A Pichia pastoris single-cell biosensor for detection of enzymatically produced methanol.

We conducted single-cell analyses of the methylotrophic yeast Pichia pastoris to develop a biosensor for the detection of methanol produced by heterologous enzymes. In this biosensor, methanol and its subsequent metabolism induce expression of a gene encoding a fluorescent protein that was placed under the control of a methanol-inducible promoter. Using quantitative analyses of fluorescence microscopy images, a methanol-inducible promoter and a host strain were selected, and preculture and assay conditions were optimized to improve the methanol detection limit. Fluorescence-activated cell sorting (FACS) analysis of the distribution and geometric mean of cellular fluorescence intensity against various concentrations of methanol revealed a detection limit of 2.5 μM. Finally, this biosensor was applied to evaluate the activity of a heterologously expressed pectin methylesterase (PME). The cellular fluorescence intensity was proportional to the copy number of the PME expression cassette, the protein level, and the enzyme activity. This biosensor can be used for high-throughput screening of single cells harboring high methanol-producing activity, and thereby, the development of a bioconversion process using methanol-producing enzymes.