Presentation Information
[2Biocat-13]Enhancing the biosynthetic capacity of Saccharomyces cerevisiae through synergistic point and structural mutagenesis
○Ryosuke Yamada1 (1. Osaka Metropolitan University (Japan))
Keywords:
Carotenoid,Metabolic engineering,Point and structural mutageneses,Protein production,Saccharomyces cerevisiae
[Background]
The yeast Saccharomyces cerevisiae has long been recognized as a cornerstone of industrial biotechnology due to its Generally Recognized as Safe status and its robust performance in large-scale fermentation processes. These characteristics make it a valuable host for the production of diverse proteins and high-value chemicals through metabolic engineering. In previous work, I developed a novel mutagenesis strategy that enables the simultaneous introduction of both point mutations and large-scale structural mutations into the yeast genome. By leveraging this dual-action approach, it becomes possible to effectively explore a broader sequence space, leading to the significant improvement of various functions in yeast. To further push the boundaries of S. cerevisiae as a high-performance production platform, there is a pressing need to develop strains capable of sustaining high levels of heterologous protein expression. In the present study, I aimed to construct novel mutant strains with enhanced protein production capacities by employing the aforementioned mutagenesis strategy.
[Results]
To facilitate the identification of high-performing candidates, the fluorescence intensity of Green Fluorescent Protein (GFP) was utilized as a quantitative phenotypic indicator. Initially, point and structural mutations were systematically introduced into the strain YPH499/pEUPGGFP, which constitutively expresses GFP. Following a rigorous selection process based on fluorescence profiles, I successfully isolated a prominent mutant designated as YPH499/pEUPGGFP/Mu10G39. This specific strain exhibited a 2.5-fold increase in GFP fluorescence compared to the parental strain, suggesting a fundamental shift in its biosynthetic capabilities. To evaluate the versatility and industrial potential of this platform, the GFP expression plasmid was cured, and a heterologous carotenoid biosynthesis pathway was introduced via the plasmid pEU20-Beta3. The resulting modified strain, YPH499Mu10G39/pEU20Beta3, achieved a carotenoid yield of 6.74 mg/g-dry cell weight after 72 hours of cultivation, representing a 2.9-fold increase over the parental control. To elucidate the molecular mechanisms underlying these phenotypic improvements, a comprehensive transcriptome analysis was performed. The data suggested that the superior production performance of YPH499Mu10G39 was the result of a coordinated upregulation of energy metabolism, increased availability of amino acid precursors, enhanced ribosomal biogenesis, and improved cellular stress tolerance. Supporting these findings, the intracellular ATP content of the YPH499Mu10G39 strain was measured at 4.1-fold higher than that of the parental strain at the 72-hour mark. This significant increase in energy availability likely provides the metabolic driving force necessary for high-intensity biosynthesis.
[Conclusion]
This study demonstrates the successful development of a robust platform strain, YPH499Mu10G39, through the strategic application of point and structural mutagenesis. The results highlight the strain's exceptional potential for high-yield protein and chemical production, positioning it as a versatile and efficient host for future metabolic engineering applications in the bio-based economy.
The yeast Saccharomyces cerevisiae has long been recognized as a cornerstone of industrial biotechnology due to its Generally Recognized as Safe status and its robust performance in large-scale fermentation processes. These characteristics make it a valuable host for the production of diverse proteins and high-value chemicals through metabolic engineering. In previous work, I developed a novel mutagenesis strategy that enables the simultaneous introduction of both point mutations and large-scale structural mutations into the yeast genome. By leveraging this dual-action approach, it becomes possible to effectively explore a broader sequence space, leading to the significant improvement of various functions in yeast. To further push the boundaries of S. cerevisiae as a high-performance production platform, there is a pressing need to develop strains capable of sustaining high levels of heterologous protein expression. In the present study, I aimed to construct novel mutant strains with enhanced protein production capacities by employing the aforementioned mutagenesis strategy.
[Results]
To facilitate the identification of high-performing candidates, the fluorescence intensity of Green Fluorescent Protein (GFP) was utilized as a quantitative phenotypic indicator. Initially, point and structural mutations were systematically introduced into the strain YPH499/pEUPGGFP, which constitutively expresses GFP. Following a rigorous selection process based on fluorescence profiles, I successfully isolated a prominent mutant designated as YPH499/pEUPGGFP/Mu10G39. This specific strain exhibited a 2.5-fold increase in GFP fluorescence compared to the parental strain, suggesting a fundamental shift in its biosynthetic capabilities. To evaluate the versatility and industrial potential of this platform, the GFP expression plasmid was cured, and a heterologous carotenoid biosynthesis pathway was introduced via the plasmid pEU20-Beta3. The resulting modified strain, YPH499Mu10G39/pEU20Beta3, achieved a carotenoid yield of 6.74 mg/g-dry cell weight after 72 hours of cultivation, representing a 2.9-fold increase over the parental control. To elucidate the molecular mechanisms underlying these phenotypic improvements, a comprehensive transcriptome analysis was performed. The data suggested that the superior production performance of YPH499Mu10G39 was the result of a coordinated upregulation of energy metabolism, increased availability of amino acid precursors, enhanced ribosomal biogenesis, and improved cellular stress tolerance. Supporting these findings, the intracellular ATP content of the YPH499Mu10G39 strain was measured at 4.1-fold higher than that of the parental strain at the 72-hour mark. This significant increase in energy availability likely provides the metabolic driving force necessary for high-intensity biosynthesis.
[Conclusion]
This study demonstrates the successful development of a robust platform strain, YPH499Mu10G39, through the strategic application of point and structural mutagenesis. The results highlight the strain's exceptional potential for high-yield protein and chemical production, positioning it as a versatile and efficient host for future metabolic engineering applications in the bio-based economy.
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