Biology subjects

Lahtvee, P.-J.

Publications and source records attributed to Lahtvee, P.-J..

11 recordsLinked to original sources

bioRxiv2026

Engineering Stable Hydrogels with Polydisperse Yeast Exopolysaccharides for Embedding Cancer Spheroids

Polysaccharides are often used to mimic physiological environments such as for cancer research models. However, established polysaccharides can display limited long-term stability and high batch-to-batch variability. To overcome this, biomanufactured polysaccharides are increasingly utilized in biomaterials. Here, we produced and characterized Rhodotorula toruloides yeast exopolysaccharides (EPS) and used it to engineer hydrogel for culturing cancer cells. Yeast fermentation of glucose, mannose, and xylose yielded varying EPS amounts (1.68, 1.44, and 0.48 g/L, respectively) with similar compositions, suggesting a common biosynthetic pathway. The glucose-derived EPS characterization identified multiple linkage types and three molecular weight fractions (1.75, 30.0, and 1000 kDa), and its solutions exhibited Newtonian behavior, indicating minimal chain-chain interactions. Solubilizing this polydisperse EPS with polyethylene glycol diacrylate and UV-crosslinking it enabled the engineering of semi-interpenetrating polymer network hydrogel that efficiently embedded cancer spheroids. Our study introduces an integrated biomanufacturing strategy to generate stable and consistent biomaterials, applicable for tissue engineering. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/703759v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@110d079org.highwire.dtl.DTLVardef@e6e390org.highwire.dtl.DTLVardef@662540org.highwire.dtl.DTLVardef@17afd5_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Acidification by nitrogen metabolism triggers extracellular biopolymer production in an oleaginous yeast

The oleaginous yeast Rhodotorula toruloides is a natural producer of lipids and carotenoids. However, its potential as a producer of extracellular biopolymers remains unexplored. Hence, we aimed to evaluate the R. toruloides CCT0783 for extracellular biopolymer production. We found that the carbon-to-nitrogen ratio influenced exopolysaccharide (EPS) production, reaching 5.84 {+/-} 0.45 g/L under glucose-rich conditions. We characterized the crude biopolymer using FTIR and GC-MS, identifying polysaccharide peaks, wherein EPS consisted of (%) glucose (88.83 {+/-} 4.87), galactose (5.50 {+/-} 1.50), mannose (4.80 {+/-} 1.57), and xylose and arabinose (0.87 {+/-} 0.04) monomers. The dried EPS also contained a fractional presence of protein (1.0%). Interestingly, inorganic, but not organic, nitrogen metabolism was associated with the acidification of the culture environment and simultaneous EPS production. A comparison between non-buffered and buffered media indicated that EPS was produced when the final pH was around or below 2, which was observed in the former but not in the latter. Finally, a comparative bioinformatics analysis allowed us to map a putative EPS biosynthesis and transport pathways, as well as regulators of intracellular pH maintenance. In conclusion, our study demonstrates R. toruloides potential as an extracellular microbial biopolymer producer, enabling its development as a cell factory. ImportanceMicrobial biopolymers are extensively investigated for their impact on the environment and health. However, developing a biotechnology process for producing such biopolymers remains challenging despite their potential for valuable applications. Considering this opportunity, we investigated the oleaginous yeast Rhodotorula toruloides as a producer of extracellular biopolymers. Our study describes the conditions of exopolysaccharide (EPS) biosynthesis in R. toruloides. This extracellular biopolymer could have a wide range of applications, from gelling agents in pharmaceuticals to emulsifiers in the food industry. Furthermore, our comparative bioinformatics analysis results could be useful for metabolic engineering and developing Rhodotorula cell factories.

microbiology↗

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↗

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↗

bioRxiv2022

A conundrum of r-protein stability: unbalanced stoichiometry of r-proteins during stationary phase in Escherichia coli

Bacterial ribosomes are composed of three ribosomal RNA (rRNA) and over 50 ribosomal protein (r-protein) molecules. R-proteins are essential for ribosome assembly, structural stability, and also participate in almost all ribosome functions. Ribosomal components are present in stoichiometric amounts in the mature 70S ribosomes during the exponential and early stationary growth phases. Ribosomes are degraded in stationary phase, however, the stability and fate of r-proteins during stationary growth phase are not known. In this study, we report a quantitative analysis of ribosomal components during the extended stationary phase in E.coli. We show that: (a) the quantity of ribosomes per cell mass decreases in the stationary phase. (b) 70S ribosomes contain r-proteins in stoichiometric amounts. (c) 30S subunits are degraded faster than 50S. (d) In total proteome, the quantity of 21 r-proteins decreases during 14 days (short-lived r-proteins) concomitantly with the reduction of cellular RNA. (e) 30 r-proteins are stable and form a pool of free r-proteins (stable r-proteins). Thus r-proteins are present in non-stochiometric amounts in the proteome of E. coli during the extended stationary phase. IMPORTANCERibosome degradation has been extensively described from the viewpoint of its main component - rRNA. Here we aim to complement our knowledge by quantitatively analyzing r-protein degradation and stability in the ribosomes as well as in the whole-cell proteome during stationary phase in E. coli. R-proteins are considered to be very stable in the proteome. Here we show that a specific set of r-proteins are rapidly degraded after release from the rRNA. The degradation of r-proteins is an intriguing new aspect of r-protein metabolism in bacteria.

molecular biology↗

bioRxiv2021

Metabolism Control in 3D Printed Living Materials

The three-dimensional printing of cells offers an attractive opportunity to design and develop innovative biotechnological applications, such as the fabrication of biosensors or modular bioreactors. Living materials (LMs) are cross-linked polymeric hydrogel matrices containing cells, and recently, one of the most deployed LMs consists of F127-bis-urethane methacrylate (F127-BUM). The material properties of F127-BUM allow reproducible 3D printing and stability of LMs in physiological environments. These materials are permissible for small molecules like glucose and ethanol. However, no information is available for oxygen, which is essential-- for example, towards the development of aerobic bioprocesses using microbial cell factories. To address this challenge, we investigated the role of oxygen as a terminal electron acceptor in the budding yeasts respiratory chain and determined its permissibility in LMs. We quantified the ability of cell-retaining LMs to utilize oxygen and compared it with cells in suspension culture. We found that the cells ability to consume oxygen was heavily impaired inside LMs, indicating that the metabolism mostly relied on fermentation instead of respiration. To demonstrate an application of these 3D printed LMs, we evaluated a comparative brewing process. The analysis showed a significantly higher (3.7%) ethanol production using 3D printed LMs than traditional brewing, indicating an efficient control of the metabolism. Towards molecular and systems biology studies using LMs, we developed a highly reliable method to isolate cells from LMs for flow cytometry and further purified macromolecules (proteins, RNA, and DNA). Our results show the application of F127-BUM-based LMs for microaerobic processes and envision the development of diverse bioprocesses using versatile LMs in the future.

bioengineering↗

bioRxiv2020

Characterization of potassium, sodium and their interactions effects in yeasts

Biotechnology requires efficient microbial cell factories. The budding yeast Saccharomyces cerevisiae is an important cell factory but for a sustainable use of natural resources more diverse cellular attributes are essential. Here, we benchmarked non-conventional yeasts Kluyveromyces marxianus (KM) and Rhodotorula toruloides (RT) against the extensively characterized strains of S. cerevisiae, CEN.PK and W303. We developed a computational method for the characterization of cell/vacuole volumes and observed an inverse relationship between the maximal growth rate and the median cell volume that was responsive to monovalent cations. We found that the supplementation of certain K+ concentrations to CEN.PK cultures containing 1.0 M Na+ increased the specific growth rate by four-fold with a parabolic shift in the median cell/vacuole volumes. The impairment of ethanol and acetate utilization in CEN.PK, acetate in W303, at the higher K+/Na+ concentrations implied an interference in the metabolic pathways required for their consumption. In RT cultures, the supplementation of K+/Na+ induced a trade-off in glucose utilization but alleviated cellular aggregates formation where specified cationic concentrations increased the beta-carotene yield by 60% compared with the reference. Our comparative analysis of cell/vacuole volumes using exponential phase cultures showed that the median volumes decreased the most for KM and the least for RT in response to studied cations. Noteworthy for the implication in aging research using yeasts, the vacuole to cell volume ratio increased with the increase in cell volume for W303 and KM, but not for CEN.PK and RT. ImportanceFor designing efficient bioprocesses characterization of microbial cell factories in the relevant culture environment is important. The control of cell volume in response to salt stress is crucial for the productivity of microbial cell factories. We developed an open source computational method for the analysis of optical microscopy images that allowed us to quantify changes in cell/vacuole volumes in response to common salts in yeasts. Our study provides a framework for appreciating the role of cellular/organellar volumes in response to changing physiological environment. Our analysis showed that K+/Na+ interactions could be used for improving the cellular fitness of CEN.PK and increasing the productivity of beta-carotene in R. toruloides, which is a commercially important antioxidant and a valuable additive in foods.

bioengineering↗

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↗

bioRxiv2020

Physical confinement impacts cellular phenotype within living materials

Additive manufacturing allows three-dimensional printing of polymeric materials together with cells, creating living materials for applications in biomedical research and biotechnology. However, understanding the cellular phenotype within living materials is lacking and a key limitation for their wider application. Herein, we present an approach to characterize the cellular phenotype within living materials. We immobilized the budding yeast Saccharomyces cerevisiae in three different photocross-linkable triblock polymeric hydrogels containing F127-bis-urethane methacrylate, F127-dimethacrylate, or poly(alkyl glycidyl ether)-dimethacrylate. Using optical and scanning electron microscopy, we showed that hydrogels based on these polymers were stable under physiological conditions, but yeast colonies showed differences in the interaction within the living materials. We found that the physical confinement, imparted by compositional and structural properties of the hydrogels, impacted the cellular phenotype by reducing the size of cells in living materials compared with suspension cells. These properties also contributed to the differences in immobilization patterns, growth of colonies, and colony coatings. We observed that a composition-dependent degradation of polymers was likely possible by cells residing in the living materials. In conclusion, our investigation highlights the need for a holistic understanding of the cellular response within hydrogels to facilitate the synthesis of application-specific polymers and the design of advanced living materials in the future.

bioengineering↗

bioRxiv2020

Benchmarking accuracy and precision of intensity-based absolute quantification of protein abundances in Saccharomyces cerevisiae

Protein quantification via label-free mass spectrometry (MS) has become an increasingly popular method for determining genome-wide absolute protein abundances. A known caveat of this approach is the poor technical reproducibility, i.e. how consistent the estimations are when the same sample is measured repeatedly. Here, we measured proteomics data for Saccharomyces cerevisiae with both biological and inter-batch technical triplicates, to analyze both accuracy and precision of protein quantification via MS. Moreover, we analyzed how these metrics vary when applying different methods for converting MS intensities to absolute protein abundances. We found that a simple normalization and rescaling approach performs as accurately yet more precisely than methods that rely on external standards. Additionally, we show that inter-batch reproducibility is worse than biological reproducibility for all evaluated methods. These results subsequently serve as a benchmark for assessing MS data quality for protein quantification, whilst also underscoring current limitations in this approach.

bioinformatics↗

bioRxiv2020

Proteome overabundance enables respiration but limitation onsets carbon overflow

Central carbon metabolism produces energy and precursor metabolites for biomass in heterotrophs. Carbon overflow yields metabolic byproducts and, here, we examined its dependency on nutrient and growth using the unicellular eukaryotic model organism Saccharomyces cerevisiae. We performed quantitative proteomics analysis together with metabolic modeling and found that proteome overabundance enabled respiration, and variation in energy efficiency caused distinct composition of biomass at different carbon to nitrogen ratio and growth rate. Our results showed that ceullar resource allocation for ribosomes was determinative of growth rate, but energy constrains on protein synthesis incepted carbon overflow by prioritizing abundance of ribosomes and glycolysis over mitochondria. We proved that glycolytic efficiency affected energy metabolism by making a trade-off between low and high energy production pathways. Finally, we summarized cellular energy budget underlying nutrient-responsive and growth rate-dependent carbon overflow, and suggested implications of results for bioprocesses and pathways relevant in cancer metabolism in humans.

systems biology↗