Phosphoketolase mutants and their use in the production of 3-hydroxypropionic acid

Abstract

The present application provides phosphoketolase mutants, related products thereof and applications thereof in production of acetyl phosphate, acetyl-CoA and / or 3-hydroxypropionic acid. The phosphoketolase mutants provided by the present application have high catalytic capacity for both six-carbon ketose and five-carbon ketose, and can catalyze the conversion of six-carbon ketose and five-carbon ketose into acetyl phosphate with high efficiency, and then efficiently generate subsequent products acetyl-CoA and 3-hydroxypropionic acid, thereby having great application potential in production of acetyl-CoA and products (such as 3-hydroxypropionic acid) taking acetyl-CoA as a precursor.

Description

Technical Field

[0001] The present 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 in order 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 brewer's yeast 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 carbon loss problem 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), ultimately achieving 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 its 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 program is a new strategy for the effective utilization of PK enzymes. This strategy 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, various techniques have been applied to investigate the substrate specificity of enzymes. Given that the PK pathway generates 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 developing 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-mentioned 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 with 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 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 or the fusion protein as described in the second aspect.

[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 recombinant Saccharomyces cerevisiae or recombinant Pichia pastoris, preferably recombinant Saccharomyces cerevisiae.

[0018] In a sixth aspect, the present invention provides the use of the phosphoketolase mutant described in the first aspect, the fusion protein described in the second aspect, the enzyme or enzyme composition described in the third aspect, the polynucleotide described in the fourth aspect and / or the nucleic acid construct, recombinant vector, recombinant microorganism or in vitro recombinant cell 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 a seventh aspect, the present invention provides a method for producing acetyl phosphate, acetyl-CoA and / or 3-hydroxypropionic acid, which uses the phosphoketolase mutant described in the first aspect, the fusion protein described in the second aspect, the enzyme or enzyme composition described in the third aspect, the polynucleotide described in the fourth aspect and / or the nucleic acid construct, recombinant vector, recombinant microorganism or in vitro recombinant cell described in the fifth aspect, and uses 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, the fusion protein described in the second aspect, the enzyme agent or enzyme composition described in the third aspect, or the recombinant microorganism or in vitro recombinant cell described in the fifth aspect, ketose is catalyzed to produce acetyl phosphate, the obtained acetyl phosphate is optionally further converted to produce acetyl-CoA, and the acetyl-CoA is optionally further converted to produce 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 extensive experimental screening. The phosphoketolase mutant of the present invention has broad substrate specificity. In particular, it can catalyze the conversion of six-carbon ketoses (e.g., fructose-6-phosphate) into acetyl phosphate with a much higher efficiency than the wild-type phosphoketolase from which it is derived, thereby efficiently generating the subsequent products acetyl-CoA and 3-hydroxypropionic acid, while not affecting the catalytic ability for five-carbon ketoses (e.g., 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 for xylulose-5-phosphate is comparable to that of the wild-type phosphoketolase (i.e., both are stronger), while its catalytic ability for six-carbon ketoses (e.g., 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 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 illustrated by the accompanying figures, and these exemplary illustrations do not limit the embodiments. The word "exemplary" is used herein to mean "serving as an example, example, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

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

[0031] Figure 2 The fold changes of enzyme activities of phosphoketolase mutants E229A, D263A, and E262A towards fructose-6-phosphate (F6P) and xylulose-5-phosphate (X5P) compared with wild-type phosphoketolase (WT) are shown;

[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 towards fructose-6-phosphate (F6P) and xylulose-5-phosphate (X5P) compared to the wild-type phosphoketolase (WT);

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

[0034] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0035] Unless explicitly defined 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 chemical reaction. In this context, it 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 with reference to the 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 enterocolitica phosphoketolase (abbreviated as xPK, whose amino acid sequence is shown in SEQ ID NO: 1), a series of xPK mutants were designed by making amino acid substitutions at specific sites based on the amino acid sequence shown in SEQ ID NO: 1; then, the encoding nucleic acids of the xPK mutants were codon-optimized to enable their expression in yeast and chemically synthesized.

[0040] It is known in the art that establishing a linear relationship between mutation screening and growth rate based on growth rate and colony size can be used to simply and effectively perform preliminary screening of mutations. In Saccharomyces cerevisiae, cell growth rate can be partially restored by introducing different copies of the enzyme phosphoketolase (PK) when the glycolytic pathway (EMP) is blocked. Therefore, to achieve growth-coupled screening, we selected the Saccharomyces cerevisiae strain CEN.PK 113-11C (available from the following reference: 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 to construct strain QJ1 (whose genotype is pfk1Δpfk2ΔXI3::(TEF1-PTA-PGI1t)his3Δ1::(TDH3p-Transhydrogenase-HMG1t)gpp1Δgpp2Δ) for mutant library construction and mutant screening. The codon-optimized nucleic acid coding sequences of the designed xPK mutants were constructed into expression plasmids, and the expression plasmids were transformed into strain QJ1 to construct the 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 clone size and the results were summarized. All possible mutants were then subjected to in vitro protein expression and purification. The plasmid 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 protocol was as follows: a single colony was inoculated into 4 ml of Luria-Bertani (LB) medium containing ampicillin; then, the culture was shaken at 220 rpm at 37°C overnight; then, the bacterial solution was transferred to 800 ml of Luria-Bertani (LB) medium containing ampicillin and cultured under the same conditions until the OD600 reached 0.6-0.8; then, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM to induce protein expression for 16-18 hours; for protein purification, the cells were disrupted 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 an in vitro enzyme activity assay. Specifically, the enzyme activity of the xPK mutant in catalyzing xylulose 5-phosphate (X5P) or fructose 6-phosphate (F6P) was first evaluated by enzyme-linked ultraviolet spectrophotometry and end-point colorimetry. During the operation, each 75 μL reaction system included 30 mM KH2PO4, 5 mM MgCl2 (pH = 7.5), 120 mM ribose-5-phosphate (R5P) or F6P and 1 mM cofactor TPP. When measuring the enzyme activity of xPK for X5P, it is necessary to add more than 2 U 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 enzyme activity assays using F6P as a substrate, after adding a certain amount of xPK mutant protein, the reaction was incubated 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. The mixture was then centrifuged at 2300 g for 5 minutes, and 200 μL of the supernatant was measured spectrophotometrically at 505 nm. Quantitative analysis results were obtained using a lithium acetyl phosphate standard solution.

[0044] For enzyme activity assays using R5P as a substrate, first add appropriate amounts of Rpe and Rpi, followed by incubation at 37°C for 10 minutes. Then, add a certain amount of xPK mutant protein, and the subsequent steps are 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: 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 2 Figure 1 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 in vitro enzymatic activity assays, the inventors selected three mutant proteins: V705I, E229A, and H546A, which exhibited significantly enhanced enzymatic activity compared to wild-type xPK. The specific enzymatic activity assays of these three xPK mutants and wild-type xPK for 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 activity of these three xPK mutants for F6P was several times higher than that of wild-type xPK. Meanwhile, their catalytic activity for X5P remained comparable to that of wild-type xPK. These results indicate that these three mutant proteins possess high enzymatic activity for both X5P and F6P, and their combined enzymatic activity is significantly higher than that of 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. Based on the enzyme activity assay results in 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, which has a shorter pathway, was further verified.

[0053] In this example, a specifically modified Saccharomyces cerevisiae strain CEN.PK 113-11C (genotype: ACC1p::TEF1p MCRC pM2M XI3::(TDH3p-xPK-ADH1t-TEF1-PTA-PGI1t)Med2*432YMth1Δ231, 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 3-hydroxypropionic acid production verification strain.

[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 stronger promoter was used to replace the native promoter of ACC1, which is key for catalyzing the conversion of acetyl-CoA to 3-HP, to enhance its expression. Finally, a plasmid expressing the C- and N-termini of the MCR gene, which is already capable of producing 3-HP, was introduced.

[0055] Recombinant expression plasmids for the wild-type xPK enzyme and the three xPK mutants screened in Example 1 were introduced into the aforementioned Saccharomyces cerevisiae to integrate their nucleic acid coding sequences into the Saccharomyces cerevisiae genome. The resulting strains were named QJH2, QJH3, QJH4, and QJH5, respectively. A Saccharomyces cerevisiae strain without any expression plasmids was also used as a negative control, designated QJH1. After 72 hours of fermentation, the 3-hydroxypropionic acid content in the fermentation broth was measured.

[0056] 3-Hydroxypropionic acid was detected by high performance liquid chromatography (HPLC) at a temperature of 65°C. The HPLC system used was manufactured by Shimadzu Corporation of Japan and connected to an Aminex HPX-87H chromatographic column manufactured by Bio-Rad Corporation of the United States. Specific detection conditions included using 0.5 mM H2SO4 as the mobile phase, a flow rate of 0.4 mL / 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 indicate that the xPK mutant of the present invention can significantly increase the supply of acetyl-CoA in Saccharomyces cerevisiae compared to its wild-type enzyme (i.e., has significantly higher enzyme activity), thereby significantly increasing the production 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 various 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 amino acid sequence of the phosphoketolase mutant is 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, recombinant vector or recombinant microorganism comprising the polynucleotide according to claim 4.

6. The nucleic acid construct, recombinant vector or recombinant microorganism according to claim 5, characterized in that: The recombinant microorganism is a recombinant yeast strain.

7. The nucleic acid construct, recombinant vector or recombinant microorganism according to claim 6, characterized in that: The recombinant yeast strain is a recombinant Saccharomyces cerevisiae or a recombinant Pichia pastoris strain.

8. 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 or recombinant microorganism according to any one of claims 5 to 7 in producing acetyl phosphate, acetyl-CoA and / or 3-hydroxypropionic acid using ketose as a reaction substrate.

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

10. The use according to claim 9, characterized in that The ketose is xylulose-5-phosphate and / or fructose-6-phosphate.

11. A method for producing acetyl phosphate, acetyl-CoA 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 or enzyme composition according to claim 3, the polynucleotide according to claim 4 and / or the nucleic acid construct, recombinant vector or recombinant microorganism according to any one of claims 5 to 7, and uses ketose as a reaction substrate to produce acetyl phosphate, acetyl-CoA and / or 3-hydroxypropionic acid.

12. The method according to claim 11, characterized in that The ketose is selected from the group consisting of: xylulose 5-phosphate, fructose 6-phosphate, sedulose 7-phosphate and / or erythrose 4-phosphate.

13. The method according to claim 12, characterized in that The ketose is xylulose-5-phosphate and / or fructose-6-phosphate.

14. The method according to any one of claims 11 to 13, characterized in that: The method comprises: in the presence 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, or the recombinant microorganism according to any one of claims 5 to 7, catalyzing the reaction of ketose to generate acetyl phosphate.

15. The method according to claim 14, characterized in that The resulting acetyl phosphate is converted into acetyl-CoA by phosphate acetyltransferase.

16. The method according to claim 15, characterized in that The obtained acetyl-CoA is converted into 3-hydroxypropionic acid under the action of acetyl-CoA carboxylase or malonyl-CoA reductase.

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