Recent lbnl_bs_bse items

Advanced pathways for hydrogen production: a collective view from a technical experts meeting

Thu, 14 May 2026 00:00:00 +0000

The current status of advanced water splitting pathways (using photoelectrochemical, biological and thermochemical platforms) toward viable technologies to produce renewable and sustainable hydrogen is assessed in a virtual international meeting. Hydrogen is an essential fuel and feedstock that can be produced in multiple ways to meet requirements for technological sectors that include energy storage, transportation, petroleum refining, and ammonia synthesis. To consider the future state of hydrogen manufacturing, a team of experts has assembled and examined three emerging hydrogen production technologies – photoelectrochemical, biological, and thermochemical. Each of these emerging technologies holds significant long-term potential for cost reduction while lowering industrial emissions associated with conventional methods of hydrogen manufacture ( e.g. , steam methane reforming) by using sunlight and renewable resources as primary sources of energy and feedstock, respectively....

Contrasting effects of glutamate and branched-chain amino acid metabolism on acid tolerance in a Castellaniella isolate from acidic groundwater

Thu, 7 May 2026 00:00:00 +0000

ABSTRACT Groundwater acidification co-occurring with nitrate pollution is a common, global environmental health hazard. Denitrifying bacteria have been leveraged for the in situ removal of nitrate in groundwater. However, co-existing stressors—such as low pH—reduce the efficacy of biological removal processes. Castellaniella sp. str. MT123 is a complete denitrifier that was isolated from acidic, nitrate-contaminated groundwater. The strain grows robustly by nitrate respiration at pH < 6.0, completely reducing nitrate to dinitrogen gas. Genomic analyses of MT123 revealed few previously characterized acid tolerance genes. Thus, we utilized a combination of proteomics, metabolomics, and competitive mutant fitness to characterize the genetic mechanisms of MT123 acclimation to growth under mildly acidic conditions. We found that glutamate accumulation is critical in the acid acclimation of MT123, possibly through consumption of intracellular protons via glutamate decarboxylation...

Quantitative Dissection of Agrobacterium Virulence to Generate a Synthetic Ti Plasmid

Fri, 24 Apr 2026 00:00:00 +0000

Agrobacterium is not only a costly plant pathogen but is also an essential tool for plant transformation. Though Agrobacterium-mediated transformation (AMT) has been heavily studied, its polygenic nature and complex transcriptional regulation make identification of the genetic basis of transformational efficiency difficult through traditional genetic and bioinformatic approaches. Here, we use a bottom-up synthetic approach to systematically engineer the tumor-inducing plasmid (pTi), wherein the majority of virulence machinery is encoded. Using a validated toolkit to control Agrobacterium gene expression in planta, we perform a quantitative dissection of AMT to investigate the contributions of critical vir-genes at different expression levels. We construct a synthetic pTi capable of transient plant and stable fungal transformation and characterize bottlenecks and solutions for complex polygenic synthetic pTi designs. Our reductionist approach...

Causes and consequences of experimental variation in Nicotiana benthamiana transient expression

Tue, 14 Apr 2026 00:00:00 +0000

Infiltration of Agrobacterium tumefaciens into Nicotiana benthamiana has become a foundational technique in plant biology, enabling efficient delivery of transgenes in planta with technical ease, robust signal, and relatively high throughput. Despite transient expression’s prevalence in disciplines such as synthetic biology, little work has been done to describe and address the variability inherent in this system, a concern for experiments that rely on highly quantitative readouts. In a comprehensive analysis of N. benthamiana agroinfiltration experiments, we model sources of variability that affect transient expression. Our findings emphasize the need to validate normalization methods under the specific conditions of each study, as distinct normalization schemes do not always reduce variation either within or between experiments. Using a dataset of 1915 plants collected over three years, we develop a model of variation in N. benthamiana transient expression, using power analysis...

Statistical design of experiments for production and purification of vanillin and aminophenols from commercial lignin

Thu, 9 Apr 2026 00:00:00 +0000

Production and purification of vanillin from lignin using ion exchange, and subsequent conversion to aminophenol ionic liquid precursors.

Lignin is a complex polyphenolic substance that collectively represents the largest renewable source of aromatic carbon on Earth. Despite low yield and purity from depolymerized lignin, bio-derived vanillin (4-hydroxy-3-methoxybenzaldehyde) is a desirable molecule in the food/beverage and fragrance industries. To maximize vanillin yield from commercially-available softwood lignin, a series of oxidation reactions were conducted using a Box-Behnken statistical design. By varying time, temperature and O ₂ pressure, optimal conditions were selected using the Response Surface Method, and a maximum vanillin yield of 5.3 wt% was achieved. Flash chromatography of depolymerized lignin was investigated by comparing anion exchange and reversed-phase resins and fractions were characterized by GC-MS, GPC-HPLC, FT-IR and NMR. Anion exchange...

Functionalized benzylamines from commercial kraft lignin

Thu, 9 Apr 2026 00:00:00 +0000

Benzylamines are key intermediates in pharmaceuticals, agrochemicals, and polymers, but their conventional production relies on benzyl chloride - a petroleum-derived compound with high toxicity and energy demands. Lignin, accounting for up to 30% of plant biomass, is the largest renewable source of aromatic carbon on Earth. However, its highly complex and recalcitrant structure poses a major barrier to efficient conversion into high-value chemicals. Here, we developed a catalytic approach to convert commercial kraft lignin into phenolic benzylamines through selective depolymerization and subsequent functionalization. We systematically evaluated the effects of three alcohol solvents, formic acid (FA), and a ruthenium-on‑carbon (Ru/C) catalyst on monophenol yield and selectivity. Up to 6.5 wt% monophenol yield was achieved using methanol (MeOH), FA, and Ru/C at 300 °C for 2 h. Quantum thermodynamic simulations based on the COSMO-RS model confirmed the superior solvation...

Genetic variations and their interaction with thirdhand smoke exposure on anxiety and memory in Collaborative Cross mice

Mon, 6 Apr 2026 00:00:00 +0000

Thirdhand smoke (THS) is linked to adverse health effects, but the effect of genetic variations on behavioral outcomes is poorly understood. To investigate this, we assessed anxiety- and memory-related behaviors in 820 mice from 21 strains of the genetically diverse Collaborative Cross (CC) mouse that were exposed to THS from 4 through 10 weeks of age. Anxiety was evaluated with a light/dark box assay with a previously established risk score system. Females were generally more sensitive: THS reduced anxiety risk in strains CC013, CC019, and CC051, but increased risk in CC036 and CC061, while males showed no significant effects. Memory was tested using passive avoidance: impairments were observed in both sexes in CC016 and CC019, with sex-dependent effects in CC002 and CC051. A genome-wide association study identified 2,347 SNPs associated with anxiety and 1,568 SNPs with memory, with 32 and 85 SNPs, respectively, interacting with THS exposure. Enrichment analyses revealed distinct...

Feedstock-efficient conversion through hydrogen and formate-driven metabolism in Escherichia coli

Tue, 24 Mar 2026 00:00:00 +0000

Product yields for biomanufacturing processes are often constrained by the tight coupling of cellular energy generation and carbon metabolism in sugar-based fermentation systems. To overcome this limitation, we engineered Escherichia coli to utilize hydrogen gas (H₂) and formate (HCOO^-) as alternative sources of energy and reducing equivalents, thereby decoupling energy generation from carbon metabolism. This approach enabled precise suppression of decarboxylative oxidation during acetate growth, with 86.6 ± 1.6 % of electrons from hydrogen gas (via soluble hydrogenase from Cupriavidus necator H16) and 98.4 ± 3.6 % of electrons from formate (via formate dehydrogenase from Pseudomonas sp. 101) offsetting acetate oxidation. Hydrogen gas supplementation led to a titratable and stoichiometric reduction in CO₂ evolution in acetate-fed cultures. Metabolomic analysis suggests that this metabolic decoupling redirects carbon...

Maximizing long-term biohydrogen production with Clostridium thermocellum for high solids conversion of lignocellulosic biomass

Wed, 18 Mar 2026 00:00:00 +0000

Novel reactor design and sequencing fed-batch operation enhance biohydrogen production from lignocellulosic biomass to 24.87 L H 2 /L, achieving over 95% solubilization of cellulose and hemicellulose. Biological hydrogen production from lignocellulosic biomass sustainably couples organic waste reduction with renewable energy generation. Efficient conversion is challenged by the structural complexity of lignocellulose and resulting recalcitrance to enzymatic degradation. Clostridium thermocellum natively breaks down biomass with highly effective hemi-/cellulases systems ( i.e. , cellulosomes) and generates hydrogen in anaerobic cultivation, creating a compelling platform for lignocellulosic biohydrogen production. Achieving commercially viable production rates requires balancing high biomass loading and throughput against uniform mixing conditions required for enzyme dispersion, pH and temperature control, and efficient hydrogen and metabolite removal in continuous operation....

Machine Learning-Driven Solvent Screening for Biobased 2,3-Butanediol Extraction

Fri, 13 Mar 2026 00:00:00 +0000

Biobased 2,3-butanediol (2,3-BDO) is a valuable biomass-derived chemical due to its versatility in being transformed into a wide variety of products. However, the separation and purification of 2,3-BDO from fermentation broth remain a significant challenge owing to its high boiling point and hydrophilic nature. Herein, we developed a machine learning (ML)-based screening workflow that uses molecular calculations as training data and requires only a small number of experimental measurements for validation to identify alternative solvent candidates for the liquid–liquid extraction (LLE) of 2,3-BDO from aqueous solution. In particular, 130 density functional theory (DFT) calculations with the implicit solvation method not only built a correlation between the computational partition coefficient and the experimental distribution coefficient of 2,3-BDO but also parameterized an Extra-Trees ML model to screen the distribution coefficient for a wider range of 6717 organic solvents. The...

Automated Strain Construction for Biosynthetic Pathway Screening in Yeast

Tue, 3 Mar 2026 00:00:00 +0000

Automation accelerates the Design-Build-Test-Learn (DBTL) cycle for synthetic biology; however, most strain construction pipelines lack robotic integration. Here, we present the workflow design and source code for a modular, integrated protocol that automates the Build step in Saccharomyces cerevisiae. We programmed the Hamilton Microlab VANTAGE to integrate off-deck hardware via its central robotic arm, enabling automated steps that increased throughput to 2,000 transformations per week. We developed a user interface with the Hamilton VENUS software to support on-demand parameter customization. As a proof of concept, we screened a gene library in an engineered yeast strain producing verazine, a key intermediate in the biosynthesis of steroidal alkaloids. Our pipeline rapidly identified pathway bottlenecks and genes that enhanced verazine production by 2.0- to 5-fold. This technical note provides resources for synthetic biologists designing yeast workflows for biofoundries...

33 Unresolved Questions in Nanoscience and Nanotechnology

Tue, 3 Mar 2026 00:00:00 +0000

Significant advances in science and engineering often emerge at the intersections of disciplines. Nanoscience and nanotechnology are inherently interdisciplinary, uniting researchers from chemistry, physics, biology, medicine, materials science, and engineering. This convergence has fostered novel ways of thinking and enabled the development of materials, tools, and technologies that have transformed both basic and applied research, as well as how we address critical societal challenges. In this Nano Focus, we pose and explore 33 questions whose answers could profoundly impact fields such as energy, electronics, the environment, optics, and medicine. These questions highlight the need for deeper foundational understanding, improved tools and techniques, and innovative applications─each with significant societal relevance. Together, they represent a global call-to-action for the scientific community.

Rapid Evaluation of Amine-Functionalized Solvents for Biomass Deconstruction Using High-Throughput Screening and One-Pot Enzymatic Saccharification

Tue, 17 Feb 2026 00:00:00 +0000

Efficient and sustainable pretreatment of lignocellulosic biomass is critical for biofuel and biochemical production, yet its optimization is often hindered by slow, labor-intensive experimental methods. Here, we report the first demonstration of a custom-built, miniaturized, high-throughput screening platform integrated with one-pot enzymatic saccharification, enabling parallel evaluation of solvent type, feedstock, and temperature with minimal material use and high reproducibility. As a proof-of-concept, the HTX platform was used to screen five amine-functionalized solvents, including isopropanolamine, butylamine, N-methylbutylamine, ethanolamine, and ethanolamine acetate across three bioenergy crops (sorghum, poplar, and switchgrass) and pretreatment temperatures ranging from 80 to 140 °C. Vacuum drying successfully removed more than 99% of the solvents from the pretreated biomass, eliminating the need for water washing prior to saccharification. Isopropanolamine and N-methylbutylamine...

Automation and machine learning drive rapid optimization of isoprenol production in Pseudomonas putida

Tue, 10 Feb 2026 00:00:00 +0000

Advances in genome engineering have improved our ability to perturb microbial metabolic networks, yet bioproduction campaigns often struggle with parsing complex metabolic datasets to efficiently enhance product titers. We address this challenge by coupling laboratory automation with machine learning to systematically optimize the production of isoprenol, a sustainable aviation fuel precursor, in Pseudomonas putida. The simultaneous downregulation through CRISPR interference of combinations of up to four gene targets, guided by machine learning, permitted us to increase isoprenol titer 5-fold in six consecutive design-build-test-learn cycles. Moreover, machine learning enabled us to swiftly explore a vast experimental design space of 800,000 possible combinations by strategically recommending approximately 400 priority constructs. High-throughput proteomics allowed us to validate CRISPRi downregulation and identify biological mechanisms driving production increases. Our work demonstrates...

Functional delineation of the luminal epithelial microenvironment in breast using cell-based screening in combinatorial microenvironments

Tue, 10 Feb 2026 00:00:00 +0000

Microenvironment signals are potent determinants of cell fate and arbiters of tissue homeostasis, however understanding how different microenvironment factors coordinately regulate cellular phenotype has been experimentally challenging. Here we used a high-throughput microenvironment microarray comprised of 2640 unique pairwise signals to identify factors that support proliferation and maintenance of primary human mammary luminal epithelial cells. Multiple microenvironment factors that modulated luminal cell number were identified, including: HGF, NRG1, BMP2, CXCL1, TGFB1, FGF2, PDGFB, RANKL, WNT3A, SPP1, HA, VTN, and OMD. All of these factors were previously shown to modulate luminal cell numbers in painstaking mouse genetics experiments, or were shown to have a role in breast cancer, demonstrating the relevance and power of our high-dimensional approach to dissect key microenvironmental signals. RNA-sequencing of primary epithelial and stromal cell lineages identified the cell...

Cost impact of hexose-to-pentose sugar ratios for biomanufacturing

Wed, 4 Feb 2026 00:00:00 +0000

Central to the long-term vision for biomanufacturing is the ability to deconstruct plant cell walls to sugars that microbes can convert to products. Aside from glucose, the most abundant sugar in biomass is xylose, a pentose sugar. Industrially relevant microbes have been engineered to co-ferment xylose and glucose. Most nth plant technoeconomic analyses (TEAs) assume similar consumption rates and product yields for both sugars, but in reality, xylose is consumed more slowly. Feedstocks can be selected, or engineered, to alter the glucan-to-xylan ratio (GXR) but no TEAs have quantified the impact of this strategy systematically. This study explores the cost impacts of varying the glucan-to-xylan ratio (GXR) from 1.9 to 6.7 for co-fermenting glucose and xylose to ethanol and bisabolene. The minimum selling prices (MSPs) for both products decrease as the GXR increases, with the largest reductions at shorter residence times. For instance, with an increase in GXR from 1.9 to 6.7,...

Engineered Production of Hydroxycinnamoyl Tyramine Conjugates Limits the Growth of the Pathogen Pseudomonas syringae in Arabidopsis

Tue, 27 Jan 2026 00:00:00 +0000

Hydroxycinnamoyl tyramine conjugates are phenolamides produced by plants in response to pathogen attack and biotic stresses. Their proposed mechanisms of action include cytotoxicity towards pathogens, cell wall reinforcement to restrict pathogen proliferation, and signaling activity to trigger general stress responses. Here, we engineered the production of the tyramine conjugates p-coumaroyltyramine (CT) and feruloyltyramine (FT) in Arabidopsis to gain insight into their mode of action. Co-expression of feedback-insensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and tyrosine decarboxylase increased tyramine content. Additional expression of tyramine hydroxycinnamoyltransferase led to de-novo production of CT and FT, which were found as soluble and cell-wall-bound forms. FT was associated with lignin in stems. The growth of pathogenic Pseudomonas syringae was reduced in rosettes of the Arabidopsis CT- and FT-producing lines compared to wild type. These lines also...

Advances in ionic liquid recycling for lignocellulosic biomass pretreatment

Wed, 21 Jan 2026 00:00:00 +0000

In this review, we provide a comprehensive assessment of recent advances in ionic liquid recycling technologies. Ionic liquids (ILs) are promising solvents for the pretreatment of lignocellulosic biomass due to their ability to disrupt cellulose, hemicellulose, and lignin structures. However, large-scale implementation requires the development of efficient recovery and recycling methods. This review provides a comprehensive analysis of the recyclability potential of ILs used in biomass pretreatment, emphasizing their mechanisms, recent innovations, and ongoing challenges. We begin by discussing the structural diversity and tunability of ILs, which underlie their effectiveness in biomass deconstruction. The distinct roles of IL anions and cations in dissolving specific biomass components are systematically presented and compared. Advances in IL recycling techniques, including antisolvent precipitation methods, membrane separation, and distillation, are critically examined, with...

A highly active Burkholderia polyketoacyl-CoA thiolase for production of triacetic acid lactone

Thu, 15 Jan 2026 00:00:00 +0000

Triacetic acid lactone (TAL) is a versatile platform chemical traditionally biosynthesized via decarboxylative Claisen condensation by 2-pyrone synthase. However, this route is limited by poor efficiency and dependence on malonyl-CoA. Here, we show that non-decarboxylative Claisen condensation by polyketoacyl-CoA thiolases offers a more efficient alternative. Through mining homologs of a previously reported enzyme from Cupriavidus necator, we identify five thiolases with TAL production activity. One candidate, BktBbr from Burkholderia sp. RF2-non_BP3, exhibits approximately 30-fold higher activity in vitro and supports 30-fold higher TAL titers in Escherichia coli compared to the original enzyme. Fed-batch fermentation achieves titers up to 2.8 g L⁻¹. Structural analysis of BktBbr co-crystallized with CoA esters guides rational engineering to further enhance performance. Our discovery of a highly active thiolase establishes an alternative enzymatic route to produce TAL efficiently,...

YAP1 dysfunction promotes molecular properties linked to breast cancer susceptibility.

Tue, 13 Jan 2026 00:00:00 +0000

YAP1 is a co-transcription factor that promotes malignant and stem cell properties in cancer. We previously found that YAP1 dysregulation is associated with aging in human mammary epithelia. With increased age, YAP1 expression changes in luminal epithelial cells, the prospective breast cancer cell of origin. Because age is a significant risk factor for breast cancer, we tested if YAP1 dysregulation acted early in cancer progression by conferring cellular states associated with increased cancer susceptibility. Here we find, that with increased age and genetic risk for developing cancer, human breast tissues showed significantly increased YAP1 expression and cultured primary human mammary epithelial cells (HMEC) showed significantly increased expression of both YAP1 and its transcriptional targets. Increased YAP1 expression in cultured HMEC induced gene expression changes associated with increased cancer susceptibility such as genes associated with: stem cell fate, increased telomerase...

Electronic Structure of the Au Nanoparticle-TiO2 Heterojunction: Influence of Nanoparticle Size, Shape, Oxygen Vacancies, and Temperature

Mon, 12 Jan 2026 00:00:00 +0000

The electronic structure of the gold nanoparticle-titanium dioxide (AuNP-TiO₂) heterojunction plays a critical role for charge transfer and recombination dynamics that underpin its photocatalytic function. However, building a representative model to capture the key physics remains a significant challenge. Here, we investigate the influence of the AuNP size and shape, as well as oxygen vacancy (V_O) defects at the anatase-phase TiO₂ (101) surface and the temperature of the heterojunction, on its interfacial electronic properties. Using density functional theory (DFT), we compare the closed-shell Au₂₀ and open-shell Au₁₉ clusters interfaced with pristine and V_O defect TiO₂ surfaces. We find that the presence of a V_O defect transforms pure TiO₂ from a p-type to an n-type semiconductor, reversing the interfacial band bending from downward to upward. For the heterosystem, density of states...

Predictive CRISPR-mediated gene downregulation for enhanced production of sustainable aviation fuel precursor in Pseudomonas putida

Tue, 16 Dec 2025 00:00:00 +0000

CRISPR interference (CRISPRi) has emerged as a valuable tool for redirecting metabolic flux to enhance bioproduction. However, its application is often constrained by two challenges: (i) rationally identifying effective gene targets for downregulation and (ii) efficiently constructing multiplexed CRISPRi systems. In this study, we address both challenges by integrating a computational prioritization tool with a versatile assembly method for building multiplexed CRISPRi systems. FluxRETAP (Flux-Reaction Target Prioritization) accurately identified gene targets whose knockdown led to substantial increase of isoprenol titers in Pseudomonas putida KT2440, outperforming a conventional non-computational, pathway-guided target selection. The highest isoprenol titer of nearly 1.5 g/L was achieved by knocking down PP_4118 (a gene encoding α-ketoglutarate dehydrogenase). The use of VAMMPIRE (Versatile Assembly Method for MultiPlexing CRISPRi-mediated downREgulation) enabled accurate...

Engineered Accumulation of Protocatechuate in Corn Biomass to Enhance Biomanufacturing

Tue, 16 Dec 2025 00:00:00 +0000

The in-planta accumulation of coproducts in crops can enhance the value of lignocellulosic biomass and facilitate a sustainable bioeconomy. Corn stover represents a major renewable source of lignocellulose for the production of advanced biofuels and bioproducts. In this study, we engineered corn with a bacterial gene encoding a dehydroshikimate dehydratase (QsuB) to overproduce protocatechuate (DHBA). Transgenic corn lines accumulate up to 2.9% DHBA on a dry weight basis in leaf and stem biomass. DHBA occurs in the form of glucosides that are extractable from biomass using aqueous methanol as the solvent. The analysis of lignin did not show any evidence for the incorporation of DHBA; however, an increase in the lignin syringyl to guaiacyl ratio and a higher relative abundance of p-coumarate groups compared with total lignin units were observed in QsuB-modified corn. Alkaline hydrolysates prepared from QsuB corn were enriched in DHBA compared to the hydrolysates obtained...

Engineering Polyketide Stereocenters with Ketoreductase Domain Exchanges

Mon, 8 Dec 2025 00:00:00 +0000

Polyketide synthases (PKSs) are versatile biosynthetic megasynthases capable of producing a diverse range of natural products with many applications, including in pharmaceuticals. The stereochemical precision of PKSs makes them a powerful tool for engineering tailored, unnatural polyketides; however, modifying the stereocenters of a PKS product while maintaining production levels remains a significant challenge. In this study, we systematically tested and evaluated strategies for ketoreductase (KR) domain exchanges, the domain responsible for setting stereocenters of polyketide products. After first optimizing the method for KR exchanges, we then performed 44 KR domain exchanges on three different PKSs to obtain high production of all four stereoisomers in vivo. By testing both one- and two-module PKS systems, we investigated how downstream modules process intermediates with altered stereochemistry and found that the configuration of the α-substituents was critical for gatekeeping...

Engineered plants for the production of the antioxidants arbutin and gallate

Mon, 8 Dec 2025 00:00:00 +0000

The shikimate pathway is a crucial metabolic route for the biosynthesis of numerous valuable chemicals. In this study, we engineered the shikimate pathway in plants via expression of microbial enzymes to produce the two important antioxidants gallate and arbutin. The engineered pathways utilize the aromatics protocatechuate and 4-hydroxybenzoate as metabolic intermediates. Through transient expression in Nicotiana benthamiana leaves, we first identified biosynthetic routes for the production of gallate from either chorismate or 3-dehydroshikimate. Gallate production was then achieved in Arabidopsis using a genetic background that overproduces protocatechuate and via expression of a mutated version of the 4-hydroxybenzoate hydroxylase PobA from Pseudomonas sp. Arbutin production was obtained in Arabidopsis using a genetic background that overproduces 4-hydroxybenzoate and via expression of the monooxygenase MNX1 from Candida parapsilosis. The best Arabidopsis transgenic lines accumulated...

Cooperative Research and Development Agreement (CRADA) Final Report - Develop High-Throughput Workflows for Whole-Genome Sequencing and Insertion Site Screening

Tue, 2 Dec 2025 00:00:00 +0000

Cooperative Research and Development Agreement (CRADA) Final Report - Develop High-Throughput Workflows for Whole-Genome Sequencing and Insertion Site Screening

Biomanufacturing from gaseous C1 feedstocks: A perspective on opportunities and challenges

Tue, 2 Dec 2025 00:00:00 +0000

Single-carbon (C1) substrates including carbon dioxide, carbon monoxide, and methane are abundantly available from natural and anthropogenic sources and present potential feedstocks for biomanufacturing. Utilizing these C1 gas feedstocks in bioprocesses for sustainable production of chemicals and fuels could prove pivotal in removing excess carbon from the atmosphere. This perspective describes the spectrum and sources of CO2, CO, and CH4 and examines emerging opportunities in microbial bioconversion and bioelectrochemical processes for these feedstocks. We discuss existing challenges in bioprocess development that currently restrict the commercialization of C1 biomanufacturing technologies. We detail different aerobic and anaerobic bioconversion approaches for C1 feedstocks employing pure and mixed cultures and examine the suitability of each scenario for producing specific molecules. Beyond strain engineering and bioprocess constraints, we address often overlooked factors that...

Optimal production of Phanerochaete chrysosporium manganese peroxidases and Trametes sp. C30 laccase hybrid Lac131 in Aspergillus niger for lignin bioconversion

Tue, 2 Dec 2025 00:00:00 +0000

BackgroundIncorporating the production of related ligninolytic enzymes into industrial filamentous fungus Aspergillus niger will enhance the bioconversion of lignocelluloses to various chemical products.ResultsIn this study, transgenic expression of Phanerochaete chrysosporium manganese peroxidases (mnps) and Trametes sp. C30 laccase hybrid Lac131 (lac131) were examined and optimized in A. niger 11414 prtT∆ strain. Five mnps (mnp1, mnp2, mnp3, mnp4, and mnp5) and lac131 genes were expressed separately or in combination. The transgenic strain containing the entire mnp2 genomic coding sequence (gmnp2) exhibited the highest mnP activity among the five mnp over-expression strains in the modified minimal medium (mMM) with addition of 5 g/L bovine hemoglobin (bHg). We examined the effects of hemin and bHg on mnP production in the gmnp2 strain cultures and found that at least 1 g/L bHg was required, while hemin was not. Culture conditions for mnP production were further optimized...

Perspectives for artificial intelligence in bioprocess automation

Tue, 2 Dec 2025 00:00:00 +0000

Recent advances in artificial intelligence (AI) have rapidly changed the lab automation landscape, promoting self-driving laboratories (SDLs) that enable autonomous scientific discovery. These trends are increasingly applied in bioprocess development, yet bioprocessing faces unique challenges - biological complexity, regulatory and safety requirements, and multiscale experimentation - that distinguish it from other automation domains. Rather than pursuing full autonomy, we foresee that hybrid SDLs, combining AI-driven decision-making with sustained human oversight, represent the most practical near-term trajectory. This review examines three interconnected perspectives: (i) hybrid human-machine decision-making for bioprocessing; (ii) laboratory design considerations in the era of AI; and (iii) scale-up challenges when transitioning from screening to manufacturing. We highlight critical gaps in data standardization and the required community efforts necessary...

Comparison of the efficacy of a biocompatible and a distillable solvent for pretreatment of mixed bioenergy feedstocks

Mon, 1 Dec 2025 00:00:00 +0000

Biomass deconstruction is a crucial step in the production of lignocellulosic biofuels and bioproducts. However, identifying and selecting an optimal pretreatment solvent that enhances enzymatic saccharification while being cost-efficient and ensuring sustainability remains a challenge. In this study, we compare the effectiveness of the biocompatible ionic liquid cholinium lysinate ([Ch][Lys]) against the distillable solvent ethanolamine, when used for the pretreatment of mixed bioenergy feedstocks, including poplar, switchgrass, and sorghum. [Ch][Lys] was used at a concentration of 10 % wt. in a one-pot configuration without biomass washing and ethanolamine was used in a concentrated form and removed with a vacuum oven, before performing enzymatic hydrolysis and microbial conversion. Our results show that ethanolamine pretreatment consistently enhances the glucose yield across various biomass types compared to [Ch][Lys], with improvements ranging from 22.0 % to 52.7 %....

Biosensor-driven strain engineering reveals key cellular processes for maximizing isoprenol production in Pseudomonas putida

Tue, 18 Nov 2025 00:00:00 +0000

Synthetic biology generates vast combinatorial designs, yet high-throughput analytical methods to screen them are poorly matched to interrogate this search space. We address this challenge by developing a biosensor-driven, growth-coupled selection strategy in Pseudomonas putida for isoprenol, a potential aviation fuel precursor. We found and characterized a noncanonical signaling pathway, revealing a functional and physical complex between a hybrid histidine kinase and an alcohol dehydrogenase, whose activity is tuned by heterodimerization. Leveraging this biosensor in a pooled CRISPRi library selection, we identified key host limitations. Iterative combinatorial strain engineering derived from these hits yielded a 36-fold titer increase to ~900 milligrams per liter. Integrated omics analysis revealed that metabolic rewiring toward amino acid catabolism was crucial for this improvement. This observation was found to be beneficial by technoeconomic analysis. Our modular...

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

Tue, 18 Nov 2025 00:00:00 +0000

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...

Differences in GenBank and RefSeq annotations may affect genomics data interpretation for Pseudomonas putida KT2440

Tue, 18 Nov 2025 00:00:00 +0000

Annotations of genomic features are cornerstone data that support routine workflows in conventional omics analyses in Pseudomonas putida KT2440 and other organisms. The GenBank and the RefSeq versions of the annotated KT2440 genome are two popular resources widely cited in the literature; yet, they originate from distinct prediction pipelines and possess potentially different biological information that is often overlooked. In this study, we systematically compared the features present in these resources and found that approximately 16% of the total of KT2440 open reading frames (ORFs) show differences in their predicted genomic positions across GenBank and RefSeq, despite sharing equivalent locus tag codes. Furthermore, we show that these discrepancies can affect the results of high-throughput analyses by processing a collection of RNAseq expression data sets utilizing both annotations. Our findings provide a comprehensive overview of the current state of available resources...

Semi-automated biofoundry workflows for sequence coevolution-guided isoprene synthase engineering

Tue, 21 Oct 2025 00:00:00 +0000

Biofoundries serve as transformative platforms for accelerating the engineering of enzymes and microorganisms toward biomanufacturing. In this study, we developed scalable enzyme engineering workflows tailored for biofoundry applications, focusing on isoprene synthase (IspS) - a critical rate-limiting enzyme in the isoprene biosynthesis. By integrating computational mutation design based on sequence coevolution analysis and laboratory automation, we conducted three rounds of site-directed mutagenesis and screening. Approximately 100 genetic mutants were synthesized per round and these workflows can be easily scaled up to thousands without extensive optimization. Moreover, this approach enabled the rapid identification of IspS variants with up to 4.5-fold improvement in catalytic efficiency and simultaneously enhanced thermostability. Additionally, introducing the engineered IspS into Methylococcus capsulatus Bath improved methane-to-isoprene bioconversion, achieving a titer of...

The La Silla Schmidt Southern Survey

Wed, 8 Oct 2025 00:00:00 +0000

We present the La Silla Schmidt Southern Survey (LS4), a new wide-field, time-domain survey to be conducted with the 1 m ESO Schmidt telescope. The 268 megapixel LS4 camera mosaics 32 2k × 4k fully depleted CCDs, providing a ∼20 deg2 field of view with 1″ pixel−1 resolution. The LS4 camera will have excellent performance at longer wavelengths: in a standard 45 s exposure the expected 5σ limiting magnitudes in g, i, z are ∼21.5, ∼20.9, and ∼20.3 mag (AB), respectively. The telescope design requires a novel filter holder that fixes different bandpasses over each quadrant of the detector. Two quadrants will have i band, while the other two will be g and z band with color information obtained by dithering targets across the different quadrants. The majority (90%) of the observing time will be used to conduct a public survey that monitors the extragalactic sky at both moderate (3 days) and high (1 day) cadence, as well as focused observations within the Galactic plane and bulge. Alerts...

RB-TnSeq elucidates dicarboxylic-acid-specific catabolism in β-proteobacteria for improved plastic monomer upcycling

Tue, 7 Oct 2025 00:00:00 +0000

Dicarboxylic acids are key components of many polymers and plastics, making them a target for both engineered microbial degradation and sustainable bioproduction. In this study, we generated a comprehensive data set of functional evidence for the genetic basis of dicarboxylic and fatty acid metabolism using randomly barcoded transposon sequencing (RB-TnSeq). We identified four β-proteobacteria that displayed robust growth with dicarboxylic acid sole carbon source and cultured their mutant libraries with dicarboxylic and fatty acids with carbon chain lengths from C3 to C12. The resulting fitness data suggested that dicarboxylic and fatty acid metabolisms are largely distinct, and different sets of β-oxidation genes are required for catabolizing dicarboxylic versus fatty acids of the same carbon chain lengths. In addition, we identified transcriptional regulators and transporters with strong fitness phenotypes related to dicarboxylic acid utilization. In Ralstonia sp. UNC404CL21Col...

FluxRETAP: a REaction TArget Prioritization genome-scale modeling technique for selecting genetic targets

Tue, 7 Oct 2025 00:00:00 +0000

MOTIVATION: Metabolic engineering is rapidly evolving as a result of new advances in synthetic biology tools and automation platforms that enable high throughput strain construction, as well as the development of machine learning tools (ML) for biology. However, selecting genetic engineering targets that effectively guide the metabolic engineering process is still challenging. ML can provide predictive power for synthetic biology, but current technical limitations prevent the independent use of ML approaches without previous biological knowledge. RESULTS: Here, we present FluxRETAP, a simple and computationally inexpensive method that leverages the prior mechanistic knowledge embedded in genome-scale models for suggesting targets for genetic overexpression, downregulation or deletion, with the final goal of increasing the production of a desired metabolite. This method can provide a list of desirable engineering targets that can be combined with current ML pipelines. FluxRETAP...

Biosensor-Driven Strain Engineering Reveals Key Cellular Processes for Maximizing Isoprenol Production in Pseudomonas putida

Thu, 25 Sep 2025 00:00:00 +0000

ABSTRACT

Synthetic biology tools have accelerated the generation of simple mutants, but combinatorial testing remains challenging. High-throughput methods struggle translating from proof-of-principle molecules to advanced bioproducts. We address this challenge with a biosensor-driven strategy for enhanced isoprenol production in Pseudomonas putida , a key precursor for sustainable aviation fuel and platform chemicals. This biosensor leverages P. putida ’s native response to short-chain alcohols via a previously uncharacterized hybrid histidine kinase signaling cascade. Refactoring the biosensor for a conditional growth-based selection enabled identification of competing cellular processes with a ∼16,500-member CRISPRi-library. An iterative combinatorial strain engineering approach yielded an integrated P. putida strain producing ∼900 mg/L isoprenol in glucose minimal medium, a 36-fold increase. Ensemble -omics analysis revealed metabolic rewiring, including amino acid...

Differences in GenBank and RefSeq annotations may affect genomics data interpretation for Pseudomonas putida KT2440

Thu, 25 Sep 2025 00:00:00 +0000

Annotations of genomic features are cornerstone data that support routine workflows in conventional omics analyses in Pseudomonas putida KT2440 and other organisms. The GenBank and the RefSeq versions of the annotated KT2440 genome are two popular resources widely cited in the literature; yet, they originate from distinct prediction pipelines and possess potentially different biological information that is often overlooked. In this study, we systematically compared the features present in these resources and found that approximately 16% of the total of KT2440 ORFs show differences in their predicted genomic positions across GenBank and RefSeq, despite sharing equivalent locus tag codes. Furthermore, we show that these discrepancies can affect the results of high-throughput analyses by processing a collection of RNAseq expression datasets utilizing both annotations. Our findings provide a comprehensive overview of the current state of available resources for genomics research in...

Novel origin of replication for environmentally isolated Pantoea strain enables expression of heterologous proteins, pathways and products

Thu, 25 Sep 2025 00:00:00 +0000

Plasmids isolated or characterized from environmental samples serve as a resource that can be used to develop genetic tools for characterizing recently isolated or less-studied microbes. In this report, we leveraged sequences from a previously characterized groundwater plasmidome and developed a screen to identify novel plasmid origins. Putative origin sequences were used to construct a barcoded plasmid library, which contained both known and newly predicted origins. This library was tested against a panel of representative bacterial strains and led to the identification of 3 novel origins that putatively replicate in gram-negative bacteria not previously associated with these origin sequences. We empirically validated one of the newly identified origins, 6911, to be functional in both the model bacterial strain, Escherichia coli BW25113, as well as in Pantoea sp. MT58, a fast growing and metal tolerant, environmentally important bacterium from the widespread Pantoea genus. We...

A large-scale screening campaign of putative carbohydrate-active enzymes reveals a novel xylanase from anaerobic gut fungi

Mon, 8 Sep 2025 00:00:00 +0000

The genomes of anaerobic gut fungi (AGF) encode a diverse array of carbohydrate-active enzymes (CAZymes), yet exceedingly few of these enzymes have been experimentally validated or expressed in heterologous systems. Here, we developed a predictive bioinformatic pipeline to annotate novel putative CAZymes from anaerobic fungi and validate their activity through large-scale heterologous expression in Escherichia coli. A total of 173 fungal proteins from Piromyces finnis associated with biomass degradation were synthesized and expressed in E. coli, and 9.8% were soluble with expression levels exceeding 5% of the total proteome using high-throughput proteomic screening. Among these 17 heterologously expressed proteins, analysis with AlphaFold and FoldSeek predicted 13 multi-functional proteins containing catalytic domains fused with repetitive fungal dockerins, and half of the substrate predictions were experimentally validated. One promising enzyme, celsome_012,...

Cost of Deconstruction Depots for Diversified, Waste-Based Lignocellulosic Sugars Using Distillable Solvents

Mon, 8 Sep 2025 00:00:00 +0000

Transitioning to a bioeconomy that makes use of low-emission and waste feedstocks requires greater flexibility to accommodate seasonal variations and mitigate long-term storage challenges, such as material loss and fire risk. To achieve this goal, biomass deconstruction technologies must efficiently handle diverse feedstocks. Here, we assess the cost of using butylaminea distillable solventto deconstruct 22 different biomass feedstocks: 7 herbaceous, 9 woody, 4 food processing residues, and 2 blends. Lignocellulosic sugar production costs, based on current empirical data, range from $1.3 to 6.1/kg, suggesting that substantial improvements are required to compete with conventional sugars. The high solvent loading (850 g/kg of whole slurry) is a process bottleneck. Lowering the solvent loading to 59 g/kg of whole slurry, demonstrated in an L-scale reactor using poplar biomass, reduces the minimum sugar selling price by 33%. Solvent loading and recovery, solid loading, sugar yield,...

Inactivation of BSL-2 and BSL-3 human pathogens using FATHHOME’s Trinion Disinfector: A rapid and eco-friendly ozone-based dry disinfection approach

Tue, 26 Aug 2025 00:00:00 +0000

The role of personal protective equipment (PPE) in protecting against exposure to infectious agents and toxic chemicals is well-established. However, the global surge in PPE demand during the pandemic exposed challenges, including shortages and environmental impacts from disposable waste. Developing effective, scalable, and sustainable decontamination methods for the reuse of PPE is essential. Ozone has emerged as a promising, eco-friendly disinfectant due to its strong oxidative properties, rapid action, and residue-free breakdown into oxygen. This study evaluates the effectiveness of the FATHHOME Trinion Disinfector, an innovative ozone-based dry sterilization device, for inactivating pathogens on PPE materials, such as not resistant to oil 95 (N95) masks and face shields. The device's bactericidal performance was tested against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Enterococcus durans, Enterococcus faecalis, and Saccharomyces...

Multi-scale computational screening and mechanistic insights of cyclic amines as solvents for improved lignocellulosic biomass processing

Mon, 25 Aug 2025 00:00:00 +0000
A computational screening workflow for the efficient deconstruction of cellulose, lignin and hemicellulose fractions of lignocellulosic biomass using cyclic amines as solvents. Lignocellulosic biomass is a promising feedstock for production of affordable fuels and chemicals from renewable resources. Effective solubilization and subsequent deconstruction of its cellulose, hemicellulose, and lignin fractions is essential for the viability of future biorefineries. This study used quantum chemistry-based equilibrium thermodynamics methods to evaluate the potential of 650 cyclic amines to solubilize cellulose, hemicellulose, and lignin. The activity coefficients of solvent - biopolymer interactions were predicted using the COSMO-RS (COnductor-like Screening MOdel for Real Solvents) method and used to identify cyclic amines that can efficiently dissolve and extract selective fractions of biopolymers during biomass pretreatment. Among the 650 cyclic amines, 1-piperazineethanmaine was...

From bench to biofactory: high-throughput technologies and automated workflows to accelerate biomanufacturing

Mon, 18 Aug 2025 00:00:00 +0000
Microbial production of target molecules has advanced significantly in recent years driven by innovations in enzyme engineering, DNA synthesis, and genomic editing. However, to access the massive potential of microbial production, a vast parametric space remains to be investigated to optimize these biobased processes for a robust bioeconomy. Here, we review the current state of the art, some key challenges and possible solutions. We see a critical role of automation, high-throughput technologies, self-driving and cloud labs, and data management to enable Artificial Intelligence/Machine Learning and mechanistic models to overcome the design space challenges and accelerate the development of novel bio-based solutions. Accurate models will expedite the development and scale-up of engineered microbes for a range of final products from many starting materials.

Storage-Induced Collapse of Lignin Macromolecular Structure and Its Impacts on the Biorefinery

Wed, 13 Aug 2025 00:00:00 +0000
Lignin plays a vital role in the economics of biorefineries, serving as a source of process energy and a feedstock for sustainable fuels and chemical production. While understanding lignin's chemical composition is crucial, emerging evidence suggests that a more comprehensive understanding of its macromolecular structure is critical to explaining its complex behavior in the biorefinery. This study investigated the collapse of the lignin network in corn stover feedstock after harvest and storage as a result of the microbial digestion of hemicellulose. Fluorescence microscopy was used to detect the collapse of lignin by the changes in lignin's fluorescence lifetime, anisotropy, and the number of effective emitters. Our in situ microscopic results revealed lignin's coil-globule transition phenomena, which was only previously predicted by molecular dynamics modeling of extracted lignin in solvent. This collapse of lignin macromolecular structure was supported by results from NMR,...

The importance of ester cleavage in the butylamine pretreatment of hybrid poplar

Tue, 29 Jul 2025 00:00:00 +0000
Butylamine is an effective in-and-out pretreatment solvent for hybrid poplar. It penetrates the cell walls and breaks ester cross-linkages, facilitating lignin release and improving enzymatic digestion. This work explores the “in-and-out” pretreatment of hybrid poplar with butylamine as a distillable protic solvent and reagent. The butylamine solvent can be removed by vacuum distillation with >95% solvent removal in all cases, providing a valuable scheme for efficient solvent recovery and recycling. Running the reaction with neat butylamine at 140 °C for 3 hours results in high yields of monosaccharides (90% glucose and 71% xylose) after enzymatic digestion, and a good tolerance to water content with no significant reduction in glucose yield up to an 8 : 1 water : butylamine ratio. We investigate the mechanisms of this pretreatment using powder X-ray diffraction, thermogravimetric analysis, fluorescence microscopy, elemental analysis, solid state and solution state nuclear...

Engineering controllable alteration of malonyl-CoA levels to enhance polyketide production

Tue, 29 Jul 2025 00:00:00 +0000
Heterologous expression of polyketide synthase (PKS) genes in Escherichia coli has enabled the production of various valuable natural and synthetic products. However, the limited availability of malonyl-CoA (M-CoA) in E. coli remains a substantial impediment to high-titer polyketide production. Here we address this limitation by disrupting the native M-CoA biosynthetic pathway and introducing an orthogonal pathway comprising a malonate transporter and M-CoA ligase, enabling efficient M-CoA biosynthesis under malonate supplementation. This approach substantially increases M-CoA levels, enhancing fatty acid and polyketide titers while reducing the promiscuous activity of PKSs toward undesired acyl-CoA substrates. Subsequent adaptive laboratory evolution of these strains provides insights into M-CoA regulation and identifies mutations that further boost M-CoA and polyketide production. This strategy improves E. coli as a host for polyketide biosynthesis and advances understanding...

High-Throughput Microfluidic Electroporation (HTME): A Scalable, 384-Well Platform for Multiplexed Cell Engineering

Tue, 29 Jul 2025 00:00:00 +0000
Electroporation-mediated gene delivery is a cornerstone of synthetic biology, offering several advantages over other methods: higher efficiencies, broader applicability, and simpler sample preparation. Yet, electroporation protocols are often challenging to integrate into highly multiplexed workflows, owing to limitations in their scalability and tunability. These challenges ultimately increase the time and cost per transformation. As a result, rapidly screening genetic libraries, exploring combinatorial designs, or optimizing electroporation parameters requires extensive iterations, consuming large quantities of expensive custom-made DNA and cell lines or primary cells. To address these limitations, we have developed a High-Throughput Microfluidic Electroporation (HTME) platform that includes a 384-well electroporation plate (E-Plate) and control electronics capable of rapidly electroporating all wells in under a minute with individual control of each well. Fabricated using scalable...

Fast growth and high-titer bioproduction from renewable formate via metal-dependent formate dehydrogenase in Escherichia coli

Tue, 29 Jul 2025 00:00:00 +0000
Microbial bioproduction using one-carbon (C1) feedstocks has the potential to decarbonize the manufacturing of materials, fuels, and chemicals. Formate is a promising C1 feedstock, and the realization of industrial, formatotrophic platform organisms is a key goal for C1-based bioproduction. So far, a major limitation for synthetic formatotrophy has been slow energy supply due to slow formate dehydrogenase activity. Here, we implement a fast, metal-dependent formate dehydrogenase complex in a synthetic formatotrophic Escherichia coli utilizing the reductive glycine pathway. After a short-term evolution, we demonstrate formatotrophic growth of E. coli with a doubling time of less than 4.5 h, comparable to the fastest natural formatotrophs. To further explore the potential of a formate-based bioeconomy, this strain is engineered to produce mevalonate, as well as the terpenoid and aviation fuel precursor isoprenol, using formate we generate directly from the electrochemical reduction...

Merging the computational design of chimeric type I polyketide synthases with enzymatic pathways for chemical biosynthesis

Tue, 29 Jul 2025 00:00:00 +0000
Synthetic biology offers the promise of manufacturing chemicals more sustainably than petrochemistry. Yet, both the rate at which biomanufacturing can synthesize these molecules and the net chemical accessible space are limited by existing pathway discovery methods, which can often rely on arduous literature searches. Here, we introduce BioPKS pipeline, an automated retrobiosynthesis tool combining multifunctional type I polyketide synthases (PKSs) and monofunctional enzymes via two complementary tools: RetroTide and DORAnet. Monofunctional enzymes are valuable for carefully decorating a substrate’s carbon backbone while PKSs are unique in their ability to iteratively catalyze carbon-carbon bond formation reactions, thereby expanding carbon backbones in a predictable fashion. We evaluate the performance of BioPKS pipeline using a previously reported set of 155 biomanufacturing candidates, achieving exact synthetic designs for 93 compounds and generating chemically similar pathways...

The mevalonate pathway of isoprenoid biosynthesis supports metabolic flexibility in Mycobacterium marinum

Tue, 29 Jul 2025 00:00:00 +0000
Isoprenoids are a diverse class of natural products that are essential in all domains of life. Most bacteria synthesize isoprenoids through either the methylerythritol phosphate (MEP) pathway or the mevalonate (MEV) pathway, while a small subset encodes both pathways, including the pathogen Mycobacterium marinum (Mm). It is unclear whether the MEV pathway is functional in Mm, or why Mm encodes seemingly redundant metabolic pathways. Here we show that the MEP pathway is essential in Mm while the MEV pathway is dispensable in culture, with the ΔMEV mutant having no growth defect in axenic culture but a competitive growth defect compared to WT Mm. We found that the MEV pathway does not play a role in ex vivo or in vivo infection but does play a role in survival of peroxide stress. Metabolite profiling revealed that modulation of the MEV pathway causes compensatory changes in the concentration of MEP intermediates DOXP and CDP-ME, suggesting that the MEV pathway...

Hybrid biological-chemical strategy for converting polyethylene into a recyclable plastic monomer using engineered Corynebacterium glutamicum

Mon, 28 Jul 2025 00:00:00 +0000
Converting polyethylene (PE) into valuable materials, particularly ones that are better for the environment than the incumbent plastics, not only helps mitigate environmental issues caused by plastic waste but also alleviates the long-standing problem of microbial fermentation competing with food supplies. However, the inherent robustness of PE due to its strong carbon-carbon bonds and high molecular weight necessitates harsh decomposition conditions, resulting in diverse decomposition outcomes that present significant challenges for downstream applications, especially for bioconversion. In this study, we demonstrate a hybrid biological-chemical conversion process for PE, converting its decomposition products, namely short-chain diacids, into a monomer, β-keto-δ-lactone (BKDL), for highly recyclable polydiketoenimine plastics using engineered Corynebacterium glutamicum. Since BKDL synthesis requires a substantial supply of malonyl-CoA, we employed an alternative biosynthesis pathway...

MITRE Domain Specific Language (DSL) for synthetic biology workflows: CRADA Final Report

Wed, 16 Jul 2025 00:00:00 +0000
MITRE Domain Specific Language (DSL) for synthetic biology workflows: CRADA Final Report

TCF High Efficiency Anaerobic Electroporation (CRADA Final Report)

Wed, 16 Jul 2025 00:00:00 +0000
TCF High Efficiency Anaerobic Electroporation (CRADA Final Report)

Abstraction hierarchy to define biofoundry workflows and operations for interoperable synthetic biology research and applications

Wed, 16 Jul 2025 00:00:00 +0000
Lack of standardization in biofoundries limits the scalability and efficiency of synthetic biology research. Here, we propose an abstraction hierarchy that organizes biofoundry activities into four interoperable levels: Project, Service/Capability, Workflow, and Unit Operation, effectively streamlining the Design‑Build‑Test‑Learn (DBTL) cycle. This framework enables more modular, flexible, and automated experimental workflows. It improves communication between researchers and systems, supports reproducibility, and facilitates better integration of software tools and artificial intelligence. Our approach lays the foundation for a globally interoperable biofoundry network, advancing collaborative synthetic biology and accelerating innovation in response to scientific and societal challenges.

Advanced Algal Biofoundries for the Production of Polyurethane Precursors (CRADA Final Report)

Wed, 16 Jul 2025 00:00:00 +0000
Advanced Algal Biofoundries for the Production of Polyurethane Precursors (CRADA Final Report)

Consecutive low-frequency shifts in A/T content denote nucleosome positions across microeukaryotes

Tue, 15 Jul 2025 00:00:00 +0000
Nucleosomes are the basic repeating unit, each spanning ≈150bp, that structures DNA in the nucleus and their positions have major consequences on gene activity. Here, through analyzing DNA signatures across 1117 microeukaryote genomes, we discovered ≈150bp shifts in A/T content associated with nucleosome organization. Often consecutively arrayed across the genome, A/T peaks were enriched surrounding transcriptional start sites in specific clades. Most nucleosomes (both in vitro and in vivo) across eukaryotes aligned with A/T peaks, even in the presence of DNA modifications. Using artificial intelligence-based approaches, we describe DNA features associated with nucleosomes and construct a deep learning (DL) model for improved nucleosome occupancy prediction. Using this model, we found that ≈70% of "random" transfer DNA inserts from an in vivo fungal RB-TDNAseq library avoided DL predicted nucleosome-bound regions. This study reveals a eukaryote-wide strategy...

Capacitive response of biological membranes

Tue, 15 Jul 2025 00:00:00 +0000
We present a minimal model to analyze the capacitive response of a biological membrane subjected to a step voltage via blocking electrodes. Through a perturbative analysis of the underlying electrolyte transport equations, we show that the leading-order relaxation of the transmembrane potential is governed by a capacitive timescale, τ_{C}=λ_{D}L/D(2+Γδ^{M}/L/4+Γδ^{M}/λ_{D}), where λ_{D} is the Debye screening length, L is the electrolyte width, Γ is the ratio of the permittivity of the electrolyte to the membrane, δ^{M} is the membrane thickness, and D is the ionic diffusivity. This timescale is considerably shorter than the traditional RC timescale λ_{D}L/D for a bare electrolyte due to the membrane's low permittivity and finite thickness. Beyond the linear regime, however, salt diffusion in the bulk electrolyte drives a secondary, nonlinear relaxation process of the transmembrane potential over a longer timescale τ_{L}=L^{2}/4π^{2}D. A simple equivalent-circuit model accurately...

Author Correction: Machine learning-led semi-automated medium optimization reveals salt as key for flaviolin production in Pseudomonas putida

Tue, 15 Jul 2025 00:00:00 +0000
Author Correction: Machine learning-led semi-automated medium optimization reveals salt as key for flaviolin production in Pseudomonas putida

The oleaginous yeast Rhodosporidium toruloides engineered for biomass hydrolysate-derived (E)-α-bisabolene production

Tue, 15 Jul 2025 00:00:00 +0000
The oleaginous yeast Rhodosporidiumtoruloides has been exploited for many bioproducts, including several terpenes, owing to its oleaginous nature and biomass inhibitor tolerance. Here, we built upon previous (E)-α-bisabolene work by iteratively stacking the complete mevalonate pathway from Saccharomyces cerevisiae onto a multicopy bisabolene synthase parent strain. Metabolomics and proteomics verified heterologous pathway expression and identified metabolic bottlenecks at three intermediate steps, with candidate feedback-resistant mevalonate kinases screening improving titers 15%. Subtle differences in codon optimization, and preliminary attenuation of competing flux toward lipids resulted in 6-fold, 7-fold higher titers relative to controls, respectively. Media optimization led to modest improvements, with zinc identified as the most promising at 10% titer improvement. Ultimately, high-performance strains were cultivated with corn-stover biomass hydrolysate in microtiter plates...

Non-DNA radiosensitive targets that initiate persistent behavioral deficits in rats exposed to space radiation

Tue, 15 Jul 2025 00:00:00 +0000
Predicting future CNS risks for astronauts during deep-space missions will rely substantially on ground-based rodent data with space-relevant ions and behaviors. For rats, the accumulated evidence indicates that less densely ionizing radiation, such as ⁴He and ¹²C ions, induce behavior deficits at lower doses than densely ionizing ions, such as ⁴⁸Ti and ⁵⁶Fe. However, this observation conflicts with standard somatic radiobiology, in which densely ionizing ions are generally more effective than less densely ionizing ions, and where the DNA/nucleus is the accepted target for radiation-induced tumorigenesis, cytogenetic aberrations, genetic mutations, and reproductive cell death. To gain deeper insight into the subcellular nature of the radiation targets for behavior risks, we compared the effects of dose, fluence, and linear energy transfer (LET) of ⁴He and ⁵⁶Fe particles using existing datasets for four distinct...

Recent Advances in the Use of Ionic Liquids and Deep Eutectic Solvents for Lignocellulosic Biorefineries and Biobased Chemical and Material Production

Tue, 15 Jul 2025 00:00:00 +0000
Biorefineries, which process biomass feedstocks into valuable (bio)products, aim to replace fossil fuel-based refineries to produce energy and chemicals, reducing environmental and health hazards, including climate change, and supporting a sustainable economy. In particular, lignocellulose-based biorefineries, utilizing the most abundant renewable feedstock on Earth, have significant potential to supply sustainable energy, chemicals and materials. Ionic liquids (ILs, organic salts with low melting temperatures) and deep eutectic solvents (DESs, mixtures with eutectic points lower than the ideal mixture) are capable of dissolving some of the key lignocellulose polymers, and even the whole biomass. Furthermore, they have intrinsic advantages over molecular solvents, including safer usage profiles and high tunability, which allow tailored physicochemical properties. Such properties provide unique opportunities for the development of new processes that could unlock the full potential...

Solvent Screening for Separation Processes Using Machine Learning and High-Throughput Technologies

Mon, 14 Jul 2025 00:00:00 +0000
As the chemical industry shifts toward sustainable practices, there is a growing initiative to replace conventional fossil-derived solvents with environmentally friendly alternatives such as ionic liquids (ILs) and deep eutectic solvents (DESs). Artificial intelligence (AI) plays a key role in the discovery and design of novel solvents and the development of green processes. This review explores the latest advancements in AI-assisted solvent screening with a specific focus on machine learning (ML) models for physicochemical property prediction and separation process design. Additionally, this paper highlights recent progress in the development of automated high-throughput (HT) platforms for solvent screening. Finally, this paper discusses the challenges and prospects of ML-driven HT strategies for green solvent design and optimization. To this end, this review provides key insights to advance solvent screening strategies for future chemical and separation processes.

Regulatory orchestration of FK506 biosynthesis in Streptomyces tsukubaensis NRRL 18488 revealed through systematic analysis

Fri, 11 Jul 2025 00:00:00 +0000
Streptomyces tsukubaensis NRRL 18488, the primary producer of the immunosuppressant FK506, was analyzed to elucidate regulatory features of secondary metabolism. Completion of its 7.9-Mb linear genome enabled accurate re-annotation of the FK506 biosynthetic gene cluster (BGC). Transcriptome analysis during BGC activation revealed major transcriptional shifts from primary to secondary metabolism, especially in genes involved in FK506 biosynthesis and lysine metabolism. Primary transcriptome mapping identified 1,225 transcription units and uncovered post-transcriptional regulation of allylmalonyl-CoA production, a key FK506 precursor. Ribosome profiling demonstrated that AT-rich codons reduce translational efficiency in S. tsukubaensis, with pronounced ribosome pausing at the TTA codon within the FK506 BGC. Substituting this codon relieved pausing and improved FK506 production. Together, these integrative genomic, transcriptomic, and translatomic analyses highlight...

Evolution-guided tolerance engineering of Pseudomonas putida KT2440 for production of the aviation fuel precursor isoprenol

Wed, 9 Jul 2025 00:00:00 +0000
Isoprenol (3-methyl-3-buten-1-ol) is a precursor to aviation fuels and other commodity chemicals and can be microbially synthesized from renewable carbon streams. Its production has been demonstrated in Pseudomonas putida KT2440 but its titers, rates, and yields have yet to reach commercially viable levels, potentially due to toxicity to the bacterial chassis. We hypothesized that utilization of Tolerization Adaptive Laboratory Evolution (TALE) would generate P. putida hosts more tolerant to isoprenol and suitable for enhanced production phenotypes. Here, we performed a comprehensive TALE campaign using three strains, the wild-type and two strains lacking subsets of known isoprenol catabolism and transport functions in quadruplicate independently evolved lineages. Several evolved clones from each starting strain displayed robust growth (up to 0.2 h^-1) at 8 g/L of isoprenol, where starting strains could not grow. Whole genome resequencing of the 12 independent strain...

Comparison between two different approaches for the deconstruction of lignocellulosic feedstocks using alkanolamine-based solvents

Tue, 1 Jul 2025 00:00:00 +0000
Schematic depiction of the approach used to screen different pretreatment solvents on different biomass feedstocks.

Enabling malic acid production from corn-stover hydrolysate in Lipomyces starkeyi via metabolic engineering and bioprocess optimization

Wed, 25 Jun 2025 00:00:00 +0000
BackgroundLipomyces starkeyi is an oleaginous yeast with a native metabolism well-suited for production of lipids and biofuels from complex lignocellulosic and waste feedstocks. Recent advances in genetic engineering tools have facilitated the development of L. starkeyi into a microbial chassis for biofuel and chemical production. However, the feasibility of redirecting L. starkeyi lipid flux away from lipids and towards other products remains relatively unexplored. Here, we engineer the native metabolism to produce malic acid by introducing the reductive TCA pathway and a C4-dicarboxylic acid transporter to the yeast.ResultsHeterogeneous expression of two genes, the Aspergillus oryzae malate transporter and malate dehydrogenase, enabled L. starkeyi malic acid production. Overexpression of a third gene, the native pyruvate carboxylase, allowed titers to reach approximately 10 g/L during shaking flasks cultivations, with production of malic acid inhibited at pH values less...

Quantitative dissection of Agrobacterium T-DNA expression in single plant cells reveals density-dependent synergy and antagonism

Mon, 23 Jun 2025 00:00:00 +0000
Agrobacterium pathogenesis, which involves transferring T-DNA into plant cells, is the cornerstone of plant genetic engineering. As the applications that rely on Agrobacterium increase in sophistication, it becomes critical to achieve a quantitative and predictive understanding of T-DNA expression at the level of single plant cells. Here we examine if a classic Poisson model of interactions between pathogens and host cells holds true for Agrobacterium infecting Nicotiana benthamiana. Systematically challenging this model revealed antagonistic and synergistic density-dependent interactions between bacteria that do not require quorum sensing. Using various approaches, we studied the molecular basis of these interactions. To overcome the engineering constraints imposed by antagonism, we created a dual binary vector system termed ‘BiBi’, which can improve the efficiency of a reconstituted complex metabolic pathway in a predictive fashion. Our findings illustrate how combining theoretical...

Emergence and disruption of cooperativity in a denitrifying microbial community

Wed, 4 Jun 2025 00:00:00 +0000
Anthropogenic perturbations to the nitrogen cycle, primarily through use of synthetic fertilizers, is driving an unprecedented increase in the emission of nitrous oxide (N2O), a potent greenhouse gas and an ozone depleting substance, causing urgency in identifying the sources and sinks of N2O. Microbial denitrification is a primary contributor to biotic production of N2O in anoxic regions of soil, marine systems, and wastewater treatment facilities. Here, through comprehensive genome analysis, we show that pathway partitioning is a ubiquitous mechanism of complete denitrification within microbial communities. We have investigated mechanisms and consequences of process partitioning of denitrification through detailed physiological characterization and kinetic modeling of a synthetic community of Rhodanobacter thiooxydans FW510-R12 and Acidovorax sp. GW101-3H11. We have discovered that these two bacterial isolates, from a heavily nitrate (NO3-) contaminated superfund site, complete...

Ultrafast Formation of Charge Transfer Trions at Molecular‐Functionalized 2D MoS2 Interfaces

Tue, 3 Jun 2025 00:00:00 +0000
Abstract: In this work, we investigate trion dynamics occurring at the heterojunction between organometallic molecules and a monolayer transition metal dichalcogenide (TMD) with transient electronic sum frequency generation (tr‐ESFG) spectroscopy. By pumping at 2.4 eV with laser pulses, we have observed an ultrafast hole transfer, succeeded by the emergence of charge‐transfer trions. This observation is facilitated by the cancellation of ground state bleach and stimulated emission signals due to their opposite phases, making tr‐ESFG especially sensitive to the trion formation dynamics. The presence of charge‐transfer trion at molecular functionalized TMD monolayers suggests the potential for engineering the local electronic structures and dynamics of specific locations on TMDs and offers a potential for transferring unique electronic attributes of TMD to the molecular layers.

Long-Term, Non-Invasive Detection of Low-Dose Ionizing Radiation Exposure

Tue, 3 Jun 2025 00:00:00 +0000
Ionizing radiation induces complex changes in cells and tissues. The conventional approach to biological dosimetry has been to integrate physical and clinical measurements to optimize dose assessment. Molecular biodosimetry is an effective strategy to monitor radiation exposure and hematologic, cytogenetic, protein and transcript-based approaches have been developed to increase dose estimation accuracy. However, these approaches are invasive, time-consuming, have limited effectiveness over time, and importantly do not accurately inform on low dose radiation exposures. Therefore, novel, non-invasive, biomarkers are required that can overcome these limitations. We developed a pipeline that employs Fourier transform infrared (FTIR) spectroscopy in the mid-infrared spectrum to identify a signature of low dose ionizing radiation exposure in mouse ear pinnae over time. Two cohorts of C57BL/6J and one cohort of BALB/c mice were followed for ninety days after total body X-ray exposures...

A Reexamination of CO2 Reduction with Fe2S2 Hydrogenase Mimics: Lessons in Using a Hydrogen Evolution Reaction Catalyst for CO2 to Formate Catalysis

Tue, 3 Jun 2025 00:00:00 +0000
Recent reports show [FeFe] hydrogenase mimics are active for the electrochemical reduction of CO2 to formate (HCOO−). Herein, the electrochemical reduction of CO2 with the [FeFe] hydrogenase mimic [Fe2(μ‐pdt)(CO)6, 1, where pdt = propane‐1,3‐dithiolate] in acetonitrile is reported. In the presence of the weak acid, methanol (MeOH), 1 reduces CO2 to both CO (Faradaic Efficiency maximum [FEmax] of 16 ± 6%) and HCOO− (FEmax = 20%) and produces H2 (FEmax = 56 ± 4%). Without added MeOH, 1 reacts with adventitious water to form H2 (FEmax = 85 ± 1%), HCOO− (FEmax = 7.8%), and CO (FEmax = 7 ± 3%) with CO32− being detected by infrared spectroscopy. Product formation is potential dependent: more negative potentials increases selectivity for HCOO− over CO. The first reduction of 1 forms a pdt‐bridged dimer, 2. However, the reduction of 2 at the potentials required for electrochemical CO2 reduction leads to two new species. Using density functional theory, and infrared spectroelectrochemistry...

Ultrafast Formation of Charge Transfer Trions at Molecular‐Functionalized 2D MoS2 Interfaces

Tue, 3 Jun 2025 00:00:00 +0000
In this work, we investigate trion dynamics occurring at the heterojunction between organometallic molecules and a monolayer transition metal dichalcogenide (TMD) with transient electronic sum frequency generation (tr-ESFG) spectroscopy. By pumping at 2.4 eV with laser pulses, we have observed an ultrafast hole transfer, succeeded by the emergence of charge-transfer trions. This observation is facilitated by the cancellation of ground state bleach and stimulated emission signals due to their opposite phases, making tr-ESFG especially sensitive to the trion formation dynamics. The presence of charge-transfer trion at molecular functionalized TMD monolayers suggests the potential for engineering the local electronic structures and dynamics of specific locations on TMDs and offers a potential for transferring unique electronic attributes of TMD to the molecular layers.

A Resilient Platform for the Discrete Functionalization of Gold Surfaces Based on N‑Heterocyclic Carbene Self-Assembled Monolayers

Tue, 3 Jun 2025 00:00:00 +0000
We describe the synthesis and characterization of a versatile platform for gold functionalization, based on self-assembled monolayers (SAMs) of distal-pyridine-functionalized N-heterocyclic carbenes (NHC) derived from bis(NHC) Au(I) complexes. The SAMs are characterized using polarization-modulation infrared reflectance-absorption spectroscopy, surface-enhanced Raman spectroscopy, and X-ray photoelectron spectroscopy. The binding mode is examined computationally using density functional theory, including calculations of vibrational spectra and direct comparisons to the experimental spectroscopic signatures of the monolayers. Our joint computational and experimental analyses provide structural information about the SAM binding geometries under ambient conditions. Additionally, we examine the reactivity of the pyridine-functionalized SAMs toward H₂SO₄ and W(CO)₅(THF) and verify the preservation of the introduced functionality at the interface. Our...

Ensemble Detection of DNA Engineering Signatures

Tue, 3 Jun 2025 00:00:00 +0000
Synthetic biology is creating genetically engineered organisms at an increasing rate for many potentially valuable applications, but this potential comes with the risk of misuse or accidental release. To begin to address this issue, we have developed a system called GUARDIAN that can automatically detect signatures of engineering in DNA sequencing data, and we have conducted a blinded test of this system using a curated Test and Evaluation (T&E) data set. GUARDIAN uses an ensemble approach based on the guiding principle that no single approach is likely to be able to detect engineering with perfect accuracy. Critically, ensembling enables GUARDIAN to detect sequence inserts in 13 target organisms with a high degree of specificity that requires no subject matter expert (SME) review.

Section-level genome sequencing and comparative genomics of Aspergillus sections Cavernicolus and Usti

Thu, 22 May 2025 00:00:00 +0000
The genus Aspergillus is diverse, including species of industrial importance, human pathogens, plant pests, and model organisms. Aspergillus includes species from sections Usti and Cavernicolus, which until recently were joined in section Usti, but have now been proposed to be non-monophyletic and were split by section Nidulantes, Aenei and Raperi. To learn more about these sections, we have sequenced the genomes of 13 Aspergillus species from section Cavernicolus (A. cavernicola, A. californicus, and A. egyptiacus), section Usti (A. carlsbadensis, A. germanicus, A. granulosus, A. heterothallicus, A. insuetus, A. keveii, A. lucknowensis, A. pseudodeflectus and A. pseudoustus), and section Nidulantes (A. quadrilineatus, previously A. tetrazonus). We compared these genomes with 16 additional species from Aspergillus to explore their genetic diversity, based on their genome content, repeat-induced point mutations (RIPs), transposable elements, carbohydrate-active enzyme (CAZyme)...

Alternate routes to acetate tolerance lead to varied isoprenol production from mixed carbon sources in Pseudomonas putida

Tue, 20 May 2025 00:00:00 +0000
Lignocellulose is a renewable resource for the production of a diverse array of platform chemicals, including the biofuel isoprenol. Although this carbon stream provides a rich source of sugars, other organic compounds, such as acetate, can be used by microbial hosts. Here, we examined the growth and isoprenol production in a Pseudomonas putida strain pre-tolerized ("PT") background where its native isoprenol catabolism pathway is deleted, using glucose and acetate as carbon sources. We found that PT displays impaired growth in minimal medium containing acetate and often fails to grow in glucose-acetate medium. Using a mutant recovery-based approach, we generated tolerized strains that overcame these limitations, achieving fast growth and isoprenol production in the mixed carbon feed. Changes in the glucose and acetate assimilation routes, including an upregulation in PP_0154 (SpcC, succinyl-CoA:acetate CoA-transferase) and differential expression of the gluconate assimilation...

Retrobiosynthesis of unnatural lactams via reprogrammed polyketide synthase

Mon, 19 May 2025 00:00:00 +0000
Engineered polyketide synthases (PKSs) have great potential as biocatalysts. These unnatural enzymes are capable of synthesizing molecules that are either not amenable to biosynthesis or are extremely challenging to access chemically. PKSs can thus be a powerful platform to expand the chemical landscape beyond the limits of conventional metabolic engineering. Here we employ a retrobiosynthesis approach to design and construct PKSs to produce δ-valerolactam (VL) and three enantiopure α-substituted VL analogues that have no known biosynthetic route. We introduce the engineered PKSs and pathways for various malonyl-CoA derivatives into Pseudomonas putida and use proteomics, metabolomics and culture condition optimization to improve the production of our target compounds. These α-substituted VLs are polymerized into polyamides (nylon-5) or converted into their N-acryloyl derivatives. RAFT polymerization produces bio-derived polymers with potential biomedical applications. Overall,...

Recent developments of oleaginous yeasts toward sustainable biomanufacturing

Mon, 19 May 2025 00:00:00 +0000
Oleaginous yeast are remarkably versatile organisms, distinguished by their natural capacities to accumulate high levels of neutral lipids and broad substrate range. With recent growing interests in engineering non-model organisms as superior biomanufacturing platforms, oleaginous yeasts have emerged as promising chassis for oleochemicals, terpenoids, organic acids, and other valuable products. Advancement in systems biology along with genetic tool development have significantly expanded our understanding of the metabolism in these species and enabled engineering efforts to produce biofuels and bioproducts from diverse feedstocks. This review examines the latest technical advances in oleaginous yeast research toward sustainable biomanufacturing. We cover recent developments in systems biology-enabled metabolism understanding, genetic tools, feedstock utilization, and strain engineering approaches for the production of various valuable chemicals.

Understanding the impacts of inorganic species in woody biomass for preprocessing and pyrolysis–A review

Tue, 13 May 2025 00:00:00 +0000
Woody biomass represents an abundant resource for sustainable biofuels, biochemicals, and bioproducts. Technologies for converting woody biomass have been established for decades, and research consistently highlights the critical role of inorganic species and ash plays in feedstock handling and conversion processes, including equipment plugging, corrosion, and catalyst deactivation. A thorough understanding of the variability, transport behavior, and downstream impact of inorganic species in woody biomass is essential for defining feedstock quality specifications and developing effective management strategies for conversion processes. This review compiles critical information in five main sections: 1) inorganic species concentration in woody biomass, based on anatomical fractions and their sources of variability; 2) technique features for quantifying inorganic elemental chemical analysis; 3) impacts of inorganic species on biomass preprocessing; 4) impacts of inorganic species...

A polyketide-based biosynthetic platform for diols, amino alcohols and hydroxy acids

Tue, 13 May 2025 00:00:00 +0000
Medium- and branched-chain diols and amino alcohols are important industrial solvents, polymer building blocks, cosmetics and pharmaceutical ingredients, yet biosynthetically challenging to produce. Here we present an approach that uses a modular polyketide synthase (PKS) platform for the efficient production of these compounds. This platform takes advantage of a versatile loading module from the rimocidin PKS and nicotinamide adenine dinucleotide phosphate-dependent terminal thioester reductases. Reduction of the terminal aldehyde with alcohol dehydrogenases enables the production of diols, oxidation enables the production of hydroxy acids and specific transaminases allow the production of various amino alcohols. Furthermore, replacement of the malonyl-coenzyme A-specific acyltransferase in the extension module with methyl- or ethylmalonyl-coenzyme A-specific acyltransferase enables the production of branched-chain diols, amino alcohols and carboxylic acids in high titres. Use...

Characterization of Kariya (Hildegardia barteri (Mast.) Kosterm) Seed Oil Fatty Acid Methyl Ester Prepared from Basic Catalytic Transesterification

Tue, 13 May 2025 00:00:00 +0000
The rising global energy demand, alongside concerns regarding environmental deterioration due to the use of fossil fuels, has spurred extensive investigation into renewable energy alternatives. Biomass-derived biodiesel, especially from lesser-known oil sources, emerges as a promising option. This research focuses on analyzing the fatty acid methyl esters (FAMEs) derived from Kariya (Hildegardia barteri (Mast.) Kosterm) seed oil through basic catalytic transesterification using gas chromatography–flame ionization detector (GC–FID) analysis, assessing its potential as a biodiesel feedstock. Oil extraction from Kariya seeds was carried out using three solvents (n-hexane, ethanol, and a 1:1 blend of hexane and ethanol), followed by transesterification with methanol. Gas chromatography–mass spectrometry (GC–MS) and GC–FID analyses were utilized to identify and quantify FAMEs in the resulting biodiesel. The results revealed various FAMEs, including methyl myristate, methyl palmitate,...

Section-level genome sequencing and comparative genomics of Aspergillus sections Cavernicolus and Usti.

Thu, 8 May 2025 00:00:00 +0000
The genus Aspergillus is diverse, including species of industrial importance, human pathogens, plant pests, and model organisms. Aspergillus includes species from sections Usti and Cavernicolus, which until recently were joined in section Usti, but have now been proposed to be non-monophyletic and were split by section Nidulantes, Aenei and Raperi. To learn more about these sections, we have sequenced the genomes of 13 Aspergillus species from section Cavernicolus (A. cavernicola, A. californicus, and A. egyptiacus), section Usti (A. carlsbadensis, A. germanicus, A. granulosus, A. heterothallicus, A. insuetus, A. keveii, A. lucknowensis, A. pseudodeflectus and A. pseudoustus), and section Nidulantes (A. quadrilineatus, previously A. tetrazonus). We compared these genomes with 16 additional species from Aspergillus to explore their genetic diversity, based...

Modulation of Active Site Picosecond Dynamics in Mutant Forms of a Thermophilic Alcohol Dehydrogenase (HT-ADH)

Thu, 8 May 2025 00:00:00 +0000
Modulation of Active Site Picosecond Dynamics in Mutant Forms of a Thermophilic Alcohol Dehydrogenase (HT-ADH)

Atacama Large Aperture Submillimeter Telescope (AtLAST) science: Resolving the hot and ionized Universe through the Sunyaev-Zeldovich effect

Thu, 1 May 2025 00:00:00 +0000
An omnipresent feature of the multi-phase "cosmic web" - the large-scale filamentary backbone of the Universe - is that warm/hot (≳ 10 ⁵ K) ionized gas pervades it. This gas constitutes a relevant contribution to the overall universal matter budget across multiple scales, from the several tens of Mpc-scale intergalactic filaments, to the Mpc intracluster medium (ICM), all the way down to the circumgalactic medium (CGM) surrounding individual galaxies from ∼ 1 kpc up to their respective virial radii (∼ 100 kpc). The study of the hot baryonic component of cosmic matter density represents a powerful means for constraining the intertwined evolution of galactic populations and large-scale cosmological structures, for tracing the matter assembly in the Universe and its thermal history. To this end, the Sunyaev-Zeldovich (SZ) effect provides the ideal observational tool for measurements out to the beginnings of structure formation. The SZ effect is caused by the scattering...

Author Correction: A map of the rubisco biochemical landscape

Thu, 1 May 2025 00:00:00 +0000
Correction to: Naturehttps://doi.org/10.1038/s41586-024-08455-0 Published online 22 January 2025 In the version of the article initially published, the affiliations of Hana A. Chang (Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, CA, USA) and Ron Milo (Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, Israel) were incorrect and have now been amended in the HTML and PDF versions of the article.

Machine learning-led semi-automated medium optimization reveals salt as key for flaviolin production in Pseudomonas putida

Fri, 25 Apr 2025 00:00:00 +0000
Although synthetic biology can produce valuable chemicals in a renewable manner, its progress is still hindered by a lack of predictive capabilities. Media optimization is a critical, and often overlooked, process which is essential to obtain the titers, rates and yields needed for commercial viability. Here, we present a molecule- and host-agnostic active learning process for media optimization that is enabled by a fast and highly repeatable semi-automated pipeline. Its application yielded 60% and 70% increases in titer, and 350% increase in process yield in three different campaigns for flaviolin production in Pseudomonas putida KT2440. Explainable Artificial Intelligence techniques pinpointed that, surprisingly, common salt (NaCl) is the most important component influencing production. The optimal salt concentration is very high, comparable to seawater and close to the limits that P. putida can tolerate. The availability of fast Design-Build-Test-Learn (DBTL) cycles allowed...

In planta production of the nylon precursor beta-ketoadipate

Tue, 22 Apr 2025 00:00:00 +0000
Beta-ketoadipate (βKA) is an intermediate of the βKA pathway involved in the degradation of aromatic compounds in several bacteria and fungi. Beta-ketoadipate also represents a promising chemical for the manufacturing of performance-advantaged nylons. We established a strategy for the in planta synthesis of βKA via manipulation of the shikimate pathway and the expression of bacterial enzymes from the βKA pathway. Using Nicotiana benthamiana as a transient expression system, we demonstrated the efficient conversion of protocatechuate (PCA) to βKA when plastid-targeted bacterial-derived PCA 3,4-dioxygenase (PcaHG) and 3-carboxy-cis,cis-muconate cycloisomerase (PcaB) were co-expressed with 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (AroG) and 3-dehydroshikimate dehydratase (QsuB). This metabolic pathway was reconstituted in Arabidopsis by introducing a construct (pAtβKA) with stacked pcaG, pcaH, and pcaB genes into a PCA-overproducing genetic background that expresses AroG...

Corn stover variability drives differences in bisabolene production by engineered Rhodotorula toruloides

Tue, 8 Apr 2025 00:00:00 +0000
Microbial conversion of lignocellulosic biomass represents an alternative route for production of biofuels and bioproducts. While researchers have mostly focused on engineering strains such as Rhodotorula toruloides for better bisabolene production as a sustainable aviation fuel, less is known about the impact of the feedstock heterogeneity on bisabolene production. Critical material attributes like feedstock composition, nutritional content, and inhibitory compounds can all influence bioconversion. Further, the given feedstocks can have a marked influence on selection of suitable pretreatment and hydrolysis technologies, optimizing the fermentation conditions, and possibly even modifying the microorganism's metabolic pathways, to better utilize the available feedstock. This work aimed to examine and understand how variations in corn stover batches, anatomical fractions, and storage conditions impact the efficiency of bisabolene production by R. toruloides. All of these represent...

Enzymatic cleavage of model lignin dimers depends on pH, enzyme, and bond type

Mon, 7 Apr 2025 00:00:00 +0000
Lignin is composed of phenylpropanoid monomers linked by ether and carbon-carbon bonds to form a complex heterogeneous structure. Bond-specific studies of lignin-modifying enzymes (LMEs; e.g., laccases and peroxidases) are limited by the polymerization of model lignin substrates and repolymerization of cleavage products. Here we present a high throughput platform to screen LME activities on four tagged model lignin compounds that represent the β-O-4’, β-β’, 5–5’, and 4-O-5’ linkages in lignin. We utilized nanostructure-initiator mass spectrometry (NIMS) and model lignin compounds with tags containing perfluorinated and cationic moieties, which effectively limit polymerization and condensation of the substrates and their degrading products. Sub-microliter sample droplets were printed on the NIMS chip with a novel robotics method. This rapid platform enabled characterization of LMEs across a range of pH 3–10 and relative quantification of modified (typically oxidized), cleaved,...

Corynebacterium glutamicum as an Efficient Omnivorous Microbial Host for the Bioconversion of Lignocellulosic Biomass

Wed, 2 Apr 2025 00:00:00 +0000
Corynebacterium glutamicum has been successfully employed for the industrial production of amino acids and other bioproducts, partially due to its native ability to utilize a wide range of carbon substrates. We demonstrated C. glutamicum as an efficient microbial host for utilizing diverse carbon substrates present in biomass hydrolysates, such as glucose, arabinose, and xylose, in addition to its natural ability to assimilate lignin-derived aromatics. As a case study to demonstrate its bioproduction capabilities, L-lactate was chosen as the primary fermentation end product along with acetate and succinate. C. glutamicum was found to grow well in different aromatics (benzoic acid, cinnamic acid, vanillic acid, and p-coumaric acid) up to a concentration of 40 mM. Besides, ¹³C-fingerprinting confirmed that carbon from aromatics enter the primary metabolism via TCA cycle confirming the presence of β-ketoadipate pathway in C. glutamicum....

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

Wed, 2 Apr 2025 00:00:00 +0000
Efficient conversion of pentose sugars remains a significant barrier to the replacement of petroleum-derived chemicals with plant biomass-derived bioproducts. While the oleaginous yeast Rhodosporidiumtoruloides (also known as Rhodotorulatoruloides) has a relatively robust native metabolism of pentose sugars compared to other wild yeasts, faster assimilation of those sugars will be required for industrial utilization of pentoses. To increase the rate of pentose assimilation in R.toruloides, we leveraged previously reported high-throughput fitness data to identify potential regulators of pentose catabolism. Two genes were selected for further investigation, a putative transcription factor (RTO4_12978, Pnt1) and a homolog of a glucose transceptor involved in carbon catabolite repression (RTO4_11990). Overexpression of Pnt1 increased the specific growth rate approximately twofold early in cultures on xylose and increased the maximum specific growth by 18% while decreasing accumulation...

Corrigendum: Corynebacterium glutamicum as an efficient omnivorous microbial host for the bioconversion of lignocellulosic biomass

Wed, 2 Apr 2025 00:00:00 +0000
[This corrects the article DOI: 10.3389/fbioe.2022.827386.].

Microbial Pathways for Cost-Effective Low-Carbon Renewable Indigoidine

Wed, 2 Apr 2025 00:00:00 +0000
Indigoidine is a bioadvantaged platform molecule with diverse applications, including use as a textile dye, biotransistor, biosolar cell, biosensor, and food coloring. There are multiple microbial hosts and carbon sources that can be used and optimized for its production, yet there is limited guidance for which options have the greatest commercial potential. Here, we consider five different host microbes and combine genome-scale metabolic models with techno-economic and lifecycle assessment models. Pseudomonas putida currently outperforms synthetic indigo production and other indigoidine-producing hosts, using glucose, xylose, and lignin-derived aromatics to produce indigoidine at a minimum selling price of $2.9/kg and a greenhouse gas (GHG) footprint of 3.5 kgCO_2e/kg. Optimizing pathways-achieving 90% of the theoretical indigoidine yield from sugars and aromatics-can reduce costs 6-7-fold and GHG emissions 3-10-fold. From a cost perspective, microbes that co-utilize...

Non-canonical d-xylose and l-arabinose metabolism via d-arabitol in the oleaginous yeast Rhodosporidium toruloides

Wed, 2 Apr 2025 00:00:00 +0000
R. toruloides is an oleaginous yeast, with diverse metabolic capacities and high tolerance for inhibitory compounds abundant in plant biomass hydrolysates. While R. toruloides grows on several pentose sugars and alcohols, further engineering of the native pathway is required for efficient conversion of biomass-derived sugars to higher value bioproducts. A previous high-throughput study inferred that R. toruloides possesses a non-canonical l-arabinose and d-xylose metabolism proceeding through d-arabitol and d-ribulose. In this study, we present a combination of genetic and metabolite data that refine and extend that model. Chiral separations definitively illustrate that d-arabitol is the enantiomer that accumulates under pentose metabolism. Deletion of putative d-arabitol-2-dehydrogenase (RTO4_9990) results in > 75% conversion of d-xylose to d-arabitol, and is growth-complemented on pentoses by heterologous xylulose kinase expression. Deletion of putative d-ribulose kinase...

Spatiotemporal dynamics of ionic reorganization near biological membrane interfaces

Tue, 18 Mar 2025 00:00:00 +0000
Electrical signals in excitable cells involve spatially localized ionic fluxes through ion channels and pumps on cellular lipid membranes. Common approaches to understand how these fluxes spread assume that the membrane and the surrounding electrolyte comprise an equivalent circuit of capacitors and resistors, which ignores the localized nature of transmembrane ion transport, the resulting ionic gradients and electric fields, and their spatiotemporal relaxation. Here, we consider a model of localized ion pumping across a lipid membrane, and use theory and simulation to investigate how the electrochemical signal propagates spatiotemporally in and out of plane along the membrane. The localized pumping generates long-ranged electric fields with three distinct scaling regimes along the membrane: a constant potential near-field region, an intermediate monopolar region, and a far-field dipolar region. Upon sustained pumping, the monopolar region expands radially in plane with a steady...

High-Throughput Large-Scale Targeted Proteomics Assays for Quantifying Pathway Proteins in Pseudomonas putida KT2440

Tue, 18 Mar 2025 00:00:00 +0000
Targeted proteomics is a mass spectrometry-based protein quantification technique with high sensitivity, accuracy, and reproducibility. As a key component in the multi-omics toolbox of systems biology, targeted liquid chromatography-selected reaction monitoring (LC-SRM) measurements are critical for enzyme and pathway identification and design in metabolic engineering. To fulfill the increasing need for analyzing large sample sets with faster turnaround time in systems biology, high-throughput LC-SRM is greatly needed. Even though nanoflow LC-SRM has better sensitivity, it lacks the speed offered by microflow LC-SRM. Recent advancements in mass spectrometry instrumentation significantly enhance the scan speed and sensitivity of LC-SRM, thereby creating opportunities for applying the high speed of microflow LC-SRM without losing peptide multiplexing power or sacrificing sensitivity. Here, we studied the performance of microflow LC-SRM relative to nanoflow LC-SRM by monitoring 339...

Cooperative Research and Development Agreement (CRADA) Final Report - MITRE Domain Specific Language (DSL) for synthetic biology workflows

Thu, 13 Mar 2025 00:00:00 +0000
Cooperative Research and Development Agreement (CRADA) Final Report - MITRE Domain Specific Language (DSL) for synthetic biology workflows

Machine Learning Enabled Process Optimization for Pharmacologically Relevant Dependent Variables

Wed, 12 Mar 2025 00:00:00 +0000
Final Report