CN110699373B - Uridine diphosphate glucose high-yield strain and application thereof - Google Patents

Abstract

UDPG dependent glycosyltransferases (UGTs) can catalyze glycosylation modification of various compounds, but the expensive and poorly stable sugar donor UDPG (uridine diphosphate glucose) is a major factor limiting the large-scale application of UGTs. In view of the limitations of the existing solving measures, the invention adopts an open source throttling strategy to modify the pyrimidine synthesis path in the escherichia coli, change the carbon source flow and the metabolism path of the UDPG, construct the UDPG high-yield strain and verify the application feasibility of the strain. Meanwhile, the high-efficiency biosynthesis of the natural non-nutritive sweetener rebaudioside KA is realized.

Description

Uridine diphosphate glucose high-yield strain and application thereof

Technical Field

The invention belongs to the technical fields of natural product biosynthesis and microbial metabolism engineering and food chemical industry, and particularly relates to a method for improving the content of endogenous UDPG in escherichia coli, a UDPG high-yield strain obtained by using the method and application of the UDPG high-yield strain.

Background

Glycosylation modification is one of the important means to improve or alter the physical properties and biological activity of natural products. Compared with a chemical method, the enzymatic glycosylation modification has the advantages of mild reaction conditions, good selectivity, environmental protection and the like, and is concerned by academia and industry. UDP-glucose dependent glycosyltransferases (UGTs, EC 2.4.1. X) are a class of enzymes that use uridine diphosphate glucose (UDPG) as a glycosyl donor, widely occurring in plants, animals and microorganisms, in vast numbers (Bock KW. Biochem Phacol, 2016, 99:11-17). UGTs can catalyze the site-directed glycosylation modification of natural and non-natural compounds in a regioselective and stereoselective manner, and can be used as an effective tool enzyme in various fields such as fine chemical engineering, drug development, food and beverage, daily chemicals, pesticides and the like (Lairson LL, et al, annu Rev Biochem,2007,77:521-555;Bak S,et al.Plant Physiol,2008,148:1295-1308;Chen HY,et al.Plant J2017,89:195-203).

The large consumption of expensive and poorly stable UDP-glucose (UDPG) is a major limiting factor for the industrialized application of UGTs, so that UGTs are limited to laboratory-scale applications at present. In order to solve the problem of the source of UGTs glycosyl donor UDPG, and reduce the cost of UGTs catalytic compound glycosylation modification, researchers have developed a series of attempts in escherichia coli hosts. 1) An in vitro regeneration system is constructed, and the dosage of exogenous UDPG is reduced. An in-vitro One-pot (One-pot) reaction system is the most commonly used UDPG regeneration strategy, namely, the glycosylation modification reaction catalyzed by UGTs is compatible and coupled with the UDPG synthesis reaction catalyzed by sucrose synthase, so that the cyclic supply of the UDPG is realized. Sayaka et al coupled sucrose synthase AtSUS-1 of Arabidopsis thaliana with glycosyltransferase CaUGT2 to achieve glycosylation of curcumin by one-pot method, increase glycosylation modification efficiency by 7 times, and decrease exogenous UDPG by 90% (-)

K, et al Biotechnol Adv,2016, 34:88-111). However, the UDPG regeneration reaction system still needs to be added with a small amount of UDPG, and has poor stability. 2) The UDP-glucose metabolic pathway is modified, and the synthesis amount of endogenous UDPG is increased. The metabolic engineering is utilized to modify the UDPG biosynthesis way of a host, the endogenous content is improved, and a whole cell reaction and a fermentation method are adopted to carry out glycosylation modification of a substrate, so that the method is one of glycosylation modification strategies widely used in recent years. In E.coli, the overexpression of UDPG synthesis-related enzymes such as UDPG pyrophosphorylase (galU) and phosphoglucomutase (pgm) is a common strategy for increasing the synthesis amount of endogenous UDPG, but the effect is not obvious enough. Therefore, sohng group of Korean university overexpressed galU in E.coli BL21 (DE 3) since 2013, knocked out glucose-6-phosphate isomerase (pgi), glucose-6-phosphate dehydrogenase (zwf) and UDP-glucose hydrolase (ushA) genes, and constructed an engineering strain E.coli MA3F. Through over-expression of related UGT in E.coli MA3F, glycosylation modification of apigenin and baicalein is realized, but the substrate concentration is lower, and the conversion is incomplete (thin NH et al processBiochem,2013, 48:1744-1748). 3) An exogenous UDPG synthesis way is introduced, so that the content of endogenous UDPG is improved. Fei Wenli et al introduced sucrose phosphorylase (BasP) and bifidobacterium bifidum UDP-glucose pyrophosphorylase (ugpA) genes into E.coli, and coupled with glycosyltransferase EUGT11 constructed a whole cell catalyst to achieve efficient synthesis of rebaudioside D, but the substrate concentration was to be increased (Fei Liwen et al. Food and fermentation industries, 2018, 44:1-7). />

Rebaudioside KA is a novel tetracyclic diterpenoid derived from stevia rebaudiana (Stevia rebaudiana) and isolated, purified and identified in 2014 by MA.Ibrahim (Ibrahim M A, et al J Nat Prod,2014, 77:1231-1235). The sweetness of the Lai Bao Digan KA is about 300 times that of the sucrose, but the calorific value is less than 1% of that of the sucrose, and the Lai Bao Digan KA is pure in sweetness and free of obvious aftertaste and bitter taste, and is an ideal natural sweetener. The subject group adopts UDP-glycosyltransferase Oled coupled sucrose synthase of Streptomyces antibioticus to carry out site-directed glycosylation modification of rubusoside in the early stage, thereby realizing selective biosynthesis of rebaudioside KA (CN 107164435A). However, a large amount of exogenous UDPG still needs to be added in the reaction, and the industrialized application cost is high. The invention uses the reaction as a model reaction to verify the application feasibility of the UDPG high-yield escherichia coli, and simultaneously realizes the high-efficiency and low-cost biosynthesis of the rebaudioside KA.

Disclosure of Invention

The invention aims to reconstruct an endogenous UDPG metabolic pathway of escherichia coli, and on the basis of fully analyzing the endogenous UDPG metabolic flow of the escherichia coli, the 'open source-throttle' scheme is adopted to design and reconstruct the biosynthesis and metabolic pathway of the UDPG, thereby eliminating the use of exogenous UDPG in the catalysis reaction of UDPG dependent glycosyltransferase and constructing and obtaining the strain with high yield of the UDPG. Because the synthesis and decomposition of the endogenous UDPG of the escherichia coli are in dynamic change processes, the result of strain transformation is difficult to monitor by measuring the content of the UDPG, the invention takes the glycosyltransferase Oled to catalyze the rubusoside to synthesize the rebaudioside KA as a model reaction, verifies the feasibility of the synthesis and metabolic pathway transformation of the UDPG, screens the high-yield strain of the UDPG, and prepares the KA on a large scale on the basis.

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

a construction method of uridine diphosphate glucose high-yield strain is provided, which modifies the strain synthesizing uridine diphosphate glucose and overexpresses related key enzymes in pyrimidine synthesis pathway of the strain. The key enzyme for pyrimidine synthesis is one or more selected from orotic acid pyrophosphorylase pyrE, orotic acid nucleotide decarboxylase pyrF and uracil nucleotide kinase pyrH. Single over-expression or multiple co-expression may be achieved by episomal plasmid and/or genomic site-directed integration. The gene sequence of pyrE is shown as SEQ ID NO. 1, the amino acid sequence is shown as SEQ ID NO. 2, the gene sequence of pyrF is shown as SEQ ID NO. 3, and the amino acid sequence is shown as SEQ ID NO. 4. The pyrH gene sequence is shown as SEQ ID NO. 5, and the amino acid sequence is shown as SEQ ID NO. 6.

The method also comprises the step of modifying the carbon source flow, namely knocking out the glucose 6-phosphate isomerase gene pgi, so as to prevent the isomerization of the glucose 6-phosphate into fructose 6-phosphate and promote the glucose 6-phosphate to enter a UDPG synthesis path. The pgi gene sequence is shown in SEQ ID NO. 7.

The method of the invention further comprises inhibiting the endogenous UDPG dehydrogenase gene, namely knocking out, and avoiding the conversion of the endogenous UDPG into UDP-gluconic acid. The ugd gene sequence is shown in SEQ ID NO. 8.

Another object of the invention is to provide a high-yield strain of UDPG, obtained by the method of the invention.

The UDPG high-yield strain is obtained by constructing the method singly or in combination, and is preferably an escherichia coli engineering strain obtained by modifying an escherichia coli endogenous UDPG biosynthesis pathway.

The invention also aims to provide application of the uridine diphosphate glucose high-yield strain, wherein sucrose synthase and uridine diphosphate glucose dependent glycosyltransferase are co-expressed in the uridine diphosphate glucose high-yield strain or the uridine diphosphate glucose dependent glycosyltransferase is expressed singly, and a biocatalyst is constructed for efficient glycosylation modification of compounds.

For this reason, the present invention is illustrated with respect to the synthesis of rebaudioside KA, mogrosides, glycosylated daidzein.

In order to achieve the above object, the present invention adopts the following research contents:

1) And adopting a co-expression vector to carry out serial expression on sucrose synthase and glycosyltransferase OleD to obtain engineering strains E.coli C & 1-E.coli C &8, and evaluating the UDPG synthesis and regeneration efficiency of 8 engineering bacteria by the yield of the rice Bao Digan KA.

2) Integrating sucrose synthase into the genome of escherichia coli, introducing an OleD gene by using an expression vector to obtain an engineering strain E.coli C &9, and evaluating the synthesis and regeneration efficiency of UDPG.

3) The expression vector is adopted to respectively overexpress and co-express orotic acid pyrophosphorylase (pyrE), decarboxylase (pyrF) and uracil nucleotide kinase (pyrH) in escherichia coli, and engineering strains E.coli CP &1, CP &2, CP &3 and CP &4 are respectively obtained after sucrose synthase and OleD genes are introduced. The synthesis and regeneration efficiency of UDPG were evaluated.

4) The copy numbers of pyrE, pyrF and pyrH genes in the genome of escherichia coli are increased through homologous recombination to respectively obtain engineering bacteria E.coli C & 13-C &15, and sucrose synthase and OleD genes are introduced to obtain engineering strains E.coli CP & 5-CP &7. The synthesis and regeneration efficiency of UDPG was measured.

5) Knocking out glucose 6-phosphate isomerase (pgi) genes of escherichia coli to obtain engineering strains E.coli CP &8 and E.coli CP &9, introducing sucrose synthase and OleD genes, and constructing E.coli CPM &1 and E.coli CPM &2. The synthesis and regeneration efficiency of UDPG was measured.

6) Knocking out UDPG dehydrogenase genes (ugd) of escherichia coli to obtain engineering strains E.coli CPDeltaU &1 and E.coli CPDeltaU &2, and introducing sucrose synthase and OleD genes to construct E.coli CPMDeltaU &1 and E.coli CPMDeltaU &2. The synthesis and regeneration efficiency of UDPG was measured.

7) Optimizing E.coli CPM delta U &2 catalytic synthesis reaction conditions of rebaudioside KA, verifying the amplification feasibility of an endogenous UDPG regeneration and circulation system, and not adding exogenous UDPG in all the condition optimization processes disclosed by the invention, wherein the specific optimization steps are as follows: different reaction temperatures (25-55 ℃) are examined, preferably 40 ℃; examining different reaction pH values (5.5-10.0), preferably pH 7.5; different sucrose concentrations (500-900 g/L), preferably 800g/L, are examined; examining different rubusoside concentrations (2.5-25 g/L), preferably 22.5g/L; the concentration of the cells was examined at different levels (25-125 g/L), preferably 100mg/mL.

Under the preferable reaction condition, the reaction system is amplified to 0.5L, and the 16-hour yield of the rebaudioside KA is 95.7 percent, so that the invention adopts the UDPG high-yield strain to construct a novel biosynthesis path of the rebaudioside KA.

8) On the basis of the UDPG high-yield strain, the reliability and the general applicability of the UDPG high-yield strain are further verified by constructing a glycosyltransferase OleD single-enzyme whole-cell catalyst, a mogroside IIIE glycosylation modification, a glycosyltransferase YjiC and sucrose synthase coexpression and other modes.

Based on an open source-throttling strategy, the invention obtains the UDPG metabolic pathway reconstruction strain by designing and reforming the endogenous UDPG biosynthesis and metabolic pathway of the escherichia coli and optimizing the reaction conditions, and the strain can realize the efficient regeneration and circulation of the endogenous UDPG and can be used for the donor UDPG of different glycosyltransferase catalytic reactions.

In addition, under the condition of completely avoiding the use of exogenous UDPG, the high-yield escherichia coli strain of the UDPG can realize the efficient synthesis of rebaudioside KA by 22.5g/L rubusoside, and the conversion rate of the rubusoside reaches 95.7%. Therefore, the invention establishes a novel technology for biosynthesis of rebaudioside KA without exogenous UDPG, can realize high-efficiency low-cost synthesis of the natural sweetener rebaudioside KA, and has wide industrialized application prospect.

The invention has the following advantages:

1) The high-yield escherichia coli strain disclosed by the invention can increase the content of endogenous UDPG, realize the cyclic regeneration of the endogenous UDPG, and provide a UDPG sugar donor for the glycosylation reaction catalyzed by the UDPG dependent glycosyltransferase.

2) Compared with a UDPG regeneration system and a strain reported in the literature, the UDPG high-yield escherichia coli strain disclosed by the invention can completely avoid the use of exogenous UDPG, and does not influence the catalysis efficiency of UDPG dependent glycosyltransferase.

3) The novel biological preparation process of the natural sweetener rebaudioside KA disclosed by the invention completely eliminates the use of exogenous UDPG and has huge industrial application prospect.

In conclusion, the high-yield escherichia coli strain disclosed by the invention can realize high-efficiency biosynthesis and cyclic regeneration of endogenous UDPG, and can provide a sufficient UDPG sugar donor for the glycosylation reaction catalyzed by the UDPG dependent glycosyltransferase. The chassis strain and the solution are provided for solving the problem of the cost of the UDPG in the large-scale application of the UDPG dependent glycosyltransferase, and the application value is huge. In addition, the novel natural sweetener rebaudioside KA biosynthesis process disclosed by the invention is simple to operate, low in cost and wide in market application prospect.

Drawings

FIG. 1 is a HPLC analysis result of E.coli CP & 1-CP &4 catalyzed synthesis of rebaudioside KA.

FIG. 2 shows the HPLC analysis result (16 hours of reaction) of rebaudioside KA in a 0.5L production system.

FIG. 3 is a purity analysis result of rebaudioside KA.

Detailed Description

The following examples illustrate specific steps of the invention, but are not limited to the scope of the examples.

The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art unless otherwise indicated.

The invention will now be described in further detail with reference to the following specific examples, which are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

In the following examples, various processes and methods, which are not described in detail, are conventional methods well known in the art.

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.

EXAMPLE 1 endogenous UDPG circulating Strain construction

The co-expression vectors pETDuet-1 and pACYCDeut-1 are adopted, and the co-expression plasmids of Arabidopsis thaliana and Vigna radiata sucrose synthase genes (sus) and glycosyltransferase OleD genes (OleD) are respectively constructed by means of enzyme cutting sites Nco I/Hind III and Nde I/Xho I according to different sequences of inserting two multiple cloning sites: pEaT-GT, pEGT-aT, pAaT-GT, pAGT-aT, pEvT-GT, pEGT-vT, pAvT-GT, pAGT-vT. And respectively transforming the co-expression plasmids into escherichia coli BL21 (DE 3) to obtain engineering strains E.coli C &1, E.coli C &2, E.coli C &3, E.coli C &4, E.coli C &5, E.coli C &6, E.coli C &7 and E.coli C &8. The Arabidopsis thaliana sucrose synthase gene sequence is shown as SEQ ID NO. 9, and the amino acid sequence is shown as SEQ ID NO. 10. The gene sequence of the Vigna radiata sucrose synthase is shown as SEQ ID NO. 11, and the amino acid sequence is shown as SEQ ID NO. 12. The OleD gene sequence is shown in SEQ ID NO. 13, and the OleD amino acid sequence is shown in SEQ ID NO. 14.

After induction of expression, SDS-PAGE analyzes protein expression of sucrose synthase and glycosyltransferase Oled. The results show that sucrose synthase and Oled can be expressed in soluble form in 8 strains, and the ratio of sucrose synthase to Oled to soluble total protein is shown in Table 1.

TABLE 1 soluble expression of sucrose synthase and glycosyltransferase Oled

/>

Embodiment 2 Activity of sucrose synthase and yield determination of rebaudioside KA

After the engineering bacteria in the example 1 are induced to express and then resuspended by sodium phosphate buffer solution, the engineering bacteria are crushed by ultrasound under the low temperature condition and centrifuged to prepare the corresponding crude enzyme solution. And (3) measuring the activity of sucrose synthase in the E.coli C & 1-E.coli C &8 crude enzyme liquid by adopting a DNS (3, 5-dinitrosalicylic acid) method. The results showed that the sucrose synthase activity units (IU) in the E.coli C &1 to E.coli C &8 crude enzyme solutions were 0.159,0.162,0.145,0.133,0.08,0.11,0.06,0.09, respectively.

The yield of rebaudioside KA in the present invention is an indicator of the level of endogenous UDPG. The engineering bacteria in example 1 were induced to express and then resuspended in a 25g/L ratio in M9 medium (pH 7.0) to obtain the corresponding whole cell catalyst. In a 50mL whole-cell catalytic reaction system, rubusoside is 2g/L, sucrose is 600g/L, and the reaction is carried out for 18 hours at 30 ℃ in a shaking way. After the reaction is finished, heating to remove protein, adding an equal volume of methanol for treatment, performing HPLC analysis on filtrate obtained by centrifugal filtration, and detecting the yield of the rebaudioside KA to judge the content of endogenous UDPG and the recycling condition.

HPLC detection conditions: HPLC model: agilent 1260; chromatographic column: YMC-Pack ODS-A (250 mm. Times.4.6 mm,5 μm); mobile phase: ultrapure water A (containing 1%o of formic acid) and acetonitrile B (containing 1%o of formic acid); column temperature: 35 ℃; detection wavelength: 210nm; sample injection amount: 10. Mu.L; analysis time: 25min; flow rate: 1mL/min; elution gradient: 10 to 90 percent of B.

HPLC analysis results of catalytic synthesis of rebaudioside KA by using E.coli CP & 1-CP &4 are shown in FIG. 1. The comparison results of the catalytic efficiencies of E.coli C & 1-E.coli C &8 are shown in Table 2, 8 engineering bacteria can realize the circulation of endogenous UDPG, but the yield of rebaudioside KA is lower than 35% due to the lower content of endogenous UDPG in escherichia coli, and the large-scale application cannot be realized.

TABLE 2 comparison of catalytic efficiencies of strains E.coli C & 1-E.coli C &8

EXAMPLE 3 genomic integration of sucrose synthase

The Arabidopsis thaliana sucrose synthase gene is integrated into the genome of E.coli BL21 (DE 3) by utilizing a lambda-Red homologous recombination method, and the OleD gene is introduced by means of an expression vector pET22b (+) to obtain an engineering strain E.coli C &9. After induction of expression, 50ml of whole cell catalytic system was constructed as in example 2. HPLC detection showed only 17.3% yield of rebaudioside KA. In combination with examples 2 and 3, E.coli C &1 containing the free co-expression vector pEaT-GT was optimally catalyzed, so pEaT-GT was selected to evaluate the effect of UDPG metabolic pathway modification.

Example 4 engineering of the UDP biosynthetic pathway

In E.coli, orotate pyrophosphorylase (pyrE) catalyzes the synthesis of orotate nucleotides from 5-phosphoribosyl-1-pyrophosphate (PRPP) and orotate, which in turn is decarboxylated by decarboxylase (pyrF) to UMP. UMP can form UDP under the catalysis of uracil nucleotide kinase (pyrH). UDP can form UTP, and ultimately UDPG, under the catalysis of nucleoside diphosphate kinase. Furthermore, UDP can be directly used to synthesize UDPG under the catalysis of sucrose synthase. Therefore, increasing the synthesis amount of UDP is important to increase the content of endogenous UDPG.

The single-enzyme expression plasmid pC-E, pC-F for pyrE and pyrF and the double-enzyme co-expression plasmid pC-E-F were constructed using the co-expression vector pCDFDeut-1 with the aid of the cleavage sites Nco I/Hind III and Nde I/Xho I. The pyrH expression plasmid pA-H was constructed using the cleavage sites Nde I/Xho I and pACYCDeet-1. Co-transforming pC-E, pC-F and pA-H into E.coli BL21 to obtain engineering strains E.coli C &10, C &11 and C &12. On the basis, co-expression plasmids pEaT-GT are respectively introduced to construct engineering strains E.coli CP &1, CP &2 and CP &3. The pC-E-F, pA-H and pEaT-GT are jointly introduced into E.coli BL21 to obtain engineering bacteria E.coli CP &4.

After the engineering bacteria induce expression, the analysis result of SDS-PAGE shows that the soluble expression quantity of pyrE, pyrF, pyrH in E.coli CP & 1-CP &4 is more than 5%. To evaluate the effect of UDP increase on endogenous UDPG content, a whole cell catalytic system was constructed as in example 2. The results show that the efficiency of the catalytic synthesis of rebaudioside KA by e.coli CP & 1-CP &4 is 44.8%, 54.5%, 42.1% and 30.8%, respectively. The result shows that the increase of UDP biosynthesis can significantly increase endogenous UDPG content and provide a sugar donor for glycosyltransferase.

EXAMPLE 5 genomic integration of pyrE, pyrF and pyrH

As pyrE, pyrF and pyrH are all enzymes endogenous to Escherichia coli, the coding genes of pyrE, pyrF and pyrH are respectively inserted into the coding genes of three enzymes in E.coli BL21 chromosome by adopting a lambda-Red homologous recombination method so as to increase the copy number and obtain engineering bacteria E.coli C & 13-C &15. On the basis, co-expression plasmids pEaT-GT are respectively introduced to obtain engineering strains E.coli CP & 5-CP &7. A whole cell catalytic system was constructed according to embodiment 2 to determine the content of endogenous UDPG, the yields of rebaudioside KA were 25.8%, 29.0% and 21.5%, respectively. The results show that the content of endogenous UDPG in E.coli CP & 5-CP &7 is obviously lower than that of E.coli CP & 1-CP &4. Thus, the present invention selects the episomal plasmid to engineer the UDPG biosynthetic pathway.

Example 6 6 knockout of glucose phosphate isomerase

In order to further increase the content of endogenous UDPG, the invention knocks out a glucose isomerase 6-phosphate gene (pgi) on the basis of an E.coli C &10 strain over-expressing pyrE and an E.coli C &11 strain over-expressing pyrF to change the carbon source flow of the escherichia coli so as to avoid isomerization of glucose into fructose 6-phosphate. A50 bp sequence upstream and downstream of the pgi gene was selected as homology arm based on the genomic sequence of E.coli BL21 (DE 3) (GenBank: NC-012971.2), and a 20bp amplification primer was added to the 3' end. The upstream primer pgi-homo-F:5'-CGCTACAATCTTCCAAAGTCACAATTCTCAAAATCAGAAGAGTATTGCTAGTGTAGGCTGGAGCTGCTTC-3' (SEQ ID NO: 15) and the downstream primer pgi-homo-R:5'-GTTGCCGGATGCGGCGTGAACGCCTTATCCGGCCTACATATCGACGATGAATGGGAATTAGCCATGGTCC-3' (SEQ ID NO: 16). And (3) knocking out pgi genes in E.coli C &10 and E.coli C &11 respectively by adopting a lambda-Red homologous recombination method to obtain engineering strains E.coli CP &8 and E.coli CP &9, and introducing co-expression plasmids pEaT-GT to construct E.coli CPM &1 and E.coli CPM &2 respectively.

The whole cell catalytic system was constructed according to embodiment 2 with yields of 60.8% and 70.1% for e.coll CPM &1 and e.coll CPM &2 catalyzed rebaudioside KA synthesis, respectively. The results indicate that the endogenous UDPG content of E.coli is significantly increased after pgi gene knockout.

Example 7 knockout of UDPG dehydrogenase Gene (ugd)

Examples 1-6 are all based on an "open source" strategy to increase biosynthesis of endogenous UDPG in E.coli, but UDPG can be converted in vivo to UDP-gluconic acid by UDPG dehydrogenase, reducing the content of endogenous UDPG. Therefore, the invention knocks out UDPG dehydrogenase gene (ugd) on the basis of E.coli CP &8 and E.coli CP &9 to reduce the consumption of UDPG. A ugd knockout primer was designed and synthesized according to the method of example 6, (upstream primer ugd-homo-F:5'-CGCAAGTAACAAAAGACAATCAGGGCGTAAATAGCCCTGATAACAGGATGGTGTAGGCTGGAGCTGCTTC-3' (SEQ ID NO: 17)), and downstream primer ugd-homo-R:5'-GATGCTAAAAACATCATGATTCACAGTTAAGTTAATTCTGAGAGCATGAAATGGGAATTAGCCATGGTCC-3' (SEQ ID NO: 18)) were subjected to knockout of the ugd genes of E.coli CP &8 and E.coli CP &9 by lambda-Red homologous recombination to obtain engineering strains E.coli CP ΔU &1 and E.coli CP ΔU &2, respectively, and E.coli CPM ΔU &1 and E.coli CPM ΔU &2 were constructed by introducing the coexpression plasmid pEaT-GT.

The whole cell catalytic system was constructed as in example 2 with yields of 81% and 99.2% for e.coll CPM Δu &1 and e.coll CPM Δu &2 catalytic rebaudioside KA synthesis, respectively. The result shows that the content of the endogenous UDPG of the escherichia coli is effectively improved through an open source throttling strategy, and an escherichia coli strain E.coli CP delta U &2 with high yield of UDPG is constructed. Under the condition of no exogenous UDPG, E.coli CPM delta U &2 expressing glycosyltransferase Oled can catalyze 2.5g/L rubusoside to be converted into rebaudioside KA, and the substrate concentration is far higher than that reported in the literature.

Embodiment 8 E. optimal temperature for the Synthesis of rebaudioside KA by coll CPM ΔU &2

As a novel whole-cell catalyst, the invention examines the reaction conditions of E.coli CPM delta U &2 catalyzed synthesis of rebaudioside KA. Reaction temperature investigation: in a 50mL whole-cell catalytic reaction system, the concentration of rubusoside is 10g/L and 12.5g/L, the reaction temperature is 25-55 ℃, the reaction pH is 7.0, and the reaction time is 18 hours. After the reaction, the yield of rebaudioside KA was detected by HPLC, and the HPLC method is described in example 2. As a result, as shown in Table 3, the optimal temperature for the E.coli CPM ΔU &2 catalyzed synthesis of rebaudioside KA was 40 ℃, but rubusoside was not completely converted.

TABLE 3 influence of temperature on rebaudioside KA biosynthesis

Embodiment 9 E.optimal pH for the Synthesis of rebaudioside KA by coll CPM ΔU &2

Reaction pH investigation: in a 50mL whole-cell catalytic reaction system, the concentration of rubusoside is 12.5g/L, the reaction temperature is 40 ℃, the reaction pH is 5.5-9.0, and the reaction time is 18 hours. After the reaction, the yield of rebaudioside KA was detected by HPLC, and the HPLC method is described in example 2. As a result, as shown in Table 4, the E.coli CPM. DELTA.U &2 catalyzed synthesis of rebaudioside KA had an optimal pH of 7.5 and a yield of 90.1%.

TABLE 4 pH influence on rebaudioside KA biosynthesis

EXAMPLE 10 optimal sucrose concentration and cell amount

Optimum sucrose concentration study: in a 50mL whole-cell catalytic reaction system, the concentration of rubusoside is 12.5g/L, the concentration of sucrose is 500-900 g/L, the reaction temperature is 40 ℃, the reaction pH is 7.5, and the reaction time is 18 hours. After the reaction, the yield of rebaudioside KA was detected by HPLC, and the HPLC method is described in example 2. As a result, as shown in Table 5, the optimal sucrose concentration for E.coli CPM. DELTA.U &2 catalyzed synthesis of rebaudioside KA was 800g/L with substantially complete substrate conversion.

TABLE 5 influence of sucrose concentration on rebaudioside KA biosynthesis

Investigation of the optimal cell quantity: in a 50mL whole-cell catalytic reaction system, the concentration of rubusoside is 12.5-25 g/L, the reaction temperature is 40 ℃, the reaction pH is 7.5, the concentration of thallus is 25-125 g/L, and the reaction time is 18 hours. After the reaction, the yield of rebaudioside KA was detected by HPLC, and the HPLC method is described in example 2. As a result, as shown in Table 6, the optimal cell concentration for E.coli CPM. DELTA.U &2 catalyzed synthesis of rebaudioside KA was 100g/L, and the yield of 22.5g/L rubusoside to rebaudioside KA was greater than 95%.

TABLE 6 influence of the cell amount on rebaudioside KA biosynthesis

Embodiment 11 preparation of rebaudioside KA

Under the optimal reaction conditions, the reaction system of E.coli CPM delta U &2 for catalyzing the rubusoside to synthesize the rebaudioside KA is amplified to 0.5L. The HPLC analysis results are shown in FIG. 2. The results show that the conversion rate of rebaudioside KA reaches the plateau and reaches 95.4% after 16 hours of reaction. Purification according to the method of CN107164435A gave rebaudioside KA with purity greater than 99.5% (purity analysis result is shown in FIG. 3) with recovery of 80.2%. This result further verifies the feasibility of the UDPG high producing strain.

Embodiment 12 Oled catalytic synthesis of rebaudioside KA

And (3) transforming the OleD gene into E.coli CPDeltaU &2 by using pET22b (+) to obtain an engineering strain E.coli CPM DeltaU &3. After induction of the table, a whole cell catalytic system will be constructed according to the optimal catalytic conditions of E.coli CPM. DELTA.U &2, but with a substrate concentration reduced to 1g/L. HPLC detection showed 99.6% yield of rebaudioside KA.

Example 13 other uses of UDPG high-producing Strain

Because glycosyltransferase Oled can catalyze the fixed-point glycosylation modification of mogroside IIIE to synthesize mogroside IIIE-Glu, the invention utilizes UDPG high-yield strain E.coll CPM delta U &2 expressing Oled as a whole-cell catalyst to synthesize mogroside IIIE-Glu under the condition of no addition of exogenous UDPG, and the reaction formula is shown as follows.

50ml of reaction system: mogroside IIIE is 5g/L, sucrose is 800g/L, the reaction temperature is 40 ℃, the reaction pH is 7.5, the cell concentration is 100g/L, and the reaction is carried out for 18 hours. The yield of mogroside IIIE-Glu is 98.8% which is obviously higher than 85.6% reported in the literature (Li Chengfei, et al, university of Chinese medicine, university of medical science, 2019, 50:222-229). The result shows that the UDPG high-yield strain constructed by the invention has good universal applicability.

Example 14 other uses of UDPG high-producing Strain

Glycosyltransferase YjiC from Bacillus licheniformis catalyzes the synthesis of glycosylated daidzein by site-directed glycosylation modification of daidzein (hydroxy group 7) (Pandey PR, et al mol cells.2014, 37:172-177). The invention constructs a sucrose synthase gene co-expression plasmid pE-Y-At of YjiC gene and Arabidopsis thaliana by using pETDuet-1, and introduces E.coli CP delta U &2 to obtain engineering strain E.coli YAt. E.coli YAt is used as a whole-cell catalyst, and the synthesis condition of glycosylated daidzein is examined under the condition of no exogenous UDPG addition, and the reaction formula is shown as follows.

5ml reaction system: daidzein is 1g/L, sucrose is 800g/L, the reaction temperature is 40 ℃, the reaction pH is 7.5, the concentration of thallus is 100g/L, the concentration of DMSO is 5% (v/v), and the reaction time is 18 hours. The yield of glycosylated daidzein was 99.0% significantly higher than 62% reported in the literature (substrate concentration of only 0.05 g/L) (Pandey PR, et al mol cells.2014, 37:172-177). The result shows that the UDPG high-yield strain constructed by the invention can be applied to different UDPG dependent glycosyltransferases.

Sequence listing

<110> university of Chinese medical science

<120> uridine diphosphate glucose high-yield strain and application thereof

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 642

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 1

atgaaaccat atcagcgcca gtttattgaa tttgcgctta gcaagcaggt gttaaagttt 60

ggcgagttta cgctgaaatc cgggcgcaaa agcccctatt tcttcaacgc cgggctgttt 120

aataccgggc gcgacctggc actgttaggc cgtttttacg ctgaagcatt ggtggattct 180

ggcattgagt tcgatctgct gtttggccct gcttacaaag ggatcccgat tgcgaccact 240

accgccgtgg cgctggcgga gcatcatgac cttgacctgc cgtactgctt taaccgcaaa 300

gaggcaaaag accacggtga aggcggcaat ctggttggta gcgcgttaca aggacgcgta 360

atgctggtag atgatgtgat caccgccgga acggcgattc gcgaatcgat ggagattatt 420

caggctaatg gcgcgacgct tgctggcgta ttgatttcgc ttgatcgtca ggaacggggg 480

cgcggcgaga tttcggcgat tcaggaagtt gagcgtgatt acaactgcaa agtgatctct 540

atcatcaccc tgaaagatct gattgcctat ctggaagaga agccggaaat ggcggaacat 600

ctggcggcgg ttaaggccta tcgcgaagag tttggcgttt aa 642

<210> 3

<211> 213

<212> PRT

<213> Escherichia coli (Escherichia coli)

<400> 3

Met Lys Pro Tyr Gln Arg Gln Phe Ile Glu Phe Ala Leu Ser Lys Gln

1 5 10 15

Val Leu Lys Phe Gly Glu Phe Thr Leu Lys Ser Gly Arg Lys Ser Pro

20 25 30

Tyr Phe Phe Asn Ala Gly Leu Phe Asn Thr Gly Arg Asp Leu Ala Leu

35 40 45

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

50 55 60

Asp Leu Leu Phe Gly Pro Ala Tyr Lys Gly Ile Pro Ile Ala Thr Thr

65 70 75 80

Thr Ala Val Ala Leu Ala Glu His His Asp Leu Asp Leu Pro Tyr Cys

85 90 95

Phe Asn Arg Lys Glu Ala Lys Asp His Gly Glu Gly Gly Asn Leu Val

100 105 110

Gly Ser Ala Leu Gln Gly Arg Val Met Leu Val Asp Asp Val Ile Thr

115 120 125

Ala Gly Thr Ala Ile Arg Glu Ser Met Glu Ile Ile Gln Ala Asn Gly

130 135 140

Ala Thr Leu Ala Gly Val Leu Ile Ser Leu Asp Arg Gln Glu Arg Gly

145 150 155 160

Arg Gly Glu Ile Ser Ala Ile Gln Glu Val Glu Arg Asp Tyr Asn Cys

165 170 175

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

180 185 190

Glu Lys Pro Glu Met Ala Glu His Leu Ala Ala Val Lys Ala Tyr Arg

195 200 205

Glu Glu Phe Gly Val

210

<210> 3

<211> 738

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 3

atgacgttaa ctgcttcatc ttcttcccgc gctgttacga attctcctgt ggttgttgcc 60

cttgattatc ataatcgtga tgacgcgctg tcctttgtcg acaagatcga cccacgcgat 120

tgtcgtctga aggtcggcaa agagatgttt acattgtttg ggccacagtt tgtgcgcgaa 180

cttcaacagc gtggttttga tatctttctt gacctgaaat tccacgatat tcctaacact 240

gcagcgcacg ctgtcgctgc tgcagctgac ttaggcgtgt ggatggtgaa tgttcatgcc 300

tctggtgggg cgcgtatgat gaccgcagcg cgtgaggcac tggttccgtt tggcaaagat 360

gcaccgcttt tgattgctgt gacagtgttg accagcatgg aagccagcga cctggtcgat 420

cttggcatga cactgtcacc tgcagattat gcagaacgtc tggcggcact gacgcaaaaa 480

tgtggccttg atggtgtggt gtgttctgct caggaagctg tgcgctttaa acaggtattc 540

ggtcaggagt tcaaactggt tacgccgggc attcgtccgc aggggagtga agctggtgac 600

cagcgccgca ttatgacgcc agaacaggcg ttgtcggctg gtgttgatta tatggtgatt 660

ggtcgcccgg taacgcaatc ggtagatcca gcgcagacgc tgaaagcgat caacgcctct 720

ttacagcgga gtgcatga 738

<210> 4

<211> 245

<212> PRT

<213> Escherichia coli (Escherichia coli)

<400> 4

Met Thr Leu Thr Ala Ser Ser Ser Ser Arg Ala Val Thr Asn Ser Pro

1 5 10 15

Val Val Val Ala Leu Asp Tyr His Asn Arg Asp Asp Ala Leu Ser Phe

20 25 30

Val Asp Lys Ile Asp Pro Arg Asp Cys Arg Leu Lys Val Gly Lys Glu

35 40 45

Met Phe Thr Leu Phe Gly Pro Gln Phe Val Arg Glu Leu Gln Gln Arg

50 55 60

Gly Phe Asp Ile Phe Leu Asp Leu Lys Phe His Asp Ile Pro Asn Thr

65 70 75 80

Ala Ala His Ala Val Ala Ala Ala Ala Asp Leu Gly Val Trp Met Val

85 90 95

Asn Val His Ala Ser Gly Gly Ala Arg Met Met Thr Ala Ala Arg Glu

100 105 110

Ala Leu Val Pro Phe Gly Lys Asp Ala Pro Leu Leu Ile Ala Val Thr

115 120 125

Val Leu Thr Ser Met Glu Ala Ser Asp Leu Val Asp Leu Gly Met Thr

130 135 140

Leu Ser Pro Ala Asp Tyr Ala Glu Arg Leu Ala Ala Leu Thr Gln Lys

145 150 155 160

Cys Gly Leu Asp Gly Val Val Cys Ser Ala Gln Glu Ala Val Arg Phe

165 170 175

Lys Gln Val Phe Gly Gln Glu Phe Lys Leu Val Thr Pro Gly Ile Arg

180 185 190

Pro Gln Gly Ser Glu Ala Gly Asp Gln Arg Arg Ile Met Thr Pro Glu

195 200 205

Gln Ala Leu Ser Ala Gly Val Asp Tyr Met Val Ile Gly Arg Pro Val

210 215 220

Thr Gln Ser Val Asp Pro Ala Gln Thr Leu Lys Ala Ile Asn Ala Ser

225 230 235 240

Leu Gln Arg Ser Ala

245

<210> 5

<211> 726

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 5

atggctacca atgcaaaacc cgtctataaa cgcattctgc ttaagttgag tggcgaagct 60

ctgcagggca ctgaaggctt cggtattgat gcaagcatac tggatcgtat ggctcaggaa 120

atcaaagaac tggttgaact gggtattcag gttggtgtgg tgattggtgg gggtaacctg 180

ttccgtggcg ctggtctggc gaaagcgggt atgaaccgcg ttgtgggcga ccacatgggg 240

atgctggcga ccgtaatgaa cggcctggca atgcgtgatg cactgcaccg cgcctatgtg 300

aacgctcgtc tgatgtccgc tattccattg aatggcgtgt gcgacagcta cagctgggca 360

gaagctatca gcctgttgcg caacaaccgt gtggtgatcc tctccgccgg tacaggtaac 420

ccgttcttta ccaccgactc agcagcttgc ctgcgtggta tcgaaattga agccgatgtg 480

gtgctgaaag caaccaaagt tgacggcgtg tttaccgctg atccggcgaa agatccaacc 540

gcaaccatgt acgagcaact gacttacagc gaagtgctgg aaaaagagct gaaagtcatg 600

gacctggcgg ccttcacgct ggctcgtgac cataaattac cgattcgtgt tttcaatatg 660

aacaaaccgg gtgcgctgcg ccgtgtggta atgggtgaaa aagaagggac tttaatcacg 720

gaataa 726

<210> 6

<211> 241

<212> PRT

<213> Escherichia coli (Escherichia coli)

<400> 6

Met Ala Thr Asn Ala Lys Pro Val Tyr Lys Arg Ile Leu Leu Lys Leu

1 5 10 15

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

20 25 30

Ile Leu Asp Arg Met Ala Gln Glu Ile Lys Glu Leu Val Glu Leu Gly

35 40 45

Ile Gln Val Gly Val Val Ile Gly Gly Gly Asn Leu Phe Arg Gly Ala

50 55 60

Gly Leu Ala Lys Ala Gly Met Asn Arg Val Val Gly Asp His Met Gly

65 70 75 80

Met Leu Ala Thr Val Met Asn Gly Leu Ala Met Arg Asp Ala Leu His

85 90 95

Arg Ala Tyr Val Asn Ala Arg Leu Met Ser Ala Ile Pro Leu Asn Gly

100 105 110

Val Cys Asp Ser Tyr Ser Trp Ala Glu Ala Ile Ser Leu Leu Arg Asn

115 120 125

Asn Arg Val Val Ile Leu Ser Ala Gly Thr Gly Asn Pro Phe Phe Thr

130 135 140

Thr Asp Ser Ala Ala Cys Leu Arg Gly Ile Glu Ile Glu Ala Asp Val

145 150 155 160

Val Leu Lys Ala Thr Lys Val Asp Gly Val Phe Thr Ala Asp Pro Ala

165 170 175

Lys Asp Pro Thr Ala Thr Met Tyr Glu Gln Leu Thr Tyr Ser Glu Val

180 185 190

Leu Glu Lys Glu Leu Lys Val Met Asp Leu Ala Ala Phe Thr Leu Ala

195 200 205

Arg Asp His Lys Leu Pro Ile Arg Val Phe Asn Met Asn Lys Pro Gly

210 215 220

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

225 230 235 240

Glu

<210> 7

<211> 1650

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 7

atgaaaaaca tcaatccaac gcagaccgct gcctggcagg cactacagaa acacttcgat 60

gaaatgaaag acgttacgat cgccgatctt tttgctaaag acggcgatcg tttttctaag 120

ttctccgcaa ccttcgacga tcagatgctg gtggattact ccaaaaaccg catcactgaa 180

gagacgctgg cgaaattaca ggatctggcg aaagagtgcg atctggcggg cgcgattaag 240

tcgatgttct ctggcgagaa gatcaaccgc actgaaaacc gcgccgtgct gcacgtagcg 300

ctgcgtaacc gtagcaatac cccgattttg gttgatggca aagacgtaat gccggaagtc 360

aacgcggtgc tggagaagat gaaaaccttc tcagaagcga ttatttccgg tgagtggaaa 420

ggttataccg gcaaagcaat cactgacgta gtgaacatcg ggatcggcgg ttctgacctc 480

ggcccataca tggtgaccga agctctgcgt ccgtacaaaa accacctgaa catgcacttt 540

gtttctaacg tcgatgggac tcacatcgcg gaagtgctga aaaaagtaaa cccggaaacc 600

acgctgttcc tggtagcatc taaaaccttc accactcagg aaactatgac caacgcccat 660

agcgcgcgtg actggttcct gaaagcggca ggtgatgaaa aacacgttgc aaaacacttt 720

gcggcgcttt ccaccaatgc caaagccgtt ggcgagtttg gtattgatac tgccaacatg 780

ttcgagttct gggactgggt tggcggccgt tactctttgt ggtcagcgat tggcctgtcg 840

attgttctct ccatcggctt tgataacttc gttgaactgc tttccggcgc acacgcgatg 900

gacaagcatt tctccaccac gcctgccgag aaaaacctgc ctgtactgct ggcgctgatt 960

ggcatctggt acaacaattt ctttggtgcg gaaactgaag cgattctgcc gtatgaccag 1020

tatatgcacc gtttcgcggc gtacttccag cagggcaata tggagtccaa cggtaagtat 1080

gttgaccgta acggtaacgt tgtggattac cagactggcc cgattatctg gggtgaacca 1140

ggcactaacg gtcagcacgc gttctaccag ctgatccacc agggaaccaa aatggtaccg 1200

tgcgatttca tcgctccggc tatcacccat aacccgctct ctgatcatca ccagaaactg 1260

ctgtctaact tcttcgccca gaccgaagcg ctggcgtttg gtaaatcccg cgaagtggtt 1320

gagcaggaat atcgtgatca gggtaaagat ccggcaacgc ttgactacgt ggtgccgttc 1380

aaagtattcg aaggtaaccg cccgaccaac tccatcctgc tgcgtgaaat cactccgttc 1440

agcctgggtg cgttgattgc gctgtatgag cacaaaatct ttactcaggg cgtgatcctg 1500

aacatcttca ccttcgacca gtggggcgtg gaactgggta aacagctggc gaaccgtatt 1560

ctgccagagc tgaaagatga taaagaaatc agcagccacg atagctcgac caatggtctg 1620

attaaccgct ataaagcgtg gcgcggttaa 1650

<210> 8

<211> 1167

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 8

atgaaaatca ccatttccgg tactggctat gtcggcttgt caaacgggct tctaatcgca 60

caaaatcatg aggttgtggc attagatatt ttaccgtcac gcgttgctat gctgaatgat 120

cggatatctc ctattgttga taaggaaatt cagcagtttt tgcaatcaga taaaatacac 180

tttaatgcca cattagataa aaatgaagcc taccgggatg ctgattatgt catcatcgcc 240

actccaaccg actatgatcc taaaactaat tatttcaata catccagtgt agaatcagta 300

attaaagacg tagttgagat aaatccttat gcggttatgg tgatcaaatc aacggttccc 360

gttggtttta ccgcagcgat gcataagaaa tatcgcactg aaaatattat attctccccg 420

gaatttctcc gtgagggtaa agccctttac gataatctcc atccttcacg tattgtcatc 480

ggtgagcgtt cagaacgcgc agaacgtttt gctgctctgt tacaggaagg agcgattaag 540

caaaatatcc cgaccctgtt taccgactcc actgaagcag aagcgattaa actttttgca 600

aacacctacc tggcgatgcg cgtggcgtac tttaacgaac tggatagcta tgcagaaagt 660

ttaggtctga attcccgtca aataatcgaa ggcgtttgtc tcgacccacg tattggcaac 720

cattacaaca atccgtcgtt tggttatggt ggttattgtc tgccgaaaga taccaagcag 780

ttactggcga actaccagtc tgtgccgaat aacctgatct cggcaattgt cgatgctaac 840

cgcacgcgta aagattttat tgccgatgcc attttgtcac gcaagccgca agtggtgggt 900

atttatcgtc tgattatgaa gagcggttca gacaacttcc gcgcgtcttc cattcagggg 960

attatgaaac gtatcaaggc gaaaggcgtt gaagtgatca tttacgagcc ggtgatgaaa 1020

gaagactcat tcttcaactc tcgcctggaa cgtgatctcg ccactttcaa acaacaagcc 1080

gatgtcatta tttccaaccg tatggcagaa gagcttaaag atgtggcaga taaggtctac 1140

acccgcgatc tctttggcag cgactaa 1167

<210> 9

<211> 2427

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 9

atggccaatg cagagcgcat gatcacacgt gtgcacagtc aacgtgaacg tctgaacgag 60

accctggtta gcgagcgcaa cgaggttctg gcactgttaa gccgcgtgga agcaaagggc 120

aaaggcatcc tgcagcaaaa ccagatcatt gccgagtttg aagccctgcc ggaacagacc 180

cgtaagaagc tggagggtgg cccgttcttt gatctgctga aaagcaccca ggaagcaatt 240

gtgttacctc cgtgggtggc actggcagtt cgtccgcgtc cgggcgtttg ggaatacctg 300

cgtgtgaatc tgcatgcact ggtggttgag gagctgcagc ctgccgaatt cctgcatttc 360

aaggaagaac tggtggatgg cgttaaaaac ggtaatttta cattagagct ggactttgaa 420

ccgtttaatg ccagcattcc gcgcccgacc ctgcataaat atatcggtaa cggcgtggat 480

tttctgaatc gccatctgag cgccaagctg tttcatgaca aggagagctt actgcctctg 540

ctgaaatttc tgcgtctgca tagtcaccag ggcaagaacc tgatgctgag cgaaaagatc 600

caaaatctga acaccctgca gcacaccctg cgtaaagccg aggaatatct ggccgaactg 660

aagagcgaaa ccctgtatga ggagtttgag gccaagttcg aggagatcgg cctggagcgt 720

ggctggggtg acaacgcaga acgtgtgctg gacatgattc gtctgctgct ggacctgctg 780

gaggcaccgg atccgtgcac actggagaca ttcctgggcc gcgtgccgat ggttttcaat 840

gtggtgattc tgagcccgca cggctacttt gcacaggaca acgttctggg ttatccggat 900

acaggtggcc aagtggtgta cattctggat caggtgcgtg ccttagagat cgagatgctg 960

cagcgcatta aacagcaggg cctgaatatc aaaccgcgca tcctgatcct gacccgtctg 1020

ttacctgatg ccgtgggcac aacctgcggt gaacgcctgg aacgcgtgta tgatagcgaa 1080

tactgtgaca ttctgcgtgt gccgttccgt acagagaaag gcatcgtgcg taaatggatt 1140

agccgcttcg aggtttggcc ttacctggaa acctacaccg aggatgcagc agtggagtta 1200

agcaaagagc tgaacggcaa gccggacctg attattggca actacagcga cggcaacctg 1260

gtggccagcc tgttagccca caaattaggt gtgacccagt gtaccatcgc ccacgcactg 1320

gaaaagacaa aatacccgga cagcgatatc tactggaaaa agttagatga taaatatcac 1380

ttcagctgcc agtttaccgc cgacatcttt gccatgaacc acaccgattt tattatcaca 1440

agcacattcc aggaaatcgc aggcagtaaa gagaccgttg gccagtacga gagccataca 1500

gcctttacac tgcctggcct gtatcgtgtt gtgcacggca tcgatgtgtt tgatcctaaa 1560

tttaacattg ttagcccggg tgcagacatg agtatctact tcccgtacac cgaggagaag 1620

cgccgtctga ccaagtttca cagtgaaatc gaggaactgc tgtacagtga cgtggagaac 1680

aaggagcatc tgtgcgtgtt aaaggataag aaaaaaccga tcttatttac aatggcacgc 1740

ctggatcgcg tgaagaatct gagtggcctg gttgagtggt atggcaaaaa tacccgcctg 1800

cgcgaactgg ccaatctggt tgtggttggt ggcgaccgtc gtaaagaaag caaggacaac 1860

gaggagaagg ccgagatgaa gaaaatgtac gatctgatcg aagagtataa gctgaatggc 1920

cagtttcgct ggatcagcag tcagatggac cgtgtgcgca atggcgaact gtatcgctac 1980

atttgtgaca caaagggcgc attcgttcag ccggcactgt atgaggcctt cggcctgaca 2040

gtggtggaag ccatgacctg cggcctgccg acctttgcaa cctgcaaagg cggcccggca 2100

gaaatcatcg ttcatggcaa gagcggcttc catatcgatc cgtatcatgg tgaccaggcc 2160

gccgatacac tggcagactt ttttaccaaa tgtaaagagg atccgagcca ctgggatgag 2220

attagcaagg gtggtctgca gcgcatcgaa gaaaaataca cctggcagat ctacagccaa 2280

cgtctgctga ccctgaccgg tgtgtatggt ttctggaaac acgtgagtaa tctggaccgt 2340

ctggaagccc gccgttacct ggaaatgttc tatgcactga aatatcgccc gctggcacaa 2400

gccgttcctc tggcacaaga cgattaa 2427

<210> 10

<211> 808

<212> PRT

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 10

Met Ala Asn Ala Glu Arg Met Ile Thr Arg Val His Ser Gln Arg Glu

1 5 10 15

Arg Leu Asn Glu Thr Leu Val Ser Glu Arg Asn Glu Val Leu Ala Leu

20 25 30

Leu Ser Arg Val Glu Ala Lys Gly Lys Gly Ile Leu Gln Gln Asn Gln

35 40 45

Ile Ile Ala Glu Phe Glu Ala Leu Pro Glu Gln Thr Arg Lys Lys Leu

50 55 60

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

65 70 75 80

Val Leu Pro Pro Trp Val Ala Leu Ala Val Arg Pro Arg Pro Gly Val

85 90 95

Trp Glu Tyr Leu Arg Val Asn Leu His Ala Leu Val Val Glu Glu Leu

100 105 110

Gln Pro Ala Glu Phe Leu His Phe Lys Glu Glu Leu Val Asp Gly Val

115 120 125

Lys Asn Gly Asn Phe Thr Leu Glu Leu Asp Phe Glu Pro Phe Asn Ala

130 135 140

Ser Ile Pro Arg Pro Thr Leu His Lys Tyr Ile Gly Asn Gly Val Asp

145 150 155 160

Phe Leu Asn Arg His Leu Ser Ala Lys Leu Phe His Asp Lys Glu Ser

165 170 175

Leu Leu Pro Leu Leu Lys Phe Leu Arg Leu His Ser His Gln Gly Lys

180 185 190

Asn Leu Met Leu Ser Glu Lys Ile Gln Asn Leu Asn Thr Leu Gln His

195 200 205

Thr Leu Arg Lys Ala Glu Glu Tyr Leu Ala Glu Leu Lys Ser Glu Thr

210 215 220

Leu Tyr Glu Glu Phe Glu Ala Lys Phe Glu Glu Ile Gly Leu Glu Arg

225 230 235 240

Gly Trp Gly Asp Asn Ala Glu Arg Val Leu Asp Met Ile Arg Leu Leu

245 250 255

Leu Asp Leu Leu Glu Ala Pro Asp Pro Cys Thr Leu Glu Thr Phe Leu

260 265 270

Gly Arg Val Pro Met Val Phe Asn Val Val Ile Leu Ser Pro His Gly

275 280 285

Tyr Phe Ala Gln Asp Asn Val Leu Gly Tyr Pro Asp Thr Gly Gly Gln

290 295 300

Val Val Tyr Ile Leu Asp Gln Val Arg Ala Leu Glu Ile Glu Met Leu

305 310 315 320

Gln Arg Ile Lys Gln Gln Gly Leu Asn Ile Lys Pro Arg Ile Leu Ile

325 330 335

Leu Thr Arg Leu Leu Pro Asp Ala Val Gly Thr Thr Cys Gly Glu Arg

340 345 350

Leu Glu Arg Val Tyr Asp Ser Glu Tyr Cys Asp Ile Leu Arg Val Pro

355 360 365

Phe Arg Thr Glu Lys Gly Ile Val Arg Lys Trp Ile Ser Arg Phe Glu

370 375 380

Val Trp Pro Tyr Leu Glu Thr Tyr Thr Glu Asp Ala Ala Val Glu Leu

385 390 395 400

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

405 410 415

Asp Gly Asn Leu Val Ala Ser Leu Leu Ala His Lys Leu Gly Val Thr

420 425 430

Gln Cys Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Pro Asp Ser

435 440 445

Asp Ile Tyr Trp Lys Lys Leu Asp Asp Lys Tyr His Phe Ser Cys Gln

450 455 460

Phe Thr Ala Asp Ile Phe Ala Met Asn His Thr Asp Phe Ile Ile Thr

465 470 475 480

Ser Thr Phe Gln Glu Ile Ala Gly Ser Lys Glu Thr Val Gly Gln Tyr

485 490 495

Glu Ser His Thr Ala Phe Thr Leu Pro Gly Leu Tyr Arg Val Val His

500 505 510

Gly Ile Asp Val Phe Asp Pro Lys Phe Asn Ile Val Ser Pro Gly Ala

515 520 525

Asp Met Ser Ile Tyr Phe Pro Tyr Thr Glu Glu Lys Arg Arg Leu Thr

530 535 540

Lys Phe His Ser Glu Ile Glu Glu Leu Leu Tyr Ser Asp Val Glu Asn

545 550 555 560

Lys Glu His Leu Cys Val Leu Lys Asp Lys Lys Lys Pro Ile Leu Phe

565 570 575

Thr Met Ala Arg Leu Asp Arg Val Lys Asn Leu Ser Gly Leu Val Glu

580 585 590

Trp Tyr Gly Lys Asn Thr Arg Leu Arg Glu Leu Ala Asn Leu Val Val

595 600 605

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

610 615 620

Glu Met Lys Lys Met Tyr Asp Leu Ile Glu Glu Tyr Lys Leu Asn Gly

625 630 635 640

Gln Phe Arg Trp Ile Ser Ser Gln Met Asp Arg Val Arg Asn Gly Glu

645 650 655

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

660 665 670

Leu Tyr Glu Ala Phe Gly Leu Thr Val Val Glu Ala Met Thr Cys Gly

675 680 685

Leu Pro Thr Phe Ala Thr Cys Lys Gly Gly Pro Ala Glu Ile Ile Val

690 695 700

His Gly Lys Ser Gly Phe His Ile Asp Pro Tyr His Gly Asp Gln Ala

705 710 715 720

Ala Asp Thr Leu Ala Asp Phe Phe Thr Lys Cys Lys Glu Asp Pro Ser

725 730 735

His Trp Asp Glu Ile Ser Lys Gly Gly Leu Gln Arg Ile Glu Glu Lys

740 745 750

Tyr Thr Trp Gln Ile Tyr Ser Gln Arg Leu Leu Thr Leu Thr Gly Val

755 760 765

Tyr Gly Phe Trp Lys His Val Ser Asn Leu Asp Arg Leu Glu Ala Arg

770 775 780

Arg Tyr Leu Glu Met Phe Tyr Ala Leu Lys Tyr Arg Pro Leu Ala Gln

785 790 795 800

Ala Val Pro Leu Ala Gln Asp Asp

805

<210> 11

<211> 2445

<212> DNA

<213> mung bean (Vigna radiata)

<400> 11

atgtctactc agccaaaacc aaaacttggt aggcttccca gtatcagaga tcgtgttgaa 60

gacactctct ctgctcatcg taacgaactc atttctcttc tctccaggta tgtggctcag 120

gggaaaggga ttttgcaacc tcataactta attgatgaac ttgacaacat ccctggggac 180

gatcaagcaa agttggatct taaaaatggc cccttcggtg aaattatcag ggcagcacag 240

gaagccatag ttttgcctcc ttttgtggca ataggagttc gtccaagacc tggtgtttgg 300

gaatatgtcc gtgttaatgt ctctgaactg agcgtggagc aattaagcgt tgctgaatat 360

ctgagcttta aggaggaact tgtggatgga aagaataatg acagttttgt actggaactt 420

gattttgagc cattcaatgc ctcattccct cgtccatccc gttcatcatc cattggcaat 480

ggtgtccaat ttctcaatcg ccacctttca tcaattatgt tccgcaacaa agattccttg 540

gaccccttgc ttgatttcct ccgagctcac aagtataagg gccatgctct gatgttaaat 600

gacagaatac agaacatttc caaactccag actgctctgg ccaagactga ggattatctt 660

aataagattc cacgtgatac accttattca gagtttgaat acgagttaca aggaatgggc 720

tttgagagag gatggggtga tactgctgaa agggtattgg aaacgatgca tctgctactg 780

gatattcttc aggctcctga tccttctacc ctagagactt ttcttggcag agtgccaatg 840

gtattcaatg ttgttatatt gtctcctcat ggcttcttcg gacaagccaa tgtcttgggt 900

ttgcctgaca ctggtgggca ggttgtttat atactagatc aagtgcgtgc ccttgagaat 960

gagatgctcc ttcggattaa gaaacaagga ttagatttca ctcctagaat tctgattgtt 1020

accaggctaa tacccgatgc aaaggggaca acttgcaacc aacgactaga aagagtcagt 1080

ggtaccgagc acactcatat tttgcgagtt ccattcagat cagagtcagg aactctccgt 1140

aaatggattt caaggtttga tgtgtggcct tatctagaga cttattcaga ggacgttgcc 1200

agtgaaattg ctgctgagtt acaaggctat cctgacttca tcattggaaa ctacagtgat 1260

gggaatcttg ttgcatcttt attggcttat aaaatgggag ttacacagtg caccatcgct 1320

catgcacttg agaagacaaa atatccagat tcagatatat actggaagaa atttgatgac 1380

aaatatcact tttcatgcca gttcactgct gatttaatag ccatgaataa cgctgatttt 1440

atcatcacca gtacctacca agagattgca ggaacgaaaa atacagttgg acagtatgag 1500

agccacactg gttttactct tcctgggctg tacagggttg tacatggcat tgatgttttt 1560

gatcccaagt tcaatattgt ttctcctgga gcagatatgt caatatattt cccctactct 1620

gaaaagcaga acaggcttac atccctgcac ggttccattg aacagctact gtatgatcca 1680

acacagactg atgactacat tggaacactg aaagacaagt caaagcccat aattttctca 1740

atggcaaggc tagacagagt gaagaacatg acaggattgg ttgaattatt tggtaagagc 1800

agcaaattga gggagctggt caaccttgtt gtagtagctg gttacattga tgtaaagaag 1860

tccagcgaca gagaagaaat tgcagaaatt gagaagatgc acgctctcat caaagagtac 1920

aacttaaacg gtgattttcg ttggattgct gctcaaacaa atagggcacg taacggggag 1980

ctgtatcgct acatagcaga cacacaaggt gctttcgttc agcctgcatt ctatgaggct 2040

tttgggctta cagttgtgga agccatgacc tgtggactcc ccacatttgc tactagcaat 2100

ggtggtccag ctgagatcat cgaacatggt atatcaggat ttcacattga tccttatcac 2160

cctgatcagg cttcagagct attggttgaa ttcttccaaa cctgcaagac tgacccaagc 2220

cattggaaga aaatatctga tggtggtctt aaaagaattt atgaaagcta cacttggaag 2280

atttattccg aaaggctctt gaccttggcg ggagtttaca gtttctggaa atacgtttcc 2340

aaattggaga ggcgtgaaac tcgacgatat cttgagatgt tctatatcct caagttccgt 2400

gatttggcaa aatctgtgcc cctagcaaaa gatgatgcaa gttaa 2445

<210> 12

<211> 814

<212> PRT

<213> mung bean (Vigna radiata)

<400> 12

Met Ser Thr Gln Pro Lys Pro Lys Leu Gly Arg Leu Pro Ser Ile Arg

1 5 10 15

Asp Arg Val Glu Asp Thr Leu Ser Ala His Arg Asn Glu Leu Ile Ser

20 25 30

Leu Leu Ser Arg Tyr Val Ala Gln Gly Lys Gly Ile Leu Gln Pro His

35 40 45

Asn Leu Ile Asp Glu Leu Asp Asn Ile Pro Gly Asp Asp Gln Ala Lys

50 55 60

Leu Asp Leu Lys Asn Gly Pro Phe Gly Glu Ile Ile Arg Ala Ala Gln

65 70 75 80

Glu Ala Ile Val Leu Pro Pro Phe Val Ala Ile Gly Val Arg Pro Arg

85 90 95

Pro Gly Val Trp Glu Tyr Val Arg Val Asn Val Ser Glu Leu Ser Val

100 105 110

Glu Gln Leu Ser Val Ala Glu Tyr Leu Ser Phe Lys Glu Glu Leu Val

115 120 125

Asp Gly Lys Asn Asn Asp Ser Phe Val Leu Glu Leu Asp Phe Glu Pro

130 135 140

Phe Asn Ala Ser Phe Pro Arg Pro Ser Arg Ser Ser Ser Ile Gly Asn

145 150 155 160

Gly Val Gln Phe Leu Asn Arg His Leu Ser Ser Ile Met Phe Arg Asn

165 170 175

Lys Asp Ser Leu Asp Pro Leu Leu Asp Phe Leu Arg Ala His Lys Tyr

180 185 190

Lys Gly His Ala Leu Met Leu Asn Asp Arg Ile Gln Asn Ile Ser Lys

195 200 205

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

210 215 220

Arg Asp Thr Pro Tyr Ser Glu Phe Glu Tyr Glu Leu Gln Gly Met Gly

225 230 235 240

Phe Glu Arg Gly Trp Gly Asp Thr Ala Glu Arg Val Leu Glu Thr Met

245 250 255

His Leu Leu Leu Asp Ile Leu Gln Ala Pro Asp Pro Ser Thr Leu Glu

260 265 270

Thr Phe Leu Gly Arg Val Pro Met Val Phe Asn Val Val Ile Leu Ser

275 280 285

Pro His Gly Phe Phe Gly Gln Ala Asn Val Leu Gly Leu Pro Asp Thr

290 295 300

Gly Gly Gln Val Val Tyr Ile Leu Asp Gln Val Arg Ala Leu Glu Asn

305 310 315 320

Glu Met Leu Leu Arg Ile Lys Lys Gln Gly Leu Asp Phe Thr Pro Arg

325 330 335

Ile Leu Ile Val Thr Arg Leu Ile Pro Asp Ala Lys Gly Thr Thr Cys

340 345 350

Asn Gln Arg Leu Glu Arg Val Ser Gly Thr Glu His Thr His Ile Leu

355 360 365

Arg Val Pro Phe Arg Ser Glu Ser Gly Thr Leu Arg Lys Trp Ile Ser

370 375 380

Arg Phe Asp Val Trp Pro Tyr Leu Glu Thr Tyr Ser Glu Asp Val Ala

385 390 395 400

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

405 410 415

Asn Tyr Ser Asp Gly Asn Leu Val Ala Ser Leu Leu Ala Tyr Lys Met

420 425 430

Gly Val Thr Gln Cys Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr

435 440 445

Pro Asp Ser Asp Ile Tyr Trp Lys Lys Phe Asp Asp Lys Tyr His Phe

450 455 460

Ser Cys Gln Phe Thr Ala Asp Leu Ile Ala Met Asn Asn Ala Asp Phe

465 470 475 480

Ile Ile Thr Ser Thr Tyr Gln Glu Ile Ala Gly Thr Lys Asn Thr Val

485 490 495

Gly Gln Tyr Glu Ser His Thr Gly Phe Thr Leu Pro Gly Leu Tyr Arg

500 505 510

Val Val His Gly Ile Asp Val Phe Asp Pro Lys Phe Asn Ile Val Ser

515 520 525

Pro Gly Ala Asp Met Ser Ile Tyr Phe Pro Tyr Ser Glu Lys Gln Asn

530 535 540

Arg Leu Thr Ser Leu His Gly Ser Ile Glu Gln Leu Leu Tyr Asp Pro

545 550 555 560

Thr Gln Thr Asp Asp Tyr Ile Gly Thr Leu Lys Asp Lys Ser Lys Pro

565 570 575

Ile Ile Phe Ser Met Ala Arg Leu Asp Arg Val Lys Asn Met Thr Gly

580 585 590

Leu Val Glu Leu Phe Gly Lys Ser Ser Lys Leu Arg Glu Leu Val Asn

595 600 605

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

610 615 620

Glu Glu Ile Ala Glu Ile Glu Lys Met His Ala Leu Ile Lys Glu Tyr

625 630 635 640

Asn Leu Asn Gly Asp Phe Arg Trp Ile Ala Ala Gln Thr Asn Arg Ala

645 650 655

Arg Asn Gly Glu Leu Tyr Arg Tyr Ile Ala Asp Thr Gln Gly Ala Phe

660 665 670

Val Gln Pro Ala Phe Tyr Glu Ala Phe Gly Leu Thr Val Val Glu Ala

675 680 685

Met Thr Cys Gly Leu Pro Thr Phe Ala Thr Ser Asn Gly Gly Pro Ala

690 695 700

Glu Ile Ile Glu His Gly Ile Ser Gly Phe His Ile Asp Pro Tyr His

705 710 715 720

Pro Asp Gln Ala Ser Glu Leu Leu Val Glu Phe Phe Gln Thr Cys Lys

725 730 735

Thr Asp Pro Ser His Trp Lys Lys Ile Ser Asp Gly Gly Leu Lys Arg

740 745 750

Ile Tyr Glu Ser Tyr Thr Trp Lys Ile Tyr Ser Glu Arg Leu Leu Thr

755 760 765

Leu Ala Gly Val Tyr Ser Phe Trp Lys Tyr Val Ser Lys Leu Glu Arg

770 775 780

Arg Glu Thr Arg Arg Tyr Leu Glu Met Phe Tyr Ile Leu Lys Phe Arg

785 790 795 800

Asp Leu Ala Lys Ser Val Pro Leu Ala Lys Asp Asp Ala Ser

805 810

<210> 13

<211> 1248

<212> DNA

<213> Streptomyces (Streptomyces)

<400> 13

atgaccaccc agaccactcc cgcccacatc gccatgttct ccatcgccgc ccacggccat 60

gtgaacccca gcctggaggt gatccgtgaa ctcgtcgccc gcggccaccg ggtcacgtac 120

gccattccgc ccgtcttcgc cgacaaggtg gccgccaccg gcgcccggcc cgtcctctac 180

cactccaccc tgcccggccc cgacgccgac ccggaggcat ggggaagcac cctgctggac 240

aacgtcgaac cgttcctgaa cgacgcgatc caggcgctcc cgcagctcgc cgatgcctac 300

gccgacgaca tccccgatct cgtcctgcac gacatcacct cctacccggc ccgcgtcctg 360

gcccgccgct ggggcgtccc ggcggtctcc ctctccccga acctcgtcgc ctggaagggt 420

tacgaggagg aggtcgccga gccgatgtgg cgcgaacccc ggcagaccga gcgcggacgg 480

gcctactacg cccggttcga ggcatggctg aaggagaacg ggatcaccga gcacccggac 540

acgttcgcca gtcatccgcc gcgctccctg gtgctcatcc cgaaggcgct ccagccgcac 600

gccgaccggg tggacgaaga cgtgtacacc ttcgtcggcg cctgccaggg agaccgcgcc 660

gaggaaggcg gctggcagcg gcccgccggc gcggagaagg tcgtcctggt gtcgctcggc 720

tcggcgttca ccaagcagcc cgccttctac cgggagtgcg tgcgcgcctt cgggaacctg 780

cccggctggc acctcgtcct ccagatcggc cggaaggtga cccccgccga actgggggag 840

ctgccggaca acgtggaggt gcacgactgg gtgccgcagc tcgcgatcct gcgccaggcc 900

gatctgttcg tcacccacgc gggcgccggc ggcagccagg aggggctggc caccgcgacg 960

cccatgatcg ccgtaccgca ggccgtcgac cagttcggca acgccgacat gctccaaggg 1020

ctcggcgtcg cccggaagct ggcgaccgag gaggccaccg ccgacctgct ccgcgagacc 1080

gccctcgctc tggtggacga cccggaggtc gcgcgccggc tccggcggat ccaggcggag 1140

atggcccagg agggcggcac ccggcgggcg gccgacctca tcgaggccga actgcccgcg 1200

cgccacgagc ggcaggagcc ggtgggcgac cgacccaacg gtgggtga 1248

<210> 14

<211> 415

<212> PRT

<213> Streptomyces (Streptomyces)

<400> 14

Met Thr Thr Gln Thr Thr Pro Ala His Ile Ala Met Phe Ser Ile Ala

1 5 10 15

Ala His Gly His Val Asn Pro Ser Leu Glu Val Ile Arg Glu Leu Val

20 25 30

Ala Arg Gly His Arg Val Thr Tyr Ala Ile Pro Pro Val Phe Ala Asp

35 40 45

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

50 55 60

Pro Gly Pro Asp Ala Asp Pro Glu Ala Trp Gly Ser Thr Leu Leu Asp

65 70 75 80

Asn Val Glu Pro Phe Leu Asn Asp Ala Ile Gln Ala Leu Pro Gln Leu

85 90 95

Ala Asp Ala Tyr Ala Asp Asp Ile Pro Asp Leu Val Leu His Asp Ile

100 105 110

Thr Ser Tyr Pro Ala Arg Val Leu Ala Arg Arg Trp Gly Val Pro Ala

115 120 125

Val Ser Leu Ser Pro Asn Leu Val Ala Trp Lys Gly Tyr Glu Glu Glu

130 135 140

Val Ala Glu Pro Met Trp Arg Glu Pro Arg Gln Thr Glu Arg Gly Arg

145 150 155 160

Ala Tyr Tyr Ala Arg Phe Glu Ala Trp Leu Lys Glu Asn Gly Ile Thr

165 170 175

Glu His Pro Asp Thr Phe Ala Ser His Pro Pro Arg Ser Leu Val Leu

180 185 190

Ile Pro Lys Ala Leu Gln Pro His Ala Asp Arg Val Asp Glu Asp Val

195 200 205

Tyr Thr Phe Val Gly Ala Cys Gln Gly Asp Arg Ala Glu Glu Gly Gly

210 215 220

Trp Gln Arg Pro Ala Gly Ala Glu Lys Val Val Leu Val Ser Leu Gly

225 230 235 240

Ser Ala Phe Thr Lys Gln Pro Ala Phe Tyr Arg Glu Cys Val Arg Ala

245 250 255

Phe Gly Asn Leu Pro Gly Trp His Leu Val Leu Gln Ile Gly Arg Lys

260 265 270

Val Thr Pro Ala Glu Leu Gly Glu Leu Pro Asp Asn Val Glu Val His

275 280 285

Asp Trp Val Pro Gln Leu Ala Ile Leu Arg Gln Ala Asp Leu Phe Val

290 295 300

Thr His Ala Gly Ala Gly Gly Ser Gln Glu Gly Leu Ala Thr Ala Thr

305 310 315 320

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

325 330 335

Met Leu Gln Gly Leu Gly Val Ala Arg Lys Leu Ala Thr Glu Glu Ala

340 345 350

Thr Ala Asp Leu Leu Arg Glu Thr Ala Leu Ala Leu Val Asp Asp Pro

355 360 365

Glu Val Ala Arg Arg Leu Arg Arg Ile Gln Ala Glu Met Ala Gln Glu

370 375 380

Gly Gly Thr Arg Arg Ala Ala Asp Leu Ile Glu Ala Glu Leu Pro Ala

385 390 395 400

Arg His Glu Arg Gln Glu Pro Val Gly Asp Arg Pro Asn Gly Gly

405 410 415

<210> 15

<211> 70

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

cgctacaatc ttccaaagtc acaattctca aaatcagaag agtattgcta gtgtaggctg 60

gagctgcttc 70

<210> 16

<211> 70

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

gttgccggat gcggcgtgaa cgccttatcc ggcctacata tcgacgatga atgggaatta 60

gccatggtcc 70

<210> 17

<211> 70

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

cgcaagtaac aaaagacaat cagggcgtaa atagccctga taacaggatg gtgtaggctg 60

gagctgcttc 70

<210> 18

<211> 70

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

gatgctaaaa acatcatgat tcacagttaa gttaattctg agagcatgaa atgggaatta 60

gccatggtcc 70

Claims (9)

1. The construction method of the uridine diphosphate glucose high-yield strain is characterized in that the uridine diphosphate glucose synthesis strain is modified, related key enzymes in pyrimidine synthesis pathway, namely orotic acid pyrophosphorylase pyrE, orotic acid nucleotide decarboxylase pyrF or uracil nucleotide kinase pyrH are singly and over-expressed in an episomal mode, the gene sequence of pyrE is shown as SEQ ID NO. 1, the gene sequence of pyrF is shown as SEQ ID NO. 3, the gene sequence of pyrH is shown as SEQ ID NO. 5, and the construction method specifically comprises the following steps:

(1) Adopting a pETDuet-1 co-expression vector, constructing a co-expression plasmid pEaT-GT of Arabidopsis thaliana sucrose synthase gene and glycosyltransferase OleD gene by virtue of enzyme cutting sites, inserting the sucrose synthase gene into an Nco I/HindIII site, inserting the OleD gene into an Nde I/Xho I site, wherein the Arabidopsis thaliana sucrose synthase gene sequence is shown as SEQ ID NO 9, and the OleD gene sequence is shown as SEQ ID NO 13;

(2) Constructing a single-enzyme expression plasmid pC-E of pyrE by using a co-expression vector pCDFDeut-1 and using an enzyme cleavage site Nco I/Hind III; construction of a single enzyme expression plasmid pC-F for pyrF by means of the cleavage site Nde I/Xho I; constructing pyrH expression plasmid pA-H by using restriction enzyme sites Nde I/Xho I; and respectively converting pC-E, pC-F or pA-H into E.coli BL21 to obtain engineering strains.

2. The method of claim 1, further comprising inhibiting the glucose 6-phosphate isomerization pathway and the endogenous uridine diphosphate glucose metabolic pathway.

3. The method of claim 2, wherein the inhibiting of the glucose 6-phosphate isomerization pathway is by knocking out the glucose 6-phosphate isomerase gene pgi, thereby preventing isomerization of glucose 6-phosphate to fructose 6-phosphate and promoting glucose 6-phosphate to enter the uridine diphosphate glucose synthesis pathway.

4. The method according to claim 2, wherein the inhibition of the endogenous uridine diphosphate glucose metabolic pathway is a knock-out of a uridine diphosphate glucose dehydrogenase gene, avoiding the conversion of endogenous uridine diphosphate glucose to UDP-gluconic acid.

5. A high-yield strain of uridine diphosphate glucose, characterized in that the strain obtained is constructed by the method according to any one of claims 1-4.

6. The use of a high-producing strain of uridine diphosphate glucose according to claim 5, characterized in that a sucrose synthase and a uridine diphosphate glucose-dependent glycosyltransferase are co-expressed in the high-producing strain of uridine diphosphate glucose, a biocatalyst is constructed for efficient glycosylation modification of compounds.

7. The use according to claim 6, characterized in that said uridine diphosphate glucose-producing strain is E.coli.

8. The use according to claim 6, characterized in that said uridine diphosphate glucose high-producing strain synthesizes rebaudioside KA, mogroside or glycosylated daidzein as a biocatalyst.

9. The use according to claim 8, characterized in that the uridine diphosphate glucose high-producing strain is used as a biocatalyst to synthesize rebaudioside KA under the following reaction conditions: the concentration of the substrate rubusoside is 2.5-25 g/L; the concentration of sucrose is 500-900 g/L; the reaction temperature is 25-55 ℃; the pH of the reaction is 5.5-10; the concentration of the bacterial cells is 25 g/L-125 g/L.

Images