Engineering of hyaluronic acid synthases from Streptococcus equi subsp. zooepidemicus and Pasteurella multocida towards improved HA chain length and titer

Hyaluronan (hyaluronic acid; HA) is the non-sulfated glycosaminoglycan product of HA synthases that can vary in length from 103-107 Daltons. Depending on polymer length, concentration and localization, HA possesses variable physicochemical properties that serve useful in pharmaceutical, biomedical and cosmetic applications worth billions ($) in commercial valuation worldwide. A deeper molecular understanding of the HA polymerization control by the synthase machinery can and will facilitate the tuned production of HA, according to the intended application. The general objective of this doctoral investigation, therefore, is to implement protein engineering principles to Class I and Class II HA synthases, with supplication of computational modeling, to improve HA production by means of polymer length and quantity. The Class I HAS from Streptococcus zooepidemicus (seHAS) was first subjected to protein engineering. seHAS was recombinantly expressed in three microbial hosts (E. coli, B. subtilis and S. cerevisiae) and could direct HA synthesis in all three hosts. E. coli was selected for enzyme engineering due to easy handling and quick doubling time. Fluorescent epitope tagging confirmed the presence of seHAS and localization in the outer membrane of the Gram-negative host, contrary to the inner membrane reported in literature. Of the many screening systems attempted to be established, the agarose gel electrophoresis screening platform was the most reliable and could discriminate between empty vector controls, wild type and seHAS variants. Screening of the site-saturation mutagenesis libraries (of the conserved Cys226, Cys262, and Cys281 and polar membrane residues Lys48 and Glu327) and one random mutagenesis library (1392 error-prone PCR variants) failed to identify one seHAS variant with improved chain length specificity. However, alternative positive results were discovered. Site-saturation mutagenesis variants (K48L and K48E) produced consistently monodispersed low molecular weight (LMW; 1 MDa) HA with polydispersity lower than that of seHAS-WT. Homology model analysis hinted at the potential role of HA-HAS interaction in the control of HA polymerization. The discovery of these new positions bifurcates into another dimension of HA, which is chain polydispersity. A better understanding of these product-enzyme interactions can provide clues for production of monodispersed HA. The second protein engineering involved the Class II HAS from Pasteurella multocida (pmHAS). The knowledge-gaining directed evolution (KnowVolution) approach successfully improved the enzymatic activity of the membrane-associated pmHAS. Two screening systems were simultaneously employed to detect improvements in enzymatic output: agarose gel electrophoresis for chain length and the CTAB turbidimetric assay for HA titer. With CTAB, absorbance values of HA synthesized by pmHAS-expressing E. coli BL21 GOLD (DE3) cells were at least 5-fold higher than that of the baseline (empty vector control). Through KnowVolution, seven improved epPCR variants out of 1392 were identified, eight prospective beneficial positions from these variants were saturated and the most beneficial amino acid substitutions (T40L, V59M and T104A) were recombined to generate the final variant (pmHAS-VF). Production of HA up to 4.7 MDa and with a two-fold improvement in mass-based total turnover number over wild type was achieved. This is the first case of a Class II HA synthase directed evolution and an example of a simultaneous dual property improvement through protein engineering. The most complete and validated model to date of pmHAS32-703 was also generated to gain molecular insight into the improved properties. The substitutions in pmHAS-VF are located at the N-terminal domain, away from either glycosyltransferase active sites of pmHAS, suggesting their non-catalytic role. Molecular dynamics simulations reveal the improved flexibility of the N-terminal region allowing it to swing from the GlcNAc-transferase domain to the GlcA-transferase domain. This suggests a newly found importance of the N-terminal domain in HA synthesis. Overall, the ability to synthesize longer HA polymers at higher output brings promise to improved HA production. In the last chapter entitled, “Differential Hyaluronic Acid Synthesis”, methods for production of either HMW HA or LMW HA were investigated. HA synthesis using purified pmHAS was slow despite the addition of the tetrasaccharide synthesis initiator (HA4) and produced only LMW HA. HA synthesis with sonicated pmHAS-expressing E. coli cell lysate was faster, was HA4-independent and produced LMW HA. When pmHAS-expressing E. coli cells were subjected to freeze-thaw and lysozyme treatment, polydispersed HMW HA were generated. More importantly, the strain E. coli BL21 GOLD (DE3) expressing pmHAS was demonstrated to autonomously produce HA, suggesting that this strain naturally possesses metabolic pathways to synthesize the precursors: UDP-GlcA and UDP-GlcNAc. Moreover, the addition of nucleotide sugar precursors prompts the production of fewer but longer HA polymers. When the in vitro synthesized HA products were boiled for 5 min, up to 97% of protein contaminants (detected by BCA assay and Image J analysis) could be precipitated out of solution, albeit at the expense of HA chain depolymerization. This work not only illustrates that cell disruption influences HA production but also offers quick and inexpensive methods to generate semi-purified LMW or HMW HA. In these investigations, protein engineering and computational modeling were employed complementarily to dissect both Class I seHAS and Class II pmHAS, not only having industrial relevance in mind, but to also contribute to the existing knowledge of HAS biology. Ultimately, protein engineering is a powerful tool for tuning HA production with respect to polymer length, quantity and size distribution

Indigenous Geographies: Research as Reconciliation

Employing a reflexive and co-constructed narrative analysis, this article explores our experiences as a non-Indigenous doctoral student and a First Nations research assistant working together within the context of a community-based participatory Indigenous geography research project. Our findings revealed that within the research process there were experiences of conflict, and opportunities to reflect upon our identity and create meaningful relationships. While these experiences contributed to an improved research process, at a broader level, we suggest that they also represented our personal stories of reconciliation. In this article, we share these stories, specifically as they relate to reconciliatory processes of re-education and cultural regeneration. We conclude by proposing several policy recommendations to support research as a pathway to reconciliation in Canada

Plant-Derived Sucrose Is a Key Element in the Symbiotic Association between Trichoderma virens and Maize Plants1[C][W]

Fungal species belonging to the genus Trichoderma colonize the rhizosphere of many plants, resulting in beneficial effects such as increased resistance to pathogens and greater yield and productivity. However, the molecular mechanisms that govern the recognition and association between Trichoderma and their hosts are still largely unknown. In this report, we demonstrate that plant-derived sucrose (Suc) is an important resource provided to Trichoderma cells and is also associated with the control of root colonization. We describe the identification and characterization of an intracellular invertase from Trichoderma virens (TvInv) important for the mechanisms that control the symbiotic association and fungal growth in the presence of Suc. Gene expression studies revealed that the hydrolysis of plant-derived Suc in T. virens is necessary for the up-regulation of Sm1, the Trichoderma-secreted elicitor that systemically activates the defense mechanisms in leaves. We determined that as a result of colonization of maize (Zea mays) roots by T. virens, photosynthetic rate increases in leaves and the functional expression of tvinv is crucial for such effect. In agreement, the steady-state levels of mRNA for Rubisco small subunit and the oxygen-evolving enhancer 3-1 were increased in leaves of plants colonized by wild-type T. virens. We conclude that during the symbiosis, the sucrolytic activity in the fungal cells affects the sink activity of roots, directing carbon partitioning toward roots and increasing the rate of photosynthesis in leaves. A discussion of the role of Suc in controlling the fungal proliferation on roots and its pivotal role in the coordination of plant-microbe associations is provided