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 609. Differing Genotypic Contexts Between E. coli and A. baumannii Modulate the Role of blaADC-7 in the Development of Antibiotic Collateral Sensitivity

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S284-S285
Author(s):  
Erin McClure ◽  
Julia Newman ◽  
Nikhil Krishnan ◽  
Joseph Rutter ◽  
Andrea M Hujer ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Fold Increase ◽  
Protein Product ◽  
Coli Strain ◽  
Fourth Generation ◽  
Clinical Strain ◽  
Health Crisis ◽  
E Coli ◽  
Collateral Sensitivity ◽  
Resistance Evolution

Abstract Background Antibiotic resistance is a global health crisis. While persistent drug discovery of novel antibiotics has previously been relied upon to thwart resistance, evolution inevitably perseveres. While genes conferring antibiotic resistance have previously been characterized, it is unclear how varying genetic contexts can change the antibiotic resistance phenotype a given gene confers. Methods The DH10B strain of E. coli was transformed with a blaADC-7 plasmid. In 12 evolutionary replicates, the modified E. coli strain and a clinical strain of A. baumannii containing the same resistance gene were passaged daily for 10 days on cefepime gradient agar plates with gradually increasing concentrations of cefepime. MICs of cefepime and a diverse set of 15 other drugs were determined for the parental strains and after the final passage passage. MIC of cefepime after intermediary passages were determined for select replicates. Lastly the blaADC-7 gene after the final passage was sequenced. Results At the end of 10 passages, collateral sensitivity in A. baumannii was observed to tigecycline and fosfomycin in 5 and 6 replicates respectively, out of 12 total. 4 out of 12 E. coli replicates displayed collateral sensitivity to minocycline (Figure 1). In the third E. coli replicate, Sanger sequencing revealed a novel S286R mutation in blaADC-7 appearing in passage seven which preceded a several log fold increase in the MIC of cefepime (Figures 2 and 3). No additional mutations were found in the other evolutionary replicates. Conclusion Patterns of resistance varied among antibiotics of the same class, (e.g., tetracyclines, fourth-generation cephalosporins) in both E. coli and A. baumannii; however, A. baumannii expressed less widespread collateral resistance than E. coli. A previously undiscovered S286R mutation in blaADC-7 coincided with a pronounced increased in resistance to cefepime. Further studies are required to determine whether this mutation gives rise to a structural change in the protein product. Given that no other mutations were found, resistance to cefepime and subsequent collateral resistance to other antibiotics may have developed due to epigenetic changes or mutations outside the blaADC-7 genes. Indeed, future experiments with whole-genome sequencing may reveal such changes. Disclosures All authors: No reported disclosures.

scholarly journals Ethanol production by Escherichia coli from detoxified lignocellulosic teak wood hydrolysates with high concentration of phenolic compounds

2021 ◽  
Author(s):  
Estefanía Sierra-Ibarra ◽  
Jorge Alcaraz-Cienfuegos ◽  
Alejandra Vargas-Tah ◽  
Alberto Rosas-Aburto ◽  
Ángeles Valdivia-López ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Phenolic Compounds ◽  
Ethanol Production ◽  
Enzymatic Saccharification ◽  
Coli Strain ◽  
High Concentration ◽  
E Coli ◽  
Thermochemical Pretreatment ◽  
Teak Wood ◽  
Ethanologenic Escherichia Coli

Abstract Teak wood residues were subjected to thermochemical pretreatment, enzymatic saccharification, and detoxification to obtain syrups with a high concentration of fermentable sugars for ethanol production with the ethanologenic Escherichia coli strain MS04. Teak is a hardwood, and thus a robust deconstructive pretreatment was applied followed by enzymatic saccharification. The resulting syrup contained 60 g L−1 glucose, 18 g L−1 xylose, 6 g L−1 acetate, less than 0.1 g L−1 of total furans, and 12 g L−1 of soluble phenolic compounds (SPC). This concentration of SPC is toxic to E. coli, and thus two detoxification strategies were assayed: 1) treatment with Coriolopsis gallica laccase followed by addition of activated carbon and 2) overliming with Ca(OH)2. These reduced the phenolic compounds by 40 and 76%, respectively. The detoxified syrups were centrifuged and fermented with E. coli MS04. Cultivation with the over-limed hydrolysate showed a 60% higher volumetric productivity (0.45 gETOH L−1 h−1). The bioethanol/sugars yield was over 90% in both strategies.

scholarly journals Glycylcyclines bind to the high-affinity tetracycline ribosomal binding site and evade Tet(M)- and Tet(O)-mediated ribosomal protection.

1996 ◽  
Vol 40 (9) ◽  
pp. 2226-2228 ◽  
Author(s):  
J Bergeron ◽  
M Ammirati ◽  
D Danley ◽  
L James ◽  
M Norcia ◽  
...  
Keyword(s):  
Binding Site ◽  
Tetracycline Resistance ◽  
Fold Increase ◽  
Coli Strain ◽  
Resistance Factors ◽  
E Coli ◽  
High Affinity ◽  
High Affinity Binding Site ◽  
Free Translation ◽  
70S Ribosome

N,N-dimethylglycylamido (DMG) derivatives of 6-demethyl-6-deoxytetracycline and doxycycline bind 5-fold more effectively than tetracycline to the tetracycline high-affinity binding site on the Escherichia coli 70S ribosome, which correlates with a 10-fold increase in potency for inhibition of E. coli cell-free translation. The potencies of DMG-doxycycline and DMG-6-demethyl-6-deoxytetracycline were unaffected by the ribosomal tetracycline resistance factors Tet(M) and Tet(O) in cell-free translation assays and whole-cell bioassays with a conditional Tet(M)-producing E. coli strain.

Evaluation of thioesterases from Acinetobacter baylyi for production of free fatty acids

2017 ◽  
Vol 63 (4) ◽  
pp. 321-329 ◽  
Author(s):  
Rahul Ukey ◽  
William E. Holmes ◽  
Rakesh Bajpai ◽  
Andrei Y. Chistoserdov
Keyword(s):  
Fatty Acids ◽  
Free Fatty Acids ◽  
Vaccenic Acid ◽  
Fold Increase ◽  
Coli Strain ◽  
Wax Ester ◽  
Acinetobacter Baylyi ◽  
E Coli ◽  
Storage Lipids ◽  
Ester Biosynthesis

Acinetobacter baylyi is one of few Gram-negative bacteria capable of accumulating storage lipids in the form of triacylglycerides and wax esters, which makes it an attractive candidate for production of lipophilic products, including biofuel precursors. Thioesterases play a significant dual role in the triacylglyceride and wax ester biosynthesis by either providing or removing acyl-CoA from this pathway. Therefore, 4 different thioesterase genes were cloned from Acinetobacter baylyi ADP1 and expressed in Escherichia coli to investigate their contribution to free fatty acids (FFAs) accumulation. Overexpression of the genes tesA′ (a leaderless form of the gene tesA) and tesC resulted in increased accumulation of FFAs when compared with the host E. coli strain. Overexpression of tesA′ showed a 1.87-fold increase in production of long-chain fatty acids (C16 to C18) over the host strain. Unlike TesC and the other investigated thioesterases, the TesA′ thioesterase also produced shorter chain FFAs (e.g., myristic acid) and unsaturated FFAs (e.g., cis-vaccenic acid (18:1Δ11)). A comparison of the remaining 3 A. baylyi ADP1 thioesterases (encoded by the tesB, tesC, and tesD genes) revealed that only the strain containing the tesC gene produced statistically higher levels of FFAs over the control, suggesting that it possesses the acyl-ACP thioesterase activity. Both E. coli strains containing the tesB and tesD genes produced levels of FFAs similar to those of the plasmid-free control E. coli strain, which indicates that TesB and TesD lack the acyl-ACP thioesterase activity.

Transport of escherichia coli through soil to groundwater traced by randomly amplified polymorphic DNA (RAPD)

1997 ◽  
Vol 35 (11-12) ◽  
pp. 351-357 ◽  
Author(s):  
R. Rothmaier ◽  
A. Weidenmann ◽  
K. Botzenhart
Keyword(s):  
Negative Impact ◽  
Random Amplified Polymorphic Dna ◽  
Coli Strain ◽  
Soil Samples ◽  
Test Field ◽  
Liquid Manure ◽  
E Coli ◽  
Rapd Pattern ◽  
Geological Situation ◽  
Different Strains

Isolates (50) of E. coli obtained from liquid manure (20 bovine, 20 porcine) were genotyped using random amplified polymorphic DNA (RAPD). Typing revealed 9 and 14 different strains in bovine and porcine liquid manure respectively with no strains in common. One porcine strain, showing a simple RAPD pattern, was subcultured and spread on a test field (1.5l/m2 at 1010 cfu/l) in a drinking water protection zone with loamy to sandy sediments in the Donauried area, Baden-Wurttemberg. Soil samples and groundwaters were collected at monthly intervals October 1994 – June 1995 during which 114 E. coli isolates were recovered. The first occurrence and maximum concentration of E. coli in soil samples taken from more than 20cm depth was in January 1995, declining rapidly with depth and time. All isolates from soil and only one from groundwater showed the RAPD pattern of the spread E. coli strain. The results could not demonstrate a severe negative impact of the spreading of liquid manure on the bacteriological quality of the groundwater in the given geological situation. The distinct strain patterns found in different kinds of liquid manure suggest that genotyping of E. coli by RAPD may be an adequate tool for tracing sources of faecal contamination.

scholarly journals Regulation of Expression of the TIR-Containing Protein C Gene of the Uropathogenic Escherichia coli Strain CFT073

Pathogens ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 549
Author(s):  
Julia Ittensohn ◽  
Jacqueline Hemberger ◽  
Hannah Griffiths ◽  
Maren Keller ◽  
Simone Albrecht ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein C ◽  
Interleukin 1 ◽  
Coli Strain ◽  
Uropathogenic Escherichia Coli ◽  
Reporter Construct ◽  
Regulation Of Expression ◽  
E Coli ◽  
Strain Cft073 ◽  
Myeloid Differentiation Factor

The uropathogenic Escherichia coli strain CFT073 causes kidney abscesses in mice Toll/interleukin-1 receptor domain-containing protein C (TcpC) dependently and the corresponding gene is present in around 40% of E. coli isolates of pyelonephritis patients. It impairs the Toll-like receptor (TLR) signaling chain and the NACHT leucin-rich repeat PYD protein 3 inflammasome (NLRP3) by binding to TLR4 and myeloid differentiation factor 88 as well as to NLRP3 and caspase-1, respectively. Overexpression of the tcpC gene stopped replication of CFT073. Overexpression of several tcpC-truncation constructs revealed a transmembrane region, while its TIR domain induced filamentous bacteria. Based on these observations, we hypothesized that tcpC expression is presumably tightly controlled. We tested two putative promoters designated P1 and P2 located at 5′ of the gene c2397 and 5′ of the tcpC gene (c2398), respectively, which may form an operon. High pH and increasing glucose concentrations stimulated a P2 reporter construct that was considerably stronger than a P1 reporter construct, while increasing FeSO4 concentrations suppressed their activity. Human urine activated P2, demonstrating that tcpC might be induced in the urinary tract of infected patients. We conclude that P2, consisting of a 240 bp region 5′ of the tcpC gene, represents the major regulator of tcpC expression.

scholarly journals Novel Bacterial Production of Two Different Bioactive Forms of Human Stem-Cell Factor

2021 ◽  
Vol 22 (12) ◽  
pp. 6361
Author(s):  
Eunyoung Lee ◽  
Michelle Novais de Paula ◽  
Sangki Baek ◽  
Huynh Kim Khanh Ta ◽  
Minh Tan Nguyen ◽  
...  
Keyword(s):  
Stem Cell ◽  
Ion Exchange ◽  
Stem Cell Factor ◽  
Transmembrane Domain ◽  
Coli Strain ◽  
Exchange Chromatography ◽  
Soluble Form ◽  
Human Stem Cell ◽  
E Coli ◽  
Human Stem Cell Factor

Human stem-cell factor (hSCF) stimulates the survival, proliferation, and differentiation of hematopoietic cells by binding to the c-Kit receptor. Various applications of hSCF require the efficient and reliable production of hSCF. hSCF exists in three forms: as two membrane-spanning proteins hSCF248 and hSCF229 and truncated soluble N-terminal protein hSCF164. hSCF164 is known to be insoluble when expressed in Escherichia coli cytoplasm, requiring a complex refolding procedure. The activity of hSCF248 has never been studied. Here, we investigated novel production methods for recombinant hSCF164 and hSCF248 without the refolding process. To increase the solubility of hSCF164, maltose-binding protein (MBP) and protein disulfide isomerase b’a’ domain (PDIb’a’) tags were attached to the N-terminus of hSCF164. These fusion proteins were overexpressed in soluble form in the Origami 2(DE3) E. coli strain. These solubilization effects were enhanced at a low temperature. His-hSCF248, the poly-His tagged form of hSCF248, was expressed in a highly soluble form without a solubilization tag protein, which was unexpected because His-hSCF248 contains a transmembrane domain. hSCF164 was purified using affinity and ion-exchange chromatography, and His-hSCF248 was purified by ion-exchange and gel filtration chromatography. The purified proteins stimulated the proliferation of TF-1 cells. Interestingly, the EC50 value of His-hSCF248 was 1 pg/mL, 100-fold lower than 9 ng/mL hSCF164. Additionally, His-hSCF248 decreased the doubling time, increased the proportion of S and G2/M stages in the cell cycle, and increased the c-Myc expression at a 1000-fold lower concentration than hSCF164. In conclusion, His-hSCF248 was expressed in a soluble form in E. coli and had stronger activity than hSCF164. The molecular chaperone, MBP, enabled the soluble overexpression of hSCF164.

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