Method for biosynthesis of xylose

文档序号：1884839 发布日期：2021-11-26 浏览：21次中文

阅读说明：本技术 一种生物合成木糖的方法 (Method for biosynthesis of xylose ) 是由江会锋逯晓云刘玉万初斋林崔博卢丽娜于 2020-05-22 设计创作，主要内容包括：本发明提供了一种生物合成L-木糖方法,其利用醛缩酶如D-果糖-6-磷酸醛缩酶(FSA)将底物甲醛和羟基乙醛转化为L-木糖,或者可利用羟基乙醛缩合酶(GALS)、其突变体或具有催化甲醛合成羟基乙醛功能的酶如苯甲酰甲酸脱羧酶(BFD)以及醛缩酶如D-果糖-6-磷酸醛缩酶(FSA)突变体的组合将底物甲醛“一锅法”转化为L-木糖。本发明的生物合成方法具有转化率高、生产工艺简单、绿化友好、易于规模化生产等优点。(The present invention provides a process for the biosynthesis of L-xylose by converting the substrates formaldehyde and glycolaldehyde into L-xylose using an aldolase such as D-fructose-6-phosphate aldolase (FSA), or by converting the substrate formaldehyde into L-xylose "one-pot" using a combination of a glycolaldehyde condensing enzyme (GALS), a mutant thereof or an enzyme having the function of catalyzing the synthesis of glycolaldehyde from formaldehyde, such as benzoylformate decarboxylase (BFD), and an aldolase such as a D-fructose-6-phosphate aldolase (FSA) mutant. The biosynthesis method of the invention has the advantages of high conversion rate, simple production process, greening friendliness, easy large-scale production and the like.)

1. A method for biosynthesis of L-xylose comprising the steps of: step 1, formaldehyde and glycolaldehyde are synthesized into glyceraldehyde under the action of aldolase, mutant thereof or enzyme with the catalytic function; step 2, leading glyceraldehyde and glycolaldehyde to synthesize L-xylose under the action of aldolase, mutant thereof or enzyme with the catalytic function;

preferably, wherein the aldolase or mutant thereof can catalyze the synthesis of glyceraldehyde from formaldehyde and glycolaldehyde or can catalyze the synthesis of L-xylose from glycolaldehyde and glyceraldehyde, can be a D-fructose-6-phosphate aldolase (FSA) and/or a mutant thereof;

preferably, wherein the aldolases used in step 1 and step 2, mutants thereof or enzymes having such catalytic function may be the same or different; more preferably, the same enzyme or mutant thereof is used in both steps; still more preferably, both steps use D-fructose-6-phosphate aldolase (FSA) and/or mutants thereof;

preferably, the amino acid sequence of the FSA mutant comprises a mutation of the amino acid residue at a position corresponding to a129 and/or a165 of SEQ ID No. 5; preferably, the mutations at the two positions are a129T and a 165G; preferably, the amino acid sequence of the FSA mutant comprises a mutation at the a129T/a165G site.

2. The method according to claim 1, further comprising, before the first step, the steps of: step 0, synthesizing hydroxyacetaldehyde from substrate formaldehyde under the action of hydroxyacetaldehyde synthase, a mutant thereof or an enzyme with the function of catalyzing formaldehyde to synthesize hydroxyacetaldehyde;

preferably, the glycolaldehyde condensing enzyme or mutant thereof may catalyze the condensation of formaldehyde to glycolaldehyde, e.g. GALS and/or a mutant thereof; the enzyme with the function of catalyzing formaldehyde to synthesize hydroxyacetaldehyde is benzoylformate decarboxylase (BFD) and/or a mutant thereof.

More preferably, the amino acid sequence of the BFD mutant comprises a mutation of the amino acid residue at least one position corresponding to at least one of W86, N87, L109, L110, H281, Q282, a460 of SEQ ID No.1, and wherein at least one of the mutation positions is W86 or N87; also preferably, the amino acid sequence of the BFD mutant comprises the following site mutations: W86/N87, W86/N87/L109/L110, W86/N87/L109/L110/A460 or W86/N87/L109/L110/H281/Q282/A460; also preferably, wherein the mutation of the amino acid residues of W86, N87, L109, L110, H281, Q282 and a460 is W86R, N87T, L109G, L110E, H281V, Q282F and a460M, respectively; still more preferably, the amino acid sequence of the BFD mutant comprises the following site mutations: W86R/N87T, W86R/N87T/L109G/L110E, W86R/N87T/L109G/L110E/A460M or W86R/N87T/L109G/L110E/H281V/Q282F/A460M.

3. The process according to claim 1 or 2, wherein the hydroxyacetaldehyde condensing enzyme, its mutants or the enzyme having the function of catalyzing the synthesis of hydroxyacetaldehyde by formaldehyde and the aldolase, its mutants or the enzyme having the function of catalyzing the synthesis of glyceraldehyde by formaldehyde and hydroxyacetaldehyde or the synthesis of L-xylose by hydroxyacetaldehyde and glyceraldehyde used is in the form of a purified enzyme or its mutants, an enzymatic cleavage supernatant or whole cells.

4. The method according to any one of claims 1 to 3, which is carried out in a "one-pot" manner in a buffer system; preferably, the buffer may be triethanolamine buffer, MOPS buffer, HEPES buffer, phosphate buffer, Tris buffer, or acetate buffer; more preferably, the pH of the buffer may be 6.5-8.5, e.g. 7-8;

preferably, the "one-pot" reaction is carried out at 10-50 ℃, for example at 20-40 ℃, exemplary at 25 ℃ or 37 ℃;

preferably, the "one-pot" reaction is carried out for 1 to 72 hours, for example 18 to 48 hours, illustratively 24 hours;

preferably, the "one-pot" reaction is carried out under shaking conditions.

5. The process according to any of claims 1 to 4, wherein the glycolaldehyde condensing enzyme, mutant thereof or enzyme having the function of catalyzing the synthesis of glycolaldehyde from formaldehyde and aldolase, mutant thereof or enzyme having the function of catalyzing the synthesis of glyceraldehyde from formaldehyde and glycolaldehyde or catalyzing the synthesis of L-xylose from glycolaldehyde and glyceraldehyde used has a weight of 1 (1-10), such as 1 (1-8), 1 (1-5), exemplarily 1:1, 1:2, 1:3, 1:4, 1: 5.

6. Use of a hydroxyacetaldehyde synthase, mutants thereof, an enzyme or related biomaterial having the function of catalyzing the synthesis of hydroxyacetaldehyde from formaldehyde, as well as an aldolase, mutants thereof, an enzyme or related biomaterial having the function of catalyzing the synthesis of glyceraldehyde from formaldehyde and hydroxyacetaldehyde, or the synthesis of L-xylose from hydroxyacetaldehyde and glyceraldehyde, in the biosynthesis of L-xylose;

more preferably, the amino acid sequence of the BFD mutant comprises a mutation of the amino acid residue at least one position corresponding to at least one of W86, N87, L109, L110, H281, Q282, a460 of SEQ ID No.1, and wherein at least one of the mutation positions is W86 or N87; also preferably, the amino acid sequence of the BFD mutant comprises the following site mutations: W86/N87, W86/N87/L109/L110, W86/N87/L109/L110/A460 or W86/N87/L109/L110/H281/Q282/A460; also preferably, wherein the mutation of the amino acid residues of W86, N87, L109, L110, H281, Q282 and a460 is W86R, N87T, L109G, L110E, H281V, Q282F and a460M, respectively; still more preferably, the amino acid sequence of the BFD mutant comprises the following site mutations: W86R/N87T, W86R/N87T/L109G/L110E, W86R/N87T/L109G/L110E/A460M or W86R/N87T/L109G/L110E/H281V/Q282F/A460M;

preferably, wherein the aldolase or mutant thereof can catalyze the synthesis of glyceraldehyde from formaldehyde and glycolaldehyde or can catalyze the synthesis of L-xylose from glycolaldehyde and glyceraldehyde is D-fructose-6-phosphate aldolase (FSA) and/or a mutant thereof:

7. A composition comprising a hydroxyacetaldehyde synthase, a mutant thereof, an enzyme or related biomaterial having the function of catalyzing the synthesis of hydroxyacetaldehyde by formaldehyde, and an aldolase, a mutant thereof, an enzyme or related biomaterial having the function of catalyzing the synthesis of glyceraldehyde by formaldehyde and hydroxyacetaldehyde, or the synthesis of L-xylose by hydroxyacetaldehyde and glyceraldehyde;

preferably, wherein the aldolase or mutant thereof can catalyze the synthesis of glyceraldehyde from formaldehyde and glycolaldehyde or can catalyze the synthesis of L-xylose from glycolaldehyde and glyceraldehyde, is a D-fructose-6-phosphate aldolase (FSA) and/or a mutant thereof;

preferably, the ratio by weight of the glycolaldehyde condensing enzyme, mutant thereof or enzyme having the function of catalyzing the synthesis of glycolaldehyde from formaldehyde to aldolase, mutant thereof or enzyme having the function of catalyzing the synthesis of glyceraldehyde from formaldehyde and glycolaldehyde or the synthesis of L-xylose from glycolaldehyde and glyceraldehyde is 1 (1-10), such as 1 (1-8), 1 (1-5), exemplarily 1:1, 1:2, 1:3, 1:4, 1: 5.

8. Use of the composition of claim 7 for the biosynthesis of L-xylose.

9. Use according to claim 6 or 8, wherein the combination or composition is used for the synthesis of L-xylose using formaldehyde as substrate.

10. The use according to claim 6 or the composition according to claim 7, wherein the biological material related to the hydroxyacetaldehyde synthase is a nucleic acid molecule encoding an enzyme capable of expressing the hydroxyacetaldehyde synthase, a mutant thereof, or an enzyme having the function of catalyzing the synthesis of hydroxyacetaldehyde by formaldehyde, or an expression cassette, a recombinant vector, a recombinant bacterium, or a transgenic cell line containing the nucleic acid molecule;

the related biological material of the aldolase is a coding nucleic acid molecule capable of expressing the aldolase, a mutant thereof or an enzyme with the function of catalyzing formaldehyde and glycolaldehyde to synthesize glyceraldehyde or catalyzing glycolaldehyde and glyceraldehyde to synthesize L-xylose, or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule; '

Preferably, the recombinant bacterium is obtained by introducing a nucleic acid molecule which codes the glycolaldehyde condensing enzyme, a mutant thereof, an enzyme or an aldolase which has the function of catalyzing formaldehyde to synthesize glycolaldehyde, a mutant thereof, an enzyme which has the function of catalyzing formaldehyde and glycolaldehyde to synthesize glyceraldehyde, or an enzyme which has the function of catalyzing glycolaldehyde and glyceraldehyde to synthesize L-xylose into a host cell; for example, in the form of a recombinant vector into a host cell;

preferably, the recombinant vector is a bacterial plasmid, a bacteriophage, a yeast plasmid or a retroviral packaging plasmid carrying a nucleic acid molecule encoding said glycolaldehyde synthase, a mutant thereof or an enzyme having the function of catalyzing the synthesis of glycolaldehyde from formaldehyde or glycolaldehyde, or an aldolase, a mutant thereof or an enzyme having the function of catalyzing the synthesis of glyceraldehyde from formaldehyde and glycolaldehyde, or the synthesis of L-xylose from glycolaldehyde and glyceraldehyde;

preferably, the host cell may be a prokaryotic cell, such as a bacterium, more particularly e.coli, or a lower eukaryotic cell, such as a yeast cell.

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a method for biosynthesizing L-xylose.

Background

Although the rare sugar has a small content in nature, the rare sugar plays an important role in the fields of diet, health care, medicine and the like due to the potential biological activity and low toxicity. The rare sugar has sweetness of natural sugar, but can not or rarely be metabolized by the body, so the sugar can be used as a substitute of high-calorie sugar such as sucrose, for example, pentose substance xylitol, the sweetness of which is equal to 90 percent of that of sucrose, the calorie of which is 60 percent of that of sucrose, can be used as an auxiliary therapeutic agent for diabetes patients, and has physiological activities of reducing blood sugar, preventing decayed teeth, improving liver function, losing weight, alleviating diarrhea, regulating intestinal functions and the like. In addition, the rare sugar can inhibit the production of active oxygen, and for example, D-allose has a property of inhibiting the production of active oxygen as found by He Senjia et al, Japan, Xiangchuan university. They confirmed by experiments that D-allose protects against visceral ischemic disorders. In particular, the rare L-pentoses (such as L-xylose, L-ribose, etc.) can not only be used as precursors for the synthesis of compounds with different biological activities, but also have some particular efficacy themselves. For example, L-ribose has anti-tumor activity, which can increase the mortality of tumor cells, decrease the spread of tumor cells, and delay the growth of malignant tumors.

The preparation method of the rare sugar mainly comprises a chemical synthesis method and a biotransformation method. Chemical synthesis: mainly utilizes catalytic hydrogenation, addition reaction, Mitsunobu reaction, Ferrier rearrangement and BF₃·Et₂And (4) synthesizing rare sugar by methods such as peroxidation initiated by O. Macmillan et al first proposed the concept of a 2-step process for the synthesis of rare sugars in 2004. Firstly, protected hydroxyacetaldehyde is taken as a raw material, an Aldol product (alpha-Aldol dimer) with high enantioselectivity is mildly obtained under the catalysis of L-proline, and then the Aldol product is added with Lewis acid and TiCl₄、MgBr₂Under the action of the substances, the substance is used as an acceptor to perform Aldol condensation (Aldol condensation) with enol silane ether, so that the series hexose is synthesized with high yield and high enantioselectivity. However, the chemical method synthesizes diluteIn the presence of sugar, multi-step catalytic and protective reactions are needed, and the reaction conditions are harsh and complex to operate, so that the yield of rare sugar is low, a large amount of byproducts are generated, the chemical pollution is serious, and the problem of insufficient stereoselectivity generally exists. And (3) a biological conversion method: in the last two decades, Izumori et al, university of xiangchuan, have been working on the biological preparation of rare sugars, and have proposed a set of complete biological preparation strategies-Izumoring, which are suitable for all rare sugars, i.e., using D-tagatose 3-epimerase (DTE), Polyol Dehydrogenase (PDH), oxidoreductase, and aldose isomerase to perform interconversion between all monosaccharides (mainly four-carbon, five-carbon, and six-carbon sugars) and sugar alcohols to prepare various rare sugars. However, the conversion rate of some rare sugars is still very low due to the problems of enzyme activity and specificity.

The carbon resource on the earth is rich, the global formaldehyde capacity is about 6400 ten thousand tons in 2013, the global methanol capacity is about 10324 ten thousand tons, the recoverable storage capacity of natural gas is about 185.7 billion cubic meters, and the recoverable storage capacity of raw coal is about 8915.31 billion tons. Meanwhile, the enzyme catalyst is more and more favored by people due to the characteristics of high activity, high chemical selectivity and the like. Therefore, it is of great significance to explore the synthesis of rare sugars by using formaldehyde as a raw material and using enzyme as a catalyst.

Disclosure of Invention

To overcome the problems of the prior art, in one aspect, the present invention provides a method for biosynthesis of L-xylose, comprising the steps of: step 1, formaldehyde and glycolaldehyde are synthesized into glyceraldehyde under the action of aldolase, mutant thereof or enzyme with the catalytic function; and 2, synthesizing the L-xylose by glyceraldehyde and glycolaldehyde under the action of aldolase, mutant thereof or enzyme with the catalytic function.

According to an embodiment of the invention, wherein the aldolases used in step 1 and step 2, mutants thereof or enzymes having such catalytic function may be the same or different. Preferably, the same enzyme or mutant thereof is used in both steps; more preferably, both steps use D-fructose-6-phosphate aldolase (FSA) and/or mutants thereof.

According to an embodiment of the invention, it further comprises, before the first step, the steps of: and (0) synthesizing the glycolaldehyde by substrate formaldehyde under the action of glycolaldehyde condensing enzyme, mutant thereof or enzyme with the function of catalyzing formaldehyde to synthesize the glycolaldehyde.

According to an embodiment of the invention, wherein the glycolaldehyde condensing enzyme or mutant thereof may catalyze the condensation of formaldehyde to glycolaldehyde, e.g. GALS and/or mutant thereof; the enzyme with the function of catalyzing formaldehyde to synthesize hydroxyacetaldehyde can be benzoylformate decarboxylase (BFD) and/or a mutant thereof. It is understood that in the context of the present invention, reference is made to glycolaldehyde condensing enzymes, mutants thereof and enzymes having the function of catalyzing the synthesis of glycolaldehyde from formaldehyde, all having this definition.

According to an embodiment of the invention, wherein the aldolase or mutant thereof can catalyze the synthesis of glyceraldehyde from formaldehyde and glycolaldehyde or can catalyze the synthesis of L-xylose from glycolaldehyde and glyceraldehyde, can be a D-fructose-6-phosphate aldolase (FSA) and/or a mutant thereof. It is understood that in the context of the present invention, the aldolases mentioned, mutants thereof and enzymes having the function of catalyzing the synthesis of glyceraldehyde from formaldehyde and glycolaldehyde or the synthesis of L-xylose from glycolaldehyde and glyceraldehyde have this definition.

According to an embodiment of the present invention, wherein the amino acid sequence of the BFD mutant comprises a mutation of an amino acid residue in at least one position (e.g. one, two, three, four, five, six or seven positions) corresponding to at least one of W86, N87, L109, L110, H281, Q282, a460 of SEQ ID No.1, and wherein at least one of the mutation positions is W86 or N87. Preferably, the amino acid sequence of the BFD mutant comprises the following site mutations: W86/N87, W86/N87/L109/L110, W86/N87/L109/L110/A460 or W86/N87/L109/L110/H281/Q282/A460. Preferably, wherein the mutation of the amino acid residues of W86, N87, L109, L110, H281, Q282 and a460 is W86R, N87T, L109G, L110E, H281V, Q282F and a460M, respectively. More preferably, the amino acid sequence of the BFD mutant comprises the following site mutations: W86R/N87T, W86R/N87T/L109G/L110E, W86R/N87T/L109G/L110E/A460M or W86R/N87T/L109G/L110E/H281V/Q282F/A460M. It is understood that in the context of the present invention, the BFD mutants mentioned all have this definition.

According to an embodiment of the invention, wherein the amino acid sequence of the FSA mutant comprises a mutation in an amino acid residue corresponding to position a129 and/or a165 of SEQ ID No. 5. Preferably, the mutations at the two positions may be a129T and a165G, respectively. Preferably, the amino acid sequence of the FSA mutant comprises a mutation at the a129T/a165G site. It is to be understood that in the context of the present invention, the FSA mutants mentioned all have this definition.

According to an embodiment of the present invention, the glycolaldehyde condensing enzyme, mutant thereof or enzyme having the function of catalyzing the synthesis of glycolaldehyde from formaldehyde and aldolase, mutant thereof or enzyme having the function of catalyzing the synthesis of glyceraldehyde from formaldehyde and glycolaldehyde or L-xylose from glycolaldehyde and glyceraldehyde used therein may be in the form of purified enzyme or mutant thereof, enzyme cleavage supernatant or whole cell.

According to an embodiment of the invention, the process may be performed in a "one-pot" manner, possibly in a buffer system. The buffer solution can be triethanolamine buffer solution, MOPS buffer solution, HEPES buffer solution, phosphate buffer solution, Tris buffer solution, acetate buffer solution and the like. The pH of the buffer may be 6.5-8.5, for example 7-8.

According to an embodiment of the invention, the concentration of the substrate formaldehyde in the reaction system may be 0.5-30g/L, such as 1-5g/L, 1.5-3g/L, illustratively 2g/L, 2.5g/L or 3 g/L; the concentration of the decarboxylase or the mutant thereof in the reaction system may be 0.1-10mg/mL, such as 0.2-8mg/mL, 0.3-5mg/mL, 0.5-3mg/mL, illustratively 1 mg/mL; the aldolase or mutant thereof may be present in the reaction system at a concentration of 0.1 to 10mg/mL, for example, 0.2 to 8mg/mL, 0.3 to 5mg/mL, 0.5 to 5mg/mL, and illustratively 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5 mg/mL.

According to an embodiment of the present invention, the weight ratio of the glycolaldehyde condensing enzyme, mutant thereof or enzyme having the function of catalyzing the synthesis of glycolaldehyde from formaldehyde to aldolase, mutant thereof or enzyme having the function of catalyzing the synthesis of glyceraldehyde from formaldehyde and glycolaldehyde or the synthesis of L-xylose from glycolaldehyde to glyceraldehyde used is not particularly limited, and may be, for example, 1 (1-10), such as 1 (1-8), 1 (1-5), illustratively 1:1, 1:2, 1:3, 1:4, 1: 5.

According to an embodiment of the invention, the "one-pot" reaction may be carried out at 10-50 ℃, for example at 20-40 ℃, exemplarily at 25 ℃ or 37 ℃.

According to an embodiment of the invention, the "one-pot" reaction may be carried out for 1 to 72 hours, for example 18 to 48 hours, illustratively 24 hours.

According to an embodiment of the invention, the "one-pot" reaction may be carried out under shaking conditions.

In another aspect, the present invention also provides the use of the above-mentioned glycolaldehyde synthase, its mutant, enzyme having a function of catalyzing the synthesis of glycolaldehyde from formaldehyde or related biological material, and aldolase, its mutant, enzyme having a function of catalyzing the synthesis of glyceraldehyde from formaldehyde and glycolaldehyde or L-xylose from glycolaldehyde and glyceraldehyde or related biological material in the biosynthesis of L-xylose.

In still another aspect, the present invention also provides a composition comprising the above-described glycolaldehyde synthase, a mutant thereof, an enzyme having a function of catalyzing the synthesis of glycolaldehyde from formaldehyde or a related biomaterial, and an aldolase, a mutant thereof, an enzyme having a function of catalyzing the synthesis of glyceraldehyde from formaldehyde and glycolaldehyde or a function of catalyzing the synthesis of L-xylose from glycolaldehyde and glyceraldehyde, or a related biomaterial, and use of the composition for the biosynthesis of L-xylose. In the composition, the weight ratio of the glycolaldehyde condensing enzyme, the mutant thereof or the enzyme having the function of catalyzing the synthesis of glycolaldehyde from formaldehyde to aldolase, the mutant thereof or the enzyme having the function of catalyzing the synthesis of glyceraldehyde from formaldehyde and glycolaldehyde or the synthesis of L-xylose from glycolaldehyde to glyceraldehyde is not particularly limited, and may be, for example, 1 (1-10), such as 1 (1-8), 1 (1-5), illustratively 1:1, 1:2, 1:3, 1:4, 1: 5.

According to an embodiment of the present invention, the above-mentioned glycolaldehyde synthase, its mutant, enzyme having a function of catalyzing the synthesis of glycolaldehyde from formaldehyde or related biomaterial, and aldolase, its mutant, enzyme having a function of catalyzing the synthesis of glyceraldehyde from formaldehyde and glycolaldehyde or L-xylose from glycolaldehyde and glyceraldehyde or related biomaterial are used in the biosynthesis of L-xylose using formaldehyde as a substrate.

According to an embodiment of the invention, the biological material related to the glycolaldehyde condensing enzyme is a coding nucleic acid molecule capable of expressing the glycolaldehyde condensing enzyme, a mutant thereof or an enzyme having the function of catalyzing formaldehyde to synthesize glycolaldehyde, or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule.

According to an embodiment of the invention, the biological material related to the aldolase is a nucleic acid molecule encoding an enzyme capable of expressing the aldolase, a mutant thereof or an enzyme having the function of catalyzing the synthesis of glyceraldehyde from formaldehyde and glycolaldehyde or the synthesis of L-xylose from glycolaldehyde and glyceraldehyde, or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line comprising said nucleic acid molecule.

According to an embodiment of the invention, the recombinant bacterium is a recombinant bacterium obtained by introducing a nucleic acid molecule encoding the glycolaldehyde condensing enzyme, a mutant thereof, an enzyme having the function of catalyzing formaldehyde to synthesize glycolaldehyde, or an aldolase, a mutant thereof, or an enzyme having the function of catalyzing formaldehyde and glycolaldehyde to synthesize glyceraldehyde, or catalyzing glycolaldehyde and glyceraldehyde to synthesize L-xylose, into a host cell; for example, in the form of a recombinant vector, into a host cell.

Wherein the recombinant vector is a bacterial plasmid, a bacteriophage, a yeast plasmid or a retrovirus packaging plasmid carrying a nucleic acid molecule encoding the glycolaldehyde acetal, a mutant thereof, an enzyme having the function of catalyzing formaldehyde to synthesize glycolaldehyde, or an aldolase, a mutant thereof, or an enzyme having the function of catalyzing formaldehyde and glycolaldehyde to synthesize glyceraldehyde, or catalyzing glycolaldehyde and glyceraldehyde to synthesize L-xylose.

The host cell may be a prokaryotic cell, such as a bacterium, more specifically e.coli, or a lower eukaryotic cell, such as a yeast cell, among others.

In the context of the present invention, amino acids are represented by a single-letter or three-letter code and have the following meanings: a: ala (alanine); r: arg (arginine); n: asn (asparagine); d: asp (aspartic acid); c: cys (cysteine); q: gln (glutamine); e: glu (glutamic acid); g: gly (glycine); h: his (histidine); i: ile (isoleucine); l: leu (leucine); k: lys (lysine); m: met (methionine); f: phe (phenylalanine); p: pro (proline); s: ser (serine); t: thr (threonine); w: trp (tryptophan); y: tyr (tyrosine); v: val (valine).

In the context of the present invention, mutants are described in terms of their mutation at a particular residue, the position of which is determined by alignment with or reference to the wild-type enzyme sequence. In the context of the present invention, it also relates to any variant carrying these same mutations at functionally equivalent residues.

Herein, the mutation site and its substitution are expressed by the position number of the mutation site and the amino acid kind of the site, for example, W86R indicates that tryptophan at the 86 th position corresponding to SEQ ID NO:1 is mutated to arginine in alignment with SEQ ID NO: 1; N87T shows the mutation of asparagine to threonine at the position 87 corresponding to SEQ ID NO 1 in alignment with SEQ ID NO 1. In the present invention, "/" is used to indicate a combination of mutation sites, for example, "W86/N87" indicates that both tryptophan at position 86 and asparagine at position 87 are mutated, and comprises two mutation sites, and is a double mutant. By analogy, "W86/N87/L109/L110" indicates that the corresponding four sites are mutated simultaneously, and the four sites are four mutants.

Advantageous effects

The invention utilizes aldolase, mutant thereof or enzyme with the function of catalyzing formaldehyde and glycolaldehyde to synthesize glyceraldehyde or catalyzing glycolaldehyde and glyceraldehyde to synthesize L-xylose by taking formaldehyde and glycolaldehyde as substrates, and also can utilize glycolaldehyde condensing enzyme, mutant thereof or combination of the enzyme with the function of catalyzing formaldehyde to synthesize glycolaldehyde and aldolase, mutant thereof or enzyme with the function of catalyzing formaldehyde and glycolaldehyde to synthesize glyceraldehyde or catalyzing glycolaldehyde and glyceraldehyde to synthesize L-xylose by taking formaldehyde as a substrate, thereby providing a new way and a new thought for synthesizing rare sugar. The method for biologically synthesizing the L-xylose has high conversion rate (up to 65 percent), rich substrate sources and mild reaction conditions; has the advantages of simple production process, greening friendliness, easy large-scale production and the like.

Drawings

FIG. 1 shows plasmid maps of pET-28a-BFD, pET-28a-GALS and pET-28a-FSA, wherein 1a is the plasmid map of pET-28a-BFD, 1b is pET-28a-GALS, and 1c is pET-28a-FSA (A129T/A165G).

FIG. 2 shows a spectrum of HPLC detection of L-xylose.

FIG. 3 shows the conversion of L-xylose at different enzyme ratios, where FALD is the substrate formaldehyde.

FIG. 4 shows the yields of hydroxyacetaldehyde by two enzymes GALS and FLS catalyzing formaldehyde to form hydroxyacetaldehyde at different substrate concentrations, where FLS is a mutant formaldehyde-cleaving enzyme (Formolase) of benzaldehyde-cleaving enzyme (BAL) and FALD is the substrate formaldehyde.

Detailed Description

The method for biosynthesis of monosaccharides according to the present invention will be described in further detail with reference to specific examples. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.

Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods. The details of the partial molecular cloning method vary depending on the reagents, enzymes or kits provided by the supplier, and should be conducted according to the product instructions, and will not be described in detail in the examples.

Example 1BFD, GALS, FSA Gene acquisition, vector construction

The BFD (benzoyl formate decarboxylase) gene species source is Pseudomonas putida, the amino acid of the BFD gene species source is shown as SEQ ID NO.1, on the premise of not changing the BFD amino acid sequence, the codon of the wild type gene is replaced by the codon preferred (high frequency use) by Escherichia coli, after codon optimization, the gene sequence has the codon preferred by Escherichia coli, and the gene sequence is shown as SEQ ID NO. 2. The gene sequence was directly synthesized on pET-28a vector between the restriction sites NdeI and XhoI, and the recombinant plasmid was named pET-28a-BFD (see FIG. 1 a). In addition, the mutant of BFD also has the function of catalyzing 1-formaldehyde to synthesize 2-hydroxyacetaldehyde, and is listed in chinese patent application No. cn201710096307.x (publication No. CN 106916794A). GALS is a mutant of BFD, the activity of catalyzing formaldehyde to synthesize hydroxyacetaldehyde is obviously higher than that of BFD, the mutant is named as hydroxyacetaldehyde synthase, the amino acid sequence is shown as SEQ ID No.3, and the gene sequence is shown as SEQ ID No. 4. And compared with FLS (mutant formaldehyde enzyme (Formolase) of benzaldehyde lyase BAL) which is an enzyme catalyzing formaldehyde to synthesize hydroxyacetaldehyde, known in the prior art (see Siegel JB, Smith AL, Poust S, et AL, synthetic protein enzymes a novel one-carbon assembly diagnosis pathway. Proc Natl Acad Sci USA,2015,112(12): 3704-. Therefore, GALS is used in the subsequent examples.

Coli, wherein the amino acid of the FSA (D-Fructose-6-Phosphate Aldolase ) gene species is shown as SEQ ID NO.5, and the gene sequence is shown as SEQ ID NO. 6. Coli genome, and constructing into pET-28a vector, located between enzyme cutting sites NdeI and XhoI, and mutating alanine (A) at position 129 to threonine (T), and mutating alanine (A) at position 165 to glycine (G), wherein the recombinant plasmid is named pET-28a-FSA (A129T/A165G) (shown in figure 1b), the amino acid sequence is shown as SEQ ID NO.7, and the gene sequence is shown as SEQ ID NO. 8.

Example 2 expression of genes

For in vitro detection of GALS and FSA (A129T/A165G) enzyme activities, the enzyme was exogenously expressed and purified in E.coli.

(1) Coli expression type recombinant plasmids pET-28a-GALS and pET-28a-FSA (A129T/A165G) are respectively transferred into E.coli BL21(DE3) to obtain recombinant bacteria. Screening positive clones by using kanamycin-resistant plates (Kan +, 100mg/mL), and culturing overnight at 37 ℃;

(2) the single clones were picked up in 5mL of LB liquid medium (Kan +, 100mg/mL), cultured at 37 ℃ and 220rpm until OD600 became 0.6-0.8. Transferring 5mL of LB medium to 800mL of 2YT medium (Kan +, 100mg/mL), culturing at 37 deg.C and 220rpm to OD₆₀₀When the concentration is 0.6-0.8 ℃, cooling to 16 ℃, adding IPTG (isopropyl thiogalactoside) to the final concentration of 0.5mM, and carrying out induced expression for 16 h;

(3) collecting the culture bacteria liquid into a bacteria collection bottle, and centrifuging at 5500rpm for 15 min;

(4) the supernatant was discarded, and the resulting pellet was suspended in 35mL of protein buffer (50mM triethanolamine buffer, pH7.4) and poured into a 50mL centrifuge tube and stored in a freezer at-80 ℃.

EXAMPLE 3 protein purification

(1) Breaking the bacteria: the high-pressure low-temperature crusher is adopted to crush the bacteria for 2 times under the conditions of the pressure of 1200bar and the temperature of 4 ℃. Centrifuging at 10000rpm for 45min at 4 ℃;

(2) and (3) purification: filtering the supernatant with a 0.45 μm microporous membrane, and purifying by nickel affinity chromatography, which comprises the following steps:

a: column balancing: before hanging the supernatant, ddH is firstly used₂Washing 2 column volumes with O, and balancing 1 column volume of the Ni affinity chromatography column with protein buffer solution;

b: loading: the supernatant is slowly passed through a Ni affinity chromatographic column at the flow rate of 0.5mL/min, and the steps are repeated once;

c: and (3) eluting the hybrid protein: washing 1 column volume with protein buffer solution, and eluting the hybrid protein with strong binding with 50mL protein buffer solution containing 50mM and 100mM imidazole respectively;

d: eluting the target protein: the target protein was eluted with 20mL of a protein buffer containing 200mM imidazole, and the first few drops of the sample were collected and sampled for detection by 12% SDS-PAGE.

(3) Liquid changing: the collected target protein was concentrated by centrifugation (4 ℃ C., 3400rpm) using a 50mL Amicon ultrafiltration tube (30kDa, Millipore Co.) to 1 mL. Adding 15mL protein buffer solution without imidazole, concentrating to 1mL, repeating for 1 time to obtain GALS and FSA (A129T/A165G) proteins.

(4) And detecting the concentration of the concentrated protein by a Nondrop 2000 micro spectrophotometer and diluting to 10mg/mL to obtain the GALS and FSA (A129T/A165G) protein.

EXAMPLE 4 Synthesis of L-xylose

1: formaldehyde; 2: a hydroxyacetaldehyde; 3: glyceraldehyde; 4: l-xylose; GALS: a hydroxyacetaldehyde synthase; FSA (A129T/A165G): d-fructose-6-phosphate aldolase mutant.

(1) Derivatization reagent formulation (1 mL): 21.1mg of benzyloxyamine hydrochloride was dissolved in 660. mu.L of pyridine, and 300. mu.L of methanol, 40. mu. L H, was added₂O, mixing uniformly and storing at 4 ℃.

(2) Reaction conditions are as follows: 2g/L formaldehyde, 50mM triethanolamine buffer, pH7.4, 1mg/mL GALS, 1mg/mL FSA (A129T/A165G), shaking at 25 ℃ for 24 h. mu.L of sample was taken, 50. mu.L of derivatizing reagent was added, reaction was carried out at 50 ℃ for 1h, 140. mu.L of methanol was added, filtration and HPLC detection were carried out.

HPLC detection conditions: mobile phase: a: 0.1% (v/v) trifluoroacetic acid TFA; b: dissolved in 80% CH₃CN 0.095% (v/v) TFA. Elution gradient: the mobile phase B is 20-60% in 16 min. Column: X-Bridge TM C18,5 μm, 4.6X 250 mm. Flow rate: 1mL/min, detection wavelength: 215nm, column oven temperature: 35 ℃, sample introduction: 20 μ L. The results of the detection are shown in FIG. 2. After GALS and FSA (A129T/A165G) are added, xylose can be directly synthesized from formaldehyde, and the conversion rate can reach 65% under the condition of 1g/L formaldehyde (figure 3).

SEQ ID NO.1-6 are shown below:

SEQ ID NO. 1: amino acid sequence of BFD

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQASRKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEALLTNVDAANLPRPLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRHVSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSAPRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHQYDPGQYLKPGTRLISVTCDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVFDTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYFCAAGGLGFALPAAIGVQLAEPERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGALRWFAGVLEAENVPGLDVPGIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK*

SEQ ID NO. 2: BFD gene sequence:

ATGGCTTCTGTTCACGGTACCACCTACGAACTGCTGCGTCGTCAGGGTATCGACACCGTTTTCGGTAACCCGGGTTCTAACGAACTGCCGTTCCTGAAAGACTTCCCGGAAGACTTCCGTTACATCCTGGCTCTGCAGGAAGCTTGCGTTGTTGGTATCGCTGACGGTTACGCTCAGGCTTCTCGTAAACCGGCTTTCATCAACCTGCACTCTGCTGCTGGTACCGGTAACGCTATGGGTGCTCTGTCTAACGCTTGGAACTCTCACTCTCCGCTGATCGTTACCGCTGGTCAGCAGACCCGTGCTATGATCGGTGTTGAAGCTCTGCTGACCAACGTTGACGCTGCTAACCTGCCGCGTCCGCTGGTTAAATGGTCTTACGAACCGGCTTCTGCTGCTGAAGTTCCGCACGCTATGTCTCGTGCTATCCACATGGCTTCTATGGCTCCGCAGGGTCCGGTTTACCTGTCTGTTCCGTACGACGACTGGGACAAAGACGCTGACCCGCAGTCTCACCACCTGTTCGACCGTCACGTTTCTTCTTCTGTTCGTCTGAACGACCAGGACCTGGACATCCTGGTTAAAGCTCTGAACTCTGCTTCTAACCCGGCTATCGTTCTGGGTCCGGACGTTGACGCTGCTAACGCTAACGCTGACTGCGTTATGCTGGCTGAACGTCTGAAAGCTCCGGTTTGGGTTGCTCCGTCTGCTCCGCGTTGCCCGTTCCCGACCCGTCACCCGTGCTTCCGTGGTCTGATGCCGGCTGGTATCGCTGCTATCTCTCAGCTGCTGGAAGGTCACGACGTTGTTCTGGTTATCGGTGCTCCGGTTTTCCGTTACCACCAGTACGACCCGGGTCAGTACCTGAAACCGGGTACCCGTCTGATCTCTGTTACCTGCGACCCGCTGGAAGCTGCTCGTGCTCCGATGGGTGACGCTATCGTTGCTGACATCGGTGCTATGGCTTCTGCTCTGGCTAACCTGGTTGAAGAATCTTCTCGTCAGCTGCCGACCGCTGCTCCGGAACCGGCTAAAGTTGACCAGGACGCTGGTCGTCTGCACCCGGAAACCGTTTTCGACACCCTGAACGACATGGCTCCGGAAAACGCTATCTACCTGAACGAATCTACCTCTACCACCGCTCAGATGTGGCAGCGTCTGAACATGCGTAACCCGGGTTCTTACTACTTCTGCGCTGCTGGTGGTCTGGGTTTCGCTCTGCCGGCTGCTATCGGTGTTCAGCTGGCTGAACCGGAACGTCAGGTTATCGCTGTTATCGGTGACGGTTCTGCTAACTACTCTATCTCTGCTCTGTGGACCGCTGCTCAGTACAACATCCCGACCATCTTCGTTATCATGAACAACGGTACCTACGGTGCTCTGCGTTGGTTCGCTGGTGTTCTGGAAGCTGAAAACGTTCCGGGTCTGGACGTTCCGGGTATCGACTTCCGTGCTCTGGCTAAAGGTTACGGTGTTCAGGCTCTGAAAGCTGACAACCTGGAACAGCTGAAAGGTTCTCTGCAGGAAGCTCTGTCTGCTAAAGGTCCGGTTCTGATCGAAGTTTCTACCGTTTCTCCGGTTAAATAA

SEQ ID NO. 3: amino acid sequence of GALS:

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQASRKPAFINLHSAAGTGNAMGALSNARTSHSPLIVTAGQQTRAMIGVEAGETNVDAANLPRPLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRHVSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSAPRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYVFYDPGQYLKPGTRLISVTCDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVFDTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYFCAAGGLGFALPAAIGVQLAEPERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGMLRWFAGVLEAENVPGLDVPGIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK*

SEQ ID NO. 4: the gene sequence of GALS:

ATGGCTTCTGTTCACGGTACCACCTACGAACTGCTGCGTCGTCAGGGTATCGACACCGTTTTCGGTAACCCGGGTTCTAACGAACTGCCGTTCCTGAAAGACTTCCCGGAAGACTTCCGTTACATCCTGGCTCTGCAGGAAGCTTGCGTTGTTGGTATCGCTGACGGTTACGCTCAGGCTTCTCGTAAACCGGCTTTCATCAACCTGCACTCTGCTGCTGGTACCGGTAACGCTATGGGTGCTCTGTCTAACGCTCGTACCTCTCACTCTCCGCTGATCGTTACCGCTGGTCAGCAGACCCGTGCTATGATCGGTGTTGAAGCTGGTGAAACCAACGTTGACGCTGCTAACCTGCCGCGTCCGCTGGTTAAATGGTCTTACGAACCGGCTTCTGCTGCTGAAGTTCCGCACGCTATGTCTCGTGCTATCCACATGGCTTCTATGGCTCCGCAGGGTCCGGTTTACCTGTCTGTTCCGTACGACGACTGGGACAAAGACGCTGACCCGCAGTCTCACCACCTGTTCGACCGTCACGTTTCTTCTTCTGTTCGTCTGAACGACCAGGACCTGGACATCCTGGTTAAAGCTCTGAACTCTGCTTCTAACCCGGCTATCGTTCTGGGTCCGGACGTTGACGCTGCTAACGCTAACGCTGACTGCGTTATGCTGGCTGAACGTCTGAAAGCTCCGGTTTGGGTTGCTCCGTCTGCTCCGCGTTGCCCGTTCCCGACCCGTCACCCGTGCTTCCGTGGTCTGATGCCGGCTGGTATCGCTGCTATCTCTCAGCTGCTGGAAGGTCACGACGTTGTTCTGGTTATCGGTGCTCCGGTTTTCCGTTACGTTTTTTACGACCCGGGTCAGTACCTGAAACCGGGTACCCGTCTGATCTCTGTTACCTGCGACCCGCTGGAAGCTGCTCGTGCTCCGATGGGTGACGCTATCGTTGCTGACATCGGTGCTATGGCTTCTGCTCTGGCTAACCTGGTTGAAGAATCTTCTCGTCAGCTGCCGACCGCTGCTCCGGAACCGGCTAAAGTTGACCAGGACGCTGGTCGTCTGCACCCGGAAACCGTTTTCGACACCCTGAACGACATGGCTCCGGAAAACGCTATCTACCTGAACGAATCTACCTCTACCACCGCTCAGATGTGGCAGCGTCTGAACATGCGTAACCCGGGTTCTTACTACTTCTGCGCTGCTGGTGGTCTGGGTTTCGCTCTGCCGGCTGCTATCGGTGTTCAGCTGGCTGAACCGGAACGTCAGGTTATCGCTGTTATCGGTGACGGTTCTGCTAACTACTCTATCTCTGCTCTGTGGACCGCTGCTCAGTACAACATCCCGACCATCTTCGTTATCATGAACAACGGTACCTACGGTATGCTGCGTTGGTTCGCTGGTGTTCTGGAAGCTGAAAACGTTCCGGGTCTGGACGTTCCGGGTATCGACTTCCGTGCTCTGGCTAAAGGTTACGGTGTTCAGGCTCTGAAAGCTGACAACCTGGAACAGCTGAAAGGTTCTCTGCAGGAAGCTCTGTCTGCTAAAGGTCCGGTTCTGATCGAAGTTTCTACCGTTTCTCCGGTTAAATAA

SEQ ID No. 5: amino acid sequence of FSA:

MELYLDTANVAEVERLARIFPIAGVTTNPSIIAASKESIWEVLPRLQKAIGDEGILFAQTMSRDAQGMVKEAKHLRDAIPGIVVKIPVTSEGLAAIKMLKKEGITTLGTAVYSAAQGLLAALAGAKYVAPYVNRVDAQGGDGIRTVQELQALLEMHAPESMVLAASFKTPRQALDCLLAGCESITLPLDVAQQMLNTPAVESAIEKFEHDWNAAFDTTHL

SEQ ID NO. 6: gene sequence for FSA:

ATGGAACTGTATCTGGACACCGCTAACGTCGCAGAAGTCGAACGTCTGGCACGCATATTCCCGATTGCCGGGGTGACAACTAACCCGAGCATTATCGCTGCCAGCAAGGAGTCCATCTGGGAAGTGCTGCCGCGCCTTCAAAAAGCGATCGGTGATGAGGGCATTCTGTTTGCTCAGACCATGAGCCGCGACGCGCAGGGTATGGTGAAAGAAGCGAAACACCTGCGCGACGCTATTCCGGGCATTGTGGTGAAAATTCCGGTAACCTCTGAAGGTCTGGCAGCAATTAAAATGCTGAAGAAAGAAGGCATTACTACGCTGGGAACCGCAGTGTACAGCGCCGCGCAAGGATTACTGGCGGCGCTGGCTGGAGCCAAATACGTTGCTCCATACGTTAACCGCGTAGATGCCCAGGGCGGTGACGGCATTCGTACTGTACAGGAGTTGCAAGCGTTACTGGAAATGCATGCGCCAGAAAGCATGGTGCTGGCTGCCAGCTTTAAAACACCACGTCAGGCGCTGGATTGTTTGCTGGCTGGATGTGAATCCATCACACTGCCCTTAGATGTAGCGCAACAAATGCTTAACACCCCTGCGGTAGAGTCAGCTATAGAGAAGTTCGAGCACGACTGGAATGCCGCATTTGACACTACTCATCTCTAASEQ ID NO. 7: amino acid sequence of FSA (A129T/A165G):

MELYLDTSDVVAVKALSRIFPLAGVTTNPSIIAAGKKPLDVVLPQLHEAMGGQGRLFAQVMATTAEGMVNDALKLRSIIADIVVKVPVTAEGLAAIKMLKAEGIPTLGTAVYGAAQGLLSALAGAEYVAPYVNRIDAQGGSGIQTVTDLHQLLKMHAPQAKVLAASFKTPRQALDCLLAGCESITLPLDVAQQMISYPAVDAAVAKFEQDWQGAFGRTSI

SEQ ID NO. 8: gene sequence of FSA (A129T/A165G)

atggaactgtatctggatacttcagacgttgttgcggtgaaggcgctgtcacgtatttttccgctggcgggtgtgaccactaacccaagcattatcgccgcgggtaaaaaaccgctggatgttgtgcttccgcaacttcatgaagcgatgggcggtcaggggcgtctgtttgcccaggtaatggctaccactgccgaagggatggttaatgacgcgcttaagctgcgttctattattgcggatatcgtggtgaaagttccggtgaccgccgaggggctggcagctattaagatgttaaaagcggaagggattccgacgctgggaaccgcggtatatggcgcagcacaagggctgctgtcggcgctggcaggtgcggaatatgttgcgccttacgttaatcgtattgatgctcagggcggtagcggcattcagactgtgaccgacttacaccagttattgaaaatgcatgcgccgcaggcgaaagtgctggcagcgagtttcaaaaccccgcgtcaggcgctggactgcttactggcaggatgtgaatcaattactctgccactggatgtggcacaacagatgattagctatccggcggttgatgccgctgtggcgaagtttgagcaggactggcagggagcgtttggcagaacgtcgatt

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

<120> method for biosynthesis of xylose

<130> CPCN20110550

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 528

<212> PRT

<213> Pseudomonas putida

<400> 1

Met Ala Ser Val His Gly Thr Thr Tyr Glu Leu Leu Arg Arg Gln Gly

1 5 10 15

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

20 25 30

Lys Asp Phe Pro Glu Asp Phe Arg Tyr Ile Leu Ala Leu Gln Glu Ala

35 40 45

Cys Val Val Gly Ile Ala Asp Gly Tyr Ala Gln Ala Ser Arg Lys Pro

50 55 60

Ala Phe Ile Asn Leu His Ser Ala Ala Gly Thr Gly Asn Ala Met Gly

65 70 75 80

Ala Leu Ser Asn Ala Trp Asn Ser His Ser Pro Leu Ile Val Thr Ala

85 90 95

Gly Gln Gln Thr Arg Ala Met Ile Gly Val Glu Ala Leu Leu Thr Asn

100 105 110

Val Asp Ala Ala Asn Leu Pro Arg Pro Leu Val Lys Trp Ser Tyr Glu

115 120 125

Pro Ala Ser Ala Ala Glu Val Pro His Ala Met Ser Arg Ala Ile His

130 135 140

Met Ala Ser Met Ala Pro Gln Gly Pro Val Tyr Leu Ser Val Pro Tyr

145 150 155 160

Asp Asp Trp Asp Lys Asp Ala Asp Pro Gln Ser His His Leu Phe Asp

165 170 175

Arg His Val Ser Ser Ser Val Arg Leu Asn Asp Gln Asp Leu Asp Ile

180 185 190

Leu Val Lys Ala Leu Asn Ser Ala Ser Asn Pro Ala Ile Val Leu Gly

195 200 205

Pro Asp Val Asp Ala Ala Asn Ala Asn Ala Asp Cys Val Met Leu Ala

210 215 220

Glu Arg Leu Lys Ala Pro Val Trp Val Ala Pro Ser Ala Pro Arg Cys

225 230 235 240

Pro Phe Pro Thr Arg His Pro Cys Phe Arg Gly Leu Met Pro Ala Gly

245 250 255

Ile Ala Ala Ile Ser Gln Leu Leu Glu Gly His Asp Val Val Leu Val

260 265 270

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

275 280 285

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

290 295 300

Ala Ala Arg Ala Pro Met Gly Asp Ala Ile Val Ala Asp Ile Gly Ala

305 310 315 320

Met Ala Ser Ala Leu Ala Asn Leu Val Glu Glu Ser Ser Arg Gln Leu

325 330 335

Pro Thr Ala Ala Pro Glu Pro Ala Lys Val Asp Gln Asp Ala Gly Arg

340 345 350

Leu His Pro Glu Thr Val Phe Asp Thr Leu Asn Asp Met Ala Pro Glu

355 360 365

Asn Ala Ile Tyr Leu Asn Glu Ser Thr Ser Thr Thr Ala Gln Met Trp

370 375 380

Gln Arg Leu Asn Met Arg Asn Pro Gly Ser Tyr Tyr Phe Cys Ala Ala

385 390 395 400

Gly Gly Leu Gly Phe Ala Leu Pro Ala Ala Ile Gly Val Gln Leu Ala

405 410 415

Glu Pro Glu Arg Gln Val Ile Ala Val Ile Gly Asp Gly Ser Ala Asn

420 425 430

Tyr Ser Ile Ser Ala Leu Trp Thr Ala Ala Gln Tyr Asn Ile Pro Thr

435 440 445

Ile Phe Val Ile Met Asn Asn Gly Thr Tyr Gly Ala Leu Arg Trp Phe

450 455 460

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

465 470 475 480

Ile Asp Phe Arg Ala Leu Ala Lys Gly Tyr Gly Val Gln Ala Leu Lys

485 490 495

Ala Asp Asn Leu Glu Gln Leu Lys Gly Ser Leu Gln Glu Ala Leu Ser

500 505 510

Ala Lys Gly Pro Val Leu Ile Glu Val Ser Thr Val Ser Pro Val Lys

515 520 525

<210> 2

<211> 1587

<212> DNA

<213> Pseudomonas putida

<400> 2

atggcttctg ttcacggtac cacctacgaa ctgctgcgtc gtcagggtat cgacaccgtt 60

ttcggtaacc cgggttctaa cgaactgccg ttcctgaaag acttcccgga agacttccgt 120

tacatcctgg ctctgcagga agcttgcgtt gttggtatcg ctgacggtta cgctcaggct 180

tctcgtaaac cggctttcat caacctgcac tctgctgctg gtaccggtaa cgctatgggt 240

gctctgtcta acgcttggaa ctctcactct ccgctgatcg ttaccgctgg tcagcagacc 300

cgtgctatga tcggtgttga agctctgctg accaacgttg acgctgctaa cctgccgcgt 360

ccgctggtta aatggtctta cgaaccggct tctgctgctg aagttccgca cgctatgtct 420

cgtgctatcc acatggcttc tatggctccg cagggtccgg tttacctgtc tgttccgtac 480

gacgactggg acaaagacgc tgacccgcag tctcaccacc tgttcgaccg tcacgtttct 540

tcttctgttc gtctgaacga ccaggacctg gacatcctgg ttaaagctct gaactctgct 600

tctaacccgg ctatcgttct gggtccggac gttgacgctg ctaacgctaa cgctgactgc 660

gttatgctgg ctgaacgtct gaaagctccg gtttgggttg ctccgtctgc tccgcgttgc 720

ccgttcccga cccgtcaccc gtgcttccgt ggtctgatgc cggctggtat cgctgctatc 780

tctcagctgc tggaaggtca cgacgttgtt ctggttatcg gtgctccggt tttccgttac 840

caccagtacg acccgggtca gtacctgaaa ccgggtaccc gtctgatctc tgttacctgc 900

gacccgctgg aagctgctcg tgctccgatg ggtgacgcta tcgttgctga catcggtgct 960

atggcttctg ctctggctaa cctggttgaa gaatcttctc gtcagctgcc gaccgctgct 1020

ccggaaccgg ctaaagttga ccaggacgct ggtcgtctgc acccggaaac cgttttcgac 1080

accctgaacg acatggctcc ggaaaacgct atctacctga acgaatctac ctctaccacc 1140

gctcagatgt ggcagcgtct gaacatgcgt aacccgggtt cttactactt ctgcgctgct 1200

ggtggtctgg gtttcgctct gccggctgct atcggtgttc agctggctga accggaacgt 1260

caggttatcg ctgttatcgg tgacggttct gctaactact ctatctctgc tctgtggacc 1320

gctgctcagt acaacatccc gaccatcttc gttatcatga acaacggtac ctacggtgct 1380

ctgcgttggt tcgctggtgt tctggaagct gaaaacgttc cgggtctgga cgttccgggt 1440

atcgacttcc gtgctctggc taaaggttac ggtgttcagg ctctgaaagc tgacaacctg 1500

gaacagctga aaggttctct gcaggaagct ctgtctgcta aaggtccggt tctgatcgaa 1560

gtttctaccg tttctccggt taaataa 1587

<210> 3