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DOE OSTI2023

Development of sulfonated carbon-based solid-acid catalysts derived from biorefinery residues and biomass ash for xylan hydrolysis

In this study, biorefinery residues and biomass ash were used to develop sulfonated carbon-based solid-acid catalysts to enhance xylan hydrolysis. Here, metals extracted from the biomass ash were impregnated into a carbon support prepared from biorefinery residues to synthesize sulfonated ash-impregnated carbon-based catalyst. Metals present in the ash most probably are responsible for the increased acid density in the catalyst. Using this catalyst, 11.4 % xylose yield was produced, an increase of 171 % over the yield obtained from a catalyst without ash impregnation. Using a xylan-to-catalyst ratio of 1:2, 72.9 % xylose yield was obtained, comparable to using 0.5 wt% sulfuric acid under the same reaction condition. Moreover, this catalyst significantly inhibited xylose dehydration, only forming 11.3 % furfural compared to that generated using dilute acid. Hence, these results show the potential for using biomass ash as a metal source to increase the number of acid sites in solid-acid catalysts used for biomass pretreatment.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2021

Deconstruction of Woody Biomass via Protic and Aprotic Ionic Liquid Pretreatment for Ethanol Production

Ionic liquids (ILs) have emerged as important solvents for conversion of lignocellulosic feedstocks to fuels and chemicals due to their ability to enable efficient biomass deconstruction and fractionation. Woody biomass derived from forest and agricultural residues has the potential to be used for production of biofuels and its removal from forests can help mitigate disastrous wildfires in fire-prone states like California. This study evaluated woody biomass types (pine, almond, walnut, and fir) from California as potential biofuel feedstocks. The feedstocks were pretreated with the ILs cholinium lysinate ([Ch][Lys]) and ethanolamine acetate ([EOA][OAc]), followed by enzymatic hydrolysis and fermentation of lignocellulosic sugars to produce ethanol. Under optimal conditions, [EOA][OAc] pretreatment and enzymatic hydrolysis generated glucose and xylose yields in the range of 24-82 and 14-80%, respectively, while glucose and xylose yields for the [Ch][Lys] ranged between 28-83 and 23-80%, respectively. Maximum fermentable sugar was released from almond wood, and the lowest amount was from pine and fir. Further, blends of feedstocks were also explored, and a blend with a mass ratio of 2/2/1 (almond/walnut/pine) resulted in maximum glucose and xylose (>90%) yields using [Ch][Lys]. Fermentation of this hydrolysate using a C5-utilizing strain of Saccharomyces cerevisiae resulted in a maximum ethanol concentration of 17.9 g/L for mixture biomass hydrolysate, corresponding to 60.8% fermentation efficiency. This study represents the first demonstration of the use of these ILs for pretreatment of woody biomass blends that resulted in a high overall conversion efficiency for ethanol production.

09 BIOMASS FUELS↗

DOE OSTI2026

Selective designer hydrolysates from Miscanthus bioenergy crop for sustainable utilization

Designer enzymatic hydrolysates deliver targeted, optimized fermentable sugars while reducing the formation of inhibitory by-products. Herein, five distinct enzymatic hydrolysates (EH) were generated from Miscanthus biomass using alkaline-ethanol fractionation and hydrothermal-mechanical refining (HMR) pretreatment approaches. Noticeably, alkali-ethanol fractionated hemicellulose A (HMA) and hemicellulose B (HMB) hydrolysis resulted in xylose-rich enzymatic hydrolysates with 104.8 ± 5.6 g L −1 and 173.4 ± 4.6 g L −1 xylose, respectively. All five hydrolysates encompassing varied carbon to nitrogen ratios were evaluated for lipid production using an engineered oleaginous yeast, R. toruloides 880-ADS. Microbial fermentation of xylose-rich hydrolysates, viz. HMR-EH2, HMA-EH, and HMB-EH, resulted in lipid yields ranging from 0.05 to 0.12 g g −1 sugar. Compositional and 2D NMR analyses revealed that HMA-EH residues are highly lignin-enriched while preserving the non-condensed structure conducive to valorization. Overall, the findings highlighted a novel, selective trade-off approach for maximizing lipid titers from designer hydrolysates, corroborating complete resource recovery for establishing integrated biorefineries.

Singh, Shuchi [University of Illinois at Urbana-Ch↗

DOE OSTI2022

Revealing oxidative pentose metabolism in new Pseudomonas putida isolates

Abstract The Pseudomonas putida group in the Gammaproteobacteria has been intensively studied for bioremediation and plant growth promotion. Members of this group have recently emerged as promising hosts to convert intermediates derived from plant biomass to biofuels and biochemicals. However, most strains of P. putida cannot metabolize pentose sugars derived from hemicellulose. Here, we describe three isolates that provide a broader view of the pentose sugar catabolism in the P. putida group. One of these isolates clusters with the well‐characterized P. alloputida KT2440 (Strain BP6); the second isolate clustered with plant growth‐promoting strain P. putida W619 (Strain M2), while the third isolate represents a new species in the group (Strain BP8). Each of these isolates possessed homologous genes for oxidative xylose catabolism ( xylDXA ) and a potential xylonate transporter. Strain M2 grew on arabinose and had genes for oxidative arabinose catabolism ( araDXA ). A CRISPR interference (CRISPRi) system was developed for strain M2 and identified conditionally essential genes for xylose growth. A glucose dehydrogenase was found to be responsible for initial oxidation of xylose and arabinose in strain M2. These isolates have illuminated inherent diversity in pentose catabolism in the P. putida group and may provide alternative hosts for biomass conversion.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Xylanolytic metabolism is regulated by coordination of transcription factors XynR and XylR in extremely thermophilic Caldicellulosiruptorales

ABSTRACT Global transcription factors (TFs) control metabolic processes in bacteria to efficiently utilize available carbon. The orderCaldicellulosiruptoraleshas drawn interest due to the ability of its members to degrade components of lignocellulosic biomass. Regulatory reconstruction ofAnaerocellum (f. Caldicellulosiruptor) besciiidentified two major global transcription factors for xylan utilization, XynR and XylR, and the corresponding putative transcription factor binding sites. Recombinant versions of XynR (LacI family) and XylR (ROK family) were subjected to fluorescence polarization (FP) and biolayer interferometry (BLI) analysis to confirm the predicted binding sites. Four XynR sites and two XylR sites were validated, accounting for 20 of 26 genes regulated by XynR and six of seven genes regulated by XylR. Bioinformatic analysis of the individual genes controlled by the two regulators showed an inter-dependent scheme for xylan conversion; the transport of xylooligosaccharides (XOS) is dependent on XylR, while enzymes responsible for hydrolysis are controlled by both regulators. For xylose catabolism by the xylose isomerase-xylulose kinase pathway, regulation is also split, with XylR controlling xylose isomerase and XynR controlling xylokinase. The XynR/XylR regulator pair withinA. besciiis conserved in all sequenced species ofCaldicellulosiruptorales, suggesting similarities in regulating linear xylan conversion. In other xylanolytic thermophiles, XylR homologs control xylan degradation, compared to just 6 out of 26 genes forA. bescii. These results show that two separate regulatory schemes (dual repression) are coordinated byA. besciito effectively regulate the hemicellulose inventory and xylan catabolism. IMPORTANCE To take full advantage of extreme thermophiles as platform metabolic engineering microorganisms, the tools for genetic manipulation must be further developed, and strategies that exploit a better understanding of metabolic regulation need to be discerned.Anaerocellum bescii, the most studied of the extremely thermophilic fermentative anaerobic bacteria that can utilize microcrystalline cellulose, can degrade microcrystalline cellulose and hemicellulose and has been metabolically engineered to convert the resulting sugars to products such as ethanol and acetone. For xylan, in particular, two major global transcription factors (TFs), XynR and XylR, play a role in sugar metabolism, although their predicted regulatory interdependence from bioinformatics analysis has not been elucidated experimentally. Here, fluorescence polarization (FP) and biolayer interferometry (BLI) were used to explore this issue to support metabolic engineering efforts aimed at improving carbohydrate processing to industrial chemicals.

Biotechnology & Applied Microbiology↗

DOE OSTI2022

Transcriptomic Data Sets for Zymomonas mobilis 2032 during Fermentation of Ammonia Fiber Expansion (AFEX)-Pretreated Corn Stover and Switchgrass Hydrolysates

The transcriptomes of Zymomonas mobilis 2032 were captured during the fermentation of ammonia fiber expansion (AFEX)-pretreated corn stover and switchgrass hydrolysates containing different concentrations of glucose and xylose. RNA samples were collected when Z. mobilis was fermenting glucose or xylose. Here, we present transcriptome sequencing (RNA-Seq) data obtained during separate phases of glucose or xylose consumption.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

CAZymes from the thermophilic fungus Thermoascus aurantiacus are induced by C5 and C6 sugars

Abstract Background Filamentous fungi are excellent lignocellulose degraders, which they achieve through producing carbohydrate active enzymes (CAZymes). CAZyme production is highly orchestrated and gene expression analysis has greatly expanded understanding of this important biotechnological process. The thermophilic fungus Thermoascus aurantiacus secretes highly active thermostable enzymes that enable saccharifications at higher temperatures; however, the genome-wide measurements of gene expression in response to CAZyme induction are not understood. Results A fed-batch system with plant biomass-derived sugars d -xylose, l -arabinose and cellobiose established that these sugars induce CAZyme expression in T. aurantiacus . The C5 sugars induced both cellulases and hemicellulases, while cellobiose specifically induced cellulases. A minimal medium formulation was developed to enable gene expression studies of T. aurantiacus with these inducers. It was found that d-xylose and l -arabinose strongly induced a wide variety of CAZymes, auxiliary activity (AA) enzymes and carbohydrate esterases (CEs), while cellobiose facilitated lower expression of mostly cellulase genes. Furthermore, putative orthologues of different unfolded protein response genes were up-regulated during the C5 sugar feeding together with genes in the C5 sugar assimilation pathways. Conclusion This work has identified two additional CAZyme inducers for T. aurantiacus , l -arabinose and cellobiose, along with d -xylose. A combination of biochemical assays and RNA-seq measurements established that C5 sugars induce a suite of cellulases and hemicellulases, providing paths to produce broad spectrum thermotolerant enzymatic mixtures.

09 BIOMASS FUELS↗

DOE OSTI2025

Rapid monitoring of fermentations: a feasibility study on biological 2,3-butanediol production

2,3-butanediol (2,3-BDO) is an economically important platform chemical that can be produced by the fermentation of sugars using an engineered strain of Zymomonas mobilis . These fermentations require continuous monitoring and modification of fermentation conditions to maximize 2,3-BDO yields and minimize the production of the undesired coproducts glycerol and acetoin. Because of the time required for sampling and off-line chromatographic measurement of fermentation samples, the ability of fermentation scientists to modify fermentation conditions in a timely manner is limited. The goal of this study was to test if near-infrared spectroscopy (NIRS) along with multivariate statistics could reduce the time needed for this analysis and enable real-time monitoring and control of the fermentation. In this work we developed partial least squares (PLS) calibration models to predict the concentrations of glucose, xylose, 2,3-BDO, acetoin, and glycerol in fermentations via NIRS using two different spectrometers and two different spectroscopy modalities. We first evaluated the feasibility of rapid NIRS monitoring through experiments where we measured the signals from each analyte of interest and built NIRS-based PLS models using spectra from synthetic samples containing uncorrelated concentrations of these analytes. All analytes showed unique spectral signatures, and this initial modeling showed that all analytes could be detected simultaneously. We then began work with samples from laboratory fermentation experiments and tested the feasibility of regression model development across two spectral collection modalities (at-line and on-line) and two instruments: a laboratory-grade instrument and a low-cost instrument with a more limited spectral range. All modalities showed promise in the ability to monitor Z. mobilis fermentations of glucose and xylose to 2,3-BDO. The low-cost instrument displayed a lower signal-to-noise ratio than the laboratory-grade instrument, which led to comparatively lower performance overall, but still provided sufficient accuracy to monitor fermentation trends. While the ease of use of on-line monitoring systems was favored as compared to at-line systems due to the lack of sampling required and potential for automated process control, we observed some decrease in performance due to the additional complexity of the sample matrix. We have demonstrated that NIRS combined with multivariate analysis can be used for at-line and on-line monitoring of the concentrations of glucose, xylose, 2,3-BDO, acetoin, and glycerol during Z. mobilis fermentations. The decrease in signal-to-noise ratio when using a low-cost spectrometer led to greater prediction error than the laboratory-grade spectrometer for at-line monitoring. The on-line monitoring modality showed great promise for real time process control via NIRS.

09 BIOMASS FUELS↗

DOE OSTI2022

Data for A Chemical-Free Pretreatment for Biosynthesis of Bioethanol and Lipids from Lignocellulosic Biomass: An Industrially Relevant 2G Biorefinery Approach

A wide range of inorganic and organic chemicals are used during the pretreatment and enzymatic hydrolysis of lignocellulosic biomass to produce biofuels. Developing an industrially relevant 2G biorefinery process using such chemicals is challenging and requires more unit operations for downstream processing. A sustainable process has been developed to achieve industrially relevant titers of bioethanol with significant ethanol yield. The pretreatment of sorghum biomass was performed by a continuous pilot-scale hydrothermal reactor followed by disk milling. Enzymatic hydrolysis was performed without washing the pretreated biomass. Moreover, citrate buffer strength was reduced to 100-fold (50 mM to 0.5 mM) during the enzymatic hydrolysis. Enzymatic hydrolysis at 0.5 mM citrate buffer strength showed that significant sugar concentrations of 222 ± 2.3 to 241 ± 2.3 g/L (glucose + xylose) were attained at higher solids loadings of 50 to 60% (w/v). Furthermore, hydrolysates were fermented to produce bioethanol using two different xylose-fermenting Saccharomyces cerevisiae strains and a co-culture of xylose-fermenting and non-GMO yeast cultures. Bioethanol titer of 81.7 g/L was achieved with an ethanol yield of 0.48 gp/gs. Additionally, lipids were produced using the oleaginous yeast Rhodosporidium toruloides , yielding 13.2 g/L lipids with cellular lipid accumulation of 38.5% w/w from 100 g/L of sugar concentration. In summary, reducing the strength of the citrate buffer during enzymatic hydrolysis and omitting inorganic chemicals from the pretreatment process enhances the fermentability of hydrolysates and can also reduce operating costs.

Conversion↗

DOE OSTI2020

Chemical Free Two-Step Hydrothermal Pretreatment to Improve Sugar Yields from Energy Cane

Energy cane is an attractive lignocellulosic feedstock for processing into biofuels and bioproducts. A low-severity two-step hydrothermal pretreatment was investigated on energy cane for the production of monomeric sugar. Pretreatment temperature and time, in addition to the effect of disk milling, were observed for the glucose and xylose yields during hydrolysis. At residence times above 5 min in case of pretreatment at 200 °C, all of the hemicellulose was observed to be solubilized. The pretreatment condition of 200 °C for 10 min with disk milling was observed to provide the highest glucose concentration of 5.4%, and 200 °C for 5 min with disk milling provided the highest xylose concentration of 2.15%. The effect of disk milling in improving the sugar concentrations during hydrolysis was significant, especially at lower pretreatment temperatures and times. Low xylose yields at higher temperatures were attributed to the formation of degradation products at increased severity.

09 BIOMASS FUELS↗

DOE OSTI2022

A Chemical-Free Pretreatment for Biosynthesis of Bioethanol and Lipids from Lignocellulosic Biomass: An Industrially Relevant 2G Biorefinery Approach

A wide range of inorganic and organic chemicals are used during the pretreatment and enzymatic hydrolysis of lignocellulosic biomass to produce biofuels. Developing an industrially relevant 2G biorefinery process using such chemicals is challenging and requires more unit operations for downstream processing. A sustainable process has been developed to achieve industrially relevant titers of bioethanol with significant ethanol yield. The pretreatment of sorghum biomass was performed by a continuous pilot-scale hydrothermal reactor followed by disk milling. Enzymatic hydrolysis was performed without washing the pretreated biomass. Moreover, citrate buffer strength was reduced to 100-fold (50 mM to 0.5 mM) during the enzymatic hydrolysis. Enzymatic hydrolysis at 0.5 mM citrate buffer strength showed that significant sugar concentrations of 222 ± 2.3 to 241 ± 2.3 g/L (glucose + xylose) were attained at higher solids loadings of 50 to 60% (w/v). Furthermore, hydrolysates were fermented to produce bioethanol using two different xylose-fermenting Saccharomyces cerevisiae strains and a co-culture of xylose-fermenting and non-GMO yeast cultures. Bioethanol titer of 81.7 g/L was achieved with an ethanol yield of 0.48 gp/gs. Additionally, lipids were produced using the oleaginous yeast Rhodosporidium toruloides, yielding 13.2 g/L lipids with cellular lipid accumulation of 38.5% w/w from 100 g/L of sugar concentration. In summary, reducing the strength of the citrate buffer during enzymatic hydrolysis and omitting inorganic chemicals from the pretreatment process enhances the fermentability of hydrolysates and can also reduce operating costs.

Deshavath, Narendra Naik↗

DOE OSTI2022

Alicyclobacillus mali FL18 as a Novel Source of Glycosyl Hydrolases: Characterization of a New Thermophilic β-Xylosidase Tolerant to Monosaccharides

A thermo-acidophilic bacterium, Alicyclobacillus mali FL18, was isolated from a hot spring of Pisciarelli, near Naples, Italy; following genome analysis, a novel putative β-xylosidase, AmβXyl, belonging to the glycosyl hydrolase (GH) family 3 was identified. A synthetic gene was produced, cloned in pET-30a(+), and expressed in Escherichia coli BL21 (DE3) RIL. The purified recombinant protein, which showed a dimeric structure, had optimal catalytic activity at 80 °C and pH 5.6, exhibiting 60% of its activity after 2 h at 50 °C and displaying high stability (more than 80%) at pH 5.0–8.0 after 16 h. AmβXyl is mainly active on both para-nitrophenyl-β-D-xylopyranoside (K M 0.52 mM, k cat 1606 s -1 , and k cat /K M 3088.46 mM -1 ·s -1 ) and para-nitrophenyl-α-L-arabinofuranoside (K M 10.56 mM, k cat 2395.8 s -1 , and k cat /K M 226.87 mM -1 ·s -1 ). Thin-layer chromatography showed its ability to convert xylooligomers (xylobiose and xylotriose) into xylose, confirming that AmβXyl is a true β-xylosidase. Furthermore, no inhibitory effect on enzymatic activity by metal ions, detergents, or EDTA was observed except for 5 mM Cu 2+ . AmβXyl showed an excellent tolerance to organic solvents; in particular, the enzyme increased its activity at high concentrations (30%) of organic solvents such as ethanol, methanol, and DMSO. Lastly, the enzyme showed not only a good tolerance to inhibition by xylose, arabinose, and glucose, but was activated by 0.75 M xylose and up to 1.5 M by both arabinose and glucose. The high tolerance to organic solvents and monosaccharides together with other characteristics reported above suggests that AmβXyl may have several applications in many industrial fields.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Examination of Genetic Control Elements in the Phototrophic Firmicute Heliomicrobium modesticaldum

Heliomicrobium modesticaldum has been used as a model organism for the Heliobacteria, the only phototrophic family in the Firmicutes. It is a moderately thermophilic anoxygenic phototrophic bacterium that is capable of fermentative growth in the dark. The genetic manipulation of H. modesticaldum is still in its infancy. Methods to introduce genes through the use of exogenous plasmids and to delete genes from the chromosome through the use of the native CRISPR/Cas system have been developed in the last several years. To expand our genetic toolkit, it was necessary to control gene expression. In this study, we analyzed constitutive and inducible promoters developed for clostridia for their use in H. modesticaldum and further tested two reporters, adhB and lacZ, as indicators of promoter strength. Alcohol dehydrogenase (AdhB) was unsuitable as a reporter in this species due to high endogenous activity and/or low activity of the reporter, but a thermostable LacZ worked well as a reporter. A set of constitutive promoters previously reported to work in Clostridium thermocellum was found to be reliable for controlling the expression of the lacZ reporter gene in H. modesticaldum at a range of activities spanning an order of magnitude. An anhydrotetracycline-inducible promoter was created by inserting tetO operators into a strong constitutive promoter, but it was not fully repressible. The implementation of a xylose-inducible promoter resulted in complete repression of β-gal in the absence of xylose, and reliable expression tunable through the concentration of xylose added to the culture.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Glucose assimilation rate determines the partition of flux at pyruvate between lactic acid and ethanol in Saccharomyces cerevisiae

Engineered Saccharomyces cerevisiae expressing a lactic acid dehydrogenase can metab- olize pyruvate into lactic acid. However, three pyruvate decarboxylase (PDC) isozymes drive most carbon flux toward ethanol rather than lactic acid. Deletion of endoge- nous PDCs will eliminate ethanol production, but the resulting strain suffers from C 2 auxotrophy and struggles to complete a fermentation. Engineered yeast assimilating xylose or cellobiose produce lactic acid rather than ethanol as a major product with- out the deletion of any PDC genes. We report here that sugar flux, but not sensing, contributes to the partition of flux at the pyruvate branch point in S. cerevisiae express- ing the Rhizopus oryzae lactic acid dehydrogenase (LdhA). While the membrane glucose sensors Snf3 and Rgt2 did not play any direct role in the option of predominant product, the sugar assimilation rate was strongly correlated to the partition of flux at pyruvate: fast sugar assimilation favors ethanol production while slow sugar assimilation favors lactic acid. Applying this knowledge, we created an engineered yeast capable of simultaneously converting glucose and xylose into lactic acid, increasing lactic acid production to approximately 17 g L –1 from the 12 g L –1 observed during sequential consumption of sugars. This work elucidates the carbon source-dependent effects on product selection in engineered yeast.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

All You Can Eat Yeast: Substituting Hexose Transporters With AtSWEET7 Alleviates Glucose Repression, Enabling Simultaneous Utilization of Sugars in Renewable Feedstocks

Yeast sugar transporters have highly evolved for preferential glucose transport, a significant roadblock for utilizing non-glucose sugars in renewable feedstocks such as lignocellulosic biomass. To enable simultaneous transport of multiple sugars, native hexose transporters were replaced by SWEET7p from Arabidopsis thaliana in engineered Saccharomyces cerevisiae capable of fermenting xylose. Engineered S. cerevisiae exhibited reduced glucose preference, simultaneously co-fermenting glucose, mannose, fructose, and xylose both in synthetic and industrial media. Continuous culture experiments demonstrated the co-consuming phenotype and alleviation of glucose repression by engineered S. cerevisiae. In addition to hexose and pentose, the NKSW7-1 strain consumed xylitol as a carbon source. Through transcriptomic and metabolomic analysis of the NKSW7-1 strain, we show that the replacement of HXT1-7 with AtSWEET7 led to systemwide reprogramming of the central carbon metabolism. This broad transport capacity of AtSWEET7p holds promise for achieving co-consumption of all sugars in underutilized renewable feedstocks by microbial cell factory.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Cas9-Based Metabolic Engineering of Issatchenkia orientalis for Enhanced Utilization of Cellulosic Hydrolysates

Issatchenkia orientalis, exhibiting high tolerance against harsh environmental conditions, is a promising metabolic engineering host for producing fuels and chemicals from cellulosic hydrolysates containing fermentation inhibitors under acidic conditions. Although genetic tools for I. orientalis exist, they require auxotrophic mutants so that the selection of a host strain is limited. We developed a drug resistance gene (cloNAT)-based genome-editing method for engineering any I. orientalis strains and engineered I. orientalis strains isolated from various sources for xylose fermentation. Specifically, xylose reductase, xylitol dehydrogenase, and xylulokinase from Scheffersomyces stipitis were integrated into an intended chromosomal locus in four I. orientalis strains (SD108, IO21, IO45, and IO46) through Cas9-based genome editing. Furthermore, the resulting strains (SD108X, IO21X, IO45X, and IO46X) efficiently produced ethanol from cellulosic and hemicellulosic hydrolysates even though the pH adjustment and nitrogen source were not provided. As they presented different fermenting capacities, selection of a host I. orientalis strain was crucial for producing fuels and chemicals using cellulosic hydrolysates.

09 BIOMASS FUELS↗

DOE OSTI2021

Complete and efficient conversion of plant cell wall hemicellulose into high-value bioproducts by engineered yeast

Abstract Plant cell wall hydrolysates contain not only sugars but also substantial amounts of acetate, a fermentation inhibitor that hinders bioconversion of lignocellulose. Despite the toxic and non-consumable nature of acetate during glucose metabolism, we demonstrate that acetate can be rapidly co-consumed with xylose by engineered Saccharomyces cerevisiae . The co-consumption leads to a metabolic re-configuration that boosts the synthesis of acetyl-CoA derived bioproducts, including triacetic acid lactone (TAL) and vitamin A, in engineered strains. Notably, by co-feeding xylose and acetate, an enginered strain produces 23.91 g/L TAL with a productivity of 0.29 g/L/h in bioreactor fermentation. This strain also completely converts a hemicellulose hydrolysate of switchgrass into 3.55 g/L TAL. These findings establish a versatile strategy that not only transforms an inhibitor into a valuable substrate but also expands the capacity of acetyl-CoA supply in S. cerevisiae for efficient bioconversion of cellulosic biomass.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Role of glucuronoxylomannan and steryl glucosides in protecting against cryptococcosis

The development of vaccines for fungal diseases, including cryptococcosis, is an emergent line of research and development. In previous studies, we showed that a Cryptococcus mutant lacking the SGL1 gene (∆sgl1) accumulates certain glycolipids called steryl glucosides (SGs) on the fungal capsule, promoting an effective immunostimulation that totally protects the host from a secondary cryptococcal infection. However, this protection is lost when the cryptococcal capsule is absent in the ∆sgl1 background. The cryptococcal capsule is mainly composed of glucuronoxylomannan (GXM), a polysaccharide microfiber consisting of glucuronic acid, xylose, and mannose linked by glycosidic bonds forming specific triads. In this study, we engineered cells to lack each of the GXM components and tested the effect of these deletions on protection under the condition of SG accumulation. We found that glucuronic acid and xylose are required for protection, and their absence abrogates the production of IFNγ and IL-17A by γδ T cells, which are necessary stimulants for the protective phenotype of the ∆sgl1. We analyzed the structure of the GXM microfibers and found that although the deletion of SGL1 only slightly affects the size and distribution of these microfibers, it significantly changes the ratio of mannose to other components. In conclusion, this study identifies the structural modifications that the deletion of SGL1 and the consequent accumulation of SGs impart to the GXM structure of C. neoformans. This provides significant insights into the protective mechanisms mediated by SG accumulation on the capsule, with important implications for the future development of an efficacious cryptococcal vaccine.

Cryptococcus neoformans↗