Recombinant yarrowia lipolytica for synthesizing trehalose as well as construction method and application of recombinant yarrowia lipolytica

Abstract

The invention discloses recombinant yarrowia lipolytica for synthesizing trehalose as well as a construction method and application of the recombinant yarrowia lipolytica. The method comprises the following steps: by taking yarrowia lipolytica capable of synthesizing erythritol as a synthetic chassis, connecting a trehalose synthase gene and an amylase gene in series to form a surface display gene expression cassette, and transforming the yarrowia lipolytica to form recombinant yarrowia lipolytica CGMCC No.31034; then, glucose is firstly fermented by the recombinant saccharomycetes to generate erythritol, and yeast cells are separated to serve as a whole-cell catalyst to convert starch in one step to generate trehalose. After the conversion is finished, adding a nitrogen source and inorganic salt into the conversion liquid, and inoculating saccharomyces cerevisiae to decompose a small amount of miscellaneous glucose in the conversion liquid to obtain the high-purity trehalose. As yeast whole-cell transformation is adopted in the method, compared with processes such as breaking cells from escherichia coli to purify enzyme and the like, the steps are reduced, and the enzyme preparation cost is avoided.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a proprietary Yarrowia lipolytica for synthesizing trehalose and an application thereof. Background Art

[0002] Trehalose is composed of two molecules of α-glucose connected by an α-1,1 glycosidic bond. It is a non-reducing disaccharide. Trehalose is found in various animal, plant and microbial cells, especially in low-water or dehydrated cells. It has an important physiological function in maintaining cell tolerance.

[0003] Trehalose has become one of the raw materials for cosmetics due to its excellent moisturizing properties. It can be used in skin cosmetics such as facial cleansers to inhibit skin dryness; it can be used in lipsticks, mouth fresheners, and oral fragrances as a sweetener, flavor improver, and quality improver. Anhydrous trehalose can be used in skin creams, etc. as a dehydrating agent for phospholipids and enzymes. Since trehalose has very important effects in the fields of medicine, food, and cosmetics, and new effects are constantly being developed and applied, how to prepare it more economically on a large scale is of great value.

[0004] At present, the main method for producing trehalose is enzyme conversion, using E. coli for enzyme expression. The expression process is relatively complicated, and antibiotics (such as kanamycin or ampicillin) are required to maintain the screening pressure, and expensive inducers (such as IPTG) are required. In addition, the E. coli cell wall needs to be broken to release trehalose synthase. At the same time, harmful substances such as endotoxins of E. coli are also released. At the same time, E. coli is a pathogenic microorganism and is not suitable for food preparation. The whole process is relatively cumbersome.

[0005] Therefore, researching and developing a new synthetic pathway that can efficiently convert starch into trehalose has very important application value for improving production efficiency and reducing production costs. Summary of the invention

[0006] In view of the shortcomings of the prior art, the object of the present invention is to provide a proprietary Yarrowia lipolytica for synthesizing trehalose, thereby achieving efficient synthesis of trehalose.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows: A proprietary Yarrowia lipolytica for synthesizing trehalose, wherein the proprietary Yarrowia lipolytica is the Yarrowia lipolytica strain Yarrowia lipolytica CGMCC No. 31034, which was deposited on June 21, 2024 at the General Microbiology Center of China Culture Collection Administration (Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing).

[0008] Another object of the present invention is to overcome the shortcomings of the existing methods for producing trehalose and to provide a method for constructing an engineered strain of a proprietary Yarrowia lipolytica that can efficiently synthesize trehalose.

[0009] In order to achieve the above object, the technical solution of the present invention is as follows: Yarrowia lipolytica strains were used as Yarrowia lipolytica strains that can synthesize citric acid. Yarrowia lipolytica strains are good hosts for synthesizing citric acid. There are many literatures that have documented that Yarrowia lipolytica strains can synthesize citric acid using glucose or glycerol as raw materials (e.g., Cavallo et al. Yarrowia lipolytica: a model yeast for citric acid production. FEMS Yeast Research, 17, 2017, fox084). Three enzymes (amylase, saccharifying enzyme, trehalose synthase) were displayed on the surface of yeast cells that can synthesize citric acid, and the whole cells were used as enzyme catalysts to catalyze the synthesis of trehalose from soluble starch.

[0010] Specifically, the method for constructing a proprietary Yarrowia lipolytica for synthesizing trehalose is to use a yeast strain that can synthesize citric acid, such as Yarrowia lipolytica W29, as a starting strain (Zhang et al. 2019. pHselectively regulates citric acid and lipid production in Yarrowia lipolyticaW29 during nitrogen-limited growth on glucose, Journal of Biotechnology, 290,10-15), which is a model strain and can be obtained from a public strain library. Then, the gene surface display expression frame containing amylase-saccharifying enzyme and trehalose synthase is transferred into the yeast. Therefore, the yeast contains the coding sequence of amylase-saccharifying enzyme and trehalose synthase. The deoxyribonucleic acid coding sequences of these three enzymes have been made public and can be obtained free of charge from the public GenBank database. For example, the amylase coding sequence from Bacillus subtilis NA64 (SEQ ID No. 1), the saccharifying enzyme gene from Bacillus stearothermophilus (SEQ ID No. 2), the amylase gene (SEQ ID No. 1) and the saccharifying enzyme gene (SEQ ID No. 2) of Bacillus subtilis NA64 fused together to form SEQ ID No. 3, and the trehalose synthase coding sequence (TreS) (SEQ ID No. 4) from the thermoacidophilic archaeon Picroplilus torridus can be retrieved by those skilled in the art from the GenBank public database.

[0011] According to the codon preference of Yarrowia lipolytica, an expression cassette containing an α-amylase-saccharifying enzyme fusion enzyme and a trehalose synthase coding sequence is artificially synthesized, and these expression cassettes are transformed into a Yarrowia lipolytica starting strain capable of synthesizing citric acid, and yeast transformants containing both α-amylase-saccharifying enzyme and trehalose synthase expression cassettes are screened in a screening medium, and single colony clones are picked out for identification. The sequence of the α-amylase-saccharifying enzyme fusion gene expression cassette is such as SEQ ID No.5, which contains a mycophenolic acid screening gene; the sequence of the trehalose synthase gene expression cassette is such as SEQ ID No.6, which contains a sucrase screening gene. The specific screening method is detailed in Example 1.

[0012] The expression cassette containing the α-amylase-glucoamylase fusion gene contains the following DNA components in addition to the mycophenolic acid selection gene: upstream 5' end homology arm (26S rDNA) - promoter sequence (hp4d) - α-amylase-glucoamylase fusion gene - surface display protein gene (cpw130) - terminator sequence (Ter) - mycophenolic acid selection marker sequence - downstream 3' end homology arm (26SrDNA), the entire sequence is SEQ ID No. 5; The expression cassette containing the trehalose synthase gene, in addition to the sucrase screening gene, also contains the following DNA components: upstream 5' end homology arm (26S rDNA) - promoter sequence (hp4d) - surface display protein gene (Pir1) - trehalose synthase gene - terminator sequence (Ter) - sucrase screening marker sequence - downstream 3' end homology arm (26S rDNA), and the entire sequence is SEQ ID No.6.

[0013] Another object of the present invention is to provide a method for synthesizing trehalose using the proprietary Yarrowia lipolytica as described above, the method comprising the following steps: S1. Cultivate the engineered proprietary Yarrowia lipolytica strain (a strain displaying amylase-saccharifying enzyme and trehalose synthase on its surface) in a medium containing a carbon source, a nitrogen source, an inorganic salt and water, and perform oscillation or stirring fermentation culture at an initial pH value of 2.0 to 8.0 and a temperature of 25 to 35°C to synthesize citric acid first. After the fermentation, separate the bacterial liquid to obtain yeast cells and fermentation liquid, and the fermentation liquid is used to separate citric acid; wash the yeast cells with pure water three times to remove citric acid and other impurities inside and outside the cells; S2, adding a soluble starch aqueous solution to the cleaned yeast cells in S1, and converting and synthesizing trehalose at 50-80° C. The conversion time is 20-30 hours; S3, adding nitrogen source and inorganic salt to the trehalose-containing conversion solution described in S2, inoculating Saccharomyces cerevisiae for fermentation and culturing, and fermenting for 24-36 hours at a fermentation temperature of 28-32°C; S4, separating and purifying high-purity trehalose from S3.

[0014] Preferably, in step S1, the initial pH value of the fermentation is 4-7, more preferably 5.5; the initial temperature of the fermentation is 25-35°C, more preferably 28°C-32°C, most preferably 30°C.

[0015] Preferably, the culture medium for synthesizing citric acid is a culture medium containing a carbon source, a nitrogen source, an inorganic salt and water, and the culture conditions are shaking or stirring at a pH value of 2.5 to 8.0 and a temperature of 25 to 35° C. More preferably, the pH value is 5.0 to 6.0. The culture temperature is preferably 28 to 32° C., more preferably 30° C.

[0016] Preferably, the carbon source is solid glucose or liquid glucose, and the amount of the carbon source is 50 to 350 g / L; the nitrogen source is a mixture of one or more of yeast powder, yeast extract, corn syrup, diammonium phosphate, and ammonium citrate, and the amount of the nitrogen source is 5 to 25 g / L; the inorganic salt is one or more of magnesium sulfate, manganese chloride, and copper chloride, and the amount of the inorganic salt is 0 to 2.5 g / L. The above-mentioned nitrogen source dosage is the conventional dosage in this field and can be selected according to actual needs; for example, it can be: the nitrogen source addition amount is 0.2% to 4% (mass volume percentage), and it is adjusted as needed within this range. Inorganic salts generally provide trace elements for microbial growth and are used in very small amounts. The amount of inorganic salt in the present invention is 0.01 g / L to 2 g / L, and it is adjusted as needed within this range.

[0017] More preferably, the amount of the carbon source is 200-300 g / L; the amount of the nitrogen source is 5-20 g / L; the amount of the inorganic salt is 0.01-2 g / L, more preferably 0.005-2 g / L.

[0018] Preferably, in step S2, the yeast cells convert maltose into trehalose comprising the following steps: S21, adding soluble starch with a mass percentage concentration of 10% to 30% to the yeast cells, and performing conversion by shaking or stirring at a temperature of 50 to 80° C. and a pH of 3 to 8 for 20 to 30 hours; S22, separating the transformation liquid into bacterial liquid to obtain enzyme-containing yeast cells and cell transformation liquid containing trehalose; S23. The enzyme-containing yeast cells obtained in step S22 can be repeatedly subjected to steps S21 and S22. That is, the separated enzyme-containing yeast cells can also be used as enzyme catalysts to convert 5% to 30% of soluble starch again to generate trehalose. The cells can be reused 8 to 10 times.

[0019] The separation and purification method for obtaining trehalose comprises conventional steps such as filtration sterilization, concentration, ion exchange, re-concentration, decolorization, crystallization, etc.

[0020] Preferably, the yeast used in the present invention to purify the conversion liquid is Saccharomyces cerevisiae, which can grow on a culture medium with maltose and glucose as the sole carbon source, but cannot grow on a culture medium with trehalose as the sole carbon source. The conversion liquid is inoculated with the purifying yeast for fermentation and culture to obtain pure trehalose. Saccharomyces cerevisiae has a wide range of sources and is a food safety microorganism, which can be isolated from the surface of fruits such as grapes.

[0021] Compared with the existing trehalose synthesis technology, the present invention has the following beneficial effects: (1) The present invention uses the food-safe Yarrowia lipolytica as the enzyme expression host, which is permitted for use in the fermentation and synthesis of food additives. The previous technology used Escherichia coli as the expression host, and the use of Escherichia coli in the food additive fermentation industry is subject to certain restrictions; (2) The Yarrowia lipolytica surface-displayed synthetic enzyme strain used in the present invention first synthesizes citric acid, and the resulting yeast cells are used as cell catalysts to convert soluble starch into trehalose. The previous methods all used Escherichia coli to express the enzyme alone, and then crushed the cells to release the enzyme, which was then added to the conversion solution. The operation was relatively complicated and the cost was high. The enzyme-containing cells obtained by this method are by-products of citric acid fermentation, and there is basically no cost for enzyme production, and the conversion rate is as high as 80% or more; (3) The enzyme-containing Yarrowia lipolytica used in the present invention can be recycled multiple times, while the enzyme purified from Escherichia coli can only be used once, thus saving the cost of the enzyme; (4) The present invention treats the by-product glucose produced during the conversion process and the remaining substrate maltose by adding a brewer's yeast, which is allowed to be used in the fermentation synthesis food additive industry; the production of trehalose and the removal of by-products are carried out in the same reactor, further simplifying the production steps of trehalose and reducing production costs. In addition, there is no need to use chromatographic separation, thereby reducing separation costs.

[0022] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, some of the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 The figure is a schematic diagram of the expression cassette containing the α-amylase-glucoamylase fusion gene, which contains the upstream 5' end homology arm (26S rDNA) - promoter sequence (hp4d) - α-amylase-glucoamylase fusion gene - surface display protein gene (cpw130) - terminator sequence (Ter) - mycophenolic acid selection marker sequence guaB - downstream 3' end homology arm (26S rDNA); Figure 2The schematic diagram of the surface display expression cassette containing the trehalose synthase gene contains the upstream homology arm sequence (this schematic diagram is the 5' end sequence of 26S rDNA), the promoter sequence, the anchor protein gene sequence (such as the Pir1 gene), the trehalose synthase gene, the terminator sequence, the screening gene expression cassette (here is the sucrase gene) and the downstream homology arm sequence (this schematic diagram is the 3' end sequence of 26S rDNA); Figure 3 A single colony grown in the selection medium can grow in solid YNB minimal medium containing mycophenolic acid and sucrose; Figure 4 The selected transformants that can grow on a basic medium containing sucrose and mycophenolic acid are inoculated on a solid medium containing soluble starch; Figure 5 Liquid HPLC analysis of the transformation liquid of the engineered Yarrowia lipolytica cells after transformation in 50 g / L soluble starch for 25 hours; Figure 6 HPLC analysis after adding Saccharomyces cerevisiae to the transformation solution; Figure 7 The morphology and mass spectrometry analysis of trehalose isolated and purified from Saccharomyces cerevisiae purified liquid; Figure 8 This is a comparison chart of the synthesis efficiency of the products after yeast cells were used repeatedly for 6 times. DETAILED DESCRIPTION

[0025] In order to better describe the present invention, the following is further illustrated by specific examples. The methods in the following examples are conventional methods unless otherwise specified.

[0026] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art; the reagents or materials described, unless otherwise specified, are all from commercial channels.

[0027] The sequence containing the α-amylase-glucoamylase fusion gene expression cassette is as shown in SEQ ID No.5, which contains the mycophenolic acid screening gene; it sequentially contains the upstream 5' end homology arm (26S rDNA)-promoter sequence (hp4d)-α-amylase-glucoamylase fusion gene-surface display protein gene (cpw130)-terminator sequence (Ter)-mycophenolic acid screening marker sequence guaB-downstream 3' end homology arm (26S rDNA), and the arrangement of these DNA elements is shown in the schematic diagram Figure 1 It was artificially synthesized according to the sequence of SEQ ID No.5 and commissioned to Shanghai Ruimian Biotechnology Co., Ltd. for synthesis.

[0028] The sequence containing the trehalose synthase gene expression cassette is as shown in SEQ ID No. 6, and contains the sucrase screening gene; Figure 2This is a schematic diagram of the surface display expression cassette containing the trehalose synthase gene, which contains, in sequence, an upstream homology arm sequence (this schematic diagram is the 5' end sequence of 26S rDNA), a promoter sequence, an anchoring protein gene sequence (such as the Pir1 gene), a trehalose synthase gene, a terminator sequence, a screening gene expression cassette (here is the sucrase gene), and a downstream homology arm sequence (this schematic diagram is the 3' end sequence of 26S rDNA).

[0029] After the complete sequence of the gene surface display expression framework is synthesized, it is used to transform Yarrowia lipolytica that can synthesize citric acid (such as Yarrowia lipolytica W29), the transformation method is detailed in the literature published by the inventor ( Microbial Cell Factories , 2018,17:133.). The cells were cultured in a basic selection medium containing sucrose (20 g / L) and mycophenolic acid (300 μg / mL). The components of the selection medium were: yeast nitrogen base 10 g / L, ammonium sulfate 5 g / L, sucrose (20 g / L) and mycophenolic acid (300 μg / mL), agar powder 15 g / L, pH 6.0. Since Yarrowia lipolytica itself cannot utilize sucrose and is not resistant to mycophenolic acid, the transformants that can grow in a medium containing sucrose and mycophenolic acid contain the sucrase suc2 gene and the mycophenolic acid resistance gene, and thus can grow ( Figure 3 ), indicating that the genome contains SEQ ID No.5 and SEQ ID No.6. It also contains amylase-glucoamylase gene and trehalose synthase gene, thereby obtaining a transformant containing amylase-glucoamylase and trehalose synthase genes with surface display expression. One of the transformants was selected and inoculated in YPD solid medium containing soluble starch, cultured at 30 degrees for 48 hours, and iodine solution was added. A significant transparent circle was found, indicating that amylase-glucoamylase hydrolyzed starch, so that it would not turn blue when it met iodine solution, such as Figure 4 The bacteria with the largest transparent zone were selected for preservation and stored in the China General Microbiological Culture Collection Center with the preservation number of CGMCC No.31034.

[0030] The following examples describe in detail the yeast Yarrowia lipolytica Effects of CGMCC NO.31034 strain on fermentation synthesis of trehalose under different pH, temperature, substrate concentration, different nitrogen sources and concentrations.

[0031] 4.1 Proprietary Yarrowia lipolytica Yarrowia lipolytica CGMCC NO.31034 Fermentation of glucose to synthesize citric acid The proprietary Yarrowia lipolytica CGMCC NO.31034 was inoculated in a fermentation medium, which contained 200 g / L of glucose monohydrate, 8 g / L of yeast powder, 2 g / L of peptone, 3 g / L of ammonium citrate, 0.01 g / L of zinc chloride, 0.01 g / L of manganese chloride, 0.05 g / L of magnesium sulfate heptahydrate, pH 6.5, and sterilization. Fermentation was carried out in a 3-liter fermenter, containing 1.3 liters of fermentation medium, at a temperature of 30 degrees, stirring at 550 rpm, and aeration at 1 vvm. Regular sampling and detection revealed that strain CGMCC NO.31034 completely utilized glucose at 50 hours, with citric acid at 107.5 ± 5.5 g / L. Yeast cells were obtained by centrifugation, and the cells were washed 3 times with sterile water to remove citric acid. The yeast cells were used as whole-cell catalysts to catalyze maltose to synthesize trehalose.

[0032] 4.2 Effect of pH on the synthesis of trehalose 50 ml transformation medium was placed in a 250 ml shake flask. The composition was (g / L): 50 soluble starch, 10 g yeast cells obtained in 4.1 above (wet weight). The pH was adjusted to 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0, respectively, and the transformation was performed at 50°C with shaking. Samples were taken at 30 hours and the trehalose content was determined. The results were: 15.4 27.5 32.4 36.5 41.6 30.3 (g / L).

[0033] It can be preliminarily seen from the above results that yeast can synthesize trehalose under conditions of pH 3.0-8.0, but the amount of trehalose synthesized under pH 2.0 is significantly lower than that under other pH conditions. The synthesis efficiency is highest under neutral conditions of pH 7, with 41.6 g / L of trehalose converted from 50 g / L of soluble starch, and the conversion rate reaches 83.2%. It can be seen that the proprietary strain can achieve unexpected results in converting soluble starch.

[0034] 4.3 Effect of temperature on trehalose synthesis by cells The composition of the conversion solution is the same as that of 4.2 above, the pH is adjusted to 7.0, and after sterilization, the yeast obtained in 4.1 above is inoculated. Yarrowia lipolytica CGMCC NO.31034, oscillated at 50℃, 60℃, 70℃, 75℃, 80℃. Samples were taken after 30 hours of fermentation and the trehalose content was determined. The results were: 40.3 43.6 33.4 19.3 13.7 (g / L), From the above results, it can be seen that yeast can synthesize trehalose at temperatures between 50 and 80 degrees Celsius. However, the best synthesis effect is achieved at 60 degrees Celsius, with 43.6 grams per liter of trehalose being obtained from 50 grams per liter of soluble starch, and the conversion rate reaches 87.2%.

[0035] 4.4 Effect of different soluble starch concentrations on trehalose synthesis 50 ml of conversion solution was placed in a 250 ml shake flask. The ingredients were (g / L): soluble starch was 50, 100, 150, 200, 250, 300 respectively. The pH was adjusted to 7.0. After sterilization, 10 g of yeast was added. Yarrowia lipolytica CGMCCNO.31034 was fermented under shaking at 60℃. Samples were taken after 50 hours and the trehalose content was determined. The results were: 42.5 83.2 123.3 133 152.8 171.4 (g / L).

[0036] From the above results, it can be seen that the amount of synthetic trehalose is basically unchanged in the range of 50-150 g / L of substrate concentration, but as the concentration increases further, the soluble starch solution is difficult to completely dissolve, resulting in a relatively lower overall conversion rate. The conversion rate of soluble starch at 50-150 g / L exceeds 80%, but at 300 g / L the conversion rate is less than 70%.

[0037] Figure 5 This is the HPLC analysis chart after conversion at 60℃ for 50 hours at 300g / L soluble starch. It can be seen that although more trehalose is synthesized, it also contains more maltose and glucose. It may be that the high concentration of substrate inhibits the activity of the enzyme. Therefore, in actual use, the recommended substrate concentration is about 150g / L.

[0038] Example 5 Biopurification of Saccharomyces cerevisiae Containing Trehalose Conversion Solution after Conversion After trehalose is synthesized in the above Example 4, there is still a small amount of residual maltose and glucose in the fermentation broth. In order to improve the purity of trehalose in the fermentation broth for purification, brewer's yeast is added to ferment and consume glucose and maltose.

[0039] In the above-mentioned Example 4.3, the conversion liquid was obtained by reaction at 60°C, and after sterilization, Saccharomyces cerevisiae was added and fermented at 30°C for 36 hours. The components of the conversion liquid were analyzed by HPLC. The liquid phase HPLC analysis spectrum is shown as follows: Figure 6 As shown. Figure 6 It can be seen that after adding brewer's yeast, glucose and maltose can be completely decomposed, and the purity of trehalose in the fermentation liquid can be improved. At this time, the purity of trehalose is more than 95%, which is very beneficial to the separation and purification of trehalose.

[0040] It can be seen from the above results that adding cerevisiae yeast to the conversion liquid can significantly improve the purity of trehalose in the fermentation liquid, making it easier to separate it later.

[0041] Trehalose can be separated and purified from the fermentation broth by using separation methods commonly known in the art, which can be mastered by technicians through limited operational practice.

[0042] First, the bacterial liquid is separated. The methods used include centrifugation (tubular centrifuge centrifugation), ceramic membrane filtration, plate and frame filtration, etc. After obtaining the clarified fermentation liquid, it is concentrated to a refractive index of 60% and cooled. Trehalose can be crystallized and precipitated. Centrifugation is performed to obtain trehalose crystals. Secondary crystallization is then performed, and the steps are: dissolution, decolorization, ion exchange, concentration, and recrystallization. Centrifuge the crystals and dry them to obtain white trehalose. The crystals of trehalose are rectangular columns. The obtained trehalose sample and the standard product are subjected to mass spectrometry analysis. The mass spectra of the two are completely consistent, such as Figure 7 shown.

[0043] Yeast strains Yarrowia lipolytica CGMCC NO.31034 synthesizes citric acid in a fermentation medium containing glucose, and uses yeast cells obtained after bacterial liquid separation to convert soluble starch to synthesize trehalose. After the first conversion, the bacterial liquid is separated, and the obtained yeast cells are used to convert soluble starch to synthesize trehalose. After the conversion is completed, the bacterial liquid is separated again, and the obtained cells can be used again to convert and synthesize trehalose. The cells can be reused 6 times, and the relative yield still retains 85%. Figure 8 After the transformation, Saccharomyces cerevisiae was added to decompose and remove glucose according to the method described in Example 4 to improve the purity of trehalose. The cells were generally reused for more than 5-6 times.

[0044] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0045] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A proprietary strain of Yarrowia lipolytica that synthesizes trehalose from starch, It is characterized in that The proprietary Yarrowia lipolytica is Yarrowia lipolytica CGMCC No. 31034, which is preserved in the China General Microorganism Collection Center. The chassis microorganism for preparing the proprietary Yarrowia lipolytica is Yarrowia lipolytica capable of synthesizing citric acid. The proprietary Yarrowia lipolytica contains amylase-saccharifying enzyme and trehalose synthase, and the proprietary Yarrowia lipolytica can convert soluble starch into trehalose.

2. A method for constructing a proprietary Yarrowia lipolytica for synthesizing trehalose as claimed in claim 1, It is characterized in that The Yarrowia lipolytica that can synthesize citric acid is used as the chassis microorganism, and the trehalose synthase gene and the amylase-saccharifying enzyme gene are co-displayed on the surface of the chassis microorganism through the surface display carrier protein cpw130 to construct a proprietary Yarrowia lipolytica that synthesizes trehalose.

3. The method for constructing the proprietary Yarrowia lipolytica for synthesizing trehalose as claimed in claim 2, It is characterized in that The method for constructing the strain comprises the following steps: S1. Design and synthesize the amylase-saccharifying enzyme gene surface display expression cassette; S2. Design and synthesize the surface display expression cassette of trehalose synthase gene; S3, respectively transforming the above two gene surface display expression cassettes into Yarrowia lipolytica that can synthesize citric acid; S4. Screening Yarrowia lipolytica containing the above gene surface display expression cassette to obtain a proprietary Yarrowia lipolytica that synthesizes trehalose.

4. A method for synthesizing trehalose using the proprietary Yarrowia lipolytica as claimed in claim 1, It is characterized in that The synthesis process includes the following steps: S1. In a culture medium containing a carbon source, a nitrogen source, an inorganic salt and water, fermenting glucose to synthesize citric acid using the proprietary Yarrowia lipolytica, wherein the initial pH value of the fermentation is 2.0-8.0 and the initial temperature of the fermentation is 25-35° C.; S2. After the fermentation is completed, the bacterial liquid is separated to obtain the fermentation liquid containing citric acid and yeast cells; S3, using the yeast cell as a whole-cell catalyst to convert soluble starch into trehalose to obtain a conversion solution; S4, adding a nitrogen source and an inorganic salt to the conversion liquid and inoculating brewer's yeast to ferment and decompose impurities in the conversion liquid to obtain a trehalose fermentation liquid; S5. Separating and purifying the trehalose fermentation broth to obtain trehalose.

5. The method for synthesizing trehalose using a proprietary yeast Yarrowia lipolytica as claimed in claim 4, It is characterized in that In step S3, yeast cells convert soluble starch into trehalose, comprising the following steps: S31, adding soluble starch with a mass percentage concentration of 5% to 30% to the yeast cells, and performing conversion by shaking or stirring at a temperature of 50 to 80° C. and a pH of 3 to 8 for 20 to 30 hours; S32, separating the transformed bacterial solution to obtain enzyme-containing yeast cells and a transformation solution containing trehalose.

6. The method for synthesizing trehalose using a proprietary yeast Yarrowia lipolytica as claimed in claim 4, It is characterized in that In step S1, the initial pH value of the fermentation is 5.5, and the initial temperature of the fermentation is 30°C.

7. The method for synthesizing trehalose using a proprietary Yarrowia lipolytica strain as claimed in claim 4, It is characterized in that In step S1, the carbon source in the culture medium is solid glucose or liquid glucose, and the amount of the carbon source is 50 to 350 g / L; the nitrogen source is any one of yeast powder, yeast extract, corn steep liquor, corn steep liquor, diammonium hydrogen phosphate, and ammonium citrate, or a mixture thereof, and the amount of the nitrogen source is 5 to 25 g / L; the inorganic salt is one or more of magnesium sulfate, manganese chloride, and copper chloride, and the amount of the inorganic salt is 0 to 2.5 g / L.

8. The method for synthesizing trehalose using a proprietary Yarrowia lipolytica strain as claimed in claim 7, It is characterized in that In step S1, the amount of the carbon source is 200-300 g / L; the amount of the nitrogen source is 5-20 g / L; and the amount of the inorganic salt is 0.005-2 g / L.

9. The method for synthesizing trehalose using a proprietary Yarrowia lipolytica strain as claimed in claim 5, It is characterized in that In step S5, the separation and purification includes: separating the bacterial liquid to obtain a clarified fermentation liquid containing trehalose, concentrating to obtain a concentrated liquid rich in trehalose, performing a primary crystallization to obtain a crude trehalose product, re-dissolving the crude product, removing ions by ion exchange, decolorizing, concentrating, performing a secondary crystallization to obtain a refined trehalose product, and drying.

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