Biology subjects

Pinheiro, M. J.

Publications and source records attributed to Pinheiro, M. J..

4 recordsLinked to original sources

bioRxiv2023

Improving xylose consumption in Rhodotorula toruloides through heterologous expression of xylose reductase and xylulokinase

The oleaginous yeast Rhodotorula toruloides is a promising host for sustainable bioproduction due to its capacity to naturally utilize xylose present in lignocellulosic biomass, an abundant and renewable resource. However, its xylose consumption pathway is still not completely understood. To better understand the potential limitations in xylose utilization in R. toruloides, heterologous xylose reductase from Scheffersomyces stipitis, together with the native and heterologous xylulokinases from three different microorganisms (Scheffersomyces stipitis, Candida intermedia, and Escherichia coli) were overexpressed solely and in combination. The overexpression of xylulokinases showed more significant improvements in terms of xylose consumption rate compared to the single overexpression of xylose reductase. When the heterologous xylulokinase from Escherichia coli was overexpressed, the specific xylose consumption rate was improved by 66% and the maximum specific growth rate by 30% compared to the parental strain. The xylose specific consumption rate increased by 146% and the maximum specific growth rate increased by 118% when heterologous genes for xylose reductase and xylulokinase from E. coli were overexpressed together. These results suggest that the low expression of xylulokinase in R. toruloides, which has been reported previously, could limit its sugar consumption, while supporting higher lipid accumulation in this yeast.

synthetic biology↗

bioRxiv2023

Autotrophic adaptive laboratory evolution of the acetogen Clostridium autoethanogenum delivers the gas-fermenting strain LAbrini with superior growth, products, and robustness

Microbes able to convert gaseous one-carbon (C1) waste feedstocks are increasingly important to transition to the sustainable production of renewable chemicals and fuels. Acetogens are interesting biocatalysts since gas fermentation using Clostridium autoethanogenum has already been commercialised. However, most acetogen strains need complex nutrients, display slow growth, and are not robust for routine bioreactor fermentations. In this work, we used three different and independent adaptive laboratory evolution (ALE) strategies to evolve the wild-type C. autoethanogenum to grow faster, without yeast extract and to be robust in operating continuous bioreactor cultures. Multiple evolved strains with improved phenotypes were isolated on a minimal medium with one strain, named "LAbrini" (LT1), exhibiting superior performance regarding the maximum specific growth rate, product profile, and robustness in continuous cultures. Whole-genome sequencing of the evolved strains identified 25 mutations. Of particular interest are two genes that acquired seven different mutations across the three ALE strategies, potentially as a result of convergent evolution. Reverse genetic engineering of sporulation-related mutations in genes CLAU_3129 (spo0A) and CLAU_1957 recovered all three superior features of our ALE strains through triggering significant proteomic rearrangements. This work provides a robust C. autoethanogenum strain to accelerate phenotyping and genetic engineering and to better understand acetogen metabolism, which we named "LAbrini".

bioengineering↗

bioRxiv2022

Development of a dedicated Golden Gate Assembly platform (RtGGA) for Rhodotorula toruloides

Rhodotorula toruloides is a potential chassis to microbial cell factories as this yeast can metabolise different substrates into a diverse range of natural products, but the lack of efficient synthetic biology tools hinders its applicability. In this study, the modular, versatile and efficient Golden Gate DNA assembly system was adapted to the first basidiomycete, an oleaginous yeast R. toruloides (RtGGA). R. toruloides CCT 0783 was sequenced, and used for the RtGGA design. The DNA fragments were assembled with predesigned 4-nt overhangs and a library of standardized parts was created containing promoters, genes, terminators, insertional regions, and resistance genes. The library was combined to create cassettes for the characterization of promoters strength and to overexpress the carotenoid production pathway. A variety of reagents, plasmids, and strategies were used and the RtGGA proved to be robust. The RtGGA was used to build three versions of the carotenoid overexpression cassette were built by using different promoter combinations. The cassettes were transformed into R. toruloides and the three new strains were characterized. Total carotenoid concentration increased by 41%. The dedicated GGA platform fills a gap in the advanced genome engineering toolkit for R. toruloides, enabling the efficient design of complex metabolic pathways.

synthetic biology↗

bioRxiv2020

Xylose Metabolism and the Effect of Oxidative Stress on Lipid and Carotenoid Production in Rhodotorula toruloides: Insights for Future Biorefinery

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 towards 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 CO2 excretion, three distinct growth phases were identified during batch cultivations. The intracellular flux estimations correlated well with the measured protein levels and demonstrated improved NADPH regeneration, phosphoketolase activity and reduced beta-oxidation, correlating with increasing lipid yields. Light irradiation resulted in 70% higher carotenoid and 40% higher lipid yields. The presence of hydrogen peroxide did not affect the carotenoid yield but culminated in the highest lipid yield of 0.65 g/gDCW. The adapted strain showed improved fitness and 2.3-fold higher carotenoid yield than the parental strain. This work presented a holistic view of xylose conversion into microbial oil and carotenoids by R. toruloides for further cost-effective and renewable production of these molecules.

systems biology↗