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At least 55 records · Page 3
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Inducible flippase-mediated metabolic engineering of Rhodosporidium toruloides for enhanced 3-hydroxypropionic acid production from corn stover hydrolysate

Rhodosporidium toruloides has gained increasing interests as a promising non-model host organism to produce a wide range of bioproducts from lignocellulosic biomass. Increasing the bioproduct titers, rates, and yields remains a challenge, largely due to a lack of robust and well-characterized genetic tools in this host. Here we developed an inducible flippase (FLP) and flippase recognition target (FRT) system that enables genetic manipulations without the need for additional selection markers. Synthetic inducible promoters were established, enabling regulation of FLP expression and efficient antibiotic marker removal. Leveraging this system, we engineered a strain to optimize 3-hydroxypropionic acid (3HP) production. Over four rounds of iterative genomic editing to resolve pathway bottlenecks, we achieved a 3HP titer of 69.4 g/L in fed-batch fermentation - the highest level reported in yeast from lignocellulosic hydrolysates. The engineered high 3HP producing strain offers a robust platform for sustainable bio-based chemical production from lignocellulosic feedstocks.

3-hydroxypropionic acid↗
DOE OSTI2026

Understanding and Mitigating Stickiness in Biochar Produced through Acid Hydrolysis and Dehydration

Levulinic acid is a platform chemical with significant potential for conversion into a wide range of biobased chemicals and fuels. A common process for producing levulinic acid from lignocellulosic feedstocks involves acid hydrolysis and dehydration (AHDH), where hexose polymers are hydrolyzed into monomeric sugars and subsequently dehydrated to levulinic acid and formic acid in the presence of dilute sulfuric acid. However, scaling the AHDH process is challenging because of the formation of byproducts such as sticky biochar, which accumulates in continuous-flow reactors, reducing effective reaction volume and increasing process downtime. This study investigates the effect of a chemical preconditioning step on mitigating sticky biochar formation. Woody biomass was preconditioned at 170 °C with 0.26 wt % sulfuric acid for 30 min, resulting in substantial removal of hemicellulose and acid-soluble lignin. AHDH of these preconditioned solids produced biochar that did not adhere to reactor surfaces. TGA analysis confirmed that the chemical preconditioning step minimized interactions between hemicellulose-derived degradation products and lignin side chains, reducing sticky char formation. Additionally, the study observed a 6% higher yield of organic acids from softwood species compared to hardwoods, with bark content shown to negatively impact yield. These findings suggest that targeted preconditioning of lignocellulosic biomass can enhance reactor operability and improve organic acid production efficiency in AHDH processes.

biopolymers↗
DOE OSTI2025

A techno-economic assessment of carbon dioxide removal pathways via biochemical conversion of lignocellulose to biofuels and bioplastics

Biomass carbon removal and storage (BiCRS) is a promising pathway to mitigate climate change via large scale removal of atmospheric carbon dioxide (CO 2 ). We modeled several fermentation technologies, producing a variety of bioproducts from lignocellulosic feedstocks, to understand their levelized cost of CO 2 removal under multiple scenarios. Lifecycle greenhouse gas (GHG) emissions are accounted to provide cradle-to-grave estimates of carbon intensity (CI). We did not account for the avoided fossil CO 2 emissions from the use of biofuels in our CO 2 removal cost calculations, because avoided emissions do not contribute to CO 2 removal. The main products from the fermentation technologies we modeled include renewable diesel, ethanol, sustainable aviation fuel (SAF), and polyethylene (PE), with co-products including CO 2 , adipic acid, steam, and electricity. PE, depending on its end-of-life management, can serve as a form of biogenic carbon storage. PE has the potential to remove 1.2–1.5 tCO 2 per dry t-biomass, whereas biofuels have the potential to remove 0.3–0.9 tCO 2 per dry t-biomass, indicating that PE production is a more efficient method of carbon removal. We quantify costs of CO 2 removal to be $\$$60 – $\$$675 per metric tCO 2 removed across the various fermentation pathways. Under the scenarios analyzed, bioplastic production from lignocellulosic biomass is a more cost-effective route to CO 2 removal than biofuel production, with costs of CO 2 removal via bioplastics being 50–90 % lower than that of biofuels. Future research should explore the potential benefits and drawbacks of expanding bioplastic production for large-scale CO 2 removal.

09 BIOMASS FUELS↗
DOE OSTI2020

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

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

09 BIOMASS FUELS↗
DOE 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 OSTI2020

Predicted annual biomass input to maintain soil organic carbon under contrasting management

Corn (Zea mays L.) residue is an important source of lignocellulosic feedstocks for meeting the US renewable energy goals. However, there is uncertainty in how much biomass can be sustainably harvested. The objectives of this study were to (i) simulate soil organic carbon (SOC) dynamics in the 0–20, 20–30, and 0–30 cm soil depths for the 2005–2015 study period using CQESTR, a process-based C model; (ii) establish trends in SOC with residue removal and tillage management over the predictive period of 2016–2035; and (iii) recommend practices to maintain SOC stocks in a corn–soybean (Glycine max L. [Merr.]) rotation with corn residue removal in western Minnesota. The residue removal treatments in the conventional tillage since 1995 (CT 95 ) and no tillage since 1995 (NT 95 ) were initiated in 2005, and in 2006 for the no tillage since 2005 (NT 05 ). Corn residue removal treatments were none, cob removal, ~50% removal, and ~75% removal (R0, RCob, R50, and R75, respectively). Only R0 in NT 05 increased SOC (0.32 Mg C ha –1 yr –1 ) stocks in the top 30 cm soil depth by 2035 with annual aboveground biomass input of 6.30 Mg ha –1 yr –1 . It was concluded that only the NT 05 field has the potential to maintain SOC at the current rate of biomass addition, and a minimal annual aboveground C (MSC) input of 3.75 Mg C ha –1 yr –1 is required to maintain SOC until 2035. These simulation results indicate that caution should be used in removing residue especially under conventional tillage.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2021

Exploring filamentous fungi depolymerization of corn stover in the context bioenergy queuing operations

Recalcitrance of lignocellulosic feedstocks to depolymerization is a significant barrier for bioenergy production approaches that require conversion of monomeric carbohydrates to renewable energy sources. This study assesses how low-cost modifications in the feedstock supply chain can be transformed into targeted pretreatments in the context of the entire bioenergy supply chain. The aim of this research is to overcome the physiochemical barriers in corn stover that necessitate increased severity in conversion in terms of chemical loading, temperature, and residence time. Corn stover samples were inoculated with a selective (Ceriporiopsis subvermispora) and non-selective (Phaenarochaete chrysosporium) lignin degrading filamentous fungal strains, then stored aerobically to determine the working envelope for fungal pretreatment to achieve lignin degradation. Dry matter loss and gross chemical makeup of corn stover varied by the length of treatment (2 and 4 weeks) and by the moisture content of the treated corn stover samples (40 and 60%, wet basis). Dry matter loss in P. chrysosporium inoculated biomass was elevated compared to the C. subvermispora inoculated biomass; however, treatment also induced additional chemical composition changes suggestive of depolymerization. Scanning electron microscope images reveal hyphae attached within cell lumen and suggest structural changes within P. chrysosporium treated corn stover after 60% moisture storage. These results highlight that fungal treatment approaches must balance loss of convertible material with the potential for reduction in recalcitrance. Techno-economic assessment (TEA) of fungal pretreatment in a short-term queuing system indicated the viability of this approach compared to conventional queuing operations. The total queuing system cost was estimated at $\$$1.65/tonne of biomass stored. After applying the credit of $\$$1.48/tonne from energy savings in the conversion phase using fungal pretreated biomass, the total system cost was $0.80 lower than traditional biomass queueing approach. While the TEA results suggested that treating biomass with C. subvermispora is the most economically viable storage method in the designed fungal-assisted queuing system, future research should focus on additional fungal depolymerization such as those observed in the P. chrysosporium inoculated biomass.

09 BIOMASS FUELS↗
DOE OSTI2025

Enhancing the flowability of woody biomass slurries in wet biorefineries

Feeding wet lignocellulosic biomass (e.g., softwood and hardwood) slurries into high-pressure, high-temperature reactors at an industrially relevant scale presents significant challenges, such as process equipment plugging. Here, in this study, we investigate the possibility of improving the flowability of biomass slurries in an industrial scale wet biorefinery by tuning the physical and chemical characteristics of the biomass particles. To understand the effects of the chemical characteristics of woody biomass on flowability, cellulose pulp particles are produced from pulp sheets via knife milling, pelletizing, and crumbling. These cellulose pulp particles are processed for acid hydrolysis dehydration (AHDH) at one –tonne-per day pilot scale. The levulinic acid yield (a product of cellulose AHDH) and the flowability of the biomass particles are compared using market pulp, 1 mm crumbled pine softwood, a blend of 2 mm hammer-milled pine softwood with 10 wt.% bark, and 2 mm hammer-milled hardwood material with 10 wt.% bark (to represent forest residues). The results indicate that the presence of hemicellulose and lignin influences the flowability of lignocellulosic feedstocks. Crumbled wood particles show poor flowability at the pilot scale, while the presence of fine materials (less than 0.7 mm) and bark improves the flowability of biomass slurries without affecting the organic acid yields (based on C6 carbohydrate content).

09 - BIOMASS FUELS↗
DOE OSTI2023

Biomass pretreatment with distillable ionic liquids for an effective recycling and recovery approach

Ionic liquid (IL) pretreatment methods show incredible promise for the efficient conversion of lignocellulosic feedstocks to fuels and chemicals. Given their low vapor pressures, distillation-based methods of extracting ionic liquids out of biomass post-pretreatment have historically been ignored in favor of alternative methods. We demonstrate a process to distill four acetate-based ionic liquids ([EthA][OAc], [PropA][OAc], [MAEthA][OAc], and [DMAEthA][OAc]) at low pressure and high purity that overcome some disadvantages of “water washing” and “one pot” recovery methods. Out of four tested ILs, ethanolamine acetate ([EthA][OAc]) is shown to have the most agreeable conversion metrics for commercial bioconversion processes achieving 73.6% and 51.4% of theoretical glucose and xylose yields respectively and >85 % recovery rates. Our process metrics are factored into a techno-economic analysis where [EthA][OAc] distillation is compared to other recovery methods as well as ethanolamine pretreatment at both milliliter and liter scales. In conclusion, although our TEA shows [EthA][OAc] distillation underperforming against other processes, we show a step-by-step avenue to reduce sugar production cost below the wholesale dextrose price at scale.

09 BIOMASS FUELS↗
DOE OSTI2020

Effects of blending C3-C4 alcohols on motor gasoline properties and performance of spark ignition engines: A review

Supplementing petroleum fuels with sustainable and renewable alternatives is a good option for increasing the sustainability of transportation fuels. Alcohols are particularly attractive blendstocks for spark ignition engines, mainly due to their desirable fuel properties including high octane, evaporative cooling, and reduced sooting propensity. Although the use of SI engines is widespread around the world, predominately for light duty vehicles, concerns about CO2 emissions and other sustainability issues necessitate increased engine efficiencies, reduced tailpipe pollutants, and lower lifecycle carbon emissions. The intelligent blending of C2-C4 alcohols into motor gasoline is a viable method for achieving these goals. There are a multitude of ways to produce renewable alcohols such as through fermentation from first-generation feedstocks (sugar and corn) and second-generation feedstocks (lignocellulosic biomass), or by gasification and mixed alcohol fuel synthesis routes from lignocellulosic biomass. Currently ethanol is extensively used in motor gasoline fuels worldwide and although many have proposed the use of C3 and C4 alcohols in motor gasoline as an improvement over ethanol, the higher cost of production and lack of clear definition as to their benefit over ethanol when blended into motor gasoline have led to slow acceptance into the market. In this review, special emphasis is placed on the effects of blending C3 and C4 alcohols into motor gasoline in terms of physicochemical properties, volatility behavior, and engine performance when compared to ethanol blends. Furthermore, the impact of blending C3 and C4 alcohols with gasoline on emission (particulate matter, nitrogen oxides, carbon monoxide, hydrocarbons, and unregulated oxygenates) and combustion (volumetric efficiency, thermal efficiency, fuel consumption, and cold performance) characteristics is discussed. Although there are some disagreements in the literature over the effect of alcohols predominately around the type of SI engine, i.e., port fuel injected versus direct fuel injected and engine operating mode, generally it is stated that alcohols can potentially reduce soot, unburned hydrocarbons and CO emissions while increasing thermal efficiency when proper engine configuration/calibration is used. Lastly, research that must be conducted to find the optimum combination of alcohol blends and engine configurations is highlighted and discussed.

09 BIOMASS FUELS↗
DOE OSTI2025

Quantifying Impacts of Biomass Pelletization on Fast Pyrolysis Using a Single-Particle Reactor, X-ray Computed Tomography, and Computational Modeling

The pore structure and density of lignocellulosic feedstocks dictate intraparticle transport phenomena and thereby play an important role in thermochemical conversion processes such as fast pyrolysis for biofuel and biochemical production. Variations in microstructure are inherent from different biomass species and can be introduced by preprocessing techniques such as cutting and pelletization. Morphological changes also occur during conversion and lead to vastly different pore structures and behavior during pyrolysis, which impact required conversion times and product distributions. The current work presents a comprehensive comparison of fast pyrolysis of neat and pelletized pine feedstocks, which includes single-particle experiments, modeling, and 3D imaging by X-ray computed tomography (XCT). The particle-scale model included anisotropic heat and mass transport in a shrinking particle with pyrolysis reactions based on the CRECK mechanism with boundary conditions informed by reactor-scale simulations of the single-particle reactor. The models were validated by measurements of the temperature and mass loss from single-particle pyrolysis experiments of neat and pelletized pine. Quantitative analysis of XCT geometries revealed that pyrolytic conversion yielded chars with increased porosity and permeability compared to the unpyrolyzed materials, along with decreased tortuosity and anisotropy. Pelletization of the pine feedstock resulted in a much denser, less permeable material, which converted slower and produced more residual char after pyrolysis compared to neat pine. The results from particle modeling revealed that accounting for the dynamic and anisotropic heat and mass transport caused by differences in pore structure is critical to achieving agreement with experimental results. Overall, this study highlights the dramatic differences in conversion behavior imparted by pelletization and the importance of capturing microstructural attributes in computational models to guide the design and optimization of pyrolysis processes for specific biomass feedstocks.

09 BIOMASS FUELS↗
DOE OSTI2021

Coupling of Flavonoid Initiation Sites with Monolignols Studied by Density Functional Theory

Lignin recalcitrance presents a challenge for the development of a bioeconomy that employs lignocellulosic feedstocks. The efficiency of lignin deconstruction is improved by a reduction of molecular weight, and given the discovery that flavones serve as initiation sites in lignin biosynthesis, these molecular weight reductions could potentially be achieved with plant metabolic engineering to over-express flavonoids. Upon increasing the flavonoid content in lignin, the bond strengths and properties of flavonoid-monolignol linkages become increasingly important. To that end, the current work applies density functional theory calculations to elucidate the bond dissociation enthalpies (BDEs) of flavonoid-monolignol linkages, including dimers with oxidized monolignols. Specifically, the dimer bond strengths and monomer hydrogen abstraction energies for the flavonoids tricin, chrysoeriol, luteolin, apigenin, catechin, epicatechin, epigallocatechin, and epigallocatechin gallate are calculated, when coupled to seven natural and engineered monolignols. Results indicate that 4'-O-ß linkage strengths between flavonoids and monolignols are of comparable strength to inter-monolignol ß-O-4 linkages, with average flavonoid-monolignol BDEs of 70.7 kcal/mol relative to ~69.3 kcal/mol in analogous canonical monolignols. Epigallocatechin yielded the lowest 4'-O-ß bond strength of 52.3 kcal/mol when coupled to an oxidized monolignol, while the flavones overall produced lower average BDEs, relative to the flavanols. Substituents at the 3'-C and 5'-C positions on flavonoids affected the dimer linkage strengths to a greater extent than glycosylation or substituents further from the linkage. Erythro and threo stereochemistry across the flavonoid-monolignol linkage library exhibited only small energetic differences and no pronounced correlations. Taken together, the predictions from this work support the concept that higher concentrations of flavonoid initiation sites in lignin may afford linkage properties conducive to more facile lignin depolymerization.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Greenhouse Gas Emissions Associated with Pretreatment Techniques Utilized for Biosugar Production

Less carbon-intensive production of biosugars from lignocellulosic materials can lead to improved manufacturing of biofuels and bioproducts. In this study, the production of biosugars is analyzed to understand the potential of producing bioproducts from biosugars with a low carbon intensity. Life-cycle assessment is conducted on five lignocellulosic feedstocks along with seven pretreatment techniques to produce biosugars as an important platform intermediate for producing bioproducts. The production of electricity using lignin with and without heat integration was also incorporated. In addition, seven bioproducts are analyzed for the estimation of the GHG emissions budget, which represents the emissions available to convert, separate, and upgrade biosugars into bioproducts within the 70% emissions reduction target. Corn stover-deacetylation and dilute acid pretreatment (CS-DDA) provide the lowest GHG emissions for biosugar (0.03 kg CO 2 eq/kg biosugar) and thereby the highest GHG emissions budget for the case of lactic acid production (3.76 kg CO 2 eq/kg lactic acid). Natural gas and chemicals are the major contributors to all of the pretreatment techniques under study. The outcomes from this study can benefit the advancement of upcoming biobased processes that meet decarbonization targets.

Biopolymers↗
DOE OSTI2020

Solvent-induced membrane stress in biofuel production: molecular insights from small-angle scattering and all-atom molecular dynamics simulations

The disruptive effect of organic solvents on microbial membranes represents a significant challenge to the economical production of green fuels and value-added chemicals from lignocellulosic feedstocks. One route to overcoming this challenge is to engineer microbes with membranes capable of resisting organic solvent stresses. In this regard, it is useful to understand the mechanisms by which organic solvents disrupt typical biomembranes. In this study, molecular dynamics (MD) simulation, complemented by small-angle X-ray and neutron scattering (SANS/SAXS), provide a molecular-scale view of the disruption of a microbial model membrane by 1-butanol and tetrahydrofuran (THF), two common water–organic cosolvent mixtures of importance in biofuel production. Solvent interactions at the interface between the head-group and fatty acid tail regions lead to more dramatic membrane changes than interactions solely at the head-groups or tails. Although both organic solvents are found to partition into the membrane, the depth of solvent penetration into the membrane is quite different. Specifically, 1-butanol localizes near the interface between the lipid heads and tails at low concentrations, but partitions into both the head and tail regions at high concentrations. In contrast, THF, overall, partitions less than 1-butanol and prefers the lipid tail regions. Importantly, the presence of 1-butanol near the head/tail interface introduces drastic membrane changes not seen with THF. The organic solvent interactions with the lipids lead to membrane thinning and fluidization, but more so for 1-butanol than for THF. These results suggest that an aim for the future engineering of robust membranes could be to design lipid head groups that reduce the accumulation of organic solvents at the head–tail interface and that rational designs need also be cognizant of the different solvent-specific mechanisms responsible for membrane disruption.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Switchgrass Steroidal Saponins Reduce Fungal Disease but Decrease Yeast Fermentation Yield

Increasing the production of bioproducts from lignocellulosic feedstocks requires improvement in both field production and biorefinery efficiency. When plant traits arise that improve field production but decrease biofuel yield, these trade-offs can represent challenges in the entire production process. To examine trade-offs between field and production traits, we examined factors underlying switchgrass resistance to fungal rust pathogens in field conditions and factors that impede yeast fermentation in the lab using repeated measurements on a switchgrass genetic diversity panel. We found that the same switchgrass genotypes that showed high fungal pathogen resistance also showed recalcitrance to yeast fermentation. These switchgrass genotypes were mostly from the Atlantic genetic group, which had high levels of specialized metabolites of the saponin class. Among 1589 metabolites identified through metabolomics, we found that saponins were among the most likely to explain variation in both rust infection and fermentation yield using random forest feature selection, and that only four of these were sufficient to explain 57.9% of the variation in rust susceptibility. Through follow-up testing in recalcitrant biomass, we found that the bacterium Zymomonas mobilis does not suffer the same inhibition as the yeast Saccharomyces cerevisiae, and that the addition of ergosterol (thought to be the fungal cellular target of saponin inhibition) rescues yeast fermentation. Several lines of evidence point to a central role for saponins as key metabolites protecting switchgrass from fungal pathogens and interfering with yeast fermentation, underscoring an ongoing need for collaboration between plant breeders and biofuel production scientists.

VanWallendael, Acer [North Carolina State Universi↗
DOE OSTI2022

Complete Genome Sequences of Five Isolated Pseudomonas Strains that Catabolize Pentose Sugars and Aromatic Compounds Obtained from Lignocellulosic Biomass

We report on complete genome sequences of five Pseudomonas soil isolates that are capable of metabolizing pentose sugars and aromatic monomers. These complete genome sequence data provide insight into possible alternative hosts for the production of biofuels and bio-based chemicals from lignocellulosic feedstock.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides

Abstract Background Rhodosporidium toruloides has emerged as a promising host for the production of bioproducts from lignocellulose, in part due to its ability to grow on lignocellulosic feedstocks, tolerate growth inhibitors, and co-utilize sugars and lignin-derived monomers. Ent -kaurene derivatives have a diverse range of potential applications from therapeutics to novel resin-based materials. Results The Design, Build, Test, and Learn (DBTL) approach was employed to engineer production of the non-native diterpene ent -kaurene in R. toruloides . Following expression of kaurene synthase (KS) in R. toruloides in the first DBTL cycle, a key limitation appeared to be the availability of the diterpene precursor, geranylgeranyl diphosphate (GGPP). Further DBTL cycles were carried out to select an optimal GGPP synthase and to balance its expression with KS, requiring two of the strongest promoters in R. toruloides , ANT (adenine nucleotide translocase) and TEF1 (translational elongation factor 1) to drive expression of the KS from Gibberella fujikuroi and a mutant version of an FPP synthase from Gallus gallus that produces GGPP. Scale-up of cultivation in a 2 L bioreactor using a corn stover hydrolysate resulted in an ent -kaurene titer of 1.4 g/L. Conclusion This study builds upon previous work demonstrating the potential of R. toruloides as a robust and versatile host for the production of both mono- and sesquiterpenes, and is the first demonstration of the production of a non-native diterpene in this organism.

59 BASIC BIOLOGICAL SCIENCES↗