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All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Adaptive Laboratory Evolution for Enhanced Performance of Cupriavidus Necator on Formic Acid

The threat of global warming, driven by rising carbon emissions, highlights the need to decarbonize our economy. This requires innovative solutions for managing carbon waste and its effective utilization. One promising method for CO2 capture and sequestration is the electrochemical reduction of CO2 to formic acid, a soluble C1 molecule that can be used to store carbon and energy, and as a feedstock for biological conversion. Cupriavidus necator H16, a soil bacterium capable of consuming and growing on formic acid as its sole carbon and energy source, is well positioned to upgrade CO2-derived formic acid into platform chemicals and fuel precursors. To improve the performance of C. necator on formic acid, adaptive laboratory evolution (ALE), a proven tool for improving microbial fitness, has been conducted using continuous pH-stat bioreactors. The system works on the basis that consumption of formic acid raises the pH and triggers the addition of more formic acid to maintain the pH (in this case 6.7), such that formic acid is provided at the same rate as it is consumed. This system has been coupled with level control to achieve continuous fermentation where cells acquiring mutations that improve growth on formic acid become more abundant in the population, from which they can be isolated and characterized. During developmental experiments it was discovered that formic acid accumulated to inhibitory levels. It was determined that the nitrogen source, ammonium hydroxide, must be tailored to the carbon consumption to avoid formic acid accumulation. The ALE ran in three lineages for approximately 3000 hours and more than 500 generations. Evolved isolates obtained from each lineage demonstrated an increase in growth rate in conjunction with improve formate utilization compared to the parental strain when evaluated in pH-stat bioreactors. The isolates with improved performance were then subjected to whole genome sequencing to identify potentially causative mutations. Mutations in several key genes across different lineages have been found and will be evaluated individually and in combination to identify those that improve growth on formic acid. Incorporating these mutations into production strains has the potential to greatly improve formic acid conversion and further industrial decarbonization.

adaptive laboratory evolution↗
DOE OSTI2023

Phenothiazines Rapidly Induce Laccase Expression and Lignin-Degrading Properties in the White-Rot Fungus Phlebia radiata

Phlebia radiata is a widespread white-rot basidiomycete fungus with significance in diverse biotechnological applications due to its ability to degrade aromatic compounds, xenobiotics, and lignin using an assortment of oxidative enzymes including laccase. In this work, a chemical screen with 480 conditions was conducted to identify chemical inducers of laccase expression in P. radiata. Among the chemicals tested, phenothiazines were observed to induce laccase activity in P. radiata, with promethazine being the strongest laccase inducer of the phenothiazine-derived compounds examined. Secretomes produced by promethazine-treated P. radiata exhibited increased laccase protein abundance, increased enzymatic activity, and an enhanced ability to degrade phenolic model lignin compounds. Transcriptomics analyses revealed that promethazine rapidly induced the expression of genes encoding lignin-degrading enzymes, including laccase and various oxidoreductases, showing that the increased laccase activity was due to increased laccase gene expression. Finally, the generality of promethazine as an inducer of laccases in fungi was demonstrated by showing that promethazine treatment also increased laccase activity in other relevant fungal species with known lignin conversion capabilities including Trametes versicolor and Pleurotus ostreatus.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Data for Engineering and Evolution of Yarrowia lipolytica for Producing Lipids from Lignocellulosic Hydrolysates

Yarrowia lipolytica , an oleaginous yeast, shows promise for industrial fermentation due to its robust acetyl-CoA flux and well-developed genetic engineering tools. However, its lack of an active xylose metabolism restricts the conversion of cellulosic sugars to valuable products. To address this, metabolic engineering, and adaptive laboratory evolution (ALE) were applied to the Y. lipolytica PO1f strain, resulting in an efficient xylose-assimilating strain (XEV). Whole-genome sequencing (WGS) of the XEV followed by reverse engineering revealed that the amplification of the heterologous oxidoreductase pathway and a mutation in the GTPase-activating protein gene (YALI0B12100g) might be the primary reasons for improved xylose assimilation in the XEV strain. When a sorghum hydrolysate was used, the XEV strain showed superior xylose consumption and lipid production compared to its parental strain (X123). This study advances our understanding of xylose metabolism in Y. lipolytica and proposes effective metabolic engineering strategies for optimizing lignocellulosic hydrolysates.

Hydrolysate↗
DOE OSTI2024

Adaptive laboratory evolution and metabolic engineering of Cupriavidus necator for improved catabolism of volatile fatty acids

Bioconversion of high-volume waste streams into value-added products will be an integral component of the growing bioeconomy. Volatile fatty acids (VFAs) (e.g., butyrate, valerate, and hexanoate) are an emerging and promising waste-derived feedstock for microbial carbon upcycling. Cupriavidus necator H16 is a favorable host for conversion of VFAs into various bioproducts due to its diverse carbon metabolism, ease of metabolic engineering, and use at industrial scales. Here, in this study, we report that a common strategy to improve product titers in C. necator, deletion of the polyhydroxybutyrate (PHB) biosynthetic operon, results in a significant growth defect on VFA substrates. Using adaptive laboratory evolution, we identify mutations to the regulator gene phaR, the two-component response regulator-histidine kinase pair encoded by H16_A1372/H16_A1373, and the tripartite transporter assembly encoded by H16_A2296-A2298 as causative for improved growth on VFA substrates. Deletion of phaR and H16_A1373 led to significantly reduced NADH abundance accompanied by large changes to expression of genes involved in carbon metabolism, balance of electron carriers, and oxidative stress tolerance that may be responsible for improved growth of these engineered strains. These results provide insight into the role of PHB biosynthesis in carbon and energy metabolism and highlight a key role for the regulator PhaR in global regulatory networks. By combining mutations, we generated platform strains with significant growth improvements on VFAs, which can enable improved conversion of waste-derived VFA substrates to target bioproducts.

09 BIOMASS FUELS↗
DOE OSTI2022

Hybrid Solid Oxide Fuel Cell/Gas Turbine Model Development for Electric Aviation

A thermodynamic model was developed and validated to analyze a high-performance solid oxide fuel cell and gas turbine (SOFC-GT) hybrid power system for electric aviation. This study used a process simulation software package (ProMax) to study the role of SOFC design and operation on the feasibility and performance of the hybrid system. Standard modules, including compressor, turbine, heat exchanger, reforming reactor, and combustor were used from the ProMax tool suite while a custom module was created to simulate the SOFC stack. The model used an SOFC test data set as an input. Additional SOFC stack performance effects, such as pressure, temperature, and utilization of air and fuel, were added from open source data. System performance predictors were SOFC specific power, fuel-to-electricity conversion efficiency, and hybrid system efficiency. Using these input data and predictors, a static thermodynamic performance model was created that can be modified for different system configurations and operating conditions. Prior to creating the final aircraft performance model, initial demonstration models were developed to validate output results. We used the NASA SOFC model as a benchmark, which was created with their Numerical Propulsion System Simulator (NPSS) software framework. Our output results matched within 1% of both the NASA model and open source SOFC performance data. With confidence gained in the accuracy of this model, a 1-MW SOFC-GT hybrid power system was constructed for an aircraft propulsion concept. Overall hybrid system efficiencies of > 75% FTE were observed during standard 36,000 feet cruise flight conditions.

30 DIRECT ENERGY CONVERSION↗
DOE OSTI2020

DNA Methylation Patterns of Chronic Explosive Breaching in U.S. Military Warfighters

Background: Injuries from exposure to explosions rose dramatically during the Iraq and Afghanistan wars, which motivated investigation of blast-related neurotrauma. We have undertaken human studies involving military “breachers” —exposed to controlled, low-level blast during a 3-days explosive breaching course. Methods: We screened epigenetic profiles in peripheral blood samples from 59 subjects (in two separate U.S. Military training sessions) using Infinium MethylationEPIC BeadChips. Participants had varying numbers of exposures to blast over their military careers (empirically defined as high ≥ 40, and conversely, low < 39 breaching exposures). Daily self-reported physiological symptoms were recorded. Tinnitus, memory problems, headaches, and sleep disturbances are most frequently reported. Results: We identified 14 significantly differentially methylated regions (DMRs) within genes associated with cumulative blast exposure in participants with high relative to low cumulative blast exposure. Notably, NTSR1 and SPON1 were significantly differentially methylated in high relative to low blast exposed groups, suggesting that sleep dysregulation may be altered in response to chronic cumulative blast exposure. In comparing lifetime blast exposure at baseline (prior to exposure in current training), and top associated symptoms, we identified significant DMRs associated with tinnitus, sleep difficulties, and headache. Notably, we identified KCNN3, SOD3, MUC4, GALR1, and WDR45B, which are implicated in auditory function, as differentially methylated associated with self-reported tinnitus. These findings suggest neurobiological mechanisms behind auditory injuries in our military warfighters and are particularly relevant given tinnitus is not only a primary disability among veterans, but has also been demonstrated in active duty medical records for populations exposed to blast in training. Additionally, we found that differentially methylated regions associated with the genes CCDC68 and COMT track with sleep difficulties, and those within FMOD and TNXB track with pain and headache. Conclusion: Sleep disturbances, as well as tinnitus and chronic pain, are widely reported in U.S. military service members and veterans. As we have previously demonstrated, DNA methylation encapsulates lifetime exposure to blast. The current data support previous findings and recapitulate transcriptional regulatory alterations in genes involved in sleep, auditory function, and pain. These data uncovered novel epigenetic and transcriptional regulatory mechanism underlying the etiological basis of these symptoms.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Multiplexed fitness profiling by RB-TnSeq elucidates pathways for lignin-related aromatic catabolism in Sphingobium sp. SYK-6

Bioconversion of lignin-related aromatic compounds relies on robust catabolic pathways in microbes. Sphingobium sp. SYK-6 (SYK-6) is a well-characterized aromatic catabolic organism that has served as a model for microbial lignin conversion, and its utility as a biocatalyst could potentially be further improved by genome-wide metabolic analyses. To this end, we generate a randomly barcoded transposon insertion mutant (RB-TnSeq) library to study gene function in SYK-6. The library is enriched under dozens of enrichment conditions to quantify gene fitness. Several known aromatic catabolic pathways are confirmed, and RB-TnSeq affords additional detail on the genome-wide effects of each enrichment condition. Selected genes are further examined in SYK-6 or Pseudomonas putida KT2440, leading to the identification of new gene functions. The findings from this study further elucidate the metabolism of SYK-6, while also providing targets for future metabolic engineering in this organism or other hosts for the biological valorization of lignin.

09 BIOMASS FUELS↗
DOE OSTI2020

Genome‐wide quantitative trait loci detection for biofuel traits in switchgrass ( Panicum virgatum L.)

Abstract Switchgrass ( Panicum virgatum L.) has been identified as a potential feedstock for cellulosic ethanol production in United States for its high biomass yield and adaptation to marginal lands. Composition of the cell wall plays an important role in bioethanol conversion. A total of 209 pseudo‐F 1 testcross progenies obtained from a biparental cross, AP13 × VS16, were grown at three locations from 2008 to 2011. Near‐infrared spectroscopy was used to estimate cell wall composition from biomass harvested at maturity. A linkage map of the pseudo‐F 1 testcross was constructed with 8,757 SNPs developed by genotyping‐by‐sequencing. Quantitative trait loci (QTL) analysis was performed on eight lignocellulosic traits, namely, klason lignin, sugar, glucose, xylose, hexose, ethanol, hexosoic ethanol, and cell wall ethanol conversion percentage. A total of 327 QTL were recorded for the eight lignocellulosic traits. We have identified 111 major regions in the switchgrass genome that underlie these lignocellulosic traits. Scanning of the genome sequence for genes flanking the QTL peaks, we identified 45 important genes that are involved in lignin biosynthesis, carbohydrate and sugar metabolism, and other biological and cellular functions. Identification of valuable genes associated with QTL along with pleotropic effects of the significant number of QTL suggests that simultaneous selection and genetic improvement of these traits are possible using marker‐assisted selection.

Ali, Shahjahan↗
DOE OSTI2021

Chimeric cellobiohydrolase I expression, activity, and biochemical properties in three oleaginous yeast

Consolidated bioprocessing using oleaginous yeast is a promising modality for the economic conversion of plant biomass to fuels and chemicals. However, yeast are not known to produce effective biomass degrading enzymes naturally and this trait is essential for efficient consolidated bioprocessing. We expressed a chimeric cellobiohydrolase I gene in three different oleaginous, industrially relevant yeast: Yarrowia lipolytica, Lipomyces starkeyi, and Saccharomyces cerevisiae to study the biochemical and catalytic properties and biomass deconstruction potential of these recombinant enzymes. Our results showed differences in glycosylation, surface charge, thermal and proteolytic stability, and efficacy of biomass digestion. L. starkeyi was shown to be an inferior active cellulase producer compared to both the Y. lipolytica and S. cerevisiae enzymes, whereas the cellulase expressed in S. cerevisiae displayed the lowest activity against dilute-acid-pretreated corn stover. Comparatively, the chimeric cellobiohydrolase I enzyme expressed in Y. lipolytica was found to have a lower extent of glycosylation, better protease stability, and higher activity against dilute-acid-pretreated corn stover.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Downregulation of barley ferulate 5-hydroxylase dramatically alters straw lignin structure without impact on mechanical properties

Barley is a major cereal crop for temperate climates, and a diploid genetic model for polyploid wheat. Cereal straw biomass is an attractive source of feedstock for green technologies but lignin, a key determinant of feedstock recalcitrance, complicates bio-conversion processes. However, manipulating lignin content to improve the conversion process could negatively affect agronomic traits. An alternative approach is to manipulate lignin composition which influences the physical and chemical properties of straw. This study validates the function of a barley ferulate 5-hydroxylase gene and demonstrates that its downregulation using the RNA-interference approach substantially impacts lignin composition. We identified five barley genes having putative ferulate 5-hydroxylase activity. Downregulation of HvF5H1 substantially reduced the lignin syringyl/guaiacyl (S/G) ratio in straw while the lignin content, straw mechanical properties, plant growth habit, and grain characteristics all remained unaffected. Metabolic profiling revealed significant changes in the abundance of 173 features in the HvF5H1-RNAi lines. The drastic changes in the lignin polymer of transgenic lines highlight the plasticity of barley lignification processes and the associated potential for manipulating and improving lignocellulosic biomass as a feedstock for green technologies. On the other hand, our results highlight some differences between the lignin biosynthetic pathway in barley, a temperate climate grass, and the warm climate grass, rice, and underscore potential diversity in the lignin biosynthetic pathways in grasses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Mixed polyamide and polyester upcycling via chemical autoxidation and engineered Pseudomonas putida

Polyamides, such as nylons, are often used in multi-component materials, like textiles and packaging, and are accompanied with unique recycling challenges. Recently, autoxidation and bioconversion has emerged as a tandem approach for the conversion of mixed plastics waste to single products, however the fate of polyamides in these processes is unknown. Here, we optimized the autoxidation of nylon-6 and nylon-6,6 depolymerization, achieving >92 mol% nitrogen recovery from both substrates, predominantly as acetamide, and 20–27 mol% carbon recovery (not including acetamide). Experiments with 13 C-labeled acetic acid demonstrated that the carbon in acetamide was solvent derived. Autoxidation of mixed nylon-6 and poly(ethylene terephthalate) (PET) post-consumer fibers resulted in similar carbon and nitrogen recoveries from nylon, while PET was depolymerized to terephthalic acid (TPA) at >65 C-mol% recovery. Next, we engineered Pseudomonas putida KT2440 to utilize acetamide as the sole carbon and nitrogen source for growth through the constitutive expression of genes encoding amidase enzymes, including a native amidase (PP_0613) shown to be active on C 2 –C 4 amides. Heterologous chromosomal expression of amiE, encoding the amidase from P. aeruginosa, was found to be superior to PP_0613 constitutive expression in genome integrated strains. Prior engineering to enable TPA conversion to β-ketoadipate pathway intermediate protocatechuate was leveraged and combined with deletion of pcaD to produce muconolactone as a product. Finally, a stacked strain engineered for conversion of acetamide, TPA, and DCAs was evaluated on the reaction product from autoxidation of mixed post-consumer nylon and PET fibers without any supplemental nitrogen, achieving quantitative yields in the presence of supplemental carbon.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Important Roles of Thiols in Methylmercury Uptake and Translocation by Rice Plants

We report the bioaccumulation of the neurotoxin methylmercury (MeHg) in rice is a significant concern due to its potential risk to humans. Thiols have been known to affect MeHg bioavailability in microorganisms, but how thiols influence MeHg accumulation in rice plants remains unknown. Here, we investigated effects of common low-molecular-weight thiols, including cysteine (Cys), glutathione (GSH), and penicillamine (PEN), on MeHg uptake and translocation by rice plants. Results show that rice roots can rapidly take up MeHg, and this process is influenced by the types and concentrations of thiols in the system. The presence of Cys facilitated MeHg uptake by roots and translocation to shoots, while GSH could only promote MeHg uptake, but not translocation, by roots. Conversely, PEN significantly inhibited MeHg uptake and translocation to shoots. Using labeled 13 Cys assays, we also found that MeHg uptake was coupled with Cys accumulation in rice roots. Moreover, analyses of comparative transcriptomics revealed that key genes associated with metallothionein and SULTR transporter families may be involved in MeHg uptake. These findings provide new insights into the uptake and translocation of MeHg in rice plants and suggest potential roles of thiol attributes in affecting MeHg bioavailability and bioaccumulation in rice.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Simultaneous carbon catabolite repression governs sugar and aromatic co-utilization in Pseudomonas putida M2

ABSTRACT Pseudomonas putida have emerged as promising biocatalysts for the conversion of sugars and aromatic compounds obtained from lignocellulosic biomass. Understanding the role of carbon catabolite repression (CCR) in these strains is critical to optimize biomass conversion to fuels and chemicals. The CCR functioning in P. putida M2, a strain capable of consuming both hexose and pentose sugars as well as aromatic compounds, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 co-utilized sugars and aromatic compounds simultaneously; however, during cultivation with glucose and aromatic compounds ( p- coumarate and ferulate) mixture, intermediates (4-hydroxybenzoate and vanillate) accumulated, and substrate consumption was incomplete. In contrast, xylose-aromatic consumption resulted in transient intermediate accumulation and complete aromatic consumption, while xylose was incompletely consumed. Proteomics analysis revealed that glucose exerted stronger repression than xylose on the aromatic catabolic proteins. Key glucose (Eda) and xylose (XylX) catabolic proteins were also identified at lower abundance during cultivation with aromatic compounds implying simultaneous catabolite repression by sugars and aromatic compounds. Reduction of crc expression via CRISPRi led to faster growth and glucose and p -coumarate uptake in the CRISPRi strains compared to the control, while no difference was observed on xylose+ p -coumarate. The increased abundances of Eda and amino acid biosynthesis proteins in the CRISPRi strain further supported these observations. Lastly, small RNAs (sRNAs) sequencing results showed that CrcY and CrcZ homologues levels in M2, previously identified in P. putida strains, were lower under strong CCR (glucose+ p -coumarate) condition compared to when repression was absent ( p -coumarate or glucose only). IMPORTANCE A newly isolated Pseudomonas putida strain, P. putida M2, can utilize both hexose and pentose sugars as well as aromatic compounds making it a promising host for the valorization of lignocellulosic biomass. Pseudomonads have developed a regulatory strategy, carbon catabolite repression, to control the assimilation of carbon sources in the environment. Carbon catabolite repression may impede the simultaneous and complete metabolism of sugars and aromatic compounds present in lignocellulosic biomass and hinder the development of an efficient industrial biocatalyst. This study provides insight into the cellular physiology and proteome during mixed-substrate utilization in P. putida M2. The phenotypic and proteomics results demonstrated simultaneous catabolite repression in the sugar-aromatic mixtures, while the CRISPRi and sRNA sequencing demonstrated the potential role of the crc gene and small RNAs in carbon catabolite repression.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Engineering Novosphingobium aromaticivorans to produce cis,cis -muconic acid from biomass aromatics

ABSTRACT The platform chemical cis,cis- muconic acid ( cc MA) provides facile access to a number of monomers used in the synthesis of commercial plastics. It is also a metabolic intermediate in the β-ketoadipic acid pathway of many bacteria and, therefore, a current target for microbial production from abundant renewable resources via metabolic engineering. This study investigates Novosphingobium aromaticivorans DSM12444 as a chassis for the production of cc MA from biomass aromatics. The N. aromaticivorans genome predicts that it encodes a previously uncharacterized protocatechuic acid (PCA) decarboxylase and a catechol 1,2-dioxygenase, which would be necessary for the conversion of aromatic metabolic intermediates to cc MA. This study confirmed the activity of these two enzymes in vitro and compared their activity to ones that have been previously characterized and used in cc MA production. From these results, we generated one strain that is completely derived from native genes and a second that contains genes previously used in microbial engineering synthesis of this compound. Both of these strains exhibited stoichiometric production of cc MA from PCA and produced greater than 100% yield of cc MA from the aromatic monomers that were identified in liquor derived from alkaline pretreated biomass. Our results show that a strain completely derived from native genes and one containing homologs from other hosts are both capable of stoichiometric production of cc MA from biomass aromatics. Overall, this work combines previously unknown aspects of aromatic metabolism in N. aromaticivorans and the genetic tractability of this organism to generate strains that produce cc MA from deconstructed biomass. IMPORTANCE The production of commodity chemicals from renewable resources is an important goal toward increasing the environmental and economic sustainability of industrial processes. The aromatics in plant biomass are an underutilized and abundant renewable resource for the production of valuable chemicals. However, due to the chemical composition of plant biomass, many deconstruction methods generate a heterogeneous mixture of aromatics, thus making it difficult to extract valuable chemicals using current methods. Therefore, recent efforts have focused on harnessing the pathways of microorganisms to convert a diverse set of aromatics into a single product. Novosphingobium aromaticivorans DSM12444 has the native ability to metabolize a wide range of aromatics and, thus, is a potential chassis for conversion of these abundant compounds to commodity chemicals. This study reports on new features of N. aromaticivorans that can be used to produce the commodity chemical cis,cis -muconic acid from renewable and abundant biomass aromatics.

09 BIOMASS FUELS↗
DOE OSTI2026

Elucidating biodegradation of dimethyl terephthalate by two Rhodococcus strains for its valorization applications

Dimethyl terephthalate (DMT) serves as the precursor in the production of polyethylene terephthalate, polytrimethylene terephthalate, and polybutylene terephthalate. The widespread use of DMT in the polymer industry and its ubiquitous existence in end products raise alarms about its potential harm to humans and animals. DMT can enter the environment through the degradation of polymers and their end products, and cause endocrine disruption, oxidative stress, and an elevated risk of cancer. In recent years, DMT has also gained renewed interest in its potential for plastic recycling and upcycling. In this study, we identified two strains of Rhodococcus that possess DMT-degrading capabilities and utilized transcriptomic analysis and gene knockout to elucidate the mechanisms of DMT degradation. R. opacus PD630 and R. jostii RPET were found to convert up to 1 g/L DMT into mono-methyl terephthalate (MMT). A putative DMTase (RS34275) was identified for this conversion. R. jostii RPET also demonstrates the ability to convert DMT into MMT and to utilize MMT for its cellular growth via the terephthalate pathway. A putative MMTase (RS21885) as the sole enzyme was identified for the conversion of MMT into terephthalate in the RPET strain. In addition, we successfully produced lycopene and lipids from an engineered RPET strain using DMT as a substrate. Our findings will facilitate future DMT bioremediation and bio-upcycling of DMT-associated plastics, enabling the production of value-added products.

Biodegradation↗
DOE OSTI2023

Engineering transcriptional regulation of pentose metabolism in Rhodosporidium toruloides for improved conversion of xylose to bioproducts

Abstract Efficient conversion of pentose sugars remains a significant barrier to the replacement of petroleum-derived chemicals with plant biomass-derived bioproducts. While the oleaginous yeast Rhodosporidium toruloides (also known as Rhodotorula toruloides ) has a relatively robust native metabolism of pentose sugars compared to other wild yeasts, faster assimilation of those sugars will be required for industrial utilization of pentoses. To increase the rate of pentose assimilation in R. toruloides, we leveraged previously reported high-throughput fitness data to identify potential regulators of pentose catabolism. Two genes were selected for further investigation, a putative transcription factor (RTO4_12978, Pnt1) and a homolog of a glucose transceptor involved in carbon catabolite repression (RTO4_11990). Overexpression of Pnt1 increased the specific growth rate approximately twofold early in cultures on xylose and increased the maximum specific growth by 18% while decreasing accumulation of arabitol and xylitol in fast-growing cultures. Improved growth dynamics on xylose translated to a 120% increase in the overall rate of xylose conversion to fatty alcohols in batch culture. Proteomic analysis confirmed that Pnt1 is a major regulator of pentose catabolism in R. toruloides. Deletion of RTO4_11990 increased the growth rate on xylose, but did not relieve carbon catabolite repression in the presence of glucose. Carbon catabolite repression signaling networks remain poorly characterized in R. toruloides and likely comprise a different set of proteins than those mainly characterized in ascomycete fungi.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Crabtree/Warburg-like aerobic xylose fermentation by engineered Saccharomyces cerevisiae

Bottlenecks in the efficient conversion of xylose into cost-effective biofuels have limited the widespread use of plant lignocellulose as a renewable feedstock. The yeast Saccharomyces cerevisiae ferments glucose into ethanol with such high metabolic flux that it ferments high concentrations of glucose aerobically, a trait called the Crabtree/Warburg Effect. In contrast to glucose, most engineered S. cerevisiae strains do not ferment xylose at economically viable rates and yields, and they require respiration to achieve sufficient xylose metabolic flux and energy return for growth aerobically. Here, we evolved respiration-deficient S. cerevisiae strains that can grow on and ferment xylose to ethanol aerobically, a trait analogous to the Crabtree/Warburg Effect for glucose. Through genome sequence comparisons and directed engineering, we determined that duplications of genes encoding engineered xylose metabolism enzymes, as well as TKL1, a gene encoding a transketolase in the pentose phosphate pathway, were the causative genetic changes for the evolved phenotype. Reengineered duplications of these enzymes, in combination with deletion mutations in HOG1, ISU1, GRE3, and IRA2, increased the rates of aerobic and anaerobic xylose fermentation. Importantly, we found that these genetic modifications function in another genetic background and increase the rate and yield of xylose-to-ethanol conversion in industrially relevant switchgrass hydrolysate, indicating that these specific genetic modifications may enable the sustainable production of industrial biofuels from yeast. We propose a model for how key regulatory mutations prime yeast for aerobic xylose fermentation by lowering the threshold for overflow metabolism, allowing mutations to increase xylose flux and to redirect it into fermentation products.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Analysis of a preQ1-I riboswitch in effector-free and bound states reveals a metabolite-programmed nucleobase-stacking spine that controls gene regulation

Abstract Riboswitches are structured RNA motifs that recognize metabolites to alter the conformations of downstream sequences, leading to gene regulation. To investigate this molecular framework, we determined crystal structures of a preQ1-I riboswitch in effector-free and bound states at 2.00 Å and 2.65 Å-resolution. Both pseudoknots exhibited the elusive L2 loop, which displayed distinct conformations. Conversely, the Shine-Dalgarno sequence (SDS) in the S2 helix of each structure remained unbroken. The expectation that the effector-free state should expose the SDS prompted us to conduct solution experiments to delineate environmental changes to specific nucleobases in response to preQ1. We then used nudged elastic band computational methods to derive conformational-change pathways linking the crystallographically-determined effector-free and bound-state structures. Pathways featured: (i) unstacking and unpairing of L2 and S2 nucleobases without preQ1—exposing the SDS for translation and (ii) stacking and pairing L2 and S2 nucleobases with preQ1—sequestering the SDS. Our results reveal how preQ1 binding reorganizes L2 into a nucleobase-stacking spine that sequesters the SDS, linking effector recognition to biological function. The generality of stacking spines as conduits for effector-dependent, interdomain communication is discussed in light of their existence in adenine riboswitches, as well as the turnip yellow mosaic virus ribosome sensor.

59 BASIC BIOLOGICAL SCIENCES↗