Rhodotorula Toruloides Research Articles

De Novo Biosynthesis of 2-Phenylethanol by Metabolic Engineering the Oleaginous Yeast Rhodotorula toruloides.

Microbial production offers a sustainable route to plant- and chemical-based manufacturing. 2-Phenylethanol (2-PE) has been widely used in foods, flavors, and pharmaceuticals. Herein, de novo biosynthesis of 2-PE was achieved for the first time by rewiring the non-native producer Rhodotorula toruloides. Our results showed that the generation of phenylalanine (Phe) and the recycling of the amino group could be bypassed since the Ehrlich pathway outperformed the phenylacetaldehyde-dependent one in de novo biosynthesis of 2-PE when the adenosine 5'-triphosphate (ATP) citrate synthase (ACL) was inactivated. The 2-PE titer was enhanced to 151.7 mg/L in a shake flask by alleviating feedback inhibition, enhancing precursor availability and cofactor balance. Finally, the production of 2-PE was elevated to 1.06 g/L with a yield of 8.5 mg/g glucose and productivity of 8 mg/L/h in a 3 L bioreactor. Our results should shed light on the microbial production of other aromatic derivatives with R. toruloides.

Chitosan-coated manganese ferrite nanoparticles enhanced Rhodotorula toruloides carotenoid production.

The present study aims to analyze the interaction between Rhodotorula toruloides and magnetic nanoparticles and evaluate their effect on carotenoid production. The manganese ferrite nanoparticles were synthesized without chitosan (MnFe2O4) and chitosan coating (MnFe2O4-CS) by the co-precipitation method assisted by hydrothermal treatment. XRD (X-ray diffraction), Magnetometry, Dynamic Light Scattering (DLS) and FTIR (Fourier-Transform Infrared Spectroscopy), are used to characterize the magnetic nanoparticles. The crystallite size of MnFe2O4 was 16 nm for MnFe2O4 and 20 nm for MnFe2O4-CS. The magnetic saturation of MnFe2O4-CS was lower (39.6 ± 0.6 emu/g) than the same MnFe2O4 nanoparticles (42.7 ± 0.3 emu/g), which was attributed to the chitosan fraction presence. The MnFe2O4-CS FTIR spectra revealed the presence of the characteristic chitosan bands. DLS demonstrated that the average hydrodynamic diameters were 344 nm for MnFe2O4 and 167 nm for MnFe2O4-CS. A kinetic study of cell immobilization performed with their precipitation with a magnet demonstrated that interaction between magnetic nanoparticles and R. toruloides was characterized by an equilibrium time of 2h. The adsorption isotherm models (Langmuir and Freundlich) were fitted to the experimental values. The trypan blue assay was used for cell viability assessment. The carotenoid production increased to 256.2 ± 6.1 µg/g dry mass at 2.0 mg/mL MnFe2O4-CS. The use of MnFe2O4-CS to stimulate carotenoid yeast production and the magnetic separation of biomass are promising nanobiotechnological alternatives. Magnetic cell immobilization is a perspective technique for obtaining cell metabolites.

Mixed and membrane-separated culturing of synthetic cyanobacteria-yeast consortia reveals metabolic cross-talk mimicking natural cyanolichens

Metabolite exchange mediates crucial interactions in microbial communities, significantly impacting global carbon and nitrogen cycling. Understanding these chemically-mediated interactions is essential for elucidating natural community functions and developing engineered synthetic communities. This study investigated membrane-separated bioreactors (mBRs) as a novel tool to identify transient metabolites and their producers/consumers in mixed microbial communities. We compared three co-culture methods (direct mixed, 2-chamber mBR, and 3-chamber mBR) to grow a synthetic binary community of the cyanobacterium Synechococcus elongatus PCC 7942 and the fungus Rhodotorula toruloides NBRC 0880, as well as axenic S. elongatus. Despite not being natural lichen constituents, these organisms exhibited interactions resembling those in cyanolichens. S. elongatus fixed CO2 into sugars as the primary shared metabolite, while R. toruloides secreted various biochemicals, predominantly sugar alcohols, mirroring the metabolite exchange observed in natural lichens. The mBR systems successfully captured metabolite gradients and revealed rapidly consumed compounds, including TCA cycle intermediates and amino acids. Our approach demonstrated that the 2-chamber mBR optimally balanced metabolite exchange and growth dynamics. This study provides insights into cross-species metabolic interactions and presents a valuable tool for investigating and engineering synthetic microbial communities with potential applications in biotechnology and environmental science.

Cell wall alterations occurring in an evolved multi-stress tolerant strain of the oleaginous yeast Rhodotorula toruloides

The oleaginous yeast species Rhodotorula toruloides is a promising candidate for applications in circular bioeconomy due to its ability to efficiently utilize diverse carbon sources being tolerant to cellular stress in bioprocessing. Previous studies including genome-wide analyses of the multi-stress tolerant strain IST536 MM15, derived through adaptive laboratory evolution from a promising IST536 strain for lipid production from sugar beet hydrolysates, suggested the occurrence of significant modifications in the cell wall. In this study, the cell wall integrity and carbohydrate composition of those strains was characterized to gain insights into the physicochemical changes associated to the remarkable multi-stress tolerance phenotype of the evolved strain. Compared to the original strain, the evolved strain exhibited a higher proportion of glucomannans, fucogalactomannans, and chitin relative to (1→4)-linked glucans, and an increased presence of glycoproteins with short glucosamine derived oligosaccharides, which have been found to be associated to ethanol stress tolerance and physical strength of the cell wall. Furthermore, the evolved strain cells were found to be significantly smaller than the original strain and more resistant to thermal and mechanical disruption, consistent with higher proportion of beta-linked polymers instead of glycogen, conferring a more rigid and robust cell wall. These findings provide further insights into the cell wall composition of this basidiomycetous red yeast species and into the alterations occurring in a multi-stress tolerant evolved strain. This new information can guide yeast genome engineering towards more robust strains of biotechnological relevance.

Molecular Identification and Antifungal Activity of Candida chrysomelidarum and Rhodotorula toruloides Isolated from Indonesian Indigenous Fruit Flacourtia inermis Roxb. (Lobi-lobi) against Pathogenic Mold Aspergillus flavus

Aspergillus flavus is one of the main contaminating mold species in most feed products, significantly impacting human and animal health. This study aims to isolate the epiphytic yeasts from the fruit Flacourtia inermis Roxb and test their potential ability to act as biocontrol agents for A. flavus. The antagonistic test was performed using a dual culture method, and the potential yeast isolates were then identified using a combination of morphology characterization and molecular sequence analysis. From 42 yeast isolated from F. inermis fruit, 2 yeast isolates (LCA 75 and LCA 102) demonstrated the highest inhibition growth against A. flavus (32.32 and 32.30 %, respectively). The isolates LCA 75 and LCA 102 were identified as Candida chrysomelidarum and Rhodotorula toruloides, respectively. Rhodotorula toruloides, identified as one of the yeast isolates (LCA 102), is an unconventional red yeast demonstrating notable antagonistic effects against A. flavus, inhibiting its growth by 32.30 %. This yeast, also known as Rhodosporidium toruloides, R. rubescens, R. glutinis, or R. gracilis, belongs to the subphylum Pucciniomycotina within the division Basidiomycota. The significant inhibition of mycelium growth and sporulation observed in our study highlights the potential of R. toruloides, along with C. chrysomelidarum (LCA 75), as effective biocontrol agents to reduce mycotoxin contamination in chicken feed by A. flavus. This study represents a significant advancement in the management of A. flavus contamination in animal feed. The findings not only underscore the viability of yeast-based biocontrol strategies but also contribute to a safer, more sustainable approach to preventing mycotoxin proliferation in feedstocks. HIGHLIGHTS Founding Out of 42 yeast isolates, two (LCA 75 and LCA 102) demonstrated the highest inhibition growth against A. flavus, with inhibition rates of 32.32 and 32.30 %, respectively. Identified Yeasts: LCA 75 as Candida chrysomelidarum LCA 102 as Rhodotorula toruloides (alternatively referred to as Rhodosporidium toruloides, R. rubescens, R. glutinis, or R. gracilis). Characteristics of Rhodotorula toruloides Unconventional red yeast belonging to the subphylum Pucciniomycotina within Basidiomycota. Commonly associated with various plant species and found on plant surfaces, including leaves, stems, and fruits. Identified yeast isolates exhibited inhibition activities leading to reduced mycelium growth and sporulation of A. flavus. These yeast isolates have potential as biocontrol agents to prevent mycotoxin contamination in chicken feed caused by A. flavus. GRAPHICAL ABSTRACT

The Plasma Membrane H+-ATPase Promoter Driving the Expression of FADX Enables Highly Efficient Production of Punicic Acid in Rhodotorula toruloides Cultivated on Glucose and Crude Glycerol.

Punicic acid (PuA) is a conjugated fatty acid with a wide range of nutraceutical properties naturally present in pomegranate seed oil. To meet the rising demand for pomegranate seed oil, a single-cell oil enriched in PuA provides a sustainable biomass-derived alternative. This study describes the production of a PuA-enriched single-cell oil through the engineering of the red yeast Rhodotorula toruloides grown in glucose and a low-cost substrate, crude glycerol. The gene for Punica granatum fatty acid conjugase, PgFADX, was randomly integrated into the genome of R. toruloides without disrupting the carotenoid synthesis. In shake flask studies, the effects of three promoters (PPGI1, PNAR1, and PPMA1) on PuA production were evaluated. PuA titers of 105.77 mg/L and 72.81 mg/L were obtained from engineered cells expressing PgFADX from the PPMA1 promoter cultivated for 72 h in glucose and for 168 h in crude glycerol, respectively. Furthermore, the detailed lipid analysis revealed a high enrichment PuA in the triacylglycerol lipid structures, even without substantial modifications to the metabolic pathways. This report demonstrates the high potential of R. toruloides in the upcycling of a low-cost substrate, crude glycerol, into a value-added product such as PuA. The findings support the feasibility of using engineered R. toruloides for sustainable production of PuA-enriched single-cell oil.

Hydrothermal conditioning of oleaginous yeast cells to enable recovery of lipids as potential drop-in fuel precursors

BackgroundLipids produced using oleaginous yeast cells are an emerging feedstock to manufacture commercially valuable oleochemicals ranging from pharmaceuticals to lipid-derived biofuels. Production of biofuels using oleaginous yeast is a multistep procedure that requires yeast cultivation and harvesting, lipid recovery, and conversion of the lipids to biofuels. The quantitative recovery of the total intracellular lipid from the yeast cells is a critical step during the development of a bioprocess. Their rigid cell walls often make them resistant to lysis. The existing methods include mechanical, chemical, biological and thermochemical lysis of yeast cell walls followed by solvent extraction. In this study, an aqueous thermal pretreatment was explored as a method for lysing the cell wall of the oleaginous yeast Rhodotorula toruloides for lipid recovery.ResultsHydrothermal pretreatment for 60 min at 121 °C with a dry cell weight of 7% (w/v) in the yeast slurry led to a recovery of 84.6 ± 3.2% (w/w) of the total lipids when extracted with organic solvents. The conventional sonication and acid-assisted thermal cell lysis led to a lipid recovery yield of 99.8 ± 0.03% (w/w) and 109.5 ± 1.9% (w/w), respectively. The fatty acid profiles of the hydrothermally pretreated cells and freeze-dried control were similar, suggesting that the thermal lysis of the cells did not degrade the lipids.ConclusionThis work demonstrates that hydrothermal pretreatment of yeast cell slurry at 121 °C for 60 min is a robust and sustainable method for cell conditioning to extract intracellular microbial lipids for biofuel production and provides a baseline for further scale-up and process integration.Graphical abstract

Efficient sugar utilization and high tolerance to inhibitors enable Rhodotorula toruloides C23 to robustly produce lipid and carotenoid from lignocellulosic feedstock

The utilization of lignocellulosic substrates for microbial oil production by oleaginous yeasts has been evidenced as an economically viable process for industrial-scale biodiesel preparation. Efficient sugar utilization and tolerance to inhibitors are critical for lipid production from lignocellulosic substrates. This study investigated the lignocellulosic sugar utilization and inhibitor tolerance characteristics of Rhodotorula toruloides C23. The results demonstrated that C23 exhibited robust glucose and xylose assimilation irrespective of their ratios, yielding over 21 g/L of lipids and 11 mg/L of carotenoids. Furthermore, C23 exhibited high resistance and efficiently degradation towards toxic inhibitors commonly found in lignocellulosic hydrolysates. The potential molecular mechanism underlying xylose metabolism in C23 was explored, with several key enzymes and signal regulation pathways identified as potentially contributing to its superior lipid synthesis performance. The study highlights R. toruloides C23 as a promising candidate for robust biofuel and carotenoid production through direct utilization of non-detoxified lignocellulosic hydrolysates.

Dual-Regulation in Peroxisome and Cytoplasm toward Efficient Limonene Biosynthesis with Rhodotorula toruloides.

Rhodotorula toruloides is a potential workhorse for production of various value-added chemicals including terpenoids, oleo-chemicals, and enzymes from low-cost feedstocks. However, the limited genetic toolbox is hindering its metabolic engineering. In the present study, four type I and one novel type II peroxisomal targeting signal (PTS1/PTS2) were characterized and employed for limonene production for the first time in R. toruloides. The implant of the biosynthesis pathway into the peroxisome led to 111.5 mg/L limonene in a shake flask culture. The limonene titer was further boosted to 1.05 g/L upon dual-metabolic regulation in the cytoplasm and peroxisome, which included employing the acetoacetyl-CoA synthase NphT7, adding an additional copy of native ATP-dependent citrate lyase, etc. The final yield was 0.053 g/g glucose, which was the highest ever reported. The newly characterized PTSs should contribute to the expansion of genetic toolboxes forR. toruloides. The results demonstrated that R. toruloides could be explored for efficient production of terpenoids.

Cell envelope and stress-responsive pathways underlie an evolved oleaginous Rhodotorula toruloides strain multi-stress tolerance

BackgroundThe red oleaginous yeast Rhodotorula toruloides is a promising cell factory to produce microbial oils and carotenoids from lignocellulosic hydrolysates (LCH). A multi-stress tolerant strain towards four major inhibitory compounds present in LCH and methanol, was derived in our laboratory from strain IST536 (PYCC 5615) through adaptive laboratory evolution (ALE) under methanol and high glycerol selective pressure.ResultsComparative genomic analysis suggested the reduction of the original strain ploidy from triploid to diploid, the occurrence of 21,489 mutations, and 242 genes displaying copy number variants in the evolved strain. Transcriptomic analysis identified 634 genes with altered transcript levels (465 up, 178 down) in the multi-stress tolerant strain. Genes associated with cell surface biogenesis, integrity, and remodelling and involved in stress-responsive pathways exhibit the most substantial alterations at the genome and transcriptome levels. Guided by the suggested stress responses, the multi-stress tolerance phenotype was extended to osmotic, salt, ethanol, oxidative, genotoxic, and medium-chain fatty acid-induced stresses.ConclusionsThe comprehensive analysis of this evolved strain provided the opportunity to get mechanistic insights into the acquisition of multi-stress tolerance and a list of promising genes, pathways, and regulatory networks, as targets for synthetic biology approaches applied to promising cell factories, toward more robust and superior industrial strains. This study lays the foundations for understanding the mechanisms underlying tolerance to multiple stresses in R. toruloides, underscoring the potential of ALE for enhancing the robustness of industrial yeast strains.

Nitrogen starvation causes lipid remodeling in Rhodotorula toruloides

BackgroundThe oleaginous yeast Rhodotorula toruloides is a promising chassis organism for the biomanufacturing of value-added bioproducts. It can accumulate lipids at a high fraction of biomass. However, metabolic engineering efforts in this organism have progressed at a slower pace than those in more extensively studied yeasts. Few studies have investigated the lipid accumulation phenotype exhibited by R. toruloides under nitrogen limitation conditions. Consequently, there have been only a few studies exploiting the lipid metabolism for higher product titers.ResultsWe performed a multi-omic investigation of the lipid accumulation phenotype under nitrogen limitation. Specifically, we performed comparative transcriptomic and lipidomic analysis of the oleaginous yeast under nitrogen-sufficient and nitrogen deficient conditions. Clustering analysis of transcriptomic data was used to identify the growth phase where nitrogen-deficient cultures diverged from the baseline conditions. Independently, lipidomic data was used to identify that lipid fractions shifted from mostly phospholipids to mostly storage lipids under the nitrogen-deficient phenotype. Through an integrative lens of transcriptomic and lipidomic analysis, we discovered that R. toruloides undergoes lipid remodeling during nitrogen limitation, wherein the pool of phospholipids gets remodeled to mostly storage lipids. We identify specific mRNAs and pathways that are strongly correlated with an increase in lipid levels, thus identifying putative targets for engineering greater lipid accumulation in R. toruloides. One surprising pathway identified was related to inositol phosphate metabolism, suggesting further inquiry into its role in lipid accumulation.ConclusionsIntegrative analysis identified the specific biosynthetic pathways that are differentially regulated during lipid remodeling. This insight into the mechanisms of lipid accumulation can lead to the success of future metabolic engineering strategies for overproduction of oleochemicals.

Engineering non-conventional yeast Rhodotorula toruloides for ergothioneine production

BackgroundErgothioneine (EGT) is a distinctive sulfur-containing histidine derivative, which has been recognized as a high-value antioxidant and cytoprotectant, and has a wide range of applications in food, medical, and cosmetic fields. Currently, microbial fermentation is a promising method to produce EGT as its advantages of green environmental protection, mild fermentation condition, and low production cost. However, due to the low-efficiency biosynthetic process in numerous cell factories, it is still a challenge to realize the industrial biopreparation of EGT. The non-conventional yeast Rhodotorula toruloides is considered as a potential candidate for EGT production, thanks to its safety for animals and natural ability to synthesize EGT. Nevertheless, its synthesis efficiency of EGT deserves further improvement.ResultsIn this study, out of five target wild-type R. toruloides strains, R. toruloides 2.1389 (RT1389) was found to accumulate the highest EGT production, which could reach 79.0 mg/L at the shake flask level on the 7th day. To achieve iterative genome editing in strain RT1389, CRISPR-assisted Cre recombination (CACR) method was established. Based on it, an EGT-overproducing strain RT1389-2 was constructed by integrating an additional copy of EGT biosynthetic core genes RtEGT1 and RtEGT2 into the genome, the EGT titer of which was 1.5-fold increase over RT1389. As the supply of S-adenosylmethionine was identified as a key factor determining EGT production in strain RT1389, subsequently, a series of gene modifications including S-adenosylmethionine rebalancing were integrated into the strain RT1389-2, and the resulting mutants were rapidly screened according to their EGT production titers with a high-throughput screening method based on ergothionase. As a result, an engineered strain named as RT1389-3 was selected with a production titer of 267.4 mg/L EGT after 168 h in a 50 mL modified fermentation medium.ConclusionsThis study characterized the EGT production capacity of these engineered strains, and demonstrated that CACR and high-throughput screening method allowed rapid engineering of R. toruloides mutants with improved EGT production. Furthermore, this study provided an engineered RT1389-3 strain with remarkable EGT production performance, which had potential industrial application prospects.

β-Carotene production from sugarcane molasses by a newly isolated Rhodotorula toruloides L/24-26-1.

Production of carotenoids by yeast fermentation is an advantaged technology due to its easy scaling and safety. Nevertheless, carotenoid production needs an economic culture medium and other efficient yeast stains. The study aims to isolate and identify a yeast strain capable of producing carotenoids using a cost-effective substrate. A new strain was identified asRhodotorula toruloidesL/24-26-1, which can produce carotenoids at different pretreated and unpretreated sugarcane molasses concentrations (40 and 80g/L). The highest biomass concentration (18.6 ± 0.6g/L) was reached in the culture using 80g/L of hydrolyzed molasses. On the other hand, the carotenoid accumulation reached the maximum value using pretreated molasses at 40g/L (715.4 ± 15.1µg/g d.w). In this case, the β-carotene was 1.5 times higher than that on the control medium. The yeast growth in molasses was not correlated with carotenoid production. The most outstanding production of The DPPH, ABTS, and FRAP tests demonstrated the antioxidant activity of the obtained carotenogenic extracts. This research demonstrated theR. toruloidesL/24-26-1 strain biotechnological potential for carotenoid compounds. The yeast produces carotenoids with antioxidant activity in an inexpensive medium, such as sulfuric acid pretreated and unpretreated molasses.

Biomass of the oleaginous yeast Rhodotorula toruloides as feed ingredient in the diet of Arctic char (Salvelinus alpinus): impact on quality; safety and sensory outcome

ABSTRACT The aim of this study was to test the impact of replacing vegetable oils commonly used in the feed of Arctic char (Salvelinus alpinus) with biomass of the oleaginous yeast Rhodotorula toruloides. For that, a control feed containing vegetable oils and an experimental feed including yeast biomass at 15% level, were fed to Arctic char. Sensory properties of the fish were tested. The content of undesirable compounds in the new feed ingredient were evaluated. Selected heavy metals and organic pollutants were measured in the yeast biomass and the levels found were below the limits of the regulations defined by the European Union. Moisture and ash contents were similar between the two feeds. Hepatic activity of 7-ethoxyresorufin-O-deethylase was evaluated between the two fish groups and no significant differences were observed. The sensory analysis conducted on heat treated fish fillets with untrained volunteers showed no perceptible sensory difference.

Invertase production by Rhodotorula toruloides in submerged and surface adhesion on magnetic nanoparticles fermentations

The invertase production by Rhodotorula toruloides was evaluated in the submerged fermentation and surface adhesion fermentation on the chitosan-coated magnetic manganese ferrite nanoparticles (MnFe2O4-Ch). The synthesis of chitosan-coated manganese ferrite was carried out by coprecipitation in a one-step method. According to X-ray diffractograms, the manganese ferrite magnetic nanoparticles had a spinel structure, and the crystal size calculated by the Scherrer equation was 20.12 nm. FTIR verified the presence of chitosan amino groups, which were quantified spectrophotometrically in reaction with ninhydrin, obtaining 7.53 mmol/g of nanoparticles. The hydrodynamic size of MnFe2O4-Ch was analyzed by DLS and revealed the presence of agglomerates. The mean particle size, obtained through Gaussian fitting of the histogram, was 239 nm. The MnFe2O4-Ch magnetic saturation was 36.7 emu/g at 20 kOe and 300 K. In the kinetic assay, by counting free cells in the Neubauer chamber after applying an external magnetic field, it was demonstrated that equilibrium in the absorption of yeast in MnFe2O4-Ch was achieved after 2 h of incubation. Adsorption isotherm at 30 °C was fitted to the Freundlich model. The fermentation was performed in the presence and absence of MnFe2O4-Ch. Results on the gravimetric assay and cell count with and without trypan blue assay indicated that the presence of magnetic nanoparticles in the fermentation had no adverse effect on the growth and viability of R. toruloides. The highest enzymatic activity (0.86 ± 0.03 UI/mL) was observed in the presence of MnFe2O4-Ch at 1 mg/mL. Fermentation by adhesion to the surface of magnetic nanoparticles may have advantages in enzyme production by yeast.

Modularly engineering Rhodotorula toruloides for α-terpineol production.

α-Terpineol is a monoterpenoid alcohol that has been widely used in the flavor, fragrance, and pharmaceutical industries because of its sensory and biological properties. However, few studies have focused on the microbial production of α-terpineol. The oleaginous yeast Rhodotorula toruloides is endowed with a natural mevalonate pathway and is a promising host in synthetic biology and biorefinery. The primary objective of this work was to engineer R. toruloides for the direct biosynthesis of α-terpineol. The improvement in monoterpenoid production was achieved through the implementation of modular engineering strategies, which included the enhancement of precursor supply, blocking of downstream pathways, and disruption of competing pathways. The results of these three methods showed varying degrees of favorable outcomes in enhancing α-terpineol production. The engineered strain 5L6HE5, with competitive pathway disruption and increased substrate supply, reached the highest product titer of 1.5mg/L, indicating that reducing lipid accumulation is an efficient method in R. toruloides engineering for terpenoid synthesis. This study reveals the potential of R. toruloides as a host platform for the synthesis of α-terpineol as well as other monoterpenoid compounds.

Corn stover variability drives differences in bisabolene production by engineered Rhodotorula toruloides.

This study elucidates the effects of different sources of corn stover on bisabolene production by engineered Rhodotorula toruloides, highlighting the importance of understanding feedstock variability to enhance bioprocess efficiency and economic outcomes.

Raman spectroscopy online monitoring of biomass production, intracellular metabolites and carbon substrates during submerged fermentation of oleaginous and carotenogenic microorganisms

BackgroundMonitoring and control of both growth media and microbial biomass is extremely important for the development of economical bioprocesses. Unfortunately, process monitoring is still dependent on a limited number of standard parameters (pH, temperature, gasses etc.), while the critical process parameters, such as biomass, product and substrate concentrations, are rarely assessable in-line. Bioprocess optimization and monitoring will greatly benefit from advanced spectroscopy-based sensors that enable real-time monitoring and control. Here, Fourier transform (FT) Raman spectroscopy measurement via flow cell in a recirculatory loop, in combination with predictive data modeling, was assessed as a fast, low-cost, and highly sensitive process analytical technology (PAT) system for online monitoring of critical process parameters. To show the general applicability of the method, submerged fermentation was monitored using two different oleaginous and carotenogenic microorganisms grown on two different carbon substrates: glucose fermentation by yeast Rhodotorula toruloides and glycerol fermentation by marine thraustochytrid Schizochytrium sp. Additionally, the online FT-Raman spectroscopy approach was compared with two at-line spectroscopic methods, namely FT-Raman and FT-infrared spectroscopies in high throughput screening (HTS) setups.ResultsThe system can provide real-time concentration data on carbon substrate (glucose and glycerol) utilization, and production of biomass, carotenoid pigments, and lipids (triglycerides and free fatty acids). Robust multivariate regression models were developed and showed high level of correlation between the online FT-Raman spectral data and reference measurements, with coefficients of determination (R2) in the 0.94–0.99 and 0.89–0.99 range for all concentration parameters of Rhodotorula and Schizochytrium fermentation, respectively. The online FT-Raman spectroscopy approach was superior to the at-line methods since the obtained information was more comprehensive, timely and provided more precise concentration profiles.ConclusionsThe FT-Raman spectroscopy system with a flow measurement cell in a recirculatory loop, in combination with prediction models, can simultaneously provide real-time concentration data on carbon substrate utilization, and production of biomass, carotenoid pigments, and lipids. This data enables monitoring of dynamic behaviour of oleaginous and carotenogenic microorganisms, and thus can provide critical process parameters for process optimization and control. Overall, this study demonstrated the feasibility of using FT-Raman spectroscopy for online monitoring of fermentation processes.

Application of adaptive laboratory evolution to improve the tolerance of Rhodotorula strain to methanol in crude glycerol and development of an effective method for cell lysis.

Rhodotorula toruloides can utilize crude glycerol as the low-cost carbon source for lipid production, but its growth is subjected to inhibition by methanol in crude glycerol. Here, transcriptome profiling demonstrated that 1004 genes were significantly regulated in the strain R. toruloides TO2 under methanol stress. Methanol impaired the function of membrane transport and subsequently weakened the utilization of glycerol, activities of the primary metabolism and functions of nucleus and ribosome. Afterwards the tolerance of TO2 to methanol was improved by using two-round adaptive laboratory evolution (ALE). The final strain M2-ale had tolerance up to 3.5% of methanol. 1 H NMR-based metabolome analysis indicated that ALE not only improved the tolerance of M2-ale to methanol but also tuned the carbon flux towards the biosynthesis of glycerolipid-related metabolites. The biomass and lipid titer of M2-ale reached 14.63±0.45 g L-1 and 7.06±0.44 g L-1 at 96 h in the crude glycerol medium, which increased up to 17.69% and 31.39%, respectively, comparing with TO2. Afterwards, an effective method for cell lysis was developed by combining sonication and enzymatic hydrolysis (So-EnH). The lytic effect of So-EnH was validated by using confocal imaging and flow cytometry. At last, lipid recovery rate reached 95.4±2.7% at the optimized condition.

An Evolved Strain of the Oleaginous Yeast Rhodotorula toruloides, Multi-Tolerant to the Major Inhibitors Present in Lignocellulosic Hydrolysates, Exhibits an Altered Cell Envelope.

The presence of toxic compounds in lignocellulosic hydrolysates (LCH) is among the main barriers affecting the efficiency of lignocellulose-based fermentation processes, in particular, to produce biofuels, hindering the production of intracellular lipids by oleaginous yeasts. These microbial oils are promising sustainable alternatives to vegetable oils for biodiesel production. In this study, we explored adaptive laboratory evolution (ALE), under methanol- and high glycerol concentration-induced selective pressures, to improve the robustness of a Rhodotorula toruloides strain, previously selected to produce lipids from sugar beet hydrolysates by completely using the major C (carbon) sources present. An evolved strain, multi-tolerant not only to methanol but to four major inhibitors present in LCH (acetic acid, formic acid, hydroxymethylfurfural, and furfural) was isolated and the mechanisms underlying such multi-tolerance were examined, at the cellular envelope level. Results indicate that the evolved multi-tolerant strain has a cell wall that is less susceptible to zymolyase and a decreased permeability, based on the propidium iodide fluorescent probe, in the absence or presence of those inhibitors. The improved performance of this multi-tolerant strain for lipid production from a synthetic lignocellulosic hydrolysate medium, supplemented with those inhibitors, was confirmed.