Engineering of Saccharomyces cerevisiae for shikimate and malonyl-CoA pathway-derived precursor supply
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Date
2024
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Abstract
Many natural products are generated by polyketide and non-ribosomal peptide synthases (PKS and NRPS), which necessitates active acyl and peptide carry domains (ACP and PCP). This activation was enabled by the generation of two Saccharomyces cerevisiae CEN.PK2-1C variants (ST003 and ST004) harboring and functionally expressing a phosphopantetheinyl transferase (PPtase) SFPpBs from Bacillus subtilis.
These strains serve as a foundation for further metabolic engineering approaches to enhance precursor provision. The glycolysis and malonyl-CoA pathway were successfully modified, leading to improved flux towards malonyl-CoA, as confirmed by a 3-hydroxypropionic acid (3-HP) detection method and subsequent NMR confirmation. Next, to implement the genes acc^1S659A,S686A,S1157A, ald6, pdc1, and acsSe^L641P for overexpression, the generated 3-HP titers were further improved by deleting adh1 and implementing tpi1. Despite the encountered obstacles, the study successfully demonstrates the elevation of malonyl-CoA levels in yeast, yielding 3-HP titers of up to 249.66 mg L-1 in batch cultivations.
Using the initially generated PPtase strains as a basis, the genes for ARO4p^K229L, ARO7p^G141S, and AroLpEc, were integrated while simultaneously deleting aro10 and pdc5. Making use of the tyrosine ammonia-lyase (TALpHa) enzyme from Herpetosiphon aurantiacus to generate p-coumaric acid (p-CA), the successful generated yeast strains demonstrated a modified shikimate pathway with elevated phenylalanine and tyrosine levels. Results revealed varying p-CA concentrations in different yeast strains, with ST004-based strains consistently outperforming ST003-based strains, resulting in p-CA concentration of up to 130.60 mg L-1 in batch cultivation. This highlighted, although successful, a notable discrepancy between the reported literature and this work, emphasizing the significant impact of cultivation conditions on yields for both pathways, as supported by existing literature.
Finally, malonyl-CoA pathway and shikimate pathway modifications were implemented in a single yeast strain (ST027), exhibiting initial 3-HP production with eventual p-CA production at slightly higher levels than compared to solely shikimate pathway-modified strain (ST008). This suggests a potential synergistic effect between both pathways, supporting increased tyrosine production. The present thesis resulted in the optimized strains with the necessity for cultivation optimization attempts, ultimately further increasing production titers of compounds harnessing precursors from both pathways, as for instance, cryptophycins, resveratrol, or maybe aromatic phytocanabinoids.
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Keywords
Saccharomyces cerevisiae, Shikimate pathway, Malonyl-CoA, Metabolic engineering
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Biotechnologie, Saccharomyces cerevisiae