RESUMEN
Ehrlich and demethiolation pathways as two competing branches converted amino acid into alcohols. Controlling both pathways offers considerable potential for industrial applications including alcohols overproduction, flavor-quality control and developing new flavors. While how to regulate ehrlich and demethiolation pathways is still not applicable. Taking the conversion of methionine into methionol and methanethiol for example, we constructed two suppression subtractive cDNA libraries of Clonostachys rosea by using suppression subtractive hybridization (SSH) technology for screening regulators controlling the conversion. E3 ubiquitin-protein ligase gene HUWE1 screened from forward SSH library was validated to be related with the biosynthesis of end products. Overexpressing HUWE1 in C. rosea and S. cerevisiae significantly increased the biosynthesis of methanethiol and its derivatives in demethiolation pathway, while suppressed the biosynthesis of methional and methionol in ehrlich pathway. These results attained the directional regulation of both pathways by overexpressing HUWE1. Thus, HUWE1 has potential to be a key target for controlling and enhancing alcohols production by metabolic engineering.
Asunto(s)
Alcoholes/metabolismo , Aminoácidos/metabolismo , Expresión Génica , Ingeniería Metabólica , Redes y Vías Metabólicas , Ubiquitina-Proteína Ligasas/genética , Biología Computacional/métodos , Regulación Fúngica de la Expresión Génica , Biblioteca de Genes , Metionina/metabolismo , Propanoles/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sulfuros/metabolismoRESUMEN
Aroma results from the interplay of volatile organic compounds (VOCs) and the attributes of microbial-producing aromas are significantly affected by fermentation conditions. Among the VOCs, only a few of them contribute to aroma. Thus, screening and identification of the key VOCs is critical for microbial-producing aroma. The traditional method is based on gas chromatography-olfactometry (GC-O), which is time-consuming and laborious. Considering the Tuber melanosporum fermentation system as an example, a new method to screen and identify the key VOCs by combining the aroma evaluation method with principle component analysis (PCA) was developed in this work. First, an aroma sensory evaluation method was developed to screen 34 potential favorite aroma samples from 504 fermentation samples. Second, PCA was employed to screen nine common key VOCs from these 34 samples. Third, seven key VOCs were identified by the traditional method. Finally, all of the seven key VOCs identified by the traditional method were also identified, along with four others, by the new strategy. These results indicate the reliability of the new method and demonstrate it to be a viable alternative to the traditional method.
Asunto(s)
Ascomicetos/metabolismo , Técnicas Biosensibles , Fermentación , Análisis de Componente Principal , Compuestos Orgánicos Volátiles/análisis , Carbono/metabolismo , Cromatografía de Gases , Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Odorantes/análisis , TemperaturaRESUMEN
The aroma attributes of sulfurous, mushroom and earthy are the most important characteristics of the aroma of Tuber melanosporum. However, these three aroma attributes are absent in the T. melanosporum fermentation system. To improve the quality of the aroma, repeated freeze-thaw treatment (RFTT) was adopted to affect the interplay of volatile organic compounds (VOCs). Using RFTT, not only was the score on the hedonic scale of the aroma increased from the "liked slightly" to the "liked moderately" grade, but the aroma attributes of sulfurous, mushroom and earthy could also be smelled in the T. melanosporum fermentation system for the first time. A total of 29 VOCs were identified, and 9 compounds were identified as the key discriminative volatiles affected by RFTT. Amino acid analysis revealed that methionine, valine, serine, phenylalanine, isoleucine and threonine were the key substrates associated with the biosynthesis of the 9 key discriminative VOCs. This study noted that amino acid metabolism played an important role in the regulation of the aroma of the T. melanosporum fermentation system.
Asunto(s)
Ascomicetos/metabolismo , Fermentación , Congelación , Olfato , Aminoácidos/análisis , Cromatografía de Gases y Espectrometría de Masas , Humanos , Análisis de Componente Principal , Compuestos Orgánicos Volátiles/análisisRESUMEN
Truffle (Tuber spp.), also known as "underground gold," is popular in various cuisines because of its unique and characteristic aroma. Currently, truffle fruiting bodies are mostly obtained from nature and semi-artificial cultivation. However, the former source is scarce, and the latter is time-consuming, usually taking 4 to 12 years before harvest of the fruiting body. The truffle submerged fermentation process was first developed in Tang's lab as an alternative to its fruiting bodies. To the best of our knowledge, most reports of truffle submerged fermentation come from Tang's group. This review examines the current state of the truffle submerged fermentation process. First, the strategy to optimize the truffle submerged fermentation process is summarized; the final conditions yielded not only the highest reported truffle biomass but also the highest production of extracellular and intracellular polysaccharides. Second, the comparison of metabolites produced by truffle fermentation and fruiting bodies is presented, and the former were superior to the latter. Third, metabolites (i.e., volatile organic compounds, equivalent umami concentration, and sterol) derived from truffle fermentation could be regulated by fermentation process optimization. These findings indicated that submerged fermentation of truffles can be used for commercial production of biomass and metabolites as a promising alternative to generating its fruiting bodies in bioreactor.
Asunto(s)
Ascomicetos/crecimiento & desarrollo , Ascomicetos/metabolismo , Reactores Biológicos/microbiología , Biotecnología/métodos , Fermentación , Biotecnología/tendencias , Metaboloma , Polisacáridos/metabolismoRESUMEN
Tuber melanosporum is highly appreciated in culinary contexts due to its unique and characteristic aroma. T. melanosporum fermentation has been established as a promising alternative for fruiting bodies to produce volatile organic compounds (VOCs). In this work, a technique using a combination of chromatographic fingerprint similarity analysis, head-space solid phase microextraction and gas chromatography was developed to rank the significance of fermentation conditions on the VOCs profile during T. melanosporum fermentation. Omission tests indicated that the absence of major carbon source (i.e., sucrose) in the fermentation media had the most significant effect on the profile of VOCs, followed by the absence of yeast extract or peptone. Consideration of the culture conditions revealed that VOCs produced was the most significantly affected by temperature. These results indicated that it is possible to adjust the aroma of truffles via fermentation process control.
Asunto(s)
Ascomicetos/química , Cromatografía de Gases/métodos , Fermentación , Microextracción en Fase Sólida/métodos , Compuestos Orgánicos Volátiles/metabolismo , Concentración de Iones de Hidrógeno , TemperaturaRESUMEN
Tuber melanosporum, known as the black diamond of cuisine, is highly appreciated for its unique and characteristic aroma, which is mainly due to its volatile organic sulfur-containing compounds (VOSCs). In this work, by adding 5 g/L L-methionine to the fermentation medium, the activities of aminotransferase and α-ketoacid decarboxylase were significantly enhanced by 103 and 250%, respectively, while the activities of alcohol dehydrogenase and demethiolase were decreased by 277 and 39%. Then, the six VOSCs, i.e., methanethiol (MTL), dimethyl sulfide (DMS), dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), 3-(methylthio)propanal (methional), and 3-(methylthio)-1-propanol (methionol), were first detected in the submerged fermentation of T. melanosporum. These results indicated that the biosynthesis of VOSCs was triggered by aminotransferase and α-ketoacid decarboxylase. The production of methional and methionol increased with the increased concentrations of L-methionine (i.e., 5, 10, 15, and 20 g/L) before day 4 of the culture protocol, and methionol was the major product in the Ehrlich pathway. The production of MTL was significantly decreased after day 4 with a significantly increased DMDS, and DMDS was the major product of the demethiolation pathway. Compared with the demethiolation pathway with a total flux of sulfur of 11.33-24.32 µM, the Ehrlich pathway with a total flux of sulfur of 6,149-10,330 µM was considered the major pathway for the biosynthesis of VOSCs. This is the first report linking the metabolism of L-methionine to the biosynthesis of VOSCs by the Ehrlich and demethiolation pathways during the submerged fermentation of T. melanosporum.
Asunto(s)
Ascomicetos/metabolismo , Metionina/metabolismo , Azufre/metabolismo , Compuestos Orgánicos Volátiles/metabolismo , Ascomicetos/química , Fermentación , Microbiología Industrial , Compuestos Orgánicos Volátiles/químicaRESUMEN
As a potential intermediary feedstock, succinic acid takes an important place in bulk chemical productions. For the first time, a method combining Plackett-Burman design (PBD), steepest ascent method (SA), and Box-Behnken design (BBD) was developed to optimize Actinobacillus succinogenes ATCC 55618 fermentation medium. First, glucose, yeast extract, and MgCO(3) were identified to be key medium components by PBD. Second, preliminary optimization was run by SA method to access the optimal region of the key medium components. Finally, the responses, that is, the production of succinic acid, were optimized simultaneously by using BBD, and the optimal concentration was located to be 84.6 g L(-1) of glucose, 14.5 g L(-1) of yeast extract, and 64.7 g L(-1) of MgCO(3). Verification experiment indicated that the maximal succinic acid production of 52.7 ± 0.8 g L(-1) was obtained under the identified optimal conditions. The result agreed with the predicted value well. Compared with that of the basic medium, the production of succinic acid and yield of succinic acid against glucose were enhanced by 67.3% and 111.1%, respectively. The results obtained in this study may be useful for the industrial commercial production of succinic acid.
Asunto(s)
Actinobacillus/metabolismo , Reactores Biológicos/microbiología , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Modelos Biológicos , Modelos Estadísticos , Ácido Succínico/metabolismo , Actinobacillus/clasificación , Simulación por Computador , Interpretación Estadística de Datos , Especificidad de la Especie , Ácido Succínico/aislamiento & purificaciónRESUMEN
The aroma active compounds of three Tuber fruiting bodies (i.e., Tuber himalayense, Tuber indicum, and Tuber sinense) were firstly systematically evaluated by instrumental gas chromatography-olfactometry combining with quantitative analysis, aroma reconstitution, and omission tests. Twelve aroma active compounds were characterized by aroma extract dilution analysis, and 3-(methylthio) propanal, 3-methylbutanal, and 1-octen-3-ol with the highest flavor dilution (FD) factor (i.e., 1,024-2,048) were suggested as key contributors to the aroma. Odor activity value (OAV) was employed to determine the relative contribution of each compound to the aroma, and the compound with the highest FD factor also had the highest OAV (i.e., 10,234-242,951). Then, the synthetic blends of odorants (aroma reconstitution) were prepared with OAV larger than 15, and their aromas were very similar to the originals. Omission tests were carried out to verify the significance of 3-(methylthio) propanal, 1-octen-3-ol, and 3-methylbutanal as key compounds in the aroma of tested Tuber fruiting bodies.
Asunto(s)
Ascomicetos/química , Cromatografía de Gases/métodos , Aromatizantes/análisis , Cuerpos Fructíferos de los Hongos/química , Aceites Volátiles/análisisRESUMEN
The mated fermentation process for the production of lycopene by Blakeslea trispora NRRL 2895 (+) and NRRL 2896 (-) was systematically optimized in shake flasks. The ratio of the (+) to (-) strains, the lycopene cyclase inhibitors piperidine and creatinine, the trisporic acid structural analog abscisic acid, the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) precursor leucine, and the mevalonate kinase enhancer penicillin were all identified as key factors affecting lycopene biosynthesis. With an optimal ratio of 5:1 for the (+) to (-) strains and the addition of 6 g/L creatinine on day 3, the highest lycopene production was 98.1 ± 15.5 mg/L. Based on the above result, the addition of 0.1 g/L penicillin on day 4, 150 µmol/L abscisic acid on day 3 or 0.5 g/L leucine on day 4 enhanced lycopene production to 119.7 ± 17.2, 120.6 ± 12.3 and 135.2 ± 7.0 mg/L, respectively. Finally, an integrated strategy by combining the above key factors was developed, and the highest lycopene production of 156.2 ± 15.4 mg/L was obtained, which was enhanced by 134.9% comparing with its production of 66.5 ± 3.6 mg/L before the optimization process of this work. The results obtained in this study may be useful for large-scale industrial lycopene production.
Asunto(s)
Carotenoides/biosíntesis , Técnicas de Cocultivo/métodos , Modelos Biológicos , Spiroplasma/clasificación , Spiroplasma/metabolismo , Carotenoides/aislamiento & purificación , Simulación por Computador , Fermentación , Licopeno , Especificidad de la EspecieRESUMEN
The dissolved oxygen tension of 20% of air saturation, pH-shift from 4.0 to 5.5 on day 3, and a moderate shear stress (calculated as an impeller tip speed, V(tip) = 0.926 - 2.161 m/s) were identified to be the key factors in scaling-up the mated fermentation of Blakeslea trispora NRRL 2895 (+) and 2896 (-) for lycopene production from a shake flask to a stirred-tank fermenter. The maximal lycopene production of 183.3 mg/L was obtained in 7.5-L stirred-tank fermenter, and then the mated fermentation process was successfully step-wise scaled-up from 7.5- to 200-L stirred-tank fermenter. The comparability of the fermentation process was well controlled and the lycopene production was maintained during the process scale-up. Furthermore, with the integrated addition of 150 µmol/L abscisic acid on day 3, 0.5 g/L leucine and 0.1 g/L penicillin on day 4, the highest lycopene production of 270.3 mg/L was achieved in the mated fermentation of B. trispora in stirred-tank fermenter.
Asunto(s)
Reactores Biológicos , Carotenoides/biosíntesis , Mucorales/crecimiento & desarrollo , Ácido Abscísico/farmacología , Medios de Cultivo/química , Medios de Cultivo/farmacología , Leucina/farmacología , LicopenoRESUMEN
Uniform design and partial least squares regression were adopted to quantitatively analyze the effects of plant oil and fatty acid as well as their addition amount and addition time on the performance of Tuber melanosporum submerged fermentation. The regression models showed the optimal scheme was the addition of 1.2 mL soybean oil on day 9, which was validated by experiment. The maximal biomass of 25.89 +/- 1.01 g/L and extracellular polysaccharides (EPS) production of 6.51 +/- 0.68 g/L were obtained, which were enhanced by 18.5% and 86%, respectively. Palmitic acid was identified to be the key component to stimulate the cell growth and EPS biosynthesis, and stearic acid was beneficial for the production of intracellular polysaccharides. Not only the biomass but also EPS production obtained in this work are the highest reported in the batch fermentation of truffle.
Asunto(s)
Ascomicetos/efectos de los fármacos , Reactores Biológicos/microbiología , Ácidos Grasos/farmacología , Inhibidores de Crecimiento/farmacología , Aceite de Soja/farmacología , Ascomicetos/crecimiento & desarrollo , Ascomicetos/metabolismo , Biomasa , Fermentación , Ácido Palmítico/análisis , Ácido Palmítico/farmacología , Polisacáridos/metabolismo , Aceite de Soja/química , Ácidos Esteáricos/análisis , Ácidos Esteáricos/farmacología , Factores de TiempoRESUMEN
A novel method using Plackett-Burman design (PBD) coupled with Draper-Lin small composite design (SCD) and desirability function (DF) was developed to optimize Tuber melanosporum fermentation medium. Firstly, sucrose, yeast extract, peptone and Mg(2+) were identified to be key medium components by PBD. Secondly, in order to evaluate the relationships between responses and the identified key components, mathematical models were developed by SCD. Finally, by using DF, the responses were optimized simultaneously and the optimal concentration was located to be 73 g/L sucrose, 11 g/L yeast extract, 8 g/L peptone, and 46 mM Mg(2+). Under the identified optimal conditions, the maximal biomass, the production of extracellular (EPS) and intracellular (IPS) polysaccharides was 25.10 + or - 0.12 g/L, 3.88 + or - 0.23 g/L and 2.87 + or - 0.32 g/L, respectively, which agreed with the predicted values well. Compared with the basic medium, the biomass, the production of EPS and IPS was enhanced by 54.4%, 71.7% and 124.2%, respectively.
Asunto(s)
Medios de Cultivo/metabolismo , Fermentación/fisiología , Modelos Biológicos , Pholiota/fisiología , Análisis de Varianza , Biomasa , Espacio Extracelular/metabolismo , Espacio Intracelular/metabolismo , Polisacáridos/biosíntesis , Análisis de Regresión , Reproducibilidad de los ResultadosRESUMEN
For the first time, a fed-batch fermentation process of Tuber melanosporum was developed for the efficient production of bioactive mycelia and Tuber polysaccharides. Each 1.67 g/L of peptone and 8.33 g/L of yeast extract were added on day 3, 6, and 9, respectively, and sucrose was fed to maintain its concentration around 35-5 g/L when its residual level decreased to 10-5 g/L. Then, the maximal biomass, the production of extracellular polysaccharides (EPS) and intracellular polysaccharides (IPS) reached 53.72+/-2.57 g DW/L, 7.09+/-0.62 and 4.43+/-0.21 g/L, respectively. Compared with the batch culture conducted in the enriched medium, the biomass, the production of EPS and IPS were enhanced by 55.8%, 222.3% and 103.2%, respectively. Not only the cell density but also the production of EPS and IPS were the highest ever reported in truffle fermentation, and the biomass was also the highest as ever reported in mushroom fermentation.
Asunto(s)
Agaricales/metabolismo , Reactores Biológicos/microbiología , Biotecnología/métodos , Técnicas de Cultivo de Célula/métodos , Fermentación , Polisacáridos/química , Polisacáridos/metabolismo , Biomasa , Carbohidratos/química , Carbono/química , Medios de Cultivo/metabolismo , Cinética , Nitrógeno/química , Peptonas/química , Sacarosa/químicaRESUMEN
During the submerged fermentation of medicinal mushroom Chinese truffle Tuber sinense, there was no significant effect of metal ion on the cell growth and the production of intracellular polysaccharides, while metal ion and its concentration significantly affected the production of extracellular polysaccharides (EPS). By using the approach of "one-variable-at-a-time", 50 mM Mg2+ was identified to be the most favorable for EPS production, and the next was 10 mM K+. A mathematical model, constructed by response surface methodology combination with full factorial design, was applied to study the synergic effect of Mg2+ and K+. EPS production reached its peak value of 5.86 g/L under their optimal combination of 30 mM Mg2+ and 5 mM K+ predicted by the model, which was higher by 130.7% compared with the basal fermentation medium without metal ion. The validation experiment showed the experimental values agreed with the predicted values well. EPS production obtained in this work was the highest reported in the culture of T. sinense.