Search

Abstract Background Metabolic coupling of product synthesis and microbial growth is a prominent approach for maximizing production performance. Growth-coupling (GC) also helps stabilizing target production and allows the selection of superior production strains by adaptive laboratory evolution. To support the implementation of growth-coupling strain designs, we seek to identify biologically relevant, metabolic principles that enforce strong growth-coupling on the basis of reaction knockouts. Results We adapted an established bilevel programming framework to maximize the minimally guaranteed production rate at a fixed, medium growth rate. Using this revised formulation, we identified various GC intervention strategies for metabolites of the central carbon metabolism, which were examined for GC generating principles under diverse conditions. Curtailing the metabolism to render product formation an essential carbon drain was identified as one major strategy generating strong coupling of metabolic activity and target synthesis. Impeding the balancing of cofactors and protons in the absence of target production was the underlying principle of all other strategies and further increased the GC strength of the aforementioned strategies. Conclusion Maximizing the minimally guaranteed production rate at a medium growth rate is an attractive principle for the identification of strain designs that couple growth to target metabolite production. Moreover, it allows for controlling the inevitable compromise between growth coupling strength and the retaining of microbial viability. With regard to the corresponding metabolic principles, generating a dependency between the supply of global metabolic cofactors and product synthesis appears to be advantageous in enforcing strong GC for any metabolite. Deriving such strategies manually, is a hard task, due to which we suggest incorporating computational metabolic network analyses in metabolic engineering projects seeking to determine GC strain designs.

Abstract Background Thermotolerance is a highly desirable trait of microbial cell factories and has been the focus of extensive research. Yeast usually tolerate only a narrow temperature range and just two species, Kluyveromyces marxianus and Ogataea polymorpha have been described to grow at reasonable rates above 40 °C. However, the complex mechanisms of thermotolerance in yeast impede its full comprehension and the rare physiological data at elevated temperatures has so far not been matched with corresponding metabolic analyses. Results To elaborate on the metabolic network response to increased fermentation temperatures of up to 49 °C, comprehensive physiological datasets of several Kluyveromyces and Ogataea strains were generated and used for 13C-metabolic flux analyses. While the maximum growth temperature was very similar in all investigated strains, the metabolic network response to elevated temperatures was not conserved among the different species. In fact, metabolic flux distributions were remarkably irresponsive to increasing temperatures in O. polymorpha, while the K. marxianus strains exhibited extensive flux rerouting at elevated temperatures. Conclusions While a clear mechanism of thermotolerance is not deducible from the fluxome level alone, the generated data can be valued as a knowledge repository for using temperature to modulate the metabolic activity towards engineering goals.

Abstract Background Ogataea polymorpha is a thermotolerant, methylotrophic yeast with significant industrial applications. While previously mainly used for protein synthesis, it also holds promise for producing platform chemicals. O. polymorpha has the distinct advantage of using methanol as a substrate, which could be potentially derived from carbon capture and utilization streams. Full development of the organism into a production strain and estimation of the metabolic capabilities require additional strain design, guided by metabolic modeling with a genome-scale metabolic model. However, to date, no genome-scale metabolic model is available for O. polymorpha. Results To overcome this limitation, we used a published reconstruction of the closely related yeast Komagataella phaffii as a reference and corrected reactions based on KEGG and MGOB annotation. Additionally, we conducted phenotype microarray experiments to test the suitability of 190 substrates as carbon sources. Over three-quarter of the substrate use was correctly reproduced by the model and 27 new substrates were added, that were not present in the K. phaffii reference model. Conclusion The developed genome-scale metabolic model of O. polymorpha will support the engineering of synthetic metabolic capabilities and enable the optimization of production processes, thereby supporting a sustainable future methanol economy.