Aureobasidium pullulans strain for producing pullulan as well as construction method and application of aureobasidium pullulans strain

Abstract

The invention relates to an aureobasidium pullulans strain for producing pullulan as well as a construction method and application of the aureobasidium pullulans strain, and belongs to the technical field of bioengineering. According to the aureobasidium pullulans strain constructed by the invention, alpha-phosphoglucose mutase PGM and uridine diphosphate glucose pyrophosphorylase UGP are continuously overexpressed, so that the yield of pullulan produced by fermentation of aureobasidium pullulans is remarkably improved, the fermentation titer of the strain is improved, the stability is improved, and the yield of pullulan produced by fermentation of aureobasidium pullulans is improved. An excellent strain is provided for preparing the pullulan by a microbial fermentation method, the separation cost in the downstream purification process of the pullulan is reduced, and an excellent strain is provided for industrial production of the pullulan.

Description

Technical Field

[0001] The invention relates to a pullulan-producing Aureobasidium pullulans strain and a construction method and application thereof, belonging to the technical field of bioengineering. Background Art

[0002] Pullulan, also known as pullulan or budding short spore sugar, is an important metabolite of Aureobasidium pullulans. It is a natural extracellular polysaccharide with good water solubility. Pullulan is a linear polysaccharide, a high molecular weight polymer composed of maltotriose repeating units connected to each other. The maltotriose repeating units are connected by α-1,4 glycosidic bonds, and the repeating units are connected by α-1,6 glycosidic bonds. Pullulan has very good water solubility and low viscosity in aqueous solution, but it is insoluble in most organic solvents. Pullulan is non-hygroscopic, non-toxic, non-carcinogenic and edible in its natural state. The regular alternation of α-1,4 and α-1,6 glycosidic bonds of pullulan gives it excellent structural flexibility and solubility. At the same time, pullulan has very high thermal stability, is still stable at 250°C, and will decompose at temperatures above 250°C. Pullulan can form a transparent, oil-proof and oxygen-proof film, which can be used in hollow capsule shells, food, candy and other fields. Pullulan exhibits adhesion during the drying process and foam retention when dissolved in water. Due to its adhesiveness, it is used in cosmetics to promote the application of active ingredients on the body. It can be foreseen that pullulan is a new multifunctional biological product with great application prospects and development value, and has broad market prospects in the future. However, the yield of pullulan is low and cannot meet the market demand; at the same time, there are problems such as high viscosity of the fermentation liquid, low dissolved oxygen in the fermentation environment, weak metabolic capacity of the strain and high production cost when preparing pullulan by biological fermentation.

[0003] At present, the main production method of pullulan is microbial fermentation. Microbial fermentation has been increasingly valued for its advantages such as the use of renewable carbon sources, but the wild-type Aureobasidium pullulans strain has low fermentation titer and poor stability, which seriously limits the industrialization of pullulan production by fermentation. Therefore, it is necessary to construct a high-yield Aureobasidium pullulans strain to improve the production efficiency of pullulan. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a pullulan-producing Aureobasidium pullulans strain and a construction method and application thereof.

[0005] The technical solution adopted by the present invention is:

[0006] The invention discloses a pullulan-producing Aureobasidium pullulans strain, which is a Aureobasidium pullulans strain that continuously overexpresses α-phosphoglucomutase PGM and UDP-glucose pyrophosphorylase UGP.

[0007] Preferably according to the present invention, the gene sequence of the α-phosphoglucomutase PGM is SEQ ID NO.1, and the amino acid sequence of the α-phosphoglucomutase PGM is SEQ ID NO.2; the gene sequence of the uridine diphosphate glucose pyrophosphorylase UGP is SEQ ID NO.3, and the amino acid sequence of the uridine diphosphate glucose pyrophosphorylase UGP is SEQ ID NO.4.

[0008] Preferably according to the present invention, the pullulan-producing Aureobasidium sp. strain is Aureobasidium sp. S1, which was deposited in the China Center for Type Culture Collection on October 14, 2024, with the deposit address: Wuhan University, Wuhan, China, and the deposit number: CCTCC NO: M20242197.

[0009] The method for constructing the above-mentioned pullulan-producing Aureobasidium pullulans strain comprises the following steps:

[0010] (1) Construction of an overexpression vector for α-phosphoglucomutase PGM

[0011] The genome of wild-type Aureobasidium pullulans strain was used as a template to amplify the PGM gene sequence fragment by PCR reaction, and the PGM gene sequence fragment was cloned into the vector pLH454 to construct the PGM gene overexpression plasmid pFRD1.

[0012] (2) Obtaining a strain overexpressing α-phosphoglucomutase PGM

[0013] The gene overexpression plasmid pFRD1 of step (1) is transformed into the host strain Aureobasidium pullulans AP, and after screening, a strain overexpressing Aureobasidium pullulans α-phosphoglucomutase PGM is obtained;

[0014] (3) Construction of an overexpression vector for UDP-glucose pyrophosphorylase (UGP)

[0015] The genome of wild-type Aureobasidium pullulans strain was used as a template to amplify the UGP gene sequence fragment by PCR reaction, and the UGP gene sequence fragment was cloned into the vector pLH1080 to construct the UGP gene overexpression plasmid pFRD2;

[0016] (4) Obtaining a strain that co-overexpresses α-phosphoglucomutase PGM and UDP-glucose pyrophosphorylase UGP

[0017] The gene overexpression plasmid pFRD2 of step (3) is transformed into the budding Aureobasidium pullulans α-phosphoglucomutase PGM overexpression strain of step (2), and after screening, a budding Aureobasidium pullulans α-phosphoglucomutase PGM and uridine diphosphate glucose pyrophosphorylase UGP co-overexpression strain is obtained, which is a budding Aureobasidium pullulans high-yielding strain.

[0018] Preferably according to the present invention, the nucleotide sequence of the vector pLH454 in step (1) is as shown in SEQ ID NO.5.

[0019] Preferably according to the present invention, the nucleotide sequence of the vector pLH1080 in step (3) is as shown in SEQ ID NO.6.

[0020] Preferably, according to the present invention, the method for constructing the host strain Aureobasidium pullulans AP in step (2) comprises the following steps:

[0021] The plasmid pLH472 regulating Cre expression by Tet-on was transformed into a wild-type budding Aureobasidium pullulans strain, and the host strain budding Aureobasidium pullulans AP was obtained after screening. The host strain budding Aureobasidium pullulans AP can achieve the recombinant removal of the hygromycin resistance gene hph in the loxP-hph-loxP element, so that the hph marker can be repeatedly used to achieve continuous overexpression or knockout of multiple genes.

[0022] Further preferably, the nucleotide sequence of the plasmid pLH472 is shown as SEQ ID NO.7.

[0023] Application of the above-mentioned Aureobasidium pullulans strain in the production of pullulan.

[0024] The method for producing pullulan by fermentation using the above-mentioned Aureobasidium pullulans strain comprises the following steps:

[0025] The budding Aureobasidium pullulans strain is inoculated on a YPD solid culture medium plate, cultured until the mycelium covers the entire plate, the mycelium is collected and inoculated into a seed culture medium to obtain a seed liquid, the seed liquid is inoculated into a fermentation culture medium at an inoculation rate of 10-20% (V / V), and fermentation culture is performed to obtain a fermentation liquid containing pullulan.

[0026] Preferably according to the present invention, the composition and preparation method of the YPD medium are:

[0027] Glucose 20g / L, tryptone 20g / L, yeast extract 10g / L, solvent is water, high pressure sterilization at 115℃ for 20min; add 1.5% agar to the solid culture medium.

[0028] Preferably according to the present invention, the composition and preparation method of the seed culture medium are:

[0029] Sucrose 50g / L, sodium chloride 1g / L, magnesium sulfate heptahydrate 0.2g / L, anhydrous potassium hydrogen phosphate 6.3g / L, ammonium sulfate 0.6g / L, yeast extract 2g / L, the solvent is water, adjust the pH to 6.5, and sterilize at 115℃ for 30min.

[0030] Preferably according to the present invention, the composition and preparation method of the fermentation medium are:

[0031] Glucose 100g / L, sodium chloride 1g / L, magnesium sulfate heptahydrate 0.2g / L, anhydrous potassium hydrogen phosphate 6.3g / L, ammonium sulfate 0.6g / L, yeast extract 2g / L, Tween-80 0.05%, defoamer 3%, solvent is water, adjust pH to 5.5, and sterilize at 115℃ for 30min.

[0032] Beneficial effects:

[0033] 1. The budding Aureobasidium gene engineering strain of the present invention continuously overexpresses α-phosphoglucomutase PGM and uridine diphosphate glucose pyrophosphorylase UGP, which significantly improves the yield of pullulan produced by fermentation of budding Aureobasidium, improves the fermentation titer of the strain, and increases stability, provides an excellent strain for preparing pullulan by microbial fermentation, reduces the separation cost in the downstream purification process of pullulan, and provides an excellent strain for the industrial production of pullulan. The present invention overcomes the problem in the prior art that in the existing budding Aureobasidium fermentation process for producing pullulan, the fermentation titer of the strain is low and the stability is poor, resulting in an increase in the cost of the subsequent pullulan purification process and the inability to carry out industrial production.

[0034] 2. The genetically engineered strain of Aureobasidium pullulans of the present invention can be used for the production of pullulan. The strain was fermented in a 100L fermenter with a liquid volume of 60L for 3 days. HPLC analysis showed that the pullulan yield was 89.2g / L, which was 85.8% higher than the 48g / L of the host strain.

[0035] 3. The present invention transforms the plasmid pLH472 (Tet-on-cre, hph) for Tet-on regulated Cre expression into the genome of the wild-type Aureobasidium pullulans strain, and obtains Aureobasidium pullulans AP that can be induced to express Cre with doxycycline through transformant screening and hygromycin resistance gene recombination, and can achieve the recombination and removal of the hygromycin resistance gene hph in the loxP-hph-loxP element, so that the hph marker can be repeatedly used to achieve continuous overexpression or knockout of multiple genes.

[0036] 4. The present invention uses Aureobasidium pullulans as the base cell, analyzes the metabolic key points in the pullulan synthesis network, utilizes genome editing technology and large-fragment DNA assembly technology to strengthen and regulate the pullulan synthesis-related functional modules, constructs a cell factory with high pullulan production, and realizes efficient and green biomanufacturing of pullulan. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the plasmid pLH472 map.

[0038] Figure 2 It is the electrophoresis diagram of the PCR amplification product of the Cre-loxP system, where M is DNA Marker, N is the negative control, P is the positive control, and 1-4 are transformants that have successfully integrated the cre gene.

[0039] Figure 3 The double restriction enzyme electrophoresis diagram of the overexpression vector pFRD1; wherein, M is a DNA marker, and 1 is a double restriction enzyme digestion verification vector of EcoRⅠ and BamHⅠ.

[0040] Figure 4 Schematic diagram of the overexpression vector pFRD1.

[0041] Figure 5 1 is the PCR amplification electrophoresis diagram of the PGM gene transformant, wherein M is a DNA Marker, N is a negative control, P is a positive control, and 1-4 are the Aureobasidium pullulans transformants that successfully overexpress the PGM gene.

[0042] Figure 6 The double restriction enzyme electrophoresis diagram of the overexpression vector pFRD2; wherein, M is a DNA marker, and 1 is a double restriction enzyme digestion verification vector of EcoRⅠ and BamHⅠ.

[0043] Figure 7 Schematic diagram of the overexpression vector pFRD2.

[0044] Figure 8 PCR amplification electrophoresis diagram of UGP gene transformants, wherein M is DNA Marker, N is negative control, P is positive control, and 1-4 are Aureobasidium pullulans transformants that successfully overexpressed UGP gene.

[0045] Fig. 9 It is a bar graph showing the production of pullulan of the budding Pullulanase strain in a 5L fermenter.

[0046] Fig.10 This is a bar graph showing the pullulan production of the budding Pullulanase strain in a 20 L fermenter.

[0047] Fig.11This is a bar graph showing the pullulan production of the budding Pullulanase strain in a 100 L fermenter. DETAILED DESCRIPTION

[0048] For a better understanding of the present invention, the present invention is further described in detail below in conjunction with embodiments, but the scope of protection claimed by the present invention is not limited to the scope represented by the embodiments.

[0049] The raw materials used in the present invention, unless otherwise specified, are all conventional commercially available products. The methods used in the present invention, unless otherwise specified, are all conventional methods in the art. The quality of each substance used in the present invention is the quality for conventional use.

[0050] A pullulan-producing Aureobasidium pullulans genetically engineered strain, wherein the Aureobasidium pullulans genetically engineered strain is a Aureobasidium pullulans strain that continuously overexpresses α-phosphoglucomutase PGM and uridine diphosphate glucose pyrophosphorylase UGP;

[0051] Among them, the gene sequence of the α-phosphoglucomutase PGM is SEQ ID NO.1; the amino acid sequence of the α-phosphoglucomutase PGM is SEQ ID NO.2; the gene sequence of the uridine diphosphate glucose pyrophosphorylase UGP is SEQ ID NO.3; and the amino acid sequence of the uridine diphosphate glucose pyrophosphorylase UGP is SEQ ID NO.4.

[0052] In a preferred embodiment of the present invention, the pullulan-producing Aureobasidium sp. genetically engineered strain is Aureobasidium sp. S1, which was deposited in the China Center for Type Culture Collection on October 14, 2024, with a deposit address of Wuhan University, Wuhan, China, and a deposit number of CCTCC NO: M20242197.

[0053] The method for constructing the pullulan-producing Aureobasidium pullulans genetically engineered strain comprises the following steps:

[0054] (1) Construction of an overexpression vector for α-phosphoglucomutase PGM

[0055] The genome of wild-type Aureobasidium pullulans CGMCC 3.3984 strain (purchased from China General Microbiological Culture Collection Center) was used as a template to amplify the gene sequence fragment of PGM by PCR reaction, and the PCR amplification product was recovered to obtain the target fragment; the gene sequence fragment of PGM was cloned into the vector pLH454 (synthesized by Beijing BGI, SEQ ID NO.5) to construct the gene overexpression plasmid pFRD1 of PGM;

[0056] (2) Obtaining a strain overexpressing α-phosphoglucomutase PGM

[0057] The gene overexpression plasmid pFRD1 of step (1) is transformed into the host strain Aureobasidium pullulans AP, and the Aureobasidium pullulans α-phosphoglucomutase PGM overexpression strain AP-1 is obtained through transformant screening and hygromycin resistance gene recombination;

[0058] (3) Construction of an overexpression vector for UDP-glucose pyrophosphorylase (UGP)

[0059] The genome of wild-type Aureobasidium pullulans CGMCC 3.3984 strain was used as a template to amplify the gene sequence fragment of UGP by PCR reaction, and the PCR amplification product was recovered to obtain the target fragment; the gene sequence fragment of UGP was cloned into the vector pLH1080 (synthesized by Beijing BGI, SEQ ID NO.6) to construct the gene overexpression plasmid pFRD2 of UGP;

[0060] (4) Obtaining a strain that co-overexpresses α-phosphoglucomutase PGM and UDP-glucose pyrophosphorylase UGP

[0061] The gene overexpression plasmid pFRD2 of step (3) is transformed into the budding Aureobasidium pullulans α-phosphoglucomutase PGM overexpression strain AP-1 of step (2), and after transformant screening and hygromycin resistance gene recombination, the budding Aureobasidium pullulans α-phosphoglucomutase PGM and uridine diphosphate glucose pyrophosphorylase UGP co-overexpression strain S1 is obtained, which is a budding Aureobasidium pullulans genetically engineered strain producing pullulan.

[0062] The method for constructing the host strain Aureobasidium pullulans AP comprises the following steps:

[0063] The plasmid pLH472 (Tet-on-cre, hph, SEQ ID NO.7) synthesized by Beijing BGI and regulated by Tet-on Cre expression was transformed into the wild-type Aureobasidium pullulans CGMCC 3.3984 strain, and the host strain Aureobasidium pullulans AP was obtained through transformant screening and hygromycin resistance gene recombination. The host strain Aureobasidium pullulans AP can achieve the recombination removal of the hygromycin resistance gene hph in the loxP-hph-loxP element, so that the hph marker can be repeatedly used to achieve continuous overexpression or knockout of multiple genes. The specific mechanism is as follows:

[0064] An exogenous cre gene is integrated into the genome of the host strain Aureobasidium pullulans AP, and the gene is regulated and expressed by the Tet-on system. When the Aureobasidium pullulans AP is used as a starting bacterium for genetic modification, and loxP-hph-loxP is used as a screening marker, the Tet-on system can be activated by doxycycline to express the Cre recombinase, thereby realizing the recombination of the loxP-hph-loxP element and knocking out the resistance gene hph, thereby realizing continuous gene overexpression or knockout using an hph marker and achieving the ultimate goal of achieving no exogenous resistance gene residue in the genome of the engineered strain.

[0065] The application of the above-mentioned Aureobasidium pullulans genetic engineering strain in the production of pullulan.

[0066] The method for fermenting and producing pullulan using the above-mentioned Aureobasidium pullulans genetically engineered strain comprises the following steps:

[0067] The budding Aureobasidium pullulans genetically engineered strain is inoculated on a YPD solid culture medium plate, cultured until the mycelium covers the entire plate, the mycelium is collected and inoculated into a seed culture medium to obtain a seed liquid, the seed liquid is inoculated into a fermentation culture medium at an inoculation rate of 10-20% (V / V), and fermentation culture is performed to obtain a fermentation liquid containing pullulan.

[0068] Wherein, the composition and preparation method of the YPD medium are:

[0069] Glucose 20g / L, tryptone 20g / L, yeast extract 10g / L, solvent is water, autoclave at 115℃ for 20min; solid culture medium is added with 1.5% agar;

[0070] The composition and preparation method of the seed culture medium are as follows:

[0071] Sucrose 50g / L, sodium chloride 1g / L, magnesium sulfate heptahydrate 0.2g / L, anhydrous potassium hydrogen phosphate 6.3g / L, ammonium sulfate 0.6g / L, yeast extract 2g / L, solvent is water, adjust pH to 6.5, autoclave at 115℃ for 30min;

[0072] The components and preparation method of the fermentation medium are as follows:

[0073] Glucose 100g / L, sodium chloride 1g / L, magnesium sulfate heptahydrate 0.2g / L, anhydrous potassium hydrogen phosphate 6.3g / L, ammonium sulfate 0.6g / L, yeast extract 2g / L, Tween-80 0.05%, defoamer 3%, solvent is water, adjust pH to 5.5, and sterilize at 115℃ for 30min.

[0074] The primers involved in the examples have sequences as shown in Table 1:

[0075] Table 1. Primer sequences used

[0076]

[0077]

[0078] Example 1: Construction of Cre-loxP genetic operating system

[0079] The construction of the Cre-loxP genetic operating system includes the following steps:

[0080] The wild-type Aureobasidium pullulans CGMCC 3.3984 was used as the starting strain and the plasmid pLH472 ( Figure 1 , synthesized by Beijing BGI, SEQ ID NO.7), the cre gene regulated by Tet-on was integrated into the wild-type Aureobasidium pullulans CGMCC 3.3984 genome and the selection marker gene hph was recombined to obtain Aureobasidium pullulans AP. The Aureobasidium pullulans AP genome was extracted, P1 / P2 (Table 1) were designed as verification primers, and PCR was used to verify whether the Tet-on-cre element was integrated into the genome. The results are as follows Figure 2 As shown in the figure, Aureobasidium pullulans AP is the host strain for constructing a genetically engineered strain of Aureobasidium pullulans with high pullulan production.

[0081] The successful integration of the Tet-on-cre element in the budding Aureobasidium AP strain enables the budding Aureobasidium AP strain to be induced to express Cre recombinase in a culture medium containing doxycycline. The Cre recombinase can recognize the loxP site, thereby recombining the hph gene in the loxP-hph-loxP, realizing the repeated use of the hph marker, and then continuously applying the same hph marker to achieve overexpression or knockout of multiple genes.

[0082] The above-mentioned induction recombination method is: about 2000 spores of the Cre-loxP genetic operating system overexpression clones are evenly spread on a YPD solid medium containing 150 μg / mL doxycycline, cultured at 25°C until a single clone grows, and then 100 clones are randomly selected and transferred to a YPD solid medium for culture at 28°C for 24 hours, and then the clones are transferred one by one to a YPD solid medium containing 150 μg / mL hygromycin and cultured at 28°C for 72 hours, and finally the transformants with successful resistance marker induction are screened by phenotypic observation, that is, the clones that cannot grow on the YPD solid medium containing hygromycin but can grow normally on the YPD solid medium are the transformants with successful hph marker induction recombination. One of the correct clones is selected and named as budding short bacillus AP. The budding short bacillus AP is a host strain for constructing a budding short bacillus genetic engineering strain containing a Cre-loxP genetic operating system and the hygromycin resistance gene hph is recombinantly removed.

[0083] Example 2: Construction of a genetically engineered strain of Aureobasidium pullulans with high pullulan production

[0084] The construction of a genetically engineered strain of Aureobasidium pullulans with high pullulan production comprises the following steps:

[0085] (1) Construction of PGM gene overexpression vector:

[0086] In order to amplify the PGM gene sequence fragment, the amplification primers PGM-F and PGM-R (Table 1) were designed using the genome of Aureobasidium pullulans as a template. The genome of the wild-type Aureobasidium pullulans CGMCC 3.3984 strain was used as a template to recover the PGM gene sequence fragment by PCR amplification. Then, the PGM gene sequence fragment was ligated with the starting vector pLH454 (synthesized by Beijing BGI, SEQ ID NO.5) linearized by EcoRⅠ / BamHⅠ double restriction digestion using the C113-ClonExpress-MultiS One Step Cloning Kit of Novozymes. The ligation product was transformed into Escherichia coli JM109 competent cells, evenly spread on LB solid medium containing 100 μg / mL kanamycin, and inverted and cultured at 37°C overnight. Single clones were picked for colony PCR verification and plasmid EcoRⅠ / BamHⅠ double restriction digestion verification. The double restriction digestion verification results are shown in Figure 2. Figure 3 As shown, two clear bands of 965 bp and 4869 bp were obtained, and the overexpression vector pFRD1 successfully connected with the PGM gene sequence fragment was obtained. Figure 4 shown.

[0087] The gene sequence of the PGM is SEQ ID NO.1, and the amino acid sequence is SEQ ID NO.2, which contains 554 amino acids.

[0088] The LB solid medium containing 100 μg / mL kanamycin has the following components: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder, and the solvent is water. Sterilize at 121°C for 20 min. After sterilization, add kanamycin to a final concentration of 100 μg / mL when the culture is cooled to about 50°C.

[0089] (2) Obtaining strains overexpressing PGM genes:

[0090] First, use a cotton swab to wash the spores of Aureobasidium AP from the plate, inoculate them into YPD liquid medium, and culture them at 250 rpm and 28°C for 14 h. 600 When the pH value is 1, place the conical flask containing the bacterial solution on ice for 30 minutes; then centrifuge at 8000rpm for 1 minute in a 4℃ centrifuge and discard the supernatant; add 25mL sorbitol-trisodium citrate solution to gently resuspend the bacterial precipitate, and centrifuge at 8000rpm and 4℃ for 1 minute; after discarding the supernatant, continue to add 25mL sorbitol-trisodium citrate solution to gently resuspend the bacterial precipitate, and centrifuge at 8000rpm and 4℃ for 1 minute; after discarding the supernatant, add 5mL sorbitol-trisodium citrate solution to gently resuspend the bacterial precipitate, and then add 1mL enzymolysis solution, and enzymolyze at 100rpm and 30℃ for 30 minutes; centrifuge at 4000rpm and 4℃ for 5 minutes and discard the supernatant; add 25mL sorbitol solution, and centrifuge at 4000rpm and 4℃ for 5 minutes; after discarding the supernatant, add 5mL sorbitol-trisodium citrate solution to gently resuspend the bacterial precipitate, and then add 1mL enzymolysis solution, and enzymolyze at 100rpm and 30℃ for 30 minutes; centrifuge at 4000rpm and 4℃ for 5 minutes and discard the supernatant; Gently resuspend the bacterial precipitate with alcohol solution to obtain the AP competent state of budding Aureobasidium; take 100 μL of competent state to a new 2mL sterile centrifuge tube, add 10 μg of high-fidelity enzyme to amplify the linearized pFRD1 plasmid fragment, and mix evenly; then use an electroporator to perform electric shock, and quickly add 1mL PERS solution after electric shock; culture and recover at 28℃, 100rpm for 4h; after centrifugation at 4000rpm for 5min, discard 900 μL of supernatant, mix the remaining supernatant with the bacterial cells, and then plate on YPD solid medium containing corresponding resistance (containing 100 μg / mL hygromycin) for culture until transformants grow out, and screen the transformants. The transformant phenotype is insensitive to hygromycin resistance. The genome of such transformants is verified, and the verification primers P3 / P4 (Table 1) are designed. The PCR amplification results are shown in Figure 5 As shown, the transformants met the PgpdA+Ttrpc amplification positive test, and one of the correct and highest-yielding PGM overexpression clones was selected for induced recombination of the resistance marker hygromycin, thereby obtaining the Aureobasidium pullulans PGM gene overexpression strain AP-1 that was not hygromycin-resistant.

[0091] The preparation method of the sorbitol-sodium citrate solution is as follows: weigh 182.17 g of D-sorbitol and 14.7 g of trisodium citrate dihydrate, add appropriate amount of distilled water to dissolve, then add distilled water to make up to 1 L, and sterilize at 121° C. for 20 min.

[0092] The preparation method of the PERS solution is as follows: weigh 20 g of peptone, 10 g of yeast extract, and 20 g of glucose, add appropriate amount of distilled water to dissolve, then add distilled water to make the volume to 1 L, and sterilize at 121° C. for 20 min.

[0093] The preparation method of the sorbitol solution is as follows: weigh 182.17 g of D-sorbitol, add an appropriate amount of distilled water to dissolve it, then add distilled water to make the volume to 1 L, and sterilize at 121° C. for 20 min.

[0094] The preparation method of the enzymatic hydrolysate is as follows: weigh 0.5 g of lysing enzyme (Sigma Aldrich, D9515) and 0.2 g of lysing enzyme (Sigma lyzing Enzyme), add 2 mL of sterile sorbitol-sodium citrate solution to dissolve, and filter and sterilize to obtain the enzymatic hydrolysate.

[0095] The inducing recombination method comprises the following steps: about 2000 spores of transformants with a phenotype of being insensitive to hygromycin resistance are evenly spread on a YPD solid culture medium containing 150 μg / mL doxycycline, cultured at 25°C until a single clone grows out, and then 100 single clones are randomly selected and transferred to a YPD solid culture medium for culture at 28°C for 24 hours, and then the clones are transferred one by one to a YPD solid culture medium containing 150 μg / mL hygromycin and cultured at 28°C for 72 hours, and finally, transformants with successful resistance marker induction are screened through phenotypic observation, that is, clones that cannot grow on a YPD solid culture medium containing hygromycin but can grow normally on a YPD solid culture medium are transformants with successful hph marker induced recombination.

[0096] (3) Construction of UGP gene overexpression vector:

[0097] In order to amplify the UGP gene sequence fragment, the amplification primers UGP-F and UGP-R (Table 1) were designed using the genome of Aureobasidium pullulans as a template. The genome of the wild-type Aureobasidium pullulans CGMCC 3.3984 strain was used as a template to recover the UGP gene sequence fragment by PCR amplification. Then, the UGP gene sequence fragment was ligated with the starting vector pLH1080 (synthesized by Beijing BGI, SEQ ID NO.6) linearized by EcoRⅠ / BamHⅠ double restriction digestion using the C113-ClonExpress-MultiS One Step Cloning Kit of Novozymes. The ligation product was transformed into Escherichia coli JM109 competent cells, evenly spread on LB solid culture medium containing 100 μg / mL kanamycin, and inverted and cultured overnight at 37°C. Single clones were picked for colony PCR verification and plasmid EcoRⅠ / BamHⅠ double restriction digestion verification. The double restriction digestion verification results are shown in Figure 2. Figure 6 As shown, two clear bands of 1856 bp and 10352 bp were obtained, and the overexpression vector pFRD2 successfully connected to the UGP gene sequence fragment was obtained. Figure 7 shown.

[0098] The gene sequence of the UGP is SEQ ID NO.3, and the amino acid sequence is SEQ ID NO.4, which contains 516 amino acids.

[0099] (4) Obtaining strains co-overexpressing PGM and UGP genes

[0100] First, the spores of the AP-1 strain overexpressing PGM were washed off the plate with a cotton swab and inoculated into YPD liquid medium. The culture was carried out at 250 rpm and 28 °C for 14 h. 600When the pH value is 1, place the conical flask containing the bacterial solution on ice for 30 minutes; then centrifuge at 8000rpm for 1 minute in a 4℃ centrifuge and discard the supernatant; add 25mL of sorbitol-trisodium citrate solution to gently resuspend the bacterial precipitate, centrifuge at 8000rpm and 4℃ for 1 minute; discard the supernatant and continue to add 25mL of sorbitol-trisodium citrate solution to gently resuspend the bacterial precipitate, centrifuge at 8000rpm and 4℃ for 1 minute; discard the supernatant and add 5mL of sorbitol-trisodium citrate solution to gently resuspend the bacterial precipitate, then add 1mL of enzyme The solution was enzymatically hydrolyzed at 100 rpm and 30 °C for 30 min; the supernatant was discarded after centrifugation at 4000 rpm and 4 °C for 5 min; 25 mL of sorbitol solution was added, and the supernatant was centrifuged at 4000 rpm and 4 °C for 5 min; after discarding the supernatant, the bacterial precipitate was gently resuspended with 5 mL of sorbitol solution to obtain the competent state of Aureobasidium shortrum S1; 100 μL of competent state was taken to a new 2 mL sterile centrifuge tube, and 10 μg of high-fidelity enzyme amplified linearized pFRD2 plasmid fragment was added, and mixed evenly; then electroporated with an electroporator, and 1 mL was quickly added after electroporation. PERS solution; 28 ℃, 100 rpm culture recovery 4h; 4000 rpm centrifugation for 5min, discard 900 μL supernatant, mix the remaining supernatant with the bacteria and plate on YPD solid medium (containing 100 μg / mL hygromycin) containing corresponding resistance for culture until transformants grow out, and screen the transformants. The transformant phenotype is insensitive to hygromycin resistance. The genome of such transformants is verified, and the verification primers P5 / P6 (Table 1) are designed and verified. The PCR amplification results are shown in Figure 8 As shown, the transformants satisfied the PgdhA+Ttrpc amplification positive, and one of the correct and highest-yielding UGP overexpression clones was picked for induced recombination of the resistance marker hygromycin, thereby obtaining the budding Aureobasidium pullulans PGM and UGP gene overexpression strain S1 that was not hygromycin-resistant, that is, the budding Aureobasidium pullulans genetically engineered strain with high pullulan production.

[0101] The inducing recombination method is as follows: about 2000 spores of transformants with a phenotype of hygromycin resistance insensitive are evenly spread on a YPD solid culture medium containing 150 μg / mL doxycycline, cultured at 25°C until a single clone grows out, and then 100 single clones are randomly selected and transferred to a YPD solid culture medium for culture at 28°C for 24 hours, and then the clones are transferred one by one to a YPD solid culture medium containing 150 μg / mL hygromycin and cultured at 28°C for 72 hours, and finally, transformants with successful resistance marker induction are screened by phenotypic observation, that is, clones that cannot grow on a YPD solid culture medium containing hygromycin but can grow normally on a YPD solid culture medium are transformants with successful hph marker induced recombination.

[0102] The above-mentioned high-pullulan-producing Aureobasidium sp. genetically engineered strain is Aureobasidium sp. S1, which was deposited in the China Center for Type Culture Collection on October 14, 2024, with the deposit address: Wuhan University, Wuhan, China, and the deposit number: CCTCC NO: M20242197.

[0103] Example 3: Fermentation production of pullulan by genetically engineered strains of Aureobasidium pullulans

[0104] Pullulan was produced by fermentation in a 5L fermenter using the genetically engineered strain of Aureobasidium pullulans constructed in Example 2, and the specific steps are as follows:

[0105] (1) Inoculate the genetically engineered strain of Aureobasidium pullulans on a YPD solid culture medium plate and place it in an incubator at 28° C. and culture it upside down for 4 days until the bacteria cover the entire culture medium plate;

[0106] (2) collecting mycelium of the genetically engineered strain of Aureobasidium pullulans and inoculating it into a seed culture medium, and culturing it at 28° C. and 250 rpm for 16 h to obtain a seed solution;

[0107] (3) 300 mL of the seed solution was transferred to a 5-L fermenter containing 3 L of fermentation medium, and cultured at 28° C., 400 rpm, and an aeration rate of 1 vvm for 3 days to obtain a fermentation broth containing pullulan.

[0108] The fermentation broth was collected and test samples were prepared, and the pullulan content in the samples was determined by HPLC.

[0109] The preparation method of the test sample is as follows: 2 mL of the fermentation liquid that has been shaken and evenly mixed is taken, the supernatant is taken after centrifugation, diluted 40 times with a 0.5 mol / L sodium nitrate aqueous solution, filtered through a 0.22 μm inorganic filter membrane, and then stored in a liquid phase vial for HPLC detection.

[0110] The HPLC method for detecting pullulan is: Agilent high performance liquid chromatograph differential detector, SB-806HQ analytical column (300mm×8.0mm), mobile phase: 0.5mol / L sodium nitrate aqueous solution, flow rate 0.5mL / min, column temperature 30°C, wavelength 193nm, injection volume 20μL.

[0111] The results showed that compared with the starting strain Aureobasidium pullulans AP, the pullulan production of the Aureobasidium pullulans genetically engineered strain was significantly increased. Fig. 9 The pullulan yield of the starting strain Aureobasidium pullulans AP was 48 g / L, and the pullulan yield of the genetically engineered strain of Aureobasidium pullulans was 83.9 g / L, which was a significant increase of 74.8% compared with the starting strain.

[0112] Example 4: Small-scale test for the production of pullulan by fermentation of genetically engineered strains of Aureobasidium pullulans

[0113] Pullulan was produced by fermentation in a 20 L fermenter using the Aureobasidium pullulans genetically engineered strain constructed in Example 2. The specific steps are as follows:

[0114] (1) Inoculate the genetically engineered strain of Aureobasidium pullulans on a YPD solid culture medium plate and incubate it upside down in a 28°C incubator for 4 days until the bacterial cells cover the entire culture medium plate;

[0115] (2) collecting mycelium of the genetically engineered strain of Aureobasidium pullulans and inoculating it into a seed culture medium, and culturing it at 28° C. and 250 rpm for 16 h to obtain a seed solution;

[0116] (3) 2 L of seed liquid was transferred to a 20 L fermentation tank containing 12 L of fermentation medium, and cultured at 28° C., 400 rpm, and aeration volume of 1 vvm for 3 days to obtain a fermentation broth containing pullulan.

[0117] The fermentation broth was collected, and a test sample was prepared according to the method of Example 3, and the pullulan content therein was determined by HPLC. The results showed that compared with the starting strain Aureobasidium pullulans AP, the pullulan yield of the Aureobasidium pullulans genetically engineered strain was significantly increased. Fig.10 The pullulan yield of the starting strain Aureobasidium pullulans AP was 48 g / L, and the pullulan yield of the genetically engineered strain of Aureobasidium pullulans was 85.6 g / L, which was 78.3% higher than that of the starting strain, a significant increase.

[0118] Example 5: Chinese scale-up production of pullulan by fermentation of genetically engineered Aureobasidium pullulans strains

[0119] The budding Aureobasidium pullulans genetic engineering strain constructed in Example 2 was fermented in a 100 L fermenter to produce pullulan, and the specific steps were as follows:

[0120] (1) Inoculate the genetically engineered strain of Aureobasidium pullulans on a YPD solid culture medium plate and place it in an incubator at 28° C. and culture it upside down for 4 days until the bacteria cover the entire culture medium plate;

[0121] (2) collecting mycelium of the genetically engineered strain of Aureobasidium pullulans and inoculating it into a seed culture medium, and culturing it at 28° C. and 250 rpm for 16 h to obtain a seed solution;

[0122] (3) 10 L of the seed solution was transferred to a 100 L fermentation tank containing 60 L of fermentation medium, and cultured at 28° C., 250 rpm, and an aeration rate of 1 vvm for 3 days to obtain a fermentation broth containing pullulan.

[0123] The fermentation broth was collected, and a test sample was prepared according to the method of Example 3, and the pullulan content therein was determined by HPLC. The results showed that compared with the starting strain Aureobasidium pullulans AP, the pullulan yield of the Aureobasidium pullulans genetically engineered strain was significantly increased. Fig.11 The pullulan yield of the starting strain Aureobasidium pullulans AP was 48 g / L, and the pullulan yield of the genetically engineered strain of Aureobasidium pullulans was 89.2 g / L, which was 85.8% higher than that of the starting strain, a significant increase.

[0124] The budding Aureobasidium pullulans genetic engineering strain constructed by the present invention can produce a high amount of pullulan, and compared with the starting strain, the yield of pullulan is greatly improved. In a 100L fermentation tank with a liquid volume of 60L, the yield of pullulan is increased from 48g / L of the starting strain to 89.2g / L, the fermentation titer of the strain is improved, the fermentation stability is increased, a certain foundation is laid for realizing the industrial fermentation production of pullulan, and an excellent strain is provided.

Claims

1. A pullulan-producing Aureobasidium pullulans strain, characterized in that: The budding Aureobasidium pullulans strain is a budding Aureobasidium pullulans strain that continuously overexpresses α-phosphoglucomutase PGM and uridine diphosphate glucose pyrophosphorylase UGP.

2. The pullulan-producing Aureobasidium pullulans strain according to claim 1, characterized in that: The gene sequence of the α-phosphoglucomutase PGM is SEQ ID NO.1, and the amino acid sequence of the α-phosphoglucomutase PGM is SEQ ID NO.2; the gene sequence of the uridine diphosphate glucose pyrophosphorylase UGP is SEQ ID NO.3, and the amino acid sequence of the uridine diphosphate glucose pyrophosphorylase UGP is SEQ ID NO.

4.

3. The pullulan-producing Aureobasidium pullulans strain according to claim 1, characterized in that: The pullulan-producing Aureobasidium sp. strain is Aureobasidium sp. S1, which was deposited in the China Center for Type Culture Collection on October 14, 2024, with the deposit address: Wuhan University, Wuhan, China, and the deposit number: CCTCC NO: M20242197.

4. The method for constructing the pullulan-producing Aureobasidium pullulans strain according to claim 1, characterized in that: The steps include: (1) Construction of an overexpression vector for α-phosphoglucomutase PGM The genome of wild-type Aureobasidium pullulans strain was used as a template to amplify the PGM gene sequence fragment by PCR reaction, and the PGM gene sequence fragment was cloned into the vector pLH454 to construct the PGM gene overexpression plasmid pFRD1. (2) Obtaining a strain overexpressing α-phosphoglucomutase PGM The gene overexpression plasmid pFRD1 of step (1) is transformed into the host strain Aureobasidium pullulans AP, and after screening, a strain overexpressing Aureobasidium pullulans α-phosphoglucomutase PGM is obtained; (3) Construction of an overexpression vector for UDP-glucose pyrophosphorylase (UGP) The genome of wild-type Aureobasidium pullulans strain was used as a template to amplify the UGP gene sequence fragment by PCR reaction, and the UGP gene sequence fragment was cloned into the vector pLH1080 to construct the UGP gene overexpression plasmid pFRD2; (4) Obtaining a strain that co-overexpresses α-phosphoglucomutase PGM and UDP-glucose pyrophosphorylase UGP The gene overexpression plasmid pFRD2 of step (3) is transformed into the budding Aureobasidium pullulans α-phosphoglucomutase PGM overexpression strain of step (2), and after screening, a budding Aureobasidium pullulans α-phosphoglucomutase PGM and uridine diphosphate glucose pyrophosphorylase UGP co-overexpression strain is obtained, which is a budding Aureobasidium pullulans strain producing pullulan.

5. The construction method according to claim 4, characterized in that: The nucleotide sequence of the vector pLH454 in step (1) is shown in SEQ ID NO.5; Preferably, the nucleotide sequence of the vector pLH1080 in step (3) is as shown in SEQ ID NO.

6.

6. The construction method according to claim 4, characterized in that: The method for constructing the host strain Aureobasidium pullulans AP in step (2) comprises the following steps: The plasmid pLH472 regulating Cre expression by Tet-on was transformed into a wild-type budding Aureobasidium pullulans strain, and the host strain budding Aureobasidium pullulans AP was obtained after screening. The host strain budding Aureobasidium pullulans AP can achieve the recombinant removal of the hygromycin resistance gene hph in the loxP-hph-loxP element, so that the hph marker can be repeatedly used to achieve continuous overexpression or knockout of multiple genes.

7. The construction method according to claim 6, characterized in that: The nucleotide sequence of the plasmid pLH472 is shown in SEQ ID NO.

7.

8. Use of the Aureobasidium pullulans strain according to claim 1 in producing pullulan.

9. A method for producing pullulan by fermentation using the Aureobasidium pullulans strain according to claim 1, characterized in that: The steps include: The budding Aureobasidium pullulans strain is inoculated on a YPD solid culture medium plate, cultured until the mycelium covers the entire plate, the mycelium is collected and inoculated into a seed culture medium to obtain a seed liquid, the seed liquid is inoculated into a fermentation culture medium at an inoculation rate of 10-20% (V / V), and fermentation culture is performed to obtain a fermentation liquid containing pullulan.

10. The method according to claim 9, characterized in that The composition and preparation method of the seed culture medium are as follows: Sucrose 50g / L, sodium chloride 1g / L, magnesium sulfate heptahydrate 0.2g / L, anhydrous potassium hydrogen phosphate 6.3g / L, ammonium sulfate 0.6g / L, yeast extract 2g / L, solvent is water, adjust pH to 6.5, autoclave at 115℃ for 30min; Preferably, the composition and preparation method of the fermentation medium are: Glucose 100g / L, sodium chloride 1g / L, magnesium sulfate heptahydrate 0.2g / L, anhydrous potassium hydrogen phosphate 6.3g / L, ammonium sulfate 0.6g / L, yeast extract 2g / L, Tween-80 0.05%, defoamer 3%, solvent is water, adjust pH to 5.5, and sterilize at 115℃ for 30min.

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