Biology subjects

Baidoo, E. E.

Publications and source records attributed to Baidoo, E. E..

6 recordsLinked to original sources

bioRxiv2025

The mevalonate pathway of isoprenoid biosynthesis supports metabolic flexibility in Mycobacterium marinum

Isoprenoids are a diverse class of natural products that are essential in all domains of life. Most bacteria synthesize isoprenoids through either the methylerythritol phosphate (MEP) pathway or the mevalonate (MEV) pathway, while a small subset encodes both pathways, including the pathogen Mycobacterium marinum (Mm). It is unclear whether the MEV pathway is functional in Mm, or why Mm encodes seemingly redundant metabolic pathways. Here we show that the MEP pathway is essential in Mm while the MEV pathway is dispensable in culture, with the {Delta}MEV mutant having no growth defect in axenic culture but a competitive growth defect compared to WT Mm. We found that the MEV pathway does not play a role in ex vivo or in vivo infection but does play a role in survival of peroxide stress. Metabolite profiling revealed that modulation of the MEV pathway causes compensatory changes in the concentration of MEP intermediates DOXP and CDP-ME, suggesting that the MEV pathway is functional and that the pathways interact at the metabolic level. Finally, the MEV pathway is upregulated early in the shift down to hypoxia, suggesting that it may provide metabolic flexibility to this bacterium. Interestingly, we found that our complemented strains, which vary in copy number of the polyprenyl synthetase idsB2, responded differently to peroxide and UV stresses, suggesting a role for this gene as a determinant of downstream prenyl phosphate metabolism. Together, these findings suggest that MEV may serve as an anaplerotic pathway to make isoprenoids under stress conditions. ImportanceOrganisms from all domains of life utilize isoprenoids to carry out thousands of critical and auxiliary cellular processes, including signaling, membrane integrity, stress response, and host-pathogen interactions. The common precursor of all isoprenoids is synthesized via one of two biosynthetic pathways and importantly, some bacteria encode both pathways, including M. marinum. We found that only one pathway is essential in M. marinum, while the nonessential pathway may confer metabolic flexibility to help the bacterium better adapt to various environmental conditions. We also found that the polyprenyl synthetase IdsB2 plays an important role in driving such phenotypes. Further, we demonstrate metabolic interplay between both functional pathways. These insights represent the first characterization of isoprenoid biosynthesis in dual pathway-encoding mycobacteria.

microbiology↗

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

Bottlenecks in the Implementation of Genome Scale Metabolic Model Based Designs for Bioproduction from Aromatic Carbon Sources

Genome scale metabolic models (GSMM) are commonly used to identify gene deletion sets that result in growth coupling, pairing product formation with substrate utilization. While such approaches can improve strain performance beyond levels typically accessible using targeted strain engineering approaches, sustainable feedstocks often pose a challenge for GSMM-based methods due to incomplete underlying metabolic data. Specifically, we address a four-gene deletion design for the lignin-derived non-sugar carbon source, para-coumarate, that proved challenging to implement. We examine the performance of the fully implemented design for p-coumarate to glutamine, a useful biomanufacturing intermediate. In this study glutamine is then converted to indigoidine, an alternative sustainable pigment and a model heterologous product. Through omics, promoter-variation and growth characterization of a fully implemented gene deletion design, we provide evidence that aromatic catabolism in the completed design is rate-limited by fumarate hydratase activity in the citrate cycle and required careful optimization of the final fumarate hydratase protein (PP_0897) expression to achieve growth and production. A metabolic cross-feeding experiment with the completed design strain also revealed an unanticipated nutrient requirement suggesting additional functions for the fumarate hydratase protein. A double sensitivity analysis confirmed a strict requirement for fumarate hydratase activity in the strain where all genes in the growth coupling design have been implemented. While a complete implementation of the design was achieved, this study highlights the challenge of precisely inactivating metabolic reactions encoded by under-characterized proteins especially in the context of multi-gene edits.

microbiology↗

bioRxiv2021

Engineering site-selective incorporation of fluorine into natural product analogs

While bioactive compounds are commonly derived both by human design as well as from living organisms, man-made and natural products typically display very different structural characteristics. As such, a longstanding goal in the discovery of new molecular function is to develop approaches to incorporate the advantageous elements of both groups of molecules, thereby expanding the molecular space accessible for this purpose. In this work, we report the engineering a fluorine-selective enzyme that can complement mutated acyltransferase (AT) domains of a modular polyketide synthase, which are the main determinants of the identity and location of substituents on polyketides, to produce different fluorinated regioisomers of the erythromycin precursor in vitro. We further show that by engineering cell uptake of fluorinated building blocks, we can control fluorine selectivity in vivo to produce selectively fluorinated polyketides using engineered E. coli. These results demonstrate that it is possible to introduce fluorine, a key synthetic design element for drug development, selectively into the scaffold of a complex natural product and produce these analogs by microbial fermentation.

synthetic biology↗

bioRxiv2020

An iron (II) dependent oxygenase performs the last missing step of plant lysine catabolism

Due to low abundance in many staple food crops, the essential amino acid lysine must be produced industrially to meet global food supply needs. Despite intensive study, manipulation, and agricultural importance, the steps of plant lysine catabolism beyond the 2-oxoadipate (2OA) intermediate remain undescribed. Recently we described a missing step in the D-lysine catabolic pathway of the bacterium Pseudomonas putida in which 2OA is converted to D-2-hydroxyglutarate (D2HG) via hydroxyglutarate synthase (HglS), an enzyme belonging to the previously uncharacterized DUF1338 protein family. Here we solve the structure of HglS to 1.1[A] resolution in the substrate-free form and in complex with 2OA. Structural similarity to hydroxymandelate synthase suggested a successive decarboxylation and intramolecular hydroxylation mechanism forming 2HG in a Fe(II)- and O2-dependent manner, which is validated experimentally. 2OA specificity was mediated by a single arginine (R74), highly conserved across nearly all DUF1338 family proteins, including in 76% of plant enzymes. In Arabidopsis thaliana, a DUF1338 homolog is coexpressed with known lysine catabolism enzymes, and mutants show significant germination rate defects consistent with disrupted lysine catabolism. Structural and biochemical analysis of the Oryza sativa homolog FLO7 revealed identical activity to HglS despite low sequence identity. Our results suggest that nearly all DUF1338 containing enzymes likely catalyze the same biochemical reaction, exerting the same physiological function across bacteria and eukaryotes. SignificanceTo meet human demands, millions of tons of lysine are produced by bacterial fermentation annually due to its low abundance in staple crops. Here, we show the last missing step of lysine catabolism in nearly all plant endosperms is likely catalyzed by an iron-dependant DUF1338-containing enzyme homologous to the bacterial hydroxyglutarate synthase. Structural and bioninformatic analyses of DUF1338-containing enzymes showed high conservation of critical catalytic and specificity-conferring residues across multiple domains of life despite low sequence identity. These results suggest that the DUF1338 family evolved a specific physiological function within lysine catabolism across multiple domains of life.

molecular biology↗

bioRxiv2019

Chemoinformatic-guided engineering of polyketide synthases

Polyketide synthase (PKS) engineering is an attractive method to generate new molecules such as commodity, fine and specialty chemicals. A significant challenge in PKS design is engineering a partially reductive module to produce a saturated {beta}-carbon through a reductive loop exchange. In this work, we sought to establish that chemoinformatics, a field traditionally used in drug discovery, could provide a viable strategy to reductive loop exchanges. We first introduced a set of donor reductive loops of diverse genetic origin and chemical substrate structures into the first extension module of the lipomycin PKS (LipPKS1). Product titers of these engineered unimodular PKSs correlated with atom pair chemical similarity between the substrate of the donor reductive loops and recipient LipPKS1, reaching a titer of 165 mg/L of short chain fatty acids produced by Streptomyces albus J1074 harboring these engineered PKSs. Expanding this method to larger intermediates requiring bimodular communication, we introduced reductive loops of divergent chemosimilarity into LipPKS2 and determined triketide lactone production. Collectively, we observed a statistically significant correlation between atom pair chemosimilarity and production, establishing a new chemoinformatic method that may aid in the engineering of PKSs to produce desired, unnatural products.

biochemistry↗