DNA transfer between two different species mediated by heterologous cell fusion in Clostridium coculture.

Prokaryotic evolution is driven by random mutations and horizontal gene transfer (HGT). HGT occurs via transformation, transduction, or conjugation. We have previously shown that in syntrophic cocultures of Clostridium acetobutylicum and Clostridium ljungdahlii, heterologous cell fusion leads to a large-scale exchange of proteins and RNA between the two organisms. Here, we present evidence that heterologous cell fusion facilitates the exchange of DNA between the two organisms. Using selective subculturing, we isolated C. acetobutylicum cells which acquired and integrated into their genome portions of plasmid DNA from a plasmid-carrying C. ljungdahlii strain. Limiting-dilution plating and DNA methylation data based on PacBio Single-Molecule Real Time (SMRT) sequencing support the existence of hybrid C. acetobutylicum/C. ljungdahlii cells. These findings expand our understanding of multi-species microbiomes, their survival strategies, and evolution.IMPORTANCEInvestigations of natural multispecies microbiomes and synthetic microbial cocultures are attracting renewed interest for their potential application in biotechnology, ecology, and medical fields. 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. We now show that heterologous cell fusion between the two Clostridium organisms can facilitate the exchange of DNA. 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. Single-molecule real-time sequencing revealed that the erm gene was integrated into the genome in a mosaic fashion. Our data also support the persistence of hybrid C. acetobutylicum/C. ljungdahlii cells displaying hybrid DNA-methylation patterns.

Similar but distinct: The impact of biomechanical forces and culture age on the production, cargo loading, and biological efficacy of human megakaryocytic extracellular vesicles for applications in cell and gene therapies.

Megakaryocytic extracellular vesicles (MkEVs) promote the growth and megakaryopoiesis of hematopoietic stem and progenitor cells (HSPCs) largely through endogenous miR-486-5p and miR-22-3p cargo. Here, we examine the impact of biomechanical force and culture age/differentiation on the formation, properties, and biological efficacy of MkEVs. We applied biomechanical force to Mks using two methods: shake flask cultures and a syringe pump system. Force increased MkEV production in a magnitude-dependent manner, with similar trends emerging regardless of whether flow cytometry or nanoparticle tracking analysis was used for MkEV counting. Both methods produced MkEVs that were relatively depleted of miR-486-5p and miR-22-3p cargo. However, while the shake flask-derived MkEVs were correspondingly less effective in promoting megakaryocytic differentiation of HSPCs, the syringe pump-derived MkEVs were more effective in doing so, suggesting the presence of unique, unidentified miRNA cargo components. Higher numbers of MkEVs were also produced by "older" Mk cultures, though miRNA cargo levels and MkEV bioactivity were unaffected by culture age. A reduction in MkEV production by Mks derived from late-differentiating HSPCs was also noted. Taken together, our results demonstrate that biomechanical force has an underappreciated and deeply influential role in MkEV biology, though that role may vary significantly depending on the nature of the force. Given the ubiquity of biomechanical force in vivo and in biomanufacturing, this phenomenon must be grappled with before MkEVs can attain clinical relevance.

Cell-culture process optimization via model-based predictions of metabolism and protein glycosylation.

In order to meet the rising demand for biologics and become competitive on the developing biosimilar market, there is a need for process intensification of biomanufacturing processes. Process development of biologics has historically relied on extensive experimentation to develop and optimize biopharmaceutical manufacturing. Experimentation to optimize media formulations, feeding schedules, bioreactor operations and bioreactor scale up is expensive, labor intensive and time consuming. Mathematical modeling frameworks have the potential to enable process intensification while reducing the experimental burden. This review focuses on mathematical modeling of cellular metabolism and N-linked glycosylation as applied to upstream manufacturing of biologics. We review developments in the field of modeling cellular metabolism of mammalian cells using kinetic and stoichiometric modeling frameworks along with their applications to simulate, optimize and improve mechanistic understanding of the process. Interest in modeling N-linked glycosylation has led to the creation of various types of parametric and non-parametric models. Most published studies on mammalian cell metabolism have performed experiments in shake flasks where the pH and dissolved oxygen cannot be controlled. Efforts to understand and model the effect of bioreactor-specific parameters such as pH, dissolved oxygen, temperature, and bioreactor heterogeneity are critically reviewed. Most modeling efforts have focused on the Chinese Hamster Ovary (CHO) cells, which are most commonly used to produce monoclonal antibodies (mAbs). However, these modeling approaches can be generalized and applied to any mammalian cell-based manufacturing platform. Current and potential future applications of these models for Vero cell-based vaccine manufacturing, CAR-T cell therapies, and viral vector manufacturing are also discussed. We offer specific recommendations for improving the applicability of these models to industrially relevant processes.

Megakaryocyte membrane-wrapped nanoparticles for targeted cargo delivery to hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) are desirable targets for gene therapy but are notoriously difficult to target and transfect. Existing viral vector-based delivery methods are not effective in HSPCs due to their cytotoxicity, limited HSPC uptake and lack of target specificity (tropism). Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) are attractive, nontoxic carriers that can encapsulate various cargo and enable its controlled release. To engineer PLGA NP tropism for HSPCs, megakaryocyte (Mk) membranes, which possess HSPC-targeting moieties, were extracted and wrapped around PLGA NPs, producing MkNPs. In vitro, fluorophore-labeled MkNPs are internalized by HSPCs within 24 h and were selectively taken up by HSPCs versus other physiologically related cell types. Using membranes from megakaryoblastic CHRF-288 cells containing the same HSPC-targeting moieties as Mks, CHRF-wrapped NPs (CHNPs) loaded with small interfering RNA facilitated efficient RNA interference upon delivery to HSPCs in vitro. HSPC targeting was conserved in vivo, as poly(ethylene glycol)-PLGA NPs wrapped in CHRF membranes specifically targeted and were taken up by murine bone marrow HSPCs following intravenous administration. These findings suggest that MkNPs and CHNPs are effective and promising vehicles for targeted cargo delivery to HSPCs.

The role of biomechanical stress in extracellular vesicle formation, composition and activity.

Extracellular vesicles (EVs) are cornerstones of intercellular communication with exciting fundamental, clinical, and more broadly biotechnological applications. However, variability in EV composition, which results from the culture conditions used to generate the EVs, poses significant fundamental and applied challenges and a hurdle for scalable bioprocessing. Thus, an understanding of the relationship between EV production (and for clinical applications, manufacturing) and EV composition is increasingly recognized as important and necessary. While chemical stimulation and culture conditions such as cell density are known to influence EV biology, the impact of biomechanical forces on the generation, properties, and biological activity of EVs remains poorly understood. Given the omnipresence of these forces in EV preparation and in biomanufacturing, expanding the understanding of their impact on EV composition-and thus, activity-is vital. Although several publications have examined EV preparation and bioprocessing and briefly discussed biomechanical stresses as variables of interest, this review represents the first comprehensive evaluation of the impact of such stresses on EV production, composition and biological activity. We review how EV biogenesis, cargo, efficacy, and uptake are uniquely affected by various types, magnitudes, and durations of biomechanical forces, identifying trends that emerge both generically and for individual cell types. We also describe implications for scalable bioprocessing, evaluating processes inherent in common EV production and isolation methods, and propose a path forward for rigorous EV quality control.

Anaerobic fluorescent reporters for cell identification, microbial cell biology and high-throughput screening of microbiota and genomic libraries.

The lack of real-time reporters in obligate anaerobes has limited studies in gene expression, promoter characterization, library screening, population dynamics, and cell biology in these organisms. While the use of enzymatic, colorimetric, and luminescent reporters has been reported, the need for reliable anaerobic fluorescent proteins is widely acknowledged. Recently, the fluorescent proteins HaloTag, SNAP-tag and FAST have been established as reliable reporters in Clostridium spp., thus suggesting that these reporters can be adopted widely for many obligate anaerobes. With a multitude of labeling options, these anaerobic fluorescent proteins hold a great potential for screening promoters, terminators, and RBS sites, tracking population dynamics in complex multi-species co-cultures, such as microbiomes, screening libraries, and in cell biology studies of protein localization and interactions using high-resolution microscopy.

Interspecies Microbial Fusion and Large-Scale Exchange of Cytoplasmic Proteins and RNA in a Syntrophic Clostridium Coculture.

Microbial syntrophy is universal in nature, profoundly affecting the composition and function of microbiomes. We have recently reported data suggesting direct cell-to-cell interactions leading to electron and material exchange between the two microbes in the syntrophy between Clostridium ljungdahlii and C. acetobutylicum Here, transmission electron microscopy and electron tomography demonstrated cell wall and membrane fusions between the two organisms, whereby C. ljungdahlii appears to invade C. acetobutylicum pole to pole. Correlative fluorescence transmission electron microscopy demonstrated large-scale exchange of proteins. Flow cytometry analysis captured the extent and dynamic persistence of these interactions. Dividing hybrid cells were identified containing stained proteins from both organisms, thus demonstrating persistence of cells with exchanged cellular components. Fluorescence microscopy and flow cytometry of one species with stained RNA and the other tagged with a fluorescent protein demonstrated extensive RNA exchange and identified hybrid cells, some of which continued to divide, while some were in an advanced C. acetobutylicum sporulation form. These data demonstrate that cell fusion enables large-scale cellular material exchange between the two organisms. Although unanticipated and never previously reported, these phenomena are likely widely distributed in nature, have profound implications for species evolution and the function of microbial communities, and could find utility in biotechnology. They may shed new light onto little-understood phenomena, such as antibiotic heteroresistance of pathogens, pathogen invasion of human tissues, and the evolutionary trajectory and persistence of unculturable bacteria.IMPORTANCE We report that two different bacterial organisms engage in heterologous cell fusion that leads to massive exchange of cellular material, including proteins and RNA, and the formation of persistent hybrid cells. The interspecies cell fusion observed here involves a syntrophic microbial system, but these heterologous cell fusions were observed even under nonstrict syntrophic conditions, leaving open the possibility that strict syntrophy may not be necessary for interspecies cell fusion and cellular material exchange. Formation of hybrid cells that contain proteins and RNA from both organisms is unexpected and unprecedented. Such fusion events are likely widely distributed in nature, but have gone undetected. The implications are profound and may shed light onto many unexplained phenomena in human health, natural environments, evolutionary biology, and biotechnology.

Development of Strong Anaerobic Fluorescent Reporters for Clostridium acetobutylicum and Clostridium ljungdahlii Using HaloTag and SNAP-tag Proteins.

One of the biggest limitations in the study and engineering of anaerobic Clostridium organisms is the lack of strong fluorescent reporters capable of strong and real-time fluorescence. Recently, we developed a strong fluorescent reporter system for Clostridium organisms based on the FAST protein. Here, we report the development of two new strong fluorescent reporter systems for Clostridium organisms based on the HaloTag and SNAP-tag proteins, which produce strong fluorescent signals when covalently bound to fluorogenic ligands. These new fluorescent reporters are orthogonal to the FAST ligands and to each other, allowing for simultaneous labeling and visualization. We used HaloTag and SNAP-tag to label the strictly anaerobic organisms Clostridium acetobutylicum and Clostridium ljungdahlii We have also identified a new strong promoter for protein expression in C. acetobutylicum, based on the phosphotransacetylase gene (pta) from C. ljungdahlii Furthermore, the HaloTag and the SNAP-tag, in combination with the previously described FAST system, were successfully used to measure cell populations in bacterial mixed cultures and showed the simultaneous orthogonal labeling of HaloTag and SNAP-tag together with the FAST protein reporter. Finally, we show the expression of recombinant fusion protein of FAST and the ZapA division protein (from C. acetobutylicum) in C. ljungdahlii. The availability of multiple strong fluorescent reporters is a major addition to the genetic toolkit of Clostridium and other anaerobes that will lead to better understanding of these unique organisms.IMPORTANCE Up to this point, assays and methods involving fluorescent reporter proteins were unavailable or limited in Clostridium organisms and other strict anaerobes. Green fluorescent protein (GFP), mCherry, and flavin-binding proteins (and their derivatives) have been used only in a few clostridia with limited success and yielded low fluorescence compared to aerobic microbial systems. Recently, we reported a new strong fluorescent reporter system based on the FAST protein as a first step in expanding the fluorescence-based reporters for Clostridium and other anaerobic microbial platforms. Additional strong orthogonal fluorescent proteins, with distinct emission spectra are needed to allow for (i) multispecies tracking within the growing field of microbial cocultures and microbiomes, (ii) protein localization and tracking in anaerobes, and (iii) identification and development of natural and synthetic promoters, ribosome-binding sites (RBS), and terminators for optimal protein expression in anaerobes. Here, we present two new strong fluorescent reporter systems based on the HaloTag and SNAP-tag proteins.

Direct cell-to-cell exchange of matter in a synthetic Clostridium syntrophy enables CO2 fixation, superior metabolite yields, and an expanded metabolic space.

In microbial fermentations at least 33% of the sugar-substrate carbon is lost as CO2 during pyruvate decarboxylation to acetyl-CoA, with the corresponding electrons lost in the form of H2. Previous attempts to reduce this carbon and electron loss focused on engineering of a single organism. In nature, most microorganisms live in complex communities where syntrophic interactions result in superior resource utilization. Here, we show that a synthetic syntrophy consisting of the solventogen Clostridium acetobutylicum, which converts simple and complex carbohydrates into a variety of chemicals, and the acetogen C. ljungdahlii which fixes CO2, achieved carbon recoveries into C2-C4 alcohols almost to the limit of substrate-electron availability, with minimal H2 and CO2 release. The syntrophic co-culture produced robust metabolic outcomes over a broad range of starting population ratios of the two organisms. We show that direct cell-to-cell interactions and material exchange among the two microbes enabled unforeseen rearrangements in the metabolism of the individual species that resulted in the production of non-native metabolites, namely isopropanol and 2,3-butanediol. This was accomplished by pathway-specific alterations of gene expression brought about by one organism on the other, and vice versa. While some of these gene-expression alterations can be explained by the exchange of metabolites that induce specific gene expression patterns, others, as demonstrated by co-culture setup in a transwell system, cannot. The latter, for now, would be attributed to complex direct physical interactions among the two organisms, thus providing a glimpse of the potential microbial complexity of simple or multicomponent microbiomes. Such direct material-transfer phenomena have not been documented in the literature. Furthermore, our study shows that syntrophic cultures offer a flexible platform for metabolite production with superior carbon recovery that can also be applied to electron-enhanced fermentations enabling even higher carbon recoveries.

Building cellular pathways and programs enabled by the genetic diversity of allo-genomes and meta-genomes.

Engineering pathways, programs and traits that require interactions of many, often unknown, genes requires advanced engineering strategies in the context of synthetic biology. Such strategies derive from three basic requirements: a suitably enlarged gene pool compared to the parent strain; a method of identification and incorporation of genetic-element interactions to generate the multigenic pathway or trait; and a process of selection of individuals from diverse strain populations that benefit the desirable pathway or trait. We review potential methods utilized in such advanced engineering strategies, emphasizing methods that explore the genomic diversity of allogeneic DNA (the allogenome) or the metagenome. We also propose a modular iterative approach for developing multigenic cellular traits.

Exploring the heterologous genomic space for building, stepwise, complex, multicomponent tolerance to toxic chemicals.

Modern bioprocessing depends on superior cellular traits, many stemming from unknown genes and gene interactions. Tolerance to toxic chemicals is such an industrially important complex trait, which frequently limits the economic feasibility of producing commodity chemicals and biofuels. Chemical tolerance encompasses both improved cell viability and growth under chemical stress. Building upon the success of our recently reported semisynthetic stress response system expressed off plasmid pHSP (Heat Shock Protein), we probed the genomic space of the solvent tolerant Lactobacillus plantarum to identify genetic determinants that impart solvent tolerance in combination with pHSP. Using two targeted enrichments, one for superior viability and one for better growth under ethanol stress, we identified several beneficial heterologous DNA determinants that act synergistically with pHSP. In separate strains, a 209% improvement in survival and an 83% improvement in growth over previously engineered strains based on pHSP were thus generated. We then developed a composite phenotype of improved growth and survival by combining the identified L. plantarum genetic fragments. This demonstrates the concept for a sequential, iterative assembly strategy for building multigenic traits by exploring the synergistic effects of genetic determinants from a much broader genomic space. The best performing strain produced a 3.7-fold improved survival under 8% ethanol stress, as well as a 32% increase in growth under 4% ethanol. This strain also shows significantly improved tolerance to n-butanol. Improved solvent production is rarely examined in tolerance engineering studies. Here, we show that our system significantly improves ethanol productivity in a Melle-Boinot-like fermentation process.

Proteomic analyses of the phase transition from acidogenesis to solventogenesis using solventogenic and non-solventogenic Clostridium acetobutylicum strains.

The fermentation carried out by the solvent-producing bacterium, Clostridium acetobutylicum, is characterized by two distinct phases: acidogenic and solventogenic phases. Understanding the cellular physiological changes occurring during the phase transition in clostridial fermentation is important for the enhanced production of solvents. To identify protein changes upon entry to stationary phase where solvents are typically produced, we herein analyzed the proteomic profiles of the parental wild type C. acetobutylicum strains, ATCC 824, the non-solventogenic strain, M5 that has lost the solventogenic megaplasmid pSOL1, and the synthetic simplified alcohol forming strain, M5 (pIMP1E1AB) expressing plasmid-based CoA-transferase (CtfAB) and aldehyde/alcohol dehydrogenase (AdhE1). A total of 68 protein spots, corresponding to 56 unique proteins, were unambiguously identified as being differentially present after the phase transitions in the three C. acetobutylicum strains. In addition to changes in proteins known to be involved in solventogenesis (AdhE1 and CtfB), we identified significant alterations in enzymes involved in sugar transport and metabolism, fermentative pathway, heat shock proteins, translation, and amino acid biosynthesis upon entry into the stationary phase. Of these, four increased proteins (AdhE1, CAC0233, CtfB and phosphocarrier protein HPr) and six decreased proteins (butyrate kinase, ferredoxin:pyruvate oxidoreductase, phenylalanyl-tRNA synthetase, adenylosuccinate synthase, pyruvate kinase and valyl-tRNA synthetase) showed similar patterns in the two strains capable of butanol formation. Interestingly, significant changes of several proteins by post-translational modifications were observed in the solventogenic phase. The proteomic data from this study will improve our understanding on how cell physiology is affected through protein levels patterns in clostridia.

Toward a semisynthetic stress response system to engineer microbial solvent tolerance.

UNLABELLED: Strain tolerance to toxic metabolites is an important trait for many biotechnological applications, such as the production of solvents as biofuels or commodity chemicals. Engineering a complex cellular phenotype, such as solvent tolerance, requires the coordinated and tuned expression of several genes. Using combinations of heat shock proteins (HSPs), we engineered a semisynthetic stress response system in Escherichia coli capable of tolerating high levels of toxic solvents. Simultaneous overexpression of the HSPs GrpE and GroESL resulted in a 2-fold increase in viable cells (CFU) after exposure to 5% (vol/vol) ethanol for 24 h. Co-overexpression of GroESL and ClpB on coexisting plasmids resulted in 1,130%, 78%, and 25% increases in CFU after 24 h in 5% ethanol, 1% n-butanol, and 1% i-butanol, respectively. Co-overexpression of GrpE, GroESL, and ClpB on a single plasmid produced 200%, 390%, and 78% increases in CFU after 24 h in 7% ethanol, 1% n-butanol, or 25% 1,2,4-butanetriol, respectively. Overexpression of other autologous HSPs (DnaK, DnaJ, IbpA, and IbpB) alone or in combinations failed to improve tolerance. Expression levels of HSP genes, tuned through inducible promoters and the plasmid copy number, affected the effectiveness of the engineered stress response system. Taken together, these data demonstrate that tuned co-overexpression of GroES, GroEL, ClpB, and GrpE can be engaged to engineer a semisynthetic stress response system capable of greatly increasing the tolerance of E. coli to solvents and provides a starting platform for engineering customized tolerance to a wide variety of toxic chemicals. IMPORTANCE: Microbial production of useful chemicals is often limited by the toxicity of desired products, feedstock impurities, and undesired side products. Improving tolerance is an essential step in the development of practical platform organisms for production of a wide range of chemicals. By overexpressing autologous heat shock proteins in Escherichia coli, we have developed a modular semisynthetic stress response system capable of improving tolerance to ethanol, n-butanol, and potentially other toxic solvents. Using this system, we demonstrate that a practical stress response system requires both tuning of individual gene components and a reliable framework for gene expression. This system can be used to seek out new interacting partners to improve the tolerance phenotype and can be used in the development of more robust solvent production strains.

Metabolic flux analysis of embryonic stem cells using three distinct differentiation protocols and comparison to gene expression patterns.

Metabolic flux analysis (MFA) was performed on mouse embryonic stem cells cultured under three distinct differentiation conditions: classical embryoid body formation (EB), and on surfaces coated with either gelatin (GEL) or matrigel (MAT). MFA was based on 15 metabolic reactions and eight transport steps, and was carried out based on measurements of four substrates and/or metabolites: glucose, lactate, glutamine, and glutamate. Fluxes representing biomass production remained fairly constant for all three culture conditions with at most a 40% variation. In contrast, major temporal variations, up to 500%, were observed for all other major central metabolic fluxes across all culture conditions. Fluxes were compared to gene-expression patterns measured by microarray analysis. Particularly interesting is the correlation between the metabolic fluxes with expression patterns of the corresponding genes of the pyruvate to lactate reaction, whereby the genes for several isoforms of the lactate dehydrogenase enzyme were examined. The patterns of this flux were notably different in the EB cultures compared to the GEL and MAT cultures and reflected differences in oxygen and nutrient transport in EB vs. the GEL and MAT cultures. Another novel finding of this study is an event occurring between Days 4 and 5 of differentiation, which was identified by a notable change in both the metabolic fluxes and gene-expression patterns. This suggests that metabolic patterns can be used as effective beacons of changes in differentiating stem cells. Overall, and qualitatively, core metabolic fluxes, under the three culture conditions examined, correlated well with gene-expression patterns.

A genomic-library based discovery of a novel, possibly synthetic, acid-tolerance mechanism in Clostridium acetobutylicum involving non-coding RNAs and ribosomal RNA processing.

We generated a genomic library from sheared Clostridium acetobutylicum ATCC 824 DNA, whereby inserts can be expressed in both directions from the thiolase promoter, P(thl). Serial transfer of library-bearing C. acetobutylicum cultures exposed to increasing butyrate concentrations enriched for inserts containing fragments of rRNA genetic loci. The selected library inserts were placed so that antisense (to the rRNAs) non-coding RNAs (ncRNAs) would be transcribed from P(thl). Different enriched inserts imparted similar butyrate-tolerance characteristics. A minimal tolerance fragment (RDNA7) was identified as the 16S-rRNA promoter region. Expressed on plasmid pRD7 off P(thl), RDNA7 can produce putative ncRNAs termed ncRNA(RD7). C. acetobutylicum 824(pRD7) showed superior resistance to butyrate and other carboxylic acids. Transcriptional analysis of butyrate stress identified 120 differentially expressed genes between 824(pRD7) and 824(pSOS95del). The few upregulated genes included the ffh gene of the putative signal recognition particle (SRP) system. Northern analysis of ncRNA(RD7) and corresponding antisense RNAs demonstrated multiple ncRNA(RD7) molecules in 824(pRD7). Several corresponding antisense RNA molecules were identified both in 824(pRD7) and 824(pSOS95del), but at much higher levels in 824(pRD7). Northern analysis of 16S rRNA expression suggested complex RDNA7-dependent rRNA processing. Our data suggest that by hybridizing against unprocessed rRNA precursors, ncRNA(RD7) alters rRNA processing, and these alterations result in acid tolerance, possibly through a mechanism involving the Ffh protein.

Metabolic engineering of Clostridium acetobutylicum M5 for highly selective butanol production.

To improve butanol selectivity, Clostridium acetobutylicum M5(pIMP1E1AB) was constructed by adhE1-ctfAB complementation of C. acetobutylicum M5, a derivative strain of C. acetobutylicum ATCC 824, which does not produce solvents due to the lack of megaplasmid pSOL1. The gene products of adhE1-ctfAB catalyze the formation of acetoacetate and ethanol/butanol with acid re-assimilation in solventogenesis. Effects of the adhE1-ctfAB complementation of M5 were studied by batch fermentations under various pH and glucose concentrations, and by flux balance analysis using a genome-scale metabolic model for this organism. The metabolically engineered M5(pIMP1E1AB) strain was able to produce 154 mM butanol with 9.9 mM acetone at pH 5.5, resulting in a butanol selectivity (a molar ratio of butanol to total solvents) of 0.84, which is much higher than that (0.57 at pH 5.0 or 0.61 at pH 5.5) of the wild-type strain ATCC 824. Unlike for C. acetobutylicum ATCC 824, a higher level of acetate accumulation was observed during fermentation of the M5 strain complemented with adhE1 and/or ctfAB. A plausible reason for this phenomenon is that the cellular metabolism was shifted towards acetate production to compensate reduced ATP production during the largely growth-associated butanol formation by the M5(pIMP1E1AB) strain.

Comparative transcriptional analysis of embryoid body versus two-dimensional differentiation of murine embryonic stem cells.

Understanding the process of ex vivo embryonic stem (ES) cell differentiation is important for generating higher yields of desirable cell types or lineages and for understanding fundamental aspects of ES differentiation. We used DNA microarray analysis to investigate the differentiation of mouse ES cells cultured under three differentiation conditions. Embryoid body (EB) formation was compared to differentiation on surfaces coated with either gelatin (GEL) or matrigel (MAT). Based on the transcriptional patterns of a list of literature-based "stemness" genes, ES cell differentiation on the two coated surfaces appeared similar but not identical to EB differentiation. A notable difference was the GEL and MAT upregulation but EB downregulation of nine such stemness genes, which are related to cell adhesion and epithelial differentiation. Further, GEL and MAT differentiation showed higher expression of bone formation-related genes (Spp1, Csf1, Gsn, Bmp8b, Crlf1). Gene ontology analysis shows an increase in the expression of genes related to migration and cell structure in all three conditions. Overall, GEL and MAT conditions resulted in a more similar to each other transcriptional profile than to the EB condition, and such differences are apparently related to higher nutrient and metabolite gradients and limitations in the EB versus the GEL or MAT cultures.

Dynamics of genomic-library enrichment and identification of solvent tolerance genes for Clostridium acetobutylicum.

A Clostridium acetobutylicum ATCC 824 genomic library was constructed using randomly sheared DNA. Library inserts conferring increased tolerance to 1-butanol were isolated using two protocols. Protocol I utilized a single round of butanol challenges in batch culture, while protocol II, which gave clearly superior outcomes, was based on the serial transfer of stationary-phase cultures into progressively higher butanol concentrations. DNA microarray analysis made a high-resolution assessment of the dynamic process of library enrichment possible for the first time. Protocol I yielded a library insert containing the entire coding region of the gene CAC0003 (which codes for a protein of unknown function) but also several DNA fragments containing promoter regions. Protocol II enabled the successful identification of DNA fragments containing several intact genes conferring preferential growth under conditions of butanol stress. Since expression using the employed library is possible only from natural promoters, among the enriched genes, we identified 16 genes that constitute the first cistron of a transcriptional unit. These genes include four transcriptional regulators (CAC0977, CAC1463, CAC1869, and CAC2495). After subcloning plasmids carrying the CAC0003 and CAC1869 genes, strains 824(pCAC0003) and 824(pCAC1869) exhibited 13% and an 81% increases, respectively, in butanol tolerance relative to the plasmid control strain. 824(pCAC1869) consistently grew to higher cell densities in challenged and unchallenged cultures and exhibited prolonged metabolism. Our serial enrichment approach provided a more detailed understanding of the dynamic process of library enrichment under conditions of selective growth. Further characterization of the genes identified in this study will likely enhance our understanding of the complex phenotype of solvent tolerance.

Diffusion, mixing, and associated dye effects in DNA-microarray hybridizations.

Typical DNA microarrays utilize diffusion of dye-labeled cDNA probes followed by sequence-specific hybridization to immobilized targets. Here we experimentally estimated the distance typical probes travel during static 16-h hybridizations. Probes labeled with Cy3 and Cy5 were individually introduced to opposite sides of a microarray with minimal convective mixing. Oppositely labeled probes diffused across the initial front separating the two solutions, generating a zone with both dyes present. Diffusion-distance estimates for Cy3- and Cy5-labeled cDNAs were 3.8 mm and 2.6 mm, respectively, despite having almost identical molecular masses. In separate 16-h hybridization experiments with oppositely labeled probes premixed, arrays that were continuously mixed had 15-20% higher signal intensities than arrays hybridized statically. However, no change was observed in the Cy3/Cy5 signal intensity ratio between continuously mixed and static hybridizations. This suggests that the observed dye bias in diffusion-distance estimates results from differences in the detection limits of Cy3 and Cy5-labeled cDNA, a potential concern for array data on low-abundance transcripts. Our conservative diffusion-distance estimates indicate that replicate targets >7.6 mm apart will not compete for scarce probes. Also, raising the microarray gap height would delay the onset of diffusion-limited hybridization by increasing the amount of available probe.