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991.
Clementina Dellomonaco Carlos Rivera Paul Campbell Ramon Gonzalez 《Applied and environmental microbiology》2010,76(15):5067-5078
Although lignocellulosic sugars have been proposed as the primary feedstock for the biological production of renewable fuels and chemicals, the availability of fatty acid (FA)-rich feedstocks and recent progress in the development of oil-accumulating organisms make FAs an attractive alternative. In addition to their abundance, the metabolism of FAs is very efficient and could support product yields significantly higher than those obtained from lignocellulosic sugars. However, FAs are metabolized only under respiratory conditions, a metabolic mode that does not support the synthesis of fermentation products. In the work reported here we engineered several native and heterologous fermentative pathways to function in Escherichia coli under aerobic conditions, thus creating a respiro-fermentative metabolic mode that enables the efficient synthesis of fuels and chemicals from FAs. Representative biofuels (ethanol and butanol) and biochemicals (acetate, acetone, isopropanol, succinate, and propionate) were chosen as target products to illustrate the feasibility of the proposed platform. The yields of ethanol, acetate, and acetone in the engineered strains exceeded those reported in the literature for their production from sugars, and in the cases of ethanol and acetate they also surpassed the maximum theoretical values that can be achieved from lignocellulosic sugars. Butanol was produced at yields and titers that were between 2- and 3-fold higher than those reported for its production from sugars in previously engineered microorganisms. Moreover, our work demonstrates production of propionate, a compound previously thought to be synthesized only by propionibacteria, in E. coli. Finally, the synthesis of isopropanol and succinate was also demonstrated. The work reported here represents the first effort toward engineering microorganisms for the conversion of FAs to the aforementioned products.Concerns about climate change and the depletion and cost of petroleum resources have ignited interest in the establishment of a bio-based industry (5, 49, 61), and the conceptual model of a biorefinery has emerged (27, 28, 45). Given its abundance in nature, the carbohydrate portion of edible crops such as sugarcane, sugar beet, maize (corn), and sorghum is currently used as the primary feedstock in the biological production of fuels and chemicals (12, 49, 52). Although the use of nonedible lignocellulosic sugars has been proposed as an efficient and sustainable avenue to the aforementioned processes, the availability of fatty acid (FA)-rich feedstocks and recent progress in the development of oil-accumulating organisms make FAs an attractive alternative. Edible oil-rich crops such as rapeseed, sunflower, soybean, and palm are currently available and widely used as feedstocks for chemical conversion to biodiesel (6), while oleaginous algae and nonedible FA-rich crops along with industrial by-products are receiving greater attention as longer-term alternatives. These nonedible FA-rich feedstocks are presently generated in large amounts and can be exploited for the biological production of fuels and chemicals (14, 22, 51, 56, 57). Unfortunately, microbial platforms to enable this are at present almost absent.FAs not only are abundant but also offer several advantages when used for fuel and chemical production. For example, their metabolism to the key intermediate metabolite acetyl coenzyme A (acetyl-CoA) is very efficient, as it results in 100% carbon recovery (Fig. (Fig.1).1). Since many fuels and chemicals can be derived from acetyl-CoA, high yields can be realized if FAs are used as the carbon source. In contrast, sugar metabolism generates one molecule of carbon dioxide (or formate) per molecule of acetyl-CoA, limiting the yield of products derived from acetyl-CoA (Fig. (Fig.1).1). The product yield advantage of FAs over sugars is also supported by the more highly reduced nature of their carbon atoms. Table Table11 provides a comparison of maximum theoretical yields, on both weight and carbon bases, for the production of biofuels and biochemicals from FAs and lignocellulosic sugars. Maximum theoretical yields have been calculated from stoichiometry based on the pathways shown in Fig. Fig.11 for the utilization of FAs and glucose, the synthesis of products, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation. The stoichiometric coefficients were obtained by conducting elemental balances on carbon, hydrogen, and oxygen, and an ATP balance was also included in the analysis. As an example, when production of biofuels (e.g., ethanol and butanol) is considered, utilization of FAs (e.g., palmitic acid [C16:0]) as a substrate returns product yields 2.7-fold (wt/wt) or 1.4-fold (C/C) higher than those for sugars (calculations are provided for glucose but are equally valid for other lignocellulosic sugars). Although the current prices of feedstocks on a weight basis are comparable (lower than 20¢/pound), the data reported in Fig. S1a in the supplemental material show that the price per carbon for glucose derived from corn is remarkably higher. Regardless of the basis used for calculations (i.e., weight or carbon basis), when maximum theoretical yields and costs of FA and sugar feedstocks are accounted for, the advantages of using FAs are remarkable (see Fig. S1b in the supplemental material).Open in a separate windowFIG. 1.Pathways engineered in E. coli for the conversion of fatty acids to fuels (red) and chemicals (green). Also shown is the catabolism of fatty acids via the β-oxidation pathway (orange) and of glucose through the Embden-Meyerhof-Parnas pathway (blue). Relevant reactions are represented by the names of the genes coding for the enzymes (E. coli genes unless otherwise specified in parentheses as follows: C. acetobutylicum, ca; C. beijerinckii, cb): aceA, isocitrate lyase; aceB, malate synthase A; adc, acetoacetate decarboxylase (ca); ackA, acetate kinase; adh, secondary alcohol dehydrogenase (cb); adhE, acetaldehyde/alcohol dehydrogenase; adhE2, secondary alcohol dehydrogenase (ca); atoA and atoD, acetyl-CoA:acetoacetyl-CoA transferase; atoB, acetyl-CoA acetyltransferase; bcd, butyryl-CoA dehydrogenase (ca); crt, crotonase (ca); etfAB, electron transfer flavoprotein (ca); fadA, 3-ketoacyl-CoA thiolase; fadB, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase; fadD, acyl-CoA synthetase; fadE, acyl-CoA dehydrogenase; hbd, β-hydroxybutyryl-CoA dehydrogenase (ca); icd, isocitrate dehydrogenase; pta, phosphate acetyltransferase; sdhABCD, succinate dehydrogenase; scpA, methylmalonyl-CoA mutase; scpB, methylmalonyl-CoA decarboxylase; scpC, propionyl-CoA:succinate CoA transferase; sucA, 2-oxoglutarate dehydrogenase; sucB, dihydrolipoyltranssuccinylase; and sucCD, succinyl-CoA synthetase. Abbreviations: 2[H] = NADH = FADH2 = QH2 = H2; P/O, amount of ATP produced per oxygen consumed in the oxidative phosphorylation.
Open in a separate windowaStoichiometry is based on the pathways shown in Fig. Fig.11 for the utilization of FAs and glucose, the synthesis of products, the TCA cycle, and oxidative phosphorylation. For the synthesis of biochemicals, CO2 fixation via the Wood-Ljungdahl pathway (50) (2CO2 + ATP + 8[H] → acetyl-CoA) or the carboxylation of phosphoenolpyruvate (54) (phosphoenolpyruvate + CO2 → oxaloacetate + ATP) were also considered (not shown in Fig. Fig.1).1). The stoichiometric coefficients were obtained by conducting elemental balances on carbon, hydrogen, and oxygen. An ATP balance was also included in the analysis for the reactions shown in italics. All other reactions represent ATP-generating pathways. Every acetyl-CoA oxidized through the TCA cycle generates three NADH, one reduced flavin adenine dinucleotide (FADH2), and one ATP equivalent. Eleven ATPs can be generated from the oxidation of the NADH and FADH2 produced in the TCA cycle (two and three ATPs per FADH2 and NADH, respectively) via coupling between the electron transfer chain and oxidative phosphorylation.Despite the aforementioned advantages, biological conversion of FA-rich feedstocks has been exploited only for the production of polyhydroxyalkanoates (46, 47), with no report to date of organisms engineered for the conversion of FAs to fuels and chemicals (see the text in the supplemental material for more details).Escherichia coli is one of the most amenable organisms to industrial applications and has been engineered for biofuel production (52). The utilization of FAs in E. coli is mediated by enzymes encoded by the fad regulon and the ato operon (11) (Fig. (Fig.1).1). Products of the fad regulon mediate the transport, acylation, and β-oxidation of medium-chain (C7 to C11) and long-chain (C12 to C18) FAs. Two additional enzymes encoded by the atoD-atoA and atoB genes (part of the atoDAEB operon) are also required for the growth of E. coli on short-chain (C4 to C6) FAs (25). The expression of the fad regulon and ato operon is controlled by FadR (fadR) and AtoC (atoC), respectively (44).While advantageous, the high degree of reduction of carbon in FAs also poses a metabolic challenge because their average degree of reduction per carbon is higher than in biomass. Therefore, the incorporation of fatty acids into cell mass generates reducing equivalents (Fig. (Fig.1)1) and hence requires the presence of an external electron acceptor. That is, the aforementioned pathways are active only in the respiratory metabolism of FAs, which leads to the synthesis of cell mass and carbon dioxide but no other metabolic product. Therefore, fuel and chemical production from FAs requires the engineering of a respiro-fermentative metabolic mode that would support the synthesis of fermentative products during respiratory metabolism of FAs. To this end, we metabolically engineered native and heterologous pathways for the efficient catabolism of FAs and the synthesis of fuels and chemicals in E. coli. Biofuels, commodity chemicals, and polymer building blocks were chosen as model products to illustrate the feasibility of the proposed approach. 相似文献
TABLE 1.
Comparison of maximum theoretical yields for the production of biofuels and biochemicals from fatty acids (palmitic acid) and lignocellulosic sugars (glucose)Pathway stoichiometry for the synthesis of the specified product from glucose (C6H12O6) or palmitic acid (C16H32O2)a | Maximum yield (wt basis/C basis) |
---|---|
Biofuels | |
Ethanol (C2H6O) | |
C6H12O6 → 2C2H6O + 2CO2 | 0.51/0.67 |
C16H32O2 → 23/3C2H6O + 2/3CO2 | 1.38/0.96 |
C16H32O2 + 51/7H2O → 53/7C2H6O + 6/7CO2 + 8/7[H]; 8/7[H] + 2/7O2 → 4/7H2O | 1.36/0.95 |
Butanol (C4H10O) | |
C6H12O6 → C4H10O + 2CO2 +H2O | 0.41/0.67 |
C16H32O2 + 7/2H2O → 53/14C4H10O + 6/7CO2 + 8/7[H]; 8/7[H] + 2/7O2 → 4/7H2O | 1.10/0.95 |
Biochemicals | |
Acetate (C2H4O2) | |
C6H12O6 + 2H2O → 3C2H4O2 | 1.00/1.00 |
C16H32O2 + 7H2O + 7CO2 → 23/2C2H4O2 | 2.70/1.44 |
Acetone (C3H6O) | |
C6H12O6 → 3/2C3H6O + 3/2CO2 + 3/2H2O | 0.48/0.75 |
C16H32O2 + 5/4H2O + 5/4CO2 → 23/4C3H6O | 1.30/1.08 |
Isopropanol (C3H8O) | |
C6H12O6 → 4/3C3H8O + 2CO2 + 2/3H2O | 0.44/0.67 |
C16H32O2 + 40/9H2O → 46/9C3H8O + 2/3CO2 | 1.20/0.96 |
Succinate (C4H6O4) | |
C6H12O6 + 6/7CO2 → 12/7C4H6O4 + 6/7H2O | 1.12/1.14 |
C16H32O2 + 152/17CO2 + 86/17H2O → 106/17C4H6O4 + 80/17[H]; 80/17[H] + 20/17O2 → 40/17H2O | 2.87/1.56 |
Propionate (C3H6O2) | |
C6H12O6 → 12/7C3H6O2 + 6/7CO2 + 6/7H2O | 0.70/0.86 |
C16H32O2 + 262/83CO2 + 370/83H2O → 530/83C3H6O2 + 216/83[H]; 216/83[H] + 54/83O2 → 108/83H2O | 1.81/1.20 |
992.
Marta S. Dardanelli Hamid Manyani Sergio González-Barroso Miguel A. Rodríguez-Carvajal Antonio M. Gil-Serrano Maria R. Espuny Francisco Javier López-Baena Ramon A. Bellogín Manuel Megías Francisco J. Ollero 《Plant and Soil》2010,328(1-2):483-493
In this work we studied how biotic and abiotic stresses can alter the pattern of flavonoids exuded by Osumi soybean roots. A routine method was developed for the detection and characterization of the flavonoids present in soybean root exudates using HPLC-MS/MS. Then, a systematic screening of the flavonoids exuded under biotic stress, the presence of a plant growth promoting rhizobacterium, and salt stress was carried out. Results obtained indicate that the presence of Chryseobacterium balustinum Aur9 or 50 mM NaCl changes qualitatively the pattern of flavonoids exuded when compared to control conditions. Thus, in the presence of C. balustinum Aur9, soybean roots did not exude quercetin and naringenin and, under salt stress, flavonoids daidzein and naringenin could not be detected. Soybean root exudates obtained under saline conditions showed a diminished capacity to induce the expression of the nodA gene in comparison to the exudates obtained in the absence of salt. Moreover, lipochitooligosaccharides (LCOs) were not detected or weakly detected when Sinorhizobium fredii SMH12 was grown in the exudates obtained under salt stress conditions or under salt stress in the presence of C. balustinum Au9, respectively. 相似文献
993.
994.
Matrix metalloproteases (MMPs) cleave native collagen at a single site despite the fact that collagen contains more than one scissile bond that can, in principle, be cleaved. For peptide bond hydrolysis to occur at one specific site, MMPs must (1) localize to a region near the unique scissile bond, (2) bind residues at the catalytic site that form the scissile bond, and (3) hydrolyze the corresponding peptide bond. Prior studies suggest that for some types of collagen, binding of noncatalytic MMP domains to amino acid sequences in the vicinity of the true cleavage site facilitates the localization of collagenases. In the present study, our goal was to determine whether binding to the catalytic site also plays a role in determining MMP specificity. To investigate this, we computed the conformational free energy landscape of Type III collagen at each potential cleavage site. The free energy profiles suggest that although all potential cleavage sites sample unfolded states at relatively low temperatures, the true cleavage site samples structures that are complementary to the catalytic site. By contrast, potential cleavage sites that are not cleaved sample states that are relatively incompatible with the MMP active site. Furthermore, our findings point to a specific role for arginine residues in modulating the structural stability of collagen near the collagenase cleavage site. These data imply that locally unfolded potential cleavage sites in Type III collagen sample distinct unfolded ensembles, and that the region about the true collagenase cleavage site samples states that are most complementary to the MMP active site. Proteins 2010. © 2009 Wiley‐Liss, Inc. 相似文献
995.
Alba Carreras Mauricio Rojas Theodora Tsapikouni Josep M Montserrat Daniel Navajas Ramon Farré 《Respiratory research》2010,11(1):91
Background
The aim was to test the hypothesis that the blood serum of rats subjected to recurrent airway obstructions mimicking obstructive sleep apnea (OSA) induces early activation of bone marrow-derived mesenchymal stem cells (MSC) and enhancement of endothelial wound healing.Methods
We studied 30 control rats and 30 rats subjected to recurrent obstructive apneas (60 per hour, lasting 15 s each, for 5 h). The migration induced in MSC by apneic serum was measured by transwell assays. MSC-endothelial adhesion induced by apneic serum was assessed by incubating fluorescent-labelled MSC on monolayers of cultured endothelial cells from rat aorta. A wound healing assay was used to investigate the effect of apneic serum on endothelial repair.Results
Apneic serum showed significant increase in chemotaxis in MSC when compared with control serum: the normalized chemotaxis indices were 2.20 ± 0.58 (m ± SE) and 1.00 ± 0.26, respectively (p < 0.05). MSC adhesion to endothelial cells was greater (1.75 ± 0.14 -fold; p < 0.01) in apneic serum than in control serum. When compared with control serum, apneic serum significantly increased endothelial wound healing (2.01 ± 0.24 -fold; p < 0.05).Conclusions
The early increases induced by recurrent obstructive apneas in MSC migration, adhesion and endothelial repair suggest that these mechanisms play a role in the physiological response to the challenges associated to OSA.996.
Background
Zipf''s law states that the relationship between the frequency of a word in a text and its rank (the most frequent word has rank , the 2nd most frequent word has rank ,…) is approximately linear when plotted on a double logarithmic scale. It has been argued that the law is not a relevant or useful property of language because simple random texts - constructed by concatenating random characters including blanks behaving as word delimiters - exhibit a Zipf''s law-like word rank distribution.Methodology/Principal Findings
In this article, we examine the flaws of such putative good fits of random texts. We demonstrate - by means of three different statistical tests - that ranks derived from random texts and ranks derived from real texts are statistically inconsistent with the parameters employed to argue for such a good fit, even when the parameters are inferred from the target real text. Our findings are valid for both the simplest random texts composed of equally likely characters as well as more elaborate and realistic versions where character probabilities are borrowed from a real text.Conclusions/Significance
The good fit of random texts to real Zipf''s law-like rank distributions has not yet been established. Therefore, we suggest that Zipf''s law might in fact be a fundamental law in natural languages. 相似文献997.
998.
The Cariaco Basin off the Venezuelan coast in the Caribbean Sea is the world's largest truly marine body of anoxic water. The first rRNA survey of microbial eukaryotes in this environment revealed a number of novel lineages, but sampled only a fraction of the entire diversity. The goal of this study was to significantly improve recovery of protistan rRNA from the Basin. This was achieved by a systematic application of multiple PCR primer sets and substantially larger sequencing efforts. We focused on the most diverse habitat in the basin, anoxic waters approximately 100m below the oxic-anoxic interface, and detected novel lineages that escaped the single PCR primer approach. All clones obtained proved unique. A 99% sequence similarity cut-off value combined these clones into operational taxonomic units (OTUs), over 75% of which proved novel. Some of these OTUs form deep branches within established protistan groups. Others signify discovery of novel protistan lineages that appear unrelated to any known microeukaryote. Surprisingly, even this large-scale multi-primer rRNA approach still missed a substantial part of the samples' rRNA diversity. The overlap between the species lists obtained with different primers is low, with only 4% of OTUs shared by all three libraries, and the number of species detected only once is large (55%). This strongly indicates that, at least in anoxic environments, protistan diversity may be much larger than is commonly thought. A single sample appears to contain thousands of largely novel protistan species. Multiple PCR primer combinations may be needed to capture these species. 相似文献
999.
Background
The magnetoencephalograms (MEGs) are mainly due to the source currents. However, there is a significant contribution to MEGs from the volume currents. The structure of the anatomical surfaces, e.g., gray and white matter, could severely influence the flow of volume currents in a head model. This, in turn, will also influence the MEGs and the inverse source localizations. This was examined in detail with three different human head models. 相似文献1000.
Juan J Grau Ramon Palmero Maribel Marmol Jose Domingo-Domenech Mariano Monzo Jose Fuster Oscar Vidal Constantino Fondevila Juan C Garcia-Valdecasas 《World journal of surgical oncology》2006,4(1):1-9