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DOE OSTI2022

Protein-based models offer mechanistic insight into complex nickel metalloenzymes

There are ten nickel enzymes found across biological systems, each with a distinct active site and reactivity that spans reductive, oxidative, and redox–neutral processes. We focus on the reductive enzymes, which catalyze reactions that are highly germane to the modern-day climate crisis: [NiFe] hydrogenase, carbon monoxide dehydrogenase, acetyl coenzyme A synthase, and methyl coenzyme M reductase. The current mechanistic understanding of each enzyme system is reviewed along with existing knowledge gaps, which are addressed through the development of protein-derived models, as described here. Finally, this opinion is intended to highlight the advantages of using robust protein scaffolds for modeling multiscale contributions to reactivity and inspire the development of novel artificial metalloenzymes for other small molecule transformations.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2021

SIRT1 reduces epigenetic and non-epigenetic changes to maintain the quality of postovulatory aged oocytes in mice

Highlights: • The intensity of H3K9ac and H3K4me2/3 increased in postovulatory aged oocytes. • SIRT1 overexpression inhibited the increase of H3K9ac and H3K4me2/3. • SIRT1 overexpression ameliorated aging-induced epigenetic and non-epigenetic changes in oocytes. Postovulatory oocyte aging has a major influence on the development potential of embryos. Many antioxidants can delay oocyte aging by regulating the expression of SIRT1. However, there is a lack of knowledge on SIRT1 function in postovulatory oocyte aging. In vitro transcribed RNA of Sirt1 was injected into fresh oocytes to investigate the function of SIRT1 during postovulatory oocyte aging. In the present study, SIRT1 was found to be down-regulated in aged oocytes compared with fresh oocytes. Meanwhile the intensity of acetylation of H3K9 (H3K9ac) and H3K4 methylation increased in postovulatory aged oocytes. After the oocytes were injected with SIRT1 and aged for 12 h, the intensity of H3K9ac and H3K4 methylation markedly decreased compared with controls. Furthermore, SIRT1 overexpression also reduced the aging-induced oocyte morphological changes and reactive oxygen species accumulation, maintained the spindle normal morphology and attenuated the aging-associated abnormalities of mitochondrial function. The role of SIRT1 in protecting oocyte aging was diminished when oocytes with overexpressed SIRT1 were cultured with SIRT1 inhibitor EX-527. Briefly, these present results show that SIRT1 not only reduced the non-epigenetic changes such as abnormal oocyte morphology, ROS accumulation, spindle defects and mitochondrial dysfunctions but also regulated the epigenetic changes in order to maintain the quality of postovulatory aged oocytes.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

MIER3 induces epithelial-mesenchymal transition and promotes breast cancer cell aggressiveness via forming a co-repressor complex with HDAC1/HDAC2/Snail

Highlights: • The expression of MIER3 was correlated with poor outcome of breast cancer patients. • MIER3 induced EMT and promoted cell migration. • MIER3 interacted with HDAC1/HDAC2 and Snail to form a repressive complex. • The repressive complex bound to E-cadherin promoter and reduced its acetylation. Breast cancer is one of the most frequently diagnosed cancers and the leading cause of cancer death in women. MIER3 (Mesoderm induction early response 1, family member3) is considered as a potential oncogene for breast cancer. However, the role of MIER3 in breast cancer remain largely unknown. The expression of MIER3 was detected and the relationship between its expression and clinicopathological characteristics was also analyzed. The effect of MIER3 on proliferation and migration of breast cancer cells was detected in vitro and in vivo. Western blot, IF, and Co-IP were employed to detect the relationship between MIER3, HDAC1, HDAC2, and Snail. ChIP assay was performed to determine the binding of MIER3/HDAC1/HDAC2/Snail complex to the promoter of E-cadherin. In this study, we found that MIER3 was upregulated in breast cancer tissue and closely associated with poor prognosis of patients. MIER3 could promote the proliferation, migration, and epithelial-mesenchymal transition (EMT) of breast cancer cells. Further studies showed that MIER3 interacted with HDAC1/HDAC2 and Snail to form a repressive complex which could bind to E-cadherin promoter and was related to its deacetylation. Our study concluded that MIER3 was involved in forming a co-repressor complex with HDAC1/HDAC2/Snail to promote EMT by silencing E-cadherin.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

Are Methanol-Derived Foliar Methyl Acetate Emissions a Tracer of Acetate-Mediated Drought Survival in Plants?

Upregulation of acetate fermentation in plants has recently been described as an evolutionarily conserved drought survival strategy, with the amount of acetate produced directly correlating to survival. However, destructive measurements are required to evaluate acetate-linked drought responses, limiting the temporal and spatial scales that can be studied. Here, 13C-labeling studies with poplar (Populus trichocarpa) branches confirmed that methyl acetate is produced in plants from the acetate-linked acetylation of methanol. Methyl acetate emissions from detached leaves were strongly stimulated during desiccation, with total emissions decreasing with the leaf developmental stage. In addition, diurnal methyl acetate emissions from whole physiologically active poplar branches increased as a function of temperature, and light-dark transitions resulted in significant emission bursts lasting several hours. During experimental drought treatments of potted poplar saplings, light-dark methyl acetate emission bursts were eliminated while strong enhancements in methyl acetate emissions lasting > 6 days were observed with their initiation coinciding with the suppression of transpiration and photosynthesis. The results suggest that methyl acetate emissions represent a novel non-invasive tracer of acetate-mediated temperature and drought survival response in plants. The findings may have important implications for the future understanding of acetate-mediated drought responses to transcription, cellular metabolism, and hormone signaling, as well as its associated changes in carbon cycling and water use from individual plants to whole ecosystems.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Screening of alkali-assisted storage conditions to define the operational window of deacetylation within storage systems in the bioenergy supply chain

Overcoming biomass heterogeneity and associated recalcitrance to thermal, chemical, and enzymatic depolymerization is a necessary but challenging aspect of valorizing lignocellulosic biomass to fuels and chemicals. Here, this study explores how this recalcitrance can be reduced during the supply chain unit operation of storage, which is required for seasonally harvested agricultural residues to maintain constant throughput at a biorefinery. In this work, partial alkali pretreatment was performed just prior to corn stover entering storage, which had the benefit of providing a high pH environment entering storage such that soluble sugars were preserved and saponified acetyl groups in hemicellulose. This work investigated a range of viable conditions where saponification and preservation occur simultaneously by varying the moisture content (40% and 60%) and concentrations of sodium hydroxide (low and high) during aerobic and anaerobic storage. Anaerobic conditions preserved overall dry matter below 5% in the three scenarios evaluated, and the highest alkali loading solubilized up to 15% lignin, 18% xylan, and 50% of acetate, meanwhile doubling the extractable components. Scanning electron microscopy images highlighted potential physical impacts including cell-wall disruption near vascular bundles, pitting within parenchyma cells, and cell-wall distortion. Techno-economic assessment indicated that this storage approach and associated logistics system is economically competitive with a conventional approach using low-moisture bales.

09 BIOMASS FUELS↗

DOE OSTI2020

High–level β–carotene production from xylose by engineered Saccharomyces cerevisiae without overexpression of a truncated HMG1 (t HMG1 )

Beta-carotene is a natural pigment and health-promoting metabolite, and has been widely used in the nutraceutical, feed, and cosmetic industries. Here, we engineered a GRAS yeast Saccharomyces cerevisiae to produce beta-carotene from xylose, the sec- ond most abundant and inedible sugar component of lignocellulose biomass. Specifically, a beta-carotene biosynthetic pathway containing crtYB, crtI, and crtE from Xanthophyllomyces dendrorhous was introduced into a xylose-fermenting S. cerevisiae. Here, the resulting strain produced beta-carotene from xylose at a titer threefold higher than from glucose. Interestingly, overexpression of tHMG1, which has been reported as a critical genetic perturbation to enhance metabolic fluxes in the mevalonate pathway and beta-carotene production in yeast when glucose is used, did not further improve the production of beta-carotene from xylose. Through fermentation profiling, metabolites analysis, and transcriptional studies, we found the advantages of using xylose as a carbon source, instead of glucose, for beta-carotene production to be a more respiratory feature of xylose consumption, a larger cytosolic acetyl!CoA pool, and an upregulated expression level of rate-limiting genes in the beta-carotene!producing pathway, including ACS1 and HMG1. As a result, 772.8 mg/L of beta-carotene was ob- tained in a fed-batch bioreactor culture with xylose feeding. Considering the in- evitable large scale production of xylose when cellulosic biomass!based bioeconomy is implemented, our results suggest xylose utilization is a promising strategy for overproduction of carotenoids and other isoprenoids in engineered S. cerevisiae.

09 BIOMASS FUELS↗

DOE OSTI2021

Engineering Clostridium cellulovorans for highly selective n ‐butanol production from cellulose in consolidated bioprocessing

Abstract Cellulosic n ‐butanol from renewable lignocellulosic biomass has gained increased interest. Previously, we have engineered Clostridium cellulovorans , a cellulolytic acidogen, to overexpress the bifunctional butyraldehyde/butanol dehydrogenase gene adh E2 from C. acetobutylicum for n ‐butanol production from crystalline cellulose. However, butanol production by this engineered strain had a relatively low yield of approximately 0.22 g/g cellulose due to the coproduction of ethanol and acids. We hypothesized that strengthening the carbon flux through the central butyryl‐CoA biosynthesis pathway and increasing intracellular NADH availability in C. cellulovorans adh E2 would enhance n ‐butanol production. In this study, thiolase ( thl A CA ) from C. acetobutylicum and 3‐hydroxybutyryl‐CoA dehydrogenase ( hbd CT ) from C. tyrobutyricum were overexpressed in C. cellulovorans adh E2 to increase the flux from acetyl‐CoA to butyryl‐CoA. In addition, ferredoxin‐NAD(P) + oxidoreductase ( fnr ), which can regenerate the intracellular NAD(P)H and thus increase butanol biosynthesis, was also overexpressed. Metabolic flux analyses showed that mutants overexpressing these genes had a significantly increased carbon flux toward butyryl‐CoA, which resulted in increased production of butyrate and butanol. The addition of methyl viologen as an electron carrier in batch fermentation further directed more carbon flux towards n ‐butanol biosynthesis due to increased reducing equivalent or NADH. The engineered strain C. cellulovorans adh E2‐ fnr CA ‐ thl A CA ‐ hbd CT produced n ‐butanol from cellulose at a 50% higher yield (0.34 g/g), the highest ever obtained in batch fermentation by any known bacterial strain. The engineered C. cellulovorans is thus a promising host for n ‐butanol production from cellulosic biomass in consolidated bioprocessing.

Bao, Teng↗

DOE OSTI2021

Cloning and characterization of a panel of mitochondrial targeting sequences for compartmentalization engineering in Saccharomyces cerevisiae

Mitochondrion is generally considered as the most promising subcellular organelle for compartmentalization engineering. Much progress has been made in reconstituting whole metabolic pathways in the mitochondria of yeast to harness the precursor pools (i.e., pyruvate and acetyl-CoA), bypass competing pathways, and minimize transportation limitations. However, only a few mitochondrial targeting sequences (MTSs) have been characterized (i.e., MTS of COX4), limiting the application of compartmentalization engineering for multigene biosynthetic pathways in the mitochondria of yeast. In the present study, based on the mitochondrial proteome, a total of 20 MTSs were cloned and the efficiency of these MTSs in targeting heterologous proteins, including the Escherichia coli FabI and enhanced green fluorescence protein (EGFP) into the mitochondria was evaluated by growth complementation and confocal microscopy. After systematic characterization, six of the well!performed MTSs were chosen for the colocalization of complete biosynthetic pathways into the mitochondria. As proof of concept, the full alpha-santalene biosynthetic pathway consisting of 10 expression cassettes capable of converting acety coA to alpha-santalene was compartmentalized into the mitochondria, leading to a 3.7 fold improvement in the production of alpha-santalene. Furthermore, the newly characterized MTSs should contribute to the expanded metabolic engineering and synthetic biology toolbox for yeast mitochondrial compartmentalization engineering.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Activity-based protein profiling of chitin catabolism

Microbial catabolism of chitin, a highly abundant component of polymeric organic matter worldwide, is a fundamental cog in the carbon and nitrogen cycles that govern CO2 emissions, nutrient dynamics in aquatic systems, and soil vitality. Despite this critical biogeochemical function, we have a poor understanding of the synergy between various microbial chitinases and key accessory enzymes involved in chitin catabolism. We designed and synthesized activity-based probes (ABPs) for profiling active chitinolytic enzymes by modifying the chitin derivatives N-acetyl glucosamine and chitotriose. The ABPs were used to determine the active complement of chitinolytic enzymes produced over time by the soil bacterium Cellvibrio japonicus treated with various C substrates. This approach allowed us to demonstrate the utility of these ABPs in determining the intricate synergy between chitinases and other glycoside hydrolases that microbes employ to catabolize chitin. The strategy can be used to gain molecular level insights that can be used to better understand microbial roles in soil C and N cycling in the face of a changing climate.

Zegeye, Elias↗

DOE OSTI2020

Metribuzin resistance via enhanced metabolism in a multiple herbicide resistant Lolium rigidum population

Abstract BACKGROUND The photosystem II (PSII)‐inhibiting herbicides are important for Australian farmers to control Lolium rigidum Gaud. and other weed species in trazine tolerant (TT)‐canola fields. A L. rigidum population (R) collected from a TT‐canola field from Western Australia showed multiple resistance to PSII, acetyl‐coenzyme A carboxylase (ACCase) and acetolactate synthase (ALS) inhibitors. The mechanisms of multiple resistance in this R population were determined. RESULTS The R population showed a low‐level (about 3.0‐fold) resistance to the PSII‐inhibiting herbicides metribuzin and atrazine. Sequencing of the psbA gene revealed no differences between the R and susceptible (S) sequences. Furthermore, [ 14 C]‐metribuzin experiments found no significant difference in metribuzin foliar uptake and translocation between the R and S plants. However, [ 14 C]‐metribuzin metabolism in R plants was 2.3‐fold greater than in S plants. The cytochrome P450 monooxygenase inhibitor piperonyl butoxide (PBO) enhanced plant mortality response to metribuzin and atrazine in both R and S populations. In addition, multiple resistance to ALS and ACCase inhibitors are due to known resistance mutations in ALS and ACCase genes. CONCLUSION The results demonstrate that enhanced metribuzin metabolism likely involving cytochrome P450 monooxygenase contributes to metribuzin resistance in Lolium rigidum. This is the first report of metabolic resistance to the PSII‐inhibiting herbicide metribuzin in Australian Lolium rigidum . © 2020 Society of Chemical Industry

Ma, Hongju↗

DOE OSTI2025

Tailoring a 3D Covalent Organic Framework Toward Facile Functionalization

Three-dimensional covalent organic frameworks (3D COFs) are notably crystalline and stable, but their architectures and monomer structures make them difficult to functionalize. Here, a new strategy is presented to render COF-300 and other imine-linked frameworks amenable to facile functionalization in the last step of synthesis. By reducing the imine linkages to secondary amines and appending them with acetyl halide groups, the linkages are converted to electrophiles that can be readily reacted with nucleophilic guests. This route yields eight new COF-300 derivatives, bearing chloroacetyl, bromoacetyl, azide, cyano, amino, hydroxyl, methoxy, or thiomethyl groups appended to the inter-monomer linkages. The new materials are characterized through solid-state NMR, infrared spectroscopy, and powder X-ray diffraction, among other techniques, finding that the reported linkage transformations proceed to complete conversion while retaining the crystallinity of the materials. Microcrystal electron diffraction (microED) data are used to solve the evacuated structure of the amine-linked framework COF-300-AR for the first time, providing conclusive evidence of this framework's guest-induced phase change, along with the structure of the new framework COF-300-NH 2 . Finally, COF-300-NH 2 is shown to have significantly improved adsorption capacity for CO 2 and perfluoroalkyl substances (PFAS), highlighting the benefits of this synthetic strategy for the generation of customized adsorbents.

COF-300↗

DOE OSTI2021

Major involvement of two laccase genes in conidial pigment biosynthesis in Aspergillus oryzae

Wild-type strains of Aspergillus oryzae develop yellow, yellow-green, green, or brown conidia. Previous reports suggested that the conidiation initiates with the biosynthesis of a yellow pigment YWA1 from acetyl-CoA by a polyketide synthase encoded by wA (AO090102000545). This is followed by the conversion to other pigment by a laccase encoded by yA (AO090011000755). Based on orthologous pathways in other aspergilli, it is reasonable to hypothesize that in addition to yA, AO090102000546 encoding laccase and AO090005000332 encoding Ayg1-like hydrolase play a role in A. oryzae conidial pigment biosynthesis. However, the involvement of these two genes in conidial pigmentation remains unclear. In this study, we tested this hypothesis by assessing the conidial colors of both disruption and overexpression mutants to verify whether AO090102000546 and AO090005000332 were associated with the conidial pigmentation. Here, observation of single, double, and triple disruptants of these three genes suggested that conidial pigments were synthesized by two laccase genes, AO090011000755 and AO090102000546, whereas Ayg1-like hydrolase gene AO090005000332 was proven to have no obvious association with the synthesis. This was corroborated by observing the phenotype of each overexpression mutant. Interestingly, AO090005000332 overexpression mutant produced smoky yellow-green conidia, different from the wild-type strain. Thus, the AO090005000332-encoded protein is likely to maintain the enzymatic activity. However, the expression level was observed to be one-third of that of AO090102000546 and one-seventh of that of AO090011000755. Consequently, apparent lack of obvious contribution of AO090005000332 to conidial pigmentation could be attributed to its low expression level. Expression analysis indicated similar profiles in several wild-type strains.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

Unlocking plant-microbial interactions in deep Mollisols in the Midwestern US: Linking depth gradients in roots, microbial activity, and soil carbon in agroecosystems

Deep-rooted plants may build soil carbon (C) stocks, but most research has focused on shallow soils, leaving gaps in our understanding of how shifts in the balance between decomposition and C inputs drive soil C accumulation with depth. Thus, our objectives were to: (1) link depth gradients in root biomass with microbial activity and soil C stocks down to 1 m, and (2) examine the potential of simple C inputs to prime soil C across depths. To this end, we dug 5 quantitative soil pits in Argiudolls under mature perennial miscanthus plots in the SoyFACE Farm (Champaign-Urbana, IL). We added 13 C labeled glucose to our soils to determine the fate of simple C inputs with depth. We found that fine root biomass, total soil C, mineral-associated organic C (MAOC), particulate organic C (POC), and microbial activity (as measured by potential enzyme activity) declined with depth. POC declined more rapidly than MAOC, resulting in an increase in the ratio of MAOC-to-POC. Root biomass, enzyme activity (either acid phosphatase or n-acetyl-glucosaminadase) activity, and microbial respiration explained 74% and 38% of the variability in soil total C and MAOC, respectively, while POC was dependent on root biomass and microbial respiration (47%). Although the incorporation of simple 13 C inputs into MAOC was similar across depths, these inputs led to greater net MAOC losses in shallow soils than in deeper soils between 50 and 100 cm. The divergent impact of simple C inputs across depths may suggest that MAOC in shallow soils is more susceptible to priming losses, while C inputs into deep soils may instead be more persistent. Collectively, our results suggest that depth gradients in soil C stocks represents a balance between inputs, decomposition, and microbial necromass production and that increases in root C inputs by deep-rooted plants may have the potential to build stable MAOC.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

A quantitative assessment of (bacterio)chlorophyll assignments in the cryo-EM structure of the Chloracidobacterium thermophilum reaction center

Chlorophylls and bacteriochlorophylls are the primary pigments used by photosynthetic organisms for light-harvesting, energy transfer, and electron transfer. Many molecular structures of (bacterio)chlorophyll-containing protein complexes are available, some of which contain mixtures of different (bacterio)chlorophyll types. Differentiating these, which sometimes are structurally similar, is challenging but is required for leveraging structural data to gain functional insight. The reaction center complex from Chloroacidobacterium thermophilum has a hybrid (bacterio)chlorophyll antenna system containing both chlorophyll a and bacteriochlorophyll a molecules. The recent availability of its cryogenic electron microscopy (cryo-EM) structure provides an opportunity for a quantitative analysis of their identities, and chemical environments. Here, we describe a theoretical basis for differentiating chlorophyll a and bacteriochlorophyll a in a cryo-EM map, and apply the approach to the experimental cryo-EM maps of the (bacterio)chlorophyll sites of the chloroacidobacterial reaction center. The comparison reveals that at ~2.2-Å resolution, chlorophyll a and bacteriochlorophyll a are easily distinguishable, but the orientation of the bacteriochlorophyll a acetyl moiety is not; however, the latter can confidently be assigned by identifying a hydrogen bond donor from the protein environment. Furthermore, this study reveals the opportunities and challenges in assigning (bacterio)chlorophyll types in structural biology, the accuracy of which is vital for downstream investigations.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Targeted metabolomic analysis identifies increased serum levels of GABA and branched chain amino acids in canine diabetes

Introduction Dogs with naturally occurring diabetes mellitus represent a potential model for human type 1 diabetes, yet signifcant knowledge voids exist in terms of the pathogenic mechanisms underlying the canine disorder. Untargeted metabolomic studies from a limited number of diabetic dogs identifed similarities to humans with the disease. Objective To expand and validate earlier metabolomic studies, identify metabolites that difer consistently between diabetic and healthy dogs, and address whether certain metabolites might serve as disease biomarkers. Methods Untargeted metabolomic analysis via liquid chromatography-mass spectrometry was performed on serum from diabetic (n=15) and control (n=15) dogs. Results were combined with those of our previously published studies using identical methods (12 diabetic and 12 control dogs) to identify metabolites consistently diferent between the groups in all 54 dogs. Thirty-two candidate biomarkers were quantifed using targeted metabolomics. Biomarker concentrations were compared between the groups using multiple linear regression (corrected P<0.0051 considered signifcant). Results Untargeted metabolomics identifed multiple persistent diferences in serum metabolites in diabetic dogs compared with previous studies. Therefore, targeted metabolomics showed increases in gamma amino butyric acid, valine, leucine, isoleucine, citramalate, and 2-hydroxyisobutyric acid in diabetic versus control dogs while indoxyl sulfate, N-acetyl-L-aspartic acid, kynurenine, anthranilic acid, tyrosine, glutamine, and tauroursodeoxycholic acid were decreased. Conclusion Several of these fndings parallel metabolomic studies in both human diabetes and other animal models of this disease. Given recent studies on the role of GABA and branched chain amino acids in human diabetes, the increase in serum concentrations in canine diabetes warrants further study of these metabolites as potential biomarkers, and to identify similarity in mechanisms underlying this disease in humans and dogs.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

Deacetylation and Mechanical Refining Pathway for the Bioconversion of Sugarcane Bagasse

Advancing lignocellulose biorefining is imperative for the deployment of cellulosic (2G) biofuels. This work investigates the tailoring of the alkaline deacetylation and mechanical refining (DMR) pathway for the bioconversion of sugarcane bagasse. Experiments are conducted at laboratory and pilot scales, varying the pretreatment conditions (70–92 °C; 48–100 g NaOH /kg) and the mechanical refining technologies (PFI and disk refining). The pretreatments selectively solubilize acetyl groups (> 86%) and lignin (10–63%) while mostly preserving structural carbohydrates in the solid phase. Enzymatic hydrolysis generates hydrolysates of clean sugars (glucose and xylose), with sugar yields increasing up to 81% for glucose and 89% for xylose in response to delignification and mechanical refining. Biochemical methane potential assays reveal specific methane productions of up to 568 NmL CH₄ gVS⁻¹ for alkaline liquor monodigestion and 344 NmL CH₄ gVS⁻¹ for co-digestion with sugarcane vinasse from the conventional (1G) sugarcane ethanol, indicating a strong potential for bioenergy recovery from this process stream. Synergies are identified in integrating 1G ethanol, 2G DMR processing of bagasse, and anaerobic co-digestion of 1G vinasse and 2G DMR alkaline liquor. This technology enables sugarcane biorefineries to enhance the co-production of ethanol, methane, and concentrated streams of CO 2 .

09 BIOMASS FUELS↗

DOE OSTI2022

Genome sequence, phylogenetic analysis, and structure-based annotation reveal metabolic potential of Chlorella sp. SLA-04

Algae are a broad class of photosynthetic eukaryotes that are phylogenetically and physiologically diverse. Most of the phylogenetic diversity has been inferred from 18S rDNA sequencing since there are only a few complete genomes available in public databases. Here we use ultra-long-read Nanopore sequencing to determine a gapless, telomere-to-telomere complete genome sequence of Chlorella sp. SLA-04, previously described as Chlorella sorokiniana SLA-04. Chlorella sp. SLA-04 is a green alga that grows to high cell density in a wide variety of environments - high and neutral pH, high and low alkalinity, and high and low salinity. SLA-04's ability to grow in high pH and high alkalinity media without external CO 2 supply is favorable for large-scale algal biomass production. Phylogenetic analysis performed using ribosomal DNA and conserved protein sequences consistently reveal that Chlorella sp. SLA-04 forms a distinct lineage from other strains of Chlorella sorokiniana. We complement traditional genome annotation methods with high throughput structural predictions and demonstrate that this approach expands functional prediction of the SLA-04 proteome. Genomic analysis of the SLA-04 genome identifies the genes capable of utilizing TCA cycle intermediates to replenish cytosolic acetyl-CoA pools for lipid production. We also identify a complete metabolic pathway for sphingolipid anabolism that may allow SLA-04 to readily adapt to changing environmental conditions and facilitate robust cultivation in mass production systems. Altogether, this work clarifies the phylogeny of Chlorella sp. SLA-04 within Trebouxiophyceae and demonstrates how structural predictions can be used to improve annotation beyond sequencebased methods.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Engineering and evolution of Yarrowia lipolytica for producing lipids from lignocellulosic hydrolysates

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.

60 APPLIED LIFE SCIENCES↗