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Christophe Delye - One of the best experts on this subject based on the ideXlab platform.
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universal pcr assays detecting mutations in Acetyl Coenzyme a carboxylase or acetolactate synthase that endow herbicide resistance in grass weeds
2011Co-Authors: Christophe Delye, Fanny Pernin, Séverine MichelAbstract:Delye C, Pernin F & Michel S (2011). ‘Universal’ PCR assays detecting mutations in Acetyl-Coenzyme A carboxylase or acetolactate synthase that endow herbicide resistance in grass weeds. Weed Research51, 353–362. Summary Herbicides inhibiting Acetyl-Coenzyme A carboxylase (ACCase) or acetolactate synthase (ALS) are key for grass weed control. Yet, numerous cases of resistance have evolved. Using the derived cleaved amplified polymorphic sequence method, we developed molecular assays to detect amino acid replacements at the seven ACCase codons (1781, 1999, 2027, 2041, 2078, 2088 and 2096) and at two ALS codons (197 and 574) known to play a role in herbicide resistance in grass weeds. For each codon, one assay detecting all known amino acid replacements endowing herbicide resistance was developed. The nine assays were successfully used to genotype ACCase and ALS in 39 grass species. Their flexible design enables easy detection of new mutations at the targeted codons. Because they can be implemented with basic molecular biology facilities and no previous knowledge of the ACCase or ALS sequence of the grass weed of interest, these assays are tools of choice to easily detect resistance caused by alteration(s) of ACCase or ALS in such species.
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geographical variation in resistance to Acetyl Coenzyme a carboxylase inhibiting herbicides across the range of the arable weed alopecurus myosuroides black grass
2010Co-Authors: Christophe Delye, Jean-philippe Guillemin, Séverine Michel, Bruno Chauvel, Aurelie Berard, Dominique Brunel, Fabrice Dessaint, Valerie Le CorreAbstract:Summary • The geographical structure of resistance to herbicides inhibiting Acetyl-Coenzyme A carboxylase (ACCase) was investigated in the weed Alopecurus myosuroides (black-grass) across its geographical range to gain insight into the process of plant adaptation in response to anthropogenic selective pressures occurring in agricultural ecosystems. • We analysed 297 populations distributed across six countries in A. myosuroides’ main area of occupancy. The frequencies of plants resistant to two broadly used ACCase inhibitors and of seven mutant, resistant ACCase alleles were assessed using bioassays and genotyping, respectively. • Most of the resistance was not endowed by mutant ACCase alleles. Resistance and ACCase allele distribution patterns were characterized by mosaicism. The prevalence of resistance and of ACCase alleles differed among countries. • Resistance clearly evolved by redundant evolution of a set of resistance alleles or genes, most of which remain unidentified. Resistance in A. myosuroides was shaped by variation in the herbicide selective pressure at both the individual field level and the national level.
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Complex genetic control of non-target-site-based resistance to herbicides inhibiting Acetyl-Coenzyme A carboxylase and acetolactate-synthase in Alopecurus myosuroides Huds.
2010Co-Authors: Cécile Petit, Bastien Duhieu, Karelle Boucansaud, Christophe DelyeAbstract:The genetic control of non-target-site-based resistance (NTSR) to three herbicides inhibiting Acetyl-Coenzyme A carboxylase (ACCase) and one inhibiting acetolactate-synthase (ALS) was investigated in Alopecurus myosuroides (black-grass). Hundred controlled pairings were used to determine the minimum number of loci involved in NTSR to each herbicide and their associated resistance pattern. Resistant parental plants survived herbicide doses ranging from 1 to 12 times the field rate. In a single plant, NTSR to a given herbicide involved at least one to three dominant loci or one recessive locus. Accumulation of up to at least three NTSR loci in a single plant could be necessary to confer resistance. Most NTSR loci endowing resistance to one herbicide did not confer detectable resistance to any other herbicide assayed. This is the first study demonstrating that NTSR is a quantitative trait. It also revealed three nested levels of complexity in NTSR: the loci, which can confer resistance at the whole plant level depending on the herbicide and on the dose used; the individual plants, which can accumulate various sets of NTSR loci via sexual reproduction; and the populations, which are made of various frequencies of genotypes each containing different numbers and combinations of NTSR loci.
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Status of black-grass (Alopecurus myosuroides) resistance to Acetyl-Coenzyme A carboxylase inhibitors in France
2007Co-Authors: Christophe Delye, Yosra Menchari, Jean-philippe Guillemin, Annick Matejicek, Séverine Michel, Christine Camilleri, Bruno ChauvelAbstract:We assessed the contributions of target site- and non-target site-based resistance to herbicides inhibiting Acetyl-Coenzyme A carboxylase (ACC) in Alopecurus myosuroides (black grass). A total of 243 A. myosuroides populations collected across France were analysed using herbicide sensitivity bioassay (24 300 seedlings analysed) and ACC genotyping (13 188 seedlings analysed). Seedlings resistant to at least one ACC-inhibiting herbicide were detected in 99.2% of the populations. Mutant, resistant ACC allele(s) were detected in 56.8% of the populations. Among the five resistant ACC alleles known in A. myosuroides, alleles containing an isoleucine-to-leucine substitution at codon 1781 were predominant (59.5% of the plants containing resistant ACC alleles). Comparison of the results from herbicide sensitivity bioassays with genotyping indicated that more than 75% of the plants resistant to ACC-inhibiting herbicides in France would be resistant via increased herbicide metabolism. Analysis of herbicide application records suggested that in 15.9% of the populations studied, metabolism-based resistance to ACC-inhibiting herbicides was mostly selected for by herbicides with other modes of action. Our study revealed the importance of non-target site-based resistance in A. myosuroides. Using herbicides with alternative modes of action to control populations resistant to ACC-inhibiting herbicides, the recommended management approach, may thus be jeopardised by the widespread occurrence of metabolism-based resistance mechanisms conferring broad-spectrum cross-resistance
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molecular bases for sensitivity to Acetyl Coenzyme a carboxylase inhibitors in black grass
2005Co-Authors: Christophe Delye, Annick Matejicek, Séverine Michel, Xiaoqi Zhang, Stephen B PowlesAbstract:In grasses, residues homologous to residues Ile-1,781 and Ile-2,041 in the carboxyl-transferase (CT) domain of the chloroplastic Acetyl-Coenzyme A (CoA) carboxylase (ACCase) from the grass weed black-grass (Alopecurus myosuroides [Huds.]) are critical determinants for sensitivity to two classes of ACCase inhibitors, aryloxyphenoxypropionates (APPs) and cyclohexanediones. Using natural mutants of black-grass, we demonstrated through a molecular, biological, and biochemical approach that residues Trp-2,027, Asp-2,078, and Gly-2,096 are also involved in sensitivity to ACCase inhibitors. In addition, residues Trp-2,027 and Asp-2,078 are very likely involved in CT activity. Using three-dimensional modeling, we found that the side chains of the five residues are adjacent, located at the surface of the inside of the cavity of the CT active site, in the vicinity of the binding site for APPs. Residues 1,781 and 2,078 are involved in sensitivity to both APPs and cyclohexanediones, whereas residues 2,027, 2,041, and 2,096 are involved in sensitivity to APPs only. This suggests that the binding sites for these two classes of compounds are overlapping, although distinct. Comparison of three-dimensional models for black-grass wild-type and mutant CTs and for CTs from organisms with contrasted sensitivity to ACCase inhibitors suggested that inhibitors fitting into the cavity of the CT active site of the chloroplastic ACCase from grasses to reach their active sites may be tight. The three-dimensional shape of this cavity is thus likely of high importance for the efficacy of ACCase inhibitors.
Paul A Lindahl - One of the best experts on this subject based on the ideXlab platform.
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the tunnel of Acetyl Coenzyme a synthase carbon monoxide dehydrogenase regulates delivery of co to the active site
2005Co-Authors: Xiangshi Tan, Huaykeng Loke, Shawn B Fitch, Paul A LindahlAbstract:The effect of [CO] on Acetyl-CoA synthesis activity of the isolated α subunit of Acetyl-Coenzyme A synthase/carbon monoxide dehydrogenase from Moorella thermoacetica was determined. In contrast to the complete α2β2 enzyme where multiple CO molecules exhibit strong cooperative inhibition, α was weakly inhibited, apparently by a single CO with KI = 1.5 ± 0.5 mM; other parameters include kcat = 11 ± 1 min-1 and KM = 30 ± 10 μM. The α subunit lacked the previously described “majority” activity of the complete enzyme but possessed its “residual” activity. The site affording cooperative inhibition may be absent or inoperative in isolated α subunits. Ni-activated α rapidly and reversibly accepted a methyl group from CH3−Co3+FeSP affording the equilibrium constant KMT = 10 ± 4, demonstrating the superior nucleophilicity of αred relative to Co1+FeSP. CO inhibited this reaction weakly (KI = 540 ± 190 μM). NiFeC EPR intensity of α developed in accordance with an apparent Kd = 30 μM, suggesting that the state exhibit...
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Acetyl-Coenzyme A synthase: the case for a Ni_p^0-based mechanism of catalysis
2004Co-Authors: Paul A LindahlAbstract:Acetyl-CoA synthase (also known as carbon monoxide dehydrogenase) is a bifunctional Ni-Fe-S-containing enzyme that catalyzes the reversible reduction of CO_2 to CO and the synthesis of Acetyl-Coenzyme A from CO, CoA, and a methyl group donated by a corrinoid iron-sulfur protein. The active site for the latter reaction, called the A-cluster, consists of an Fe_4S_4 cubane bridged to the proximal Ni site (Ni_p), which is bridged in turn to the so-called distal Ni site. In this review, evidence is presented that Ni_p achieves a zero-valent state at low potentials and during catalysis. Ni_p appears to be the metal to which CO and methyl groups bind and then react to form an Acetyl-Ni_p intermediate. Methyl group binding requires reductive activation, where two electrons reduce some site on the A-cluster. The coordination environment of the distal Ni suggests that it could not be stabilized in redox states lower than 2+. The rate at which the [Fe_4S_4]^2+ cubane is reduced is far slower than that at which reductive activation occurs, suggesting that the cubane is not the site of reduction. An intriguing possibility is that Ni_p^2+ might be reduced to the zero-valent state. Reinforcing this idea are Ni-organometallic complexes in which the Ni exhibits analogous reactivity properties when reduced to the zero-valent state. A zero-valent Ni stabilized exclusively with biological ligands would be remarkable and unprecedented in biology.
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methylation of carbon monoxide dehydrogenase from clostridium thermoaceticum and mechanism of Acetyl Coenzyme a synthesis
1997Co-Authors: David P Barondeau, Paul A LindahlAbstract:Carbon monoxide dehydrogenase from Clostridium thermoaceticum was methylated such that all bound methyl groups could subsequently react with CO and Coenzyme A (or OH-) to yield Acetyl-Coenzyme A (Acetyl-CoA) (or acetate). Methyl groups could not bind enzyme lacking the labile Ni of the A-cluster, but could bind such samples after incubation in aqueous Ni2+, a process known to reinsert the labile Ni and reactivate the enzyme. Bound methyl groups inhibited the ability of 1,10-phenanthroline to remove the labile Ni, and the amount bound approximately correlated with the amount of labile Ni. This is strong evidence that the methyl group used in Acetyl-CoA synthesis binds the labile Ni. Evidence is presented that a redox site (called the D site) other than the spin-coupled metals that define the A-cluster must be reduced before methylation can occur. Both methyl and Acetyl intermediates appear to be EPR-silent. The Acetyl intermediate reacted slowly with OH- to yield acetate and rapidly with CoAS- to yield ace...
Valerie Le Corre - One of the best experts on this subject based on the ideXlab platform.
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geographical variation in resistance to Acetyl Coenzyme a carboxylase inhibiting herbicides across the range of the arable weed alopecurus myosuroides black grass
2010Co-Authors: Christophe Delye, Jean-philippe Guillemin, Séverine Michel, Bruno Chauvel, Aurelie Berard, Dominique Brunel, Fabrice Dessaint, Valerie Le CorreAbstract:Summary • The geographical structure of resistance to herbicides inhibiting Acetyl-Coenzyme A carboxylase (ACCase) was investigated in the weed Alopecurus myosuroides (black-grass) across its geographical range to gain insight into the process of plant adaptation in response to anthropogenic selective pressures occurring in agricultural ecosystems. • We analysed 297 populations distributed across six countries in A. myosuroides’ main area of occupancy. The frequencies of plants resistant to two broadly used ACCase inhibitors and of seven mutant, resistant ACCase alleles were assessed using bioassays and genotyping, respectively. • Most of the resistance was not endowed by mutant ACCase alleles. Resistance and ACCase allele distribution patterns were characterized by mosaicism. The prevalence of resistance and of ACCase alleles differed among countries. • Resistance clearly evolved by redundant evolution of a set of resistance alleles or genes, most of which remain unidentified. Resistance in A. myosuroides was shaped by variation in the herbicide selective pressure at both the individual field level and the national level.
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nucleotide variability at the Acetyl Coenzyme a carboxylase gene and the signature of herbicide selection in the grass weed alopecurus myosuroides huds
2004Co-Authors: Christophe Delye, Séverine Michel, Cecile Straub, Valerie Le CorreAbstract:Acetyl Coenzyme A carboxylase (ACCase) is the target of highly effective herbicides. We investigated the nucleotide variability of the ACCase gene in a sample of 18 black-grass (Alopecurus myosuroides [Huds.]) populations to search for the signature of herbicide selection. Sequencing 3,396 bp encompassing ACCase herbicide-binding domain in 86 individuals revealed 92 polymorphisms, which formed 72 haplotypes. The ratio of nonsynonymous versus synonymous substitutions was very low, in agreement with ACCase being a vital metabolic enzyme. Within black grass, most nonsynonymous substitutions were related to resistance to ACCase-inhibiting herbicides. Differentiation between populations was strong, in contrast to expectations for an allogamous, annual plant. Significant H tests revealed recent hitchhiking events within populations. These results were consistent with recent and local positive selection. We propose that, although they have only been used since at most 15 black-grass generations, ACCase-inhibiting herbicides have exerted a positive selection targeting resistant haplotypes that has been strong enough to have a marked effect upon ACCase nucleotide diversity. A minimum-spanning network of nonrecombinant haplotypes revealed multiple, independent apparitions of resistance-associated mutations. This study provides the first evidence for the signature of ongoing, recent, pesticide selection upon variation at the gene encoding the targeted enzyme in natural plant populations.
Séverine Michel - One of the best experts on this subject based on the ideXlab platform.
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universal pcr assays detecting mutations in Acetyl Coenzyme a carboxylase or acetolactate synthase that endow herbicide resistance in grass weeds
2011Co-Authors: Christophe Delye, Fanny Pernin, Séverine MichelAbstract:Delye C, Pernin F & Michel S (2011). ‘Universal’ PCR assays detecting mutations in Acetyl-Coenzyme A carboxylase or acetolactate synthase that endow herbicide resistance in grass weeds. Weed Research51, 353–362. Summary Herbicides inhibiting Acetyl-Coenzyme A carboxylase (ACCase) or acetolactate synthase (ALS) are key for grass weed control. Yet, numerous cases of resistance have evolved. Using the derived cleaved amplified polymorphic sequence method, we developed molecular assays to detect amino acid replacements at the seven ACCase codons (1781, 1999, 2027, 2041, 2078, 2088 and 2096) and at two ALS codons (197 and 574) known to play a role in herbicide resistance in grass weeds. For each codon, one assay detecting all known amino acid replacements endowing herbicide resistance was developed. The nine assays were successfully used to genotype ACCase and ALS in 39 grass species. Their flexible design enables easy detection of new mutations at the targeted codons. Because they can be implemented with basic molecular biology facilities and no previous knowledge of the ACCase or ALS sequence of the grass weed of interest, these assays are tools of choice to easily detect resistance caused by alteration(s) of ACCase or ALS in such species.
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geographical variation in resistance to Acetyl Coenzyme a carboxylase inhibiting herbicides across the range of the arable weed alopecurus myosuroides black grass
2010Co-Authors: Christophe Delye, Jean-philippe Guillemin, Séverine Michel, Bruno Chauvel, Aurelie Berard, Dominique Brunel, Fabrice Dessaint, Valerie Le CorreAbstract:Summary • The geographical structure of resistance to herbicides inhibiting Acetyl-Coenzyme A carboxylase (ACCase) was investigated in the weed Alopecurus myosuroides (black-grass) across its geographical range to gain insight into the process of plant adaptation in response to anthropogenic selective pressures occurring in agricultural ecosystems. • We analysed 297 populations distributed across six countries in A. myosuroides’ main area of occupancy. The frequencies of plants resistant to two broadly used ACCase inhibitors and of seven mutant, resistant ACCase alleles were assessed using bioassays and genotyping, respectively. • Most of the resistance was not endowed by mutant ACCase alleles. Resistance and ACCase allele distribution patterns were characterized by mosaicism. The prevalence of resistance and of ACCase alleles differed among countries. • Resistance clearly evolved by redundant evolution of a set of resistance alleles or genes, most of which remain unidentified. Resistance in A. myosuroides was shaped by variation in the herbicide selective pressure at both the individual field level and the national level.
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Status of black-grass (Alopecurus myosuroides) resistance to Acetyl-Coenzyme A carboxylase inhibitors in France
2007Co-Authors: Christophe Delye, Yosra Menchari, Jean-philippe Guillemin, Annick Matejicek, Séverine Michel, Christine Camilleri, Bruno ChauvelAbstract:We assessed the contributions of target site- and non-target site-based resistance to herbicides inhibiting Acetyl-Coenzyme A carboxylase (ACC) in Alopecurus myosuroides (black grass). A total of 243 A. myosuroides populations collected across France were analysed using herbicide sensitivity bioassay (24 300 seedlings analysed) and ACC genotyping (13 188 seedlings analysed). Seedlings resistant to at least one ACC-inhibiting herbicide were detected in 99.2% of the populations. Mutant, resistant ACC allele(s) were detected in 56.8% of the populations. Among the five resistant ACC alleles known in A. myosuroides, alleles containing an isoleucine-to-leucine substitution at codon 1781 were predominant (59.5% of the plants containing resistant ACC alleles). Comparison of the results from herbicide sensitivity bioassays with genotyping indicated that more than 75% of the plants resistant to ACC-inhibiting herbicides in France would be resistant via increased herbicide metabolism. Analysis of herbicide application records suggested that in 15.9% of the populations studied, metabolism-based resistance to ACC-inhibiting herbicides was mostly selected for by herbicides with other modes of action. Our study revealed the importance of non-target site-based resistance in A. myosuroides. Using herbicides with alternative modes of action to control populations resistant to ACC-inhibiting herbicides, the recommended management approach, may thus be jeopardised by the widespread occurrence of metabolism-based resistance mechanisms conferring broad-spectrum cross-resistance
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molecular bases for sensitivity to Acetyl Coenzyme a carboxylase inhibitors in black grass
2005Co-Authors: Christophe Delye, Annick Matejicek, Séverine Michel, Xiaoqi Zhang, Stephen B PowlesAbstract:In grasses, residues homologous to residues Ile-1,781 and Ile-2,041 in the carboxyl-transferase (CT) domain of the chloroplastic Acetyl-Coenzyme A (CoA) carboxylase (ACCase) from the grass weed black-grass (Alopecurus myosuroides [Huds.]) are critical determinants for sensitivity to two classes of ACCase inhibitors, aryloxyphenoxypropionates (APPs) and cyclohexanediones. Using natural mutants of black-grass, we demonstrated through a molecular, biological, and biochemical approach that residues Trp-2,027, Asp-2,078, and Gly-2,096 are also involved in sensitivity to ACCase inhibitors. In addition, residues Trp-2,027 and Asp-2,078 are very likely involved in CT activity. Using three-dimensional modeling, we found that the side chains of the five residues are adjacent, located at the surface of the inside of the cavity of the CT active site, in the vicinity of the binding site for APPs. Residues 1,781 and 2,078 are involved in sensitivity to both APPs and cyclohexanediones, whereas residues 2,027, 2,041, and 2,096 are involved in sensitivity to APPs only. This suggests that the binding sites for these two classes of compounds are overlapping, although distinct. Comparison of three-dimensional models for black-grass wild-type and mutant CTs and for CTs from organisms with contrasted sensitivity to ACCase inhibitors suggested that inhibitors fitting into the cavity of the CT active site of the chloroplastic ACCase from grasses to reach their active sites may be tight. The three-dimensional shape of this cavity is thus likely of high importance for the efficacy of ACCase inhibitors.
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nucleotide variability at the Acetyl Coenzyme a carboxylase gene and the signature of herbicide selection in the grass weed alopecurus myosuroides huds
2004Co-Authors: Christophe Delye, Séverine Michel, Cecile Straub, Valerie Le CorreAbstract:Acetyl Coenzyme A carboxylase (ACCase) is the target of highly effective herbicides. We investigated the nucleotide variability of the ACCase gene in a sample of 18 black-grass (Alopecurus myosuroides [Huds.]) populations to search for the signature of herbicide selection. Sequencing 3,396 bp encompassing ACCase herbicide-binding domain in 86 individuals revealed 92 polymorphisms, which formed 72 haplotypes. The ratio of nonsynonymous versus synonymous substitutions was very low, in agreement with ACCase being a vital metabolic enzyme. Within black grass, most nonsynonymous substitutions were related to resistance to ACCase-inhibiting herbicides. Differentiation between populations was strong, in contrast to expectations for an allogamous, annual plant. Significant H tests revealed recent hitchhiking events within populations. These results were consistent with recent and local positive selection. We propose that, although they have only been used since at most 15 black-grass generations, ACCase-inhibiting herbicides have exerted a positive selection targeting resistant haplotypes that has been strong enough to have a marked effect upon ACCase nucleotide diversity. A minimum-spanning network of nonrecombinant haplotypes revealed multiple, independent apparitions of resistance-associated mutations. This study provides the first evidence for the signature of ongoing, recent, pesticide selection upon variation at the gene encoding the targeted enzyme in natural plant populations.
Barbara U Kozak - One of the best experts on this subject based on the ideXlab platform.
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engineering cytosolic Acetyl Coenzyme a supply in saccharomyces cerevisiae pathway stoichiometry free energy conservation and redox cofactor balancing
2016Co-Authors: Harmen M Van Rossum, Jack T Pronk, Barbara U Kozak, Antonius J A Van MarisAbstract:Saccharomyces cerevisiae is an important industrial cell factory and an attractive experimental model for evaluating novel metabolic engineering strategies. Many current and potential products of this yeast require Acetyl Coenzyme A (Acetyl-CoA) as a precursor and pathways towards these products are generally expressed in its cytosol. The native S. cerevisiae pathway for production of cytosolic Acetyl-CoA consumes 2 ATP equivalents in the Acetyl-CoA synthetase reaction. Catabolism of additional sugar substrate, which may be required to generate this ATP, negatively affects product yields. Here, we review alternative pathways that can be engineered into yeast to optimize supply of cytosolic Acetyl-CoA as a precursor for product formation. Particular attention is paid to reaction stoichiometry, free-energy conservation and redox-cofactor balancing of alternative pathways for Acetyl-CoA synthesis from glucose. A theoretical analysis of maximally attainable yields on glucose of four compounds (n-butanol, citric acid, palmitic acid and farnesene) showed a strong product dependency of the optimal pathway configuration for Acetyl-CoA synthesis. Moreover, this analysis showed that combination of different Acetyl-CoA production pathways may be required to achieve optimal product yields. This review underlines that an integral analysis of energy coupling and redox-cofactor balancing in precursor-supply and product-formation pathways is crucial for the design of efficient cell factories.
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engineering Acetyl Coenzyme a supply functional expression of a bacterial pyruvate dehydrogenase complex in the cytosol of saccharomyces cerevisiae
2014Co-Authors: Barbara U Kozak, Jack T Pronk, Harmen M Van Rossum, Marijke A H Luttik, Michiel Akeroyd, Kirsten R Benjamin, Simon De Vries, Jeanmarc Daran, Antonius J A Van MarisAbstract:The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic Acetyl Coenzyme A (Acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic Acetyl-CoA via Acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of an ATP-independent pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic Acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required simultaneous expression of E. faecalis genes encoding its E1α, E1β, E2, and E3 subunits, as well as genes involved in lipoylation of E2, and addition of lipoate to growth media. A strain lacking ACS that expressed these E. faecalis genes grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs(+) reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial microorganisms is a promising option for minimizing the energy costs of precursor supply in Acetyl-CoA-dependent product pathways. Importance: Genetically engineered microorganisms are intensively investigated and applied for production of biofuels and chemicals from renewable sugars. To make such processes economically and environmentally sustainable, the energy (ATP) costs for product formation from sugar must be minimized. Here, we focus on an important ATP-requiring process in baker's yeast (Saccharomyces cerevisiae): synthesis of cytosolic Acetyl Coenzyme A, a key precursor for many industrially important products, ranging from biofuels to fragrances. We demonstrate that pyruvate dehydrogenase from the bacterium Enterococcus faecalis, a huge enzyme complex with a size similar to that of a ribosome, can be functionally expressed and assembled in the cytosol of baker's yeast. Moreover, we show that this ATP-independent mechanism for cytosolic Acetyl-CoA synthesis can entirely replace the ATP-costly native yeast pathway. This work provides metabolic engineers with a new option to optimize the performance of baker's yeast as a "cell factory" for sustainable production of fuels and chemicals.
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engineering Acetyl Coenzyme a supply functional expression of a bacterial pyruvate dehydrogenase complex in the cytosol of saccharomyces cerevisiae
2014Co-Authors: Barbara U Kozak, Jack T Pronk, Marijke A H Luttik, Michiel Akeroyd, Kirsten R Benjamin, Simon De Vries, Jeanmarc Daran, Harmen M Van Rossum, Liang Wu, Antonius J A Van MarisAbstract:The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic Acetyl Coenzyme A (Acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic Acetyl-CoA via Acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of an ATP-independent pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic Acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required simultaneous expression of E. faecalis genes encoding its E1?, E1?, E2, and E3 subunits, as well as genes involved in lipoylation of E2, and addition of lipoate to growth media. A strain lacking ACS that expressed these E. faecalis genes grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs+ reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial microorganisms is a promising option for minimizing the energy costs of precursor supply in Acetyl-CoA-dependent product pathways.