Biology subjects

Shih, P. M.

Publications and source records attributed to Shih, P. M..

At least 19 recordsLinked to original sources

bioRxiv2026

Machine learning guided design of strong transcriptional activation domains

Artificial intelligence has accelerated the design of structured proteins, but it remains difficult to design functional intrinsically disordered proteins because of weak structural constraints and poorly defined sequence-to-function relationships1. Activation domains (ADs), transcription factor regions that recruit coactivators, are an excellent test case for the de novo design of intrinsically disordered sequences. ADs are one of the oldest groups of functional intrinsically disordered sequences2 and designed ADs have applications in synthetic biology, biotechnology, and agriculture. We computationally generated 500,000 candidate sequences that resembled known ADs and predicted AD function with extant machine learning models, including three neural networks. From high-scoring sequences, we selected 18,750 for experimental validation in yeast. Surprisingly, >90% of the 16,770 well-measured sequences function as ADs. Sequences predicted to be active by multiple models were stronger, motivating development of a fast and accurate consensus AD predictor, MADRIGAL. A number of our designed ADs retain functionality across fungi, plants, and human cells. This work illustrates a generalizable paradigm for incorporating sequence-to-function models into the design loop to efficiently generate functional intrinsically disordered sequences.

synthetic biology↗

bioRxiv2026

Genome-scale mapping of plant transcription factor effector domains reveals conserved regulatory principles across eukaryotes

Eukaryotic life spans species that diverged over a billion years ago, yet our molecular view of gene regulation remains heavily skewed toward Opisthokonta model systems, particularly yeast and humans. The degree to which the protein features governing transcriptional regulation are conserved across eukaryotes or have diversified within individual eukaryotic supergroups remains unresolved. To provide a distinct evolutionary reference point, we used a massively parallel reporter assay in plant cells to identify transcriptional effector domains and define their sequence constraints, measuring ~26,000 protein fragments tiling nearly all Arabidopsis transcription factors (1,859 proteins). Comparison with the activity of the same peptide sequences in yeast revealed that activation is governed by a mixture of deeply conserved biochemical features and lineage-dependent functional differences across widely divergent eukaryotic lineages. Likewise, plant repression domains are governed by flexible sequence grammars that extend beyond conserved repression motifs. Together, these results reveal both broadly conserved and clade-specific principles governing eukaryotic transcriptional regulation.

plant biology↗

bioRxiv2026

De novo Rubisco design with protein language models

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) fixes the majority of carbon dioxide globally but is challenged with low specificity for CO2 versus O2 and low catalytic efficiencies. Traditional engineering efforts have remained difficult because folding, assembly, specificity, and catalysis are tightly coupled, hampering efforts to explore sequence space. Therefore, we leveraged recent advances in protein large language models (PLMs) to generate sequences beyond those observed in nature, using both ProGen-2 that was fine-tuned on a limited dataset of non-Form I Rubiscos and an ESM-2 discriminator. With this approach, we generated 5.6 million novel Rubisco-like sequences and identified 21 highly diverse candidates predicted to be active that occupy regions of Rubisco phylogenetic space not previously observed in nature. Six designs were soluble in Escherichia coli, and five were shown to produce quantifiable 3PGA. One design produced an apparent CO2/O2 specificity estimate beyond the range of the natural representative Rubiscos assayed. We also solved the crystal structure of one de novo design that reproduced the predicted dimer and active-site geometry with sub-angstrom C agreement. Sequence-only generation followed by independent structural filtering therefore recovered soluble, active Rubiscos from regions of sequence space that are not represented in genomic databases. Together, these results establish a scalable strategy for accessing previously unexplored Rubisco sequence space, providing a broadly accessible path toward generating de novo Rubiscos that may have activity and specificity parameters needed to address longstanding limitations in biological carbon fixation.

biochemistry↗

bioRxiv2026

A toolkit for programmable transcriptional engineering across eukaryotic kingdoms

Chromatin is essential for eukaryotic life. Tens of thousands of chromatin regulator (CR) proteins exist in eukaryotic genomes that are predicted to modulate chromatin states; however, their molecular functions remain largely untested experimentally. Here, we construct a library of over 300 full-length CRs from humans, plants, yeast, protozoa, and virus, each fused to DNA-binding domains, and test their direct effect on transcriptional repression and activation in plants and human cells. We discover CRs with cross-kingdom functionality when transferred across eukaryotes, including CRs that outperform existing tools for programmable transcriptional repression and activation in plants and human cells. Using pooled CRISPR screens, we demonstrate a suite of CRISPR repressors that titrate gene expression at intermediate levels. Finally, we identify RCOR1 and MTA2 as universal eukaryotic repressors that retain repressive activity in plants, yeast, and human cells. Our toolkit advances synthetic eukaryotic engineering and expands our understanding of CR functionality across eukaryotes.

synthetic biology↗

bioRxiv2025

Diversity-driven biochemical survey reveals dimeric structural origin of rubisco

Rubisco is the entry point of nearly all organic carbon into the biosphere and is present in all domains of life. Despite its global importance, biochemical studies of this enzyme superfamily have been limited to a relatively narrow set of subclades. Recent advances in metagenomics have dramatically reshaped our understanding of both microbial and rubisco diversity; however, biochemical characterization of these sequences has not kept pace with the exponential growth in sequence data. To better survey the functional and structural diversity of rubisco, we systematically sampled and synthesized a library of diverse rubisco sequences with an emphasis on clades that have previously not been characterized. Our updated phylogenetic analysis reveals that many deep-branching rubiscos assemble as dimers, supporting a dimeric origin for the superfamily -- in contrast to the ecologically dominant hexadecameric form I. Additionally, we discover and structurally characterize the largest rubisco described to date, originating from a cryptic, early-branching subclade with novel structural folds that have previously not been observed in the rubisco superfamily. By integrating biochemical data with an updated phylogenetic framework, we propose a revised nomenclature for the rubisco protein family that reflects current insights and will better accommodate future discoveries.

biochemistry↗

bioRxiv2025

Active learning enables discovery of transcriptional activators across fungal evolutionary space

Biological discovery and design are increasingly being guided by predictive models in place of costly experimentation. However, existing datasets are often biased by overrepresentation from model organisms, leading to failures in evolutionary studies of non-model species. We present a hybrid framework that leverages high-throughput molecular assays and active learning to quantify biological properties across evolutionary space. We focus on transcriptional activators, which contain activation domains (ADs) that promote gene expression. ADs are intrinsically disordered and poorly conserved, which limits their study using comparative genomics. Here, we developed ADhunter, a high-capacity regression model that outperforms state-of-theart algorithms in identifying and quantifying the strength of transcriptional activators. Model uncertainty was used to guide evolutionary sampling across 7.8 million proteins from 2,400 fungal genomes. We functionally characterized 9,836 ADs from 1,071 fungal genomes, providing a 15.5-fold expansion in genome representation compared to existing datasets. Comprehensive sampling from non-model genomes improved model generalizability and provides the first functional annotation for 3,416 proteins from 670 non-model fungi. Model interpretability analysis aligns with the biophysical model of AD function and reveals novel, underrepresented protein codes, highlighting the importance of sampling from non-model organisms to build evolutionarily robust models for predicting biological properties.

genomics↗

bioRxiv2025

Carbon-Efficient Conversion through Hydrogen and Formate-Driven Metabolism in Escherichia coli

Product yields for biomanufacturing processes are often constrained by the tight coupling of cellular energy generation and carbon metabolism in sugar-based fermentation systems. To overcome this limitation, we engineered Escherichia coli to utilize hydrogen gas (H2) and formate (HCOO-) as alternative sources of energy and reducing equivalents, thereby decoupling energy generation from carbon metabolism. This approach enabled precise suppression of decarboxylative oxidation during acetate growth, with 86.6 {+/-} 1.6% of electrons from hydrogen gas (via soluble hydrogenase from Cupriavidus necator H16) and 98.4 {+/-} 3.6% of electrons from formate (via formate dehydrogenase from Pseudomonas sp. 101) offsetting acetate oxidation. Hydrogen gas supplementation led to a titratable and stoichiometric reduction in CO2 evolution in acetate-fed cultures. Metabolomic analysis suggests that this metabolic decoupling redirects carbon flux through the glyoxylate shunt, partially bypassing two decarboxylative steps in the TCA cycle. We demonstrated the utility of this strategy by applying it to mevalonate biosynthesis, where formate supplementation during glucose fermentation increased titers by 57.6% in our best-performing strain. Flux balance analysis further estimated that 99.0 {+/-} 2.8% of electrons from formate were used to enhance mevalonate production. These findings highlight a broadly applicable strategy for enhancing biomanufacturing efficiency by leveraging external reducing power to optimize feedstock and energy use. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=65 SRC="FIGDIR/small/657096v2_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@12f77dborg.highwire.dtl.DTLVardef@165d6c1org.highwire.dtl.DTLVardef@f6302forg.highwire.dtl.DTLVardef@d59bb3_HPS_FORMAT_FIGEXP M_FIG C_FIG Created in BioRender. Panich, J. (2025) https://BioRender.com/0tmtp94

synthetic biology↗

bioRxiv2024

Predictable modulation of plant root development using engineered cytokinin regulators

Achieving precise control over quantitative developmental phenotypes is a key objective in plant biology. Recent advances in synthetic biology have enabled tools to reprogram entire developmental pathways; however, the complexity of designing synthetic genetic programs and the inherent interactions between various signaling processes remains a critical challenge. Here, we leverage Type-B response regulators to modulate the expression of genes involved in cytokinin-dependent growth and development processes. We rationally engineered these regulators to modulate their transcriptional activity (i.e., repression or activation) and potency while reducing their sensitivity to cytokinin. By localizing the expression of these engineered transcription factors using tissue-specific promoters, we can predictably tune cytokinin-regulated traits. As a proof of principle, we deployed this synthetic system in Arabidopsis thaliana to either decrease or increase the number of lateral roots. The simplicity and modularity of our approach makes it an ideal system for controlling other developmental phenotypes of agronomic interest in plants.

plant 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

Bacterial Form I' rubisco has smaller carbon isotope fractionation than its Form I counterpart

Form I rubiscos evolved in Cyanobacteria [≥]2.5 billion years ago and are enzymatically unique due to the presence of small subunits (RbcS) that cap both ends of an octameric large subunit (RbcL) rubisco assembly to form a hexadecameric (L8S8) holoenzyme. Although RbcS was previously thought to be integral to Form I rubisco stability, the recent discovery of a closely related sister clade of octameric rubiscos (Form I; L8) demonstrates that the enzyme complex assembles without small subunits (Banda et al. 2020). Rubisco also displays a kinetic isotope effect (KIE) where the 3PG product is depleted in 13C relative to 12C. In Cyanobacteria only two Form I KIE measurements exist, making interpretation of bacterial carbon isotope data difficult. To aid comparison, we measured in vitro the KIEs of Form I (Candidatus Promineofilum breve) and Form I (Synechococcus elongatus PCC 6301) rubiscos and found the KIE to be smaller in the L8 rubisco (16.25 {+/-} 1.36{per thousand} vs. 22.42 {+/-} 2.37{per thousand} respectively). Therefore, while small subunits may not be necessary for protein stability, they may affect the KIE. Our findings may provide insight into the function of RbcS and allow more refined interpretation of environmental carbon isotope data.

evolutionary biology↗

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

The trans-regulatory landscape of gene networks in plants

The effector domains of transcription factors play a key role in controlling gene expression; however, their functional nature is poorly understood, hampering our ability to explore this fundamental dimension of gene regulatory networks. To map the trans-regulatory landscape in a complex eukaryote, we systematically characterized the putative effector domains of over 400 Arabidopsis thaliana transcription factors for their capacity to modulate transcription. We demonstrate that transcriptional effector activity can be integrated into gene regulatory networks capable of elucidating the functional dynamics underlying gene expression patterns. We further show how newly characterized domains can enhance genome engineering efforts and reveal how plant transcriptional activators share regulatory features conserved across distantly related eukaryotes. Our results provide a framework to systematically characterize the regulatory role of transcription factors at a genome-scale in order to understand the transcriptional wiring of biological systems.

plant 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↗

bioRxiv2022

A dolabralexin-deficient mutant provides insight into specialized diterpenoid metabolism in maize (Zea mays).

Two major groups of maize (Zea mays) specialized metabolites, termed kauralexins and dolabralexins, serve as known or predicted diterpenoid defenses against pathogens, herbivores, and other environmental stressors. To consider physiological roles of the recently discovered dolabralexin pathway, we examined dolabralexin structural diversity, tissue-specificity, and stress-elicited production in a defined biosynthetic pathway mutant. Metabolomics analyses support a larger number of dolabralexin pathway products than previously known. We identified dolabradienol as a previously undetected pathway metabolite and characterized its enzymatic production. Transcript and metabolite profiling showed that dolabralexin biosynthesis and accumulation predominantly occurs in primary roots and shows quantitative variation across genetically diverse inbred lines. Generation and analysis of CRISPR-Cas9-derived loss-of- function Kaurene Synthase-Like 4 (Zmksl4) mutants demonstrated dolabralexin production deficiency, thus supporting ZmKSL4 as the diterpene synthase responsible for the conversion of geranylgeranyl pyrophosphate precursors into dolabradiene and downstream pathway products. Zmksl4 mutants further display altered root-to-shoot ratios and root architecture in response to water deficit, consistent with an interactive role of maize dolabralexins in plant vigor during abiotic stress.

plant biology↗

bioRxiv2022

Evolution of Carbon Isotope Fractionation in Cyanobacteria

The history of Earths carbon cycle reflects trends in atmospheric composition convolved with the evolution of photosynthesis. Fortunately, key parts of the carbon cycle have been recorded in the carbon isotope ratios of sedimentary rocks. The dominant model used to interpret this record as a proxy for ancient atmospheric CO2 is based on carbon isotope fractionations of modern photoautotrophs, and longstanding questions remain about how their evolution might have impacted the record. We tested the intersection of environment and evolution by measuring both biomass ({varepsilon}p) and enzymatic ({varepsilon}Rubisco) carbon isotope fractionations of a cyanobacterial strain (Synechococcus elongatus PCC 7942) solely expressing a putative ancestral Form 1B rubisco dating to >>1 Ga. This strain, nicknamed ANC, grows in ambient pCO2 and displays larger {varepsilon}p values than WT, despite having a much smaller {varepsilon}Rubisco (17.23 {+/-} 0.61{per thousand} vs. 25.18 {+/-} 0.31{per thousand} respectively). Measuring both enzymatic and biomass fractionation revealed a surprising result--ANC {varepsilon}p exceeded ANC {varepsilon}Rubisco in all conditions tested, contradicting prevailing models of cyanobacterial carbon isotope fractionation. However, these models were corrected by accounting for cyanobacterial physiology, notably the CO2 concentrating mechanism (CCM). Our model suggested that additional fractionating processes like powered inorganic carbon uptake systems contribute to {varepsilon}p, and this effect is exacerbated in ANC. Understanding the evolution of rubisco and the CCM is therefore critical for interpreting the carbon isotope record. Large fluctuations in that record may reflect the evolving efficiency of carbon fixing metabolisms in addition to changes in atmospheric CO2. Significance StatementEarth scientists rely on chemical fossils like the carbon isotope record to derive ancient atmospheric CO2 concentrations, but interpretation of this record is calibrated using modern organisms. We tested this assumption by measuring the carbon isotope fractionation of a reconstructed ancestral rubisco enzyme (>1 billion years old) in vivo and in vitro. Our results contradicted prevailing models of carbon flow in Cyanobacteria, but our data could be rationalized if light-driven uptake of CO2 is taken into account. Our study showed that the carbon isotope record tracks both the evolution of photosynthesis physiology as well as changes in atmospheric CO2, highlighting the value of considering both evolution and physiology for comparative biological approaches to understanding Earths history.

evolutionary 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

Discovery of photosynthesis genes through whole-genome sequencing of acetate-requiring mutants of Chlamydomonas reinhardtii

Large-scale mutant libraries have been indispensable for genetic studies, and the development of next-generation genome sequencing technologies has greatly advanced efforts to analyze mutants. In this work, we sequenced the genomes of 660 Chlamydomonas reinhardtii acetate-requiring mutants, part of a larger photosynthesis mutant collection previously generated by insertional mutagenesis with a linearized plasmid. We identified 554 insertion events from 509 mutants by mapping the plasmid insertion sites through paired-end sequences, in which one end aligned to the plasmid and the other to a chromosomal location. Nearly all (96%) of the events were associated with deletions, duplications, or more complex rearrangements of genomic DNA at the sites of plasmid insertion, and 1405 genes in total were affected. Functional annotations of these genes were enriched in those related to photosynthesis, signaling, and tetrapyrrole synthesis as would be expected from a library enriched for photosynthesis mutants. Systematic manual analysis of the disrupted genes for each mutant generated a list of 273 higher-confidence candidate photosynthesis genes, and we experimentally validated two genes that are essential for photoautotrophic growth, CrLPA3 and CrPSBP4. The inventory of candidate genes includes 55 genes from a phylogenomically defined set of conserved genes in green algae and plants. Altogether, 68 candidate genes encode proteins with previously characterized functions in photosynthesis in Chlamydomonas, land plants, and/or cyanobacteria, 15 genes encode proteins previously shown to have functions unrelated to photosynthesis, and 190 genes encode proteins without any functional annotation, signifying that our results connect a function related to photosynthesis to these previously unknown proteins. This mutant library, with genome sequences that reveal the molecular extent of the chromosomal lesions and resulting higher-confidence candidate genes, represents a rich resource for gene discovery and protein functional analysis in photosynthesis.

plant biology↗

bioRxiv2021

Phototrophy and carbon fixation in Chlorobi postdate the rise of oxygen

While most productivity on the surface of the Earth today is fueled by oxygenic photosynthesis, during the early parts of Earth history it is thought that anoxygenic photosynthesis--using compounds like ferrous iron or sulfide as electron donors--drove most global carbon fixation. Anoxygenic photosynthesis is still performed by diverse bacteria in niche environments today. Of these, the Chlorobi (formerly green sulfur bacteria) are often interpreted as being particularly ancient and are frequently proposed to have fueled the biosphere early in Earth history before the rise of oxygenic photosynthesis. Here, we perform comparative genomic, phylogenetic, and molecular clock analyses to determine the antiquity of the Chlorobi and their characteristic phenotypes. We show that contrary to common assumptions, the Chlorobi clade is relatively young, with anoxygenic phototrophy, carbon fixation via the rTCA pathway, and iron oxidation all significantly postdating the rise of oxygen ~2.3 billion years ago. The Chlorobi therefore could not have fueled the Archean biosphere, but instead represent a relatively young radiation of organisms which likely acquired the capacity for anoxygenic photosynthesis and other traits via horizontal gene transfer sometime after the evolution of oxygenic Cyanobacteria.

evolutionary biology↗