scholarly journals Increasing ATP turnover boosts productivity of 2,3-butanediol synthesis in Escherichia coli

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Simon Boecker ◽  
Björn-Johannes Harder ◽  
Regina Kutscha ◽  
Stefan Pflügl ◽  
Steffen Klamt
Keyword(s):  
Escherichia Coli ◽  
Superior Performance ◽  
High Yield ◽  
Synthesis Rate ◽  
Specific Substrate ◽  
Substrate Uptake ◽  
Control Strain ◽  
Producer Strain ◽  
High Productivity ◽  
Microaerobic Conditions

Abstract Background The alcohol 2,3-butanediol (2,3-BDO) is an important chemical and an Escherichia coli producer strain was recently engineered for bio-based production of 2,3-BDO. However, further improvements are required for realistic applications. Results Here we report that enforced ATP wasting, implemented by overexpressing the genes of the ATP-hydrolyzing F1-part of the ATPase, leads to significant increases of yield and especially of productivity of 2,3-BDO synthesis in an E. coli producer strain under various cultivation conditions. We studied aerobic and microaerobic conditions as well as growth-coupled and growth-decoupled production scenarios. In all these cases, the specific substrate uptake and 2,3-BDO synthesis rate (up to sixfold and tenfold higher, respectively) were markedly improved in the ATPase strain compared to a control strain. However, aerobic conditions generally enable higher productivities only with reduced 2,3-BDO yields while high product yields under microaerobic conditions are accompanied with low productivities. Based on these findings we finally designed and validated a three-stage process for optimal conversion of glucose to 2,3-BDO, which enables a high productivity in combination with relatively high yield. The ATPase strain showed again superior performance and finished the process twice as fast as the control strain and with higher 2,3-BDO yield. Conclusions Our results demonstrate the high potential of enforced ATP wasting as a generic metabolic engineering strategy and we expect more applications to come in the future.

scholarly journals Rational Metabolic Engineering of Escherichia Coli for High-yield L-serine Production

2020 ◽  
Author(s):  
Chenyang Wang ◽  
Junjun Wu ◽  
Lei Wang ◽  
Xiaojia Chen ◽  
Qinyu Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Regulatory Mechanism ◽  
Feedback Inhibition ◽  
Random Mutagenesis ◽  
Phosphoglycerate Kinase ◽  
High Yield ◽  
High Productivity ◽  
Yield Production ◽  
Pharmaceutical Industries

Abstract Background: L-serine is widely used in the food, cosmetic and pharmaceutical industries, and direct fermentation of L-serine from glucose is an attractive technique. However, L-serine producers have historically been developed via classical random mutagenesis due to the complicated metabolic network and regulatory mechanism of L-serine production, leading to un-optimal productivity and yield of L-serine and thus limiting its large-scale industrial production. Result: In this study, a high-yield and high-productivity Escherichia coli strain was constructed by a defined genetic modification methodology for L-serine production. First, L-serine-mediated feedback inhibition was removed and L-serine biosynthetic pathway genes (serAfr, serC and serB) associated with phosphoglycerate kinase (pgk) were overexpressed. Secondly, L-serine conversion pathway was further examined by introducing a glyA mutation (K229G) and deleting other degrading enzymes based on deletion of initial sdaA. Finally, the L-serine transport system was rationally engineered to reduce the uptake and accelerate the export of L-serine. The optimally engineered strain produced 35 g/L L-serine with a productivity of 0.98 g/L/h and yield of 0.42 g/g glucose in a 5-L fermenter, the highest productivity and yield of L-serine from glucose reported to date. Transcriptome and intermediate metabolite were analyzed to further understand the regulatory mechanism of L-serine production. Conclusion: These results demonstrated that combined metabolic and bioprocess engineering strategies can improve L-serine productivity and yield, thus providing basic principles for rationally designing of high-yield production strains and paving the way for towards a simple and economical process for industrial L-serine production.

scholarly journals Escherichia coli metabolism under short-term repetitive substrate dynamics: Adaptation and trade-offs

2020 ◽  
Author(s):  
Eleni Vasilakou ◽  
Mark C. M. van Loosdrecht ◽  
S. Aljoscha Wahl
Keyword(s):  
Escherichia Coli ◽  
Uptake Rate ◽  
Large Scale ◽  
Oxygen Availability ◽  
Specific Substrate ◽  
Substrate Uptake ◽  
Uptake System ◽  
Average Growth ◽  
Stress Changes ◽  
Energy Spilling

AbstractBackgroundMicrobial metabolism is highly dependent on the environmental conditions. Especially, the substrate concentration, as well as oxygen availability, determine the metabolic rates. In large-scale bioreactors, microorganisms encounter dynamic conditions in substrate and oxygen availability (mixing limitations), which influence their metabolism and subsequently their physiology. Earlier, single substrate pulse experiments were not able to explain the observed physiological changes generated under large-scale industrial fermentation conditions.ResultsIn this study we applied a repetitive feast-famine regime in an aerobic Escherichia coli culture in a time-scale of seconds. The regime was applied for several generations, allowing cells to adapt to the (repetitive) dynamic environment. The observed response was highly reproducible over the cycles, indicating that cells were indeed fully adapted to the regime. We observed an increase of the specific substrate and oxygen consumption (average) rates during the feast-famine regime, compared to a steady-state (chemostat) reference environment. The increased rates at same (average) growth rate led to a reduced biomass yield (30% lower). Interestingly, this drop was not followed by increased by-product formation, pointing to the existence of energy-spilling reactions and/or less effective ATP synthesis. During the feast-famine cycle, the cells rapidly increased their uptake rate. Within 10 seconds after the beginning of the feeding, the substrate uptake rate was higher (4.68 μmol/gCDW/s) than reported during batch growth (3.3 μmol/gCDW/s). The high uptake led to an accumulation of several intracellular metabolites, during the feast phase, accounting for up to 34 % of the carbon supplied. Although the metabolite concentrations changed rapidly, the cellular energy charge remained unaffected, suggesting well-controlled balance between ATP producing and ATP consuming reactions. The role of inorganic polyphosphate as an energy buffer is discussed.ConclusionsThe adaptation of the physiology and metabolism of Escherichia coli under substrate dynamics, representative for large-scale fermenters, revealed the existence of several cellular mechanisms coping with stress. Changes in the substrate uptake system, storage potential and energy-spilling processes resulted to be of great importance. These metabolic strategies consist a meaningful step to further tackle reduced microbial performance, observed under large-scale cultivations.

Breeding for improved productivity, multiple resistance and wide adaptation in chickpea (Cicer arietinum L.)

2004 ◽  
Vol 2 (3) ◽  
pp. 181-187 ◽  
Author(s):  
S. S. Yadav ◽  
J. Kumar ◽  
Neil C. Turner ◽  
Jens Berger ◽  
Robert Redden ◽  
...  
Keyword(s):  
Cicer Arietinum ◽  
Central India ◽  
Superior Performance ◽  
Yield Potential ◽  
High Yield ◽  
Multiple Resistance ◽  
Supplemental Irrigation ◽  
High Productivity ◽  
Wide Adaptation ◽  
Disease Resistances

Chickpea (Cicer arietinum L.) is an important crop for developed as well as underdeveloped countries, especially those in the Indian sub-continent that contribute more than 60% to both the global area and global production. The harsh environmental conditions under which chickpeas are generally grown impose restrictions on the expression of genetic yield potential. In the present study, a number of different breeding approaches for the development of genotypes possessing multiple resistances to different biotic and abiotic stresses, coupled with enhanced productivity are reported. In one study, 90 genetically diverse genotypes (35 medium-sized desi types, 35 bold-seeded desi types, 10 medium-sized kabuli types and 10 bold-seeded kabuli types) were tested in several locations in the 2000–2002 seasons, under rainfed (dryland) conditions and with supplemental irrigation. The bold-seeded desi genotypes gave superior performance in the rainfed environment, while the bold-seeded kabuli genotypes outyielded the other cultivars under supplemental irrigation. From crosses between accessions from geographically diverse sources, crosses between lines carrying multiple disease resistances, and crosses between the cultivated chickpea and the wild species, C. reticulatum, 23 selections were tested for yield and resistance to multiple stresses at various locations in northern and central India. From the crosses between geographically diverse parents, six high-yielding kabuli genotypes with wide adaptation and drought tolerance were identified. Pyramiding genes for multiple resistances proved useful in identifying eight lines possessing multiple disease resistance. Introgressing wild genes generated nine genotypes with high yield potential, resistance to soil-borne diseases and adaptation to water-limited environments. We conclude that high productivity, multiple resistance and wide adaptability can be achieved simultaneously by using potentially complementary approaches.

scholarly journals 1671. Diagnosis and management of Escherichia coli bacteriuria at a Veterans Affairs hospital

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S821-S821
Author(s):  
Niyati H Shah ◽  
Brooke K Decker ◽  
Brooke K Decker ◽  
Gaetan Sgro ◽  
Monique Y Boudreaux-Kelly ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Treatment ◽  
High Yield ◽  
Comorbid Conditions ◽  
Provider Education ◽  
Transmitted Infection ◽  
Physician Practices ◽  
E Coli ◽  
Diagnosis And Management ◽  
Tract Infections

Abstract Background The IDSA recommends against screening for and treating ASB in all patients except for those pregnant or undergoing urologic procedures. Nevertheless, antibiotic treatment of ASB is widespread. We conducted a retrospective analysis of physician practices in diagnosis and management of Escherichia coli (E. coli) ASB in a male Veteran population, and compared outcomes in ASB patients treated or not treated with antibiotics. Methods Patients with an E. coli positive urine culture during an ED visit or inpatient admission from 01/2017 to 12/2017 were screened. Patients admitted to the intensive care unit or diagnosed with a sexually transmitted infection, pyelonephritis, prostatitis, or epididymitis/orchitis were excluded. A total of 163 patients were included. Demographics, clinical comorbidities and severity of illness, and outcomes were compared in ASB patients managed with or without antibiotics. ANOVA and Chi-square or Fisher’s exact tests were utilized for comparing measurements. Results ASB was present in 92/163 patients. The majority (74%) of these patients were given antibiotics. Regardless of qSOFA score or alternate infection, there were no significant differences in outcomes between ASB patients treated or not treated with antibiotics: 3-month mortality (15% vs 21%; p = 0.53), emergence of newly resistant bacterial pathogens (7% vs 13%; p = 0.43), recurrent urinary tract infections (61% vs 50%; p = 0.72), clearance of urinary pathogens (75% vs 58%; p = 0.45), length of hospital stay (7 vs 6 days, p = 0.67). Factors that were predictive of physician treatment of ASB included patient comorbid conditions such as benign prostatic hyperplasia, pyuria, and the absence of hematuria. The incidence of adverse events with antibiotic treatment of ASB was low. Conclusion The rate of antibiotic treatment of E. coli ASB in male veterans is high. Outcomes do not differ among ASB patients managed with or without antibiotics. Future studies examining outcomes in patients prescribed antibiotics for multiple episodes of ASB may yield differences, particularly in emergence of resistant pathogens. Focusing on patients with comorbid conditions who are not critically ill would be a high yield target for provider education to reduce ASB treatment. Disclosures All Authors: No reported disclosures

PCR for the Specific Detection of an Escherichia coli O157:H7 Laboratory Control Strain

2015 ◽  
Vol 78 (9) ◽  
pp. 1738-1744 ◽  
Author(s):  
MICHAEL KNOWLES ◽  
DOMINIC LAMBERT ◽  
GEORGE HUSZCZYNSKI ◽  
MARTINE GAUTHIER ◽  
BURTON W. BLAIS
Keyword(s):  
Escherichia Coli ◽  
Pathogenic Bacteria ◽  
Control Measure ◽  
Positive Control ◽  
Food Microbiology ◽  
Escherichia Coli O157 ◽  
Control Strain ◽  
E Coli ◽  
Signature Sequences ◽  
Materials Used

Control strains of bacterial pathogens such as Escherichia coli O157:H7 are commonly processed in parallel with test samples in food microbiology laboratories as a quality control measure to assure the satisfactory performance of materials used in the analytical procedure. Before positive findings can be reported for risk management purposes, analysts must have a means of verifying that pathogenic bacteria (e.g., E. coli O157:H7) recovered from test samples are not due to inadvertent contamination with the control strain routinely handled in the laboratory environment. Here, we report on the application of an in-house bioinformatic pipeline for the identification of unique genomic signature sequences in the development of specific oligonucleotide primers enabling the identification of a common positive control strain, E. coli O157:H7 (ATCC 35150), using a simple PCR procedure.

Potential Public Health Significance of Non-Escherichia coli Coliforms in Food

1979 ◽  
Vol 42 (2) ◽  
pp. 161-163 ◽  
Author(s):  
ROBERT M. TWEDT ◽  
BRENDA K. BOUTIN
Keyword(s):  
Public Health ◽  
Escherichia Coli ◽  
Diarrheal Disease ◽  
Health Hazard ◽  
High Yield ◽  
Potential Health ◽  
E Coli ◽  
Potential Health Hazard ◽  
Health Significance

Several coliform species other than Escherichia coli are often associated with and possibly responsible for acute and chronic diarrheal disease. Recent evidence suggests that non-Escherichia coli coliforms may be capable of colonizing the human intestine and producing enterotoxin(s) in high-yield. Whether these organisms are newly capable of causing disease because of infestation with extrachromosomal factors mediating pathogenicity or simply because of inherent pathogenic capabilities that have gone unrecognized, they pose a potential health hazard. Food, medical, and public health microbiologists should be aware that the non-E. coli coliforms contaminating foods may be potential enteropathogens. This possibility may make determination of their pathogenic capabilities even more important than identification of their taxonomic characteristics.

scholarly journals Aerobic Growth of Escherichia coli Is Reduced, and ATP Synthesis Is Selectively Inhibited when Five C-terminal Residues Are Deleted from the ϵ Subunit of ATP Synthase

2015 ◽  
Vol 290 (34) ◽  
pp. 21032-21041 ◽  
Author(s):  
Naman B. Shah ◽  
Thomas M. Duncan
Keyword(s):  
Escherichia Coli ◽  
Atp Synthase ◽  
Atp Hydrolysis ◽  
Atp Synthesis ◽  
Intrinsic Rate ◽  
Synthesis Rate ◽  
Final Segment ◽  
Rotary Catalysis

F-type ATP synthases are rotary nanomotor enzymes involved in cellular energy metabolism in eukaryotes and eubacteria. The ATP synthase from Gram-positive and -negative model bacteria can be autoinhibited by the C-terminal domain of its ϵ subunit (ϵCTD), but the importance of ϵ inhibition in vivo is unclear. Functional rotation is thought to be blocked by insertion of the latter half of the ϵCTD into the central cavity of the catalytic complex (F1). In the inhibited state of the Escherichia coli enzyme, the final segment of ϵCTD is deeply buried but has few specific interactions with other subunits. This region of the ϵCTD is variable or absent in other bacteria that exhibit strong ϵ-inhibition in vitro. Here, genetically deleting the last five residues of the ϵCTD (ϵΔ5) caused a greater defect in respiratory growth than did the complete absence of the ϵCTD. Isolated membranes with ϵΔ5 generated proton-motive force by respiration as effectively as with wild-type ϵ but showed a nearly 3-fold decrease in ATP synthesis rate. In contrast, the ϵΔ5 truncation did not change the intrinsic rate of ATP hydrolysis with membranes. Further, the ϵΔ5 subunit retained high affinity for isolated F1 but reduced the maximal inhibition of F1-ATPase by ϵ from >90% to ∼20%. The results suggest that the ϵCTD has distinct regulatory interactions with F1 when rotary catalysis operates in opposite directions for the hydrolysis or synthesis of ATP.

scholarly journals Cooperative Roles of Nitric Oxide-Metabolizing Enzymes To Counteract Nitrosative Stress in Enterohemorrhagic Escherichia coli

2019 ◽  
Vol 87 (9) ◽  
Author(s):  
Takeshi Shimizu ◽  
Akio Matsumoto ◽  
Masatoshi Noda
Keyword(s):  
Nitric Oxide ◽  
Escherichia Coli ◽  
Stationary Phase ◽  
Nitrosative Stress ◽  
Enterohemorrhagic Escherichia Coli ◽  
The Other ◽  
Anaerobic Growth ◽  
Bacterial Cells ◽  
Metabolizing Enzymes ◽  
Microaerobic Conditions

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) has at least three enzymes, NorV, Hmp, and Hcp, that act independently to lower the toxicity of nitric oxide (NO), a potent antimicrobial molecule. This study aimed to reveal the cooperative roles of these defensive enzymes in EHEC against nitrosative stress. Under anaerobic conditions, combined deletion of all three enzymes significantly increased the NO sensitivity of EHEC determined by the growth at late stationary phase; however, the expression of norV restored the NO resistance of EHEC. On the other hand, the growth of Δhmp mutant EHEC was inhibited after early stationary phase, indicating that NorV and Hmp play a cooperative role in anaerobic growth. Under microaerobic conditions, the growth of Δhmp mutant EHEC was inhibited by NO, indicating that Hmp is the enzyme that protects cells from NO stress under microaerobic conditions. When EHEC cells were exposed to a lower concentration of NO, the NO level in bacterial cells of Δhcp mutant EHEC was higher than those of the other EHEC mutants, suggesting that Hcp is effective at regulating NO levels only at a low concentration. These findings of a low level of NO in bacterial cells with hcp indicate that the NO consumption activity of Hcp was suppressed by Hmp at a low range of NO concentrations. Taken together, these results show that the cooperative effects of NO-metabolizing enzymes are regulated by the range of NO concentrations to which the EHEC cells are exposed.

High Yield of Active STb Enterotoxin from a Fusion Protein (MBP-STb) Expressed in Escherichia coli

1993 ◽  
Vol 4 (4) ◽  
pp. 275-281 ◽  
Author(s):  
C.E. Handl ◽  
J. Harel ◽  
J.I. Flock ◽  
J.D. Dubreuil
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
High Yield
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