scholarly journals Deletion of Pgi Gene in E. coli Increases Tolerance to Furfural and 5-Hydroxymethyl Furfural in Media Containing Glucose-Xylose Mixture

2020 ◽  
Author(s):  
Syed Bilal Jilani ◽  
Chandra Dev ◽  
Danish Eqbal ◽  
Kamran Jawed ◽  
Rajendra Prasad ◽  
...  
Keyword(s):  
Pentose Phosphate ◽  
Sugar Concentration ◽  
Glucose Load ◽  
Yield Strain ◽  
E Coli ◽  
Genetic Manipulations ◽  
Hydroxymethyl Furfural ◽  
Treatment Conditions ◽  
Lower Glucose ◽  
Lignocellulosic Sugars

Abstract Background: Furfural and 5-hydroxymethyl furfural (5-HMF) are key furan inhibitors that are generated due to breakdown of lignocellulosic sugars at high temperature and acidic treatment conditions. Both furfural and 5-HMF act in a synergistic manner to inhibit microbial metabolism and resistance to both is a desirable characteristic for efficient conversion of lignocellulosic carbon to ethanol. Genetic manipulations targeted toward increasing cellular NADPH pools have successfully imparted tolerance against furfural and 5-HMF. In present study, deletion of pgi gene as a strategy to augment carbon flow through pentose phosphate pathway (PPP) was studied in ethanologenic Escherichia coli strain SSK101 to impart tolerance towards either furfural or 5-HMFor both inhibitors together.Results: A key gene of EMP pathway, pgi, was deleted in an ethanologenic E. coli strain SSK42 to yield strain SSK101. In presence of 1 g/L furfural in minimal AM1 media, the rate of biomass formation for strain SSK101 was up to 1.9-fold higher as compared to parent SSK42 strain, and it was able to clear furfural in half the time. Tolerance to inhibitor was associated with glucose as carbon source and not xylose, and the tolerance advantage of SSK101 was neutralized in LB media. Bioreactor studies were performed under binary stress of furfural and 5-HMF (1 g/L each) and different glucose concentrations in a glucose-xylose mixture with final sugar concentration of 5.5%, mimicking major components of dilute acid treated biomass hydrolysate. In the mixture having 6 g/L and 12 g/L glucose, SSK101 strain produced ~ 18 g/L and 20 g/L ethanol, respectively. Interestingly, the maximum ethanol productivity was better at lower glucose load with 0.46 g/(L.h) between 96–120 h, as compared to higher glucose load where it was 0.33 g/(L.h) between 144–168 h. Importantly, parent strain SSK42 did not exhibit significant metabolic activity under similar conditions of inhibitor load and sugar concentration.Conclusions: E. coli strain SSK101 with pgi deletion had enhanced tolerance against both furfural and 5-HMF, which was associated with presence of glucose in media. Strain SSK101 also had improved fermentation characteristics under both hyperosmotic as well as binary stress of furfural and 5-HMF in media containing glucose-xylose mixture.

scholarly journals Deletion of pgi gene in E. coli increases tolerance to furfural and 5-hydroxymethyl furfural in media containing glucose-xylose mixture

2020 ◽  
Author(s):  
Syed Bilal Jilani ◽  
Chandra Dev ◽  
Danish Eqbal ◽  
Kamran Jawed ◽  
Rajendra Prasad ◽  
...  
Keyword(s):  
Pentose Phosphate ◽  
Sugar Concentration ◽  
Glucose Load ◽  
Yield Strain ◽  
E Coli ◽  
Genetic Manipulations ◽  
Hydroxymethyl Furfural ◽  
Treatment Conditions ◽  
Lower Glucose ◽  
Lignocellulosic Sugars

Abstract Background: Furfural and 5-hydroxymethyl furfural (5-HMF) are key furan inhibitors that are generated due to breakdown of lignocellulosic sugars at high temperature and acidic treatment conditions. Both furfural and 5-HMF act in a synergistic manner to inhibit microbial metabolism and resistance to both is a desirable characteristic for efficient conversion of lignocellulosic carbon to ethanol. Genetic manipulations targeted toward increasing cellular NADPH pools have successfully imparted tolerance against furfural and 5-HMF. In present study, deletion of pgi gene as a strategy to augment carbon flow through pentose phosphate pathway (PPP) was studied in ethanologenic Escherichia coli strain SSK101 to impart tolerance towards either furfural or 5-HMFor both inhibitors together.Results: A key gene of EMP pathway, pgi, was deleted in an ethanologenic E. coli strain SSK42 to yield strain SSK101. In presence of 1 g/L furfural in minimal AM1 media, the rate of biomass formation for strain SSK101 was up to 1.9-fold higher as compared to parent SSK42 strain, and it was able to clear furfural in half the time. Tolerance to inhibitor was associated with glucose as carbon source and not xylose, and the tolerance advantage of SSK101 was neutralized in LB media. Bioreactor studies were performed under binary stress of furfural and 5-HMF (1 g/L each) and different glucose concentrations in a glucose-xylose mixture with final sugar concentration of 5.5%, mimicking major components of dilute acid treated biomass hydrolysate. In the mixture having 6 g/L and 12 g/L glucose, SSK101 strain produced ~18 g/L and 20 g/L ethanol, respectively. Interestingly, the maximum ethanol productivity was better at lower glucose load with 0.46 g/(L.h) between 96-120 h, as compared to higher glucose load where it was 0.33 g/(L.h) between 144-168 h. Importantly, parent strain SSK42 did not exhibit significant metabolic activity under similar conditions of inhibitor load and sugar concentration.Conclusions: E. coli strain SSK101 with pgi deletion had enhanced tolerance against both furfural and 5-HMF, which was associated with presence of glucose in media. Strain SSK101 also had improved fermentation characteristics under both hyperosmotic as well as binary stress of furfural and 5-HMF in media containing glucose-xylose mixture.

scholarly journals Enhancing Terpene Yield from Sugars via Novel Routes to 1-Deoxy-d-Xylulose 5-Phosphate

2014 ◽  
Vol 81 (1) ◽  
pp. 130-138 ◽  
Author(s):  
James Kirby ◽  
Minobu Nishimoto ◽  
Ruthie W. N. Chow ◽  
Edward E. K. Baidoo ◽  
George Wang ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Xylose Reductase ◽  
Pentose Phosphate ◽  
Terpene Biosynthesis ◽  
Content Type ◽  
E Coli ◽  
Pathway Expression ◽  
Terpene Synthesis ◽  
Selection For

ABSTRACTTerpene synthesis in the majority of bacterial species, together with plant plastids, takes place via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. The first step of this pathway involves the condensation of pyruvate and glyceraldehyde 3-phosphate by DXP synthase (Dxs), with one-sixth of the carbon lost as CO2. A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C5sugars to terpenes and also circumvent regulatory mechanisms that control Dxs, but there is no enzyme known that can convert a sugar into its 1-deoxy equivalent. Employing a selection for complementation of adxsdeletion inEscherichia coligrown on xylose as the sole carbon source, we uncovered two candidate nDXP genes. Complementation was achieved either via overexpression of the wild-typeE. coliyajOgene, annotated as a putative xylose reductase, or via various mutations in the nativeribBgene.In vitroanalysis performed with purified YajO and mutant RibB proteins revealed that DXP was synthesized in both cases from ribulose 5-phosphate (Ru5P). We demonstrate the utility of these genes for microbial terpene biosynthesis by engineering the DXP pathway inE. colifor production of the sesquiterpene bisabolene, a candidate biodiesel. To further improve flux into the pathway from Ru5P, nDXP enzymes were expressed as fusions to DXP reductase (Dxr), the second enzyme in the DXP pathway. Expression of a Dxr-RibB(G108S) fusion improved bisabolene titers more than 4-fold and alleviated accumulation of intracellular DXP.

PBP4 and PBP5 are involved in regulating exopolysaccharide synthesis during Escherichia coli biofilm formation

Microbiology ◽  
2021 ◽  
Vol 167 (3) ◽  
Author(s):  
Sathi Mallick ◽  
Shanti Kiran ◽  
Tapas Kumar Maiti ◽  
Anindya S. Ghosh
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Type Species ◽  
Pentose Phosphate ◽  
Bacterial Cells ◽  
Surface Adhesion ◽  
Content Type ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Link Type

Escherichia coli low-molecular-mass (LMM) Penicillin-binding proteins (PBPs) help in hydrolysing the peptidoglycan fragments from their cell wall and recycling them back into the growing peptidoglycan matrix, in addition to their reported involvement in biofilm formation. Biofilms are external slime layers of extra-polymeric substances that sessile bacterial cells secrete to form a habitable niche for themselves. Here, we hypothesize the involvement of Escherichia coli LMM PBPs in regulating the nature of exopolysaccharides (EPS) prevailing in its extra-polymeric substances during biofilm formation. Therefore, this study includes the assessment of physiological characteristics of E. coli CS109 LMM PBP deletion mutants to address biofilm formation abilities, viability and surface adhesion. Finally, EPS from parent CS109 and its ΔPBP4 and ΔPBP5 mutants were purified and analysed for sugars present. Deletions of LMM PBP reduced biofilm formation, bacterial adhesion and their viability in biofilms. Deletions also diminished EPS production by ΔPBP4 and ΔPBP5 mutants, purification of which suggested an increased overall negative charge compared with their parent. Also, EPS analyses from both mutants revealed the appearance of an unusual sugar, xylose, that was absent in CS109. Accordingly, the reason for reduced biofilm formation in LMM PBP mutants may be speculated as the subsequent production of xylitol and a hindrance in the standard flow of the pentose phosphate pathway.

scholarly journals Computational Design and Analysis of Modular Cells for Large Libraries of Exchangeable Product Synthesis Modules

2021 ◽  
Author(s):  
Sergio Garcia ◽  
Cong T Trinh
Keyword(s):  
Renewable Resources ◽  
Plant Biomass ◽  
New Product ◽  
Computational Design ◽  
Computational Method ◽  
E Coli ◽  
Genetic Manipulations ◽  
Endogenous Production ◽  
Target Molecules ◽  
Rapid Generation

Microbial metabolism can be harnessed to produce a large library of useful chemicals from renewable resources such as plant biomass. However, it is laborious and expensive to create microbial biocatalysts to produce each new product. To tackle this challenge, we have recently developed modular cell (ModCell) design principles that enable rapid generation of production strains by assembling a modular (chassis) cell with exchangeable production modules to achieve overproduction of target molecules. Previous computational ModCell design methods are limited to analyze small libraries of around 20 products. In this study, we developed a new computational method,named ModCell-HPC, capable of designing modular cells for large libraries with hundredths of products with a highly-parallel and multi-objective evolutionary algorithm. We demonstrated ModCell-HPC to design Escherichia coli modular cells towards a library of 161 endogenous production modules. From these simulations, we identified E. coli modular cells with few genetic manipulations that can produce dozens of molecules in a growth-coupled manner under different carbons sources. These designs revealed key genetic manipulations at the chassis and module levels to accomplish versatile modular cells. Furthermore, we used ModCell-HPC to identify design features that allow an existing modular cell to be re-purposed towards production of new molecules. Overall, ModCell-HPC is a useful tool towards more efficient and generalizable design of modular cells to help reduce research and development cost in biocatalysis.

Finding Minimum Reaction Cuts of Metabolic Networks Under a Boolean Model Using Integer Programming and Feedback Vertex Sets

2012 ◽  
pp. 774-791
Author(s):  
Takeyuki Tamura ◽  
Kazuhiro Takemoto ◽  
Tatsuya Akutsu
Keyword(s):  
Integer Programming ◽  
Metabolic Network ◽  
Drug Targets ◽  
Metabolic Networks ◽  
Boolean Model ◽  
Pentose Phosphate ◽  
Minimum Size ◽  
E Coli ◽  
Potential Applications ◽  
Vertex Set

In this paper, the authors consider the problem of, given a metabolic network, a set of source compounds and a set of target compounds, finding a minimum size reaction cut, where a Boolean model is used as a model of metabolic networks. The problem has potential applications to measurement of structural robustness of metabolic networks and detection of drug targets. They develop an integer programming-based method for this optimization problem. In order to cope with cycles and reversible reactions, they further develop a novel integer programming (IP) formalization method using a feedback vertex set (FVS). When applied to an E. coli metabolic network consisting of Glycolysis/Glyconeogenesis, Citrate cycle and Pentose phosphate pathway obtained from KEGG database, the FVS-based method can find an optimal set of reactions to be inactivated much faster than a naive IP-based method and several times faster than a flux balance-based method. The authors also confirm that our proposed method works even for large networks and discuss the biological meaning of our results.

scholarly journals Complete Genome Sequences of the Escherichia coli Donor Strains ST18 and MFDpir

2020 ◽  
Vol 9 (45) ◽  
Author(s):  
Simon A. Jackson ◽  
Bridget J. Fellows ◽  
Peter C. Fineran
Keyword(s):  
Escherichia Coli ◽  
Complete Genome ◽  
Nanopore Sequencing ◽  
Genome Sequences ◽  
Content Type ◽  
E Coli ◽  
Genetic Manipulations ◽  
Complete Genomes

ABSTRACT Escherichia coli ST18 and MFDpir are donors commonly used to transfer oriTRP4-containing plasmids to diverse bacteria via conjugation. ST18 and MFDpir were constructed via multiple genetic manipulations involving several E. coli strains. Here, we used Illumina and Nanopore sequencing to determine the complete genomes of these widely used strains.

Finding Minimum Reaction Cuts of Metabolic Networks Under a Boolean Model Using Integer Programming and Feedback Vertex Sets

2010 ◽  
Vol 1 (1) ◽  
pp. 14-31 ◽  
Author(s):  
Takeyuki Tamura ◽  
Kazuhiro Takemoto ◽  
Tatsuya Akutsu
Keyword(s):  
Integer Programming ◽  
Metabolic Network ◽  
Drug Targets ◽  
Metabolic Networks ◽  
Boolean Model ◽  
Pentose Phosphate ◽  
Minimum Size ◽  
E Coli ◽  
Potential Applications ◽  
Vertex Set

In this paper, the authors consider the problem of, given a metabolic network, a set of source compounds and a set of target compounds, finding a minimum size reaction cut, where a Boolean model is used as a model of metabolic networks. The problem has potential applications to measurement of structural robustness of metabolic networks and detection of drug targets. They develop an integer programming-based method for this optimization problem. In order to cope with cycles and reversible reactions, they further develop a novel integer programming (IP) formalization method using a feedback vertex set (FVS). When applied to an E. coli metabolic network consisting of Glycolysis/Glyconeogenesis, Citrate cycle and Pentose phosphate pathway obtained from KEGG database, the FVS-based method can find an optimal set of reactions to be inactivated much faster than a naive IP-based method and several times faster than a flux balance-based method. The authors also confirm that our proposed method works even for large networks and discuss the biological meaning of our results.

Overexpression of an oxidoreductase YghA confers tolerance against furfural in ethanologenic Escherichia coli strain SSK42

2021 ◽  
Author(s):  
S. Bilal Jilani ◽  
Rajendra Prasad ◽  
Syed Shams Yazdani
Keyword(s):  
Fold Increase ◽  
Microbial Metabolism ◽  
Coli Strain ◽  
Production Costs ◽  
E Coli ◽  
Hydroxymethyl Furfural ◽  
Hexose Sugars ◽  
Pentose Sugar ◽  
Efficient Fermentation ◽  
Ethanologenic Escherichia Coli

Furfural is a common furan inhibitor formed due to dehydration of pentose sugar like xylose and acts as an inhibitor of microbial metabolism. Overexpression of NADH specific FucO and deletion of NADPH specific YqhD had been a successful strategy in the past in conferring tolerance against furfural in E. coli , which highlight the importance of oxidoreductases in conferring tolerance against furfural. In a screen consisting of various oxidoreductases, dehydrogenases, and reductases, we identified yghA gene as an overexpression target to confer tolerance against furfural. YghA preferably used NADH as a cofactor and had apparent K m value of 0.03 mM against furfural. In presence of 1 g L −1 furfural and 10% xylose (wt/vol), yghA overexpression in an ethanologenic E. coli strain SSK42 resulted in a 5.3-fold increase in ethanol titers as compared to the control strain with an efficiency of ∼97%. YghA also exhibited activity against the lesser toxic inhibitor 5-hydroxymethyl furfural that is formed due to dehydration of hexose sugars and thus is a formidable target for overexpression in ethanologenic strain for fermentation of sugars in biomass hydrolysate. IMPORTANCE Lignocellulosic biomass represents an inexhaustible source of carbon for second-generation biofuels. Thermo-acidic pretreatment of biomass is performed to loosen the lignocellulosic fibers and make the carbon bioavailable for microbial metabolism. The pretreatment process also results in the formation of inhibitors that inhibit microbial metabolism and increase production costs. Furfural is a potent furan inhibitor that increases the toxicity of other inhibitors present in the hydrolysate. Thus it is desirable to engineer furfural tolerance in E. coli for efficient fermentation of hydrolysate sugars.

scholarly journals A comparison of DNA repair and survival of Escherichia coli O157:H7 following exposure to both low- and medium-pressure UV irradiation

2007 ◽  
Vol 5 (3) ◽  
pp. 407-415 ◽  
Author(s):  
J. L. Zimmer-Thomas ◽  
R. M. Slawson ◽  
P. M. Huck
Keyword(s):  
Dna Repair ◽  
Drinking Water ◽  
Water Treatment ◽  
Uv Irradiation ◽  
Drinking Water Treatment ◽  
Uv Exposure ◽  
E Coli ◽  
Medium Pressure ◽  
Treatment Conditions ◽  
Low Levels

DNA repair and survival of pathogenic E. coli O157:H7 was investigated following exposure to ultraviolet (UV) radiation from both low-pressure (LP) and medium-pressure (MP) lamps. This study included irradiation at UV doses used in drinking water treatment and lower doses indicative of potential treatment problems. Immediately following UV exposure, an average log inactivation of 4.5 or greater was observed following all tested doses of LP (5, 8, 20 and 40 mJ/cm2) or MP UV (5 and 8 mJ/cm2) indicating the sensitivity of E. coli O157:H7 to UV irradiation. Following conditions conducive to repair, maximum photo repair occurred rapidly within 30 minutes after low doses (5 and 8 mJ/cm2) of LP UV. The rate of repair was much higher than reported previously in non-pathogenic E. coli (which occurred within 2 hours). In contrast to LP UV, limited photo repair of E. coli O157:H7 was observed following MP UV exposure at reduced doses (5 and 8 mJ/cm2). At these lower doses, low levels of light independant repair were observed following LP UV, but not following exposure of MP UV irradiation. This study indicates that MP UV may enhance UV disinfection of E. coli O157:H7 by reducing the ability to repair following non-ideal treatment conditions. Following doses used in drinking water treatment (20 and 40 mJ/cm2), low levels of photo repair following LP UV were evident.

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