Biology subjects

Shih, P.

Publications and source records attributed to Shih, P..

16 recordsLinked to original sources

bioRxiv2026

Form I and II Rubiscos Exhibit Temperature Dependent Carbon Kinetic Isotope Effects

Nearly all carbon on Earth today is fixed by the enzyme ribulose-1,5-bisphopshate carboxylase/oxygenase ( rubisco), which converts carbon dioxide (CO2) to sugar phosphates. All rubiscos measured thus far display a kinetic isotope effect (KIE) where 12CO2 is fixed at a faster rate than 13CO2. The relationship between rubiscos KIE and the carbon isotope composition of plants, algae, and organic matter is central to many fields in the Earth sciences, plant biology, and biochemistry. Currently, all applications assume that the KIE does not vary with temperature. Here, we examine this assumption experimentally with in vitro KIE measurements of two rubiscos from phylogenetically distinct host organisms and rubisco protein clades - a Form I rubisco from the plant, Spinacia oleracea (spinach) and a Form II rubisco from the bacterium Rhodosprillium rubrum. We that find that both KIEs decrease linearly by [~]4.5{per thousand} from 10-35{degrees}C with statistically indistinguishable slopes. We place these results into biological and geologic contexts by comparing them to observed variations in the carbon isotope composition of modern terrestrial plants and marine organic carbon, the geologic carbon isotope record, and rubiscos biochemistry. We show that the measured temperature dependencies are sufficiently large to impact our interpretations of the enzymatic processes that drive variations in rubisco KIEs, as well as applications of stable carbon isotopes in the Earth and biological sciences. Significance StatementThe carbon isotope composition of plants, algae, and organic matter are interpreted with models that assume the kinetic isotope effect of the carbon-fixing enzyme rubisco is temperature-independent, even though temperature varies by tens of degrees across the Earth today and in the past. Here, we demonstrate that the kinetic isotope effect of rubisco is temperature-dependent, suggesting that some of this isotopic variation may be due to intrinsic enzyme properties alone. In addition, though the rubiscos we measured are from diverse organisms (plant vs. bacteria), their KIEs show statistically indistinguishable temperature dependencies. This data forms the basis for future thermodynamic models on rubisco biochemistry.

biochemistry↗

bioRxiv2026

CO2 fixation mediated by the carbon concentrating mechanism enables a rapid response to nitrogen deprivation in cyanobacteria

Cyanobacteria are leading biomass producers of the ocean whose ecological success relies on their ability to respond to dynamic availability of nutrients like CO2 and nitrogen, which require distinct adaptive mechanisms. To survive nitrogen deprivation, cyanobacteria undergo a reversible transition to a dormant mode. Under low CO2 levels, a CO2 concentrating mechanism (CCM) supports their CO2 fixation. While the CCM and nitrogen assimilation have been shown to share some regulatory pathways, how the CCM impacts the response to nitrogen deprivation remains underexplored. In this study, by using mutants of the coastal cyanobacteria Synechococcus sp. PCC 7002 lacking a CCM component, we show that the high rate of carbon fixation mediated by the CCM tunes the speed of the nitrogen deprivation response in {beta}-cyanobacteria. We first show that CCM mutants are deficient in inducing their typical nitrogen deprivation response under atmospheric CO2. However, at higher CO2 concentrations, the CCM mutants induce the nitrogen deprivation response. By combining Rubisco kinetics modeling with measurement of the response speed to nitrogen in various CO2 concentrations, we show that the speed of the nitrogen deprivation response increases linearly with Rubiscos carboxylation rate. We further reveal that the regulation of nitrogen response by the CCM is also present in the distantly related freshwater cyanobacteria Synechococcus elongatus PCC 7942, suggesting a widespread role of this regulation across {beta}-cyanobacteria. This study demonstrates that CO2 fixation by the cyanobacterial CCM is a key regulator of the nitrogen deprivation response, favoring a rapid response to dynamic environments.

plant biology↗

bioRxiv2026

Unfiltered: A rapid method for Agrobacterium-mediated transformation of Basidiomycete and Ascomycete yeasts

Agrobacterium-mediated transformation (AMT) is a critical method for genetic manipulation of non-model fungi, yet it remains a laborious and inefficient technique. When cocultured in acetosyringone-supplemented induction medium, Agrobacterium transfers DNA directly into yeast cells using its virulence machinery. Membrane filters are commonly used to support the co-culture of yeast and Agrobacterium on agar plates, however some reports demonstrate that these filters are unnecessary for specific yeast species. Here we confirm across diverse budding yeasts that membrane filters are not necessary for effective AMT. Concentrating the cells via centrifugation and "spotting" the cell pellet directly onto the induction medium proved effective. This reduces hands-on time to 15 minutes and eliminates filter cost. In the oleaginous yeast, Rhodotorula toruloides, this simplified method increases transformation efficiency by 66% to 2,500 transformants per 106 recipient cells. We further optimized the Agrobacterium:Rhodotorula cell ratio and culture resuspension volume to achieve more than 200,000 CFU per transformation representing a 2-3 fold improvement over previously implemented protocols. This spot-plating method was successfully applied to seven yeast species, including one for which genetic transformation has not previously been reported, Botryozyma nematodophila. This approach highlights the broad applicability of the spot-plating method across diverse yeast systems. Furthermore, this method could facilitate high-throughput transformation workflows that are critical for genome-scale functional studies.

microbiology↗

bioRxiv2025

Binary vector origin predictably determines Agrobacterium-mediated transformation outcome across eukaryotic kingdom

Agrobacterium-mediated transformation (AMT) is the primary means of genetic engineering in plants and many fungi, but the factors that control transformation outcomes--efficiency, transgene insertion number, and transgene integrity--remain poorly characterized. Although transformation outcomes dictate an events potential utility in both industrial and academic contexts, AMT remains largely unoptimized for these metrics. Here, we systematically analyze the impact of the transgene-harboring binary vector on transformation outcomes across plant and fungal species. Through a comparison of different plasmid origin of replication (ORI) families and engineered copy number variants, our results reveal that the ORI family--not plasmid copy number--dictates T-DNA insertion number, backbone inclusion, and transformation efficiency, while plasmid copy number tuning alters efficiency without changing ORI family-specific signatures. Independent of plasmid copy number across kingdoms, the most widely used pVS1 ORI-based vectors (e.g. pCambia) result in significantly more insertions per transformant and high levels of transgene silencing compared to the less-utilized pSa ORI family, which enriches for more uniform single insertion events. Furthermore, we demonstrate that ORI-dependent transformation outcomes in yeast predictably reflect those in Arabidopsis. Together, these results lay the foundation for future binary vector design aimed at achieving more predictable, controllable, and optimized transformation outcomes across diverse eukaryotic hosts.

plant biology↗

bioRxiv2025

Causes and consequences of experimental variation in Nicotiana benthamiana transient expression

Infiltration of Agrobacterium tumefaciens into Nicotiana benthamiana has become a foundational technique in plant biology, enabling efficient delivery of transgenes in planta with technical ease, robust signal, and relatively high throughput. Despite transient expressions prevalence in disciplines such as synthetic biology, little work has been done to describe and address the variability inherent in this system, a concern for experiments that rely on highly quantitative readouts. In a comprehensive analysis of N. benthamiana agroinfiltration experiments, we model sources of variability that affect transient expression. Our findings emphasize the need to validate normalization methods under the specific conditions of each study, as distinct normalization schemes do not always reduce variation either within or between experiments. Using a dataset of 1,915 plants collected over three years, we develop a model of variation in N. benthamiana transient expression, using power analysis to determine the number of individual plants required for a given effect size. Drawing on our longitudinal data, these findings inform practical guidelines for minimizing variability through strategic experimental design and power analysis, providing a foundation for more robust and reproducible use of N. benthamiana in quantitative plant biology and synthetic biology applications.

plant biology↗

bioRxiv2025

Multiplexed profiling of transcriptional regulators in plant cells

Transcriptional regulators play key roles in plant growth, development, and environmental responses; however, understanding how their regulatory activity is encoded at the protein level has been hindered by a lack of multiplexed large-scale methods to characterize protein libraries in planta. Here, we present ENTRAP-seq (Enrichment of Nuclear Trans-elements Reporter Assay in Plants), a high-throughput method that introduces protein-coding libraries into plant cells to drive a nuclear magnetic sorting-based reporter, enabling multiplexed measurement of regulatory activity from thousands of protein variants. Using ENTRAP-seq and machine learning, we screened 1,495 plant viruses and identified hundreds of novel putative transcriptional regulatory domains found in structural proteins and enzymes not associated with gene regulation. In addition, we combined ENTRAP-seq with machine-guided design to engineer the activity of a plant transcription factor in a semi-rational fashion. Our findings demonstrate how scalable protein function assays deployed in planta will enable the characterization of natural and synthetic coding diversity in plants.

plant biology↗

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↗

bioRxiv2024

Giving it a fair shake: A simple, fast, & efficient method to extract amatoxins from the death cap mushroom, Amanita phalloides

1.1.The death cap mushroom, Amanita phalloides, is well known for containing amatoxins such as alpha- and beta-amanitin, which inhibit eukaryotic RNA polymerase II. While these toxins have been used in research for almost a century, they have recently garnered attention for their role in drug-antibody conjugates. The amatoxins are still largely extracted from wild mushrooms, which cannot be made to fruit in the lab. We propose simplified extraction methods that could reduce hazardous exposures to dust and expedite sample analysis without sacrificing accuracy. We recently developed a Lateral Flow Immunoassay (LFIA), for which we identified that sample maceration was not needed to extract the amatoxins and that the incubation time for extraction could be accomplished in 1 minute. In this current work, we hypothesized that these same extraction adjustments-minimal tissue maceration and reduced incubation time-could be transferable to instrumental detection methods. To test the need for sample maceration, we utilized three different techniques: 1) traditional mortar and pestle, 2) a similarly disruptive method of bead beating, and 3) no grinding, but rather hand shaking dried mushroom tissue in extraction buffer. In addition, we performed the solvent extraction step at varying times to observe if more time allows for more toxin to be removed from the tissue. Lastly, we utilized two comparable solvent evaporation methods (rotovap or speedvac) to establish if multiple samples could be processed simultaneously, thus improving sample throughput. We adjusted aspects of the typical extraction protocol, which resulted in a rapid (1 min) incubation step, along with minimal sample handling (no grinding) of the dried mushroom tissue. We present an extraction protocol that saves time, reduces equipment contamination, and minimizes risk to the researcher. The impact of this faster, safer method may help produce these important toxins faster, for both research and medical use.

microbiology↗

bioRxiv2024

Cyanobacteria from marine oxygen deficient zones encode both form I and form II rubiscos

Cyanobacteria are highly abundant in the marine photic zone and primary drivers of the conversion of inorganic carbon to biomass. To date, all studied Cyanobacterial lineages encode carbon fixation machinery hinged upon form I rubisco enzymes within a CO2-concentrating carboxysome. Here, we report that the AMZ IB lineage of Prochlorococcus from global oxygen deficient zones (ODZs) harbor both form I and form II rubisco enzymes, the latter of which are typically non-carboxysomal and possess biochemical properties tuned towards low oxygen environments. Our analyses reveal that these cyanobacterial form II enzymes are functional in vitro and were likely acquired via lateral gene transfer from proteobacteria. Global metagenomic read recruitment demonstrates that Prochlorococcus with form II rubisco are essentially restricted to ODZs in the Eastern Tropical Pacific, suggesting that acquisition may confer an advantage specifically under low-O2 conditions. Populations of AMZ IB Prochlorococcus express both forms of rubisco in situ, with the highest form II rubisco expression at depths where both oxygen and light are particularly low, possibly as a mechanism to increase the efficiency of photoautotrophy under energy limitation. Our findings expand the diversity of carbon fixation configurations in the microbial world and may have implications for the overall capacity of ODZs to sequester carbon.

microbiology↗

bioRxiv2024

Expression of a mammalian RNA demethylase increases flower number and floral stem branching in Arabidopsis thaliana

RNA methylation plays a central regulatory role in plant biology and is a relatively new target for plant improvement efforts. In nearly all cases, perturbation of the RNA methylation machinery results in deleterious phenotypes. However, a recent landmark paper reported that transcriptome-wide use of the human RNA demethylase FTO substantially increased the yield of rice and potatoes. Here, we have performed the first independent replication of those results and broader transferability of the trait, demonstrating increased flower and fruit count in the model species Arabidopsis thaliana. We also performed RNA-seq of our FTO-transgenic plants, which we analyzed in conjunction with previously-published datasets to detect several previously-recognized patterns in the functional and structural classification of the upregulated and downregulated genes. From these, we present mechanistic hypotheses to explain these surprising results with the goal of spurring more widespread interest in this promising new approach to plant engineering.

plant biology↗

bioRxiv2023

Mapping the biochemical landscape of rubisco

Rubisco is the primary CO2 fixing enzyme of the biosphere yet has slow kinetics. The roles of evolution and chemical mechanism in constraining the sequence landscape of rubisco remain debated. In order to map sequence to function, we developed a massively parallel assay for rubisco using an engineered E. coli where enzyme function is coupled to growth. By assaying >99% of single amino acid mutants across CO2 concentrations, we inferred enzyme velocity and CO2 affinity for thousands of substitutions. We identified many highly conserved positions that tolerate mutation and rare mutations that improve CO2 affinity. These data suggest that non-trivial kinetic improvements are readily accessible and provide a comprehensive sequence-to-function mapping for enzyme engineering efforts.

biochemistry↗

bioRxiv2023

Plant-based production of diverse human milk oligosaccharides

Human milk oligosaccharides (HMOs) are a diverse class of carbohydrates that aid in the health and development of infants. The vast health benefits of HMOs have made them a commercial target for microbial production; however, producing the [~]130 structurally diverse HMOs at scale has proven difficult. Here, we produce a vast diversity of HMOs by leveraging the robust carbohydrate anabolism of plants. This diversity includes high value HMOs, such as lacto-N-fucopentaose I, that have not yet been commercially produced using state-of-the-art microbial fermentative processes. HMOs produced in transgenic plants provided strong bifidogenic properties, indicating their ability to serve as a prebiotic supplement. Technoeconomic analyses demonstrate that producing HMOs in plants provides a path to the large-scale production of specific HMOs at lower prices than microbial production platforms. Our work demonstrates the promise in leveraging plants for the cheap and sustainable production of HMOs.

plant biology↗

bioRxiv2023

Systematic identification of transcriptional activator domains from non-transcription factor proteins in plants and yeast

Transcription factors promote gene expression via trans-regulatory activation domains. Although whole genome scale screens in model organisms (e.g. human, yeast, fly) have helped identify activation domains from transcription factors, such screens have been less extensively used to explore the occurrence of activation domains in non-transcription factor proteins, such as transcriptional coactivators, chromatin regulators and some cytosolic proteins, leaving a blind spot on what role activation domains in these proteins could play in regulating transcription. We utilized the activation domain predictor PADDLE to mine the entire proteomes of two model eukaryotes, Arabidopsis thaliana and Saccharomyces cerevisiae (1). We characterized 18,000 fragments covering predicted activation domains from >800 non-transcription factor genes in both species, and experimentally validated that 89% of proteins contained fragments capable of activating transcription in yeast. Peptides with similar sequence composition show a broad range of activities, which is explained by the arrangement of key amino acids. We also annotated hundreds of nuclear proteins with activation domains as putative coactivators; many of which have never been ascribed any function in plants. Furthermore, our library contains >250 non-nuclear proteins containing peptides with activation domain function across both eukaryotic lineages, suggesting that there are unknown biological roles of these peptides beyond transcription. Finally, we identify and validate short, universal eukaryotic activation domains that activate transcription in both yeast and plants with comparable or stronger performance to state-of-the-art activation domains. Overall, our dual host screen provides a blueprint on how to systematically discover novel genetic parts for synthetic biology that function across a wide diversity of eukaryotes. Significance StatementActivation domains promote transcription and play a critical role in regulating gene expression. Although the mapping of activation domains from transcription factors has been carried out in previous genome-wide screens, their occurrence in non-transcription factors has been less explored. We utilize an activation domain predictor to mine the entire proteomes of Arabidopsis thaliana and Saccharomyces cerevisiae for new activation domains on non-transcription factor proteins. We validate peptides derived from >750 non-transcription factor proteins capable of activating transcription, discovering many potentially new coactivators in plants. Importantly, we identify novel genetic parts that can function across both species, representing unique synthetic biology tools.

genetics↗

bioRxiv2023

Cynipid wasps systematically reprogram host metabolism and restructure cell walls in developing galls

Many insects have evolved the ability to manipulate plant growth to generate extraordinary structures called galls in which insect larva can develop while being sheltered within and feeding on the plant. In particular, Cynipid (Hymenoptera: Cynipidae) wasps have evolved to form some of the most morphologically complex galls known and generate an astonishing array of gall shapes, colors, and sizes. However, the biochemical basis underlying these remarkable cellular and developmental transformations remains poorly understood. A key determinant in plant cellular development is the deposition of the cell wall to dictate the physical form and physiological function of newly developing cells, tissues, and organs. However, it is unclear to what degree cell walls are restructured to initiate and support the formation of new gall tissue. Here, we characterize the molecular alterations underlying gall development using a combination of metabolomic, histological, and biochemical techniques to elucidate how leaf cells are reprogrammed to form galls. Strikingly, gall development involves an exceptionally coordinated spatial deposition of lignin and xylan to form de novo gall vasculature. Our results highlight how Cynipid wasps can radically change the metabolite profile and restructure the cell wall to enable the formation of galls, providing new insights into the mechanism of gall induction and the extent to which plants can be entirely reprogrammed to form novel structures and organs.

plant biology↗

bioRxiv2020

Functional analysis of the fatty acid and alcohol metabolism of Pseudomonas putida using RB-TnSeq

ABSTRACTWith its ability to catabolize a wide variety of carbon sources and a growing engineering toolkit, Pseudomonas putida KT2440 is emerging as an important chassis organism for metabolic engineering. Despite advances in our understanding of this organism, many gaps remain in our knowledge of the genetic basis of its metabolic capabilities. These gaps are particularly noticeable in our understanding of both fatty acid and alcohol catabolism, where many paralogs putatively coding for similar enzymes co-exist making biochemical assignment via sequence homology difficult. To rapidly assign function to the enzymes responsible for these metabolisms, we leveraged Random Barcode Transposon Sequencing (RB-TnSeq). Global fitness analyses of transposon libraries grown on 13 fatty acids and 10 alcohols produced strong phenotypes for hundreds of genes. Fitness data from mutant pools grown on varying chain length fatty acids indicated specific enzyme substrate preferences, and enabled us to hypothesize that DUF1302/DUF1329 family proteins potentially function as esterases. From the data we also postulate catabolic routes for the two biogasoline molecules isoprenol and isopentanol, which are catabolized via leucine metabolism after initial oxidation and activation with CoA. Because fatty acids and alcohols may serve as both feedstocks or final products of metabolic engineering efforts, the fitness data presented here will help guide future genomic modifications towards higher titers, rates, and yields.IMPORTANCE To engineer novel metabolic pathways into P. putida, a comprehensive understanding of the genetic basis of its versatile metabolism is essential. Here we provide functional evidence for the putative roles of hundreds of genes involved in the fatty acid and alcohol metabolism of this bacterium. These data provide a framework facilitating precise genetic changes to prevent product degradation and channel the flux of specific pathway intermediates as desired.Competing Interest StatementJ.D.K. has financial interests in Amyris, Lygos, Demetrix, Napigen, Maple Bio, and Apertor Labs. C.B.E has a financial interest in Perlumi Chemicals.View Full Text

microbiology↗

bioRxiv2018

Hydrogen-based metabolism - An ancestral trait in lineages sibling to the Cyanobacteria

The metabolic machinery from which microbial aerobic respiration evolved is tightly linked to the origins of oxygenic Cyanobacteria (Oxyphotobacteria). Even though the majority of Oxyphotobacteria are photoautotrophs and can use carbohydrates with oxygen (O2) as the electron acceptor, all are fermenters under dark anoxic conditions. Studies suggest that the ancestor of Oxyphotobacteria may have used hydrogen (H2) as an electron donor and that two types of NiFe hydrogenases are essential for its oxidation. Melainabacteria and Sericytochromatia, close phylogenetic neighbors to Oxyphotobacteria comprise fermentative and aerobic representatives, or organisms capable of both. Margulisbacteria (candidate divisions RBX-1 and ZB3) and Saganbacteria (candidate division WOR-1), a novel cluster of bacteria phylogenetically related to Melainabacteria, Sericytochromatia and Oxyphotobacteria may further constrain the metabolic platform in which oxygenic photosynthesis and aerobic respiration arose. Here, we predict the metabolisms of Margulisbacteria and Saganbacteria from new and published metagenome-assembled genomes (MAGs) and single amplified genomes (SAGs), and compare them to their phylogenetic neighbors. Sediment-associated Margulisbacteria are predicted to have a fermentation-based metabolism featuring a variety of hydrogenases, a nitrogenase for nitrogen (N2) fixation, and electron bifurcating complexes involved in cycling of ferredoxin and NAD(P)H. Overall, the genomic features suggest the capacity for metabolic fine-tuning under strictly anoxic conditions. In contrast, the genomes of Margulisbacteria from the ocean ecosystem encode an electron transport chain that supports aerobic growth. Similarly, some Saganbacteria genomes encode various hydrogenases, and others may have the ability to use O2 under certain conditions via a putative novel type of heme copper O2 reductase. Like Melainabacteria and Sericytochromatia, Margulisbacteria and Saganbacteria have diverse energy metabolisms capable of fermentation, and aerobic or anaerobic respiration. In summary, our findings support the hypothesis that the ancestor of these groups was an anaerobe in which fermentation and H2 metabolism were central metabolic features. Our genomic data also suggests that contemporary lineages sibling to the Oxyphotobacteria may have acquired the ability to use O2 as a terminal electron acceptor under certain environmental conditions.

microbiology↗