Hyaluronic acid (HA) is a biopolymer with valuable applications in the pharmaceutical and cosmetic industries. The molecular weight of HA is important for its rheological, biological and commercial properties. Currently, high molecular weight HA is extracted from animal sources. Recent pandemic outbreaks of viruses H5N1 and H1N1 (avian and swine influenza) have raised concerns regarding the safety of animal-derived pharmaceuticals; making fermentation the preferred source of HA. Throughout this study, the mechanism of molecular weight control of HA polymerisation in S. zooepidemicus was investigated. While several aspects are still unknown, it was found that levels of activated monomers, (UDP-sugars) have a fundamental role in molecular weight control. High levels of activated sugars strongly correlated with high molecular weight. Throughout rational strain engineering, several strains harbouring a collection of genes were engineered using S. zooepidemicus as a host microorganism. Five genes of the so-called has operon were cloned into a nisin inducible plasmid and transformed into S. zooepidemicus. Several significant changes were observed in HA molecular weight. In order to understand those changes as a complex system, rather than isolated parts of the cell, a systems approach was undertaken. The main goal of this approach was to examine the structure and dynamics of cellular functions using global measurements on changes in proteins or metabolite concentrations, in response to genetic perturbations. As part of the systems biotechnology approach, methods for proteins and metabolites harvesting were optimised. Harvesting metabolites and proteins in microorganisms is a non-trivial process (Chapter 4 and 5). Encapsulated bacteria presents additional challenges since capsular polysaccharides interfere with extraction and downstream analysis. In terms of metabolite harvesting, several studies have reported rapid turnovers in low abundant intracellular sugar metabolites. Four different protocols for cellular harvesting were tested. The best method found was centrifugation, which allowed for efficient medium removal and enabled quantification of the broadest range of sugar metabolites. Unlike observations for other microbes, changes in metabolite pools due to a delay of extraction by centrifugation were not observed. Our hypothesis was that the capsule itself isolates the cells from their surroundings and still supports them with nutrients during cellular harvesting (Chapter 3). Protein harvesting also proved to be technically challenging (Chapter 5), especially when fluorescent dyes were employed for protein visualisation(Chapter 6). Despite this, using hyaluronidase to remove the HA capsule the first reference map for S. zooepidemicus was completed. Besides giving insight into the most abundantly expressed proteins, as well as facilitating the design of better diagnostics and treatments for streptococcal infections, our reference map can be used to engineer superior production strains (Chapter 5). In Chapter 6, proteins involved in MW control were separated using 2D differential in gel electrophoresis (DIGE) and identified by mass spectrometry. Moreover, the wild-type was compared with both the empty vector control strain and a high molecular-weight producing strain harbouring phosphoglucoisomerase (pgi). An enzyme involved in controlling the levels of the intracellular pool of one of the activated sugars, UDP-N-acetylglucosamide-1-carboxivinyltransferase, was down-regulated in the control line. Overexpression of UDP-N-Acetylglucosamide 1-carboxivinyltransferase, decreased both the concentration of activated precursors and HA molecular weight. Gene knockout of one of the copies of that particular gene could potentially guide us to further improvement of the strain. By overexpressing all the genes involved in the HA pathway (Appendix A) we have pushed gene expression as far as possible and the next option was to look at process optimisation. Several studies have found that culture conditions affect HA molecular weight: higher molecular weight is produced under aerobic conditions and when using maltose as the carbon source. To overcome the lower sugar uptake and growth rate observed under maltose, a two stage batch fermentation process was conducted. By feeding glucose when cell growth was stopped through amino acid auxotrophy, we achieved high molecular weight HA production in stationary phase. Using engineered strains, HA >5 MDa was obtained (Chapter 7). This thesis represents a good example of systems biotechnology for strain improvement and is a step forward in understanding the mechanism of molecular weight control of HA production by bacterial fermentation.
| Identifer | oai:union.ndltd.org:ADTP/280765 |
| Creators | Esteban Marcellin |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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