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At least 109 records · Page 6

Strangers in a foreign land: ‘Yeastizing’ plant enzymes

Abstract Expressing plant metabolic pathways in microbial platforms is an efficient, cost‐effective solution for producing many desired plant compounds. As eukaryotic organisms, yeasts are often the preferred platform. However, expression of plant enzymes in a yeast frequently leads to failure because the enzymes are poorly adapted to the foreign yeast cellular environment. Here, we first summarize the current engineering approaches for optimizing performance of plant enzymes in yeast. A critical limitation of these approaches is that they are labour‐intensive and must be customized for each individual enzyme, which significantly hinders the establishment of plant pathways in cellular factories. In response to this challenge, we propose the development of a cost‐effective computational pipeline to redesign plant enzymes for better adaptation to the yeast cellular milieu. This proposition is underpinned by compelling evidence that plant and yeast enzymes exhibit distinct sequence features that are generalizable across enzyme families. Consequently, we introduce a data‐driven machine learning framework designed to extract ‘yeastizing’ rules from natural protein sequence variations, which can be broadly applied to all enzymes. Additionally, we discuss the potential to integrate the machine learning model into a full design‐build‐test cycle.

59 BASIC BIOLOGICAL SCIENCES↗

Acetate-based biological platforms: Bridging carbon dioxide utilization and high-value bioproduct production in oleaginous yeasts

Acetate is emerging as a promising two-carbon substrate in the circular bioeconomy, bridging the gap between single-carbon sources and high-value biofuels and bioproducts. This review examines the key pathways for acetate production, including the electrochemical reduction of carbon dioxide, syngas fermentation, and biological acetogenesis. It focuses on acetate metabolism in oleaginous yeasts, such as Yarrowia lipolytica and Rhodotorula toruloides, which efficiently convert acetate-derived acetyl-CoA units into diverse bioproducts such as lipids, fatty alcohols, triacetic acid lactone, and carotenoids. Recent advances in metabolic engineering, transcriptomics, and metabolic flux analysis have improved the understanding of acetate assimilation in these organisms, thereby increasing their potential for industrial applications. In addition, the feasibility of a biological gas-to-liquid platform that utilizes acetate as a central intermediate for scalable biomanufacturing is discussed. Integrating acetate utilization with sustainable production strategies offers a promising path to advance the bio-based economy. Using acetate as a versatile metabolic intermediate enables the conversion of industrial emissions into biofuels and bioproducts while avoiding the energetic and toxicity constraints associated with direct fermentation of gaseous substrates.

59 BASIC BIOLOGICAL SCIENCES↗

Xylose metabolic engineering of Issatchenkia orientalis for 3-hydroxypropionic acid production from cellulosic hydrolysate without nutrient supplementation

Bioconversion of lignocellulosic biomass offers a promising alternative to petroleum-based chemical production. However, inefficient xylose utilization and toxic compounds in cellulosic hydrolysate limit microbial fermentation, as the hydrolysate contains substantial amounts of xylose in addition to glucose. To address these challenges, we engineered Issatchenkia orientalis to produce 3-hydroxypropionic acid (3-HP) directly from sorghum hydrolysate under low-pH conditions. A heterologous xylose utilization pathway consisting of XYL1, XYL2, and XYL3 from Scheffersomyces stipitis was introduced into an engineered 3-HP producing strain, enabling efficient conversion of xylose to 3-HP. The engineered strain produced 46.8 g/L 3-HP from sorghum hydrolysate without nutrient supplementation. To eliminate the lag phase under low-pH conditions, fermentation was conducted at pH 6.0 for the first three days, after which pH control was discontinued and in situ 3-HP accumulation buffered the culture. This partial pH control strategy increased 3-HP productivity by 55% from 0.20 to 0.31 g/L∙h, while maintaining low-pH conditions. Introducing an additional copy of XYL2 further increased 3-HP titer to 53.5 g/L and the yield by 33%, from 0.30 to 0.40 g/g sugars, with pH reaching 4.5 at the end of fermentation. This represents one of the highest reported 3-HP titers and yields from cellulosic hydrolysate without additional nutrient supplementation. This work demonstrates a nutrient-independent and low-pH bioprocess for upgrading lignocellulosic hydrolysate into 3-HP, highlighting the industrial potential of engineered xylose-utilizing I. orientalis for sustainable production of platform chemicals from renewable feedstocks.

3-Hydroxypropionic acid↗

Design of diverse, functional mitochondrial targeting sequences across eukaryotic organisms using variational autoencoder

Mitochondria play a key role in energy production and metabolism, making them a promising target for metabolic engineering and disease treatment. However, despite the known influence of passenger proteins on localization efficiency, only a few protein-localization tags have been characterized for mitochondrial targeting. To address this limitation, we leverage a Variational Autoencoder to design novel mitochondrial targeting sequences. In silico analysis reveals that a high fraction of the generated peptides (90.14%) are functional and possess features important for mitochondrial targeting. We characterize artificial peptides in four eukaryotic organisms and, as a proof-of-concept, demonstrate their utility in increasing 3-hydroxypropionic acid titers through pathway compartmentalization and improving 5-aminolevulinate synthase delivery by 1.62-fold and 4.76-fold, respectively. Moreover, we employ latent space interpolation to shed light on the evolutionary origins of dual-targeting sequences. Overall, our work demonstrates the potential of generative artificial intelligence for both fundamental research and practical applications in mitochondrial biology.

59 BASIC BIOLOGICAL SCIENCES↗

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

The oleaginous yeast Rhodosporidium toruloides has been exploited for many bioproducts, including several terpenes, owing to its oleaginous nature and biomass inhibitor tolerance. Here, we built upon previous (E)-a-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 at 300g/L total sugar, achieving 20.8g/L bisabolene, the highest reported titer in the literature. A 2L glucose minimal medium bioreactor achieved 19.3 g/L bisabolene and a literature-high productivity of 0.11 g/L/h.

60 APPLIED LIFE SCIENCES↗

Omics-driven onboarding of the carotenoid producing red yeast Xanthophyllomyces dendrorhous CBS 6938

Transcriptomics is a powerful approach for functional genomics and systems biology, yet it can also be used for genetic part discovery. Here, we derive constitutive and light-regulated promoters directly from transcriptomics data of the basidiomycete red yeast Xanthophyllomyces dendrorhous CBS 6938 (anamorph Phaffia rhodozyma) and use these promoters with other genetic elements to create a modular synthetic biology parts collection for this organism. X. dendrorhous is currently the sole biotechnologically relevant yeast in the Tremellomycete class-it produces large amounts of astaxanthin, especially under oxidative stress and exposure to light. Thus, we performed transcriptomics on X. dendrorhous under different wavelengths of light (red, green, blue, and ultraviolet) and oxidative stress. Differential gene expression analysis (DGE) revealed that terpenoid biosynthesis was primarily upregulated by light through crtI, while oxidative stress upregulated several genes in the pathway. Further gene ontology (GO) analysis revealed a complex survival response to ultraviolet (UV) where X. dendrorhous upregulates aromatic amino acid and tetraterpenoid biosynthesis and downregulates central carbon metabolism and respiration. The DGE data was also used to identify 26 constitutive and regulated genes, and then, putative promoters for each of the 26 genes were derived from the genome. Simultaneously, a modular cloning system for X. dendrorhous was developed, including integration sites, terminators, selection markers, and reporters. Each of the 26 putative promoters were integrated into the genome and characterized by luciferase assay in the dark and under UV light. The putative constitutive promoters were constitutive in the synthetic genetic context, but so were many of the putative regulated promoters. Notably, one putative promoter, derived from a hypothetical gene, showed ninefold activation upon UV exposure. Thus, this study reveals metabolic pathway regulation and develops a genetic parts collection for X. dendrorhous from transcriptomic data. Therefore, this study demonstrates that combining systems biology and synthetic biology into an omics-to-parts workflow can simultaneously provide useful biological insight and genetic tools for nonconventional microbes, particularly those without a related model organism. This approach can enhance current efforts to engineer diverse microbes.

60 APPLIED LIFE SCIENCES↗

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

ABSTRACT 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 acute 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 is functional and that the pathways interact at the metabolic level. Finally, the MEV pathway is upregulated early in the shift down to hypoxia, suggesting that it may provide metabolic flexibility to this bacterium. Interestingly, we found that our complemented strains, which vary in copy number of the polyprenyl synthetase idsB2 , responded differently to peroxide and UV stresses, suggesting a role for this gene as a determinant of downstream prenyl phosphate metabolism. Together, these findings suggest that MEV may serve as an anaplerotic pathway to make isoprenoids under stress conditions. IMPORTANCE Organisms from all domains of life utilize isoprenoids to carry out thousands of critical and auxiliary cellular processes, including signaling, maintaining membrane integrity, stress response, and host-pathogen interactions. The common precursor of all isoprenoids is synthesized via one of two biosynthetic pathways. Importantly, some bacteria encode both pathways, including M. marinum . We found that only one pathway is essential in M. marinum , while the nonessential pathway may confer metabolic flexibility to help the bacterium better adapt to various environmental conditions. We also found that the polyprenyl synthetase IdsB2 plays an important role in driving such phenotypes. Further, we demonstrate metabolic interplay between both functional pathways. These insights represent the first characterization of isoprenoid biosynthesis in dual pathway-encoding mycobacteria.

Qabar, Christine M. [Department of Plant and Micro↗

Integrating CO2 Electrolysis with Gas Fermentation to Produce Valuable Fuels and Chemicals

Many industrial activities squander CO2, decreasing process yield. We envision a future where this waste carbon is instead captured, upgraded, and valorized directly at the point of emission. Within the CO2 Reduction and Upgrading Consortium (a collaboration of seven US national laboratories and industrial partners), we are pursuing this goal by developing and de-risking new technologies for low temperature CO2 electrolysis, coupled with biological upgrading of intermediates into more valuable compounds. One such process involves electrocatalytic reduction of CO2 to generate carbon monoxide (CO). As both a carbon and energy source, CO represents an attractive feedstock for microbial upgrading by certain syngas-fermenting species, such as the autotrophic bacterium Clostridium autoethanogenum. Our team has developed new genetic tools and optimized cultivation techniques to enhance C. autoethanogenum as a platform host for the biological conversion of syngas into value-added products. For example, we have created novel CRISPR-based genetic engineering techniques to build new, genome-reduced, platform strains of C. autoethanogenum with improved growth rates. Further, we ve introduced heterologous biochemical pathways into C. autoethanogenum to enable the production of high-value compounds from syngas, such as the isoprenoid precursor mevalonic acid. With the tools of electrochemistry and synthetic biology, there is virtually no limit to the spectrum of products that could be sustainably manufactured from CO2.

09 BIOMASS FUELS↗

Self-Assembling Cell-Free Systems for Scalable Bioconversion

This project focused on developing cell-free systems to directly express multi-enzyme catalysts and perform CO2 bioconversions for industrial chemical production. The use of cell-free expression (CFE) systems derived from bacterial lysates is emerging as a promising approach for biomanufacturing. CFEs are genetically programmable, permit the expression of toxic enzymes, and allow for rapid prototyping of metabolic pathways. Research Contributions: 1. Understanding the Area Investigated: This research advances the understanding of cell-free systems by demonstrating their capability to perform complex multi-enzyme reactions. By directly expressing multi-gene systems, CFEs avoid the high costs and inefficiencies associated with producing and purifying enzymes for multi-step pathways. 2. Technical Effectiveness and Economic Feasibility: The project successfully engineered a CFE-based multienzyme biocatalyst for the de novo synthesis of serine and glycine from CO2 equivalents (formate and bicarbonate) and ammonia. This method achieved a 30% conversion rate of formate into these industrially important amino acids. Additionally, an 8-gene CFE biocatalyst was developed to produce malate, conserving 43% of carbon that would otherwise be lost as CO2. This approach has the potential to reach higher carbon efficiency than microbial production. 3. Public Benefit: The cell-free production of chemicals like serine, glycine, and malate using electrochemically generated formate could significantly reduce CO2 emissions. For example, satisfying the global malate market with this method could avoid approximately 400,000 tons of CO2 emissions annually. This work demonstrates the potential of CFE systems to produce platform chemicals, contributing to environmental sustainability and reducing reliance on petrochemicals. Future Prospects: The CFE-based biocatalyst process could be extended to produce a variety of chemicals, including other industrial di-acids, aromatics, terpenes, alcohols, and polymers. This project showcases the capabilities of cell-free expression systems for prototyping carbon-conserving pathways and sustainably bioproducing platform chemicals, marking a significant step towards economically-viable industrial processes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Synthetic Fungal Strains for Solar System Exploration and Colonization

Solar system exploration and eventual colonization efforts are constrained by limits on the mass of material that can embark from Earth. Thus, creative use of the resources available in situ could reduce mission costs and extend the scope of such activities. To that end, we are developing synthetic fungal strains to produce specialized materials from the resources found throughout the solar system. A primary goal is to develop a suite of Saccharomyces cerevisiae strains to serve as generic production chassis for synthetic metabolic pathways. These strains must perform consistently upon challenge by unique conditions including exposure to microgravity, cosmic radiation, the rigors of launch and re-entry, and long-term stasis. Presently, we are establishing systematic datasets profiling epigenetic, transcriptional, translational and metabolic states of S. cerevisiae under relevant operating conditions. These will deepen our understanding of the physiological changes associated with space travel and enable rational engineering of optimal production strains.

Biotechnology↗

Microbes display broad diversity in cobamide preferences

ABSTRACT Cobamides, the vitamin B 12 (cobalamin) family of cofactors, are used by most organisms but produced by only a fraction of prokaryotes, and are thus considered key shared nutrients among microbes. Cobamides are structurally diverse, with multiple different cobamides found in most microbial communities. The ability to use different cobamides has been tested for several bacteria and microalgae, and nearly all show preferences for certain cobamides. This approach is limited by the commercial unavailability of cobamides other than cobalamin. Here, we have extracted and purified seven commercially unavailable cobamides to characterize bacterial cobamide preferences based on growth in specific cobamide-dependent conditions. The tested bacteria include engineered strains of Escherichia coli , Sinorhizobium meliloti , and Bacillus subtilis expressing native or heterologous cobamide-dependent enzymes, cultured under conditions that functionally isolate specific cobamide-dependent processes such as methionine synthesis. Comparison of these results to those of previous studies of diverse bacteria and microalgae revealed that a broad diversity of cobamide preferences exists not only across different organisms but also between different cobamide-dependent metabolic pathways within the same organism. The microbes differed in the cobamides that support growth most efficiently, cobamides that do not support growth, and the minimum cobamide concentrations required for growth. The latter differ by up to four orders of magnitude across organisms from different environments and by up to 20-fold between cobamide-dependent enzymes within the same organism. Given that cobamides are shared, required for use of specific growth substrates, and essential for central metabolism in certain organisms, cobamide preferences likely impact community structure and function. IMPORTANCE Nearly all bacteria are found in microbial communities with tens to thousands of other species. Molecular interactions such as metabolic cooperation and competition are key factors underlying community assembly and structure. Cobamides, the vitamin B 12 family of enzyme cofactors, are one such class of nutrients, produced by only a minority of prokaryotes but required by most microbes. A unique aspect of cobamides is their broad diversity, with nearly 20 structural forms identified in nature. Importantly, this structural diversity impacts growth as most bacteria that have been tested show preferences for specific cobamide forms. We measured cobamide-dependent growth in several model bacteria and compared the results to those of previous analyses of cobamide preference. We found that cobamide preferences vary widely across bacteria, showing the importance of characterizing these aspects of cobamide biology to understand the impact of cobamides on microbial communities.

Mok, Kenny C. (ORCID:0000000252276987)↗

Genetically pliable green algae for bioproduction of modified fatty acids, nutritional therapeutic oils, and biopharmaceuticals

Homologous recombination (HR) is an essential tool for complex metabolic engineering in yeast, but transgene integration into plant and green algal nuclear genomes predominantly occurs by non-homologous end-joining. Species of the closely related, oleaginous trebouxiophytes Auxenochlorella and Prototheca, are unusual among the green algae in that HR is the favored mechanism for DNA integration into the nuclear genome. This property enables locus-specific targeting of gene cassettes encoding multiple enzymes for manipulating existing biochemical pathways or introducing new functions. Genetic malleability, and regulatory approval for human consumption, coupled with robust fermentation performance at industrial scale, establishes Auxenochlorella and Prototheca as prime candidates for algal production of biochemicals and biomaterials. The examples presented here highlight strain improvement and engineering for synthesis of hydroxylated fatty acids for biomaterials, structured triglycerides resembling human milk fat for infant nutrition, very-long-chain mono- and polyunsaturated fatty acids with nutraceutical or therapeutic potential, and cannabinoids for pharmacological applications.

Moseley, Jeffrey L. [University of California, Ber↗

Metabolic engineering strategies for producing decanoic acid and related oleochemicals: 1-decanol, 2-nonanone, and poly(3-hydroxydecanoate) in Escherichia coli

Medium-chain (mc-) oleochemicals are an important class of renewable chemicals with broad industrial applications; however, their sustainable microbial production remains challenging. In this study, we developed a versatile metabolic engineering and fed-batch strategy to produce C 10 -oleochemicals in Escherichia coli. Central to this approach is an engineered mc-acyl-ACP thioesterase Cl FatB3-tr-D10S with C 10 species accounting for around 70% of the total fatty acids produced. To expand product diversity, we established a decanoyl-CoA pool through co-expression of fadD, enabling downstream conversion into multiple product classes. Through pathway tuning, enzyme bioprospecting, strain engineering and fermentation optimization strategies, we demonstrated selective production of 1-decanol, 2-nonanone and poly(3-hydroxydecanoate) (C 10 -PHA). Production of decanoic acid and 1-decanol were achieved by optimizing expression of Cl fatB3-tr-D10S and, Mt fadD6 and Ma acr, respectively. Leveraging β-oxidation enabled the production of β-ketoacyl-CoA intermediates, which were converted to 2-nonanone via heterologous Mlu fadE, Vf fadB and Ps fadM expression. Additionally, expression of phaJ2 and phaC2 facilitated the conversion of decanoyl-CoA pool into C 10 -PHA homopolymer. Altogether, this work demonstrates a versatile and tunable platform for medium-chain oleochemical production.

1-Decanol↗

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

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 assembly of CRISPRi constructs containing up to five sgRNA arrays, reducing context dependency and achieving uniform, position-independent gene downregulation. The integration of FluxRETAP and VAMMPIRE has the potential to advance metabolic engineering by rapidly identifying CRISPRi-mediated knockdowns and knockdown combinations that enhance bioproduction titers, with potential applicability to other microbial systems.

CRISPR interference↗

Advances in genetic tools for metabolic engineering of non-conventional yeasts

Non-conventional yeasts are emerging as powerful alternatives to Saccharomyces cerevisiae for metabolic engineering, owing to their innate stress tolerance, broad substrate utilization, and distinctive metabolic capabilities. These attributes position them as promising chassis for producing biofuels, pharmaceuticals, and specialty chemicals. This review synthesizes recent advances in genetic toolkits for four such species—Pichia kudriavzevii (Issatchenkia orientalis), Starmerella bombicola, Debaryomyces hansenii, and Pachysolen tannophilus—highlighting progress across plasmid architectures (episomal and integrative), identification of autonomously replicating sequences and centromeric elements, and the development of safe-harbor genomic loci. We summarize promoter and terminator libraries enabling tunable expression, the expansion of auxotrophic and antifungal selection markers with recycling strategies, and the rapid adaptation of CRISPR-based systems (Cas9 and Cas12a) with optimized guide RNA expression, multiplex editing, and approaches that enhance homologous recombination (e.g., KU70/80 disruption). We also review landing-pad platforms for modular, repeated integrations and transposon-based tools (e.g., piggyBac) that facilitate multigene pathway assembly. Collectively, these innovations are accelerating design-build-test-learn cycles and enabling precise, scalable engineering of non-conventional yeasts. Remaining challenges—including limited species-specific episomal systems, variable transformation efficiencies, genome-stability concerns, and alternative codon usage—define clear priorities for future toolkit development. Together, these advances and open needs chart a path toward robust, sustainable biomanufacturing using diverse non-conventional yeast chassis.

59 BASIC BIOLOGICAL SCIENCES↗

Tools for genetic engineering and gene expression control in Novosphingobium aromaticivorans and Rhodobacter sphaeroides

ABSTRACT Alphaproteobacteria have a variety of cellular and metabolic features that provide important insights into biological systems and enable biotechnologies. For example, some species are capable of converting plant biomass into valuable biofuels and bioproducts that have the potential to contribute to the sustainable bioeconomy. Among the Alphaproteobacteria, Novosphingobium aromaticivorans , Rhodobacter sphaeroides , and Zymomonas mobilis show promise as organisms that can be engineered to convert extracted plant lignin or sugars into bioproducts and biofuels. Genetic manipulation of these bacteria is needed to introduce engineered pathways and modulate expression of native genes with the goal of enhancing bioproduct output. Although recent work has expanded the genetic toolkit for Z. mobilis , N. aromaticivorans and R. sphaeroides still need facile, reliable approaches to deliver genetic payloads to the genome and to control gene expression. Here, we expand the platform of genetic tools for N. aromaticivorans and R. sphaeroides to address these issues. We demonstrate that Tn 7 transposition is an effective approach for introducing engineered DNA into the chromosome of N. aromaticivorans and R. sphaeroides . We screen a synthetic promoter library to identify isopropyl β-D-1-thiogalactopyranoside-inducible promoters with regulated activity in both organisms (up to ~15-fold induction in N. aromaticivorans and ~5-fold induction in R. sphaeroides ). Combining Tn 7 integration with promoters from our library, we establish CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference systems for N. aromaticivorans and R. sphaeroides (up to ~10-fold knockdown in N. aromaticivorans and R. sphaeroides ) that can target essential genes and modulate engineered pathways. We anticipate that these systems will greatly facilitate both genetic engineering and gene function discovery efforts in these species and other Alphaproteobacteria. IMPORTANCE It is important to increase our understanding of the microbial world to improve health, agriculture, the environment, and biotechnology. For example, building a sustainable bioeconomy depends on the efficient conversion of plant material to valuable biofuels and bioproducts by microbes. One limitation in this conversion process is that microbes with otherwise promising properties for conversion are challenging to genetically engineer. Here we report genetic tools for Novosphingobium aromaticivorans and Rhodobacter sphaeroides that add to the burgeoning set of tools available for genome engineering and gene expression in Alphaproteobacteria. Our approaches allow straightforward insertion of engineered pathways into the N. aromaticivorans or R. sphaeroides genome and control of gene expression by inducing genes with synthetic promoters or repressing genes using CRISPR interference. These tools can be used in future work to gain additional insight into these and other Alphaproteobacteria and to aid in optimizing yield of biofuels and bioproducts.

Hall, Ashley N.↗

Development of emerging model microorganisms: Megasphaera elsdenii for biomass and organic acid upgrading to fuels and chemicals

The metabolic diversity of microorganisms in nature represents a largely untapped source of valuable compounds that are difficult or impossible to produce in the limited number of available model systems. Efforts to produce longer-chain alcohols, such as hexanol, in organisms like Escherichia coli have met with limited success; production of C6 and larger products remains low, highlighting the challenges of extending chain elongation pathways beyond a single cycle. Megasphaera elsdenii naturally condenses acetyl-CoA to efficiently generate C4–C8 organic acids, making it a promising candidate for producing fuels and chemicals from lactate and plant-derived carbohydrates. This high native flux through the chain elongation pathway offers the potential for higher yields and titers of medium-chain products, such as hexanol, compared to conventional hosts. Recent advances—most notably the development of a transformation method for M. elsdenii—have further opened the organism to detailed physiological studies and bioengineering. While full development of M. elsdenii as a hexanol-producing platform was not achieved, significant progress was made in understanding its metabolism and building foundational genetic tools for future engineering.

60 APPLIED LIFE SCIENCES↗

Carbon source–driven metabolic and regulatory remodeling defines phenomic states in Lipomyces starkeyi

Lipomyces is a genus of oleaginous yeasts with potential for contributing to reliable biomanufacturing supply chains. However, progress in advanced strain designs and engineering efforts are still constrained by a lack of understanding of the underlying molecular drivers of Lipomyces phenotypes. To address this gap, we collected a suite of multi-omic data to dissect how carbon source availability reshapes the metabolic network, lipid allocation, and regulatory architecture of Lipomyces starkeyi. We observed that glucose promotes biosynthetic and proliferative processes supported by abundant energy and carbon intermediates, xylose enhances redox-balancing mechanisms centered on the pentose phosphate pathway, and glycerol activates respiratory metabolism, ß-oxidation, and the glyoxylate cycle. Lipid species distributions remained consistent in both nitrogen replete and depleted conditions across the carbon sources, indicating robust production mechanisms. Regulatory protein identification and network analysis revealed glycerol-driven respiratory growth favors regulatory programs integrating stress tolerance, redox balance, and lipid-associated metabolism, whereas xylose growth activates compensatory transcriptional responses aimed at maintaining mitochondrial function. Nitrogen limitation modulates the strength of these responses but does not fundamentally alter their direction, reinforcing carbon source as the dominant driver of regulatory architecture. Taken together, this data enhances the understanding of Lipomyces molecular rearrangements and provides a foundation for further development of predictive phenotypic tools in this genus.

Biotechnology↗