Biology subjects

Baidoo, E.

Publications and source records attributed to Baidoo, E..

11 recordsLinked to original sources

bioRxiv2026

Distinct Strategies for Engineering Bioenergy Traits in Sorghum Drive Divergent Functional Shifts in the Rhizosphere Microbiome

Engineering bioenergy crops for enhanced biomass digestibility and high-value co-products is a promising approach to sustainable biofuel production. However, the impact of these engineered crops on the belowground microbiome remains poorly understood. In this study, we characterized the composition and functional responses of the belowground microbiome associated with transgenic sorghum lines: one with reduced cell wall crosslinking (AT10) and another with elevated protocatechuic acid (PCA) production (Qsub). The lines were grown under field conditions with four irrigation levels, and samples were collected twice during the growing season. We found that the two transgenic types drove a subtle but significant divergence in bacterial communities, both compositionally and functionally. Community-wide functional profiling showed that AT10 rhizospheres were consistently depleted in genes associated with mobile genetic elements, while Qsub rhizospheres were enriched in TonB-dependent transport and vanillate monooxygenase, an enzyme that funnels lignin-derived monomers toward PCA degradation, alongside reduced pectin- and cellulose-degradation genes. Genome-resolved analysis linked these community-level shifts to specific taxa: in Qsub, the enriched MAGs were taxonomically diverse, ranging from genome-reduced Patescibacteria with an episymbiotic lifestyle to taxa enriched in lignin-associated auxiliary activity enzymes. In AT10, the depleted Azospirillaceae MAGs carried unusually high mobile genetic element content, consistent with the community-wide pattern. Collectively, these findings offer new insights into how genetic modifications aimed at improving bioenergy traits shapes the composition and function of the rhizosphere microbiome, with potential downstream consequences for soil carbon cycling.

plant biology↗

bioRxiv2023

Ensemble and Iterative Engineering for Maximized Bioconversion to the Blue Pigment, Indigoidine from Non-Canonical Sustainable Carbon Sources

While many heterologous molecules can be produced via microbial bioconversion processes, maximizing their titers, rates, and yields from lignin-derived carbon streams remains challenging. Growth coupling can not only increase titers and yields but also shift the production period from stationary phase to growth phase. These methods for designing growth-coupling strains however require multi-gene edits for implementation which may be perceived as impractical. Here, we computationally evaluated 4,114 potential solutions for growth coupling para-coumarate to indigoidine production and prototype two cut sets in Pseudomonas putida KT2440. We used adaptive laboratory evolution (ALE) on the initial triple deletion strain to restore growth on p-CA. Using X-ray tomography on this post-ALE strain we revealed increased cell density and decreased cell volume. Proteomics identified upregulated peroxidases that mitigate reactive oxygen species formation. Nine iterative stepwise modifications further informed by model-guided and rational approaches realized a growth coupled strain that produced 7.3 g/L indigoidine at 77% MTY in para-coumarate minimal medium. These ensemble strategies provide a blueprint for producing target molecules at high product titers, rates, and yields. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/532821v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@f0754aorg.highwire.dtl.DTLVardef@a45d24org.highwire.dtl.DTLVardef@df0596org.highwire.dtl.DTLVardef@1e7e140_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2021

Engineering sorghum for higher 4-hydroxybenzoic acid content

Lignocellulosic biomass represents a renewable source of sugars for the manufacturing of bioproducts such as biofuels. The high cost associated with deconstruction of plant biomass to simple sugars remains one of the challenges preventing the deployment of economically sustainable advanced bioproducts. The accumulation in-planta of value-added coproducts such as platform chemicals can improve the economics of biofuels. Among other crops, sorghum is an ideal bioenergy feedstock due to its low input requirements, efficient nitrogen recycling, and high water use efficiency and biomass yields. In this work, we engineered sorghum to overproduce the valuable chemical 4-hydroxybenzoic acid (4-HBA) by co-expressing plastid-targeted versions of Escherichia coli chorismate pyruvate-lyase (UbiC) and feedback-resistant 3-deoxy-D-arabino-heptulonate-7-phosphate synthase (AroG*). Two independent lines containing the dual aroG*-ubiC construct were selected for characterization in the T2 generation. Using liquid chromatography-mass spectrometry, analysis of methanolic extracts obtained from biomass samples revealed an accumulation of 4-HBA in the two transgenic lines (corresponding to 1.56% and 1.72% dry weight, respectively), with 4-HBA glucose conjugates representing major forms. Measurements of biomass composition and several agronomic traits showed no significant difference between transgenic and wild-type control plants grown under controlled environment. This work demonstrates the transferability of the ubiC engineering approach to sorghum; generated lines will be useful to assess the agronomic performance of modified 4-HBA-rich sorghum under natural conditions.

plant biology↗

bioRxiv2020

A bimodular PKS platform that expands the biological design space

Traditionally engineered to produce novel bioactive molecules, Type I modular polyketide synthases (PKSs) could be engineered as a new biosynthetic platform for the production of de novo fuels, commodity chemicals, and specialty chemicals. Previously, our investigations manipulated the first module of the lipomycin PKS to produce short chain ketones, 3-hydroxy acids, and saturated, branched carboxylic acids. Building upon this work, we have expanded to multi-modular systems by engineering the first two modules of lipomycin to generate unnatural polyketides as potential biofuels and specialty chemicals in Streptomyces albus. First, we produce 20.6 mg/L of the ethyl ketone, 4,6 dimethylheptanone through a reductive loop exchange in LipPKS1 and a ketoreductase knockouts in LipPKS2. We then show that an AT swap in LipPKS1 and a reductive loop exchange in LipPKS2 can produce the potential fragrance 3-isopropyl-6-methyltetrahydropyranone. Highlighting the challenge of maintaining product fidelity, in both bimodular systems we observed side products from premature hydrolysis in the engineered first module and stalled dehydration in reductive loop exchanges. Collectively, our work expands the biological design space and moves the field closer to the production of “designer” biomolecules.HighlightsEngineered lipomycin module 1 and module 2 to produce unnatural polyketides as valuable bio-based chemicalsA reductive loop swap and ketoreductase knockout used to produce 20 mg/mL of a novel ethyl ketone, a gasoline replacementAn acyltransferase swap and reductive loop swap successfully produced δ-lactone, a potential fragrant compoundIncomplete reduction and premature hydrolysis observed in engineered modulesCompeting Interest StatementJ.D.K. has a financial interest in Amyris, Lygos, Demetrix, Napigen, Maple Bio, Berkeley Brewing Sciences, Ansa Biotech and Apertor Labs.View Full Text

synthetic biology↗

bioRxiv2019

Identification, characterization, and application of a highly sensitive lactam biosensor from Pseudomonas putida

Caprolactam is an important polymer precursor to nylon traditionally derived from petroleum and produced on a scale of 5 million tons per year. Current biological pathways for the production of caprolactam are inefficient with titers not exceeding 2 mg/L, necessitating novel pathways for its production. As development of novel metabolic routes often require thousands of designs and result in low product titers, a highly sensitive biosensor for the final product has the potential to rapidly speed up development times. Here we report a highly sensitive biosensor for valerolactam and caprolactam from Pseudomonas putida KT2440 which is >1000x more sensitive to exogenous ligand than previously reported sensors. Manipulating the expression of the sensor oplR (PP_3516) substantially altered the sensing parameters, with various vectors showing Kd values ranging from 700 nM (79.1 g/L) to 1.2 mM (135.6 mg/L). Our most sensitive construct was able to detect in vivo production of caprolactam above background at ~6 g/L. The high sensitivity and range of OplR is a powerful tool towards the development of novel routes to the biological synthesis of caprolactam. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/700484v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@ff5b8corg.highwire.dtl.DTLVardef@d365aaorg.highwire.dtl.DTLVardef@b79b18org.highwire.dtl.DTLVardef@27732f_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2019

Host engineering for improved valerolactam production in Pseudomonas putida

Pseudomonas putida is a promising bacterial chassis for metabolic engineering given its ability to metabolize a wide array of carbon sources, especially aromatic compounds derived from lignin. However, this omnivorous metabolism can also be a hindrance when it can naturally metabolize products produced from engineered pathways. Herein we show that P. putida is able to use valerolactam as a sole carbon source, as well as degrade caprolactam. Lactams represent important nylon precursors, and are produced in quantities exceeding one million tons per year[1]. To better understand this metabolism we use a combination of Random Barcode Transposon Sequencing (RB-TnSeq) and shotgun proteomics to identify the oplBA locus as the likely responsible amide hydrolase that initiates valerolactam catabolism. Deletion of the oplBA genes prevented P. putida from growing on valerolactam, prevented the degradation of valerolactam in rich media, and dramatically reduced caprolactam degradation under the same conditions. Deletion of oplBA, as well as pathways that compete for precursors L-lysine or 5-aminovalerate, increased the titer of valerolactam from undetectable after 48 hours of production to ~90 mg/L. This work may serve as a template to rapidly eliminate undesirable metabolism in non-model hosts in future metabolic engineering efforts.

synthetic biology↗

bioRxiv2018

Methyl ketone production by Pseudomonas putida is enhanced by plant-derived amino acids

Plant biomass is an attractive source of renewable carbon for conversion to biofuels and bio-based chemicals. Conversion strategies often use a fraction of the total biomass, focusing on sugars from cellulose and hemicellulose. Strategies that use plant components such as plant-derived aromatics and amino acids have the potential to improve the efficiency of overall biomass conversion. Pseudomonas putida is a promising host for biomass conversion for its ability to metabolize a wide variety of organic compounds, including aromatics derived from lignin. P. putida was engineered to produce medium chain methyl ketones, which are promising diesel blendstocks and potential platform chemicals, from glucose and lignin-related aromatics, 4-hydroxybenzoate (4-HB) and protocatechuate (PCA). Unexpectedly, P. putida methyl ketone production was enhanced 2-to 5-fold compared to sugar controls when Arabidopsis thaliana hydrolysates derived from engineered plants that overproduce 4-HB and PCA, while E. coli production was lowered in these hydrolysates. This enhancement was more pronounced (~7-fold increase) with hydrolysates derived from non-engineered switchgrass (Panicum virgatum L.) suggesting it did not arise from overproduction of 4-HB and PCA. Global proteomic analysis of the methyl ketone-producing P. putida suggested that plant-derived amino acids may be the source of this enhancement. Mass spectrometry-based measurements of plant-derived amino acids demonstrated a high correlation between methyl ketone production and amino acid concentration in plant hydrolysates. Amendment of glucose-containing minimal media with a defined mixture of amino acids similar to those found in the hydrolysates studied led to a 9-fold increase in methyl ketone titer (1.1 g/L).

bioengineering↗

bioRxiv2018

Massively parallel fitness profiling reveals multiple novel enzymes in Pseudomonas putida lysine metabolism

The lysine metabolism of Pseudomonas putida can produce multiple important commodity chemicals and is implicated in rhizosphere colonization. However, despite intensive study, the biochemical and genetic links between lysine metabolism and central metabolism remain unresolved in P. putida. Here, we leverage Random Barcode Transposon Sequencing (RB-TnSeq), a genome-wide assay measuring the fitness of thousands of genes in parallel, to identify multiple novel enzymes in both L- and D-lysine metabolism. We first describe three pathway enzymes that catabolize 2-aminoadipate (2AA) to 2-ketoglutarate (2KG) connecting D-lysine to the TCA cycle. One of these enzymes, PP_5260, contains a DUF1338 domain, a family without a previously described biological function. We demonstrate PP_5260 converts 2-oxoadipate (2OA) to 2-hydroxyglutarate (2HG), a novel biochemical reaction. We expand on recent work showing that the glutarate hydroxylase, CsiD, can co-utilize both 2OA and 2KG as a co-substrate in the hydroxylation of glutarate. Finally we demonstrate that the cellular abundance of D- and L-lysine pathway proteins are highly sensitive to pathway specific substrates. This work demonstrates the utility of RB-TnSeq for discovering novel metabolic pathways in even well-studied bacteria.\n\nImportanceO_LIP. putida is an attractive host for metabolic engineering as its lysine metabolism can be utilized for the production of multiple important commodity chemicals.\nC_LIO_LIWe demonstrate the first biochemical evidence of a bacterial 2OA catabolic pathway to central metabolites.\nC_LIO_LIDUF1338 proteins are widely dispersed across many kingdoms of life. Here we demonstrate the first biochemical evidence of function for a member of this protein family.\nC_LI

microbiology↗

bioRxiv2018

Hopanoid composition mediates bacterial ethanol tolerance

Hopanoids are abundant membrane lipids found in diverse bacterial lineages, but their physiological roles are not well understood. The ethanol fermenter Zymomonas mobilis features the highest measured concentration of hopanoids, leading to the hypothesis that these lipids can protect against bacterial solvent toxicity. However, the lack of genetic tools for manipulating hopanoid composition in vivo has limited their further functional analysis. Because of polyploidy (> 50 genome copies per cell), we found that disruptions of essential hopanoid biosynthesis (hpn) genes in Z. mobilis act as genetic knockdowns, reliably modulating the abundance of different hopanoid species. Using a set of hpn transposon mutants, we demonstrate that both reduced hopanoid content and modified hopanoid head group composition mediate growth and survival in ethanol. In contrast, the amount of hopanoids, but not their polar group composition, contributes to fitness at low pH. Spectroscopic analysis of model membranes showed that hopanoids protect against several ethanol-driven phase transitions in membrane structure, including lipid interdigitation and bilayer dissolution. We propose that hopanoids act through a combination of hydrophobic and inter-lipid hydrogen bonding interactions to stabilize bacterial membranes against solvent stress.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=196 HEIGHT=200 SRC=\"FIGDIR/small/443473v2_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (57K):\norg.highwire.dtl.DTLVardef@119f9adorg.highwire.dtl.DTLVardef@5701bcorg.highwire.dtl.DTLVardef@330bfaorg.highwire.dtl.DTLVardef@1574d44_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2018

GONST2 transports GDP-Mannose for sphingolipid glycosylation in the Golgi apparatus of Arabidopsis

The Golgi lumen is the site of many different glycosylation events, including cell wall polysaccharide biosynthesis and lipid glycosylation. Transporters are necessary for the import of the substrates required for glycosylation (nucleotide sugars) from the cytosol where they are synthesized. Plants use four GDP-linked sugars to glycosylate macromolecules: GDP-L-Fucose, GDP-D-Mannose, GDP-L-Galactose and GDP-D-Glucose. Of the predicted fifty-one members of the nucleotide sugar transporter/triose phosphate transporter family in Arabidopsis, only four appear to contain the conserved motif needed for the transport of GDP-linked sugars, GOLGI LOCALIZED NUCLEOTIDE SUGAR TRANSPORTER (GONST) 1-4. Previously, we have demonstrated that GONST1 provides GDP-D-Mannose for glycosylation of a class of sphingolipids, the glycosylinositolphosphorylceramides (GIPCs). Here, we characterize its closest homologue, GONST2, and conclude that it also specifically provides substrate for GIPC glycosylation. Expression of GONST2 driven by the GONST1 promoter is able to rescue the severe growth phenotype of gonst1. Loss of GONST2 exacerbates the gonst1 constitutive hypersensitive response, as well as the reduced cell wall cellulose content. The gonst2 mutant grows normally under standard conditions, but has enhanced resistance to the powdery mildew-causing fungus Golovinomyces orontii.

plant biology↗

bioRxiv2017

Rhodosporidium toruloides: A new platform organism for conversion of lignocellulose into terpene biofuels and bioproducts

BackgroundEconomical conversion of lignocellulosic biomass into biofuels and bioproducts is central to the establishment of a robust bioeconomy. This requires a conversion host that is able to both efficiently assimilate the major lignocellulose-derived carbon sources and divert their metabolites toward specific bioproducts.\n\nResultsIn this study, the carotenogenic yeast Rhodosporidium toruloides was examined for its ability to convert lignocellulose into two non-native sesquiterpenes with biofuel (bisabolene) and pharmaceutical (amorphadiene) applications. We found that R. toruloides can efficiently convert a mixture of glucose and xylose from hydrolyzed lignocellulose into these bioproducts, and unlike many conventional production hosts, its growth and productivity were enhanced in lignocellulosic hydrolysates relative to purified substrates. This organism was demonstrated to have superior growth in corn stover hydrolysates prepared by two different pretreatment methods, one using a novel biocompatible ionic liquid (IL) choline -ketoglutarate, which produced 261 mg/L of bisabolene at bench-scale, and the other using an alkaline pretreatment, which produced 680 mg/L of bisabolene in a high gravity fed-batch bioreactor. Interestingly, R. toruloides was also observed to assimilate p-coumaric acid liberated from acylated grass lignin in the IL hydrolysate, a finding we verified with purified substrates. R. toruloides was also able to consume several additional compounds with aromatic motifs similar to lignin monomers, suggesting that this organism may have the metabolic potential to convert depolymerized lignin streams alongside lignocellulosic sugars.\n\nConclusionsThis study highlights the natural compatibility of R. toruloides with bioprocess conditions relevant to lignocellulosic biorefineries and demonstrates its ability to produce non-native terpenes.

bioengineering↗