Metabolic engineering of microbial hosts for the biosynthesis of high molecular weight hyaluronan

• Metabolic Engineering von mikrobiellen Wirten zur Biosynthese von hochmolekularem Hyaluronan

Schulte, Sandra; Blank, Lars Mathias (Thesis advisor); Elling, Lothar (Thesis advisor)

1. Auflage. - Aachen : Apprimus (2018)
Book, Dissertation / PhD Thesis

In: Applied microbiology 6
Page(s)/Article-Nr.: 1 Online-Ressource (IX, 124 Seiten) : Illustrationen

Dissertation, RWTH Aachen University, 2018

Abstract

The demand for hyaluronan (HA), a linear polysaccharide consisting of glucuronic acid and Nacetylglucosamine, as ingredient in medical and cosmetic products rises steadily. Especially the niche of high molecular weight (MW) HA requires attention, as isolation from animal sources prevails here. The traditional HA production processes are neither efficient nor environmentally friendly, therefore, the present work aimed at establishing a microbial host for high MW hyaluronan production. By synthetic biology means, the use of the alternative carbon sources sucrose and N-acetylglucosamine, directly feeding into the precursor synthesis pathways, was aimed at. Two strategies were explored for the production of high MW HA. 1. Saccharomyces cerevisiae, the baker’s yeast, was used as a eukaryotic host to evaluate if eukaryotic HA synthases, which were reported to produce high MW HA, can be used for chassis construction. 2. The streptococcal HA synthases were revisited for high MW HA synthesis using Lactococcus lactis as host. To produce high MW HA in S. cerevisiae one not only requires a functional HA synthase, but also a pathway to the precursor UDP-glucuronic acid (UDP-GlcA). Here, the streptococcal pathway was investigated. In addition, to enable the use of the alternative carbon sources sucrose and Nacetylglucosamine, synthetic pathways were elucidated that allow a partial orthogonality to the native yeast metabolism. The envisaged yeast chassis hence carries two modules for HA precursor synthesis and the HA synthase itself. S. cerevisiae turned out to be not suitable for recombinant HA production. Though one HA synthase showed activity in vitro, no HA could be detected in vivo. By using yeGFP-fusionconstructs it was shown that the synthases do not localize correctly within the cells. Fluorescencewas observed in intracellular compartments, but not at the plasma membrane. Therefore, the synthetic pathway leading to the HA precursor UDP-N-acetylglucosamine was not investigated indetail, as S. cerevisiae possesses a native pathway for its biosynthesis. As for the biosynthesis of UDP-GlcA, a sucrose negative chassis was successfully constructed that can be used for further applications. Functionality of the sucrose synthase and subsequent production of UDP-GlcA could not be shown. The HA produced by the Streptococcus zooepidemicus HA synthase is well characterized and is commercially available. Notably, the synthesis potential of other streptococcal synthases is notfully explored and hence here first the available genetic inventory was searched for microbial HA synthases. It turned out that despite the vast increase in genetic information, only two additional HA synthases could be identified, underlining the previously recognized scarcity of this protein in bacteria. The tested HA synthases indeed produced HA of different MW both in vitro and in vivo; that of Streptococcus pyogenes was much lower than that of Streptococcus uberis and Streptococcus parauberis. However, the production of truly high MW requires further investigation. The results of this thesis do not only contribute to the ongoing effort of high MW HA synthesis, but also to the concept of chassis construction including the use of alternative carbon sources.