Methylotrophic bacterium-based molecular sensor for the detection of low concentrations of methanol

Methylotrophic bacterium Methylorubrum extorquens is a promising microorganism for the production of value-added compounds from methanol. This study focused on the development of a single-cell level biosensor system that detects methanol by using the intrinsic regulatory machinery which responds to the presence of methanol in this bacterium. A green fluorescent protein (GFP) gene located downstream of the promoter region of the serine glyoxylate aminotransferase gene (P_sga) or the methanol dehydrogenase subunit 1 precursor gene (P_mxaF) was inserted into the chromosome of M. extorquens wild-type strain AM1. The expression of GFP upon methanol exposure was measured by spectrofluorometer and fluorescence-activated cell sorting (FACS). The strain harboring P_sga-gfp emitted fluorescence only when methanol was supplied to the culture medium, while the other strain harboring P_mxaF-gfp showed high basal fluorescence even in the absence of methanol. The fluorescence intensity of the P_sga-gfp strain depended on a methanol concentration higher than 25 μM, and the sensitivity and dose-dependency of this strain were much higher than previous systems using Escherichia coli. The methanol-sensing properties of the engineered M. extorquens strain were comparable to those of a methylotrophic yeast-based biosensor, suggesting the usefulness of methylotrophic microorganisms as platforms for single-cell sensing of C₁ compounds. The constructed methanol sensor strain, coupled with flow cytometry techniques, provides a high-throughput and highly sensitive screening method for the selection of functional methanol-producing enzymes.