Biology subjects

Pearson, A. N.

Publications and source records attributed to Pearson, A. N..

14 recordsLinked to original sources

bioRxiv2025

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

Dicarboxylic acids are key components of many polymers and plastics, making them a target for both engineered microbial degradation and sustainable bioproduction. In this study, we generated a comprehensive dataset of functional evidence for the genetic basis of dicarboxylic and fatty acid metabolism using randomly-barcoded transposon sequencing (RB-TnSeq). We identified four {beta}-proteobacteria that displayed robust growth with dicarboxylic acid sole carbon sources and cultured their mutant libraries with dicarboxylic and fatty acids with carbon chain lengths from C3 to C12. The resulting fitness data suggested that dicarboxylic and fatty acid metabolisms are largely distinct, and different sets of {beta}-oxidation genes are required for catabolizing dicarboxylic versus fatty acids of the same carbon chain lengths. Additionally, we identified transcriptional regulators and transporters with strong fitness phenotypes related to dicarboxylic acid utilization. In Ralstonia sp. UNC404CL21Col (R. CL21), we deleted two transcriptional repressors to improve its utilization of short chain dicarboxylic acids. We exploited the diacid-utilizing catabolism of R. CL21 to upcycle a mock mixture of the dicarboxylic acids produced when polyethylene is oxidized. After introducing a heterologous indigoidine production pathway, this engineered Rastonia produced 0.56 {+/-} 0.02 g/L indigoidine from a mixture of dicarboxylic acids as a carbon source, demonstrating the potential of R. CL21 to upcycle plastics waste to products derived from tricarboxylic acid (TCA) cycle intermediates. ImportanceUpcycling the carbon in plastic wastes to value-added products is a promising approach to address the plastics waste and climate crises, and dicarboxylic acid metabolism is an important facet of several approaches. Improving our understanding of the genetic basis of this metabolism has the potential to uncover new enzymes and genetic parts for engineered pathways involving dicarboxylic acids. Our dataset is the most comprehensive interrogation of dicarboxylic acid catabolism to date, and this work will be of utility to researchers interested in both plastics bioproduction and upcycling applications.

microbiology↗

bioRxiv2025

Identification and characterization of shifted GU wobble pairs resulting from alternative protonation of RNA

RNA can serve as an enzyme, small molecule sensor, and vaccine, and it may have been a conduit for the origin of life. Despite these profound functions, RNA is thought to have quite limited molecular diversity. A pressing question, therefore, is whether RNA can adopt novel molecular states that enhance its function. Covalent modifications of RNA have been demonstrated to augment biological function, but much less is known about non-covalent alterations such as novel protonated or tautomeric forms. Conventionally, a G*U wobble has the U shifted into the major groove. We used a cheminformatic approach to identify four structural families of shifted G*U wobbles in which the G instead resides in the major groove of RNA, which requires alternative tautomeric states of either base, or an anionic state of the U. We provide experimental support for these shifted G*U wobbles via the potent, and unconventional, in vivo reactivity of the U with dimethylsulfate (DMS) in three organisms. These shifted wobbles may play important functional roles and could serve as drug targets. Our cheminformatics approach is general and can be applied to identify alternative protonation states in other RNA motifs, as well as in DNA and proteins. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/630957v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1921d65org.highwire.dtl.DTLVardef@1c8124borg.highwire.dtl.DTLVardef@28fbddorg.highwire.dtl.DTLVardef@af8d20_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

SLC4A11 mediates ammonia import and promotes cancer stemness in hepatocellular carcinoma

End stage liver disease is marked by portal hypertension, systemic elevations in ammonia, and development of hepatocellular carcinoma (HCC). While these clinical consequences of cirrhosis are well described, it remains poorly understood whether hepatic insufficiency and the accompanying elevations in ammonia contribute to HCC carcinogenesis. Using preclinical models, we discovered that ammonia entered the cell through the transporter SLC4A11 and served as a nitrogen source for amino acid and nucleotide biosynthesis. Elevated ammonia promoted cancer stem cell properties in vitro and tumor initiation in vivo. Enhancing ammonia clearance reduced HCC stemness and tumor growth. In patients, elevations in serum ammonia were associated with an increased incidence of HCC. Taken together, this study forms the foundation for clinical investigations using ammonia lowering agents as potential therapies to mitigate HCC incidence and aggressiveness.

cancer biology↗

bioRxiv2023

Development of Corynebacterium glutamicum as a monoterpene production platform

Monoterpenes are commonly known for their role in the flavors and fragrances industry and are also gaining attention for other uses like insect repellant and as potential renewable fuels for aviation. Corynebacterium glutamicum, a Generally Recognized as Safe microbe, has been a choice organism in industry for the annual million ton-scale bioproduction of amino acids for more than 50 years; however, efforts to produce monoterpenes in C. glutamicum have remained relatively limited. In this study, we report a further expansion of the C. glutamicum biosynthetic repertoire through the development and optimization of a mevalonate-based monoterpene platform. In the course of our plasmid design iterations, we increased flux through the mevalonate-based bypass pathway, measuring isoprenol production as a proxy for monoterpene precursor abundance and demonstrating the highest reported titers in C. glutamicum to date at nearly 1500 mg/L. Our designs also evaluated the effects of backbone, promoter, and GPP synthase homolog origin on monoterpene product titers. Monoterpene production was further improved by disrupting competing pathways for isoprenoid precursor supply and by implementing a biphasic production system to prevent volatilization. With this platform, we achieved 321.1 mg/L of geranoids, 723.6 mg/L of 1,8-cineole, and 227.8 mg/L of linalool. Furthermore, we determined that C. glutamicum first oxidizes geraniol through an aldehyde intermediate before it is asymmetrically reduced to citronellol. Additionally, we demonstrate that the aldehyde reductase, AdhC, possesses additional substrate promiscuity for acyclic monoterpene aldehydes. HighlightsO_LIDesign of a mevalonate-based monoterpene production platform in C. glutamicum C_LIO_LIHighest production titers of geranoids, eucalyptol, and linalool reported in C. glutamicum to date C_LIO_LIIdentification of citronellal as an intermediate in the reduction of geraniol to citronellol by C. glutamicum C_LI

synthetic biology↗

bioRxiv2023

Genetically refactored Agrobacterium-mediated transformation

Members of Agrobacterium are costly plant pathogens while also essential tools for plant transformation. Though Agrobacterium-mediated transformation (AMT) has been heavily studied, its polygenic nature and its complex transcriptional regulation make identifying the genetic basis of transformational efficiency difficult through traditional genetic and bioinformatic approaches. Here we use a bottom-up synthetic approach to systematically refactor the tumor-inducing plasmid, wherein the majority of AMT machine components are encoded, into a minimal set of genes capable of plant and fungal transformation that is both controllable and orthogonal to its environment. We demonstrate that engineered vectors can be transferred to new heterologous bacteria, enabling them to transform plants. Our reductionist approach demonstrates how bottom-up engineering can be used to dissect and elucidate the genetic underpinnings of complex biological traits, and may lead to the development of strains of bacteria more capable of transforming recalcitrant plant species of societal importance.

microbiology↗

bioRxiv2023

Characterization and diversification of AraC/XylS family regulators guided by transposon sequencing

In this study, we explored the development of engineered inducible systems. Publicly available data from previous transposon sequencing assays were used to identify regulators of metabolism in Pseudomonas putida KT2440. For the AraC-family regulators (AFRs) represented in this data, we posited AFR/promoter/inducer groupings. Eleven promoters were characterized for a response to their proposed inducers in P. putida, and the resultant data were used to create and test nine two-plasmid sensor systems in E. coli. Several of these were further developed into a palette of single-plasmid inducible systems. From these experiments, we observed an unreported inducer response from a previously characterized AFR, demonstrated that the addition of a P. putida transporter improved the sensor dynamics of an AFR in E. coli, and identified an uncharacterized AFR with a novel potential inducer specificity. Finally, targeted mutations in an AFR, informed by structural predictions, enabled further diversification of these inducible plasmids. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/550116v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@fb1ee2org.highwire.dtl.DTLVardef@a88f1dorg.highwire.dtl.DTLVardef@4bbd5borg.highwire.dtl.DTLVardef@7cdbfc_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Foldy: a web application for interactive protein structure analysis

Foldy is a cloud-based application that allows non-expert scientists to easily access and utilize advanced AI-based structural biology tools, including AlphaFold and DiffDock. Built on Kubernetes, it can be deployed by universities, departments, and labs without requiring hardware resources, but can also be configured to utilize available computers. Foldy enables scientists to predict the structure of proteins and complexes up to 3000 amino acids, visualize Pfam annotations, and dock ligands with AutoDock Vina and DiffDock. Our manuscript describes the user interface and deployment considerations of Foldy, as well as some of our applications. By democratizing access to sophisticated AI-based tools, Foldy can facilitate life science research and promote the wider adoption of structural bioinformatics tools. Our work demonstrates that even the most advanced tools can be made accessible to a broad audience through user-friendly platforms like Foldy, and we believe it will be a valuable resource for researchers across scientific disciplines. The public structures available on the Lawrence Berkeley Labs Foldy deployment can be viewed at https://foldy.lbl.gov. Author SummaryFoldy is a cloud-based application that enables scientists to use AI-based structural biology tools such as AlphaFold and DiffDock without software expertise. Built on Kubernetes, it can be set up by universities, departments, and labs with no need for hardware resources. Foldy can predict the structure of proteins and complexes up to 3000 amino acids, visualize Pfam annotations, and dock ligands with AutoDock Vina and DiffDock. Our public structures can be viewed at https://foldy.lbl.gov. Our manuscript highlights the user interface, deployment considerations, and product applications of Foldy. Its an accessible solution for researchers who are not software experts and can handle the traffic of thousands of users and hundreds of thousands of protein structures and docked ligands. This makes advanced AI-based tools more widely available, paving the way for accelerating life science research. By developing an easy-to-use platform, our work demonstrates that even the most sophisticated AI-based tools can be made accessible to a wide audience. Foldy enables more scientists to draw from the rapidly growing field of structural biology, making it a valuable tool for researchers across scientific disciplines. We look forward to its adoption by the scientific community.

bioinformatics↗

bioRxiv2023

The pGinger family of expression plasmids

The pGinger suite of expression plasmids comprises 43 plasmids that will enable precise constitutive and inducible gene expression in a wide range of gram-negative bacterial species. Constitutive vectors are composed of 16 synthetic constitutive promoters upstream of RFP, with a broad host range BBR1 origin and a kanamycin resistance marker. The family also has seven inducible systems (Jungle Express, Psal/NahR, Pm/XylS, Prha/RhaS, LacO1/LacI, LacUV5/LacI, and Ptet/TetR) controlling RFP expression on BBR1/kanamycin plasmid backbones. For four of these inducible systems (Jungle Express, Psal/NahR, LacO1/LacI, and Ptet/TetR), we created variants that utilize the RK2 origin and spectinomycin or gentamicin selection. Relevant RFP expression and growth data have been collected in the model bacterium Escherichia coli as well as Pseudomonas putida. All pGinger vectors are available via the Joint BioEnergy Institute (JBEI) Public Registry.

synthetic biology↗

bioRxiv2022

Engineering Pseudomonas putida KT2440 for chain length tailored free fatty acid and oleochemical production

Despite advances in understanding the metabolism of Pseudomonas putida KT2440, a promising bacterial host for producing valuable chemicals from plant-derived feedstocks, a strain capable of producing free fatty acid-derived chemicals has not been developed. Guided by functional genomics, we engineered P. putida to produce medium- and long-chain free fatty acids (FFAs) to titers of up to 670 mg/L, paving the road for the production of high-value oleochemicals and biofuels from cheap feedstocks, such as plant biomass, using this host. Additionally, by taking advantage of the varying substrate preferences of paralogous native fatty acyl-CoA ligases, we employed a strategy to control FFA chain length that resulted in a P. putida strain specialized in producing medium-chain FFAs. Finally, we demonstrate the production of oleochemicals in these strains by synthesizing medium-chain fatty acid methyl esters, compounds useful as biodiesel blending agents, in various media including sorghum hydrolysate at titers greater than 300 mg/L.

synthetic biology↗

bioRxiv2021

Nitrogen metabolism in Pseudomonas putida: functional analysis using random barcode transposon sequencing

Pseudomonas putida KT2440 has long been studied for its diverse and robust metabolisms, yet many genes and proteins imparting these growth capacities remain uncharacterized. Using pooled mutant fitness assays, we identified genes and proteins involved in the assimilation of 52 different nitrogen containing compounds. To assay amino acid biosynthesis, 19 amino acid drop- out conditions were also tested. From these 71 conditions, significant fitness phenotypes were elicited in 672 different genes including 100 transcriptional regulators and 112 transport-related proteins. We divide these conditions into 6 classes, and propose assimilatory pathways for the compounds based on this wealth of genetic data. To complement these data, we characterize the substrate range of three promiscuous aminotransferases relevant to metabolic engineering efforts in vitro. Furthermore, we examine the specificity of five transcriptional regulators, explaining some fitness data results and exploring their potential to be developed into useful synthetic biology tools. In addition, we use manifold learning to create an interactive visualization tool for interpreting our BarSeq data, which will improve the accessibility and utility of this work to other researchers. IMPORTANCEUnderstanding the genetic basis of P. putidas diverse metabolism is imperative for us to reach its full potential as a host for metabolic engineering. Many target molecules of the bioeconomy and their precursors contain nitrogen. This study provides functional evidence linking hundreds of genes to their roles in the metabolism of nitrogenous compounds, and provides an interactive tool for visualizing these data. We further characterize several aminotransferases, lactamases, and regulators--which are of particular interest for metabolic engineering.

microbiology↗

bioRxiv2021

The Great Deceiver: miR-2392's Hidden Role in Driving SARS-CoV-2 Infection

MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene regulation that have a major impact on many diseases and provides an exciting avenue towards antiviral therapeutics. From patient transcriptomic data, we have discovered a circulating miRNA, miR-2392, that is directly involved with SARS-CoV-2 machinery during host infection. Specifically, we show that miR-2392 is key in driving downstream suppression of mitochondrial gene expression, increasing inflammation, glycolysis, and hypoxia as well as promoting many symptoms associated with COVID-19 infection. We demonstrate miR-2392 is present in the blood and urine of COVID-19 positive patients, but not detected in COVID-19 negative patients. These findings indicate the potential for developing a novel, minimally invasive, COVID-19 detection method. Lastly, using in vitro human and in vivo hamster models, we have developed a novel miRNA-based antiviral therapeutic that targets miR-2392, significantly reduces SARS-CoV-2 viability in hamsters and may potentially inhibit a COVID-19 disease state in humans.

systems biology↗

bioRxiv2020

Characterization of an ECF56-family sigma factor from Streptomyces venezuelae reveals a highly conserved regulome

Bacteria often possess alternative sigma factors that initiate the transcription of specific genes under environmental stresses, the largest and most diverse group being the extracytoplasmic function (ECF) sigma factors. The regulation of ECF activity is crucial for ensuring the distinct transcription of stress responsive genes only occurs under the appropriate conditions. While most ECFs are comprised of only the core {sigma}2 and {sigma}4 regions, a unique form of ECF sigma factor regulation also contains a C-terminal extension bearing homology to the NTF2 superfamily of protein domains. While previous work has shown that this NTF2 domain can affect transcriptional activity in vivo in ECF41 and ECF42, its role in the newly classified ECF56 subgroup is unknown. In this work, we show that truncation of the C-terminus of the ECF56 sigma factor SVEN_4562 of Streptomyces venezuelae upregulates its activity in a hybrid assay. Through transcriptomics in S. venezuelae, we found that this truncated ECF56 sigma factor has a highly conserved promoter sequence in vivo. Bioinformatic assays illustrated that deep branches of the Actinobacteria phylum contained putative ECF56 promoter motifs identical to those found in the S. venezuelae ECF56 regulon. We validated these findings through ex situ hybrid assays illustrating that truncated ECF56 sigma factors from phylogenetically diverse Actinobacteria activate transcription from these promoters. Importantly, our work shows that the genetic infrastructure of the ECF56 family of sigma factors is highly conserved and performs important functions yet to be understood in Actinobacteria. ImportanceMost ECF sigma-factors rely on anti-sigma factor regulation; in contrast, the unique classes of ECF sigma-factors that contain a C-terminal extension are thought to respond directly to an environmental signal. Here we show that the cis-acting regulatory element of the ECF56 regulon is likely highly conserved in many Actinobacteria, with exact nucleotide level conservation over ~2 billion years of evolution. The high conservation of this genetic architecture, as well as a conserved gene content within the regulon, strongly point to a specialized and important role in Actinobacteria biology.

microbiology↗

bioRxiv2019

Leveraging host metabolism for bisdemethoxycurcumin production in Pseudomonas putida

Pseudomonas putida is a saprophytic bacterium with robust metabolisms and strong solvent tolerance making it an attractive host for metabolic engineering and bioremediation. Due to its diverse carbon metabolisms, its genome encodes an array of proteins and enzymes that can be readily applied to produce valuable products. In this work we sought to identify design principles and bottlenecks in the production of type III polyketide synthase (T3PKS)-derived compounds in P. putida. T3PKS products are widely used as nutraceuticals and medicines and often require aromatic starter units, such as coumaroyl-CoA, which is also an intermediate in the native coumarate catabolic pathway of P. putida. Using a randomly barcoded transposon mutant (RB-TnSeq) library, we assayed gene functions for a large portion of aromatic catabolism, confirmed known pathways, and proposed new annotations for two aromatic transporters. The 1,3,6,8-tetrahydroxynapthalene synthase of Streptomyces coelicolor (RppA), a microbial T3PKS, was then used to rapidly assay growth conditions for increased T3PKS product accumulation. The feruloyl/coumaroyl CoA synthetase (Fcs) of P. putida was used to supply coumaroyl-CoA for the curcuminoid synthase (CUS) of Oryza sativa, a plant T3PKS. We identified that accumulation of coumaroyl-CoA in this pathway results in extended growth lag times in P. putida. Deletion of the second step in coumarate catabolism, the enoyl-CoA hydratase-lyase (Ech), resulted in increased production of the type III polyketide bisdemethoxycurcumin.

microbiology↗

bioRxiv2019

Identification, characterization, and application of a highly sensitive lactam biosensor from Pseudomonas putida

Caprolactam is an important polymer precursor to nylon traditionally derived from petroleum and produced on a scale of 5 million tons per year. Current biological pathways for the production of caprolactam are inefficient with titers not exceeding 2 mg/L, necessitating novel pathways for its production. As development of novel metabolic routes often require thousands of designs and result in low product titers, a highly sensitive biosensor for the final product has the potential to rapidly speed up development times. Here we report a highly sensitive biosensor for valerolactam and caprolactam from Pseudomonas putida KT2440 which is >1000x more sensitive to exogenous ligand than previously reported sensors. Manipulating the expression of the sensor oplR (PP_3516) substantially altered the sensing parameters, with various vectors showing Kd values ranging from 700 nM (79.1 g/L) to 1.2 mM (135.6 mg/L). Our most sensitive construct was able to detect in vivo production of caprolactam above background at ~6 g/L. The high sensitivity and range of OplR is a powerful tool towards the development of novel routes to the biological synthesis of caprolactam. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/700484v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@ff5b8corg.highwire.dtl.DTLVardef@d365aaorg.highwire.dtl.DTLVardef@b79b18org.highwire.dtl.DTLVardef@27732f_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗