Biology subjects

Yoshikuni, Y.

Publications and source records attributed to Yoshikuni, Y..

17 recordsLinked to original sources

bioRxiv2026

CRISPRi-assisted E. coli strains increase success rate of burdensome construct cloning

Genetic constructs meant for metabolic engineering in nonmodel microbes often use similar genetic parts to those familiar to E. coli work. The typical workflow is to clone these parts into plasmids in E. coli before they are transferred to the nonmodel host or its genome. In many cases, the metabolic burden of these constructs is stronger in the E. coli cloning phase of the workflow than in the eventual host, possibly resulting in mutation or other failure during cloning. Here, we apply generic knockdown of a range of popular expression systems, using CRISPR interference, by targeting guide RNAs to either promoters or RBSs that are commonly used in metabolic engineering. Generic targeting of a constitutive promoter series, combined with genome integration of CRISPR components, allows the use of only one or a few specific cloning strains to achieve strong knockdown of a wide range of constructs. Further, we present a recombinase-based workflow for easily adding guide RNAs with custom targets, so that users can knock down any desired promoter or ORF. Together, this group of strains comprises easy-to-use cloning strains meant for increasing success rates of difficult or burdensome cloning reactions, ultimately allowing more ambitious genetic constructs to reach their intended context.

synthetic biology↗

bioRxiv2026

A pooled screening approach reveals bacterial chemoreceptors for short-chain carboxylic acids

Bacterial chemotaxis is a key process in host colonization and virulence, mediated by large repertoires of chemoreceptors. Despite their physiological and ecological importance, mapping these chemoreceptors to their cognate metabolic ligands remains a major challenge due to the vast number of potential interactions. To address this, we developed a pooled screening assay that enriches functional chemoreceptors from a gene library. Using this approach, we identified a previously uncharacterized group of chemoreceptors in Pseudomonas species with Cache_3-Cache_2 domains that sense short-chain C3 carboxylic acids. Sequence and computational structural analyses revealed that these chemoreceptors exhibit domain features similar to a recently reported C1 formate chemoreceptor despite substantial sequence divergence. Functional assays of representative chemoreceptors confirmed robust chemotactic responses to C3 carboxylic acids, with limited responses to formate. Integrating structural and molecular dynamics analyses suggests that increased binding pocket size and altered flexibility, relative to the formate chemoreceptor, facilitate recognition of larger C3 carboxylic acid ligands. Together, our approach provides a simple and scalable framework for mapping ligand-chemoreceptor interactions and enables systematic characterization across diverse metabolites.

microbiology↗

bioRxiv2025

Crystal Structure of 3-Hydroxypropionyl-CoA Synthetase (ADP-forming) from Nitrosopumilus maritimus

The 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle in thaumarchaeota contributes significantly to global organic carbon fixation as the most energetically efficient aerobic carbon fixation pathway. The thaumarchaeal 3-Hydroxypropionyl-CoA Synthetase (ADP-forming; Nmar_1309) is crucial to this efficiency, utilizing ATP to ADP catalysis. This first reported structure of Nmar_1309 reveals a homodimer with a unique subdomain organization ([3-4-1-2-5]) and a distinct linker between subdomains 4 and 1. The presence of bound substrates including 3-hydroxypropionate, non-hydrolyzable ATP (ADPNP), and a phosphate suggests an intermediate state mimicking a reaction step immediately preceding the formation of a 3-hydroxypropionyl-phosphohistidine. Conformational differences were observed between the two chains of the homodimer, likely influenced by the binding of a single ADPNP molecule in one chain. Phylogenetic analysis suggests that while 4HB synthetases may have evolved earlier in the evolutionary timeline, 3HP synthetases in Thaumarchaeota may have occurred after the Great Oxygenation Event. These structural data provide further characterization of the 3HP/4HB cycle and, in conjunction with the structure of 4-hydroxybutyryl-CoA synthetase, Nmar_0206, provide baseline structures of the key ADP-forming Acyl-CoA synthetases within this pathway.

biophysics↗

bioRxiv2025

MAESTRO uncovers tandem paralog dynamics as a core driver of fungal stress adaptation

Understanding how fungi adapt to diverse stresses is critical for mitigating emerging drug resistance and harnessing their robustness for biotechnology. Pichia kudriavzevii is a stress-tolerant and intrinsically drug-resistant yeast, with dual industrial and clinical importance. We analyzed 170 strains using MAESTRO, a machine-learning-assisted GWAS pipeline optimized for small cohorts. MAESTRO identified biologically meaningful features and revealed that copy number variation (CNV) of tandem paralogs (TPs) is a core mechanism of multi-stress adaptation. TPs were recurrently linked to tolerance of industrial inhibitors (HMF, phenolics, heat) and antifungal drugs (fluconazole, azoles), and deletion of the TP pair gene4260/gene4261 confirmed pleiotropic effects across stresses. These findings support a TP CNV model where recombination-driven TP CNVs and gene fusions enable rapid stress adaptation. Importantly, our results suggest that antifungal resistance can arise through co-option of mechanisms originally evolved for environmental stressors, raising a One Health concern about the environmental origins of drug-resistant pathogens.

genomics↗

bioRxiv2025

Deciphering the altered conformational states of bifunctional thaumarchaeal crotonyl-CoA hydratase and 3-hydroxypropionyl-CoA dehydratase from Nitrosopumilus maritimus

The thaumarchaeal 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle represents one of the most efficient mechanisms for CO2 fixation discovered to date. Within this cycle, the enzyme encoded by Nmar_1308 from Nitrosopumilus maritimus SCM1 plays a crucial role due to its dual functionality as both a crotonyl-CoA hydratase (CCAH) and a 3-hydroxypropionyl-CoA dehydratase (3HPD). Although the importance of a bifunctional enzyme for lowering the cost of biosynthesis, the details of structural dynamics are still missing. Here, in addition to our cryogenic temperature structures, we determined the first ambient temperature structures of the Nmar_1308 protein by Serial Femtosecond X-ray Crystallography (SFX). The determined structures capture previously unobserved conformational dynamics of the Nmar_1308 protein, providing invaluable information for future synthetic biology applications.

biochemistry↗

bioRxiv2024

CRAGE-RB-PI-seq enables transcriptional profiling of rhizobacteria during plant-root colonization

Plant roots release a wide array of metabolites into the rhizosphere, shaping microbial communities and their functions. While metagenomics has expanded our understanding of these communities, little is known about the physiology of their members in host environments. Transcriptome analysis via RNA sequencing is a common approach to learning more, but its use has been challenging because plant RNA masks bacterial transcripts. To overcome this, we developed randomly-barcoded promoter-library insertion sequencing (RB-PI-seq) and combined it with chassis-independent recombinase assisted genome engineering (CRAGE), using Pseudomonas simiae WCS417 as a model rhizobacterium. This method enables targeted amplification of barcoded transcripts, bypassing plant RNA interference and allowing measurement of thousands of promoter activities during root colonization. Our analysis revealed time-resolved dynamics of promoter activities, highlighting early transcriptional reprogramming as a key determinant of successful colonization. Additionally, we discovered that transcriptional activation of xanthine dehydrogenase and a lysozyme inhibitor are crucial for evading plant immune system defenses. This framework is scalable to other bacterial species and provides new opportunities for understanding rhizobacterial gene regulation in native environments.

microbiology↗

bioRxiv2024

Synthetic genomes unveil the effects of synonymous recoding

Engineering the genetic code of an organism provides the basis for (i) making any organism safely resistant to natural viruses and (ii) preventing genetic information flow into and out of genetically modified organisms while (iii) allowing the biosynthesis of genetically encoded unnatural polymers1-4. Achieving these three goals requires the reassignment of multiple of the 64 codons nature uses to encode proteins. However, synonymous codon replacement--recoding--is frequently lethal, and how recoding impacts fitness remains poorly explored. Here, we explore these effects using whole-genome synthesis, multiplexed directed evolution, and genome-transcriptome-translatome-proteome co-profiling on multiple recoded genomes. Using this information, we assemble a synthetic Escherichia coli genome in seven sections using only 57 codons to encode proteins. By discovering the rules responsible for the lethality of synonymous recoding and developing a data-driven multi-omics-based genome construction workflow that troubleshoots synthetic genomes, we overcome the lethal effects of 62,007 synonymous codon swaps and 11,108 additional genomic edits. We show that synonymous recoding induces transcriptional noise including new antisense RNAs, leading to drastic transcriptome and proteome perturbation. As the elimination of select codons from an organisms genetic code results in the widespread appearance of cryptic promoters, we show that synonymous codon choice may naturally evolve to minimize transcriptional noise. Our work provides the first genome-scale description of how synonymous codon changes influence organismal fitness and paves the way for the construction of functional genomes that provide genetic firewalls from natural ecosystems and safely produce biopolymers, drugs, and enzymes with an expanded chemistry.

synthetic biology↗

bioRxiv2024

Functional plasticity of HCO3- uptake and CO2 fixation in Cupriavidus necator H16

Uptake and fixation of CO2 are central to strategies for CO2-based biomanufacturing. Cupriavidus necator H16 has emerged as a promising industrial host for this purpose. Despite its prominence, the ability to engineer C. necator inorganic carbon uptake and fixation is underexplored. Here, we test the role of endogenous and heterologous genes on C. necator inorganic carbon metabolism. Deletion of one of the four carbonic anhydrases in C. necator, {beta}-carbonic anhydrase can, had the most deleterious effect on C. necator autotrophic growth. Replacement of this native uptake system with several classes of dissolved inorganic carbon (DIC) transporters from Cyanobacteria and chemolithoautotrophic bacteria recovered autotrophic growth and supported higher cell densities compared to wild-type (WT) C. necator in saturating CO2 in batch culture. Several heterologous strains with Halothiobacillus neopolitanus DAB2 (hnDAB2) expressed from the chromosome in combination with diverse rubisco homologs grew in CO2 equally or better than the wild-type strain. Our experiments suggest that the primary role of Can carbonic anhydrase during autotrophic growth is for bicarbonate accumulation to support anaplerotic metabolism, and an array of DIC transporters can complement this function. This work demonstrates flexibility in HCO3- uptake and CO2 fixation in C. necator, providing new pathways for CO2-based biomanufacturing. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/593039v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@83c601org.highwire.dtl.DTLVardef@13ca34forg.highwire.dtl.DTLVardef@5b7f8org.highwire.dtl.DTLVardef@1710e48_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Label-free structural imaging of plant roots and microbes using third- harmonic generation microscopy

Root biology is pivotal in addressing global challenges including sustainable agriculture and climate change. However, roots have been relatively understudied among plant organs, partly due to the difficulties in imaging root structures in their natural environment. Here we used microfabricated ecosystems (EcoFABs) to establish growing environments with optical access and employed nonlinear multimodal microscopy of third-harmonic generation (THG) and three-photon fluorescence (3PF) to achieve label-free, in situ imaging of live roots and microbes at high spatiotemporal resolution. THG enabled us to observe key plant root structures including the vasculature, Casparian strips, dividing meristematic cells, and root cap cells, as well as subcellular features including nuclear envelopes, nucleoli, starch granules, and putative stress granules. THG from the cell walls of bacteria and fungi also provides label-free contrast for visualizing these microbes in the root rhizosphere. With simultaneously recorded 3PF fluorescence signal, we demonstrated our ability to investigate root-microbe interactions by achieving single-bacterium tracking and subcellular imaging of fungal spores and hyphae in the rhizosphere.

plant biology↗

bioRxiv2024

Multi-substrate specificity shaped the complex evolution of the aminotransferase family across the tree of life

Aminotransferases (ATs) are an ancient enzyme family that play central roles in core nitrogen metabolism essential to all organisms. However, many of the AT enzyme functions remain poorly defined, limiting our fundamental understanding of the nitrogen metabolic networks that exist in different organisms. Here we traced the deep evolutionary history of the AT family by analyzing AT enzymes from 90 species spanning the tree of life (ToL). We found that each organism has maintained a relatively small and constant number of ATs. Mapping the distribution of ATs across the ToL uncovered that many essential AT reactions are carried out by taxon-specific AT enzymes due to wide-spread non-orthologous gene displacements. This complex evolutionary history explains the difficulty of homology-based AT functional prediction. Biochemical characterizations of diverse aromatic ATs further revealed their broad substrate specificity, unlike other core metabolic enzymes that evolved to catalyze specific reactions today. Interestingly, however, we found that these AT enzymes that diverged over billion years share common signatures of multi-substrate specificity by employing different non-conserved active site residues. These findings illustrate that AT evolution had leveraged their inherent substrate promiscuity to maintain a small yet distinct set of multi-functional AT enzymes in different taxa. This evolutionary history of versatile ATs likely contributed to the establishment of robust and diverse nitrogen metabolic networks that exist throughout the ToL. The study provides a critical foundation to systematically determine diverse AT functions and underlying nitrogen metabolic networks across the ToL. Significance StatementThe ToL-wide analyses of the ubiquitous aminotransferases (AT) family revealed that the broad substrate promiscuity of ATs, which is unusual for core metabolic enzymes, allowed recruitment of distinct, non-orthologous ATs to carry out essential AT reactions in different taxa but without increasing their copy numbers. Some distantly related ATs were also found to exhibit a common signature of multi-substrate specificity by employing different non-conserved active site residues. The versatile evolutionary trajectory of the promiscuous AT enzyme family likely led to biochemical diversity of the robust nitrogen metabolic networks that exist among various extant organisms.

biochemistry↗

bioRxiv2023

Crystal Structure of 4-Hydroxybutyryl-CoA Synthetase (ADP-forming): A Key Enzyme in the Thaumarchaeal Hydroxypropionate/Hydroxybutyrate cycle

The 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle from ammonia-oxidizing Thaumarchaeota is currently considered the most energy-efficient aerobic carbon fixation pathway. The Nitrosopumilus maritimus 4-hydroxybutyryl-CoA synthetase (ADP-forming; Nmar_0206) represents one of several enzymes from this cycle that exhibit increased efficiency over crenarchaeal counterparts. This enzyme reduces energy requirements on the cell, reflecting thaumarchaeal success in adapting to low-nutrient environments. Here we show the structure of Nmar_0206 from Nitrosopumilus maritimus SCM1, which reveals a highly conserved interdomain linker loop between the CoA-binding and ATP-grasp domains. Phylogenetic analysis suggests the widespread prevalence of this loop and highlights both its underrepresentation within the PDB and structural importance within the (ATP-forming) acyl- CoA synthetase (ACD) superfamily. This linker is shown to have a possible influence on conserved interface interactions between domains, thereby influencing homodimer stability. These results provide a structural basis for the energy efficiency of this key enzyme in the modified 3HP/4HB cycle of Thaumarchaeota.

biophysics↗

bioRxiv2022

Proteome capacity constraints favor respiratory ATP generation

Cells face competing metabolic demands. These include efficient use of both limited substrates and limited proteome capacity, as well as flexibility to deal with different environments. Flexibility requires spare enzyme capacity, which is proteome inefficient. ATP generation can occur via fermentation or respiration. Fermentation is much less substrate-efficient, but often assumed to be more proteome efficient 1-3, thereby favoring fast-growing cells engaging in aerobic glycolysis 4-8. Here, however, we show that mitochondrial respiration is actually more proteome-efficient than aerobic glycolysis. Instead, aerobic glycolysis arises from cells maintaining the flexibility to grow also anaerobically. These conclusions emerged from an unbiased assessment of metabolic regulatory mechanisms, integrating quantitative metabolomics, proteomics, and fluxomics, of two budding yeasts, Saccharomyces cerevisiae and Issatchenkia orientalis, the former more fermentative and the latter respiratory. Their energy pathway usage is largely explained by differences in proteome allocation. Each organisms proteome allocation is remarkably stable across environmental conditions, with metabolic fluxes predominantly regulated at the level of metabolite concentrations. This leaves extensive spare biosynthetic capacity during slow growth and spare capacity of their preferred bioenergetic machinery when it is not essential. The greater proteome-efficiency of respiration is also observed in mammals, with aerobic glycolysis occurring in yeast or mammalian cells that maintain a fermentation-capable proteome conducive to both aerobic and anaerobic growth.

systems biology↗

bioRxiv2021

Structural Insights into Bifunctional Thaumarchaeal Crotonyl-CoA Hydratase and 3-Hydroxypropionyl-CoA Dehydratase from Nitrosopumilus maritimus

The ammonia-oxidizing thaumarchaeal 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle is one of the most energy-efficient CO2 fixation cycles discovered thus far. The protein encoded by Nmar_1308 (from Nitrosopumilus maritimus SCM1) is a promiscuous enzyme that catalyzes two essential reactions within the thaumarchaeal 3HP/4HB cycle, functioning as both a crotonyl-CoA hydratase (CCAH) and 3- hydroxypropionyl-CoA dehydratase (3HPD). In performing both hydratase and dehydratase activities, Nmar_1308 reduces the total number of enzymes necessary for CO2 fixation in Thaumarchaeota, reducing the overall cost for biosynthesis. Here, we present the first high-resolution crystal structure of this bifunctional enzyme with key catalytic residues in the thaumarchaeal 3HP/4HB pathway.

biochemistry↗

bioRxiv2020

Structural adaptation of oxygen tolerance in 4-hydroxybutyrl-CoA dehydratase, a key enzyme of archaeal carbon fixation

Autotrophic microorganisms that convert inorganic carbon into organic matter were key players in the evolution of life on Earth. As the early atmosphere became oxygenated, microorganisms needed to develop mechanisms for oxygen protection, especially those relying on enzymes containing oxygen-sensitive metal clusters (e.g., Fe-S). Here we investigated how 4-hydroxybutyryl-CoA dehydratase (4HBD) - the key enzyme of the 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle for CO2-fixation - adapted as conditions shifted from anoxic to oxic. 4HBD is found in both anaerobic bacteria and aerobic ammonia-oxidizing archaea (AOA). The oxygen-sensitive bacterial 4HBD and oxygen-tolerant archaeal 4HBD share 59 % amino acid identity. To examine the structural basis of oxygen tolerance in archaeal 4HBD, we determined the atomic resolution structure of the enzyme. Two tunnels providing access to the canonical [4Fe-4S] cluster in oxygen-sensitive bacterial 4HBD were closed with four conserved mutations found in all aerobic AOA and other archaea. Further biochemical experiments and molecular dynamics simulations support our findings that restricting access to the active site is the key to oxygen tolerance, explaining how active site evolution drove a major evolutionary transition. Significance statementAutotrophy (primary production) was the first life strategy on Earth. Before photosynthesis (using solar energy to fix carbon dioxide), life relied on chemical reactions for energy. These chemosynthetic reactions are present in all domains of life, including archaea possessing the most energy-efficient carbon fixation pathway - the 3-hydroxypropionate/4-hydroxybutyrate cycle. This efficiency results from enzyme modifications, including enhanced enzyme stability and catalysis of multiple reactions. We reveal the first structure of aerobic 4-hydroxybutyryl-CoA dehydratase (4HBD) from ammonia-oxidizing archaea. These archaea are among the most abundant organisms on the planet, and their 4HBD active site evolved oxygen tolerance to support aerobic metabolism. This modification can provide further insight into enzyme evolution on early earth, as photosynthesis developed and began oxygenating the atmosphere.

evolutionary biology↗

bioRxiv2019

A plant host enables the heterologous production and combinatorial study of fungal lignin-degrading enzymes

Lignin has significant potential as an abundant and renewable source for commodity chemicals yet remains vastly underutilized. Efforts towards engineering a biochemical route to the valorization of lignin are currently limited by the lack of a suitable heterologous host for the production of lignin-degrading enzymes. Here, we show that expression of fungal genes in Nicotiana benthamiana enables production of members from seven major classes of enzymes associated with lignin degradation (23 of 35 tested) in soluble form for direct use in lignin activity assays. We combinatorially characterized a subset of these enzymes in the context of model lignin dimer oxidation, revealing that fine-tuned coupling of peroxide-generators to peroxidases results in more extensive C-C bond cleavage compared to direct addition of peroxide. Comparison of peroxidase isoform activity revealed that the extent of C-C bond cleavage depends on peroxidase identity, suggesting that peroxidases are individually specialized in the context of lignin oxidation. We anticipate the use of N. benthamiana as a platform to rapidly produce a diverse array of fungal lignin-degrading enzymes will facilitate a better understanding of their concerted role in nature and unlock their potential for lignin valorization, including within the plant host itself.

bioengineering↗

bioRxiv2019

Coupled inter-subunit dynamics enable the fastest CO2-fixation by reductive carboxylases

Enoyl-CoA carboxylases/reductases (ECRs) belong to the most efficient CO2-fixing enzymes described to date. However, the molecular mechanisms underlying ECRs extraordinary catalytic activity on the level of the protein assembly remain elusive. Here we used a combination of ambient temperature X-ray Free Electron Laser (XFEL) and cryogenic synchrotron experiments to study the structural organization of the ECR from Kitasatospora setae. K. setae ECR is a homo-tetramer that differentiates into a dimer of dimers of open- and closed-form subunits in the catalytically active state. Using molecular dynamics simulations and structure-based mutagenesis, we show that catalysis is synchronized in K. setae ECR across the pair of two dimers. This conformational coupling of catalytic domains is conferred by individual amino acids to achieve high CO2-fixation rates. Our results provide unprecedented insights into the dynamic organization and synchronized inter- and intra-subunit communications of this remarkably efficient CO2-fixing enzyme during catalysis. Significance StatementFixation of CO2 offers real potential for reaching negative CO2 emissions in bioenergy, and bioproduct utilization. The capture and conversion of atmospheric CO2 remains a challenging task. Existing biological systems can be exploited and optimized for this use. Bacterial enoyl-CoA carboxylases/reductases (ECRs) encompass the fastest CO2-fixing enzymes found in nature to date. However, the mechanisms underlying ECRs extraordinary catalytic activity remain elusive. Our structural, computational, and biochemical results elucidate the dynamic structural organization of the ECR complex and describe how coupled motions of catalytic domains in the ECR tetramer drive carboxylation. This mechanistic understanding is critical for engineering highly efficient CO2-fixing biocatalysts for bioenergy and bioproduct applications.

biophysics↗

bioRxiv2018

Validating genome-wide CRISPR-Cas9 function in the non-conventional yeast Yarrowia lipolytica

Genome-wide mutational screens are central to understanding the genetic underpinnings of evolved and engineered phenotypes. The widespread adoption of CRISPR-Cas9 genome editing has enabled such screens in many organisms, but identifying functional sgRNAs still remains a challenge. To address this limitation, we developed a methodology to quantify the cutting efficiency of each sgRNA in a genome-scale library in the biotechnologically important yeast Yarrowia lipolytica. Screening in the presence and absence of native DNA repair enabled high-throughput quantification of sgRNA function leading to the identification of high efficiency sgRNAs that cover 94% of genes. Library validation enhanced the classification of essential genes by identifying inactive guides that create false negatives and mask the effects of successful disruptions. Quantification of guide effectiveness also creates a dataset from which functional determinants of CRISPR-Cas9 can be identified. Finally, application of the library identified mutations that led to high lipid accumulation and eliminated pseudohyphal morphology.

synthetic biology↗