In this issue, we recommend an article Algal Research: Rapid screening of high-protein Auxenochlorella pyrenoidosa mutant by an integrated system of atmospheric and room temperature plasma mutagenesis and high-throughput microbial microdroplet culture. In this study, the Atmospheric Room Temperature Plasma Mutagenesis (ARTP) and the high-throughput Microbial Microdroplet Culture System (MMC) were used to quickly select protein nuclear mutant strains with high protein, which provided promising candidate bacteria for the production of alternative protein by heterotrophic fermentation.
Figure 1 Breeding process of high-protein mutant strains of chlorella pyrenoidosa
As the global population grows, so does the demand for food, intensifying the need for new sources of protein in reduced arable land and ethical concerns about traditional meat production. Microalgae, as a promising alternative protein source, are rich in proteins, amino acids, polyunsaturated fatty acids (PUFAs), vitamins, and minerals. However, under heterotrophic culture conditions, microalgae generally has a low protein content (<40% dry weight) due to reduced photosynthesis-dependent protein synthesis, limiting their potential as an alternative protein source. To overcome these challenges, the development of novel microalgae strains with naturally high protein content is essential for efficient large-scale production of protein by heterotrophic fermentation.
The researchers used ARTP mutagenesis to obtain aureus protein A4-1 as the original algal strain, a new round of ARTP mutagenesis was conducted, after 15 seconds of irradiation, transferred to a shake bottle for culture. Cells in the log growth phase were diluted to the OD450nm values 0.6–0.8 and transferred to the MMC system for further culture.
In the MMC system, 50 droplets were initially generated, and the running time of each generation was 24-46 hours. The absorbance value of the droplets at 450 nm was detected in real time to characterize the growth of the algal strain. After three rounds of culture, the best droplet (No.28) were selected. The whole experimental process has only take 116 hours, which was a significant improvement over the traditional tablet system (Figure 2A). The droplets were monoclonal sorted, and the 23 strains were obtained, of which four strains (MMC-1,7,8 and 11) with high growth rates or high biomass concentration were selected for subsequent analysis (Figure 2B).
Figure 2 Droplets growth in MMC and cells grown in monoclonal microplates
Cell growth and biomass production of the four selected mutants (MMC-1,7,8,11) grown in a 250 mL shake flask were shown in Figure 3A. All mutants had a growth pattern similar to the A4-1 algal strain, undergoing an initial lag phase followed by rapid entry into the exponential growth phase. MMC-7 reached the highest biomass concentration of 8.21 g / L, which was 8.49% higher than the 7.57 g / L of the A4-1 strain (p <0.05). At the same time, all four mutants visually showed a golden yellow color similar to that of the A4-1 algal strain, no chlorophyll b was detected, and chlorophyll a only accounted for 1% of the wild type (WT) (Figure 3B).
Figure 3 Cell Growth and pigment composition of the mutant strain and original strain A4-1 in shake flasks
Biochemical component analysis showed that the protein content was increased by 12% to 40% in the four mutants (Figure 4). The MMC-8 mutant showed the highest protein content (63.26% dry weight) and the lowest starch content (8.59% dry weight), up 40.11% and 56.24% lower than the starter strain A4-1. In addition, MMC-8 also performed well in protein quality, and its amino acid content (44.35% dry weight) and score (95) were higher than A4-1.
Figure 4 Biochemical composition of mutant and original strain A4-1
Figure 5 Essential amino acid index and amino acid composition of series mutant strains and original strain A4-1
This study demonstrated the potential of the ARTP-MMC system as a powerful high-throughput screening platform that not only improving protein synthesis efficiency but also reducing starch content and tilted carbon allocation toward protein synthesis, important for improving biological protein production efficiency and sustainability.
Link: https://doi.org/10.1016/j.algal.2024.103509
In this issue, we recommend an article Algal Research: Rapid screening of high-protein Auxenochlorella pyrenoidosa mutant by an integrated system of atmospheric and room temperature plasma mutagenesis and high-throughput microbial microdroplet culture. In this study, the Atmospheric Room Temperature Plasma Mutagenesis (ARTP) and the high-throughput Microbial Microdroplet Culture System (MMC) were used to quickly select protein nuclear mutant strains with high protein, which provided promising candidate bacteria for the production of alternative protein by heterotrophic fermentation.
Figure 1 Breeding process of high-protein mutant strains of chlorella pyrenoidosa
As the global population grows, so does the demand for food, intensifying the need for new sources of protein in reduced arable land and ethical concerns about traditional meat production. Microalgae, as a promising alternative protein source, are rich in proteins, amino acids, polyunsaturated fatty acids (PUFAs), vitamins, and minerals. However, under heterotrophic culture conditions, microalgae generally has a low protein content (<40% dry weight) due to reduced photosynthesis-dependent protein synthesis, limiting their potential as an alternative protein source. To overcome these challenges, the development of novel microalgae strains with naturally high protein content is essential for efficient large-scale production of protein by heterotrophic fermentation.
The researchers used ARTP mutagenesis to obtain aureus protein A4-1 as the original algal strain, a new round of ARTP mutagenesis was conducted, after 15 seconds of irradiation, transferred to a shake bottle for culture. Cells in the log growth phase were diluted to the OD450nm values 0.6–0.8 and transferred to the MMC system for further culture.
In the MMC system, 50 droplets were initially generated, and the running time of each generation was 24-46 hours. The absorbance value of the droplets at 450 nm was detected in real time to characterize the growth of the algal strain. After three rounds of culture, the best droplet (No.28) were selected. The whole experimental process has only take 116 hours, which was a significant improvement over the traditional tablet system (Figure 2A). The droplets were monoclonal sorted, and the 23 strains were obtained, of which four strains (MMC-1,7,8 and 11) with high growth rates or high biomass concentration were selected for subsequent analysis (Figure 2B).
Figure 2 Droplets growth in MMC and cells grown in monoclonal microplates
Cell growth and biomass production of the four selected mutants (MMC-1,7,8,11) grown in a 250 mL shake flask were shown in Figure 3A. All mutants had a growth pattern similar to the A4-1 algal strain, undergoing an initial lag phase followed by rapid entry into the exponential growth phase. MMC-7 reached the highest biomass concentration of 8.21 g / L, which was 8.49% higher than the 7.57 g / L of the A4-1 strain (p <0.05). At the same time, all four mutants visually showed a golden yellow color similar to that of the A4-1 algal strain, no chlorophyll b was detected, and chlorophyll a only accounted for 1% of the wild type (WT) (Figure 3B).
Figure 3 Cell Growth and pigment composition of the mutant strain and original strain A4-1 in shake flasks
Biochemical component analysis showed that the protein content was increased by 12% to 40% in the four mutants (Figure 4). The MMC-8 mutant showed the highest protein content (63.26% dry weight) and the lowest starch content (8.59% dry weight), up 40.11% and 56.24% lower than the starter strain A4-1. In addition, MMC-8 also performed well in protein quality, and its amino acid content (44.35% dry weight) and score (95) were higher than A4-1.
Figure 4 Biochemical composition of mutant and original strain A4-1
Figure 5 Essential amino acid index and amino acid composition of series mutant strains and original strain A4-1
This study demonstrated the potential of the ARTP-MMC system as a powerful high-throughput screening platform that not only improving protein synthesis efficiency but also reducing starch content and tilted carbon allocation toward protein synthesis, important for improving biological protein production efficiency and sustainability.
Link: https://doi.org/10.1016/j.algal.2024.103509
