Recombinant bacteria for producing D-psicose by fermentation and preparation method thereof

Abstract

The present application discloses a recombinant bacterium for producing D-psicose by fermentation and a preparation method thereof, and belongs to the field of food chemical industry. The technical problem to be solved by the present application is: how to obtain a recombinant bacterium that uses methanol as a raw material for bacterial growth and glucose as a raw material to synthesize D-psicose. By subjecting the recipient bacteria to a purposeful transformation, the microbial strain is provided with the ability to accumulate microbial biomass using methanol and synthesize D-psicose using D-glucose, while preventing the use of D-glucose for microbial growth so that D-glucose is completely converted into D-psicose, thereby improving substrate utilization efficiency. The recombinant polymorphic Hansen yeast provided in the present application can realize the use of methanol and other compounds as raw materials for microbial growth, and after the microbial growth reaches a certain biomass, D-psicose is synthesized using D-glucose as a raw material. And then establish a corresponding technical route for synthesizing D-psicose.

Description

Technical Field

[0001] The present application belongs to the field of genetic engineering technology, and specifically relates to a recombinant bacterium for producing D-psicose by fermentation and a preparation method thereof. Background Art

[0002] D-allulose is a safe and healthy rare monosaccharide that can be widely used in the food, beverage, medicine and other industries. As a low-calorie sweetener, allulose is 70% as sweet as sucrose, but has only 0.3% of the energy of sucrose. D-allulose not only has moderate sweetness and extremely low calories, but also has multiple pharmacological effects such as lowering blood sugar response, reducing liver fat production, maintaining weight, anti-inflammatory, neuroprotective and immunosuppressive. Because of its special nutritional and biological functions, its preparation method has received more and more attention from researchers. The content of D-allulose in nature is extremely small, and it is generally found in foods such as wheat and fruit. Therefore, it cannot be prepared by natural extraction.

[0003] Traditional methods for producing D-psicose include chemical and biological methods, with chemical synthesis being the main method used initially. Bilik et al. found that in an acidic aqueous solution, D-fructose can be converted into D-psicose under the catalytic action of molybdate ions. In 1997, Donald et al. prepared D-psicose by chemical synthesis of 1,2:4,5-di-O-isopropyl-β-D-pyranose. In addition, D-psicose can also be synthesized by boiling ethanol and triethylamine. With in-depth research, it was found that chemical synthesis has complex purification steps, produces chemical waste and worthless by-products, and the sweetness of chemically synthesized sweeteners is often not pure enough, so they are not widely used.

[0004] The biosynthesis method uses natural raw materials that exist in large quantities in nature and uses biological enzymes as catalysts to prepare allulose, which is not only conducive to reducing the cost of industrial production, but also in line with the current green and environmentally friendly production principles. At present, the main method is the biotransformation method using enzymes as catalysts. Taking isomerase as an example, the thermal stability of the identified wild-type enzymes is generally low. In order to meet the needs of industrial applications, the directional mining, thermal stability analysis and modification of isomerases with different properties have begun to be widely studied; at the same time, during use, the preservation of enzymes is an extremely important link. Being able to extend the storage life of enzymes and maintain stability is the basis for their commercial application. At present, the biocatalysts that have been discovered and characterized for the biosynthesis of D-psicose are D-psicose-3-epimerase (DPEase) and D-tagatose-3-epimerase, and in most cases, D-fructose is selected to produce D-psicose through the catalytic conversion of these two enzymes.

[0005] Japan is the first country in the world to research and develop allulose. In 2012, Matsutani Chemical Industry of Japan has sold rare sugar syrup with a certain proportion of D-allulose nationwide, which is favored by consumers; in 2015, South Korea's CJ cooperated with Anderson of the United States to launch the AllSweet brand, mainly selling raw materials such as syrup and powdered sugar containing a certain proportion of D-allulose to North America and other places. At the same time, British Tate & Lyle Dolcia Prima low-calorie allulose syrup entered the market. Domestic research on allulose started late, and Jiangnan University was one of the earliest scientific research institutions to start research. Its research covers gene mining and strain development, enzyme immobilization, and separation and purification of D-allulose. In terms of the published technology, the enzymes and enzyme expression effects developed by the Jiangnan University team are not lower than the data published by large foreign companies, and are basically synchronized with the international advanced level. A number of related research and development patents have been applied for. However, the mainstream production method using diastereomerase in the current market has the following defects: (1) the cost of raw materials, sucrose and glucose are relatively high; (2) it requires a complex product separation process and expensive production equipment.

[0006] Methanol is a low-cost, easily available one-carbon organic raw material. Its energy density is higher than that of raw materials such as glucose. It can also provide more reducing power for biosynthesis, thereby improving the conversion rate of biomanufacturing. It is one of the ideal raw materials for biomanufacturing. The preparation of methanol is mainly synthetic. At present, the industry mainly uses carbon monoxide pressure catalytic hydrogenation to synthesize methanol, in which the raw material carbon monoxide can come from processed products such as coal, oil, and natural gas. On the other hand, the use of carbon dioxide hydrogenation coupled with water electrolysis to produce methanol is a potential and efficient methanol synthesis route. As a cheap, easily available, and environmentally friendly renewable carbon-one resource, the resource utilization of carbon dioxide can not only reduce carbon dioxide emissions, but also provide a technical route for green preparation, which is of great significance to green and sustainable development. Establishing a route for synthesizing D-psicose using methanol as a raw material for microbial growth and glucose as a raw material has great economic and social value. The designed synthetic route can efficiently synthesize D-psicose using glucose as a raw material through a UDP-dependent pathway. Summary of the invention

[0007] The technical problem to be solved by the present application is: how to obtain a recombinant bacterium that uses methanol as a raw material for bacterial growth and uses glucose as a raw material to synthesize D-psicose.

[0008] In order to solve the above technical problems, the present application provides a recombinant Hansenula polymorpha, wherein the recombinant Hansenula polymorpha comprises the following characteristics:

[0009] A1), containing a gene encoding hexolose kinase and / or containing a substance that regulates the expression of the hexolose kinase gene or regulates the activity or content of the hexolose kinase;

[0010] A2), containing a gene encoding UDP-glucose pyrophosphorylase and / or containing a substance that regulates the expression of the UDP-glucose pyrophosphorylase gene or regulates the activity or content of UDP-glucose pyrophosphorylase;

[0011] A3), containing a gene encoding NDP-hexose-3-epimerase and / or containing a substance that regulates the expression of the NDP-hexose-3-epimerase gene or regulates the activity or content of NDP-hexose-3-epimerase;

[0012] A4), containing a gene encoding NDP glycohydrolase and / or containing a substance that regulates the expression of the NDP glycohydrolase gene or regulates the activity or content of NDP glycohydrolase;

[0013] A5), containing the gene encoding inorganic pyrophosphatase and / or containing substances that regulate the expression of inorganic pyrophosphatase gene or regulate the activity or content of inorganic pyrophosphatase;

[0014] A6) containing a gene encoding acid phosphatase and / or containing a substance that regulates the expression of the acid phosphatase gene or regulates the activity or content of acid phosphatase;

[0015] A7), does not contain 6-phosphofructokinase 1 gene;

[0016] A8), does not contain 6-phosphofructokinase 2 gene;

[0017] A9) does not contain the 6-phosphate glucose dehydrogenase gene.

[0018] Furthermore, in the recombinant Hansenula polymorpha, the regulation in A1)-A6) can be promotion, upregulation or improvement.

[0019] Furthermore, the recombinant Hansenula polymorpha also has the following characteristics:

[0020] A10) Containing substances that regulate the expression of fructose bisphosphate aldolase gene or the activity or content of fructose bisphosphate aldolase;

[0021] A11), containing substances that regulate the expression of fructose-1,6-bisphosphatase gene or the activity or content of fructose-1,6-bisphosphatase;

[0022] A12) Containing substances that regulate the expression of transketolase genes or regulate the activity or content of transketolase;

[0023] A13) containing substances that regulate the expression of transaldolase genes or regulate the activity or content of transaldolase;

[0024] A14) Substances that regulate the expression of dihydroxyacetone synthase gene or the activity or content of dihydroxyacetone synthase;

[0025] A15) Contains substances that regulate the expression of dihydroxyacetone kinase gene or regulate the activity or content of dihydroxyacetone kinase.

[0026] Furthermore, in the recombinant Hansenula polymorpha, the substances A1)-A6) and A10)-A15) can be promoters, and the promoter can be P MOX , whose nucleotide sequence is SEQ ID No.2.

[0027] The present application also provides a method for preparing the above-mentioned recombinant Hansenula polymorpha, the method comprising:

[0028] M1), introducing the above-mentioned coding gene of hexolose kinase and / or a substance containing a substance regulating the expression of the hexolose kinase gene or regulating the activity or content of the hexolose kinase;

[0029] M2), introducing the above-mentioned UDP-glucose pyrophosphorylase coding gene and / or a substance containing a substance that regulates the expression of the UDP-glucose pyrophosphorylase gene or regulates the activity or content of UDP-glucose pyrophosphorylase;

[0030] M3), introducing the above-mentioned NDP-hexose-3-epimerase encoding gene and / or containing substances that regulate the expression of the NDP-hexose-3-epimerase gene or regulate the activity or content of the NDP-hexose-3-epimerase;

[0031] M4), introducing the above-mentioned NDP glycohydrolase coding gene and / or a substance containing a substance that regulates the expression of the NDP glycohydrolase gene or regulates the activity or content of the NDP glycohydrolase;

[0032] M5), introducing the above-mentioned inorganic pyrophosphatase coding gene and / or containing substances that regulate the expression of inorganic pyrophosphatase gene or regulate the activity or content of inorganic pyrophosphatase;

[0033] M6), introducing the above-mentioned acid phosphatase coding gene and / or a substance containing a substance that regulates the expression of the acid phosphatase gene or regulates the activity or content of the acid phosphatase;

[0034] M7), knocking out the above-mentioned 6-phosphofructokinase 1 gene;

[0035] M8), knocking out the above-mentioned 6-phosphofructokinase 2 gene;

[0036] M9), knocking out the above-mentioned 6-phosphate glucose dehydrogenase gene.

[0037] Furthermore, the method further comprises:

[0038] M10), the wild-type promoter that drives the expression of the fructose bisphosphate aldolase gene was knocked out and replaced with the PMOX ;

[0039] M11), the wild-type promoter that drives the expression of the fructose-1,6-bisphosphatase gene was knocked out and replaced with the P MOX ;

[0040] M12), the wild-type promoter that drives the expression of the transketolase gene is knocked out and replaced with the P MOX ;

[0041] M13), the wild-type promoter that drives the expression of the transaldolase gene is knocked out and replaced with the P MOX ;

[0042] M14), the wild-type promoter that drives the expression of the dihydroxyacetone synthase gene is knocked out and replaced with the P MOX ;

[0043] M7), the wild-type promoter that drives the expression of the dihydroxyacetone kinase gene is knocked out and replaced with the P MOX .

[0044] Furthermore, in the present application, the wild-type promoter for initiating the expression of the fructose bisphosphate aldolase gene is a DNA molecule having a nucleotide sequence of SEQ ID No.1.

[0045] Furthermore, in the present application, the wild-type promoter for initiating the expression of the fructose-1,6-bisphosphatase gene is a DNA molecule having a nucleotide sequence of SEQ ID No.3.

[0046] Furthermore, in the present application, the wild-type promoter for initiating the expression of the above-mentioned transketolase gene is a DNA molecule having a nucleotide sequence of SEQ ID No.4.

[0047] Furthermore, in the present application, the wild-type promoter for initiating the expression of the above-mentioned transaldolase gene is a DNA molecule having a nucleotide sequence of SEQ ID No.5.

[0048] Furthermore, in the present application, the wild-type promoter for initiating the expression of the above-mentioned dihydroxyacetone synthase gene is a DNA molecule having a nucleotide sequence of SEQ ID No.6.

[0049] Furthermore, in the present application, the wild-type promoter for initiating the expression of the above-mentioned dihydroxyacetone kinase gene is a DNA molecule with a nucleotide sequence of SEQ ID No.7.

[0050] Furthermore, in the present application, the ketokinase may be derived from humans (Homo sapiens).

[0051] Further, in the present application, the UDP-glucose pyrophosphorylase may be derived from Arabidopsis thaliana ( Arabidopsis thaliana ).

[0052] Further, in the present application, the NDP-hexose-3-epimerase may be derived from Streptomyces ( Streptomyces sp. .).

[0053] Further, in the present application, the NDP glycohydrolase may be derived from rat ( Norwegian rat ).

[0054] Further, in the present application, the inorganic pyrophosphatase may be derived from Escherichia coli ( E. coli) .

[0055] Further, in the present application, the acid phosphatase may be derived from Aspergillus ( Aspergillus sp. .).

[0056] Furthermore, in the present application, the ketokinase may be any of the following:

[0057] A1-1), a protein having an amino acid sequence of SEQ ID No. 9;

[0058] A1-2), a protein having more than 80% identity with the protein shown in A8-1) and with hexokinokinase after substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in A1-1);

[0059] A1-3), a fusion protein having the function of the hexokinases obtained by connecting a tag to the N-terminus and / or C-terminus of the protein in A1-1) or A1-2).

[0060] Further, in the present application, the UDP-glucose pyrophosphorylase may be any of the following:

[0061] A2-1), a protein having an amino acid sequence of SEQ ID No. 11;

[0062] A2-2), a protein obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in A2-1) that has more than 80% identity with the protein shown in A9-1) and is different from UDP-glucose pyrophosphorylase;

[0063] A2-3), a fusion protein having the function of the UDP-glucose pyrophosphorylase obtained by connecting a tag to the N-terminus and / or C-terminus of the protein in A2-1) or A2-2).

[0064] Further, in the present application, the NDP-hexose-3-epimerase may be any of the following:

[0065] A3-1), a protein having an amino acid sequence of SEQ ID No. 13;

[0066] A protein having more than 80% identity with the protein shown in A8-1) and NDP-hexose-3-epimerase obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in A3-1);

[0067] A3-3), a fusion protein having the function of the NDP-hexose-3-isomerase obtained by connecting a tag to the N-terminus and / or C-terminus of the protein in A3-1) or A3-2).

[0068] Further, in the present application, the NDP glycohydrolase may be any of the following:

[0069] A4-1), a protein having an amino acid sequence of SEQ ID No. 15;

[0070] A4-2), a protein obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in A4-1) having more than 80% identity with the protein shown in A11-1) and having NDP glycohydrolase;

[0071] A4-3), a fusion protein having the function of the NDP glycohydrolase obtained by connecting a tag to the N-terminus and / or C-terminus of the protein in A4-1) or A4-2).

[0072] Furthermore, in the present application, the inorganic pyrophosphatase may be any of the following:

[0073] A5-1), protein with amino acid reference sequence number NP_418647.1 (09-MAR-2022) (NCBI Reference Sequence: NP_418647.1);

[0074] A5-2), a protein obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in A5-1) that has 80% or more identity with the protein shown in A12-1) and is different from inorganic pyrophosphatase;

[0075] A5-3), a fusion protein having the inorganic pyrophosphatase function obtained by connecting a tag to the N-terminus and / or C-terminus of the protein in A5-1) or A5-2).

[0076] Furthermore, in the present application, the acid phosphatase may be any of the following:

[0077] A6-1), a protein having an amino acid sequence of SEQ ID No. 17;

[0078] A6-2), a protein obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in A6-1) that has 80% or more identity with the protein shown in A13-1) and is similar to acid phosphatase;

[0079] A6-3), a fusion protein having the acid phosphatase function obtained by connecting a tag to the N-terminus and / or C-terminus of the protein in A6-1) or A6-2).

[0080] Further, in the present application, the 6-phosphofructokinase 1 is any one of the following:

[0081] A7-1), protein with NCBI Reference Sequence Number XP_013936214.1 (14-SEP-2015);

[0082] A7-2), a protein having more than 80% identity with the protein shown in A7-1) and having 6-phosphofructokinase 1 obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in A7-1);

[0083] A7-3), a fusion protein having the function of 6-phosphofructokinase 1 obtained by connecting a tag to the N-terminus and / or C-terminus of the protein in A7-1) or A7-2).

[0084] Further, in the present application, the 6-phosphofructokinase 2 is any one of the following:

[0085] A8-1), protein with NCBI Reference Sequence Number XP_013933492.1 (14-SEP-2015);

[0086] A8-2), a protein obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in A8-1) that has more than 80% identity with the protein shown in A8-1) and is different from 6-phosphofructokinase 2;

[0087] A8-3), a fusion protein having the function of 6-phosphofructokinase 2 obtained by connecting a tag to the N-terminus and / or C-terminus of the protein in A8-1) or A8-2).

[0088] Furthermore, in the present application, the 6-phosphate glucose dehydrogenase is any one of the following:

[0089] A9-1), protein with NCBI Reference Sequence Number XP_018211730.1 (05-FEB-2020);

[0090] A9-2), a protein obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in A9-1) that has 80% or more identity with the protein shown in A9-1) and is different from 6-phosphate glucose dehydrogenase;

[0091] A9-3), a fusion protein having the function of 6-phosphate glucose dehydrogenase obtained by connecting a tag to the N-terminus and / or C-terminus of the protein in A9-1) or A9-2).

[0092] Furthermore, in the present application, the fructose bisphosphate aldolase is any one of the following:

[0093] A10-1), protein with NCBI Reference Sequence Number XP_013935471.1 (14-SEP-2015);

[0094] A10-2), a protein having more than 80% identity with the protein shown in A10-1) and fructose bisphosphate aldolase obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in A10-1);

[0095] A10-3), a fusion protein having the function of fructose bisphosphate aldolase obtained by connecting a tag to the N-terminus and / or C-terminus of the protein in A10-1) or A10-2);

[0096] Furthermore, in the present application, the fructose-1,6-bisphosphatase is any one of the following:

[0097] A11-1), protein with NCBI Reference Sequence Number XP_013935103.1 (14-SEP-2015);

[0098] A11-2), a protein obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in A11-1) that has more than 80% identity with the protein shown in A11-1) and is different from fructose-1,6-bisphosphatase;

[0099] A11-3), a fusion protein having the fructose-1,6-bisphosphatase function obtained by connecting a tag to the N-terminus and / or C-terminus of the protein in A11-1) or A11-2);

[0100] Furthermore, in the present application, the transketolase is any one of the following:

[0101] A12-1), protein with NCBI Reference Sequence Number XP_013934677.1 (14-SEP-2015);

[0102] A12-2), the amino acid sequence shown in A12-1) obtained by substitution and / or deletion and / or addition of amino acid residues has more than 80% identity with the protein shown in A12-1) and is a protein of transketolase;

[0103] A12-3), a fusion protein having the transketolase function obtained by connecting a tag to the N-terminus and / or C-terminus of the protein in A12-1) or A12-2);

[0104] Further, in the present application, the transaldolase is any one of the following:

[0105] A13-1), protein with NCBI Reference Sequence Number XP_013937091.1 (14-SEP-2015);

[0106] A13-2), the amino acid sequence shown in A13-1) is obtained by substitution and / or deletion and / or addition of amino acid residues, and has more than 80% identity with the protein shown in A13-1) and is a protein of transaldolase;

[0107] A13-3), a fusion protein having the transaldolase function obtained by connecting a tag to the N-terminus and / or C-terminus of the protein in A13-1) or A13-2);

[0108] Further, in the present application, the alcohol oxidase is any one of the following:

[0109] A14-1), protein with NCBI Reference Sequence Number XP_013934137.1 (14-SEP-2015);

[0110] A14-2), a protein having more than 80% identity with the protein shown in A14-1) and alcohol oxidase after substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in A14-1);

[0111] A14-3), a fusion protein having the alcohol oxidase function obtained by connecting a tag to the N-terminus and / or C-terminus of the protein in A14-1) or A14-2);

[0112] Further, in the present application, the dihydroxyacetone synthase is any one of the following:

[0113] A15-1), protein with NCBI Reference Sequence Number XP_013936414.1 (14-SEP-2015);

[0114] A15-2), a protein obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in A15-1) that has more than 80% identity with the protein shown in A15-1) and is different from dihydroxyacetone synthase;

[0115] A15-3), a fusion protein having the dihydroxyacetone synthase function obtained by connecting a tag to the N-terminus and / or C-terminus of the protein in A15-1) or A15-2);

[0116] Further, in the present application, the dihydroxyacetone kinase is any one of the following:

[0117] A16-1), protein with NCBI Reference Sequence Number XP_013934963.1 (14-SEP-2015);

[0118] A16-2), a protein having more than 80% identity with the protein shown in A16-1) and having the same identity as dihydroxyacetone kinase as obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in A16-1);

[0119] A16-3), a fusion protein having the dihydroxyacetone kinase function obtained by connecting a tag to the N-terminus and / or C-terminus of the protein in A16-1) or A16-2);

[0120] Furthermore, in the present application, the coding gene of the hexokinokinase is a DNA molecule whose nucleotide sequence is SEQ ID No. 8;

[0121] Furthermore, in the present application, the coding gene of the UDP-glucose pyrophosphorylase is a DNA molecule having a nucleotide sequence of SEQ ID No. 10;

[0122] Further, in the present application, the coding gene of the NDP-hexose-3-epimerase is a DNA molecule whose nucleotide sequence is SEQ ID No.12;

[0123] Further, in the present application, the coding gene of the NDP glycohydrolase is a DNA molecule whose nucleotide sequence is SEQ ID No. 14;

[0124] Further, in the present application, the inorganic pyrophosphatase encoding gene is a DNA molecule whose nucleotide sequence is the NCBI reference sequence number NC_000913.3 (449652..4449122) (-);

[0125] Further, in the present application, the acid phosphatase encoding gene is a DNA molecule having a nucleotide sequence of SEQ ID No. 16;

[0126] Further, in the present application, the coding gene of 6-phosphofructokinase 1 is a DNA molecule whose nucleotide sequence is reference sequence number XM_014080739.1 (14-SEP-2015);

[0127] Further, in the present application, the coding gene of 6-phosphofructokinase 2 is a DNA molecule whose nucleotide sequence is reference sequence number XM_014078017.1 (14-SEP-2015);

[0128] Furthermore, in the present application, the gene encoding 6-phosphoglucose dehydrogenase is a DNA molecule whose nucleotide sequence is a reference sequence number of XM_018354349.1 (05-FEB-2020).

[0129] Further, in the present application, the gene encoding the fructose bisphosphate aldolase is a DNA molecule whose nucleotide sequence is a reference sequence number of XM_014079996.1 (14-SEP-2015);

[0130] Further, in the present application, the gene encoding the fructose-1,6-bisphosphatase is a DNA molecule whose nucleotide sequence is a reference sequence number of XM_014079628.1 (14-SEP-2015);

[0131] Further, in the present application, the transketolase encoding gene is a DNA molecule whose nucleotide sequence is a reference sequence number of XM_014079202.1 (14-SEP-2015);

[0132] Further, in the present application, the transaldolase encoding gene is a DNA molecule whose nucleotide sequence is a reference sequence number of XM_014081616.1 (14-SEP-2015);

[0133] Further, in the present application, the alcohol oxidase encoding gene is a DNA molecule whose nucleotide sequence is a reference sequence number of XM_014078662.1 (14-SEP-2015);

[0134] Further, in the present application, the coding gene of the dihydroxyacetone synthase is a DNA molecule whose nucleotide sequence is reference sequence number XM_014080939.1 (14-SEP-2015);

[0135] Further, in the present application, the coding gene of the dihydroxyacetone kinase is a DNA molecule whose nucleotide sequence is a reference sequence number of XM_014079488.1 (14-SEP-2015);

[0136] The present application also provides the use of a composition in constructing the above-mentioned recombinant Hansenula polymorpha, wherein the composition contains the above-mentioned P MOX And / or the above-mentioned coding gene for hexoketol kinase and / or the coding gene for UDP-glucose pyrophosphorylase and / or the coding gene for NDP-hexose-3-isomerase and / or the coding gene for NDP sugar hydrolase and / or the coding gene for inorganic pyrophosphatase and / or the coding gene for acid phosphatase and / or a substance for knocking out the above-mentioned 6-phosphofructokinase 1 gene and / or a substance for 6-phosphofructokinase 2 gene and / or a substance for 6-phosphoglucose dehydrogenase gene.

[0137] Furthermore, in the application, the substance may be a substance that knocks out the corresponding gene through homologous recombination.

[0138] In some embodiments of the present application, the substance can be a targeting fragment.

[0139] In some embodiments of the present application, the targeting fragment for knocking out the above-mentioned 6-phosphofructokinase 1 gene is pfkAup-G418-pfkAdown, and its nucleotide sequence is shown in Table 2.

[0140] In some embodiments of the present application, the targeting fragment for knocking out the above-mentioned 6-phosphofructokinase 2 gene is pfkBup-G418-pfkBdown, and its nucleotide sequence is shown in Table 2.

[0141] In some embodiments of the present application, the targeting fragments for knocking out the above-mentioned 6-phosphate glucose dehydrogenase gene are G6PD-up and G6PD-down, and their nucleotide sequences are shown in Table 2.

[0142] The present application also provides the use of the recombinant Hansenula polymorpha in the preparation of D-psicose.

[0143] The present application also provides a bacterial agent, which contains the above-mentioned recombinant Hansenula polymorpha or a culture containing the above-mentioned recombinant Hansenula polymorpha.

[0144] The term "culture" refers to a liquid or solid product (all materials in the culture container) that has a microbial community after artificial inoculation and culture. That is, the product obtained by growing and / or amplifying microorganisms, which can be a biologically pure culture of microorganisms, or contain a certain amount of culture medium, metabolites or other components produced during the culture process.

[0145] The present application also provides a method for preparing D-psicose, which comprises using the above-mentioned recombinant Hansenula polymorpha and / or bacterial agent to prepare D-psicose using D-glucose as a substrate.

[0146] Further, the method includes amplifying (growing) the recombinant Hansenula polymorpha using methanol as a carbon source.

[0147] Furthermore, the recombinant Hansenula polymorpha cannot be amplified (grown) using D-glucose as a carbon source.

[0148] In some embodiments of the present application, the recombinant Hansenula polymorpha is amplified (grown) in a growth medium containing methanol.

[0149] In some embodiments of the present application, the growth medium components and final concentrations are as follows: Na 2 HPO 4 :25 mM, KH 2 PO 4 :25 mM, NH 4 Cl: 50 mM, Na 2 SO 4 :5 mM, MgSO 4 : 2 mM, methanol: 3 g / 100 mL, yeast powder: 0.5 g / 100 mL), trace elements: 50 μM FeCl 3 , 20 μM CaCl 2 , 10 μM MnCl 2 , 10 μM ZnSO 4 , CoCl 2 、NiCl 2 、Na 2 MO 4 、Na 2 SeO 3 and H 3 BO 3 2 μM each; the rest is water, adjusted to pH 8.0.

[0150] In some embodiments of the present application, the recombinant Hansenula polymorpha uses a transformation medium as a reaction substrate to prepare D-psicose. The composition of the transformation medium is as follows: Na 2 HPO 4:25 mM, KH 2 PO 4 :25 mM, NH 4 Cl: 50 mM, D-glucose: 20 g / 100 mL, trace elements: 50 μM FeCl 3 , 20 μM CaCl 2 , 10 μM MnCl 2 , 10 μM ZnSO 4 , CoCl 2 、NiCl 2 、Na 2 MO 4 、Na 2 SeO 3 and H 3 BO 3 2 μM each, the rest was water.

[0151] The method for constructing a recombinant Hansenula polymorpha strain provided in the present application includes 1), 2) and 3):

[0152] 1) Introducing into biological cells the coding genes of related proteins or increasing or up-regulating or promoting the expression of the coding genes of related proteins or increasing or up-regulating or promoting the activity or content of related proteins, wherein the related proteins include: dihydroxyacetone synthase, dihydroxyacetone kinase, fructose bisphosphate aldolase, fructose-1,6-bisphosphatase, transketolase and / or transaldolase.

[0153] 2) Knock out the 6-phosphofructokinase 1 gene, 6-phosphofructokinase 2 gene and / or 6-phosphoglucose dehydrogenase gene in biological cells, and down-regulate, reduce or inhibit their expression.

[0154] 3) Introducing into biological cells a gene encoding hexolose kinase, a gene encoding UDP-glucose pyrophosphorylase, a gene encoding NDP-hexose-3-isomerase, a gene encoding NDP glycohydrolase, a gene encoding inorganic pyrophosphatase and / or a gene encoding acid phosphatase, and / or enhancing or increasing the expression of a gene encoding hexolose kinase, a gene encoding UDP-glucose pyrophosphorylase, a gene encoding NDP-hexose-3-isomerase, a gene encoding NDP glycohydrolase, a gene encoding inorganic pyrophosphatase and / or a gene encoding acid phosphatase.

[0155] In the method described in the present application, method 1) provides or enhances the ability of the microbial strain to utilize methanol to accumulate microbial biomass. Method 3) provides the microorganism with the ability to synthesize D-psicose from D-glucose. Method 2) prevents the ability of utilizing D-glucose for microbial growth and accumulation of microbial biomass, thereby completely converting D-glucose into D-psicose and improving substrate utilization efficiency.

[0156] The beneficial technical effects achieved by this application are as follows:

[0157] The recombinant Hansenula polymorpha provided in the present application can use methanol and other compounds as raw materials for microbial growth, and after the microbial growth reaches a certain biomass, use D-glucose as raw material to synthesize D-psicose. And then establish the corresponding technical route for synthesizing D-psicose. BRIEF DESCRIPTION OF THE DRAWINGS

[0158] Figure 1 For the growth of different strains in different culture media;

[0159] Figure 2 The D-psicose production and conversion rate of different strains. DETAILED DESCRIPTION

[0160] The present application is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present application, not for limiting the scope of the present application. The examples provided below can be used as a guide for further improvements by ordinary technicians in the technical field, and do not constitute a limitation of the present application in any way.

[0161] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0162] Among them, Hansenula polymorpha ( Hansenula polymorpha ) DL01 strain is an American ATCC strain, number 26012;

[0163] Plasmid pPIC9k was a product of Beijing Zhuangmeng Company, catalog number ZK922;

[0164] The yeast genome extraction kit is a product of Tiangen Biochemical Technology Co., Ltd., with the product catalog number DP307;

[0165] High-fidelity TransStart FastPfu DNA polymerase was from Beijing Quanshijin Biotechnology Co., Ltd., product catalog number AP221;

[0166] Escherichia coli DH5α competent cells were from Beijing Quanshijin Biotechnology Co., Ltd., and the product catalog is CD201;

[0167] pPICZA is a product of Beijing Zhuangmeng International Bio-Gene Technology Co., Ltd., with the catalog number ZK923.

[0168] Example 1. Construction of recombinant Hansenula polymorpha engineered strain DL13

[0169] In this example, a basic bacterium DL13 was prepared that can use methanol as a raw material for bacterial growth and can convert D-glucose into D-psicose. The preparation method of the strain is as follows, and the primers used are shown in Table 3.

[0170] (1) Enhancing the expression of fructose bisphosphate aldolase gene by promoter replacement

[0171] From Hansenula polymorpha ( Hansenula polymorpha )DL01, and the promoter P of the fructose bisphosphate aldolase gene (also referred to as FBA gene) in the strain FBA (SEQ ID No.1) was replaced by the strong promoter P MOX (SEQ ID No. 2), and recombinant Hansenula polymorpha DL02 was obtained, and the specific steps are as follows:

[0172] (1-a) Construction of plasmid pPICd1:

[0173] Hansenula polymorpha ( Hansenula polymorpha ) Extraction of genomic DNA from DL01 and PCR amplification of the MOX promoter gene cluster. The genomic DNA of Hansenula polymorpha DL01 was extracted using a yeast genome extraction kit. The extracted genomic total DNA was used as a template, MOX-01F and MOX-01R were used as primers, and the promoter P was amplified using high-fidelity TransStart FastPfu DNA polymerase. MOX The MOX fragment is a DNA molecule with a nucleotide sequence of 3080-7257 positions of the pPICd1 plasmid sequence (5'-3'), wherein the 3080-5079 positions of the pPICd1 plasmid sequence are promoter P MOX The sequence of the moxT terminator sequence is at positions 7075-7257. The target fragment is recovered by agarose gel electrophoresis.

[0174] The CA fragment was artificially synthesized to obtain the pUC57-CA plasmid containing the Cre enzyme gene. The CA fragment is a DNA molecule with a nucleotide sequence of the 1st to 1403rd position of the pPICd1 plasmid sequence (5'-3'), wherein the 1st to 1056th position of the pPICd1 plasmid sequence is the Cre enzyme gene, and the 1144th to 1403rd position is the aox1T terminator sequence. The CA fragment was amplified by PCR using the pUC57-CA plasmid as a template and CA-F and CA-R as primers using high-fidelity TransStart FastPfu DNA polymerase, and the CA fragment was recovered by agarose gel electrophoresis.

[0175] Primers PIC-MOX-01U and PIC-MOX-01D were used to PCR amplify the AG fragment containing the G418 resistance gene with plasmid pPIC9k as a template, and the resistance fragment AG was recovered. The resistance fragment AG is a DNA molecule with a nucleotide sequence of positions 1391-3079 of the pPICd1 plasmid sequence (5'-3'), wherein the pPICd1 plasmid sequence has lox66 sequences at positions 1411-1444, G418 resistance genes at positions 1865-2680, and lox71 sequences at positions 3026-3059.

[0176] The primers PIC-MOX-01F and PIC-MOX-01R were used to amplify the PIC fragment by PCR with the plasmid pPIC9k as the template, and the large fragment PIC of the vector was recovered. The large fragment PIC of the vector is a DNA molecule with the nucleotide sequence of the 7258-11406th position and the 1st-20th position of the pPICd1 plasmid sequence (the pPICd1 plasmid is a circular DNA).

[0177] The MOX fragment, CA fragment, AG fragment and PIC fragment were ligated by Gibson assembly method (Gibson DG, Young L, et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. methods. 2009; 6 (5): 343-345) to obtain ligation products. The ligation products were ligated with CaCl 2 The competent cells of E. coli DH5α were transformed by the method. The cells were evenly spread on LB plates containing ampicillin and cultured at 37°C overnight. The clones were selected and the clones that could amplify the target fragment were identified and sequenced using primers CA-R / MOX-01R. The positive clones were selected to extract the plasmids and the obtained positive plasmids were named pPICd1. The sequence (5'-3') of the pPICd1 plasmid is shown in Table 1.

[0178] Table 1 Sequence of pPICd1 plasmid (5'-3')

[0179]

[0180] (1-b) Construction of plasmid pPICd1-FBA:

[0181] Using FBA-01F and FBA-01R as primers, Hansenula polymorpha ( Hansenula polymorpha)DL01 genomic DNA was used as a template and high-fidelity TransStart FastPfu DNA polymerase (Beijing Quanshijin Biotechnology Co., Ltd., product catalog number AP221) was used to amplify the FBA fragment containing the FBA gene by PCR, and the target fragment was recovered by agarose gel electrophoresis.

[0182] The primers PIH-01F (pPICd1 plasmid 5050-5079) and PIH-01R (pPICd1 plasmid 7075-7097) were used to amplify the PIH fragment by PCR with the plasmid pPICd1 as a template, and the large vector fragment PIH was recovered. The above FBA fragment and the PIH fragment were ligated by the Gibson assembly method to obtain a ligation product. The ligation product was transformed into Escherichia coli DH5α competent cells with reference to the operation of (1-a), and clones were selected. The clones that can amplify the target fragment were identified and sequenced using primers PIH-F1 / FBA-01R, and the positive clones were selected to extract the plasmids. The obtained positive plasmids were named pPICd1-FBA. The structure of pPICd1-FBA is: the fragment between the 5080-7074th positions of the pPICd1 plasmid (the small fragment between the 5080-7074th positions of the pPICd1 plasmid) is replaced by the fructose bisphosphate aldolase gene, and the other nucleotide sequences of the pPICd1 plasmid remain unchanged. The fructose bisphosphate aldolase gene in pPICd1-FBA is expressed by the promoter P MOX (Promoter P MOX The nucleotide sequence is a DNA molecule with the nucleotide sequence of the 3080-5079th position of the pPICd1 plasmid sequence, that is, SEQ ID No. 2) to initiate transcription, and the moxT terminator (the nucleotide sequence is a DNA molecule with the nucleotide sequence of the 7075-7257th position of the pPICd1 plasmid sequence) to terminate transcription, and fructose bisphosphate aldolase can be expressed.

[0183] The reference sequence of fructose bisphosphate aldolase is XP_013935471.1, 14-SEP-2015. The reference sequence of fructose bisphosphate aldolase gene is NC_027863.1 (1128399..1129478), 14-SEP-2015.

[0184] (1-c) Construction of strain DL02:

[0185] The preparation of Hansenula electroporation competent cells was as follows: Hansenula strain DL01 was picked from the YPD medium plate, transferred to 5 mL of liquid YPD medium, and cultured at 37°C overnight. Then 5 mL of overnight bacterial liquid was inoculated into 300 mL of liquid YPD medium and cultured at 37°C until OD 600nmThe culture solution was harvested at 1.2-1.5 (about 6 hours), centrifuged at 3000 g and 4°C for 10 min, and sterilized water was added to wash the precipitate. Then 50 mL of pH 7.5 washing solution was added to resuspend the precipitate, and the culture was cultured at 37°C and 100-200 r / min on a shaker for 15 min to obtain the culture solution. After that, the culture solution was centrifuged at 3000 g and 4°C for 10 min, and the precipitate was resuspended in 200 mL of ice-cold STM solution, and then centrifuged at 3000 g and 4°C for 10 min, and the precipitate was resuspended in 100 mL of ice-cold STM solution, and then centrifuged at 3000 g and 4°C for 10 min. Finally, the precipitate was resuspended in 1 mL of ice-cold STM solution, and 60 μL was dispensed into each tube and frozen at -80°C for later use.

[0186] Among them, the components and preparation of the cleaning solution are as follows: 50 mM phosphate buffer (containing 0.988 g of dipotassium hydrogen phosphate and 0.091 g of potassium dihydrogen phosphate, fixed to 1 L) was dissolved and the pH was adjusted to 7.5, the volume was made up to 200 mL with water, and sterilized at 115 °C for 30 min; the components and preparation of the STM solution are as follows: 92.421 g of sucrose, 1.21 g of Tris, 0.203 g of magnesium chloride, the pH was adjusted to 7.5, the volume was made up to 1 L with water, and sterilized at 115 °C for 30 min.

[0187] The pPICd1-FBA plasmid was amplified using primers FBA-R1 / FBA-01R to obtain the linearized pPICd1-FBA fragment.

[0188] The preparation method and electroporation conditions of Hansenula electroporation competent cells are as follows: 60 μL of electroporation Hansenula DL01 competent cells were mixed with 1-2 μg of linearized pPICd1-FBA fragment and transferred to an ice-precooled 2 mL electroporation cup, placed on ice for 5 minutes, and electroporated using a Gene Pulser Xcell electroporator. The electroporation conditions were: 1500V, 50 mV, 129Ω.

[0189] After electroporation, quickly add 1 mL of ice-cold YPD medium and mix up and down 3 times, then incubate at 37°C for 2 hours, take 100-300 μL and spread on YPD plates containing 0.1 mg / mL G418, and incubate at 37°C for 3-4 days. Pick clones from the plate, transfer to 5 mL of YPD medium, incubate at 37°C overnight, identify with primers PIH-F1 / FBA-01R, and the amplified fragment of 3kb (gene1kb) is a positive clone. After methanol treatment (i.e., the positive colony is cultured in YPD medium containing 2% methanol overnight, then streaked and picked a single clone, the following "methanol treatment" is the same operation), FBA is frozen at -80°C. The strain is named DL02.

[0190] Among them, the amino acid reference sequence number of fructose bisphosphate aldolase is XP_013935471.1 (14-SEP-2015), and the reference sequence number of fructose bisphosphate aldolase gene is XM_014079996.1 (14-SEP-2015).

[0191] (2) Enhancement of fructose-1,6-bisphosphatase gene expression by promoter replacement

[0192] From Hansenula polymorpha ( Hansenula polymorpha )DL02, and the promoter P of the fructose-1,6-bisphosphatase gene (also referred to as Fpb1 gene) in the strain was fpb1 (SEQ ID No.3) was replaced by the strong promoter P MOX (SEQ ID No. 2), and recombinant Hansenula polymorpha DL03 was obtained, and the specific steps are as follows:

[0193] (2-a) Construction of plasmid pPICd1-Fpb1

[0194] Using Fpb1-01F and Fpb1-01R as primers, the expression of Hansenula polymorpha ( Hansenula polymorpha )DL01 genomic DNA was used as a template and the Fpb1 fragment containing the Fpb1 gene was amplified by PCR using high-fidelity TransStart FastPfu DNA polymerase. The target fragment was recovered by agarose gel electrophoresis.

[0195] Using primers PIH-01F and PIH-01R, plasmid pPICd1 was used as a template to PCR amplify the PIH fragment and recover the large vector fragment PIH. The above Fpb1 fragment and the PIH fragment were ligated using the Gibson assembly method to obtain a ligation product. The ligation product was transformed into Escherichia coli DH5α competent cells according to the operation of (1-a). The clones that could amplify the target fragment were identified and sequenced using primers PIH-F1 / Fpb1-01R. The positive clones were selected to extract the plasmids. The obtained positive plasmids were named pPIH-Fpb1. The structure of pPICd1-Fpb1 is as follows: the fragment between positions 5080-7074 of the pPICd1 plasmid (the small fragment between positions 5080-7074 of the pPICd1 plasmid) was replaced with the fructose-1,6-bisphosphatase gene, and the other nucleotide sequences of the pPICd1 plasmid were kept unchanged. The fructose-1,6-bisphosphatase gene in pPICd1-Fpb1 is driven by the promoter P MOX The transcription is initiated and terminated by the moxT terminator, and fructose-1,6-bisphosphatase can be expressed.

[0196] Among them, the amino acid reference sequence number of fructose-1,6-bisphosphatase is XP_013935103.1 (14-SEP-2015), and the reference sequence number of fructose-1,6-bisphosphatase gene is XM_014079628.1 (14-SEP-2015).

[0197] (2-b) Construction of strain DL03

[0198] The pPICd1-Fpb1 plasmid was amplified with primers Fpb1-R1 / Fpb1-01R to obtain the linearized pPIH-Fpb1 plasmid, which was then electroporated into strain DL02. The electroporation process was carried out according to the "Construction of strain DL02" in (1-c). After electroporation, the concentration of G418 in the YPD plate was 0.2 mg / mL. The primers PIH-F1 / Fpb1-01R were used to identify the positive clones with a 3 kb amplified fragment. After methanol treatment, the clones were frozen at -80°C. The strain was named DL03.

[0199] (3) Enhancing the expression of the transketolase gene by promoter replacement

[0200] From Hansenula polymorpha ( Hansenula polymorpha )DL03, the promoter of the transketolase gene (also referred to as Tktl1 gene) in the strain was tktl1 (SEQ ID No.4) was replaced by the strong promoter P MOX (SEQ ID No. 2), and recombinant Hansenula polymorpha DL04 was obtained, and the specific steps are as follows:

[0201] (3-a) Construction of plasmid pPICd1-Tktl1:

[0202] Using Tktl1-01F and Tktl1-01R as primers, the polymorpha Hansenula ( Hansenula polymorpha )DL01 genomic DNA was used as a template and the Tktl1 fragment containing the Tktl1 gene was amplified by PCR using high-fidelity TransStart FastPfu DNA polymerase. The target fragment was recovered by agarose gel electrophoresis.

[0203] Using primers PIH-01F and PIH-01R, plasmid pPICd1 was used as a template to PCR amplify the PIH fragment and recover the large vector fragment PIH. The above Tktl1 fragment and the PIH fragment were ligated using the Gibson assembly method to obtain a ligation product. The ligation product was transformed into Escherichia coli DH5α competent cells with reference to the operation of (1-a). The clones that could amplify the target fragment were identified and sequenced using primers PIH-F1 / Tktl1-01R. The positive clones were selected to extract the plasmids. The obtained positive plasmids were named pPIH-Tktl1. The structure of pPICd1-Tktl1 is as follows: the fragment between positions 5080-7074 of the pPICd1 plasmid (the small fragment between positions 5080-7074 of the pPICd1 plasmid) was replaced with the transketolase gene, and the other nucleotide sequences of the pPICd1 plasmid were kept unchanged. The transketolase gene in pPICd1-Tktl1 is driven by the promoter P MOX Transcription is initiated and terminated by the moxT terminator, allowing the expression of transketolase.

[0204] Among them, the amino acid reference sequence number of transketolase is XP_013934677.1 (14-SEP-2015), and the reference sequence number of transketolase gene is XM_014079202.1 (14-SEP-2015).

[0205] (3-b) Construction of strain DL04

[0206] The pPICd1-Tktl1 plasmid was amplified with primers Tktl1-R1 / Tktl1-01R to obtain the linearized pPIH-Tktl1 plasmid, which was then electroporated into strain DL03. The electroporation process was carried out according to the "Construction of strain DL02" in (1-c). After electroporation, the concentration of G418 in the YPD plate was 0.2 mg / mL. The primers PIH-F1 / Tktl1-R1 were used to identify the positive clones, and the primers PIH-F / Tktl1-01R were used to identify the positive clones, and the amplified fragments were 4 kb. After methanol treatment, the clones were frozen at -80°C. The strain was named DL04.

[0207] (4) Enhancing the expression of the transaldolase gene by promoter replacement

[0208] From Hansenula polymorpha ( Hansenula polymorpha )DL04, the promoter of the transaldehyde gene (also referred to as Taldo1 gene) in this strain was Taldo1 (SEQ ID No.5) was replaced by the strong promoter P MOX (SEQ ID No. 2), and recombinant Hansenula polymorpha DL05 was obtained, and the specific steps are as follows:

[0209] (4-a) Construction of plasmid pPICd1-Taldo1:

[0210] Using Taldo1-01F and Taldo1-01R as primers, the expression of Hansenula polymorpha ( Hansenula polymorpha )DL01 genomic DNA was used as a template and the Taldo1 fragment containing the Taldo1 gene was amplified by PCR using high-fidelity TransStart FastPfu DNA polymerase. The target fragment was recovered by agarose gel electrophoresis.

[0211] Using primers PIH-01F and PIH-01R, plasmid pPICd1 was used as a template to PCR amplify the PIH fragment and recover the large vector fragment PIH. The above Taldo1 fragment and the PIH fragment were ligated using the Gibson assembly method to obtain a ligation product. The ligation product was transformed into Escherichia coli DH5α competent cells according to the operation of (1-a). The clones that can amplify the target fragment were identified and sequenced using primers PIH-F1 / Taldo1-01R. The positive clones were selected to extract the plasmids. The obtained positive plasmids were named pPIH-Taldo1. The structure of pPICd1-Taldo1 is as follows: the fragment between positions 5080-7074 of the pPICd1 plasmid (the small fragment between positions 5080-7074 of the pPICd1 plasmid) is replaced by the transaldolase gene, and the other nucleotide sequences of the pPICd1 plasmid remain unchanged. The transaldolase gene in pPICd1-Taldo1 is driven by the promoter P MOX Transcription is initiated and terminated by the moxT terminator, allowing the expression of transaldehyde.

[0212] Among them, the amino acid reference sequence number of transaldolase is XP_013937091.1 (14-SEP-2015), and the reference sequence number of the transaldolase gene is XM_014081616.1 (14-SEP-2015).

[0213] (4-b) Construction of strain DL05

[0214] The pPICd1-Taldo1 plasmid was amplified with primers Taldo1-R1 / Taldo1-01R to obtain the linearized pPIH-Taldo1 plasmid, which was then electroporated into strain DL04. The electroporation process was carried out according to the "Construction of strain DL02" in (1-c). After electroporation, the concentration of G418 in the YPD plate was 0.2 mg / mL. The primers PIH-F2 / Taldo1-R1 were used to identify the positive clones with a 3 kb amplified fragment. After methanol treatment, the clones were frozen at -80°C. The strain was named DL05.

[0215] (5) Enhancing the expression of dihydroxyacetone synthase gene by promoter replacement

[0216] From Hansenula polymorpha ( Hansenula polymorpha )DL05, and the promoter of the dihydroxyacetone synthase gene (also referred to as DAS gene) in the strain was DAS (SEQ ID No.6) was replaced by the strong promoter P MOX (SEQ ID No. 2), and recombinant Hansenula polymorpha DL07 was obtained, and the specific steps are as follows:

[0217] (5-a) Construction of plasmid pPICd1-DAS:

[0218] Using DAS-01F and DAS-01R as primers, the polymorpha Hansenula ( Hansenula polymorpha )DL01 genomic DNA was used as a template and the DAS fragment containing the DAS gene was amplified by PCR using high-fidelity TransStart FastPfu DNA polymerase. The target fragment was recovered by agarose gel electrophoresis.

[0219] Using primers PIH-01F and PIH-01R, plasmid pPICd1 was used as a template to PCR amplify the PIH fragment and recover the large vector fragment PIH. The above DAS fragment and PIH fragment were ligated using the Gibson assembly method to obtain a ligation product. The ligation product was transformed into Escherichia coli DH5α competent cells according to the operation of (1-a). The clones that could amplify the target fragment were identified and sequenced using primers PIH-F1 / DAS-01R. The positive clones were selected to extract the plasmids. The obtained positive plasmids were named pPIH-DAS. The structure of pPICd1-DAS is as follows: the fragment between positions 5080-7074 of the pPICd1 plasmid (the small fragment between positions 5080-7074 of the pPICd1 plasmid) was replaced with the dihydroxyacetone synthase gene, and the other nucleotide sequences of the pPICd1 plasmid were kept unchanged. The dihydroxyacetone synthase encoding gene in pPICd1-DAS is driven by the promoter P MOX Transcription is initiated and terminated by the moxT terminator, and dihydroxyacetone synthase can be expressed.

[0220] Among them, the amino acid reference sequence number of dihydroxyacetone synthase is XP_013936414.1 (14-SEP-2015), and the reference sequence number of dihydroxyacetone synthase gene is XM_014080939.1 (14-SEP-2015).

[0221] (5-b) Construction of strain DL07

[0222] The pPICd1-DAS plasmid was amplified with primers DAS-R1 / DAS-01R to obtain the linearized pPIH-DAS plasmid, which was then electroporated into strain DL05. The electroporation process was carried out according to the "Construction of strain DL02" in (1-c). After electroporation, the concentration of G418 in the YPD plate was 0.2 mg / mL. The primers PIH-F2 / DAS-R1 were used to identify the positive clones with a 4.1 kb amplified fragment. After methanol treatment, the clones were frozen at -80°C. The strain was named DL07.

[0223] (6) Enhancing the expression of dihydroxyacetone kinase gene by promoter replacement

[0224] From Hansenula polymorpha ( Hansenula polymorpha )DL07, and the promoter of the dihydroxyacetone kinase gene (also referred to as DHAK gene) in the strain was DHAK (SEQ ID No.7) was replaced by the strong promoter P MOX (SEQ ID No. 2), and recombinant Hansenula polymorpha DL08 was obtained, and the specific steps are as follows:

[0225] (6-a) Construction of plasmid pPICd1-DHAK:

[0226] Using DHAK-01F and DHAK-01R as primers, the polymorpha Hansenula ( Hansenula polymorpha )DL01 genomic DNA was used as a template and the DHAK fragment containing the DHAK gene was amplified by PCR using high-fidelity TransStart FastPfu DNA polymerase. The target fragment was recovered by agarose gel electrophoresis.

[0227] Using primers PIH-01F and PIH-01R, plasmid pPICd1 was used as a template to PCR amplify the PIH fragment and recover the large vector fragment PIH. The above DHAK fragment was ligated with the PIH fragment using the Gibson assembly method to obtain a ligation product. The ligation product was transformed into Escherichia coli DH5α competent cells according to the operation of (1-a). The clones that could amplify the target fragment were identified and sequenced using primers PIH-F1 / DHAK-01R. The positive clones were selected to extract the plasmids. The obtained positive plasmids were named pPIH-DHAK. The structure of pPICd1-DHAK is as follows: the fragment between positions 5080-7074 of the pPICd1 plasmid (the small fragment between positions 5080-7074 of the pPICd1 plasmid) is replaced with the dihydroxyacetone kinase gene, and the other nucleotide sequences of the pPICd1 plasmid remain unchanged. The dihydroxyacetone kinase encoding gene in pPICd1-DHAK is driven by the promoter P MOX Transcription is initiated and terminated by the moxT terminator, and dihydroxyacetone kinase can be expressed.

[0228] Among them, the amino acid reference sequence number of dihydroxyacetone kinase is XP_013934963.1 (14-SEP-2015), and the reference sequence number of dihydroxyacetone kinase gene is XM_014079488.1 (14-SEP-2015).

[0229] (6-b) Construction of strain DL08

[0230] The pPICd1-DHAK plasmid was amplified with primers DHAK-R1 / DHAK-01R to obtain the linearized pPIH-DHAK plasmid, which was then electroporated into strain DL07. The electroporation process was carried out according to the "Construction of strain DL02" in (1-c). After electroporation, the concentration of G418 in the YPD plate was 0.2 mg / mL. The primers PIH-F2 / DHAK-R1 were used to identify the positive clones with a 3.8 kb amplified fragment. After methanol treatment, the clones were frozen at -80°C. The strain was named DL08.

[0231] (7) Construction of DL09 strain

[0232] (7-1) Introducing the gene encoding hexokinases into DL08

[0233] From Hansenula polymorpha ( Hansenula polymorpha ) DL08 starts from people (Homo sapiens) The hexokinokinase gene (SEQ ID No. 8, also referred to as HkhkB gene) was introduced into DL08 to obtain the recombinant Hansenula polymorpha DL081, and the specific steps are as follows:

[0234] (7-1a) Construction of plasmid pPICd1-HkhkB:

[0235] Artificial human ( Homo sapiens ) to obtain the pUC57-HkhkB plasmid containing the HkhkB gene. Using the pUC57-HkhkB plasmid as a template and HkhkB-01F and HkhkB-01R as primers, the HkhkB fragment containing the HkhkB gene was amplified using high-fidelity TransStart FastPfu DNA polymerase PCR, and the target fragment was recovered by agarose gel electrophoresis.

[0236] Using primers PIH-01F and PIH-01R, plasmid pPICd1 was used as a template to PCR amplify the PIH fragment and recover the large vector fragment PIH. The above HkhkB fragment and the PIH fragment were ligated using the Gibson assembly method to obtain a ligation product. The ligation product was transformed into Escherichia coli DH5α competent cells with reference to the operation of (1-a). The clones that can amplify the target fragment were identified and sequenced using primers PIH-F1 / HkhkB-01R. The positive clones were selected to extract the plasmids, and the positive plasmids were named pPIH-HkhkB. The structure of pPIH-HkhkB is as follows: the fragment between positions 5080-7074 of the pPICd1 plasmid (the small fragment between positions 5080-7074 of the pPICd1 plasmid) is replaced by the hexokinases kinase gene (SEQ ID No. 8), and the other nucleotide sequences of the pPICd1 plasmid remain unchanged. The hexokinases kinase gene in pPIH-HkhkB is driven by the promoter P MOX The transcription is initiated and terminated by the moxT terminator to express hexokinases, wherein the amino acid sequence of hexokinases is SEQ ID No.9.

[0237] (7-1b) Construction of strain DL081

[0238] The pPICd1-HkhkB plasmid was amplified with primers HL1 and HL2 to obtain the linearized pPIH-HkhkB plasmid, which was then electrotransformed into strain DL08. The electrotransformation process was carried out according to the "Construction of strain DL02" in (1-c). After electrotransformation, the concentration of G418 in the YPD plate was 0.2 mg / mL. The primers PIH-F1 / HkhkB-01R were used to identify the positive clones with a fragment of 2.7 kb. After methanol treatment, the clones were frozen at -80°C. The strain was named DL081.

[0239] (7-2) Introducing the gene encoding UDP-glucose pyrophosphorylase into DL081

[0240] Starting from Hansenula polymorpha DL08, the UDP-glucose pyrophosphorylase gene (SEQ ID No. 10, also referred to as Augp gene) from Arabidopsis thaliana was introduced into DL081 to obtain recombinant Hansenula polymorpha DL082. The specific steps are as follows:

[0241] (7-2a) Construction of plasmid pPICd1-Augp:

[0242] The UDP-glucose pyrophosphorylase gene of Arabidopsis thaliana was artificially synthesized to obtain the pUC57-Augp plasmid containing the Augp gene. The Augp fragment containing the Augp gene was amplified by PCR using the pUC57-Augp plasmid as a template and Augp-01F and Augp-01R as primers using high-fidelity TransStart FastPfu DNA polymerase, and the target fragment was recovered by agarose gel electrophoresis.

[0243] Using primers PIH-01F and PIH-01R, plasmid pPICd1 was used as a template to PCR amplify the PIH fragment and recover the large vector fragment PIH. The above-mentioned Augp fragment and the PIH fragment were ligated using the Gibson assembly method to obtain a ligation product. The ligation product was transformed into Escherichia coli DH5α competent cells with reference to the operation of (1-a). The clones that could amplify the target fragment were identified and sequenced using primers PIH-F1 / Augp-01R. The positive clones were selected to extract the plasmids. The obtained positive plasmids were named pPIH-Augp. The structure of pPIH-Augp is as follows: the fragment between positions 5080-7074 of the pPICd1 plasmid (the small fragment between positions 5080-7074 of the pPICd1 plasmid) was replaced with the UDP-glucose pyrophosphorylase gene (SEQ ID No.10), and the other nucleotide sequences of the pPICd1 plasmid were kept unchanged. The UDP-glucose pyrophosphorylase gene in pPIH-Augp is driven by the promoter P MOX Initiate transcription, terminate transcription by moxT terminator, and express UDP-glucose pyrophosphorylase.

[0244] (7-2b) Construction of strain DL082

[0245] The pPICd1-Augp plasmid was amplified with UL1 / UL2 primers to obtain the linearized pPIH-Augp plasmid, which was then electroporated into the strain DL081. The electroporation process was carried out according to the "Construction of strain DL02" in (1-c). After electroporation, the G418 concentration in the YPD plate was 0.2 mg / mL. The primers PIH-F1 / Augp-01R were used for identification, and the amplified fragment of 2.7 kb was a positive clone. After methanol treatment, it was frozen at -80°C. The strain was named DL082.

[0246] (7-3) Introducing the gene encoding NDP-hexose-3-epimerase into DL082

[0247] Starting from Hansenula polymorpha DL082, NDP-hexose-3-epimerase (SEQ ID No. 12, also referred to as Snh3e gene) from Streptomyces sp. was introduced into DL082 to obtain recombinant Hansenula polymorpha DL083. The specific steps are as follows:

[0248] (7-3a) Construction of plasmid pPICd1-Snh3e:

[0249] The NDP-hexose-3-epimerase gene of Streptomyces sp. was artificially synthesized to obtain the pUC57-Snh3e plasmid containing the Snh3e gene. The Snh3e fragment containing the Snh3e gene was amplified by PCR using the pUC57-Snh3e plasmid as a template and Snh3e-01F and Snh3e-01R as primers using high-fidelity TransStart FastPfu DNA polymerase, and the target fragment was recovered by agarose gel electrophoresis.

[0250] Using primers PIH-01F and PIH-01R, plasmid pPICd1 was used as a template to PCR amplify the PIH fragment, and the large vector fragment PIH was recovered. The above Snh3e fragment and the PIH fragment were ligated using the Gibson assembly method to obtain a ligation product. The ligation product was transformed into Escherichia coli DH5α competent cells according to the operation of (1-a), and the clones that can amplify the target fragment were identified and sequenced using primers PIH-F1 / Snh3e-01R. The positive clones were selected to extract the plasmids, and the obtained positive plasmids were named pPIH-Snh3e. The structure of pPIH-Snh3e is: the fragment between positions 5080-7074 of the pPICd1 plasmid (the small fragment between positions 5080-7074 of the pPICd1 plasmid) was replaced with the NDP-hexose-3-epimerase gene (SEQ ID No. 12), and the other nucleotide sequences of the pPICd1 plasmid were kept unchanged. The NDP-hexose-3-epimerase gene in pPIH-Snh3e is driven by the promoter P MOX The transcription is initiated and terminated by the moxT terminator, thereby expressing NDP-hexose-3-epimerase, wherein the amino acid sequence of NDP-hexose-3-epimerase is SEQ ID No.13.

[0251] (7-3b) Construction of strain DL083

[0252] The pPICd1-Snh3e plasmid was amplified with EL1 / EL2 primers to obtain the linearized pPIH-Snh3e plasmid, which was then electroporated into the strain DL082. The electroporation process was carried out according to the "Construction of strain DL02" in (1-c). After electroporation, the concentration of G418 in the YPD plate was 0.2 mg / mL. The primers PIH-F1 / Snh3e-01R were used to identify the positive clones with a 2.7 kb amplified fragment. After methanol treatment, the clones were frozen at -80°C. The strain was named DL083.

[0253] (7-4) Introducing the gene encoding NDP glycohydrolase into DL083

[0254] Starting from Hansenula polymorpha DL083, NDP glycohydrolase (SEQ ID No. 14, also referred to as Rnudt22 gene) from rat (Rattusnorvegicus) was introduced into DL083 to obtain recombinant Hansenula polymorpha DL084. The specific steps are as follows:

[0255] (7-4a) Construction of plasmid pPICd1-Rnudt22:

[0256] The NDP glycohydrolase gene of rat (Rattus norvegicus) was artificially synthesized to obtain the pUC57-Rnudt22 plasmid containing the Rnudt22 gene. The Rnudt22 fragment containing the Rnudt22 gene was amplified by PCR using the pUC57-Rnudt22 plasmid as a template and Rnudt22-01F and Rnudt22-01R as primers using high-fidelity TransStart FastPfu DNA polymerase, and the target fragment was recovered by agarose gel electrophoresis.

[0257] Using primers PIH-01F and PIH-01R, plasmid pPICd1 was used as a template to PCR amplify the PIH fragment and recover the large vector fragment PIH. The above Rnudt22 fragment and the PIH fragment were ligated using the Gibson assembly method to obtain a ligation product. The ligation product was transformed into Escherichia coli DH5α competent cells according to the operation of (1-a). The clones that could amplify the target fragment were identified and sequenced using primers PIH-F1 / Rnudt22-01R. The positive clones were selected to extract the plasmids. The obtained positive plasmids were named pPIH-Rnudt22. The structure of pPIH-Rnudt22 is as follows: the fragment between positions 5080-7074 of the pPICd1 plasmid (the small fragment between positions 5080-7074 of the pPICd1 plasmid) was replaced with the NDP glycohydrolase gene, and the other nucleotide sequences of the pPICd1 plasmid were kept unchanged. The NDP glycohydrolase gene in pPIH-Rnudt22 is driven by the promoter P MOX The transcription is initiated and terminated by the moxT terminator, thereby expressing NDP glycohydrolase, wherein the amino acid sequence of NDP glycohydrolase is SEQ ID No.15.

[0258] (7-4b) Construction of strain DL084

[0259] The pPICd1-Rnudt22 plasmid was amplified with primers NL1 / NL2 to obtain the linearized pPIH-Rnudt22 plasmid, which was then electroporated into strain DL083. The electroporation process was carried out according to the "Construction of strain DL02" in (1-c). After electroporation, the concentration of G418 in the YPD plate was 0.2 mg / mL. The primers PIH-F1 / Rnudt22-01R were used to identify the positive clones with a 2.7 kb amplified fragment. After methanol treatment, the clones were frozen at -80°C. The strain was named DL084.

[0260] (7-5) Introducing the gene encoding inorganic pyrophosphatase into DL084

[0261] Starting from Hansenula polymorpha DL084, an inorganic pyrophosphatase (reference sequence number NC_000913.3 (4449652..4449122) (-), also referred to as Epaa gene) from Escherichia coli (E. coli) was introduced into DL084 to obtain recombinant Hansenula polymorpha DL085. The specific steps are as follows:

[0262] (7-5a) Construction of plasmid pPICd1-Epaa:

[0263] The inorganic pyrophosphatase gene of Escherichia coli (E. coli) was artificially synthesized to obtain the pUC57-Epaa plasmid containing the Epaa gene. The Epaa fragment containing the Epaa gene was amplified by PCR using the pUC57-Epaa plasmid as a template and Epaa-01F and Epaa-01R as primers using high-fidelity TransStartFastPfu DNA polymerase, and the target fragment was recovered by agarose gel electrophoresis.

[0264] Using primers PIH-01F and PIH-01R, and plasmid pPICd1 as a template, PCR amplified the PIH fragment, and recovered the large vector fragment PIH. The above-mentioned Epaa fragment and the PIH fragment were ligated using the Gibson assembly method to obtain a ligation product. The ligation product was transformed into Escherichia coli DH5α competent cells with reference to the operation of (1-a), and the clones that could amplify the target fragment were identified and sequenced using primers PIH-F1 / Epaa-01R. The positive clones were selected to extract the plasmids, and the obtained positive plasmids were named pPIH-Epaa. The structure of pPIH-Epaa is as follows: the fragment between positions 5080-7074 of the pPICd1 plasmid (the small fragment between positions 5080-7074 of the pPICd1 plasmid) is replaced with the inorganic pyrophosphatase gene, and the other nucleotide sequences of the pPICd1 plasmid remain unchanged. The inorganic pyrophosphatase gene in pPIH-Epaa is driven by the promoter P MOX The transcription is initiated and terminated by the moxT terminator, and inorganic pyrophosphatase can be expressed. The amino acid reference sequence number of the inorganic pyrophosphatase is NP_418647.1, 09-MAR-2022.

[0265] (7-5b) Construction of strain DL085

[0266] The pPICd1-Epaa plasmid was amplified with AL1 / AL2 primers to obtain the linearized pPIH-Epaa plasmid, which was then electroporated into the strain DL084. The electroporation process was carried out according to the "Construction of strain DL02" in (1-c). After electroporation, the concentration of G418 in the YPD plate was 0.2 mg / mL. The primers PIH-F1 / Epaa-01R were used to identify the positive clones with a 2.7 kb amplified fragment. After methanol treatment, the clones were frozen at -80°C. The strain was named DL085.

[0267] (8) Introducing the gene encoding acid phosphatase into DL085

[0268] Starting from Hansenula polymorpha DL085, the acid phosphatase gene (SEQ ID No. 16, also referred to as Aapp gene) from Aspergillus sp. was introduced into DL085 to obtain recombinant Hansenula polymorpha DL09. The specific steps are as follows:

[0269] (8-a) Construction of plasmid pPICd1-Aapp:

[0270] The acid phosphatase gene of Aspergillus sp. was artificially synthesized to obtain the pUC57-Aapp plasmid containing the Aapp gene. The Aapp fragment containing the Aapp gene was amplified by PCR using the pUC57-Aapp plasmid as a template and Aapp-01F and Aapp-01R as primers using high-fidelity TransStart FastPfu DNA polymerase, and the target fragment was recovered by agarose gel electrophoresis.

[0271] Using primers PIH-01F and PIH-01R, and plasmid pPICd1 as a template, PCR amplified the PIH fragment, and recovered the large vector fragment PIH. The above Aapp fragment and the PIH fragment were ligated using the Gibson assembly method to obtain a ligation product. The ligation product was transformed into Escherichia coli DH5α competent cells with reference to the operation of (1-a), and the clones that could amplify the target fragment were identified and sequenced using primers PIH-F1 / Aapp-01R. The positive clones were selected to extract the plasmids, and the obtained positive plasmids were named pPIH-Aapp. The structure of pPIH-Aapp is as follows: the fragment between positions 5080-7074 of the pPICd1 plasmid (the small fragment between positions 5080-7074 of the pPICd1 plasmid) is replaced with the acid phosphatase gene, and the other nucleotide sequences of the pPICd1 plasmid remain unchanged. The acid phosphatase gene in pPIH-Aapp is driven by the promoter P MOX The transcription is initiated and terminated by the moxT terminator, and acid phosphatase can be expressed, wherein the amino acid sequence of the acid phosphatase is SEQ ID No.17.

[0272] (8-b) Construction of strain DL09

[0273] The pPICd1-Aapp plasmid was amplified with primers PL1 / PL2 to obtain the linearized pPIH-Aapp plasmid, which was then electroporated into strain DL085. The electroporation process was carried out according to the "Construction of strain DL02" in (1-c). After electroporation, the concentration of G418 in the YPD plate was 0.2 mg / mL. The primers PIH-F1 / Aapp-01R were used to identify the positive clones with a 2.7 kb amplified fragment. After methanol treatment, the clones were frozen at -80°C. The strain was named DL09.

[0274] (9) Knockout of 6-phosphofructokinase 1 gene

[0275] From Hansenula polymorpha ( Hansenula polymorpha ) Starting from DL09, the 6-phosphofructokinase 1 gene (also referred to as pfkA gene) of DL09 was knocked out to obtain the DL09 mutant DL11. The specific steps are as follows:

[0276] Using pfkA-up and pfkA-down as primers and plasmid pPICd1 as template, the high-fidelity TransStartFastPfu DNA polymerase was used to amplify the target fragment pfkAup-G418-pfkAdown by fusion PCR, and the fragment was recovered by gel. The target fragment pfkAup-G418-pfkAdown contains the upstream sequence of the pfkA gene of Hansenula polymorpha (1-70), the lox66-G418 resistance gene-lox71 gene cluster (71-1766), and the downstream sequence of the pfkA gene (1767-1836).

[0277] The fragment pfkAup-G418-pfkAdown was electro-transformed into strain DL09. The electro-transformation process was carried out according to the "Construction of strain DL02" in (1-c). After electro-transformation, the concentration of G418 in the YPD plate was 0.2 mg / mL. The primers pfkA-F / pfkA-R were used to identify that the amplified fragment of 3.4 kb was a positive clone. After methanol treatment, it was frozen at -80℃. The strain was named DL11. Using PfkA-F / PfkA-R as primers, PCR amplification of strain DL11 was positive for 1.6 kb, proving that the pfkA gene was knocked out.

[0278] Among them, the amino acid reference sequence number of 6-phosphofructokinase 1 is XP_013936214.1 (14-SEP-2015), and the reference sequence number of 6-phosphofructokinase 1 gene is XM_014080739.1 (14-SEP-2015).

[0279] (10) Knockout of 6-phosphofructokinase 2 gene

[0280] From Hansenula polymorpha ( Hansenula polymorpha ) Starting from DL11, the 6-phosphofructokinase 2 gene (also referred to as pfkB gene) of DL11 was knocked out to obtain the mutant DL12 of DL11. The specific steps are as follows:

[0281] Using pfkB-up and pfkB-down as primers and plasmid pPICd1 as template, the high-fidelity TransStartFastPfu DNA polymerase was used to amplify the target fragment pfkBup-G418-pfkBdown by fusion PCR, and the fragment was recovered by gel. The fragment sequentially contains the upstream sequence of the pfkB gene of Hansenula polymorpha (1-70), the lox66-G418 resistance gene-lox71 gene cluster (71-1766), and the downstream sequence of the pfkB gene (1767-1836).

[0282] The fragment pfkBup-G418-pfkBdown was electro-transformed into strain DL11. The electro-transformation process was carried out according to the "Construction of strain DL02" in (1-c). After electro-transformation, the concentration of G418 in the YPD plate was 0.2 mg / mL. The primers pfkB-F / pfkB-R were used to identify that the amplified fragment of 3.4 kb was a positive clone. After methanol treatment, it was frozen at -80℃. The strain was named DL12. Using PfkB-F / PfkB-R as primers, PCR amplification of strain DL12 was positive for 1.6 kb, proving that the pfkB gene was knocked out.

[0283] Among them, the amino acid reference sequence number of 6-phosphofructokinase 2 is XP_013933492.1 (14-SEP-2015), and the reference sequence number of its 6-phosphofructokinase 2 gene is XM_014078017.1 (14-SEP-2015).

[0284] (11) Knockout of 6-phosphate glucose dehydrogenase gene

[0285] From Hansenula polymorpha ( Hansenula polymorpha ) Starting from DL12, the 6-phosphate glucose dehydrogenase gene (also referred to as G6PD gene) of DL12 was knocked out to obtain the DL12 mutant DL13. The specific steps are as follows:

[0286] Using G6PD-up and G6PD-down as primers and plasmid pPICd1 as template, the high-fidelity TransStartFastPfu DNA polymerase was used to amplify the target fragment G6PDup-G418-G6PDdown by fusion PCR, and the fragment was recovered by gel. The fragment sequentially contains the upstream sequence of the G6PD gene of Hansenula polymorpha (1-70), the lox66-G418 resistance gene-lox71 gene cluster (71-1766), and the downstream sequence of the G6PD gene (1767-1836).

[0287] The fragment G6PDup-G418 -G6PDdown was electrotransformed into strain DL12. The electrotransformation process was carried out according to the "Construction of strain DL02" in (1-c). After electrotransformation, the concentration of G418 in the YPD plate was 0.2 mg / mL. The primers G6PD-F / G6PD-R were used to identify that the amplified fragment of 3.4 kb was a positive clone. After methanol treatment, it was frozen at -80℃. The strain was named DL13. Using G6PD-F / G6PD-R as primers, PCR amplification of strain DL13 was positive for 1.6 kb, proving that the G6PD gene was knocked out.

[0288] Among them, the amino acid reference sequence number of 6-phosphate glucose dehydrogenase is XP_018211730.1 (05-FEB-2020), and the reference sequence number of 6-phosphate glucose dehydrogenase gene is XM_018354349.1 (05-FEB-2020).

[0289] Table 2: Sequences of targeting fragments

[0290]

[0291] Table 3. List of primer sequences used in this application

[0292]

[0293] Example 2: Construction of recombinant Hansenula polymorpha engineered strain DL21

[0294] Construction of DL21: Using DL01 as the starting strain, molecular biological operations were performed according to steps (7) to (11) in Example 1. The reagents and primer sequences used were exactly the same.

[0295] Example 3: Preparation of D-psicose using recombinant Hansenula polymorpha engineered strain DL13

[0296] In this example, the recombinant engineered strain of Hansenula polymorpha DL13 was used as a fermentation cell. The cell grows with methanol as a carbon source and can convert D-glucose into D-psicose.

[0297] (1) Growth medium composition.

[0298] The components and final concentrations of the growth medium are as follows: Na 2 HPO 4 :25 mM, KH 2 PO 4 :25 mM, NH 4 Cl: 50 mM, Na 2 SO 4 :5 mM, MgSO 4 : 2 mM, methanol: 3 g / 100 mL, yeast powder: 0.5 g / 100 mL), trace elements: 50 μM FeCl 3 , 20 μM CaCl 2 , 10 μM MnCl 2 , 10 μM ZnSO 4 , CoCl 2 、NiCl 2 、Na 2 MO 4 、Na 2 SeO 3 and H 3 BO 3 2 μM each; the rest is water, adjusted to pH 8.0.

[0299] (2) Transformation medium composition: Na 2 HPO 4 :25 mM, KH 2 PO 4 :25 mM, NH 4 Cl: 50 mM, D-glucose: 20 g / 100 mL, trace elements: 50 μM FeCl 3 , 20 μM CaCl 2 , 10 μM MnCl 2 , 10 μM ZnSO 4 , CoCl 2 、NiCl 2 、Na 2 MO 4 、Na 2 SeO 3 and H 3 BO 3 2 μM each, the rest was water.

[0300] (3) Bacterial culture and enzyme induction

[0301] The wild-type strain DL01, engineered strain DL08, engineered strain DL09, engineered strain DL21 and engineered strain DL13 cultured overnight were inoculated into a shake flask (500 mL Erlenmeyer flask) containing 200 mL of growth medium at 1% inoculation volume and cultured in a Shanghai Zhicheng shaker at 30 °C and 200 rpm for 3 days. The cells were collected and the OD of the bacterial solution was detected. 600nm The results showed that when the methanol utilization related targets were strengthened, the OD values ​​of strains DL08, DL09 and DL13 600nm 18.95±2.36, 20.84±1.55, and 20.03±1.97, respectively, which were significantly higher than those of DL01 and DL21, and the growth could reach the level of using glucose as the carbon source ( Figure 1 ).

[0302] (4) Cultivation of bacteria in a medium with glucose as the carbon source

[0303] The wild-type strain DL01, engineered strain DL08, engineered strain DL09, engineered strain DL21 and engineered strain DL13 cultured overnight were inoculated at 1% in a shake flask (500 mL Erlenmeyer flask) containing 200 mL of glucose growth medium (methanol in the growth medium was replaced with the same mass of glucose) and cultured in a Shanghai Zhicheng shaker at 30 °C and 200 rpm for 3 days. The OD 600nm The results showed that when the glycolysis pathway was blocked, strains DL13 and DL21 could not use glucose to grow and could not consume glucose ( Figure 1 ).

[0304] Figure 1 The growth of different strains in different culture media. Figure 1 Methanol in the middle refers to methanol growth medium; glucose refers to glucose growth medium. The vertical axis OD 600 The OD value of the bacterial culture solution after 3 days of culture is 600nm light absorption value.

[0305] (5) Whole-cell catalysis of D-psicose

[0306] The cells collected in step (3) were resuspended in a shake flask containing 20 mL of transformation medium, and the cell concentration was fixed at 20 OD (i.e., OD 600nmThe concentration of D-psicose was 20. If the amount of bacteria was insufficient, multiple bottles were combined. After culturing at 30°C for 20 hours, the supernatant was centrifuged and filtered. The content of D-psicose was detected by HPLC. HPLC used a Hi-Plex Ca column (300 mm×7.7 mm, 8μm); the mobile phase was ultrapure water, the flow rate was 0.5mL / min; the column temperature was 78°C; the detector was a differential refractometer; the temperature was 40°C. D-psicose standard was purchased from Shanghai Yuanye Biotechnology (Cat. No.: S48276). The results are as follows Figure 2 As shown, the results showed that the D-psicose production of wild-type strain DL01 and engineered strain DL08 was 0, the D-psicose production of engineered strain DL09 was 28.08±2.33g / L, the D-psicose production of engineered strain DL13 was 105.34±5.92g / L, and the conversion rate was 78.73% (psicose production (g) / glucose consumption (g)), while the D-psicose production of DL21 was 95.5±3.95g / L, and the conversion rate was 65.5% (psicose production (g) / glucose consumption (g)). This shows that strain DL13, which blocks the glycolysis pathway and enhances methanol utilization, has good characteristics for producing D-psicose. However, the production of DL21 was lower than that of DL13, and the biomass in the growth induction stage was lower than that of DL13.

[0307] The present application has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present application, and without the need to carry out unnecessary experimental conditions, the present application can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present application provides specific embodiments, it should be understood that further improvements can be made to the present application. In a word, according to the principles of the present application, the present application is intended to include any changes, uses or improvements to the present application, including departure from the disclosed scope in the present application and changes made with conventional techniques known in the art.

Claims

1. A recombinant Hansenula polymorpha, wherein the recombinant Hansenula polymorpha comprises the following characteristics: A1), contains the gene encoding hexoketokinase; A2), containing the gene encoding UDP-glucose pyrophosphorylase; A3), containing the gene encoding NDP-hexose-3-epimerase; A4), containing the coding gene of NDP sugar hydrolase; A5), contains the gene encoding inorganic pyrophosphatase; A6), contains the gene encoding acid phosphatase; A7), does not contain 6-phosphofructokinase 1 gene; A8), does not contain 6-phosphofructokinase 2 gene; A9), does not contain 6-phosphate glucose dehydrogenase gene; The ketokinase is any one of the following: A1-1), a protein having an amino acid sequence of SEQ ID No. 9; A1-2), a fusion protein having the function of the hexokinases obtained by connecting a tag to the N-terminus and / or C-terminus of the protein in A1-1); The UDP-glucose pyrophosphorylase is any one of the following: A2-1), a protein having an amino acid sequence of SEQ ID No. 11; A2-2), a fusion protein having the function of UDP-glucose pyrophosphorylase obtained by connecting a tag to the N-terminus and / or C-terminus of the protein in A2-1); The NDP-hexose-3-epimerase is any one of the following: A3-1), a protein having an amino acid sequence of SEQ ID No. 13; A3-2), a fusion protein having the function of the NDP-hexose-3-epimerase obtained by connecting a tag to the N-terminus and / or C-terminus of the protein in A3-1; The NDP glycohydrolase is any one of the following: A4-1), a protein having an amino acid sequence of SEQ ID No. 15; A4-2), a fusion protein having the function of the NDP glycohydrolase obtained by connecting a tag to the N-terminus and / or C-terminus of the protein in A4-1); The inorganic pyrophosphatase is any one of the following: A5-1), the protein with amino acid reference sequence number NP_418647.1; A5-2), a fusion protein having the inorganic pyrophosphatase function obtained by connecting a tag to the N-terminus and / or C-terminus of the protein in A5-1) 2; The acid phosphatase is any of the following: A6-1), a protein having an amino acid sequence of SEQ ID No. 17; A6-2), a fusion protein having the acid phosphatase function obtained by connecting a tag to the N-terminus and / or C-terminus of the protein described in A6-1) 2; The 6-phosphofructokinase 1 is a protein with the NCBI reference sequence number XP_013936214.1; The 6-phosphofructokinase 2 is a protein with the NCBI reference sequence number XP_013933492.1; The 6-phosphate glucose dehydrogenase is a protein with the NCBI reference sequence number XP_018211730.

1.

2. The recombinant Hansenula polymorpha according to claim 1, characterized in that: The recombinant Hansenula polymorpha also has the following characteristics: A10) Containing substances that regulate the expression of fructose bisphosphate aldolase gene or the activity or content of fructose bisphosphate aldolase; A11), containing substances that regulate the expression of fructose-1,6-bisphosphatase gene or the activity or content of fructose-1,6-bisphosphatase; A12) Containing substances that regulate the expression of transketolase genes or regulate the activity or content of transketolase; A13) containing substances that regulate the expression of transaldolase genes or regulate the activity or content of transaldolase; A14) Substances that regulate the expression of dihydroxyacetone synthase gene or the activity or content of dihydroxyacetone synthase; A15) Substances that regulate the expression of dihydroxyacetone kinase gene or the activity or content of dihydroxyacetone kinase; The substance described in A10)-A15) is the promoter P MOX , whose nucleotide sequence is SEQ ID No.

2.

3. A method for preparing the recombinant Hansenula polymorpha according to claim 1 or 2, the method comprising: M1), introducing the gene encoding the hexokinases described in claim 1; M2), introducing the gene encoding the UDP-glucose pyrophosphorylase described in claim 1; M3), introducing the gene encoding the NDP-hexose-3-epimerase described in claim 1; M4), introducing the gene encoding the NDP sugar hydrolase described in claim 1; M5), introducing the gene encoding the inorganic pyrophosphatase described in claim 1; M6), introducing the gene encoding the acid phosphatase described in claim 1; M7), knocking out the 6-phosphofructokinase 1 gene described in claim 1; M8), knocking out the 6-phosphofructokinase 2 gene described in claim 1; M9), knocking out the 6-phosphate glucose dehydrogenase gene described in claim 1.

4. A method for preparing the recombinant Hansenula polymorpha according to claim 1 or 2, the method comprising: M1), introducing the gene encoding the hexolose kinase as claimed in claim 1 and a substance containing a substance for regulating the expression of the hexolose kinase gene or regulating the activity or content of the hexolose kinase; M2), introducing the gene encoding the UDP-glucose pyrophosphorylase described in claim 1 and a substance containing a substance that regulates the expression of the UDP-glucose pyrophosphorylase gene or regulates the activity or content of the UDP-glucose pyrophosphorylase; M3), introducing the gene encoding the NDP-hexose-3-epimerase described in claim 1 and a substance containing a substance that regulates the expression of the NDP-hexose-3-epimerase gene or regulates the activity or content of the NDP-hexose-3-epimerase; M4), introducing the gene encoding the NDP glycohydrolase described in claim 1 and a substance containing a substance that regulates the expression of the NDP glycohydrolase gene or regulates the activity or content of the NDP glycohydrolase; M5), introducing the gene encoding the inorganic pyrophosphatase described in claim 1 and a substance containing a substance that regulates the expression of the inorganic pyrophosphatase gene or regulates the activity or content of the inorganic pyrophosphatase; M6), introducing the gene encoding the acid phosphatase described in claim 1 and a substance containing a substance that regulates the expression of the acid phosphatase gene or regulates the activity or content of the acid phosphatase; M7), knocking out the 6-phosphofructokinase 1 gene described in claim 1; M8), knocking out the 6-phosphofructokinase 2 gene described in claim 1; M9), knocking out the 6-phosphate glucose dehydrogenase gene described in claim 1; M1)-M6) are promoters P MOX , whose nucleotide sequence is SEQ ID No.

2.

5. The method according to claim 3 or 4, characterized in that: The method further comprises: M10), knocking out the wild-type promoter that drives the expression of the fructose bisphosphate aldolase gene described in claim 2 and replacing it with the P described in claim 2 MOX ; M11), knocking out the wild-type promoter that drives the expression of the fructose-1,6-bisphosphatase gene described in claim 2 and replacing it with the P described in claim 2 MOX ; M12), knocking out the wild-type promoter that drives the expression of the transketolase gene described in claim 2 and replacing it with the promoter described in claim 2 MOX ; M13), knocking out the wild-type promoter that drives the expression of the transaldolase gene described in claim 2 and replacing it with the P described in claim 2 MOX ; M14), knocking out the wild-type promoter that drives the expression of the dihydroxyacetone synthase gene described in claim 2 and replacing it with the promoter described in claim 2 MOX ; M7), knocking out the wild-type promoter that drives the expression of the dihydroxyacetone kinase gene described in claim 2 and replacing it with the P described in claim 2 MOX .

6. Use of a composition in constructing the recombinant Hansenula polymorpha according to claim 1 or 2, wherein the composition contains a promoter P MOX , a gene encoding hexoketose kinase as claimed in claim 1, a gene encoding UDP-glucose pyrophosphorylase, a gene encoding NDP-hexose-3-epimerase, a gene encoding NDP sugar hydrolase, a gene encoding inorganic pyrophosphatase, a gene encoding acid phosphatase, a substance for knocking out the 6-phosphofructokinase 1 gene as claimed in claim 1, a substance for knocking out the 6-phosphofructokinase 2 gene as claimed in claim 1, and a substance for knocking out the 6-phosphoglucose dehydrogenase gene as claimed in claim 1; The promoter P MOX The nucleotide sequence is SEQ ID No.

2.

7. Use of the recombinant Hansenula polymorpha according to claim 1 or 2 in the preparation of D-psicose.

8. A bacterial agent, characterized in that: The bacterial agent contains the recombinant Hansenula polymorpha according to claim 1 or 2 or a culture containing the recombinant Hansenula polymorpha according to claim 1 or 2.

9. A method for preparing D-psicose, characterized in that: The method comprises using the recombinant Hansenula polymorpha described in claim 1 or 2 and / or the bacterial agent described in claim 8 to prepare D-psicose using D-glucose as a substrate.

10. The method according to claim 9, characterized in that The method includes amplifying the recombinant Hansenula polymorpha using methanol as a carbon source.

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