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At least 163 records · Page 9
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Generation of a chromosome-scale genome assembly of the insect-repellent terpenoid-producing Lamiaceae species, Callicarpa americana

Plants exhibit wide chemical diversity due to the production of specialized metabolites that function as pollinator attractants, defensive compounds, and signaling molecules. Lamiaceae (mints) are known for their chemodiversity and have been cultivated for use as culinary herbs, as well as sources of insect repellents, health-promoting compounds, and fragrance. We report the chromosome-scale genome assembly of Callicarpa americana L. (American beautyberry), a species within the early-diverging Callicarpoideae clade of Lamiaceae, known for its metallic purple fruits and use as an insect repellent due to its production of terpenoids. Using long-read sequencing and Hi-C scaffolding, we generated a 506.1-Mb assembly spanning 17 pseudomolecules with N50 contig and N50 scaffold sizes of 7.5 and 29.0 Mb, respectively. In all, 32,164 genes were annotated, including 53 candidate terpene synthases and 47 putative clusters of specialized metabolite biosynthetic pathways. Our analyses revealed 3 putative whole-genome duplication events, which, together with local tandem duplications, contributed to gene family expansion of terpene synthases. Kolavenyl diphosphate is a gateway to many of the bioactive terpenoids in C. americana; experimental validation confirmed that CamTPS2 encodes kolavenyl diphosphate synthase. Syntenic analyses with Tectona grandis L. f. (teak), a member of the Tectonoideae clade of Lamiaceae known for exceptionally strong wood resistant to insects, revealed 963 collinear blocks and 21,297 C. americana syntelogs. Access to the C. americana genome provides a road map for rapid discovery of genes encoding plant-derived agrichemicals and a key resource for understanding the evolution of chemical diversity in Lamiaceae.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Flavonoids naringenin chalcone, naringenin, dihydrotricin, and tricin are lignin monomers in papyrus

Recent studies demonstrate that several polyphenolic compounds produced from beyond the canonical monolignol biosynthetic pathways can behave as lignin monomers, participating in radical coupling reactions and being incorporated into lignin polymers. Here, we show various classes of flavonoids, the chalconoid naringenin chalcone, the flavanones naringenin and dihydrotricin, and the flavone tricin, incorporated into the lignin polymer of papyrus (Cyperus papyrus L.) rind. These flavonoids were released from the rind lignin by Derivatization Followed by Reductive Cleavage (DFRC), a chemical degradative method that cleaves the β-ether linkages, indicating that at least a fraction of each was integrated into the lignin as β-ether-linked structures. Due to the particular structure of tricin and dihydrotricin, whose C-3' and C-5' positions at their B-rings are occupied by methoxy groups, these compounds can only be incorporated into the lignin through 4'–O–β bonds. However, naringenin chalcone and naringenin have no substituents at these positions and can therefore form additional carbon–carbon linkages, including 3'– or 5'–β linkages that form phenylcoumaran structures not susceptible to cleavage by DFRC. Furthermore, Nuclear Magnetic Resonance analysis indicated that naringenin chalcone can also form additional linkages through its conjugated double bond. The discovery expands the range of flavonoids incorporated into natural lignins, further broadens the traditional definition of lignin, and enhances the premise that any phenolic compound present at the cell wall during lignification could be oxidized and potentially integrated into the lignin structure, depending only on its chemical compatibility. This study indicates that papyrus lignin has a unique structure, as it is the only lignin known to date that integrates such a diversity of phenolic compounds from different classes of flavonoids. This discovery will open up new ways to engineer and design lignins with specific properties and for enhanced value.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Beyond Solanaceae: incorporation of feruloyltyramine and feruloyloctopamine into Cannabaceae lignins

The ferulic acid amides, feruloyltyramine and feruloyloctopamine, have been widely reported as integral constituents in the lignins in several species of Solanaceae in which they function as authentic lignin monomers. In the present study, we demonstrate that these ferulic acid amides are likewise incorporated into the lignins of species within Cannabaceae, including hemp (Cannabis sativa), hops (Humulus lupulus), and European nettle tree (Celtis australis). Structural analyses using derivatization followed by reductive cleavage (DFRC) and two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy revealed that these ferulic acid amides are incorporated via 4−O- and 8−O-ether linkages, as well as through 8−5′ linkages forming phenylcoumaran structures. Examination of a broad phylogenetic range of plant families demonstrated the absence of these ferulic acid amides from the lignins of all families studied except Solanaceae and Cannabaceae. Given the distant phylogenetic relationship between Solanaceae and Cannabaceae, the recruitment of these ferulic acid amides as lignin monomers in both lineages likely constitutes a case for convergent evolution at the level of lignin biosynthetic pathways. The significance of these ferulic acid amides lies in their unique role as the sole nitrogen-containing phenolic compounds known to participate in lignin formation.

Cannabaceae↗
DOE OSTI2020

Genomics-enabled analysis of specialized metabolism in bioenergy crops: Current progress and challenges

Plants produce a staggering diversity of specialized small molecule metabolites that play vital roles in mediating environmental interactions and stress adaptation. This chemical diversity derives from dynamic biosynthetic pathway networks that are often species-specific and operate under tight spatiotemporal and environmental control. A growing divide between demand and environmental challenges in food and bioenergy crop production have intensified research on these complex metabolite networks and their contribution to crop fitness. High-throughput omics technologies provide access to ever-increasing data resources for investigating plant metabolism. However, the efficiency of using such system-wide data to decode the gene and enzyme functions controlling specialized metabolism has remained limited; due largely to the recalcitrance of many plants to genetic approaches and the lack of ‘user-friendly’ biochemical tools for studying the diverse enzyme classes involved in specialized metabolism. With emphasis on terpenoid metabolism in the bioenergy crop switchgrass as an example, this review aims to illustrate current advances and challenges in the application of DNA synthesis and synthetic biology tools for accelerating the functional discovery of genes, enzymes and pathways in plant specialized metabolism. These technologies have accelerated knowledge development on the biosynthesis and physiological roles of diverse metabolite networks across many ecologically and economically important plant species and can provide resources for application to precision breeding and natural product metabolic engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Compensatory Guaiacyl Lignin Biosynthesis at the Expense of Syringyl Lignin in 4CL1 -Knockout Poplar -Knockout Poplar

The lignin biosynthetic pathway is highly conserved in angiosperms, yet pathway manipulations give rise to a variety of taxon-specific outcomes. Knockout of lignin-associated 4-coumarate:CoA ligases (4CLs) in herbaceous species mainly reduces guaiacyl (G) lignin and enhances cell wall saccharification. In this work we show that CRISPR-knockout of 4CL1 in poplar (Populus tremula x alba) preferentially reduced syringyl (S) lignin, with negligible effects on biomass recalcitrance. Concordant with reduced S-lignin was downregulation of ferulate 5-hydroxylases (F5Hs). Lignification was largely sustained by 4CL5, a low-affinity paralog of 4CL1 typically with only minor xylem expression or activity. Levels of caffeate, the preferred substrate of 4CL5, increased in line with significant upregulation of caffeoyl shikimate esterase1. Upregulation of caffeoyl-CoA O-methyltransferase1 and downregulation of F5Hs are consistent with preferential funneling of 4CL5 products toward G-lignin biosynthesis at the expense of S-lignin. Thus, transcriptional and metabolic adaptations to 4CL1-knockout appear to have enabled 4CL5 catalysis at a level sufficient to sustain lignification. Finally, genes involved in sulfur assimilation, the glutathione-ascorbate cycle, and various antioxidant systems were upregulated in the mutants, suggesting cascading responses to perturbed thioesterification in lignin biosynthesis.

09 BIOMASS FUELS↗
DOE OSTI2023

Crystal structure of MbnF: an NADPH-dependent flavin monooxygenase from Methylocystis strain SB2

Methanobactins (MBs) are ribosomally produced and post-translationally modified peptides (RiPPs) that are used by methanotrophs for copper acquisition. The signature post-translational modification of MBs is the formation of two heterocyclic groups, either an oxazolone, pyrazinedione or imidazolone group, with an associated thioamide from an X -Cys dipeptide. The precursor peptide (MbnA) for MB formation is found in a gene cluster of MB-associated genes. The exact biosynthetic pathway of MB formation is not yet fully understood, and there are still uncharacterized proteins in some MB gene clusters, particularly those that produce pyrazinedione or imidazolone rings. One such protein is MbnF, which is proposed to be a flavin monooxygenase (FMO) based on homology. To help to elucidate its possible function, MbnF from Methylocystis sp. strain SB2 was recombinantly produced in Escherichia coli and its X-ray crystal structure was resolved to 2.6 Å resolution. Based on its structural features, MbnF appears to be a type A FMO, most of which catalyze hydroxylation reactions. Preliminary functional characterization shows that MbnF preferentially oxidizes NADPH over NADH, supporting NAD(P)H-mediated flavin reduction, which is the initial step in the reaction cycle of several type A FMO enzymes. It is also shown that MbnF binds the precursor peptide for MB, with subsequent loss of the leader peptide sequence as well as the last three C-terminal amino acids, suggesting that MbnF might be needed for this process to occur. Finally, molecular-dynamics simulations revealed a channel in MbnF that is capable of accommodating the core MbnA fragment minus the three C-terminal amino acids.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Generation of a highly attenuated strain of Pseudomonas aeruginosa for commercial production of alginate

Alginate is an important polysaccharide that is commonly used as a gelling agent in foods, cosmetics and healthcare products. Currently, all alginate used commercially is extracted from brown seaweed. However, with environmental changes such as increasing ocean temperature and the increasing number of biotechnological uses of alginates with specific properties, there is an emerging need for more reliable and customizable sources of alginate. An alternative to seaweed for alginate production is Pseudomonas aeruginosa, a common Gram-negative bacterium that can form alginate containing biofilms. However, P. aeruginosa is an opportunistic pathogen that can cause life-threatening infections in immunocompromised patients. Therefore, we sought to engineer a non-pathogenic P. aeruginosa strain that is safe for commercial production of alginate. Using a homologous recombination strategy, we sequentially deleted five key pathogenicity genes from the P. aeruginosa chromosome, resulting in the marker-free strain PGN5. Intraperitoneal injection of mice with PGN5 resulted in 0% mortality, while injection with wild-type P. aeruginosa resulted in 95% mortality, providing evidence that the systemic virulence of PGN5 is highly attenuated. Importantly, PGN5 produces large amounts of alginate in response to overexpression of MucE, an activator of alginate biosynthesis. The alginate produced by PGN5 is structurally identical to alginate produced by wildtype P. aeruginosa, indicating that the alginate biosynthetic pathway remains functional in this modified strain. The genetic versatility of P. aeruginosa will allow us to further engineer PGN5 to produce alginates with specific chemical compositions and physical properties to meet different industrial and biomedical needs.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Development of genetic tools for heterologous protein expression in a pentose‐utilizing environmental isolate of Pseudomonas putida

Abstract Pseudomonas putida has emerged as a promising host for the conversion of biomass‐derived sugars and aromatic intermediates into commercially relevant biofuels and bioproducts. Most of the strain development studies previously published have focused on P. putida KT2440, which has been engineered to produce a variety of non‐native bioproducts. However, P. putida is not capable of metabolizing pentose sugars, which can constitute up to 25% of biomass hydrolysates. Related P. putida isolates that metabolize a larger fraction of biomass‐derived carbon may be attractive as complementary hosts to P. putida KT2440. Here we describe genetic tool development for P. putida M2, a soil isolate that can metabolize pentose sugars. The functionality of five inducible promoter systems and 12 ribosome binding sites was assessed to regulate gene expression. The utility of these expression systems was confirmed by the production of indigoidine from C6 and C5 sugars. Chromosomal integration and expression of non‐native genes was achieved by using chassis‐independent recombinase‐assisted genome engineering (CRAGE) for single‐step gene integration of biosynthetic pathways directly into the genome of P. putida M2. These genetic tools provide a foundation to develop hosts complementary to P. putida KT2440 and expand the ability of this versatile microbial group to convert biomass to bioproducts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Phycocyanobilin biosynthesis in Galdieria sulphuraria requires isomerization of phycoerythrobilin synthesized by bilin reductases

Phycobiliproteins are essential components of the light-harvesting antennae in cyanobacteria and red algae, requiring covalently bound open-chain tetrapyrrole chromophores (bilins) for proper function. In the red alga Galdieria sulphuraria , the primary chromophore is phycocyanobilin (PCB), despite the apparent presence of only biosynthetic genes for phycoerythrobilin (PEB) biosynthesis (PEBA and PEBB). This observation suggests the presence of an alternative, atypical biosynthetic pathway for PCB. In this study, we confirmed the presence of PEB:PCB isomerase activity in an enriched protein fraction from G. sulphuraria . To further investigate this unusual pathway, we combined in silico analyses with biochemical assays. Phylogenetic analyses confirmed the placement of the G. sulphuraria ferredoxin-dependent bilin reductases within the PEBA and PEBB lineages, typically associated with PEB synthesis, whereas the related red alga Cyanidioschyzon merolae was found to contain only PCYA. This gene distribution presents a functional paradox. G. sulphuraria harbors PEB biosynthesis genes but no detectable PEB chromophores and lacks known PCB-synthesizing enzymes despite containing PCB. Functional characterization of recombinant GsPEBA (G. sulphuraria PEBA) and GsPEBB ( G. sulphuraria PEBB) confirmed their roles in PEB synthesis, demonstrating that these enzymes have not evolved to synthesize PCB or act as isomerases despite their phylogenetic placement. In contrast, Cm PCYA ( C. merolae PCYA) catalyzed direct PCB formation from biliverdin. Together, these findings reveal an atypical isomerase-based pathway for PCB biosynthesis in G. sulphuraria , expanding our understanding of bilin metabolism and providing new insight into the evolutionary flexibility of photosynthetic pigment biosynthesis in Rhodophyta.

14 SOLAR ENERGY↗
DOE OSTI2021

Cytochrome P450–catalyzed biosynthesis of furanoditerpenoids in the bioenergy crop switchgrass ( Panicum virgatum L.)

Specialized diterpenoid metabolites are important mediators of plant-environment interactions in monocot crops. To understand metabolite functions in plant environmental adaptation that ultimately can enable crop improvement strategies, a deeper knowledge of the underlying species-specific biosynthetic pathways is required. Furthermore, we report the genomics-enabled discovery of five cytochrome P450 monooxygenases (CYP71Z25-CYP71Z29) that form previously unknown furanoditerpenoids in the monocot bioenergy crop Panicum virgatum (switchgrass). Combinatorial pathway reconstruction showed that CYP71Z25-CYP71Z29 catalyze furan ring addition directly to primary diterpene alcohol intermediates derived from distinct class II diterpene synthase products. Transcriptional co-expression patterns and the presence of select diterpenoids in switchgrass roots support the occurrence of P450-derived furanoditerpenoids in planta. Integrating molecular dynamics, structural analysis and targeted mutagenesis identified active site determinants that contribute to the distinct catalytic specificities underlying the broad substrate promiscuity of CYP71Z25-CYP71Z29 for native and non-native diterpenoids.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Compilation and utilization of a sorghum transcriptome compendium for gene regulatory network analysis and crop trait engineering

Sorghum bicolor (Sorghum) is a drought and heat tolerant C4 grass crop used to produce grain, forage, biofuels, and other bioproducts. Genetic improvement of sorghum hybrid crops is aided by a large and diverse germplasm, sorghum's diploid inbreeding genetics, and a relatively small genome that has facilitated genomic research. Over the past 20 years, the sorghum research community characterized the cytogenetic and recombinant landscapes of sorghum's 10 chromosomes, sequenced and annotated the sorghum genome, and used that information to identify genes/alleles that modulate flowering time, plant height, seed shattering, and other important traits. More recently, >1000 RNA-seq transcriptome profiles were collected from 15 sorghum genotypes to help understand the genetic basis of variation in growth and development of sorghum stems, tillers, roots, and leaves, and the regulation of biosynthetic pathways that produce epicuticular wax, dhurrin, and RFOs, compounds that contribute to sorghum's resilience. Transcriptome studies were designed to identify differentially expressed genes that are co-expressed during development or in response to a treatment to enable construction of gene regulatory networks. Co-expression and network analysis identified transcription factors and their cognate binding sites in target gene promoters and signaling pathways that modulate gene regulatory networks providing gene editing targets for further trait optimization. RNA-seq data from >20 experiments targeting sorghum organs, tissues, cell types, developmental stages, and responses to environmental conditions (i.e., diel, day-length, shading, water-deficit, temperature) has been compiled in a sorghum transcriptome compendium. The goal of this resource paper is to describe compendium content, accessibility, and a compendium data analysis pipeline and to illustrate the types of information that can be derived from the compendium with a focus on the elucidation of gene regulatory networks useful for guiding the improvement of sorghum traits through gene editing.

RNA-seq↗
DOE OSTI2025

A plant-specific cytochrome b 5 –like protein is essential for phytosterol biosynthesis

Sterols are essential isoprenoid derivatives that contribute to membrane structure and function. In plants, they also serve as precursors to phytohormones and specialized metabolites important for development, defense, and health. Although the sterol biosynthetic pathway is considered well-characterized, we report the discovery of a plant-specific cytochrome b 5 –like protein, CB5LP, as a critical component of phytosterol biosynthesis. Loss of CB5LP in Arabidopsis causes embryonic defects, seedling lethality, and accumulation of 14α-methyl-sterols, with reduced levels of sitosterol and stigmasterol—indicating a defect in sterol 14α-demethylation. TurboID-based proximity labeling and in vitro assays show that CB5LP physically and functionally interacts with CYP51, a cytochrome P450 enzyme catalyzing this demethylation step. Unlike canonical cytochrome b 5 proteins, CB5LP has a reversed topology and is exclusive to plants, acting as an evolutionarily distinct electron donor. This discovery reveals an uncharacterized redox partnership essential for sterol biosynthesis and highlights a promising target for the development of selective herbicide.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

MbnC Is Not Required for the Formation of the N-Terminal Oxazolone in the Methanobactin from Methylosinus trichosporium OB3b

A number of environmental and medical applications have been proposed for MBs, including bioremediation of toxic metals and nanoparticle formation, as well as the treatment of copper- and iron-related diseases. However, before MBs can be modified and optimized for any specific application, the biosynthetic pathway for MB production must be defined.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

maranasgroup/scRBA

Saccharomyces cerevisiae is an important model organism and a workhorse in bioproduction. Here, we reconstructed a compact and tractable genome-scale resource balance analysis (RBA) model (i.e., named scRBA) to analyze metabolic fluxes and proteome allocation in a computationally efficient manner. Resource capacity models such as scRBA provide the quantitative means to identify bottlenecks in biosynthetic pathways due to enzyme, compartment size, and/or ribosome availability limitations. ATP maintenance rate and in vivo apparent turnover numbers (kapp) were regressed from metabolic flux and protein concentration data to capture observed physiological growth yield and proteome efficiency and allocation, respectively. Estimated parameter values were found to vary with oxygen and nutrient availability. Overall, this work (i) provides condition-specific model parameters to recapitulate phenotypes corresponding to different extracellular environments, (ii) alludes to the enhancing effect of substrate channeling and post-translational activation on in vivo enzyme efficiency in glycolysis and electron transport chain, and (iii) reveals that the Crabtree effect is underpinned by specific limitations in mitochondrial proteome capacity and secondarily ribosome availability rather than overall proteome capacity.

Dinh, Hoang↗
DOE OSTI2025

genomeocean: a pretrained microbial genome foundational model (genomeoceanLLM) v1.0

We present Genomeocean, a foundational genome language model that represents the microbial genome sequences from complex environmental samples. By training on a large, diverse metagenomic dataset, Genomeocean learns species-specific sequence composition and can generate long, realistic open reading frames (ORFs). Our model employs a Byte-pair-encoding (BPE) tokenization strategy, allowing it to efficiently process large genomic datasets and generate long sequences up to 50kb. We demonstrate that fine-tuning Genomeocean can generate novel gene clusters encoding biosynthetic pathways, showcasing its ability to model both fundamental and complex biological processes. Our work establishes Genomeocean as a powerful tool for understanding microbial genome biology and paves the way for its application in a range of fields, from synthetic biology to microbiome research.

Wang, Zhong [Lawrence Berkeley National Laboratory↗
DOE OSTI2023

Metabolic engineering to improve production of 3-hydroxypropionic acid from corn-stover hydrolysate in Aspergillus species

Fuels and chemicals derived from non-fossil sources are needed to lessen human impacts on the environment while providing a healthy and growing economy. 3-hydroxypropionic acid (3-HP) is an important chemical building block that can be used for many products. Biosynthesis of 3-HP is possible; however, low production is typically observed in those natural systems. Biosynthetic pathways have been designed to produce 3-HP from a variety of feedstocks in different microorganisms. In this study, the 3-HP β-alanine pathway consisting of aspartate decarboxylase, β-alanine-pyruvate aminotransferase, and 3-hydroxypropionate dehydrogenase from selected microorganisms were codon optimized for Aspergillus species and placed under the control of constitutive promoters. The pathway was introduced into Aspergillus pseudoterreus and subsequently into Aspergillus niger, and 3-HP production was assessed in both hosts. A. niger produced higher initial 3-HP yields and fewer co-product contaminants and was selected as a suitable host for further engineering. Proteomic and metabolomic analysis of both Aspergillus species during 3-HP production identified genetic targets for improvement of flux toward 3-HP including pyruvate carboxylase, aspartate aminotransferase, malonate semialdehyde dehydrogenase, succinate semialdehyde dehydrogenase, oxaloacetate hydrolase, and a 3-HP transporter. Overexpression of pyruvate carboxylase improved yield in shake-flasks from 0.09 to 0.12 C-mol 3-HP C-mol -1 glucose in the base strain expressing 12 copies of the β-alanine pathway. Deletion or overexpression of individual target genes in the pyruvate carboxylase overexpression strain improved yield to 0.22 C-mol 3-HP C-mol -1 glucose after deletion of the major malonate semialdehyde dehydrogenase. Further incorporation of additional β-alanine pathway genes and optimization of culture conditions (sugars, temperature, nitrogen, phosphate, trace elements) for 3-HP production from deacetylated and mechanically refined corn stover hydrolysate improved yield to 0.48 C-mol 3-HP C-mol -1 sugars and resulted in a final titer of 36.0 g/L 3-HP. The results of this study establish A. niger as a host for 3-HP production from a lignocellulosic feedstock in acidic conditions and demonstrates that 3-HP titer and yield can be improved by a broad metabolic engineering strategy involving identification and modification of genes participated in the synthesis of 3-HP and its precursors, degradation of intermediates, and transport of 3-HP across the plasma membrane.

09 BIOMASS FUELS↗
DOE OSTI2023

Harnessing redox proteomics to study metabolic regulation and stress response in lignin-fed Rhodococci

Abstract Background Rhodococci are studied for their bacterial ligninolytic capabilities and proclivity to accumulate lipids. Lignin utilization is a resource intensive process requiring a variety of redox active enzymes and cofactors for degradation as well as defense against the resulting toxic byproducts and oxidative conditions. Studying enzyme expression and regulation between carbon sources will help decode the metabolic rewiring that stymies lignin to lipid conversion in these bacteria. Herein, a redox proteomics approach was applied to investigate a fundamental driver of carbon catabolism and lipid anabolism: redox balance. Results A consortium of Rhodococcus strains was employed in this study given its higher capacity for lignin degradation compared to monocultures. This consortium was grown on glucose vs. lignin under nitrogen limitation to study the importance of redox balance as it relates to nutrient availability. A modified bottom–up proteomics workflow was harnessed to acquire a general relationship between protein abundance and protein redox states. Global proteomics results affirm differential expression of enzymes involved in sugar metabolism vs. those involved in lignin degradation and aromatics metabolism. As reported previously, several enzymes in the lipid biosynthetic pathways were downregulated, whereas many involved in β-oxidation were upregulated. Interestingly, proteins involved in oxidative stress response were also upregulated perhaps in response to lignin degradation and aromatics catabolism, which require oxygen and reactive oxygen species and generate toxic byproducts. Enzymes displaying little-to-no change in abundance but differences in redox state were observed in various pathways for carbon utilization (e.g., β‑ketoadipate pathway), lipid metabolism, as well as nitrogen metabolism (e.g., purine scavenging/synthesis), suggesting potential mechanisms of redox-dependent regulation of metabolism. Conclusions Efficient lipid production requires a steady carbon and energy flux while balancing fundamental requirements for enzyme production and cell maintenance. For lignin, we theorize that this balance is difficult to establish due to resource expenditure for enzyme production and stress response. This is supported by significant changes to protein abundances and protein cysteine oxidation in various metabolic pathways and redox processes.

09 BIOMASS FUELS↗
DOE OSTI2022

Data for Metabolic Engineering of Oleaginous Yeast Rhodotorula toruloides for Overproduction of Triacetic Acid Lactone

The plant-sourced polyketide triacetic acid lactone (TAL) has been recognized as a promising platform chemical for the biorefinery industry. However, its practical application was rather limited due to low natural abundance and inefficient cell factories for biosynthesis. Here, we report the metabolic engineering of oleaginous yeast Rhodotorula toruloides for TAL overproduction. We first introduced a 2-pyrone synthase gene from Gerbera hybrida (GhPS) into R. toruloides and investigated the effects of different carbon sources on TAL production. We then systematically employed a variety of metabolic engineering strategies to increase the flux of acetyl-CoA by enhancing its biosynthetic pathways and disrupting its competing pathways. We found that overexpression of ATP-citrate lyase (ACL1) improved TAL production by 45% compared to the GhPS overexpressing strain, and additional overexpression of acetyl-CoA carboxylase (ACC1) further increased TAL production by 29%. Finally, we characterized the resulting strain I12-ACL1-ACC1 using fed-batch bioreactor fermentation in glucose or oilcane juice medium with acetate supplementation and achieved a titer of 28 or 23 g/L TAL, respectively. This study demonstrates that R. toruloides is a promising host for the production of TAL and other acetyl-CoA-derived polyketides from low-cost carbon sources.

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