CN118909815A - Method for improving saccharomyces cerevisiae protein yield by optimizing protein translation and extension process - Google Patents

Abstract

The present invention discloses a method for optimizing the protein translation extension process to increase the protein yield of saccharomyces cerevisiae, and belongs to the technical field of microbial genetic engineering. The present invention improves the global protein synthesis ability of saccharomyces cerevisiae by regulating the translation extension factor in the translation initiation process, so that the protein content of saccharomyces cerevisiae is increased from 45.63g/100g to 48.06g/100g, and the protein yield is increased from 3.23g/L to 3.46g/L, providing technical support for low-cost, green single-cell protein production, and having broad application prospects in the field of alternative proteins.

Description

A method for optimizing protein translation elongation process to increase protein yield in Saccharomyces cerevisiae

Technical Field

The invention relates to a method for optimizing the protein translation extension process to improve the protein yield of brewer's yeast, belonging to the technical field of microbial genetic engineering.

Background Art

Due to the sharp increase in the global population, there is a huge protein gap between the supply of protein and the expected protein demand in 2050. In order to meet the increased protein demand due to population growth, the land development area of traditional planting and animal husbandry has increased significantly, resulting in a large amount of greenhouse gas emissions and loss of biodiversity. In the future, the existing food production strategy will not be able to meet people's demand for protein. Therefore, finding more sustainable, more accessible and healthier sources of protein has become the focus of current food research.

Microbial protein, namely single cell protein (SCP), has become a promising source of protein. It can be obtained through fermentation and cultivation of industrial and agricultural waste. Single cell protein is a complex cytoplasmic mass containing protein and other components such as carbohydrates, fats, nucleic acids, etc. Its main biological sources include yeast, bacteria and algae, etc. It is considered to be an emerging source of protein for human or animal consumption. Compared with traditional proteins, microbial protein raw materials have a wide range of sources. Industrial and agricultural waste can be used as raw materials for the production of single cell protein. The production efficiency is high and the cycle is short. It can be mass-produced using existing fermentation technology and the microorganisms reproduce quickly. It is environmentally friendly. The production of microbial single cell protein is carried out in a bioreactor, which saves land resources and is not restricted by environmental and climatic conditions. On November 23, 2023, the National Health Commission issued an announcement on the approval of yeast protein as a new food raw material, which explained that yeast protein is made from brewer's yeast, which is cultured, fermented, and centrifuged to obtain the raw material of the bacteria, and is made through processes such as nucleic acid removal, centrifugation, enzymolysis, extraction, purification, separation, sterilization, and drying. Its main nutrients are protein (≥70.0g/100g), fat, dietary fiber, and water. Therefore, yeast protein can be used as an ideal new alternative protein with the characteristics of wide sources, easy access, and environmental friendliness.

At present, the breeding work to improve protein production is relatively mature. Commonly used irrational breeding strategies include ARTP mutagenesis, laboratory adaptive evolution, heavy ion radiation mutagenesis, etc., based on which the performance of many strains has been improved. However, traditional breeding methods are cumbersome to operate, it is difficult to improve their own protein synthesis ability, and there are defects such as poor genetic stability.

Summary of the invention

Technical issues:

The present invention overcomes the shortcomings of existing breeding technologies and provides a rational design method for regulating the protein synthesis process. By regulating the extension process of cerevisiae protein translation, efficient synthesis of cerevisiae single-cell protein can be achieved, thereby improving the production efficiency and yield of cerevisiae protein.

Technical solution:

The present invention provides a method for increasing the protein yield of Saccharomyces cerevisiae by optimizing the protein translation elongation process, that is, increasing the translation rate of proteins by overexpressing eukaryotic translation elongation factors, thereby increasing the global protein synthesis capacity in Saccharomyces cerevisiae. The invention is of great significance for promoting the global synthesis of Saccharomyces cerevisiae proteins and the application of alternative proteins.

The invention provides a method for improving the yield of saccharomyces cerevisiae protein by optimizing the protein translation extension process, which is to integrate one or more genes of Tef1, Eft1, Yef3 and Anb1 into the saccharomyces cerevisiae genome.

In one embodiment, the method is to integrate the Tef1, Eft1, Yef3 or Anb1 gene into the genomic Gal80 site.

In one embodiment, the method comprises the steps of:

(1) Connecting the gene Tef1, Eft1, Yef3 or Anb1 to the plasmid pUMRI-B-△GAL80 to obtain the recombinant plasmids pUMRI-△GAL80-TEF1, pUMRI-△GAL80-EFT1, pUMRI-△GAL80-YEF3, and pUMRI-△GAL80-ANB1, respectively;

(2) The recombinant plasmid constructed in step (1) is linearized and integrated into the chromosome of Saccharomyces cerevisiae by homologous recombination.

In one embodiment, the Saccharomyces cerevisiae is not limited to BY4741

The present invention also provides a recombinant Saccharomyces cerevisiae, wherein one or more genes of Tef1, Eft1, Yef3 and Anb1 derived from Saccharomyces cerevisiae BY4741 are integrated into the genome of the recombinant Saccharomyces cerevisiae.

In one embodiment, the nucleotide sequence of the Tef1 gene is shown as SEQ ID NO.1; the nucleotide sequence of the Eft1 gene is shown as SEQ ID NO.2; the nucleotide sequence of the Yef3 gene is shown as SEQ ID NO.3; and the nucleotide sequence of the Anb1 gene is shown as SEQ ID NO.4.

In one embodiment, the Saccharomyces cerevisiae includes but is not limited to BY4741.

The invention also provides application of the recombinant cerevisiae yeast in protein production.

In one embodiment, the use is to ferment the recombinant Saccharomyces cerevisiae in a culture medium for a period of time.

In one embodiment, the culture medium contains: tryptone (20 g/L), yeast extract (10 g/L), and galactose (20 g/L).

In one embodiment, the fermentation is carried out at 28-32°C, especially at 30°C.

In one embodiment, the fermentation is for at least 48 hours.

In one embodiment, the recombinant Saccharomyces cerevisiae is first activated in a culture medium containing tryptone, yeast extract, and glucose, and then fermented.

In one embodiment, the engineered yeast is based on the wild-type yeast BY4741 as a starting strain.

Beneficial effects:

The present invention uses wild-type BY4741 as the starting strain, screens different translation elongation factors for enhancing the global protein synthesis ability of Saccharomyces cerevisiae, and determines its effect on the protein yield of Saccharomyces cerevisiae through fermentation verification, and determines that overexpression of eukaryotic translation elongation factor Tef1 can enhance the synthesis of global protein in Saccharomyces cerevisiae. Compared with the original strain, the protein content of the engineered bacteria increased from 45.63g/100g to 48.06g/100g, and the protein yield increased from 3.23g/L to 3.46g/L.

The present invention improves the global protein synthesis ability of Saccharomyces cerevisiae by regulating the translation elongation factor in the translation initiation process, thereby achieving efficient synthesis of Saccharomyces cerevisiae protein. The method can provide a reference for the large-scale production of yeast protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the effect of overexpression of eukaryotic elongation factors on protein content in Saccharomyces cerevisiae.

FIG2 shows the effect of overexpression of eukaryotic elongation factors on the biomass of Saccharomyces cerevisiae.

FIG3 shows the effect of overexpression of eukaryotic elongation factors on protein production in Saccharomyces cerevisiae.

DETAILED DESCRIPTION

The culture medium involved in the following examples is:

YPD liquid culture medium: 20 g/L glucose, 20 g/L tryptone, 10 g/L yeast extract, sterilized by high pressure steam at 115°C for 30 min; when preparing solid plates, add 15 g/L agar powder on this basis.

YPG liquid culture medium: 20 g/L galactose, 20 g/L tryptone, 10 g/L yeast extract, sterilized by high pressure steam at 115°C for 30 min; when preparing solid plates, add 15 g/L agar powder on this basis.

20mg/mL G418 sulfate mother solution: 0.4g G418 sulfate powder is dissolved in 20mL sterile water, filtered and sterilized using a sterilized 0.22μm water syringe filter in a clean bench, dispensed into brown centrifuge tubes, and stored at -20℃. When used, add 10μL of mother solution to every 1mL of culture medium, with a working concentration of 200μg/mL, for the preparation of YPD-G418 and YPG-G418 liquid culture medium.

LB liquid medium: NaCl 10g/L, tryptone 10g/L, yeast extract 5g/L, sterilized at 120℃ for 20min. When preparing solid plates, add 15g/L agar powder.

The final working concentration of ampicillin was 100 μg/mL; the final working concentration of kanamycin sulfate was 50 μg/mL; and the final working concentration of G418 was 200 μg/mL.

The biochemical materials involved in the following embodiments are as follows:

The wild-type strain of Saccharomyces cerevisiae BY4741 was preserved in our laboratory, and the competent Escherichia coli TOP10 was purchased from Novagen, USA.

The yeast integrative plasmid pUMRI-B-ΔGAL80 was disclosed in the paper "De novo production of hydroxytyrosol by metabolic engineering of Saccharomyces cerevisiae".

The methods involved in the following embodiments are:

Determination of protein content: Collect bacterial cells in the fermentation broth, dry to constant weight, and determine the protein quality by Kjeldahl method, referring to GB 5009.5-2016 "National Food Safety Standard Determination of Protein in Food".

Unless otherwise specified, the "protein content" mentioned in the specific embodiments refers to the amount of protein per unit cell (g _protein /100g _{bacterial cells} ).

Unless otherwise specified, the "protein yield" mentioned in the specific implementation mode refers to the concentration of protein in each L of fermentation liquid (g/L). The determination method is: collect bacterial cells in the fermentation liquid, wash and dry to constant weight, and determine the protein content therein by Kjeldahl nitrogen determination method with reference to GB5009.5-2016 "National Food Safety Standard Determination of Protein in Food", and calculate the protein yield according to the following formula: protein yield (g _protein /L _{fermentation liquid} ) = (protein content × bacterial mass) ÷ (100 × fermentation liquid volume).

Unless otherwise specified, the "microorganism mass" mentioned in the specific implementation mode refers to the mass of the microorganism cells in the fermentation broth after centrifugation and washing and drying to a constant weight.

The strains, plasmids, translation elongation factors and primers involved in the following examples are shown in Tables 1 to 4.

Table 1: Translation elongation factors and their functions

Table 2: Primer list

Table 3: Plasmid list

Table 4: List of strains

Basic operations in molecular biology:

(1) E. coli plasmid extraction, genome extraction, PCR product purification, and gel recovery were performed according to the instructions of the corresponding kit unless otherwise specified.

(2) Amplify the DNA fragment using PCR technology, using different reaction systems and procedures according to experimental requirements. The reaction system refers to Table 4, and the reaction procedure refers to Table 5.

Table 5: PCR reaction system

Table 6: PCR reaction program

The pUMRI-B-ΔGAL80 involved in the following examples is disclosed in the following paper: Liu YJ, Liu H, Hu HT, et al. De Novo Production of Hydroxytyrosol by Metabolic Engineering of Saccharomyces cerevisiae [J]. Journal of Agricultural and Food Chemistry, 2022, 70(24): 7490-7499.

Example 1: Strengthening the protein translation elongation process to increase the protein translation rate

1. Construction of yeast strain PUMRI-△GAL80-TEF1

The Tef1 fragment (nucleotide sequence as shown in SEQ ID NO.1) was amplified from the whole genome of wild-type yeast BY4741 by PCR, and the primers were TEF1-F and TEF1-R. The Tef1 fragment was connected to the plasmid pUMRI-B-△GAL80 to obtain the recombinant plasmid pUMRI-△GAL80-TEF1. The recombinant plasmid pUMRI-△GAL80-TEF1 was cut with SfiI, linearized and integrated into the Gal80 site of BY4741 to obtain the yeast engineering strain PUMRI-△GAL80-TEF1. The primer nucleotide sequences are shown in Table 2, the PCR reaction system is shown in Table 5, and the PCR reaction program is shown in Table 6.

Example 2: Construction of yeast strain PUMRI-ΔGAL80-EFT1

The Eft1 fragment (nucleotide sequence as shown in SEQ ID NO.2) was amplified from the whole genome of wild-type yeast BY4741 by PCR, and the primers were EFT1-F and EFT1-R. The Eft1 fragment was connected to the plasmid pUMRI-B-△GAL80 to obtain the recombinant plasmid pUMRI-△GAL80-EFT1. The recombinant plasmid pUMRI-△GAL80-EFT1 was cut with SfiI, linearized and integrated into the Gal80 site of BY4741 to obtain the yeast engineering strain PUMRI-△GAL80-EFT1. The primer nucleotide sequences are shown in Table 2, the PCR reaction system is shown in Table 5, and the PCR reaction program is shown in Table 6.

Example 3: Construction of yeast strain PUMRI-ΔGAL80-YEF3

The Yef3 fragment (nucleotide sequence as shown in SEQ ID NO.3) was amplified from the whole genome of wild-type yeast BY4741 by PCR, with primers YEF3-F and YEF3-R, and the Yef3 fragment was connected to the plasmid pUMRI-B-△GAL80 to obtain the recombinant plasmid pUMRI-△GAL80-YEF3. The recombinant plasmid pUMRI-△GAL80-YEF3 was digested with SfiI, linearized and integrated into the Gal80 site of BY4741 to obtain the yeast engineering strain PUMRI-△GAL80-YEF3. The primer nucleotide sequences are shown in Table 2, the PCR reaction system is shown in Table 5, and the PCR reaction program is shown in Table 6.

Example 4: Construction of yeast strain PUMRI-ΔGAL80-ANB1

The Anb1 fragment (nucleotide sequence is shown in SEQ ID NO.4) was amplified from the whole genome of wild-type yeast BY4741 by PCR, and the primers were ANB1-F and ANB1-R. The Anb1 fragment was connected to the plasmid pUMRI-B-△GAL80 to obtain the recombinant plasmid pUMRI-△GAL80-ANB1. The recombinant plasmid pUMRI-△GAL80-ANB1 was cut with SfiI, linearized and integrated into the Gal80 site of BY4741 to obtain the yeast engineering strain PUMRI-△GAL80-ANB1. The primer nucleotide sequences are shown in Table 2, the PCR reaction system is shown in Table 5, and the PCR reaction program is shown in Table 6.

Example 5: Construction of yeast strain PUMRI-B-ΔGAL80

The plasmid pUMRI-B-△GAL80 was transformed into the Saccharomyces cerevisiae BY4741 strain to obtain the yeast engineering strain PUMRI-B-△GAL80.

Example 6: Production of Yeast Protein

The strains constructed in Examples 1 to 5 were inoculated in YPD liquid medium, activated at 30°C and 250r·min ^-1 , and then inoculated in 50mL YPG liquid medium at a ratio of 2% (v/v), and cultured at 30°C and 250r·min ^-1 for 48h. 10mL of fermentation broth was taken into a weighed 15mL centrifuge tube, centrifuged at 8000r·min ^-1 for 10min, the culture medium was discarded, the precipitate was suspended with sterile water and centrifuged again, washed twice and centrifuged, the supernatant was discarded, and placed in a 105°C oven to constant weight to detect the dry weight (DCW) of the bacteria. The remaining fermentation broth was collected by centrifugation at 8000r·min ^-1 for 10min, washed twice with distilled water and dried, and its protein content (g/100g) was determined by Kjeldahl nitrogen determination method, and strains with excellent protein content and production capacity were screened.

The fermentation results are shown in Figures 1 to 3, where the protein content (48.06g/100g) and protein yield (3.46g/L) of the strain PUMRI-△GAL80-TEF1 were significantly higher than those of the control strain BY4741 (protein content 45.63g/100g, protein yield 3.23g/L). Gene Tef1, as a eukaryotic translation elongation factor 1, is activated after binding to guanosine triphosphate (GTP) and binds to aminoacyl-tRNA, allowing aminoacyl-tRNA to bind to the A site. Overexpression of Tef1 can accelerate the process of translation elongation and speed up the translation rate.

Therefore, by regulating the protein translation elongation process, the global protein synthesis rate of Saccharomyces cerevisiae can be accelerated, which is beneficial to the efficient synthesis of yeast proteins and greatly improves the production efficiency and yield of Saccharomyces cerevisiae proteins.

Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims (10)

1. A recombinant Saccharomyces cerevisiae, characterized in that one or more genes of Tef1, Eft1, Yef3, and Anb1 are integrated into the genome. 2. The recombinant Saccharomyces cerevisiae according to claim 1, characterized in that the Saccharomyces cerevisiae includes but is not limited to BY4741. 3. The recombinant brewer's yeast according to claim 1 or 2, characterized in that the nucleotide sequence of the Tef1 gene is shown in SEQ ID NO.1; the nucleotide sequence of the Eft1 gene is shown in SEQ ID NO.2; the nucleotide sequence of the Yef3 gene is shown in SEQ ID NO.3; and the nucleotide sequence of the Anb1 gene is shown in SEQ ID NO.4. 4. A method for increasing the protein yield of Saccharomyces cerevisiae by optimizing the protein translation elongation process, characterized in that one or more genes of Tef1, Eft1, Yef3, and Anb1 are integrated into the Saccharomyces cerevisiae genome. 5. The method according to claim 4, characterized in that the Tef1, Eft1, Yef3 or Anb1 gene is integrated into the genomic Gal80 site. 6. The method according to claim 4 or 5, characterized in that the cerevisiae yeast includes but is not limited to BY4741. 7. Use of the recombinant Saccharomyces cerevisiae according to any one of claims 1 to 3 in protein production. 8. The use according to claim 7, characterized in that the recombinant Saccharomyces cerevisiae is fermented in a culture medium for a period of time. 9. The use according to claim 8, characterized in that the recombinant Saccharomyces cerevisiae is first activated in a culture medium containing tryptone, yeast extract and glucose, and then fermented. 10. The method according to any one of claims 7 to 9, characterized in that the fermentation is carried out at 28 to 32°C for at least 48 hours.