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Xylitol is an increasingly popular functional food additive, and the newly isolated yeast Wickerhamomyces anomalus WA has shown extensive substrate utilization capability, with the ability to grow on hexose (d-galactose, d-glucose, d-mannose, l-fructose, and d-sorbose) and pentose (d-xylose and l-arabinose) substrates, as well as high tolerance to xylose at concentrations of up to 300 g/L. Optimal xylitol fermentation conditions were achieved at 32 °C, 140 rpm, pH 5.0, and initial cell concentration OD 600 of 2.0, with YP (yeast extract 10 g/L, peptone 20 g/L) as the optimal nitrogen source. Xylitol yield increased from 0.61 g/g to 0.91 g/g with an increase in initial substrate concentration from 20 g/L to 180 g/L. Additionally, 20 g/L glycerol was found to be the optimal co-substrate for xylitol fermentation, resulting in an increase in xylitol yield from 0.82 g/g to 0.94 g/g at 140 rpm, enabling complete conversion of xylose to xylitol., Competing Interests: Declaration of competing interest None of authors have any conflicts of interest to report., (Copyright © 2024 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

The present study examines the impact of nitrogen sources (yeast extract, ammonium sulfate peptone, ammonium nitrate, urea, and sodium nitrate), salt solution (0.5 g/L MgSO 4 , 0.5 g/L KH 2 PO 4 , 0.3 g/L CaCl 2 ), trace elements solution (0.1 g/L CuSO 4 , 0.1 g/L FeSO 4 , 0.02 g/L MnCl 2 , 0.02 g/L ZnSO 4 ), operational parameters (temperature, aeration, agitation, initial pH and xylose concentration) and co- substrate supplementation (glucose, fructose, maltose, sucrose, and glycerol) on xylitol biosynthesis by Candida tropicalis ATCC 13803 using synthetic xylose. The significant medium components were identified using the Plackett Burman design followed by central composite designs to obtain the optimal concentration for the critical medium components in shaker flasks. Subsequently, the effect of operational parameters was examined using the One Factor At a Time method, followed by the impact of five co-substrates on xylitol biosynthesis in a 1 L bioreactor. The optimal media components and process parameters are as follows: peptone: 12.68 g/L, yeast extract: 6.62 g/L, salt solution (0.5 g/L MgSO 4 , 0.5 g/L KH 2 PO 4 , and 0.3 g/L CaCl 2 ): 1.23 X (0.62 g/L, 0.62 g/L, and 0.37 g/L respectively), temperature: 30 °C, pH: 6, agitation: 400 rpm, aeration: 1 vvm, and xylose: 50 g/L. Optimization studies resulted in xylitol yield and productivity of 0.71 ± 0.004 g/g and 1.48 ± 0.018 g/L/h, respectively. Glycerol supplementation (2 g/L) further improved xylitol yield (0.83 ± 0.009 g/g) and productivity (1.87 ± 0.020 g/L/h) by 1.66 and 3.12 folds, respectively, higher than the unoptimized conditions thus exhibiting the potential of C. tropicalis ATCC 13803 being used for commercial xylitol production.

Areca nut husk is the most promising alternative source of low-cost raw materials because it contains a considerable amount of five-carbon monosaccharide sugar in the form of xylose. This polymeric sugar can be isolated and transformed into a value-added chemical using fermentation. To extract sugars from areca nut husk fibers, preliminary pretreatment, such as dilute acid hydrolysis (H 2 SO 4 ), was performed. The hemicellulosic hydrolysate of areca nut husk can produce xylitol through fermentation, but toxic components inhibit the growth of microorganisms. To overcome this, a series of detoxification treatments, including pH adjustment, activated charcoal, and ion exchange resin, were carried out to reduce the concentration of inhibitors in the hydrolysate. This study reports a remarkable 99% removal of inhibitors in the hemicellulosic hydrolysate. Subsequently, a fermentation process using Candida tropicalis (MTCC6192) was executed with the detoxified hemicellulosic hydrolysate of areca nut husk, yielding an optimum xylitol yield of 0.66 g/g. This study concludes that detoxification techniques like pH adjustment, activated charcoal, and ion exchange resins are the most economical and effective methods for eliminating toxic compounds in hemicellulosic hydrolysates. Therefore, the medium derived after detoxification from areca nut hydrolysate may be considered to have significant potential for xylitol production.

In this study, the potential of bagasse pith (the waste of sugar and paper industry) was investigated for bio-xylitol production for the first time. Xylose-rich hydrolysate was prepared using 8% dilute sulfuric acid, at 120 °C for 90 min. Then, the acid-hydrolyzed solution was detoxified by individual overliming (OL), active carbon (AC), and their combination (OL+AC). The amounts of reducing sugars and inhibitors (furfural and hydroxyl methyl furfural) were measured after acid pre-treatment and detoxification process. Thereafter, xylitol was produced from detoxified hydrolysate by Rhodotorula mucilaginosa yeast. Results showed that after acid hydrolysis, the sugar yield was 20%. Detoxification by overliming and active carbon methods increased the reducing sugar content up to 65% and 36% and decreased the concentration of inhibitors to >90% and 16%, respectively. Also, combined detoxification caused an increase in the reducing sugar content (>73%) and a complete removal of inhibitors. The highest productivity of xylitol (0.366 g/g) by yeast was attained after the addition of 100 g/l non-detoxified xylose-rich hydrolysate into fermentation broth after 96 h, while the xylitol productivity enhanced to 0.496 g/g after adding the similar amount of xylose-rich hydrolysate detoxified by combined method (OL+AC2.5%)., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Production of potential value-added products from different lignocellulosic biomass is becoming more common due to the availability of the feedstocks in abundance and the environment- friendly nature of the microbial production process. Due to the large array of its applications in the pharmaceutical and food sectors, xylitol is considered as potential value-added compound for production. In this study, organic waste samples were collected from various habitats and screened for potential yeast isolates for xylitol production. Among 124 tested isolates, Candida tropicalis K2 showed the highest potential for xylitol production as well as inhibitors tolerance (Furfural, 5-hydroxymethyl furfural and acetic acid) phenotypes. C. tropicalis K2 produced 90 g/L of xylitol in batch fermentation (100 g/L xylose supplemented with 20 g/L of glycerol as co-substrate) with the yield and productivity of 0.90 g/g and 1.5 g/L.h, respectively, at pH 5.5 and 30°C temperature. Together, >10% higher xylitol yield was achieved when glycerol was used as a co-substrate with pure xylose. Moreover, with non-detoxified corncob and Albizia pod hydrolysates, C. tropicalis K2 isolate produced 0.62 and 0.69 g/g of xylitol yields and 1.04 and 0.75 g/L.h xylitol productivities, respectively. Thus, C. tropicalis K2 isolate could be considered as promising candidate for xylitol production from different lignocellulosic biomass. HIGHLIGHTS K2 isolate was screened from natural sites of biomass degradation and characterized for xylitol production.Non-detoxified Candia tropicalis pod and corncob hydrolysates were explored for xylitol production using selected Albizia K2 isolate.A maximum of 0.90 g/g yield and 1.07 g/L.h xylitol productivity was achieved with pure xylose.A >10% increase in xylitol yield was achieved using glycerol as a co-substrate.C. tropicalis K2 isolate.A maximum of 0.90 g/g yield and 1.07 g/L.h xylitol productivity was achieved with pure xylose.A >10% increase in xylitol yield was achieved using glycerol as a co-substrate.

Purpose: Erythritol is a valuable compound as sweetener and chemical material however cannot be fermented from the abundant substrate xylose., Methods: The strain Trichosporonoides oedocephalis ATCC 16958 was employed to produce polyols including xylitol and erythritol by metabolic engineering approaches., Results: The introduction of a substrate-specific ribose-5-phosphate isomerase endowed T. oedocephalis with xylose-assimilation activity to produce xylitol, and eliminated glycerol production simultaneously. A more value-added product, erythritol was produced by further introducing a homologous xylulose kinase. The carbon flux was redirected from xylitol to erythritol by adding high osmotic pressure. The production of erythritol was improved to 46.5 g/L in flasks by fermentation adjustment, and the process was scaled up in a 5-L fermentor, with a 40 g/L erythritol production after 120 h, and a time-space yield of 0.56 g/L/h., Conclusion: This study demonstrated the potential of T. oedocephalis in the synthesis of multiple useful products from xylose., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Lignocellulosic biomasses are extensively used by researchers to produce a variety of renewable bioproducts. This research described an environment-friendly technique of xylitol production by an adapted strain of Candida tropicalis from areca nut hemicellulosic hydrolysate, produced through enzymatic hydrolysis. To enhance the activity of xylanase enzymes, lime and acid pretreatment was conducted to make biomass more amenable for saccharification. To improve the efficiency of enzymatic hydrolysis, saccharification parameters like xylanase enzyme loading were varied. Results exposed that the highest yield (g/g) of reducing sugar, about 90%, 83%, and 15%, were achieved for acid-treated husk (ATH), lime-treated husk (LTH), and raw husk (RH) at an enzyme loading of 15.0 IU/g. Hydrolysis was conducted at a substrate loading of 2% (w/V) at 30 °C, 100 rpm agitation, for 12 h hydrolysis time at pH 4.5 to 5.0. Subsequently, fermentation of xylose-rich hemicellulose hydrolysate was conducted with pentose utilizing the yeast Candida tropicalis to produce xylitol. The optimum concentration of xylitol was obtained at about 2.47 g/L, 3.83 g/L, and 5.88 g/L, with yields of approximately 71.02%, 76.78%, and 79.68% for raw fermentative hydrolysate (RFH), acid-treated fermentative hydrolysate (ATFH), and lime-treated fermentative gydrolysate (LTFH), respectively. Purification and crystallization were also conducted to separate xylitol crystals, followed by characterization like X-ray diffraction (XRD) and scanning electron microscopy (SEM) analysis. Results obtained from crystallization were auspicious, and about 85% pure xylitol crystal was obtained., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

This study explores utilizing pineapple peel (PP) hydrolysate as a promising carbon source for xylitol production, covering scopes from the pre-treatment to the fermentation process. The highest xylose concentration achieved was around 20 g/L via mild acid hydrolysis (5% nitric acid, 105 °C, 20-min residence time) with a solid loading of 10%. Two sets fermentability experiments were carried out of varying pH levels in synthetic media that includes acetic acid as the main inhibitors and hydrolysate supplemented with diverse nitrogen source. The results revealed that pH 7 exhibited the highest xylitol production, yielding 0.35 g/g. Furthermore, urea was found to be a highly promising and cost-effective substitute for yeast extract, as it yielded a comparable xylitol production of 0.31 g/g with marginal difference of only 0.01 g/g compared to yeast extract further highlights the viability of urea as the preferred option for reducing xylitol production cost. The absence of a significant difference between the synthetic media and hydrolysate, with only a marginal variance of 0.35 to 0.32 g/g, implies that acetic acid is indeed the primary constraint in xylitol production using PP hydrolysate. The study sheds light on PP biomass's potential for xylitol production, aligning economic benefits with environmental sustainability and waste management., (© 2023. The Author(s).)

Background: Xylitol has a wide range of applications in the pharmaceuticals, cosmetic, food and beverage industry. Microbial xylitol production reduces the risk of contamination and is considered as environment friendly and sustainable compared to the chemical method. In this study, random mutagenesis and genetic engineering approaches were employed to develop Candida tropicalis strains with reduced xylitol dehydrogenase (XDH) activity to eliminate co-substrate requirement for corn cob-based xylitol-ethanol biorefinery., Results: The results suggest that when pure xylose (10% w/v) was fermented in bioreactor, the Ethyl methane sulfonate (EMS) mutated strain (C. tropicalis K2M) showed 9.2% and XYL2 heterozygous (XYL2/xyl2Δ::FRT) strain (C. tropicalis K21D) showed 16% improvement in xylitol production compared to parental strain (C. tropicalis K2). Furthermore, 1.5-fold improvement (88.62 g/L to 132 g/L) in xylitol production was achieved by C. tropicalis K21D after Response Surface Methodology (RSM) and one factor at a time (OFAT) applied for media component optimization. Finally, corncob hydrolysate was tested for xylitol production in biorefinery mode, which leads to the production of 32.6 g/L xylitol from hemicellulosic fraction, 32.0 g/L ethanol from cellulosic fraction and 13.0 g/L animal feed., Conclusions: This work, for the first time, illustrates the potential of C. tropicalis K21D as a microbial cell factory for efficient production of xylitol and ethanol via an integrated biorefinery framework by utilising lignocellulosic biomass with minimum waste generation., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Xylitol, a sugar substitute, is widely used in various food formulations and finds a steady global market. In this study, xylitol crystals were produced from corncob by fermentation (as an alternative to the chemical catalytic process) by a GRAS yeast Pichia caribbica MTCC 5703 and characterized in detail for their purity and presence of any possible contaminant that may adversely affect mammalian cell growth and proliferation. The acute and chronic oral toxicity trials demonstrated no gross pathological changes with average weekly weight gain in female Wistar rats at high xylitol loading (LD 50  > 10,000 mg/kg body weight). The clinical chemistry analysis supported the evidence of no dose-dependent effect by analyzing blood biochemical parameters. The finding suggests the possible application of the crystals (> 98% purity) as a food-grade ingredient for commercial manufacture pending human trials., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Quinoa straw is rich in hemicellulose, and it could be hydrolyzed into xylose. It is a promising energy resource alternative that acts as a potential low-cost material for producing xylitol. In this study, quinoa straw was used as a substrate subjected to the hydrolysis of dilute sulfuric acid solution. Based on the production of xylose and inhibitors during hydrolysis, the optimal conditions for the hydrolysis of hemicellulose in quinoa straw were determined. Detoxification was performed via activated carbon adsorption. The optimal detoxification conditions were determined on the basis of major inhibitor concentrations in the hydrolysate. When the addition of activated carbon was 3% at 30 °C for 40 min, the removal of formic acid, acetic acid, furfural, and 5-HMF could reach 66.52%, 64.54%, 88.31%, and 89.44%, respectively. In addition to activated carbon adsorption, vacuum evaporation was further conducted to perform two-step detoxification. Subsequently, the detoxified hydrolysate was used for xylitol fermentation. The yield of xylitol reached 0.50 g/g after 96 h of fermentation by Candida tropicalis (CICC 1779). It is 1.2-fold higher than that obtained through the sole vacuum evaporation method. This study validated the feasibility of xylitol production from quinoa straw via a biorefinery process.

Xylitol (C 5 H 12 O 5 ), an amorphous sugar alcohol of crystalline texture has received great interest on the global market due to its numerous applications in different industries. In addition to its high anticariogenic and sweetening properties, characteristics such as high solubility, stability and low glycemic index confer xylitol its fame in the food and odontological industries. Moreover, it also serves as a building-block in the production of polymers. As a result of the harmful effects of the chemical production of xylitol, the biotechnological means of producing this polyol have evolved over the decades. In contrast to the high consumption of energy, long periods of purification, specialized equipment and high production cost encountered during its chemical synthesis, the biotechnological production of xylitol offers advantages both to the economy and the environment. Non-Saccharomyces yeast strains, also termed as nonconventional, possess the inherent capacity to utilize D-xylose as a sole carbon source, unlike Saccharomyces species., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Six different yeasts were used to study their metabolism of glucose and xylose, and mainly their capacity to produce ethanol and xylitol. The strains used were Candida guilliermondii, Debaryomyces hansenii, Saccharomyces cerevisiae, Kluyveromyces marxianus, Meyerozyma guilliermondii and Clavispora lusitaniae, four isolated from a rural mezcal fermentation facility. All of them produced ethanol when the substrate was glucose. When incubated in a medium containing xylose instead of glucose, only K. marxianus and M. guilliermondii were able to produce ethanol from xylose. On the other hand, all of them could produce some xylitol from xylose, but the most active in this regard were K. marxianus, M. guilliermondii, C. lusitaniae, and C. guilliermondii with the highest amount of xylitol produced. The capacity of all strains to take up glucose and xylose was also studied. Xylose, in different degrees, produced a redox imbalance in all yeasts. Respiration capacity was also studied with glucose or xylose, where C. guilliermondii, D. hansenii, K. marxianus and M. guilliermondii showed higher cyanide resistant respiration when grown in xylose. Neither xylose transport nor xylitol production were enhanced by an acidic environment (pH 4), which can be interpreted as the absence of a proton/sugar symporter mechanism for xylose transport, except for C. lusitaniae. The effects produced by xylose and their magnitude depend on the background of the studied yeast and the conditions in which these are studied., (Copyright © 2022. Published by Elsevier B.V.)

The present study demonstrates a novel strategy involving two-step fermentation of lignocellulosic hydrolysate for the integrated production of ethanol and xylitol using a newly isolated yeast strain, Candida sojae JCM 1644. The isolated strain was characterised by its carbohydrate assimilation efficiency and tolerance towards inhibitors generated during pretreatment and fermentation of lignocellulosic biomass. In brief, the study comprised alkali treatment of Brassica juncea followed by its saccharification with cellulase consortia. An isolated strain was used for the co-production of xylitol and ethanol from sugar hydrolysate, and several parameters were systematically optimised for maximum co-production of ethanol and xylitol. Out of total glucose (149.72 g/L) and xylose (84.21 g/L) present in biomass hydrolysate, a product yield of 0.45 g/g (ethanol) and 0.62 g/g (xylitol) was achieved for a two-step fermentation process, which was 15.57% and 11.78% higher than the yield achieved for ethanol and xylitol, respectively, in a one-step fermentation process., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Xylitol is pentahydroxy sugar alcohol, existing in very trace amount in fruits and vegetables, and finds varied application in industries like food, pharmaceuticals, confectionaries, etc. and is of prime importance to health. Owing to its trace occurrence in nature and considerable increase in market demand that exceeds availability, alternate production through biotechnological and chemical approach is in process. Biochemical production involves substrates like lignocellulosic biomasses and industrial effluents and is an eco-friendly process with high dependency on physico-chemical parameters. Although the chemical processes are faster, high yielding and economical, they have a great limitation as usage of toxic chemicals and thus need to be regulated and replaced by an environment friendly approach. Microbes play a major role in xylitol production through a biotechnological process towards the development of a sustainable system. Major microbes working on assimilation of xylose for production of xylitol include Candida tropicalis, Candida maltose, Bacillus subtilis, Debaromyces hansenii, etc. The present review reports all probable microbial xylitol production biochemical pathways encompassing diverse bioprocesses involved in uptake and conversion of xylose sugars from agricultural residues and industrial effluents. A comprehensive report on xylitol occurrence and biotechnological production processes with varied substrates has been encompassed. KEY POINTS: • Xylitol from agro-industrial waste • Microbial xylose assimilation., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Xylose, the most abundant pentose sugar of the hemicellulosic fraction of lignocellulosic biomass, has to be utilized rationally for the commercial viability of biorefineries. An effective pre-treatment strategy for the release of xylose from the biomass and an appropriate microbe of the status of an Industrial strain for the utilization of this pentose sugar are key challenges which need special attention for the economic success of the biomass value addition to chemicals. Xylitol and xylonic acid, the alcohol and acid derivatives of xylose are highly demanded commodity chemicals globally with plenty of applications in the food and pharma industries. This review emphasis on the natural and metabolically engineered strains utilizing xylose and the progressive and innovative fermentation strategies for the production and subsequent recovery of the above said chemicals from pre-treated biomass medium., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Xylitol is widely used in the food and pharmaceutical industries as a valuable commodity product. Biotechnological production of xylitol from lignocellulosic biomass by microorganisms is a promising alternative option to chemical synthesis or bioconversion from D-xylose. In this study, four metabolic mutants of Aspergillus niger were constructed and evaluated for xylitol accumulation from D-xylose and lignocellulosic biomass. All mutants had strongly increased xylitol production from pure D-xylose, beechwood xylan, wheat bran and cotton seed hulls compared to the reference strain, but not from several other feed stocks. The triple mutant ΔladAΔxdhAΔsdhA showed the best performance in xylitol production from wheat bran and cotton seed hulls. This study demonstrated the large potential of A. niger for xylitol production directly from lignocellulosic biomass by metabolic engineering., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Exorbitant outputs of waste xylose mother liquor (WXML) and corncob residue from commercial-scale production of xylitol create environmental problems. To reduce the wastes, a Saccharomyces cerevisiae strain tolerant to WXML was conferred with abilities to express the genes of xylose reductase, a xylose-specific transporter and enzymes of the pentose phosphate pathway. This strain showed a high capacity to produce xylitol from xylose in WXML with glucose as a co-substrate. Additionally, a simultaneous saccharification and fermentation (SSF) process was designed to use corncob residues and cellulase instead of directly adding glucose as a co-substrate. Xylitol titer and the productivity were, respectively, 91.0 g l -1 and 1.26 ± 0.01 g l -1  h -1 using 20% WXML, 55 g DCW l -1 delignified corncob residues and 11.8 FPU g cellulose cellulase at 35° during fermentation. This work demonstrates the promising strategy of SSF to exploit waste products to xylitol fermentation process.-1 cellulase at 35° during fermentation. This work demonstrates the promising strategy of SSF to exploit waste products to xylitol fermentation process., (© 2021 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Cofactor availability is often a rate-limiting factor in the bioconversion of xylose to xylitol. The overexpression of pentose phosphate pathway genes and the deletion of Embden-Meyerhof-Parnas pathway genes can modulate the glucose metabolic flux and increase the intracellular NADPH supply, enabling Escherichia coli cells to produce xylitol from corncob hydrolysates. The effects of zwf and/or gnd overexpression and pfkA , pfkB , and/or pgi deletion on the intracellular redox environment and xylitol production were examined. The NADPH-enhanced strain 2bpgi produced 162 g/L xylitol from corncob hydrolysates after a 76 h fed-batch fermentation in a 15 L bioreactor, which was 13.3% greater than the 143 g/L xylitol produced by the IS5-d control strain. Additionally, the xylitol productivity and xylitol yield per glucose for 2bpgi were 2.13 g/L/h and 2.50 g/g, respectively. Thus, the genetic modifications in 2bpgi significantly enhanced NADPH regeneration, making 2bpgi a potentially useful strain for the industrial-scale production of xylitol from detoxified corncob hydrolysates.

We report improved NADPH flux and xylitol biosynthesis in engineered E. coli. Xylitol is produced from xylose via an NADPH dependent reductase. We utilize 2-stage dynamic metabolic control to compare two approaches to optimize xylitol biosynthesis, a stoichiometric approach, wherein competitive fluxes are decreased, and a regulatory approach wherein the levels of key regulatory metabolites are reduced. The stoichiometric and regulatory approaches lead to a 20-fold and 90-fold improvement in xylitol production, respectively. Strains with reduced levels of enoyl-ACP reductase and glucose-6-phosphate dehydrogenase, led to altered metabolite pools resulting in the activation of the membrane bound transhydrogenase and an NADPH generation pathway, consisting of pyruvate ferredoxin oxidoreductase coupled with NADPH dependent ferredoxin reductase, leading to increased NADPH fluxes, despite a reduction in NADPH pools. These strains produced titers of 200 g/L of xylitol from xylose at 86% of theoretical yield in instrumented bioreactors. We expect dynamic control over the regulation of the membrane bound transhydrogenase as well as NADPH production through pyruvate ferredoxin oxidoreductase to broadly enable improved NADPH dependent bioconversions or production via NADPH dependent metabolic pathways., (Copyright © 2021 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Background: Xylitol is a five-carbon sugar alcohol that has numerous beneficial health properties. It has almost the same sweetness as sucrose but has lower energy value compared to the sucrose. Metabolism of xylitol is insulin independent and thus it is an ideal sweetener for diabetics. It is widely used in food products, oral and personal care, and animal nutrition as well. Here we present a two-stage strategy to produce bio-xylitol from D-xylose using a recombinant Pichia pastoris expressing a heterologous xylose reductase gene. The recombinant P. pastoris cells were first generated by a low-cost, standard procedure. The cells were then used as a catalyst to make the bio-xylitol from D-xylose., Results: Pichia pastoris expressing XYL1 from P. stipitis and gdh from B. subtilis demonstrated that the biotransformation was very efficient with as high as 80% (w/w) conversion within two hours. The whole cells could be re-used for multiple rounds of catalysis without loss of activity. Also, the cells could directly transform D-xylose in a non-detoxified hemicelluloses hydrolysate to xylitol at 70% (w/w) yield., Conclusions: We demonstrated here that the recombinant P. pastoris expressing xylose reductase could transform D-xylose, either in pure form or in crude hemicelluloses hydrolysate, to bio-xylitol very efficiently. This biocatalytic reaction happened without the external addition of any NAD(P)H, NAD(P) + , and auxiliary substrate as an electron donor. Our experimental design & findings reported here are not limited to the conversion of D-xylose to xylitol only but can be used with other many oxidoreductase reactions also, such as ketone reductases/alcohol dehydrogenases and amino acid dehydrogenases, which are widely used for the synthesis of high-value chemicals and pharmaceutical intermediates.

The present study focuses on operational parameters for the efficient acid catalyzed rice straw pretreatment process for xylitol production. 75.77 % xylose yield was attained when the 24 h presoaked rice straw (≤10 mm or ≤ 15 mm) in 1.5 % (v/v) H 2 SO 4 was pretreated in the same reactor at 121 °C for 30 min. Neutralization with barium hydroxide produced insoluble salt and noticeably reduced HMF and furfurals. Xylitol yield of 0.6 g/g of xylose, was achieved by fermenting rice straw hydrolysate medium with C. tropicalis MTCC 6192. This two-step process of production of xylitol from xylose rich hydrolysate is much simpler and produced minimal inhibitors including organic acids such as acetic acid. This process is modified for upscaling at optimized parameters and will simultaneously minimize the pollution problem caused by rice straw and is also promising for commercial scale., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The aim of this study was to evaluate the effect of aqueous solutions of deep eutectic solvent, Cholinium Chloride:Urea ([Ch]Cl:U) at 50 wt% and 20 wt%, using different molar ratios (1:1, 2:1 and 1:2) on the enzymatic hydrolysis of xylan for xylose production and its subsequent bioconversion into xylitol using a recombinant yeast strain. The lowest xylan conversion into xylose (45%) was obtained using 1:2 [Ch]Cl:U molar ratio. On the other hand, the 1:1 [Ch]Cl:U molar ratio, at 20 wt% in water, improved this conversion, achieving the highest xylose yield (81.4%). The xylitol production was then optimized with [Ch]Cl:U (1:1) at 20 wt% by simultaneous saccharification and fermentation process, attaining 23.67 g/L, corresponding to 66.04% of xylitol yield. This study reveals the possibility of using xylan solubilized in DES aqueous solutions directly for xylitol production, thus assembling a one-step process., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A novel two-stage fermentation strategy was applied to produce xylitol and ethanol from the whole acid-pretreated corn cob slurry. The acid-pretreated corn cob was used without filtration and detoxification by the two-stage fermentation with the robust Kluyveromyces marxianus CICC 1727-5. In the first stage, xylose in the slurry after dilute acid pretreatment of lignocellulosic biomass was used to produce xylitol under micro-aeration conditions. In the second stage, simultaneous saccharification fermentation was carried out, and the ethanol was produced from glucose releasing from the solid. Important parameters, such as aeration rate, cellulase loading during xylose utilization and SSF fermentation were studied for best performance. The two-stage fermentation strategy removed the inhibition of glucose on xylose, and little xylose was left in the fermentation broth. Under the optimized condition, the maximum ethanol and xylitol concentration were 52 g/L and 24.2 g/L corresponding to the yield of 0.41 g/g and 0.82 g/g, respectively., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Xylitol is a commercially important chemical with multiple applications in the food and pharmaceutical industries. According to the US Department of Energy, xylitol is one of the top twelve platform chemicals that can be produced from biomass. The chemical method for xylitol synthesis is however, expensive and energy intensive. In contrast, the biological route using microbial cell factories offers a potential cost-effective alternative process. The bioprocess occurs under ambient conditions and makes use of biocatalysts and biomass which can be sourced from renewable carbon originating from a variety of cheap waste feedstocks., Result: In this study, biotransformation of xylose to xylitol was investigated using Yarrowia lipolytica, an oleaginous yeast which was firstly grown on a glycerol/glucose for screening of co-substrate, followed by media optimisation in shake flask, scale up in bioreactor and downstream processing of xylitol. A two-step medium optimization was employed using central composite design and artificial neural network coupled with genetic algorithm. The yeast amassed a concentration of 53.2 g/L xylitol using pure glycerol (PG) and xylose with a bioconversion yield of 0.97 g/g. Similar results were obtained when PG was substituted with crude glycerol (CG) from the biodiesel industry (titer: 50.5 g/L; yield: 0.92 g/g). Even when xylose from sugarcane bagasse hydrolysate was used as opposed to pure xylose, a xylitol yield of 0.54 g/g was achieved. Xylitol was successfully crystallized from PG/xylose and CG/xylose fermentation broths with a recovery of 39.5 and 35.3%, respectively., Conclusion: To the best of the author's knowledge, this study demonstrates for the first time the potential of using Y. lipolytica as a microbial cell factory for xylitol synthesis from inexpensive feedstocks. The results obtained are competitive with other xylitol producing organisms.

Recent advances in biomass conversion technologies have shown a promising future toward fermentation during xylitol production. Xylitol is one of the top 12 renewable added-value chemicals that can be obtained from biomass according to US Department of Energy (USDOE). Currently, xylitol accounts for approximately US$823.6 million of annual sales in the market, and this amount is expected to reach US$1.37 billion by 2025. This high demand has been achieved owing to the chemical conversion of hemicellulosic hydrolysates from different lignocellulosic biomasses, which is a costly and non-ecofriendly process. Xylose-rich hemicellulosic hydrolysates are the major raw materials for xylitol production through either chemical or biotechnological routes. Economic production of a clean hemicellulosic hydrolysate is one of the major bottlenecks for xylitol production on the commercial scale. Advancements in biotechnology, such as the isolation of novel microorganisms, genetic manipulation of xylose metabolizing strains, and modifications in the fermentation process, can enhance the economic feasibility of xylitol production on the large scale. Furthermore, xylitol production in integrated biorefineries can be even more economic, given the readily available raw materials and the co-use of steam, electricity, and water, among others. Exploring new biotechnology techniques in integrated biorefineries would open new markets and opportunities for sustainable xylitol production to fulfill the market's growing demands for this sugar alcohol. This article is a review of the advancements reported in the whole biotechnological process for xylitol production, and involve pretreatment technologies, hemicellulosic hydrolysate preparation, xylose conversion into xylitol, and product recovery. Special attention is devoted to current metabolic engineering strategies to improve this bioprocess, as well as to the importance of xylitol production processes in biorefineries.

This work deals with the development of an improved process for xylitol production from corn cob hydrolysate by biotechnological routes emphasizing the detoxification of corncob acid hydrolysate. The acid hydrolysate obtained by acid hydrolysis of corn cob was concentrated and detoxified by activated charcoal, membrane process and ion exchange resin process. The resultant partially purified corncob hydrolysate was used in fermentation. The fermentation of acid hydrolysate containing 56.5 g/L xylose was carried out in a 14 L fermenter at pH 4.5 for 48 h with 150 rpm stirring rate at 30 °C. A xylitol yield of 62% was achieved from the partially purified acid hydrolysate medium during fermentation using Candida tropicalis MTCC 6192. The purity of xylitol was increased to 92-94% upon downstream processing of carbonation, subsequently ion exchange process and activated charcoal., (Copyright © 2019. Published by Elsevier Ltd.)

In this study, recombinant E. coli BL21(DE3)/pCDFDuet-1-XR-GDH harboring xylose reductase (XR) and glucose dehydrogenase (GDH) were immobilized and applied for the production of xylitol from xylose mother liquor (XML). Various immobilization methods were screened and the cross-linking approach with diatomite and polyetherimide as the raw materials and glutaraldehyde as the cross-linking agent was the optimal one, and the recovery activity reached of 80.3% after immobilization. The half-life of immobilized cells was 1.52 times to that of free cells. Batch experiments showed that the enzyme activity of immobilized cells remained 70.5% of the initial activity after 10 batches and the space-time yield of xylitol reached of 11.5 g/(L h). The production of xylitol from xylose mother liquor by immobilized E. coli cells containing xylose reductase and glucose dehydrogenase was reported for the first time, which paved a foundation for industrial production of xylitol from waste xylose mother liquor., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

This study investigates ultrasonic enhancement of xylitol production from sugarcane bagasse using C. tropicalis MTCC 184 immobilized on PU foam. Initial xylitol yield of 0.53 g/g xylose improved to 0.65 g/g of xylose (in 36 h fermentation) after optimization of medium and fermentation parameters. Optimum values of experimental parameters for maximum xylitol were: yeast extract = 5.78 g/L, (NH 4 ) 2 SO 4  = 3.22 g/L, KH 2 PO 4  = 0.58 g/L, MgSO 4 ·7H 2 O = 0.57 g/L and temperature = 29.3 °C, initial pH = 6.2, agitation rate = 151 rpm and initial xylose concentration = 20.9 g/L. Application of 37 kHz sonication @10% duty cycle during fermentation at optimum conditions resulted in marked intensification of fermentation kinetics. Xylitol yield of 0.66 g/g of xylose has been obtained in ultrasound-assisted fermentation in just 15 h. Fitting of time profiles of substrates and products to kinetic model has highlighted actual physical mechanisms underlying 2-fold faster kinetics induced by sonication., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In this work, the industrial Saccharomyces cerevisiae PE-2 strain, presenting innate capacity for xylitol accumulation, was engineered for xylitol production by overexpression of the endogenous GRE3 gene and expression of different xylose reductases from Pichia stipitis. The best-performing GRE3-overexpressing strain was capable to produce 148.5 g/L of xylitol from high xylose-containing media, with a 0.95 g/g yield, and maintained close to maximum theoretical yields (0.89 g/g) when tested in non-detoxified corn cob hydrolysates. Furthermore, a successful integrated strategy was developed for the production of xylitol from whole slurry corn cob in a presaccharification and simultaneous saccharification and fermentation process (15% solid loading and 36 FPU) reaching xylitol yield of 0.93 g/g and a productivity of 0.54 g/L·h. This novel approach results in an intensified valorization of lignocellulosic biomass for xylitol production in a fully integrated process and represents an advance towards a circular economy., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In the present study, recombinant strains were constructed for xylitol production by cloning and expressing the novel xylitol dehydrogenase (xdh) and alcohol dehydrogenase (adh) genes in E. coli BL21 (DE3) from Gluconobacter thailandicus CGMCC1.3748. The optimum pH, temperature, specific activity and kinetic parameters were further investigated for purified XDH. The co-culture of G. thailandicus (30 g/L), BL21-xdh (20 g/L) and BL21-adh (20 g/L) produced 34.34 g/L of xylitol after 48 h in the presence of 40 g/L d-arabitol and 2% ethanol. The concentration of xylitol produced in this co-biotransformation was found to be 2.7-folds higher than the xylitol yield of G. thailandicus alone, while the yield was increased by 4.8% when compared to that of G. thailandicus mixed with BL21-xdh under the similar experimental conditions., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The present study demonstrates utilization of secondary agricultural wastes for xylitol production. The highest xylan-to-xylose (70%) conversion was achieved using dilute nitric acid as catalyst followed by resin treatment. Results show that resin treatment efficiently removed nitrate salt (70%), phenolic content and 5-HMF (70%). Highest xylitol yield (85%) was achieved during fermentation using Candida tropicalis MTCC 6192 from the neutralized hemicellulosic hydrolysate medium. Good recovery (>15%) was achieved from corncob with 85% xylose to xylitol conversion during fermentation. This two-step process for transformation of agri-waste to xylitol is much simpler and it could possibly be considered for up scaling after process optimization parameters., (Copyright © 2017. Published by Elsevier Ltd.)

Recent years have seen an increase in the use of lignocellulosic materials in the development of bioproducts. Because sisal fiber is a low cost raw material and is readily available, this work aimed to evaluate its hemicellulose fraction for the simultaneous production of xylitol and ethanol. The sisal fiber presented a higher hemicellulose content than other frequently-employed biomasses, such as sugarcane bagasse. A pretreatment with dilute acid and low temperatures was conducted in order to obtain the hemicellulose fraction. The highest xylose contents (0.132 g·g -1 of sisal fiber) were obtained at 120 °C with 2.5% ( v / v ) of sulfuric acid. The yeast Candida tropicalis CCT 1516 was used in the fermentation. In the sisal fiber hemicellulose hydrolysate, the maximum production of xylitol (0.32 g·g -1 ) and of ethanol (0.27 g·g -1 ) was achieved in 60 h. Thus, sisal fiber presents as a potential biomass for the production of ethanol and xylitol, creating value with the use of hemicellulosic liquor without detoxification and without the additional steps of alkaline pretreatment., Competing Interests: The authors declare no conflict of interest.

This study evaluated the possibility of using rapeseed straw hemicellulosic hydrolysate as a fermentation medium for xylitol production. Two yeast strains, namely Debaryomyces hansenii and Candida guilliermondii, were used for this bioconversion process and their performance to convert xylose into xylitol was compared. Additionally, different strategies were evaluated for the hydrolysate detoxification before its use as a fermentation medium. Assays in semi-defined media were also performed to verify the influence of hexose sugars on xylose metabolism by the yeasts. C. guilliermondii exhibited higher tolerance to toxic compounds than D. hansenii. Not only the toxic compounds present in the hydrolysate affected the yeast's performance, but glucose also had a negative impact on their performance. It was not necessary to completely eliminate the toxic compounds to obtain an efficient conversion of xylose into xylitol, mainly by C. guilliermondii (Y P/S =0.55g/g and 0.45g/g for C. guilliermondii and D. hansenii, respectively)., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Inefficient transport of xylose into Saccharomyces cerevisiae is a major hurdle for production of xylitol, a natural sweetener with five carbons. To facilitate the xylose transport and hence increase xylose conversion to xylitol, the araE gene encoding an arabinose:H + symporter (AraE) from Bacillus subtilis and the XYL1 gene from Scheffersomyces stipitis were expressed in Saccharomyces cerevisiae EBY.VW4000, a hxt null mutant. The resulting strain of EXHA exhibited 4.1 fold increases in xylose consumption rate and xylitol productivity, relative to the control strain without AraE. Also, overexpression of AraE in wild type S. cerevisiae D452-2 having all hexose transporters and the XYL1 gene increased both xylose consumption and xylitol production considerably. In a glucose-limited fed-batch culture with intermittent addition of xylose, the DXXA strain with multiple copies of araE and XYL1 produced 177.8g/L xylitol with 2.47g/L-h productivity, which were 26.9 and 17.6 times higher than those for a batch culture of the DX strain expressing the XYL1 gene only, respectively. It was concluded that B. subtilis AraE might be a potent xylose transporter and conferred much higher xylose-consuming and xylitol-producing abilities to S. cerevisiae., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Phlebia sp. MG-60 is a white-rot fungus that produces ethanol with high efficiency from lignocellulosic biomass without additional enzymes. Through engineering of this powerful metabolic pathway for fermentation in Phlebia sp. MG-60, chemical compounds other than ethanol could be produced. Here, we demonstrate sugar accumulation from unbleached hardwood kraft pulp and conversion of xylose to xylitol by pyruvate decarboxylase (pdc)-negative Phlebia sp. MG-60. We isolated Phlebia sp. strain MG-60-P2 from protoplasts to unify the protoplast phenotypes of the regenerated strains. Homologous recombination achieved a stable pdc-knockout line, designated KO77. The KO77 line produced traces of ethanol, but accumulated xylitol from xylose or glucose from unbleached hardwood kraft pulp. These metabolic changes in the pdc-knockout strain reflect the potential of metabolic engineering in Phlebia sp. MG-60 for direct production of chemical compounds from lignocellulosic biomass., (Copyright © 2017. Published by Elsevier Ltd.)

Komagataella pastoris was cultivated in glucose/xylose mixtures for production of chitin-glucan complex (CGC), a cell-wall polysaccharide. The culture preferred glucose as substrate for growth, resulting in high biomass yields (0.46-0.54g/g). After glucose depletion, xylose was consumed but no cell growth was observed, indicating K. pastoris was unable to use it for growth. Interestingly, concomitant with xylose consumption, xylitol synthesis was noticed, reaching a maximum concentration of 7.64g/L, with a yield on xylose of 0.52g/g. Lower CGC production was reached as the xylose content was increased in the substrate mixtures, due to the lower biomass production. Moreover, cultivation in the presence of xylitol resulted in CGC enriched in chitin with higher molecular weight. These results suggest the possibility of using K. pastoris for the co-production of CGC and xylitol using glucose/xylose-rich substrates. It may also be a strategy to tailor CGC composition and average molecular weight., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Xylitol production from xylose by the yeast Candida magnoliae TISTR 5663 was enhanced by supplementing the fermentation medium with furfural (300mg/L) and glucose (3g/L with an initial mass ratio of glucose to xylose of 1:10) together under oxygen limiting conditions. In the presence of furfural and glucose, the final concentration of xylitol was unaffected relative to control cultures but the xylitol yield on xylose increased by about 5%. Supplementation of the culture medium with glucose alone at an initial concentration of 3g/L, stimulated the volumetric and specific rates of xylose consumption and the rate of xylitol production from xylose. In a culture medium containing 30g/L xylose, 300mg/L furfural and 3g/L glucose, the volumetric production rate of xylitol was 1.04g/L h and the specific production rate was 0.169g/g h. In the absence of furfural and glucose, the volumetric production rate of xylitol was ∼35% lower and the specific production rate was nearly 30% lower. In view of these results, xylose-containing lignocellulosic hydrolysates contaminated with furfural can be effectively used for producing xylitol by fermentation so long as the glucose-to-xylose mass ratio in the hydrolysate does not exceed 1:10 and the furfural concentration is ≤300mg/L., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Xylitol is a non-fermentable sugar alcohol used as sweetener. Corynebacterium glutamicum ATCC13032 was metabolically engineered for xylitol production from the lignocellulosic pentose sugars xylose and arabinose. Direct conversion of xylose to xylitol was achieved through the heterologous expression of NAD(P)H-dependent xylose reductase (xr) gene from Rhodotorula mucilaginosa. Xylitol synthesis from arabinose was attained through polycistronic expression of l-arabinose isomerase (araA), d-psicose 3 epimerase (dpe) and l-xylulose reductase (lxr) genes from Escherichia coli, Agrobacterium tumefaciens and Mycobacterium smegmatis, respectively. Expression of xr and the synthetic araA-dpe-lxr operon under the control of IPTG-inducible Ptac promoter enabled production of xylitol from both xylose and arabinose in the mineral (CGXII) medium with glucose as carbon source. Additional expression of a pentose transporter (araTF) gene enhanced xylitol production by about four-fold compared to the parent strain. The constructed strain Cg-ax3 produced 6.7±0.4g/L of xylitol in batch fermentations and 31±0.5g/L of xylitol in fed-batch fermentations with a specific productivity of 0.28±0.05g/g cdw/h. The strain Cg-ax3 was also validated for xylitol production from pentose rich, acid pre-treated liquor of sorghum stover (SAPL) and the results were comparable in both SAPL (27±0.3g/L) and mineral medium (31±0.5g/L)., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Cotton stalk is a widely distributed and abundant lignocellulosic waste found in Turkey. Because of its rich xylose content, it can be a promising source for the production of xylitol. Xylitol can be produced by chemical or biotechnological methods. Because the biotechnological method is a simple process with great substrate specificity and low energy requirements, it is more of an economic alternative for the xylitol production. This study aimed to use cotton stalk for the production of xylitol with Candida tropicalis Kuen 1022. For this purpose, the combined effects of different oxygen concentration, inoculum level and substrate concentration were investigated to obtain high xylitol yield and volumetric xylitol production rate. Candida tropicalis Kuen 1022 afforded different concentrations of xylitol depending on xylose concentration, inoculum level, and oxygen concentration. The optimum xylose, yeast concentration, and airflow rate for cotton stalk hydrolysate were found as 10.41 g L(-1), 0.99 g L(-1), and 1.02 vvm, respectively, and under these conditions, xylitol yield and volumetric xylitol production rate were obtained as 36% and 0.06 g L(-1) hr(-1), respectively. The results of this study show that cotton stalk can serve as a potential renewable source for the production of xylitol.

The yeast Candida guilliermondii FTI 20037 is well-known for its ability to produce xylitol from xylose. Recently, this strain was found to produce greater than 5% (w/v) ethanol from glucose. This level of ethanol is typically not exceeded by wild-type strains of other native pentose-fermenting yeasts. This prompted the current study to examine the ability of C. guilliermondii FTI 20037 to utilize and ferment high concentrations of each of the hexoses commonly found in lignocellulosic hydrolysates. In defined media, FTI 20037 fermented 14.4%-25.9% (w/v) of glucose, mannose or galactose individually to ethanol in concentrations ranging from 6% to 9.3% (w/v). Fermentation was completed within 36 h (for glucose) to 100 h (for galactose). In 25.9% (w/v) glucose, FTI 20037 produced 9.3% (w/v) ethanol within 40 h. FTI 20037 produced xylitol exclusively when xylose was given as the sole carbon source. The strain utilized arabinose poorly. Under the same fermentation conditions, an industrial Saccharomyces cerevisiae strain produced slightly higher levels of ethanol [9.9% (w/v)] from 25.0% (w/v) glucose. Another pentose-fermenting yeast Pachysolen tannophilus also fermented high concentrations of glucose and mannose to produce relatively high peak ethanol concentrations; however, this yeast required considerably longer to completely consume these hexoses. The ability of FTI 20037 to produce high level of ethanol rapidly from glucose is remarkable. To our knowledge, this is the first known instance of a non-modified native xylose-fermenting yeast strain able to produce such high levels of ethanol from glucose as rapidly as S. cerevisiae in a defined medium., (Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The yeast Candida tropicalis DSM 7524 produces xylitol, a natural, low-calorie sweetener, by fermentation of xylose. In order to increase xylitol production rate during the submerged fermentation process, some parameters-substrate (xylose) concentration, pH, aeration rate, temperature and fermentation strategy-have been optimized. The maximum xylitol yield reached at 60-80 g/L initial xylose concentration, pH 5.5 at 37 °C was 83.66% (w/w) on consumed xylose in microaerophilic conditions (kLa = 2·h(-1)). Scaling up on 3 L fermenter, with a fed-batch strategy, the best xylitol yield was 86.84% (w/w), against a 90% of theoretical yield. The hyper-acidophilic behaviour of C. tropicalis makes this strain particularly promising for industrial application, due to the possibility to work in non-sterile conditions.

Three transporter genes including Kluyveromyces marxianus aquaglyceroporin gene (KmFPS1), Candida intermedia glucose/xylose facilitator gene (CiGXF1) or glucose/xylose symporter gene (CiGXS1) were over-expressed in K. marxianus YZJ017 to improve xylitol production at elevated temperatures. The xylitol production of YZJ074 that harbored CiGXF1 was improved to 147.62g/L in Erlenmeyer flask at 42°C. In fermenter, 99.29 and 149.60g/L xylitol were produced from 99.55 and 151.91g/L xylose with productivity of 4.14 and 3.40g/L/h respectively at 42°C. Even at 45°C, YZJ074 could produce 101.30g/L xylitol from 101.41g/L xylose with productivity of 2.81g/L/h. Using fed-batch fermentation through repeatedly adding non-sterilized substrate directly, YZJ074 could produce 312.05g/L xylitol which is the highest yield reported to date. The engineered strains YZJ074 which can produce xylitol at elevated temperatures is an excellent foundation for xylitol bioconversion., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Several recombinant Kluyveromyces marxianus strains were constructed through overexpressing the Neurospora crassa xylose reductase genes. YZJ015, which maintained the original xylitol dehydrogenase gene, produced xylitol with the highest productivity (1.49 g L(-1) h(-1)) from 100 g L(-1) xylose at 42 °C. Even at 45 °C, YZJ015 was still able to produce 60.03 g L(-1) xylitol from 100 g L(-1) xylose with a productivity of 1.25 g L(-1)h(-1). In addition, for 20 rounds of cell recycling at 42 °C, YZJ015 produced 71.35 g L(-1) xylitol from 100 g L(-1) xylose with a productivity of 4.43 g L(-1) h(-1) per cycle. YZJ017, in which the xylitol dehydrogenase gene was disrupted, produced 100.02 g L(-1) xylitol at a yield of 1.01 g g(-1) from 100 g L(-1) xylose with 40 g L(-1) glycerol as co-substrate at 42 °C. These engineered strains provide an excellent foundation for xylitol production at elevated temperatures., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Escherichia coli engineered to uptake xylose while metabolizing glucose was previously shown to produce high levels of xylitol from a mixture of glucose and xylose when expressing NADPH-dependent xylose reductase from Candida boidinii (CbXR) (Cirino et al., Biotechnol Bioeng. 2006;95:1167-1176). We then described the effects of deletions of key metabolic pathways (e.g., Embden-Meyerhof-Parnas and pentose phosphate pathway) and reactions (e.g., transhydrogenase and NADH dehydrogenase) on resting-cell xylitol yield (Y RPG: moles of xylitol produced per mole of glucose consumed) (Chin et al., Biotechnol Bioeng. 2009;102:209-220). These prior results demonstrated the importance of direct NADPH supply by NADP+-utilizing enzymes in central metabolism for driving heterologous NADPH-dependent reactions. This study describes strain modifications that improve coupling between glucose catabolism (oxidation) and xylose reduction using two fundamentally different strategies. We first examined the effects of deleting the phosphofructokinase (pfk) gene(s) on growth-uncoupled xylitol production and found that deleting both pfkA and sthA (encoding the E. coli-soluble transhydrogenase) improved the xylitol Y RPG from 3.4 ± 0.6 to 5.4 ± 0.4. The second strategy focused on coupling aerobic growth on glucose to xylitol production by deleting pgi (encoding phosphoglucose isomerase) and sthA. Impaired growth due to imbalanced NADPH metabolism (Sauer et al., J Biol Chem. 2004;279:6613-6619) was alleviated upon expressing CbXR, resulting in xylitol production similar to that of the growth-uncoupled precursor strains but with much less acetate secretion and more efficient utilization of glucose. Intracellular nicotinamide cofactor levels were also quantified, and the magnitude of the change in the NADPH/NADP+ ratio measured from cells consuming glucose in the absence vs. presence of xylose showed a strong correlation to the resulting Y RPG., (Copyright © 2011 American Institute of Chemical Engineers (AIChE).)

The interaction of D-xylose isomerase purified from two sources with Mn2+ and D-xylose or the competitive inhibitor xylitol has been examined by nuclear magnetic resonance. A greater paramagnetic effect of enzyme-bound Mn2+ on the alpha anomer of D-xylose than on the beta anomer was observed, providing independent evidence for the specificity of D-xylose isomerase for the alpha anomeric form of D-xylose. The exchange rate of alpha-D-xylose into the ternary complex, determined from the normalized paramagnetic contribution to the transverse relaxation rate (1/fT2p) of the carbon 1 proton of alpha-D-xylose, exceeds Vmax for the enzymatic reaction by 3 orders of magnitude. The amount of xylitol necessary to displace alpha-D-xylose from the substrate-enzyme-Mn2+ complex is consistent with the Km value for alpha-D-xylose and the inhibitor constant Ki for xylitol previously determined by the methods of enzyme kinetics. These results suggest that the NMR experiments observe complexes of D-xylose isomerase which are kinetically and thermodynamically competent to participate in catalysis. From the frequency dependence of the paramagnetic contribution to the longitudinal relaxation rate (1/T1p) of the carbon 1 proton of alpha-D-xylose, the correlation time (tauc) which modulates the dipolar interaction between enzyme-bound Mn2+ and alpha-D-xylose has been determined (5.1 x 1o(-10) s). From these observations a range of calculated distances between enzyme-bound Mn2+ and the carbon 1 proton of alpha-D-xylose (9.1 +/- 0.7 A) has been found. The enzyme-bound Mn2+ has comparable effects on the carbon 1, carbon 2, and carbon 5 protons of alpha-D-xylose, suggesting that these protons of the enzyme-bound substrate are equidistant from the bound Mn2+. A similar distance (9.4 +/- 0.7 A) between the enzyme-bound Mn2+ and the terminal methylene protons of xylitol, an analog of the open chain intermediate in the reaction, has been determined. The results of the present substrate relaxation and previous water relaxation studies suggest that two small ligands such as water molecules or a large portion of the protein intervene between the bound metal ion and the bound substrate in the active ternary complex.

Mortlock, R. P. (Michigan State University, East Lansing), and W. A. Wood. Metabolism of pentoses by Aerobacter aerogenes. II. Mechanism of acquisition of kinase, isomerase, and dehydrogenase activity. J. Bacteriol. 88:845-849. 1964.-Aerobacter aerogenes PRL-R3 possesses the genetic information to synthesize, in the presence of the appropriate inducer, at least three members of the family of ketopentokinases, two members of the family of pentitol dehydrogenases, and two members of the family of aldopentose isomerases. That is, d-xylulokinase, d-ribulokinase, l-ribulokinase, ribitol dehydrogenase, d-arabitol dehydrogenase, d-xylose (--> d-xylulose) isomerase, and l-arabinose (--> l-ribulose) isomerase activities were detectable within 4 hr after addition of inducer. The possibility that mutation and selection are involved in the formation of l-xylulokinase, l-arabitol dehydrogenase, d-arabinose (--> d-ribulose) isomerase and d-lyxose (--> d-xylulose) isomerase could not be eliminated, because 11 hr or more of incubation after the addition of inducer were required before the appearance of these enzyme activities. d-Xylulokinase activity was induced in less than 2 hr when d-xylose or d-arabitol were inducers, but 45 hr were required for the appearance of activity when xylitol was the inducer, and 83 hr were required when d-lyxose was the inducer. Likewise, the time required for induction of ribitol dehydrogenase was 2 hr for ribitol, 12 hr for d-arabinose, and 45 hr for xylitol. The time required for the appearance of enzyme activity correlated with the time required for the beginning of cell growth and substrate utilization.

Mortlock, R. P. (Michigan State University, East Lansing), D. D. Fossitt, D. H. Petering, and W. A. Wood. Metabolism of pentoses and pentitols by Aerobacter aerogenes. III. Physical and immunological properties of pentitol dehydrogenases and pentulokinases. J. Bacteriol. 89:129-135. 1965.-Four pentulokinases and three pentitol dehydrogenases were purified from Aerobacter aerogenes PRL-R3, and the properties of the enzymes within each family were compared. d-Ribulokinase was purified from cells grown on ribitol, d-arabitol, and xylitol, and from a mutant constitutive for ribitol dehydrogenase; d-xylulokinase, from cells grown on d-xylose and xylitol; l-ribulokinase, from cells grown on l-arabinose; and l-xylulokinase, after growth on l-xylose. Similarly, ribitol dehydrogenase was purified after growth on ribitol, d-arabinose, and xylitol, and from the ribitol dehydrogenase-constitutive mutant. d-Arabitol dehydrogenase was obtained after growth on d-arabitol or d-xylose, and xylitol dehydrogenase was obtained after growth on xylitol. Except for l-xylulokinase, which also had a different S(20) value, the pentulokinases had identical pH optima, K(m) for the ketopentose, and sedimentation constants, and fractionated identically by a number of procedures. These kinases could be distinguished only by their substrate and immunological specificity. Ribitol dehydrogenase and d-arabitol dehydrogenase could be distinguished by several properties, but the properties of xylitol dehydrogenase were always similar to ribitol dehydrogenase. In all cases, individual kinases or dehydrogenases produced by different inducers had identical properties. These data constitute evidence against multiple forms of the same enzyme being produced by different inducers and against the dehydrogenase family containing essentially identical proteins differing only at the active site. For the kinases, three of the four appear to differ only at the active site.