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.

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.

Seasonal Variation In The Composition Of Epicuticular Waxes Of Quevcus Robur Leaves

1992 ◽  
Vol 47 (11-12) ◽  
pp. 800-806 ◽  
Author(s):  
P.-G. Gülz ◽  
E. Müller
Keyword(s):  
Fatty Acids ◽  
Climatic Factors ◽  
Dry Weight ◽  
Vegetation Period ◽  
Wax Ester ◽  
Primary Alcohols ◽  
Mean Values ◽  
Leaf Surface Area ◽  
Ester Biosynthesis ◽  
Genetically Determined

The epicuticular leaf waxes of Quercus robur were analyzed continuously over a two years vegetation period with preparation every week from April to November. The folded leaflets in buds have waxes quite different in yield and composition from those of mature leaves. They contain homologous series of hydrocarbons, wax esters, primary alcohols, fatty acids and triterpenoids from the beginning, but not aldehydes. After leaf unfolding a dynamic biosynthesis of alcohols, aldehydes and fatty acids is observed in May and June. Wax content is doubled per dry weight or in cm2 leaf surface area and 80-fold per one leaf in that time. During leaf development tetracosanol becomes the dominant epicuticular wax component comprising ca. 40% of the wax. In both years of the study a reactivation of wax ester biosynthesis is observed in October and November. Esters with chain length C36 and C38 increased particularly. From July to November the wax composition remained nearly constant within mean values and their standard deviations. Within the two years studied most values concerning wax composition are reproducible and are therefore genetically determined. In spring the growing processes are influenced by climatic factors

scholarly journals Multiple Pathways for Triacylglycerol Biosynthesis in Streptomyces coelicolor

2008 ◽  
Vol 74 (9) ◽  
pp. 2573-2582 ◽  
Author(s):  
Ana Arabolaza ◽  
Eduardo Rodriguez ◽  
Silvia Altabe ◽  
Hector Alvarez ◽  
Hugo Gramajo
Keyword(s):  
Mutant Strain ◽  
De Novo ◽  
Streptomyces Coelicolor ◽  
Lipid Production ◽  
Wax Ester ◽  
Acinetobacter Baylyi ◽  
Triple Mutant ◽  
Storage Lipids ◽  
Tag Biosynthesis

ABSTRACT The terminal reaction in triacylglyceride (TAG) biosynthesis is the esterification of diacylglycerol (DAG) with a fatty acid molecule. To study this reaction in Streptomyces coelicolor, we analyzed three candidate genes (sco0958, sco1280, and sco0123) whose products significantly resemble the recently identified wax ester synthase/acyl-coenzyme A (CoA):DAG acyltransferase (DGAT) from Acinetobacter baylyi. The deletion of either sco0123 or sco1280 resulted in no detectable decrease in TAG accumulation. In contrast, the deletion of sco0958 produced a dramatic reduction in neutral lipid production, whereas the overexpression of this gene yielded a significant increase in de novo TAG biosynthesis. In vitro activity assays showed that Sco0958 mediates the esterification of DAG using long-chain acyl-CoAs (C14 to C18) as acyl donors. The Km and V max values of this enzyme for myristoyl-CoA were 45 μM and 822 nmol mg−1 min−1, respectively. Significantly, the triple mutant strain was not completely devoid of storage lipids, indicating the existence of alternative TAG-biosynthetic routes. We present strong evidence demonstrating that the residual production of TAG in this mutant strain is mediated, at least in part, by an acyl-CoA-dependent pathway, since the triple mutant still exhibited DGAT activity. More importantly, there was substantial phospholipid:DGAT (PDAT) activity in the wild type and in the triple mutant. This is the first time that a PDAT activity has been reported for bacteria, highlighting the extreme metabolic diversity of this industrially important soil microorganism.

Overproduction of free fatty acids in E. coli: Implications for biodiesel production

2008 ◽  
Vol 10 (6) ◽  
pp. 333-339 ◽  
Author(s):  
Xuefeng Lu ◽  
Harmit Vora ◽  
Chaitan Khosla
Keyword(s):  
Fatty Acids ◽  
Free Fatty Acids ◽  
Biodiesel Production ◽  
E Coli

Factors affecting the deterioration of stored flaxseed including the potential of insect infestation

1991 ◽  
Vol 71 (2) ◽  
pp. 327-335 ◽  
Author(s):  
N. D. G. White ◽  
D. S. Jayas
Keyword(s):  
Fatty Acids ◽  
Seed Germination ◽  
Free Fatty Acids ◽  
Low Frequency ◽  
Fold Increase ◽  
Factors Affecting ◽  
Linum Usitatissimum L ◽  
Group A ◽  
Insect Reproduction ◽  
Ground Flaxseed

Flaxseed (Linum usitatissimum L. 'McGregor') stored at 10, 20, 30, 40, and 50 °C and 35, 50, 60, and 70% relative humidity (RH) for up to 12 mo deteriorated in quality in < 1–3 mo at the two highest temperatures, although the seed was stored "dry" (≤ 10% moisture content, MC). Initial fatty acid values (FAV) of 41.1 mg KOH 100 g−1 dry seed (0.51% free fatty acids in oil) rarely increased more than 1.5-fold over 12 mo at 10 or 20 °C and up to 10% MC, or at 30 °C and 7 to 8% MC. Using FAV as a storage quality-loss criterion, flaxseed at 8–9% MC could be stored for 6 mo at 30 °C, 1–2 mo at 40 °C, or a few weeks at 50 °C with less than a 1.5-fold increase. A twofold increase in FAV was correlated to a discoloured or charred appearance of seeds and a rapid loss in seed germination. Seed germination did not decrease during 12 mo at 10 or 20 °C and 70% RH, or at 30 °C and 60% RH. The fungi Aspergillus glaucus group, A. candidus Link, and Penicillium spp. infected seed at some temperatures and relative humidities with low frequency by 6 mo, and A. flavus Link also occurred at 12 mo. Visible microflora were absent after 6 mo on seed at 40 and 50 °C. The beetles Oryzaephilus mercator (Fauvel), O. surinamensis L., Tribolium castaneum (Herbst), and T. confusum J. duVal survived and produced some larvae on both whole and ground flaxseed in 2 mo. McGregor was slightly more suitable for insect reproduction than NorMan or NorLin. Five other insect species could not survive. Extensive infestation of flaxseed by stored-product insects is unlikely. Key words: Flaxseed, storage, free fatty acids, germination, microflora, insects

scholarly journals Genome-wide targets identification by CRISPRi-Omics for high-titer production of free fatty acids in Escherichia coli

2020 ◽  
Author(s):  
Lixia Fang ◽  
Jie Fan ◽  
Congya Wang ◽  
Yingxiu Cao ◽  
Hao Song
Keyword(s):  
Escherichia Coli ◽  
Fatty Acids ◽  
Free Fatty Acids ◽  
Metabolic Networks ◽  
High Titer ◽  
Cell Structure ◽  
Cell Factory ◽  
Microbial Cell Factory ◽  
Cell Potential ◽  
E Coli

AbstractTo construct a superior microbial cell factory for chemical synthesis, a major challenge is to fully exploit cell potential via identifying and engineering beneficial gene targets in the sophisticated metabolic networks. Here, we develop an approach that integrates CRISPR interference (CRISPRi) to readily modulate genes expression and omics analyses to identify potential targets in multiple cellular processes, enabling systematical discovery of beneficial chromosomal gene targets that can be engineered to optimize free fatty acids (FFAs) production in Escherichia coli. We identify 56 beneficial genes via synergistic CRISPRi-Omics strategy, including 46 novel targets functioning in cell structure and division, and signaling transduction that efficiently facilitate FFAs production. Upon repressing ihfA and overexpressing aidB and tesA’ in E. coli, the recombinant strain LihfA-OaidB results in a FFAs titer of 21.6 g L-1 in fed-batch fermentation, which, to our best knowledge, is the maximum FFAs titer by the recombinant E. coli reported to date.

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.

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 Lipid-induced insulin resistance does not impair insulin access to skeletal muscle

2015 ◽  
Vol 308 (11) ◽  
pp. E1001-E1009 ◽  
Author(s):  
Cathryn M. Kolka ◽  
Joyce M. Richey ◽  
Ana Valeria B. Castro ◽  
Josiane L. Broussard ◽  
Viorica Ionut ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Skeletal Muscle ◽  
Fatty Acids ◽  
Free Fatty Acids ◽  
Fold Increase ◽  
Canine Model ◽  
Hepatic Insulin Resistance ◽  
Lipid Infusion ◽  
Peripheral Insulin Resistance ◽  
Induce Insulin Resistance

Elevated plasma free fatty acids (FFA) induce insulin resistance in skeletal muscle. Previously, we have shown that experimental insulin resistance induced by lipid infusion prevents the dispersion of insulin through the muscle, and we hypothesized that this would lead to an impairment of insulin moving from the plasma to the muscle interstitium. Thus, we infused lipid into our anesthetized canine model and measured the appearance of insulin in the lymph as a means to sample muscle interstitium under hyperinsulinemic euglycemic clamp conditions. Although lipid infusion lowered the glucose infusion rate and induced both peripheral and hepatic insulin resistance, we were unable to detect an impairment of insulin access to the lymph. Interestingly, despite a significant, 10-fold increase in plasma FFA, we detected little to no increase in free fatty acids or triglycerides in the lymph after lipid infusion. Thus, we conclude that experimental insulin resistance induced by lipid infusion does not reduce insulin access to skeletal muscle under clamp conditions. This would suggest that the peripheral insulin resistance is likely due to reduced cellular sensitivity to insulin in this model, and yet we did not detect a change in the tissue microenvironment that could contribute to cellular insulin resistance.

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