Method for preparing single-cell protein based on methanol

Abstract

The invention belongs to the technical field of fermentation engineering, and relates to a preparation method of single-cell protein, in particular to a method for preparing single-cell protein based on methanol as a unique carbon source. According to the method for preparing the single-cell protein based on the methanol, a yarrowia lipolytica strain is used as a fermentation strain, a strain capable of adapting to high-concentration methanol is screened through adaptive laboratory induction optimization, key enzymes and genes in the cell wall synthesis process are recognized, and the protein content is increased by weakening cell wall synthesis; and the fermentation process is optimized, so that the yield and the quality of the protein can be effectively improved, and high-level production of the single-cell protein by taking methanol as a unique carbon source is successfully realized.

Description

Technical Field

[0001] The invention belongs to the technical field of fermentation engineering, and relates to a method for preparing single-cell protein, in particular to a method for preparing single-cell protein based on methanol as the sole carbon source. Background Art

[0002] In recent years, the main goal of green biomanufacturing has been to build microbial cell factories through synthetic biology technology and produce valuable products using renewable raw materials. At present, the biomanufacturing industry is highly dependent on sugars as the main raw material, which leads to vicious competition in the food and feed industries. Therefore, the development of non-grain and low-cost alternative raw materials to replace sugars is crucial for the sustainable development of biomanufacturing.

[0003] As a non-food raw material, methanol is considered to be an ideal carbon source for the next generation due to its abundant reserves and low price. At the same time, the progress in synthesizing methanol through carbon dioxide hydrogenation has also opened up a way for renewable methanol production. Moreover, compared with traditional carbon sources such as glucose and glycerol, methanol has stronger reducibility and contains higher energy, which will be beneficial to cell growth and biosynthesis.

[0004] To meet the global demand for animal-derived protein, the world needs to produce 1.25 billion tons of meat and dairy products each year. However, due to the inefficiency of converting feed into meat and dairy products, increasing the production of meat and dairy products will not be able to sustainably meet the growing demand for protein. Although artificial meat and plant-based meat substitutes are currently developing rapidly, the higher production costs and poorer taste make them less competitive than animal protein produced through traditional animal husbandry. Therefore, there is still a need to find and develop new protein resources in this field.

[0005] Currently, single-cell protein (SCP) is a potential alternative. Single-cell protein is produced in algae (protein content is about 60-70%), fungi (protein content is about 30-50%) and bacteria (protein content is about 50-80%). The unique ability to utilize substrates (such as CO2 or methane) and the high growth rate of these microorganisms make the production process of SCP more efficient and sustainable than traditional agriculture. Yarrowia lipolytica has become an ideal host for the production of SCP due to its good growth characteristics and high protein content. However, the traditional Yarrowia lipolytica has limited protein production and high production costs under the culture conditions of methanol as the only carbon source. Therefore, it is of positive significance in the art to develop an optimized process that can efficiently use methanol as the only carbon source to produce single-cell protein. Summary of the invention

[0006] To this end, the technical problem to be solved by the present invention is to provide a method for preparing single-cell protein based on methanol, which can efficiently utilize methanol as a raw material to produce single-cell protein, so as to solve the problem in the prior art that the utilization rate of methanol by microorganisms is low, thereby affecting the single-cell protein yield.

[0007] In order to solve the above technical problems, the method for preparing single cell protein based on methanol according to the present invention comprises the following steps:

[0008] (1) taking a Yarrowia lipolytica strain for induction culture;

[0009] (2) continuing to genetically modify the Yarrowia lipolytica strain, knocking out its gene PAS_chr4_0305, and obtaining a genetically engineered strain;

[0010] (3) Using the genetically engineered strain to carry out fermentation culture and collecting the fermentation product to obtain.

[0011] Specifically, in the method for preparing single-cell protein based on methanol, in step (1), the induction culture step includes the step of sequentially placing the Yarrowia lipolytica strain in an induction culture medium containing different contents of methanol for induction culture;

[0012] Preferably, in the induction culture step, the methanol content of the induction culture medium is gradually increased;

[0013] Preferably, the Yarrowia lipolytica strain includes Yarrowia lipolytica, CICC32291.

[0014] Specifically, in the method for preparing single cell protein based on methanol, in step (1), the induction culture step comprises:

[0015] placing the Yarrowia lipolytica strain in a first induction medium containing 1-3 wt % methanol for a first induction culture;

[0016] Continuing to place the Yarrowia lipolytica strain in a second induction medium containing 4-7 wt % methanol for a second induction culture;

[0017] The Yarrowia lipolytica strain is then placed in a third induction medium containing 8-12 wt % methanol for a third induction culture.

[0018] Specifically, in the method for preparing single cell protein based on methanol, in step (1):

[0019] The temperature of the first induction culture step is 28-30°C; and / or,

[0020] The temperature of the second induction culture step is 30-33°C; and / or,

[0021] The temperature of the third induction culture step is 34-37°C.

[0022] Specifically, in the method for preparing single-cell protein based on methanol, in the step (1), the first induction medium, the second induction medium and / or the third induction medium independently include Delft alkaline salt medium;

[0023] Preferably, the Delft alkaline salt medium comprises the following components: ammonium chloride 0.5-2 g / L, dipotassium hydrogen phosphate 0.7-3 g / L, L-cysteine ​​0.3-0.8 g / L, magnesium sulfate heptahydrate 0.05-0.08 g / L, ferrous sulfate heptahydrate 0.005-0.009 g / L, biotin 0.025-0.045 mg / L, calcium chloride dihydrate 0.002-0.006 g / L, boric acid 0.0001-0.0004 g / L, zinc sulfate heptahydrate 0.0001-0.0004 g / L, and copper sulfate pentahydrate 0.0005-0.0009 g / L.

[0024] Specifically, in the method for preparing single-cell protein based on methanol, in step (1), the first induction culture, the second induction culture, and the third induction culture steps independently include the step of proliferating and subculturing the Yarrowia lipolytica strain.

[0025] Specifically, in the method for preparing single cell protein based on methanol, in step (2), the PAS_chr4_0305 gene knockout step comprises:

[0026] To construct PAS_chr4_0305 donor DNA, the upstream and downstream fragments of the PAS_chr4_0305 gene were amplified from the host genome using the following primers PAS_chr4_0305-up-F / PAS_chr4_0305-up-R and PAS_chr4_0305-down-F / PAS_chr4_0305-down-R, respectively; the upstream and downstream fragments were fused and amplified by fusion PCR to prepare the PAS_chr4_0305 donor;

[0027] The PAS_chr4_0305 donor was transformed into competent cells, and the cells were cultured to preserve the strain.

[0028] Specifically, in the method for preparing single-cell protein based on methanol, in the step (3), the fermentation medium of the fermentation culture step includes the following components: 10-25 g / L methanol, 10-15 g / L yeast extract, 20-35 g / L peptone, 1-3 g / L magnesium sulfate, 5-20 g / L sulfuric acid, 2-5 g / L potassium dihydrogen phosphate, 2-5 g / L potassium hydrogen phosphate, 0.01-0.02 g / L ferric chloride, 0.01-0.02 g / L manganese sulfate, 0.01-0.02 g / L zinc sulfate, 2-4 mg / L copper sulfate, and 2-4 mg / L sodium molybdate.

[0029] Specifically, in the method for preparing single-cell protein based on methanol, in step (3), the conditions of the fermentation culture step include: the fermentation temperature is 34-37°C;

[0030] Preferably, when the bacterial OD is less than 300, the ventilation speed is controlled at 0.6-1vvm;

[0031] Preferably, when the bacterial OD is greater than 300 and less than 500, the ventilation speed is controlled at 0.8-1.2 vvm;

[0032] Preferably, when the bacterial cell OD is greater than 500, the ventilation rate is controlled at 1.8-2.4 vvm.

[0033] Specifically, the method for preparing single-cell protein based on methanol, in the step (3), further comprises the step of carrying out seed culture of the genetically engineered strain;

[0034] Preferably, the seed culture medium in the seed culture step comprises: 10-25 g / L yeast powder, 20-40 g / L peptone, and 20-30 g / L glucose.

[0035] The invention also discloses the single-cell protein prepared by the method for preparing the single-cell protein based on methanol.

[0036] The present invention also discloses the use of the single-cell protein prepared by the method for preparing single-cell protein based on methanol and its processed products for preparing animal feed products.

[0037] The present invention also discloses a method for inducing a strain for preparing single-cell protein based on methanol, which is characterized by comprising the steps of inducing the strain and carrying out genetic modification according to the methods described in steps (1) and (2) in the aforementioned method for preparing single-cell protein based on methanol;

[0038] Preferably, the characteristics of the induced culture and genetic modification are the same as the defined characteristics in the aforementioned method for preparing single-cell protein based on methanol.

[0039] The method for preparing single-cell protein based on methanol described in the present invention uses the Yarrowia lipolytica strain as the fermentation strain, screens the strain that can adapt to high-concentration methanol through adaptive laboratory induction optimization, identifies key enzymes and genes in the cell wall synthesis process, increases the protein content by weakening the cell wall synthesis, and optimizes the fermentation process, which can effectively improve the protein yield and quality, and successfully achieves high-level production of single-cell protein using methanol as the sole carbon source.

[0040] The method for preparing single-cell protein based on methanol described in the present invention can improve the methanol conversion performance of the strain through adaptive laboratory induced optimization and identification of key enzymes and genes in the cell wall synthesis process. The single-cell protein yield of the overall process is significantly improved, and the protein content is significantly increased.

[0041] The method for preparing single-cell protein based on methanol of the present invention has the advantages of low production cost, high production efficiency, environmental friendliness, etc., and has broad application prospects in the fields of food, feed, medicine, etc. DETAILED DESCRIPTION

[0042] The present invention provides a method for preparing single-cell protein based on methanol. The method can efficiently utilize methanol as a raw material to produce single-cell protein and effectively increase the yield of single-cell protein by inducing and modifying a fermentation strain and optimizing a fermentation process.

[0043] The present invention provides a method for preparing single cell protein based on methanol, comprising the following steps:

[0044] (1) taking a Yarrowia lipolytica strain for induction culture;

[0045] (2) continuing to genetically modify the Yarrowia lipolytica strain, knocking out its gene PAS_chr4_0305, and obtaining a genetically engineered strain;

[0046] (3) Using the genetically engineered strain to carry out fermentation culture and collecting the fermentation product to obtain.

[0047] In some feasible embodiments, in step (1), the induction culture step comprises the step of sequentially placing the Yarrowia lipolytica strain in an induction medium containing different contents of methanol for induction culture;

[0048] Preferably, in the induction culture step, the methanol content of the induction culture medium is gradually increased;

[0049] Preferably, the Yarrowia lipolytica strain includes Yarrowia lipolytica, CICC32291.

[0050] In some feasible embodiments, in step (1), the induction culture step comprises:

[0051] placing the Yarrowia lipolytica strain in a first induction medium containing 1-3 wt % methanol for a first induction culture;

[0052] Continuing to place the Yarrowia lipolytica strain in a second induction medium containing 4-7 wt % methanol for a second induction culture;

[0053] The Yarrowia lipolytica strain is then placed in a third induction medium containing 8-12 wt % methanol for a third induction culture.

[0054] In some feasible implementation schemes, in step (1):

[0055] The temperature of the first induction culture step is 28-30°C; and / or,

[0056] The temperature of the second induction culture step is 30-33°C; and / or,

[0057] The temperature of the third induction culture step is 34-37°C.

[0058] In some feasible embodiments, in step (1), the first induction medium, the second induction medium and / or the third induction medium independently comprise Delft alkaline salt medium;

[0059] As an exemplary embodiment, the Delft alkaline salt medium includes the following components: ammonium chloride 0.5-2 g / L, dipotassium hydrogen phosphate 0.7-3 g / L, L-cysteine ​​0.3-0.8 g / L, magnesium sulfate heptahydrate 0.05-0.08 g / L, ferrous sulfate heptahydrate 0.005-0.009 g / L, biotin 0.025-0.045 mg / L, calcium chloride dihydrate 0.002-0.006 g / L, boric acid 0.0001-0.0004 g / L, zinc sulfate heptahydrate 0.0001-0.0004 g / L, and copper sulfate pentahydrate 0.0005-0.0009 g / L.

[0060] In some feasible embodiments, in step (1), the first induction culture, the second induction culture, and the third induction culture steps independently include the steps of multiplying and subculturing the Yarrowia lipolytica strain.

[0061] As an exemplary embodiment, in step (1), the induction culture step comprises:

[0062] The Yarrowia lipolytica strain is added to a Delft alkaline salt medium containing 2 wt % methanol, and cultured at 28-30° C. and 150-250 rpm; when the bacterial OD exceeds 0.8, the bacterial liquid is inoculated into a fresh Delft alkaline salt medium containing 2% methanol for continued culture, and the culture is continuously subcultured for 20-30 times;

[0063] Continue to take the bacterial solution and inoculate it into a fresh Delft alkaline salt medium containing 5 wt% methanol, and culture it at 30-33°C and 150-250 rpm; when the bacterial OD exceeds 1.2, take the bacterial solution and inoculate it into a fresh Delft alkaline salt medium containing 5% methanol and continue to culture it, and pass it for 20-30 times;

[0064] Continue to take the bacterial liquid and inoculate it into fresh Delft alkaline salt medium containing 10% methanol, and culture it at 34-37°C and 150-250rpm; when the bacterial OD exceeds 1.5, take the bacterial liquid and inoculate it into fresh Delft alkaline salt medium containing 10% methanol and continue to culture, and continuously subculture for 20-30 times.

[0065] In some feasible embodiments, in step (2), the PAS_chr4_0305 gene knockout step comprises:

[0066] To construct PAS_chr4_0305 donor DNA, the upstream and downstream fragments of the PAS_chr4_0305 gene were amplified from the host genome using the following primers PAS_chr4_0305-up-F / PAS_chr4_0305-up-R and PAS_chr4_0305-down-F / PAS_chr4_0305-down-R, respectively;

[0067] The upstream and downstream fragments were fused and amplified by fusion PCR to prepare the PAS_chr4_0305 donor;

[0068] The PAS_chr4_0305 donor was transformed into competent cells, and the cells were cultured to preserve the strain.

[0069] In some feasible embodiments, in the step (3), the fermentation medium of the fermentation culture step includes the following components: methanol 10-25 g / L, yeast extract 10-15 g / L, peptone 20-35 g / L, magnesium sulfate 1-3 g / L, sulfuric acid 5-20 g / L, potassium dihydrogen phosphate 2-5 g / L, dipotassium hydrogen phosphate 2-5 g / L, ferric chloride 0.01-0.02 g / L, manganese sulfate 0.01-0.02 g / L, zinc sulfate 0.01-0.02 g / L, copper sulfate 2-4 mg / L, sodium molybdate 2-4 mg / L.

[0070] In some feasible embodiments, in step (3), the conditions of the fermentation culture step include: the fermentation temperature is 34-37°C;

[0071] Preferably, when the bacterial cell OD is less than 300, the ventilation speed is controlled to 0.6-1vvm, preferably compressed air is used for ventilation;

[0072] Preferably, when the bacterial OD is greater than 300 and less than 500, the ventilation speed is controlled to 0.8-1.2 vvm, and preferably a mixed gas of air and pure oxygen is used for ventilation, such as an air:pure oxygen volume ratio of 2:1;

[0073] Preferably, when the bacterial cell OD is greater than 500, the ventilation rate is controlled to 1.8-2.4 vvm, and preferably a mixed gas of air and pure oxygen is used for ventilation, such as an air:pure oxygen volume ratio of 1:2.

[0074] In some feasible embodiments, the step (3) further comprises the step of carrying out seed culture of the genetically engineered strain;

[0075] Preferably, the seed culture medium in the seed culture step comprises: 10-25 g / L yeast powder, 20-40 g / L peptone, and 20-30 g / L glucose.

[0076] In order to facilitate the understanding of the present invention, the present invention will be further described below in conjunction with examples. It should be understood that the following examples are only for a better understanding of the present invention and do not mean that the present invention is limited to the following examples.

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the invention belongs. The term "and / or" as may be used herein includes any and all combinations of one or more of the associated listed items.

[0078] In the following examples of the present invention, as an exemplary scheme, the Yarrowia lipolytica was purchased from the China Industrial Microbiological Culture Collection Center, with the collection number: CICC 32291.

[0079] In the following embodiments of the present invention, the detection method involved includes testing of bacterial OD, dry weight, yield and crude protein content, and the specific method includes:

[0080] (1) OD detection

[0081] In the following examples of the present invention, the term "OD value" refers to the OD value of the fermentation broth, which is obtained by ultraviolet spectrophotometry, specifically: using an ultraviolet spectrophotometer (Alpha-1106, Shanghai PUYUAN Instrument Co., Ltd.) to detect the OD value of the fermentation broth at 600nm;

[0082] (2) Calculation method of dry cell weight: unit: g / L

[0083] Weigh 1.00L of fermentation broth, centrifuge at 8000 rpm for 10 min, add 1L of water to obtain the bacterial cells and resuspend them evenly, centrifuge again at 8000 rpm for 10 min to obtain wet bacterial cells, dry them at 110°C to constant weight, and weigh their mass to be m1 g (accurate to 4 decimal places);

[0084] Dry weight of bacteria = m1 / 1g / L;

[0085] (3) Crude protein content: unit: %

[0086] According to the national standard GB / T 6432-2018 "Determination of crude protein in feed by Kjeldahl method", the crude protein content (mass fraction) is obtained through digestion, ammonia distillation and titration.

[0087] Example 1 Adaptive Laboratory Evolution

[0088] The Yarrowia lipolytica (CICC 32291) was inoculated into a Delft alkaline salt medium containing 2 wt % methanol, wherein the formula was: 0.5 g / L ammonium chloride, 0.7 g / L dipotassium hydrogen phosphate, 0.3 g / L L-cysteine, 0.05 g / L magnesium sulfate heptahydrate, 0.005 g / L ferrous sulfate heptahydrate, 0.025 mg / L biotin, 0.002 g / L calcium chloride dihydrate, 0.0001 g / L boric acid, 0.0001 g / L zinc sulfate heptahydrate, and 0.0005 g / L copper sulfate pentahydrate.

[0089] The culture was carried out at 28°C and 200 rpm. When the bacterial OD exceeded 0.8, the bacterial liquid was inoculated into a fresh Delft alkaline salt medium containing 2 wt% methanol for continued cultivation. The above induction culture steps were repeated for 30 consecutive passages.

[0090] Continue to take the above bacterial solution and inoculate it into a fresh Delft alkaline salt medium containing 5wt% methanol, and culture it at 33°C and 200rpm. When the OD exceeds 1.2, take the bacterial solution and inoculate it into a fresh Delft alkaline salt medium containing 5wt% methanol and continue to culture it, and pass it for 30 times.

[0091] Continue to take the bacterial liquid and inoculate it into fresh Delft alkaline salt medium containing 10wt% methanol, and culture it at 35°C and 200rpm. When the bacterial OD exceeds 1.5, take the bacterial liquid and inoculate it into fresh Delft alkaline salt medium containing 10wt% methanol and continue to culture it. After 30 consecutive subcultures, the strain is preserved.

[0092] Example 2 Adaptive Laboratory Evolution

[0093] The Yarrowia lipolytica (CICC 32291) was inoculated into a Delft alkaline salt medium containing 1 wt% methanol, wherein the formula was: 1 g / L ammonium chloride, 2 g / L dipotassium hydrogen phosphate, 0.5 g / L L-cysteine, 0.06 g / L magnesium sulfate heptahydrate, 0.007 g / L ferrous sulfate heptahydrate, 0.035 mg / L biotin, 0.004 g / L calcium chloride dihydrate, 0.0003 g / L boric acid, 0.0003 g / L zinc sulfate heptahydrate, and 0.0007 g / L copper sulfate pentahydrate.

[0094] The culture was carried out at 29°C and 150 rpm. When the bacterial OD exceeded 0.8, the bacterial liquid was inoculated into fresh Delft alkaline salt medium containing 1 wt% methanol for further culture. The above induction culture steps were repeated for 30 consecutive subcultures.

[0095] Continue to take the above bacterial solution and inoculate it into a fresh Delft alkaline salt medium containing 4 wt % methanol, and culture it at 30°C and 150 rpm. When the OD exceeds 1.2, take the bacterial solution and inoculate it into a fresh Delft alkaline salt medium containing 4 wt % methanol and continue to culture it, and pass it for 30 times in a row.

[0096] Continue to take the bacterial liquid and inoculate it into fresh Delft alkaline salt medium containing 8wt% methanol, and culture it at 34°C and 150rpm. When the bacterial OD exceeds 1.5, take the bacterial liquid and inoculate it into fresh Delft alkaline salt medium containing 8wt% methanol and continue to culture it. After 30 consecutive subcultures, the strain is preserved.

[0097] Example 3 Adaptive Laboratory Evolution

[0098] The Yarrowia lipolytica (CICC 32291) was inoculated into a Delft alkaline salt medium containing 3 wt % methanol, the composition of which was: 2 g / L ammonium chloride, 3 g / L dipotassium hydrogen phosphate, 0.8 g / L L-cysteine, 0.08 g / L magnesium sulfate heptahydrate, 0.009 g / L ferrous sulfate heptahydrate, 0.045 mg / L biotin, 0.006 g / L calcium chloride dihydrate, 0.0004 g / L boric acid, 0.0004 g / L zinc sulfate heptahydrate, and 0.0009 g / L copper sulfate pentahydrate.

[0099] The culture was carried out at 30°C and 250 rpm. When the bacterial OD exceeded 0.8, the bacterial liquid was inoculated into fresh Delft alkaline salt medium containing 3 wt% methanol for continued cultivation. The above induction culture steps were repeated for 30 consecutive passages.

[0100] Continue to take the above bacterial solution and inoculate it into fresh Delft alkaline salt medium containing 7wt% methanol, and culture it at 32°C and 250rpm. When the OD exceeds 1.2, take the bacterial solution and inoculate it into fresh Delft alkaline salt medium containing 7wt% methanol and continue to culture it, and pass it for 30 times.

[0101] Continue to take the bacterial liquid and inoculate it into fresh Delft alkaline salt medium containing 12wt% methanol, and culture it at 37°C and 250rpm. When the bacterial OD exceeds 1.5, take the bacterial liquid and inoculate it into fresh Delft alkaline salt medium containing 12wt% methanol and continue to culture it. After 30 consecutive subcultures, the strain is preserved.

[0102] Example 4

[0103] In this example, the strain after induction optimization in Example 1 was treated to weaken cell wall synthesis, that is, PAS_chr4_0305 gene was knocked out, which specifically includes the following steps.

[0104] In this embodiment, the PCR operation in each operation step can be amplified by conventional means in the art, and the operation of PCR amplification based on the following designed primers is a conventional method in the art.

[0105] In this embodiment, the PCR system involved in the following operation includes: 10 μl of amplification buffer, the formula is: 50mmol / L KCl, 10mmol / L Tris-HCl (pH 8.4 at room temperature), 1.5mmol / LMgCl2, 100μg / mL bovine serum albumin (BSA); 200μmol / L of each of the four substrates (dATP, dCTP, dGTP, dTTP); 10-100pmol of primers; 0.1-2μg of template DNA; 2.5μg of high-fidelity DNA polymerase; add purified water to 100μl. The concentrations of primers and templates in the PCR system in the following operation steps are different, and the amounts of other reagents can be consistent.

[0106] In this embodiment, the PCR procedure involved in the following conventional PCR operation includes:

[0107] (1) Denaturing at 94-98°C for 20-30 seconds to heat the double-stranded DNA template to dissociate it;

[0108] (2) Annealing at 50-65°C for 20-40 seconds to bind the short DNA molecules of the primer to the flanking regions of the target DNA;

[0109] (3) Extension at 68-72°C for 25-45 seconds, DNA polymerase extends the 3' end of the primer along the template chain;

[0110] (4) Repeat steps (1-3) 25-35 times to obtain target DNA copies.

[0111] Construct PAS_chr4_0305 donor DNA; use primers PAS_chr4_0305-up-F / PAS_chr4_0305-up-R and PAS_chr4_0305-down-F / PAS_chr4_0305-down-R to amplify the upstream and downstream fragments of the PAS_chr4_0305 gene (as shown in Table 1 below) from the Yarrowia lipolytica genome; fuse the upstream and downstream fragments and amplify by fusion PCR to prepare the PAS_chr4_0305 donor.

[0112] Specifically, the amplification includes raw materials: DNA template, primer, high-fidelity DNA polymerase and dNTP, and the process includes three consecutive steps: (1) 94-98°C and keep for 20-30 seconds to heat the double-stranded DNA template to dissociate it; (2) 50-65°C for 20-40 seconds to anneal, and the short DNA molecule of the primer is combined with the flanking region of the target DNA; (3) 68-72°C for 25-45 seconds to extend, and the DNA polymerase extends the 3' end of the primer along the template chain. These steps are repeated 25-35 times to obtain a copy of the target DNA.

[0113] Among them, the construction of plasmid pPICZ-Cas9-gPNSII-3 uses primers gPNSII-3-1F / gPNSII-3-1R and gPNSII-3-2F / gPNSII-3-2R (containing Cas9 and GUT1-gRNA controlled by the bidirectional promoter P) to obtain Cas part and gRNA part by PCR amplification (HTX1 series) as a template. The two parts were fused by Gibson assembly to produce plasmid pPICZ-Cas9-gPNSII-3. Transformation was screened on LB agar plates containing 50μg / mL Zeocin. In this part of the PCR system, the primer concentration involved was 20pmol and the template was 0.2μg, and the remaining reagents referred to the aforementioned PCR system scheme.

[0114] Continue using primers gPNSI-2-1F / gPNSI-2-1R, gPNSI-2-2F / gPNSI-2-2R, gPAS_chr4_0305-1F / gPAS_chr4_0305-1R

[0115] and gPAS_chr4_0305-2F / gPAS_chr4_0305-2R to construct plasmids pPICZ-Cas9-gPNSI-2 and pPICZ-Cas9-gPAS_chr4_0305.

[0116] To construct the GDH1 donor DNA, primers PNSII-3-up-F / PNSII-3-up-R, PAOX1-F / PAOX1-R, GDH1-F / GDH1-R, TADH2(GDH1)-F / TADH2(GDH1)-R and PNSII-3-down-F / PNSII-3-down-R were used to amplify the upstream part, the promoter part, the GDH1 part, the terminator part and the downstream part from the Yarrowia lipolytica genome (see ALL-PAS_chr4_0305 in Table 1 below). Then, these 5 fragments were fused by fusion PCR to obtain the GDH1 donor DNA cassette. In this part of the PCR system, the primer concentration involved was 40 pmol, the template was 0.8 μg, and the other reagents were referred to the aforementioned PCR system scheme.

[0117] For GLN1 donor DNA, the upstream part, promoter part, GLN1 part, terminator part and downstream part were amplified from the genome of Pinus truncatula using primers PNSI-2-up-F / PNSI-2-up-R, PDAS2-F / PDAS2-R, GLN1-F / GLN1-R, TADH2(GLN1)-F / TADH2(GLN1)-R and PNSI-2-down-F / PNSI-2-down-R. The five fragments were then fused by fusion PCR to generate a GLN1 donor DNA cassette. In this part of the PCR system, the primer concentration involved was 90 pmol and the template was 1.8 μg. The remaining reagents were referred to the aforementioned PCR system scheme.

[0118] To construct the PAS_chr4_0305 donor DNA, the upstream and downstream fragments of the PAS_chr4_0305 gene were amplified from the P. pastoris genome using primers PAS_chr4_0305-up-F / PAS_chr4_0305-up-R and PAS_chr4_0305-down-F / PAS_chr4_0305-down-R, respectively. The upstream and downstream fragments were then fused and amplified by fusion PCR to prepare the PAS_chr4_0305 donor cassette.

[0119] Finally, the gRNA expression plasmid and the corresponding donor DNA were co-transformed into competent cells of Yarrowia lipolytica, and the transformed cells were grown on YPD plates containing 100 μg / mL Zeocin for 3 days, and the strains were preserved to obtain the desired transformed strains.

[0120] In this embodiment, the relevant primers involved are shown in Table 1 below.

[0121] Table 1 Primer list

[0122]

[0123]

[0124] Example 5 Fermentation Optimization

[0125] The modified strain obtained in Example 4 was used for seed culture and fermentation amplification culture.

[0126] Prepare seed culture medium as follows: 10 g / L yeast powder, 20 g / L peptone, 20 g / L glucose, sterilize at 121°C for 20 minutes.

[0127] The bacterial strain preserved after weakening cell wall synthesis is inoculated into the seed culture medium at an inoculation rate of 5% and grown until the OD is greater than 1.5, which is the first-level seed solution.

[0128] Prepare fermentation medium as follows: 25 g / L methanol, 15 g / L yeast extract, 35 g / L peptone, 3 g / L magnesium sulfate, 20 g / L sulfuric acid, 5 g / L potassium dihydrogen phosphate, 5 g / L dipotassium hydrogen phosphate, 0.02 g / L ferric chloride, 0.02 g / L manganese sulfate, 0.02 g / L zinc sulfate, 4 mg / L copper sulfate, 4 mg / L sodium molybdate, and the balance is water; sterilize at 121°C for 20 minutes.

[0129] Inoculate the grown first-grade seed liquid into the prepared fermentation medium at a rate of 5%, control the fermentation temperature at 35°C, and perform the fermentation according to the following procedure:

[0130] When OD is less than 300, use compressed air with a ventilation volume of 0.6 vvm;

[0131] When OD was greater than 300 and less than 500, a mixed gas (air: pure oxygen at a ratio of 2:1) was introduced at a ventilation rate of 0.8 vvm;

[0132] When OD was greater than 500, a mixed gas (air: pure oxygen at a ratio of 1:2) was introduced at a ventilation rate of 1.8 vvm.

[0133] During the fermentation process, the OD of the fermentation liquid was tested every hour. When the OD of the fermentation liquid did not increase for three consecutive hours, the fermentation was stopped. After the tank was stopped, the dry weight and crude protein content of the bacteria were tested.

[0134] In this embodiment, the entire fermentation process lasts for 34 hours. When the tank is stopped, the OD is 214, the dry weight is 58 g / L, and the crude protein content is 65%.

[0135] Example 6

[0136] The fermentation process of the single-cell protein described in this embodiment is the same as that in the aforementioned embodiment 5, the only difference is that the fermentation medium is prepared to include: 25 g / L methanol, 15 g / L yeast extract, 35 g / L peptone, 3 g / L magnesium sulfate, 20 g / L sulfuric acid, 5 g / L potassium dihydrogen phosphate, 5 g / L dipotassium hydrogen phosphate, 0.02 g / L ferric chloride, 0.02 g / L manganese sulfate, 0.02 g / L zinc sulfate, 4 mg / L copper sulfate, 4 mg / L sodium molybdate, and the balance is water; 121° C., sterilize for 20 minutes.

[0137] In this embodiment, the entire fermentation process lasts for 30 hours. When the tank is stopped, the OD is 240, the dry weight is 70 g / L, and the crude protein content is 66%.

[0138] Example 7

[0139] The fermentation process of the single-cell protein described in this embodiment is the same as that in the aforementioned embodiment 5, the only difference is that the fermentation medium is prepared to include: 10 g / L methanol, 12 g / L yeast extract, 20 g / L peptone, 2 g / L magnesium sulfate, 12 g / L sulfuric acid, 3 g / L potassium dihydrogen phosphate, 2 g / L dipotassium hydrogen phosphate, 0.015 g / L ferric chloride, 0.01 g / L manganese sulfate, 0.015 g / L zinc sulfate, 2 mg / L copper sulfate, 3 mg / L sodium molybdate, and the balance is water; 121° C., sterilize for 20 minutes.

[0140] In this embodiment, the entire fermentation process lasts for 32 hours. When the tank is stopped, the OD is 224, the dry weight is 62 g / L, and the crude protein content is 67%.

[0141] Example 8

[0142] The fermentation process of the single-cell protein described in this embodiment is the same as that in the aforementioned embodiment 5, the only difference is that the fermentation medium is prepared to include: 18 g / L methanol, 10 g / L yeast extract, 28 g / L peptone, 1 g / L magnesium sulfate, 12 g / L sulfuric acid, 2 g / L potassium dihydrogen phosphate, 4 g / L dipotassium hydrogen phosphate, 0.01 g / L ferric chloride, 0.03 g / L manganese sulfate, 0.01 g / L zinc sulfate, 3 mg / L copper sulfate, 2 mg / L sodium molybdate, and the balance is water; 121° C., sterilize for 20 minutes.

[0143] In this embodiment, the entire fermentation process lasts for 33 hours. When the tank is stopped, the OD is 231, the dry weight is 64 g / L, and the crude protein content is 68%.

[0144] Comparative Example 1

[0145] The fermentation process of the single-cell protein in this comparative example is the same as that in the aforementioned Example 5, the only difference being that the Yarrowia lipolytica strain induced and cultured in Example 1 is directly used to carry out the fermentation process of Example 5 without genetic modification.

[0146] In this comparative example, the entire fermentation process lasted for 62 hours. When the tank was stopped, the OD was 112, the dry weight was 37 g / L, and the crude protein content was 43%.

[0147] Comparative Example 2

[0148] The fermentation process of the single-cell protein in this comparative example is the same as that in the aforementioned Example 5, the only difference is that the Yarrowia lipolytica (CICC 32291) is directly used to carry out the genetic modification in Example 4 and then the fermentation process in Example 5 is carried out, without the need for strain induction culture.

[0149] In this comparative example, the entire fermentation process lasted for 46 h. When the tank was stopped, the OD was 168, the dry weight was 46 g / L, and the crude protein content was 51%.

[0150] Comparative Example 3

[0151] The fermentation process of the single cell protein in this comparative example is the same as that in the aforementioned Example 5, the only difference is that during the induction culture of Yarrowia lipolytica (CICC 32291) in Example 1, the entire induction culture process is optimized using a Delft alkaline salt medium containing 2 wt %.

[0152] In this comparative example, the entire fermentation process lasted for 41 hours. When the tank was stopped, the OD was 202, the dry weight was 52 g / L, and the crude protein content was 60%.

[0153] Comparative Example 4

[0154] The fermentation process of the single cell protein in this comparative example is the same as that in the aforementioned Example 5, the only difference is that during the induction culture of Yarrowia lipolytica (CICC 32291) in Example 1, the entire induction culture process is optimized using a Delft alkaline salt medium containing 5wt%.

[0155] In this comparative example, the entire fermentation process lasted for 39 hours. When the tank was stopped, the OD was 213, the dry weight was 56 g / L, and the crude protein content was 62%.

[0156] Comparative Example 5

[0157] The fermentation process of the single cell protein in this comparative example is the same as that in the aforementioned Example 5, the only difference is that during the induction culture of Yarrowia lipolytica (CICC 32291) in Example 1, the entire induction culture process is optimized using a Delft alkaline salt medium containing 10 wt %.

[0158] In this comparative example, the entire fermentation process lasted for 37 hours. When the tank was stopped, the OD was 216, the dry weight was 58 g / L, and the crude protein content was 63%.

[0159] Comparative Example 6

[0160] The fermentation process of the single-cell protein in this comparative example is the same as that in the aforementioned Example 5, the only difference being that the fermentation medium composition includes: 10 g / L methanol, 10 g / L yeast extract, and 35 g / L peptone.

[0161] In this comparative example, the entire fermentation process lasted for 46 h. When the tank was stopped, the OD was 198, the dry weight was 52 g / L, and the crude protein content was 63%.

[0162] In summary, the method for producing single-cell protein based on methanol described in the present invention can effectively improve the yield and quality of protein by inducing the culture of the strain, identifying the key enzymes and genes in the cell wall synthesis process, and optimizing the fermentation conditions. Not only is the methanol conversion rate high, but the single-cell protein yield is also significantly improved, and the protein content is significantly increased.

[0163] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for preparing single cell protein based on methanol, characterized in that: The steps include: (1) taking a Yarrowia lipolytica strain for induction culture; (2) continuing to genetically modify the Yarrowia lipolytica strain, knocking out its gene PAS_chr4_0305, and obtaining a genetically engineered strain; (3) Using the genetically engineered strain to carry out fermentation culture and collecting the fermentation product to obtain.

2. The method for preparing single cell protein based on methanol according to claim 1, characterized in that: In the step (1), the induction culture step comprises the step of sequentially placing the Yarrowia lipolytica strain in an induction culture medium containing different contents of methanol for induction culture; Preferably, in the induction culture step, the methanol content of the induction culture medium is gradually increased.

3. The method for preparing single cell protein based on methanol according to claim 1 or 2, characterized in that: In the step (1), the induction culture step comprises: placing the Yarrowia lipolytica strain in a first induction medium containing 1-3 wt % methanol for a first induction culture; Continuing to place the Yarrowia lipolytica strain in a second induction medium containing 4-7 wt % methanol for a second induction culture; The Yarrowia lipolytica strain is then placed in a third induction medium containing 8-12 wt % methanol for a third induction culture.

4. The method for preparing single cell protein based on methanol according to claim 3, characterized in that: In the step (1): The temperature of the first induction culture step is 28-30°C; and / or, The temperature of the second induction culture step is 30-33°C; and / or, The temperature of the third induction culture step is 34-37°C.

5. The method for preparing single cell protein based on methanol according to claim 3 or 4, characterized in that: In the step (1), the first induction medium, the second induction medium and / or the third induction medium independently comprise Delft alkaline salt medium; Preferably, the Delft alkaline salt medium comprises the following components: ammonium chloride 0.5-2g / L, dipotassium hydrogen phosphate 0.7-3g / L, L-cysteine ​​0.3-0.8g / L, magnesium sulfate heptahydrate 0.05-0.08g / L, ferrous sulfate heptahydrate 0.005-0.009g / L, biotin 0.025-0.045mg / L, calcium chloride dihydrate 0.002-0.006g / L, boric acid 0.0001-0.0004g / L, zinc sulfate heptahydrate 0.0001-0.0004g / L, and copper sulfate pentahydrate 0.0005-0.0009g / L; Preferably, in step (1), the first induction culture, the second induction culture and the third induction culture steps independently include the step of multiplying and subculturing the Yarrowia lipolytica strain.

6. The method for preparing single cell protein based on methanol according to any one of claims 1 to 5, characterized in that: In the step (2), the PAS_chr4_0305 gene knockout step comprises: To construct PAS_chr4_0305 donor DNA, the upstream and downstream fragments of the PAS_chr4_0305 gene were amplified from the host genome using the following primers PAS_chr4_0305-up-F / PAS_chr4_0305-up-R and PAS_chr4_0305-down-F / PAS_chr4_0305-down-R, respectively; the upstream and downstream fragments were fused and amplified by fusion PCR to prepare the PAS_chr4_0305 donor; The PAS_chr4_0305 donor was transformed into competent cells, and the cells were cultured to preserve the strain.

7. The method for preparing single cell protein based on methanol according to any one of claims 1 to 6, characterized in that: In the step (3), the fermentation medium of the fermentation culture step includes the following components: 10-25 g / L methanol, 10-15 g / L yeast extract, 20-35 g / L peptone, 1-3 g / L magnesium sulfate, 5-20 g / L sulfuric acid, 2-5 g / L potassium dihydrogen phosphate, 2-5 g / L dipotassium hydrogen phosphate, 0.01-0.02 g / L ferric chloride, 0.01-0.02 g / L manganese sulfate, 0.01-0.02 g / L zinc sulfate, 2-4 mg / L copper sulfate, and 2-4 mg / L sodium molybdate.

8. The method for preparing single cell protein based on methanol according to any one of claims 1 to 7, characterized in that: In step (3), the conditions of the fermentation culture step include: the fermentation temperature is 34-37°C; Preferably, when the bacterial OD is less than 300, the ventilation speed is controlled at 0.6-1vvm; Preferably, when the bacterial OD is greater than 300 and less than 500, the ventilation speed is controlled at 0.8-1.2 vvm; Preferably, when the bacterial cell OD is greater than 500, the ventilation rate is controlled at 1.8-2.4 vvm.

9. The method for preparing single cell protein based on methanol according to any one of claims 1 to 8, characterized in that: The step (3) further includes the step of carrying out seed culture of the genetically engineered strain; Preferably, the seed culture medium in the seed culture step comprises: 10-25 g / L yeast powder, 20-40 g / L peptone, and 20-30 g / L glucose.

10. A method for inducing a strain for preparing single-cell protein based on methanol, characterized in that: The method comprises the steps of inducing strain culture and genetic modification according to steps (1) and (2) in the method for preparing single-cell protein based on methanol according to any one of claims 1 to 9; Preferably, the characteristics of the induced culture and genetic modification are the same as those defined in claims 1-9.

Images