Genome-scale metabolic modeling reveals metabolic trade-offs associated with lipid production in Rhodotorula toruloides.

Rhodotorula toruloides is a non-conventional, oleaginous yeast able to naturally accumulate high amounts of microbial lipids. Constraint-based modeling of R. toruloides has been mainly focused on the comparison of experimentally measured and model predicted growth rates, while the intracellular flux patterns have been analyzed on a rather general level. Hence, the intrinsic metabolic properties of R. toruloides that make lipid synthesis possible are not thoroughly understood. At the same time, the lack of diverse physiological data sets has often been the bottleneck to predict accurate fluxes. In this study, we collected detailed physiology data sets of R. toruloides while growing on glucose, xylose, and acetate as the sole carbon source in chemically defined medium. Regardless of the carbon source, the growth was divided into two phases from which proteomic and lipidomic data were collected. Complemental physiological parameters were collected in these two phases and altogether implemented into metabolic models. Simulated intracellular flux patterns demonstrated the role of phosphoketolase in the generation of acetyl-CoA, one of the main precursors during lipid biosynthesis, while the role of ATP citrate lyase was not confirmed. Metabolic modeling on xylose as a carbon substrate was greatly improved by the detection of chirality of D-arabinitol, which together with D-ribulose were involved in an alternative xylose assimilation pathway. Further, flux patterns pointed to metabolic trade-offs associated with NADPH allocation between nitrogen assimilation and lipid biosynthetic pathways, which was linked to large-scale differences in protein and lipid content. This work includes the first extensive multi-condition analysis of R. toruloides using enzyme-constrained models and quantitative proteomics. Further, more precise kcat values should extend the application of the newly developed enzyme-constrained models that are publicly available for future studies.

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

Rhodotorula toruloides is a potential chassis for 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 (RtGGA) was adapted to the first basidiomycete, an oleaginous yeast R. toruloides. R. toruloides CCT 0783 was sequenced, and used for the GGA 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 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.

Induction of resistance mechanisms in Rhodotorula toruloides for growth in sugarcane hydrolysate with high inhibitor content.

The oleaginous yeast Rhodotorula toruloides is a potential lipid producer for biodiesel production. However, this yeast shows growth inhibition due to harmful compounds when cultivated in hemicellulose hydrolysate. Here, we present a comparative analysis of colony selection and heterologous adaptive laboratory enhancement (ALE) strategies for obtaining robust strains. We implemented these ALE strategies for R. toruloides in a culture medium containing sugarcane hemicellulose hydrolysate. Our comparison study showed that the strain obtained with heterogeneous ALE strategy (Rth) reached a µmax of 55% higher than the parental strain. It also exhibited higher biomass production (6.51 g/l) and lipid content (60%). ALE with colony selection strategy (Rtc) had a fitness gain in terms of shortening of the lag phase (9 h) when compared to Rth and parental strain (11.67, 12.33 h, respectively). When cultivated in Eucalyptus urograndis hemicellulose hydrolysate, the Rth strain achieved a high lipid content, 64%. Kinetics studies showed a strong effect of acetic acid as a repressor of xylose consumption during R. toruloides cultivation.Key points• Distinct adaptive laboratory strategies resulted in strains with different physiologies.• Heterologous adaptive laboratory enhancement provided the best results (fitness gain of 55% in µmax).• The Rth strain achieved a lipid content of 64.3% during cultivation in eucalyptus hemicellulose hydrolysate.

Screening and Growth Characterization of Non-conventional Yeasts in a Hemicellulosic Hydrolysate.

Lignocellulosic biomass is an attractive raw material for the sustainable production of chemicals and materials using microbial cell factories. Most of the existing bioprocesses focus on second-generation ethanol production using genetically modified Saccharomyces cerevisiae, however, this microorganism is naturally unable to consume xylose. Moreover, extensive metabolic engineering has to be carried out to achieve high production levels of industrially relevant building blocks. Hence, the use of non-Saccharomyces species, or non-conventional yeasts, bearing native metabolic routes, allows conversion of a wide range of substrates into different products, and higher tolerance to inhibitors improves the efficiency of biorefineries. In this study, nine non-conventional yeast strains were selected and screened on a diluted hemicellulosic hydrolysate from Birch. Kluyveromyces marxianus CBS 6556, Scheffersomyces stipitis CBS 5773, Lipomyces starkeyi DSM 70295, and Rhodotorula toruloides CCT 7815 were selected for further characterization, where their growth and substrate consumption patterns were analyzed under industrially relevant substrate concentrations and controlled environmental conditions in bioreactors. K. marxianus CBS 6556 performed poorly under higher hydrolysate concentrations, although this yeast was determined among the fastest-growing yeasts on diluted hydrolysate. S. stipitis CBS 5773 demonstrated a low growth and biomass production while consuming glucose, while during the xylose-phase, the specific growth and sugar co-consumption rates were among the highest of this study (0.17 h-1 and 0.37 g/gdw*h, respectively). L. starkeyi DSM 70295 and R. toruloides CCT 7815 were the fastest to consume the provided sugars at high hydrolysate conditions, finishing them within 54 and 30 h, respectively. R. toruloides CCT 7815 performed the best of all four studied strains and tested conditions, showing the highest specific growth (0.23 h-1), substrate co-consumption (0.73 ± 0.02 g/gdw*h), and xylose consumption (0.22 g/gdw*h) rates. Furthermore, R. toruloides CCT 7815 was able to produce 10.95 ± 1.37 gL-1 and 1.72 ± 0.04 mgL-1 of lipids and carotenoids, respectively, under non-optimized cultivation conditions. The study provides novel information on selecting suitable host strains for biorefinery processes, provides detailed information on substrate consumption patterns, and pinpoints to bottlenecks possible to address using metabolic engineering or adaptive evolution experiments.

Effect of Ink and Pretreatment Conditions on Bioethanol and Biomethane Yields from Waste Banknote Paper.

Waste banknote paper is a residue from the banking industry that cannot be recycled due to the presence of ink, microbial load and special coating that provides protection against humidity. As a result, waste banknote paper ends up being burned or buried, which brings environmental impacts, mainly caused by the presence of heavy metals in its composition. To minimize the environmental impacts that come from the disposal of waste banknote paper, this study proposes to produce value-added products (bioethanol and biogas) from waste banknote paper. For this, the effect of ink and pretreatment conditions on bioethanol and biomethane yields were analyzed. Waste banknote paper provided by the Central Bank of Iran was used. The raw material with ink (WPB) and without ink (WPD) was pretreated using sulfuric acid at different concentrations (1%, 2%, 3%, and 4%) and the nitrogen explosive decompression (NED) at different temperatures (150 °C, 170 °C, 190 °C, and 200 °C). The results show that the use of NED pretreatment in WPD resulted in the highest glucose concentration of all studies (13 ± 0.19 g/L). The acid pretreatment for WPB showed a correlation with the acid concentration. The highest ethanol concentration was obtained from the fermentation using WPD pretreated with NED (6.36 ± 0.72 g/L). The maximum methane yields varied between 136 ± 5 mol/kg TS (2% acid WPB) and 294 ± 4 mol/kg TS (3% acid WPD). Our results show that the presence of ink reduces bioethanol and biogas yields and that the chemical-free NED pretreatment is more advantageous for bioethanol and biogas production than the acid pretreatment method. Waste banknote paper without ink is a suitable feedstock for sustainable biorefinery processes.

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 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 CO2 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 ß-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/gDCW. 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.

C/N ratio and carbon source-dependent lipid production profiling in Rhodotorula toruloides.

Microbial oils are lipids produced by oleaginous microorganisms, which can be used as a potential feedstock for oleochemical production. The oleaginous yeast Rhodotorula toruloides can co-produce microbial oils and high-value compounds from low-cost substrates, such as xylose and acetic acid (from hemicellulosic hydrolysates) and raw glycerol (a byproduct of biodiesel production). One step towards economic viability is identifying the best conditions for lipid production, primarily the most suitable carbon-to-nitrogen ratio (C/N). Here, we aimed to identify the best conditions and cultivation mode for lipid production by R. toruloides using various low-cost substrates and a range of C/N ratios (60, 80, 100, and 120). Turbidostat mode was used to achieve a steady state at the maximal specific growth rate and to avoid continuously changing environmental conditions (i.e., C/N ratio) that inherently occur in batch mode. Regardless of the carbon source, higher C/N ratios increased lipid yields (up to 60% on xylose at a C/N of 120) but decreased the specific growth rate. Growth on glycerol resulted in the highest specific growth and lipid production (0.085 g lipids/gDW*h) rates at C/Ns between 60 and 100. We went on to study lipid production using glycerol in both batch and fed-batch modes, which resulted in lower specific lipid production rates compared with turbisdostat, however, fed batch is superior in terms of biomass production and lipid titers. By combining the data we obtained in these experiments with a genome-scale metabolic model of R. toruloides, we identified targets for improvements in lipid production that could be carried out either by metabolic engineering or process optimization.

Lipid production from hemicellulose with Lipomyces starkeyi in a pH regulated fed-batch cultivation.

This study investigated lipid production from the hemicellulosic fraction of birch wood by the oleaginous yeast Lipomyces starkeyi. Birch wood chips were thermochemically pretreated by hot water extraction, and the liquid phase, containing 45.1 g/l xylose as the major sugar, 13.1 g/l acetic acid and 4.7 g/l furfural, was used for cultivations of L. starkeyi CBS1807. The hydrolysate strongly inhibited yeast growth; the strain could only grow in medium containing 30% hydrolysate at pH 6. At pH 5, growth stopped already upon the addition of about 10% hydrolysate. In fed-batch cultures fed with hydrolysate or a model xylose-acetic acid mixture, co-consumption of xylose and acetic acid was observed, which resulted in a pH increase. This phenomenon was utilized to establish a pH-stat fed-batch cultivation in which, after an initial feeding, hydrolysate or model mixture was connected to the pH-regulation system of the bioreactor. Under these conditions we obtained growth and lipid production in cultures grown on either xylose or glucose during the batch phase. In cultivations fed with model mixture, a maximum lipid content of 60.5% of the cell dry weight (CDW) was obtained; however, not all xylose was consumed. When feeding hydrolysate, growth was promoted and carbon sources were completely consumed, resulting in higher CDW with maximum lipid content of 51.3%. In both cultures the lipid concentration was 8 g/l and a lipid yield of 0.1 g/g carbon source was obtained. Lipid composition was similar in all cultivations, with C18:1 and C16:0 being the most abundant fatty acids. Copyright © 2016 John Wiley & Sons, Ltd.

High concentrations of dried sorghum stalks as a biomass feedstock for single cell oil production by Rhodosporidium toruloides.

BACKGROUND: Environmental crisis and concerns for energy security have made the research for renewable fuels that will substitute the usage of fossil fuels an important priority. Biodiesel is a potential substitute for petroleum, but its feasibility is hindered by the utilization of edible vegetable oil as raw material, which is responsible for a large fraction of the production cost and fosters the food versus fuel competition. Microbial oils are an interesting alternative as they do not compete with food production, and low cost renewable materials could serve as raw materials during cultivation of microorganisms. Sweet sorghum is an excellent candidate as substrate for microbial oil production, as it possesses high photosynthetic activity yielding high amounts of soluble and insoluble carbohydrates, and does not require high fertilization and irrigation rates. RESULTS: Initially the ability of sweet sorghum to fully support yeast growth, both as a carbon and nitrogen source was evaluated. It was found that addition of an external nitrogen source had a negative impact on single cell oil (SCO) production yields, which has a positive effect on the process economics. Subsequently the effect of the presence of a distinct saccharification step on SCO was examined. The presence of an enzymatic saccharification step prior to SCO production improved the production of SCO, especially in high solid concentrations. Removal of solids was also investigated and its positive effect on SCO production was also demonstrated. When juice from 20% w/w enzymatically liquefied sweet sorghum was used as the raw material, SCO production was 13.77 g/L. To the best of our knowledge this is one of the highest SCO titers reported in the literature when renewable raw materials were utilized. CONCLUSIONS: The use of sweet sorghum at high solid concentrations as a feedstock for the efficient production of SCO by Rhodosporidium toruloides was demonstrated. Moreover, addition of enzymes not only led to liquefaction of sweet sorghum and permitted liquid fermentation, but also enhanced lipid production by 85.1% and 15.9% when dried stalks or stalk juice was used, respectively.

Sodium citrate and potassium phosphate as alternative adsorption buffers in hydrophobic and aromatic thiophilic chromatographic purification of plasmid DNA from neutralized lysate.

The number of studies on gene therapy using plasmid vectors (pDNA) has increased in recent years. As a result, the demand for preparations of pDNA in compliance with recommendations of regulatory agencies (EMEA, FDA) has also increased. Plasmid DNA is often obtained through fermentation of transformed Escherichia coli and purification by a series of unit operations, including chromatography. Hydrophobic interaction chromatography (HIC) and thiophilic aromatic chromatography (TAC), both using ammonium sulfate buffers, are commonly employed with success. This work was aimed at studying the feasibility of utilizing alternative salts in the purification of pDNA from neutralized lysate with phenyl-agarose (HIC) and mercaptopyrimidine-agarose (TAC) adsorbents. Their selectivity toward sc pDNA was evaluated through adsorption studies using 1.5 mol/L sodium citrate and 2.0 mol/L potassium phosphate as adsorption buffers. Chromatography with mercaptopyrimidine-agarose adsorbent and 1.5 mol/L sodium citrate was able to recover 91.1% of the pDNA with over 99.0% removal of gDNA and endotoxin. This represents a potential alternative for the primary recovery of sc pDNA. However, the most promising result was obtained using 2.0 mol/L potassium phosphate buffer and a mercaptopyrimidine-agarose column. In a single chromatographic step, this latter buffer/adsorbent system recovered 68.5% of the pDNA with 98.8% purity in accordance with the recommendations of regulatory agencies with regard to RNA and endotoxin impurity.