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 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.