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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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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 OSTI2021

The Role of Sugar Signaling in Regulating Plant Fatty Acid Synthesis

Photosynthates such as glucose, sucrose, and some of their derivatives play dual roles as metabolic intermediates and signaling molecules that influence plant cell metabolism. Such sugars provide substrates for de novo fatty acid (FA) biosynthesis. However, compared with the well-defined examples of sugar signaling in starch and anthocyanin synthesis, until recently relatively little was known about the role of signaling in regulating FA and lipid biosynthesis. Recent research progress shows that trehalose 6-phosphate and 2-oxoglutarate (2-OG) play direct signaling roles in the regulation of FA biosynthesis by modulating transcription factor stability and enzymatic activities involved in FA biosynthesis. Specifically, mechanistic links between sucrose non-fermenting−1–related protein kinase 1 (SnRK1)–mediated trehalose 6-phosphate (T6P) sensing and its regulation by phosphorylation of WRI1 stability, diacylglycerol acyltransferase 1 (DGAT1) enzyme activity, and of 2-OG–mediated relief of inhibition of acetyl-CoA carboxylase (ACCase) activity by protein PII are exemplified in detail in this review.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

New Insights Into Lignification via Network and Multi-Omics Analyses of Arogenate Dehydratase Knock-Out Mutants in Arabidopsis thaliana

Multiple Arabidopsis arogenate dehydratase (ADT) knock-out (KO) mutants, with phenotypes having variable lignin levels (up to circa 70% reduction), were studied to investigate how differential reductions in ADTs perturb its overall plant systems biology. Integrated "omics" analyses (metabolome, transcriptome, and proteome) of wild type (WT), single and multiple ADT KO lines were conducted. Transcriptome and proteome data were collapsed into gene ortholog (GO) data, with this allowing for enzymatic reaction and metabolome cross-comparisons to uncover dominant or likely metabolic biosynthesis reactions affected. Network analysis of enzymes-highly correlated to stem lignin levels-deduced the involvement of novel putative lignin related proteins or processes. These included those associated with ribosomes, the spliceosome, mRNA transport, aminoacyl tRNA biosynthesis, and phosphorylation. While prior work helped explain lignin biosynthesis regulation at the transcriptional level, our data here provide support for a new hypothesis that there are additional post-transcriptional and translational level processes that need to be considered. These findings are anticipated to lead to development of more accurate depictions of lignin/phenylpropanoid biosynthesis models in situ, with new protein targets identified for further biochemical analysis and/or plant bioengineering. Additionally, using KEGG defined functional categorization of proteomics and transcriptomics analyses, we detected significant changes to glucosinolate, a-linolenic acid, nitrogen, carotenoid, aromatic amino acid, phenylpropanoid, and photosynthesis-related metabolic pathways in ADT KO mutants. Metabolomics results also revealed that putative carotenoid and galactolipid levels were generally increased in amount, whereas many glucosinolates and phenylpropanoids (including flavonoids and lignans) were decreased in the KO mutants.

Arabidopsis, arogenate dehydratases, lignin, multi↗
DOE OSTI2023

Wounding rapidly alters transcription factor expression, hormonal signaling, and phenolic compound metabolism in harvested sugarbeet roots

Injuries sustained by sugarbeet (Beta vulgaris L.) roots during harvest and postharvest operations seriously reduce the yield of white sugar produced from stored roots. Although wound healing is critically important to reduce losses, knowledge of these processes is limited for this crop as well as for roots in other species. To better understand the metabolic signals and changes that occur in wounded roots, dynamic changes in gene expression were determined by RNA sequencing and the activity of products from key genes identified in this analysis were determined in the 0.25 to 24 h following injury. Nearly five thousand differentially expressed genes that contribute to a wide range of cellular and molecular functions were identified in wounded roots. Highly upregulated genes included transcription factor genes, as well as genes involved in ethylene and jasmonic acid (JA) biosynthesis and signaling and phenolic compound biosynthesis and polymerization. Enzyme activities for key genes in ethylene and phenolic compound biosynthesis and polymerization also increased due to wounding. Results indicate that wounding causes a major reallocation of metabolism in sugarbeet taproots. Although both ethylene and JA are likely involved in triggering wound responses, the greater and more sustained upregulation of ethylene biosynthesis and signaling genes relative to those of JA, suggest a preeminence of ethylene signaling in wounded sugarbeet roots. Changes in gene expression and enzymes involved in phenolic compound metabolism additionally indicate that barriers synthesized to seal off wounds, such as suberin or lignin, are initiated within the first 24 h after injury.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

The yeast ALA synthase C‐terminus positively controls enzyme structure and function

Abstract 5‐Aminolevulinic acid synthase (ALAS) is a pyridoxal 5′‐phosphate (PLP)‐dependent enzyme that catalyzes the first and rate‐limiting step of heme biosynthesis in α‐proteobacteria and several non‐plant eukaryotes. All ALAS homologs contain a highly conserved catalytic core, but eukaryotes also have a unique C‐terminal extension that plays a role in enzyme regulation. Several mutations in this region are implicated in multiple blood disorders in humans. In Saccharomyces cerevisiae ALAS (Hem1), the C‐terminal extension wraps around the homodimer core to contact conserved ALAS motifs proximal to the opposite active site. To determine the importance of these Hem1 C‐terminal interactions, we determined the crystal structure of S. cerevisiae Hem1 lacking the terminal 14 amino acids (Hem1 ΔCT). With truncation of the C‐terminal extension, we show structurally and biochemically that multiple catalytic motifs become flexible, including an antiparallel β‐sheet important to Fold‐Type I PLP‐dependent enzymes. The changes in protein conformation result in an altered cofactor microenvironment, decreased enzyme activity and catalytic efficiency, and ablation of subunit cooperativity. These findings suggest that the eukaryotic ALAS C‐terminus has a homolog‐specific role in mediating heme biosynthesis, indicating a mechanism for autoregulation that can be exploited to allosterically modulate heme biosynthesis in different organisms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Adaptive laboratory evolution of methylotrophic Escherichia coli enables synthesis of all amino acids from methanol-derived carbon

Recent attempts to create synthetic Escherichia coli methylotrophs identified that de novo biosynthesis of amino acids, in the presence of methanol, presents significant challenges in achieving autonomous methylotrophic growth. Previously engineered methanol-dependent strains required co-utilization of stoichiometric amounts of co-substrates and methanol. As such, these strains could not be evolved to grow on methanol alone. In this work, we have explored an alternative approach to enable biosynthesis of all amino acids from methanol-derived carbon in minimal media without stoichiometric coupling. First, we identified that biosynthesis of threonine was limiting the growth of our methylotrophic E. coli. To address this, we performed adaptive laboratory evolution to generate a strain that grew efficiently in minimal medium with methanol and threonine. Methanol assimilation and growth of the evolved strain were analyzed and, interestingly, we found that the evolved strain synthesized all amino acids, including threonine, from methanol-derived carbon. Here, the evolved strain was then further engineered through overexpression of an optimized threonine biosynthetic pathway. We show that the resulting methylotrophic E. coli strain has a methanol-dependent growth phenotype with homoserine as co-substrate. In contrast to previous methanol-dependent strains, co-utilization of homoserine is not stoichiometrically linked to methanol assimilation. As such, future engineering of this strain and successive adaptive evolution could enable autonomous growth on methanol as the sole carbon source.

13C-labeling analysis↗
DOE OSTI2021

Endothelial cells control vascular smooth muscle cell cholesterol levels by regulating 24-dehydrocholesterol reductase expression

Highlights: • Suspended endothelial and vascular smooth muscle cells organize to form spherical vascular organoids in hanging drops. • Vascular smooth muscle cells downregulate the expression of metabolism regulating genes in presence of endothelial cells. • Endothelial cells decrease the levels of 24-dehydrocholesterol reductase and cholesterol in vascular smooth muscle cells. • Diminished cholesterol biosynthesis inhibits vascular smooth muscle cell activity. Communication of vascular cells is essential for the control of organotypic functions of blood vessels. In this context, vascular endothelial cells (EC) act as potent regulators of vascular smooth muscle cell (VSMC) functions such as contraction and relaxation. However, the impact of ECs on the gene expression pattern of VSMCs is largely unknown. Here, we investigated changes of the VSMC transcriptome by utilizing 3D human vascular organoids organized as a core of VSMCs enclosed by a monolayer of ECs. Microarray-based analyses indicated that interaction with ECs for 48 h down-regulates expression of genes in VSMCs controlling rate-limiting steps of the cholesterol biosynthesis such as HMGCR, HMGCS1, DHCR24 and DHCR7. Protein analyses revealed a decrease in the abundance of DHCR24 (24-dehydrocholesterol reductase) and lower cholesterol levels in VSMCs co-cultured with ECs. On the functional level, the blockade of the DHCR24 activity impaired adhesion, migration and proliferation of VSMCs. Collectively, these findings indicate that ECs have the capacity to instruct VSMCs to shut down the expression of DHCR24 thereby limiting their cholesterol biosynthesis, which may support their functional steady state.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Gene targets for engineering osmotolerance in Caldicellulosiruptor bescii

Abstract Background Caldicellulosiruptor bescii , a promising biocatalyst being developed for use in consolidated bioprocessing of lignocellulosic materials to ethanol, grows poorly and has reduced conversion at elevated medium osmolarities. Increasing tolerance to elevated fermentation osmolarities is desired to enable performance necessary of a consolidated bioprocessing (CBP) biocatalyst. Results Two strains of C. bescii showing growth phenotypes in elevated osmolarity conditions were identified. The first strain, ORCB001, carried a deletion of the FapR fatty acid biosynthesis and malonyl-CoA metabolism repressor and had a severe growth defect when grown in high-osmolarity conditions—introduced as the addition of either ethanol, NaCl, glycerol, or glucose to growth media. The second strain, ORCB002, displayed a growth rate over three times higher than its genetic parent when grown in high-osmolarity medium. Unexpectedly, a genetic complement ORCB002 exhibited improved growth, failing to revert the observed phenotype, and suggesting that mutations other than the deleted transcription factor (the fruR/cra gene) are responsible for the growth phenotype observed in ORCB002. Genome resequencing identified several other genomic alterations (three deleted regions, three substitution mutations, one silent mutation, and one frameshift mutation), which may be responsible for the observed increase in osmolarity tolerance in the fruR / cra -deficient strain, including a substitution mutation in dnaK , a gene previously implicated in osmoresistance in bacteria. Differential expression analysis and transcription factor binding site inference indicates that FapR negatively regulates malonyl-CoA and fatty acid biosynthesis, as it does in many other bacteria. FruR/Cra regulates neighboring fructose metabolism genes, as well as other genes in global manner. Conclusions Two systems able to effect tolerance to elevated osmolarities in C. bescii are identified. The first is fatty acid biosynthesis. The other is likely the result of one or more unintended, secondary mutations present in another transcription factor deletion strain. Though the locus/loci and mechanism(s) responsible remain unknown, candidate mutations are identified, including a mutation in the dnaK chaperone coding sequence. These results illustrate both the promise of targeted regulatory manipulation for osmotolerance (in the case of fapR ) and the challenges (in the case of fruR/cra ).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Engineering Clostridium cellulovorans for highly selective n ‐butanol production from cellulose in consolidated bioprocessing

Abstract Cellulosic n ‐butanol from renewable lignocellulosic biomass has gained increased interest. Previously, we have engineered Clostridium cellulovorans , a cellulolytic acidogen, to overexpress the bifunctional butyraldehyde/butanol dehydrogenase gene adh E2 from C. acetobutylicum for n ‐butanol production from crystalline cellulose. However, butanol production by this engineered strain had a relatively low yield of approximately 0.22 g/g cellulose due to the coproduction of ethanol and acids. We hypothesized that strengthening the carbon flux through the central butyryl‐CoA biosynthesis pathway and increasing intracellular NADH availability in C. cellulovorans adh E2 would enhance n ‐butanol production. In this study, thiolase ( thl A CA ) from C. acetobutylicum and 3‐hydroxybutyryl‐CoA dehydrogenase ( hbd CT ) from C. tyrobutyricum were overexpressed in C. cellulovorans adh E2 to increase the flux from acetyl‐CoA to butyryl‐CoA. In addition, ferredoxin‐NAD(P) + oxidoreductase ( fnr ), which can regenerate the intracellular NAD(P)H and thus increase butanol biosynthesis, was also overexpressed. Metabolic flux analyses showed that mutants overexpressing these genes had a significantly increased carbon flux toward butyryl‐CoA, which resulted in increased production of butyrate and butanol. The addition of methyl viologen as an electron carrier in batch fermentation further directed more carbon flux towards n ‐butanol biosynthesis due to increased reducing equivalent or NADH. The engineered strain C. cellulovorans adh E2‐ fnr CA ‐ thl A CA ‐ hbd CT produced n ‐butanol from cellulose at a 50% higher yield (0.34 g/g), the highest ever obtained in batch fermentation by any known bacterial strain. The engineered C. cellulovorans is thus a promising host for n ‐butanol production from cellulosic biomass in consolidated bioprocessing.

Bao, Teng↗
DOE OSTI2026

Nitrogen Status Rewires Transcriptional Regulation of Dhurrin, a Dual‐Purpose Defense Metabolite in Sorghum bicolor

Dhurrin, a cyanogenic glucoside, plays an important role in Sorghum bicolor physiology and defense. The concentration of dhurrin in sorghum is influenced by both nitrogen status and stage of plant organ development. While nitrogen resupply activates the expression of genes for dhurrin biosynthesis, the molecular mechanisms underlying this regulation remain unclear. In this study, we investigated the transcriptional response of sorghum to nitrogen resupply following growth under nitrogen-limiting conditions. Using a time-course design, we measured hydrogen cyanide potential (HCNp), growth, and nitrate content at 0-, 2-, 6-, 12-, 24-, 36-, 48-, and 60-h after resupply and collected tissue for RNAseq analysis in parallel for analysis of gene expression and construction of gene regulatory networks (GRNs). HCNp (mg g −1 DW) increased significantly in leaf and stem tissues following nitrogen resupply, with increases in the leaf partially driven by continued declines in controls under ongoing nitrogen stress. Expression of the dhurrin pathway genes was upregulated in leaves from 24 h after nitrogen resupply, with diel expression patterns observable over the remaining time points. No upregulation was observed in roots or stems, suggesting that developmental context overrides environmental cues. GRN analysis identified candidate transcription factors regulating dhurrin biosynthesis genes, including members of the MYB, bZIP, and GARP-type transcription factor families. Some of these candidate transcription factors may be involved in relieving senescence-associated suppression of dhurrin biosynthesis and link nitrogen signaling to pathway activation. These findings provide new insight into the nitrogen-responsive regulation of dhurrin in sorghum, highlighting candidate regulators for future functional characterization.

S. bicolor↗
DOE OSTI2021

Regulation of dhurrin pathway gene expression during Sorghum bicolor development

Plant defence models evaluate the costs and benefits of resource investments at different stages in the life cycle. Poor understanding of the molecular regulation of defence deployment and remobilization hampers accuracy of the predictions. Cyanogenic glucosides, such as dhurrin are phytoanticipins that release hydrogen cyanide upon bio-activation. In this study, RNA-seq was used to investigate the expression of genes involved in the biosynthesis, bio-activation and recycling of dhurrin in Sorghum bicolor . Genes involved in dhurrin biosynthesis were highly expressed in all young developing vegetative tissues (leaves, leaf sheath, roots, stems), tiller buds and imbibing seeds and showed gene specific peaks of expression in leaves during diel cycles. Genes involved in dhurrin bio-activation were expressed early in organ development with organ-specific expression patterns. Genes involved in recycling were expressed at similar levels in the different organ during development, although post-floral initiation when nutrients are remobilized for grain filling, expression of GSTL1 decreased > tenfold in leaves and NITB2 increased > tenfold in stems. Results are consistent with the establishment of a pre-emptive defence in young tissues and regulated recycling related to organ senescence and increased demand for nitrogen during grain filling. This detailed characterization of the transcriptional regulation of dhurrin biosynthesis, bioactivation and remobilization genes during organ and plant development will aid elucidation of gene regulatory networks and signalling pathways that modulate gene expression and dhurrin levels. In-depth knowledge of dhurrin metabolism could improve the yield, nitrogen use efficiency and stress resilience of Sorghum .

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Chlamydomonas cells transition through distinct Fe nutrition stages within 48 h of transfer to Fe-free medium

Low iron (Fe) bioavailability can limit the biosynthesis of Fe-containing proteins, which are especially abundant in photosynthetic organisms, thus negatively affecting global primary productivity. Understanding cellular coping mechanisms under Fe limitation is therefore of great interest. For this paper, we surveyed the temporal responses of Chlamydomonas ( Chlamydomonas reinhardtii ) cells transitioning from an Fe-rich to an Fe-free medium to document their short and long-term adjustments. While slower growth, chlorosis and lower photosynthetic parameters are evident only after one or more days in Fe-free medium, the abundance of some transcripts, such as those for genes encoding transporters and enzymes involved in Fe assimilation, change within minutes, before changes in intracellular Fe content are noticeable, suggestive of a sensitive mechanism for sensing Fe. Promoter reporter constructs indicate a transcriptional component to this immediate primary response. With acetate provided as a source of reduced carbon, transcripts encoding respiratory components are maintained relative to transcripts encoding components of photosynthesis and tetrapyrrole biosynthesis, indicating metabolic prioritization of respiration over photosynthesis. In contrast to the loss of chlorophyll, carotenoid content is maintained under Fe limitation despite a decrease in the transcripts for carotenoid biosynthesis genes, indicating carotenoid stability. These changes occur more slowly, only after the intracellular Fe quota responds, indicating a phased response in Chlamydomonas, involving both primary and secondary responses during acclimation to poor Fe nutrition.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Computational design and engineering of an Escherichia coli strain producing the nonstandard amino acid para -aminophenylalanine

Introducing heterologous pathways into host cells constitutes a promising strategy for synthesizing nonstandard amino acids (nsAAs) to enable the production of proteins with expanded chemistries. However, this strategy has proven challenging, as the expression of heterologous pathways can disrupt cellular homeostasis of the host cell. Here, we sought to optimize the heterologous production of the nsAA para -aminophenylalanine (pAF) in Escherichia coli . First, we incorporated a heterologous pAF biosynthesis pathway into a genome-scale model of E. coli metabolism and computationally identified metabolic interventions in the host’s native metabolism to improve pAF production. Next, we explored different approaches of imposing these flux interventions experimentally and found that the upregulation of flux in the chorismate biosynthesis pathway through the elimination of feedback inhibition mechanisms could significantly raise pAF titers (~20-fold) while maintaining a reasonable pAF production-growth rate trade-off. Overall, this study provides a promising strategy for the biosynthesis of nsAAs in engineered cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Structure-Based Prototyping of Allosteric Inhibitors of Human Uridine/Cytidine Kinase 2 (UCK2)

Pyrimidine nucleotide biosynthesis in humans is a promising chemotherapeutic target for infectious diseases caused by RNA viruses. Because mammalian cells derive pyrimidine ribonucleotides through a combination of de novo biosynthesis and salvage, combined inhibition of dihydroorotate dehydrogenase (DHODH; the first committed step in de novo pyrimidine nucleotide biosynthesis) and uridine/cytidine kinase 2 (UCK2; the first step in salvage of exogenous nucleosides) strongly attenuates viral replication in infected cells. However, while several pharmacologically promising inhibitors of human DHODH are known, to date there are no reports of medicinally viable leads against UCK2. Here we use structure-based drug prototyping to identify two classes of promising leads that non-competitively inhibit UCK2 activity. In the process we have identified a hitherto unknown allosteric site at the inter-subunit interface of this homotetrameric enzyme. By reducing the kcat of human UCK2 without altering its KM, these new inhibitors have the potential to enable systematic dialing of the fractional inhibition of pyrimidine salvage to achieve the desired antiviral effect with minimal host toxicity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Engineered Membrane Vesicle Production via oprF or oprI Deletion Has Distinct Phenotypic Effects in Pseudomonas putida

Membrane vesicle (MV) production is a natural phenomenon in Gram-negative bacteria and represents an emerging synthetic biology tool for the secretion of biomolecules or bioproducts. Manipulation of membrane components has proven successful in enhancing MV production. However, the impact of membrane disruptions on strain fitness and protein composition warrants further investigation for the use of MVs in industrial bioprocesses. Here, we identify and characterize two genetic engineering strategies for inducing hypervesiculation-deletion of genes for the outer membrane porin OprF or the lipoprotein OprI-in the commonly used platform Pseudomonas putida KT2440. Deletion of oprI generated up to a 1.5-fold increase in MVs, larger MVs with a greater proportion of outer membrane proteins, and no significant impact on strain fitness compared to wild type. In contrast, deletion of oprF, relative to wild type, generated up to a 4-fold increase in MVs but diminished growth, permeabilized membranes, and increased cytosolic protein packaging. Both hypervesiculation phenotypes increased nontargeted and MV-targeted mNeonGreen extracellular signal by up to 6-fold, demonstrating vesiculation as a mechanism for protein secretion. Despite increased blebbing of MVs from gene deletions, proteins involved in membrane biosynthesis were not elevated relative to wild type. Overexpression of gpsA, which initiates glycerophospholipid biosynthesis, in the ΔoprF background improved the membrane integrity by 37% and maintained MV formation, highlighting the importance of membrane biosynthesis in restoring the membrane in hypervesiculating strains. Together, this study provides genetic engineering strategies with corresponding phenotypic outcomes toward providing a synthetic biology toolset for MV deployment in P. putida.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Engineering a universal and efficient platform for terpenoid synthesis in yeast

Engineering microbes for the production of valuable natural products is often hindered by the regulation of native competing metabolic networks in host. This is particularly evident in the case of terpenoid synthesis in yeast, where the canonical terpenoid precursors are tightly coupled to the biosynthesis of sterols essential for yeast viability. One way to circumvent this limitation is by engineering product pathways less connected to the host native metabolism. Here, we introduce a two-step isopentenol utilization pathway (IUP) in Saccharomyces cerevisiae to augment the native mevalonate pathway by providing a shortcut to the synthesis of the common terpenoid precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). As such, the IUP was capable of elevating the IPP/DMAPP pool by 147-fold compared with the native pathway. We further demonstrate that cofeeding isoprenol and prenol enhances geranyl diphosphate (GPP) content for monoterpene biosynthesis. More importantly, we established a synthetic three-step route for efficient synthesis of di-and tetraterpene precursor geranylgeranyl diphosphate (GGPP), circumventing the competition with farnesyl diphosphate (FPP) for sterol biosynthesis and elevating the GGPP level by 374-fold. We combine these IUP-supported precursor-forming platforms with downstream terpene synthases to harness their potential and improve the production of industrially relevant terpenoids by several fold. In conclusion, our exploration provides a universal and effective platform for supporting terpenoid synthesis in yeast.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Engineering peroxisomal biosynthetic pathways for maximization of triterpene production in Yarrowia lipolytica

Constructing efficient cell factories for product synthesis is frequently hampered by competing pathways and/or insufficient precursor supply. This is particularly evident in the case of triterpenoid biosynthesis in Yarrowia lipolytica, where squalene biosynthesis is tightly coupled to cytosolic biosynthesis of sterols essential for cell viability. Here, we addressed this problem by reconstructing the complete squalene biosynthetic pathway, starting from acetyl-CoA, in the peroxisome, thus harnessing peroxisomal acetyl-CoA pool and sequestering squalene synthesis in this organelle from competing cytosolic reactions. This strategy led to increasing the squalene levels by 1,300-fold relatively to native cytosolic synthesis. Subsequent enhancement of the peroxisomal acetyl-CoA supply by two independent approaches, 1) converting cellular lipid pool to peroxisomal acetyl-CoA and 2) establishing an orthogonal acetyl-CoA shortcut from CO 2 -derived acetate in the peroxisome, further significantly improved local squalene accumulation. Using these approaches, we constructed squalene-producing strains capable of yielding 32.8 g/L from glucose, and 31.6 g/L from acetate by employing a cofeeding strategy, in bioreactor fermentations. Our findings provide a feasible strategy for protecting intermediate metabolites that can be claimed by multiple reactions by engineering peroxisomes in Y. lipolytica as microfactories for the production of such intermediates and in particular acetyl-CoA-derived metabolites.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Bacterium-enabled transient gene activation by artificial transcription factors for resolving gene regulation in maize

Abstract Understanding gene regulatory networks is essential to elucidate developmental processes and environmental responses. Here, we studied regulation of a maize (Zea mays) transcription factor gene using designer transcription activator-like effectors (dTALes), which are synthetic Type III TALes of the bacterial genus Xanthomonas and serve as inducers of disease susceptibility gene transcription in host cells. The maize pathogen Xanthomonas vasicola pv. vasculorum was used to introduce 2 independent dTALes into maize cells to induced expression of the gene glossy3 (gl3), which encodes a MYB transcription factor involved in biosynthesis of cuticular wax. RNA-seq analysis of leaf samples identified, in addition to gl3, 146 genes altered in expression by the 2 dTALes. Nine of the 10 genes known to be involved in cuticular wax biosynthesis were upregulated by at least 1 of the 2 dTALes. A gene previously unknown to be associated with gl3, Zm00001d017418, which encodes aldehyde dehydrogenase, was also expressed in a dTALe-dependent manner. A chemically induced mutant and a CRISPR-Cas9 mutant of Zm00001d017418 both exhibited glossy leaf phenotypes, indicating that Zm00001d017418 is involved in biosynthesis of cuticular waxes. Bacterial protein delivery of dTALes proved to be a straightforward and practical approach for the analysis and discovery of pathway-specific genes in maize.

59 BASIC BIOLOGICAL SCIENCES↗