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

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 <i>Escherichia coli</i> growth as an indicator, based on sulfur assimilation following the decarboxylation of l-cysteic acid, a taurine precursor compound. The <i>E. coli</i> used has a double deletion mutation of <i>cysA</i> (sulfate/thiosulfate ABC transporter) and <i>ssuD</i> (FMNH₂-dependent alkanesulfonate monooxygenase) genes. If needed, an additional defect in enzyme genes, such as <i>cysC</i> (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 <i>Yarrowia lipolytica</i> 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 <i>mscK</i> (mechanosensitive channel) missense mutations enable external sulfate above a specific concentration to enter the cell.