Construction method of yarrowia lipolytica strain capable of efficiently utilizing formic acid

Abstract

The invention discloses a construction method of a yarrowia lipolytica strain capable of efficiently utilizing formic acid, which comprises the following steps: by taking yarrowia lipolytica Po1f as an original strain, firstly, overexpressing a formic acid utilization key pathway-reductive glycine pathway (rGly) related gene through a genetic engineering means, and constructing a recombinant strain Po1f-rGly; then, a laboratory shake flask adaptive evolution (SFC) strategy with formic acid as driving force is adopted, the formic acid concentration is increased through yeast powder assisted transition and gradient, and an evolved strain is obtained through directed evolution. Compared with an original strain, the strain realizes breakthrough transformation from formic acid inutilization to efficient formic acid assimilation. Through a strategy of combining genetic engineering and adaptive evolution, the formic acid nutritional type yarrowia lipolytica with industrial application potential is successfully constructed, and a novel microbial chassis is provided for biological refinery and single-cell protein production based on formic acid substrates.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of bioengineering, and particularly relates to a method for realizing growth of Yarrowia lipolytica strains with formic acid as the sole carbon source. BACKGROUND

[0002] Yarrowia lipolytica is a non-conventional oleaginous yeast with great potential for industrial production, which has good stress resistance and can grow in various adverse environments such as high osmotic pressure and low pH. In addition, Yarrowia lipolytica also has a wide substrate utilization spectrum, and can grow using carbon sources such as sugars, oils and alkanes. Formic acid, as an important intermediate product of CO2 reduction, is a key bridge for indirect CO2 fixation by microorganisms. However, the utilization rate and assimilation efficiency of Yarrowia lipolytica for formic acid are not ideal, and formic acid is toxic to cells, which is not conducive to the growth of cells under high concentration conditions. Therefore, it is particularly important to develop Yarrowia lipolytica strains that can efficiently utilize formic acid.

[0003] Adaptive evolution, as an efficient phenotype-oriented strain modification strategy, can simulate natural selection pressure to directionally screen strains with enhanced target traits, and particularly shows significant advantages in improving substrate utilization efficiency and enhancing stress resistance. In recent studies on improving the tolerance and utilization rate of yeast formic acid, Zhang Yanfei et al. screened strains that could tolerate 1M formate salt by using SC medium (synthetic complete medium) with gradually increasing formate salt concentration. Liu Zihu et al. used glucose as an auxiliary carbon source for adaptive domestication strategy, and realized the efficient growth of the strain under the mixed carbon source of 20g / L glucose and 50g / L formate salt in MM medium, with the highest OD 600 6.88. However, this still cannot realize the growth of Yarrowia lipolytica with formate as the sole carbon source. If the formic acid assimilation pathway of Yarrowia lipolytica is strengthened in advance, combined with adaptive evolution, it can theoretically realize the growth of Yarrowia lipolytica with formic acid as the sole carbon source. SUMMARY

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of the present application is to provide a method for realizing growth of Yarrowia lipolytica strains with formic acid as the sole carbon source.

[0005] The purpose of the present application is realized by the following technical scheme:

[0006] A method for constructing Yarrowia lipolytica strains that can efficiently utilize formic acid is provided, which is based on Yarrowia lipolytica as the host strain, and overexpresses c-1-tetrahydrofolate synthetase (MIS1), aminomethyltransferase (GcvT), glycine cleavage system P protein (GcvP) and glycine cleavage system H protein (GcvH) in the genome to obtain a strain Po1f-rGly with a strengthened reductive glycine cycle.

[0007] Subsequently, the engineering strain Po1f-rGly was subjected to three-stage adaptive evolution, which was a three-stage of yeast powder assisted transition, single formic acid carbon source screening, and gradient concentration increasing.

[0008] The NCBI-GeneID of the c-1-tetrahydrofolate synthetase (MIS1) is 2907923; the NCBI-GeneID of the aminomethyltransferase (GcvT) is 2907779; the NCBI-GeneID of the glycine cleavage system P protein (GcvP) is 2905991; and the NCBI-GeneID of the glycine cleavage system H protein (GcvH) is 2912864.

[0009] The gene sequences of the c-1-tetrahydrofolate synthetase (MIS1), the aminomethyltransferase (GcvT), the glycine cleavage system P protein (GcvP), and the glycine cleavage system H protein (GcvH) from Yarrowia lipolytica Po1f are shown in SEQ. No. 1-4.

[0010] Preferably, the Yarrowia lipolytica is Yarrowia lipolytica Po1f.

[0011] During the adaptive evolution, the formate concentration in the yeast powder assisted transition stage is 200 mM, and then the concentration is increased from 100 mM to 200 mM when formic acid is used as the only carbon source for domestication.

[0012] During the yeast powder assisted transition stage, 0.5 g / L of yeast powder is added to assist the growth of formic acid.

[0013] During the adaptive evolution, each generation is cultured to the stable growth phase.

[0014] During the adaptive evolution, the Yarrowia lipolytica is subcultured for more than 3 generations, and the OD 600 When there is no longer change, the next stage is entered.

[0015] Specifically, the steps include the following:

[0016] (1) The TEF-MIS1-CYC1t, TEF-GcvT-tCYC1, PDC1p-GcvP-TDH2t, and pGPD-GcvH-TXPR2 expression frames are constructed by the method of overlap extension PCR, and then the four expression frames are linked with the 113 plasmid by the method of multi-fragment cloning and transformed into E. coli DH5α; the correct plasmid is obtained by enzyme digestion, and the gene recombinant fragment is transformed into the host strain to obtain the strain Po1f-rGly.

[0017] (2) Adaptive evolution pre-culture: the engineered strain obtained in step (1) is inoculated in YPF medium with formic acid as carbon source for culture, and the strain is used as seed liquid in adaptive evolution when it enters stationary phase;

[0018] (3) Adaptive evolution: the seed liquid in step (2) is centrifuged to collect the bacterial cells, which are washed twice and then subcultured in minimal medium containing yeast powder and 100 mM formate for a certain time (initial OD 600 0.1-0.2); after multiple subcultures in the medium with the same formic acid concentration, if the OD 600 of the strain does not change after continuous subculture for more than 3 generations, the strain is transferred to minimal medium containing only 100 mM formate for culture (initial OD 600 0.1-0.2), and whether it can grow with formate as the sole carbon source is observed; if not, it is transferred back to the medium with additional yeast powder for further culture; similarly, multiple subcultures are continued in the minimal formic acid medium with 100 mM formate concentration until the OD 600 of the strain does not change after continuous subculture for more than 3 generations, the strain is transferred to minimal medium containing only 200 mM formate for subculture (initial OD 600 0.1-0.2); after adaptive evolution for 150-250 days, a strain capable of stable growth with high-concentration formic acid (preferably 200 mM) as the sole carbon source is obtained.

[0019] Further, in step (2): the YPF medium contains 20 g / L sodium formate, 20 g / L peptone, and 10 g / L yeast powder.

[0020] Further, in step (3): the washing is with sterile physiological saline;

[0021] The minimal formic acid medium with added yeast powder is a minimal formic acid medium containing 0.5 g / L yeast powder and 200 mM formate.

[0022] The biomass of the strain obtained after adaptive evolution can reach 1.8-2.5 in the minimal medium with 200 mM formate concentration.

[0023] The above method, step (1), step (2), and step (3) described the common culture conditions are 30°C, and the shaking speed is 180-200 rpm.

[0024] A Yarrowia lipolytica strain resistant to high formic acid concentration and capable of using formic acid as the sole carbon source is prepared by the above method.

[0025] The Yarrowia lipolytica strain resistant to high formic acid concentration can tolerate 1M concentration of formate in a minimal medium with formic acid as the only carbon source, while the starting strain Po1f cannot grow in a minimal medium with 100mM formate as the only carbon source.

[0026] Advantages:

[0027] The present application combines rational design (genetic engineering) and non-rational screening (adaptive evolution), and realizes the growth of the strain with formic acid as the only carbon source by targeting and strengthening the core pathway of formic acid metabolism through genetic engineering, and balancing the initial growth demand and the final single carbon source utilization target by adopting a three-stage evolution scheme of "yeast powder assisted transition → single formic acid carbon source screening → gradient concentration increasing". As a key intermediate product of CO2 reduction, the strain of the present application can directly grow with formic acid as the only carbon source, providing an efficient microbial chassis for indirect fixation of CO2, production of single cell protein, oil or chemicals, and meeting the needs of sustainable biological manufacturing under the "double carbon" target. At the same time, it also provides a universal strategy for the modification of other strains with difficult-to-utilize carbon sources. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is the metabolic map of the genetically engineered strain Po1f-rGly of the present application;

[0029] Figure 2 is a comparison chart of the growth curves of YIrGly and ScrGly;

[0030] Figure 3 is a process chart of the adaptive evolution of Po1f-rGly;

[0031] Figure 4 is an adaptive evolution chart of 003-rGly;

[0032] Figure 5 is an adaptive evolution chart of Po1f strain;

[0033] Figure 6 is a growth curve of the FXH001 strain in a minimal medium with different concentrations of formate (with formate as the only carbon source);

[0034] Figure 7 is the formate consumption of the FXH001 strain in a minimal medium with different concentrations of formate;

[0035] Figure 8 is a comparison of formate consumption of FXH001 and Po1f-rGly (containing 1g / L glucose and 200mM formate);

[0036] Figure 9Comparison of growth of FXH001 and Po1f-rGly on YPF solid medium containing 1 M formate concentration. DETAILED DESCRIPTION

[0037] The technical solutions of the present application are described in detail below through specific examples. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0038] Example 1. Strengthening of the reductive glycine cycle of Yarrowia lipolytica Po1f

[0039] Since the reductive glycine pathway of S. cerevisiae has been constructed to enhance the assimilation of formic acid, in order to compare the effects of constructing genes of different origins, plasmids 113-GPD-TEF-MIS1-GcvT-GcvP-GcvH (Po1f) and 113-GPD-TEF-MIS1-GcvT-GcvP-GcvH (S288C) were constructed. The specific steps are as follows:

[0040] The genes c-1-tetrahydrofolate synthetase (MIS1), aminomethyltransferase (GcvT), glycine cleavage system P protein (GcvP), and glycine cleavage system H protein (GcvH) were amplified from the genomes of Yarrowia lipolytica Po1f and S. cerevisiae S288C by polymerase chain reaction (PCR) technology.

[0041] Among them, the genes MIS1 and GcvT form the TEF-MIS1-CYC1t and TEF-GcvT-tCYC1 expression frames with the TEF promoter and CYC terminator. The gene GcvP forms the PDC1p-GcvP-TDH2t expression frame with the PDC1 promoter and TDH terminator. The gene GcvH forms the pGPD-GcvH-TXPR2 expression frame with the GPD promoter and terminator TXPR. Through the method of multi-fragment cloning, the four expression frames are linked with the 113 plasmid and transformed into E. coli DH5α to construct 113-GPD-TEF-MIS1-GcvT-GcvP-GcvH (Po1f) and plasmid 113-GPD-TEF-MIS1-GcvT-GcvP-GcvH (S288C).

[0042] The 113-GPD-TEF-MIS1-GcvT-GcvP-GcvH recombinant plasmid was digested with Not I restriction endonuclease, and the digested band was recovered by agarose gel electrophoresis to obtain the recovered fragment containing the target gene.

[0043] The specific steps for constructing the strain Po1f-rGly are as follows:

[0044] 1. Preparation of Yarrowia lipolytica competence (freshly prepared)

[0045] (1) Take Yarrowia lipolytica YPD plate from -80℃ low temperature preservation refrigerator, and culture at 30℃ for 24h; (2) select single point with good growth, inoculate in YPD test tube, and culture overnight at 30℃, then inoculate in 100mL fresh YPD flask with 1% inoculation amount for further culture; (3) when OD 600 =1.0-1.2, divide 50mL bacterial solution into one sterilized EP tube, centrifuge at 4200r / min for 8min, and remove supernatant; (4) resuspend bacterial slurry with 50mL sterilized water, centrifuge at 4200r / min for 8min, and remove supernatant; (5) add 50mL 0.1mol / L lithium acetate to resuspend bacterial slurry, place at room temperature for 1h, centrifuge at 4200r / min for 8min, and remove supernatant; (6) wash bacterial cells with 2mL 0.1mol / L lithium acetate again, then add 2mL 0.1mol / L lithium acetate to resuspend cells, and divide 50μL into one sterilized 1.5mL EP tube, ready for use.

[0046] 2. Boil 10μL single-stranded salmon sperm DNA in 100℃ boiling water for 10min;

[0047] 3. Take recovered fragment GPD-TEF-MIS1-GcvT-GcvP-GcvH of transformed target gene, mix with single-stranded salmon sperm DNA of the above step, and slowly add to 50μL Yarrowia lipolytica competence cells, mix gently, and then culture at 30℃ and 150r / min air bath for 15min;

[0048] 4. Add 720μL 40% polyethylene glycol 4000 solution and 90μL 1mol / L dithiothreitol to the mixture, mix gently, and then culture at 30℃ and 225r / min shaking speed for 60min;

[0049] 5. Heat shock at 39℃ for 10min, centrifuge at 4200r / min for 10min, and remove supernatant;

[0050] 6. Add 1mL 0.1mol / L lithium acetate to resuspend bacterial cells, place at room temperature for 5min, then coat two uracil and leucine deficient plates, one plate is coated with 100μL transformation liquid, and the other plate is coated with 100μL enriched transformation liquid, and then culture at 30℃ for 2-3 days.

[0051] Single colonies were picked for colony PCR verification, and the Yarrowia lipolytica genome was extracted for genome PCR verification. Finally, the strains that passed PCR verification were inoculated in YPD test tubes for pre-culture, and were labeled as YIrGly for overexpression of the endogenous reductive glycine pathway in Po1f, and ScrGly for overexpression of the reductive glycine pathway from S. cerevisiae in Po1f.

[0052] Subsequently, the test tubes were inoculated with 5% inoculation amount in minimal formic acid medium containing 0.5 g / L yeast powder, and the expression effects of genes from different sources were compared. As shown in FIG. 1, the biomass of YIrGly was significantly greater than that of ScrGly, and the OD Figure 2 600 at 72 h reached 0.530, which was 10% more than that of ScrGly. Therefore, YIrGly overexpressing the endogenous reductive glycine pathway gene was selected for subsequent adaptive evolution, and was labeled as Po1f-rGly.

[0053] Preferably, the genes of c-1-tetrahydrofolate synthetase (MIS1), aminomethyltransferase (GcvT), glycine cleavage system P protein (GcvP), and glycine cleavage system H protein (GcvH) from Yarrowia lipolytica Po1f are used, and the sequences thereof are shown in SEQ. No. 1-4.

[0054] Example 2: Adaptive evolution of Po1f-rGly

[0055] 1. Adaptive evolution process

[0056] (1) Po1f-rGly liquid stored at -80°C was taken out for thawing, inoculated on a YPD solid plate (20 g / L glucose, 20 g / L peptone, 10 g / L yeast powder, 20 g / L agar, pH natural), and cultured at 30°C to isolate single colonies; a single colony was picked and inoculated in YPD liquid medium, and cultured at 30°C, 200 r / min for 72 h. (2) The YPD culture activated liquid was centrifuged at 5000 rpm for 5 min to collect the bacterial cells, the supernatant was discarded, and the bacterial cells were washed twice with sterile normal saline (5000 rpm, 5 min) and resuspended in YPF liquid medium (10 g / L yeast powder, 20 g / L peptone, 20 g / L formic acid), and cultured at 30°C, 180 r / min for 48 h until the stable phase to serve as a seed liquid. (3) 2 mL of the seed liquid was taken in a sterile EP tube, centrifuged at 5000 rpm for 5 min, washed twice, and resuspended in 50 mL of minimal formic acid medium (0.5 g / L yeast powder, 100 mM sodium formate, initial OD 600 0.1-0.2), and cultured at 30°C, 180 r / min. OD 600 was measured every 24 h; at the stable phase (OD 600 ​Glycerol stock preparation - 1st passage. (4) Continue to passage at 1:50 inoculation ratio, under the same conditions (30°C, 180 r / min; 0.5 g / L yeast extract, 100 mM formate in minimal formate medium; initial OD 600 0.1) with 5000 rpm centrifugation, twice physiological saline washing, and fresh medium inoculation. (5) When the strain grows stably in 100 mM formate medium containing yeast extract, remove the yeast extract and keep only 100 mM formate, continue to passage according to the same procedure. (6) After the strain adapts to the environment without yeast extract, increase the formate concentration to 200 mM, continue to passage culture, and other conditions are the same as above.

[0057] 2. Adaptive evolution results

[0058] Through the evolution of staged gradient pressure passage, in the minimal medium containing 0.5 g / L yeast extract and 100 mM formate, the OD 600 of the strain increased steadily from the initial 0.1-0.2 to 1.22 at the 20th generation, and was maintained at 1.15-1.25 at the 21st-22nd generations; then the yeast extract was removed, and only 100 mM formate was used as the carbon source, and the OD 600 of the first generation decreased to 0.324, but rose to 1.30 at the 7th generation, and was stable at 1.30-1.40 at the 8th-9th generations; finally, the formate concentration was increased to 200 mM, and the OD 600 of the first generation was 0.587, jumped to 2.012 at the 10th generation, and was stable at about 2.12 at the 11th-13th generations, indicating that the strain has successfully adapted to the high-concentration formate environment and significantly enhanced the biomass production capacity. Figure 3

[0059] The above process was repeated several times in different batches, and strains adapted to the high-concentration formate environment were obtained, and the OD 600 was stable at above 1.8 when the formate concentration was increased to 200 mM. Any of the strains was selected and named as FXH001, and its formate utilization performance was investigated.

[0060] In order to reflect the repeatability of the technology, under the same conditions, another Yarrowia lipolytica engineering strain Y003 (which has no difference with the original strain in formate utilization) in the laboratory was used to construct 003-rGly, and the above process was repeated, and strains adapted to the high-concentration formate environment were also obtained, and the OD 600 was stable at above 1.8 when the formate concentration was increased to 200 mM. Figure 4

[0061] ​​Meanwhile, to demonstrate the superiority of the combined strategy, we also domesticated the original strain using the same process as the aforementioned adaptive evolution. The results showed that even after 20 passages, the original strain still could not achieve growth using formic acid as the sole carbon source. Figure 5 ).

[0062] Example 3. Formic acid utilization performance of evolved strains

[0063] 1. Determination of growth curve

[0064] During the bacterial culture process, samples were taken every 24 hours to detect the growth status of the strain, i.e., OD. 600 The OD of the yeast was determined by colorimetry. 1 mL of fermentation broth was placed in a 1.5 mL centrifuge tube, diluted appropriately, and the absorbance was measured at 600 nm using a UV spectrophotometer with a 0.5 cm path length cuvette. The absorbance was then multiplied by the corresponding dilution factor to obtain the OD value. 600 The growth curves of FXH001 in minimum culture media with different formate concentrations (50 mM-1.5 M) were determined. Figure 6 As shown, the strain exhibited the best growth at a formate concentration of 200 mM, reaching its highest OD value at 192 h. 600 The OD value reached 1.92. At a formate concentration of 1.5 M, the strain's OD... 600 It can also grow from 0.031 to 0.248.

[0065] 2. FXH001 Formic Acid Consumption Capacity Test

[0066] Formate concentration was determined using an Agilent 1100 HPLC system (Agilent, China) equipped with a bio-Rad Aminex HPx-87H column (Bio-Red). FXH001 was cultured in a minimal medium containing 200 mM formate, and formate consumption was observed based on growth. When the growth rate decreased, 50 mM formate was added until growth completely ceased. The final formate concentration in the medium was then measured. Figure 7 As shown, FXH001 can utilize up to 264.49 mM formate. Since Po1f-rGly cannot utilize formate as its sole carbon source, the formate utilization capacity of the two strains before and after acclimation was compared in a minimal medium containing 1 g / L glucose and 200 mM formate. The results are as follows... Figure 8 As shown, Po1f-rGly consumed only 70.5 mM sodium formate in 120 hours, while PXH001 consumed all the formate in the culture medium in just 96 hours.

[0067] 3. Plate test for the formic acid tolerance of bacterial strains

[0068] Take the culture after FXH001 and Po1f-rGly fermentation broth, adjust the OD of each strain 600 = 1, after gradient dilution of different times, take 3 μL respectively on the YPF solid medium containing 1M formate, 30℃ culture, compare the growth of different strains before and after domestication, as shown in Figure 9 Due to the high concentration of formic acid, the Po1f-rGly strain strengthened by reducing glycine can grow, but it is still greatly inhibited, while the strain adapted by adaptive evolution grows well.

[0069] 4. Analysis of formic acid tolerance and utilization ability of FXH001

[0070] The results show that the strains adapted by formic acid exhibit significant growth advantage in the minimal medium with formate as the only carbon source, and their tolerance mechanism and metabolic ability are comprehensively improved. Even at an extreme concentration of 1.5M, it can achieve a growth breakthrough from OD600 0.031 to 0.248, while the control strain Po1f dies in a high-concentration formate environment due to proton dynamics leakage. Through the strategy of strengthening the formic acid assimilation pathway combined with adaptive evolution, FXH001 not only realizes the efficient consumption of 264.49mM formate (3.75 times higher than the consumption of 70.5mM by Po1f-rGly strain), but also shows significant growth advantage in solid medium containing 1.5M formate, while the unadapted strain Po1f-rGly is still severely inhibited. The study confirms that the combined strategy successfully constructs a functional strain that can not only tolerate high-concentration formate but also use it as the only carbon source. Its rapid metabolic characteristics (complete consumption of 200mM formate in 96h) and strong proton homeostasis maintenance ability provide a new type of chassis cell with application potential for industrial-grade formic acid bioconversion system.

[0071] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. A method for constructing a formic acid-efficient lipophilic yeast strain, characterized in that, The enhanced formic acid assimilation pathway is based on Yersinia lipolytica, which is an engineered strain obtained by overexpressing the genes c-1-tetrahydrofolate synthase (MIS1), aminomethyltransferase (GcvT), glycine cleavage system P protein (GcvP), and glycine cleavage system H protein (GcvH) in its genome.

2. The method for constructing a formic acid-efficient lipophilic yeast strain according to claim 1, characterized in that, The engineered strain underwent a three-stage adaptive evolution process, which consisted of yeast extract-assisted transition, screening with a single formic acid carbon source, and gradient concentration escalation.

3. The method for constructing a formic acid-efficient lipophilic yeast strain according to claim 1, characterized in that, The gene sequence of c-1-tetrahydrofolate synthase (MIS1) is shown in SEQ. NO.1, the gene sequence of aminomethyltransferase (GcvT) is shown in SEQ. NO.2, the gene sequence of glycine cleavage system P protein (GcvP) is shown in SEQ. NO.3, and the gene sequence of glycine cleavage system H protein (GcvH) is shown in SEQ. NO.

4.

4. The method for constructing a formic acid-efficient lipophilic yeast strain according to claim 1, characterized in that, The *Yersinia lipolytica* strain is *Yersinia lipolytica* Po1f (… Yarrowia lipolytica Po1f).

5. The method for constructing a formic acid-efficient lipophilic yeast strain according to claim 1, characterized in that, Expression cassettes TEF-MIS1-CYC1t, TEF-GcvT-tCYC1, PDC1p-GcvP-TDH2t, and pGPD-GcvH-TXPR2 were constructed using overlap-extended PCR. Subsequently, the four expression cassettes were ligated to plasmid 113 and transformed into E. coli DH5α using a multi-fragment cloning method. The correctly sequenced plasmid was digested with enzymes to obtain recombinant gene fragments, which were then transformed into the host bacterium to obtain strain Po1f-rGly.

6. The method for constructing a formic acid-efficient lipophilic yeast strain according to claim 2, characterized in that, During adaptive evolution, the formate concentration ranged from 200 mM during the transition phase assisted by yeast powder. Subsequently, when formic acid was used as the sole carbon source for domestication, the concentration was initially increased from 100 mM to 200 mM after the growth stabilized.

7. The method for constructing a formic acid-efficient lipophilic yeast strain according to claim 2, characterized in that, During the yeast powder-assisted transition stage, 0.5 g / L of yeast powder was added to assist formic acid growth.

8. The method for constructing a formic acid-efficient lipophilic yeast strain according to claim 2, characterized in that, In adaptive evolution, each generation is cultured until it enters a stable growth phase.

9. The method for constructing a formic acid-efficient lipophilic yeast strain according to claim 2, characterized in that, In adaptive evolution, the OD (Oxytoxin Oxytoxin) of Yersinia lipophila after three or more generations of subculturing... 600 When things stop changing, proceed to the next stage.

10. A *Yarrowia lipolytica* strain tolerant to high formic acid concentrations and capable of using formic acid as the sole carbon source, characterized in that... The strain was prepared by any one of claims 1 to 9.

Images