Pichia pastoris strain with high-temperature resistance and high methanol conversion rate and use thereof

Abstract

A Pichia pastoris strain with high-temperature resistance and a high methanol conversion rate. By mean of using a protoplast fusion method, the strain with a high methanol utilization rate, Pichia pastoris HTX-33 (CGMCC NO. 25207) is fused with a high-temperature-resistant Pichia manshurica strain screened from rotten wood from the vineyards of Yuanshi Vineyard in Yinchuan, Ningxia Province to obtain a hybrid strain that is resistant to high temperatures and efficiently utilizes methanol. The hybrid strain is identified as a Pichia pastoris strain and can achieve high biomass growth and high methanol utilization at 37°C by means of using methanol as the sole carbon source. The strain has the advantages of being green, environmentally-friendly, pollution-free, high in expression rate, low in cost, etc.

Description

A high-temperature-resistant Pichia yeast strain with high methanol conversion rate and its application Technical Field

[0001] The invention belongs to the field of microorganisms and biotechnology, and particularly relates to a high-temperature-resistant and high-methanol conversion rate Pichia pastoris strain and an application thereof. Background Art

[0002] To meet global demand for animal-based protein, the world will need to produce 1.25 billion tons of meat and dairy products annually. However, due to the inefficiency of converting feed into meat and dairy products, increasing meat and dairy production will not be able to sustainably meet humanity's growing demand for protein. Although artificial meat and plant-based meat substitutes are currently developing rapidly, their high production costs and poor taste make them less competitive than animal protein produced by traditional livestock farming. Therefore, it remains necessary to find and develop new protein resources. Single-cell proteins (SCPs) produced by algae (protein content of approximately 60-70%), fungi (protein content of approximately 30-50%), and bacteria (protein content of approximately 50-80%) are a potential protein alternative. The unique substrate utilization ability (such as CO2 or methane) and high growth rate of these microorganisms make SCP production more efficient and sustainable than traditional agriculture.

[0003] The concentration of CO₂ in the global atmosphere is increasing at an alarming rate, and climate change and the greenhouse effect have seriously affected human survival and development. The increasingly serious environmental crisis has intensified the need to shift from a traditional petrochemical economy to sustainable feedstocks. Microbial assimilation of one-carbon (C₁) compounds has emerged as a promising approach to mitigating climate change. Methanol is considered an ideal C₁ resource for the chemical industry and biomanufacturing because it can not only be produced from low-quality coal but also can be produced in large quantities through electrocatalytic conversion of CO₂ and hydrogen (electrolysis of water), which is considered one of the most promising approaches to achieving global carbon neutrality. Compared with gaseous C₁ compounds, methanol is easier to transport and store as a liquid. In addition, methanol has a high degree of reduction, which can provide a greater driving force for product biosynthesis. Therefore, its cheap and abundant source, flexible production process, compatibility with current transportation and fermentation infrastructure, and high reduction potential make methanol an extremely attractive carbon source for SCP biosynthesis.

[0004] Pichia pastoris is a naturally occurring microorganism that can utilize methanol as its sole carbon and energy source, making it an ideal host for methanol bioconversion. In particular, P. pastoris is an important industrial methanolic yeast, GRAS (Generally Recognized as Safe) certified by the US Food and Drug Administration (FDA) and widely used for recombinant protein production. For decades, researchers have used P. pastoris to ferment methanol for the production of SCP. However, the low methanol conversion rate in P. pastoris is a key limitation to efficient SCP production. Furthermore, because the fermentation temperature of P. pastoris is actually higher than its growth temperature (28-30°C), its intolerance to higher temperatures often reduces substrate conversion efficiency and increases cooling costs in industrial production processes. Therefore, developing a P. pastoris strain that is resistant to high temperatures and can efficiently utilize methanol is key to achieving the economical production of SCP using methanol as a feedstock.

[0005] Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, the present invention provides a high-temperature-resistant, high-methanol-conversion strain, Pichia pastoris SHTX-33. SHTX-33 is derived by fusing the high-methanol-utilizing strain P. pastoris HTX-33 (CGMCC No. 25207) with a high-temperature-resistant Pichia manshurica strain, obtained from decaying wood in the vineyards of Yuanshi Winery in Yinchuan, Ningxia. P. pastoris SHTX-33 is capable of achieving high biomass growth and high methanol utilization at 37°C using methanol as the sole carbon source. It offers advantages such as being environmentally friendly, pollution-free, with a high expression rate and low cost.

[0007] The present invention provides a Pichia pastoris strain (Pichia pastoris) SHTX-33 with high temperature resistance and high methanol conversion rate, whose deposit number is CGMCC NO. 27346. The strain was deposited on May 15, 2023, at the General Microbiology Center of the China Culture Collection Administration (CGMCC for short), and the depository address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its classification name is Pichia pastoris.

[0008] The present invention further provides the use of the Pichia pastoris strain in the production of microbial protein or feed.

[0009] Preferably, wherein the Pichia strain is fermented using methanol as the sole carbon and energy source.

[0010] Specifically, the volume ratio of methanol added to the culture medium is 0.3-0.6%.

[0011] In a specific embodiment, the culture medium uses Delft basic salt medium as the basic medium, and its components are: 7.5g / L ammonium sulfate, 14.4g / L potassium dihydrogen phosphate, 0.5g / L magnesium sulfate heptahydrate, the initial pH value of the culture medium is 6.5, and it is sterilized at 121°C for 20 minutes before use.

[0012] In one embodiment, the culture conditions for fermentation are 30-40° C. Preferably, the culture conditions for fermentation are 37° C. and 200-240 rpm.

[0013] The present invention provides a method for culturing the Pichia pastoris strain, wherein methanol is used as a carbon source and an energy source to ferment the Pichia pastoris strain.

[0014] Specifically, the culture conditions for fermentation are 30-40° C. Preferably, the culture conditions for fermentation are 37° C. and 200-240 rpm.

[0015] The beneficial effects of the present invention compared with the prior art are:

[0016] The P. pastoris SHTX-33 provided by the present invention is robust and can achieve high methanol conversion at 37°C. Experimental results show that the growth of the P. pastoris SHTX-33 provided by the present invention at 33°C-37°C and Delft+0.5% (v / v) methanol is significantly better than that of the parent P. pastoris HTX-33, and under shake flask culture conditions, the methanol utilization rate of the P. pastoris SHTX-33 provided by the present invention at 35°C or 37°C and Delft+0.5% (v / v) methanol is significantly better than that of the parent P. pastoris HTX-33, and its methanol utilization rate is increased by 18.45% and 24.1% compared with that of P. pastoris HTX-33. Therefore, the strain culture method of the present invention is simple, the late growth is rapid, the adaptability is strong, and the tolerance is high. It can solve the low methanol utilization rate and the strain's intolerance to high temperatures in the current industrial fermentation process, reduce fermentation costs, and has good prospects for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG1 is a schematic diagram of the protoplast fusion and screening process of the P. pastoris SHTX-33 strain provided by the present invention.

[0018] FIG2 is a growth curve diagram of P. pastoris SHTX-33 provided by the present invention and its parent P. pastoris HTX-33 under the conditions of 33° C. and Delft+0.5% (v / v) methanol.

[0019] FIG3 is a growth curve diagram of P. pastoris SHTX-33 provided by the present invention and its parent P. pastoris HTX-33 under the conditions of 35° C. and Delft+0.5% (v / v) methanol.

[0020] FIG4 is a growth curve diagram of P. pastoris SHTX-33 provided by the present invention and its parent P. pastoris HTX-33 under the conditions of 37° C., Delft+0.5% (v / v) methanol.

[0021] Figure 5 shows the maximum OD values ​​of P. pastoris SHTX-33 provided by the present invention and its parent P. pastoris HTX-33 at 33°C, 35°C, 37°C, Delft + 0.5% (v / v) methanol. 600 value.

[0022] Biomaterial deposit information

[0023] The strain HTX-33, whose preservation number is CGMCC NO.25207, is classified as Pichia pastoris. It was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on June 29, 2022. The address of the preservation unit is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0024] The strain SHTX-33, whose preservation number is CGMCC NO.27346, is classified as Pichia pastoris. It was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on May 15, 2023. The address of the preservation unit is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0025] Example 1: Parental origin of P. pastoris SHTX-33

[0026] Source of high temperature resistant parent: rotten wood samples were collected from rotten wood in the vineyard of Yuanshi Winery in Yinchuan, Ningxia. The samples were divided into test tubes containing 3mL YPD culture medium, shaken and cultured at 40°C and 220rpm for 48 hours, the obtained bacterial solution was diluted and spread on the YPD plate, and continued to be cultured in a 40°C incubator for 48 hours to obtain a monoclonal strain. Yeast monoclonal strains were selected from them and the obtained yeast monoclonal strains were inoculated into 250mL shake flasks containing 100mL YPD culture medium, shaken and cultured at 40°C and 220rpm for 24 hours to prepare seed liquid. The bacteria were collected by low temperature and low speed centrifugation (4°C, 5000rpm, 5min), and the bacteria were resuspended with YPD culture medium at an initial OD600 =0.25, plated in a 48-well plate, with eight replicates of each sample, and 1 mL added to each well. YPD medium was used as a blank control, and the growth curve of the strain was measured at 40°C and 800 rpm using an automated microbial growth curve analyzer. Growth of selected monoclonal strains was evaluated, and a 40°C-resistant yeast strain was identified. The strain was identified as Pichia manshurica (PM).

[0027] Parental source of high methanol utilization: Pick a single clone of wild-type P. pastoris X-33 and inoculate it into a 250 mL shake flask containing 100 mL Delft + 2% (w / v) glycerol medium. Shake and culture at 30°C and 220 rpm for 24 hours to prepare seed liquid. Collect the cells by low-temperature low-speed centrifugation (4°C, 5000 rpm, 5 minutes), resuspend the cells in Delft medium, and use the initial OD 600 =0.01 was inoculated into a 250 mL shake flask containing 100 mL of Delft + 0.5% (v / v) methanol medium and continued to be cultured at 33°C and 220 rpm. Each time in the logarithmic phase, a 1:100 transfer was performed to fresh Delft + 0.5% (v / v) methanol medium, with three replicates each time. The above process was repeated for approximately 350 generations (approximately 100 days) to domesticate a stable bacterial colony with high methanol conversion efficiency. After dilution plating and growth verification, a Pichia pastoris strain with high methanol utilization was screened and ultimately named HTX-33.

[0028] Example 2: Protoplast fusion and screening of P. pastoris SHTX-33 strain

[0029] The fusion and screening process of P. pastoris SHTX-33 is shown in Figure 1. Under aerobic conditions, single clones of P. pastoris HTX-33 and PM were inoculated into 250 mL shake flasks containing 100 mL of YPD medium. Enrichment cultures were performed at 33°C, 220 rpm, and 40°C, 220 rpm, respectively. Samples were taken at mid-logarithmic phase. A 1 mL sample was placed in a 10 mL sterile centrifuge tube and centrifuged at 3000 rpm for 5 minutes at room temperature. The supernatant was discarded. Wash with sterile water and centrifuge twice, add an equal volume of mercaptoethanol, treat at 28 ° C for 10 minutes, centrifuge at 2000 rpm for 10 minutes at room temperature to remove the supernatant, then add 2 mL of 2% snail lysozyme, and perform enzymolysis at 38 ° C for 2 hours. The protoplast-bacterial mixture is collected by low-temperature low-speed centrifugation (4 ° C, 2000 rpm, 10 minutes), the supernatant is removed and washed twice with ST hypertonic buffer, and the protoplasts are resuspended in STC hypertonic buffer at 4 ° C. The bacterial suspension of P. pastoris HTX-33 protoplasts is inactivated at 70 ° C for 40 minutes; the bacterial suspension of PM protoplasts is irradiated under a 20W ultraviolet lamp at a vertical distance of 25 cm for 30 minutes to inactivate the parent. Equal volumes of inactivated parental protoplasts were mixed and centrifuged at low speed (4°C, 2000 rpm, 10 minutes). The mixture was washed twice with STC hypertonic buffer, then an equal volume of PEG cosolvent was added and incubated at 30°C in a water bath for 30 minutes. The mixture was centrifuged (4°C, 2000 rpm, 10 minutes), resuspended (in STC hypertonic buffer), and plated onto YPD hypertonic regeneration plates. After incubation at 37°C for 5-7 days, 200 monoclonal strains with large, plump colonies were selected and transferred to Delft minimal salts medium with methanol as the sole carbon source. High-throughput screening was performed at 37°C and 700 rpm. Only four monoclonal strains were found to grow in methanol medium. The four monoclonal strains were inoculated into 250 mL shake flasks containing 100 mL Delft + 0.5% (v / v) methanol medium and cultured at 37°C and 220 rpm for 48 h to prepare seed solution. The solution was diluted with fresh Delft + 0.5% (v / v) methanol medium to an initial OD of 600 =0.01 was inoculated into 100 mL of Delft + 0.5% (v / v) methanol medium and cultured at 37°C with shaking at 220 rpm. Three replicates were set up for each group, and their growth was measured. After comparison, a strain with high methanol conversion efficiency was identified as Pichia pastoris and named SHTX-33.

[0030] Example 3: Determination of P. pastoris SHTX-33 Growth

[0031] In this experiment, the growth of P. pastoris SHTX-33 was evaluated using a fully automatic microbial growth curve analyzer. The determination method was the same as in Example 1. Monoclonal colonies of P. pastoris SHTX-33 and the parent P. pastoris HTX-33 were picked and inoculated into a 250 mL shake flask containing 100 mL Delft + 0.5% (v / v) methanol medium, and cultured at 37 ° C, 220 rpm and 33 ° C, 220 rpm for 24 hours to prepare seed liquid. Dilute with fresh Delft + 0.5% (v / v) methanol medium to the initial OD 600 =0.25, then inoculated into 48-well plates, with 8 replicates per sample and 1 mL added to each well. Delft medium was used as a blank control, and the growth curves of the strains at 33°C, 35°C, and 37°C were measured (see Figures 2, 3, and 4).

[0032] From the results, it can be seen that as the temperature increases, the growth of P.pastoris SHTX-33 and its parent P.pastoris HTX-33 in Delft+0.5% (v / v) methanol medium is inhibited. However, under the same temperature conditions, the growth of SHTX-33 is significantly better than that of its parent HTX-33. The growth curve represented by SHTX-33 in the growth curve is always higher than that of its parent HTX-33 (see Figures 2, 3, and 4). And as the temperature increases, the lag phase of SHTX-33 and its parent HTX-33 in Delft+0.5% (v / v) methanol medium gradually shortens, while at the same temperature, the lag phase of SHTX-33 during growth is shorter than that of HTX-33, and it begins to enter the logarithmic growth phase earlier. Compare the maximum OD values ​​of SHTX-33 and its parent HTX-33. 600 (See Figure 5), it was found that as the temperature increased, the maximum OD values ​​of SHTX-33 and its parent HTX-33 in Delft+0.5% (v / v) methanol medium increased. 600 All of them gradually decreased, but under the same temperature conditions, the maximum OD of SHTX-33 600 Specifically, under the conditions of 33°C, Delft + 0.5% (v / v) methanol, the maximum OD of SHTX-33 compared with the parent HTX-33 was significantly higher than that of the parent HTX-33. 600 Increased by 19.67%; at 35 ° C, Delft + 0.5% (v / v) methanol conditions, SHTX-33 compared to the parent HTX-33, its maximum OD 600 Increased by 25.25%; at 37 ° C, Delft + 0.5% (v / v) methanol conditions, SHTX-33 compared to the parent HTX-33, its maximum OD600 An increase of 9.18%. The above results show that P.pastoris SHTX-33 is robust and has high tolerance to temperature. It can achieve high biomass growth under the same conditions, and it grows rapidly in the later stage and has strong adaptability. It can solve the problem of strains not being able to tolerate high temperatures in the current industrial fermentation process, reduce fermentation costs, and has good prospects for industrial application. However, when using 48-well plates to measure growth, there is a possibility of methanol volatilization, and the amount of methanol volatilization will also increase with increasing temperature. Therefore, using 48-well plates can only qualitatively analyze the growth conditions and temperature tolerance of P.pastoris SHTX-33 and its parent P.pastoris HTX-33 under the same conditions, and cannot truly reflect its methanol to biomass conversion rate. Therefore, in the subsequent experiment to determine the methanol to biomass conversion rate of P.pastoris SHTX-33, we will use a shake flask experiment and cover the shake flask with a sealing film for measurement to prevent the volatilization of methanol from affecting the experimental results.

[0033] Example 4: Determination of the conversion rate of methanol to biomass of P. pastoris SHTX-33 As described in Example 3, in order to avoid evaporation of methanol, we used a method of covering the shake flask with a sealing film to determine the methanol utilization rate. Monoclonal colonies of P. pastoris SHTX-33 and the parent P. pastoris HTX-33 were picked and inoculated into 250 mL shake flasks containing 100 mL Delft + 0.5% (v / v) methanol medium, and shaken and cultured at 37°C, 220 rpm and 33°C, 220 rpm for 24 hours to prepare seed liquid. Dilute with fresh Delft + 0.5% (v / v) methanol medium to the initial OD 600 =0.05, inoculated into 250 mL shake flasks containing 100 mL of Delft + 0.5% (v / v) methanol medium, and cultured at 33°C, 35°C, and 37°C at 220 rpm. Three replicates were set up for each group. After the methanol was consumed, the cells were centrifuged, washed, centrifuged, and dried to obtain their dry weight (DCW), and the methanol utilization rate was calculated. The results are shown in Table 1.

[0034] Table 1. Conversion rates of methanol to biomass of P. pastoris SHTX-33 and its parent P. pastoris HTX-33 at different temperatures and in the presence of Delft + 0.5% (v / v) methanol

[0035] As can be seen from the table, the methanol utilization rate of P.pastoris SHTX-33 is higher than that of its parent, P.pastoris HTX-33, and the difference becomes more obvious as the temperature increases. Analysis shows that at 33°C and Delft + 0.5% (v / v) methanol, there is no significant difference in methanol utilization between P.pastoris SHTX-33 and its parent, P.pastoris HTX-33. At 35°C and Delft + 0.5% (v / v) methanol, the methanol utilization rate of P.pastoris SHTX-33 increases by 18.45% compared to its parent, P.pastoris HTX-33. At 37°C and Delft + 0.5% (v / v) methanol, the methanol utilization rate of P.pastoris SHTX-33 increases by 24.1% compared to its parent, P.pastoris HTX-33. These data indicate that P. pastoris SHTX-33 is robust and has a high methanol conversion rate. Under the same conditions, it can achieve a higher methanol to biomass conversion rate, which can solve the problem of low methanol conversion rate of strains in current industrial fermentation processes and has good prospects for industrial application.

Claims

1. A high temperature resistant and high methanol conversion rate Pichia pastoris strain SHTX-33, characterized in that: Its accession number is CGMCC NO.27346.

2. Use of the Pichia pastoris strain as claimed in claim 1 in the production of microbial protein or feed.

3. The use according to claim 2, characterized in that The Pichia yeast strain is fermented using methanol as the sole carbon and energy source.

4. The use according to claim 3, characterized in that The volume ratio of methanol added to the fermentation medium is 0.3-0.6%.

5. The use according to claim 4, characterized in that The culture medium uses Delft basic salt medium as the basic culture medium, and its components include 7.5 g / L ammonium sulfate, 14.4 g / L potassium dihydrogen phosphate, and 0.5 g / L magnesium sulfate heptahydrate. The initial pH value of the culture medium is 6.5, and the culture medium is sterilized at 121° C. for 20 minutes before use.

6. The use according to any one of claims 3 to 5, characterized in that The fermentation temperature is 30-40°C.

7. The use according to any one of claims 3 to 5, characterized in that The fermentation culture conditions were 37°C and 200-240 rpm.

8. A method for culturing the Pichia pastoris strain according to claim 1, characterized in that The Pichia strain was fermented with methanol as a carbon and energy source.

9. The method according to claim 8, characterized in that The fermentation temperature is 30-40°C.

10. The method according to claim 8, characterized in that The fermentation culture conditions were 37°C and 200-240 rpm.

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