Creating Useful Chemicals with Genetically Modified Yeast

Jim Crocker
10th September, 2024

Creating Useful Chemicals with Genetically Modified Yeast

The metabolic engineering strategy for producing (R)-citramalate in the budding yeast (Saccharomyces cerevisiae) involved introducing a heterologous synthesis pathway (blue arrows) while simultaneously deleting key genes (red) to increase the cytosolic supply of its precursors, pyruvate and acetyl-CoA.

Image adapted from: Mitsui et al. / CC BY (Source)

Key Findings

  • The study by RIKEN focused on enhancing (R)-citramalate production in the yeast Saccharomyces cerevisiae
  • Researchers inhibited the transport of key metabolites to mitochondria to increase their cytosolic concentrations
  • The engineered yeast strain produced 16.5 mM (R)-citramalate, the highest reported production to date
The budding yeast, Saccharomyces cerevisiae, shows remarkable resilience to organic acids and alcohols, making it an ideal candidate for producing various compounds in toxic environments. A recent study conducted by RIKEN focused on enhancing the production of (R)-citramalate, a precursor for methyl methacrylate and a metabolic intermediate for higher alcohols, using this hardy yeast[1]. This study aimed to overcome the challenge of compartmentalized intracellular metabolites, specifically acetyl-CoA, which limits its availability in the cytosol for efficient (R)-citramalate synthesis. In Escherichia coli, previous research demonstrated the production of higher alcohols using a 2-keto acid-based pathway, which efficiently converts glucose to 1-propanol and 1-butanol[2]. However, S. cerevisiae presents a unique challenge due to its organelles that compartmentalize metabolites, restricting the cytosolic pool of acetyl-CoA and pyruvate. To address this, the RIKEN team inhibited the transport of these metabolites to the mitochondria, aiming to increase their cytosolic concentrations for enhanced (R)-citramalate production. The study employed a multi-faceted approach to achieve this goal. First, the researchers constructed a heterologous pathway to supply cytosolic acetyl-CoA. This involved expressing enzymes from other organisms known to produce acetyl-CoA directly in the cytosol. Additionally, they attempted to export (R)-citramalate from the cells by expressing a heterologous dicarboxylate transporter gene, which would facilitate the removal of the synthesized product and potentially drive the reaction forward by reducing intracellular (R)-citramalate concentrations. The effectiveness of these strategies was evaluated by measuring (R)-citramalate production in engineered yeast strains. By combining these positive approaches, the researchers constructed a final strain that produced 16.5 mM (R)-citramalate in batch culture flasks. This represents the highest reported production of (R)-citramalate by recombinant S. cerevisiae to date. The findings of this study build upon earlier research that explored metabolic pathways and enzyme activities in yeast. For instance, the expression of heterologous phosphoketolase enzymes in S. cerevisiae demonstrated the potential to redirect carbon flux towards acetyl-CoA synthesis, thereby reducing carbon loss[3]. Similarly, the modulation of mitochondrial pyruvate carriers, as shown in another study, highlighted the importance of controlling pyruvate transport to adapt cellular metabolism to nutrient availability[4]. These insights were instrumental in designing the strategies employed in the current study to enhance cytosolic acetyl-CoA availability. Furthermore, the study also ties into the broader context of metabolic engineering for biofuel production. The directed evolution of citramalate synthase (CimA) from Methanococcus jannaschii in E. coli, which bypassed threonine biosynthesis to produce higher alcohols, showcased the potential of optimizing enzyme activities for improved production yields[2]. By applying similar principles of pathway optimization and enzyme engineering, the RIKEN team successfully enhanced (R)-citramalate production in yeast. In conclusion, the study conducted by RIKEN represents a significant advancement in metabolic engineering, demonstrating the highest (R)-citramalate production in recombinant S. cerevisiae. By inhibiting mitochondrial transport of key metabolites and constructing a heterologous pathway for cytosolic acetyl-CoA supply, the researchers effectively addressed the challenge of metabolite compartmentalization. This work not only expands our understanding of yeast metabolism but also paves the way for future efforts in bio-based chemical production.

BiotechGeneticsBiochem

References

Main Study

1) Production of (R)-citramalate by engineered Saccharomyces cerevisiae.

Published 9th September, 2024

https://doi.org/10.1016/j.mec.2024.e00247

Related Studies

2) Directed evolution of Methanococcus jannaschii citramalate synthase for biosynthesis of 1-propanol and 1-butanol by Escherichia coli.

https://doi.org/10.1128/AEM.02046-08

3) Functional expression and evaluation of heterologous phosphoketolases in Saccharomyces cerevisiae.

https://doi.org/10.1186/s13568-016-0290-0

4) Regulation of mitochondrial pyruvate uptake by alternative pyruvate carrier complexes.

https://doi.org/10.15252/embj.201490197


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