# Truncation of LPD1 promoter and adaptive evolution increase cytosolic acetyl-CoA supply in yeast

## TL;DR

Researchers increased cytosolic acetyl-CoA in yeast by modifying a gene promoter and evolving the cells, improving production of valuable compounds.

## Contribution

A novel strategy combining promoter truncation and adaptive evolution to boost cytosolic acetyl-CoA levels in yeast.

## Key findings

- Deleting SUT526 in the LPD1 promoter reduced TCA cycle entry and increased cytosolic acetyl-CoA.
- Adaptive evolution restored growth and enhanced acetyl-CoA synthetase activity and squalene production.
- The approach redirects carbon flux to improve biosynthesis of acetyl-CoA-derived compounds.

## Abstract

Acetyl-CoA is a central metabolic intermediate that serves as a key precursor for the biosynthesis of high-value compounds such as terpenoids. However, its compartmentalization within Saccharomyces cerevisiae limits its availability in the cytosol, constraining production of cytosol-derived metabolites. In this study, we aimed to redirect carbon flux toward cytosolic acetyl-CoA synthesis by reducing entry into the tricarboxylic acid cycle. To achieve this, we attenuated LPD1 expression by deleting the noncoding RNA SUT526, which is located within the LPD1 promoter region and overlaps an upstream regulatory element. This intervention impaired cell growth and hindered the utilization of non-fermentable carbon sources such as ethanol. To address this limitation, adaptive laboratory evolution was performed in ethanol-based medium, leading to rapid recovery of growth and extended cell viability. The evolved strains exhibited enhanced acetyl-CoA synthetase activity and elevated squalene production, suggesting an increased cytosolic acetyl-CoA supply. These improvements reflect enhanced flux through acetyl-CoA-dependent biosynthetic pathways. This work presents a targeted strategy for modulating central carbon metabolism to increase cytosolic acetyl-CoA supply, providing a framework for efficient production of acetyl-CoA derived compounds in yeast.

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