CN113699195B - Method for biosynthesis of xylose - Google Patents

Method for biosynthesis of xylose Download PDF

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CN113699195B
CN113699195B CN202010444222.8A CN202010444222A CN113699195B CN 113699195 B CN113699195 B CN 113699195B CN 202010444222 A CN202010444222 A CN 202010444222A CN 113699195 B CN113699195 B CN 113699195B
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江会锋
逯晓云
刘玉万
初斋林
崔博
卢丽娜
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Tianjin National Synthetic Biotechnology Innovation Center Co ltd
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Abstract

The present invention provides a method for biosynthesis of L-xylose, which converts the substrates formaldehyde and hydroxyacetaldehyde into L-xylose using an aldolase such as D-fructose-6-phosphate aldolase (FSA), or can convert the substrate formaldehyde into L-xylose using a "one-pot" method using a combination of a hydroxyacetaldehyde condensing enzyme (GALS), a mutant thereof or an enzyme having a function of catalyzing formaldehyde synthesis to hydroxyacetaldehyde such as benzoylformate decarboxylase (BFD), and an aldolase such as D-fructose-6-phosphate aldolase (FSA) mutant. The biosynthesis method has the advantages of high conversion rate, simple production process, friendly greening, easy mass production and the like.

Description

Method for biosynthesis of xylose
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a method for biosynthesis of L-xylose.
Background
Rare sugar has a small content in nature, but plays an important role in the fields of diet, health care, medicine and the like due to the potential bioactivity and low toxicity. The rare sugar usually has the sweetness of natural sugar but can not or seldom be metabolized by the organism, so that the rare sugar can be used as a substitute of high-calorie sugar such as sucrose, for example, pentose substance xylitol, the sweetness of which is equivalent to 90 percent of that of sucrose and the calorie of which is 60 percent of that of sucrose, can be used as an auxiliary therapeutic agent for diabetics, and has physiological activities of reducing blood sugar, preventing decayed teeth, improving liver function, reducing weight, relieving diarrhea, regulating intestinal tract function and the like. Furthermore, rare sugars can inhibit the production of active oxygen, for example, D-allose has been found by university of Japan, university of Ipomoea He Senjian, et al to have the property of inhibiting the production of active oxygen. They confirmed by experiments that D-allose can protect against visceral ischemic disorders. In particular, rare L-pentoses (e.g., L-xylose, L-ribose, etc.) can be used not only as precursors for the synthesis of compounds with different biological activities, but also as such have some special efficacy. For example, L-ribose has anti-tumor activity, which can increase the mortality of tumor cells, reduce the spread of tumor cells, and delay the growth of malignant tumors.
The preparation method of rare sugar mainly comprises a chemical synthesis method and a biological conversion method. Synthesizing by a chemical method: mainly uses catalytic hydrogenation, addition reaction, mitsunobu reaction, ferrier rearrangement and BF 3 ·Et 2 O-initiated peroxidation, etc. Macmillan et al, in 2004, first proposed the concept of 2-step synthesis of rare sugars. They firstly take protected hydroxy acetaldehyde as raw material, gently obtain high enantioselectivity Aldol product (alpha-Aldol dimer) under the catalysis of L-proline, then make them pass through Lewis acid and TiCl 4 、MgBr 2 Aldol condensation (Aldol condensation) is carried out on the enol silane ether serving as a receptor under the action of the substances, so that the series hexoses are synthesized with high yield and high enantioselectivity. However, the chemical method for synthesizing rare sugar requires multi-step catalytic and protective reactions, and has the problems of harsh reaction conditions, complex operation, low yield of rare sugar, more byproducts, serious chemical pollution and insufficient stereoselectivity. Bioconversion process: izumori et al, university of Japan, xiangchuan, have been devoted to the last two decadesIn the biological preparation of rare sugar, a complete biological preparation strategy-Izumoring method applicable to all rare sugar is provided, namely D-tagatose 3-epimerase (DTE), polyol dehydrogenase (polyol dehydrogenase, PDH), oxidoreductase and aldose isomerase are utilized to carry out the mutual conversion between all monosaccharides (mainly four-carbon sugar, five-carbon sugar and six-carbon sugar) and sugar alcohol to prepare various rare sugar. However, the conversion rate of some rare sugars is still low due to enzyme activity and specificity problems.
The global carbon-resource is rich, the global formaldehyde productivity in 2013 is about 6400 ten thousand tons, the global methanol productivity is about 10324 ten thousand tons, the natural gas can be stored for about 185.7 trillion cubic meters, and the raw coal can be stored for about 8915.31 hundred million tons. Meanwhile, enzyme catalysts are increasingly favored by people due to the characteristics of high activity, high chemical selectivity and the like. Therefore, it is important to search for synthesis of rare sugars using formaldehyde as a raw material and an enzyme as a catalyst.
Disclosure of Invention
In order to overcome the problems in 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 hydroxy aldehyde are synthesized into glyceraldehyde under the action of aldolase, mutant or enzyme with the catalytic function; step 2, glyceraldehyde and hydroxyaldehyde are synthesized into L-xylose under the action of aldolase, mutant or enzyme with the catalytic function.
According to an embodiment of the invention, wherein the aldolase or mutant thereof may catalyze formaldehyde and hydroxyaldehyde to glyceraldehyde or may catalyze hydroxyaldehyde and glyceraldehyde to L-xylose, may be D-fructose-6-phosphate aldolase (FSA) and/or a mutant thereof.
According to an embodiment of the present invention, wherein the aldolases, mutants thereof or enzymes having this catalytic function used in step 1 and step 2 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 a mutant thereof.
According to an embodiment of the invention, it further comprises the following steps before the first step: step 0, synthesizing the formaldehyde serving as a substrate into the hydroxy-acetaldehyde under the action of hydroxy-acetaldehyde condensing enzyme, mutant or enzyme with the function of catalyzing formaldehyde to synthesize hydroxy-acetaldehyde.
According to an embodiment of the invention, wherein the hydroxyaldehyde condensing enzyme or mutant thereof may catalyze the condensation of formaldehyde to hydroxyaldehyde, e.g. GALS and/or mutants thereof; the enzyme having a function of catalyzing formaldehyde to synthesize hydroxyacetaldehyde may be a Benzoyl Formate Decarboxylase (BFD) and/or a mutant thereof. It is to be understood that in the context of the present invention, all references to the hydroxyaldehyde condensation enzyme, mutants thereof and enzymes having the function of catalyzing the synthesis of formaldehyde to hydroxyaldehyde have this definition.
According to an embodiment of the invention, wherein the aldolase or mutant thereof may catalyze formaldehyde and hydroxyaldehyde to glyceraldehyde or may catalyze hydroxyaldehyde and glyceraldehyde to L-xylose, may be D-fructose-6-phosphate aldolase (FSA) and/or a mutant thereof. It is to be understood that in the context of the present invention, the mentioned aldolases, mutants thereof and enzymes having the function of catalyzing the synthesis of glyceraldehyde from formaldehyde and hydroxyaldehyde or catalyzing the synthesis of L-xylose from hydroxyaldehyde and glyceraldehyde have this definition.
According to an embodiment of the invention, wherein the amino acid sequence of the BFD mutant comprises a mutation of an amino acid residue at least one position (e.g. one, two, three, four, five, six or seven positions) in W86, N87, L109, L110, H281, Q282, a460 corresponding to SEQ ID No.1, and wherein at least one mutation position is W86 or N87. Preferably, the amino acid sequence of the BFD mutant comprises the following site mutations: W86/N87, W86/N87/L109/L110/A460 or W86/N87/L109/L110/H281/Q282/A460. Preferably, wherein the mutations of the amino acid residues of W86, N87, L109, L110, H281, Q282 and a460 are 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, W R/N87T/L109G/L110E, W R/N87T/L109G/L110E/A460M or W86R/N87T/L109G/L110E/H281V/Q282F/A460M. It is to be understood that in the context of the present invention, all mentioned BFD mutants have this definition.
According to an embodiment of the invention, wherein the amino acid sequence of the FSA mutant comprises a mutation of the amino acid residues at positions a129 and/or a165 corresponding to SEQ ID No. 5. Preferably, the mutations at the two sites may be a129T and a165G, respectively. Preferably, the amino acid sequence of the FSA mutant comprises a site mutation of A129T/A165G. It is to be understood that in the context of the present invention, all mentioned FSA mutants have this definition.
According to an embodiment of the present invention, wherein the aldolase, mutant thereof or enzyme having a function of catalyzing the synthesis of aldol from formaldehyde and aldolase, mutant thereof or enzyme having a function of catalyzing the synthesis of glyceraldehyde from formaldehyde and aldol or catalyzing the synthesis of L-xylose from aldol and glyceraldehyde used may be in the form of a purified enzyme or mutant thereof, an enzyme lysate or whole cells.
According to embodiments of the invention, the process may be performed in a "one-pot" manner, which may be performed in a buffer system. The buffer may be triethanolamine buffer, MOPS buffer, HEPES buffer, phosphate buffer, tris buffer, acetate buffer, etc. The pH of the buffer may be from 6.5 to 8.5, for example from 7 to 8.
According to embodiments of the present invention, the concentration of the substrate formaldehyde in the reaction system may be 0.5-30g/L, for example 1-5g/L,1.5-3g/L, illustratively 2g/L, 2.5g/L or 3g/L; the concentration of the decarboxylase or mutant thereof in the reaction system may be 0.1-10mg/mL, for example 0.2-8mg/mL,0.3-5mg/mL,0.5-3mg/mL, and exemplary 1mg/mL; the aldolase or mutant thereof may be present in the reaction system at a concentration of 0.1-10mg/mL, for example 0.2-8mg/mL,0.3-5mg/mL,0.5-5mg/mL, illustratively 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL.
According to an embodiment of the present invention, wherein the weight ratio of the hydroxyaldehyde condensation enzyme, mutant thereof or enzyme having a function of catalyzing formaldehyde to synthesize hydroxyaldehyde to aldolase, mutant thereof or enzyme having a function of catalyzing formaldehyde to synthesize glyceraldehyde or catalyzing hydroxyaldehyde to synthesize L-xylose is not particularly limited, for example, may be 1 (1-10), such as 1 (1-8), 1 (1-5), illustratively 1:1, 1:2, 1:3, 1:4, 1:5.
According to embodiments of the present invention, the "one pot" reaction may be carried out at 10-50 ℃, e.g. 20-40 ℃, illustratively 25 ℃ or 37 ℃.
According to embodiments of the present 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 embodiments of the present invention, the "one pot" reaction may be performed under shaking conditions.
In another aspect, the present invention also provides the use of the above-described hydroxyaldehyde synthase, mutant thereof, enzyme having a function of catalyzing formaldehyde to synthesize hydroxyaldehyde or related biological material, aldolase, mutant thereof, enzyme having a function of catalyzing formaldehyde and hydroxyaldehyde to synthesize glyceraldehyde or catalyzing hydroxyaldehyde and glyceraldehyde to synthesize L-xylose or a combination of related biological materials for biosynthesis of L-xylose.
In a further aspect, the invention also provides a composition comprising the above-described hydroxyaldehyde synthase, a mutant thereof, an enzyme or related biological material having a function of catalyzing formaldehyde to synthesize hydroxyaldehyde, and aldolase, a mutant thereof, an enzyme or related biological material having a function of catalyzing formaldehyde and hydroxyaldehyde to synthesize glyceraldehyde or catalyzing hydroxyaldehyde and glyceraldehyde to synthesize L-xylose, and the use of the composition for biosynthesis of L-xylose. In the composition, the weight ratio of the hydroxyaldehyde condensation enzyme, a mutant thereof or an enzyme having a function of catalyzing formaldehyde to synthesize hydroxyaldehyde to the aldolase, a mutant thereof or an enzyme having a function of catalyzing formaldehyde to synthesize glyceraldehyde or catalyzing hydroxyaldehyde to synthesize L-xylose is not particularly limited, and may be, for example, 1 (1-10), for example, 1 (1-8), 1 (1-5), and exemplified by 1:1, 1:2, 1:3, 1:4, 1:5.
According to an embodiment of the present invention, the above-mentioned hydroxyaldehyde synthase, mutant thereof, enzyme having a function of catalyzing formaldehyde to synthesize hydroxyaldehyde or related biological material, aldolase, mutant thereof, enzyme having a function of catalyzing formaldehyde and hydroxyaldehyde to synthesize glyceraldehyde or catalyzing hydroxyaldehyde and glyceraldehyde to synthesize L-xylose or a combination of related biological materials are used for biosynthesis of L-xylose with formaldehyde as a substrate.
According to an embodiment of the invention, the relevant biological material of the hydroxyaldehyde condensation enzyme is a nucleic acid molecule encoding the hydroxyaldehyde condensation enzyme, a mutant thereof or an enzyme having the function of catalyzing formaldehyde synthesis, or an expression cassette, recombinant vector, recombinant bacterium or transgenic cell line comprising the nucleic acid molecule.
According to an embodiment of the invention, the relevant biological material of 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 hydroxyaldehyde or the synthesis of L-xylose from hydroxyaldehyde, or an expression cassette, recombinant vector, recombinant bacterium or transgenic cell line containing said nucleic acid molecule.
According to an embodiment of the present invention, the recombinant bacterium is a recombinant bacterium obtained by introducing a nucleic acid molecule encoding the hydroxyaldehyde condensation enzyme, a mutant thereof, or an enzyme having a function of catalyzing formaldehyde to synthesize hydroxyaldehyde, or aldolase, a mutant thereof, or an enzyme having a function of catalyzing formaldehyde and hydroxyaldehyde to synthesize glyceraldehyde, or hydroxyaldehyde and glyceraldehyde to synthesize L-xylose into a host cell; for example, in the form of a recombinant vector.
Wherein the recombinant vector is a bacterial plasmid, bacteriophage, yeast plasmid or retrovirus packaging plasmid carrying a nucleic acid molecule encoding the hydroxyaldehyde condensation enzyme, a mutant thereof or an enzyme or aldolase having a function of catalyzing formaldehyde to synthesize hydroxyaldehyde, a mutant thereof or an enzyme having a function of catalyzing formaldehyde and hydroxyaldehyde to synthesize glyceraldehyde or catalyzing hydroxyaldehyde and glyceraldehyde to synthesize L-xylose.
Wherein the host cell may be a prokaryotic cell, such as a bacterium, more particularly e.g.E.coli, or a lower eukaryotic cell, such as a yeast cell.
In the context of the present invention, amino acids are represented by single-letter or three-letter codes, having 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 substitution thereof are expressed by the position number of the mutation site and the amino acid type of the site, for example W86R means that tryptophan at the 86 th position corresponding to SEQ ID NO.1 is mutated to arginine in comparison with SEQ ID NO. 1; N87T represents an asparagine mutation to threonine at position 87 corresponding to SEQ ID NO.1 in comparison 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 the double mutant comprises two mutation sites. By analogy, "W86/N87/L109/L110" means that the corresponding four sites are mutated simultaneously, and are four mutants.
Advantageous effects
The invention utilizes aldolase, mutant or enzyme with the function of catalyzing formaldehyde and hydroxy aldehyde to synthesize glyceraldehyde or catalyzing hydroxy aldehyde and glyceraldehyde to synthesize L-xylose, takes formaldehyde and hydroxy aldehyde as substrates to synthesize L-xylose, can also utilize the combination of aldolase, mutant or enzyme with the function of catalyzing formaldehyde to synthesize hydroxy aldehyde and aldolase, mutant or enzyme with the function of catalyzing formaldehyde and hydroxy aldehyde to synthesize glyceraldehyde or catalyzing hydroxy aldehyde and glyceraldehyde to synthesize L-xylose, takes formaldehyde as substrates to synthesize L-xylose, and provides a new way and a new idea for synthesizing rare sugar. The method for synthesizing L-xylose has high conversion rate (up to 65%), rich substrate sources and mild reaction conditions; has the advantages of simple production process, green and environment-friendly effect, easy mass production and the like.
Drawings
FIG. 1 shows plasmid maps of pET-28a-BFD, pET-28a-GALS and pET-28a-FSA, where 1a is the plasmid map of pET-28a-BFD, 1b is the plasmid map of pET-28a-GALS, and 1c is the plasmid map of 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 various enzyme ratios, wherein FALD is the substrate formaldehyde.
FIG. 4 shows the yields of formaldehyde to hydroxyacetaldehyde for two enzymes GALS and FLS at different substrate concentrations, where FLS is a mutant formaldehyde enzyme (formase) of benzaldehyde lyase (BAL) and FALD is the substrate formaldehyde.
Detailed Description
The method of 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 illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.
Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods. Some molecular cloning method details vary from reagent, enzyme or kit provider to kit provider, and should be performed according to 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 is Pseudomonas putida in source, the amino acid is shown as SEQ ID NO.1, the codon of the wild type gene is replaced by the codon favored by Escherichia coli (used at high frequency) on the premise of not changing the amino acid sequence of BFD, and after the codon is optimized, the gene sequence has the preferred codon of Escherichia coli, and the gene sequence is shown as SEQ ID NO. 2. The gene sequence is directly synthesized on a pET-28a vector and is positioned between the restriction enzyme sites NdeI and XhoI, and the recombinant plasmid is named pET-28a-BFD (shown in figure 1 a). In addition, the mutant of BFD also has the function of catalyzing 1-formaldehyde to synthesize 2-hydroxy-acetaldehyde, and the mutant of BFD is listed in Chinese patent application No. CN201710096307.X (publication No. CN 106916794A). GALS is a mutant of BFD, has obviously higher activity of catalyzing formaldehyde to synthesize the hydroxy acetaldehyde than BFD, is named as hydroxy acetaldehyde synthase, has an amino acid sequence shown as SEQ ID NO.3 and a gene sequence shown as SEQ ID NO. 4. And compared with FLS (mutant formaldehyde enzyme of benzaldehyde lyase BAL) known in the prior art (see Siegel JB, smith AL, poust S, et AL computing protein design enables a novel one-carbon assimilation path.Proc Natl Acad Sci USA,2015,112 (12): 3704-3709, and PoustS, pietty J, bar-Even A, et AL mechanical analysis of an engineered enzyme that catalyzes the formose reaction.chemBiochem,2015,16 (13): 1950-1954)), GALS can specifically catalyze formaldehyde to glycolaldehyde, and as the substrate concentration increases, the catalytic efficiency of both enzymes increases, but the catalytic effect of GALS is more pronounced, which is about 13 times that of FLS (as shown in FIG. 4). Thus, the following examples were performed using GALS.
The FSA (D-Fructose-6-Phosphate Aldolase ) gene has the source of E.coli, the amino acid of which is shown as SEQ ID NO.5, and the gene sequence of which is shown as SEQ ID NO. 6. The FSA gene is obtained from E.coli genome by PCR, and is constructed on a pET-28a vector, is positioned between NdeI and XhoI of enzyme cutting sites, and is used for mutating alanine (A) at 129 th site into threonine (T), meanwhile, the alanine (A) at 165 th site is mutated into glycine (G), the recombinant plasmid is named pET-28a-FSA (A129T/A165G) (shown in figure 1 b), the amino acid sequence of the recombinant plasmid 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 the GALS, FSA (A129T/A165G) enzyme activity, the enzyme was exogenously expressed and purified in E.coli.
(1) E.coli expression type recombinant plasmids pET-28a-GALS and pET-28a-FSA (A129T/A165G) are respectively transferred into E.coli BL21 (DE 3) to obtain recombinant bacteria. Positive clone selection (kan+, 100 mg/mL) was performed using kanamycin resistance plates, and cultured overnight at 37 ℃;
(2) Monoclonals were selected into 5mL LB liquid medium (Kan+, 100 mg/mL), and cultured at 37℃and 220rpm until OD600 was 0.6-0.8. The bacterial solution in 5mL LB medium was transferred to 800mL 2YT medium (Kan+, 100 mg/mL), and cultured at 37℃and 220rpm to OD 600 When the concentration is 0.6-0.8, cooling to 16 ℃, adding IPTG to a final concentration of 0.5mM, and inducing expression for 16h;
(3) Collecting the culture solution into a fungus collecting bottle, and centrifuging at 5500rpm for 15min;
(4) The supernatant was discarded, and the resulting bacterial pellet was suspended in 35mL of a protein buffer (50 mM triethanolamine buffer, pH 7.4), poured into a 50mL centrifuge tube, and stored at-80 ℃.
EXAMPLE 3 protein purification
(1) And (3) breaking bacteria: the sterilization is carried out 2 times under the pressure of 1200bar and the temperature of 4 ℃ by adopting a high-pressure low-temperature crusher. Centrifuging at 10000rpm for 45min at 4deg.C;
(2) Purifying: filtering the supernatant with a 0.45 μm microporous filter membrane, and purifying by nickel affinity chromatography, wherein the specific steps are as follows:
a: column balance: before hanging supernatant, ddH is used 2 O washing 2 column volumes, and balancing 1 column volume of the Ni affinity chromatography column by using a protein buffer solution;
b: loading: passing the supernatant through Ni affinity chromatography column slowly at a flow rate of 0.5mL/min, repeating for one time;
c: eluting the hybrid protein: washing 1 column volume with protein buffer, and eluting the bound impurity protein with 50mL of protein buffer containing 50mM and 100mM of imidazole respectively;
d: eluting the target protein: the target protein was eluted with 20mL of 200mM imidazole in protein buffer, and the first few samples were collected and subjected to 12% SDS-PAGE.
(3) Liquid replacement: the collected target protein was concentrated by centrifugation (4 ℃ C., 3400 rpm) using a 50mL Amicon ultrafiltration tube (30 kDa, millipore Co.) to 1mL. 15mL of imidazole-free protein buffer was added, concentrated to 1mL, and repeated 1 time to give GALS, FSA (A129T/A165G) protein.
(4) Detecting the concentration of the concentrated protein by using a Nondrop 2000 micro-spectrophotometer and diluting to 10mg/mL to obtain GALS and FSA (A129T/A165G) proteins.
EXAMPLE 4 Synthesis of L-xylose
1: formaldehyde; 2: hydroxy acetaldehyde; 3: glyceraldehyde; 4: l-xylose; GALS: a hydroxyaldehyde synthase; FSA (A129T/A165G): d-fructose-6-phosphate aldolase mutant.
(1) Derivatization reagent formulation (1 mL): 21.1mg of benzoxamine hydrochloride was dissolved in 660. Mu.L of pyridine and 300. Mu.L of methanol, 40. Mu. L H were added 2 Mixing, and storing at 4deg.C.
(2) Reaction conditions: 2G/L formaldehyde, 50mM triethanolamine buffer, pH7.4, 1mg/mL GALS,1mg/mL FSA (A129T/A165G), and shaking reaction at 25℃for 24h. 10. Mu.L of the sample was taken, 50. Mu.L of the derivatizing reagent was added, reacted at 50℃for 1 hour, 140. Mu.L of methanol was added, and the mixture was filtered and detected by HPLC.
HPLC detection conditions: mobile phase: a:0.1% (v/v) trifluoroacetic acid TFA; b: dissolved in 80% CH 3 CN 0.095% (v/v) TFA. Elution gradient: the mobile phase B accounts for 20-60% within 16 min. Column: X-Bridge TM C18,5 μm, 4.6X1250 mm. Flow rate: 1mL/min, detection wavelength: 215nm, column oven temperature: 35 ℃, sample injection amount: 20. Mu.L. The detection results are shown in FIG. 2. Xylose can be directly synthesized from formaldehyde after addition of GALS and FSA (A129T/A165G), and the conversion rate can reach 65% under the condition of 1G/L formaldehyde (figure 3).
SEQ ID NOS.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: gene sequence of GALS:
ATGGCTTCTGTTCACGGTACCACCTACGAACTGCTGCGTCGTCAGGGTATCGACACCGTTTTCGGTAACCCGGGTTCTAACGAACTGCCGTTCCTGAAAGACTTCCCGGAAGACTTCCGTTACATCCTGGCTCTGCAGGAAGCTTGCGTTGTTGGTATCGCTGACGGTTACGCTCAGGCTTCTCGTAAACCGGCTTTCATCAACCTGCACTCTGCTGCTGGTACCGGTAACGCTATGGGTGCTCTGTCTAACGCTCGTACCTCTCACTCTCCGCTGATCGTTACCGCTGGTCAGCAGACCCGTGCTATGATCGGTGTTGAAGCTGGTGAAACCAACGTTGACGCTGCTAACCTGCCGCGTCCGCTGGTTAAATGGTCTTACGAACCGGCTTCTGCTGCTGAAGTTCCGCACGCTATGTCTCGTGCTATCCACATGGCTTCTATGGCTCCGCAGGGTCCGGTTTACCTGTCTGTTCCGTACGACGACTGGGACAAAGACGCTGACCCGCAGTCTCACCACCTGTTCGACCGTCACGTTTCTTCTTCTGTTCGTCTGAACGACCAGGACCTGGACATCCTGGTTAAAGCTCTGAACTCTGCTTCTAACCCGGCTATCGTTCTGGGTCCGGACGTTGACGCTGCTAACGCTAACGCTGACTGCGTTATGCTGGCTGAACGTCTGAAAGCTCCGGTTTGGGTTGCTCCGTCTGCTCCGCGTTGCCCGTTCCCGACCCGTCACCCGTGCTTCCGTGGTCTGATGCCGGCTGGTATCGCTGCTATCTCTCAGCTGCTGGAAGGTCACGACGTTGTTCTGGTTATCGGTGCTCCGGTTTTCCGTTACGTTTTTTACGACCCGGGTCAGTACCTGAAACCGGGTACCCGTCTGATCTCTGTTACCTGCGACCCGCTGGAAGCTGCTCGTGCTCCGATGGGTGACGCTATCGTTGCTGACATCGGTGCTATGGCTTCTGCTCTGGCTAACCTGGTTGAAGAATCTTCTCGTCAGCTGCCGACCGCTGCTCCGGAACCGGCTAAAGTTGACCAGGACGCTGGTCGTCTGCACCCGGAAACCGTTTTCGACACCCTGAACGACATGGCTCCGGAAAACGCTATCTACCTGAACGAATCTACCTCTACCACCGCTCAGATGTGGCAGCGTCTGAACATGCGTAACCCGGGTTCTTACTACTTCTGCGCTGCTGGTGGTCTGGGTTTCGCTCTGCCGGCTGCTATCGGTGTTCAGCTGGCTGAACCGGAACGTCAGGTTATCGCTGTTATCGGTGACGGTTCTGCTAACTACTCTATCTCTGCTCTGTGGACCGCTGCTCAGTACAACATCCCGACCATCTTCGTTATCATGAACAACGGTACCTACGGTATGCTGCGTTGGTTCGCTGGTGTTCTGGAAGCTGAAAACGTTCCGGGTCTGGACGTTCCGGGTATCGACTTCCGTGCTCTGGCTAAAGGTTACGGTGTTCAGGCTCTGAAAGCTGACAACCTGGAACAGCTGAAAGGTTCTCTGCAGGAAGCTCTGTCTGCTAAAGGTCCGGTTCTGATCGAAGTTTCTACCGTTTCTCCGGTTAAATAA
SEQ ID NO.5: amino acid sequence of FSA:
MELYLDTANVAEVERLARIFPIAGVTTNPSIIAASKESIWEVLPRLQKAIGDEGILFAQTMSRDAQGMVKEAKHLRDAIPGIVVKIPVTSEGLAAIKMLKKEGITTLGTAVYSAAQGLLAALAGAKYVAPYVNRVDAQGGDGIRTVQELQALLEMHAPESMVLAASFKTPRQALDCLLAGCESITLPLDVAQQMLNTPAVESAIEKFEHDWNAAFDTTHL
SEQ ID NO.6: gene sequence of 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, improvement, etc. 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 Tianjin Industrial biotechnology, national academy of sciences
<120> a 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
<211> 528
<212> PRT
<213> artificial sequence
<400> 3
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 Arg Thr Ser His Ser Pro Leu Ile Val Thr Ala
85 90 95
Gly Gln Gln Thr Arg Ala Met Ile Gly Val Glu Ala Gly Glu 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 Val Phe 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 Met 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> 4
<211> 1587
<212> DNA
<213> artificial sequence
<400> 4
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 acgctcgtac ctctcactct ccgctgatcg ttaccgctgg tcagcagacc 300
cgtgctatga tcggtgttga agctggtgaa 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
gttttttacg 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 ctacggtatg 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> 5
<211> 220
<212> PRT
<213> Escherichia coli
<400> 5
Met Glu Leu Tyr Leu Asp Thr Ala Asn Val Ala Glu Val Glu Arg Leu
1 5 10 15
Ala Arg Ile Phe Pro Ile Ala Gly Val Thr Thr Asn Pro Ser Ile Ile
20 25 30
Ala Ala Ser Lys Glu Ser Ile Trp Glu Val Leu Pro Arg Leu Gln Lys
35 40 45
Ala Ile Gly Asp Glu Gly Ile Leu Phe Ala Gln Thr Met Ser Arg Asp
50 55 60
Ala Gln Gly Met Val Lys Glu Ala Lys His Leu Arg Asp Ala Ile Pro
65 70 75 80
Gly Ile Val Val Lys Ile Pro Val Thr Ser Glu Gly Leu Ala Ala Ile
85 90 95
Lys Met Leu Lys Lys Glu Gly Ile Thr Thr Leu Gly Thr Ala Val Tyr
100 105 110
Ser Ala Ala Gln Gly Leu Leu Ala Ala Leu Ala Gly Ala Lys Tyr Val
115 120 125
Ala Pro Tyr Val Asn Arg Val Asp Ala Gln Gly Gly Asp Gly Ile Arg
130 135 140
Thr Val Gln Glu Leu Gln Ala Leu Leu Glu Met His Ala Pro Glu Ser
145 150 155 160
Met Val Leu Ala Ala Ser Phe Lys Thr Pro Arg Gln Ala Leu Asp Cys
165 170 175
Leu Leu Ala Gly Cys Glu Ser Ile Thr Leu Pro Leu Asp Val Ala Gln
180 185 190
Gln Met Leu Asn Thr Pro Ala Val Glu Ser Ala Ile Glu Lys Phe Glu
195 200 205
His Asp Trp Asn Ala Ala Phe Asp Thr Thr His Leu
210 215 220
<210> 6
<211> 663
<212> DNA
<213> Escherichia coli
<400> 6
atggaactgt atctggacac cgctaacgtc gcagaagtcg aacgtctggc acgcatattc 60
ccgattgccg gggtgacaac taacccgagc attatcgctg ccagcaagga gtccatctgg 120
gaagtgctgc cgcgccttca aaaagcgatc ggtgatgagg gcattctgtt tgctcagacc 180
atgagccgcg acgcgcaggg tatggtgaaa gaagcgaaac acctgcgcga cgctattccg 240
ggcattgtgg tgaaaattcc ggtaacctct gaaggtctgg cagcaattaa aatgctgaag 300
aaagaaggca ttactacgct gggaaccgca gtgtacagcg ccgcgcaagg attactggcg 360
gcgctggctg gagccaaata cgttgctcca tacgttaacc gcgtagatgc ccagggcggt 420
gacggcattc gtactgtaca ggagttgcaa gcgttactgg aaatgcatgc gccagaaagc 480
atggtgctgg ctgccagctt taaaacacca cgtcaggcgc tggattgttt gctggctgga 540
tgtgaatcca tcacactgcc cttagatgta gcgcaacaaa tgcttaacac ccctgcggta 600
gagtcagcta tagagaagtt cgagcacgac tggaatgccg catttgacac tactcatctc 660
taa 663
<210> 7
<211> 220
<212> PRT
<213> artificial sequence
<400> 7
Met Glu Leu Tyr Leu Asp Thr Ser Asp Val Val Ala Val Lys Ala Leu
1 5 10 15
Ser Arg Ile Phe Pro Leu Ala Gly Val Thr Thr Asn Pro Ser Ile Ile
20 25 30
Ala Ala Gly Lys Lys Pro Leu Asp Val Val Leu Pro Gln Leu His Glu
35 40 45
Ala Met Gly Gly Gln Gly Arg Leu Phe Ala Gln Val Met Ala Thr Thr
50 55 60
Ala Glu Gly Met Val Asn Asp Ala Leu Lys Leu Arg Ser Ile Ile Ala
65 70 75 80
Asp Ile Val Val Lys Val Pro Val Thr Ala Glu Gly Leu Ala Ala Ile
85 90 95
Lys Met Leu Lys Ala Glu Gly Ile Pro Thr Leu Gly Thr Ala Val Tyr
100 105 110
Gly Ala Ala Gln Gly Leu Leu Ser Ala Leu Ala Gly Ala Glu Tyr Val
115 120 125
Ala Pro Tyr Val Asn Arg Ile Asp Ala Gln Gly Gly Ser Gly Ile Gln
130 135 140
Thr Val Thr Asp Leu His Gln Leu Leu Lys Met His Ala Pro Gln Ala
145 150 155 160
Lys Val Leu Ala Ala Ser Phe Lys Thr Pro Arg Gln Ala Leu Asp Cys
165 170 175
Leu Leu Ala Gly Cys Glu Ser Ile Thr Leu Pro Leu Asp Val Ala Gln
180 185 190
Gln Met Ile Ser Tyr Pro Ala Val Asp Ala Ala Val Ala Lys Phe Glu
195 200 205
Gln Asp Trp Gln Gly Ala Phe Gly Arg Thr Ser Ile
210 215 220
<210> 8
<211> 660
<212> DNA
<213> artificial sequence
<400> 8
atggaactgt atctggatac ttcagacgtt gttgcggtga aggcgctgtc acgtattttt 60
ccgctggcgg gtgtgaccac taacccaagc attatcgccg cgggtaaaaa accgctggat 120
gttgtgcttc cgcaacttca tgaagcgatg ggcggtcagg ggcgtctgtt tgcccaggta 180
atggctacca ctgccgaagg gatggttaat gacgcgctta agctgcgttc tattattgcg 240
gatatcgtgg tgaaagttcc ggtgaccgcc gaggggctgg cagctattaa gatgttaaaa 300
gcggaaggga ttccgacgct gggaaccgcg gtatatggcg cagcacaagg gctgctgtcg 360
gcgctggcag gtgcggaata tgttgcgcct tacgttaatc gtattgatgc tcagggcggt 420
agcggcattc agactgtgac cgacttacac cagttattga aaatgcatgc gccgcaggcg 480
aaagtgctgg cagcgagttt caaaaccccg cgtcaggcgc tggactgctt actggcagga 540
tgtgaatcaa ttactctgcc actggatgtg gcacaacaga tgattagcta tccggcggtt 600
gatgccgctg tggcgaagtt tgagcaggac tggcagggag cgtttggcag aacgtcgatt 660

Claims (7)

1. A method of biosynthesis of L-xylose comprising the steps of:
step 0, synthesizing a substrate formaldehyde into hydroxy aldehyde under the action of a hydroxy aldehyde condensing enzyme mutant;
step 1, formaldehyde and hydroxy aldehyde are synthesized into glyceraldehyde under the action of aldolase mutant;
step 2, synthesizing L-xylose by glyceraldehyde and hydroxyaldehyde under the action of aldolase mutant;
the hydroxy acetaldehyde condensing enzyme mutant is GALS, and the amino acid sequence of the GALS is shown in SEQ ID NO. 3;
wherein both steps 1) and 2) use a D-fructose-6-phosphate aldolase (FSA) mutant;
the amino acid sequence of the FSA mutant is the amino acid sequence obtained by mutating A at 129 th position of SEQ ID NO.7 into T and mutating A at 165 th position into G;
the method is carried out in a one-pot manner;
wherein the weight ratio of GALS to D-fructose-6-phosphate aldolase (FSA) mutant is 1:1-1:4.
2. The method according to claim 1, wherein the GALS and D-fructose-6-phosphate aldolase (FSA) mutants are in the form of purified enzymes, enzyme cleavage supernatants or whole cells.
3. The method according to any one of claims 1-2, said "one pot method" being performed in a buffer system.
4. A method according to claim 3, wherein the buffer is a triethanolamine buffer, a MOPS buffer, a HEPES buffer, a phosphate buffer, a Tris buffer or an acetate buffer; the pH value of the buffer solution is 6.5-8.5.
5. A process according to claim 3, wherein the "one pot process" is carried out at a temperature of from 10 to 50 ℃.
6. A process according to claim 3, wherein the "one pot process" is carried out for 1 to 72 hours.
7. Use of a composition comprising a hydroxyaldehyde condensation enzyme mutant GALS having an amino acid sequence as shown in SEQ ID No.3 and a D-fructose-6-phosphate aldolase (FSA) mutant having an amino acid sequence as obtained by mutating a129 th a to T and a165 th a to G of SEQ ID No.7 in a weight ratio of GALS to FSA mutant of 1:1 to 1:4 for catalyzing the biosynthesis of L-xylose by formaldehyde "one pot".
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