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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 649 records · Page 36
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Structural and functional analyses explain Pea KAI2 receptor diversity and reveal stereoselective catalysis during signal perception

KAI2 proteins are plant α/β hydrolase receptors which perceive smoke-derived butenolide signals and endogenous, yet unidentified KAI2-ligands (KLs). The number of functional KAI2 receptors varies among species and KAI2 gene duplication and sub-functionalization likely plays an adaptative role by altering specificity towards different KLs. Legumes represent one of the largest families of flowering plants and contain many agronomic crops. Prior to their diversification, KAI2 underwent duplication resulting in KAI2A and KAI2B. Here we demonstrate that Pisum sativum KAI2A and KAI2B are active receptors and enzymes with divergent ligand stereoselectivity. KAI2B has a higher affinity for and hydrolyses a broader range of substrates including strigolactone-like stereoisomers. We determine the crystal structures of PsKAI2B in apo and butenolide-bound states. The biochemical, structural, and mass spectra analyses of KAI2s reveal a transient intermediate on the catalytic serine and a stable adduct on the catalytic histidine, confirming its role as a bona fide enzyme. Our work uncovers the stereoselectivity of ligand perception and catalysis by diverged KAI2 receptors and proposes adaptive sensitivity to KAR/KL and strigolactones by KAI2B.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Rewiring the specificity of extracytoplasmic function sigma factors

Significance Bacterial phenotypes require the concerted expression of multiple genes, usually coordinated by a transcriptional regulator. Although the functions of many genes in sequenced bacterial genomes can be inferred, the regulatory networks that coordinate their expression are only known in a few model systems. Using a bioinformatic and experimental approach, we solve the DNA-specificity code of extracytoplasmic function sigma factors (ECF σs), a major class of bacterial regulators. We develop and use a high-stringency pipeline to predict the genes regulated by 67% of ECF σs in >10,000 species, providing a comprehensive look at the role of a broadly distributed family of gene regulatory proteins. This conceptual and computational framework is potentially applicable to other bacterial regulators.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Cross talk between RNA N6‐methyladenosine methyltransferase‐like 3 and miR‐186 regulates hepatoblastoma progression through Wnt/β‐catenin signalling pathway

Abstract Objectives N6‐methyladenosine (m6A) is a ubiquitous epigenetic RNA modification that plays a pivotal role in tumour development and metastasis. In this study, we aimed to investigate the expression profiling, clinical significance, biological function and the regulation of m6A‐related genes in hepatoblastoma (HB). Materials and Methods The mRNA and protein expression levels of m6A‐related genes were analysed using Gene Expression Omnibus (GEO) and tissue microarray (TMA) cohort. Kaplan‐Meier analysis was performed to evaluate the prognostic value of m6A‐related genes in HB. Knockdown of m6A‐related genes was conducted to analyse its function on cell proliferation, migration and invasion. Furthermore, bioinformatics analysis and experimental verification were used to explore the potential molecular mechanism and signalling pathway. Results We found that most m6A‐related genes were significantly upregulated in HB tumour tissues. High levels of methyltransferase‐like 3 (METTL3, P = .013), YTHDF2 ( P = .037) and FTO ( P = .032) indicated poor clinical outcomes, and the upregulation of METTL3 was an independent prognostic factor in HB patients. Functional assays showed that knockdown of METTL3 could dramatically suppress the proliferation, migration and invasion of HB cells. In addition, METTL3 was identified to be a direct target of microRNA‐186 (miR‐186). Consistently, miR‐186 was low expressed in HB tumour tissues. Moreover, overexpression of miR‐186 significantly inhibited cell aggressive phenotype both in vitro and in vivo, while the inhibitory effect could be reversed by METTL3 overexpression. Mechanism study indicated that miR‐186/METTL3 axis contributed to the progression of HB via the Wnt/β‐catenin signalling pathway. Conclusions M6A‐related genes were frequently dysregulated in HB. miR‐186/METTL3/Wnt/β‐catenin axis might serve as novel therapeutic targets and prognostic biomarkers in HB.

Cui, Xichun↗
DOE OSTI2021

Predictive Models of Genetic Redundancy in Arabidopsis thaliana

Abstract Genetic redundancy refers to a situation where an individual with a loss-of-function mutation in one gene (single mutant) does not show an apparent phenotype until one or more paralogs are also knocked out (double/higher-order mutant). Previous studies have identified some characteristics common among redundant gene pairs, but a predictive model of genetic redundancy incorporating a wide variety of features derived from accumulating omics and mutant phenotype data is yet to be established. In addition, the relative importance of these features for genetic redundancy remains largely unclear. Here, we establish machine learning models for predicting whether a gene pair is likely redundant or not in the model plant Arabidopsis thaliana based on six feature categories: functional annotations, evolutionary conservation including duplication patterns and mechanisms, epigenetic marks, protein properties including posttranslational modifications, gene expression, and gene network properties. The definition of redundancy, data transformations, feature subsets, and machine learning algorithms used significantly affected model performance based on holdout, testing phenotype data. Among the most important features in predicting gene pairs as redundant were having a paralog(s) from recent duplication events, annotation as a transcription factor, downregulation during stress conditions, and having similar expression patterns under stress conditions. We also explored the potential reasons underlying mispredictions and limitations of our studies. This genetic redundancy model sheds light on characteristics that may contribute to long-term maintenance of paralogs, and will ultimately allow for more targeted generation of functionally informative double mutants, advancing functional genomic studies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Functional characterization of 1-aminocyclopropane-1-carboxylic acid oxidase gene in Arabidopsis thaliana and its potential in providing flood tolerance

Ethylene is a phytohormone that has gained importance through its role in stress tolerance and fruit ripening. In our study we evaluated the functional potential of the enzyme involved in ethylene biosynthesis of plants called ACC (aminocyclopropane-1-carboxylic acid) oxidase which converts precursor ACC to ethylene. Studies on ethylene have proven that it is effective in improving the flood tolerance in plants. Thus our goal was to understand the potential of ACC oxidase gene overexpression in providing flood tolerance in transgenic plants. ACC oxidase gene was PCR amplified and inserted into the pBINmgfp5-er vector, under the control of a constitutive Cauliflower Mosaic Virus promoter. GV101 strain of Agrobacterium tumefaciens containing recombinant pBINmgfp5-er vector (referred herein as pBIN-ACC) was used for plant transformation by the ‘floral dip’ method. The transformants were identified through kanamycin selection and grown till T3 (third transgenic) generation. The flood tolerance was assessed by placing both control and transgenic plants on deep plastic trays filled with tap water that covered the soil surface. Our result shows that wild-type Arabidopsis could not survive more than 20 days under flooding while the transgenic lines survived 35 days, suggesting development of flood tolerance with overexpression of ACC oxidase. Further molecular studies should be done to elucidate the role and pathways of ACC oxidase and other phytohormones involved in the development of flood adaptation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

The function of the PduJ microcompartment shell protein is determined by the genomic position of its encoding gene

Bacterial microcompartments (MCPs) are complex organelles that consist of metabolic enzymes encapsulated within a protein shell. Here, we investigate the function of the PduJ MCP shell protein. PduJ is 80% identical in amino acid sequence to PduA and both are major shell proteins of the 1,2-propanediol (1,2-PD) utilization (Pdu) MCP of Salmonella . Prior studies showed that PduA mediates the transport of 1,2-PD (the substrate) into the Pdu MCP. Surprisingly, however, results presented here establish that PduJ has no role 1,2-PD transport. The crystal structure revealed that PduJ was nearly identical to that of PduA and, hence, offered no explanation for their differential functions. Interestingly, however, when a pduJ gene was placed at the pduA chromosomal locus, the PduJ protein acquired a new function, the ability to mediate 1,2-PD transport into the Pdu MCP. To our knowledge, these are the first studies to show that that gene location can determine the function of a MCP shell protein. We propose that gene location dictates protein-protein interactions essential to the function of the MCP shell.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Functional Redundancy in Soil Microbial Community Based on Metagenomics Across the Globe

Understanding the contribution of soil microbial communities to ecosystem processes is critical for predicting terrestrial ecosystem feedbacks under changing climate. Our current understanding lacks a consistent strategy to formulate the linkage between microbial systems and ecosystem processes due to the presumption of functional redundancy in soil microbes. Here we present a global soil microbial metagenomic analysis to generalize patterns of microbial taxonomic compositions and functional potentials across climate and geochemical gradient. Our analyses show that soil microbial taxonomic composition varies widely in response to climate and soil physicochemical gradients, while microbial functional attributes based on metagenomic gene abundances are redundant. Among 17 climate zones, microbial taxonomic compositions were more distinct than functional potentials, as climate and edaphic properties showed more significant influence on microbial taxonomic compositions than on functional potentials. Microbial taxonomies formed a larger and more complex co-occurrence network with more module structures than functional potentials. Functional network was strongly inter-connected among different categories, whereas taxonomic network was more positively interactive in the same taxonomic groups. This study provides strong evidence to support the hypothesis of functional redundancy in soil microbes, as microbial taxonomic compositions vary to a larger extent than functional potentials based on metagenomic gene abundances in terrestrial ecosystems across the globe.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

RNA interference in vivo in Schistosoma japonicum: Establishing and optimization of RNAi mediated suppression of gene expression by long dsRNA in the intra-mammalian life stages of worms

Schistosomes are parasitic platyhelminths that threaten over 600 million people globally. In recent years, RNA interference (RNAi) has been widely used as a molecular tool in research into the genomic function of parasites. We aim to develop effective protocols for application of RNAi technology in the intra-mammalian life stages of Schistosoma japonicum. In this work, the expression of the parasite gene encoding cathepsin B1 (SjCB1) was targeted by exposing the worms to 10 μg of long dsRNA dissolved in 0.1 ml of 0.7% NaCl injected into the tail vein of infected mice. This method was effective and specific for eliciting SjCB1 gene suppression in both male and female adult worms in vivo (>79.4% in male and >91.5% in female knockdown relative to control). In 60 cercaria infected mice, RNAi suppression of gene expression was best achieved by using 10 μg of target dsRNA for at least 4 days. The recommended procedure for interference producing long-term suppression was an injection of dsRNA on the first day of infection with booster injections administered every 4 days for up to 26 days. Long-term suppression of three published functional genes (peroxiredoxin-1, mago nashi, insulin receptor) in S. japonicum provided more information about the role of the expression of these genes in producing particular phenotypes. The protocols described here may be more convenient, economical and applicable, than currently available technology and have contributed to the observation of more phenotypes during worm development from schistosomula to adult. These approaches may promote and facilitate further studies into functional schistosome genomics.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

Transcriptome divergence during leaf development in two contrasting switchgrass (Panicum virgatum L.) cultivars

The genetics and responses to biotic stressors of tetraploid switchgrass (Panicum virgatum L.) lowland cultivar ‘Kanlow’ and upland cultivar Summer are distinct and can be exploited for trait improvement. In general, there is a paucity of data on the basal differences in transcription across tissue developmental times for switchgrass cultivars. Here, the changes in basal and temporal expression of genes related to leaf functions were evaluated for greenhouse grown ‘Kanlow’, and ‘Summer’ plants. Three biological replicates of the 4 th leaf pooled from 15 plants per replicate were harvested at regular intervals beginning from leaf emergence through senescence. Increases and decreases in leaf chlorophyll and N content were similar for both cultivars. Likewise, multidimensional scaling (MDS) analysis indicated both cultivar-independent and cultivar-specific gene expression. Cultivar-independent genes and gene-networks included those associated with leaf function, such as growth/senescence, carbon/nitrogen assimilation, photosynthesis, chlorophyll biosynthesis, and chlorophyll degradation. However, many genes encoding nucleotide-binding leucine rich repeat (NB-LRRs) proteins and wall-bound kinases associated with detecting and responding to environmental signals were differentially expressed. Several of these belonged to unique cultivar-specific gene co-expression networks. Analysis of genomic resequencing data provided several examples of NB-LRRs genes that were not expressed and/or apparently absent in the genomes of Summer plants. It is plausible that cultivar (ecotype)-specific genes and gene-networks could be one of the drivers for the documented differences in responses to leaf-borne pathogens between these two cultivars. Incorporating broad resistance to plant pathogens in elite switchgrass germplasm could improve sustainability of biomass production under low-input conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Genetic correlation network prediction of forest soil microbial functional organization

Soil ecological functions are largely determined by the activities of soil microorganisms, which, in turn, are regulated by relevant interactions between genes and their corresponding pathways. Therefore, the genetic network can theoretically elucidate the functional organization that supports complex microbial community functions, although this has not been previously attempted. We generated a genetic correlation network based on 5421 genes derived from metagenomes of forest soils, identifying 7191 positive and 123 negative correlation relationships. This network consisted of 27 clusters enriched with sets of genes within speci fi c functions, represented with corresponding cluster hubs. The clusters revealed a hierarchical architecture, re fl ecting the functional organization in the soil metagenomes. Positive correlations mapped functional associations, whereas negative correlations often mapped regulatory processes. The potential functions of uncharacterized genes were predicted based on the functions of located clusters. The global genetic correlation network highlights the functional organization in soil metagenomes and provides a resource for predicting gene functions. We anticipate that the genetic correlation network may be exploited to comprehensively decipher soil microbial commu

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

Kinetic modeling of microbial growth, enzyme activity, and gene deletions: An integrated model of β-glucosidase function in Cellvibrio japonicus

Understanding the complex growth and metabolic dynamics in microorganisms requires advanced kinetic models containing both metabolic reactions and enzymatic regulation to predict phenotypic behaviors under different conditions and perturbations. Most current kinetic models lack gene expression dynamics and are separately calibrated to distinct media, which consequently makes them unable to account for genetic perturbations or multiple substrates. This challenge limits our ability to gain a comprehensive understanding of microbial processes towards advanced metabolic optimizations that are desired for many biotechnology applications. Here, we present an integrated computational and experimental approach for the development and optimization of mechanistic kinetic models for microbial growth and metabolic and enzymatic dynamics. Our approach integrates growth dynamics, gene expression, protein secretion, and gene-deletion phenotypes. We applied this methodology to build a dynamic model of the growth kinetics in batch culture of the bacterium Cellvibrio japonicus grown using either cellobiose or glucose media. The model parameters were inferred from an experimental data set using an evolutionary computation method. The resulting model was able to explain the growth dynamics of C. japonicus using either cellobiose or glucose media and was also able to accurately predict the metabolite concentrations in the wild-type strain as well as in β-glucosidase gene deletion mutant strains. We validated the model by correctly predicting the non-diauxic growth and metabolite consumptions of the wild-type strain in a mixed medium containing both cellobiose and glucose, made further predictions of mutant strains growth phenotypes when using cellobiose and glucose media, and demonstrated the utility of the model for designing industrially-useful strains. Importantly, the model is able to explain the role of the different β-glucosidases and their behavior under genetic perturbations. Here, this integrated approach can be extended to other metabolic pathways to produce mechanistic models for the comprehensive understanding of enzymatic functions in multiple substrates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Exploring drought-responsive crucial genes in Sorghum

Drought severely affects global food production. Sorghum is a typical drought-resistant model crop. Based on RNA-seq data for Sorghum with multiple time points and the gray correlation coefficient, this paper firstly selects candidate genes via mean variance test and constructs weighted gene differential co-expression networks (WGDCNs); then, based on guilt-by-rewiring principle, the WGDCNs and the hidden Markov random field model, drought-responsive crucial genes are identified for five developmental stages respectively. Enrichment and sequence alignment analysis reveal that the screened genes may play critical functional roles in drought responsiveness. A multilayer differential co-expression network for the screened genes reveals that Sorghum is very sensitive to pre-flowering drought. Furthermore, a crucial gene regulatory module is established, which regulates drought responsiveness via plant hormone signal transduction, MAPK cascades, and transcriptional regulations. The proposed method can well excavate crucial genes through RNA-seq data, which have implications in breeding of new varieties with improved drought tolerance.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

Comparative Transcriptomics Analyses across Species, Organs, and Developmental Stages Reveal Functionally Constrained lncRNAs

The functionality of long noncoding RNAs (lncRNAs) is disputed. In general, lncRNAs are under weak selective pressures, suggesting that the majority of lncRNAs may be nonfunctional. However, although some surveys showed negligible phenotypic effects upon lncRNA perturbation, key biological roles were demonstrated for individual lncRNAs. Most lncRNAs with proven functions were implicated in gene expression regulation, in pathways related to cellular pluripotency, differentiation, and organ morphogenesis, suggesting that functional lncRNAs may be more abundant in embryonic development, rather than in adult organs. To test this hypothesis, we perform a multidimensional comparative transcriptomics analysis, across five developmental time points (two embryonic stages, newborn, adult, and aged individuals), four organs (brain, kidney, liver, and testes), and three species (mouse, rat, and chicken). We find that, overwhelmingly, lncRNAs are preferentially expressed in adult and aged testes, consistent with the presence of permissive transcription during spermatogenesis. LncRNAs are often differentially expressed among developmental stages and are less abundant in embryos and newborns compared with adult individuals, in agreement with a requirement for tighter expression control and less tolerance for noisy transcription early in development. For differentially expressed lncRNAs, we find that the patterns of expression variation among developmental stages are generally conserved between mouse and rat. Moreover, lncRNAs expressed above noise levels in somatic organs and during development show higher evolutionary conservation, in particular, at their promoter regions. Thus, we show that functionally constrained lncRNA loci are enriched in developing organs, and we suggest that many of these loci may function in an RNA-independent manner.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Embryotoxic and pharmacologic potency ranking of six azoles in the rat whole embryo culture by morphological and transcriptomic analysis

Differential gene expression analysis in the rat whole embryo culture (WEC) assay provides mechanistic insight into the embryotoxicity of test compounds. In our study, we hypothesized that comparative analysis of the transcriptomes of rat embryos exposed to six azoles (flusilazole, triadimefon, ketoconazole, miconazole, difenoconazole and prothioconazole) could lead to a better mechanism-based understanding of their embryotoxicity and pharmacological action. For evaluating embryotoxicity, we applied the total morphological scoring system (TMS) in embryos exposed for 48 h. The compounds tested showed embryotoxicity in a dose-response fashion. Functional analysis of differential gene expression after 4 h exposure at the ID{sub 10} (effective dose for 10% decreased TMS), revealed the sterol biosynthesis pathway and embryonic development genes, dominated by genes in the retinoic acid (RA) pathway, albeit in a differential way. Flusilazole, ketoconazole and triadimefon were the most potent compounds affecting the RA pathway, while in terms of regulation of sterol function, difenoconazole and ketoconazole showed the most pronounced effects. Dose-dependent analysis of the effects of flusilazole revealed that the RA pathway related genes were already differentially expressed at low dose levels while the sterol pathway showed strong regulation at higher embryotoxic doses, suggesting that this pathway is less predictive for the observed embryotoxicity. A similar analysis at the 24-hour time point indicated an additional time-dependent difference in the aforementioned pathways regulated by flusilazole. In summary, the rat WEC assay in combination with transcriptomics could add a mechanistic insight into the embryotoxic potency ranking and pharmacological mode of action of the tested compounds. - Highlights: • Embryonic exposure to azoles revealed concentration-dependent malformations. • Transcriptomics could enhance the mechanistic knowledge of embryotoxicants. • Retinoic acid gene set identifies early embryotoxic responses to azoles. • Toxic versus pharmacologic potency determines functional efficacy.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

multiPhATE: bioinformatics pipeline for functional annotation of phage isolates

To address the need for improved phage annotation tools that scale, we created an automated throughput annotation pipeline: multiple-genome Phage Annotation Toolkit and Evaluator (multiPhATE). multiPhATE is a throughput pipeline driver that invokes an annotation pipeline (PhATE) across a user-specified set of phage genomes. This tool incorporates a de novo phage gene calling algorithm and assigns putative functions to gene calls using protein-, virus- and phage-centric databases. multiPhATE’s modular construction allows the user to implement all or any portion of the analyses by acquiring local instances of the desired databases and specifying the desired analyses in a configuration file. We demonstrate multiPhATE by annotating two newly sequenced Yersinia pestis phage genomes. Within multiPhATE, the PhATE processing pipeline can be readily implemented across multiple processors, making it adaptable for throughput sequencing projects. Software documentation assists the user in configuring the system.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Structural and functional analyses reveal the contributions of the C- and N-lobes of Argonaute protein to selectivity of RNA target cleavage

Some gene transcripts have cellular functions as regulatory noncoding RNAs. For example, ~23-nucleotide (nt)–long siRNAs are loaded into Argonaute proteins. The resultant ribonucleoprotein assembly, the RNA-induced silencing complex (RISC), cleaves RNAs that are extensively base-paired with the loaded siRNA. To date, base complementarity is recognized as the major determinant of specific target cleavage (or slicing), but little is known about how Argonaute inspects base pairing before cleavage. A hallmark of Argonaute proteins is their bilobal structure, but despite the significance of this structure for curtailing slicing activity against mismatched targets, the molecular mechanism remains elusive. Here, our structural and functional studies of a bilobed yeast Argonaute protein and its isolated catalytic C-terminal lobe (C-lobe) revealed that the C-lobe alone retains almost all properties of bilobed Argonaute: siRNA-duplex loading, passenger cleavage/ejection, and siRNAdependent RNA cleavage. A 2.1 Å–resolution crystal structure revealed that the catalytic C-lobe mirrors the bilobed Argonaute in terms of guide-RNA recognition and that all requirements for transitioning to the catalytically active conformation reside in the C-lobe. Nevertheless, we found that in the absence of the N-terminal lobe (N-lobe), target RNAs are scanned for complementarity only at positions 5–14 on a 23-nt guide RNA before endonucleolytic cleavage, thereby allowing for some off-target cleavage. Of note, acquisition of an N-lobe expanded the range of the guide RNA strand used for inspecting target complementarity to positions 2–23. To summarize, these findings offer clues to the evolution of the bilobal structure of catalytically active Argonaute proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Multi-Omics integration can be used to rescue metabolic information for some of the dark region of the Pseudomonas putida proteome

In every omics experiment, genes or their products are identified for which even state of the art tools are unable to assign a function. In the biotechnology chassis organism Pseudomonas putida, these proteins of unknown function make up 14% of the proteome. This missing information can bias analyses since these proteins can carry out functions which impact the engineering of organisms. As a consequence of predicting protein function across all organisms, function prediction tools generally fail to use all of the types of data available for any specific organism, including protein and transcript expression information. Additionally, the release of Alphafold predictions for all Uniprot proteins provides a novel opportunity for leveraging structural information. We constructed a bespoke machine learning model to predict the function of recalcitrant proteins of unknown function in Pseudomonas putida based on these sources of data, which annotated 1079 terms to 213 proteins. Among the predicted functions supplied by the model, we found evidence for a significant overrepresentation of nitrogen metabolism and macromolecule processing proteins. These findings were corroborated by manual analyses of selected proteins which identified, among others, a functionally unannotated operon that likely encodes a branch of the shikimate pathway.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Functional overexpression and characterization of lipogenesis-related genes in the oleaginous yeast Yarrowia lipolytica

Single cell oil (SCO) is an attractive energy source due to scalability, utilization of low-cost renewable feedstocks, and type of product(s) made. Engineering strains capable of producing high lipid titers and yields is crucial to the economic viability of these processes. However, lipid synthesis in cells is a complex phenomenon subject to multiple layers of regulation, making gene target identification a challenging task. In this study, we aimed to identify genes in the oleaginous yeast Yarrowia lipolytica whose overexpression enhances lipid production by this organism. Here, to this end, we examined the effect of the overexpression of a set of 44 native genes on lipid production in Y. lipolytica, including those involved in glycerolipid synthesis, fatty acid synthesis, central carbon metabolism, NADPH generation, regulation, and metabolite transport and characterized each resulting strain’s ability to produce lipids growing on both glucose and acetate as a sole carbon source. Our results suggest that a diverse subset of genes was effective at individually influencing lipid production in Y. lipolytica, sometimes in a substrate-dependent manner. The most productive strain on glucose overexpressed the diacylglycerol acyltransferase DGA2 gene, increasing lipid titer, cellular content, and yield by 236, 165, and 246 %, respectively, over our control strain. On acetate, our most productive strain overexpressed the acylglycerol-phosphate acyltransferase SLC1 gene, with a lipid titer, cellular content, and yield increase of 99, 91, and 151 %, respectively, over the control strain. Aside from genes encoding enzymes that directly catalyze the reactions of lipid synthesis, other ways by which lipogenesis was increased in these cells include overexpressing the glycerol-3-phosphate dehydrogenase (GPD1) gene to increase production of glycerol head groups and overexpressing the 6-phosphogluconolactonase (SOL3) gene from the oxidative pentose phosphate pathway to increase NADPH availability for fatty acid synthesis. Taken together, our study demonstrates that the overall kinetics of microbial lipid synthesis is sensitive to a wide variety of factors. Fully optimizing a strain for single cell oil processes could involve manipulating and balancing many of these factors, and, due to mechanistic differences by which each gene product investigated here impacts lipid synthesis, there is a high likelihood that many of these genes will work synergistically to further increase lipid production when simultaneously overexpressed.

59 BASIC BIOLOGICAL SCIENCES↗