Bumblebee candida pseudohyaloformis engineering bacteria, construction method and application thereof

Abstract

The application belongs to the field of genetic engineering and biological fermentation technology, and relates to a Bombus candidus Candida albicans engineering bacterium and a construction method and application thereof. An ADH1 gene of the Bombus candidus Candida albitis engineering bacterium is knocked out. The ability of the engineering bacterium to utilize linoleic acid is enhanced. By using the engineering bacterium, the yield can be improved, the fermentation time can be shortened, and the production cost can be reduced in the process of fermenting and synthesizing sophorolipids, thereby bringing actual value to industrialized large-scale production and application.

Description

Technical Field

[0001] This application belongs to the fields of genetic engineering and bio-fermentation technology, and relates to an engineered strain of bumblebee Candida albicans, its construction method and application. Background Technology

[0002] Sophorolipids synthesized by microorganisms, in addition to possessing the surface tension-reducing, foaming, and emulsifying properties of general surfactants, also exhibit environmental friendliness, 100% biodegradability, mildness, low irritation, and certain antibacterial properties. Therefore, sophorolipids, as biosurfactants, have significant potential value in industries such as daily chemical detergents, home care, and skincare, and have received increasing attention in recent years.

[0003] Currently, research on improving the ability of microorganisms to synthesize sophorolipids focuses on modifying key genes in their direct metabolic pathways. Although the ability of modified sophorolipids to synthesize sophorolipids is significantly improved compared to wild strains, the production cost of sophorolipids remains high, and further research is needed on key genes in the side metabolic pathways that affect its synthesis.

[0004] In view of the above, this application is hereby submitted. Summary of the Invention

[0005] This application provides one or more embodiments of an engineered bumblebee *Candida albicans* strain, its construction method, and its application. The technical solutions include the following:

[0006] One or more embodiments of this application provide an engineered strain of *Candida bumblebee*, wherein the ADH1 gene of the engineered strain of *Candida bumblebee* is knocked out.

[0007] In some embodiments of this application, the ADH1 gene encodes an enzyme with the amino acid sequence shown in SEQ ID No. 2.

[0008] In some embodiments of this application, the nucleotide sequence of the ADH1 gene is shown in SEQ ID No. 1.

[0009] In some embodiments of this application, the chassis bacteria of the engineered bumblebee Candida albicans include the wild-type strain numbered ATCC 22214.

[0010] One or more embodiments of this application also provide a method for constructing the engineered bumblebee Candida albicans, the method comprising the steps of providing bumblebee Candida albicans to be modified and knocking out the ADH1 gene.

[0011] One or more embodiments of this application provide a method for producing sophorolipid, the method comprising a step of fermentation with *Candida bacillus*; wherein the *Candida bacillus* is the engineered *Candida bacillus* strain.

[0012] In some embodiments of this application, during the fermentation process, citric acid is used to adjust the pH of the fermentation system to 2.2-3.

[0013] In some embodiments of this application, the bumblebee Candida albicans is fermented into the logarithmic phase, and then the fermentation system is adjusted to a pH of 2.2-3 with citric acid.

[0014] In some embodiments of this application, fermentation satisfies one or more of the following conditions:

[0015] (1) The fermentation medium comprises 0.2%–0.4% (w / v) yeast extract, 7%–9% (w / v) linoleic acid, 9%–11% (w / v) glucose, 0.05%–0.15% (w / v) KH₂PO₄, 0.05%–0.15% (w / v) Na₂HPO₄•12H₂O, 0.04%–0.06% (w / v) MgSO₄•7H₂O, and water; and,

[0016] (2) Incubate at 25℃~30℃ and 200rpm~250rpm for 6 to 8 days.

[0017] One or more embodiments of this application provide a method for producing sophorolipid, the method comprising a step of fermentation with *Candida bacillus*; wherein, during the fermentation process, citric acid is used to adjust the pH of the fermentation system to 2.2-3.

[0018] In some embodiments of this application, the bumblebee Candida albicans is fermented into the logarithmic phase, and then the fermentation system is adjusted to a pH of 2.2-3 with citric acid.

[0019] In some embodiments of this application, fermentation satisfies one or more of the following conditions:

[0020] (1) The fermentation medium comprises 0.2%–0.4% (w / v) yeast extract, 7%–9% (w / v) linoleic acid, 9%–11% (w / v) glucose, 0.05%–0.15% (w / v) KH₂PO₄, 0.05%–0.15% (w / v) Na₂HPO₄•12H₂O, 0.04%–0.06% (w / v) MgSO₄•7H₂O, and water; and,

[0021] (2) Incubate at 25℃~30℃ and 200rpm~250rpm for 4 to 8 days.

[0022] One or more embodiments of this application provide a method for producing a product containing sophorolipids, the method comprising the following steps:

[0023] Sophorolipids are produced using the aforementioned production method; and,

[0024] Preparation of products containing sophorolipids;

[0025] Optionally, the product containing sophorolipids can be a daily chemical detergent, a household care product, or a beauty and skin care product.

[0026] Details of one or more embodiments of this application are set forth in the following description, and other features, objects, and advantages of this application will become apparent from the specification and its claims. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This application relates to the sophorolipid metabolism side pathway and regulatory targets.

[0029] Figure 2 The results of plate screening of gene knockout strains;

[0030] Figure 3 To measure the yield of sophorolipids produced by fermentation of ΔADH1, ΔMFE-2 and ΔFADH strains;

[0031] Figure 4 The effect of citric acid on the yield and synthesis rate of sophorolipids produced by the strain;

[0032] Figure 5 The effects of lactic acid and acetic acid on the yield and synthesis rate of sophorolipids produced by the strain were investigated. Detailed Implementation

[0033] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0035] the term

[0036] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0037] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0038] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0039] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0040] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0041] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0042] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0043] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0044] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0045] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0046] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0047] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0048] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass-volume percentage.

[0049] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0050] A first aspect of this application provides an engineered strain of *Candida bumblebee*, wherein the ADH1 gene of the engineered strain is knocked out. The ADH1 gene can be completely or partially knocked out, inactivated, etc., to render the engineered strain lacking ω-hydroxy fatty acid dehydrogenase, thus preventing it from performing ω-oxidative metabolism. See [link to previous section]. Figure 1 .

[0051] This application provides a genetically engineered strain that efficiently utilizes long-chain fatty acids (linoleic acid as a substrate). Experiments have shown that the strain constructed in this application has an enhanced ability to utilize linoleic acid compared to the wild strain.

[0052] In some embodiments, the ADH1 gene encodes an enzyme with an amino acid sequence as shown in SEQ ID No. 2, or has at least 90% (90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%) homology with SEQ ID No. 2.

[0053] In some embodiments, the nucleotide sequence of the ADH1 gene is as shown in SEQ ID No. 1, or the sequence has at least 90% (90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%) homology with SEQ ID No. 1.

[0054] The embodiments of this application do not specifically limit the substrate of *Candida bumblebee*, which can be the wild-type strain numbered ATCC22214, or other substrate strains that have been identified as *Candida bumblebee* through species identification.

[0055] A second aspect of the embodiments of this application provides a method for constructing the engineered bumblebee Candida albicans, the method comprising the steps of providing bumblebee Candida albicans to be modified and knocking out the ADH1 gene.

[0056] In some implementations, the construction method includes the following steps:

[0057] S1. Vector construction; includes the following steps:

[0058] A1. Extract the genome of *Candida bumblebee*, use the genomic DNA as a template and ADH1F / ADH1R as primers to amplify the ADH1 gene by PCR. The sequence is SEQ ID No. 1. The ADH1 gene fragment is purified by agarose gel electrophoresis and gel cutting.

[0059] A2. Synthesize the hygromycin resistance gene (HPH) sequence containing a strong promoter and terminator of Candida albicans, the sequence being SEQ ID No. 3;

[0060] A3. The purified ADH1 gene fragment was ligated into a T-vector using TA cloning. The ligation solution was then transformed into JM109 competent cells, plated, positive transformants were selected, amplified, cultured, and the Tv-ADH1 plasmid was extracted.

[0061] A4. Using Tv-ADH1 plasmid as a template and rADH1F / rADH1R as primers, reverse PCR was used to obtain a linearized vector with partial deletion of the ADH1 gene. The linearized vector was purified by agarose gel electrophoresis and gel cutting.

[0062] A5. Using the synthesized hygromycin resistance (HPH) gene sequence as a template, and InADH1F / InADH1R as primers, the HPH gene fragment with homologous arms was amplified by PCR, and the PCR product was purified by agarose gel electrophoresis and gel cutting.

[0063] A6. By seamless cloning, the linearized vector obtained in A4 and the HPH gene fragment with homologous arms in A5 were ligated. The ligation solution was used to transform JM109 competent cells, plated, positive transformants were selected, amplified and cultured, and the plasmid TA-AD-HP-AD-TA was extracted.

[0064] A7. Using TA-AD-HP-AD-TA plasmid as a template and ADH1F / ADH1R as primers, PCR amplification was performed, followed by agarose gel electrophoresis detection and gel purification to obtain the ADH1 gene linear knockout cassette (AD-HP-AD).

[0065] S2. Preparation of competent cells of *Candida bumblebee* and construction of ADH1 gene knockout strain;

[0066] Competent cells of *Candida bumblebee* were prepared using the lithium acetate method. The ADH1 gene linear knockout cassette (AD-HP-AD) was transformed into competent cells via electroporation. The transformation solution was plated on YPD plates containing 0.5 ppm hygromycin. Colonies that grew normally on the YPD plates containing hygromycin were selected for YPD liquid culture, and genomic DNA was extracted. Using the extracted genomic DNA as a template and ADH1F / ADH1R as primers, PCR amplification was performed. The amplified DNA fragments were detected by agarose gel electrophoresis. If the ADH1 gene linear knockout cassette (AD-HP-AD) was obtained, it indicated that the colony was an ADH1 gene knockout strain.

[0067] To compare the effects of the ADH1 gene, the multifunctional oxidase (MFE-2) gene, and the hydroxy fatty acid oxidase (FADH) gene on the synthesis of sophorolipids, this application also constructed MFE-2 and FADH gene knockout strains. The vector construction and gene knockout strain construction methods are the same as those described in S1 and S2 above: the primers used to amplify the MFE-2 gene fragment and the FADH gene fragment are MFE-2F / MFE-2R and FADHF / FADHR, respectively; the primers used for reverse PCR are rMFE-2F / rMFE-2R and rFADHF / rFADHR, respectively; the primers used to amplify the HPH gene fragment with homologous arms are InMFE-2F / IInMFE-2R and InFADHF / InFADHR, respectively; and the primers used to amplify the MFE-2 gene linear knockout cassette (AD-MF-AD) and the FADH gene linear knockout cassette (AD-FA-AD), respectively.

[0068] A third aspect of this application provides a method for producing sophorolipids, the method comprising a step of fermentation with *Candida blakeana*; wherein the *Candida blakeana* is an engineered strain of *Candida blakeana*.

[0069] In some embodiments of this application, during fermentation, the pH of the fermentation system is adjusted to 2.2–3 using citric acid (e.g., 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, optionally 2.2–2.8, and more preferably 2.5). This fermentation method effectively and significantly shortens the fermentation time required to reach maximum sophorolipid yield, thus accelerating sophorolipid synthesis.

[0070] A fourth aspect of this application provides a method for producing sophorolipid, the method comprising a step of fermentation with *Candida bumblebee*; wherein, during the fermentation process, citric acid is used to adjust the pH of the fermentation system to 2.2-3 (e.g., 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, optionally 2.2-2.8, and further optionally 2.5).

[0071] In the production methods provided in the third and fourth aspects of the embodiments of this application, the fermentation of *Candida bumblebee* enters the logarithmic growth phase, and then the fermentation system is adjusted to a pH of 2.2–3 with citric acid, approximately 24–72 hours after fermentation (24h, 26h, 28h, 30h, 32h, 34h, 36h, 38h, 40h, 42h, 44h, 46h, 48h, 50h, 52h, 54h, 56h, 58h, 60h, 62h, 64h, 66h, 68h, 70h, 72h). Optionally, it is added during the mid-logarithmic growth phase. After 72 hours of cultivation, i.e., entering the late logarithmic growth phase, the fermentation broth automatically decreases to a lower pH, and the addition of citric acid has little effect on accelerating the accumulation and synthesis of sophorolipids.

[0072] Another major factor affecting the production cost of sophorolipids is the fermentation time. Sophorolipids are a secondary metabolite that microorganisms begin to synthesize after reaching the logarithmic growth phase, and it often takes 7 days or even longer to reach the maximum accumulation. However, there are few reports on how to effectively intervene in the fermentation process and accelerate the synthesis and accumulation of sophorolipids.

[0073] The production method provided in this application can increase the yield while shortening the fermentation time and reducing the production cost during the microbial fermentation synthesis of sophorolipids, bringing practical value to large-scale industrial production and application.

[0074] In the production methods provided in the third and fourth aspects of the embodiments of this application, it is understood that fermentation is carried out under suitable conditions, such as in the presence of the substrate linoleic acid. In some embodiments of this application, fermentation satisfies one or more of the following conditions:

[0075] (1) The fermentation medium comprises 0.2%–0.4% (w / v) yeast extract, 7%–9% (w / v) linoleic acid, 9%–11% (w / v) glucose, 0.05%–0.15% (w / v) KH₂PO₄, 0.05%–0.15% (w / v) Na₂HPO₄•12H₂O, 0.04%–0.06% (w / v) MgSO₄•7H₂O, and water; and,

[0076] (2) Incubate at 25℃~30℃ and 200rpm~250rpm for 4 to 8 days.

[0077] The fermentation medium contains, for example, yeast powder concentrations of 0.2%, 0.25%, 0.3%, 0.35%, and 0.4%; linoleic acid concentrations of 7%, 7.2%, 7.4%, 7.6%, 7.8%, 8%, 8.2%, 8.4%, 8.8%, and 9%; glucose concentrations of 9%, 9.2%, 9.4%, 9.6%, 9.8%, 10%, 10.2%, 10.4%, 10.8%, and 11%; and KH₂PO₄ concentrations of 0.05%, 0.06%, and 0. The concentrations of Na2HPO4•12H2O are, for example, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, and 0.15%, respectively. The concentrations of MgSO4•7H2O are, for example, 0.04%, 0.045%, 0.05%, 0.055%, and 0.06%.

[0078] Fermentation temperatures, for example, are 25℃, 25.5℃, 26℃, 26.5℃, 27℃, 27.5℃, 28℃, 28.5℃, 29℃, 29.5℃, and 30℃; fermentation speeds, for example, are 200 rpm, 205 rpm, 210 rpm, 215 rpm, 220 rpm, 225 rpm, 230 rpm, 235 rpm, 240 rpm, 245 rpm, and 250 rpm; and fermentation times, for example, are 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, and 8 days.

[0079] In the production methods provided in the third and fourth aspects of the embodiments of this application, fermentation can be carried out in an Erlenmeyer flask, for example, in a 500mL Erlenmeyer flask, wherein the content of the fermentation medium can be 150mL to 300mL, for example, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300mL, preferably 200mL to 250mL.

[0080] In the production methods provided in the third and fourth aspects of the embodiments of this application, citric acid is added to the reaction system in the form of a citric acid solution. Optionally, the concentration of citric acid in the citric acid solution is 0.05M to 2M, for example, 0.05M, 0.1M, 0.15M, 0.2M, 0.4M, 0.6M, 0.8M, 1.0M, 1.2M, 1.4M, 1.6M, 1.8M, or 2M; alternatively, it is 0.5M to 1.5M; and further alternatively, it is 1M.

[0081] In the production methods provided in the third and fourth aspects of this application, *Candida bumblebee* is inoculated in the form of a seed culture. The preparation method of the seed culture includes, but is not limited to: picking a single colony of *Candida bumblebee*, inoculating it into YPD seed culture medium, culturing it, and preparing the seed culture. In this application embodiment, the preparation of the seed culture can be carried out in a test tube containing YPD seed culture medium, for example, in a test tube containing 10 mL of YPD seed culture medium. Optionally, the culture conditions may include: a temperature of 25℃~30℃ (e.g., 25℃, 25.5℃, 26℃, 26.5℃, 27℃, 27.5℃, 28℃, 28.5℃, 29℃, 29.5℃, 30℃); and a rotation speed of 200 rpm~250 rpm (e.g., 200 rpm, 205 rpm, 210 rpm, 215 rpm, 220 rpm, 225 rpm, 230 rpm). 0 rpm, 235 rpm, 240 rpm, 245 rpm, 250 rpm; culture to OD600 is 1.0 to 1.5 (e.g., 1, 1.1, 1.2, 1.3, 1.4, 1.5, optionally 1), taking approximately 24 to 48 hours (e.g., 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 ​​hours). Optionally, the seed culture is inoculated into the fermentation medium at an amount of 2.5% to 5% (v / v) (e.g., 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%).

[0082] A fifth aspect of this application provides a method for producing a product containing sophorolipids, the method comprising the following steps:

[0083] Sophorolipids are produced using the aforementioned production method; and,

[0084] Prepare products containing sophorolipids.

[0085] This application does not specifically limit the types of products, including but not limited to daily chemical detergents, household care products, or beauty and skin care products. For example, dishwashing liquid, shower gel, laundry detergent, cleaning soap, shampoo, facial cleanser, makeup remover, etc.

[0086] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0087] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0088] Example 1

[0089] I. Preparation of linear knockout cassettes for ADH1, MFE-2, and FADH genes

[0090] A1. Extract the genome of *Candida bumblebee*. Using the genomic DNA as a template, amplify the ADH1, MFE-2, and FADH genes by PCR using primers ADH1F / ADH1R, MFE-2F / MFE-2R, and FADHF / FADHR as shown in Table 1. The sequences are SEQ ID No. 1, SEQ ID No. 4, and SEQ ID No. 5, respectively. The three gene fragments were purified by agarose gel electrophoresis and gel cutting.

[0091] A2. Synthesize the hygromycin resistance gene (HPH) sequence containing a strong promoter and terminator of Candida albicans, the sequence being SEQ ID No. 3;

[0092] A3. By TA cloning, the purified ADH1, MFE-2 and FADH gene fragments were ligated into T-vectors, and the ligation solution was transformed into JM109 competent cells. The cells were plated, positive transformants were selected, amplified and cultured, and Tv-ADH1, Tv-MFE-2 and Tv-FADH plasmids were extracted.

[0093] A4. Using Tv-ADH1, Tv-MFE-2 and Tv-FADH plasmids as templates, and rADH1F / rADH1R, rMFE-2F / rMFE-2R and rFADHF / rFADHR in Table 1 as primers, reverse PCR was used to obtain linearized vectors with partial sequence deletions. Agarose gel electrophoresis and gel cutting were used to purify each linearized vector.

[0094] A5. Using the synthesized hygromycin resistance (HPH) gene sequence as a template, the HPH gene fragment with homologous arms was amplified by PCR using InADH1F / InADH1R, InMFE-2F / InMFE-2R and InFADHF / InFADHR in Table 1, respectively. The PCR products were then purified by agarose gel electrophoresis and gel cutting.

[0095] A6. By seamless cloning, the linearized vectors obtained in A4 and the corresponding HPH gene fragments in A5 were ligated. The ligation solution was used to transform JM109 competent cells, plated, positive transformants were selected, amplified and cultured, and circular plasmids ΔTA-ADH1, ΔTA-MFE-2 and ΔTA-FADH containing the knockout cassette sequences of each gene were extracted.

[0096] A7. Using the circular plasmids obtained in A6 as templates, and the corresponding ADH1F / ADH1R, MFE-2F / MFE-2R, and FADHF / FADHR primers in Table 1 as primers, the linear knockout cassettes of the ADH1 gene (AD-HP-AD), MFE-2 gene (AD-MF-AD), and FADH gene (AD-FA-AD) were purified by PCR amplification and agarose gel electrophoresis.

[0097] II. Preparation and electroporation of competent cells of *Candida albicans* (bumblebee) to construct gene knockout strains

[0098] A single colony of *S. bombicola* (ATCC 22214) was inoculated into a 250 mL shake flask containing 25 mL of YPD medium and cultured at 30 °C and 300 rpm for 18 hours.

[0099] Inoculate the culture medium from the previous step into a 250 mL shake flask containing 50 mL of YPD medium at a rate of 2% (v / v), and culture the cells at 30 °C and 120 rpm until the OD600 value is between 1 and 2.

[0100] Transfer the bacterial culture into a 50mL centrifuge tube, centrifuge at 3000g and 4℃ for 5 minutes to collect the bacterial cells, then resuspend the precipitate in 50mL of sterile water cooled on ice, centrifuge at 3000g and 4℃ for 5 minutes, and discard the supernatant; then resuspend the precipitate in 50mL of sterile water cooled on ice and centrifuge again.

[0101] Resuspend the bacterial pellet in 4 mL of ice-cold 1 M sterile sorbitol solution, centrifuge at 3000 g and 4 °C for 5 minutes, and discard the supernatant.

[0102] The precipitate was suspended in 4 mL of freshly prepared 0.1 M lithium acetate solution (3500 μL water, 400 μL 1 M lithium acetate, 200 μL 1 M DTT), and left at room temperature for 15 minutes. Then, it was centrifuged at 3000 g and 4 °C for 5 minutes and the supernatant was discarded.

[0103] Resuspend the bacterial pellet in 4 mL of ice-cold 1 M sterile sorbitol solution, centrifuge at 3000 g and 4 °C for 5 minutes, and discard the supernatant. Resuspend the bacterial cells in 1 M sorbitol solution, place on ice, and use as soon as possible.

[0104] Pipette 50 μL of the yeast suspension into a centrifuge tube, add 2.5 mg of DNA solution for transformation to the yeast suspension, mix well, and pre-cool on ice for 5 minutes.

[0105] The mixture was transferred to an electroporation cup with a 0.2 cm gap and placed on ice for 5 minutes. Subsequently, a 5 ms pulse with a 2.5 kV pulse was applied to the mixture using a MicroPulser (Bio-Rad).

[0106] Remove the electroporation vessel and immediately add ice-cold 1M sorbitol. Gently mix and transfer to a 1.5 mL centrifuge tube. Incubate the mixture at 30°C for 1 hour. Spread 200 μL of the mixture onto a selective medium and incubate at 30°C for approximately 1 week. For the selective medium, use YPD plates containing 1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose, and 500 ppm hygromycin.

[0107] Single colonies from hygromycin YPD plates were selected for liquid amplification culture, followed by extraction of genomic DNA. Using the genomic DNA as a template, PCR amplification was performed using primers listed in Table 1, and the target engineered strain was verified by agarose gel electrophoresis.

[0108] Figure 2 The results of plating the electroporation mixture onto 500ppm hygromycin YPD plates showed that gene knockout strains were successfully obtained: ΔADH1, ΔMFE-2, and ΔFADH.

[0109] III. Fermentation production of sophorolipids using ΔADH1, ΔMFE-2, and ΔFADH strains

[0110] Single colonies of the engineered strain and wild-type strain obtained in step 2 were picked and inoculated into test tubes containing 10 mL of YPD seed medium. They were cultured at 30°C and 225 rpm until OD600 = 1.0 (approximately 24-48 h). Then, 5% (v / v) of the inoculum was inoculated into 500 mL shake flasks containing 250 mL of fermentation medium and cultured at 30°C and 225 rpm for 7 days.

[0111] The fermentation medium consisted of 0.3% (w / v) yeast extract, 8% (w / v) linoleic acid, 10% (w / v) glucose, 0.1% KH2PO4, 0.1% (w / v) Na2HPO4•12H2O, 0.05% (w / v) MgSO4•7H2O, and water.

[0112] IV. Detection of Sophorolipid Production

[0113] The residual sugar content and sophorolipid content in the culture medium were determined.

[0114] (1) Determination of residual sugar content

[0115] The glucose concentration in the culture medium was determined using an SBA-40E biosensor analyzer. The fermentation broth was centrifuged, and the supernatant was collected. The supernatant was filtered through a 0.45 μm filter membrane, and the filtrate was diluted to an appropriate factor. 25 μL of the filtrate was injected. The residual glucose content of the fermentation broth (g / L) = n × m / 200 (n is the instrument reading, and m is the dilution factor).

[0116] (2) Determination of sophorolipid content

[0117] The total sugar content in the culture medium was determined using the anthrone method. Then, based on the ratio between the molecular weights of sophorolipids and glucose (i.e., 1.91g of sophorolipids is equivalent to 1g of glucose), the content of sophorolipids could be calculated.

[0118] Total sophorolipid content: Take 500 μL of fermentation broth, add 1 mL of ethanol for extraction, shake and mix at room temperature, and incubate at 12000 r·min. -1 Centrifuge for 10 minutes, measure the total sugar concentration in the supernatant, subtract the residual sugar in the supernatant to obtain the glucose content in sophorolipids, and then calculate the total sophorolipid content.

[0119] Figure 3 The results showed that when linoleic acid was used as a hydrophobic substrate, the amount of sophorolipid synthesized by the ΔADH1 strain was significantly higher than that of the ΔFADH strain, and extremely significantly higher than that of the wild strain and the ΔMFE-2 strain.

[0120] V. Effects of Acid Type on the Fermentation Production of Sophorolipids by ΔADH1, ΔFADH, and Wild Strains

[0121] 1. The effect of citric acid

[0122] Single colonies of the ΔADH1, ΔFADH, and wild-type strains obtained in step two were inoculated into test tubes containing 10 mL of YPD seed culture medium. The culture was carried out at 30°C and 225 rpm until OD600 = 1 (approximately 24–48 h). Then, 5% (v / v) of the culture medium was inoculated into 500 mL shake flasks containing 250 mL of fermentation medium. The culture was carried out at 30°C and 225 rpm for 48 h. 1 M citric acid was added to adjust the pH of the fermentation broth to 2.5, and fermentation continued for 5 days. Samples were taken every 24 h to detect the yield of sophorolipids, using the same method as in step four.

[0123] The fermentation medium consisted of 0.3% (w / v) yeast extract, 8% (w / v) linoleic acid, 10% (w / v) glucose, 0.1% KH2PO4, 0.1% Na2HPO4•12H2O, 0.05% MgSO4•7H2O, and water.

[0124] Figure 4 The results showed that after 48 hours of fermentation, the group with added citric acid not only had a higher total yield of sophorolipids than the group without added citric acid, but also a significantly shorter time required to reach the maximum yield.

[0125] 2. Effects of lactic acid and acetic acid on the fermentation production of sophorolipids by strain ΔADH1

[0126] The fermentation production method is the same as "1. Effect of citric acid", except that when the fermentation culture reaches 48h, 1M lactic acid and 1M acetic acid are added to adjust the pH of the fermentation broth to 2.5. The method for detecting sophorolipids is the same as in step four.

[0127] Figure 5 The results showed that after fermentation for 48 hours, adding lactic acid and acetic acid to adjust the pH of the fermentation broth to 2.5 had no significant effect on the amount of sophorolipid produced by the ΔADH1 strain or the time required. This indicates that the main reason why citric acid can increase the yield and synthesis rate of sophorolipid is not due to the effect of pH, but rather because it affects the activity of key enzymes in the glucose glycolysis metabolic pathway.

[0128] SEQ ID No.1 (Molecular type: DNA; Organism: Candida bombicola; Length: 1882 bp)

[0129]

[0130] SEQ ID No. 2 (Molecular type: Protein; Organism: *S. bombicola*; Length: 368 aa):

[0131] MLTWPLITSSLRSVTNKFTISRHLSKMVQGTMKAAVIKEFGKPIEVCQVPIPQPKKDEILIKVAYSGICGTDHHAWKGDWPAKPNLPFIPGHEGVGRVVEVGEEVDHLKKGDLVGVPWLYNACSHCEQCFAGWETLCKNQNNAGYSVNGTLAEYVIGDPNYVARVPEGVDLAKVAPVLCAGVTV YKGLKVTEAKAGQWVLVSGLGLGQLGVQFAKAMGFNVIGVDIDDAKLETAKKLGATYAFNGMTQDIGKEIHNAVGGVHGCIVTAVGRAAFGALKAIRQRGTMVCIGIPPGDFPLDIFNTVMNGITVRGSIVGTRIDMMEALEFFRMGKIENHVQLDTIDNIDKVMNLMDQGKLPGRVVFDFTK.

[0132] SEQ ID No. 3 (Molecular type: DNA; Organism: Artificially synthesized; Length: 1753 bp):

[0133]

[0134] SEQ ID No. 4 (Molecular type: DNA; Organism: Candida bombicola; Length: 2886 bp):

[0135]

[0136] SEQ ID No. 5 (Molecular type: DNA; Organism: Candida bombicola; Length: 1344bp):

[0137]

[0138] Table 1

[0139]

[0140] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples 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.

[0141] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for producing sophorolipid, characterized in that, The production method includes a fermentation step using *Candida albicans*, a bumblebee yeast; wherein... The bumblebee Candida is an engineered bumblebee Candida, the ADH1 gene of which is knocked out, and the chassis bacteria of which are wild-type bacteria numbered ATCC 22214. During fermentation, citric acid is used to adjust the pH of the fermentation system to 2.2–3; After the bumblebee Candida albicans ferments into mid-log phase, the fermentation system is adjusted to pH 2.2–3 with citric acid. Fermentation must meet the following conditions: The fermentation medium consisted of 0.2%–0.4% (w / v) yeast extract, 7%–9% (w / v) linoleic acid, 9%–11% (w / v) glucose, 0.05%–0.15% (w / v) KH2PO4, 0.05%–0.15% (w / v) Na2HPO4•12H2O, 0.04%–0.06% (w / v) MgSO4•7H2O, and water.

2. The method for producing sophorolipids according to claim 1, characterized in that, The ADH1 gene encodes an enzyme with the amino acid sequence shown in SEQ ID No.

2.

3. The method for producing sophorolipids according to claim 2, characterized in that, The nucleotide sequence of the ADH1 gene is shown in SEQ ID No.

1.

4. The method for producing sophorolipids according to claim 1, characterized in that, During fermentation, citric acid is used to adjust the pH of the fermentation system to 2.2–2.

8.

5. The method for producing sophorolipids according to claim 1, characterized in that, The citric acid is added to the reaction system in the form of a citric acid solution.

6. The method for producing sophorolipids according to claim 5, characterized in that, The concentration of citric acid in the citric acid solution is 0.05M to 2M.

7. The method for producing sophorolipid according to claim 6, characterized in that, The concentration of citric acid in the citric acid solution is 0.5M to 1.5M.

8. The method for producing sophorolipids according to claim 1, characterized in that, The bumblebee Candida albicans was inoculated with a seed culture.

9. The method for producing sophorolipid according to claim 8, characterized in that, The method for preparing the seed liquid includes: picking a single colony of *Candida bacillus*, inoculating it into YPD seed culture medium, culturing it, and preparing the seed liquid.

10. The method for producing sophorolipids according to claim 9, characterized in that, The cultivation conditions include: temperature of 25℃~30℃; rotation speed of 200rpm~250rpm; and cultivation until OD600 is 1.0~1.

5.

11. The method for producing sophorolipids according to claim 9, characterized in that, The seed culture was inoculated into the fermentation medium at a rate of 2.5% to 5% (v / v).

12. The method for producing sophorolipid according to any one of claims 1 to 11, characterized in that, Fermentation should meet the following conditions: ferment at 25℃~30℃ and 200rpm~250rpm for 4 to 8 days.

13. A method for producing a product containing sophorolipids, characterized in that, The production method includes the following steps: Sophorolipids are produced using the production method according to any one of claims 1 to 12; and, Prepare products containing sophorolipids.

14. The method for producing the product containing sophorolipids according to claim 13, characterized in that, The products containing sophorolipids are daily chemical detergents, household care products, or beauty and skin care products.

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