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Valencene, a kind of sesquiterpenoid with a citrus flavor, is mainly found in Valencia orange and is commonly used in cosmetics and food additives, as well as industrial synthetic nootkatone. In this study, synthetic biology was used to create a Saccharomyces cerevisiae cell factory to produce valencene. Fistly, valencene synthase gene (CnVS) from Callitropsis nootkatensis was inserted into the chromosome of the chassis strain YTT-T5. The resulting strain VAL-01 could produce 1.1 mg·L-1 valencene. Protein fusion technique was used, different valencene synthases were compared and the copy number of key genes was adjusted, yielding valencene to 436.4 mg·L-1. Then, knocking-out the transcription factor ROX1 resulted in valencene improvement by 17.4%. Moreover, the induction system of galactose was regulated, transcription factor PDR3 and INO2 were overexpressed. The engineered strain VAL-10 could produce 2 798.6 mg·L-1 valencene by high cell density fermentation method (nearly 2 500 times higher than VAL-01). This study provides a basis for green production of valencene.
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Patchoulol is an important sesquiterpenoid in the volatile oil of Pogostemon cablin, and is also considered to be the main contributing component to the pharmacological efficacy and fragrance of P. cablin oil, which has antibacterial, antitumor, antioxidant, and other biological activities. Currently, patchoulol and its essential oil blends are in high demand worldwide, but the traditional plant extraction method has many problems such as wasting land and polluting the environment. Therefore, there is an urgent need for a new method to produce patchoulol efficiently and at low cost. To broaden the production method of patchouli and achieve the heterologous production of patchoulol in Saccharomyces cerevisiae, the patchoulol synthase(PS) gene from P. cablin was codon optimized and placed under the inducible strong promoter GAL1 to transfer into the yeast platform strain YTT-T5, thereby obtaining strain PS00 with the production of(4.0±0.3) mg·L~(-1) patchoulol. To improve the conversion rate, this study used protein fusion method to fuse SmFPS gene from Salvia miltiorrhiza with PS gene, leading to increase the yield of patchoulol to(100.9±7.4) mg·L~(-1) by 25-folds. By further optimizing the copy number of the fusion gene, the yield of patchoulol was increased by 90% to(191.1±32.7) mg·L~(-1). By optimizing the fermentation process, the strain was able to achieve a patchouli yield of 2.1 g·L~(-1) in a high-density fermentation system, which was the highest yield so far. This study provides an important basis for the green production of patchoulol.
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Saccharomyces cerevisiae/metabolismo , Sesquiterpenos/metabolismo , Pogostemon , Óleos Voláteis/metabolismoRESUMO
Monoterpenes are widely used in cosmetics, food, medicine, agriculture and other fields. With the development of synthetic biology, it is considered as a potential way to create microbial cell factories to produce monoterpenes. Engineering Saccharomyces cerevisiae to produce monoterpenes has been a research hotspot in synthetic biology. In S. cerevisiae, the production of geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) is catalyzed by a bifunctional enzyme farnesyl pyrophosphate synthetase(encoded by ERG20 gene) which is inclined to synthesize FPP essential for yeast growth. Therefore, reasonable control of FPP synthesis is the basis for efficient monoterpene synthesis in yeast cell factories. In order to achieve dynamic control from GPP to FPP biosynthesis in S. cerevisiae, we obtained a novel chassis strain HP001-pERG1-ERG20 by replacing the ERG20 promoter of the chassis strain HP001 with the promoter of cyclosqualene cyclase(ERG1) gene. Further, we reconstructed the metabolic pathway by using GPP and neryl diphosphate(NPP), cis-GPP as substrates in HP001-pERG1-ERG20. The yield of GPP-derived linalool increased by 42.5% to 7.6 mg·L~(-1), and that of NPP-derived nerol increased by 1 436.4% to 8.3 mg·L~(-1). This study provides a basis for the production of monoterpenes by microbial fermentation.
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Fermentação , Geraniltranstransferase/genética , Monoterpenos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Ginsenoside Rh_2 is a rare active ingredient in precious Chinese medicinal materials such as Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma, and Panacis Quinquefolii Radix. It has important pharmacological activities such as anti-cancer and improving human immunity. However, due to the extremely low content of ginsenoside Rh_2 in the source plants, the traditional way of obtaining it has limitations. This study intended to apply synthetic biological technology to develop a cell factory of Saccharomyces cerevisiae to produce Rh_2 by low-cost fermentation. First, we used the high protopanaxadiol(PPD)-yielding strain LPTA as the chassis strain, and inserted the Panax notoginseng enzyme gene Pn1-31, together with yeast UDP-glucose supply module genes[phosphoglucose mutase 1(PGM1), α-phosphoglucose mutase(PGM2), and uridine diphosphate glucose pyrophosphorylase(UGP1)], into the EGH1 locus of yeast chromosome. The engineered strain LPTA-RH2 produced 17.10 mg·g~(-1) ginsenoside Rh_2. This strain had low yield of Rh_2 while accumulated much precursor PPD, which severely restricted the application of this strain. In order to further improve the production of ginsenoside Rh_2, we strengthened the UDP glucose supply module and ginsenoside Rh_2 synthesis module by engineered strain LPTA-RH2-T. The shaking flask yield of ginsenoside Rh_2 was increased to 36.26 mg·g~(-1), which accounted for 3.63% of the dry weight of yeast cells. Compared with those of the original strain LPTA-RH2, the final production and the conversion efficiency of Rh_2 increased by 112.11% and 65.14%, respectively. This study provides an important basis for further obtaining the industrial-grade cell factory for the production of ginsenoside Rh_2.
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Humanos , Fermentação , Ginsenosídeos , Panax/genética , Panax notoginseng , Saccharomyces cerevisiae/genética , Uridina Difosfato GlucoseRESUMO
OBJECTIVE@#With the rapid development of sleep medicine, there are various methods for detecting sleep diseases. This study compared the correlation between the lightweight watch-type sleep monitor (Actiwatch) and the "gold standard" polysomnography (PSG) in the Chinese population, in order to provide a basis for clinical application.@*METHODS@#From August 2018 to December 2019, 121 subjects who simultaneously performed sleep breathing monitoring (PSG) and wearing a watch-type sleep monitor (Actiwatch) in the Sleep Center of Peking University People's Hospital were enrolled. All subjects received PSG and Actiwatch at the same time, and filled out the sleep diary next morning. Monitoring indicators were collected for linear correlation analysis and paired t test to compare the differences.@*RESULTS@#Under low sensitivity conditions, the correlation coefficient of total sleep time (TST) between PSG and Actiwatch was 0.53 (P < 0.05). Paired t test analysis showed that there was no significant difference between the TSTs of Actiwatch and PSG (t=-0.890, P=0.36). According to age stratification, the smaller the age, the stronger the correlation between the TSTs of Actiwatch and PSG, and the coefficient could be up to 0.92 (P < 0.05). Paired t test showed that there was no significant difference between them (t=-1.057, P=0.35). According to the stratification by diagnosis, the correlation coefficient between the TSTs of Actiwatch and PSG in normal PSG group could be as high as 0.79 (P < 0.05), the results of paired t test showed that there was no significant difference between the TSTs of Actiwatch and PSG in normal PSG group (t=-0.784, P=0.44).@*CONCLUSION@#As a wearable home recorder, when the analysis parameters of Actiwatch were set as low sensitivity, PSG and Actiwatch had the highest TST correlation. The younger the age, the stronger correlation between the TSTs of Actiwatch and PSG. The PSG and Actiwatch subjects with normal PSG presentation had a higher TST correlation.
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Humanos , Actigrafia , Polissonografia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Sono , Transtornos do Sono-Vigília , TempoRESUMO
This study engineered β-carotene ketolase CrtW and β-carotene hydroxylase CrtZ to improve biosynthesis of astaxanthin in Escherichia coli. Firstly, crtW was randomly mutated to increase CrtW activities on conversion from β-carotene to astaxanthin. A crtW* mutant with A6T, T105A and L239M mutations has improved 5.35-fold astaxanthin production compared with the wild-type control. Secondly, the expression levels of crtW* and crtZ on chromosomal were balanced by simultaneous modulation RBS regions of their genes using RBS library. The strain RBS54 selected from RBS library, directed the pathway exclusively towards the desired product astaxanthin as predominant carotenoid (99%). Lastly, the number of chromosomal copies of the balanced crtW-crtZ cassette from RBS54 was increased using a Cre-loxP based technique, and a strain with 30 copies of the crtW*-crtZ cassette was selected. This final strain DL-A008 had a 9.8-fold increase of astaxanthin production compared with the wild-type control. Fed-batch fermentation showed that DL-A008 produced astaxanthin as predominant carotenoid (99%) with a specific titer of 0.88 g·L without addition of inducer. In conclusion, through constructing crtW mutation, balancing the expression levels between crtW* and crtZ, and increasing the copy number of the balanced crtW*-crtZ cassette, the activities of β-carotene ketolase and β-carotene hydroxylase were improved for conversion of β-carotene to astaxanthin with higher efficiency. The series of conventional and novel metabolic engineering strategies were designed and applied to construct the astaxanthin hetero-producer strain of E. coli, possibly offering a general approach for the construction of stable hetero-producer strains for other natural products.
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Ginsenoside F1 is a rare ginsenoside in medicinal plants such as Panax ginseng,P. notogingseng and P. quinquefolius. It has strong pharmacological activities of anti-tumor,anti-oxidation and anti-aging. In order to directly produce ginsenoside F1 by using inexpensive raw materials such as glucose,we integrated the codon-optimized P.ginseng dammarenediol-Ⅱ synthase(Syn Pg DDS),P.ginseng protopanaxadiol synthase(Syn Pg PPDS),P. ginseng protopanaxatriol synthase(Syn Pg PPTS) genes and Arabidopsis thaliana cytochrome P450 reductase(At CPR1) gene into triterpene chassis strain BY-T3. The transformant BY-PPT can produce protopanaxatriol. Then we integrated the Sacchromyces cerevisiae phosphoglucomutase 1(PGM1),phosphoglucomutase 2(PGM2) and UDP-glucose pyrophosphorylase 1(UGP1) genes into chassis strain BY-PPT. The UDP-glucose supply module increased UDP-glucose production by 8. 65 times and eventually reached to 44. 30 mg·L-1 while confirmed in the transformant BY-PPT-GM. Next,we integrated the UDPglucosyltransferase Pg3-29 gene which can catalyze protopanaxatriol to produce ginsenoside F1 into chassis strain BY-PPT-GM. The transformant BY-F1 produced a small amount of ginsenoside F1 which was measured as 0. 5 mg·L-1. After the fermentation process was optimized,the titer of ginsenoside F1 could be increased by 900 times to 450. 5 mg·L-1. The high-efficiency UDP-glucose supply module in this study can provide reference for the construction of cell factories for production of saponin,and provide an important basis for further obtaining high-yield ginsenoside yeast cells.
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Ginsenosídeos/metabolismo , Glucose , Panax , Saccharomyces cerevisiae/metabolismo , Uridina Difosfato GlucoseRESUMO
Objective:To observe the effects of recipes for tonifying kidney and replenishing Qi, Zuoguiwan (ZG) and Yiqi Congming Tang(YQ) on memory capacity, expressions of learning and memory-related genes expression, and explore the changes in relevant epigenetic modification enzymes. Method:SD male rats with natural aging (24 months old) were used as animal models and randomly divided into aged control group, aged ZG group(12.12 g·kg-1), aged YQ group(10.18 g·kg-1), aged compound group(11.15 g·kg-1) and aged antagonist RU38486 group(5×10-3g·kg-1). Another 5 months old male SD rats were included as the young control group. Morris water maze method was used to observe the spatial learning and memory ability of the rats. The co-localizations of histone deacetylase 2 (HDAC2) and methyl CpG binding protein 2 (MeCP2) in hippocampus of rats in each group were observed by laser confocal microscope. The changes in expressions of glucocorticoid receptor (GR), synapsin1(Syn-1), HDAC2, and histone acetyltransferase 1(HAT1) proteins in hippocampus of each group were detected by Western blot, and mRNA expression of HDAC2 was detected by Real-time fluorescence quantitative polymerase Chain reaction (Real-time PCR). At the same time, the effects of ZG, YQ and compound decoction in alleviating the above-mentioned abnormal changes were observed. Result:Compared with the young control group(control group), the latency of the aged control group was significantly prolonged (PPPPPPPConclusion:ZG group, YQ group, and compound group can improve the spatial learning and memory abilities of aged rats by increasing the expression of learning-memory-associated protein GR and epigenetic modification enzyme HAT1, and reducing the expression of HDAC2 and the co-localization of HDAC2 protein and MeCP2 in the nucleus.
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Dammarenediol-Ⅱ is an important precursor in the biosynthesis pathway of ginsenosides which are the main active components of Panax quinquefolius and Panax ginseng. For constructing a dammarenediol- Ⅱ-producing cell factory, the triterpenoid precursors of yeast are improved significantly by the modular pathway engineering strategy on the basis of an MVA optimized strain. The strain overexpressing Salvia miltiorrhiza SmFPS and Arabidopsis thaliana AtSQS2 could yield 67.4 mg·g−1 squalene, accounting for about 6.74% of cell dry weight. In our further work, an Arabidopsis thaliana 2,3-oxidosqualene synthase AtSQE2 was found to be able to increase the downstream lanosterol yield by 22-fold, reaching 47.9 mg·g−1. Then, regulating dammarenediol-Ⅱ synthase gene expression, using anti-sense RNA technology for regulation of ERG7 in the ergosterol pathway, and optimizing fermentation process were successively performed. Finally, the synthesis flux of triterpenes was increased to 10 g·L−1 for the first time, and we constructed an efficient cell factory that can produce 15 g·L−1 dammarenediol-Ⅱ, which lays a solid foundation of industrial synthesis of dammarane-type ginsenosides.
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Hederagenin is an effective constituent of many medical plants, such as Clematidis Radix, and has a wide range of applications in anti-tumor, anti-inflammatory, antidepressant, hepatoprotective antibacterial, et al. In order to obtain the efficient production of yeast cells for hederagenin,we successfully cloned and screened out a P450 gene MdMA02 from Malus×domestica which can catalyze oleanolic acid C-23 oxidation with our developed plug and play platform. Its amino acid homology is only 32% as compared to characterized CYP72A68v2. By transforming MdMA02 to the oleanolic acid-producing strain BY-OA, a hederagenin-producing strain was constructed and hederagenin's titer could achieve 101 mg·L⁻¹ using high cell density fermentation, which was 337 times higher than in shake flasks culturing. This study provides a basis for further research on promoting the creation of oleanane-type pentacyclic triterpenoids biosynthetic pathway analysis and relative cell factories construction.
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Cucurbitadienol has anti-inflammation, anti-cancer activities, and acts as a precursor of traditional Chinese medicine active ingredients mogroside and cucurbitacine. For construction of a Sacchromyces cerevisiae cell factory for production of cucurbitadienol, we firstly cloned a cucurbitadienol synthase (CBS) gene from Siraitia grosvenorii. Then, through heterologous expression of CBS in the triterpenoid chassis strain WD-2091, the engineered strain could produced 27.44 mg•L ⁻¹ cucurbitadienol, which was determined by GC-MS. Further regulation of CBS expression led to cucurbitadienol's titer increasing by 202.07% and reaching 82.89 mg•L ⁻¹ in the shake flask fermentation and 1 724.10 mg•L ⁻¹ in the high cell density fermentation. Our research promotes the cucurbitane-type tetracyclic triterpenoids synthesis pathway analysis progress and provides the basis for further obtaining cell factories for production of cucurbitadienol tetracyclic triterpenoids.
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<p><b>OBJECTIVE</b>To study the effect of obesity on pulmonary function in newly diagnosed asthmatic children of different age groups.</p><p><b>METHODS</b>Two hundred and ninety-four children with newly diagnosed asthma were classified into preschool-age (<6 years) and school-age (6 to 12.5 years) groups. They were then classified into obese, overweight, and normal-weight subgroups based on their body mass index (BMI). All the children underwent pulmonary function tests, including large airway function tests [forced vital capacity (FVC%) and forced expiratory volume in one second (FEV1%)] and small airway function tests [maximal expiratory flow at 25% of vital capacity (MEF25%), maximal expiratory flow at 50% of vital capacity (MEF50%), and maximal expiratory flow at 75% of vital capacity (MEF75%)].</p><p><b>RESULTS</b>The school-age group showed lower FEV1%, MEF25%, and MEF50% than the preschool-age group (P<0.05) after adjustment for sex and BMI. The normal-weight children in the school-age group had lower FEV1%, MEF25%, and MEF50% compared with their counterparts in the preschool-age group (P<0.05). The overweight children in the school-age group showed lower FVC% and MEF50% than those in the preschool-age group. However, all the pulmonary function parameters showed no significant differences between the obese children in the preschool-age and school-age groups. In the preschool-age group, FVC%, FEV1%, and MEF75% of the obese children were lower than those of the normal-weight children. In the school-age group, only FVC% and FEV1% showed differences between the obese and normal-weight children (P<0.05).</p><p><b>CONCLUSIONS</b>The effect of obesity on the pulmonary function varies with age in children with asthma, and the effect is more obvious in those of preschool age.</p>
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Criança , Pré-Escolar , Humanos , Fatores Etários , Asma , Volume Expiratório Forçado , Pulmão , ObesidadeRESUMO
Nerolidol is an important constituent of terpenoid essential oil and has excellent anti-tumor, anti-bacterial, and anti-oxidative properties. For realizing heterogenous production of nerolidol, our research firstly integrated the codon-optimized Actinidia sinensis nerolidol synthase gene (NES) into the terpenoid chassis strain FPP-001, and obtained NES-001 that could produce 2.71 mg•L⁻¹ nerolidol. Then, the N-terminal of the NES was fused with FPS by linker peptide GGGS. With this strategy, nerolidol production improved by 59.80-fold, reaching 162.07 mg•L⁻¹. Finally, by introduction of auxotrophic marker TRP1 in NES-002, the resulting strain NES-003 could produce 1 711.53 mg•L⁻¹ by high cell density fermentation method. This study provides the basis for the fermentative production of nerolidol and other sesquiterpenoids.
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Serotonin (5-hydroxytryptamine, 5-HT) is an endogenous molecule playing key roles in life activity. In the treatment of metabolic diseases such as obesity, the traditional paradigm of appetite suppression by serotonin in the central nervous system has some limitations. In recent years, great progress has been made in the study of the regulation of energy metabolism by peripheral serotonin system. Recent studies revealed that peripheral serotonin plays an important role in regulation of adipogenesis and energy expenditure in adipose tissues, insulin secretion in pancreatic β-cell and glycogen synthesis in liver, etc. This review summarizes the recent advances in the function of serotonin in the peripheral organs of energy metabolism. We propose that peripheral serotonin system may serve as an attractive new therapeutic target for the treatment of metabolic diseases in the near future.
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Objective To investigate the mechanism of 3-phosphoinositide-dependent protein kinase 1(PDK1)poly-ubiquitination.Methods Co-immunoprecipitation(Co-IP)and Western blot(WB)were used to analyze poly-ubiquitination of PDK1.It was confirmed that ubiquitin ligase smad ubiquitylation regulatory factor 1(Smurf1)inprove PDK1 poly-ubiquitination within MEF cells,site-directed mutagenesis and WB before PDK1 poly-ubiquitination sites were determined.Results We found that PDK1 could undergoes poly-ubiquitination,ubiquitin ligase Smurf1 was found to be a direct E3 ligase for PDK1 poly-ubiquitination and might rely on the ubiquitin ligase Smurf 1 K699 site activity.K304 was PDK1 poly-ubiquitination modification site point.Conclusion The ubiquitin ligase Smurf1 can promate poly-ubiquitination of PDK1.
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Lupane-type triterpenoids, such as betulinic acid, are derived from lupeol and have excellent properties in anti-HIV, anti-cancer activities and so on. For realizing heterogenous production of lupane-type triterpenoids, our research firstly integrated all the seven genes in the MVA pathway in Saccharomyces cerevisiae to increase the supply of squalene (triterpenoids universal precursor) in a single step using the DNA assembler method. Next, cell factories for production of lupeol was constructed by integrating Arabidopsis thaliana lupeol synthetic gene (AtLUP) into chromosome of triterpenoid chassis strain. Results showed that the MVA pathway, about 20 kb nucleotide length, could be assembled in one-pot process and the doubled MVA pathway could significantly improve squalene by 500-fold, reaching 354.00 mg•L⁻¹. NK2-LUP was obtained by introducing AtLUP gene on chromosome, and could produce 8.23 mg•L⁻¹ lupeol. This study supports the possibility of large-scale biosynthetic pathway assembly in S.cerevisiae and lays the foundation of obtaining cell factories for production of lupan-type triterpenoids at the same time.
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<p><b>OBJECTIVE</b>To study the skin prick test (SPT) reactivity to house dust mite allergens in overweight and normal weight children with allergic asthma before and after standard subcutaneous specific immunotherapy.</p><p><b>METHODS</b>Two hundred and fifteen children with allergic asthma who had positive SPT responses to Dermatophagoides pteronyssinus (DP) and Dermatophagoides farinae (DF) were enrolled. According to the weight index, they were classified into overweight (n=63) and normal weight groups (n=152). Skin indices (SI) to DP and DF were compared between the two groups at 6 months and 1 year after standard subcutaneous specific immunotherapy.</p><p><b>RESULTS</b>The overweight group had a significantly larger histamine wheal diameter than the normal weight group after controlling the variation in testing time (P<0.05). After controlling the variation in weights, there were significant differences in the SIs to DP and DF before specific immunotherapy and at 6 months and 1 year after specific immunotherapy. At 6 months and 1 year after specific immunotherapy, the SIs to DP and DF were significantly reduced in both groups (P<0.05), and the overweight group had greater decreases in the SIs to DP and DF than the normal weight group.</p><p><b>CONCLUSIONS</b>The overweight children with allergic asthma have stronger responses to histamine than the normal weight patients. Specific immunotherapy can reduce the reactivity to dust mite allergens in children with allergic asthma. Within one year after specific immunotherapy, the overweight children with allergic asthma have a significantly greater decrease in the reactivity to dust mite allergens than the normal weight patients.</p>
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Adolescente , Animais , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Asma , Alergia e Imunologia , Terapêutica , Dermatophagoides farinae , Alergia e Imunologia , Dermatophagoides pteronyssinus , Alergia e Imunologia , Imunoterapia , Sobrepeso , Alergia e Imunologia , Pyroglyphidae , Alergia e Imunologia , Testes CutâneosRESUMO
For microbial production of lycopene, the lycopene synthetic genes from Pantoea agglomerans were integrated into Saccharomyces cerevisiae strain BY4742, to obtain strain ZD-L-000 for production of 0.17 mg · L(-1) lycopene. Improving supplies of isoprenoid precursors was then investigated for increasing lycopene production. Four key genes were chosen to be overexpressed, inclu- ding truncated 3-hydroxy-3-methylglutaryl-CoA reductase gene (tHMG1), which is the major rate-limiting enzyme in the mevalonate (MVA) pathway, a mutated global regulatory factor gene (upc2.1), a fusion gene of FPP synthase (ERG20) and endogenous GGPP synthase (BTS1), which is a key enzyme in the diterpenoid synthetic pathway, and GGPP synthase gene (SaGGPS) from Sulfolobus acidocaldarius. Over-expression of upc2.1 could not improve lycopene production, while over-expression of tHMGI , BTS1-ERG20 and SaGGPS genes led to 2-, 16. 9- and20. 5-fold increase of lycopene production, respectively. In addition, three effective genes, tHMG1, BTS1-ERG20 and SaGGPS, were integrated into rDNA sites of ZD-L-000, resulting in strain ZD-L-201 for production of 13.23 mg · L(-1) lycopene, which was 77-fold higher than that of the parent strain. Finally, two-phase extractive fermentation was performed. The titer of lycopene increased 10-fold to 135.21 mg · L(-1). The engineered yeast strains obtained in this work provided the basis for fermentative production of lycopene.
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Proteínas de Bactérias , Genética , Metabolismo , Vias Biossintéticas , Carotenoides , Genes Sintéticos , Engenharia Genética , Pantoea , Genética , Saccharomyces cerevisiae , Genética , MetabolismoRESUMO
<p><b>OBJECTIVE</b>To optimize the synthetic pathway and fermentation process of yeast cell factories for production of oleanoic acid.</p><p><b>METHOD</b>Using the DNA assembler method, one copy of Glycyrrhiza glabra beta-amyrin synthase (GgbAS), Medicago truncatula oleanolic acid synthase (MtOAS) and Arabidopsis thaliana cytochrome P450 reductase 1 (AtCPR1) genes were introduced into Saccharomyces cerevisiae strain BY-OA, resulting in strain BY-20A. YPD medium with different glucose concentration were then used to cultivate strain BY-2OA.</p><p><b>RESULT</b>Increasing gene copies of GgbAS, MtOAS and AtCPR1 resulted in increased beta-amyrin and oleanolic acid production. The strain BY-2OA produced 136.5 mg x L(-1) beta-amyrin and 92.5 mg x L(-1) oleanolic acid, which were 54% and 30% higher than the parent strain BY-OA. Finally, the titer of oleanolic acid increased to 165.7 mg x L(-1) when cultivated in YPD medium with 40 mg x L(-1) glucose.</p><p><b>CONCLUSION</b>Production of oleanoic acid increased significantly in the yeast strain BY-2OA, which can provide the basis for creating an alternative way for production of oleanoic acid in place of extraction from plant sources.</p>
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Biomassa , Biotecnologia , Métodos , Relação Dose-Resposta a Droga , Fermentação , Glucose , Farmacologia , Ácido Oleanólico , Saccharomyces cerevisiae , Biologia Celular , MetabolismoRESUMO
<p><b>OBJECTIVE</b>To determine the effects of 5-hydroxymethyl furfural (5-HMF), an extract of Rehmannia glutinosa Libosch, on several down-regulated signaling molecules involved in learning and memory in hippocampal neurons.</p><p><b>METHODS</b>After cultured for 7 days, primary hippocampal neurons were divided into 5 groups: normal, corticosterone model, RU38486, 5-HMF, and donepezil group. Neuron survival rates were calculated 24 h later using SYTO13-PI double-fluorescence staining and an 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. β-galactosidase activity was also assayed. Protein expressed by the glucocorticoid receptor (GCR), brainderived neurotrophic factor (BDNF), and N-methyl-D-aspartate receptor 2B (NR2B), as well as phosphorylationcyclic adenosine monophosphate (cAMP) response element binding protein (p-CREB), phosphorylation-extracellular signal-regulated kinase (p-ERK), and phosphorylation-synapsin (p-synapsin) were quantified with Western blot.</p><p><b>RESULTS</b>Hippocampal neuron survival rates and the above-mentioned proteins were dramatically decreased (P<0.05), β-galactosidase activity was significantly increased in the model group. but the effect was reversed by 5-HMF, RU38486, and to a lesser extent by donepezil (P<0.05).</p><p><b>CONCLUSION</b>5-HMF extracts from the Chinese herb Rehmannia glutinosa Libosch could protect hippocampal neurons from glucocorticoid injury and from down-regulated signaling molecules in the GCR-BDNF-NR2B-p-ERK-p-CREB-p-synapsin signal transduction pathway.</p>