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All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

L–malic acid production from xylose by engineered Saccharomyces cerevisiae

L-malic acid is widely used in the food, chemical, and pharmaceutical industries. Here, we report on production of malic acid from xylose, the second most abundant sugar in lignocellulosic hydrolysates, by engineered Saccharomyces cerevisiae. To enable malic acid production in a xylose-assimilating S. cerevisiae, we overexpressed PYC1 and PYC2, coding for pyruvate carboxylases, a truncated MDH3 coding for malate dehydrogenase, and SpMAE1, coding for a Schizosaccharomyces pombe malate transporter. Additionally, both the ethanol and glycerol-producing pathways were blocked to enhance malic acid production. The resulting strain produced malic acid from both glucose and xylose, but it produced much higher titers of malic acid from xylose than glucose. Interestingly, the engineered strain had higher malic acid yield from lower concentrations (10 g L –1 ) of xylose, with no ethanol production, than from higher xylose concentrations (20 and 40 g L –1 ). As such, a fed-batch culture maintaining xylose concentrations at low levels was conducted and 61.2 g L –1 of malic acid was produced, with a productivity of 0.32 g L –1 h. Furthermore, these results represent successful engineering of S. cerevisiae for the production of malic acid from xylose, confirming that that xylose offers the efficient production of various biofuels and chemicals by engineered S. cerevisiae.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

High–level β–carotene production from xylose by engineered Saccharomyces cerevisiae without overexpression of a truncated HMG1 (t HMG1 )

Beta-carotene is a natural pigment and health-promoting metabolite, and has been widely used in the nutraceutical, feed, and cosmetic industries. Here, we engineered a GRAS yeast Saccharomyces cerevisiae to produce beta-carotene from xylose, the sec- ond most abundant and inedible sugar component of lignocellulose biomass. Specifically, a beta-carotene biosynthetic pathway containing crtYB, crtI, and crtE from Xanthophyllomyces dendrorhous was introduced into a xylose-fermenting S. cerevisiae. Here, the resulting strain produced beta-carotene from xylose at a titer threefold higher than from glucose. Interestingly, overexpression of tHMG1, which has been reported as a critical genetic perturbation to enhance metabolic fluxes in the mevalonate pathway and beta-carotene production in yeast when glucose is used, did not further improve the production of beta-carotene from xylose. Through fermentation profiling, metabolites analysis, and transcriptional studies, we found the advantages of using xylose as a carbon source, instead of glucose, for beta-carotene production to be a more respiratory feature of xylose consumption, a larger cytosolic acetyl!CoA pool, and an upregulated expression level of rate-limiting genes in the beta-carotene!producing pathway, including ACS1 and HMG1. As a result, 772.8 mg/L of beta-carotene was ob- tained in a fed-batch bioreactor culture with xylose feeding. Considering the in- evitable large scale production of xylose when cellulosic biomass!based bioeconomy is implemented, our results suggest xylose utilization is a promising strategy for overproduction of carotenoids and other isoprenoids in engineered S. cerevisiae.

09 BIOMASS FUELS↗
DOE OSTI2020

Enhanced 2'-Fucosyllactose production by engineered Saccharomyces cerevisiae using xylose as a co-substrate

2'-Fucosyllactose (2'-FL), a human milk oligosaccharide with confirmed benefits for infant health, is a promising infant formula ingredient. Although Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum, and Bacillus subtilis have been engineered to produce 2'-FL, their titers and productivities need be improved for economic production. Glucose along with lactose have been used as substrates for producing 2'-FL, but accu- mulation of by-products due to overflow metabolism of glucose hampered efficient production of 2' -FL regardless of a host strain. To circumvent this problem, we used xylose, which is the second most abundant sugar in plant cell wall hydrolysates and is metabolized through oxidative metabolism, for the production of 2'-FL by engi- neered yeast. Specifically, we modified an engineered S. cerevisiae strain capable of assimilating xylose to pro- duce 2' -FL from a mixture of xylose and lactose. First, a lactose transporter (Lac12) from Kluyveromyces lactis was introduced. Second, a heterologous 2'-FL biosynthetic pathway consisting of enzymes Gmd, WcaG, and WbgL from Escherichia coli was introduced. Third, we adjusted expression levels of the heterologous genes to maximize 2'-FL production. The resulting engineered yeast produced 25.5 g/L of 2'-FL with a volumetric productivity of 0.35 g/L∙h in a fed-batch fermentation with lactose and xylose feeding to mitigate the glucose repression. Interestingly, the major location of produced 2'-FL by the engineered yeast can be changed using different culture media. While 72% of the produced 2'-FL was secreted when a complex medium was used, 82% of the produced 2'-FL remained inside the cells when a minimal medium was used. In conclusion, as yeast extract is already used as food and animal feed ingredients, 2'-FL enriched yeast extract can be produced cost-effectively using the 2'-FL-accumu- lating yeast cells.

2′ -fucosyllactose↗
DOE OSTI2024

Advances in Engineering Nucleotide Sugar Metabolism for Natural Product Glycosylation in Saccharomyces cerevisiae

Glycosylation is a ubiquitous modification present across all of biology, affecting many things such as physicochemical properties, cellular recognition, subcellular localization, and immunogenicity. Nucleotide sugars are important precursors needed to study glycosylation and produce glycosylated products. Saccharomyces cerevisiae is a potentially powerful platform for producing glycosylated biomolecules, but it lacks nucleotide sugar diversity. Nucleotide sugar metabolism is complex, and understanding how to engineer it will be necessary to both access and study heterologous glycosylations found across biology. This review overviews the potential challenges with engineering nucleotide sugar metabolism in yeast from the salvage pathways that convert free sugars to their associated UDP-sugars to de novo synthesis where nucleotide sugars are interconverted through a complex metabolic network with governing feedback mechanisms. Finally, recent examples of engineering complex glycosylation of small molecules in S. cerevisiae are explored and assessed.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Controlling circuitry underlies the growth optimization of Saccharomyces cerevisiae

Microbial growth emerges from coordinated synthesis of various cellular components from limited resources. In Saccharomyces cerevisiae, cyclic AMP (cAMP)-mediated signaling is shown to orchestrate cellular metabolism; however, it remains unclear quantitatively how the controlling circuit drives resource partition and subsequently shapes biomass growth. Here we combined experiment with mathematical modeling to dissect the signaling-mediated growth optimization of S. cerevisiae. We showed that, through cAMP-mediated control, the organism achieves maximal or nearly maximal steady-state growth during the utilization of multiple tested substrates as well as under perturbations impairing glucose uptake. However, the optimal cAMP concentration varies across cases, suggesting that different modes of resource allocation are adopted for varied conditions. Under settings with nutrient alterations, S. cerevisiae tunes its cAMP level to dynamically reprogram itself to realize rapid adaptation. Moreover, to achieve growth maximization, cells employ additional regulatory systems such as the GCN2-mediated amino acid control. This study establishes a systematic understanding of global resource allocation in S. cerevisiae, providing insights into quantitative yeast physiology as well as metabolic strain engineering for biotechnological applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Phosphoproteome Response to Dithiothreitol Reveals Unique Versus Shared Features of Saccharomyces cerevisiae Stress Responses

To cope with sudden changes in the external environment, the budding yeast Saccharomyces cerevisiae orchestrates a multifaceted response that spans many levels of physiology. Several studies have interrogated the transcriptome response to endoplasmic reticulum (ER) stress and the role of regulators such as the Ire1 kinase and Hac1 transcription factors. However, less is known about responses to ER stress at other levels of physiology. Here, we used quantitative phosphoproteomics and computational network inference to uncover the yeast phosphoproteome response to the reducing agent dithiothreitol (DTT) and the upstream signaling network that controls it. We profiled wild-type cells and mutants lacking IRE1 or MAPK kinases MKK1 and MKK2, before and at various times after DTT treatment. In addition to revealing downstream targets of these kinases, our inference approach predicted new regulators in the DTT response, including cell-cycle regulator Cdc28 and osmotic-response kinase Rck2, which we validated computationally. Our results also revealed similarities and surprising differences in responses to different stress conditions, especially in the response of protein kinase A targets. Furthermore, these results have implications for the breadth of signaling programs that can give rise to common stress response signatures.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Data for Controlling Circuitry Underlies the Growth Optimization of Saccharomyces cerevisiae

Microbial growth emerges from coordinated synthesis of various cellular components from limited resources. In Saccharomyces cerevisiae , cyclic AMP (cAMP)-mediated signaling is shown to orchestrate cellular metabolism; however, it remains unclear quantitatively how the controlling circuit drives resource partition and subsequently shapes biomass growth. Here we combined experiment with mathematical modeling to dissect the signaling-mediated growth optimization of S. cerevisiae . We showed that, through cAMP-mediated control, the organism achieves maximal or nearly maximal steady-state growth during the utilization of multiple tested substrates as well as under perturbations impairing glucose uptake. However, the optimal cAMP concentration varies across cases, suggesting that different modes of resource allocation are adopted for varied conditions. Under settings with nutrient alterations, S. cerevisiae tunes its cAMP level to dynamically reprogram itself to realize rapid adaptation. Moreover, to achieve growth maximization, cells employ additional regulatory systems such as the GCN2-mediated amino acid control. This study establishes a systematic understanding of global resource allocation in S. cerevisiae , providing insights into quantitative yeast physiology as well as metabolic strain engineering for biotechnological applications.

Conversion↗
DOE OSTI2020

Xylose Assimilation for the Efficient Production of Biofuels and Chemicals by Engineered Saccharomyces cerevisiae

Microbial conversion of plant biomass into fuels and chemicals offers a practical solution to global concerns over limited natural resources, environmental pollution, and climate change. Pursuant to these goals, researchers have put tremendous efforts and resources towards engineering the yeast Saccharomyces cerevisiae to efficiently convert xylose, the second most abundant sugar in lignocellulosic biomass, into various fuels and chemicals. Here, we summarize recent advances in metabolic engineering of yeast to address bottlenecks on xylose assimilation, and to enable simultaneous co-utilization of xylose and other carbon sources in lignocellulosic hydrolysates. We also highlight distinct characteristics of xylose metabolism that can be harnessed to produce advanced biofuels and chemicals. Although many challenges remain, recent research investments have facilitated efficient fermentation of xylose and simultaneous co-consumption of xylose and glucose. In particular, understanding of xylose-induced metabolic rewiring in engineered yeast has encouraged use of xylose as a carbon source for producing various non-ethanol bioproducts. Furthermore, to boost the lignocellulosic biomass-based bioeconomy, much attention is expected to promote xylose-utilizing efficiency via reprogramming cellular regulatory networks, to attain robust co-fermentation of xylose and other cellulosic carbon sources under industrial conditions, and to exploit the advantageous traits of yeast xylose metabolism for producing diverse fuels and chemicals.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Aim18p and Aim46p are chalcone isomerase domain–containing mitochondrial hemoproteins in Saccharomyces cerevisiae

Chalcone isomerases (CHIs) have well-established roles in the biosynthesis of plant flavonoid metabolites. Saccharomyces cerevisiae possesses two predicted CHI-like proteins, Aim18p (encoded by YHR198C) and Aim46p (YHR199C), but it lacks other enzymes of the flavonoid pathway, suggesting that Aim18p and Aim46p employ the CHI fold for distinct purposes. Here, we demonstrate using proteinase K protection assays, sodium carbonate extractions, and crystallography that Aim18p and Aim46p reside on the mitochondrial inner membrane and adopt CHI folds, but they lack select active site residues and possess an extra fungal-specific loop. Consistent with these differences, Aim18p and Aim46p lack CHI activity and also the fatty acid–binding capabilities of other CHI-like proteins, but instead bind heme. We further show that diverse fungal homologs also bind heme and that Aim18p and Aim46p possess structural homology to a bacterial hemoprotein. Collectively, our work reveals a distinct function and cellular localization for two CHI-like proteins, introduces a new variation of a hemoprotein fold, and suggests that ancestral CHI-like proteins were hemoproteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Emergence of Orchestrated and Dynamic Metabolism of Saccharomyces cerevisiae

Microbial metabolism is a fundamental cellular process that involves many biochemical events and is distinguished by its emergent properties. While the molecular details of individual reactions have been increasingly elucidated, it is not well understood how these reactions are quantitatively orchestrated to produce collective cellular behaviors. Here we developed a coarse-grained, systems, and dynamic mathematical framework, which integrates metabolic reactions with signal transduction and gene regulation to dissect the emergent metabolic traits of Saccharomyces cerevisiae. Our framework mechanistically captures a set of characteristic cellular behaviors, including the Crabtree effect, diauxic shift, diauxic lag time, and differential growth under nutrient-altered environments. It also allows modular expansion for zooming in on specific pathways for detailed metabolic profiles. Furthermore, this study provides a systems mathematical framework for yeast metabolic behaviors, providing insights into yeast physiology and metabolic engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Saccharomyces cerevisiae CellWall Remodeling in the Absence of Knr4 and Kre6 Revealed by Nano-FourierTransform Infrared Spectroscopy

The cell wall integrity (CWI) signaling pathway regulates yeast cell wall biosynthesis, cell division, and responses to external stress. The cell wall, comprised of a dense network of chitin, β-1,3- and β-1,6- glucans, and mannoproteins, is very thin, <100 nm. Alterations in cell wall composition may activate the CWI pathway. Saccharomyces cerevisiae, a model yeast, was used to study the role of individual wall components in altering the structure and biophysical properties of the yeast cell wall. Near-field Fourier transform infrared spectroscopy (nano-FT-IR) was used for the first direct, spectrochemical identification of cell wall composition in a background (wild-type) strain and two deletion mutants from the yeast knock-out collection: kre6Δ and knr4Δ. Killer toxin resistant 6 (Kre6) is an integral membrane protein required for biosynthesis of β-1,6-glucan, while Knr4 is a cell signaling protein involved in the control of cell wall biosynthesis, in particular, biosynthesis and deposition of chitin. Complementary spectral data were obtained with far-field (FF)-FT-IR, in transmission, and with attenuated total reflectance (ATR) spectromicroscopy with 3 –10 μm wavelength-dependent spatial resolution. The FF-FT-IR spectra of cells and spectra of isolated cell wall components showed that components of the cell body dominated transmission spectra and were still evident in ATR spectra. In contrast, the nano-FT-IR at ~25 nm spatial resolution could be used to characterize the yeast wall chemical structure. Our results show that the β-1,6-glucan content is decreased in kre6Δ, while all glucan content is decreased in the knr4Δ cell wall. The latter may be thinner than in wild type, since not only are mannan and chitin detectable by nano-FT-IR, but also lipid membranes and protein, indicative of cell interior.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Comparison of stress tolerance mechanisms between Saccharomyces cerevisiae and the multistress-tolerant Pichia kudriavzevii

Yeasts play a vital role in both research and industrial biomanufacturing. Saccharomyces cerevisiae has been extensively utilized as a model system. However, its application is often constrained by limited tolerance to the diverse stress conditions encountered in bioprocesses. These challenges have driven increasing interest in nonconventional, multistress-tolerant yeasts as alternative biomanufacturing hosts. This review highlights Pichia kudriavzevii as a promising nonconventional yeast for industrial applications. Unlike S. cerevisiae, P. kudriavzevii exhibits exceptional tolerance to high temperatures, elevated concentrations of furanic and phenolic inhibitors, osmotic stress, salinity, and extreme pH. These traits make it an attractive candidate for industrial processes without requiring extensive genetic modifications to enhance stress resistance. As a result, P. kudriavzevii has emerged as a flagship species for advancing bioeconomy. Despite its industrial potential, the molecular mechanisms underlying P. kudriavzevii's superior stress tolerance remain poorly understood. This review compiles current knowledge on P. kudriavzevii and compares its stress tolerance mechanisms with those of S. cerevisiae, providing insights into its innate resilience. By expanding our understanding of nonconventional yeasts, this review aims to facilitate their broader adoption as robust microbial platforms for industrial biomanufacturing.

Frousnoon, Thasneem Banu↗
DOE OSTI2026

Engineering of 2‐ketoacid Decarboxylases for Production of Isobutanol and Other Fusel Alcohols in Saccharomyces cerevisiae

Isobutanol is a fusel alcohol that can be produced microbially for use as a biofuel or upgraded into sustainable aviation fuel (SAF). A key enzyme in the isobutanol biosynthetic pathway is 2-ketoacid decarboxylase (KDC), which irreversibly decarboxylates 2-ketoisovalerate (KIV) to yield isobutyraldehyde. However, many previously characterized KDC enzymes also act promiscuously on other 2-ketoacids, (e.g., pyruvate) to produce a related aldehyde (e.g., acetaldehyde). This unwanted side reaction is especially important when isobutanol is produced in Saccharomyces cerevisiae (S. cerevisiae) because it leads to pyruvate being diverted to ethanol. In order to make S. cerevisiae a strict isobutanologen, a KDC enzyme that is specific for KIV must be deployed. In this study, we used a combination of cell-based and in vitro enzyme assays to investigate KDC substrate specificity, characterizing a large set of homologs for KIV, pyruvate, and phenylpyruvate (PPV) activity. A diverse range of substrate specificities was discovered, and some previously uncharacterized KDCs were revealed to have high KIV activity and low pyruvate activity. Multi-site saturation mutagenesis (SSM) of one of these KDCs identified mutants with increased KIV activity, while maintaining low levels of pyruvate activity. In a KIV bioconversion experiment, bioprospected and engineered KDCs allowed similar KIV consumption to when using the previously characterized Lactococcus lactis KdcA, though with some ethanol also produced. The KDCs identified here show promise for production of isobutanol and other alcohols derived from 2-ketoacids, and the dataset of newly characterized KDCs can inform future efforts to understand and engineer substrate specificity in KDCs.

2-ketoacid decarboxylase↗
DOE OSTI2020

Production of xylose enriched hydrolysate from bioenergy sorghum and its conversion to β-carotene using an engineered Saccharomyces cerevisiae

A new bioprocess has been created that allows for producing β-carotene from the xylose portion of bioenergy sorghum. Bioenergy sorghum was pretreated in a pilot-scale continuous hydrothermal reactor followed by disc refining. Xylose was extracted using low-severity dilute acid hydrolysis. A xylose yield of 64.9% (17.4 g/L) was obtained by hydrolyzing at 120 °C for 5 min with 2% sulfuric acid. The xylose-enriched syrup was separated and concentrated to either 32 g xylose/L (medium-concentrated hydrolysate, MCB) or 66 g xylose/L (high-concentrated hydrolysate, HCB). The non- (NCB), medium-, and high-concentrated xylose syrup were neutralized and fermented to β-carotene using Saccharomyces cerevisiae strain SR8B, which had been engineered for xylose utilization and β-carotene production. In HCB, MCB, and NCB cultures, the yeast produced β-carotene titers of 114.50 mg/L, 93.56 mg/L, and 82.50 mg/L, which corresponds to specific yeast biomass productions of 7.32 mg/g DCW, 8.10 mg/g DCW, and 8.29 mg/g DCW, respectively.

09 BIOMASS FUELS↗
DOE OSTI2020

Data from Enhanced 2′-Fucosyllactose Production by Engineered Saccharomyces cerevisiae using Xylose as a Co-Substrate

2′-Fucosyllactose (2′-FL), a human milk oligosaccharide with confirmed benefits for infant health, is a promising infant formula ingredient. Although Escherichia coli , Saccharomyces cerevisiae , Corynebacterium glutamicum , and Bacillus subtilis have been engineered to produce 2′-FL, their titers and productivities need be improved for economic production. Glucose along with lactose have been used as substrates for producing 2′-FL, but accumulation of by-products due to overflow metabolism of glucose hampered efficient production of 2′-FL regardless of a host strain. To circumvent this problem, we used xylose, which is the second most abundant sugar in plant cell wall hydrolysates and is metabolized through oxidative metabolism, for the production of 2′-FL by engineered yeast. Specifically, we modified an engineered S. cerevisiae strain capable of assimilating xylose to produce 2′-FL from a mixture of xylose and lactose. First, a lactose transporter (Lac12) from Kluyveromyces lactis was introduced. Second, a heterologous 2′-FL biosynthetic pathway consisting of enzymes Gmd, WcaG, and WbgL from E. coli was introduced. Third, we adjusted expression levels of the heterologous genes to maximize 2′-FL production. The resulting engineered yeast produced 25.5 g/L of 2′-FL with a volumetric productivity of 0.35 g/L∙h in a fed-batch fermentation with lactose and xylose feeding to mitigate the glucose repression. Interestingly, the major location of produced 2′-FL by the engineered yeast can be changed using different culture media. While 72% of the produced 2′-FL was secreted when a complex medium was used, 82% of the produced 2′-FL remained inside the cells when a minimal medium was used. As yeast extract is already used as food and animal feed ingredients, 2′-FL enriched yeast extract can be produced cost-effectively using the 2′-FL-accumulating yeast cells.

Conversion↗
DOE OSTI2022

Functional Divergence in a Multi-gene Family Is a Key Evolutionary Innovation for Anaerobic Growth in Saccharomyces cerevisiae

The amplification and diversification of genes into large multi-gene families often mark key evolutionary innovations, but this process often creates genetic redundancy that hinders functional investigations. When the model budding yeast Saccharomyces cerevisiae transitions to anaerobic growth conditions, the cell massively induces the expression of seven serine/threonine-rich anaerobically-induced cell wall mannoproteins (anCWMPs): TIP1, TIR1, TIR2, TIR3, TIR4, DAN1, and DAN4. Here, we show that these genes likely derive evolutionarily from a single ancestral anCWMP locus, which was duplicated and translocated to new genomic contexts several times both prior to and following the budding yeast whole genome duplication (WGD) event. Based on synteny and their phylogeny, we separate the anCWMPs into four gene subfamilies. To resolve prior inconclusive genetic investigations of these genes, we constructed a set of combinatorial deletion mutants to determine their contributions toward anaerobic growth in S. cerevisiae. We found that two genes, TIR1 and TIR3, were together necessary and sufficient for the anCWMP contribution to anaerobic growth. Overexpressing either gene alone was insufficient for anaerobic growth, implying that they encode non-overlapping functional roles in the cell during anaerobic growth. We infer from the phylogeny of the anCWMP genes that these two important genes derive from an ancient duplication that predates the WGD event, whereas the TIR1 subfamily experienced gene family amplification after the WGD event. Taken together, the genetic and molecular evidence suggests that one key anCWMP gene duplication event, several auxiliary gene duplication events, and functional divergence underpin the evolution of anaerobic growth in budding yeasts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Data for L-Malic Acid Production from Xylose by Engineered Saccharomyces cerevisiae

L-malic acid is widely used in the food, chemical, and pharmaceutical industries. Here, we report on production of malic acid from xylose, the second most abundant sugar in lignocellulosic hydrolysates, by engineered Saccharomyces cerevisiae . To enable malic acid production in a xylose-assimilating S. cerevisiae , we overexpressed PYC1 and PYC2, coding for pyruvate carboxylases, a truncated MDH3 coding for malate dehydrogenase, and SpMAE1, coding for a Schizosaccharomyces pombe malate transporter. Additionally, both the ethanol- and glycerol-producing pathways were blocked to enhance malic acid production. The resulting strain produced malic acid from both glucose and xylose, but it produced much higher titers of malic acid from xylose than glucose. Interestingly, the engineered strain had higher malic acid yield from lower concentrations (10 g L‒1) of xylose, with no ethanol production, than from higher xylose concentrations (20 and 40 g L‒1). As such, a fed-batch culture maintaining xylose concentrations at low levels was conducted and 61.2 g L‒1 of malic acid was produced, with a productivity of 0.32 g L‒1 h. These results represent successful engineering of S. cerevisiae for the production of malic acid from xylose, confirming that that xylose offers the efficient production of various biofuels and chemicals by engineered S. cerevisiae .

Conversion↗
DOE OSTI2021

A novel d-xylose isomerase from the gut of the wood feeding beetle Odontotaenius disjunctus efficiently expressed in Saccharomyces cerevisiae

Carbohydrate rich substrates such as lignocellulosic hydrolysates remain one of the primary sources of potentially renewable fuel and bulk chemicals. The pentose sugar d-xylose is often present in significant amounts along with hexoses. Saccharomyces cerevisiae can acquire the ability to metabolize d-xylose through expression of heterologous d-xylose isomerase (XI). This enzyme is notoriously difficult to express in S. cerevisiae and only fourteen XIs have been reported to be active so far. We cloned a new d-xylose isomerase derived from microorganisms in the gut of the wood-feeding beetle Odontotaenius disjunctus. Although somewhat homologous to the XI from Piromyces sp. E2, the new gene was identified as bacterial in origin and the host as a Parabacteroides sp. Expression of the new XI in S. cerevisiae resulted in faster aerobic growth than the XI from Piromyces on d-xylose media. The d-xylose isomerization rate conferred by the new XI was also 72% higher, while absolute xylitol production was identical in both strains. Interestingly, increasing concentrations of xylitol (up to 8 g L –1 ) appeared not to inhibit d-xylose consumption. The newly described XI displayed 2.6 times higher specific activity, 37% lower KM for d-xylose, and exhibited higher activity over a broader temperature range, retaining 51% of maximal activity at 30 °C compared with only 29% activity for the Piromyces XI.

59 BASIC BIOLOGICAL SCIENCES↗