RESUMO
Glycolic acid is an important chemical product widely used in various fields, including cosmetics, detergents, textiles, and more. Currently, microbial production of glycolic acid has disadvantages such as poor genetic stability, low yield, and high cost. Additionally, whole-cell catalytic production of glycolic acid typically requires the addition of relatively expensive sorbitol as a carbon source, which limits its industrial production. To develop an industrially applicable method for glycolic acid production, this study used ethylene glycol as a substrate to screen the glycolic acid-producing strains through whole-cell catalysis, obtaining a Rhodotorula sp. capable of producing glycolic acid. The strain was then subjected to UV mutagenesis and high throughput screening, and the positive mutant strain RMGly-20 was obtained. After optimization in shake flasks, the glycolic acid titer of RMGly-20 reached 17.8 g/L, a 10.1-fold increase compared to the original strain. Using glucose as the carbon source and employing a fed-batch culture in a 5 L fermenter, strain RMGly-20 produced 61.1 g/L of the glycolic acid. This achievement marks the preliminary breeding of a genetically stable glycolic acid-producing strain using a cheap carbon source, providing a new host for the biosynthesis of glycolic acid and promoting further progress toward industrial production.
Assuntos
Fermentação , Glicolatos , Rhodotorula , Glicolatos/metabolismo , Rhodotorula/metabolismo , Rhodotorula/genética , Microbiologia Industrial/métodos , Etilenoglicol/metabolismo , MutagêneseRESUMO
Plastics have become an indispensable material in many fields of human activities, with production increasing every year; however, most of the plastic waste is still incinerated or landfilled, and only 10% of the new plastic is recycled even once. Among all plastics, polyethylene terephthalate (PET) is the most produced polyester worldwide; ethylene glycol (EG) is one of the two monomers released by the biorecycling of PET. While most research focuses on bacterial EG metabolism, this work reports the ability of Saccharomyces cerevisiae and nine other common laboratory yeast species not only to consume EG, but also to produce glycolic acid (GA) as the main by-product. A two-step bioconversion of EG to GA by S. cerevisiae was optimized by a design of experiment approach, obtaining 4.51 ± 0.12 g l-1 of GA with a conversion of 94.25 ± 1.74% from 6.21 ± 0.04 g l-1 EG. To improve the titer, screening of yeast biodiversity identified Scheffersomyces stipitis as the best GA producer, obtaining 23.79 ± 1.19 g l-1 of GA (yield 76.68%) in bioreactor fermentation, with a single-step bioprocess. Our findings contribute in laying the ground for EG upcycling strategies with yeasts.
Assuntos
Biodiversidade , Etilenoglicol , Fermentação , Glicolatos , Glicolatos/metabolismo , Etilenoglicol/metabolismo , Reatores Biológicos/microbiologia , Leveduras/metabolismo , Leveduras/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
Ethylene glycol (EG) is an industrially important two-carbon diol used as a solvent, antifreeze agent, and building block of polymers such as poly(ethylene terephthalate) (PET). Recently, the use of EG as a starting material for the production of bio-fuels or bio-chemicals is gaining attention as a sustainable process since EG can be derived from materials not competing with human food stocks including CO2, syngas, lignocellulolytic biomass, and PET waste. In order to design and construct microbial process for the conversion of EG to value-added chemicals, microbes capable of catabolizing EG such as Escherichia coli, Pseudomonas putida, Rhodococcus jostii, Ideonella sakaiensis, Paracoccus denitrificans, and Acetobacterium woodii are candidates of chassis for the construction of synthetic pathways. In this mini-review, we describe EG catabolic pathways and catabolic enzymes in these microbes, and further review recent advances in microbial conversion of EG to value-added chemicals by means of metabolic engineering. KEY POINTS: ⢠Ethylene glycol is a potential next-generation feedstock for sustainable industry. ⢠Microbial conversion of ethylene glycol to value-added chemicals is gaining attention. ⢠Ethylene glycol-utilizing microbes are useful as chassis for synthetic pathways.
Assuntos
Etilenoglicol , Engenharia Metabólica , Etilenoglicol/metabolismo , Redes e Vias Metabólicas , Bactérias/metabolismo , Pseudomonas putida/metabolismo , Biocombustíveis , Escherichia coli/metabolismo , Escherichia coli/genéticaRESUMO
Ethylene glycol (EG) is a widely used industrial chemical with manifold applications and also generated in the degradation of plastics such as polyethylene terephthalate. Rhodococcus jostii RHA1 (RHA1), a potential biocatalytic chassis, grows on EG. Transcriptomic analyses revealed four clusters of genes potentially involved in EG catabolism: the mad locus, predicted to encode mycofactocin-dependent alcohol degradation, including the catabolism of EG to glycolate; two GCL clusters, predicted to encode glycolate and glyoxylate catabolism; and the mft genes, predicted to specify mycofactocin biosynthesis. Bioinformatic analyses further revealed that the mad and mft genes are widely distributed in mycolic acid-producing bacteria such as RHA1. Neither ΔmadA nor ΔmftC RHA1 mutant strains grew on EG but grew on acetate. In resting cell assays, the ΔmadA mutant depleted glycolaldehyde but not EG from culture media. These results indicate that madA encodes a mycofactocin-dependent alcohol dehydrogenase that initiates EG catabolism. In contrast to some mycobacterial strains, the mad genes did not appear to enable RHA1 to grow on methanol as sole substrate. Finally, a strain of RHA1 adapted to grow ~3× faster on EG contained an overexpressed gene, aldA2, predicted to encode an aldehyde dehydrogenase. When incubated with EG, this strain accumulated lower concentrations of glycolaldehyde than RHA1. Moreover, ecotopically expressed aldA2 increased RHA1's tolerance for EG further suggesting that glycolaldehyde accumulation limits growth of RHA1 on EG. Overall, this study provides insights into the bacterial catabolism of small alcohols and aldehydes and facilitates the engineering of Rhodococcus for the upgrading of plastic waste streams.IMPORTANCEEthylene glycol (EG), a two-carbon (C2) alcohol, is produced in high volumes for use in a wide variety of applications. There is burgeoning interest in understanding and engineering the bacterial catabolism of EG, in part to establish circular economic routes for its use. This study identifies an EG catabolic pathway in Rhodococcus, a genus of bacteria well suited for biocatalysis. This pathway is responsible for the catabolism of methanol, a C1 feedstock, in related bacteria. Finally, we describe strategies to increase the rate of degradation of EG by increasing the transformation of glycolaldehyde, a toxic metabolic intermediate. This work advances the development of biocatalytic strategies to transform C2 feedstocks.
Assuntos
Proteínas de Bactérias , Etilenoglicol , Rhodococcus , Rhodococcus/metabolismo , Rhodococcus/genética , Etilenoglicol/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Glicolatos/metabolismo , Glioxilatos/metabolismo , Álcool Desidrogenase/metabolismo , Álcool Desidrogenase/genética , PeptídeosRESUMO
Single-carbon (C1) biorefinery plays a key role in the consumption of global greenhouse gases and a circular carbon economy. Thereby, we have focused on the valorization of C1 compounds (e.g. methanol, formaldehyde, and formate) into multicarbon products, including bioplastic monomers, glycolate, and ethylene glycol. For instance, methanol, derived from the oxidation of CH4, can be converted into glycolate, ethylene glycol, or erythrulose via formaldehyde and glycolaldehyde, employing C1 and/or C2 carboligases as essential enzymes. Escherichia coli was engineered to convert formate, produced from CO via CO2 or from CO2 directly, into glycolate. Recent progress in the design of biotransformation pathways, enzyme discovery, and engineering, as well as whole-cell biocatalyst engineering for C1 biorefinery, was addressed in this review.
Assuntos
Carbono , Metanol , Metanol/metabolismo , Carbono/metabolismo , Dióxido de Carbono/metabolismo , Etilenoglicol/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Formiatos/metabolismo , Formaldeído/metabolismo , Glicolatos/metabolismoRESUMO
Microbial fermentation processes are expected to play an important role in reducing dependence on fossil-based raw materials for the production of everyday chemicals. In order to meet the growing demand for biotechnological products in the future, alternative carbon sources that do not compete with human nutrition must be exploited. The chemical conversion of the industrially emitted greenhouse gas CO2 into microbially utilizable platform chemicals such as methanol represents a sustainable strategy for the utilization of an abundant carbon source and has attracted enormous scientific interest in recent years. A relatively new approach is the microbial synthesis of products from the C2-compound ethylene glycol, which can also be synthesized from CO2 and non-edible biomass and, in addition, can be recovered from plastic waste. Here we summarize the main chemical routes for the synthesis of methanol and ethylene glycol from sustainable resources and give an overview of recent metabolic engineering work for establishing natural and synthetic microbial assimilation pathways. The different metabolic routes for C1 and C2 alcohol-dependent bioconversions were compared in terms of their theoretical maximum yields and their oxygen requirements for a wide range of value-added products. Assessment of the process engineering challenges for methanol and ethylene glycol-based fermentations underscores the theoretical advantages of new synthetic metabolic routes and advocates greater consideration of ethylene glycol, a C2 substrate that has received comparatively little attention to date.
Assuntos
Dióxido de Carbono , Metanol , Humanos , Dióxido de Carbono/metabolismo , Etilenoglicol/metabolismo , Biotecnologia , Carbono/metabolismo , Engenharia MetabólicaRESUMO
Values of cell membranes permeability coefficients for water and molecules of cryoprotective agents (CPAs) are the necessary characteristics for developing physical-mathematical models describing mass transfer processes through cell membranes in order to predict optimal cell cooling rates. We carried out a comparative analysis of the permeability coefficients of mouse oocyte membranes for molecules of water, ethylene glycol (EG), propane-1,2-diol (1,2-PD) and dimethyl sulfoxide (Me2SO), determined by applying the classical Kedem-Katchalsky model, which considers only the penetration of non-electrolyte molecules (water and CPA) through the membrane, and the model developed by us, which takes into account the transmembrane transfer of ions and the associated changes in the transmembrane electric potential. We shown that calculations based on the developed modified model provide lower values of the permeability coefficients of the oocyte membrane for water and CPA molecules. What is important that the obtained by our modified model permeability coefficients for water molecules do not depend on the type of cryoprotectant, while the application of the classical model both in our studies and works of other authors always gave different values of these coefficients in solutions with different cryoprotectants. Our modified model also makes it possible to determine the dynamics of the transmembrane electric potential of the cell under the conditions of transmembrane mass transfer and the duration of the membrane being influenced by the changes in electric potential, that is a parameter that can directly affect the viability of cells.
Assuntos
Criopreservação , Oócitos , Animais , Camundongos , Permeabilidade da Membrana Celular , Criopreservação/métodos , Crioprotetores/farmacologia , Crioprotetores/metabolismo , Dimetil Sulfóxido/farmacologia , Dimetil Sulfóxido/metabolismo , Etilenoglicol/farmacologia , Etilenoglicol/metabolismo , Oócitos/metabolismo , Permeabilidade , Água/metabolismo , FemininoRESUMO
Microbial overproduction of aromatic chemicals has gained considerable industrial interest and various metabolic engineering approaches have been employed in recent years to address the associated challenges. So far, most studies have used sugars (mostly glucose) or glycerol as the primary carbon source. In this study, we used ethylene glycol (EG) as the main carbon substrate. EG could be obtained from the degradation of plastic and cellulosic wastes. As a proof of concept, Escherichia coli was engineered to transform EG into L-tyrosine, a valuable aromatic amino acid. Under the best fermentation condition, the strain produced 2 g/L L-tyrosine from 10 g/L EG, outperforming glucose (the most common sugar feedstock) in the same experimental conditions. To prove the concept that EG can be converted into different aromatic chemicals, E. coli was further engineered with a similar approach to synthesize other valuable aromatic chemicals, L-phenylalanine and p-coumaric acid. Finally, waste polyethylene terephthalate (PET) bottles were degraded using acid hydrolysis and the resulting monomer EG was transformed into L-tyrosine using the engineered E. coli, yielding a comparable titer to that obtained using commercial EG. The strains developed in this study should be valuable to the community for producing valuable aromatics from EG.
Assuntos
Escherichia coli , Etilenoglicol , Escherichia coli/genética , Escherichia coli/metabolismo , Etilenoglicol/metabolismo , Engenharia Metabólica/métodos , Glucose/metabolismo , Tirosina/genética , Tirosina/metabolismo , Carbono/metabolismo , FermentaçãoRESUMO
Calcium oxalate (CaOx) nephrolithiasis is a prevalent disorder linked to metabolism. Examining metabolic alterations could potentially give an initial understanding of the origins of CaOx nephrolithiasis. This study aims to determine gut metabolic biomarkers differentiating CaOx nephrolithiasis utilizing untargeted and targeted metabolomics. CaOx nephrolithiasis model rats were built by 1% ethylene glycol administration. Histologic staining and renal function measurement revealed the presence of crystals in the lumen of the renal tubules, the renal injury and interstitial fibrosis in CaOx rats, demonstrating that the models of CaOx were established successfully. Hematoxylin & eosin (H&E) staining showed that CaOx group had inflammation and damage in the ileal tissue. Immunofluorescence and PCR results displayed that the tight junction proteins, ZO-1 and Occludin levels were decreased in the ileal tissues of the CaOx group. The untargeted metabolomic analysis revealed that 269 gut metabolites were differentially expressed between the CaOx group and the control group. Meanwhile, bile secretion, the main metabolic pathway in CaOx nephrolithiasis, was identified. Following, five significant bile acid metabolites were selected utilizing the targeted bile acid metabolomics, including Hyodeoxycholic acid (HDCA), Glycohyodeoxycholic acid (GHDCA), Nor-Deoxycholic Acid, omega-muricholic acid, and Taurolithocholic acid. Among these metabolites, HDCA and GHDCA presented the highest predictive accuracy with AUC = 1 to distinguish the CaOx group from the control group. As a result of network pharmacology, target genes of HDCA and GHDCA in CaOx nephrolithiasis were enriched in oxidative stress and apoptosis pathways. Conclusively, our study provides insight into bile acids metabolic changes related to CaOx nephrolithiasis. Although alterations in biochemical pathways indicate a complex pathology in CaOx rats, bile acid changes may serve as biomarkers of CaOx nephrolithiasis.
Assuntos
Oxalato de Cálcio , Cálculos Renais , Ratos , Animais , Oxalato de Cálcio/metabolismo , Etilenoglicol/toxicidade , Etilenoglicol/metabolismo , Ácidos e Sais Biliares/metabolismo , Cálculos Renais/metabolismo , Rim/metabolismo , MetabolômicaRESUMO
Ethylene glycol is an attractive two-carbon alcohol substrate for biochemical product synthesis as it can be derived from CO2 or syngas at no sacrifice to human food stocks. Here, we disclose a five-step synthetic metabolic pathway enabling the carbon-conserving biosynthesis of the versatile platform molecule 2,4-dihydroxybutyric acid (DHB) from this compound. The linear pathway chains ethylene glycol dehydrogenase, D-threose aldolase, D-threose dehydrogenase, D-threono-1,4-lactonase, D-threonate dehydratase and 2-oxo-4-hydroxybutyrate reductase enzyme activities in succession. We screen candidate enzymes with D-threose dehydrogenase and D-threonate dehydratase activities on cognate substrates with conserved carbon-centre stereochemistry. Lastly, we show the functionality of the pathway by its expression in an Escherichia coli strain and production of 1 g L-1 and 0.8 g L-1 DHB from, respectively, glycolaldehyde or ethylene glycol.
Assuntos
Etilenoglicol , Engenharia Metabólica , Humanos , Etilenoglicol/metabolismo , Redes e Vias Metabólicas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Hidroliases/metabolismo , Oxirredutases/metabolismoRESUMO
An efficient cofactor regeneration system has been developed to provide a hydride source for the preparation of optically pure alcohols by carbonyl reductase-catalyzed asymmetric reduction. This system employed a novel glucose dehydrogenase (BcGDH90) from Bacillus cereus HBL-AI. The gene encoding BcGDH90 was found through the genome-wide functional annotation. Homology-built model study revealed that BcGDH90 was a homo-tetramer, and each subunit was composed of ßD-αE-αF-αG-ßG motif, which was responsible for substrate binding and tetramer formation. The gene of BcGDH90 was cloned and expressed in Escherichia coli. The recombinant BcGDH90 exhibited maximum activity of 45.3 U/mg at pH 9.0 and 40 °C. BcGDH90 showed high stability in a wide pH range of 4.0-10.0 and was stable after the incubation at 55 °C for 5 h. BcGDH90 was not a metal ion-dependent enzyme, but Zn2+ could seriously inhibit its activity. BcGDH90 displayed excellent tolerance to 90% of acetone, methanol, ethanol, n-propanol, and isopropanol. Furthermore, BcGDH90 was applied to regenerate NADPH for the asymmetric biosynthesis of (S)-(+)-1-phenyl-1,2-ethanediol ((S)-PED) from hydroxyacetophenone (2-HAP) with high concentration, which increased the final efficiency by 59.4%. These results suggest that BcGDH90 is potentially useful for coenzyme regeneration in the biological reduction.
Assuntos
Oxirredutases do Álcool , Glucose 1-Desidrogenase , Glucose 1-Desidrogenase/metabolismo , Oxirredutases do Álcool/metabolismo , Álcoois/metabolismo , Escherichia coli/metabolismo , Solventes/metabolismo , Etilenoglicol/metabolismoRESUMO
Biological recycling of PET waste has been extensively investigated recently to tackle plastic waste pollution, and ethylene glycol (EG) is one of the main building blocks recovered from this process. Wild-type Yarrowia lipolytica IMUFRJ 50682 can be a biocatalyst to biodepolymerize PET. Herein, we report its ability to perform oxidative biotransformation of EG into glycolic acid (GA): a higher value-added chemical with varied industrial applications. We found that this yeast tolerates high EG concentrations (up to 2 M) based on maximum non-inhibitory concentration (MNIC) tests. Whole-cell biotransformation assays using resting yeast cells showed GA production uncoupled to cell growth metabolism, and 13 C nuclear magnetic resonance (NMR) analysis confirmed GA production. Moreover, higher agitation speed (450 vs. 350 rpm) resulted in a 1.12-fold GA production improvement (from 352 to 429.5 mM) during Y. lipolytica cultivation in bioreactors after 72 h. GA was constantly accumulated in the medium, suggesting that this yeast may also share an incomplete oxidation pathway (i.e., it is not metabolized to carbon dioxide) as seen in acetic acid bacterial group. Additional assays using higher chain-length diols (1,3-propanediol, 1,4-butanediol, and 1,6-hexanediol) revealed that C4 and C6 diols were more cytotoxic, suggesting that they underwent different pathways in the cells. We found that this yeast consumed extensively all these diols, however, 13 C NMR analysis from supernatant identified solely the presence of 4-hydroxybutanoic acid from 1,4-butanediol, along with GA from EG oxidation. Findings reported herein reveal a potential route for PET upcycling to a higher value-added product.
Assuntos
Etilenoglicol , Yarrowia , Etilenoglicol/metabolismo , Yarrowia/metabolismo , Biotransformação , Etilenos/metabolismoRESUMO
Ethylene glycol (EG) is a promising next generation feedstock for bioprocesses. It is a key component of the ubiquitous plastic polyethylene terephthalate (PET) and other polyester fibers and plastics, used in antifreeze formulations, and can also be generated by electrochemical conversion of syngas, which makes EG a key compound in a circular bioeconomy. The majority of biotechnologically relevant bacteria assimilate EG via the glycerate pathway, a wasteful metabolic route that releases CO2 and requires reducing equivalents as well as ATP. In contrast, the recently characterized ß-hydroxyaspartate cycle (BHAC) provides a more efficient, carbon-conserving route for C2 assimilation. Here we aimed at overcoming the natural limitations of EG metabolism in the industrially relevant strain Pseudomonas putida KT2440 by replacing the native glycerate pathway with the BHAC. We first prototyped the core reaction sequence of the BHAC in Escherichia coli before establishing the complete four-enzyme BHAC in Pseudomonas putida. Directed evolution on EG resulted in an improved strain that exhibits 35% faster growth and 20% increased biomass yield compared to a recently reported P. putida strain that was evolved to grow on EG via the glycerate pathway. Genome sequencing and proteomics highlight plastic adaptations of the genetic and metabolic networks in response to the introduction of the BHAC into P. putida and identify key mutations for its further integration during evolution. Taken together, our study shows that the BHAC can be utilized as 'plug-and-play' module for the metabolic engineering of two important microbial platform organisms, paving the way for multiple applications for a more efficient and carbon-conserving upcycling of EG in the future.
Assuntos
Pseudomonas putida , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Plásticos/metabolismo , Etilenoglicol/metabolismo , Polietilenotereftalatos/metabolismo , Carbono/metabolismoRESUMO
Urolithiasis is a common urological disorder, which causes considerable morbidity in both genders at all age groups worldwide. Though treatment options such as diuretics and non-invasive techniques to disintegrate the deposits are available, but often they are found less effective in the clinics. In this work, we planned to investigate the ameliorative effects of daidzin against the ethylene glycol (EG)-induced urolithiasis in rats. The male albino rats were distributed into four groups (n = 6) as control (group I), urolithiasis induced by the administration of 0.75% EG (group II), urolithiasis induced rats treated with 50 mg/kg of daidzin (group III), and urolithiasis rats treated with standard drug 750 mg/kg of cystone (group IV). The urine volume, pH, and total protein in the urine were assessed. The activities of marker enzymes in both plasma and kidney tissues were analyzed using assay kits. The levels of kidney function markers such as calcium, oxalate, urea, creatinine, uric acid, magnesium, BUN, and phosphorous were estimated using assay kits. The status of antioxidants and inflammatory cytokines were also examined using kits. The renal tissues were examined by histopathological analysis. Our results revealed that the daidzin treatment effectively decreased the urine pH and protein level and increased the urine volume in the urolithiasis rats. Daidzin decreased the calcium, oxalate, uric acid, and urea, creatinine, and BUN levels and also improved the magnesium and phosphorus in the urolithiasis rats. The activities of AST, ALT, ALP, GGT, and LDH were effectively reduced by the daidzin in both serum and renal tissue. Daidzin also reduced the inflammatory marker and increased the antioxidant levels. Histopathology results also proved the therapeutic effects of daidzin. Together, our results displayed that daidzin is effective in the amelioration of EG-induced urolithiasis in rats.
Assuntos
Rim , Urolitíase , Feminino , Masculino , Ratos , Antioxidantes/metabolismo , Cálcio/metabolismo , Creatinina , Etilenoglicol/efeitos adversos , Etilenoglicol/metabolismo , Rim/metabolismo , Magnésio/metabolismo , Oxalatos/efeitos adversos , Oxalatos/metabolismo , Extratos Vegetais/farmacologia , Ureia , Ácido Úrico/metabolismo , Ácido Úrico/farmacologia , Urolitíase/induzido quimicamente , Urolitíase/tratamento farmacológico , Urolitíase/metabolismo , AnimaisRESUMO
Poly(ethylene terephthalate) (PET)-degrading bacterium Ideonella sakaiensis produces hydrolytic enzymes that convert PET, via mono(2-hydroxyethyl) terephthalate (MHET), into the monomeric compounds, terephthalic acid (TPA), and ethylene glycol (EG). Understanding PET metabolism is critical if this bacterium is to be engineered for bioremediation and biorecycling. TPA uptake and catabolism in I. sakaiensis have previously been studied, but EG metabolism remains largely unexplored despite its importance. First, we identified two alcohol dehydrogenases (IsPedE and IsPedH) and one aldehyde dehydrogenase (IsPedI) in I. sakaiensis as the homologs of EG metabolic enzymes in Pseudomonas putida KT2440. IsPedE and IsPedH exhibited EG dehydrogenase activities with Ca2+ and a rare earth element (REE) Pr3+, respectively. We further found an upregulated dehydrogenase gene when the bacterium was grown on EG, whose gene product (IsXoxF) displays a minor EG dehydrogenase activity with Pr3+. IsPedE displayed a similar level of activity toward various alcohols. In contrast, IsPedH was more active toward small alcohols, whereas IsXoxF was the opposite. Structural analysis with homology models revealed that IsXoxF had a larger catalytic pocket than IsPedE and IsPedH, which could accommodate relatively bulkier substrates. Pr3+ regulated the protein expression of IsPedE negatively; IsPedH and IsXoxF were positively regulated. Taken together, these results indicated that the combination of IsPedH and IsXoxF complements the function of IsPedE in the presence of REEs. IsPedI exhibited dehydrogenase activity toward various aldehydes with the highest activity toward glycolaldehyde. This study demonstrated a unique alcohol oxidation pathway of I. sakaiensis, which could be efficient in EG utilization. KEY POINTS: ⢠IsPedH and IsXoxF complement IsPedE function in the presence of REEs. ⢠IsPedI displayed the highest dehydrogenase activity toward glycolaldehyde. ⢠Unique alcohol oxidation pathway of I. sakaiensis identified for EG utilization.
Assuntos
Etilenoglicol , Polietilenotereftalatos , Polietilenotereftalatos/metabolismo , Etilenoglicol/metabolismo , Etilenos , Oxirredutases/genética , Hidrolases/metabolismoRESUMO
This study aimed to examine the effect of vitrification on the expression of genes that are crucial for porcine early embryo development; cathepsin B (CTSB), growth differentiation factor 9 (GDF9), caudal type homeobox 2 (CDX2), and OCT-4, which play an important role in the maintenance of embryonic cell pluripotency. Their gene expression was investigated in expanded blastocysts (day 6-7) derived from in vitro matured oocytes. The quantitative real-time PCR method was used to assess the amount of relative specific transcripts in 20 vitrified (treatment group) and 32 fresh non-vitrified (control group) blastocysts. Vitrification was performed using 7.5% dimethyl sulfoxide (DMSO) plus 7.5% ethylene glycol (EG), and in the final step, 15% DMSO plus 15% EG and a 0.5 M sucrose solution and cryotop as a vitrification device. The blastocysts were warmed in 1 M, 0.5 M, and 0.25 M sucrose solution and kept in a culture medium for six hours before their fixation and further qPCR analysis. A significant upregulation in the targeted genes CTSB (p<.006), GDF9 (p<.04), and CDX2 (p<.003) was observed in the vitrified embryos compared to the fresh control group. Interestingly, the OCT-4 mRNA expression level was not affected by vitrification and remained comparable to that of the fresh non-vitrified embryos. In summary, the results of this pilot study showed, that vitrification induced substantial alteration in the expression of CTSB, GDF9, and CDX2 genes but did not influence the expression of OCT-4 gene in porcine in vitro derived blastocysts. Our data on the expression of developmentally important genes in vitrified porcine blastocyst may facilitate: (1) future improvements in culture conditions and/or cryopreservation protocol and (2) understanding the mechanism(s) of cryoinjuries inducing compromised post-thaw embryo development followed by the poor pregnancy outcome after blastocyst transfer.
Assuntos
Dimetil Sulfóxido , Vitrificação , Animais , Blastocisto/fisiologia , Criopreservação/métodos , Dimetil Sulfóxido/metabolismo , Dimetil Sulfóxido/farmacologia , Etilenoglicol/metabolismo , Etilenoglicol/farmacologia , Feminino , Oócitos , Projetos Piloto , Gravidez , Sacarose/metabolismo , Sacarose/farmacologia , SuínosRESUMO
The platform chemical ethylene glycol (EG) is used to manufacture various commodity chemicals of industrial importance, but largely remains synthesized from fossil fuels. Although several novel metabolic pathways have been reported for its bioproduction in model organisms, none has been reported for gas-fermenting, non-model acetogenic chassis organisms. Here, we describe a novel, synthetic biochemical pathway to convert acetate into EG in the industrially important gas-fermenting acetogen,Clostridium autoethanogenum. We not only developed a computational workflow to design and analyze hundreds of novel biochemical pathways for EG production but also demonstrated a successful pathway construction in the chosen host. The EG production was achieved using a two-plasmid system to bypass unfeasible expression levels and potential toxic enzymatic interactions. Although only a yield of 0.029 g EG/g fructose was achieved and therefore requiring further strain engineering efforts to optimize the designed strain, this work demonstrates an important proof-of-concept approach to computationally design and experimentally implement fully synthetic metabolic pathways in a metabolically highly specific, non-model host organism.
Assuntos
Clostridium , Etilenoglicol , Clostridium/genética , Clostridium/metabolismo , Etilenoglicol/metabolismo , Engenharia Metabólica , Redes e Vias Metabólicas/genética , PlasmídeosRESUMO
Poly(ethylene terephthalate) (PET) is a commonly used synthetic plastic; however, its nonbiodegradability results in a large amount of waste accumulation that has a negative impact on the environment. Recently, a PET-degrading bacterium, Ideonella sakaiensis 201-F6 strain, was isolated, and the enzymes involved in PET digestion, PET hydrolase (PETase), and mono(2-hydroxyethyl) terephthalic acid (MHET) hydrolase (MHETase) were identified. Despite the great potentials of I. sakaiensis in bioremediation and biorecycling, approaches to studying this bacterium remain limited. In this study, to enable the functional analysis of PETase and MHETase genes in vivo, we have developed a gene disruption system in I. sakaiensis. The pT18mobsacB-based disruption vector harboring directly connected 5'- and 3'-flanking regions of the target gene for homologous recombination was introduced into I. sakaiensis cells via conjugation. First, we deleted the orotidine 5'-phosphate decarboxylase gene (pyrF) from the genome of the wild-type strain, producing the ΔpyrF strain with 5-fluoroorotic acid (5-FOA) resistance. Next, using the ΔpyrF strain as a parent strain and pyrF as a counterselection marker, we disrupted the genes for PETase and MHETase. The growth of both Δpetase and Δmhetase strains on terephthalic acid (TPA; one of the PET hydrolytic products) was comparable to that of the parent strain. However, these mutant strains dramatically decreased the growth level on PET to that on a no-carbon source. Moreover, the Δpetase strain completely abolished PET degradation capacity. These results demonstrate that PETase and MHETase are essential for I. sakaiensis metabolism of PET. IMPORTANCE The poly(ethylene terephthalate) (PET)-degrading bacterium Ideonella sakaiensis possesses two unique enzymes able to serve in PET hydrolysis. PET hydrolase (PETase) hydrolyzes PET into mono(2-hydroxyethyl) terephthalic acid (MHET), and MHET hydrolase (MHETase) hydrolyzes MHET into terephthalic acid (TPA) and ethylene glycol (EG). These enzymes have attracted global attention, as they have potential to be used for bioconversion of PET. Compared to many in vitro studies, including biochemical and crystal structure analyses, few in vivo studies have been reported. Here, we developed a targeted gene disruption system in I. sakaiensis, which was then applied for constructing Δpetase and Δmhetase strains. Growth of these disruptants revealed that PETase is the sole enzyme responsible for PET degradation in I. sakaiensis, while PETase and MHETase play essential roles in its PET assimilation.
Assuntos
Proteínas de Bactérias/genética , Burkholderiales/genética , Burkholderiales/metabolismo , Hidrolases/genética , Polietilenotereftalatos/metabolismo , Proteínas de Bactérias/metabolismo , Etilenoglicol/metabolismo , Genes Bacterianos , Hidrolases/metabolismo , Hidrólise , Engenharia Metabólica , Ácidos Ftálicos/metabolismo , ReciclagemRESUMO
BACKGROUND: A considerable challenge in the development of bioprocesses for producing chemicals and fuels has been the high cost of feedstocks relative to oil prices, making it difficult for these processes to compete with their conventional petrochemical counterparts. Hence, in the absence of high oil prices in the near future, there has been a shift in the industry to produce higher value compounds such as fragrances for cosmetics. Yet, there is still a need to address climate change and develop biotechnological approaches for producing large market, lower value chemicals and fuels. RESULTS: In this work, we study ethylene glycol (EG), a novel feedstock that we believe has promise to address this challenge. We engineer Escherichia coli (E. coli) to consume EG and examine glycolate production as a case study for chemical production. Using a combination of modeling and experimental studies, we identify oxygen concentration as an important metabolic valve in the assimilation and use of EG as a substrate. Two oxygen-based strategies are thus developed and tested in fed-batch bioreactors. Ultimately, the best glycolate production strategy employed a target respiratory quotient leading to the highest observed fermentation performance. With this strategy, a glycolate titer of 10.4 g/L was reached after 112 h of production time in a fed-batch bioreactor. Correspondingly, a yield of 0.8 g/g from EG and productivity of 0.1 g/L h were measured during the production stage. Our modeling and experimental results clearly suggest that oxygen concentration is an important factor in the assimilation and use of EG as a substrate. Finally, our use of metabolic modeling also sheds light on the intracellular distribution through central metabolism, implicating flux to 2-phosphoglycerate as the primary route for EG assimilation. CONCLUSION: Overall, our work suggests that EG could provide a renewable starting material for commercial biosynthesis of fuels and chemicals that may achieve economic parity with petrochemical feedstocks while sequestering carbon dioxide.
Assuntos
Reatores Biológicos/microbiologia , Escherichia coli/metabolismo , Etilenoglicol/metabolismo , Fermentação , Glicolatos/metabolismo , Engenharia Metabólica/métodos , Escherichia coli/genética , Formiatos/metabolismo , Glucose/metabolismo , Ácidos Glicéricos/metabolismo , Redes e Vias Metabólicas/genética , Oxigênio/metabolismo , Xilose/metabolismoRESUMO
The use of enzymatic catalysts is an alternative to chemical catalysts as they can help to obtain products with less environmental impact, considered sustainable within the concept of green chemistry. The optimization, kinetic, lipase reuse, and scale-up of enzymatic production of ethylene glycol oleate in the batch mode were carried out using the NS 88011 lipase in a solvent-free system. For the optimization step, a 23 Central Composite Design was used and the optimized condition for the ethylene glycol oleate production, with conversions above 99%, was at 70 °C, 600 rpm, substrates molar ratio of 1:2, 1 wt% of NS 88011 in 32 H of reaction. Kinetic tests were also carried out with different amounts of enzyme, and it showed that by decreasing the amount of the enzyme, the conversion also decreases. The lipase reuse showed good conversions until the second cycle of use, after which it had a progressive reduction reaching 83% in the fourth cycle of use. The scale-up (ninefold increase) showed promising results, with conversion above 99%, achieving conversions similar to small-scale reactions. Therefore, this work proposed an environmentally safe route to produce an emollient ester using a low-cost biocatalyst in a solvent-free system.