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DOE OSTI2025

Understanding and Harnessing the Robustness of Undomesticated Yarrowia lipolytica Strains for Biosynthesis of Designer Bioesters (Final Report)

This project seeks to elucidate and harness the exceptional robustness of novel and undomesticated Y. lipolytica isolates, which were identified from a genetic diversity screening for compatibility with bioenergy development. Bioenergy-relevant isolates were further developed as microbial platforms for efficient conversion of undetoxified biomass hydrolysates into designer bio-esters continuously recovered by solvent extraction. The project has three major goals. Goal 1. Elucidate and enhance the endogenous metabolism of Y. lipolytica for superior growth, sugar utilization, and lipid accumulation in undetoxified biomass hydrolysates under hypoxic conditions. Goal 2. Understand and enhance the underlying mechanism of exceptional tolerance of Y. lipolytica to organic solvents. Goal 3. Elucidate and rewire endogenous metabolism of the most robust Y. lipolytica strains for effective conversion of accumulating lipids to designer bio-esters. Significant progress has been made toward completing all research goals. We elucidated and optimized the robustness of Y. lipolytica by utilizing mixed C5 and C6 sugars in switchgrass hydrolysates (SGH) for lipid production (Aim 1). We conducted extensive omics analysis to investigate how genetic diversity among Yarrowia strains, derived from natural isolates or developed through adaptive laboratory evolution, influences lipid production when utilizing SGH. In Aim 2, novel mechanisms and underlying genetics were discovered that enabled Yarrowia strains to thrive in cultures containing high ionic liquid (IL) concentrations. Amongst other novel findings, it was found that sterols strengthened cell membranes to confer IL toxicity resistance, specifically via increased ergosterol content upon exposure to IL. In Aim 3, mechanistic studies elucidated how Y. lipolytica utilized intracellular lipids and alkanes/alkenes, leading to our discovery of novel enzymes and pathways for making short-chain esters. Most notably, thermostable chloramphenicol transferases were repurposed to function as alcohol acetyltransferases in Y. lipolytica, as well as the Gram-negative and Gram-positive bacteria Escherichia coli and thermophile Clostridium thermocellum, respectively.

09 BIOMASS FUELS↗

DOE OSTI2025

MarK, a Novosphingobium aromaticivorans kinase required for catabolism of multiple aromatic monomers

The aromatic compounds used in a variety of industrial products are currently obtained from nonrenewable petroleum sources. Alternatively, the plant polymer lignin is an abundant renewable source of aromatics, and its depolymerization generates a variety of products that can include acetovanillone, a vanillin derivative containing an acetyl side chain. The Alphaproteobacterium Novosphingobium aromaticivorans DSM12444 can metabolize several chemically modified aromatics in deconstructed lignin, but not acetovanillone. In this work, adaptive laboratory evolution identified a single amino acid change in the previously uncharacterized gene product Saro_1862 that is necessary and sufficient for N. aromaticivorans growth with acetovanillone as a sole growth substrate, as well as other aromatic monomers not metabolized by wild-type cells. We show that a glutamate (E) to lysine (K) substitution at amino acid residue 16 of Saro_1862 results in a ~1600-fold increase in the rate of ATP-dependent acetovanillone phosphorylation. We also find that recombinant Saro_1862 E16K phosphorylates several other aromatic compounds in vitro , defining the first reported catalytic activity for the widespread UPF0261 protein domain contained in Saro_1862. Thus, we propose naming Saro_1862 MarK, for multiple aromatic kinase. A 1.57 Å crystal structure of MarK E16K predicts that the E16K substitution lies in a potential ATP binding site, suggesting how this amino acid change increased catalytic activity. A search for homologs of MarK and other proteins required for acetovanillone degradation predicts that this pathway for aromatic metabolism exists throughout the bacterial phylogeny.

Novosphingobium↗

DOE OSTI2025

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

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 of M-CoA metabolism in microbial systems.

Klass, Sarah H↗

DOE OSTI2024

Application of functional genomics for domestication of novel non-model microbes

Abstract With the expansion of domesticated microbes producing biomaterials and chemicals to support a growing circular bioeconomy, the variety of waste and sustainable substrates that can support microbial growth and production will also continue to expand. The diversity of these microbes also requires a range of compatible genetic tools to engineer improved robustness and economic viability. As we still do not fully understand the function of many genes in even highly studied model microbes, engineering improved microbial performance requires introducing genome-scale genetic modifications followed by screening or selecting mutants that enhance growth under prohibitive conditions encountered during production. These approaches include adaptive laboratory evolution, random or directed mutagenesis, transposon-mediated gene disruption, or CRISPR interference (CRISPRi). Although any of these approaches may be applicable for identifying engineering targets, here we focus on using CRISPRi to reduce the time required to engineer more robust microbes for industrial applications. One-Sentence Summary The development of genome scale CRISPR-based libraries in new microbes enables discovery of genetic factors linked to desired traits for engineering more robust microbial systems.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

A small number of point mutations confer formate tolerance in Shewanella oneidensis

ABSTRACT Microbial electrosynthesis (MES) is a sustainable approach to chemical production from CO 2 and clean electricity. However, limitations in electron transfer efficiency and gaps in understanding of electron transfer pathways in MES systems prevent full realization of this technology. Shewanella oneidensis could serve as an MES biocatalyst because it has a well-studied, efficient transmembrane electron transfer pathway. A key first step in MES in this organism could be CO 2 reduction to formate. However, we report that wild-type S. oneidensis does not tolerate high levels of formate. In this work, we created and characterized formate-tolerant strains of S. oneidensis for further engineering and future use in MES systems through adaptive laboratory evolution. Two different point mutations in a gene encoding a predicted sodium-dependent bicarbonate transporter and a DUF2721-containing protein separately confer formate tolerance to S. oneidensis . The mutations were further evaluated to understand their role in improving formate tolerance. We also show that the wild-type and mutant versions of the putative sodium-dependent bicarbonate transporter improve formate tolerance of Zymomonas mobilis , indicating the potential of transferring this formate tolerance phenotype to other organisms. IMPORTANCE Shewanella oneidensis is a bacterium with a well-studied, efficient extracellular electron transfer pathway. This capability could make this organism a suitable host for microbial electrosynthesis using CO 2 or formate as feedstocks. However, we report here that formate is toxic to S. oneidensis , limiting the potential for its use in these systems. In this work, we evolve several strains of S. oneidensis that have improved formate tolerance, and we investigate some mutations that confer this phenotype. The phenotype is confirmed to be attributed to several single point mutations by transferring the wild-type and mutant versions of each gene to the wild-type strain. Finally, the formate tolerance mechanism of one variant is studied using structural modeling and expression in another host. This study, therefore, presents a simple method for conferring formate tolerance to bacterial hosts.

Cross, Megan C. Gruenberg (ORCID:0000000291589900)↗

DOE OSTI2024

raogroupuiuc/yl_variantcalling

NGS analysis for mutation analysis in Yarrowia lipolytica evolved strains. Yarrowia lipolytica, an oleaginous yeast, shows promise for industrial fermentation due to its robust acetyl-CoA flux and well-developed genetic engineering tools. However, its lack of an active xylose metabolism restricts the conversion of cellulosic sugars to valuable products. To address this, metabolic engineering, and adaptive laboratory evolution (ALE) were applied to the Y. lipolytica PO1f strain, resulting in an efficient xylose-assimilating strain (XEV). Whole-genome sequencing (WGS) of the XEV followed by reverse engineering revealed that the amplification of the heterologous oxidoreductase pathway and a mutation in the GTPase-activating protein gene (YALI0B12100g) might be the primary reasons for improved xylose assimilation in the XEV strain. When a sorghum hydrolysate was used, the XEV strain showed superior xylose consumption and lipid production compared to its parental strain (X123). This study advances our understanding of xylose metabolism in Y. lipolytica and proposes effective metabolic engineering strategies for optimizing lignocellulosic hydrolysates.

Deewan, Anshu↗

DOE OSTI2026

RNAseq data for P. putida with vanillate

Illumina sequencing reads from RNA sequencing of vanillate-utilizing strains of Pseudomonas putida, described in Evolution and engineering of pathways for aromatic O-demethylation in Pseudomonas putida KT2440 by A. Bleem, et al. (2024)

Adaptive laboratory evolution↗

DOE OSTI2026

From Cryogenic to High Temperatures: A Microstructural Evolution of δ-phase Pu-Ga Alloys Investigated by Time-of-Flight High-resolution Neutron Diffraction

Phase stability and phase transformations in binary Pu alloys are critical for multiple applications. Long-term phase stability affects mechanical properties, microstructure, corrosion behavior, and structural integrity. A clear and detailed understanding of phase transformations and phase stability mechanisms is necessary to assess the behavior and response to unexpected stimuli. Gaining advanced knowledge on the behavior of Pu and Pu alloys at a variety of temperatures, pressures, and time will ensure better predictions and control on evolution outside laboratory measurements.

36 MATERIALS SCIENCE↗

DOE OSTI2026

Applied genomics for industrially relevant microalgal strain development & optimization: characterizing genotype-phenotype relationships towards scalable environment-enhancing energy systems

Multiple planetary boundaries considered a safe operating space for humanity have been exceeded in recent years, with twice as many boundaries transgressed in 2023 as in 2009. Bioenergy systems are unique in that they can interact with and improve many of the transgressed boundaries directly, including multiple geochemical cycles, water and land use, and climate change. Among bioenergy systems, microalgae-based environment enhancing energy (E 2 -energy) are promising bioenergy systems for drop-in biofuels, valuable materials, chemicals and therapeutics, while making deep emission cuts and remediating wastewater, all without competing for agricultural resources.

09 BIOMASS FUELS↗

DOE OSTI2026