Phosphate transketolase mutant and application thereof in production of 3-hydracrylic acid

Abstract

The invention provides a phosphoketolase mutant, a related product of the phosphoketolase mutant and application of the phosphoketolase mutant in production of acetyl phosphate, acetyl coenzyme A and / or 3-hydracrylic acid. The phosphoketolase mutant provided by the invention has relatively high catalytic capability on hexanediose and pentose, and can catalyze hexanediose and pentose to be converted into acetyl phosphoric acid at relatively high efficiency, so that subsequent products acetyl coenzyme A and 3-hydracrylic acid are efficiently generated; therefore, the method has great application potential in production of acetyl coenzyme A and products (such as 3-hydracrylic acid) taking acetyl coenzyme A as a precursor substance.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a phosphoketolase mutant and an application thereof in producing 3-hydroxypropionic acid. Background Art

[0002] Researchers have been committed to exploring and utilizing phosphoketolase (PK) from various sources to complete the downstream steps of the pentose phosphate pathway to achieve the goal of efficient utilization of acetyl-CoA and improve carbon utilization efficiency. For example, Liu Quanli and other researchers successfully changed the metabolic pathway of Saccharomyces cerevisiae by modifying phosphoketolase, thereby significantly increasing the production of soy isoflavones to 85.4 mg / L. In addition, Qin Ning and other researchers effectively solved the problem of carbon loss by creating a yeast strain capable of fermenting mixed carbohydrates; this achievement was achieved by introducing PK and phosphotransacetylase (PTA) and blocking the glycolysis pathway (EMP), and ultimately achieved the production of 2.68 g / L of free fatty acids under shake flask conditions.

[0003] However, existing studies have shown that the enzymatic activity of PK enzymes from different sources is generally low, which has become a major obstacle to their widespread application. Although the substrate specificity of phosphoketolase (PK) from various sources varies, their products all contain acetyl phosphate. Researchers such as Jiang Huifeng found that by mutating PK enzymes, the catalytic ability of the enzyme for a variety of ketoses can be enhanced. This discovery indicates that the development of an efficient screening procedure is a new strategy for the effective use of PK enzymes, which aims to screen out mutants that can effectively utilize other ketones without weakening the inherent catalytic activity of PK enzymes.

[0004] Due to the limited understanding of the functional structure of PK enzymes, a variety of techniques have been applied to study the substrate specificity of enzymes. Given that the PK pathway can produce acetyl-CoA, which has a direct impact on cell growth, growth-coupled selection provides a straightforward and effective strategy to overcome the challenges encountered in the development of screening technologies. Summary of the invention

[0005] In response to the needs and problems existing in the prior art, the present invention provides a phosphoketolase mutant having broad substrate specificity and high phosphoketolase activity, its related products and its use in the production of acetyl phosphate, acetyl-CoA and / or 3-hydroxypropionic acid, as well as a method for producing acetyl phosphate, acetyl-CoA and / or 3-hydroxypropionic acid using the phosphoketolase mutant and its related products.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a phosphoketolase mutant having an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

[0008] The above phosphoketolase mutants are all obtained by amino acid substitution based on the wild-type phosphoketolase shown in SEQ ID NO: 1, as follows:

[0009] In the amino acid sequence shown in SEQ ID NO:1, the valine residue at position 705 is replaced with an isoleucine residue (i.e., V705I mutation), thereby obtaining the amino acid sequence shown in SEQ ID NO:2;

[0010] In the amino acid sequence shown in SEQ ID NO:1, the glutamic acid residue at position 229 is replaced with an alanine residue (i.e., E229A mutation), thereby obtaining the amino acid sequence shown in SEQ ID NO:3;

[0011] In the amino acid sequence shown in SEQ ID NO: 1, the histidine residue at position 546 is replaced by an alanine residue (ie, H546A mutation), thereby obtaining the amino acid sequence shown in SEQ ID NO: 4.

[0012] In a second aspect, the present invention provides a fusion protein, which is a protein obtained by fusing the phosphoketolase mutant described in the first aspect with a protein tag.

[0013] In a feasible embodiment, the protein tag is any one or more of Poly-Arg, Poly-His, FLAG, c-myc, and HA.

[0014] In a third aspect, the present invention provides an enzyme agent or an enzyme composition, which comprises the phosphoketolase mutant as described in the first aspect or the fusion protein as described in the second aspect.

[0015] In a fourth aspect, the present invention provides a polynucleotide encoding the phosphoketolase mutant as described in the first aspect above or the fusion protein as described in the second aspect above.

[0016] In a fifth aspect, the present invention provides a nucleic acid construct, a recombinant vector, a recombinant microorganism or an in vitro recombinant cell comprising the polynucleotide as described in the fourth aspect above.

[0017] In a feasible embodiment, the recombinant microorganism is a recombinant yeast, such as a recombinant Saccharomyces cerevisiae or a recombinant Pichia pastoris, preferably a recombinant Saccharomyces cerevisiae.

[0018] In a sixth aspect, the present invention provides the use of the phosphoketolase mutant as described in the first aspect, the fusion protein as described in the second aspect, the enzyme agent or enzyme composition as described in the third aspect, the polynucleotide as described in the fourth aspect and / or the nucleic acid construct, recombinant vector, recombinant microorganism or in vitro recombinant cell as described in the fifth aspect in the production of acetyl phosphate, acetyl-CoA and / or 3-hydroxypropionic acid using ketose as a reaction substrate.

[0019] In a feasible embodiment, the ketose is selected from: xylulose 5-phosphate, fructose 6-phosphate, sedulose 7-phosphate and / or erythrose 4-phosphate, preferably selected from: xylulose 5-phosphate and / or fructose 6-phosphate.

[0020] In the seventh aspect, the present invention provides a method for producing acetyl phosphate, acetyl-CoA and / or 3-hydroxypropionic acid, the method using the phosphoketolase mutant as described in the first aspect, the fusion protein as described in the second aspect, the enzyme agent or enzyme composition as described in the third aspect, the polynucleotide as described in the fourth aspect and / or the nucleic acid construct, recombinant vector, recombinant microorganism or in vitro recombinant cell as described in the fifth aspect, and using ketose as a reaction substrate to produce acetyl phosphate, acetyl-CoA and / or 3-hydroxypropionic acid.

[0021] In a feasible embodiment, the ketose is selected from: xylulose 5-phosphate, fructose 6-phosphate, sedulose 7-phosphate and / or erythrose 4-phosphate, preferably selected from: xylulose 5-phosphate and / or fructose 6-phosphate.

[0022] In a preferred embodiment, the method comprises:

[0023] In the presence of the phosphoketolase mutant described in the first aspect above, the fusion protein described in the second aspect above, the enzyme agent or enzyme composition described in the third aspect above, or the recombinant microorganism or in vitro recombinant cell described in the fifth aspect above, ketose is catalyzed to generate acetyl phosphate, and the obtained acetyl phosphate is optionally further converted to generate acetyl-CoA, and the acetyl-CoA is optionally further converted to generate 3-hydroxypropionic acid;

[0024] Further preferably, the acetyl phosphate is converted into acetyl-CoA under the action of phosphate acetyltransferase;

[0025] Further preferably, the acetyl-CoA is converted into 3-hydroxypropionic acid under the action of acetyl-CoA carboxylase or malonyl-CoA reductase.

[0026] Beneficial Effects

[0027] The inventors obtained the phosphoketolase mutant of the present invention through scientific design and a large number of experimental screenings; the phosphoketolase mutant of the present invention has a wide range of substrate specificity, in particular, it can catalyze the conversion of six-carbon ketose (such as fructose 6-phosphate) into acetyl phosphate with a much higher efficiency than the wild-type phosphoketolase from which it is derived, and then efficiently generate the subsequent products acetyl-CoA and 3-hydroxypropionic acid, while not affecting the catalytic ability to five-carbon ketose (such as xylulose 5-phosphate); that is, compared with the wild-type phosphoketolase shown in SEQ ID NO: 1, the catalytic ability of the phosphoketolase mutant of the present invention to xylulose 5-phosphate is comparable to that of the wild-type phosphoketolase (that is, both are stronger), while its catalytic ability to six-carbon ketose (such as fructose 6-phosphate) is much higher than that of the wild-type enzyme.

[0028] In other words, the phosphoketolase mutant of the present invention has a high catalytic ability for both six-carbon ketose (e.g., fructose-6-phosphate) and five-carbon ketose (e.g., xylulose-5-phosphate), and thus has a great application potential in the production of acetyl-CoA and products with acetyl-CoA as a precursor (e.g., 3-hydroxypropionic acid). BRIEF DESCRIPTION OF THE DRAWINGS

[0029] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0030] Figure 1 The fold changes of the enzyme activities of the phosphoketolase mutants V705I, P235A, L504M, Q259L, and E223K for fructose-6-phosphate (F6P) and xylulose-5-phosphate (X5P) are shown compared with the wild-type phosphoketolase (WT);

[0031] Figure 2 Shown are the fold changes in the enzyme activities of the phosphoketolase mutants E229A, D263A, and E262A for fructose-6-phosphate (F6P) and xylulose-5-phosphate (X5P) compared to the wild-type phosphoketolase (WT);

[0032] Figure 3 Shown are the fold changes in the enzyme activities of the phosphoketolase mutants H546A, Q316A, I210A, Y500A, F503A, N547A, H315A, and H55A for fructose-6-phosphate (F6P) and xylulose-5-phosphate (X5P) compared to the wild-type phosphoketolase (WT);

[0033] Figure 4The results show a comparison of the ability of strain QJH2 integrated with the wild-type phosphoketolase expression vector and strain QJH3-5 integrated with the phosphoketolase mutants V705I, E229A, and H546A to produce 3-hydroxypropionic acid. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution in the embodiment of the present invention will be clearly and completely described below. Obviously, the described embodiment is a part of the embodiment of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Unless explicitly stated otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0036] The term "enzyme activity" refers to the ability of an enzyme to catalyze a certain chemical reaction, and in this context, specifically refers to the efficiency of an enzyme in converting a reaction substrate into a product, and is used interchangeably with the term "catalytic ability".

[0037] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0038] Example 1: Design and screening of phosphoketolase mutants

[0039] In this example, based on the Enterobacterium leucomemorans phosphoketolase (xPK for short, whose amino acid sequence is shown in SEQ ID NO: 1), a series of xPK mutants were designed by replacing amino acids at specific sites based on the amino acid sequence shown in SEQ ID NO: 1; then, the encoding nucleic acid of the xPK mutant was codon-optimized to enable its expression in yeast, and chemically synthesized.

[0040] It is known in the art that a linear relationship between mutation screening and growth rate can be established based on growth rate and clone size, which can be used to simply and effectively achieve preliminary screening of mutations. When the glycolytic pathway (EMP) of Saccharomyces cerevisiae is blocked, the cell growth rate can be partially restored by introducing different copies of phosphoketolase (PK). Therefore, in order to achieve growth-coupled screening, we selected the Saccharomyces cerevisiae strain CEN.PK 113-11C (which can be obtained by referring to the following literature: Qin et al., Flux regulation through glycolysis and respiration is balanced by inositol pyrophosphates in yeast, Cell (2023), https: / / doi.org / 10.1016 / j.cell.2023.01.014) as the starting strain, knocked out PFK1 / 2 and introduced PTA and TH, and constructed the strain QJ1 (whose genotype is pfk1Δpfk2ΔXI3::(TEF1-PTA-PGI1t)his3Δ1::(TDH3p-Transhydrogenase-HMG1t)gpp1Δgpp2Δ) for the construction of the mutant library and the screening of mutants. The codon-optimized nucleic acid coding sequence of the designed xPK mutant was constructed into an expression plasmid, and the expression plasmid was transformed into the strain QJ1 to construct a mutant library. The entire mutant library contains approximately 10^5 clones, which can fully cover the screening process.

[0041] First, a preliminary screening was performed based on the clone size and the results of the preliminary screening were summarized. All possible mutants were subjected to in vitro protein expression and purification. The plasmid used for protein expression was constructed using pET-28a(+) and E. coli DH5α, and the protein was expressed using the BL21(DE3) strain. A 6xHis tag was attached to the N-terminus of the protein. The protein purification scheme is as follows: a single colony is inoculated into 4 ml of Luria-Bertani (LB) medium containing ampicillin; then, the culture is shaken at 220 rpm overnight at 37°C; then, the bacterial solution is transferred to 800 ml of Luria-Bertani (LB) medium containing ampicillin and cultured under the same conditions until OD600 reaches 0.6-0.8; then, isopropyl β-D-1-thiogalactoside (IPTG) is added to a final concentration of 0.1 mM to induce protein expression for 16-18 hours; for protein purification, cells are broken using a high-pressure homogenizer (JNBIO) and then purified using an AKTA protein purification system equipped with a nickel column.

[0042] Then, the purified mutant protein was subjected to in vitro enzyme activity assay. Specifically, the enzyme activity of the xPK mutant catalyzing xylulose 5-phosphate (X5P) or fructose 6-phosphate (F6P) was first evaluated by enzyme-linked ultraviolet spectrophotometry and endpoint colorimetry. During the operation, each 75 μL reaction system included 30 mmol KH2PO4, 5 mmol MgCl2 (pH = 7.5), 120 mmol ribose-5-phosphate (R5P) or F6P and 1 mmol cofactor TPP. When measuring the enzyme activity of xPK for X5P, it is necessary to add more than 2U of Rpe and Rpi, and use R5P as a substrate. The enzyme concentration was determined using Bradford assay reagent (Pierce), and bovine serum albumin (Bio-Rad) was used as a standard curve for determining protein concentration.

[0043] For the enzyme activity assay with F6P as substrate, after adding a certain amount of xPK mutant protein, the reaction was allowed to proceed at 37°C for 15 minutes. Subsequently, 75 μL of the reaction mixture was mixed with 75 μL of NH2OH-HCl (2 M, pH 6.5) at room temperature to stop the reaction. After 10 minutes, 50 μL of Cl3CCOOH (0.92 M), HCl (4 M) and FeCl3*6H2O (0.185 M in 0.1 M HCl) were added. Then, the mixture was centrifuged at 2300 g for 5 minutes, and 200 μL of the supernatant was measured at 505 nm using a spectrophotometer. Quantitative analysis results were obtained using lithium acetyl phosphate standard solution.

[0044] For enzyme activity assay using R5P as substrate, appropriate amounts of Rpe and Rpi were first added, followed by incubation at 37° C. for 10 minutes. Then, a certain amount of xPK mutant protein was added, and the subsequent steps were the same as described above.

[0045] Based on the effectiveness of the primary screening mutants for X5P and / or F6P determined above, secondary screening is performed on the primary screening mutants. After the above two screenings, the following mutants were obtained, namely: V705I, P235A, L504M, Q259L, E223K, E229A, D263A, E262A, H546A, Q316A, I210A, Y500A, F503A, N547A, H315A and H55A; the above mutants were named according to the amino acid mutations at specific positions in the sequence as shown in SEQ ID NO: 1, for example, V705I means that the valine (V) at position 705 of the sequence as shown in SEQ ID NO: 1 is replaced by alanine (A), E229A means that the glutamic acid (E) at position 229 of the sequence as shown in SEQ ID NO: 1 is replaced by alanine (A), and H546A means that the histidine (H) at position 546 of the sequence as shown in SEQ ID NO: 1 is replaced by alanine (A).

[0046] The fold changes of the in vitro enzyme activities of mutants V705I, P235A, L504M, Q259L, and E223K for X5P and F6P relative to wild-type xPK are shown in Figure 1 As shown, the fold changes of the in vitro enzyme activities of mutants E229A, D263A and E262A for X5P and F6P relative to wild-type xPK are shown in Figure 2 The fold changes of the in vitro enzyme activities of mutants H546A, Q316A, I210A, Y500A, F503A, N547A, H315A and H55A for X5P and F6P relative to wild-type xPK are shown in Figure 3 shown.

[0047] according to Figure 1-Figure 3 Based on the results of in vitro enzyme activity assays, the inventors selected three mutant proteins with significantly improved enzyme activity compared with the wild-type xPK, namely V705I, E229A and H546A. The results of the specific enzyme activity assays of these three xPK mutants and the wild-type xPK for catalyzing F6P and X5P are shown in Table 1 below.

[0048] Table 1

[0049] name F6P specific enzyme activity X5P specific enzyme activity xP 0.064±0.001 0.899±0.023 xPK-1 0.186±0.025 0.881±0.047 xPK-2 0.167±0.101 0.921±0.014 xPK-3 0.205±0.097 0.895±0.009

[0050] because Figure 1-Figure 3 As shown in Table 1, the catalytic ability of the three xPK mutants for F6P is several times higher than that of the wild-type xPK, and at the same time, their catalytic ability for X5P can maintain a level comparable to that of the wild-type xPK. This result shows that the three mutant proteins have high enzyme activity for both X5P and F6P, and their comprehensive enzyme activity is significantly higher than that of the wild-type xPK.

[0051] Example 2: Phosphoketolase mutants can improve the production efficiency of 3-hydroxypropionic acid

[0052] xPK is generally used in production applications using acetyl-CoA as a precursor. According to the enzyme activity assay results of Example 1, it is expected that the three mutants of the present invention can effectively increase the supply level of acetyl-CoA. To further verify this effect, the production of 3-hydroxypropionic acid with a shorter pathway is further used for verification.

[0053] In this example, a specifically modified Saccharomyces cerevisiae strain CEN.PK 113-11C (whose genotype is ACC1p::TEF1p MCRC pM2M XI3::(TDH3p-xPK-ADH1t-TEF1-PTA-PGI1t)Med2*432YMth1Δ231, which was constructed according to the corresponding method described in the following literature: Ning Qin et al., Increased CO2 fixation enables high carbon-yield production of 3-hydroxypropionic acid in yeast, Nature Communications, https: / / doi.org / 10.1038 / s41467-024-45557-9) was used as a production verification strain for 3-hydroxypropionic acid.

[0054] First, Med2*432Y and Mth1Δ231 were introduced into the Saccharomyces cerevisiae strain CEN.PK 113-11C to inhibit the glycolytic pathway, creating an environment that more effectively reflects the effects of the mutations; second, a strong promoter was used to replace the native promoter of ACC1 to increase its expression, which is key to catalyzing the conversion of acetyl-CoA to 3-HP. Finally, a plasmid expressing the C- and N-termini of the MCR gene was introduced, which was already able to produce 3-HP.

[0055] The wild-type xPK enzyme and the recombinant expression plasmids of the three xPK mutants screened in Example 1 were respectively introduced into the above-mentioned Saccharomyces cerevisiae to integrate their nucleic acid coding sequences into the genome of Saccharomyces cerevisiae, and the obtained strains were named QJH2, QJH3, QJH4, and QJH5, respectively; at the same time, a Saccharomyces cerevisiae strain without any expression plasmid was set as a negative control, which was named QJH1. After 72 hours of fermentation, the content of 3-hydroxypropionic acid in the fermentation broth was determined.

[0056] The detection of 3-hydroxypropionic acid adopts high performance liquid chromatography (HPLC) at a detection temperature of 65°C; the high performance liquid chromatography system used is produced by Shimadzu Corporation of Japan and is connected to an Aminex HPX-87H chromatographic column produced by Bio-Rad Corporation of the United States; the specific detection conditions include: using 0.5mM H2SO4 as the mobile phase, a flow rate of 0.4mL / min, and a total analysis time of 36 minutes.

[0057] The results are as follows Figure 4 As shown; Figure 4 The results showed that compared with the yeast strain QJH1 without any expression plasmid, the production of 3-hydroxypropionic acid in the QJH2 strain integrated with the wild-type xPK enzyme was significantly improved (increased by about 80%, P=0.00262); compared with the QJH2 strain integrated with the wild-type xPK enzyme, the production of 3-hydroxypropionic acid in the recombinant yeast strains QJH3, QJH4, and QJH5 expressing the three xPK mutants was significantly improved (increased by about 46%, P values ​​were 0.000872, 0.000354, and 0.000764, respectively).

[0058] The above results show that the xPK mutant of the present invention can significantly increase the supply of acetyl-CoA in Saccharomyces cerevisiae compared with its wild-type enzyme (i.e., has significantly higher enzyme activity), thereby significantly increasing the yield of 3-hydroxypropionic acid, and has high application potential and value.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

[0060] Sequences covered in this article:

[0061] SEQ ID NO:1

[0062] MADFDSKEYLELVDKWWRATNYLSAGMIFLKSNPLFSVTNTPIKAEDVKVKPIGHWGTISGQTFLYAHANRLINKYGLNMFYVGGPGHGGQVMVTNAYLDGAYTEDYPEITQDIEGMSHLFKRFSFPGGIGSHMTAQTPGSLHEGGELGYSLSHAFGAVLDNPDQVAFAVVGDGEAETGPSMASWHSIKFLNAKNDGAVLPVLDLNGFKISNPTIFSRMSDEEITKFFEGLGYSPRFIENDDIHDYATYHQLAANILDQAIEDIQAIQNDARENGKYQDGEIPAWPVIIARLPKGWGGPTHDASNNPIENSFRAHQVPLPLEQHDLATLPEFEDWMNSYKPEELFNADGSLKDELKAIAPKGDKRMSANPITNGGADRSDLKLPNWREFANDINDDTRGKEFADSKRNMDMATLSNYLGAVSQLNPTRFRFFGPDETMSNRLWGLFNVTPRQWMEEIKEPQDQLLSPTGRIIDSQLSEHQAEGWLEGYTLTGRVGIFASYESFLRVVDTMVTQHFKWLRHASEQAWRNDYPSLNLIATSTAFQQDHNGYTHQDPGMLTHLAEKKSNFIREYLPADGNSLLAVQERAFSERHKVNLLIASKQPRQQWFTVEEAEVLANEGLKIIDWASTAPSSDVDITFASAGTEPTIETLAALWLINQAFPDVKFRYVNVVELLRLQKKSEPNMNDERELSAEEFNKYFQADTPVIFGFHAYENLIESFFFERKFTGDVYVHGYREDGDITTTYDMRVYSHLDRFHQAKEAAEILSANGKIDQAAADTFIAKMDDTLAKHFQVTRNEGRDIEEFTDWTWSPLK

[0063] SEQ ID NO:2

[0064] MADFDSKEYLELVDKWWRATNYLSAGMIFLKSNPLFSVTNTPIKAEDVKVKPIGHWGTISGQTFLYAHANRLINKYGLNMFYVGGPGHGGQVMVTNAYLDGAYTEDYPEITQDIEGMSHLFKRFSFPGGIGSHMTAQTPGSLHEGGELGYSLSHAFGAVLDNPDQVAFAVVGDGEAETGPSMASWHSIKFLNAKNDGAVLPVLDLNGFKISNPTIFSRMSDEEITKFFEGLGYSPRFIENDDIHDYATYHQLAANILDQAIEDIQAIQNDARENGKYQDGEIPAWPVIIARLPKGWGGPTHDASNNPIENSFRAHQVPLPLEQHDLATLPEFEDWMNSYKPEELFNADGSLKDELKAIAPKGDKRMSANPITNGGADRSDLKLPNWREFANDINDDTRGKEFADSKRNMDMATLSNYLGAVSQLNPTRFRFFGPDETMSNRLWGLFNVTPRQWMEEIKEPQDQLLSPTGRIIDSQLSEHQAEGWLEGYTLTGRVGIFASYESFLRVVDTMVTQHFKWLRHASEQAWRNDYPSLNLIATSTAFQQDHNGYTHQDPGMLTHLAEKKSNFIREYLPADGNSLLAVQERAFSERHKVNLLIASKQPRQQWFTVEEAEVLANEGLKIIDWASTAPSSDVDITFASAGTEPTIETLAALWLINQAFPDVKFRYVNVVELLRLQKKSEPNMNDERELSAEEFNKYFQADTPIIFGFHAYENLIESFFFERKFTGDVYVHGYREDGDITTTYDMRVYSHLDRFHQAKEAAEILSANGKIDQAAADTFIAKMDDTLAKHFQVTRNEGRDIEEFTDWTWSPLK

[0065] SEQ ID NO:3

[0066] MADFDSKEYLELVDKWWRATNYLSAGMIFLKSNPLFSVTNTPIKAEDVKVKPIGHWGTISGQTFLYAHANRLINKYGLNMFYVGGPGHGGQVMVTNAYLDGAYTEDYPEITQDIEGMSHLFKRFSFPGGIGSHMTAQTPGSLHEGGELGYSLSHAFGAVLDNPDQVAFAVVGDGEAETGPSMASWHSIKFLNAKNDGAVLPVLDLNGFKISNPTIFSRMSDEEITKFFAGLGYSPRFIENDDIHDYATYHQLAANILDQAIEDIQAIQNDARENGKYQDGEIPAWPVIIARLPKGWGGPTHDASNNPIENSFRAHQVPLPLEQHDLATLPEFEDWMNSYKPEELFNADGSLKDELKAIAPKGDKRMSANPITNGGADRSDLKLPNWREFANDINDDTRGKEFADSKRNMDMATLSNYLGAVSQLNPTRFRFFGPDETMSNRLWGLFNVTPRQWMEEIKEPQDQLLSPTGRIIDSQLSEHQAEGWLEGYTLTGRVGIFASYESFLRVVDTMVTQHFKWLRHASEQAWRNDYPSLNLIATSTAFQQDHNGYTHQDPGMLTHLAEKKSNFIREYLPADGNSLLAVQERAFSERHKVNLLIASKQPRQQWFTVEEAEVLANEGLKIIDWASTAPSSDVDITFASAGTEPTIETLAALWLINQAFPDVKFRYVNVVELLRLQKKSEPNMNDERELSAEEFNKYFQADTPVIFGFHAYENLIESFFFERKFTGDVYVHGYREDGDITTTYDMRVYSHLDRFHQAKEAAEILSANGKIDQAAADTFIAKMDDTLAKHFQVTRNEGRDIEEFTDWTWSPLK

[0067] SEQ ID NO:4

[0068] MADFDSKEYLELVDKWWRATNYLSAGMIFLKSNPLFSVTNTPIKAEDVKVKPIGHWGTISGQTFLYAHANRLINKYGLNMFYVGGPGHGGQVMVTNAYLDGAYTEDYPEITQDIEGMSHLFKRFSFPGGIGSHMTAQTPGSLHEGGELGYSLSHAFGAVLDNPDQVAFAVVGDGEAETGPSMASWHSIKFLNAKNDGAVLPVLDLNGFKISNPTIFSRMSDEEITKFFEGLGYSPRFIENDDIHDYATYHQLAANILDQAIEDIQAIQNDARENGKYQDGEIPAWPVIIARLPKGWGGPTHDASNNPIENSFRAHQVPLPLEQHDLATLPEFEDWMNSYKPEELFNADGSLKDELKAIAPKGDKRMSANPITNGGADRSDLKLPNWREFANDINDDTRGKEFADSKRNMDMATLSNYLGAVSQLNPTRFRFFGPDETMSNRLWGLFNVTPRQWMEEIKEPQDQLLSPTGRIIDSQLSEHQAEGWLEGYTLTGRVGIFASYESFLRVVDTMVTQHFKWLRHASEQAWRNDYPSLNLIATSTAFQQDANGYTHQDPGMLTHLAEKKSNFIREYLPADGNSLLAVQERAFSERHKVNLLIASKQPRQQWFTVEEAEVLANEGLKIIDWASTAPSSDVDITFASAGTEPTIETLAALWLINQAFPDVKFRYVNVVELLRLQKKSEPNMNDERELSAEEFNKYFQADTPVIFGFHAYENLIESFFFERKFTGDVYVHGYREDGDITTTYDMRVYSHLDRFHQAKEAAEILSANGKIDQAAADTFIAKMDDTLAKHFQVTRNEGRDIEEFTDWTWSPLK。

Claims

1. A phosphoketolase mutant, characterized in that The phosphoketolase mutant has an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:

4.

2. A fusion protein, which is a protein obtained by fusing the phosphoketolase mutant according to claim 1 with a protein tag.

3. An enzyme agent or enzyme composition, comprising the phosphoketolase mutant according to claim 1 or the fusion protein according to claim 2.

4. A polynucleotide encoding the phosphoketolase mutant according to claim 1, or encoding the fusion protein according to claim 2.

5. A nucleic acid construct, a recombinant vector, a recombinant microorganism or an in vitro recombinant cell comprising the polynucleotide according to claim 4; Preferably, the recombinant microorganism is a recombinant yeast strain, more preferably a recombinant Saccharomyces cerevisiae or a recombinant Pichia pastoris strain.

6. Use of the phosphoketolase mutant according to claim 1, the fusion protein according to claim 2, the enzyme agent or enzyme composition according to claim 3, the polynucleotide according to claim 4 and / or the nucleic acid construct, recombinant vector, recombinant microorganism or in vitro recombinant cell according to claim 5 in producing acetyl phosphate, acetyl-CoA and / or 3-hydroxypropionic acid using ketose as a reaction substrate.

7. The use according to claim 6, characterized in that: The ketose is selected from: xylulose 5-phosphate, fructose 6-phosphate, sedulose 7-phosphate and / or erythrose 4-phosphate, preferably selected from: xylulose 5-phosphate and / or fructose 6-phosphate.

8. A method for producing acetyl phosphate, acetyl coenzyme A and / or 3-hydroxypropionic acid, characterized in that: The method uses the phosphoketolase mutant according to claim 1, the fusion protein according to claim 2, the enzyme agent or enzyme composition according to claim 3, the polynucleotide according to claim 4 and / or the nucleic acid construct, recombinant vector, recombinant microorganism or in vitro recombinant cell according to claim 5, and uses ketose as a reaction substrate to produce acetyl phosphate, acetyl coenzyme A and / or 3-hydroxypropionic acid.

9. The method according to claim 8, characterized in that The ketose is selected from: xylulose 5-phosphate, fructose 6-phosphate, sedulose 7-phosphate and / or erythrose 4-phosphate, preferably selected from: xylulose 5-phosphate and / or fructose 6-phosphate.

10. The method according to claim 8 or 9, characterized in that: The method comprises: in the presence of the phosphoketolase mutant according to claim 1 or the fusion protein according to claim 2 or the enzyme agent or enzyme composition according to claim 3 or the recombinant microorganism or in vitro recombinant cell according to claim 5, ketose is catalyzed to generate acetyl phosphate, the obtained acetyl phosphate is optionally further converted to generate acetyl-CoA, and the acetyl-CoA is optionally further converted to generate 3-hydroxypropionic acid; Preferably, the acetyl phosphate is converted into acetyl-CoA under the action of phosphate acetyltransferase; Preferably, the acetyl-CoA is converted into 3-hydroxypropionic acid under the action of acetyl-CoA carboxylase or malonyl-CoA reductase.

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