Beverly Fu

All Publications

• CUPID-seq enables highly multiplexed amplicon sequencing via combinatorial in-line dual indexing. bioRxiv : the preprint server for biology Fu, B., Porter, R. L., Shi, H., Ea, A. C., Espeleta, A. M., Ambat, A., Relman, D. A., Huang, K. C., Xue, K. S. 2026

  Abstract

  Targeted amplicon sequencing is widely used to profile genetic variation in defined genomic regions. In microbial ecology, for example, amplicon sequencing of the 16S and 18S ribosomal RNA genes has been transformative for characterizing microbial communities. However, on high-capacity sequencing platforms with patterned flow cells, throughput is constrained by the requirement for unique dual indexes (UDIs), which increases primer costs and limits the number of samples that can be pooled per sequencing run. Here, we introduce CUPID-seq (Combinatorial, Unique, Phased, In-line Dual-indexed sequencing), a highly multiplexed amplicon-sequencing strategy that increases scalability through combinatorial indexing across two rounds of PCR. CUPID-seq introduces phased, in-line UDIs during Round 1 gene-specific amplification, enabling multiple samples to share the same Illumina UDI during Round 2 PCR while remaining uniquely identifiable. This design reduces upfront costs by up to 85% and reduces library preparation time and reagent use by up to 40%. We develop and validate CUPID-seq primers targeting the 16S V4 region and provide a computational workflow for demultiplexing in-line indexes. Although optimized here for 16S-based profiling, CUPID-seq can be readily adapted to other user-defined amplicons. By reducing cost and increasing multiplexing capacity, CUPID-seq enables users to leverage high-throughput sequencing platforms more effectively across diverse biological contexts.

  View details for DOI 10.64898/2026.05.20.726713

  View details for PubMedID 42239137

  View details for PubMedCentralID PMC13228431

• Discovery of a New Class of Aminoacyl Radical Enzymes Expands Nature's Known Radical Chemistry. Journal of the American Chemical Society Fu, B., Yang, H., Kountz, D. J., Lundahl, M. N., Beller, H. R., Broderick, W. E., Broderick, J. B., Hoffman, B. H., Balskus, E. P. 2024

  Abstract

  Radical enzymes, including the evolutionarily ancient glycyl radical enzyme (GRE) family, catalyze chemically challenging reactions that are involved in a myriad of important biological processes. All GREs possess an essential, conserved backbone glycine that forms a stable catalytically essential alpha-carbon radical. Through close examination of the GRE family, we unexpectedly identified hundreds of noncanonical GRE homologues that encode either an alanine, serine, or threonine in place of the catalytic glycine residue. Contrary to a long-standing belief, we experimentally demonstrate that these aminoacyl radical enzymes (AAREs) form stable alpha-carbon radicals on the three cognate residues when activated by partner activating enzymes. The previously unrecognized AAREs are widespread in microbial genomes, highlighting their biological importance and potential for exhibiting new reactivity. Collectively, these studies expand the known radical chemistry of living systems while raising questions about the evolutionary emergence of the AAREs.

  View details for DOI 10.1021/jacs.4c10348

  View details for PubMedID 39392720

• A cellular platform for production of C₄ monomers CHEMICAL SCIENCE Davis, M. A., Yu, V., Fu, B., Wen, M., Koleski, E. J., Silverman, J., Berdan, C. A., Nomura, D. K., Chang, M. C. Y. 2023

  View details for DOI 10.1039/d3sc02773b

  View details for Web of Science ID 001081057900001

• Mechanistic Studies of a Skatole-Forming Glycyl Radical Enzyme Suggest Reaction Initiation via Hydrogen Atom Transfer JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Fu, B., Nazemi, A., Levin, B. J., Yang, Z., Kulik, H. J., Balskus, E. P. 2022; 144 (25): 11110-11119

  Abstract

  Gut microbial decarboxylation of amino acid-derived arylacetates is a chemically challenging enzymatic transformation which generates small molecules that impact host physiology. The glycyl radical enzyme (GRE) indoleacetate decarboxylase from Olsenella uli (Ou IAD) performs the non-oxidative radical decarboxylation of indole-3-acetate (I3A) to yield skatole, a disease-associated metabolite produced in the guts of swine and ruminants. Despite the importance of IAD, our understanding of its mechanism is limited. Here, we characterize the mechanism of Ou IAD, evaluating previously proposed hypotheses of: (1) a Kolbe-type decarboxylation reaction involving an initial 1-e- oxidation of the carboxylate of I3A or (2) a hydrogen atom abstraction from the α-carbon of I3A to generate an initial carbon-centered radical. Site-directed mutagenesis, kinetic isotope effect experiments, analysis of reactions performed in D2O, and computational modeling are consistent with a mechanism involving initial hydrogen atom transfer. This finding expands the types of radical mechanisms employed by GRE decarboxylases and non-oxidative decarboxylases, more broadly. Elucidating the mechanism of IAD decarboxylation enhances our understanding of radical enzymes and may inform downstream efforts to modulate this disease-associated metabolism.

  View details for DOI 10.1021/jacs.1c13580

  View details for Web of Science ID 000819328900001

  View details for PubMedID 35704859

  View details for PubMedCentralID PMC9248008

• Elucidation of an anaerobic pathway for metabolism of L-carnitine-derived γ-butyrobetaine to trimethylamine in human gut bacteria PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rajakovich, L. J., Fu, B., Bollenbach, M., Balskus, E. P. 2021; 118 (32)

  Abstract

  Trimethylamine (TMA) is an important gut microbial metabolite strongly associated with human disease. There are prominent gaps in our understanding of how TMA is produced from the essential dietary nutrient l-carnitine, particularly in the anoxic environment of the human gut where oxygen-dependent l-carnitine-metabolizing enzymes are likely inactive. Here, we elucidate the chemical and genetic basis for anaerobic TMA generation from the l-carnitine-derived metabolite γ-butyrobetaine (γbb) by the human gut bacterium Emergencia timonensis We identify a set of genes up-regulated by γbb and demonstrate that the enzymes encoded by the induced γbb utilization (bbu) gene cluster convert γbb to TMA. The key TMA-generating step is catalyzed by a previously unknown type of TMA-lyase enzyme that utilizes a putative flavin cofactor to catalyze a redox-neutral transformation. We identify additional cultured and uncultured host-associated bacteria that possess the bbu gene cluster, providing insights into the distribution of anaerobic γbb metabolism. Lastly, we present genetic, transcriptional, and metabolomic evidence that confirms the relevance of this metabolic pathway in the human gut microbiota. These analyses indicate that the anaerobic pathway is a more substantial contributor to TMA generation from l-carnitine in the human gut than the previously proposed aerobic pathway. The discovery and characterization of the bbu pathway provides the critical missing link in anaerobic metabolism of l-carnitine to TMA, enabling investigation into the connection between this microbial function and human disease.

  View details for DOI 10.1073/pnas.2101498118

  View details for Web of Science ID 000685043400019

  View details for PubMedID 34362844

  View details for PubMedCentralID PMC8364193

• Discovery of C-C bond-forming and bond-breaking radical enzymes: enabling transformations for metabolic engineering CURRENT OPINION IN BIOTECHNOLOGY Fu, B., Balskus, E. P. 2020; 65: 94-101

  Abstract

  Radical enzymes catalyze some of the most chemically challenging CC bond-forming and bond-breaking reactions. Advances in DNA sequencing have accelerated the discovery of radical enzymes from microbes, including radical S-adenosylmethionine (rSAM) enzymes, glycyl radical enzymes (GREs), and diiron enzymes. These enzymes catalyze various reactions that yield products of industrial relevance (e.g. aromatics, hydrocarbons, and natural product derivatives), making their incorporation into engineered metabolic pathways enticing. Elucidating the mechanisms of radical enzymes that cleave and construct CC bonds will enable further enzyme discovery and engineering efforts.

  View details for DOI 10.1016/j.copbio.2020.02.003

  View details for Web of Science ID 000589902000014

  View details for PubMedID 32171888

  View details for PubMedCentralID PMC7670169

• Cholesterol Metabolism by Uncultured Human Gut Bacteria Influences Host Cholesterol Level CELL HOST & MICROBE Kenny, D. J., Plichta, D. R., Shungin, D., Koppel, N., Hall, A., Fu, B., Vasan, R. S., Shaw, S. Y., Vlamakis, H., Balskus, E. P., Xavier, R. J. 2020; 28 (2): 245-+

  Abstract

  The human microbiome encodes extensive metabolic capabilities, but our understanding of the mechanisms linking gut microbes to human metabolism remains limited. Here, we focus on the conversion of cholesterol to the poorly absorbed sterol coprostanol by the gut microbiota to develop a framework for the identification of functional enzymes and microbes. By integrating paired metagenomics and metabolomics data from existing cohorts with biochemical knowledge and experimentation, we predict and validate a group of microbial cholesterol dehydrogenases that contribute to coprostanol formation. These enzymes are encoded by ismA genes in a clade of uncultured microorganisms, which are prevalent in geographically diverse human cohorts. Individuals harboring coprostanol-forming microbes have significantly lower fecal cholesterol levels and lower serum total cholesterol with effects comparable to those attributed to variations in lipid homeostasis genes. Thus, cholesterol metabolism by these microbes may play important roles in reducing intestinal and serum cholesterol concentrations, directly impacting human health.

  View details for DOI 10.1016/j.chom.2020.05.013

  View details for Web of Science ID 000560392200013

  View details for PubMedID 32544460

  View details for PubMedCentralID PMC7435688

• Structural and Biochemical Studies of Substrate Selectivity in <i>Ascaris suum</i> Thiolases BIOCHEMISTRY Blaisse, M. R., Fu, B., Chang, M. C. Y. 2018; 57 (22): 3155-3166

  Abstract

  Thiolases are a class of carbon-carbon bond forming enzymes with important applications in biotechnology and metabolic engineering as they provide a general method for the condensation of two acyl coenzyme A (CoA) substrates. As such, developing a greater understanding of their substrate selectivity would expand our ability to engineer the enzymatic or microbial production of a broad range of small-molecule targets. Here, we report the crystal structures and biochemical characterization of Acat2 and Acat5, two biosynthetic thiolases from Ascaris suum with varying selectivity toward branched compared to linear compounds. The structure of the Acat2-C91S mutant bound to propionyl-CoA shows that the terminal methyl group of the substrate, representing the α-branch point, is directed toward the conserved Phe 288 and Met 158 residues. In Acat5, the Phe ring is rotated to accommodate a hydroxyl-π interaction with an adjacent Thr side chain, decreasing space in the binding pocket and possibly accounting for its strong preference for linear substrates compared to Acat2. Comparison of the different Acat thiolase structures shows that Met 158 is flexible, adopting alternate conformations with the side chain rotated toward or away from a covering loop at the back of the active site. Mutagenesis of residues in the covering loop in Acat5 with the corresponding residues from Acat2 allows for highly increased accommodation of branched substrates, whereas the converse mutations do not significantly affect Acat2 substrate selectivity. Our results suggest an important contribution of second-shell residues to thiolase substrate selectivity and offer insights into engineering this enzyme class.

  View details for DOI 10.1021/acs.biochem.7b01123

  View details for Web of Science ID 000434893900009

  View details for PubMedID 29381332

• Discovery and Engineering of Pathways for Production of <i>α</i>-Branched Organic Acids JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Blaisse, M. R., Dong, H., Fu, B., Chang, M. C. Y. 2017; 139 (41): 14526-14532

  Abstract

  Cell-based synthesis offers many opportunities for preparing small molecules from simple renewable carbon sources by telescoping multiple reactions into a single fermentation step. One challenge in this area is the development of enzymatic carbon-carbon bond forming cycles that enable a modular disconnection of a target structure into cellular building blocks. In this regard, synthetic pathways based on thiolase enzymes to catalyze the initial carbon-carbon bond forming step between acyl coenzyme A (CoA) substrates offer a versatile route for biological synthesis, but the substrate diversity of such pathways is currently limited. In this report, we describe the identification and biochemical characterization of a thiolase-ketoreductase pair involved in production of branched acids in the roundworm, Ascaris suum, that demonstrates selectivity for forming products with an α-methyl branch using a propionyl-CoA extender unit. Engineering synthetic pathways for production of α-methyl acids in Escherichia coli using these enzymes allows the construction of microbial strains that produce either chiral 2-methyl-3-hydroxy acids (1.1 ± 0.2 g L-1) or branched enoic acids (1.12 ± 0.06 g L-1) in the presence of a dehydratase at 44% and 87% yield of fed propionate, respectively. In vitro characterization along with in vivo analysis indicates that the ketoreductase is the key driver for selectivity, forming predominantly α-branched products even when paired with a thiolase that highly prefers unbranched linear products. Our results expand the utility of thiolase-based pathways and provide biosynthetic access to α-branched compounds as precursors for polymers and other chemicals.

  View details for DOI 10.1021/jacs.7b07400

  View details for Web of Science ID 000413503300032

  View details for PubMedID 28990776