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Engineering of Ogataea polymorpha strains with ability for high-temperature alcoholic fermentation of cellobiose
(Oxford University Press, 2024-02-22) Vasylyshyn, Roksolana; Dmytruk, Olena; Sibirny, Andriy; Ruchala, Justyna
Successful conversion of cellulosic biomass into biofuels requires organisms capable of efficiently utilizing xylose as well as cellodextrins and glucose. Ogataea (Hansenula) polymorpha is the natural xylose-metabolizing organism and is one of the most thermotolerant yeasts known, with a maximum growth temperature above 50°C. Cellobiose-fermenting strains, derivatives of an improved ethanol producer from xylose O. polymorpha BEP/cat8∆, were constructed in this work by the introduction of heterologous genes encoding cellodextrin transporters (CDTs) and intracellular enzymes (β-glucosidase or cellobiose phosphorylase) that hydrolyze cellobiose. For this purpose, the genes gh1-1 of β-glucosidase, CDT-1m and CDT-2m of cellodextrin transporters from Neurospora crassa and the CBP gene coding for cellobiose phosphorylase from Saccharophagus degradans, were successfully expressed in O. polymorpha. Through metabolic engineering and mutagenesis, strains BEP/cat8∆/gh1-1/CDT-1m and BEP/cat8∆/CBP-1/CDT-2mAM were developed, showing improved parameters for high-temperature alcoholic fermentation of cellobiose. The study highlights the need for further optimization to enhance ethanol yields and elucidate cellobiose metabolism intricacies in O. polymorpha yeast. This is the first report of the successful development of stable methylotrophic thermotolerant strains of O. polymorpha capable of coutilizing cellobiose, glucose, and xylose under high-temperature alcoholic fermentation conditions at 45°C.
Surowe dane badawcze do artykułu: Efficient production of bacterial antibiotics aminoriboflavin and roseoflavin in eukaryotic microorganisms, yeasts
(BMC, Spinger Nature, 2023-07-20) Dmytruk, Kostyantyn; Ruchala, Justyna; Fayura, Liubov; Chrzanowski, Grzegorz; Dmytruk, Olena; Tsyrulnyk, Andriy; Andreieva, Yuliia; Fedorovych, Daria; Motyka, Olena; Mattanovich, Diethard; Marx, Hans; Sibirny, Andriy
Background
Actinomycetes Streptomyces davaonensis and Streptomyces cinnabarinus synthesize a promising broad-spectrum antibiotic roseoflavin, with its synthesis starting from flavin mononucleotide and proceeding through an immediate precursor, aminoriboflavin, that also has antibiotic properties. Roseoflavin accumulation by the natural producers is rather low, whereas aminoriboflavin accumulation is negligible. Yeasts have many advantages as biotechnological producers relative to bacteria, however, no recombinant producers of bacterial antibiotics in yeasts are known.
Results
Roseoflavin biosynthesis genes have been expressed in riboflavin- or FMN-overproducing yeast strains of Candida famata and Komagataella phaffii. Both these strains accumulated aminoriboflavin, whereas only the latter produced roseoflavin. Aminoriboflavin isolated from the culture liquid of C. famata strain inhibited the growth of Staphylococcus aureus (including MRSA) and Listeria monocytogenes. Maximal accumulation of aminoriboflavin in shake-flasks reached 1.5 mg L− 1 (C. famata), and that of roseoflavin was 5 mg L− 1 (K. phaffii). Accumulation of aminoriboflavin and roseoflavin by K. phaffii recombinant strain in a bioreactor reached 22 and 130 mg L− 1, respectively. For comparison, recombinant strains of the native bacterial producer S. davaonensis accumulated near one-order less of roseoflavin while no recombinant producers of aminoriboflavin was reported at all.
Conclusions
Yeast recombinant producers of bacterial antibiotics aminoriboflavin and roseoflavin were constructed and evaluated.
Biota grzybów związana z siewkami dębu (2020-2022)
(2024) Behnke-Borowczyk, Jolanta
Razem czy osobno – różnorodność zbiorowisk grzybów a sposób odnowienia dębu.
Regeneracja dębu (Quercus spp.) powstaje na drodze naturalnej lub sztucznej. W przypadku regeneracji sztucznej wyróżniamy siew lub sadzenie sadzonek przez człowieka. Sprzymierzeńcami w powstaniu naturalnego odnowienia są zwierzęta. Szczególną rolę w tym procesie odgrywają ptaki. W Europie, sójki euroazjatyckie są głównymi rozsiewaczami nasion dębu. Rozprzestrzenianie nasion przez ptaki odgrywa kluczową rolę w tworzeniu nowych siedlisk dla roślin, umożliwiając im osiedlanie się w różnych obszarach w tym siedliskach nieleśnych i w tradycyjnych krajobrazach rolniczych. Wpływ zróżnicowania zbiorowisk roślinnych na mikrobiotę roślin oraz funkcje troficzne grzybów w relacjach z roślinami są obszarami badawczymi, które wymagają głębszej analizy. Celem było określenie zależności między miejscem wzrostu sadzonek dębu a różnorodnością zbiorowisk grzybów towarzyszącym korzenią z uwzględnieniem funkcji pełnionej przez poszczególne taksony w środowisku. Analizę taksonomiczną zbiorowisk przeprowadzono w oparciu o region ITS1, z wykorzystaniem sekwencjonowania systemem ILLUMINA. Bibliotekę OTU stworzono w oparciu o baze referencyjną NCBI.
Surowe dane badawcze do artykułu "Non-targeting siRNA-mediated responses are associated with apoptosis in chemotherapy-induced senescent skin cancer cells"
(Chemico-Biological Interactions, 2023-01-05) Betlej, Gabriela; Błoniarz, Dominika; Lewińska, Anna; Wnuk, Maciej
It is widely accepted that siRNA transfection can promote some off-target effects in the genome; however, little is known about how the cells can respond to the presence of non-viral dsRNA. In the present study, non-targeting control siRNA (NTC-siRNA) was used to evaluate its effects on the activity of pathogen and host-derived nucleic acid-associated signaling pathways such as cGAS-STING, RIG-I, MDA5 and NF-κB in A431 skin cancer cells and BJ fibroblasts. NTC-siRNA treatment promoted cytotoxicity in cancer cells. Furthermore, NTC-siRNA-treated doxorubicin-induced senescent cancer cells were more prone to apoptotic cell death compared to untreated doxorubicin-induced senescent cancer cells. NTC-siRNA stimulated the levels of NF-κB, APOBECs, ALY, LRP8 and phosphorylated STING that suggested the involvement of selected components of nucleic acid sensing pathways in NTC-siRNA-mediated cell death response in skin cancer cells. NTC-siRNA-mediated apoptosis in cancer cells was not associated with IFN-β-based pro-inflammatory response and TRDMT1-based adaptive response. In contrast, in NTC-siRNA-treated fibroblasts, an increase in the levels of RIG-I and IFN-β was not accompanied by affected cell viability. We propose that the use of NTC-siRNA in genetic engineering may provoke a number of unexpected effects that should be carefully monitored. In our experimental settings, NTC-siRNA promoted the elimination of doxorubicin-induced senescent cancer cells that may have implications in skin cancer therapies.
Dane do artykułu "Mutation Status and Glucose Availability Affect the Response to Mitochondria-Targeted Quercetin Derivative in Breast Cancer Cells"
(Cancers (Basel) MDPI, 2023-11-28) Przybylski, Paweł; Lewińska, Anna; Rzeszutek, Iwona; Błoniarz, Dominika; Moskal, Aleksandra; Betlej, Gabriela; Deręgowska, Anna; Cybularczyk-Cecotka, Martyna ; Szmatoła, Tomasz ; Litwinienko, Grzegorz; Wnuk, Maciej
Mitochondria, the main cellular power stations, are important modulators of redox-sensitive signaling pathways that may determine cell survival and cell death decisions. As mitochondrial function is essential for tumorigenesis and cancer progression, mitochondrial targeting has been proposed as an attractive anticancer strategy. In the present study, three mitochondria-targeted quercetin derivatives (mitQ3, 5, and 7) were synthesized and tested against six breast cancer cell lines with different mutation and receptor status, namely ER-positive MCF-7, HER2-positive SK-BR-3, and four triple-negative (TNBC) cells, i.e., MDA-MB-231, MDA-MB-468, BT-20, and Hs 578T cells. In general, the mito-quercetin response was modulated by the mutation status. In contrast to unmodified quercetin, 1 µM mitQ7 induced apoptosis in breast cancer cells. In MCF-7 cells, mitQ7-mediated apoptosis was potentiated under glucose-depleted conditions and was accompanied by elevated mitochondrial superoxide production, while AMPK activation-based energetic stress was associated with the alkalization of intracellular milieu and increased levels of NSUN4. Mito-quercetin also eliminated doxorubicin-induced senescent breast cancer cells, which was accompanied by the depolarization of mitochondrial transmembrane potential. Limited glucose availability also sensitized doxorubicin-induced senescent breast cancer cells to apoptosis. In conclusion, we show an increased cytotoxicity of mitochondria-targeted quercetin derivatives compared to unmodified quercetin against breast cancer cells with different mutation status that can be potentiated by modulating glucose availability.