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TY - THES AU - Wefelmeier, Katrin TI - Exploring the methylotrophic yeast Ogataea polymorpha as a production platform for valuable chemicals from C1 compounds; 1. Auflage VL - 35 PB - RWTH Aachen University VL - Dissertation CY - Aachen M1 - RWTH-2024-07371 SN - 398555224X T2 - Applied microbiology SP - 1 Online-Ressource : Illustrationen PY - 2024 N1 - Druckausgabe: 2024. - Auch veröffentlicht auf dem Publikationsserver der RWTH Aachen University N1 - Dissertation, RWTH Aachen University, 2024 AB - The question of how we can build a sustainable society that respects planetary boundaries and provides a livelihood for future generations is one of the most pressing challenges facing humanity. It is clear that the linear produce-use-waste society, which is largely dependent on fossil resources, cannot persist. Therefore, alternative solutions for a more sustainable production of industrially relevant products are urgently needed. A circular bioeconomy that relies on microorganisms to produce industrially relevant products has been proposed as a promising way forward. However, the establishment of a bioeconomy is only desirable if it is based on raw materials that do not increase the pressure on ecosystems or compete with the food industry. One carbon (C1) molecules are particularly promising microbial substrates, as they can be derived from CO<sub>2</sub> and green hydrogen and therefore have the potential to be sustainable feedstocks that do not require the use of additional arable land. Among the C1 molecules, methanol and formate represent an attractive substrate group, as they are miscible with water and can therefore be easily supplemented in conventional fermentation processes. This work investigates the potential of the native methylotrophic yeast Ogataea polymorpha as a chassis organism for the conversion of methanol and formate into industrially relevant products. Since O. polymorpha is a non-conventional yeast, the diversity of genetic tools that is available for standard model microorganisms does not yet exist. Therefore, this work extended the genetic toolbox for O. polymorpha by a set of well-characterized promoters and terminators that can be used for methanol-based cultivation approaches. These genetic elements were used to engineer the yeast to produce a range of different products from methanol. Lactate was selected as the first potential product to assess the feasibility of producing low molecular weight biochemicals from methanol. Through strain engineering and adaptive laboratory evolution, an O. polymorpha strain was obtained that produced lactate at g/L scale and showed an increased growth rate on methanol. Methanol toxicity was identified to be a major obstacle to the cultivation of O. polymorpha at laboratory scale. As a result, methanol feeding strategies for strains engineered to produce malate from methanol were further refined through the use of an automated methanol feeding system for shake flasks. Further, the malate export across the plasma membrane was identified as a key variable impacting malate production from methanol in O. polymorpha. To investigate the variety of chemicals that may be produced by O. polymorpha using methanol as a carbon source, the yeast was further modified to produce acetone and isoprene as additional proof of concept molecules. Lastly, the use of formate as a novel C1 substrate for O. polymorpha was explored. The native C1 metabolism of O. polymorpha was characterized in more detail and formate-assimilating enzymes were introduced into the peroxisomes of the yeast. Although growth with formate as the sole carbon source could not be demonstrated, it was shown that the supply of low concentrations of formate as a co-substrate can have a positive effect on biomass formation, likely due to the additional electrons that the oxidation of formate to CO<sub>2</sub> can provide for cellular metabolism. These findings therefore provide a valuable knowledge base for the future development of methylotrophic yeast strains that could contribute to a circular bioeconomy based on C1 molecules. LB - PUB:(DE-HGF)11 ; PUB:(DE-HGF)3 DO - DOI:10.18154/RWTH-2024-07371 UR - https://publications.rwth-aachen.de/record/990508 ER -