Development of Methanol Sensor Based on Sol-Gel Drop-Coating Co3O4·CdO·ZnO Nanoparticles Modified Gold-Coated µ-Chip by Electro-Oxidation Process

Herein, novel Co3O4·CdO·ZnO-based tri-metallic oxide nanoparticles (CCZ) were synthesized by a simple solution method in basic phase. We have used Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD), X-ray photoelectron spectroscopy (XPS), Field Emission Scanning Electron Microscope (FESEM), Dynamic Light Scattering (DLS), Tunneling Electron Microscopy (TEM), and Energy-Dispersive Spectroscopy (EDS) techniques to characterize the CCZ nanoparticles. XRD, TEM, DLS, and FESEM investigations have confirmed the tri-metallic nanoparticles’ structure, while XPS and EDS analyses have shown the elemental compositions of the CCZ nanoparticles. Later, a Au/μ-Chip was modified with the CCZ nanoparticles using a conducting binder, PEDOT: PSS (poly(3,4-ethylenedioxythiophene) polystyrene sulfonate) in a sol-gel system, and dried completely in air. Then, the CCZ/Au/μ-Chip sensor was used to detect methanol (MeOH) in phosphate buffer solution (PBS). Outstanding sensing performance was achieved for the CCZ/Au/μ-Chip sensor, such as excellent sensitivity (1.3842 µAµM−1cm−2), a wide linear dynamic range of 1.0 nM–2.0 mM (R2 = 0.9992), an ultra-low detection limit (32.8 ± 0.1 pM at S/N = 3), a fast response time (~11 s), and excellent reproducibility and repeatability. This CCZ/Au/μ-Chip sensor was further applied with appropriate quantification results in real environmental sample analyses.

HBsAg Production in Methanol Controlled P. pastoris GS115 MutS Bioreactor Process

When producing recombinant proteins with Pichia pastoris, cultivation parameters, such as induction temperature, dissolved oxygen level and residual methanol concentration play a crucial role in product biosynthesis and subsequent purification, therefore to maximize protein yields, the optimization of these parameters is imperative. Two different Pichia pastoris cultivation strategies for HBsAg VLP production in a 5 L stirred-tank bioreactor and the influence of different cultivation parameters on product yield were investigated. Residual methanol concentrations were controlled at low (>0.01 g/L), medium (1.5-2.0 g/L) and high (5.0-6.0 g/L) levels using a PI-based feed rate control algorithm based on the online methanol sensor signal. Product was purified using a novel and rapid purification method including steps of ammonium sulfate precipitation, size-exclusion chromatography and hydrophobic interaction chromatography. Employing an in-situ methanol sensor probe, the PI-based methanol feed rate control algorithm provided residual methanol concentration control with an average deviation of ±0.4 g/L from set-point value. Employing a cultivation protocol with an increased methanol concentration controlled at 6.0 g/L and a reduced DO level below 10 %, resulting in a final dry cell biomass concentration of 140 g/L and purified HBsAg VLPs yield of 186 mg/L. Developed purification method proved advantageous to other described methods, as it did not include time consuming extraction and centrifugation steps.

First person – Shin Ohsawa

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Shin Ohsawa is co-first author on ‘The methanol sensor Wsc1 and MAPK Mpk1 suppress degradation of methanol-induced peroxisomes in methylotrophic yeast’, published in JCS. Shin conducted the research described in this article while a PhD student in Yasuyoshi Sakai's lab at Kyoto University, Japan. He is now a postdoc in the lab of Marc Bühler at the Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland, investigating sensing and signaling mechanisms involved in gene expression and cellular dynamics in yeast.

Methanol sensor Wsc1 and MAP kinase suppress degradation of methanol-induced peroxisomes in methylotrophic yeast

In nature, methanol is produced during the hydrolysis of pectin in plant cell walls. Methanol shows circadian dynamics on plant leaves to which methanol-utilizing phyllosphere microorganisms adapt. In the methylotrophic yeast Komagataella phaffii (Pichia pastoris), the plasma membrane protein KpWsc1 senses environmental methanol concentrations, and transmits the information to induce genes for methanol metabolism together with huge peroxisomes. In this study, we show that KpWsc1 and its downstream MAPK negatively regulate pexophagy in the presence of >0.15% methanol. Although KpMpk1 was not necessary for expression of methanol-inducible genes and peroxisome biogenesis, KpMpk1, KpRlm1 and a phosphatase were found suppress pexophagy by controlling phosphorylation level of KpAtg30, the key factor of pexophagy. We reveal at the molecular level how the single methanol sensor KpWsc1 commits the cell to peroxisome synthesis and degradation according to the methanol concentration, and discuss the physiological significance of regulating pexophagy for survival in the phyllosphere.