Clarification of the xanthan biosynthesis mechanism in Xanthomonas campestris pv. campestris B100 by mass spectrometry-based proteome analysis

Xanthan is a bacterial polysaccharide commonly used in various industrial applications, particularly as a food additive. It is composed of a repeating unit in which two β1,4-linked and β1,2-linked D-glucose molecules form a cellulose-like backbone containing a side chain of β1,4-linked D-mannose, D-glucuronic acid, and β1,2-linked D-mannose.<br /><br /> In the first project, the proteome profiling of Xanthomonas campestris pv. campestris over the time of cultivation was considered. The consumption of nutrition and the amount of synthesized xanthan were particularly interesting. The reference database indicates that 2416 proteins were detected throughout the study, accounting for 54.75% of the proteome. Fluctuations in nitrate levels seemed to impact the quantity of most proteins related to nitrogen metabolism, except for Gdh and GlnA. The levels of proteins involved in sugar nucleotide metabolism remained constant throughout all stages of growth. Except for GumD, GumB, and GumC, the gum proteins exhibited no notable variations during the study. GumD, the primary enzyme forming the xanthan-repeating unit, peaked during the early stationary phase but declined during the late stationary phase. GumB and GumC, responsible for exporting xanthan, experienced a significant increase during the stationary phase.<br /><br /> More understanding of the structure of the xanthan synthesis mechanism within Xcc is needed. Consequently, the second project aims to use an LC-MS-based approach to find potential protein complexes. This technique gathers additional insights from a protein dataset analyzed using data-independent acquisition (DIA). It is crucial to validate the method by testing it on established protein complexes before its actual use. The method shows a linear relationship for most subunits within the RNA-polymerase, the ATPase, and the Ribosome. These findings suggest that a linear relationship could suggest possible complex formation or interaction. The subsequent phase involves applying this technique to study xanthan biosynthesis and recognize potential protein complexes. Investigating the xanthan synthesis mechanism identified two observable clusters in the gum proteins correlation matrix. A complex of GumB and GumC, already considered in the literature, and a combination of GumH, J, and I, which is unknown, could be identified. The method shown here provides easy and fast information about possible protein complexes in a metabolic pathway. It could be proven by investigating known protein complexes and was used further to enlighten the structure of the xanthan biosynthesis mechanism.<br /><br /> II In the third project, the proteome of Xcc was studied under the "methionine effect." A significant reduction in the exopolysaccharide productivity of Xcc cells in the presence of L-methionine characterizes this effect. The proteome investigation during methionine treatment provides valuable information about regulating the xanthan biosynthesis mechanism. In addition to the proteome analysis, a phosphoproteome analysis was conducted to further explore the "methionine effect." The method was adapted to accommodate increased culture volumes, and a phenol solution called TRIzol was used to extract sufficient protein for phosphopeptide enrichment. The proteome analysis identified 2070 proteins, with 305 showing significant differences during methionine treatment. Pathway enrichment analysis revealed a significant decrease in cysteine, methionine, and sulfur metabolism, consistent with previous observations of gene transcription rates. Carbon metabolism, related to xanthan biosynthesis through the Glycolysis/Gluconeogenesis and the Pentose phosphate pathway, showed either no abundance changes or an increase in the abundance. Notably, the Gum proteins exhibited abundance changes during methionine treatment, with only GumD showing significant differences. GumD catalyzes the initial enzymatic step in xanthan biosynthesis, making it a potential candidate for regulating xanthan synthesis. The findings of the proteome and phosphoproteome analyses suggest that methionine treatment enhances carbon metabolism in response to the presence of methionine. These findings contribute to a better understanding of the biochemical response to the "methionine effect" and complement previous observations.<br /><br /> This thesis achieved essential steps for elucidating and understanding xanthan biosynthesis. It generated further knowledge about the xanthan synthesis pathway and identified bottlenecks and optimization strategies of xanthan production. These insights offer opportunities for upcoming projects and optimization of industrial xanthan production.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Bielefeld

Year

2025

Author dc:creator

• Struck, Ben

Repository record source_url

https://pub.uni-bielefeld.de/record/3003407

OAI identifier oai:identifier

oai:pub.uni-bielefeld.de:3003407