d-Arabitol production by a high arabitol-producing yeast, Zygosaccharomyces sp. Gz-5 isolated from miso.

d-Arabitol, an alternative sweetener to sugar, has low calorie content, high sweetness, low glycemic index, and insulin resistance-improving ability. In this study, d-arabitol-producing yeast strains were isolated from various commercial types of miso, and strain Gz-5 was selected among these strains. Phylogenetic tree analysis of the internal transcribed spacer sequence revealed that strain Gz-5 was distinct from Zygosaccharomyces rouxii, a major fermenting yeast of miso. The strain, identified as Zygosaccharomyces sp. Gz-5, grew better than other Z. rouxii in 150 g/L NaCl and produced 114 g/L d-arabitol from 295 g/L glucose in a batch culture for 8 days (0.386 g/g-consumed glucose). In a fed-batch culture, the yeast produced 133 g/L d-arabitol for 14 days, and the total d-arabitol amount increased by 1.75-fold. These results indicated that Zygosaccharomyces sp. Gz-5, a non-genetically modified strain, has excellent potential for the industrial production of d-arabitol.

Comparative Conformational Analysis of Acyclic Sugar Alcohols Ribitol, Xylitol and d-Arabitol by Solution NMR and Molecular Dynamics Simulations.

Ribitol (C5H12O5) is an acyclic sugar alcohol that was recently identified in O-mannose glycan on mammalian α-dystroglycan. The conformation and dynamics of acyclic sugar alcohols such as ribitol are dependent on the stereochemistry of the hydroxyl groups; however, the dynamics are not fully understood. To gain insights into the conformation and dynamics of sugar alcohols, we carried out comparative analyses of ribitol, d-arabitol and xylitol by a crystal structure database search, solution NMR analysis and molecular dynamics (MD) simulations. The crystal structures of the sugar alcohols showed a limited number of conformations, suggesting that only certain stable conformations are prevalent among all possible conformations. The three-bond scholar coupling constants and exchange rates of hydroxyl protons were measured to obtain information on the backbone torsion angle and possible hydrogen bonding of each hydroxyl group. The 100 ns MD simulations indicate that the ribitol backbone has frequent conformational transitions with torsion angles between 180∘ and ±60∘, while d-arabitol and xylitol showed fewer conformational transitions. Taking our experimental and computational data together, it can be concluded that ribitol is more flexible than d-arabitol or xylitol, and the flexibility is at least in part defined by the configuration of the OH groups, which may form intramolecular hydrogen bonds.

Development of an Integrated Bioprocess System for Bioethanol and Arabitol Production from Sugar Beet Cossettes.

Research background: An innovative integrated bioprocess system for bioethanol production from raw sugar beet cossettes (SBC) and arabitol from remaining exhausted sugar beet cossettes (ESBC) was studied. This integrated three-stage bioprocess system is an example of the biorefinery concept to maximise the use of raw SBC for the production of high value-added products such as sugar alcohols and bioethanol. Experimental approach: The first stage of the integrated bioprocess system was simultaneous sugar extraction from SBC and its alcoholic fermentation to produce bioethanol in an integrated bioreactor system (vertical column bioreactor and stirred tank bioreactor) containing a high-density suspension of yeast Saccharomyces cerevisiae (30 g/L). The second stage was the pretreatment of ESBC with dilute sulfuric acid to release fermentable sugars. The resulting liquid hydrolysate of ESBC was used in the third stage as a nutrient medium for arabitol production by non-Saccharomyces yeasts (Spathaspora passalidarum CBS 10155 and Spathaspora arborariae CBS 11463). Results and conclusions: The obtained results show that the efficiency of bioethanol production increased with increasing temperature and prolonged residence time in the integrated bioreactor system. The maximum bioethanol production efficiency (87.22 %) was observed at a time of 60 min and a temperature of 36 °C. Further increase in residence time (above 60 min) did not result in the significant increase of bioethanol production efficiency. Weak acid hydrolysis was used for ESBC pretreatment and the highest sugar yield was reached at 200 °C and residence time of 1 min. The inhibitors of the weak acid pretreatment were produced below bioprocess inhibition threshold. The use of the obtained liqiud phase of ESBC hydrolysate for the production of arabitol in the stirred tank bioreactor under constant aeration clearly showed that S. passalidarum CBS 10155 with 8.48 g/L of arabitol (YP/S=0.603 g/g and bioprocess productivity of 0.176 g/(L.h)) is a better arabitol producer than Spathaspora arborariae CBS 10155. Novelty and scientific contribution: An innovative integrated bioprocess system for the production of bioethanol and arabitol was developed based on the biorefinery concept. This three-stage bioprocess system shows great potential for maximum use of SBC as a feedstock for bioethanol and arabitol production and it could be an example of a sustainable 'zero waste' production system.

Arabinose as an overlooked sugar for microbial bioproduction of chemical building blocks.

The circular economy is anticipated to bring a disruptive transformation in manufacturing technologies. Robust and industrial scalable microbial strains that can simultaneously assimilate and valorize multiple carbon substrates are highly desirable, as waste bioresources contain substantial amounts of renewable and fermentable carbon, which is diverse. Lignocellulosic biomass (LCB) is identified as an inexhaustible and alternative resource to reduce global dependence on oil. Glucose, xylose, and arabinose are the major monomeric sugars in LCB. However, primary research has focused on the use of glucose. On the other hand, the valorization of pentose sugars, xylose, and arabinose, has been mainly overlooked, despite possible assimilation by vast microbial communities. The present review highlights the research efforts that have explicitly proven the suitability of arabinose as the starting feedstock for producing various chemical building blocks via biological routes. It begins by analyzing the availability of various arabinose-rich biorenewable sources that can serve as potential feedstocks for biorefineries. The subsequent section outlines the current understanding of arabinose metabolism, biochemical routes prevalent in prokaryotic and eukaryotic systems, and possible products that can be derived from this sugar. Further, currently, exemplar products from arabinose, including arabitol, 2,3-butanediol, 1,2,3-butanetriol, ethanol, lactic acid, and xylitol are discussed, which have been produced by native and non-native microbial strains using metabolic engineering and genome editing tools. The final section deals with the challenges and obstacles associated with arabinose-based production, followed by concluding remarks and prospects.

Non-canonical D-xylose and L-arabinose metabolism via D-arabitol in the oleaginous yeast Rhodosporidium toruloides.

R. toruloides is an oleaginous yeast, with diverse metabolic capacities and high tolerance for inhibitory compounds abundant in plant biomass hydrolysates. While R. toruloides grows on several pentose sugars and alcohols, further engineering of the native pathway is required for efficient conversion of biomass-derived sugars to higher value bioproducts. A previous high-throughput study inferred that R. toruloides possesses a non-canonical L-arabinose and D-xylose metabolism proceeding through D-arabitol and D-ribulose. In this study, we present a combination of genetic and metabolite data that refine and extend that model. Chiral separations definitively illustrate that D-arabitol is the enantiomer that accumulates under pentose metabolism. Deletion of putative D-arabitol-2-dehydrogenase (RTO4_9990) results in > 75% conversion of D-xylose to D-arabitol, and is growth-complemented on pentoses by heterologous xylulose kinase expression. Deletion of putative D-ribulose kinase (RTO4_14368) arrests all growth on any pentose tested. Analysis of several pentose dehydrogenase mutants elucidates a complex pathway with multiple enzymes mediating multiple different reactions in differing combinations, from which we also inferred a putative L-ribulose utilization pathway. Our results suggest that we have identified enzymes responsible for the majority of pathway flux, with additional unknown enzymes providing accessory activity at multiple steps. Further biochemical characterization of the enzymes described here will enable a more complete and quantitative understanding of R. toruloides pentose metabolism. These findings add to a growing understanding of the diversity and complexity of microbial pentose metabolism.

Development of an enzymatic cascade to systematically utilize lignocellulosic monosaccharide.

BACKGROUND: The fermentation valorization of two main lignocellulosic monosaccharides, glucose and xylose, is extensively developed; however, it is restricted by limited yield and process complexity. An in vitro enzymatic cascade reaction can be an alternative approach. RESULTS: In this study, a three-stage, five-enzyme cascade was developed to convert pretreated biomass to valuable chemicals. First, a ribose-5-phosphate isomerase B mutant isomerized xylose to d-xylulose with high substrate specificity, and a d-arabinose dehydrogenase continued to reduce d-xylulose to d-arabitol. Simultaneously, glucose was utilized for the coenzyme regeneration catalyzed by a glucose dehydrogenase, generating useful gluconic acid and achieving 73% of total conversion rate after 36 h. Then, six kinds of pretreated biomass lignocellulose were hydrolyzed by cellulase and hemicellulase, and corn cob was identified as the initial substrate for providing the highest monosaccharide content. A 65% conversion rate of the lignocellulosic xylose was obtained after 24 h. CONCLUSIONS: This study presents a proof of concept to convert main lignocellulosic monosaccharides systematically by an enzymatic cascade at stoichiometric ratio. © 2022 Society of Chemical Industry.

Improved fed-batch processes with Wickerhamomyces anomalus WC 1501 for the production of D-arabitol from pure glycerol.

BACKGROUND: D-Arabitol, a five-carbon sugar alcohol, represents a main target of microbial biorefineries aiming to valorize cheap substrates. The yeast Wickerhamomyces anomalus WC 1501 is known to produce arabitol in a glycerol-based nitrogen-limited medium and preliminary fed-batch processes with this yeast were reported to yield 18.0 g/L arabitol. RESULTS: Fed-batch fermentations with W. anomalus WC 1501 were optimized using central composite design (CCD). Dissolved oxygen had not a significant effect, while optimum values were found for glycerol concentration (114.5 g/L), pH (5.9), and temperature (32.5 °C), yielding 29 g/L D-arabitol in 160 h, a conversion yield of 0.25 g of arabitol per g of consumed glycerol, and a volumetric productivity of 0.18 g/L/h. CCD optimal conditions were the basis for further improvement, consisting in increasing the cellular density (3✕), applying a constant feeding of glycerol, and increasing temperature during production. The best performing fed-batch fermentations achieved 265 g/L D-arabitol after 325 h, a conversion yield of 0.74 g/g, and a volumetric productivity of 0.82 g/L/h. CONCLUSION: W. anomalus WC 1501 confirmed as an excellent producer of D-arabitol, exhibiting a remarkable capability of transforming pure glycerol. The study reports among the highest values ever reported for microbial transformation of glycerol into D-arabitol, in terms of arabitol titer, conversion yield, and productivity.

Cloning, expression, and characterization of an arabitol dehydrogenase and coupled with NADH oxidase for effective production of L-xylulose.

A novel arabitol dehydrogenase (ArDH) gene was cloned from a bacterium named Aspergillus nidulans and expressed heterologously in Escherichia coli. The purified ArDH exhibited the maximal activity in pH 9.5 Tris-HCl buffer at 40 °C, showed Km and Vmax of 1.2 mg/mL and 9.1 U/mg, respectively. The ArDH was used to produce the L-xylulose and coupled with the NADH oxidase (Nox) for the regeneration of NAD+. In further optimization, a high conversion of 84.6% in 8 hours was achieved under the optimal conditions: 20 mM of xylitol, 100 µM NAD+ in pH 9.0 Tris-HCl buffer at 30 °C. The results indicated the coupling system with cofactor regeneration provides a promising approach for L-xylulose production from xylitol.

Optimization of xylitol production from xylose by a novel arabitol limited co-producing Barnettozyma populi NRRL Y-12728.

Xylitol is a widely marketed sweetener with good functionality and health-promoting properties. It can be synthetized by many yeast species in a one-step reduction of xylose. Arabinose is a common contaminant found in xylose and there is ongoing interest in finding biocatalysts that selectively produce xyltiol. From a screen of 99 yeasts, Barnettozyma populi Y-12728 was found to selectively produce xylitol from both mixed sugars and corn stover hemicellulosic hydrolysate. Here, fermentation conditions for xylitol production from xylose by B. populi were optimized. The medium for xylitol production was optimized through response surface methodology. The yeast produced 31.2 ± 0.4 g xylitol from xylose (50 g L-1) in 62 h using the optimized medium. The optimal pH for xylitol production was 6.0. Glucose (10 g L-1), acetic acid (6.0 g L-1), HMF (4 mM) and ethanol (2.0 g L-1) inhibited the xylitol production. The glucose inhibition was entirely mitigated by using a 2-stage aeration strategy, indicating that the yeast was inhibited by ethanol produced from glucose under low aeration. This culture strategy will greatly benefit xylitol production from hemicellulosic hydrolysates, which often contain glucose. This is the first report on optimization of xylitol production by a Barnettozyma species.

Combined mutagenesis and metabolic regulation to enhance D-arabitol production from Candida parapsilosis.

D-Arabitol is an important pentitol that is widely used in the food, pharmaceutical and chemical industries. It is mainly produced by yeasts during the biotransformation of glucose. To obtain strains with high D-arabitol production, Candida parapsilosis was mutated using atmospheric and room temperature plasma (ARTP). Among the screened mutants, mutant A6 had the highest yield at 32.92 g/L, a 53.98% increase compared with the original strain (21.38 g/L). Furthermore, metabolic regulators were added to the medium to improve D-arabitol production. Pyrithioxin dihydrochloride increased D-arabitol production by 34.4% by regulating glucose-6-phosphate dehydrogenase, and 4-methylpyrazole increased D-arabitol production by 77.4% compared with the control group by inhibiting alcohol dehydrogenase activity. Amphotericin B and Triton X-100 increased D-arabitol production by 23.8% and 42.2% by improving the membrane permeability and dissolved oxygen content, respectively. This study may provide important implications for obtaining high-yield D-arabitol strains.

Isolation, identification and screening of yeasts towards their ability to assimilate biodiesel-derived crude glycerol: microbial production of polyols, endopolysaccharides and lipid.

AIMS: To assess the ability of various newly isolated or belonging in official collections yeast strains to convert biodiesel-derived glycerol (Gly) into added-value compounds. METHODS AND RESULTS: Ten newly isolated yeast strains belonging to Debaryomyces sp., Naganishia uzbekistanensis, Rhodotorula sp. and Yarrowia lipolytica, isolated from fishes, metabolized Gly under nitrogen limitation. The aim of the study was to identify potential newly isolated microbial candidates that could produce single-cell oil (SCO), endopolysaccharides and polyols when these micro-organisms were grown on biodiesel-derived Gly. As controls producing SCO and endopolysaccharides were the strains Rhodotorula glutinis NRRL YB-252 and Cryptococcus curvatus NRRL Y-1511. At initial Gly (Gly0 ) ≈40 g l-1 , most strains presented remarkable dry cell weight (DCW) production, whereas Y. lipolytica and Debaryomyces sp. produced non-negligible quantities of mannitol and arabitol (Ara). Five strains were further cultivated at increasing Gly0 concentrations. Rhodotorula glutinis NRRL YB-252 produced 7·2 g l-1 of lipid (lipid in DCW value ≈38% w/w), whereas Debaryomyces sp. FMCC Y69 in batch-bioreactor experiment with Gly0 ≈80 g l-1 , produced 30-33 g l-1 of DCW and ~30 g l-1 of Ara. At shake-flasks with Gly0 ≈125 g l-1 , Ara of ~48 g l-1 (conversion yield of polyol on Gly consumed ≈0·62 g g-1 ) was achieved. Cellular lipids of all yeasts contained in variable concentrations oleic, palmitic, stearic and linoleic acids. CONCLUSIONS: Newly isolated, food-derived and non-previously studied yeast isolates converted biodiesel-derived Gly into several added-value metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: Alternative ways of crude Gly valorization through yeast fermentations were provided and added-value compounds were synthesized.

Screening of new yeast Pichia manchurica for arabitol production.

Arabitol has several applications in food and pharmaceutical industries as a natural sweetener, dental caries inhibitor, and texturing agent. Newly isolated yeast strains from seawater, sugarcane plantation soil samples, and Zygosaccharomyces rouxii 2635 from MTCC were tested for arabitol production. The yield of arabitol was found to be higher in seawater isolate (24.6 g L-1 ) compared to two soil isolates (22.5 g L-1 ) and Z. rouxii (19.4 g L-1 ). Based on ITS 26S rDNA sequence analysis, the seawater isolate was identified as Pichia manchurica. In the present study, the effect of different substrates, trace elements, nitrogen sources, pH, and temperature on arabitol production was examined. Three different carbon sources viz. glucose, arabinose, and galactose were studied. Glucose was determined to be the best substrate for arabitol production (27.6 g L-1 ) followed by arabinose (13.7 g L-1 ) and galactose (7.7 g L-1 ). Maximum production of arabitol was observed at pH 6.0 (34.7 g L-1 ). In addition, arabitol production was high (35.7 g L-1 ) at temperature of 30 °C. Among the different concentrations of ammonium sulfate tested (3, 4.5, 6, 7.5, and 9 g L-1 ) concentration of 6 g L-1 resulted in higher arabitol Individual metal ions had no effect on arabitol production by this strain as compared to control. Results obtained in this study identify ways for improved arabitol production with natural isolates using microbial processes.

Understanding Functional Roles of Native Pentose-Specific Transporters for Activating Dormant Pentose Metabolism in Yarrowia lipolytica.

Pentoses, including xylose and arabinose, are the second most prevalent sugars in lignocellulosic biomass that can be harnessed for biological conversion. Although Yarrowia lipolytica has emerged as a promising industrial microorganism for production of high-value chemicals and biofuels, its native pentose metabolism is poorly understood. Our previous study demonstrated that Y. lipolytica (ATCC MYA-2613) has endogenous enzymes for d-xylose assimilation, but inefficient xylitol dehydrogenase causes Y. lipolytica to assimilate xylose poorly. In this study, we investigated the functional roles of native sugar-specific transporters for activating the dormant pentose metabolism in Y. lipolytica By screening a comprehensive set of 16 putative pentose-specific transporters, we identified two candidates, YALI0C04730p and YALI0B00396p, that enhanced xylose assimilation. The engineered mutants YlSR207 and YlSR223, overexpressing YALI0C04730p and YALI0B00396p, respectively, improved xylose assimilation approximately 23% and 50% in comparison to YlSR102, a parental engineered strain overexpressing solely the native xylitol dehydrogenase gene. Further, we activated and elucidated a widely unknown native l-arabinose assimilation pathway in Y. lipolytica through transcriptomic and metabolic analyses. We discovered that Y. lipolytica can coconsume xylose and arabinose, where arabinose utilization shares transporters and metabolic enzymes of some intermediate steps of the xylose assimilation pathway. Arabinose assimilation is synergistically enhanced in the presence of xylose, while xylose assimilation is competitively inhibited by arabinose. l-Arabitol dehydrogenase is the rate-limiting step responsible for poor arabinose utilization in Y. lipolytica Overall, this study sheds light on the cryptic pentose metabolism of Y. lipolytica and, further, helps guide strain engineering of Y. lipolytica for enhanced assimilation of pentose sugars.IMPORTANCE The oleaginous yeast Yarrowia lipolytica is a promising industrial-platform microorganism for production of high-value chemicals and fuels. For decades since its isolation, Y. lipolytica has been known to be incapable of assimilating pentose sugars, xylose and arabinose, that are dominantly present in lignocellulosic biomass. Through bioinformatic, transcriptomic, and enzymatic studies, we have uncovered the dormant pentose metabolism of Y. lipolytica Remarkably, unlike most yeast strains, which share the same transporters for importing hexose and pentose sugars, we discovered that Y. lipolytica possesses the native pentose-specific transporters. By overexpressing these transporters together with the rate-limiting d-xylitol and l-arabitol dehydrogenases, we activated the dormant pentose metabolism of Y. lipolytica Overall, this study provides a fundamental understanding of the dormant pentose metabolism of Y. lipolytica and guides future metabolic engineering of Y. lipolytica for enhanced conversion of pentose sugars to high-value chemicals and fuels.

Production of arabitol from enzymatic hydrolysate of soybean flour by Debaryomyces hansenii fermentation.

Arabitol is a low-calorie sugar alcohol with anti-cariogenic properties. Enzymatic hydrolysate of soybean flour is a new renewable biorefinery feedstock containing hexose, pentose, and organic nitrogen sources. Arabitol production by Debaryomyces hansenii using soybean flour hydrolysate was investigated. Effects of medium composition, operating conditions, and culture stage (growing or stationary phase) were studied. Production was also compared at different culture volumes to understand the effect of dissolved oxygen concentration (DO). Main factors examined for medium composition effects were the carbon to nitrogen concentration ratio (C/N), inorganic (ammonium) to organic nitrogen ratio (I/O-N), and sugar composition. Arabitol yield increased with increasing C/N ratio and a high I/O-N (0.8-1.0), suggesting higher yield at stationary phase of low pH (3.5-4.5). Catabolite repression was observed, with the following order of consumption: glucose > fructose > galactose > xylose > arabinose. Arabitol production also favored hexoses and, among hexoses, glucose. DO condition was of critical importance to arabitol production and cell metabolism. The yeast consumed pentoses (xylose and arabinose) only at more favorable DO conditions. Finally, arabitol was produced in fermentors using mixed hydrolysates of soy flour and hulls. The process gave an arabitol yield of 54%, volumetric productivity of 0.90 g/L-h, and specific productivity of 0.031 g/g-h.

Production of D-arabitol from D-xylose by the oleaginous yeast Rhodosporidium toruloides IFO0880.

The sugar alcohol D-arabitol is one of the Department of Energy's top twelve bio-based building block chemicals. In this study, we found that the oleaginous yeast Rhodosporidium toruloides IFO0880 produces D-arabitol during growth on xylose in nitrogen-rich medium. Efficient xylose utilization was a prerequisite for extracellular D-arabitol production. During growth in complex media, R. toruloides produced 22 ± 2, 32 ± 2, and 49 ± 2 g/L D-arabitol from 70, 105, and 150 g/L xylose, respectively. In addition, we found that R. toruloides could potentially be used for the co-production of lipids and D-arabitol from xylose. These results demonstrate that R. toruloides can be used to produce multiple value-added chemicals from xylose.

The Ability of a Novel Strain Scheffersomyces (Syn. Candida) shehatae Isolated from Rotten Wood to Produce Arabitol.

Arabitol is a polyalcohol which has about 70% of the sweetness of sucrose and an energy density of 0.2 kcal/g. Similarly to xylitol, it can be used in the food and pharmaceutical industries as a natural sweetener, a texturing agent, a dental caries reducer, and a humectant. Biotechnological production of arabitol from sugars represents an interesting alternative to chemical production. The yeast Scheffersomyces shehatae strain 20BM-3 isolated from rotten wood was screened for its ability to produce arabitol from L-arabinose, glucose, and xylose. This isolate, cultured at 28°C and 150 rpm, secreted 4.03 ± 0.00 to 7.97 ± 0.67 g/l of arabitol from 17-30 g/l of L-arabinose assimilated from a medium containing 20-80 g/l of this pentose with yields of 0.24 ± 0.00 to 0.36 ± 0.02 g/g. An optimization study demonstrated that pH 4.0, 32°C, and a shaking frequency of 150 rpm were the optimum conditions for arabitol production by the investigated strain. Under these conditions, strain 20BM-3 produced 6.2 ± 0.17 g/l of arabitol from 17.5 g/l of arabinose after 4 days with a yield of 0.35 ± 0.01 g/g. This strain also produced arabitol from glucose, giving much lower yields, but did not produce it from xylose. The new strain can be successfully used for arabitol production from abundantly available sugars found in plant biomass.

Production of arabitol by yeasts: current status and future prospects.

Arabitol belongs to the pentitol family and is used in the food industry as a sweetener and in the production of human therapeutics as an anticariogenic agent and an adipose tissue reducer. It can also be utilized as a substrate for chemical products such as arabinoic and xylonic acids, propylene, ethylene glycol, xylitol and others. It is included on the list of 12 building block C3-C6 compounds, designated for further biotechnological research. This polyol can be produced by yeasts in the processes of bioconversion or biotransformation of waste materials from agriculture, the forest industry (l-arabinose, glucose) and the biodiesel industry (glycerol). The present review discusses research on native yeasts from the genera Candida, Pichia, Debaryomyces and Zygosaccharomyces as well as genetically modified strains of Saccharomyces cerevisiae which are able to utilize biomass hydrolysates to effectively produce L- or D-arabitol. The metabolic pathways of these yeasts leading from sugars and glycerol to arabitol are presented. Although the number of reports concerning microbial production of arabitol is rather limited, the research on this topic has been growing for the last several years, with researchers looking for new micro-organisms, substrates and technologies.

Sustainable bio-manufacturing of D-arabitol through combinatorial engineering of Zygosaccharomyces rouxii, bioprocess optimization and downstream separation.

Biosynthesis of D-arabitol, a high value-added platform chemical, from renewable carbon sources provides a sustainable and eco-friendly alternative to the chemical industry. Here, a robust brewing yeast, Zygosaccharomyces rouxii, capable of naturally producing D-arabitol was rewired through genome sequencing-based metabolic engineering. The recombinant Z. rouxii obtained by reinforcing the native D-xylulose pathway, improving reductive power of the rate-limiting step, and inhibiting the shunt pathway, produced 73.61% higher D-arabitol than the parent strain. Subsequently, optimization of the fermentation medium composition for the engineered strain provided 137.36 g/L D-arabitol, with a productivity of 0.64 g/L/h in a fed-batch experiment. Finally, the downstream separation of D-arabitol from the complex fermentation broth using an ethanol precipitation method provided a purity of 96.53%. This study highlights the importance of D-xylulose pathway modification in D-arabitol biosynthesis, and pave a complete and efficient way for the sustainable manufacturing of this value-added compound from biosynthesis to preparation.

Production of arabitol from glycerol by immobilized cells of Wickerhamomyces anomalus WC 1501.

Polyalcohols such as arabitol are among the main targets of biorefineries aiming to upcycle wastes and cheap substrates. In previous works Wickerhamomyces anomalus WC 1501 emerged as an excellent arabitol producer utilizing glycerol. Arabitol production by this strain is not growth associated, therefore, in this study, pre-grown cells were entrapped in calcium alginate beads (AB) and utilized for glycerol transformation to arabitol. Flasks experiments aimed to assess the medium composition (i.e., the concentration of inorganic and organic nitrogen sources and phosphates) and to establish the appropriate carrier-to-medium proportion. In flasks, under the best conditions of ammonium limitation and the carrier:medium ratio of 1:3 (w/v), 82.7 g/L glycerol were consumed in 168 h, yielding 31.2 g/L arabitol, with a conversion of 38% and volumetric productivity of 186 mg/mL/h. The process with immobilized cells was transferred to laboratory scale bioreactors with different configurations: stirred tank (STR), packed bed (PBR), fluidized bed (FBR), and airlift (ALR) bioreactors. The STR experienced oxygen limitation due to the need to maintain low stirring to preserve AB integrity and performed worse than flasks. Limitations in diffusion and mass transfer of oxygen and/or nutrients characterized also the PBR and the FBR and were partially relieved only in ALR, where 89.4 g/L glycerol were consumed in 168 h, yielding 38.1 g/L arabitol, with a conversion of 42% and volumetric productivity of 227 mg/mL/h. When the ALR was supplied with successive pulses of concentrated glycerol to replenish the glycerol as it was being consumed, 117 g/L arabitol were generated in 500 h, consuming a total of 285 g/L glycerol, with a 41% and 234 mg/L/h. The study strongly supports the potential of W. anomalus WC 1501 for efficient glycerol-to-arabitol conversion using immobilized cells. While the yeast shows promise by remaining viable and active for extended periods, further optimization is required, especially regarding mixing and oxygenation. Improving the stability of the immobilization process is also crucial for reusing pre-grown cells in multiple cycles, reducing dead times, biomass production costs, and enhancing the economic feasibility of the process.

Isolation, screening, and characterization of the newly isolated osmotolerant yeast Wickerhamomyces anomalus BKK11-4 for the coproduction of glycerol and arabitol.

This study explored the isolation and screening of an osmotolerant yeast, Wickerhamomyces anomalus BKK11-4, which is proficient in utilizing renewable feedstocks for sugar alcohol production. In batch fermentation with high initial glucose concentrations, W. anomalus BKK11-4 exhibited notable production of glycerol and arabitol. The results of the medium optimization experiments revealed that trace elements, such as H3BO3, CuSO4, FeCl3, MnSO4, KI, H4MoNa2O4, and ZnSO4, did not increase glucose consumption or sugar alcohol production but substantially increased cell biomass. Osmotic stress, which was manipulated by varying initial glucose concentrations, influenced metabolic outcomes. Elevated glucose levels promoted glycerol and arabitol production while decreasing citric acid production. Agitation rates significantly impacted the kinetics, enhancing glucose utilization and metabolite production rates, particularly for glycerol, arabitol, and citric acid. The operational pH dictated the distribution of the end metabolites, with glycerol production slightly reduced at pH 6, while arabitol production remained unaffected. Citric acid production was observed at pH 6 and 7, and acetic acid production was observed at pH 7. Metabolomic analysis using GC/MS identified 29 metabolites, emphasizing the abundance of sugar/sugar alcohols. Heatmaps were generated to depict the variations in metabolite levels under different osmotic stress conditions, highlighting the intricate metabolic dynamics occurring post-glucose uptake, affecting pathways such as the pentose phosphate pathway and glycerolipid metabolism. These insights contribute to the optimization of W. anomalus BKK11-4 as a whole-cell factory for desirable products, demonstrating its potential applicability in sustainable sugar alcohol production from renewable feedstocks.