No / Streptococcus equi and Streptococcus zooepidemicus are major etiological agents of upper and lower airway disease in horses. Despite the considerable animal suffering and economic burden associated with these diseases, the factors that contribute to the virulence of these equine pathogens have not been extensively investigated. Here we demonstrate the presence of a homologue of the Streptococcus pneumoniae PsaA protein in both of these equine pathogens. Inhibition of signal peptide processing by the antibiotic globomycin confirmed the lipoprotein nature of the mature proteins, and surface exposure was confirmed by their release from intact cells by mild trypsinolysis. / Project grant 056042 from The Wellcome Trust.

Hyaluronic acid (HA) is a high value biopolymer that has numerous biomedical and cosmetic applications. It is currently derived from two sources, namely animal tissues and bacterial fermentation (Fong Chong et al. 2005). The molecular weight (Mw) of HA can vary from several hundred thousand dalton (Da) to approximately 8 MDa (Widner et al. 2005). High Mw HA has surgical applications, and therefore constitutes a major component of the lucrative HA market. The current need is largely met by extraction from animal tissues, e.g., rooster comb and bovine vitreous humor (Shiedlin et al. 2004). However, the potential of contamination with adventitious agents (e.g., viruses) have raised regulatory concerns regarding the use of animal extracts in pharmaceutical products. Moreover, with recent reports of zoonotic diseases (e.g., bovine spongiform encephalitis and avian influenza virus), pharmaceutical companies are moving towards microbial HA sources. Although HA obtained from bacterial fermentation does not have the problem of viral contamination, this approach has not yet resulted in a process where HA of sufficiently high Mw for surgical applications can be derived. While attempts have been made to produce higher Mw HA through cross-linking, cross-linked HA is undesirable for certain medical procedures (e.g., ophthalmic applications) which requires a natural polymer with a short half-life. Nevertheless, due to its availability and the relative ease of purification, bacterial fermentation has the potential of replacing extraction from animal tissues as a preferred commercial source of HA. This thesis presents a good example of a metabolic engineering study where modern techniques (e.g., molecular biology, fermentation and omics technologies) are used to explain complex cell metabolism. The hypothesis for this study was that the precursors to HA, i.e., UDP-glucuronic acid and UDP-N-acetylglucosamine and consequently the genes involved in precursor generation, are important for HA Mw. However, environmental manipulation, e.g., anaerobic versus aerobic or glucose versus maltose, often results in large global changes in metabolite concentration and enzyme activities. This makes it impossible to resolve issues related to Mw control. Classical statistical methods do not provide a meaningful inference as the number of explanatory variables always exceeds the number of independent observations. Hence, it is difficult to distinguish between causative and accidental correlation. This work first examined the influence of manipulation of metabolite concentrations in the hyaluronan pathway to find an explanation for the mechanism of Mw control. To achieve this, the five essential genes of the hyaluronan synthesis (has) operon in Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) were first examined. These genes are involved in two pathways which lead to the production of either UDP-glucuronic acid or UDP-N-acetylglucosamine. Overexpression of genes involved in UDP-glucuronic acid biosynthesis decreased HA Mw, while overexpression of genes involved in UDP-N-acetylglucosamine biosynthesis increased HA Mw. The Mw variation generated provided a stepping stone for further understanding of Mw control of HA. The highest Mw observed was achieved with combined overexpression of pgi and glmU. This study proved that there is a positive correlation between UDP-N-acetylglucosamine and Mw. The first model for HA Mw control based on the concentration of activated sugar precursors is described in this study (Chapter 3). This correlation observed led to the hypothesis that high Mw HA can be achieved when an appropriate balance of the two HA precursor is maintained. Three genes in the two precursor pathways are not found in the has operon of S. zooepidemicus. To obtain a complete overview of all genes in the HA pathway, these genes were also examined using overexpression studies. Individual overexpression of these genes had negligible effects on HA Mw and production. Despite the positive correlation previously observed between UDP-N-acetylglucosamine and Mw, sequential overexpression of genes involved in the UDP-N-acetylglucosamine precursor pathway did not increase Mw of HA produced. This is surprising since the highest pool of UDP-N-acetylglucosamine was achieved in this case. This suggests that a threshold effect is present in the correlation between UDP-N-acetylglucosamine and Mw. This threshold effect may be defined by a balance between the two precursors. To investigate this phenomenon further, the precursor ratio was also manipulated by co-metabolising glucose and N-acetylglucosamine. Similar to the previous experiment, a significant increase in UDP-N-acetylglucosamine levels was observed despite only a marginal increase in Mw (Chapter 4). Surprisingly, an increase in Mw was observed with the introduction of a plasmid in S. zooepidemicus. This plasmid effect was studied on a global scale using transcriptome and proteome analysis to understand the changes occurring in the system. The increase in Mw due to the plasmid effect is independent of the functions, i.e., nisin promoter or antibiotic resistance, encoded in the plasmid. A gene involved in UDP-N-acetylglucosamine production, UDP-N-acetylglucosamide 1-carboxivinyltransferase (murA), was significantly down-regulated in both the plasmid bearing strain and the high Mw strain (pgi). In addition, overexpression of murA decreased both the concentration of activated sugar precursors and HA Mw. There was however no evidence of down-regulation of murA in the plasmid containing strain from transcriptomics data. This suggests that control is exerted either at the translation level or by protein degradation (Chapter 5). This thesis contributes and represents an ongoing effort to understand the elusive mechanism of Mw control of HA.

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.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2006. / Härtill 4 uppsatser.

This master thesis focuses on the effect of cultivation parameters on hyaluronic acid synthesis during its biotechnological production. The cultivation parameters were temperature of the cultivation, aeration, agitation, carbon source and addition of phosphatidylcholine. Changes in molecular weight and yield of hyaluronic acid, growth of biomass and medium viscosity were observed. From the obtained data we learned, that the specific growth rate greatly impacts final characteristics of hyaluronic acid. Under suboptimal temperatures a high molecular weight polymer was synthesised along with comparable yields from the control cultivation. High temperatures (40 °C) caused a dip in molecular weight. The next cultivation parameters were aeration and agitation. The highest molecular weights were obtained in cultivations with high agitation rates and intensive aeration, specifically 1 vvm/800 rpm and 2 vvm/800 rpm. Agitation had a bigger influence on molecular weight than aeration. When the carbon source was changed from sucrose to glucose, the lowest molecular weight and yield were obtained. Addition of phosphatidylcholine with concentration 160 mg/l had a positive effect on the cultivation, where the molecular weight grew and biomass growth was higher.

This diploma thesis is focused on development of a fast method for site-directed gene mutagenesis in Streptococcus zooepidemicus based on the mechanism of natural competence. Several genes were selected based on experimental data which highly probably influence hyaluronic acid synthesis. The deletion of the selected genes from genomic DNA was performed as proof of concept, and the resulting recombinant strains were characterized regarding changes of hyaluronic acid precursor concentrations (glucuronic acid and N-acetylglucosamin) in time of cultivation and the end production of hyaluronic acid.

Hyaluronic acid (HA) is an important substance, which is mostly used in pharmaceutical and cosmetic industry. This substance is commonly found in the human body. HA is one of the factors contributing to virulence of microorganisms. Some bacterial strains produce hyaluronic acid in the form of a mucoid capsule that encapsulates the cell to protect bacteria against the immune system of the host organism. One of the main producers is the bacterial strain Streptococcus equi subsp. zooepidemicus. Contipro a.s. uses the strain CO4A to produce hyaluronic acid in large scale. The production strain was obtained by random mutagenesis by UV light. The aim of the work was to study changes in the genome, which led to a significant increase in hyaluronic acid production, using DNA microarray and real-time PCR (qPCR). The genome of the strain CO4A was sequenced and compared to reference ATCC35246 [1]. The size of the genome is 2,167,251 bp and 83 relevant variants (59 SNV and 34 indels) have been identified. Variants in coding regions were annotated and amino acid sequence changes were determined. In SNV mutations there was a change in the amino acid sequence in 45 cases. The change was identified in every case of indel mutations. The expression level of selected groups of genes was monitored in both strains by the method of DNA microarrays. A cascade of increased expression level of amino sugar metabolism genes leading to the synthesis of UDP-N-acetyl glucosamine was observed in strain CO4A (the increase in expression level of these genes compared to ATCC35246 was on average 28 %). Subsequently, the expression of selected genes was verified by qPCR. There was no significant difference in the expression level of the has operon genes of both strains. The effect of supplementation of the culture medium with N-acetylglucosamine (GlcNAc), which is one of the precursors of HA synthesis, was also studied by qPCR. A positive effect of the supplementation of the culture medium with external GlcNAc in the CO4A strain has been recorded. Also, the supplementation has positive effect on the yield of HA from the medium (increase in yield was on average by 17 %). GlcNAc has been shown to have a positive effect on the yield of HA in ATCC35246 strain as well (increase in yield was 9 % on average), but no significant changes in the expression levels were found in selected groups of genes in ATCC35246.