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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.

D-Arabitol is an important functional sugar alcohol, which can be used in the preparation of foods, chemicals, and medicines. Despite biological production of D-arabitol from low-cost substrates has recently been the focus of research, low yield of this technology has limited its large-scale exploitation. Optimization of this bioprocess could be a promising option to improve the yield of D-arabitol. In this study, one-factor-at-a-time (OFAT) strategy and Box-Behnken design (BBD) were used to increase D-arabitol production by Metschnikowia reukaufii CICC 31,858 through optimizing the fermentation conditions and medium composition. The OFAT optimization provided the optimal conditions for temperature, agitation speed, and fermentation time of 30℃, 220 rpm, and 6 days, respectively. Likewise, the optimum concentrations of peptone, ammonium sulfate, KH2PO4, MgSO4·7H2O, and fumaric acid in the fermentation medium were (g/L) 7.5, 1, 2, 0.5, and 7.5, respectively. Under these optimum conditions, 80.43 g/L of D-arabitol was produced from 200 g/L of glucose, with a productivity of 0.56 g/L/h. The BBD optimization with three important components of fermentation medium (KH2PO4, MgSO4·7H2O, and fumaric acid) showed that the predicted titer of D-arabitol varied from 47.21 to 89.27 g/L, and the actual titer of D-arabitol ranged from 47.36 to 89.83 g/L. The optimum concentrations (g/L) of KH2PO4, MgSO4·7H2O, and fumaric acid in the fermentation medium were found to be 1.0, 0.5, and 4.7 g/L, respectively. Under the optimum conditions, 92.45 g/L of D-arabitol was finally produced with the yield and productivity of 0.46 g/g and 0.64 g/L/h, respectively.

Key message The interaction network and pathway map uncover the potential crosstalk between sugar and hormone metabolisms as a possible reason for leaf senescence in P. ternata . Pinellia ternata , an environmentally sensitive medicinal plant, undergoes leaf senescence twice a year, affecting its development and yield. Understanding the potential mechanism that delays leaf senescence could theoretically decrease yield losses. In this study, a typical senescent population model was constructed, and an integrated analysis of transcriptomic and metabolomic profiles of P. ternata was conducted using two early leaf senescence populations and two stay-green populations. The result showed that two key gene modules were associated with leaf senescence which were mainly enriched in sugar and hormone signaling pathways, respectively. A network constructed by unigenes and metabolisms related to the obtained two pathways revealed that several compounds such as d -arabitol and 2MeScZR have a higher significance ranking. In addition, a total of 130 hub genes in this network were categorized into 3 classes based on connectivity. Among them, 34 hub genes were further analyzed through a pathway map, the potential crosstalk between sugar and hormone metabolisms might be an underlying reason of leaf senescence in P. ternata . These findings address the knowledge gap regarding leaf senescence in P. ternata , providing candidate germplasms for molecular breeding and laying theoretical basis for the realization of finely regulated cultivation in future.

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.

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. Graphical Abstract

Abstract 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.

Background Functional sugar alcohols have been widely used in the food, medicine, and pharmaceutical industries for their unique properties. Among these, erythritol is a zero calories sweetener produced by the yeast Yarrowia lipolytica. However, in wild-type strains, erythritol is produced with low productivity and yield and only under high osmotic pressure together with other undesired polyols, such as mannitol or d-arabitol. The yeast is also able to catabolize erythritol in non-stressing conditions. Results Herein, Y. lipolytica has been metabolically engineered to increase erythritol production titer, yield, and productivity from glucose. This consisted of the disruption of anabolic pathways for mannitol and d-arabitol together with the erythritol catabolic pathway. Genes ZWF1 and GND encoding, respectively, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were also constitutively expressed in regenerating the NADPH2 consumed during erythritol synthesis. Finally, the gene RSP5 gene from Saccharomyces cerevisiae encoding ubiquitin ligase was overexpressed to improve cell thermoresistance. The resulting strain HCY118 is impaired in mannitol or d-arabitol production and erythritol consumption. It can grow well up to 35 °C and retain an efficient erythritol production capacity at 33 °C. The yield, production, and productivity reached 0.63 g/g, 190 g/L, and 1.97 g/L·h in 2-L flasks, and increased to 0.65 g/g, 196 g/L, and 2.51 g/L·h in 30-m3 fermentor, respectively, which has economical practical importance. Conclusion The strategy developed herein yielded an engineered Y. lipolytica strain with enhanced thermoresistance and NADPH supply, resulting in a higher ability to produce erythritol, but not mannitol or d-arabitol from glucose. This is of interest for process development since it will reduce the cost of bioreactor cooling and erythritol purification.

Liamocins are polyol lipids produced by the fungus Aureobasidium pullulans , and have selective antibacterial activity against Streptococcus species. Liamocins produced by A. pullulans strain NRRL 50380 on sucrose medium have a d -mannitol head group ester-linked to 3,5-dihydroxydecanoate acyl chains, three or four of which are joined together by 1,5-polyester bonds (liamocins Man-A1 and Man-B1), and similar 3′- O -acetylated analogs (Man-A2 and Man-B2). However, other types of liamocins are produced depending on the choice of strain and growth conditions. In the current study, growth on different polyols, but not sugars, resulted in considerable structural variation, including liamocins with d -galactitol (dulcitol), d -sorbitol (glucitol), d - and l -arabitol, d -xylitol, l -threitol and glycerol head groups. The head groups of liamocins produced on arabitol were shown to be entirely composed of d -arabitol. These liamocin variants were structurally characterized by NMR and MS, and tested for antibacterial activity. The new liamocin variants also had selective activity against Streptococcus . Liamocin structural variants are novel antibacterials against Streptococcus sp. that merit further investigation.

Rhodosporidium toruloides is an oleaginous yeast capable of producing a variety of biofuels and bioproducts from diverse carbon sources. Despite numerous studies showing its promise as a platform microorganism, little is known about its metabolism and physiology. In this work, we investigated the central carbon metabolism in R. toruloides IFO0880 using transcriptomics and metabolomics during growth on glucose, xylose, acetate, or soybean oil. These substrates were chosen because they can be derived from plants. Significant changes in gene expression and metabolite concentrations were observed during growth on these four substrates. We mapped these changes onto the governing metabolic pathways to better understand how R. toruloides reprograms its metabolism to enable growth on these substrates. One notable finding concerns xylose metabolism, where poor expression of xylulokinase induces a bypass leading to arabitol production. Collectively, these results further our understanding of central carbon metabolism in R. toruloides during growth on different substrates. They may also help guide the metabolic engineering and development of better models of metabolism for R. toruloides.Key points• Gene expression and metabolite concentrations were significantly changed.• Reduced expression of xylulokinase induces a bypass leading to arabitol production.• R. toruloides reprograms its metabolism to allow growth on different substrates.

Lactic acid is the monomeric unit of polylactide (PLA), a bioplastic widely used in the packaging, automotive, food, and pharmaceutical industries. Previously, the yeast Komagataella phaffii was genetically modified for the production of lactate from glycerol. For this, the bovine L-lactate dehydrogenase- (LDH)-encoding gene was inserted and the gene encoding the pyruvate decarboxylase (PDC) was disrupted, resulting in the GLp strain. This showed a yield of 67% L-lactic acid and 20% arabitol as a by-product in batches with oxygen limitation. Following up on these results, the present work endeavored to perform a detailed study of the metabolism of this yeast, as well as perturbing arabitol synthesis in an attempt to increase lactic acid titers. The GLp strain was cultivated in a glycerol-limited chemostat at different dilution rates, confirming that the production of both lactic acid and arabitol is dependent on the specific growth rate (and consequently on the concentration of the limiting carbon source) as well as on the oxygen level. Moreover, disruption of the gene encoding arabitol dehydrogenase (ArDH) was carried out, resulting in an increase of 20% in lactic acid and a 50% reduction in arabitol. This study clarifies the underlying metabolic reasons for arabitol formation in K. phaffii and points to ways for improving production of lactic acid using K. phaffii as a biocatalyst.