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DOE OSTI2024

RNA language models predict mutations that improve RNA function

Structured RNA lies at the heart of many central biological processes, from gene expression to catalysis. RNA structure prediction is not yet possible due to a lack of high-quality reference data associated with organismal phenotypes that could inform RNA function. We present GARNET (Gtdb Acquired RNa with Environmental Temperatures), a new database for RNA structural and functional analysis anchored to the Genome Taxonomy Database (GTDB). GARNET links RNA sequences to experimental and predicted optimal growth temperatures of GTDB reference organisms. Using GARNET, we develop sequence- and structure-aware RNA generative models, with overlapping triplet tokenization providing optimal encoding for a GPT-like model. Leveraging hyperthermophilic RNAs in GARNET and these RNA generative models, we identify mutations in ribosomal RNA that confer increased thermostability to the Escherichia coli ribosome. The GTDB-derived data and deep learning models presented here provide a foundation for understanding the connections between RNA sequence, structure, and function.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Human protein synthesis requires aminoacyl-tRNA pivoting during proofreading

Abstract Rigorous studies have characterized the aa-tRNA selection mechanism in bacteria, which is essential for maintaining translational fidelity. Recent investigations have identified critical distinctions in humans, such as the requirement of subunit rolling and a tenfold slower proofreading step. Although these studies captured key intermediates involved in tRNA selection, they did not elucidate the transitions of aa-tRNA between intermediates. Through diverse structure-based simulations, we simulated 1856 aa-tRNA accommodation events into the human ribosomal A site. Here we show the requirement for a distinct ~30° pivoting of aa-tRNA about the anticodon stem within the accommodation corridor. This pivoting is crucial for navigating the crowded accommodation corridor, which becomes more constrained due to subunit rolling. Subunit rolling-dependent crowding increases the steric contributions of the accommodation corridor during aa-tRNA accommodation, consistent with the tenfold reduction in the rate of proofreading. Furthermore, we show that eEF1A interacts with the accommodating aa-tRNA through conserved basic residues, limiting premature aa-tRNA dissociation from the A site. These findings provide a structural description of the human aa-tRNA selection process and demonstrate that the aa-tRNA alignment relative to the ribosomal catalytic sites is a critical determinant of translational fidelity.

Science & Technology - Other Topics↗

DOE OSTI2019

CRAGE enables rapid activation of biosynthetic gene clusters in undomesticated bacteria

Advances in the prediction of biosynthetic gene clusters (BGCs) from genomic and metagenomic data have made the large-scale identification of candidate secondary metabolite biosynthesis pathways routine. However, most predicted BGCs cannot be successfully expressed in the limited range of expression hosts currently available, which prevents the identification and characterization of their respective products. Here we show that parallelized use of a phylogenetically and physiologically diverse panel of purpose-engineered chassis strains offers a versatile strategy for the rapid activation of BGCs. We domesticated an ensemble of 25 diverse ?-proteobacteria from 11 different genera by integrating a unified landing pad that enables efficient introduction of large BGCs into their genome. We deployed this chassis-independent recombinase-assisted genome engineering (CRAGE) strategy to express several previously posited but experimentally elusive non-ribosomal peptide synthase (NRPS) and NRPSpolyketide synthase (PKS) hybrid BGCs from Photorhabdus luminescens. Successful activation of 6 BGCs identified 21 products, including several with likely roles in host-microbe interactions. Taken together, we illustrate how the regulatory and physiological diversity naturally present across bacteria can be harnessed for the exploration of novel bioproducts and provide a sizable panel of domesticated chassis strains to enable this strategy.

applied microbiology, genetic engineering, industr↗

DOE OSTI2024

CryoTRANS: predicting high-resolution maps of rare conformations from self-supervised trajectories in cryo-EM

Cryogenic electron microscopy (cryo-EM) has revolutionized structural biology, enabling efficient determination of structures at near-atomic resolutions. However, a common challenge arises from the severe imbalance among various conformations of vitrified particles, leading to low-resolution reconstructions in rare conformations due to a lack of particle images in these quasi-stable states. We introduce CryoTRANS, a method that predicts high-resolution maps of rare conformations by constructing a self-supervised pseudo-trajectory between density maps of varying resolutions. This trajectory is represented by an ordinary differential equation parameterized by a deep neural network, ensuring retention of detailed structures from high-resolution density maps. By leveraging a single high-resolution density map, CryoTRANS significantly improves the reconstruction of rare conformations and has been validated on four real-world datasets: alpha-2-macroglobulin, actin-binding protein complexes, SARS-CoV-2 spike glycoprotein, and the 70S ribosome. CryoTRANS can also predict high-resolution structures in cryogenic electron tomography maps using a high-resolution cryo-EM map.Cryogenic electron microscopy (cryo-EM) has revolutionized structural biology, enabling efficient determination of structures at near-atomic resolutions. However, a common challenge arises from the severe imbalance among various conformations of vitrified particles, leading to low-resolution reconstructions in rare conformations due to a lack of particle images in these quasi-stable states. We introduce CryoTRANS, a method that predicts high-resolution maps of rare conformations by constructing a self-supervised pseudo-trajectory between density maps of varying resolutions. This trajectory is represented by an ordinary differential equation parameterized by a deep neural network, ensuring retention of detailed structures from high-resolution density maps. By leveraging a single high-resolution density map, CryoTRANS significantly improves the reconstruction of rare conformations and has been validated on four real-world datasets: alpha-2-macroglobulin, actin-binding protein complexes, SARS-CoV-2 spike glycoprotein, and the 70S ribosome. CryoTRANS can also predict high-resolution structures in cryogenic electron tomography maps using a high-resolution cryo-EM map.

47 OTHER INSTRUMENTATION↗

DOE OSTI2025

Cell-free synthetic biology for natural product biosynthesis and discovery

Natural products have applications as biopharmaceuticals, agrochemicals, and other high-value chemicals. However, there are challenges in isolating natural products from their native producers (e.g. bacteria, fungi, plants). In many cases, synthetic chemistry or heterologous expression must be used to access these important molecules. The biosynthetic machinery to generate these compounds is found within biosynthetic gene clusters, primarily consisting of the enzymes that biosynthesise a range of natural product classes (including, but not limited to ribosomal and nonribosomal peptides, polyketides, and terpenoids). Cell-free synthetic biology has emerged in recent years as a bottom-up technology applied towards both prototyping pathways and producing molecules. Recently, it has been applied to natural products, both to characterise biosynthetic pathways and produce new metabolites. This review discusses the core biochemistry of cell-free synthetic biology applied to metabolite production and critiques its advantages and disadvantages compared to whole cell and/or chemical production routes. Specifically, we review the advances in cell-free biosynthesis of ribosomal peptides, analyse the rapid prototyping of natural product biosynthetic enzymes and pathways, highlight advances in novel antimicrobial discovery, and discuss the rising use of cell-free technologies in industrial biotechnology and synthetic biology.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2017

Structures of the peptide-modifying radical SAM enzyme SuiB elucidate the basis of substrate recognition

Posttranslational modification of ribosomally synthesized peptides provides an elegant means for the production of biologically active molecules known as RiPPs (ribosomally synthesized and posttranslationally modified peptides). Although the leader sequence of the precursor peptide is often required for turnover, the exact mode of recognition by the modifying enzymes remains unclear for many members of this class of natural products. In this report, we have used X-ray crystallography and computational modeling to examine the role of the leader peptide in the biosynthesis of a homolog of streptide, a recently identified peptide natural product with an intramolecular lysine–tryptophan cross-link, which is installed by the radical S -adenosylmethionine (SAM) enzyme, StrB. We present crystal structures of SuiB, a close ortholog of StrB, in various forms, including apo SuiB, SAM-bound SuiB, and a complex of SuiB with SAM and its peptide substrate, SuiA. Although the N-terminal domain of SuiB adopts a typical RRE (RiPP recognition element) motif, which has been implicated in precursor peptide recognition, we observe binding of the leader peptide in the catalytic barrel rather than the N-terminal domain. Computational simulations support a mechanism in which the leader peptide guides posttranslational modification by positioning the cross-linking residues of the precursor peptide within the active site. Together the results shed light onto binding of the precursor peptide and the associated conformational changes needed for the formation of the unique carbon–carbon cross-link in the streptide family of natural products.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2018

Crystal structure of the DENR-MCT-1 complex revealed zinc-binding site essential for heterodimer formation

The density-regulated protein (DENR) and the malignant T cell-amplified sequence 1 (MCT-1/MCTS1) oncoprotein support noncanonical translation initiation, promote translation reinitiation on a specific set of mRNAs with short upstream reading frames, and regulate ribosome recycling. DENR and MCT-1 form a heterodimer, which binds to the ribosome. In this work, we determined the crystal structure of the heterodimer formed by human MCT-1 and the N-terminal domain of DENR at 2.0-Å resolution. The structure of the heterodimer reveals atomic details of the mechanism of DENR and MCT-1 interaction. Four conserved cysteine residues of DENR (C34, C37, C44, C53) form a classical tetrahedral zinc ion-binding site, which preserves the structure of the DENR’s MCT-1–binding interface that is essential for the dimerization. Substitution of all four cysteines by alanine abolished a heterodimer formation. Our findings elucidate further the mechanism of regulation of DENR-MCT-1 activities in unconventional translation initiation, reinitiation, and recycling.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Cryogenic single-molecule fluorescence annotations for electron tomography reveal in situ organization of key proteins in Caulobacter

Superresolution fluorescence microscopy and cryogenic electron tomography (CET) are powerful imaging methods for exploring the subcellular organization of biomolecules. Superresolution fluorescence microscopy based on covalent labeling highlights specific proteins and has sufficient sensitivity to observe single fluorescent molecules, but the reconstructions lack detailed cellular context. CET has molecular-scale resolution but lacks specific and nonperturbative intracellular labeling techniques. Here, we describe an imaging scheme that correlates cryogenic single-molecule fluorescence localizations with CET reconstructions. Our approach achieves single-molecule localizations with an average lateral precision of 9 nm, and a relative registration error between the set of localizations and CET reconstruction of ∼30 nm. We illustrate the workflow by annotating the positions of three proteins in the bacterium Caulobacter crescentus : McpA, PopZ, and SpmX. McpA, which forms a part of the chemoreceptor array, acts as a validation structure by being visible under both imaging modalities. In contrast, PopZ and SpmX cannot be directly identified in CET. While not directly discernable, PopZ fills a region at the cell poles that is devoid of electron-dense ribosomes. We annotate the position of PopZ with single-molecule localizations and confirm its position within the ribosome excluded region. We further use the locations of PopZ to provide context for localizations of SpmX, a low-copy integral membrane protein sequestered by PopZ as part of a signaling pathway that leads to an asymmetric cell division. Our correlative approach reveals that SpmX localizes along one side of the cell pole and its extent closely matches that of the PopZ region.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2024

Gcn2 structurally mimics and functionally repurposes the HisRS enzyme for the integrated stress response

Protein kinase Gcn2 attenuates protein synthesis in response to amino acid starvation while stimulating translation of a transcriptional activator of amino acid biosynthesis. Gcn2 activation requires a domain related to histidyl-tRNA synthetase (HisRS), the enzyme that aminoacylates tRNA His . While evidence suggests that deacylated tRNA binds the HisRS domain for kinase activation, ribosomal P-stalk proteins have been implicated as alternative activating ligands on stalled ribosomes. We report crystal structures of the HisRS domain ofChaetomium thermophilumGcn2 that reveal structural mimicry of both catalytic (CD) and anticodon-binding (ABD) domains, which in authentic HisRS bind the acceptor stem and anticodon loop of tRNA His . Elements for forming histidyl adenylate and aminoacylation are lacking, suggesting that Gcn2 HisRS was repurposed for kinase activation, consistent with mutations in the CD that dysregulate yeast Gcn2 function. Substituting conserved ABD residues well positioned to contact the anticodon loop or that form a conserved ABD–CD interface impairs Gcn2 function in starved cells. Mimicry in Gcn2 HisRS of two highly conserved structural domains for binding both ends of tRNA—each crucial for Gcn2 function—supports that deacylated tRNAs activate Gcn2 and exemplifies how a metabolic enzyme is repurposed to host new local structures and sequences that confer a novel regulatory function.

Science & Technology - Other Topics↗

DOE OSTI2015

The La-related protein 1-specific domain repurposes HEAT-like repeats to directly bind a 5'TOP sequence

La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5'TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5' UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5'TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. A putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. Ultimately, these studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signalling to ribosome biogenesis.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Revisiting the growth rate hypothesis: Towards a holistic stoichiometric understanding of growth

The growth rate hypothesis (GRH) posits that variation in organismal stoichiometry (C:P and N:P ratios) is driven by growth-dependent allocation of P to ribosomal RNA. The GRH has found broad but not uniform support in studies across diverse biota and habitats. We synthesise information on how and why the tripartite growth-RNA-P relationship predicted by the GRH may be uncoupled and outline paths for both theoretical and empirical work needed to broaden the working domain of the GRH. We found strong support for growth to RNA (r 2 = 0.59) and RNA-P to P (r 2 = 0.63) relationships across taxa, but growth to P relationships were relatively weaker (r 2 = 0.09). Together, the GRH was supported in ~50% of studies. Mechanisms behind GRH uncoupling were diverse but could generally be attributed to physiological (P accumulation in non-RNA pools, inactive ribosomes, translation elongation rates and protein turnover rates), ecological (limitation by resources other than P), and evolutionary (adaptation to different nutrient supply regimes) causes. These factors should be accounted for in empirical tests of the GRH and formalised mathematically to facilitate a predictive understanding of growth.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2018

Physiological and proteomic analyses of Fe(III)-reducing co-cultures of Desulfotomaculum reducens MI-1 and Geobacter sulfurreducens PCA

We established Fe(III)-reducing co-cultures of two species of metal-reducing bacteria, the Gram-positive Desulfotomaculum reducens MI-1 and the Gram-negative Geobacter sulfurreducens PCA. Co-cultures were given pyruvate, a substrate that D. reducens can ferment and use as electron donor for Fe(III) reduction. G. sulfurreducens, which does not utilize pyruvate, relied upon products of pyruvate oxidation by D. reducens (acetate, H2) for use as electron donor in the co-culture. Fe(III) was reduced more quickly in co-cultures relative to pure cultures, and powder X-ray diffraction evidence suggests that both organisms contribute to Fe(III) reduction. Global comparative proteomic analysis was performed in order to observe differential protein abundance during co-culture versus pure culture growth. Proteins previously associated with Fe(III) reduction in G. sulfurreducens, namely c-type cytochromes and type IV pili proteins, were significantly increased in abundance in co-cultures relative to pure cultures. Dreducens ribosomal proteins were significantly increased in co-cultures, likely a reflection of faster growth rates observed for D. reducens cells while in co-culture. Furthermore, we developed multiple reaction monitoring (MRM) assays to quantitate specific biomarker peptides. The assays were validated in pure and co-cultures, and protein abundance ratios from targeted MRM and global proteomic analysis correlate strongly.

Otwell, Annie E.↗

DOE OSTI2012

Extensive gene flow over Europe and possible speciation

Biogeographical patterns and large-scale genetic structure have been little studied in ectomycorrhizal (EM) fungi, despite the ecological and economic importance of EM symbioses. We coupled population genetics and phylogenetic approaches to understand spatial structure in fungal populations on a continental scale. Using nine microsatellite markers, we characterized gene flow among 16 populations of the widespread EM basidiomycete Laccaria amethystina over Europe (i.e. over 2900 km). We also widened our scope to two additional populations from Japan (104 km away) and compared them with European populations through microsatellite markers and multilocus phylogenies, using three nuclear genes (NAR, G6PD and ribosomal DNA) and two mitochondrial ribosomal genes. European L. amethystina populations displayed limited differentiation (average FST = 0.041) and very weak isolation by distance (IBD). This panmictic European pattern may result from effective aerial dispersal of spores, high genetic diversity in populations and mutualistic interactions with multiple hosts that all facilitate migration. The multilocus phylogeny based on nuclear genes confirmed that Japanese and European specimens were closely related but clustered on a geographical basis. By using microsatellite markers, we found that Japanese populations were strongly differentiated from the European populations (FST = 0.416), more than expected by extrapolating the European pattern of IBD. Population structure analyses clearly separated the populations into two clusters, i.e. European and Japanese clusters. We discuss the possibility of IBD in a continuous population (considering some evidence for a ring species over the Northern Hemisphere) vs. an allopatric speciation over Eurasia, making L. amethystina a promising model of intercontinental species for future studies.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2018

Amplification and adaptation of centromeric repeats in polyploid switchgrass species

Centromeres in most higher eukaryotes are composed of long arrays of satellite repeats from a single satellite repeat family. Why centromeres are dominated by a single satellite repeat and how the satellite repeats originate and evolve are among the most intriguing and long-standing questions in centromere biology. We identified eight satellite repeats in the centromeres of tetraploid switchgrass (Panicum virgatum). Seven repeats showed characteristics associated with classical centromeric repeats with monomeric lengths ranging from 166 to 187 bp. Interestingly, these repeats share an 80-bp DNA motif. We demonstrate that this 80-bp motif may dictate translational and rotational phasing of the centromeric repeats with the cenH3 nucleosomes. The sequence of the last centromeric repeat, Pv156, is identical to the 5S ribosomal RNA genes. We demonstrate that a 5S ribosomal RNA gene array was recruited to be the functional centromere for one of the switchgrass chromosomes. Our findings reveal that certain types of satellite repeats, which are associated with unique sequence features and are composed of monomers in mono-nucleosomal length, are favorable for centromeres. Centromeric repeats may undergo dynamic amplification and adaptation before the centromeres in the same species become dominated by the best adapted satellite repeat.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Quantitative Proteome Profiling Reveals Cellobiose-Dependent Protein Processing and Export Pathways for the Lignocellulolytic Response in Neurospora crassa

Filamentous fungi are intensively used for producing industrial enzymes, including lignocellulases. Employing insoluble cellulose to induce the production of lignocellulases causes some drawbacks, e.g., a complex fermentation operation, which can be overcome by using soluble inducers such as cellobiose. Here, a triple β-glucosidase mutant of Neurospora crassa, which prevents rapid turnover of cellobiose and thus allows the disaccharide to induce lignocellulases, was applied to profile the proteome responses to cellobiose and cellulose (Avicel). Our results revealed a shared proteomic response to cellobiose and Avicel, whose elements included lignocellulases and cellulolytic product transporters. While the cellulolytic proteins showed a correlated increase in protein and mRNA levels, only a moderate correlation was observed on a proteomic scale between protein and mRNA levels (R 2 = 0.31). Ribosome biogenesis and rRNA processing were significantly overrepresented in the protein set with increased protein but unchanged mRNA abundances in response to Avicel. Ribosome biogenesis, as well as protein processing and protein export, was also enriched in the protein set that showed increased abundance in response to cellobiose. NCU05895, a homolog of yeast CWH43, is potentially involved in transferring a glycosylphosphatidylinositol (GPI) anchor to nascent proteins. This protein showed increased abundance but no significant change in mRNA levels. Disruption of CWH43 resulted in a significant decrease in cellulase activities and secreted protein levels in cultures grown on Avicel, suggesting a positive regulatory role for CWH43 in cellulase production. The findings should have an impact on a systems engineering approach for strain improvement for the production of lignocellulases.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2014

Benchmarking of Methods for Genomic Taxonomy

One of the first issues that emerges when a prokaryotic organism of interest is encountered is the question of what it is—that is, which species it is. The 16S rRNA gene formed the basis of the first method for sequence-based taxonomy and has had a tremendous impact on the field of microbiology. Nevertheless, the method has been found to have a number of shortcomings. In this paper, we trained and benchmarked five methods for whole-genome sequence-based prokaryotic species identification on a common data set of complete genomes: (i) SpeciesFinder, which is based on the complete 16S rRNA gene; (ii) Reads2Type that searches for species-specific 50-mers in either the 16S rRNA gene or the gyrB gene (for the Enterobacteraceae family); (iii) the ribosomal multilocus sequence typing (rMLST) method that samples up to 53 ribosomal genes; (iv) TaxonomyFinder, which is based on species-specific functional protein domain profiles; and finally (v) KmerFinder, which examines the number of cooccurring k-mers (substrings of k nucleotides in DNA sequence data). The performances of the methods were subsequently evaluated on three data sets of short sequence reads or draft genomes from public databases. In total, the evaluation sets constituted sequence data from more than 11,000 isolates covering 159 genera and 243 species. Our results indicate that methods that sample only chromosomal, core genes have difficulties in distinguishing closely related species which only recently diverged. Finally, the KmerFinder method had the overall highest accuracy and correctly identified from 93% to 97% of the isolates in the evaluations sets.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

maranasgroup/scRBA

Saccharomyces cerevisiae is an important model organism and a workhorse in bioproduction. Here, we reconstructed a compact and tractable genome-scale resource balance analysis (RBA) model (i.e., named scRBA) to analyze metabolic fluxes and proteome allocation in a computationally efficient manner. Resource capacity models such as scRBA provide the quantitative means to identify bottlenecks in biosynthetic pathways due to enzyme, compartment size, and/or ribosome availability limitations. ATP maintenance rate and in vivo apparent turnover numbers (kapp) were regressed from metabolic flux and protein concentration data to capture observed physiological growth yield and proteome efficiency and allocation, respectively. Estimated parameter values were found to vary with oxygen and nutrient availability. Overall, this work (i) provides condition-specific model parameters to recapitulate phenotypes corresponding to different extracellular environments, (ii) alludes to the enhancing effect of substrate channeling and post-translational activation on in vivo enzyme efficiency in glycolysis and electron transport chain, and (iii) reveals that the Crabtree effect is underpinned by specific limitations in mitochondrial proteome capacity and secondarily ribosome availability rather than overall proteome capacity.

Dinh, Hoang↗

DOE OSTI2013

Development of a broad-host synthetic biology toolbox for Ralstonia eutropha and its application to engineering hydrocarbon biofuel production

Background: The chemoautotrophic bacterium Ralstonia eutropha can utilize H 2 /CO 2 for growth under aerobic conditions. While this microbial host has great potential to be engineered to produce desired compounds (beyond polyhydroxybutyrate) directly from CO 2 , little work has been done to develop genetic part libraries to enable such endeavors. Results: We report the development of a toolbox for the metabolic engineering of Ralstonia eutropha H16. We have constructed a set of broad-host-range plasmids bearing a variety of origins of replication, promoters, 5' mRNA stem-loop structures, and ribosomal binding sites. Specifically, we analyzed the origins of replication pCM62 (IncP), pBBR1, pKT (IncQ), and their variants. We tested the promoters P BAD , T7, P xyls/PM , P lacUV5 , and variants thereof for inducible expression. We also evaluated a T7 mRNA stem-loop structure sequence and compared a set of ribosomal binding site (RBS) sequences derived from Escherichia coli, R. eutropha, and a computational RBS design tool. Finally, we employed the toolbox to optimize hydrocarbon production in R. eutropha and demonstrated a 6-fold titer improvement using the appropriate combination of parts. Conclusion: We constructed and evaluated a versatile synthetic biology toolbox for Ralstonia eutropha metabolic engineering that could apply to other microbial hosts as well.

59 BASIC BIOLOGICAL SCIENCES↗