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In this study, Pediococcus pentosaceus C-2-1 and C23221 contained genes encoding penocin and pediocin PA-1, mined by antiSMASH. The penocin structural gene pedA from Pediococcus pentosaceus C-2-1 was successfully expressed in Escherichia coli BL21. The presence of a 6.5 kDa recombinant penocin was confirmed by Tricine-SDS-PAGE, and the specific activity increased by 1.54-fold. The bacteriocins produced by Pediococcus pentosaceus C23221 were purified using acetic ether extraction, Sepharose Fast Flow, Sephadex G-25 gel chromatography, and reversed-phase high-performance liquid chromatography (RP-HPLC); the amino acid sequence of this bacteriocin was identical to pediocin PA-1 by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), which confirmed the expression of pediocin PA-1 gene; and the specific activity increased by 24.39-fold. The heterologous expression and purification of bacteriocins have proved the expression of pediocin-like produced by Pediococcus pentosaceus. This provides a theoretical basis for the subsequent development and application of pediocin-like.
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The bacteriocin-producing Lactiplantibacillus plantarum SL47 was isolated from conventional fermented sausages, and the bacteriocin SL47 was purified using ethyl acetate, Sephadex G-25 gel chromatography, and reversed-phase high-performance liquid chromatography (RP-HPLC). Bacteriocin SL47 was identified by HPLC-MS/MS combined with whole-genome sequencing, and the results showed it consisted of plantaricin A, J, K, and N. Further characterization analysis showed that the bacteriocin SL47 was highly thermostable (30 min, 121 °C), pH stable (2-10), sensitive to protease and exhibited broad-spectrum antibacterial ability against Gram-positive and Gram-negative bacteria. The mechanism of action showed that the bacteriocin SL47 increased cell membrane permeability, and 2 × minimum inhibitory concentration (MIC) treatment for 40 min caused apoptosis of Staphylococcus aureus F2. The count of S. aureus in the sausage that was inoculated with L. plantarum SL47 and bacteriocin SL47 decreased by about 64% and 53% of that in the initial stage, respectively. These results indicated the potential of L. plantarum SL47 and bacteriocin SL47 as a bio-preservative in meat products.
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Chrysin, a flavonoid, has been found to have been widely used in the health food field. But at present, chrysin production is hindered by the low availability of precursors and the lack of catalytic enzymes with high activity. Therefore, ZmPAL was initially screened to synthesize trans-cinnamic acid with high catalytic activity and specificity. To enhance the supply of precursors, the shikimic acid and chorismic acid pathway genes were overexpressed. Besides, the expression of the intracellular and mitochondrial carbon metabolism genes CIT, MAC1/3, CTP1, YHM2, RtME, and MDH was enhanced to increase the intracellular acetyl-CoA content. Chrysin was synthesized through a novel gene combination of ScCPR-EbFNSI-1 and PcFNSI. Finally, de novo synthesis of chrysin was achieved, reaching 41.9 mg/L, which is the highest reported concentration to date. In summary, we identified efficient enzymes for chrysin production and increased it by regulating acetyl-CoA metabolism in mitochondria and the cytoplasm, laying a foundation for future large-scale production.
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Flavonoides , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Acetilcoenzima A/metabolismo , Flavonoides/metabolismo , Engenharia MetabólicaRESUMO
Listeria monocytogenes is a common microorganism that causes food spoilage. Pediocins are some biologically active peptides or proteins encoded by ribosomes, which have a strong antimicrobial activity against L. monocytogenes. In this study, the antimicrobial activity of previously isolated P. pentosaceus C-2-1 was enhanced by ultraviolet (UV) mutagenesis. A positive mutant strain P. pentosaceus C23221 was obtained after 8 rounds of UV irradiation with increased antimicrobial activity of 1448 IU/mL, which was 8.47 folds higher than that of wild-type C-2-1. The genome of strain C23221 and wild-type C-2-1 was compared with identify the key genes for higher activity. The genome of the mutant strain C23221 consists of a chromosome of 1,742,268 bp, with 2052 protein-coding genes, 4 rRNA operons, and 47 tRNA genes, which is 79,769 bp less than the original strain. Compared with strain C-2-1, a total of 19 deduced proteins involved in 47 genes are unique to C23221 analyzed by GO database; the specific ped gene related to bacteriocin biosynthesis were detected using antiSMASH in mutant C23221, indicating mutant C23221 produced a new bacteriocin under mutagenesis conditions. This study provides genetic basis for further constituting a rational strategy to genetically engineer wild-type C-2-1 into an overproducer.
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Bacteriocinas , Pediococcus pentosaceus , Pediocinas , Pediococcus/genética , Pediococcus/metabolismo , Bacteriocinas/genética , GenômicaRESUMO
Flavonoids are natural plant products with important nutritional value, health-promoting benefits, and therapeutic potential. The use of microbial cell factories to generate flavonoids is an appealing option. The microbial biosynthesis of flavonoids is compared to the classic plant extract approach in this review, and the pharmaceutical applications were presented. This paper summarize approaches for effective flavonoid biosynthesis from microorganisms, and discuss the challenges and prospects of microbial flavonoid biosynthesis. Finally, the barriers and strategies for industrial bio-production of flavonoids are highlighted. This review offers guidance on how to create robust microbial cell factories for producing flavonoids and other relevant chemicals.
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Produtos Biológicos , Flavonoides , Flavonoides/química , Engenharia Metabólica , PlantasRESUMO
(2S)-Naringenin is an important flavonoid precursor, with multiple nutritional and pharmacological activities. Both (2S)-naringenin and other flavonoid production are hindered by poor water solubility and inhibited cell growth. To address this, we increased solubility and improved cell growth by partially glycosylating (2S)-naringenin to naringenin-7-O-glucoside, which facilitated increased extracellular secretion, by knocking out endogenous glycosyl hydrolase genes, EXG1 and SPR1, and expressing the glycosyltransferase gene (UGT733C6). Naringenin-7-O-glucoside synthesis was further improved by optimizing UDP-glucose and shikimate pathways. Then, hydrochloric acid was used to hydrolyze naringenin-7-O-glucoside to (2S)-naringenin outside the cell. Thus, our optimized Saccharomyces cerevisiae strain E32T19 produced 1184.1 mg/L (2S)-naringenin, a 7.9-fold increase on the starting strain. Therefore. we propose that glycosylation modification is a useful strategy for the efficient heterologous biosynthesis of (2S)-naringenin in S. cerevisiae.
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Flavanonas , Saccharomyces cerevisiae , Flavonoides/metabolismo , Glicosilação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
8-prenylnaringenin (8-PN) is a potent estrogen with high medicinal values. It also serves as an important precursor for many prenylated flavonoids. Microbial synthesis of 8-PN is mainly hindered by the low catalytic activity of prenyltransferases (PTS) and insufficient supply of precursors. In this work, a SfN8DT-1 from Sophora flavescens was used to improve the efficiency of (2S)-naringenin prenylation. The predicted structure of SfN8DT-1 showed that its main body is comprised of 9 α-helices and 8 loops, along with a long side chain formed by nearly 120 amino acids. SfN8DT-1 mutants with different side-chain truncated were tested in Saccharomyces cerevisiae. A mutant expressing the truncated enzyme at K62 site, designated as SfND8T-1-t62, produced the highest 8-PN titer. Molecular docking of SfN8DT-1-t62 with (2S)-naringenin and dimethylallyl diphosphate (DMAPP) showed that K185 was a potentially crucial residue. Alanine scanning within a range of 0.5 nm around these two substrates showed that the mutant K185A may decrease its affinity to substrates, which also indicated K185 was a potentially critical residue. Besides, the mutant K185W enhanced the affinity to ligands implied by the simulated saturation mutation, while the saturated mutation of K185 showed a great decrease in 8-PN production, indicating K185 is vital for the activity of SfN8DT-1. Subsequently, overexpressing the key genes of Mevalonate (MVA) pathway further improved the titer of 8-PN to 31.31 mg/L, which indicated that DMAPP supply is also a limiting factor for 8-PN synthesis. Finally, 44.92 mg/L of 8-PN was produced in a 5 L bioreactor after 120 h, which is the highest 8-PN titer reported to date.
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Dimetilaliltranstransferase , Flavanonas/biossíntese , Sophora , Dimetilaliltranstransferase/genética , Dimetilaliltranstransferase/metabolismo , Flavonoides/metabolismo , Simulação de Acoplamento Molecular , Prenilação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sophora/genética , Sophora/metabolismoRESUMO
Flavan-3-ols are a group of flavonoids that exert beneficial effects. This study aimed to enhance key metabolic processes related to flavan-3-ols biosynthesis. The engineered Saccharomyces cerevisiae strain E32 that produces naringenin from glucose was further engineered for de novo production of two basic flavan-3-ols, afzelechin (AFZ) and catechin (CAT). Through introduction of flavonoid 3-hydroxylase, flavonoid 3'-hydroxylase, dihydroflavonol 4-reductase (DFR), and leucoanthocyanidin reductase (LAR), de novo production of AFZ and CAT can be achieved. The combination of FaDFR from Fragaria × ananassa and VvLAR from Vitis vinifera was optimal. (GGGGS)2 and (EAAAK)2 linkers between DFR and LAR proved optimal for the production of AFZ and CAT, respectively. Optimization of promoters and the enhanced supply of NADPH further increased the production. By combining the best engineering strategies, the optimum strains produced 500.5 mg/L AFZ and 321.3 mg/L CAT, respectively, after fermentation for 90 h in a 5 L bioreactor. The strategies presented could be applied for a more efficient production of flavan-3-ols by various microorganisms.
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Catequina , Vitis , Flavonoides , Saccharomyces cerevisiae/genéticaRESUMO
Saccharomyces cerevisiae is one of the most important hosts in metabolic engineering. Advanced gene editing technology has been widely used in the design and construction of S. cerevisiae cell factories. With the rapid development of gene editing technology, early gene editing technologies based on recombinase and homologous recombination have been gradually replaced by new editing systems. In this review, the principle and application of gene editing technology in S. cerevisiae are summarized. Here, we first briefly describe the classical gene editing techniques of S. cerevisiae. Then elaborate the genome editing system of MegNs, ZFNs and TALENs based on endonuclease. The latest research progress is especially introduced and discussed, including the CRISPR/Cas system, multi-copy integration of heterologous metabolic pathways, and genome-scale gene editing. Finally, we envisage the application prospects and development directions of Saccharomyces cerevisiae gene editing technology.
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Edição de Genes , Saccharomyces cerevisiae , Sistemas CRISPR-Cas/genética , Endonucleases/genética , Saccharomyces cerevisiae/genética , TecnologiaRESUMO
Flavonoids have notable biological activities and have been widely used in the medicinal and chemical industries. However, single-copy integration of heterologous pathway genes limits the production of flavonoids. In this work, we designed and constructed single-step integration of multiple flavonoid (2S)-naringenin biosynthetic pathway genes in S. cerevisiae. The efficiency of the naringenin metabolic pathway gene integration into the rDNA site reached 93.7%. Subsequently, we used a high titer p-coumaric acid strain as a chassis, which eliminated feedback inhibition of tyrosine and downregulated the competitive pathway. The results indicated that increasing the supply of p-coumaric acid was effective for naringenin production. We additionally optimized the amount of donor DNA. The optimum strain produced 149.8â¯mg/L of (2S)-naringenin. The multi-copy integration of flavonoid pathway genes effectively improved (2S)-naringenin production in S. cerevisiae. We further analyzed the copy numbers and expression levels of essential genes (4CL and CHS) in the (2S)-naringenin metabolic pathway by qPCR. Higher copy numbers of the (2S)-naringenin metabolic pathway genes were associated with greater 4CL and CHS transcription, and the efficiency of naringenin production was higher. Therefore, multi-copy integration of genes in the (2S)-naringenin metabolic pathway was imperative in rewiring p-coumaric acid flux to enhance flavonoid production.
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Flavanonas , Saccharomyces cerevisiae , Vias Biossintéticas/genética , Dosagem de Genes , Engenharia Metabólica , Saccharomyces cerevisiae/genéticaRESUMO
Dihydromyricetin (DHM) is a traditional plant-extracted flavonoid with some health benefits. This study aimed to metabolically engineer the strains for DHM bioproduction. Two strains of BK-11 and BQ-21 were integrated with flavonoid 3-hydroxylase (F3H) or both F3H and flavonoid 3'-hydroxylase (F3'H). The resulting strains have expressed the enzymes of GmCPR and SlF3'5'H, and then, the promoters of INO1p and TDH1p were used to enhance further the DHM production from naringenin in Saccharomyces cerevisiae. Through multiple-copy integration, 709.6 mg/L DHM was obtained by adding 2.5 g/L naringenin in a 5 L bioreactor, implying that the synergistic effect between F3'H and flavonoid 3'5'-hydroxylase is likely to promote the DHM production. An yield of 246.4 mg/L DHM was obtained from glucose by deleting genes for branch pathways and integrating PhCHS, MsCHI, Pc4CL, and FjTAL. To our knowledge, this is the highest production reported for the de novo biosynthesis of DHM.
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BACKGROUND miR-181a is a small non-coding RNA known to be dysregulated in osteoarthritis (OA), but the role of miR-181a in human OA remains unclear. The aim of this study was to identify its function and molecular target in chondrocytes during OA pathogenesis. MATERIAL AND METHODS The function of miR-181a was assessed by gain-of-function studies in human OA chondrocytes. Potential targets of miR-181a were predicted using series of bioinformatics and intersection analysis, then confirmed by luciferase reporter assay. Gene expression was quantified using quantitative reverse transcription PCR (qRT-PCR) assays, and protein production was quantified by Western blot analysis. RESULTS The FITC apoptosis assay results indicated that the upregulation of miR-181a led to an increase of apoptosis rate in chondrocytes. Then bioinformatic analysis identified potential target sites of the miR-181a located in the 3' untranslated region of GPD1L. Dual-luciferase reporter assays results showed that GPD1L is a target gene of miR-181a. Furthermore, Western blot and qRT-PCR analysis demonstrated that miR-181a inhibited GPD1L gene expression. Increased GPD1L and decreased miRNA-181a were observed in tissues from osteoarthritis patients. Moreover, we found a highly negative correlation between miRNA-181a and GPD1L. CONCLUSIONS Our results demonstrated that miR-181a may play an important role in the pathogenesis of OA through targeting GPD1L and regulating chondrocyte apoptosis.
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Glicerolfosfato Desidrogenase/genética , MicroRNAs/genética , Osteoartrite/genética , Regiões 3' não Traduzidas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/genética , Proliferação de Células/genética , Condrócitos/metabolismo , Regulação para Baixo , Feminino , Expressão Gênica/genética , Glicerolfosfato Desidrogenase/metabolismo , Humanos , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Osteoartrite/patologia , Cultura Primária de Células , Transdução de SinaisRESUMO
BACKGROUND: The purpose of this systematic review and meta-analysis of randomized controlled trials (RCTs) was to evaluate the efficacy and safety of combined topical with intravenous tranexamic acid (TXA) versus topical, intravenous TXA alone or control for reducing blood loss after a total knee arthroplasty (TKA). METHODS: In May 2016, a systematic computer-based search was conducted in the PubMed, Embase, Cochrane Library, Web of Science, and Chinese Wanfang database. This systematic review and meta-analysis were performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement criteria. Only patients prepared for primary TKA that administration combined topical with intravenous TXA with topical TXA, intravenous (IV) TXA, or control group for reducing blood loss were included. Eligible criteria were published RCTs about combined topical with intravenous TXA with topical alone or intravenous alone. The primary endpoint was the total blood loss and need for transfusion. The complications of deep venous thrombosis (DVT) were also compiled to assess the safety of combined topical TXA with intravenous TXA. Relative risks (RRs) with 95% CIs were estimated for dichotomous outcomes, and mean differences (MDs) with 95% CIs for continuous outcomes. The Cochrane risk of bias tool was used to appraise a risk of bias. Stata 12.0 software was used for meta-analysis. RESULTS: Fifteen studies involving 1495 patients met the inclusion criteria. The pooled meta-analysis indicated that combined topical TXA with intravenous TXA can reduce the total blood loss compared with placebo with a mean of 458.66 mL and the difference is statistically significant (MDâ=â-458.66, 95% CI: -655.40 to 261.91, Pâ<â0.001). Compared with intravenous TXA, combined administrated TXA can decrease the total blood loss, and the difference is statistically significant (MDâ=â-554.03, 95% CI: -1066.21 to -41.85, Pâ=â0.034). Compared with the topical administration TXA, the pooled meta-analysis indicated that combined TXA can decrease the amount of total blood loss with mean 107.65 mL with statistically significant(MDâ=â-107.65, 95% CI: -525.55 to -239.9141.85, Pâ=â0.001). The pooled results indicated that combined topical with intravenous TXA can decrease the need for transfusion (RRâ=â0.34, 95% CI: 0.23-0.50, Pâ<â0.001). There is no significant difference between combined topical with intravenous TXA with topical or intravenous TXA (Pâ>â0.05) in terms of need for transfusion and the occurrence of DVT. CONCLUSION: Compared with topical, intravenous TXA alone or control group, combined topical with TXA, can decrease the total blood loss and subsequent need for transfusion without increasing the occurrence of DVT. The dose and timing to administration TXA is different, and more randomized controlled trials are warranted to clarify the optimal dosing and time to administration TXA.
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Antifibrinolíticos/administração & dosagem , Artroplastia do Joelho , Perda Sanguínea Cirúrgica/prevenção & controle , Ácido Tranexâmico/administração & dosagem , Administração Intravenosa , Administração Tópica , Antifibrinolíticos/uso terapêutico , Artroplastia do Joelho/efeitos adversos , Artroplastia do Joelho/métodos , Humanos , Ácido Tranexâmico/uso terapêuticoRESUMO
OBJECTIVE: To evaluate the efficacy and safety of fibrin sealant for the reduction of postoperative blood loss and transfusion requirements in patients undergoing total hip arthroplasty (THA) and total knee arthroplasty (TKA). METHODS: Electronic databases including PubMed, Embase, CENTRAL (Cochrane Controlled Trials Register), Web of Science and Google Scholar were searched from database inception to February 2016. All randomized controlled trials evaluating the efficacy and safety of topical administration of fibrin glue during primary THA or TKA were included in our meta-analysis. Transfusion requirements, total blood loss, length of hospital stay and the occurrence of infection were calculated using Stata 12.0 software. RESULTS: A total of nineteen clinical trials with 1489 patients (405 hips and 1084 knees) were finally included for meta-analysis. The results indicated that the topical administration of fibrin sealant can decrease the need for transfusion (RR = 0.33, 95%CI 0.28-0.40, P < 0.001), total blood loss (MD = -138.25, 95% CI -203.49 to -75.00), blood loss in drainage (MD -321.44, 95% CI -351.96 to -290.92, P < 0.001) and hospital stay length (MD -0.98, 95% CI -1.35 to -0.62, P < 0.001) without increasing the occurrence of infection (RR = 0.87, 95% CI 0.33 to 2.27, P = 0.775). CONCLUSION: The topical use of fibrin sealant can effectively reduce the need for transfusion, total blood loss and the volume of drainage without increasing the rate of infection.
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Artroplastia de Quadril , Artroplastia do Joelho , Adesivo Tecidual de Fibrina/administração & dosagem , Hemorragia Pós-Operatória/prevenção & controle , Administração Tópica , Transfusão de Sangue , Drenagem , Humanos , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
Microbial production of 2,3-butanediol is limited by the toxic components in the lignocellulose hydrolysate. To improve the 2,3-butanediol production via Klebsiella pneumoniae from cotton stalk hydrolysate, a method coupling a high tolerance of strain and detoxification of the hydrolysate was thus investigated in this study. The strain tolerance of K. pneumoniae to the cotton stalk hydrolysate was improved via an adaptive laboratory evolution, which involved a stepwise increase in the hydrolysate concentration in the medium. Compared with the initial strain, the resulting strain increased the biomass 3.2-fold in a medium of 20 g/L hydrolysate and produced 10.45 g/L of 2,3-butanediol at an optimal concentration of 60 g/L hydrolysate. After detoxification of cotton stalk hydrolysate, the cell metabolism of K. pneumoniae was further promoted, and the 2,3-butanediol production increased by 1.2 folds. Using fed-batch fermentation, the concentration of 2,3-butanediol reached 35.5 g/L with a yield of 0.43 g/g. The results demonstrated that the bioconversion of low-cost cotton stalk hydrolysate into 2,3-butanediol improves the economics of microbial 2,3-butanediol production.
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Butileno Glicóis/metabolismo , Microbiologia Industrial/métodos , Klebsiella pneumoniae/metabolismo , Técnicas de Cultura Celular por Lotes , Biomassa , Evolução Molecular , Fermentação , Lignina/químicaRESUMO
Endoplasmic reticulum stress (ERS)-mediated cell apoptosis has been implicated in the development of multiple diseases such as cancer, neurodegenerative diseases and ischemic reperfusion damage. Previous studies have demonstrated the adenosine-induced apoptosis in several tumor cell lines. However, the role of ERS in adenosine-induced human hepatoma HepG2 cell apoptosis remains unclear. The present study was designed to determine whether ERS is involved in adenosine-induced HepG2 cell apoptosis. The MTT assay was used to determine proliferation, and DAPI staining of cell nuclei was performed to determine cell apoptosis. The translocation of CHOP and caspase-3 was observed by immunofluorescence analysis, and the protein expression of CHOP, caspase-4 and caspase-3 was detected by Western blotting. The MTT assay demonstrated that adenosine inhibited HepG2 cell proliferation in a dose-dependent manner. DAPI staining of cell nuclei and cell cycle analysis verified cell apoptosis. The immunofluorescence assay demonstrated that adenosine induced the translocation of CHOP and of caspase-3 from the cytoplasm to the nucleus. Western blotting confirmed that CHOP, caspase-4 and caspase-3 were up-regulated in HepG2 cells after treatment with adenosine. However, JNK protein expression was not altered. These results show that ERS is involved in the adenosine-induced HepG2 cell apoptosis.