Engineering Inorganic Pyrophosphate Metabolism as a Strategy to Generate a Fluoride-Resistant Saccharomyces...

Area of Science:

• Biochemistry
• Environmental Toxicology
• Microbiology

Background:

• Fluoride is widely distributed in the environment and exhibits toxic effects on various organisms.
• The mechanisms by which fluoride alters cellular processes remain largely unknown.
• Inorganic pyrophosphatases (PPases) are crucial enzymes that hydrolyze inorganic pyrophosphate (PPi), a byproduct of ATP metabolism.

Purpose of the Study:

• To investigate the role of cytosolic inorganic pyrophosphatase (IPP1) in Saccharomyces cerevisiae's response to fluoride.
• To develop strategies for enhancing cellular tolerance to high fluoride concentrations.
• To elucidate the mechanisms of fluoride toxicity and adaptation in yeast.

Main Methods:

• Utilized a conditional yeast mutant (YPC3) with essential IPP1 gene expression.
• Generated fluoride-tolerant yeast strains through overexpression of IPP1 or its human ortholog.
• Introduced a fluoride-insensitive bacterial PPase from Streptococcus mutans to replace endogenous IPP1.

Main Results:

• Overexpression of yeast IPP1 or its human ortholog conferred increased fluoride tolerance.
• Substitution of yeast IPP1 with a bacterial fluoride-insensitive PPase also resulted in enhanced fluoride resistance.
• Yeast cells engineered for increased PPase activity could be selected on media containing high fluoride concentrations.

Conclusions:

• Maintaining adequate inorganic pyrophosphatase activity in the cytosol is essential for Saccharomyces cerevisiae to adapt to high fluoride concentrations.
• Engineering PPase activity represents a viable strategy for conferring fluoride tolerance in yeast.
• This study provides insights into cellular mechanisms of fluoride resistance and adaptation.