The use of cell factories to convert sugars from lignocellulosic biomass into chemicals in which oleochemicals and food additives, such as carotenoids, is essential for the shift toward sustainable processes. Rhodotorula toruloides is a yeast that naturally metabolises a wide range of substrates, including lignocellulosic hydrolysates, and converts them into lipids and carotenoids. In this study, xylose, the main component of hemicellulose, was used as the sole substrate for R. toruloides, and a detailed physiology characterisation combined with absolute proteomics and genome-scale metabolic models was carried out to understand the regulation of lipid and carotenoid production. To improve these productions, oxidative stress was induced by hydrogen peroxide and light irradiation and further enhanced by adaptive laboratory evolution. Based on the online measurements of growth and CO 2 excretion, three distinct growth phases were identified during batch cultivations. Majority of the intracellular flux estimations showed similar trends with the measured protein levels and demonstrated improved NADPH regeneration, phosphoketolase activity and reduced β-oxidation, correlating with increasing lipid yields. Light irradiation resulted in 70% higher carotenoid and 40% higher lipid content compared to the optimal growth conditions. The presence of hydrogen peroxide did not affect the carotenoid production but culminated in the highest lipid content of 0.65 g/g DCW. The adapted strain showed improved fitness and 2.3-fold higher carotenoid content than the parental strain. This work presents a holistic view of xylose conversion into microbial oil and carotenoids by R. toruloides, in a process toward renewable and cost-effective production of these molecules.
Microbes able to convert gaseous one-carbon (C1) waste feedstocks are of growing importance in transitioning to the biosustainable production of renewable chemicals and fuels. Acetogens are particularly interesting biocatalysts since gas fermentation usingClostridium autoethanogenumhas already been commercialised. Most non-commercial acetogen strains, however, need complex nutrients, display slow growth, and are not sufficiently robust for routine bioreactor fermentations. In this work, we used three adaptive laboratory evolution (ALE) strategies to evolve the wild-type model-acetogenC. autoethanogenumto grow faster, without complex nutrients and to be robust in operation of continuous bioreactor cultures. Seven evolved strains with improved phenotypes were isolated on minimal medium with one strain, named "LAbrini" (LT1), exhibiting superior performance in terms of the maximum specific growth rate, product profile, and robustness in continuous cultures. Differing performance of the strains between bottle batch and continuous cultures shows the importance of testing novel strains in industrially relevant continuous fermentation conditions. Interestingly, a very distinct transcriptome profile linked to a potential CO toxicity phenotype was observed in bioreactor cultures for one evolved strain. Whole-genome sequencing of the seven evolved strains identified 25 mutations with two genomic regions under stronger evolutionary pressure. Our analysis also suggests that the genotypic changes that are potentially responsible for the improved phenotypes may serve as useful candidates for metabolic engineering of cell factories. This work provides the robustC. autoethanogenumstrain LAbrini to the academic community to speed up phenotyping and genetic engineering, improve quantitative characterisation of acetogen metabolism, and facilitate the generation of high-quality steady-state datasets.
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