RESUMO
Thiamine (vitamin B1) is essential for glucose catabolism. In the yeast species, Nakaseomyces glabratus (formerly Candida glabrata) and Saccharomyces cerevisiae, the transcription factor Pdc2 (with Thi3 and Thi2) upregulates pyruvate decarboxylase (PDC) genes and thiamine biosynthetic and acquisition (THI) genes during starvation. There have not been genome-wide analyses of Pdc2 binding. Previously, we identified small regions of Pdc2-regulated genes sufficient to confer thiamine regulation. Here, we performed deletion analyses on these regions. We observed that when the S. cerevisiae PDC5 promoter is introduced into N. glabratus, it is thiamine starvation inducible but does not require the Thi3 coregulator. The ScPDC5 promoter contains a 22-bp duplication with an AT-rich spacer between the 2 repeats, which are important for regulation. Loss of the first 22-bp element does not eliminate regulation, but the promoter becomes Thi3 dependent, suggesting cis architecture can generate a Thi3-independent, thiamine starvation inducible response. Whereas many THI promoters only have 1 copy of this element, addition of the first 22-bp element to a Thi3-dependent promoter confers Thi3 independence. Finally, we performed fluorescence anisotropy and chromatin immunoprecipitation sequencing. Pdc2 and Thi3 bind to regions that share similarity to the 22-bp element in the ScPDC5 promoter and previously identified cis elements in N. glabratus promoters. Also, while Pdc2 binds to THI and PDC promoters, neither Pdc2 nor Thi3 appears to bind the evolutionarily new NgPMU3 promoter that is regulated by Pdc2. Further study is warranted because PMU3 is required for cells to acquire thiamine from environments where thiamine is phosphorylated, such as in the human bloodstream.
Assuntos
Candida glabrata , Regulação Fúngica da Expressão Gênica , Regiões Promotoras Genéticas , Piruvato Descarboxilase , Tiamina , Tiamina/metabolismo , Candida glabrata/genética , Piruvato Descarboxilase/genética , Piruvato Descarboxilase/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ligação Proteica , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Zymomonas mobilis is well known for its outstanding ability to produce ethanol with both high specific productivity and with high yield close to the theoretical maximum. The key enzyme in the ethanol production pathway is the pyruvate decarboxylase (PDC) which is converting pyruvate to acetaldehyde. Since it is widely considered that its gene pdc is essential, metabolic engineering strategies aiming to produce other compounds derived from pyruvate need to find ways to reduce PDC activity. RESULTS: Here, we present a new platform strain (sGB027) of Z. mobilis in which the native promoter of pdc was replaced with the IPTG-inducible PT7A1, allowing for a controllable expression of pdc. Expression of lactate dehydrogenase from E. coli in sGB027 allowed the production of D-lactate with, to the best of our knowledge, the highest reported specific productivity of any microbial lactate producer as well as with the highest reported lactate yield for Z. mobilis so far. Additionally, by expressing the L-alanine dehydrogenase of Geobacillus stearothermophilus in sGB027 we produced L-alanine, further demonstrating the potential of sGB027 as a base for the production of compounds other than ethanol. CONCLUSION: We demonstrated that our new platform strain can be an excellent starting point for the efficient production of various compounds derived from pyruvate with Z. mobilis and can thus enhance the establishment of this organism as a workhorse for biotechnological production processes.
Assuntos
Escherichia coli , Etanol , Ácido Láctico , Engenharia Metabólica , Piruvato Descarboxilase , Zymomonas , Zymomonas/metabolismo , Zymomonas/genética , Piruvato Descarboxilase/metabolismo , Piruvato Descarboxilase/genética , Engenharia Metabólica/métodos , Etanol/metabolismo , Ácido Láctico/metabolismo , Ácido Láctico/biossíntese , Escherichia coli/metabolismo , Escherichia coli/genética , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase/genética , Alanina/metabolismo , Ácido Pirúvico/metabolismo , FermentaçãoRESUMO
Most of kiwifruit cultivars (e.g. Actinidia chinensis cv. Donghong, "DH") were sensitive to waterlogging, thus, waterlogging resistant rootstocks (e.g. Actinidia valvata Dunn, "Dunn") were widely used for kiwifruit industry. Those different species provided ideal materials to understand the waterlogging responses in kiwifruit. Compared to the weaken growth and root activities in "DH", "Dunn" maintained the relative high root activities under the prolonged waterlogging. Based on comparative analysis, transcript levels of pyruvate decarboxylase (PDCs) and alcohol dehydrogenase (ADHs) showed significantly difference between these two species. Both PDCs and ADHs had been significantly increased by waterlogging in "DH", while they were only limitedly triggered by 2 days stress and subsided during the prolonged waterlogging in "Dunn". Thus, 19 differentially expressed transcript factors (DETFs) had been isolated using weighted gene co-expression network analysis combined with transcriptomics and transcript levels of PDCs and ADHs in waterlogged "DH". Among these DETFs, dual luciferase and electrophoretic mobility shift assays indicated AcMYB68 could bind to and trigger the activity of AcPDC2 promoter. The stable over-expression of AcMYB68 significantly up-regulated the transcript levels of PDCs but inhibited the plant growth, especially the roots. Moreover, the enzyme activities of PDC in 35S::AcMYB68 were significantly enhanced during the waterlogging response than that in wild type plants. Most interestingly, comparative analysis indicated that the expression patterns of AcMYB68 and the previously characterized AcERF74/75 (the direct regulator on ADHs) either showed no responses (AcMYB68 and AcERF74) or very limited response (AcERF75) in "Dunn". Taken together, the restricted responses of AcMYB68 and AcERF74/75 in "Dunn" endow its waterlogging tolerance.
Assuntos
Actinidia , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas , Piruvato Descarboxilase , Actinidia/genética , Actinidia/fisiologia , Actinidia/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Piruvato Descarboxilase/genética , Piruvato Descarboxilase/metabolismo , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Água/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Estresse Fisiológico , Regiões Promotoras Genéticas/genéticaRESUMO
Strawberry is one of the most popular fruits in the world, because their high fruit quality, especially with respect to the combination of aroma, flavor, color, and nutritional compounds. Pyruvate decarboxylase (PDC) is the first of two enzymes specifically required for ethanolic fermentation and catalyzes the decarboxylation of pyruvate to yield acetaldehyde and CO2. The ethanol, an important alcohol which acts as a precursor for the ester and other alcohols formation in strawberry, is produced by the PDC. The objective was found all different PDCs genes present in the strawberry genome and investigate PDC gene expression and ligand-protein interactions in strawberry fruit. Volatile organic compounds were evaluated during the development of the fruit. After this, eight FaPDC were identified with four genes that increase the relative expression during fruit ripening process. Molecular dynamics simulations were performed to analyze the behavior of Pyr and TPP ligands within the catalytic and regulatory sites of the PDC proteins. Results indicated that energy-restrained simulations exhibited minor fluctuations in ligand-protein interactions, while unrestrained simulations revealed crucial insights into ligand affinity. TPP consistently displayed strong interactions with the catalytic site, emphasizing its pivotal role in enzymatic activity. However, FaPDC6 and FaPDC9 exhibited decreased pyruvate affinity initially, suggesting unique binding characteristics requiring further investigation. Finally, the present study contributes significantly to understanding PDC gene expression and the intricate molecular dynamics underlying strawberry fruit ripening, shedding light on potential targets for further research in this critical biological pathway.
Assuntos
Fragaria , Piruvato Descarboxilase , Piruvato Descarboxilase/genética , Piruvato Descarboxilase/metabolismo , Fragaria/genética , Fragaria/metabolismo , Frutas/metabolismo , Ligantes , Proteínas de Plantas/metabolismo , Etanol/metabolismo , Piruvatos/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
Saccharomyces cerevisiae is the workhorse of fermentation industry. Upon engineering for D-lactate production by a series of gene deletions, this yeast had deficiencies in cell growth and D-lactate production at high substrate concentrations. Complex nutrients or high cell density were thus required to support growth and D-lactate production with a potential to increase medium and process cost of industrial-scale D-lactate production. As an alternative microbial biocatalyst, a Crabtree-negative and thermotolerant yeast Kluyveromyces marxianus was engineered in this study to produce high titer and yield of D-lactate at a lower pH without growth defects. Only pyruvate decarboxylase 1 (PDC1) gene was replaced by a codon-optimized bacterial D-lactate dehydrogenase (ldhA). Ethanol, glycerol, or acetic acid was not produced by the resulting strain, KMΔpdc1::ldhA. Aeration rate at 1.5 vvm and culture pH 5.0 at 30 °C provided the highest D-lactate titer of 42.97 ± 0.48 g/L from glucose. Yield and productivity of D-lactate, and glucose-consumption rate were 0.85 ± 0.01 g/g, 0.90 ± 0.01 g/(L·h), and 1.06 ± 0.00 g/(L·h), respectively. Surprisingly, D-lactate titer, productivity, and glucose-consumption rate of 52.29 ± 0.68 g/L, 1.38 ± 0.05 g/(L·h), and 1.22 ± 0.00 g/(L·h), respectively, were higher at 42 °C compared to 30 °C. Sugarcane molasses, a low-value carbon, led to the highest D-lactate titer and yield of 66.26 ± 0.81 g/L and 0.91 ± 0.01 g/g, respectively, in a medium without additional nutrients. This study is a pioneer work of engineering K. marxianus to produce D-lactate at the yield approaching theoretical maximum using simple batch process. Our results support the potential of an engineered K. marxianus for D-lactate production on an industrial scale. KEY POINTS: ⢠K. marxianus was engineered by deleting PDC1 and expressing codon-optimized D-ldhA. ⢠The strain allowed high D-lactate titer and yield under pH ranging from 3.5 to 5.0. ⢠The strain produced 66 g/L D-lactate at 30 °C from molasses without any additional nutrients.
Assuntos
Kluyveromyces , Ácido Láctico , Saccharomyces cerevisiae/metabolismo , Kluyveromyces/genética , Kluyveromyces/metabolismo , L-Lactato Desidrogenase/metabolismo , Glucose , Piruvato Descarboxilase/genética , Piruvato Descarboxilase/metabolismo , Concentração de Íons de Hidrogênio , FermentaçãoRESUMO
Understanding metabolism in the pathogen Candida glabrata is key to identifying new targets for antifungals. The thiamine biosynthetic (THI) pathway is partially defective in C. glabrata, but the transcription factor CgPdc2 upregulates some thiamine biosynthetic and transport genes. One of these genes encodes a recently evolved thiamine pyrophosphatase (CgPMU3) that is critical for accessing external thiamine. Here, we demonstrate that CgPdc2 primarily regulates THI genes. In Saccharomyces cerevisiae, Pdc2 regulates both THI and pyruvate decarboxylase (PDC) genes, with PDC proteins being a major thiamine sink. Deletion of PDC2 is lethal in S. cerevisiae in standard growth conditions, but not in C. glabrata. We uncover cryptic cis elements in C. glabrata PDC promoters that still allow for regulation by ScPdc2, even when that regulation is not apparent in C. glabrata. C. glabrata lacks Thi2, and it is likely that inclusion of Thi2 into transcriptional regulation in S. cerevisiae allows for a more complex regulation pattern and regulation of THI and PDC genes. We present evidence that Pdc2 functions independent of Thi2 and Thi3 in both species. The C-terminal activation domain of Pdc2 is intrinsically disordered and critical for species differences. Truncation of the disordered domains leads to a gradual loss of activity. Through a series of cross species complementation assays of transcription, we suggest that there are multiple Pdc2-containing complexes, and C. glabrata appears to have the simplest requirement set for THI genes, except for CgPMU3. CgPMU3 has different cis requirements, but still requires Pdc2 and Thi3 to be upregulated by thiamine starvation. We identify the minimal region sufficient for thiamine regulation in CgTHI20, CgPMU3, and ScPDC5 promoters. Defining the cis and trans requirements for THI promoters should lead to an understanding of how to interrupt their upregulation and provide targets in metabolism for antifungals.
Assuntos
Candida glabrata , Proteínas Fúngicas , Regulação Fúngica da Expressão Gênica , Piruvato Descarboxilase , Saccharomyces cerevisiae , Fatores de Transcrição , Saccharomyces cerevisiae/metabolismo , Candida glabrata/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Fúngicas/metabolismo , Piruvato Descarboxilase/genética , Tiamina/biossíntese , Carboxiliases/genética , Regiões Promotoras Genéticas , Proteínas Intrinsicamente Desordenadas/metabolismoRESUMO
Tyrosol is an important chemical in medicine and chemical industries, which can be synthesized by a four-enzyme cascade pathway constructed in our previous study. However, the low catalytic efficiency of pyruvate decarboxylase from Candida tropicalis (CtPDC) in this cascade is a rate-limiting step. In this study, we resolved the crystal structure of CtPDC and investigated the mechanism of allosteric substrate activation and decarboxylation of this enzyme toward 4-hydroxyphenylpyruvate (4-HPP). In addition, based on the molecular mechanism and structural dynamic changes, we conducted protein engineering of CtPDC to improve decarboxylation efficiency. The conversion of the best mutant, CtPDCQ112G/Q162H/G415S/I417V (CtPDCMu5), had over two-fold improvement compared to the wild-type. Molecular dynamic (MD) simulation revealed that the key catalytic distances and allosteric transmission pathways were shorter in CtPDCMu5 than in the wild type. Furthermore, when CtPDC in the tyrosol production cascade was replaced with CtPDCMu5, the tyrosol yield reached 38 g·L-1 with 99.6% conversion and 1.58 g·L-1·h-1 space-time yield in 24 h through further optimization of the conditions. Our study demonstrates that protein engineering of the rate-limiting enzyme in the tyrosol synthesis cascade provides an industrial-scale platform for the biocatalytic production of tyrosol. KEY POINTS: ⢠Protein engineering of CtPDC based on allosteric regulation improved the catalytic efficiency of decarboxylation. ⢠The application of the optimum mutant of CtPDC removed the rate-limiting bottleneck in the cascade. ⢠The final titer of tyrosol reached 38 g·L-1 in 24 h in 3 L bioreactor.
Assuntos
Álcool Feniletílico , Piruvato Descarboxilase , Piruvato Descarboxilase/genética , Piruvato Descarboxilase/metabolismo , Engenharia de Proteínas , Álcool Feniletílico/metabolismoRESUMO
The use of un-utilized feedstock and seawater for material and/or energy production using marine microbial catalysts is one potential option toward contributing to the development of a more sustainable society. Ethanol production from alginate, which is an oxidized polysaccharide present in brown seaweed, is extremely difficult due to the imbalance of reducing power in the microbial cells. Production of ethanol by such means has so far been unsuccessful using marine microbial biocatalysts. To produce ethanol from alginate, an alternative pathway consisting of a pyruvate decarboxylase gene (pdc) and an alcohol dehydrogenase II gene (adhII) derived from Zymomonas mobilis strain ZM4 was implemented into a metabolically engineered bacterium, Vibrio halioticoli, which is a representative marine alginate decomposer. No ethanol from alginate was produced in the wild-type V. halioticoli; however, the engineered V. halioticoli harboring the pdc and adhII operon (Pet operon), designated to the V. halioticoli (Pet), was able to produce 880 mg/L ethanol in maximum from 1.5% alginate for 72 h. The Pet operon also worked on the other marine alginolytic vibrios for ethanol production from alginate. This is the first case of ethanol production from alginate using marine bacterial biocatalysts under seawater-based media.
Assuntos
Alginatos , Vibrio , Humanos , Biomassa , Etanol/metabolismo , Fermentação , Polissacarídeos , Piruvato Descarboxilase/genética , Piruvato Descarboxilase/metabolismo , Vibrio/genética , Vibrio/metabolismoRESUMO
A cell population characterized by the release of glucose repression and known as [GAR+] emerges spontaneously in the yeast Saccharomyces cerevisiae. This study revealed that the [GAR+] variants exhibit retarded alcoholic fermentation when glucose is the sole carbon source. To identify the key to the altered glucose response, the gene expression profile of [GAR+] cells was examined. Based on RNA-seq data, the [GAR+] status was linked to impaired function of the Cyc8p-Tup1p complex. Loss of Cyc8p led to a decrease in the initial rate of alcoholic fermentation under glucose-rich conditions via the inactivation of pyruvate decarboxylase, an enzyme unique to alcoholic fermentation. These results suggest that Cyc8p can become inactive to attenuate alcoholic fermentation. These findings may contribute to the elucidation of the mechanism of non-genetic heterogeneity in yeast alcoholic fermentation.
Assuntos
Carbono , Saccharomyces cerevisiae , Fermentação , Glucose , Piruvato Descarboxilase/genética , Saccharomyces cerevisiae/genéticaRESUMO
Saccharomyces cerevisiae has been widely used in bioproduction. To produce a target product other than ethanol, ethanol production must be decreased to enhance target production. An ethanol non-producing yeast strain was previously constructed by knocking out pyruvate decarboxylase (PDC) genes in the ethanol synthetic pathway. However, glucose uptake by the ethanol-non-producing yeast strain was significantly decreased. In this study, dead Cas9 (dCas9) was used to reduce ethanol synthesis during 2,3-butanediol production without reduction of glucose. The binding site of guide RNA used to effectively suppress PDC1 promoter-driven red fluorescent protein expression by dCas9 was identified and applied to control PDC1 expression. The production of 2,3-butanediol rather than ethanol was improved in repetitive test tube culture. Additionally, ethanol production was decreased and 2,3-butanediol production was increased in the strain expressing dCas9 targeting the PDC1 promoter in the third round of cultivation, compared with the control strain.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Butileno Glicóis/metabolismo , Expressão Gênica , Piruvato Descarboxilase/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Saccharomyces cerevisiae has good reproductive ability in both haploid and diploid forms, a pyruvate decarboxylase plays an important role in S. cerevisiae cell metabolism. In this study, pdc1 and pdc5 double knockout strains of S. cerevisiae H14-02 (MATa type) and S. cerevisiae H5-02 (MATα type) were obtained by the Cre/loxP technique. The effects of the deletion of pdc1 and pdc5 on the metabolites of the two haploid S. cerevisiae strains were consistent. In S. cerevisiae H14-02, the ethanol conversion decreased by 30.19%, the conversion of glycerol increased by 40.005%, the concentration of acetic acid decreased by 43.54%, the concentration of acetoin increased by 12.79 times, and the activity of pyruvate decarboxylase decreased by 40.91% compared to those in the original H14 strain. The original S. cerevisiae haploid strain H14 produced a small amount of acetoin but produced very little 2,3-butanediol. However, S. cerevisiae H14-02 produced 1.420 ± 0.063 g/L 2,3-BD. This study not only provides strain selection for obtaining haploid strains with a high yield of 2,3-BD but also lays a foundation for haploid S. cerevisiae to be used as a new tool for genetic research and breeding programs.
Assuntos
Carboxiliases/genética , Piruvato Descarboxilase/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Acetoína/metabolismo , Butileno Glicóis/metabolismo , Carboxiliases/metabolismo , Etanol/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Técnicas de Inativação de Genes , Glicerol/metabolismo , Haploidia , Piruvato Descarboxilase/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Bioethanol produced by fermentative microorganisms is regarded as an alternative to fossil fuel. Bioethanol to be used as a viable energy source must be produced cost-effectively by removing expense-intensive steps such as the enzymatic hydrolysis of substrate. Consolidated bioprocessing (CBP) is believed to be a practical solution combining saccharification and fermentation in a single step catalyzed by a microorganism. Bacillus subtills with innate ability to grow on a diversity of carbohydrates seems promising for affordable CBP bioethanol production using renewable plant biomass and wastes. In this study, the genes encoding alcohol dehydrogenase from Z. mobilis (adhZ) and S. cerevisiae (adhS) were each used with Z. mobilis pyruvate decarboxylase gene (pdcZ) to create ethanologenic operons in a lactate-deficient (Δldh) B. subtilis resulting in NZ and NZS strains, respectively. The S. cerevisiae adhS caused significantly more ethanol production by NZS and therefore was used to make two other operons including one with double copies of both pdcZ and adhS and the other with a single pdcZ but double adhS genes expressed in N(ZS)2 and NZS2 strains, respectively. In addition, two fusion genes were constructed with pdcZ and adhS in alternate orientations and used for ethanol production by the harboring strains namely NZ:S and NS:Z, respectively. While the increase of gene dosage was not associated with elevated carbon flow for ethanol production, the fusion gene adhS:pdcZ resulted in a more than two times increase of productivity by strain NS:Z as compared with NZS during 48 h fermentation. The CBP ethanol production by NZS and NS:Z using potatoes resulted in 16.3 g/L and 21.5 g/L ethanol during 96 h fermentation, respectively. For the first time in this study, B. subtilis was successfully used for CBP ethanol production with S. cerevisiae alcohol dehydrogenase. The results of the study provide insights on the potentials of B. subtilis for affordable bioethanol production from inexpensive plant biomass and wastes. However, the potentials need to be improved by metabolic and process engineering for higher yields of ethanol production and plant biomass utilization.
Assuntos
Álcool Desidrogenase/genética , Bacillus subtilis/genética , Etanol/metabolismo , Engenharia Metabólica , Piruvato Descarboxilase/genética , Bacillus subtilis/metabolismo , Biomassa , Etanol/química , Fermentação/genética , Hidrólise , Ácido Láctico/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Zymomonas/enzimologia , Zymomonas/genéticaRESUMO
BACKGROUND: The current shift from a fossil-resource based economy to a more sustainable, bio-based economy requires development of alternative production routes based on utilization of biomass for the many chemicals that are currently produced from petroleum. Muconic acid is an attractive platform chemical for the bio-based economy because it can be converted in chemicals with wide industrial applicability, such as adipic and terephthalic acid, and because its two double bonds offer great versatility for chemical modification. RESULTS: We have constructed a yeast cell factory converting glucose and xylose into muconic acid without formation of ethanol. We consecutively eliminated feedback inhibition in the shikimate pathway, inserted the heterologous pathway for muconic acid biosynthesis from 3-dehydroshikimate (DHS) by co-expression of DHS dehydratase from P. anserina, protocatechuic acid (PCA) decarboxylase (PCAD) from K. pneumoniae and oxygen-consuming catechol 1,2-dioxygenase (CDO) from C. albicans, eliminated ethanol production by deletion of the three PDC genes and minimized PCA production by enhancing PCAD overexpression and production of its co-factor. The yeast pitching rate was increased to lower high biomass formation caused by the compulsory aerobic conditions. Maximal titers of 4 g/L, 4.5 g/L and 3.8 g/L muconic acid were reached with glucose, xylose, and a mixture, respectively. The use of an elevated initial sugar level, resulting in muconic acid titers above 2.5 g/L, caused stuck fermentations with incomplete utilization of the sugar. Application of polypropylene glycol 4000 (PPG) as solvent for in situ product removal during the fermentation shows that this is not due to toxicity by the muconic acid produced. CONCLUSIONS: This work has developed an industrial yeast strain able to produce muconic acid from glucose and also with great efficiency from xylose, without any ethanol production, minimal production of PCA and reaching the highest titers in batch fermentation reported up to now. Utilization of higher sugar levels remained conspicuously incomplete. Since this was not due to product inhibition by muconic acid or to loss of viability, an unknown, possibly metabolic bottleneck apparently arises during muconic acid fermentation with high sugar levels and blocks further sugar utilization.
Assuntos
Carboxiliases/metabolismo , Catecol 1,2-Dioxigenase/metabolismo , Hidroliases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ácido Sórbico/análogos & derivados , Xilose/metabolismo , Carboxiliases/genética , Catecol 1,2-Dioxigenase/genética , Clonagem Molecular , DNA Fúngico , Fermentação , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Hidroliases/genética , Hidroxibenzoatos/metabolismo , Microbiologia Industrial , Engenharia Metabólica/métodos , Redes e Vias Metabólicas , Piruvato Descarboxilase/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ácido Chiquímico/análogos & derivados , Ácido Chiquímico/metabolismo , Ácido Sórbico/isolamento & purificação , Ácido Sórbico/metabolismoRESUMO
OBJECTIVE: Zymomonas mobilis is an alpha-proteobacterium with a rapid ethanologenic pathway, involving Entner-Doudoroff (E-D) glycolysis, pyruvate decarboxylase (Pdc) and two alcohol dehydrogenase (ADH) isoenzymes. Pyruvate is the end-product of the E-D pathway and the substrate for Pdc. Construction and study of Pdc-deficient strains is of key importance for Z. mobilis metabolic engineering, because the pyruvate node represents the central branching point, most novel pathways divert from ethanol synthesis. In the present work, we examined the aerobic metabolism of a strain with partly inactivated Pdc. RESULTS: Relative to its parent strain the mutant produced more pyruvate. Yet, it also yielded more acetaldehyde, the product of the Pdc reaction and the substrate for ADH, although the bulk ADH activity was similar in both strains, while the Pdc activity in the mutant was reduced by half. Simulations with the kinetic model of Z. mobilis E-D pathway indicated that, for the observed acetaldehyde to ethanol production ratio in the mutant, the ratio between its respiratory NADH oxidase and ADH activities should be significantly higher, than the measured values. Implications of this finding for the directionality of the ADH isoenzyme operation in vivo and interactions between ADH and Pdc are discussed.
Assuntos
Zymomonas , Álcool Desidrogenase/genética , Engenharia Metabólica , Piruvato Descarboxilase/genética , Respiração , Zymomonas/genéticaRESUMO
Sugar supply is a key component of hypoxia tolerance and acclimation in plants. However, a striking gap remains in our understanding of mechanisms governing sugar impacts on low-oxygen responses. Here, we used a maize (Zea mays) root-tip system for precise control of sugar and oxygen levels. We compared responses to oxygen (21 and 0.2%) in the presence of abundant versus limited glucose supplies (2.0 and 0.2%). Low-oxygen reconfigured the transcriptome with glucose deprivation enhancing the speed and magnitude of gene induction for core anaerobic proteins (ANPs). Sugar supply also altered profiles of hypoxia-responsive genes carrying G4 motifs (sources of regulatory quadruplex structures), revealing a fast, sugar-independent class followed more slowly by feast-or-famine-regulated G4 genes. Metabolite analysis showed that endogenous sugar levels were maintained by exogenous glucose under aerobic conditions and demonstrated a prominent capacity for sucrose re-synthesis that was undetectable under hypoxia. Glucose abundance had distinctive impacts on co-expression networks associated with ANPs, altering network partners and aiding persistence of interacting networks under prolonged hypoxia. Among the ANP networks, two highly interconnected clusters of genes formed around Pyruvate decarboxylase 3 and Glyceraldehyde-3-phosphate dehydrogenase 4. Genes in these clusters shared a small set of cis-regulatory elements, two of which typified glucose induction. Collective results demonstrate specific, previously unrecognized roles of sugars in low-oxygen responses, extending from accelerated onset of initial adaptive phases by starvation stress to maintenance and modulation of co-expression relationships by carbohydrate availability.
Assuntos
Oxigênio/metabolismo , Proteínas de Plantas/genética , Açúcares/metabolismo , Transcriptoma , Zea mays/metabolismo , Anaerobiose , Glucose/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/genética , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Piruvato Descarboxilase/genética , Estresse Fisiológico , Zea mays/genéticaRESUMO
Alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) are key to the establishment of the fermentative metabolism in plants during oxygen shortage. Most of the evidence that both ADH and PDC are required for plant tolerance to hypoxia comes from experiments performed by limiting oxygen in the environment, such as by exposing plants to gaseous hypoxia or to waterlogging or submergence. However, recent experiments have shown that hypoxic niches might exist in plants grown in aerobic conditions. Here, we investigated the importance of ADH and PDC for plant growth and development under aerobic conditions, long-term waterlogging and short-term submergence. Data were collected after optimizing the software associated with a commercially-available phenotyping instrument, to circumvent problems in separation of plants and background pixels based on colour features, which is not applicable for low-oxygen stressed plants due to the low colour contrast of leaves with the brownish soil. The results showed that the growth penalty associated with the lack of functional ADH1 or both PDC1 and PDC2 is greater under aerobic conditions than in hypoxia, highlighting the importance of fermentative metabolism in plants grown under normal, aerobic conditions.
Assuntos
Álcool Desidrogenase/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Fenótipo , Piruvato Descarboxilase/metabolismo , Álcool Desidrogenase/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Hipóxia/genética , Hipóxia/metabolismo , Desenvolvimento Vegetal/fisiologia , Piruvato Descarboxilase/genéticaRESUMO
Maple syrup urine disease (MSUD) is a rare autosomal recessive inherited disorder due to defects in the branched-chain α-ketoacid dehydrogenase complex (BCKDC). MSUD varies in severity and its clinical spectrum is quite broad, ranging from mild to severe phenotypes. Thirty-three MSUD patients were recruited into this study for molecular genetic variant profiling and genotype-phenotype correlation. Except for one patient, all other patients presented with the classic neonatal form of the disease. Seventeen different variants were detected where nine were novel. The detected variants spanned across the entire BCKDHA, BCKDHB and DBT genes. All variants were in homozygous forms. The commonest alterations were nonsense and frameshift variants, followed by missense variants. For the prediction of variant's pathogenicity, we used molecular modeling and several in silico tools including SIFT, Polyphen2, Condel, and Provean. In addition, six other tools were used for the prediction of the conservation of the variants' sites including Eigen-PC, GERP++, SiPhy, PhastCons vertebrates and primates, and PhyloP100 rank scores. Herein, we presented a comprehensive characterization of a large cohort of patients with MSUD. The clinical severity of the variants' phenotypes was well correlated with the genotypes. The study underscores the importance of the use of in silico analysis of MSUD genotypes for the prediction of the clinical outcomes in patients with MSUD.
Assuntos
Análise Mutacional de DNA , Estudos de Associação Genética , Doença da Urina de Xarope de Bordo/diagnóstico , Doença da Urina de Xarope de Bordo/genética , Piruvato Descarboxilase/genética , Alelos , Criança , Pré-Escolar , Feminino , Mutação da Fase de Leitura , Homozigoto , Humanos , Lactente , Recém-Nascido , Isoleucina/genética , Leucina/genética , Masculino , Doença da Urina de Xarope de Bordo/terapia , Biologia Molecular , Mutação de Sentido Incorreto , Readmissão do Paciente , Fenótipo , Espectrometria de Massas em TandemRESUMO
Benzenoids are compounds associated with floral and fruity flavours in flowers, fruits and leaves and present a role in hormonal signalling in plants. These molecules are produced by the phenyl ammonia lyase pathway. However, some yeasts can also synthesize them from aromatic amino acids using an alternative pathway that remains unknown. Hanseniaspora vineae can produce benzenoids at levels up to two orders of magnitude higher than Saccharomyces species, so it is a model microorganism for studying benzenoid biosynthesis pathways in yeast. According to their genomes, several enzymes have been proposed to be involved in a mandelate pathway similar to that described for some prokaryotic cells. Among them, the ARO10 gene product could present benzoylformate decarboxylase activity. This enzyme catalyses the decarboxylation of benzoylformate into benzaldehyde at the end of the mandelate pathway in benzyl alcohol formation. Two homologous genes of ARO10 were found in the two sequenced H. vineae strains. In this study, nine other H. vineae strains were analysed to detect the presence and per cent homology of ARO10 sequences by PCR using specific primers designed for this species. Also, the copy number of the genes was estimated by quantitative PCR. To verify the relation of ARO10 with the production of benzyl alcohol during fermentation, a deletion mutant in the ARO10 gene of Saccharomyces cerevisiae was used. The two HvARO10 paralogues were analysed and compared with other α-ketoacid decarboxylases at the sequence and structural level.
Assuntos
Derivados de Benzeno/metabolismo , Vias Biossintéticas/genética , Hanseniaspora/genética , Piruvato Descarboxilase/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcriptoma , Benzaldeídos/metabolismo , Álcool Benzílico/metabolismo , Fermentação , Hanseniaspora/metabolismoRESUMO
Fatty acid ethyl esters (FAEEs) are fatty acid-derived molecules and serve as an important form of biodiesel. The oleaginous yeast Yarrowia lipolytica is considered an ideal host platform for the production of fatty acid-derived products due to its excellent lipid accumulation capacity. In this proof-of-principle study, several metabolic engineering strategies were applied for the overproduction of FAEE biodiesel in Y. lipolytica. Here, chromosome-based co-overexpression of two heterologous genes, namely, PDC1 (encoding pyruvate decarboxylase) and ADH1 (encoding alcohol dehydrogenase) from Saccharomyces cerevisiae, and the endogenous GAPDH (encoding glyceraldehyde-3-phosphate dehydrogenase) gene of Y. lipolytica resulted in successful biosynthesis of ethanol at 70.8 mg/L in Y. lipolytica. The engineered Y. lipolytica strain expressing the ethanol synthetic pathway together with a heterologous wax ester synthase (MhWS) exhibited the highest FAEE titer of 360.8 mg/L, which is 3.8-fold higher than that of the control strain when 2% exogenous ethanol was added to the culture medium of Y. lipolytica. Furthermore, a synthetic microbial consortium comprising an engineered Y. lipolytica strain that heterologously expressed MhWS and a S. cerevisiae strain that could provide ethanol as a substrate for the production of the final product in the final engineered Y. lipolytica strain was created in this study. Finally, this synthetic consortium produced FAEE biodiesel at a titer of 4.8 mg/L under the optimum coculture conditions.
Assuntos
Biocombustíveis/microbiologia , Ácidos Graxos/biossíntese , Engenharia Metabólica/métodos , Yarrowia/genética , Yarrowia/metabolismo , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , DNA Fúngico/genética , Escherichia coli/genética , Ésteres/química , Etanol/metabolismo , Ácidos Graxos/química , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Estudo de Prova de Conceito , Piruvato Descarboxilase/genética , Piruvato Descarboxilase/metabolismo , Energia Renovável , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Pongamia pinnata (also called Millettia pinnata), a non-edible oil yielding tree, is well known for its multipurpose benefits and acts as a potential source for medicine and biodiesel preparation. Due to increase in demand for cultivation, understanding of genetic diversity is an important parameter for further breeding and cultivation programme. Transposable elements (TEs) are a major component of plant genome but still, their evolutionary significance in Pongamia remains unexplored. In view to understand the role of TEs in genome diversity, Pongamia unigenes were screened for the presence of TE cassettes. Our analysis showed the presence of all categories of TE cassettes in unigenes with major contribution of long terminal repeat-retrotransposons towards unigene diversity. Interestingly, the insertion of some TEs was also observed in both organellar genomes. The study of insertion of TEs in coding sequence is of great interest as they may be responsible for protein diversity thereby influencing the phenotype. The present investigation confirms the exaptation phenomenon in pyruvate decarboxylase (PDC) gene where the entire exon sequence was derived from Ty3-gypsy like retrotransposon. The study of PDC protein revealed the translation of gypsy element into protein. Furthermore, the phylogenetic study confirmed the diversity in PDC gene due to insertion of the gypsy element, where the PDC genes with and without gypsy insertion were clustered separately.