Identification of the Yarrowia lipolytica cysteine sulfinic acid decarboxylase gene using a newly developed method with optimized Escherichia coli combinations of mutant alleles

TL;DR

Researchers developed a new method to identify a key enzyme gene in yeast for taurine production, which could help create sustainable fish feed.

Contribution

A novel screening method using E. coli mutants to identify CSAD genes in microorganisms was developed and successfully applied.

Findings

The Yarrowia lipolytica glutamic acid decarboxylase gene was found to have CSAD activity.

SsuD was observed to be involved in sulfur assimilation from an unknown sulfur compound.

Certain mscK mutations allow external sulfate to enter the cell at specific concentrations.

Abstract

To develop a low-cost, environmentally friendly taurine fermentation method for sustainable marine fish culture using feed derived from photosynthetically produced agricultural products, it is crucial to study cysteine sulfinic acid decarboxylase (CSAD), a key enzyme in the taurine biosynthetic pathway in applicable microorganisms. In this study, a method was devised to screen for CSAD genes using Escherichia coli growth as an indicator, based on sulfur assimilation following the decarboxylation of l-cysteic acid, a taurine precursor compound. The E. coli used has a double deletion mutation of cysA (sulfate/thiosulfate ABC transporter) and ssuD (FMNH2-dependent alkanesulfonate monooxygenase) genes. If needed, an additional defect in enzyme genes, such as cysC (adenylyl-sulfate kinase), which participates in the pathway reducing sulfate to sulfite, is also introduced. Using this method, it was demonstrated that the glutamic acid decarboxylase gene from Yarrowia lipolytica possesses CSAD activity. The identified decarboxylase was further confirmed to act on l-cysteine sulfinic acid. Additionally, two observations made during method refinement to reduce background growth in screening are discussed: that SsuD is involved in sulfur assimilation from an unknown sulfur compound and that certain mscK (mechanosensitive channel) missense mutations enable external sulfate above a specific concentration to enter the cell.

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