gtgaaaggct gggcaccgca agctaaaatt 1020

ttaaaacact ggagtaccgg cggtttagtt agccactgcg gttggaacag catgatggag 1080

ggcatgatgt ttggcgtgcc gatcatcgcc gttccgatgc atttagatca gccgtttaac 1140

gccggcttag tggaagaagc cggcgtgggt gttgaggcaa agcgtgacag cgatggcaaa 1200

attcagcgcg aagaggtggc caagagtatc aaagaagttg ttatcgagaa aacccgtgaa 1260

gacgtgcgta aaaaagcccg cgagatgggc gaaattttac gcagcaaagg tgatgagaaa 1320

atcgacgagt tagtggccga gatcagtctg ctgcgcaaaa aggccccgtg cagcatttaa 1380

<210> 23

<211> 1380

<212> DNA/RNA

<213> K53A/T181D/I194G nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 23

atggatgccc aacaaggtca taccaccacc attttaatgc tgccgtgggt tggctatggc 60

catttactgc cgtttctgga actggccaaa tctttaagtc gccgtaaact gttccacatc 120

tatttctgca gcaccagcgt ttctttagac gccatcgccc ctaaactgcc gccgagtatc 180

agtagtgacg acagtatcca gctggttgag ctgcgtctgc ctagtagtcc ggaactgccg 240

ccgcatctgc acaccaccaa tggtctgcct agccatctga tgccggcttt acaccaagct 300

tttgtgatgg ccgcacagca ctttcaagtt attctgcaga ctttagcccc gcatttactg 360

atttacgaca ttgcccaacc gtgggccccg caagttgcaa gctctttaaa cattccggcc 420

atcaacttta gcacaaccgg tgccagcatg ctgagccgta ctttacatcc gacacattac 480

ccgagcagca aattccctat cagcgaattc gtgctgcaca atcactggcg cgcaatgtat 540

gacaccgccg acggcgcact gaccgaagaa ggtcacaagg gcgaggagac tttagccaat 600

tgtctgcaca ccagttgcgg cgtggttctg gtgaacagtt ttcgcgaact ggagaccaag 660

tatattgatt atctgagcgt tctgctgaat aagaaagtgg tgccggtggg tccgctggtg 720

tatgaaccga atcaagaagg cgaagacgaa ggctatagca gcattaaaaa ttggctggat 780

aagaaagagc cgagcagcac cgttttcgtg agcttcggca ccgagtactt tccgagtaaa 840

gaagaaatgg aggagattgc ctacggtctg gaactgagcg aggtgaactt tatctgggtt 900

ctgcgctttc cgcaaggtga cagtaccagc accatcgagg acgcactgcc taaaggcttt 960

ctggagcgtg ctggtgaacg tgccatggtg gtgaaaggct gggcaccgca agctaaaatt 1020

ttaaaacact ggagtaccgg cggtttagtt agccactgcg gttggaacag catgatggag 1080

ggcatgatgt ttggcgtgcc gatcatcgcc gttccgatgc atttagatca gccgtttaac 1140

gccggcttag tggaagaagc cggcgtgggt gttgaggcaa agcgtgacag cgatggcaaa 1200

attcagcgcg aagaggtggc caagagtatc aaagaagttg ttatcgagaa aacccgtgaa 1260

gacgtgcgta aaaaagcccg cgagatgggc gaaattttac gcagcaaagg tgatgagaaa 1320

atcgacgagt tagtggccga gatcagtctg ctgcgcaaaa aggccccgtg cagcatttaa 1380

<210> 24

<211> 1380

<212> DNA/RNA

<213> K53A/T181D nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 24

atggatgccc aacaaggtca taccaccacc attttaatgc tgccgtgggt tggctatggc 60

catttactgc cgtttctgga actggccaaa tctttaagtc gccgtaaact gttccacatc 120

tatttctgca gcaccagcgt ttctttagac gccatcgccc ctaaactgcc gccgagtatc 180

agtagtgacg acagtatcca gctggttgag ctgcgtctgc ctagtagtcc ggaactgccg 240

ccgcatctgc acaccaccaa tggtctgcct agccatctga tgccggcttt acaccaagct 300

tttgtgatgg ccgcacagca ctttcaagtt attctgcaga ctttagcccc gcatttactg 360

atttacgaca ttgcccaacc gtgggccccg caagttgcaa gctctttaaa cattccggcc 420

atcaacttta gcacaaccgg tgccagcatg ctgagccgta ctttacatcc gacacattac 480

ccgagcagca aattccctat cagcgaattc gtgctgcaca atcactggcg cgcaatgtat 540

gacaccgccg acggcgcact gaccgaagaa ggtcacaaga tcgaggagac tttagccaat 600

tgtctgcaca ccagttgcgg cgtggttctg gtgaacagtt ttcgcgaact ggagaccaag 660

tatattgatt atctgagcgt tctgctgaat aagaaagtgg tgccggtggg tccgctggtg 720

tatgaaccga atcaagaagg cgaagacgaa ggctatagca gcattaaaaa ttggctggat 780

aagaaagagc cgagcagcac cgttttcgtg agcttcggca ccgagtactt tccgagtaaa 840

gaagaaatgg aggagattgc ctacggtctg gaactgagcg aggtgaactt tatctgggtt 900

ctgcgctttc cgcaaggtga cagtaccagc accatcgagg acgcactgcc taaaggcttt 960

ctggagcgtg ctggtgaacg tgccatggtg gtgaaaggct gggcaccgca agctaaaatt 1020

ttaaaacact ggagtaccgg cggtttagtt agccactgcg gttggaacag catgatggag 1080

ggcatgatgt ttggcgtgcc gatcatcgcc gttccgatgc atttagatca gccgtttaac 1140

gccggcttag tggaagaagc cggcgtgggt gttgaggcaa agcgtgacag cgatggcaaa 1200

attcagcgcg aagaggtggc caagagtatc aaagaagttg ttatcgagaa aacccgtgaa 1260

gacgtgcgta aaaaagcccg cgagatgggc gaaattttac gcagcaaagg tgatgagaaa 1320

atcgacgagt tagtggccga gatcagtctg ctgcgcaaaa aggccccgtg cagcatttaa 1380

Claims (8)

1. A method for preparing Siamenoside I is characterized in that glycoside hydrolase is used for selectively hydrolyzing mogroside V to prepare mogroside IIIE, UDP-glucose dependent glycosyltransferase and mutant thereof are used for modifying the mogroside IIIE in a site-directed glycosylation manner to synthesize the Siamenoside I.

2. The method according to claim 1, wherein the glycoside hydrolase is Exg1 derived from Saccharomyces cerevisiae having an amino acid sequence shown in SEQ ID NO 1; or the glycoside hydrolase is Asn from Aspergillus niger, and the amino acid sequence is shown in SEQ ID NO. 2.

3. The method according to claim 1, wherein the UDP-glucose dependent glycosyltransferase is derived from the glycosyltransferase UGT94-289-2 of Siraitia grosvenorii, the amino acid sequence of which is set forth in SEQ ID No. 3;

or the UDP-glucose-dependent glycosyltransferase is a UDP-glucose-dependent glycosyltransferase mutant: single-point or multi-point mutants of W16, K53, L93, M94, L97, I124, L125, T146, M150, T154, T181, T182, I194, E195, E275 and S308 in UGT94-289-2 amino acid sequence.

4. The method according to claim 3, wherein the UDP-glucose dependent glycosyltransferase mutant:

5. the method according to any one of claims 1 to 4, wherein the Siamenoside I is prepared by constructing engineering bacteria to express the glycoside hydrolase, the UDP-glucose-dependent glycosyltransferase and mutants thereof by recombination, wherein the strain is lactic acid bacteria, streptomyces, yeast or Bacillus subtilis.

6. The method according to claim 5, wherein the glycoside hydrolase and the UDP-glucose-dependent glycosyltransferase are co-expressed in the same strain or are independently expressed in different strains.

7. The method according to any one of claims 1 to 6, wherein an exogenous active sugar donor UDP-glucose is added to the reaction system; or the sucrose-sucrose synthase-UDP-glucose regeneration circulation system generates UDP-glucose; or UDP-glucose is produced by a bacterial endogenous UDP-glucose synthesis pathway; or designing a modified UDP-glucose synthetic pathway to generate UDP-glucose.

8. The method according to claim 5 or 6, wherein the reaction system is constructed with a cell extract containing a glycoside hydrolase and a UDP-glucose-dependent glycosyltransferase; or in-situ or resting cell reaction system is constructed by using thallus containing glucoside hydrolase and UDP-glucose dependent glycosyltransferase.

Images