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.

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.

Study of the polyribosyl-ribitol-phosphate precipitation mechanism by salts and organic solvents.

Precipitation has been widely applied to purification and fractionation of biological macromolecules. Several physical-chemical factors contribute to the destabilization of those solutions, such as the nature of solvent employed, presence of salts, temperature, and concentration of the macromolecule. In the case of charged biopolymers, electrostatic forces are the major contributors to their stability in solution. However, the role of each variable and the exact mechanism of precipitation are not completely understood yet. The aim of this work was to study the precipitation of polyribosyl-ribitol-phosphate (PRP, a linear homogeneous anionic biopolymer) in presence of salts and non-solvents, in order to contribute to the elucidation of its precipitation mechanism. The solvents tested (acetone, ethanol, and isopropanol) presented distinct dielectric constants. The salts used (NH4Cl, NaCl, KCl, MgCl2, and CaCl2) differ by their cations. For each salt concentration, the solvent fraction that induces precipitation was identified and the dielectric constant of the bulk solution was calculated. Precipitation always occurred at well-defined combinations of solvents and salts. At low concentration of monovalent salts, there was a linear correlation between the logarithm of the salt concentration and the inverse of the medium dielectric constant at a defined precipitation point. This is a strong indication that the stability of the solution depends almost exclusively on the balance of electrostatic forces. This behavior is compatible with the DLVO modeling of colloidal systems. When divalent salts were used, low concentrations of the counterion were sufficient to induce precipitation, due to a phenomenon called ionic condensation. Apparently, PRP precipitates when around 90% of its charges are neutralized, value that is similar to charge neutralization for DNA precipitation.

Study of the polyribosyl-ribitol-phosphate precipitation mechanism by salts and organic solvents

Precipitation has been widely applied to purification and fractionation of biological macromolecules. Several physical-chemical factors contribute to the destabilization of those solutions, such as the nature of solvent employed, presence of salts, temperature, and concentration of the macromolecule. In the case of charged biopolymers, electrostatic forces are the major contributors to their stability in solution. However, the role of each variable and the exact mechanism of precipitation are not completely understood yet. The aim of this work was to study the precipitation of polyribosyl-ribitol-phosphate (PRP, a linear homogeneous anionic biopolymer) in presence of salts and non-solvents, in order to contribute to the elucidation of its precipitation mechanism. The solvents tested (acetone, ethanol, and isopropanol) presented distinct dielectric constants. The salts used (NH4Cl, NaCl, KCl, MgCl2, and CaCl2) differ by their cations. For each salt concentration, the solvent fraction that induces precipitation was identified and the dielectric constant of the bulk solution was calculated. Precipitation always occurred at well-defined combinations of solvents and salts. At low concentration of monovalent salts, there was a linear correlation between the logarithm of the salt concentration and the inverse of the medium dielectric constant at a defined precipitation point. This is a strong indication that the stability of the solution depends almost exclusively on the balance of electrostatic forces. This behavior is compatible with the DLVO modeling of colloidal systems. When divalent salts were used, low concentrations of the counterion were sufficient to induce precipitation, due to a phenomenon called ionic condensation. Apparently, PRP precipitates when around 90% of its charges are neutralized, value that is similar to charge neutralization for DNA precipitation.

Study of the polyribosyl-ribitol-phosphate precipitation mechanism by salts and organic solvents

Precipitation has been widely applied to purification and fractionation of biological macromolecules. Several physical-chemical factors contribute to the destabilization of those solutions, such as the nature of solvent employed, presence of salts, temperature, and concentration of the macromolecule. In the case of charged biopolymers, electrostatic forces are the major contributors to their stability in solution. However, the role of each variable and the exact mechanism of precipitation are not completely understood yet. The aim of this work was to study the precipitation of polyribosyl-ribitol-phosphate (PRP, a linear homogeneous anionic biopolymer) in presence of salts and non-solvents, in order to contribute to the elucidation of its precipitation mechanism. The solvents tested (acetone, ethanol, and isopropanol) presented distinct dielectric constants. The salts used (NH4Cl, NaCl, KCl, MgCl2, and CaCl2) differ by their cations. For each salt concentration, the solvent fraction that induces precipitation was identified and the dielectric constant of the bulk solution was calculated. Precipitation always occurred at well-defined combinations of solvents and salts. At low concentration of monovalent salts, there was a linear correlation between the logarithm of the salt concentration and the inverse of the medium dielectric constant at a defined precipitation point. This is a strong indication that the stability of the solution depends almost exclusively on the balance of electrostatic forces. This behavior is compatible with the DLVO modeling of colloidal systems. When divalent salts were used, low concentrations of the counterion were sufficient to induce precipitation, due to a phenomenon called ionic condensation. Apparently, PRP precipitates when around 90% of its charges are neutralized, value that is similar to charge neutralization for DNA precipitation.

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.

Modeling of protein and phenolic compound removal from aqueous solutions by electrocoagulation.

Electrocoagulation is a technique basically applied in water and wastewater treatment, but which has a number of potential applications in polymer, protein, drug, and vaccine delivery. In this work, we correlate the current applied between the electrodes to the removal of phenolic compounds or protein from aqueous solutions, but the principle can also be applied to other biological compounds such as plant pigments and sugars. Simple and time-dependent models were developed based on the complex formation between these biological substances and the aluminium hydroxide gel phase. The models developed represent a good agreement with experimental data (R(2) as high as 0.992). Besides construction of the models, the effect of pH on the efficiency of removal of proteins and phenolic compounds was evaluated. It was found that this parameter has significant effect on the efficiency of the electrocoagulation and the maximal removal efficiency for bovine serum albumin and phenolic compound catechin was observed at pH 8.0.

Precipitation of porcine insulin with carbon dioxide.

Recent works have pointed to the use of volatile electrolytes such as carbon dioxide (CO2) dissolved in aqueous solutions as a promising alternative to the precipitating agents conventionally used for protein recovery in the food and pharmaceutical industries. In this work we investigated experimental and theoretical aspects of the precipitation of porcine insulin, a biomolecule of pharmaceutical interest, using CO2 as an acid-precipitating agent. The solubility of porcine insulin in NaHCO3 solutions in pressurized CO2 was determined as a function of temperature and pressure, with a minimum being observed close to the protein isoelectric point. A thermodynamic model was developed and successfully utilized to correlate the experimental data. Insulin was considered a polyelectrolyte in the model and its self-association reactions were also taken into account. The biological activity of insulin was maintained after precipitation with CO2, although some activity can be lost if foam is formed in the depressurization step.

Evaluation of IDA-PEVA hollow fiber membrane metal ion affinity chromatography for purification of a histidine-tagged human proinsulin.

Inabilities to process particulate material and to allow the use of high flow rates are limitations of conventional chromatography. Membranes have been suggested as matrix for affinity separation due to advantages such as allowing high flow rates and low-pressure drops. This work evaluated the feasibility of using an iminodiacetic acid linked poly(ethylenevinyl alcohol) membrane in the immobilized metal ion affinity chromatography (IMAC) purification of a human proinsulin(His)(6) of an industrial insulin production process. The screening of metal ions showed Ni(2+) as metal with higher selectivity and capacity among the Cu(2+), Ni(2+), Zn(2+) and Co(2+). The membrane showed to be equivalent to conventional chelating beads in terms of selectivity and had a lower capacity (3.68 mg/g versus 12.26 mg/g). The dynamic adsorption capacity for human proinsulin(His)(6) was unaffected by the mode of operation (dead-end and cross-flow filtration).

Evaluation of immobilized metal membrane affinity chromatography for purification of an immunoglobulin G1 monoclonal antibody.

The large scale production of monoclonal antibodies (McAbs) has gaining increased relevance with the development of the hybridoma cell culture in bioreactors creating a need for specific efficient bioseparation techniques. Conventional fixed bead affinity adsorption commonly applied for McAbs purification has the drawback of low flow rates and colmatage. We developed and evaluated a immobilized metal affinity chromatographies (IMAC) affinity membrane for the purification of anti-TNP IgG(1) mouse McAbs. We immobilized metal ions on a poly(ethylene vinyl alcohol) hollow fiber membrane (Me(2+)-IDA-PEVA) and applied it for the purification of this McAbs from cell culture supernatant after precipitation with 50% saturation of ammonium sulphate. The purity of IgG(1) in the eluate fractions was high when eluted from Zn(2+) complex. The anti-TNP antibody could be eluted under conditions causing no loss of antigen binding capacity. The purification procedure can be considered as an alternative to the biospecific adsorbent commonly applied for mouse IgG(1) purification, the protein G-Sepharose.

Gas-phase enzymatic esterification on immobilized lipases in MCM-41 molecular sieves.

Several lipolytic enzymes were immobilized in the pores of MCM-41 and Al-MCM-41 molecular sieves and used as catalysts in the gas-phase esterification of acetic acid with ethanol. The entrapment of enzymes depended on the molecular sieve and the type of enzyme used. The order of enzymatic activity for enzymes entrapped in the pores of MCM-41 and Al-MCM-41 in the esterification reaction was OF (Rhizopus niveus lipases) < FAP-15 (Rhizopus oryzae lipases) < LEX (Mucorjavanicus lipases) < PS (Pseudomonas cepacia lipases) < AK (Pseudomonas fluorescens lipases). Experiments carried out between 298 and 318 K showed no effect of temperature on catalyst yield, suggesting that the enzymes were appropriately immobilized in the pores of the molecular sieves, thus avoiding possible processes such as denaturing or autolysis.