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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.

The mechanical damage of plant tissues leads to the activation of methanol production and its release into the atmosphere. The gaseous methanol or vapors emitted by the damaged plant induce resistance in neighboring intact plants to bacterial pathogens but create favorable conditions for viral infection spread. Among the Nicotiana benthamiana methanol-inducible genes (MIGs), most are associated with plant defense and intercellular transport. Here, we characterize NbMIG21, which encodes a 209 aa protein (NbMIG21p) that does not share any homology with annotated proteins. NbMIG21p was demonstrated to contain a nucleolus localization signal (NoLS). Colocalization studies with fibrillarin and coilin, nucleolus and Cajal body marker proteins, revealed that NbMIG21p is distributed among these subnuclear structures. Our results show that recombinant NbMIG21 possesses DNA-binding properties. Similar to a gaseous methanol effect, an increased NbMIG21 expression leads to downregulation of the nuclear import of proteins with nuclear localization signals (NLSs), as was demonstrated with the GFP-NLS model protein. Moreover, upregulated NbMIG21 expression facilitates tobacco mosaic virus (TMV) intercellular transport and reproduction. We identified an NbMIG21 promoter (PrMIG21) and showed that it is methanol sensitive; thus, the induction of NbMIG21 mRNA accumulation occurs at the level of transcription. Our findings suggest that methanol-activated NbMIG21 might participate in creating favorable conditions for viral reproduction and spread.

We identified a gene, designated TRM2, responsible for methanol-inducible gene expression in the methylotrophic yeast Candida boidinii. The encoded protein Trm2p contains two C₂H₂-type zinc finger motifs near the N terminus and shows high similarity to Saccharomyces cerevisiae Adr1p and Pichia pastoris Mxr1p. A C. boidinii gene-disrupted strain (trm2Δ) could not grow on methanol or oleate, but could grow on glucose or ethanol. Trm2p was necessary for the activation of five methanol-inducible promoters tested. Trm2p was localized to the nucleus during growth on nonfermentable carbon sources, but to the cytosol during growth on glucose. A chromatin immunoprecipitation assay revealed that Trm2p specifically bound to the promoters of the alcohol oxidase gene (AOD1) and the dihydroxyacetone synthase gene in cells grown on methanol or oleate, but did not bind to these promoters in cells grown on glucose. The derepressed level of expression of AOD1, which was observed in the trm1Δ strain (the TRM1 gene encodes a transcription factor responsible for methanol-specific gene activation), was decreased in the trm1Δtrm2Δ strain to a level similar to that observed in the trm2Δ strain. These results suggest that Trm2p-dependent derepression is essential for the Trm1p-dependent methanol-specific gene activation in C. boidinii.

The endoglucanase Ⅱ of Trichoderma reesei is considered the most effective enzyme for biofinishing cotton fabrics and biostoning denim garments. However, the commercially available preparation of endoglucanase Ⅱ is usually mixed with other cellulase components, especially endoglucanase I, resulting in hydrolysis and weight loss of garments during biofinishing and biostoning. We thus isolated the endoglucanase Ⅱ gene from T. reesei to express this in Pichia pastoris, under the control of a methanol-inducible AOX1 promoter, to avoid the presence of other cellulase components. A highly expressible Mut+ transformant was selected and its expression in BMMH medium was found most suitable for the production of large amounts of the recombinant protein. Recombinant endoglucanase Ⅱ was purified to electrophoretic homogeneity, and functionally characterized by activity staining. The specific activity of recombinant endoglucanase Ⅱ was found to be 220.57 EU/mg of protein. Purified recombinant endoglucanase Ⅱ was estimated to have a molecular mass of 52.8 kDa. The increase in molecular mass was likely due to hyperglycosylation. Hyperglycosylation of recombinant endoglucanase Ⅱ secreted by P. pastoris did not change the temperature or pH optima as compared to the native protein, but did result in increased thermostability. Kinetic analysis showed that recombinant endoglucanase was most active against amorphous cellulose, such as carboxymethyl cellulose, for which it also had a high affinity.