Efficient production of polyhydroxybutyrate using lignocellulosic biomass derived from oil palm trunks by the inhibitor-tolerant strain Burkholderia ambifaria E5-3.

Lignocellulosic biomass is a valuable, renewable substrate for the synthesis of polyhydroxybutyrate (PHB), an ecofriendly biopolymer. In this study, bacterial strain E5-3 was isolated from soil in Japan; it was identified as Burkholderia ambifaria strain E5-3 by 16 S rRNA gene sequencing. The strain showed optimal growth at 37 °C with an initial pH of 9. It demonstrated diverse metabolic ability, processing a broad range of carbon substrates, including xylose, glucose, sucrose, glycerol, cellobiose, and, notably, palm oil. Palm oil induced the highest cellular growth, with a PHB content of 65% wt. The strain exhibited inherent tolerance to potential fermentation inhibitors derived from lignocellulosic hydrolysate, withstanding 3 g/L 5-hydroxymethylfurfural and 1.25 g/L acetic acid. Employing a fed-batch fermentation strategy with a combination of glucose, xylose, and cellobiose resulted in PHB production 2.7-times that in traditional batch fermentation. The use of oil palm trunk hydrolysate, without inhibitor pretreatment, in a fed-batch fermentation setup led to significant cell growth with a PHB content of 45% wt, equivalent to 10 g/L. The physicochemical attributes of xylose-derived PHB produced by strain E5-3 included a molecular weight of 722 kDa, a number-average molecular weight of 191 kDa, and a polydispersity index of 3.78. The amorphous structure of this PHB displayed a glass transition temperature of 4.59 °C, while its crystalline counterpart had a melting point of 171.03 °C. This research highlights the potential of lignocellulosic feedstocks, especially oil palm trunk hydrolysate, for PHB production through fed-batch fermentation by B. ambifaria strain E5-3, which has high inhibitor tolerance.

Optimising parameters for pilot scale steam explosion and continuous pressurised disc refining of Miscanthus and sugarcane bagasse for xylose and xylo-oligosaccharide release.

The first comparative pre-treatment study of Miscanthus (Mxg) and sugarcane bagasse (SCB) using steam explosion (SE) and pressurised disc refining (PDR) pretreatment to optimise xylose and xylo-oligosaccharide release is described. The current investigation aimed to 1) Develop optimised batch-wise steam explosion parameters for Mxg and SCB, 2) Scale from static batch steam explosion to dynamic continuous pressurised disc refining, 3) Identify, understand, and circumvent scale-up production hurdles. Optimised SE parameters released 82% (Mxg) and 100% (SCB) of the available xylan. Scaling to PDR, Miscanthus yielded 85% xylan, highlighting how robust scouting assessments for boundary process parameters can result in successful technical transfer. In contrast, SCB technical transfer was not straightforward, with significant differences observed between the two processes, 100% (SE) and 58% (PDR). This report underlines the importance of feedstock-specific pretreatment strategies to underpin process development, scale-up, and optimisation of carbohydrate release from biomass.

Spathaspora marinasilvae sp. nov., a xylose-fermenting yeast isolated from galleries of passalid beetles and rotting wood in the Amazonian rainforest biome.

Four yeast isolates were obtained from rotting wood and galleries of passalid beetles collected in different sites of the Brazilian Amazonian Rainforest in Brazil. This yeast produces unconjugated allantoid asci each with a single elongated ascospore with curved ends. Sequence analysis of the internal transcribed spacer-5.8 S region and the D1/D2 domains of the large subunit ribosomal RNA (rRNA) gene showed that the isolates represent a novel species of the genus Spathaspora. The novel species is phylogenetically related to a subclade containing Spathaspora arborariae and Spathaspora suhii. Phylogenomic analysis based on 1884 single-copy orthologs for a set of Spathaspora species whose whole genome sequences are available confirmed that the novel species represented by strain UFMG-CM-Y285 is phylogenetically close to Sp. arborariae. The name Spathaspora marinasilvae sp. nov. is proposed to accommodate the novel species. The holotype of Sp. marinasilvae is CBS 13467 T (MycoBank 852799). The novel species was able to accumulate xylitol and produce ethanol from d-xylose, a trait of biotechnological interest common to several species of the genus Spathaspora.

Enhanced Biosynthesis of d-Allulose from a d-Xylose-Methanol Mixture and Its Self-Inductive Detoxification by Using Antisense RNAs in Escherichia coli.

d-Allulose, a C-3 epimer of d-fructose, has great market potential in food, healthcare, and medicine due to its excellent biochemical and physiological properties. Microbial fermentation for d-allulose production is being developed, which contributes to cost savings and environmental protection. A novel metabolic pathway for the biosynthesis of d-allulose from a d-xylose-methanol mixture has shown potential for industrial application. In this study, an artificial antisense RNA (asRNA) was introduced into engineered Escherichia coli to diminish the flow of pentose phosphate (PP) pathway, while the UDP-glucose-4-epimerase (GalE) was knocked out to prevent the synthesis of byproducts. As a result, the d-allulose yield on d-xylose was increased by 35.1%. Then, we designed a d-xylose-sensitive translation control system to regulate the expression of the formaldehyde detoxification operon (FrmRAB), achieving self-inductive detoxification by cells. Finally, fed-batch fermentation was carried out to improve the productivity of the cell factory. The d-allulose titer reached 98.6 mM, with a yield of 0.615 mM/mM on d-xylose and a productivity of 0.969 mM/h.

Use of xylosidase 3C from Segatella baroniae to discriminate xylan non-reducing terminus substitution characteristics.

OBJECTIVE: New characterized carbohydrate-active enzymes are needed for use as tools to discriminate complex carbohydrate structural features. Fungal glycoside hydrolase family 3 (GH3) ß-xylosidases have been shown to be useful for the structural elucidation of glucuronic acid (GlcA) and arabinofuranose (Araf) substituted oligoxylosides. A homolog of these GH3 fungal enzymes from the bacterium Segatella baroniae (basonym Prevotella bryantii), Xyl3C, has been previously characterized, but those studies did not address important functional specificity features. In an interest to utilize this enzyme for laboratory methods intended to discriminate the structure of the non-reducing terminus of substituted xylooligosaccharides, we have further characterized this GH3 xylosidase. RESULTS: In addition to verification of basic functional characteristics of this xylosidase we have determined its mode of action as it relates to non-reducing end xylose release from GlcA and Araf substituted oligoxylosides. Xyl3C cleaves xylose from the non-reducing terminus of ß-1,4-xylan until occurrence of a penultimate substituted xylose. If this substitution is O2 linked, then Xyl3C removes the non-reducing xylose to leave the substituted xylose as the new non-reducing terminus. However, if the substitution is O3 linked, Xyl3C does not hydrolyze, thus leaving the substitution one-xylose (penultimate) from the non-reducing terminus. Hence, Xyl3C enables discrimination between O2 and O3 linked substitutions on the xylose penultimate to the non-reducing end. These findings are contrasted using a homologous enzyme also from S. baroniae, Xyl3B, which is found to yield a penultimate substituted nonreducing terminus regardless of which GlcA or Araf substitution exists.

Selective lignin arylation for biomass fractionation and benign bisphenols.

Lignocellulose is mainly composed of hydrophobic lignin and hydrophilic polysaccharide polymers, contributing to an indispensable carbon resource for green biorefineries1,2. When chemically treated, lignin is compromised owing to detrimental intra- and intermolecular crosslinking that hampers downstream process3,4. The current valorization paradigms aim to avoid the formation of new C-C bonds, referred to as condensation, by blocking or stabilizing the vulnerable moieties of lignin5-7. Although there have been efforts to enhance biomass utilization through the incorporation of phenolic additives8,9, exploiting lignin's proclivity towards condensation remains unproven for valorizing both lignin and carbohydrates to high-value products. Here we leverage the proclivity by directing the C-C bond formation in a catalytic arylation pathway using lignin-derived phenols with high nucleophilicity. The selectively condensed lignin, isolated in near-quantitative yields while preserving its prominent cleavable ß-ether units, can be unlocked in a tandem catalytic process involving aryl migration and transfer hydrogenation. Lignin in wood is thereby converted to benign bisphenols (34-48 wt%) that represent performance-advantaged replacements for their fossil-based counterparts. Delignified pulp from cellulose and xylose from xylan are co-produced for textile fibres and renewable chemicals. This condensation-driven strategy represents a key advancement complementary to other promising monophenol-oriented approaches targeting valuable platform chemicals and materials, thereby contributing to holistic biomass valorization.

New lactate- and pyruvate-containing polysaccharide and rhamnomannan with xylose residues from the cell wall of Rathayibacter oskolensis VKM Ac-2121T.

The cell wall of endophytic strain Rathayibacter oskolensis VKM Ac-2121T (family Microbacteriaceae, class Actinomycetes) was found to contain neutral and acidic glycopolymers. The neutral polymer is a block-type rhamnomannan partially should be substitutied by xylose residues, [→2)-α-[ß-D-Xylp-(1 â†’ 3)]-D-Manp-(1 â†’ 3)-α-D-Rhap-(1→]∼30 [→2)-α-D-Manp-(1 â†’ 3)-α-D-Rhap-(1→]∼45. The acidic polymer has branched chain, bearing lactate and pyruvate residues, →4)-α-D-[S-Lac-(2-3)-α-L-Rhap-(1 â†’ 3)]-D-Manp-(1 â†’ 3)-α-D-[4,6-R-Pyr]-D-Galp-(1 â†’ 3)-ß-D-Glcp-(1 â†’. The structures of both glycopolymers were not described in the Gram-positive bacteria to date. The glycopolymers were studied by chemical and NMR spectroscopic methods. The results of this study provide new data on diversity of bacterial glycopolymers and may prove useful in the taxonomy of the genus Rathayibacter and for understanding the molecular mechanisms of interaction between plants and plant endophytes.

Agricultural Waste Management by Production of Second-Generation Bioethanol from Sugarcane Bagasse Using Indigenous Yeast Strain.

In the wake of rapid industrialization and burgeoning transportation networks, the escalating demand for fossil fuels has accelerated the depletion of finite energy reservoirs, necessitating urgent exploration of sustainable alternatives. To address this, current research is focusing on renewable fuels like second-generation bioethanol from agricultural waste such as sugarcane bagasse. This approach not only circumvents the contentious issue of food-fuel conflicts associated with biofuels but also tackles agricultural waste management. In the present study indigenous yeast strain, Clavispora lusitaniae QG1 (MN592676), was isolated from rotten grapes to ferment xylose sugars present in the hemicellulose content of sugarcane bagasse. To liberate the xylose sugars, dilute acid pretreatment was performed. The highest reducing sugars yield was 1.2% obtained at a temperature of 121 °C for 15 min, a solid-to-liquid ratio of 1:25 (% w/v), and an acid concentration of 1% dilute acid H2SO4 that was significantly higher (P < 0.001) yield obtained under similar conditions at 100 °C for 1 h. The isolated strain was statistically optimized for fermentation process by Plackett-Burman design to achieve the highest ethanol yield. Liberated xylose sugars were completely utilized by Clavispora lusitaniae QG1 (MN592676) and gave 100% ethanol yield. This study optimizes both fermentation process and pretreatment of sugarcane bagasse to maximize bioethanol yield and demonstrates the ability of isolated strain to effectively utilize xylose as a carbon source. The desirable characteristics depicted by strain Clavispora lusitaniae shows its promising utilization in management of industrial waste like sugarcane bagasse by its conversion into renewable biofuels like bioethanol.

Genomic mining of Geobacillus stearothermophilus GF16 for xylose production from hemicellulose-rich biomasses using secreted enzymes.

The valorization of lignocellulosic biomass, derived from various bio-waste materials, has received considerable attention as a sustainable approach to improve production chains while reducing environmental impact. Microbial enzymes have emerged as key players in the degradation of polysaccharides, offering versatile applications in biotechnology and industry. Among these enzymes, glycoside hydrolases (GHs) play a central role. Xylanases, in particular, are used in a wide range of applications and are essential for the production of xylose, which can be fermented into bioethanol or find use in many other industries. Currently, fungal secretomes dominate as the main reservoir of lignocellulolytic enzymes, but thermophilic microorganisms offer notable advantages in terms of enzyme stability and production efficiency. Here we present the genomic characterization of Geobacillus stearothermophilus GF16 to identify genes encoding putative enzymes involved in lignocellulose degradation. Thermostable GHs secreted by G. stearothermophilus GF16 were investigated and found to be active on different natural polysaccharides and synthetic substrates, revealing an array of inducible GH activities. In particular, the concentrated secretome possesses significant thermostable xylanase and ß-xylosidase activities (5 ×103 U/L and 1.7 ×105 U/L, respectively), highlighting its potential for application in biomass valorization. We assessed the hemicellulose hydrolysis capabilities of various agri-food wastes using the concentrated secretome of the strain cultivated on xylan. An impressive 300-fold increase in xylose release compared to a commercially available cocktail was obtained with the secretome, underscoring the remarkable efficacy of this approach.

Deficiency of ß-xylosidase activity in Aspergillus luchuensis mut. kawachii IFO 4308.

In this study, we investigated a deleterious mutation in the ß-xylosidase gene, xylA (AkxylA), in Aspergillus luchuensis mut. kawachii IFO 4308 by constructing an AkxylA disruptant and complementation strains of AkxylA and xylA derived from A. luchuensis RIB2604 (AlxylA), which does not harbor the mutation in xylA. Only the AlxylA complementation strain exhibited significantly higher growth and substantial ß-xylosidase activity in medium containing xylan, accompanied by an increase in XylA expression. This resulted in lower xylobiose and higher xylose concentrations in the mash of barley shochu. These findings suggest that the mutation in xylA affects xylose levels during the fermentation process. Because the mutation in xylA was identified not only in the genome of strain IFO 4308 but also the genomes of other industrial strains of A. luchuensis and A. luchuensis mut. kawachii, these findings enhance our understanding of the genetic factors that affect the fermentation characteristics.

Establishment of low-cost production platforms of polyhydroxyalkanoate bioplastics from Halomonas cupida J9.

Microbial production of polyhydroxyalkanoate (PHA) is greatly restricted by high production cost arising from high-temperature sterilization and expensive carbon sources. In this study, a low-cost PHA production platform was established from Halomonas cupida J9. First, a marker-less genome-editing system was developed in H. cupida J9. Subsequently, H. cupida J9 was engineered to efficiently utilize xylose for PHA biosynthesis by introducing a new xylose metabolism module and blocking xylonate production. The engineered strain J9UΔxylD-P8xylA has the highest PHA yield (2.81 g/L) obtained by Halomonas with xylose as the sole carbon source so far. This is the first report on the production of short- and medium-chain-length (SCL-co-MCL) PHA from xylose by Halomonas. Interestingly, J9UΔxylD-P8xylA was capable of efficiently utilizing glucose and xylose as co-carbon sources for PHA production. Furthermore, fed-batch fermentation of J9UΔxylD-P8xylA coupled to a glucose/xylose co-feeding strategy reached up to 12.57 g/L PHA in a 5-L bioreactor under open and unsterile condition. Utilization of corn straw hydrolysate as the carbon source by J9UΔxylD-P8xylA reached 7.0 g/L cell dry weight (CDW) and 2.45 g/L PHA in an open fermentation. In summary, unsterile production in combination with inexpensive feedstock highlights the potential of the engineered strain for the low-cost production of PHA from lignocellulose-rich agriculture waste.

Deletion of yghZ in Escherichia coli promotes growth in presence of furfural with xylose as carbon source.

Thermo-acidic pretreatment of lignocellulosic biomass is required to make it amenable to microbial metabolism and results in generation of furfural due to breakdown of pentose sugars. Furfural is toxic to microbial metabolism and results in reduced microbial productivity and increased production costs. This study asks if deletion of yghZ gene which encodes a NADPH-dependent aldehyde reductase enzyme results in improved furfural tolerance in Escherichia coli host. The ∆yghZ strain-SSK201-was tested for tolerance to furfural in presence of 5% xylose as a carbon source in AM1 minimal medium. At 96 h and in presence of 1.0 g/L furfural, the culture harboring strain SSK201 displayed 4.5-fold higher biomass, 2-fold lower furfural concentration and 15.75-fold higher specific growth rate (µ) as compared to the parent strain SSK42. The furfural tolerance advantage of SSK201 was retained when the carbon source was switched to glucose in AM1 medium and was lost in rich LB medium. The findings have potential to be scaled up to a hydrolysate culture medium, which contains furan inhibitors and lack nutritionally rich components, under bioreactor cultivation and observe growth advantage of the ∆yghZ host. It harbors potential to generate robust industrial strains which can convert lignocellulosic carbon into metabolites of interest in a cost-efficient manner.

Xylitol fermentation characteristics with a newly isolated yeast Wickerhamomyces anomalus WA.

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 OD600 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.

Metabolic engineering of Candida tropicalis for efficient 1,2,4-butanetriol production.

1,2,4-Butanetriol serves as a precursor in the manufacture of diverse pharmaceuticals and the energetic plasticizer 1,2,4-butanetriol trinitrate. The study involved further modifications to an engineered Candida tropicalis strain, aimed at improving the production efficiency of 1,2,4-butanetriol. Faced with the issue of xylonate accumulation due to the low activity of heterologous xylonate dehydratase, we modulated iron metabolism at the transcriptional level to boost intracellular iron ion availability, thus enhancing the enzyme activity by 2.2-fold. Addressing the NADPH shortfall encountered during 1,2,4-butanetriol biosynthesis, we overexpressed pivotal genes in the NADPH regeneration pathway, achieving a 1,2,4-butanetriol yield of 3.2 g/L. The introduction of calcium carbonate to maintain pH balance led to an increased yield of 4 g/L, marking a 111% improvement over the baseline strain. Finally, the use of corncob hydrolysate as a substrate culminated in 1,2,4-butanetriol production of 3.42 g/L, thereby identifying a novel host for the conversion of corncob hydrolysate to 1,2,4-butanetriol.

Sustainable biorefinery approach by utilizing xylose fraction of lignocellulosic biomass.

Lignocellulosic biomass (LCB) has been a lucrative feedstock for developing biochemical products due to its rich organic content, low carbon footprint and abundant accessibility. The recalcitrant nature of this feedstock is a foremost bottleneck. It needs suitable pretreatment techniques to achieve a high yield of sugar fractions such as glucose and xylose with low inhibitory components. Cellulosic sugars are commonly used for the bio-manufacturing process, and the xylose sugar, which is predominant in the hemicellulosic fraction, is rejected as most cell factories lack the five­carbon metabolic pathways. In the present review, more emphasis was placed on the efficient pretreatment techniques developed for disintegrating LCB and enhancing xylose sugars. Further, the transformation of the xylose to value-added products through chemo-catalytic routes was highlighted. In addition, the review also recapitulates the sustainable production of biochemicals by native xylose assimilating microbes and engineering the metabolic pathway to ameliorate biomanufacturing using xylose as the sole carbon source. Overall, this review will give an edge on the bioprocessing of microbial metabolism for the efficient utilization of xylose in the LCB.

Maltose accumulation-induced cell death in Saccharomyces cerevisiae.

Pretreatment of lignocellulose yields a complex sugar mixture that potentially can be converted into bioethanol and other chemicals by engineered yeast. One approach to overcome competition between sugars for uptake and metabolism is the use of a consortium of specialist strains capable of efficient conversion of single sugars. Here, we show that maltose inhibits cell growth of a xylose-fermenting specialist strain IMX730.1 that is unable to utilize glucose because of the deletion of all hexokinase genes. The growth inhibition cannot be attributed to a competition between maltose and xylose for uptake. The inhibition is enhanced in a strain lacking maltase enzymes (dMalX2) and completely eliminated when all maltose transporters are deleted. High-level accumulation of maltose in the dMalX2 strain is accompanied by a hypotonic-like transcriptional response, while cells are rescued from maltose-induced cell death by the inclusion of an extracellular osmolyte such as sorbitol. These data suggest that maltose-induced cell death is due to high levels of maltose uptake causing hypotonic-like stress conditions and can be prevented through engineering of the maltose transporters. Transporter engineering should be included in the development of stable microbial consortia for the efficient conversion of lignocellulosic feedstocks.

Enhancing xylose-fermentation capacity of engineered Saccharomyces cerevisiae by multistep evolutionary engineering in inhibitor-rich lignocellulose hydrolysate.

Major progress in developing Saccharomyces cerevisiae strains that utilize the pentose sugar xylose has been achieved. However, the high inhibitor content of lignocellulose hydrolysates still hinders efficient xylose fermentation, which remains a major obstacle for commercially viable second-generation bioethanol production. Further improvement of xylose utilization in inhibitor-rich lignocellulose hydrolysates remains highly challenging. In this work, we have developed a robust industrial S. cerevisiae strain able to efficiently ferment xylose in concentrated undetoxified lignocellulose hydrolysates. This was accomplished with novel multistep evolutionary engineering. First, a tetraploid strain was generated and evolved in xylose-enriched pretreated spruce biomass. The best evolved strain was sporulated to obtain a genetically diverse diploid population. The diploid strains were then screened in industrially relevant conditions. The best performing strain, MDS130, showed superior fermentation performance in three different lignocellulose hydrolysates. In concentrated corncob hydrolysate, with initial cell density of 1 g DW/l, at 35°C, MDS130 completely coconsumed glucose and xylose, producing ± 7% v/v ethanol with a yield of 91% of the maximum theoretical value and an overall productivity of 1.22 g/l/h. MDS130 has been developed from previous industrial yeast strains without applying external mutagenesis, minimizing the risk of negative side-effects on other commercially important properties and maximizing its potential for industrial application.

Engineering the cellulolytic bacterium, Clostridium thermocellum, to co-utilize hemicellulose.

Consolidated bioprocessing (CBP) of lignocellulosic biomass holds promise to realize economic production of second-generation biofuels/chemicals, and Clostridium thermocellum is a leading candidate for CBP due to it being one of the fastest degraders of crystalline cellulose and lignocellulosic biomass. However, CBP by C. thermocellum is approached with co-cultures, because C. thermocellum does not utilize hemicellulose. When compared with a single-species fermentation, the co-culture system introduces unnecessary process complexity that may compromise process robustness. In this study, we engineered C. thermocellum to co-utilize hemicellulose without the need for co-culture. By evolving our previously engineered xylose-utilizing strain in xylose, an evolved clonal isolate (KJC19-9) was obtained and showed improved specific growth rate on xylose by ∼3-fold and displayed comparable growth to a minimally engineered strain grown on the bacteria's naturally preferred substrate, cellobiose. To enable full xylan deconstruction to xylose, we recombinantly expressed three different ß-xylosidase enzymes originating from Thermoanaerobacterium saccharolyticum into KJC19-9 and demonstrated growth on xylan with one of the enzymes. This recombinant strain was capable of co-utilizing cellulose and xylan simultaneously, and we integrated the ß-xylosidase gene into the KJC19-9 genome, creating the KJCBXint strain. The strain, KJC19-9, consumed monomeric xylose but accumulated xylobiose when grown on pretreated corn stover, whereas the final KJCBXint strain showed significantly greater deconstruction of xylan and xylobiose. This is the first reported C. thermocellum strain capable of degrading and assimilating hemicellulose polysaccharide while retaining its cellulolytic capabilities, unlocking significant potential for CBP in advancing the bioeconomy.

Engineered Saccharomyces cerevisiae as a Biosynthetic Platform of Nucleotide Sugars.

Glycosylation of biomolecules can greatly alter their physicochemical properties, cellular recognition, subcellular localization, and immunogenicity. Glycosylation reactions rely on the stepwise addition of sugars using nucleotide diphosphate (NDP)-sugars. Making these substrates readily available will greatly accelerate the characterization of new glycosylation reactions, elucidation of their underlying regulation mechanisms, and production of glycosylated molecules. In this work, we engineered Saccharomyces cerevisiae to heterologously express nucleotide sugar synthases to access a wide variety of uridine diphosphate (UDP)-sugars from simple starting materials (i.e., glucose and galactose). Specifically, activated glucose, uridine diphosphate d-glucose (UDP-d-Glc), can be converted to UDP-d-glucuronic acid (UDP-d-GlcA), UDP-d-xylose (UDP-d-Xyl), UDP-d-apiose (UDP-d-Api), UDP-d-fucose (UDP-d-Fuc), UDP-l-rhamnose (UDP-l-Rha), UDP-l-arabinopyranose (UDP-l-Arap), and UDP-l-arabinofuranose (UDP-l-Araf) using the corresponding nucleotide sugar synthases of plant and microbial origins. We also expressed genes encoding the salvage pathway to directly activate free sugars to achieve the biosynthesis of UDP-l-Arap and UDP-l-Araf. We observed strong inhibition of UDP-d-Glc 6-dehydrogenase (UGD) by the downstream product UDP-d-Xyl, which we circumvented using an induction system (Tet-On) to delay the production of UDP-d-Xyl to maintain the upstream UDP-sugar pool. Finally, we performed a time-course study using strains containing the biosynthetic pathways to produce five non-native UDP-sugars to elucidate their time-dependent interconversion and the role of UDP-d-Xyl in regulating UDP-sugar metabolism. These engineered yeast strains are a robust platform to (i) functionally characterize sugar synthases in vivo, (ii) biosynthesize a diverse selection of UDP-sugars, (iii) examine the regulation of intracellular UDP-sugar interconversions, and (iv) produce glycosylated secondary metabolites and proteins.