Autophagy-related gene ATG13 is involved in control of xylose alcoholic fermentation in the thermotolerant methylotrophic yeast Ogataea polymorpha

Abstract

Lignocellulosic biomass belongs to main sustainable renewable sources for global energy supply. One of the main challenges in the conversion of saccharified lignocellulosic biomass into bioethanol is the utilization of xylose, since lignocellulosic feedstocks contain a significant amount of this pentose. The non-conventional thermotolerant yeast Ogataea polymorpha naturally ferments xylose to ethanol at elevated temperatures (45°C). Studying the molecular mechanisms of regulation of xylose metabolism is a promising way toward increased xylose conversion to ethanol. Insertional mutagenesis was applied to yeast O. polymorpha to identify genes involved in regulation of xylose fermentation. An insertional mutant selected as 3-bromopyruvate resistant strain possessed 50% increase in ethanol production as compared to the parental strain. Increase in ethanol production was caused by disruption of an autophagy-related gene ATG13. Involvement of Atg13 in regulation of xylose fermentation was confirmed by deletion of that gene. The atg13Δ strain also produced an elevated amount of ethanol from xylose. Insertion in ATG13 gene did not disrupt HORMA domain and did not lead to defects in autophagy whereas knock out of this gene impaired autophagy process. We suggest that Atg13 plays two different functions and its role in regulation of xylose fermentation differs from that in autophagy.

Construction of advanced producers of first- and second-generation ethanol in Saccharomyces cerevisiae and selected species of non-conventional yeasts (Scheffersomyces stipitis, Ogataea polymorpha)

“…Insertional mutagenesis was used for NCYC495 strain of O. polymorpha with subsequent selection of transformants using a mineral medium supplemented with 25 mM of BrPA. Sequencing of the flanking regions revealed that the insertional cassette disrupted the ORF of a gene homologous to the S. cerevisiae autophagy-related gene ATG13 [31]. This gene encodes a regulatory subunit of the Atg1-signaling complex, stimulating Atg1 kinase activity, which is required for vesicle formation during autophagy and the cytoplasm-to-vacuole targeting pathway.…”

Peroxisomes and peroxisomal transketolase and transaldolase enzymes are essential for xylose alcoholic fermentation by the methylotrophic thermotolerant yeast, Ogataea (Hansenula) polymorpha

“…The resulting plasmid was named pUC57-5′-3′PEX3. HIS3 gene (1399 bp) involved in histidine biosynthesis was amplified using vector p∆atg13Sc [ 36 ] as a template and primers KB5 and KB6. The obtained fragment was Xba I digested and subcloned into Xba I-linearized plasmid pUC57-5′-3′PEX3.…”