scholarly journals Study on the isoprene-producing co-culture system of Synechococcus elongatus- Escherichia coli through omics analysis

2020 ◽  
Author(s):  
Hui Liu ◽  
Yujin Cao ◽  
Jing Guo ◽  
Xin Xu ◽  
Qi Long ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Culture System ◽  
Rational Design ◽  
Continuous Fermentation ◽  
Axenic Culture ◽  
Scientific Basis ◽  
Synechococcus Elongatus ◽  
Spatial And Temporal Scales ◽  
E Coli ◽  
Omics Analysis

Abstract Background: The majority of microbial fermentations are currently performed in the batch or fed-batch manner with the high process complexity, huge water consumption and so on. The continuous microbial production can contribute to the green sustainable development of the fermentation industry. The co-culture systems of photo-autotrophic and heterotrophic species can play important roles in establishing the continuous fermentation mode for the bio-based chemicals production. Results: In the present paper, the co-culture system of Synechococcus elongatus- Escherichia coli was established and put into operation stably for isoprene production. Compared with the axenic culture, the fermentation period of time was extended from 100h to 400h in the co-culture and the isoprene production was increased to 8-fold. For in depth understanding this novel system, the differential omics profiles were analyzed. The responses of BL21(DE3) to S. elongatus PCC 7942 were triggered by the oxidative pressure through the Fenton reaction and all these changes were linked with one another at different spatial and temporal scales. The oxidative stress mitigation pathways might contribute to the long-lasting fermentation process. The performance of this co-culture system can be further improved according to the fundamental rules discovered by the omics analysis. Conclusions: The isoprene-producing co-culture system of S. elongatus- E. coli was established and then analyzed by the omics methods. This study on the co-culture system of the model S. elongatus- E. coli is of significance to reveal the common interactions between photo-autotrophic and heterotrophic species without natural symbiotic relation, which could provide the scientific basis for rational design of microbial community.

scholarly journals Study on the isoprene-producing co-culture system of Synechococcus elongates–Escherichia coli through omics analysis

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Hui Liu ◽  
Yujin Cao ◽  
Jing Guo ◽  
Xin Xu ◽  
Qi Long ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Culture System ◽  
Rational Design ◽  
Continuous Fermentation ◽  
Axenic Culture ◽  
Scientific Basis ◽  
Spatial And Temporal Scales ◽  
E Coli ◽  
Omics Analysis ◽  
Symbiotic Relation

Abstract Background The majority of microbial fermentations are currently performed in the batch or fed-batch manner with the high process complexity and huge water consumption. The continuous microbial production can contribute to the green sustainable development of the fermentation industry. The co-culture systems of photo-autotrophic and heterotrophic species can play important roles in establishing the continuous fermentation mode for the bio-based chemicals production. Results In the present paper, the co-culture system of Synechococcus elongates–Escherichia coli was established and put into operation stably for isoprene production. Compared with the axenic culture, the fermentation period of time was extended from 100 to 400 h in the co-culture and the isoprene production was increased to eightfold. For in depth understanding this novel system, the differential omics profiles were analyzed. The responses of BL21(DE3) to S. elongatus PCC 7942 were triggered by the oxidative pressure through the Fenton reaction and all these changes were linked with one another at different spatial and temporal scales. The oxidative stress mitigation pathways might contribute to the long-lasting fermentation process. The performance of this co-culture system can be further improved according to the fundamental rules discovered by the omics analysis. Conclusions The isoprene-producing co-culture system of S. elongates–E. coli was established and then analyzed by the omics methods. This study on the co-culture system of the model S. elongates–E. coli is of significance to reveal the common interactions between photo-autotrophic and heterotrophic species without natural symbiotic relation, which could provide the scientific basis for rational design of microbial community.

scholarly journals Cross-Feeding between Cyanobacterium Synechococcus and Escherichia Coli in Artificial Autotrophic-Heterotrophic Co-Culture System Revealed by Integrated Omics Analysis

2021 ◽  
Author(s):  
Jiajia Ma ◽  
Taohong Guo ◽  
Lei Chen ◽  
Xinyu Song ◽  
Weiwen Zhang
Keyword(s):  
Escherichia Coli ◽  
Ascorbic Acid ◽  
Cell Growth ◽  
Carbon Metabolism ◽  
Culture System ◽  
Value Added ◽  
Ros Scavenging ◽  
E Coli ◽  
Omics Analysis ◽  
Integrated Omics

Abstract Background: The light-driven consortia consisted of sucrose-secreting cyanobacteria and heterotrophic species capable of producing valuable chemicals have recently attracted significant attention, and are considered as a promising strategy for green biomanufacturing. In a previous study (Zhang et al, 2020, Biotechnol Biofuel, 13:82), we achieved a one-step conversion of CO2 through sucrose derived from cyanobacteria to fine chemicals by constructing an artificial co-culture system consisting of sucrose-secreting Synechococcus elongateus cscB+ and 3-hydroxypropionic acid (3-HP) producing Escherichia coli ABKm. Analysis of the co-culture system showed that cyanobacterial cells were growing better than its corresponding axenic culture. To explore the underlaid mechanism and to identify the metabolic modules to further improve the co-culture system, an integrated metabolomics, transcriptomic and proteomic analysis was conducted.Results: We first explored the effect of reactive oxygen species (ROS) on cyanobacterial cell growth under co-culture system by supplementing additional ascorbic acid to scavenge ROS in CoBG-11 medium. The result showed cyanobacterial growth was obviously improved with additional 1 mM ascorbic acid under pure culture; however, cyanobacterial growth was still slower than that in the co-culture with E. coli, suggesting that the better growth of Synechococcus cscB+ might be caused by other factors more than just ROS quenching. We then investigated the intracellular metabolite levels in cyanobacteria using LC-MS based metabolomics analysis. The results showed that metabolites involved in central carbon metabolism were increased, suggesting more carbon sources were utilized by cyanobacteria in the co-culture system, which illuminating that enhanced photosynthesis attributes to the higher CO2 availability produced from co-cultivated heterotrophic partner. To further explore the interaction based on cross-feeding and metabolite exchange, quantitative transcriptomics and proteomics were applied to Synechococcus cscB+. Analysis of differentially regulated genes/proteins showed that the higher availability of carbon, nitrogen, phosphate, calcium, Cu2+, Fe3+ and co-factors was observed in co-cultivated Synechococcus cscB+ during co-cultivation, suggesting the heterotrophic partner in the system might be involved in supplementing CO2 and improving essential micronutrients necessary to maintain high photosynthetic growth of Synechococcus cscB+. Conclusion: Integrated omics analysis of the interaction mechanism between S. elongateus and E. coli showed metabolic changes such as enhanced photosynthesis, oxidative phosphorylation, essential micronutrients, and the ROS scavenging occurred at multiple levels of genes, proteins and metabolites, which might be together contributing to the better cell growth of Synechococcus cscB+ in co-cultivation. In addition, the results implicated that the co-culture system could be further improved by engineering the modules related to the ROS quenching, carbon metabolism, nitrogen metabolism, Pi transport, metal transport and co-factors biosynthesis. Finally, the light condition, which may influence the cross-feeding metabolites between phototrophic and heterotrophic species, and also affect the oxidative pressure on the E. coli strains due to the photosynthesis, could be further optimized to improve cell growth in the co-culture system, eventually leading to high productivity of value-added products.

scholarly journals In Silico Design for Systems-Based Metabolic Engineering In Escherichia Coli for The Bioconversion of Aerobic Glycerol to Succinic Acid

2020 ◽  
Author(s):  
Albert Enrique Tafur Rangel ◽  
Wendy Lorena Rios Guzman ◽  
Carmen Elvira Ojeda Cuella ◽  
Daissy Esther Mejia Perez ◽  
Ross Carlson ◽  
...  
Keyword(s):  
Machine Learning ◽  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Succinic Acid ◽  
In Silico ◽  
Rational Design ◽  
Industrial Biotechnology ◽  
E Coli ◽  
Cell Factories ◽  
Transcriptomics Data

Abstract BackgroundGlycerol has become an interesting carbon source for industrial processes as consequence of the biodiesel business growth since it has shown promising results in terms of biomass/substrate yields. Selecting the appropriate metabolic targets to build efficient cell factories and maximize the desired chemical production in as little time as possible is a major challenge in industrial biotechnology. The engineering of microbial metabolism following rational design has been widely studied. However, it is a cost-, time-, and laborious-intensive process because of the cell network complexity; thus, to be proficient is needed known in advance the effects of gene deletions.ResultsAn in silico experiment was performed to model and understand the effects of metabolic engineering over the metabolism by transcriptomics data integration. In this study, systems-based metabolic engineering to predict the metabolic engineering targets was used in order to increase the bioconversion of glycerol to succinic acid by Escherichia coli. Transcriptomics analysis suggest insights of how increase the glycerol utilization of the cell for further design efficient cell factories. Three models were used; an E. coli core model, a model obtained after the integration of transcriptomics data obtained from E. coli growing in an optimized culture media, and a third one obtained after integration of transcriptomics data obtained from E. coli after adaptive laboratory evolution experiments. A total of 2402 strains were obtained from these three models. Fumarase and pyruvate dehydrogenase were frequently predicted in all the models, suggesting that these reactions are essential to increasing succinic acid production from glycerol. Finally, using flux balance analysis results for all the mutants predicted, a machine learning method was developed to predict new mutants as well as to propose optimal metabolic engineering targets and mutants based on the measurement of importance of each knockout’s (feature’s) contribution.ConclusionsThe combination of transcriptome, systems metabolic modeling, and machine learning analyses revealed versatile molecular mechanisms involved in the utilization of glycerol. These data provide a platform to improve the prediction of metabolic engineering targets to design efficient cell factories. Our results may also work a guide platform for the selection/engineering of microorganisms for production of interesting chemical compounds.

scholarly journals Artificial modulation of cell width significantly affects the division time of Escherichia coli

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Baihui Liang ◽  
Baogang Quan ◽  
Junjie Li ◽  
Chantal Loton ◽  
Marie-Florence Bredeche ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Temporal Correlation ◽  
Cell Physiology ◽  
Bacterial Cells ◽  
Cell Width ◽  
Division Rate ◽  
Microscopy Imaging ◽  
Spatial And Temporal Scales ◽  
E Coli

Abstract Bacterial cells have characteristic spatial and temporal scales. For instance, Escherichia coli, the typical rod-shaped bacteria, always maintains a relatively constant cell width and cell division time. However, whether the external physical perturbation of cell width has an impact on cell division time remains largely unexplored. In this work, we developed two microchannel chips, namely straight channels and ‘necked’ channels, to precisely regulate the width of E. coli cells and to investigate the correlation between cell width and division time of the cells. Our results show that, in the straight channels, the wide cells divide much slower than narrow cells. In the ‘necked’ channels, the cell division is remarkably promoted compared to that in straight channels with the same width. Besides, fluorescence time-lapse microscopy imaging of FtsZ dynamics shows that the cell pre-constriction time is more sensitive to cell width perturbation than cell constriction time. Finally, we revealed a significant anticorrelation between the death rate and the division rate of cell populations with different widths. Our work provides new insights into the correlation between the geometrical property and division time of E. coli cells and sheds new light on the future study of spatial–temporal correlation in cell physiology.

scholarly journals Critical Synergistic Concentration of Lecithin Phospholipids Improves the Antimicrobial Activity of Eugenol against Escherichia coli

2017 ◽  
Vol 83 (21) ◽  
Author(s):  
Haoshu Zhang ◽  
Edward G. Dudley ◽  
Federico Harte
Keyword(s):  
Escherichia Coli ◽  
Foodborne Pathogens ◽  
Rational Design ◽  
Microbial Population ◽  
Critical Concentration ◽  
Antimicrobial Properties ◽  
Content Type ◽  
E Coli ◽  
Log Reduction ◽  
Naturally Occurring

ABSTRACT In this study, the effect of individual lecithin phospholipids on the antimicrobial properties of eugenol against Escherichia coli C600 was investigated. We tested five major phospholipids common in soy or egg lecithin (1,2-dihexadecanoyl-sn-glycero-3-phosphocholine [DPPC], 1,2-dioctadecanoyl-sn-glycero-3-phosphocholine [DSPC], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine [DPPE], 1,2-dihexadecanoyl-sn-glycero-3-phosphate [sodium salt] [DPPA], and 1,2-dihexadecanoyl-sn-glycero-3-phospho-l-serine [DPPS]) and one synthetic cationic phospholipid (1,2-dioctadecanoyl-sn-glycero-3-ethylphosphocholine [18:0 EPC]). Among the six phospholipids, DPPC, DSPC, DPPE, DPPA, and the cationic 18:0 EPC showed critical synergistic concentrations that significantly improved the inactivation effect of eugenol against E. coli after 30 min of exposure. At the critical synergistic concentration, an additional ca. 0.4 to 1.9 log reduction (ca. 0.66 to 2.17 log CFU/ml reduction) in the microbial population was observed compared to eugenol-only (control) treatments (ca. 0.25 log reduction). In all cases, increasing the phospholipid amount above the critical synergistic concentration (which was different for each phospholipid) resulted in antimicrobial properties similar to those seen with the eugenol-only (control) treatments. DPPS did not affect the antimicrobial properties of eugenol at the tested concentrations. The critical synergistic concentration of phospholipids was correlated with their critical micelle concentrations (CMC). IMPORTANCE Essential oils (EOs) are naturally occurring antimicrobials, with limited use in food due to their hydrophobicity and strong aroma. Lecithin is used as a natural emulsifier to stabilize EOs in aqueous systems. We previously demonstrated that, within a narrow critical-concentration window, lecithin can synergistically enhance the antimicrobial properties of eugenol. Since lecithin is a mixture of different phospholipids, we aimed to identify which phospholipids are crucial for the observed synergistic effect. This research studied the bioactivity of lecithin phospholipids, contributing to a rational design in using lecithin to effectively control foodborne pathogens in foods.

scholarly journals Construction and evolution of anEscherichia colistrain relying on nonoxidative glycolysis for sugar catabolism

2018 ◽  
Vol 115 (14) ◽  
pp. 3538-3546 ◽  
Author(s):  
Paul P. Lin ◽  
Alec J. Jaeger ◽  
Tung-Yun Wu ◽  
Sharon C. Xu ◽  
Abraxa S. Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Energy Metabolism ◽  
Rational Design ◽  
Minimal Medium ◽  
Global Regulation ◽  
Gene Deletions ◽  
Global Regulators ◽  
E Coli ◽  
Almost All ◽  
Tracer Experiments

The Embden–Meyerhoff–Parnas (EMP) pathway, commonly known as glycolysis, represents the fundamental biochemical infrastructure for sugar catabolism in almost all organisms, as it provides key components for biosynthesis, energy metabolism, and global regulation. EMP-based metabolism synthesizes three-carbon (C3) metabolites before two-carbon (C2) metabolites and must emit one CO2in the synthesis of the C2 building block, acetyl-CoA, a precursor for many industrially important products. Using rational design, genome editing, and evolution, here we replaced the native glycolytic pathways inEscherichia coliwith the previously designed nonoxidative glycolysis (NOG), which bypasses initial C3 formation and directly generates stoichiometric amounts of C2 metabolites. The resulting strain, which contains 11 gene overexpressions, 10 gene deletions by design, and more than 50 genomic mutations (including 3 global regulators) through evolution, grows aerobically in glucose minimal medium but can ferment anaerobically to products with nearly complete carbon conservation. We confirmed that the strain metabolizes glucose through NOG by13C tracer experiments. This redesignedE. colistrain represents a different approach for carbon catabolism and may serve as a useful platform for bioproduction.

scholarly journals A novel strategy for D-psicose and lipase co-production using a co-culture system of engineered Bacillus subtilis and Escherichia coli and bioprocess analysis using metabolomics

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Jun Zhang ◽  
Wen Luo ◽  
Zhiyuan Wang ◽  
Xiaoyan Chen ◽  
Pengmei Lv ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Bacillus Subtilis ◽  
Culture System ◽  
Fermentation Process ◽  
Tryptophan Metabolism ◽  
E Coli ◽  
Novel Strategy ◽  
By Products ◽  
Metabolomics Analysis

AbstractTo develop an economically feasible fermentation process, this study designed a novel bioprocess based on the co-culture of engineered Bacillus subtilis and Escherichia coli for the co-production of extracellular D-psicose and intracellular lipase. After optimizing the co-culture bioprocess, 11.70 g/L of D-psicose along with 16.03 U/mg of lipase was obtained; the glucose and fructose were completely utilized. Hence, the conversion rate of D-psicose reached 69.54%. Compared with mono-culture, lipase activity increased by 58.24%, and D-psicose production increased by 7.08%. In addition, the co-culture bioprocess was explored through metabolomics analysis, which included 168 carboxylic acids and derivatives, 70 organooxygen compounds, 34 diazines, 32 pyridines and derivatives, 30 benzene and substituted derivatives, and other compounds. It also could be found that the relative abundance of differential metabolites in the co-culture system was significantly higher than that in the mono-culture system. Pathway analysis revealed that, tryptophan metabolism and β-alanine metabolism had the highest correlation and played an important role in the co-culture system; among them, tryptophan metabolism regulates protein synthesis and β-alanine metabolism, which is related to the formation of metabolic by-products. These results confirm that the co-cultivation of B. subtilis and E. coli can provide a novel idea for D-psicose and lipase biorefinery, and are beneficial for the discovery of valuable secondary metabolites such as turanose and morusin.

scholarly journals Pinpointing Synechococcus Rubisco Large Subunit Sections Involved in Heterologous Holoenzyme Formation in Escherichia Coli

2021 ◽  
Author(s):  
Wei Chi Ong ◽  
Moh Lan Yap ◽  
Hann Ling Wong ◽  
Boon Hoe Lim
Keyword(s):  
Escherichia Coli ◽  
Large Subunit ◽  
Synechococcus Elongatus ◽  
Bisphosphate Carboxylase ◽  
E Coli ◽  
Successful Expression

Abstract Background: Heterologous holoenzyme formation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) have been a challenge due to limited understanding of its biogenesis. Unlike bacterial Rubiscos, eukaryotic Rubiscos are incompatible with the Escherichia coli chaperone system to fold and assemble into the functional hexadecameric conformation (L8S8), which comprise eight large subunits (RbcL) and eight small subunits (RbcS). Our previous study reported three sections (residues 248-297, 348-397 and 398-447) within the RbcL of Synechococcus elongatus PCC6301 may be important for formation of L8S8 in E. coli. Present study further examined these three sections separately, by dividing them into six sections of 25 residues (i.e. residues 248-272, 273-297, 348-372, 373-397, 398-422 and 423-447). Methods and Results: Six chimeric Rubiscos with each section within the RbcL from Synechococcus replaced by their respective counterpart sequence from Chlamydomonas reinhardtii were constructed and checked for their effect on holoenzyme formation in E. coli. Present study shows that Section 1 (residues 248-272; section of Synechococcus RbcL replaced by corresponding Chlamydomonas sequence), Section 2 (residues 273-297), Section 3 (residues 348-372) and Section 6 (residues 423-447) chimeras failed to fold and/or assemble despite successful expression of both RbcL and RbcS. Only Section 4 (residues 373-397) and 5 (residues 398-422) chimeras could form L8S8 in E. coli. Conclusion: As GroEL chaperonin mediates folding of bacterial RbcL in E. coli, residues 248-297, 348-372 and 423-447 of Synechococcus RbcL may be important for interacting with the GroEL chaperonin for successful holoenzyme formation in E. coli.

scholarly journals Displacement of Escherichia coli O157:H7 from Rumen Medium Containing Prebiotic Sugars

2002 ◽  
Vol 68 (2) ◽  
pp. 519-524 ◽  
Author(s):  
Albane de Vaux ◽  
Mark Morrison ◽  
Robert W. Hutkins
Keyword(s):  
Escherichia Coli ◽  
Culture System ◽  
Rumen Fluid ◽  
Enteric Bacteria ◽  
Fermentation Medium ◽  
Escherichia Coli O157 ◽  
Anaerobic Culture ◽  
Fluid Medium ◽  
E Coli ◽  
Cell Densities

ABSTRACT A fed-batch, anaerobic culture system was developed to assess the behavior of Escherichia coli O157:H7 in a rumen-like environment. Fermentation medium consisted of either 50% (vol/vol) raw or sterile rumen fluid and 50% phosphate buffer. Additional rumen fluid was added twice per day, and samples were removed three times per day to simulate the exiting of digesta and microbes from the rumen environment under typical feeding regimens. With both types of medium, anaerobic and enteric bacteria reached 1010 and 104 cells/ml, respectively, and were maintained at these levels for at least 5 days. When a rifampin-resistant strain of E. coli O157:H7 was inoculated into medium containing raw rumen fluid, growth did not occur. In contrast, when this strain was added to sterile rumen fluid medium, cell densities increased from 106 to 109 CFU/ml within 24 h. Most strains of E. coli O157:H7 are unable to ferment sorbitol; therefore, we assessed whether the addition of sorbitol as the only added carbohydrate could be used to competitively exclude E. coli O157:H7 from the culture system. When inoculated into raw rumen broth containing 3 g of sorbitol per liter, E. coli O157:H7 was displaced within 72 h. The addition of other competitive sugars, such as l-arabinose, trehalose, and rhamnose, to rumen medium gave similar results. However, whenever E. coli O157:H7 was grown in sterile rumen broth containing sorbitol, sorbitol-positive mutants appeared. These results suggest that a robust population of commensal ruminal microflora is required to invoke competitive exclusion of E. coli O157:H7 by the addition of “nonfermentable” sugars and that this approach may be effective as a preharvest strategy for reducing carriage of E. coli O157:H7 in the rumen.

scholarly journals Making All Parts of the 16S rRNA of Escherichia coli Accessible In Situ to Single DNA Oligonucleotides

2006 ◽  
Vol 72 (1) ◽  
pp. 733-744 ◽  
Author(s):  
L. Şafak Yilmaz ◽  
Hatice E. Ökten ◽  
Daniel R. Noguera
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Fluorescence Intensity ◽  
Rational Design ◽  
Mechanistic Model ◽  
Mechanistic Modeling ◽  
E Coli ◽  
Dna Oligonucleotides ◽  
Nucleic Acid Interactions

ABSTRACT rRNA accessibility is a major sensitivity issue limiting the design of working probes for fluorescence in situ hybridization (FISH). Previous studies empirically highlighted the accessibility of target sites on rRNA maps by grouping probes into six classes according to their brightness levels. In this study, a recently proposed mechanistic model of FISH, based on the thermodynamics of secondary nucleic acid interactions, was used to evaluate the accessibility of the 16S rRNA of Escherichia coli to fluorescein-labeled oligonucleotides when thermodynamic and kinetic barriers were eliminated. To cover the entire 16S rRNA, 109 probes were designed with an average thermodynamic affinity (ΔG o overall) of −13.5 kcal/mol. Fluorescence intensity was measured by flow cytometry, and a brightness threshold between classes 3 and 4 was used as the requirement for proof of accessibility. While 46% of the probes were above this threshold with conventional 3-h hybridizations, extending the incubation period to 96 h dramatically increased the fraction of bright probes to 86%. Insufficient thermodynamic affinity and/or fluorophore quenching was demonstrated to cause the low fluorescence intensity of the remaining 14% of the probes. In the end, it was proven that every nucleotide in the 16S rRNA of E. coli could be targeted with a bright probe and, therefore, that there were no truly inaccessible target regions in the 16S rRNA. Based on our findings and mechanistic modeling, a rational design strategy involving ΔG o overall, hybridization kinetics, and fluorophore quenching is recommended for the development of bright probes.

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