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1.
Science ; 367(6482): 1147-1151, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32139546

RESUMO

Mycobacterium tuberculosis has an unusual outer membrane that lacks canonical porin proteins for the transport of small solutes to the periplasm. We discovered that 3,3-bis-di(methylsulfonyl)propionamide (3bMP1) inhibits the growth of M. tuberculosis, and resistance to this compound is conferred by mutation within a member of the proline-proline-glutamate (PPE) family, PPE51. Deletion of PPE51 rendered M. tuberculosis cells unable to replicate on propionamide, glucose, or glycerol. Growth was restored upon loss of the mycobacterial cell wall component phthiocerol dimycocerosate. Mutants in other proline-glutamate (PE)/PPE clusters, responsive to magnesium and phosphate, also showed a phthiocerol dimycocerosate-dependent growth compromise upon limitation of the corresponding substrate. Phthiocerol dimycocerosate determined the low permeability of the mycobacterial outer membrane, and the PE/PPE proteins apparently act as solute-specific channels.


Assuntos
Amidas/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Glucose/metabolismo , Glicerol/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/genética , Transporte Biológico , Permeabilidade da Membrana Celular , Farmacorresistência Bacteriana/genética , Deleção de Genes , Lipídeos/química , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Compostos de Sulfidrila/química , Compostos de Sulfidrila/farmacologia
2.
World J Microbiol Biotechnol ; 36(3): 49, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-32157439

RESUMO

Glycerol is a by-product of biodiesel, and it has a great application prospect to be transformed to synthesize high value-added compounds. Pseudomonas chlororaphis GP72 isolated from the green pepper rhizosphere is a plant growth promoting rhizobacteria that can utilize amount of glycerol to synthesize phenazine-1-carboxylic acid (PCA). PCA has been commercially registered as "Shenqinmycin" in China due to its characteristics of preventing pepper blight and rice sheath blight. The aim of this study was to engineer glycerol utilization pathway in P. chlororaphis GP72. First, the two genes glpF and glpK from the glycerol metabolism pathway were overexpressed in GP72ANO separately. Then, the two genes were co-expressed in GP72ANO, improving PCA production from 729.4 mg/L to 993.4 mg/L at 36 h. Moreover, the shunt pathway was blocked to enhance glycerol utilization, resulting in 1493.3 mg/L PCA production. Additionally, we confirmed the inhibition of glpR on glycerol metabolism pathway in P. chlororaphis GP72. This study provides a good example for improving the utilization of glycerol to synthesize high value-added compounds in Pseudomonas.


Assuntos
Glicerol/metabolismo , Engenharia Metabólica/métodos , Pseudomonas chlororaphis/genética , Pseudomonas chlororaphis/metabolismo , Aquaporinas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Capsicum/microbiologia , China , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Regulação Bacteriana da Expressão Gênica , Técnicas de Inativação de Genes , Glicerolfosfato Desidrogenase/genética , Redes e Vias Metabólicas/genética , Fenazinas/metabolismo , Proteínas Repressoras/genética , Rizosfera
3.
Electron. j. biotechnol ; 44: 19-24, Mar. 2020. ilus, graf
Artigo em Inglês | LILACS | ID: biblio-1087631

RESUMO

BACKGROUND: Pyruvic acid (PA), a vital α-oxocarboxylic acid, plays an important role in energy and carbon metabolism. The oleaginous yeast Yarrowia lipolytica (Y. lipolytica) has considerable potential for the production of PA. An increased NaCl concentration reportedly increases the biomass and PA yield of Y. lipolytica. RESULTS: To increase the yield of PA, the NaCl-tolerant Y. lipolytica A4 mutant was produced using the atmospheric and room temperature plasma method of mutation. The A4 mutant showed growth on medium containing 160 g/L NaCl. The PA yield of the A4 mutant reached 97.2 g/L at 120 h (0.795 g/g glycerol) in a 20-L fermenter with glycerol as the sole carbon source, which was 28.9% higher than that of the parental strain. CONCLUSION: The PA yield from Y. lipolytica can be improved by increasing its NaCl tolerance.


Assuntos
Ácido Pirúvico/metabolismo , Yarrowia/genética , Yarrowia/metabolismo , Pressão Osmótica , Leveduras , Carbono/metabolismo , Cloreto de Sódio , Reatores Biológicos , Tolerância ao Sal/genética , Fermentação , Glicerol/metabolismo , Mutação
4.
Microb Cell Fact ; 19(1): 6, 2020 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-31931797

RESUMO

BACKGROUND: 1,3-propanediol (1,3-PDO) is the most widely studied value-added product that can be produced by feeding glycerol to bacteria, including Lactobacillus sp. However, previous research reported that L. reuteri only produced small amounts and had low productivity of 1,3-PDO. It is urgent to develop procedures that improve the production and productivity of 1,3-PDO. RESULTS: We identified a novel L. reuteri CH53 isolate that efficiently converted glycerol into 1,3-PDO, and performed batch co-fermentation with glycerol and glucose to evaluate its production of 1,3-PDO and other products. We optimized the fermentation conditions and nitrogen sources to increase the productivity. Fed-batch fermentation using corn steep liquor (CSL) as a replacement for beef extract led to 1,3-PDO production (68.32 ± 0.84 g/L) and productivity (1.27 ± 0.02 g/L/h) at optimized conditions (unaerated and 100 rpm). When CSL was used as an alternative nitrogen source, the activity of the vitamin B12-dependent glycerol dehydratase (dhaB) and 1,3-propanediol oxidoreductase (dhaT) increased. Also, the productivity and yield of 1,3-PDO increased as well. These results showed the highest productivity in Lactobacillus species. In addition, hurdle to 1,3-PDO production in this strain were identified via analysis of the half-maximal inhibitory concentration for growth (IC50) of numerous substrates and metabolites. CONCLUSIONS: We used CSL as a low-cost nitrogen source to replace beef extract for 1,3-PDO production in L. reuteri CH53. These cells efficiently utilized crude glycerol and CSL to produce 1,3-PDO. This strain has great promise for the production of 1,3-PDO because it is generally recognized as safe (GRAS) and non-pathogenic. Also, this strain has high productivity and high conversion yield.


Assuntos
Lactobacillus reuteri/metabolismo , Propilenoglicóis/metabolismo , Fermentação , Glicerol/metabolismo , Xarope de Milho Rico em Frutose/metabolismo , Microbiologia Industrial/métodos
5.
PLoS One ; 15(1): e0227203, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31914144

RESUMO

The mountain pine beetle, Dendroctonus ponderosae (Coleoptera: Curculionidae) is a major forest pest of pines in western North America. Beetles typically undergo a one-year life cycle with larval cold hardening in preparation for overwintering. Two-year life cycle beetles have been observed but not closely studied. This study tracks cold-hardening and preparation for overwintering by adult mountain pine beetles in their natal galleries. Adults were collected in situ between September and December 2016 for a total of nine time points during 91 days. Concentrations of 41 metabolites in these pooled samples were assessed using quantitative nuclear magnetic resonance (NMR). Levels of glycerol and proline increased significantly with lowering temperature during the autumn. Newly eclosed mountain pine beetles appear to prepare for winter by generating the same cold-tolerance compounds found in other insect larvae including mountain pine beetle, but high on-site mortality suggested that two-year life cycle adults have a less efficacious acclimation process. This is the first documentation of cold acclimation metabolite production in overwintering new adult beetles and is evidence of physiological plasticity that would allow evolution by natural selection of alternate life cycles (shortened or lengthened) under a changing climate or during expansion into new geoclimatic areas.


Assuntos
Aclimatação , Glicerol/metabolismo , Prolina/metabolismo , Gorgulhos/metabolismo , Animais , Temperatura Baixa , Resposta ao Choque Frio , Pinus/parasitologia , Estações do Ano , Gorgulhos/fisiologia
6.
Enzyme Microb Technol ; 133: 109456, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31874684

RESUMO

Dihydroxyacetone phosphate (DHAP)-dependent aldolases demonstrate important values in the production of rare ketoses due to their unique stereoselectivities. As a specific example, we developed an efficient Escherichia coli whole-cell biocatalytic cascade system in which rare ketoses were produced from abundant glycerol and catalyzed by four enzymes based on L-rhamnulose-1-phosphate aldolase (RhaD). For the semicontinuous bioconversion in which D-glyceraldehyde was continuously added, once D-glyceraldehyde was consumed, the final yields of D-sorbose and D-psicose were 15.30 g/L and 6.35 g/L, respectively. Moreover, the maximum conversion rate and productivity of D-sorbose and D-psicose were 99% and 1.11 g/L/h at 8 h, respectively. When L-glyceraldehyde was used instead of the D-isomer, the final yield of L-fructose was 16.80 g/L. Furthermore, the maximum conversion rate and productivity of L-fructose were 95% and 1.08 g/L/h at 8 h, respectively. This synthetic platform was also compatible with other various aldehydes, which allowed the production of many other high-value chemicals from glycerol.


Assuntos
Aldeído Liases/metabolismo , Escherichia coli/metabolismo , Cetoses/biossíntese , Biocatálise , Biotransformação , Frutose/metabolismo , Gliceraldeído/metabolismo , Glicerol/metabolismo , Microbiologia Industrial , Sorbose/metabolismo , Especificidade por Substrato
7.
Enzyme Microb Technol ; 132: 109437, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31731966

RESUMO

Biological production of 2,3-butanediol (2,3-BDO), a C4 platform chemical, has been studied recently, but the high cost of separation and purification before chemical conversion is substantial. To overcome this obstacle, we have conducted a study to convert 2,3-BDO to mevalonate, a terpenoid intermediate, using recombinant Pseudomonas putida and this biological process won't need the separation and purification process of 2,3-BDO. The production of mevalonate when 2,3-BDO was used as a substrate was 6.61 and 8.44 times higher than when glucose and glycerol were used as substrates under the same conditions, respectively. Lower aeration contributed to higher yields of mevalonate in otherwise identical conditions. The maximum mevalonate production on the shaking flask scale was about 2.21 g/L, in this study (product yield was 0.295, 27% of theoretical yield (1.10)). This study was the first successful attempt for mevalonate production by P. putida using 2,3-BDO as the sole carbon source and presented a new metabolic engineering tool and biological process for mevalonate synthesis.


Assuntos
Butileno Glicóis/metabolismo , Engenharia Metabólica , Ácido Mevalônico/metabolismo , Pseudomonas putida/metabolismo , Carbono/metabolismo , Glucose/metabolismo , Glicerol/metabolismo , Redes e Vias Metabólicas , Pseudomonas putida/genética
8.
Food Chem ; 305: 125479, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31505418

RESUMO

1-o-Galloylglycerol (GG) was synthesized by the enzymatic glycerolysis of propyl gallate (PG) using a food-grade lipase (Lipozyme® 435). The reaction conditions affecting the yield of GG were optimized and a yield of 76.9% ±â€¯1.2% was obtained. GG was characterized by various techniques after being separated from the reaction mixture using liquid-liquid extraction. The water solubility and hydrophilicity of GG were significantly higher than those of gallic acid (GA) and PG. The antioxidant properties, measured by the ferric reducing antioxidant power (FRAP) and hydrogen peroxide (H2O2) scavenging assays, showed that GG exhibited the highest scavenging capacity (GG > GA > PG). From the results of the 1,1-diphenyl-2-picrylhydrazyl (DPPH•) and 2,2'-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS•+) assays, GG and GA exhibited greater scavenging capacity than PG (GG = GA > PG). These results suggest that GG may be used as a water-soluble antioxidant alternative to GA for food and cosmetic applications.


Assuntos
Antioxidantes/metabolismo , Antioxidantes/farmacologia , Ácido Gálico/análogos & derivados , Glicerol/análogos & derivados , Antioxidantes/química , Ácido Gálico/química , Ácido Gálico/metabolismo , Ácido Gálico/farmacologia , Glicerol/química , Glicerol/metabolismo , Glicerol/farmacologia , Peróxido de Hidrogênio/química , Lipase/metabolismo , Extração Líquido-Líquido , Espectroscopia de Ressonância Magnética , Galato de Propila/química , Solubilidade , Temperatura
9.
J Agric Food Chem ; 68(2): 561-566, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31840510

RESUMO

2-Hydroxyphenazine (2-OH-PHZ) is an effective biocontrol antibiotic secreted by Pseudomonas chlororaphis GP72AN and is transformed from phenazine-1-carboxylic acid (PCA). PCA is the main component of the recently registered biopesticide "Shenqinmycin". Previous research showed that 2-OH-PHZ was better in controlling wheat take-all disease than PCA; however, 2-OH-PHZ production was low under natural conditions. Herein, we confirmed that PCA induced reactive oxygen species in its host P. chlororaphis GP72AN and that the addition of DTT improved PCA production by 1.8-fold, whereas the supplementation of K3[Fe(CN)6] and H2O2 increased the conversion rate of PCA to 2-OH-PHZ. Finally, a two-stage fermentation strategy combining the addition of DTT at 12 h and H2O2 at 24 h enhanced 2-OH-PHZ production. Taken together, the two-stage fermentation strategy was designed to enhance 2-OH-PHZ production for the first time, and it provided a valuable reference for the fermentation of other antibiotics.


Assuntos
Antibacterianos/biossíntese , Glicerol/metabolismo , Microbiologia Industrial/métodos , Pseudomonas chlororaphis/metabolismo , Proteínas de Bactérias/metabolismo , Meios de Cultura/química , Meios de Cultura/metabolismo , Fermentação , Peróxido de Hidrogênio/metabolismo , Fenazinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo
10.
Sci Total Environ ; 702: 134911, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31733546

RESUMO

Development of cost effective and highly efficient process for bio-based succinic acid (SA) production is a main concern for industry. The metabolically engineered Y. lipolytica strain PGC01003 was successfully used for SA production with high titre. However, this strain possesses as main drawback with a low growth rate when glycerol is used as a feedstock. Herein, gene GUT1, encoding glycerol kinase, was overexpressed in strain PGC01003 with the aim to improve glycerol uptake capacity. In the resulting strain RIY420, glycerol uptake was 13.5% higher than for the parental strain. GUT1 gene overexpression also positively influences SA production. In batch bioreactor, SA titre, yield and productivity were 32%, 39% and 143% higher, respectively, than for the parental strain PGC01003. Using a glycerol feeding strategy, SA titre, yield and productivity were further improved by 11%, 5% and 10%, respectively. Moreover, the process duration to yield the highest concentration of SA in the culture supernatant was reduced by 9%. This demonstrated the contribution of metabolically engineered strain RIY420 to lower SA process cost and increase the efficiency of bio-based SA production.


Assuntos
Glicerol/metabolismo , Ácido Succínico/metabolismo , Yarrowia/fisiologia , Transporte Biológico , Reatores Biológicos , Engenharia Metabólica , Saccharomyces cerevisiae
11.
World J Microbiol Biotechnol ; 36(1): 2, 2019 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-31811394

RESUMO

"Dirty" glycerol from biodiesel production is having a considerable environmental impact since its disposal is expensive and difficult. The increased biodiesel production in the last two decades has forced glycerol prices down, thereby making it now unprofitable for chemical companies to produce. The problem lies with the impurities of the biodiesel conversion process usually ending up within the crude glycerol fraction. These impurities are often too costly to purify with current processes, particularly for small scale producers. A wide variety of industries, including the paint, tobacco, food and pharmaceutical industries, utilize glycerol as part of their technology or products. However, the crude glycerol from biodiesel production is not of a high enough grade to be used in these industries. Biodiesel-produced crude glycerol is therefore cheap, readily available and presents itself as an attractive carbon source for industrial microbial production systems synthesizing value-added products. This mini-review will look at (a) microbial production processes which use crude glycerol to produce high-value products (product-driven research) and (b) genetic engineering of microbes which is aimed at improving microbial "dirty" glycerol utilization (substrate driven research).


Assuntos
Biocombustíveis , Fermentação , Glicerol/metabolismo , Microbiologia Industrial , Bactérias/genética , Bactérias/metabolismo , Carbono , Engenharia Genética
12.
Metabolism ; 101: 153993, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31672442

RESUMO

BACKGROUND: Therapies targeting altered activity of pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) have been proposed for hepatomas. However, the activities of these pathways in hepatomas in vivo have not been distinguished. Here we examined pyruvate entry into the tricarboxylic acid (TCA) cycle through PDH versus PC in vivo using hepatoma-bearing rats. METHODS: Hepatoma-bearing rats were generated by intrahepatic injection of H4IIE cells. Metabolism of 13C-labeled glycerol, a physiological substrate for both gluconeogenesis and energy production, was measured with 13C NMR analysis. The concentration of key metabolites and the expression of relevant enzymes were measured in hepatoma, surrounding liver, and normal liver. RESULTS: In orthotopic hepatomas, pyruvate entry into the TCA cycle occurred exclusively through PDH and the excess PDH activity compared to normal liver was attributed to downregulated pyruvate dehydrogenase kinase (PDK) 2/4. However, pyruvate carboxylation via PC and gluconeogenesis were minimal, which was linked to downregulated forkhead box O1 (FoxO1) by Akt activity. In contrast to many studies of cancer metabolism, lactate production in hepatomas was not increased which corresponded to reduced expression of lactate dehydrogenase. The production of serine and glycine in hepatomas was enhanced, but glycine decarboxylase was downregulated. CONCLUSIONS: The combination of [U-13C3]glycerol and NMR analysis enabled investigation of multiple biochemical processes in hepatomas and surrounding liver. We demonstrated active PDH and other related metabolic alterations in orthotopic hepatomas that differed substantially not only from the host organ but also from many earlier studies with cancer cells.


Assuntos
Carcinoma Hepatocelular/metabolismo , Gluconeogênese , Neoplasias Hepáticas/metabolismo , Complexo Piruvato Desidrogenase/metabolismo , Animais , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13/métodos , Carcinoma Hepatocelular/enzimologia , Ciclo do Ácido Cítrico , Glicerol/metabolismo , Fígado/enzimologia , Neoplasias Hepáticas/enzimologia , Ratos
13.
Microsc Res Tech ; 82(12): 2079-2088, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31587425

RESUMO

Microalgae have an excellent potential for producing valuable natural products, including biofuels. Therefore, it is imperative to explore and document the existing microalgal flora and utilize their potentials to cope the increasing human needs. The present work aims at exploring and characterizing newly isolated microalgae from desert Cholistan, a habitat with myriad algal diversity. Light microscopy, scanning electron microscopy, and molecular phylogenetic approaches were used for species-level identification. Characterization and growth optimization of Scendesmus sp. were analyzed under three different growth modes to determine the most favorable conditions for increasing biomass, growth rate, and lipid content. The results revealed that mixotrophic (MT) mode significantly increases photosynthetic activity, growth rate, and lipid content with glycerol as supplement carbon source. The investigated Scenedesmus dimorphous produced a maximum dry weight of 1.73 g L-1 , improved fatty acid methyl esters profile and yield lipid up to 40% of DCW (68 g L-1 ) under MT mode, which is almost double to that of photoautotrophic cultivation. The glycerol availability in medium has been identified as the critical element for boosting growth and lipid content. Thus, it can reduce the cost of biofuel production.


Assuntos
Microalgas/classificação , Fotossíntese/fisiologia , Scenedesmus/crescimento & desenvolvimento , Scenedesmus/metabolismo , Biocombustíveis/análise , Biomassa , DNA de Plantas/genética , Clima Desértico , Ácidos Graxos/análise , Glicerol/metabolismo , Microalgas/crescimento & desenvolvimento , Microalgas/isolamento & purificação , Microalgas/metabolismo , Paquistão , Filogenia , Scenedesmus/classificação , Scenedesmus/isolamento & purificação
14.
J Basic Microbiol ; 59(12): 1195-1207, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31617952

RESUMO

Klebsiella pneumoniae can naturally synthesize 3-hydroxypropionic acid (3-HP), 1,3-propanediol (1,3-PD), and 2,3-butanediol (2,3-BD) from glycerol. However, biosynthesis of these industrially important chemicals is constrained by troublesome byproducts. To clarify the influences of byproducts on 3-HP production, in this study, a total of eight byproduct-producing enzyme genes including pmd, poxB, frdB, fumC, dhaT, ilvH, adhP, and pflB were individually deleted from the K. pneumoniae genome. The resultant eight mutants presented different levels of metabolites. In 24-h shake-flask cultivation, the adhP- and pflB-deletion mutants produced 0.41 and 0.44 g/L 3-HP, respectively. Notably, the adhP and pflB double deletion mutant K. pneumoniaeΔadhPΔpflB produced 1.58 g/L 3-HP in 24-h shake-flask cultivation. When K. pneumoniaeΔadhPΔpflB was harnessed as a host strain to overexpress PuuC, a native aldehyde dehydrogenase (ALDH) catalyzing 3-hydroxypropionaldehyde (3-HPA) to 3-HP, the resulting recombinant strain K. pneumoniaeΔadhPΔpflB(pTAC-puuC) (pTAC-puuC is PuuC expression vector) generated 66.91 g/L 3-HP with a cumulative yield of 70.84% on glycerol in 60-h bioreactor cultivation. Additionally, this strain showed 2.3-, 5.1-, and 0.67-fold decrease in the concentrations of 1,3-PD, 2,3-BD, and acetic acid compared with the reference strain K. pneumoniae(pTAC-puuC). These results indicated that the byproducts exerted differential impacts on the production of 3-HP, 1,3-PD, and 2,3-BD. Although combinatorial elimination of byproduct pathways could reprogram glycerol flux, the enzyme 1,3-propanediol oxidoreductase (DhaT) that catalyzes 3-HPA to 1,3-PD and the enzymes ALDHs, especially, PuuC are most pivotal for 3-HP production. This study provides a deep understanding of how byproducts affect the production of 3-HP, 1,3-PD, and 2,3-BD in K. pneumoniae.


Assuntos
Vias Biossintéticas/fisiologia , Glicerol/metabolismo , Klebsiella pneumoniae/metabolismo , Ácido Láctico/análogos & derivados , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Reatores Biológicos , Vias Biossintéticas/genética , Butileno Glicóis/metabolismo , Expressão Gênica , Técnicas de Inativação de Genes , Klebsiella pneumoniae/genética , Ácido Láctico/metabolismo , Engenharia Metabólica , Propilenoglicóis/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
15.
BMC Genomics ; 20(1): 763, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31640564

RESUMO

BACKGROUND: One fundamental question in biology is how the evolution of eukaryotic signaling networks has taken place. "Loss of function" (lof) mutants from components of the high osmolarity glycerol (HOG) signaling pathway in the filamentous fungus Magnaporthe oryzae are viable, but impaired in osmoregulation. RESULTS: After long-term cultivation upon high osmolarity, stable individuals with reestablished osmoregulation capacity arise independently from each of the mutants with inactivated HOG pathway. This phenomenon is extremely reproducible and occurs only in osmosensitive mutants related to the HOG pathway - not in other osmosensitive Magnaporthe mutants. The major compatible solute produced by these adapted strains to cope with high osmolarity is glycerol, whereas it is arabitol in the wildtype strain. Genome and transcriptome analysis resulted in candidate genes related to glycerol metabolism, perhaps responsible for an epigenetic induced reestablishment of osmoregulation, since these genes do not show structural variations within the coding or promotor sequences. CONCLUSION: This is the first report of a stable adaptation in eukaryotes by producing different metabolites and opens a door for the scientific community since the HOG pathway is worked on intensively in many eukaryotic model organisms.


Assuntos
Adaptação Fisiológica/genética , Redes Reguladoras de Genes , Glicerol/metabolismo , Magnaporthe/fisiologia , Transdução de Sinais/genética , Dioxóis/farmacologia , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genoma Fúngico/genética , Mutação com Perda de Função , Magnaporthe/efeitos dos fármacos , Magnaporthe/genética , Magnaporthe/metabolismo , Oryza/microbiologia , Osmorregulação/genética , Doenças das Plantas/microbiologia , Pirróis/farmacologia , Estresse Salino
16.
Microb Cell Fact ; 18(1): 176, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31615519

RESUMO

BACKGROUND: Yarrowia lipolytica is an unconventional yeast with a huge industrial potential. Despite many advantages for biotechnological applications, it possesses enormous demand for oxygen, which is a bottleneck in large scale production. In this study a codon optimized bacterial hemoglobin from Vitreoscilla stercoraria (VHb) was overexpressed in Y. lipolytica for efficient growth and erythritol synthesis from glycerol in low-oxygen conditions. Erythritol is a natural sweetener produced by Y. lipolytica under high osmotic pressure and at low pH, and this process requires high oxygen demand. RESULTS: Under these conditions the VHb overexpressing strain showed mostly yeast-type cells resulting in 83% higher erythritol titer in shake-flask experiments. During a bioreactor study the engineered strain showed higher erythritol productivity (QERY = 0.38 g/l h) and yield (YERY = 0.37 g/g) in comparison to the control strain (QERY = 0.30 g/l h, YERY = 0.29 g/g). Moreover, low stirring during the fermentation process resulted in modest foam formation. CONCLUSIONS: This study showed that overexpression of VHb in Y. lipolytica allows for dynamic growth and efficient production of a value-added product from a low-value substrate.


Assuntos
Eritritol/biossíntese , Hemoglobinas , Microrganismos Geneticamente Modificados/metabolismo , Yarrowia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Reatores Biológicos , Clonagem Molecular , Fermentação , Glicerol/metabolismo , Hemoglobinas/genética , Hemoglobinas/metabolismo , Engenharia Metabólica , Oxigênio/metabolismo , Vitreoscilla/metabolismo , Yarrowia/genética , Yarrowia/metabolismo
17.
J Biotechnol ; 304: 31-37, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31421146

RESUMO

2,3-Butanediol (2,3-BD) can be produced at high titers by engineered Saccharomyces cerevisiae by abolishing the ethanol biosynthetic pathway and introducing the bacterial butanediol-producing pathway. However, production of 2,3-BD instead of ethanol by engineered S. cerevisiae has resulted in glycerol production because of surplus NADH accumulation caused by a lower degree of reduction (γ = 5.5) of 2,3-BD than that (γ = 6) of ethanol. In order to eliminate glycerol production and resolve redox imbalance during 2,3-BD production, both GPD1 and GPD2 coding for glycerol-3-phosphate dehydrogenases were disrupted after overexpressing NADH oxidase from Lactococcus lactis. As disruption of the GPD genes caused growth defects due to limited supply of C2 compounds, Candida tropicalis PDC1 was additionally introduced to provide a necessary amount of C2 compounds while minimizing ethanol production. The resulting strain (BD5_T2 nox_dGPD1,2_CtPDC1) produced 99.4 g/L of 2,3-BD with 0.5 g/L glycerol accumulation in a batch culture. The fed-batch fermentation led to production of 108.6 g/L 2,3-BD with a negligible amount of glycerol production, resulting in a high BD yield (0.462 g2,3-BD/gglucose) corresponding to 92.4 % of the theoretical yield. These results demonstrate that glycerol-free production of 2,3-BD by engineered yeast is feasible.


Assuntos
Butileno Glicóis/metabolismo , Deleção de Genes , Glicerolfosfato Desidrogenase/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Técnicas de Cultura Celular por Lotes , Candida tropicalis/enzimologia , Fermentação , Proteínas Fúngicas/genética , Engenharia Genética , Glicerol/metabolismo , Glicerol-3-Fosfato Desidrogenase (NAD+)/genética , Lactococcus lactis/enzimologia , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Piruvato Descarboxilase/deficiência , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
18.
J Ind Microbiol Biotechnol ; 46(11): 1583-1601, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31468234

RESUMO

2,3-Butanediol (2,3-BD) has great potential for diverse industries, including chemical, cosmetics, agriculture, and pharmaceutical areas. However, its industrial production and usage are limited by the fairly high cost of its petro-based production. Several bio-based 2,3-BD production processes have been developed and their economic advantages over petro-based production process have been reported. In particular, many 2,3-BD-producing microorganisms including bacteria and yeast have been isolated and metabolically engineered for efficient production of 2,3-BD. In addition, several fermentation processes have been tested using feedstocks such as starch, sugar, glycerol, and even lignocellulose as raw materials. Since separation and purification of 2,3-BD from fermentation broth account for the majority of its production cost, cost-effective processes have been simultaneously developed. The construction of a demonstration plant that can annually produce around 300 tons of 2,3-BD is scheduled to be mechanically completed in Korea in 2019. In this paper, core technologies for bio-based 2,3-BD production are reviewed and their potentials for use in the commercial sector are discussed.


Assuntos
Bactérias/metabolismo , Butileno Glicóis/metabolismo , Fermentação , Glicerol/metabolismo , Lignina/metabolismo , Engenharia Metabólica
19.
Int J Mol Sci ; 20(16)2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31434215

RESUMO

3-Iodothyronamine (T1AM) and its synthetic analog SG-2 are rapidly emerging as promising drivers of cellular metabolic reprogramming. Our recent research indicates that in obese mice a sub-chronic low dose T1AM treatment increased lipolysis, associated with significant weight loss independent of food consumption. The specific cellular mechanism of T1AM's lipolytic effect and its site of action remains unknown. First, to study the mechanism used by T1AM to gain entry into cells, we synthesized a fluoro-labeled version of T1AM (FL-T1AM) by conjugating it to rhodamine (TRITC) and analyzed its cellular uptake and localization in 3T3-L1 mouse adipocytes. Cell imaging using confocal microscopy revealed a rapid intercellular uptake of FL-T1AM into mitochondria without localization to the lipid droplet or nucleus of mature adipocytes. Treatment of 3T3-L1 adipocytes with T1AM and SG-2 resulted in decreased lipid accumulation, the latter showing a significantly higher potency than T1AM (10 µM vs. 20 µM, respectively). We further examined the effects of T1AM and SG-2 on liver HepG2 cells. A significant decrease in lipid accumulation was observed in HepG2 cells treated with T1AM or SG-2, due to increased lipolytic activity. This was confirmed by accumulation of glycerol in the culture media and through activation of the AMPK/ACC signaling pathways.


Assuntos
Tironinas/farmacologia , Células 3T3-L1 , Animais , Reprogramação Celular/efeitos dos fármacos , Glicerol/metabolismo , Células Hep G2 , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipólise/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo
20.
Bioresour Technol ; 289: 121699, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31323726

RESUMO

In this work, we propose a Mixed Integer Nonlinear Programming (MINLP) model to determine the optimal design of a poly(hydroxyalkanoate)s (PHAs) production plant configuration. The superstructure based optimization model considers different carbon sources as raw material: glycerol (crude and purified), corn starch, cassava starch, sugarcane sucrose and sugarcane molasses. The PHA extraction section includes four alternatives: the use of enzyme, solvent, surfactant-NaOCl or surfactant-chelate. Model constraints include detailed capital cost for equipment, mass and energy balances, product specifications and operating bounds on process units. The resulting MINLP model maximizes the project net present value (NPV) as objective function and it is implemented in an equation oriented environment. Optimization results show the sugarcane-enzyme option as the most promising alternative (NPV = 75.01 million USD) for PHAs production with an energy consumption of 22.56 MJ/kg PHA and a production cost of 3.02 USD/kg PHA. Furthermore, an economic sensitivity analysis is performed.


Assuntos
Poli-Hidroxialcanoatos/biossíntese , Carbono/metabolismo , Glicerol/metabolismo , Melaço , Amido/metabolismo
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