Method for synthesizing amylose through three-enzyme cascade catalysis of glucose

Abstract

The invention relates to the field of biological catalysis engineering, in particular to a method for synthesizing amylose through three-enzyme cascade catalysis of glucose. The method comprises the following steps: phosphorylating a substrate glucose into an intermediate G-6-P by using heat-resistant polyphosphate glucokinase in the presence of polyphosphoric acid; g-6-P is shifted into G-1-P by heat-resistant phosphoglucomutase, and the intermediate G-1-P is added to the non-reducing end of oligosaccharide one by one by heat-resistant alpha-glucan phosphorylase to synthesize amylose. According to the method, reaction conditions of a reaction system for synthesizing amylose by using glucose are optimized, the most suitable metal ion type and concentration, pH and buffer solution type are explored, and in the optimized reaction process for preparing amylose by catalyzing glucose in a three-enzyme cascade manner, the conversion rate is 71.93%, the yield reaches 3.5 g / L, and the space time yield is 2.33 g / L / h.

Description

Technical Field

[0001] The present invention relates to the field of biocatalytic engineering, and in particular to a method for synthesizing amylose from glucose by three-enzyme cascade catalysis. Background Art

[0002] The production of plant-derived starch is highly dependent on the photosynthetic carbon fixation pathway. Due to the low efficiency of light energy conversion and the complexity of metabolic regulation, it is difficult to break through the production capacity bottleneck.

[0003] In recent years, synthetic biology has made breakthroughs in synthesizing starch from straw. For example, an in vitro enzymatic cellulose-to-starch conversion system has been developed, achieving the first-ever conversion of non-grain cellulose to amylose. This pathway partially hydrolyzes cellulose by endoglucanase (EG) and cellobiohydrolase (CBH) to produce cellobiose. Cellobiose is then phosphorylated by cellobiose phosphorylase (CBP) to glucose and glucose-1-phosphate (G-1-P). Finally, G-1-P is polymerized onto maltooligosaccharide chains by potato alpha-glucan phosphorylase (PGP) to produce amylose. However, the actual starch yield from this pathway is only 2.1%. When cellulose is degraded into cellobiose by EG and CBH, a small amount of glucose is generated, which cannot be directly used to synthesize starch; when cellobiose undergoes phosphorolysis by CBP, 1 molecule of glucose and 1 molecule of G-1-P are generated, and G-1-P is used to synthesize starch. The amount of glucose equal to G-1-P cannot be used to synthesize starch, making the theoretical conversion rate of the entire pathway less than 50%.

[0004] Starch production relies on activated precursors, primarily phosphorylated glucose derivatives (such as G-1-P and G-6-P) and the plant-specific ADPG. Because the α-1,4-glycosidic bonds formed between these molecules require an in vitro enzymatic cellulose-to-starch conversion system, the resulting glucose cannot directly participate in starch biosynthesis and requires additional energy. To improve the efficiency of starch synthesis, glucose activation is necessary for its synthesis from glucose. Hexokinase can convert glucose to G-6-P, but this process consumes ATP, increasing the cost of artificial starch synthesis and hindering industrial production.

[0005] The key bottleneck in improving the efficiency of enzymatic cellulose conversion to starch lies in glucose activation, so it is urgent to develop a simple, low-cost and environmentally friendly biosynthetic method for synthesizing amylose from glucose. Summary of the Invention

[0006] Since glucose cannot directly participate in starch biosynthesis, the current enzymatic cellulose to starch conversion efficiency is low. Therefore, how to efficiently use glucose to synthesize amylose is the core of the enzymatic cellulose to starch conversion.

[0007] The purpose of the present invention is to provide a method for synthesizing amylose from glucose by catalyzing a three-enzyme cascade.

[0008] The method for synthesizing amylose from glucose by three-enzyme cascade catalysis according to the present invention comprises the following steps: In a buffer system, polyphosphate glucose kinase phosphorylates the substrate glucose into the intermediate G-6-P in the presence of polyphosphate; Thermostable phosphoglucomutase converts G-6-P into the intermediate G-1-P; The intermediate G-1-P is added one by one to the non-reducing end of the oligosaccharide by thermostable α-glucan phosphorylase to synthesize amylose. Wherein, the buffer system contains 1mM~5mM Mg 2+ , 1mM~20mM Co 2+ , or 1mM~50mM Mn 2+ , the pH of the buffer system is 7.75~8.0.

[0009] According to the method for synthesizing amylose from glucose by three-enzyme cascade catalysis of the present invention, the buffer system contains 5 mM Ca 2+ or Ni 2+ , 20 mM Mn 2+ , or 5 mM~8 mM Co 2+ .

[0010] According to the method for synthesizing amylose from glucose by three-enzyme cascade catalysis of the present invention, the buffer system is Tris-HCl buffer.

[0011] According to the method for synthesizing amylose from glucose by three-enzyme cascade catalysis of the present invention, the buffer system is a 50 mM Tris-HCl buffer solution with a pH of 7.75.

[0012] According to the method for synthesizing amylose from glucose by three-enzyme cascade catalysis of the present invention, the polyphosphate glucose kinase is derived from Thermobifida fusca YX The polyphosphoglucokinase (SEQ ID NO: 3) of Phaeodactylibacter sp. Thermostable phosphoglucomutase (SEQ ID NO: 2), wherein the α-glucan phosphorylase is derived from Thermotoga petrophila α-glucan phosphorylase (SEQ ID NO: 1) or a mutant thereof.

[0013] According to the method for synthesizing amylose from glucose by a three-enzyme cascade catalysis of the present invention, the substrate glucose is phosphorylated to the intermediate G-6-P by polyphosphate glucokinase in the presence of polyphosphate at 60°C; G-6-P is transformed to the intermediate G-1-P by phosphoglucomutase; and the intermediate G-1-P is added one by one to the non-reducing end of oligosaccharides by α-glucan phosphorylase to synthesize amylose.

[0014] According to the method for synthesizing amylose from glucose by a three-enzyme cascade catalysis of the present invention, the α-glucan phosphorylase mutant is obtained by subjecting the α-glucan phosphorylase with the amino acid sequence shown in SEQ ID NO: 1 to the following mutations: D774E / L775S / F776I / V777R / Y778L / T779H / Y780L / T781H / N782Q / G783W / V784C, i.e. mutant Tp αGPM3; F669S / S670A, i.e. mutant Tp αGPM5, The amino acid at position 669 mutated from Phe to Ser, the amino acid at position 670 mutated from Ser to Ala, the amino acid at position 774 mutated from Asp to Glu, the amino acid at position 775 mutated from Leu to Ser, the amino acid at position 776 mutated from Phe to Ile, the amino acid at position 777 mutated from Val to Arg, the amino acid at position 778 mutated from Tyr to Leu, the amino acid at position 779 mutated from Thr to His, and the amino acid at position 780 mutated from Tyr to Leu, amino acid 781 was mutated from Thr to His, amino acid 782 was mutated from Asn to Gln, amino acid 783 was mutated from Gly to Trp, and amino acid 784 was mutated from Val to Cys, resulting in the mutant F669S / S670A / D774E / L775S / F776I / V777R / Y778L / T779H / Y780L / T781H / N782Q / G783W / V784C, i.e., the mutant Tp αGPM7; The 485th amino acid was mutated from Arg to Lys, resulting in the mutant R485K, i.e. Tp αGPR485K; The amino acid at position 671 was mutated from Pro to Thr, the amino acid at position 672 was mutated from Pro to Thr, the amino acid at position 673 was mutated from Ala to Arg, the amino acid at position 674 was mutated from Cys to Met, and the amino acid at position 675 was mutated from Cys to Leu, resulting in the mutant P671T / P672T / A673R / C674M / C675L, i.e., the mutant Tm αGPM6; α-glucan phosphorylase Tp The 673rd amino acid of αGP was mutated from Arg to Ala, resulting in the mutant R673A. Tp αGPR673A; or The 171st amino acid was mutated from Glu to Gly to obtain the mutant E170G, i.e. Tp αGPM1.

[0015] In one embodiment of the present invention, the reaction conditions for the three-enzyme cascade catalyzed synthesis of amylose from glucose are as follows: a 200 μL reaction system contains 50 mM Tris-HCl buffer at pH 7.75, 30 mM glucose, 4 mM sodium hexametaphosphate, 20 mM Mn 2+ , 0.1 mM maltotetraose, 3.5 μM polyphosphate glucokinase, 1 μM phosphoglucomutase and 9 μM α-glucan phosphorylase, and the reaction system was carried out at 60 °C for 2 h.

[0016] Originated from Thermotoga petrophila The sequence of α-glucan phosphorylase (SEQ ID NO: 1) is: MLEKLPENLKELESLAYNLWWSWSRPAQRLWRMIDSEKWEEHRNPVKILREVSKERLEELSKDEDFIALYELTLERFTDYMEREDTWFNVNYPEWDEKIVYMCMEYGLTKALPIYSGGLGILAGDHLKSASDLGLPLIAVGLLYKHGYFTQQIDSDGRQIEIFPEYDIEELPMKPLRDEDGNQVIVEVPIDNDTVKARVFEVQVGRVKLYLLDTDFEENEDRFRKICDYLYNPEPDVRVSQEILLGIGGMKLLKTLKIKPGVIHLNEGHPAFSSLERIKSYMEEGYSFTEALEIVRQTTVFTTHTPVPAGHDRFPFDFVEKKLTKFFEGFESKELLMNLGKDEDGNFNMTYLALRTSSFINGVSKLHADVSRRMFKNVWKGVPVEEIPIEGITNGVHMGTWINREMRKLFDRYLGRVWREHTDLEGIWYGVDRIPDEELWEAHLNAKKRFIDYIRESIKRRNERLGINEPLPEISENVLIIGFARRFATYKRAVLLFSDLERLKRIVNNSERPVYIVYAGKAHPRDEGGKEFLRRIYEVSQMPDFKNKIIVLENYDIGMARLMVSGVDVWLNNPRRPMEASGTSGMKAAANGVLNASVYDGWWVEGYNGRNGWVIGDESVLPETEADDPKDAEALYELLENEIIPTYYENREKWIFMMKESIKSVAPKFSTTRMLKEYTEKFYIKGLVNREWLERRENVEKIGAWKERILKNWENVSIERIVLEDSKSVEVTVKLGDLTPNDVIVELVAGRGEGMEDLEVWKVIHIRRYRKENDLFVYTYTNGVLGHLGSPGWFYAVRVIPYHPRLPIKFLPEVPVVWKKVL。

[0017] The phosphoglucomutase sequence (SEQ ID NO:2) derived from Phaeodactylibacter sp. is as follows: MALHPQAGKTATPDQLVNIPRLITAYFTGQPDPSVREQRVSFGTSGHRGSSLNRSFNEQHILATTQAICLYRQKEGINGPVFMGIDSHALSEPAQATALEVLAANGVETMIAAGDEYTPTPAVSQAILAYNRGRAGGLADGIVITPSHNPPEDGGFKYNMTNGGPAESNVTAWIEAKANELLENGLREVKRIPFQRAMKASTTHRYDYLGAYVNGLGQVIDMDAIRSSGLEMGVDPLGGAGVHYWGHIADHYRLNLTVVDTEVDPTFRFMSLDWDGKIRMDPSSPYAMQRLIRLKDDYPVAFACDTDHDRHGIVTRSAGLMPPNHYLAVAIDYLFRHRPKWKPETGIGKTLVSSQMIDRVAARLGRKLVEVPVGFKWFVDGLFDGSLGFGGEESAGASFLDREGNAWSTDKDGIIAALLAGEITARTGKDPGEIYREFTREFGEPAYGRIDAPATPAQKDKLKKLSREQVTSSQLAGEKIEAILTEAPGNGASIGGLKAVTANGWFAARPSGTEDIYKIYAESFKGEEHLRQLQKEAQELVDRVIG。

[0018] Thermobifida fusca YX The enzyme sequence (SEQ ID NO: 3) of polyphosphate glucokinase of the present invention is as follows: MASRGRVGLGIDIGGSGIKGAPVDLDRGTLVVDPVKIATPQPATPEAVAAVVAEIVTAFADDVPQDAPLGVAFPAVIQHGVARSAANMDRSWIGTNVEELLSAVTGRRVLVVNDADAAAMAEHRYGAASGVDGVVLLTTLGTGIGTAVLVDGVLLPNTEFGHLEIDGYDAETRASASAKERENLSYKEWAEERLQRYYSVIEDLLWPDLIVVGGGVSRKADKFLPHLRLRTPIVPAKLRNTAGIVGAAVLAAERLGGDRVSA。

[0019] Advantages of the present invention: (1) The present invention is the first to use a biosynthetic method to prepare amylose using glucose as a substrate. Compared with the chemical method, this method has the advantages of simple operation, mild reaction conditions, low cost, and environmental protection.

[0020] (2) The present invention optimizes the reaction conditions of the glucose-based amylose synthesis reaction system and explores the optimal metal ion type and concentration, pH, and buffer type. After optimization, the conversion rate of the three-enzyme cascade catalyzed glucose-based amylose synthesis reaction was 71.93%, the yield reached 3.5 g / L, and the space-time yield was 2.33 g / L / h. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The synthesis route of amylose from glucose in the present application is shown, wherein Glucose: glucose; G-6-P: glucose-6-phosphate; G-1-P: glucose-1-phosphate; Amylose: amylose; PPGK: polyphosphate glucokinase; PGM: phosphoglucomutase; αGP: α-glucan phosphorylase; Figure 2 The yields of reaction systems "A", "B" and "C" for preparing amylose from glucose catalyzed by a three-enzyme cascade are shown; Figure 3 show Tk SDS-PAGE analysis of PGM and other 12 PGMs screened. (a) Figure: SDS-PAGE analysis of soluble expressed PGM, where M: protein marker; 1: Tk PGM;2: Phs PGM;3: Bb PGM;4: At PGM;5: Pa PGM;6: Ds PGM;7: Ob PGM;8: Ke PGM;9: Aa PGM;10: Prs PGM;11: Cf PGM, (b) and (c) Nib PGM and Neb SDS-PAGE analysis of PGM, where M: protein marker; 1: whole cell lysate; 2: cell pellet; 3: supernatant; 4: flow-through; Figure 4 show TkBidirectional enzyme specific activities of PGM and 10 other soluble PGMs, including: (a) PGM bidirectional enzyme specific activities detected at 37°C, the curve shows the ratio of forward enzyme specific activities at 60°C to 37°C, (b) PGM bidirectional enzyme specific activities detected at 60°C; Figure 5 show Tm SDS-PAGE analysis of αGP and other 8 screened αGPs, where (a) is the SDS-PAGE analysis of soluble expressed αGP, where M is protein marker; 1 is Bb αGP;2: Mc αGP;3: Eb αGP;4: Ar αGP;5: Tp αGP;6: Tm αGP, (b), (c), and (d): Ms αGP, Ta αGP and Cb SDS-PAGE analysis of αGP, where M: protein marker; 1: whole cell lysate; 2: cell pellet; 3: supernatant; 4: flow-through; Figure 6 show Tm Bidirectional enzyme activity of αGP and five other soluble αGPs. (a) Bidirectional enzyme activity of αGP detected at 37°C. The curve shows the ratio of the forward enzyme activity at 60°C to that at 37°C. (b) Bidirectional enzyme activity of αGP detected at 60°C. Figure 7 show Thermotoga petrophila Comparison of the bidirectional enzyme activities of the original α-glucan phosphorylase and its mutants; Figure 8 The figure shows the effect of different metal ion concentrations and types on the efficiency of the three-enzyme cascade catalyzing the production of amylose from glucose, where (a) shows the effect of 1 mM different metal ion types on the efficiency of the three-enzyme cascade catalyzing the production of amylose from glucose, (b) shows the effect of 5 mM different metal ion types on the efficiency of the three-enzyme cascade catalyzing the production of amylose from glucose, and (c) shows the effect of different concentrations of Mn 2+ Effect of the three-enzyme cascade on the efficiency of glucose to amylose production, (d) Figure shows different concentrations of Co 2+ Effects on the efficiency of the three-enzyme cascade catalyzed production of amylose from glucose; Figure 9The figure shows the effect of different buffers and pH values on the efficiency of the three-enzyme cascade catalyzing the production of amylose from glucose, wherein (a) shows the effect of different pH values on the efficiency of the three-enzyme cascade catalyzing the production of amylose from glucose, and (b) shows the effect of Tris-HCl buffers with different pH values on the efficiency of starch synthesis; Figure 10 The time course analysis of the yield and conversion rate of amylose produced by the optimized three-enzyme cascade was shown; Figure 11 Product characterization is shown. DETAILED DESCRIPTION

[0022] Test materials and reagents 1. Strains and vectors: The expression host is BL21 (DE3) Escherichia coli, and the expression plasmid vectors are pET-28a (+) and pET-30a (+).

[0023] 2. Enzymes: endonucleases, ligases.

[0024] 3. Culture medium: (1) Escherichia coli culture medium LB (1% peptone, 0.5% yeast extract, 1% NaCl, natural pH); Note: Molecular biology experimental methods not specifically described in the following examples were performed with reference to the specific methods listed in the book "Molecular Cloning Laboratory Manual" (3rd edition) by J. Sambrook, or in accordance with the kits and product instructions.

[0025] like Figure 1 As shown, the catalytic pathway for verifying amylose according to the present invention includes: the substrate glucose is phosphorylated to the intermediate G-6-P by the thermostable polyphosphate glucose kinase in the presence of polyphosphate; G-6-P is converted to G-1-P by the thermostable phosphoglucomutase, and the intermediate G-1-P is added one by one to the non-reducing end of the oligosaccharide by the thermostable α-glucan phosphorylase to synthesize amylose. Example 1. Construction of recombinant engineering bacteria producing polyphosphate glucokinase, phosphoglucomutase, and α-glucan phosphorylase and detection of amylose

[0026] The specific steps are as follows: 1. Construction of thermostable polyphosphate glucokinase (PPGK) recombinant engineering bacteria and acquisition of polyphosphate glucokinase (PPGK) (1) Inducible expression of thermostable polyphosphate glucose kinase (PPGK) Will come from Thermobifida fusca PPGK of YX F30L / R34P / T72A / V88M / A231TThe corresponding gene (ZHOU et al., 2018) was inserted between the BamHI and XholI restriction sites of the pET-28a(+) vector to construct the plasmid pET-28a(+)- tfppgk , synthesize the plasmid, and then transform the recombinant product into Escherichia coli Trans1-T1 competent cells and spread it on LB (containing 50μg / mL Kanamycin) for screening. After the sequencing is correct, the plasmid is transformed into the BL21 (DE3) Escherichia coli expression host to obtain the recombinant expression strain BL21 (DE3) (pET-28a (+) -tpαgp Positive clones were picked and inoculated into 40 mL LB liquid medium containing 50 μg / mL Kana at a final concentration, and cultured overnight in a shaking incubator at 37°C and 200 rpm.

[0027] On the second day, 4 mL of seed solution was taken from 40 mL of LB liquid medium and inoculated into 400 mL of LB liquid medium containing a final concentration of 50 μg / mL Kana, and the culture was expanded in a shaking incubator at 37°C and 200 rpm.

[0028] When the bacterial solution OD 600 When the pH value was 0.6-0.8, IPTG was added to a final concentration of 0.1 mM and cultured in a shaker at 16 °C and 200 rpm for 18 h.

[0029] After induction, the fermentation broth was centrifuged at 6000 rpm for 10 minutes at 4°C to collect the cells, and the supernatant was removed. The cell pellet was resuspended in cell lysis buffer (20 mM Tris-HCl, 0.5 M NaCl, 10 mM imidazole, pH 7.4) and centrifuged at 6000 rpm for 10 minutes to collect the cells. The supernatant was removed to remove impurities, culture medium components, dead cell debris, and other substances that may interfere with subsequent experiments. The cells were then frozen and stored at -80°C.

[0030] (2) Purification of thermostable polyphosphate glucokinase The target protein encoded by the pET-28a(+) vector carries a His tag at both the N-terminus and the C-terminus. Therefore, the PPGK protein was purified using nickel affinity chromatography, and the purified target protein was desalted by gel filtration chromatography.

[0031] (3) Determination of the enzyme activity of thermostable polyphosphate glucokinase PPGK enzyme activity unit definition: 1 U is defined as the amount of enzyme required to catalyze the production of 1 μmol of G-6-P per minute using glucose and polyphosphate as substrates, and the unit is U / mg. For the quantitative detection of G-6-P, glucose-6-phosphate dehydrogenase (G6PDH) and nicotinamide adenine dinucleotide (NAD + ) as an auxiliary reagent. In the specific reaction process, G6PDH catalyzes the reaction of G-6-P with NAD + The reaction converts G-6-P into 6-phospho-D-glucono-1,5-lactone and simultaneously generates reduced nicotinamide adenine dinucleotide (NADH). Because NADH has a characteristic absorption peak at 340 nm, its formation causes a significant change in absorbance at that wavelength. Therefore, spectrophotometric measurement of the change in absorbance at 340 nm can indirectly and accurately measure the amount of G-6-P generated, and thus determine the enzyme activity of PPGK.

[0032] The detection process consists of two main steps. The first step is to generate the target product G-6-P through an enzymatic reaction. A 200 μL reaction solution contains a final concentration of 12.5 mM glucose, 5 mM MgCl2, 1 mM sodium hexametaphosphate, and an appropriate amount of PPGK. The reaction is initiated by the addition of PPGK and incubated at 60°C for 10 minutes. The reaction is terminated by the addition of HClO4, and the pH is neutralized with KOH.

[0033] The second step reaction generates NADH. The 200 μL reaction solution contains a final concentration of 5 mM MgCl2, 6 mM NAD + , 2 U / mL G6DPH and an appropriate amount of the first step reaction product, the reaction was started by adding G6DPH, and the reaction kinetics was monitored at a wavelength of 340 nm. As the reaction proceeded, the generation of NADH would result in a decrease in the absorbance at 340 nm (OD 340 ) gradually increased. The reaction was terminated when the absorbance reached a stable state and no longer changed significantly. All reactions were measured in triplicate to ensure data accuracy and reliability.

[0034] 2. Construction of thermostable phosphoglucomutase PGM recombinant bacteria and acquisition of phosphoglucomutase PGM (1) Inducible expression of thermostable phosphoglucomutase Will come from Thermococcus kodakarensis of tkpgmThe gene (RASHID et al., 2004) was inserted between the BamHI and XholI restriction sites of the pET-28a(+) vector to construct and synthesize the plasmid. Other operations were performed according to the method of polyphosphate glucose kinase.

[0035] (2) Purification of thermostable phosphoglucomutase The target protein encoded by the pET-28a(+) vector carries a His tag at both the N-terminus and the C-terminus. Therefore, the PGM protein was purified using nickel affinity chromatography and desalted by gel filtration chromatography. Other operations refer to polyphosphate glucose kinase.

[0036] (3) Determination of the enzyme activity of thermostable phosphoglucomutase PGM catalyzes the reversible conversion of G-6-P to G-1-P. According to the requirements of the new starch synthesis pathway for PGM, the reaction direction of PGM-catalyzed conversion of G-6-P to G-1-P is defined as forward, and the reaction direction of PGM-catalyzed conversion of G-1-P to G-6-P is defined as reverse.

[0037] PGM forward reaction enzyme activity unit definition: 1 U is defined as the amount of enzyme required to catalyze the production of 1 μmol of G-1-P per minute using G-6-P as the substrate. The unit is expressed as U / mg. G-1-P generated in the reaction was measured using the high-performance anion exchange-pulsed amperometric detection (HPAEC-PAD) method.

[0038] Chromatographic conditions were as follows: A Thermo Scientific Dionex Carbopac PA200 analytical column and guard were used in this experiment. Mobile phase A was 0.1 M NaOH solution, and mobile phase B was 0.1 M NaOH solution containing 1 M sodium acetate. The column temperature was set to 30 °C, and the solute was eluted at a rate of 0.25 mL / min: the initial condition was set to 100% mobile phase A, and then mobile phase B was increased proportionally for linear gradient elution; starting from the 8th minute, the proportion of mobile phase B increased linearly from 0% to 10%; starting from the 15th minute, the proportion of mobile phase B continued to increase linearly to 20%; starting from the 25th minute, the proportion of mobile phase B increased exponentially to 100% within 2 minutes. After the elution was completed, 100% mobile phase A was used to run for 11 minutes to equilibrate the column and prepare for the next injection.

[0039] The detection process consists of two steps. The first step is to generate the target product G-1-P through an enzymatic reaction. A 200 μL reaction solution contains a final concentration of 12.5 mM G-6-P, 5 mM MgCl2, and an appropriate amount of PGM. The reaction is initiated by the addition of PGM, incubated at 60°C for 10 minutes, and terminated by incubating at 100°C in a water bath for 15 minutes.

[0040] In the second step, the sample was processed and analyzed by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The terminated sample was centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was carefully collected and filtered through a 0.22 μm microporous filter to further remove any possible particulates, prevent column clogging, and ensure sample clarity. The filtered sample was then analyzed by HPAEC-PAD, and the concentration of the target product was determined by comparison with a G-1-P standard. All reactions were performed in triplicate to ensure data accuracy and reliability.

[0041] PGM reverse reaction enzyme activity unit definition: 1 U is defined as the amount of enzyme required to catalyze the production of 1 μmol of G-6-P per minute using G-1-P as the substrate, expressed in U / mg. G-6-P generated in the reaction was measured using the high-performance anion exchange-pulsed amperometric detection (HPAEC-PAD) method, following the same procedures as for the PGM forward enzyme activity assay. All reactions were performed in triplicate to ensure data accuracy and reliability.

[0042] 3. Construction of thermostable α-glucan phosphorylase αGP recombinant engineering bacteria and acquisition of α-glucan phosphorylase αGP (1) Inducible expression of thermostable α-glucan phosphorylase agpA The gene was derived from the work of the Bibel team (BIBEL et al., 1998) and was inserted between the BamHI and NotI restriction sites of the pET-30a(+) vector to synthesize the plasmid. Other operations were performed according to the method of polyphosphate glucose kinase.

[0043] (2) Purification of thermostable α-glucan phosphorylase The target protein encoded by the pET-30a(+) vector carries a His tag at both the N-terminus and the C-terminus, so the αGP protein was purified using nickel affinity chromatography and the purified target protein was desalted by gel filtration chromatography. Tp All αGPs except αGP can be purified to a high purity by nickel affinity chromatography. Tp After nickel affinity chromatography, αGP still needs to undergo a heat treatment step to obtain a relatively pure protein. Other operations refer to polyphosphate glucose kinase.

[0044] (3) Determination of the enzyme activity of thermostable α-glucan phosphorylase αGP catalyzes the reversible phosphorolysis reaction of α-1,4-glucan. According to the demand for αGP in the new pathway for starch synthesis, the reaction direction of αGP catalyzing the addition of G-1-P to the non-reducing end of α-1,4-glucan to synthesize glycosidic bonds is defined as forward, and the reaction direction of αGP catalyzing the attack of phosphate on the non-reducing end of α-1,4-glucan to break the glycosidic bonds is defined as reverse.

[0045] αGP forward reaction enzyme activity unit definition: 1 U is defined as the amount of enzyme required to catalyze the production of 1 μmol of phosphate per minute using G-1-P as a substrate in the presence of α-1,4-glucan primer. The unit is U / mg. For quantitative phosphate determination, refer to the method proposed by Saheki et al. (SAHEKI et al., 1985) for the determination of inorganic phosphate in a mild pH range.

[0046] A 200 μL reaction solution contained maltodextrin (9 g / L final concentration), 12.5 mM G-1-P, 0.1 M sodium acetate (pH 5.5), and the appropriate amount of αGP. The reaction was initiated by the addition of αGP and incubated at 60°C for 10 min. The reaction was terminated by the addition of 800 μL of molybdate reagent (15 mM ammonium molybdate, 100 mM zinc acetate, pH 5.0), followed by the addition of 200 μL of ascorbic acid reagent (10% wt / vol, pH 5.0). The mixture was incubated at 30°C for 20 min, and the absorbance response of the reaction mixture was recorded by measuring the absorbance at 850 nm. All reactions were performed in triplicate to ensure data accuracy and reliability.

[0047] αGP reverse reaction enzyme activity unit definition: 1 U is defined as the amount of enzyme required to catalyze the production of 1 μmol of G-1-P per minute using α-1,4-glucan and phosphate as substrates. The unit is U / mg. For the quantitative detection of G-1-P, a PGM and G6PDH (glucose-6-phosphate dehydrogenase) cascade is used to convert G-1-P into NADH (reduced nicotinamide adenine dinucleotide). The formation of NADH causes a change in absorbance at 340 nm, and G-1-P production is indirectly measured spectrophotometrically (SUN et al., 2021; YOU et al., 2017).

[0048] The detection process consists of two steps. The first step is an enzymatic reaction to generate the target product, G-1-P. A 200 μL reaction solution contains a final concentration of 20 mM phosphate, 9 g / L maltodextrin, 5 mM MgCl2, and an appropriate amount of αGP. The reaction is initiated by the addition of αGP and incubated at 60°C for 10 minutes. The reaction is stopped by the addition of HClO4 and the pH is neutralized with KOH.

[0049] In the second step, PGM catalyzes the conversion of G-1-P to G-6-P, and then G6PDH uses NAD⁺ as a cofactor to oxidize G-6-P to 6-phosphogluconolactone and generate NADH. The 200 μL reaction solution contains 5 mM MgCl2, 6 mM NAD + , 2 U / mL PGM, 2 U / mL G6DPH and an appropriate amount of the first step reaction product, the reaction was started by adding G6DPH, and the reaction kinetics were monitored at a wavelength of 340 nm. 340 The reaction was terminated when the reaction reached a stable state and no longer changed. All reactions were measured in triplicate to ensure data accuracy and reliability.

[0050] 4. Detection of amylose First, an enzyme cascade reaction system for starch synthesis was constructed: a 200 μL reaction system contained 50 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer (pH 7.4), 30 mM glucose, 4 mM sodium hexametaphosphate, 5 mM MgCl2, 0.1 mM maltotetraose (M4) and appropriate amounts of PPGK, PGM and αGP. The reaction system was carried out at 60 °C for 2 h.

[0051] After the enzymatic cascade reaction, the glucan produced in the sample was treated using the Megazyme Total Starch Assay Kit, and the glucose produced was measured using the High-Performance Anion Exchange-Pulsed Amperometric Detection (HPAEC-PAD) method. All reactions were performed in triplicate to ensure data accuracy and reliability.

[0052] Chromatographic conditions were as follows: a Thermo Scientific Dionex Carbopac PA200 analytical column and guard, mobile phase A was deionized water, mobile phase B was 0.1 M NaOH solution, the column temperature was set to 30 °C, and the solute was eluted at a rate of 0.25 mL / min: the initial conditions were set to 98% mobile phase A, 2% mobile phase B, and then mobile phase B was increased proportionally for linear gradient elution; by the 8th minute, the proportion of mobile phase B was linearly increased from 2% to 5%; then the proportion of mobile phase B was increased to 100% within 17 minutes and maintained for 3 minutes; starting from the 28th minute, the proportion of mobile phase B was reduced from 100% to 2% within 8 minutes; using 98% mobile phase A, 2% mobile phase B for 4 minutes to equilibrate the column and prepare for the next injection.

[0053] like Figure 2 As shown in "A", 10 μM Thermobifida fusca PPGK from YX, Thermococcus kodakarensis Sources of PGM and Thermotoga maritima The αGP from the raw material was mixed with 50 mM HEPES buffer (pH 7.4), 30 mM glucose, 4 mM sodium hexametaphosphate, 5 mM MgCl2, and 0.1 mM maltotetraose to construct the initial reaction system "A". The enzyme cascade reaction was carried out at 60 °C for 2 h, and 0.29 g / L starch was synthesized with a conversion rate of 6.03%. Example 2: Mining and Screening of Phosphoglucomutase PGM and α-glucan phosphorylase αGP and Preparation of α-glucan phosphorylase mutant TpαGPM7

[0054] 1. Mining and screening of phosphoglucomutase Twelve phosphoglucomutase sequences were selected, as shown in Table 1.

[0055] Table 1 PGMs from 12 different sources screened Gene name Gene source Uniprot ID A0A959BHL9 A0A3D5X8C0 A0A2X0V701 A0A7J4GQ39 A0A2E1ZVD2 A0A7J0BJL5 A0A923U2U6 P38569 A0A1D8QV96 A0A928GH83 A0A386H0X2 A0A533ZIH4

[0056] The 12 PGMs screened and Thermococcus kodakarensis Source of PGM ( Tk PGM) was induced to express, and the experimental results were as follows Figure 3 As shown, except Nib PGM and Neb PGMs existed in the form of inclusion bodies. All PGMs were expressed soluble and the soluble fractions were purified by nickel affinity chromatography.

[0057] Then 10 PGMs and templates were Tk PGM was tested for bidirectional enzyme activity under different temperature conditions, such as Figure 4 However, under the test conditions of 37 ℃ and 60 ℃, no At PGM and Cf The forward or reverse enzyme activity of PGMs. By comparing the bidirectional enzyme specific activities of PGMs at 37°C and 60°C (the ratio of the forward enzyme specific activity at 60°C to the forward enzyme specific activity at 37°C is greater than 1), PGMs with good thermal stability can be screened: Phs PGM, Bb PGM, Ds PGM, Ob PGM, Ke PGM, Aa PGM, ( Figure 4 In the new artificial starch synthesis pathway, PGM is required to play the role of forward reaction. However, during the screening process, no PGM was found with a forward enzyme activity greater than the reverse enzyme activity ( Figure 4(b) Figure), therefore, the enzyme with the highest ratio of forward enzyme specific activity to reverse enzyme specific activity was selected from the above screened enzymes. Phs PGM (0.065), used for subsequent experiments.

[0058] 2. Mining and screening of α-glucan phosphorylase 9627 sequences homologous to αGP were retrieved, and 8 αGP sequences were selected, as shown in Table 2.

[0059] Table 2 αGP from eight different sources screened Gene name Gene source Uniprot ID D2C4L9 UPI001AE22C4D A0A2N2EZ06 A0A832E8H0 A0A8T4DWL2 14B UPI000989D833 A0A357CTH3 UPI0021151EF9

[0060] For 8 αGP and Tm αGP was induced to express, and the experimental results were as follows Figure 5 As shown, Ms αGP, Ta αGP and Cb αGP mainly exists in the form of inclusion bodies, and the rest of αGP is expressed in a soluble form, and the soluble components can be purified by nickel affinity chromatography. Tp αGP requires heat treatment after purification by nickel affinity chromatography to obtain a protein sample with a purity >90%.

[0061] Then the five purified αGP and template Tm αGP was subjected to bidirectional enzyme activity tests under different temperature conditions. Figure 6 As shown in Figure (a), only Tp The ratio of the forward enzyme activity of αGP at 60 ℃ to that at 37 ℃ is greater than 1. In the new artificial starch synthesis pathway, αGP is required to play the role of forward reaction. Ar αGP, Bb αGP and Tp The ratio of the specific activity of the forward enzyme to the specific activity of the reverse enzyme of αGP at 60 ℃ is >1 ( Figure 6 (b) in the figure), so choose Tp αGP was used in subsequent experiments.

[0062] Use of pathway enzymes obtained through screening Phs PGM and Tp αGP, reaction system "B" was constructed, and other conditions were the same as reaction system "A". 0.92 g / L starch was synthesized, which was higher than that of the original enzyme. Tk PGM and Tm αGP, the conversion rate increased by about 3 times to 18.87% ( Figure 2 ).

[0063] 3. α-glucan phosphorylase mutant TpPreparation of αGPM7 Design mutation primers and use plasmid pET-28a(+)- tpαgp As a template, the mutant amino acid was introduced by a point mutation kit, and the PCR product containing the mutant amino acid was used Dpn The PCR product was digested to remove the template, and the digested PCR product was transformed into Escherichia coli Tans1-T1 competent cells, and sequenced to obtain Escherichia coli containing the α-glucan phosphorylase mutant plasmid.

[0064] The recombinant plasmids were transformed into BL21 (DE3) Escherichia coli expression hosts, induced for expression, and recombinant expression strains were obtained.

[0065] At 60℃, the catalytic activity is improved. Tp The forward enzyme activity of αGPM1 is Tp αGP increased by 36.24%, Tp The reverse enzyme activity of αGPM1 is Tp αGP increased by 7.99%.

[0066] At 60 °C, the catalytic activity increased. Tp The positive enzyme activity of αGPR673A is Tp αGP increased by 50.55%, Tp The reverse enzyme activity of αGPR673A is Tp αGP increased by 6.05%.

[0067] At 60℃, the catalytic activity is improved. Tp The positive enzyme activity of αGPR485K is Tp αGP increased by 9.08%, Tp The reverse enzyme activity of αGPR485K is Tp αGP decreased by 2.72%, Tp αGPR485K compared to Tp αGP prefers to consume G-1-P to synthesize glucans.

[0068] At 60℃, the catalytic activity is greatly improved. Tm The forward enzyme activity of αGPM6 is Tm 34 times that of αGP, Tm The reverse enzyme activity of αGPM6 is Tm 37 times that of αGP.

[0069] The catalytic activity was significantly improved at 60℃. Tp The forward enzyme activity of αGPM3 is Tp αGP increased by 149.78%, Tp The reverse enzyme activity of αGPM3 is Tp αGP increased by 65.79%. Tp The forward enzyme activity of αGPM5 is Tp αGP increased by 66.25%, Tp The reverse enzyme activity of αGPM5 is Tp αGP increased by 35.81%. Tp The forward enzyme activity of αGPM7 is Tp αGP increased by 354.04%, Tp The reverse enzyme activity of αGPM7 is Tp αGP increased by 147.93%.

[0070] α-Glucan phosphorylase parent and mutants Tp The determination of the bidirectional enzyme activity of αGPM7 was carried out according to the determination of the enzyme activity of the thermostable α-glucan phosphorylase in Example 1. Figure 7 As shown. Use Tp αGPM7 participated in starch synthesis, and reaction system "C" was constructed. Under the same conditions as "B", the reaction lasted for 2 h, and 1.75 g / L starch was synthesized. The conversion rate increased from 18.87% to 35.98% ( Figure 2 ). Example 3: Optimization of reaction conditions and product identification using glucose synthesis amylose reaction system

[0071] 1. Effects of different metal ion concentrations and types on the efficiency of amylose synthesis The assay was performed at 60°C, pH 5.5-9.0 [50 mM 2-Morpholineethanesulfonic acid (MES) buffer at pH 5.5, 6.0, 6.5, 7.0; 50 mM HEPES buffer at pH 7.0, 7.5, 8.0; 50 mM Tris-HCl buffer at pH 8.0, 8.5, 9.0] Phs PGM bidirectional enzyme activity, explore the effect of different pH conditions on Phs Effect of PGM on the specific activity of bidirectional enzymes. All reactions were performed in triplicate to ensure data accuracy and reliability.

[0072] like Figure 8 As shown in Figure (a), in the artificial starch synthesis system, no additional metal ions are added or monovalent metal ions (Na + , K + 、Ag + ) and trivalent metal ions (Fe 3+ ), starch cannot be synthesized; among the divalent metal ions, only Mg is added to the reaction system. 2+ 、Zn 2+、Co 2+ 、Mn 2+ , can promote starch synthesis. It is worth noting that the addition of Co 2+ or Mn 2+ Compared with Mg 2+ , showing higher synthesis efficiency.

[0073] like Figure 8 As shown in Figure (b), even if the concentrations of monovalent metal ions and trivalent metal ions are increased to 5 mM, the artificial starch synthesis system still cannot synthesize starch. 2+ The presence of 5 mM Ca reduced the efficiency of starch synthesis. 2+ or Ni 2+ Can support starch synthesis, but not as efficiently as Mg 2+ ;5 mMCo 2+ or Mn 2+ Compared with Mg 2+ , still showed higher synthesis efficiency, and compared with 1 mM, the addition of 5 mM Co 2+ or Mn 2+ Further improved the efficiency of starch synthesis.

[0074] When 1 mM metal ions were added, Mn 2+ The efficiency of synthesizing starch is better than that of adding Co 2+ However, when 5 mM metal ions were added, the addition of Co 2+ The starch synthesis efficiency exceeds that of Mn 2+ This finding prompted further investigation of different concentrations of Mn 2+ and Co 2+ Specific impact on the efficiency of starch synthesis. Figure 8 As shown in Figure (c), 8 mMCo was added 2+ When Figure 8 In (d), when 20 mM Mn 2+ When 20 mM Mn was used, the starch synthesis efficiency was the highest, which was 3.23 g / L, which was 132% of the former. 2+ as the subsequent reaction conditions.

[0075] 2. Effects of different pH values and buffer types on the efficiency of amylose synthesis Starch yields from the enzymatic cascade reaction were measured at 60°C and pH 5.5-9.0 (50 mM MES buffer at pH 5.5, 6.0, 6.5, and 7.0; 50 mM HEPES buffer at pH 7.0, 7.5, and 8.0; and 50 mM Tris-HCl buffer at pH 8.0, 8.5, and 9.0) to investigate the effect of different pH conditions on starch synthesis efficiency. All reactions were performed in triplicate to ensure data accuracy and reliability.

[0076] Previous experimental results have confirmed that pH has an important effect on Phs The specific activity of PGM enzymes was significantly affected. Based on this finding, the effect of different pH values on starch synthesis efficiency was further studied. Figure 9 As shown in Figure (a), under the conditions of 50 mM Tris-HCl buffer at pH 8.0, the starch synthesis efficiency reached the highest level (3.39 g / L), which was 17% higher than that when HEPES buffer was used at the same pH value. This result shows that in addition to the pH value itself, the type of buffer also has an impact on the starch synthesis efficiency. Therefore, we continued to explore the effect of different concentrations of Tris-HCl buffer on starch synthesis efficiency in order to find the optimal buffer conditions, such as Figure 9 As shown in Figure (b), under the conditions of 50 mM Tris-HCl buffer at pH 7.75, the starch synthesis efficiency reached 3.44 g / L.

[0077] To use Tp The efficiency of starch synthesis by the αGPM7 mutant in the optimized reaction system was carefully determined. Figure 10 As shown in the figure, the starch synthesis reached equilibrium after 90 min. The system could utilize 30 mM glucose to synthesize 3.50 g / L starch, with a conversion rate of 71.93% and a space-time yield of 2.33 g / L / h.

[0078] 3. Product identification (1) Preparation and separation of starch The enzymatic reaction was terminated by adding HClO₄, and the pH of the reaction system was adjusted using KOH. The sample was then centrifuged at 10,000 rpm for 10 minutes to effectively remove impurities such as salt ions and proteins from the reaction system. The supernatant was collected and 95% ethanol was added at a ratio of 1:2 to promote starch precipitation. The wet starch powder was then washed once with 95% ethanol to ensure removal of any residual soluble sugars, thereby ensuring the purity of the final product. Finally, the washed starch was freeze-dried to obtain starch powder.

[0079] (2) Product characterization using iodine staining and spectrophotometry Sodium carboxymethylcellulose (CMCNa), amylose, maltodextrin, amylopectin, and artificial starch powders were systematically treated. After adding iodine solution to each sample to initiate a color reaction, the resulting color changes were observed and recorded. Subsequently, the iodine-containing sample mixtures were placed in a spectrophotometer and analyzed by spectral scanning within the 400 nm to 800 nm wavelength range.

[0080] like Figure 11 As shown, when CMCNa reacts with iodine / potassium iodide, it displays the original color of the iodine reagent (test tubes 1 and 6). Maltodextrin and amylopectin standards react with iodine / potassium iodide to produce an orange-red color (test tubes 2 and 4). In contrast, amylose standards and artificial starch react with iodine / potassium iodide to produce a blue-green color (test tubes 3 and 5). During spectral scanning, both amylose standards and artificial starch exhibit a characteristic absorption peak at 600 nm.

[0081] Based on the results of the above iodine color development reaction and spectral scanning, it is shown that the product generated in the new pathway of artificial starch synthesis is amylose.

[0082] The above embodiments are only used to understand the technical solutions of the present application and do not limit the scope of protection of the present application.

Claims

1. A method for synthesizing amylose from glucose by three-enzyme cascade catalysis, characterized in that: The method comprises the following steps: In the presence of polyphosphate in a buffer system, the substrate glucose is phosphorylated to an intermediate G-6-P by polyphosphate glucokinase, G-6-P is converted to an intermediate G-1-P by phosphoglucomutase, and the intermediate G-1-P is added one by one to the non-reducing end of the oligosaccharide by α-glucan phosphorylase to synthesize amylose, wherein the buffer system contains 1mM~5mM Mg 2+ , 1mM~20mM Co 2+ , or 1mM~50mM Mn 2+ , the pH of the buffer system is 7.75~8.

0.

2. The method for synthesizing amylose from glucose by three-enzyme cascade catalysis according to claim 1, characterized in that: The buffer system contains 20 mM Mn 2+ .

3. The method for synthesizing amylose from glucose by three-enzyme cascade catalysis according to claim 1, characterized in that: The buffer system is Tris-HCl buffer.

4. The method for synthesizing amylose from glucose by three-enzyme cascade catalysis according to claim 1, characterized in that: The buffer system is 50 mM Tris-HCl buffer at pH 7.

75.

5. The method for synthesizing amylose from glucose by three-enzyme cascade catalysis according to claim 1, characterized in that: The polyphosphate glucose kinase is derived from Thermobifida fusca YX The polyphosphate glucose kinase is derived from Phaeodactylibacter sp. The phosphoglucomutase, the α-glucan phosphorylase is derived from Thermotoga petrophila α-glucan phosphorylase.

6. The method for synthesizing amylose from glucose by three-enzyme cascade catalysis according to claim 1, characterized in that: The following reaction was carried out at 60°C: In the presence of polyphosphate, the substrate glucose is phosphorylated by polyphosphate glucokinase to the intermediate G-6-P; the G-6-P is converted to the intermediate G-1-P by phosphoglucomutase; and the intermediate G-1-P is added one by one to the non-reducing end of the oligosaccharide by α-glucan phosphorylase to synthesize amylose.

7. The method for synthesizing amylose from glucose by three-enzyme cascade catalysis according to claim 1, characterized in that: The α-glucan phosphorylase mutant is obtained by subjecting the α-glucan phosphorylase with the amino acid sequence shown in SEQ ID NO.1 to the following mutations: The amino acid at position 774 mutated from Asp to Glu, the amino acid at position 775 mutated from Leu to Ser, the amino acid at position 776 mutated from Phe to Ile, the amino acid at position 777 mutated from Val to Arg, the amino acid at position 778 mutated from Tyr to Leu, the amino acid at position 779 mutated from Thr to His, the amino acid at position 780 mutated from Tyr to Leu, the amino acid at position 781 mutated from Thr to His, the amino acid at position 782 mutated from Asn to Gln, the amino acid at position 783 mutated from Gly to Trp, and the amino acid at position 784 mutated from Val to Cys; The amino acid at position 669 mutated from Phe to Ser, and the amino acid at position 670 mutated from Ser to Ala; The amino acid at position 669 mutated from Phe to Ser, the amino acid at position 670 mutated from Ser to Ala, the amino acid at position 774 mutated from Asp to Glu, the amino acid at position 775 mutated from Leu to Ser, the amino acid at position 776 mutated from Phe to Ile, the amino acid at position 777 mutated from Val to Arg, the amino acid at position 778 mutated from Tyr to Leu, the amino acid at position 779 mutated from Thr to His, the amino acid at position 780 mutated from Tyr to Leu, the amino acid at position 781 mutated from Thr to His, the amino acid at position 782 mutated from Asn to Gln, the amino acid at position 783 mutated from Gly to Trp, and the amino acid at position 784 mutated from Val to Cys; The amino acid at position 485 was mutated from Arg to Lys; The amino acid at position 671 mutated from Pro to Thr, the amino acid at position 672 mutated from Pro to Thr, the amino acid at position 673 mutated from Ala to Arg, the amino acid at position 674 mutated from Cys to Met, and the amino acid at position 675 mutated from Cys to Leu; The amino acid at position 673 is mutated from Arg to Ala; or The 171st amino acid was mutated from Glu to Gly.

8. The method for synthesizing amylose from glucose by three-enzyme cascade catalysis according to claim 1, characterized in that: The buffer system contains 50 mM Tris-HCl buffer (pH 7.75), 30 mM glucose, 4 mM sodium hexametaphosphate, 20 mM Mn 2+ , 0.1 mM maltotetraose, 3.5 μM polyphosphate glucokinase, 1 μM phosphoglucomutase, and 9 μM α-glucan phosphorylase at 60 °C for 2 h.

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