Yarrowia lipolytica and application thereof
By developing the high-temperature resistant, low-pH, and highly efficient lipid-based carbon source-utilizing Yersinia lipophila strain ML06, the problems of tolerance and carbon source utilization efficiency of existing strains in industrial fermentation have been solved, enabling efficient and economical industrial production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-31
- Publication Date
- 2026-04-07
AI Technical Summary
Existing Yeast strains are not tolerant enough to high temperature, low pH and high concentration of organic solvents, which limits their application in industrial fermentation. They also have low efficiency in utilizing non-traditional carbon sources, resulting in high production costs and low product yields.
A new strain of Yersinia lipophila, ML06, was developed. It exhibits high temperature tolerance (≥34℃), low pH tolerance (pH 2-4), and efficient utilization of lipid carbon sources, making it suitable for industrial fermentation under high temperature and high acid conditions.
Simplify the fermentation process, reduce cooling energy consumption, broaden the sources of raw materials, improve product yield and purity, reduce production costs, and enhance the economics of industrial applications.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of microorganisms and biotechnology, and relates to a Yarrowia lipolytica strain with broad tolerance characteristics and application thereof. BACKGROUND
[0002] Yarrowia lipolytica as an unconventional yeast has become an indispensable important chassis microorganism in the field of industrial biotechnology. Due to its outstanding characteristics such as efficient secretion of proteins, natural accumulation of oil, clear metabolic pathway and GRAS (Generally Recognized as Safe) certification granted by the US Food and Drug Administration (FDA), the yeast shows great potential in large-scale biological manufacturing of enzyme preparations, organic acids, single-cell proteins, functional oils and high-value chemicals, and has a broad application prospect.
[0003] At present, a series of mature engineering strains represented by Yarrowia lipolytica PO1f and its derivative strains have been widely used in basic research and part of industrial production. These strains are usually optimized in laboratory environment, and their culture conditions (such as neutral pH, mild temperature of 28-30℃) and dependence on traditional carbon sources such as glucose can meet the conventional requirements, but they show significant limitations when facing complex and harsh industrial fermentation environment. Specifically, first, the existing strains generally lack tolerance to high temperature (such as >34℃), which not only limits their application in processes requiring high-temperature fermentation to promote product synthesis or reduce cooling costs, but also increases the huge energy consumption due to the need for heat dissipation during fermentation. Second, they lack tolerance to acidic environment (low pH), and in the production of organic acids such as citric acid, the accumulation of products will naturally cause the pH of the fermentation broth to decrease, and if the strain cannot tolerate low pH, additional alkali solution needs to be added for neutralization, which not only increases the cost but also introduces impurities. Third, the utilization efficiency of non-traditional carbon sources that are more readily available and cheaper in industry is limited, especially the lack of high-efficiency utilization of fats, which limits the conversion of cheap raw materials. Finally, the tolerance to organic solvents (such as alcohols, organic acids metabolized by themselves) that may accumulate during fermentation is weak, which affects the production intensity and final yield.
[0004] Therefore, there is an urgent need in the current field of industrial biological manufacturing to develop or construct new Yarrowia lipolytica strains with multiple tolerance characteristics. A strain that can simultaneously tolerate high temperature, low pH, high concentration of organic solvents, and can efficiently utilize oil and other cheap raw materials, will be able to directly simplify the fermentation process (such as reducing pH adjustment, reducing cooling energy consumption), broaden the source of raw materials, improve the yield and purity of products, thereby essentially reducing the overall production cost and improving the process economy. The development of such strains is of key significance to promote the application of Yarrowia lipolytica in a wider and more severe industrial scenario, and is an important direction of technological development in this field. SUMMARY
[0005] The present application aims to provide a Yarrowia lipolytica strain with high growth efficiency and efficient utilization of different carbon sources, and an application thereof.
[0006] In a first aspect, the present application provides a Yarrowia lipolytica strain, which is Yarrowia lipolytica ML06 strain, and the preservation number is CGMCC NO. 34889.
[0007] The Yarrowia lipolytica strain described above has at least one of the following characteristics:
[0008] 1) High temperature resistance; the high temperature is greater than or equal to 34℃, and in the examples, 35℃ is taken as an example; the strain can also grow normally at 25-30℃, thereby having wide industrial environmental adaptability.
[0009] 2) Acid resistance; the acid resistance is specifically the growth under the condition of pH value of 2-4, and further, the growth under the condition of pH value of 2.
[0010] 3) High efficient utilization of oil carbon source for rapid growth.
[0011] In a second aspect, the present application provides an application of the Yarrowia lipolytica strain of the first aspect in industrial fermentation.
[0012] Or, an application of the Yarrowia lipolytica strain of the first aspect in producing a target product as a fermentation chassis cell.
[0013] Or, an application of the Yarrowia lipolytica strain of the first aspect in producing a target product by industrial fermentation.
[0014] Or, an application of the Yarrowia lipolytica strain of the first aspect in preparing a recombinant microorganism for industrial fermentation.
[0015] In a third aspect, the present application provides a fermentation culture method using the Yarrowia lipolytica strain of the first aspect or a recombinant strain constructed by using the Yarrowia lipolytica strain as a chassis cell. The method comprises inoculating the Yarrowia lipolytica strain or the recombinant strain into a fermentation medium, and culturing or fermenting under high temperature and / or high acid conditions.
[0016] Further, the high temperature condition refers to a fermentation temperature greater than 34℃, for example, 35℃, 37℃ or higher. The high acid condition refers to a pH value of the fermentation system of 2 to 4. The carbon source of the fermentation medium comprises oil, such as palmitic acid, soybean oil, kitchen waste oil (kitchen waste oil), etc. In a specific embodiment, the fermentation can be carried out at pH 2, 35℃, with kitchen waste oil as the main carbon source, at which the strain ML06 can still maintain vigorous growth and the ability to synthesize target products.
[0017] In a fourth aspect, the present application provides a method for producing a target product by using the Yarrowia lipolytica of the first aspect as a host cell.
[0018] The method comprises the following steps:
[0019] 1) Knocking out the genes related to metabolic pathways in the Yarrowia lipolytica ML06 strain, or introducing an exogenous gene or gene cluster encoding a target product into the Yarrowia lipolytica ML06 strain by genetic manipulation, to construct a recombinant engineering strain.
[0020] 2) Under suitable fermentation conditions, especially under high-acid and / or high-temperature conditions, using oil as raw material or main carbon source, the recombinant engineering strain is subjected to fermentation culture, so as to produce the target product.
[0021] The present application has the beneficial effect that the ML06 strain exhibits excellent growth and metabolic activity under the stress conditions (high temperature and high acid) commonly encountered in industrial fermentation, and can be directly applied to the acid production process or reduce cooling energy consumption, and reduce the difficulty and cost of process control.
[0022] Deposit Description
[0023] Strain Name: Yarrowia lipolytica
[0024] Latin Name: Yarrowia lipolytica
[0025] Strain Number: ML06
[0026] Preservation Agency: China General Microbiological Culture Collection Center
[0027] Preservation Agency Abbreviation: CGMCC
[0028] Address: No. 3, Beichen West Road, Beijing City, Chaoyang District
[0029] Preservation Date: June 13, 2025
[0030] Preservation Center Registration Number: CGMCC No. 34889 BRIEF DESCRIPTION OF DRAWINGS
[0031] Figure 1 The colony morphology of Yarrowia lipolytica ML06.
[0032] Figure 2 The morphology of Yarrowia lipolytica ML06 under a microscope. DETAILED DESCRIPTION
[0033] The application will be further described in conjunction with the specific embodiments. The examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application.
[0034] The experimental methods in the following examples are all routine methods, unless otherwise specified, which are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.
[0035] The quantitative tests in the following examples, unless otherwise specified, are all set up with three repeated experiments, and the results are averaged.
[0036] The experimental methods used in the following examples are all routine methods, unless otherwise specified.
[0037] The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.
[0038] The percentage in the following examples, unless otherwise specified, is mass percentage.
[0039] Yarrowia lipolytica PO1f strain was purchased from Tianjin Yifang Technology Co., Ltd. (Item No. J2004).
[0040] Yarrowia lipolytica M0003 strain was isolated from environmental samples in Xinglongtai District, Panjin City, Liaoning Province.
[0041] The culture medium used is as follows:
[0042] YM culture medium: yeast powder 3.0 g, malt extract 3.0 g, glucose 10.0 g, peptone 5.0 g, water 1 L, pH 6.0.
[0043] YPD culture medium: yeast powder 10.0 g, glucose 20.0 g, peptone 20.0 g, water 1 L, pH 6.0.
[0044] YL culture medium: yeast powder 3.0 g, peptone 5.0 g, soybean oil, kitchen waste oil or palm acid containing 0.4% Bij58 emulsifier 10.0 g, esterase 0.1 g, water 1 L, pH 6.0.
[0045] Gully oil is kitchen waste oil separated, containing 98% of triglyceride.
[0046] Strain genomic DNA extraction was performed using a universal genomic DNA extraction kit (Quangen, Item No. EE101-11), which is referred to as genomic extraction kit in the following examples.
[0047] The transformation of Yarrowia lipolytica was carried out as follows: (1) Preparation of competent cells: a single colony was picked from a plate and inoculated into YPD medium for overnight culture. The next day, the culture was transferred to YPD medium and cultured to the mid-log phase. The bacterial cells were collected by centrifugation, washed with pre-cooled sterile water for 1-2 times, and resuspended in a pre-cooled sorbitol solution on ice for standby. (2) Electroporation transformation: the purified plasmid and DNA fragment were mixed with the competent cell suspension and transferred to a pre-cooled electroporation cup. An electroporator was used to perform an electric pulse under the set voltage, capacitance and resistance parameters. After the electric shock, recovery medium was immediately added to the electroporation cup, mixed well and transferred to a centrifuge tube for recovery at 28-30°C for a period of time. Finally, it was plated on YPD plates and cultured at 28-30°C for 2-4 days to grow single bacteria for identification.
[0048] Example 1, Isolation and Mutagenic Breeding of Yarrowia lipolytica ML06
[0049] 1. Isolation of the strain: Yarrowia lipolytica strain M0003 (original strain) was isolated from environmental samples in Xinglongtai District, Panjin City, Liaoning Province, and is now preserved in Micro Yuan Synthetic Biotechnology (Beijing) Co., Ltd. The M0003 strain was mutagenized using an ARTP breeding instrument (Beijing Siquingyuan Biotechnology Co., Ltd.). The specific steps are as follows: after overnight culture of the strain in YM medium at 28°C, the cells were collected by centrifugation at 8000 rpm for 20 min, washed twice with 0.85% sterile normal saline and resuspended. 10 μL of bacterial suspension was added dropwise to a sterile metal slide, which was placed on the sample table of the ARTP mutagenesis system and received plasma irradiation at a distance of 2 mm. The mutagenesis time gradient was set. After vortexing the bacterial cells, they were plated on YM agar plates, counted after incubation, and the treatment group with a lethality rate of 85% was inoculated into YM medium containing 2% (w / v) propionic acid and cultured at 30°C. During this process, the culture temperature was gradually increased to 35°C, and the culture medium pH was reduced to 2.0. After several rounds of subculture, multiple single colonies were selected from the plates, and the one with the best growth state was purified and preserved, finally named ML06 and submitted to the China General Microbiological Culture Collection Center (CGMCC) for preservation.
[0050] 2. Morphological identification of strain ML06
[0051] The strain ML06 was inoculated into YM medium plates and cultured at 28-30°C for 3 days, and the colony morphology was observed. The colony was opaque and milky white. The colony surface was dry and rough, and the edge was irregular. As the culture time prolonged, the colony gradually became wrinkled, with bumps, and showed an irregular shape (Fig. 1). Figure 1 Under a light microscope, the bacterial cells of ML06 were oval or elliptical in shape, with uneven sizes, about 3-6 μm in length and about 2-4 μm in width.Figure 2 ).
[0052] 3. ITS identification of ML06
[0053] The strain ML06 was inoculated in liquid YM medium and cultured at 28°C for 20 h. The genomic DNA of the strain ML06 was extracted using a fungal genomic DNA extraction kit. The ITS fragment was obtained by PCR amplification using the fungal universal primers ITS1 (sequence: 5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (sequence: 5'-TCCTCCGCTTATTGATATGC-3') with the genomic DNA of the strain ML06 as a template, and was sequenced. The nucleotide sequence is sequence 1.
[0054] The sequence determination and bioinformatics analysis showed that the sequence had a similarity of 100% with the ITS sequence of the Yarrowia lipolytica strain R14 and CBS 2074 in the NCBI database. The strain ML06 was preserved in the China General Microbiological Culture Collection Center on June 13, 2025, at the address of No. 1, Yihuan West Road, Beijing City, Chaoyang District, No. 3, Institute of Microbiology, Chinese Academy of Sciences, with a preservation number of CGMCC NO. 34889, and was named as ML06.
[0055] Example 2. Growth of Yarrowia lipolytica ML06 under different culture conditions
[0056] 1. Adaptability to different temperatures
[0057] After activation, the strain ML06 and the control strain PO1f were inoculated in YPD medium at an inoculation amount of 2%, and were cultured at 25°C, 30°C and 35°C, respectively, for 36 hours. The OD 600 was measured. The results are shown in Table 1. The results showed that ML06 had a more extensive temperature adaptability and could grow well under the culture condition at 35°C.
[0058] 2. Acid tolerance
[0059] After activation, the strain ML06 and the control strain PO1f were inoculated in YPD medium at an inoculation amount of 2%, and were adjusted to different pH. The OD 600 was measured after being cultured at 30°C for 36 hours. The results are shown in Table 1. The results showed that ML06 had a more extensive temperature adaptability and could grow well under the culture condition at pH 2.
[0060] Table 1. Growth of Yarrowia lipolytica M06 strain under different temperature and pH conditions
[0061]
[0062] Example 3, Yarrowia lipolytica ML06 grows in culture medium with oil as the sole carbon source
[0063] After activation of the strain ML06 and the control strain POIf, 2% inoculation was inoculated into YL medium containing palmitic acid, soybean oil, and kitchen waste oil, respectively, and cultured at 30°C for 36 hours. After adding n-hexane, the OD of the bacterial liquid part was measured after shaking and centrifugation. 600 The results are shown in Table 2. The results show that ML06 can grow better under different oil carbon sources.
[0064] Table 2. Growth of Yarrowia lipolytica M06 strain under different oil carbon sources
[0065]
[0066] Example 5, using ML06 strain as a chassis cell to produce butanediol under acidic conditions
[0067] (1) ML06 strain as a chassis cell to construct ML11: The exogenous Saccharomyces cerevisiae ICL1 gene is integrated into the upstream of the SUC1 gene of ML06 to obtain the strain ML11. The specific operation is as follows:
[0068] 1-a) The following sgRNA target sequence is designed for the upstream region of the SUC1 gene of the ML06 strain: 5'-gaggtgtgaatgagagcaag-3'. The whole gene synthesis plasmid pCRISPR-Sg has the sequence of SEQ NO. 2, which includes: 5'-sgRNA-Cas9-URA3-YlARS-ColE-Amp-3'.
[0069] 1-b) The following DNA fragment ICL is artificially synthesized, which has the sequence of SEQ NO. 3, including: 5'-Sucup-ICL1-Sucdown-3'. The homologous recombination fragment is prepared by PCR amplification using the fragment as a template and P-F / P-R as primers.
[0070] P-F sequence: 5'-atgtcattggccaatggcccgcattaccta-3'
[0071] P-R sequence: 5'-tcgtggtcgacgacggcgtacttggacgcc-3'
[0072] 1-c) The pCRISPR-Sg plasmid and the homologous recombination fragment obtained above were transformed into the target strain by electroporation method. The transformants were picked and cultured in YPD liquid medium at 30°C overnight. The transformant genome was extracted and verified by PCR method using primers N-F / N-R, and the about 3300bp target band was amplified as positive, named as strain ML11.
[0073] N-F sequence is: 5'-ccaaactttgtttggtgagtattaggtcgc-3'
[0074] N-R sequence is: 5'-ccaccggctccaacaacaacacaatcgtac-3'
[0075] 1-d) The overnight culture of ML11 was inoculated into 200mL YL medium (pH=2) containing 1% (mass percentage) kitchen waste oil and 0.005% esterase at an inoculation amount of 1%, and cultured at 35°C for 48 hours, wherein 1% kitchen waste oil was added at 24 hours, and the culture product was collected.
[0076] The culture product was centrifuged at 12000g for 10 minutes, and the supernatant was filtered through a filter membrane with a pore size of 0.22μm, and the filtrate was collected. Five flask experiments were repeated. The yield of succinic acid was determined by HPLC equipped with Aminex HPX-87H column (purchased from BioRad company) and differential detector. All samples were filtered using 0.22μm filter before loading. 5mM H2SO4 was used as mobile phase, and the analysis was carried out at a flow rate of 0.6 mL / min, and the column temperature was set to 65°C.
[0077] The results show that the yield of succinic acid produced by recombinant bacteria ML11 is 10.88±1.05g / L under the conditions of 35°C and pH2. It shows that ML06 as a chassis cell has good application prospect in the field of biological manufacturing and metabolic engineering.
[0078] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under the same parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In general, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which deviates from the scope disclosed in the present application. Some basic features can be applied within the scope of the following attached claims.
Claims
1. Yarrowia lipolytica strain ML06, with accession number CGMCC NO. 34889.
2. The application of the *Yarrowia lipolyticis* as described in claim 1 in industrial fermentation; Alternatively, the use of the Yersinia lipolytica according to claim 1 in the production of target products from industrial fermentation chassis cells; Alternatively, the application of the Yersinia lipolyticis yeast of claim 1 in the industrial fermentation production of the target product; Alternatively, the use of the Yersinia lipophila according to claim 1 in the preparation of recombinant microorganisms for industrial fermentation.
3. The application according to claim 2, characterized in that: The conditions for industrial fermentation are: containing oils, high temperature, and high acid (low pH).
4. A method for culturing or fermenting the *Yersinia lipolytica* of claim 1 or a recombinant strain constructed using it as a chassis cell, comprising the following steps: culturing or fermenting the *Yersinia lipolytica* of claim 1 or a recombinant strain constructed using it as a chassis cell under high temperature or high acid conditions.
5. A method for producing a target product using the *Yarrowia lipolytica* as the chassis cell as described in claim 1, comprising the following steps: The gene encoding the protein related to the production target product was introduced into the *Yersinia lipophila* as described in claim 1 to obtain a recombinant strain; The recombinant bacteria are synthesized using oils and other raw materials and fermented under high acid and / or high temperature conditions to produce the target product.
6. The method according to claims 1-5, characterized in that: The high-acid condition refers to a pH value of 2 in the fermentation system; the high-temperature condition refers to a temperature greater than 34°C; and the oils include palmitic acid, soybean oil, and kitchen waste oil, etc.