The potential and capability of the methylotrophic yeast Ogataea methanolica in a "methanol bioeconomy".

Efficient bioconversion of methanol, which can be generated from greenhouse gases, into valuable resources contributes to achieving climate goals and developing a sustainable economy. The methylotrophic yeast Ogataea methanolica is considered to be a suitable host for efficient methanol bioconversion because it has outstanding characteristics for the better adaptive potential to a high methanol environment (i.e., greater than 5%). This capacity represents a huge potential to construct an innovative carbon-neutral production system that converts methanol into value-added chemicals under the control of strong methanol-induced promoters. In this review, we discuss what is known about the regulation of methanol metabolism and adaptation mechanisms for 5% methanol conditions in O. methanolica in detail. We also discuss about the potential to breed "super methylotrophic yeast," which has potent growth characteristics under high methanol conditions.

Fatty acid composition of the methylotrophic yeast Komagataella phaffii grown under low- and high-methanol conditions.

In this study, we analysed the intracellular fatty acid profiles of Komagataella phaffii during methylotrophic growth. K. phaffii grown on methanol had significantly lower total fatty acid contents in the cells compared with glucose-grown cells. C18 and C16 fatty acids were the predominant fatty acids in K. phaffii, although the contents of odd-chain fatty acids such as C17 fatty acids were also relatively high. Moreover, the intracellular fatty acid composition of K. phaffii changed in response to not only carbon sources but also methanol concentrations: C17 fatty acids and C18:2 content increased significantly as methanol concentration increased, whereas C18:1 and C18:3 contents were significantly lower in methanol-grown cells. The intracellular content of unidentified compounds (Cn H2n O4 ), on the other hand, was significantly greater in cells grown on methanol. As the intracellular contents of these Cn H2n O4 compounds were significantly higher in a gene-disrupted strain for glutathione peroxidase (gpx1Δ) than in the wild-type strain, we presume that the Cn H2n O4 compounds are fatty acid peroxides. These results indicate that K. phaffii can coordinate intracellular fatty acid composition during methylotrophic growth in order to adapt to high-methanol conditions and that certain fatty acid species such as C17:0, C17:1, C17:2 and C18:2 may be related to the physiological functions by which K. phaffii adapts to high-methanol conditions.

Effects of natural deep eutectic solvents on lactic acid bacteria viability during cryopreservation.

In this study, a new method of cryopreservation of lactic acid bacteria (LAB) using natural deep eutectic solvents (NADESs) was developed. Survival rates of LAB during 24-h short-term and 180-day long-term cryostorage at - 20 °C were investigated. The results revealed that survival of Streptococcus thermophilus (S. thermophilus) in NADESs after 24 h of cryostorage was superior to survival of two other tested LAB. Moreover, survival was higher at a ratio of NADESs to S. thermophilus of 1:1 (v/v) than observed using a 4:1 (v/v) ratio. Representative freezing characteristics of five NADESs were elucidated, including thermodynamic properties and hydrogen bonding interactions after addition of water. In order to identify the protective mechanism of NADESs on cell structure and vital metabolic enzymes of S. thermophilus during cryostorage, transmission electron microscopy (TEM) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were employed and enzyme activities of both lactate dehydrogenase (LDH) and ß-galactosidase were determined. Subsequently, NADES GlyP prepared from glycerol and L-proline was demonstrated to maintain cell membrane structural integrity and significantly (p < 0.05) maintain activities of both intracellular enzymes of S. thermophilus. Moreover, NADESs could efficiently penetrate S. thermophilus cells and intracellular ß-galactosidase activity could be used to demonstrate NADESs effectiveness in maintaining S. thermophilus survival after long-term cryostorage. These results demonstrate that NADESs can be used as green cryoprotective chemical agents (CPAs) that can efficiently increase LAB viability during cryostorage. These results should have great value in the food production and probiotics industries.

Metabolic regulation adapting to high methanol environment in the methylotrophic yeast Ogataea methanolica.

Since methylotrophic yeasts such as Ogataea methanolica can use methanol as a sole carbon feedstock, they could be applied to produce valuable products from methanol, a next-generation energy source synthesized from natural gases, using genetic engineering tools. In this study, metabolite profiling of O. methanolica was conducted under glucose (Glc) and low and high methanol (L- and H-MeOH) conditions to show the adaptation mechanism to a H-MeOH environment. The yeast strain responded not only to the presence of methanol but also to its concentration based on the growth condition. Under H-MeOH conditions, O. methanolica downregulated the methanol utilization, glycolytic pathway and alcohol oxidase (AOD) isozymes and dihydroxyacetone synthase (DAS) expression compared with L-MeOH-grown cells. However, levels of energy carriers, such as ATP, were maintained to support cell survival. In H-MeOH-grown cells, reactive oxygen species (ROS) levels were significantly elevated. Along with increasing ROS levels, ROS scavenging system expression was significantly increased in H-MeOH-grown cells. Thus, we concluded that formaldehyde and H2 O2 , which are products of methanol oxidation by AOD isozymes in the peroxisome, are overproduced in H-MeOH-grown cells, and excessive ROS derived from these cells is generated in the cytosol, resulting in upregulation of the antioxidant system and downregulation of the methanol-utilizing pathway to suppress overproduction of toxic intermediates.