Engineering and characterization of Xanthomonas campestris pv. campestris towards an enhanced production of the exopolysaccharide xanthan

Steffens T (2017)
Bielefeld: Universität Bielefeld.

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Bielefeld Dissertation | English
Abstract
Xanthan is the industrially most important bacterial polysaccharide, with broad fields of application (Hublik 2012). It is produced by the Gram negative phytopathogen Xanthomonas campestris pv. campestris (Xcc). Presented in this work are three systematical, mutational approaches in order to optimize xanthan production and gain new insights in the Xcc metabolism.
In a first approach three mutant strains, carrying mutations in genes involved in the lipopolysaccharide (LPS) O-antigen biosynthesis, were used. The mutants Xcc H21012 (wxcB), Xcc H28110 (wxcK) and Xcc H20110 (wxcN) were constructed by Barbara Hötte and colleagues (1990) and in this thesis the phenotypic analyses are described. The mutant Xcc H21012 (wxcB) was characterized by the inhibition of the entire O-antigen. Moreover, the mutant phenotype was displayed through an increased xanthan production. In Xcc H28110 (wxcK) and Xcc H20110 (wxcN), no increase in xanthan production was detected. However, a novel LPS phenotype with an extended O-antigen main chain and no O-antigen branches could be detected.
For the second approach, in order to construct a Xcc strain towards enhanced xanthan production, two flagellar genes were mutated to inhibit the motility. First the structural gene fliC was mutated, then the basal flagellar gene fliM. Mutations were performed in laboratory strain Xcc B100 and in an industrial production strain, Xcc JBL007. Phenotypic analyses revealed the inhibition of motility. Furthermore, both mutational approaches resulted in enhanced xanthan producer strains. Remarkably, even the industrial production strain could be improved. Not only in production, but also in xanthan quality. The xanthan from Xcc JBL007 fliC – and Xcc JBL007 fliM – showed enhanced rheological properties. Both was shown under distinct industrial cultivation conditions. While the chemical composition did not change between xanthan by the initial or by a mutant strain, notable differences in persistence length could be measured via atomic force microscopy.
During a study with the purpose to identify sucrose related regulators, two novel regulators encoded by xcc-b100_2791 (crt1) and xcc-b100_2861 (crt2) were identified. Since they are putatively involved in Xcc carbohydrate regulation, mutants were constructed by Tobias Loka. As third approach of this thesis the mutant strains Xcc B100 crt1 – and Xcc B100 crt2 – were tested towards their cultivation characteristics and production abilities. Both mutants showed an increased xanthan yield, as compared to the initial strain Xcc B100. During the course of this work, both mutations were transferred into the production strain Xcc JBL007, after the corresponding genes in this strain were identified and sequence homologies were ensured. The xanthan production abilities of the mutant strains were then tested under industrial cultivation conditions. Both mutant strains exceeded the production of the initial strain Xcc JBL007. Results presented in this study demonstrate the possibility to further improve the xanthan production by Xcc, including the industrial xanthan production, through rational strain design.
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Steffens T. Engineering and characterization of Xanthomonas campestris pv. campestris towards an enhanced production of the exopolysaccharide xanthan. Bielefeld: Universität Bielefeld; 2017.
Steffens, T. (2017). Engineering and characterization of Xanthomonas campestris pv. campestris towards an enhanced production of the exopolysaccharide xanthan. Bielefeld: Universität Bielefeld.
Steffens, T. (2017). Engineering and characterization of Xanthomonas campestris pv. campestris towards an enhanced production of the exopolysaccharide xanthan. Bielefeld: Universität Bielefeld.
Steffens, T., 2017. Engineering and characterization of Xanthomonas campestris pv. campestris towards an enhanced production of the exopolysaccharide xanthan, Bielefeld: Universität Bielefeld.
T. Steffens, Engineering and characterization of Xanthomonas campestris pv. campestris towards an enhanced production of the exopolysaccharide xanthan, Bielefeld: Universität Bielefeld, 2017.
Steffens, T.: Engineering and characterization of Xanthomonas campestris pv. campestris towards an enhanced production of the exopolysaccharide xanthan. Universität Bielefeld, Bielefeld (2017).
Steffens, Tim. Engineering and characterization of Xanthomonas campestris pv. campestris towards an enhanced production of the exopolysaccharide xanthan. Bielefeld: Universität Bielefeld, 2017.
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