Biology subjects

PAPOUTSAKIS, E. T.

Publications and source records attributed to PAPOUTSAKIS, E. T..

11 recordsLinked to original sources

bioRxiv2026

Biological upgrading of C1-C2 products of electrocatalytic CO 2 reduction to C4-C6 carboxylates

BackgroundMicrobial chain-elongation by Clostridium kluyveri using the products (acetate and ethanol) derived from the electrocatalytic CO2 reduction reaction (CO2RR) represents a unique sustainable strategy for producing C4-C6 chemicals from CO2. However, direct integration of electrocatalytic effluents with anaerobic bioprocesses is often impeded by the physiological incompatibility between electrocatalytic product streams and microbial metabolism. Specifically, CO2RR effluents commonly contain formate, which cannot be utilized by C. kluyveri for chain elongation and therefore reduces the overall carbon efficiency of CO2 conversion to C4-C6 chemicals. Moreover, both formate and the elevated phosphate concentrations typical of electrochemical reaction solutions may inhibit microbial growth. ResultsWe show that formate at concentrations of up to 50 mM did not inhibit the growth of or the chain elongation by C. kluyveri. Based on this finding, we developed a modular two-step bioprocess. In the first step, the acetogen Clostridium ljungdahlii converts formate in CO2RR product mixtures into acetate, thereby generating additional substrates for second-step C. kluyveri-driven chain elongation, thus increasing the CO2RR carbon-conversion efficiency to C- C6 chemicals. To address the issue of C. ljungdahliis inhibition by high phosphate concentrations in electrocatalytic solutions, we explored the use of C. ljungdahlii biofilms for the first, i.e. the formate-conversion, step. C. ljungdahlii biofilms exhibit tolerance to concentrated electrolytes, enabling the conversion of up to 50 mM formate in CO2RR solutions. ConclusionsThe demonstrated two-step process constitutes the basis for the development of a robust and carbon-efficient biological process for the scalable upgrading of C1-C2 CO2RR products into higher-value C4-C6 chemicals.

synthetic biology↗

bioRxiv2025

Pseudo perfusion of Chinese Hamster Ovary (CHO) cells as a reliable platform for data generation to model and guide continuous perfusion biomanufacturing

Chinese Hamster Ovary (CHO) cell monoclonal antibody (mAb) production in continuous perfusion has witnessed a renewed interest within the biopharmaceutical industry. Widespread implementation of perfusion biomanufacturing, however, remains hindered by long process development timelines and high costs. Use of predictive scale-down platforms to generate large informative metabolic datasets and guide process development decisions is critical to decreasing a molecules time to market. While scale-down platforms based on the pseudo perfusion concept have been previously reported, they have not been rigorously validated. They are often limited by oxygen transport or insufficient metabolic characterization, reducing their role to a preliminary screening tool. Here, we report the design and validation of a pseudo perfusion platform based on a phenotype-driven approach to ascertain that the process emulates continuous perfusion characteristics and is not oxygen limited. Beyond metabolic and cell size steady state, we show that our pseudo perfusion design enables cell cycle subpopulation and intracellular antibody expression steady state. We also demonstrate that pseudo perfusion robustly predicts amino acid demands in continuous perfusion bioreactors with exceptional linear correlation across a broad range of cell-specific perfusion rates (CSPRs). When coupling the pseudo perfusion platform developed here with a workflow for metabolic characterization, we significantly augment the dimensionality and reliability of data which can be generated at this scale to gain actionable insights towards perfusion process design, ultimately reducing process development timelines and the associated costs. HighlightsResidual lactate is a key proxy for oxygen transport in scale down platform design Novel flow cytometry workflow confirms cell cycle and intracellular steady state Pseudo perfusion robustly predicts metabolic phenotypes in continuous perfusion K-means clustering analysis of nutrient rates provides insight into media design

bioengineering↗

bioRxiv2025

Enabling strong acetogenic growth on CO2 and H2: H2 solubility limits Clostridium ljungdahlii growth on CO2 and H2

Due to their ability to convert CO2, a greenhouse gas, into useful products, certain acetogenic bacterial species, such as Clostridium ljungdahlii, have been proposed as promising platform strains for renewable, carbon-negative chemical production. C. ljungdahlii, and similar acetogens, grows slowly and produce primarily acetate when grown on CO2 with H2 as the electron donor, but it grows quickly and can produce ethanol when grown on higher energy substrates, notably CO or fructose. Here, by utilizing different mixing strategies (and notably the first time use of roller bottles) to modulate the volumetric gas interfacial mass transfer coefficient (kLa), we show that, under both mixotrophic (sugar and gas utilization) and autotrophic conditions, C. ljungdahlii growth and CO2 fixation are primarily electron-limited due to the low solubility of H2 relative to CO and CO2. We demonstrate that, with sufficiently high H2 mass transfer, C. ljungdahlii can grow at similar high rates using CO2 as its sole carbon source compared to CO or fructose, a finding with significant implications for the use of acetogens in CO2-negative biomanufacturing, especially because at least 50% of CO used is oxidized and released as CO2. We also show that accumulation of fructose inhibits CO2 utilization by C. ljungdahlii under mixotrophic growth conditions, suggesting that a non-classical "catabolite repression" by fructose inhibits CO2 utilization.

bioengineering↗

bioRxiv2025

Engineering mutualism via nitrogen exchange in mixotrophic cocultures between Clostridium acetobutylicum and Clostridium ljungdahlii

We have previously shown that mixotrophic cocultures of Clostridium acetobutylicum and Clostridium ljungdahlii - using sugars and H2 as substrates - increase sugar-substrate carbon and electron conversion via CO2 and H2 capture and synthesize valuable products, such as isopropanol and 2,3-butanediol, that neither species can make independently. In this pairing, growth of C. ljungdahlii is constrained by C. acetobutylicum, since C. ljungdahlii relies on C. acetobutylicum to convert glucose into CO2, which C. ljungdahlii can use as a carbon and electron sources. However, this dependence is unilateral; C. acetobutylicums growth is not constrained by C. ljungdahlii. Consequently, population ratios between the two species can vary substantially throughout the course of fermentation and in different fermentation setups, typically with the faster growing C. acetobutylicum outcompeting C. ljungdahlii. Population ratio is an important variable because it influences metabolite yields and productivity and likely also impacts the initiation and frequency of the heterologous cell fusion events we have documented between C. acetobutylicum and C. ljungdahlii. Thus, developing methods to rationally control and maintain the population ratio are important for both biotechnological applications and fundamental study of this coculture pairing. In this study we show that the different nitrogen utilization capabilities of these two organisms enable engineering of a mutualistic mixotrophic syntrophy in which C. ljungdahlii relies on C. acetobutylicum for carbon and electrons and C. acetobutylicum relies on C. ljungdahlii for nitrogen. First, we confirm that C. ljungdahlii, but not C. acetobutylicum, can convert nitrate into biologically useful ammonium, enabling the design of a culture medium in which C. acetobutylicum can only grow in the presence of C. ljungdahlii. Second, we test different ratios of nitrate to ammonium in batch cocultures and demonstrate that rapid nitrate utilization by C. ljungdahlii prevents C. acetobutylicum from becoming nitrogen-limited at any point in batch fermentation. Finally, we show that feeding different rates of nitrate to cocultures in fed-batch mode enables control of the coculture growth rate, maintenance of stable population ratios, and higher isopropanol and butanol yields in cocultures between C. acetobutylicum and C. ljungdahlii.

bioengineering↗

bioRxiv2025

Enabling supratheoretical isopropanol yields from carbon-negative glucose fermentations with Clostridium acetobutylicum-Clostridium ljungdahlii cocultures

Synthetic microbial cocultures, which combine the unique capabilities of multiple microbes into one process, have significant potential for sustainable production of fuels and chemicals. Most studies of defined cocultures have tested relatively low cell densities in lab-scale batch cultures, not the high cell density fed-batch or continuous processes with cell retention typically required to achieve industrially-relevant volumetric productivities. Here, we explore the impact of increased cell density on isopropanol production from the syntrophic coculture of genetically-modified Clostridium acetobutylicum [CACas9 {Delta}hbd (p95ace02_atoB), with deleted 4-C metabolism expressing an acetone-formation pathway on the plasmid] with WT Clostridium ljungdahlii using first a pseudo-perfusion approach followed by perfusion culture. CACas9 {Delta}hbd (p95ace02_atoB) produces acetone without any 4-C metabolites and C. ljungdahlii converts that acetone to isopropanol. To explore the mechanism by which these cultures enable supratheoretical isopropanol yields, we first identified NADH-driven hydrogen conversion in CACas9 {Delta}hbd (p95ace02_atoB) as the thermodynamically-limiting step for acetone and thus isopropanol production. We then demonstrated the ability of C. ljungdahlii to mitigate this issue by eliminating detectable hydrogen accumulation in the coculture. Pseudo-perfusion cocultures showed that high cell densities combined with a high population fraction of C. ljungdahlii enable dramatic increases in isopropanol yields beyond the thermodynamic limitation imposed in CACas9 {Delta}hbd (p95ace02_atoB) monocultures. Finally, we demonstrate carbon-negative fermentation of glucose to isopropanol as the sole alcohol product in a perfusion bioreactor.

bioengineering↗

bioRxiv2024

Butyrate as a growth factor of Clostridium acetobutylicum

The butyrate biosynthetic pathway not only contributes to electron management and energy generation in butyrate forming bacteria, but also confers evolutionary advantages to the host by inhibiting the growth of surrounding butyrate-sensitive microbes. Proteomic data suggest that butyrate may lead to lysine butyrylation, a lesser-known post-translational modification, which might affect enzyme catalysis and thus cellular metabolism. Although high levels of butyrate induce toxic stress responses, it is not known if butyrate at non-toxic levels influences cellular processes such as growth, health, metabolism, and sporulation. Here, we show that butyrate stimulates cellular processes of Clostridium acetobutylicum, a model butyrate forming Firmicute. First, we deleted the 3-hydroxybutyryl-CoA dehydrogenase gene (hbd) from the C. acetobutylicum chromosome in order to eliminate the butyrate synthetic pathway and thus butyrate formation. For rapid genome engineering, a xylose inducible Cas9 cassette was chromosomally integrated and utilized for the one-step markerless gene deletions. The addition of non-toxic levels of butyrate revealed that butyrate has a profound effect on the growth, health, and sporulation of C. acetobutylicum. By further deleting spo0A, the gene of the master regulator of sporulation, and followed by butyrate addition experiments, we conclude that butyrate affects cellular metabolism through both Spo0A dependent and independent mechanisms. We also deleted the hbd gene from the chromosome of the asporogenous C. acetobutylicum M5 strain lacking the pSOL1 plasmid to examine the potential involvement of pSOL1 genes on the observed butyrate effects. Addition of the precursor of butyrate biosynthesis crotonate to the hbd deficient M5 strain was used to probe the role of butyrate biosynthesis pathway in electron and metabolic fluxes. Finally, we found that butyrate addition can enhance the growth of the non-butyrate forming Clostridium saccharolyticum. Our data suggest that butyrate functions as a stimulator of cellular processes, like a growth factor, in C. acetobutylicum and other Clostridium organisms, and may thus be as a modulator of microbial population dynamics. HighlightsO_LIDeployed chromosomally integrated spCas9 for markerless one-step Clostridium acetobutylicum genome engineering. C_LIO_LIDeleted 3-hydroxybutyryl-CoA dehydrogenase gene (hbd) from Clostridium acetobutylicum to elucidate the roles of butyrate in cellular processes. C_LIO_LIDemonstrated butyrate as a growth factor stimulating cellular processes in Clostridium acetobutylicum and potentially other Clostridium species. C_LIO_LISuggested butyrate as a potential modulator of microbial population based on different responses of microbes against butyrate. C_LI

bioengineering↗

bioRxiv2023

Native and engineered human megakaryocytic extracellular vesicles for targeted non-viral cargo delivery to blood stem cells

Native and engineered extracellular vesicles (EVs) generated from human megakaryocytes (huMkEVs) or from the human megakaryocytic cell line CHRF (CHEVs) interact with tropism delivering their cargo to both human and murine hematopoietic stem and progenitor cells (HSPCs). 24 hours after intravenous infusion of huMkMPs into NOD-scid IL2R{gamma}null (NSG) mice, they induced a nearly 50% increase in murine platelet counts relative to saline control, thus demonstrating the potential of these EVs, which can be stored frozen, for treating thrombocytopenias. PKH26-labeled huMkMPs or CHEVs localized to the HSPC-rich bone marrow preferentially interacting with murine HSPCs. Using engineered huMkEVs or CHEVs, their receptor-mediated tropism for HSPCs was explored to functionally deliver synthetic cargo, notably plasmid DNA coding for a fluorescent reporter, to murine HSPCs both in vitro and in vivo. These data demonstrate the potential of these EVs as a non-viral, HSPC-specific cargo vehicle for gene therapy applications to treat hematological diseases. Native and engineered human megakaryocytic extracellular vesicles for targeted non-viral cargo delivery to blood stem cells (Table of Contents): O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/536479v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@664768org.highwire.dtl.DTLVardef@d9fe13org.highwire.dtl.DTLVardef@1b795eforg.highwire.dtl.DTLVardef@1d48e2d_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical Overview: Native and engineered human megakaryocytic extracellular vesicles (huMkEVs) for provide targeted non-viral cargo delivery to blood stem cells. We demonstrate that huMkEVs as a transformational cargo-delivery system to blood stem cells (hematopoietic stem and progenitor cells, HSPCs) in NOD-scid IL2R{gamma}null (NSG) mice. Intravenous delivery of native huMkEVs enhances de novo platelet biogenesis by inducing megakaryocytic differentiation of murine HSPCs, thus demonstrating the desirable strong tropism of huMkEVs for murine HSPCs. Based on this tropism, we demonstrate that engineered huMkEVs can deliver functional plasmid-DNA cargo specifically to HSPCs.

bioengineering↗

bioRxiv2022

Polyethylene valorization by combined chemical catalysis with bioconversion by plastic-enriched microbial consortia

There are few reports of microbial deconstruction or functionalization of the recalcitrant backbone of polyolefins. However, microbes can utilize polyolefin deconstruction products, including n-alkanes. Here, we combined chemical catalysis with bioconversion to valorize polyethylene (PE) deconstruction products. High-density PE (HDPE) was deconstructed via hydrogenolysis over a ruthenium on carbon catalyst. The resulting n-alkane mixture (C4-C35) was utilized as a feedstock for microbial consortia derived from soil from local recycling plants. We found two consortia that utilized the PE-deconstruction product mix as a sole carbon source. We adapted the consortia on a commercially-available n-alkane mix to reduce the number of species present and enrich for enhanced alkane utilization. Both resulting enriched consortia utilized the PE-deconstruction product mix more effectively than the original (parent) consortia. The predominant metabolite produced by both enriched consortia was a C16-C16 wax ester. Wax esters have considerable industrial value, with the longer chain lengths (C32-C36) having the highest value. We identified two Rhodococcus aetherivorans strains that grow well on C24, indicating this species is important for the functionalization of long-chain alkanes. This work demonstrates that enriched consortia from plastic-enriched environments can be combined with chemical catalysis to valorize polyethylene. SynopsisChemical catalysis can be used to deconstruct polyethylene waste material to produce a mixture of alkanes. Enriched environmental microbial consortia can valorize these polyethylene deconstruction products via functionalization that preserves the alkane chain length thus minimizing CO2 production.

bioengineering↗

bioRxiv2022

The microRNomes of Chinese Hamster Ovary (CHO) cells and their extracellular vesicles, and how they respond to osmotic and ammonia stress

A new area of focus in Chinese Hamster Ovary (CHO) biotechnology is the role of small (exosomes) and large (microvesicles or microparticles) extracellular vesicles (EVs). CHO cells in culture exchange large quantities of proteins and RNA through these EVs, yet the content and role of these EVs remain elusive. MicroRNAs (miRs) are central to adaptive responses to stress and more broadly to changes in culture conditions. Given that EVs are highly enriched in miRs, and that EVs release large quantities of miRs both in vivo and in vitro, EVs and their miR content likely play an important role in adaptive responses. Here we report the miRNA landscape of CHO cells and their EVs under normal culture conditions and under ammonia and osmotic stress. We show that both cells and EVs are highly enriched in five miRs (among over 600 miRs) that make up about half of their total miR content, and that these highly enriched miRs differ significantly between normal and stress culture conditions. Notable is the high enrichment in mir-92a and miR-23a under normal culture conditions, in contrast to the high enrichment in let-7 family miRs (let-7c, let-7b and let-7a) under both stress conditions. The latter suggests a preserved stress-responsive function of the let-7 miR family, one of the most highly preserved miR families across species, where among other functions, let-7 miRs regulate core oncogenes, which, depending on the biological context, may tip the balance between cell cycle arrest and apoptosis. While the expected -based on their profound enrichment - important role of these highly enriched miRs remains to be dissected, our data and analysis constitute an important resource for exploring the role of miRs in cell adaptation as well as for synthetic applications.

cell biology↗

bioRxiv2021

Novel mechanism of plasmid-DNA transfer mediated by heterologous cell fusion in syntrophic coculture of Clostridium organisms

The evolution of bacteria is driven by random genetic mutations and horizontal gene transfer (HGT) of genetic material from other bacteria. HGT can occur via transformation, transduction, and conjugation. Here, we present a potential new mechanism of HGT which occurs in a syntrophic Clostridium coculture. We have previously shown that in syntrophic cocultures of Clostridium acetobutylicum and Clostridium ljungdahlii, the two organisms undergo heterologous cell fusion, which includes fusion of the peptidoglycan cell walls and membranes. Heterologous cell fusion facilitated a large-scale exchange of cytoplasmic protein and RNA between the two organisms, leading to the formation of hybrid bacterial cells containing cytoplasmic material of the two parent organisms. Here we present new evidence that cell fusion events also facilitate the exchange of plasmid DNA between the two organisms of the syntrophic coculture. Through the use of a selective subculturing process, we successfully isolated wild-type C. acetobutylicum clones which have acquired a portion of the plasmid DNA - containing the antibiotic resistance marker - from a recombinant strain of C. ljungdahlii. Fusion events led to formation of persistent aberrant hybrid cells with distinct morphogenetic characteristics. Furthermore, our data support the concept of a novel, interspecies, mechanism of acquiring antibiotic resistance. Since neither organism contains any known conjugation machinery or mechanism, these findings expand our understanding of multi-species microbiomes, their survival strategies, and evolution. IMPORTANCEInvestigations of natural multispecies microbiomes and the field of synthetic and syntrophic microbial cocultures are attracting renewed interest based on their potential application in biotechnology, ecology, and medical fields. A variety of synthetic and natural cocultures have been examined in terms of their metabolic output, but relatively few systems have been interrogated at the cellular and molecular level. Previously, we have shown the syntrophic coculture of C. acetobutylicum and C. ljungdahlii undergoes heterologous cell-to-cell fusion, which facilitates the exchange of cytoplasmic protein and RNA between the two organisms, and leads to the formation of hybrid bacterial cells. Continuing this line of investigation, we now show that heterologous cell fusion between the two Clostridium organisms can also facilitate the exchange of DNA between the two organisms. By applying selective pressures to this coculture system, we isolated clones of wild-type C. acetobutylicum which acquired the erythromycin resistance (erm) gene from the C. ljungdahlii strain carrying a plasmid with the erm gene. Fusion led to persistent hybrid cells containing DNA from both parents but with distinct properties and morphologies. Moreover, we provide evidence for a novel mechanism of acquiring antibiotic resistance mediated by the syntrophic interactions of this system. This is a major finding that may shed light on a new mechanism of bacterias ability to acquire antibiotic resistance.

microbiology↗

bioRxiv2020

Combinatorial effect of miR-486-5p & miR-22-3p mimics thrombopoietin's impact on hematopoietic stem & progenitor cells

Megakaryocytes shed and release submicron size microparticles (MkMPs), the most abundant microparticle in circulation. We have previously reported that MkMPs target peripheral-blood CD34+ hematopoietic stem/progenitor cells (HSPCs) to induce megakaryocytic differentiation and proliferation, and that small RNAs delivered to HSPCs via MkMPs play an important role in the development of this phenotype. Here, using single-molecule real-time (SMRT) RNA sequencing (RNAseq), we identify the top seven most abundant microRNAs (miRs) in MkMPs as potential candidates in mediating the effect of MkMPs on HSPCs. Using miR mimics, we demonstrate that among the seven most abundant miRs, two, miR-486-5p and miR-22-3p, are able to drive the Mk differentiation of HSPCs in the absence of thrombopoietin (TPO). The effect of these two miRs is comparable to the TPO- or MkMP-induced megakaryocytic differentiation of HSPCs, thus suggesting that these two miRs are responsible for this MkMP-induced phenotype. To probe the signaling through which MkMPs might enable this phenotype, we used kinase inhibitors of potential signaling pathways engaged in megakaryocytic differentiation. Our data suggest that MkMP-induced Mk differentiation of HSPCs is enabled through JNK and PI3K/Akt/mTOR signaling. Our data show that MkMPs activate Akt and mTOR phosphorylation. Furthermore, MkMPs downregulate PTEN expression, a direct target of miR-486-5p and a negative regulator of PI3K/Akt signaling, via JNK signaling. Taken together, our data provide a mechanistic understanding of the biological effect of MkMPs in inducing megakaryocytic differentiation of HSPCs, which, as was previously suggested, is a phenotype of potential physiological significance in stress megakaryopoiesis. Key Points O_LImiR-486-5p and miR-22-3p drive megakaryocytic differentiation in the absence of thrombopoietin. C_LIO_LIMegakaryocytic microparticles trigger megakaryocytic differentiation of CD34+ cells through JNK and PI3K/Akt/mTOR signaling. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/941104v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@4f3537org.highwire.dtl.DTLVardef@2ede40org.highwire.dtl.DTLVardef@1014d48org.highwire.dtl.DTLVardef@78cc21_HPS_FORMAT_FIGEXP M_FIG Visual Abstract C_FIG

cell biology↗