Faculty of Agriculture and Forestry

Aspergillus niger is a well-studied filamentous ascomycete fungus and one of the most used fungal species in various biotechnological sectors. Recently the research focus on A. niger has shifted towards production of biochemicals and biomaterials from plant biomass residues. However, there are still many aspects of the plant biomass conversion process by A. niger that are not known in detail, including its sugar uptake systems. Sugar uptake in fungi relies on sugar transporter (ST) proteins that control the uptake of different sugar molecules. Fungal STs are abundant and diverse in their function, which is why many of them remain uncharacterized. Identification of fungal STs for substrates like xylose can improve the industrial production of lignocellulose-based bioproducts by e.g. improving the substrate uptake rate in fungal cell factories. In this thesis, four putative xylose STs from A. niger, XltD, XltE, XltG, and XltH, were characterized both physiologically and functionally in A. niger and Saccharomyces cerevisiae, respectively, together with two previously functionally characterized xylose transporters, XltA and XltB. In addition, a fifth putative xylose transporter XltF was characterized functionally in S. cerevisiae. For the physiological characterization, A. niger 6Δxlt strain was generated by deleting xltA, xltB, xltD, xltE, xltG, and xltH genes using CRISPR/Cas9 methodology. The physiological analysis of the 6Δxlt strain revealed the presence of additional xylose transporters in the A. niger genome, which still remain to be discovered. The functional characterization of the putative A. niger xylose STs was carried out by creating six recombinant S. cerevisiae IMK1010 strains producing the STs fused with a green fluorescent protein. Functional characterization confirmed the correct localization of the STs within plasma membrane, except for XltG which accumulated inside the yeast cells. This indicated potential localization of XltG within endoplasmic reticulum and a putative role in intracellular sugar transport. Growth assays of the recombinant yeast strains demonstrated variable affinities of the A. niger STs for hexoses. The results showed XltF not to be a xylose ST, but instead a hexose ST with low-affinity for fructose and dual-affinity for glucose. The STs were further tested for their substrate specificity and affinity for xylose in a competitive assay between xylose and glucose. XltE displayed a preference for xylose over glucose identifying it as a new low-affinity xylose transporter. Although further research is needed to elucidate the roles of the studied A. niger STs, XltE is a promising candidate for enhancing xylose uptake in fungal cell factories.

The increase of antibiotic resistance is one of the major healthcare threats globally. One potential way to battle against antibiotic resistant bacterial infections is to treat them with the natural opponents of bacteria, bacteriophages, known as phage therapy. The aim of this thesis was to identify new bacteriophages against clinically notable bacterial species such as Escherichia coli, Burkholderia cepacia, Enterococcus faecalis and Enterococcus faecium. Bacteriophages were screened from various origins such as hospital sewage samples, soil samples and manure samples, collected in between 2019 and 2022. The isolated bacteriophages were then initially characterized to evaluate their potential use in phage therapy. In this thesis, two phages (fHo-Eco16, fHo-Eco17) against clinical E. coli isolate and one phage (fHo-Efa06) against clinical E. faecalis isolate were found from the recently collected Finnish hospital sewage sample pool. Both E. coli phages were classified as Felixounaviruses belonging to family of Ounavirinae and class of Caudoviricetes. Enterococcus phage fHo-Efa06 was characterized as Saphexavirus belonging to class of Caudoviricetes. Preliminary genome annotation did not reveal any characteristics of lysogenic lifecycle, or antibiotic resistance or bacterial toxin genes, which would prevent the use of phages in phage therapy. Both E. coli phages (fHo-Eco16, fHo-Eco17) showed narrow host range infecting only the primary host bacterial isolate but none of 29 other tested clinical E. coli isolates. Phage fHo-Efa06 showed relatively broad host range properties infecting nine tested E. faecalis isolates out of 20 tested E. faecalis isolates but no infection capabilities against six tested clinical E. faecium isolates. In conclusion, freshly collected hospital sewage seemed to be optimal environment to find bacteriophages against clinical bacterial isolates. Furthermore, phages fHo-Eco16, fHo-Eco17 and fHo-Efa06 did not display any strictly unsuitable properties which could prevent their use in phage therapy. In turn, to obtain the definitive certainty on the usability of the phages in therapeutic use, in-depth host range screening together with detailed functional and structural annotation for the phage genomes of fHo-Efa06, fHo-Eco16 and fHo-Eco17 should be completed.

Pests and pathogens damage crops, causing economic losses, while pesticide misuse harms the environment. Double-stranded (ds)RNA offers a safe, non-toxic, and biodegradable alternative for plant protection. Previous research established efficient dsRNA production in Pseudomonas cells using components derived from dsRNA bacteriophage phi6. The dsRNA is produced using phi6 RNA-dependent RNA polymerase (RdRp) to synthesise the strands in vivo. However, P. syringae LM2691 (the current strain used for dsRNA production) is a plant pathogen. Subsequently, there is a risk that the dsRNA preparations contain bacterial toxins. Additionally, the present dsRNA purification method is costly and non-scalable. This study aims to find a non-pathogenic Pseudomonas strain for dsRNA production, compare it with the original production strain (OPS), and optimizing dsRNA purification. To identify a non-pathogenic strain that could be used for dsRNA production, 14 environmental Pseudomonas strains underwent testing for their ability to support phi6 bacteriophage replication. Susceptibility and permissivity of the strains were tested with natural infection with phi6 bacteriophage and reverse genetics system, respectively. Only one strain could be infected and was permissive to phi6 infection. After stablishing the production system in the new strain, it exhibited faster growth than the OPS during 24 h. The new strain reached an eight-fold higher cell count than the OPS after 24 h cultivation at 23°C or 28°C. These results did not correspond to the dsRNA production, where the new strain showed a 3-fold lesser amount of total phi6-produced RNA when compared to the OPS at 28°C. The study also explored mixed-mode chromatography on Capto Core 700 medium for purifying phi6 PCs. The complexes harbouring dsRNA molecules with tobacco mosaic virus sequences were released from the bacterial cells by freezing-thawing cycles. This method preserved the integrity of the complexes, which is crucial for shielding dsRNA against RNases. Optimal purity was attained using a pH 6.5, salt-free elution buffer. While the new non-pathogenic strain identified in this study shows potential for a production system, further optimization is required for dsRNA production in the new strain. The use of mixed-mode chromatography offers a pathway for isolating polymerase complexes, potentially facilitating dsRNA delivery to plants.