PUB - Publikationen an der Universität Bielefeld

In times of scarcity of fossil resources and climate change, the need of renewable resources for the biobased economy becomes increasingly important. Within this thesis a contribution was done to achieve the aim to produce chemicals biobased and not anymore only petrochemical based. To accomplish this, the industrial relevant microorganism Corynebacterium glutamicum was engineered to utilize the hemicellulose derived pentose sugar D-xylose via an alternative pathway. This utilization was made feasible via the Weimberg pathway without loss of carbon in terms of products derived from alpha-ketoglutarate. During the first investigation two major by-products, D-xylonate and xylitol, of the new synthetic pathway were found. The latter is known as inhibitor of different microorganisms, which could be also confirmed in this thesis for C. glutamicum. The inhibition studies showed different target genes, which are partly responsible for this inhibitory effect (e.g. xylB). Interestingly, the reference strain in this thesis, a strain expressing the isomerase pathway (including the xylB gene), was already inhibited by low amounts of extracellular xylitol and accumulates the highest amount of intracellular xylitol-5-phosphate.
Within this thesis a high amount of engineered strains had to be screened and charaterized. Therefore, it was the initial aim to develop new robotic workflows and automated enzymatic assays on a robotic platform. The extended Mini Pilot Plant enables now the high-throughput microbial phenotyping with connected analytics in 384-well format. Further, it is now possible to run a complete process with separation of cell-free supernatants for analytics under sterile conditions without human interruption. Another new robotic workflow which was designed within this thesis is the miniaturized and automated repetitive batch cultivation. The developed workflow was used for fully automated adaptive laboratory evolution (ALE) of the engineered C. glutamicum strain expressing theWeimberg pathway for D-xylose utilization. The ALE process was successful and the final strain had an 260% increased maximal growth rate of max = 0.26 h-1on D-xylose as sole carbon source. The final D-xylose utilizing strain (WMB2evo) grows stable during lab-scale bioreactor operation, which demonstrates the high potential of this strain for future projects with biorefinery applications. Additionally, the occurred mutations during the ALE process were analyzed by genome sequencing and revealed about 15 potential key mutations for improved D-xylose assimilation. These found mutations can be used for rational strain engineering within further biorefinery projects. But for now the final D-xylose utilizing strain (WMB2evo) is the fastest growing C. glutamicum strain on D-xylose as sole carbon source in literature.