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1.
Sheng Wu Gong Cheng Xue Bao ; 36(6): 1126-1137, 2020 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-32597062

RESUMO

Bacitracin is a broad-spectrum cyclic peptide antibiotic, and mainly produced by Bacillus. Energy metabolism plays as a critical role in high-level production of target metabolites. In this study, Bacillus licheniformis DW2, an industrial strain for bacitracin production, was served as the original strain. First, our results confirmed that elimination of cytochrome bd oxidase branch via deleting gene cydB benefited bacitracin synthesis. Bacitracin titer and ATP content were increased by 10.97% and 22.96%, compared with those of original strain, respectively. Then, strengthening cytochrome aa3 oxidase branch via overexpressing gene qoxA was conducive to bacitracin production. Bacitracin titer and ATP content were increased by 18.97% and 34.00%, respectively. In addition, strengthening ADP synthesis supply is also proven as an effective strategy to promote intracellular ATP accumulation, overexpression of adenosine kinase DcK and adenylate kinase AdK could all improve bacitracin titers, among which, dck overexpression strain showed the better performance, and bacitracin titer was increased by 16.78%. Based on the above individual methods, a method of combining the deletion of gene cydB and overexpression of genes qoxA, dck were used to enhance ATP content of cells to 39.54 nmol/L, increased by 49.32% compared to original strain, and bacitracin titer produced by the final strain DW2-CQD (DW2ΔcydB::qoxA::dck) was 954.25 U/mL, increased by 21.66%. The bacitracin titer produced per cell was 2.11 U/CFU, increased by 11.05%. Collectively, this study demonstrates that improving ATP content was an efficient strategy to improve bacitracin production, and a promising strain B. licheniformis DW2-CQD was attained for industrial production of bacitracin.


Assuntos
Bacillus licheniformis , Bacitracina , Microbiologia Industrial , Bacillus licheniformis/metabolismo , Bacitracina/biossíntese , Metabolismo Energético/genética , Microbiologia Industrial/métodos
2.
Sheng Wu Gong Cheng Xue Bao ; 36(6): 1138-1149, 2020 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-32597063

RESUMO

Pyrroloquinoline quinone (PQQ), an important redox enzyme cofactor, has many physiological and biochemical functions, and is widely used in food, medicine, health and agriculture industry. In this study, PQQ production by recombinant Gluconobacter oxydans was investigated. First, to reduce the by-product of acetic acid, the recombinant strain G. oxydans T1 was constructed, in which the pyruvate decarboxylase (GOX1081) was knocked out. Then the pqqABCDE gene cluster and tldD gene were fused under the control of endogenous constitutive promoter P0169, to generate the recombinant strain G. oxydans T2. Finally, the medium composition and fermentation conditions were optimized. The biomass of G. oxydans T1 and G. oxydans T2 were increased by 43.02% and 38.76% respectively, and the PQQ production was 4.82 and 20.5 times higher than that of the wild strain, respectively. Furthermore, the carbon sources and culture conditions of G. oxydans T2 were optimized, resulting in a final PQQ yield of (51.32±0.899 7 mg/L), 345.6 times higher than that of the wild strain. In all, the biomass of G. oxydans and the yield of PQQ can be effectively increased by genetic engineering.


Assuntos
Gluconobacter oxydans , Microbiologia Industrial , Cofator PQQ , Fermentação , Técnicas de Inativação de Genes , Gluconobacter oxydans/genética , Gluconobacter oxydans/metabolismo , Microbiologia Industrial/métodos , Família Multigênica/genética , Organismos Geneticamente Modificados , Cofator PQQ/biossíntese , Cofator PQQ/genética , Regiões Promotoras Genéticas/genética
3.
Sheng Wu Gong Cheng Xue Bao ; 36(6): 1209-1215, 2020 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-32597070

RESUMO

Bioreactors have been central in monoclonal antibodies and vaccines manufacturing by mammalian cells in suspension culture. Numerical simulation of five impeller combinations in a stirred bioreactor was conducted, and characteristics of velocity vectors, distributions of gas hold-up, distributions of shear rate in the bioreactor using 5 impeller combinations were numerically elucidated. In addition, genetically engineered CHO cells were cultivated in bioreactor installed with 5 different impeller combinations in fed-batch culture mode. The cell growth and antibody level were directly related to the maximum shear rate in the bioreactor, and the highest viable cell density and the peak antibody level were achieved in FBMI3 impeller combination, indicating that CHO cells are sensitive to shear force produced by impeller movement when cells were cultivated in bioreactor at large scale, and the maximum shear rate would play key roles in scaling-up of bioreactor at industrial scale.


Assuntos
Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Simulação por Computador , Microbiologia Industrial , Animais , Reatores Biológicos/normas , Células CHO , Contagem de Células , Cricetinae , Cricetulus , Microbiologia Industrial/instrumentação , Microbiologia Industrial/métodos
4.
Sheng Wu Gong Cheng Xue Bao ; 36(5): 920-931, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32567275

RESUMO

The capacity for thermal tolerance is critical for industrial enzyme. In the past decade, great efforts have been made to endow wild-type enzymes with higher catalytic activity or thermostability using gene engineering and protein engineering strategies. In this study, a recently developed SpyTag/SpyCatcher system, mediated by isopeptide bond-ligation, was used to modify a rumen microbiota-derived xylanase XYN11-6 as cyclized and stable enzyme C-XYN11-6. After incubation at 60, 70 or 80 ℃ for 10 min, the residual activities of C-XYN11-6 were 81.53%, 73.98% or 64.41%, which were 1.48, 2.92 or 3.98-fold of linear enzyme L-XYN11-6, respectively. After exposure to 60-90°C for 10 min, the C-XYN11-6 remained as soluble in suspension, while L-XYN11-6 showed severely aggregation. Intrinsic and 8-anilino-1-naphthalenesulfonic acid (ANS)-binding fluorescence analysis revealed that C-XYN11-6 was more capable of maintaining its conformation during heat challenge, compared with L-XYN11-6. Interestingly, molecular cyclization also conferred C-XYN11-6 with improved resilience to 0.1-50 mmol/L Ca²âº or 0.1 mmol/L Cu²âº treatment. In summary, we generated a thermal- and ion-stable cyclized enzyme using SpyTag/SpyCatcher system, which will be of particular interest in engineering of enzymes for industrial application.


Assuntos
Endo-1,4-beta-Xilanases , Estabilidade Enzimática , Microbiologia Industrial , Microbiota , Rúmen , Animais , Ciclização , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/metabolismo , Microbiologia Industrial/métodos , Engenharia de Proteínas , Rúmen/enzimologia , Rúmen/microbiologia , Temperatura
5.
Sheng Wu Gong Cheng Xue Bao ; 36(5): 1002-1011, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32567283

RESUMO

Uridine-cytidine kinase, an important catalyst in the compensation pathway of nucleotide metabolism, can catalyze the phosphorylation reaction of cytidine to 5'-cytidine monophosphate (CMP), but the reaction needs NTP as the phosphate donor. To increase the production efficiency of CMP, uridine-cytidine kinase gene from Thermus thermophilus HB8 and polyphosphate kinase gene from Rhodobacter sphaeroides were cloned and expressed in Escherichia coli BL21(DE3). Uridine-cytidine kinase was used for the generation of CMP from cytidine and ATP, and polyphosphate kinase was used for the regeneration of ATP. Then, the D403 metal chelate resin was used to adsorb Ni²âº to form an immobilized carrier, and the immobilized carrier was specifically combined with the recombinant enzymes to form the immobilized enzymes. Finally, single-factor optimization experiment was carried out to determine the reaction conditions of the immobilized enzyme. At 30 °C and pH 8.0, 60 mmol/L cytidine and 0.5 mmol/L ATP were used as substrates to achieve 5 batches of high-efficiency continuous catalytic reaction, and the average molar yield of CMP reached 91.2%. The above method has the advantages of low reaction cost, high product yield and high enzyme utilization rate, and has good applied value for industrial production.


Assuntos
Citidina Monofosfato , Microbiologia Industrial , Fosfotransferases (Aceptor do Grupo Fosfato) , Uridina Quinase , Citidina Monofosfato/metabolismo , Escherichia coli/genética , Microbiologia Industrial/métodos , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo
6.
Gene ; 742: 144586, 2020 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-32179171

RESUMO

Pycnoporus sanguineus, an edible mushroom, produces antimicrobial and antitumor bioactive compounds and pH- and thermo- stable laccases that have multiple potential biotechnological applications. Here we reported the complete genome of the species Pycnoporus sanguineus ACCC 51,180 by using the combination of Illumina HiSeq X Ten and the PacBio sequencing technology. The represented genome is 36.6 Mb composed of 59 scaffolds with 12,086 functionally annotated protein-coding genes. The genome of Pycnoporus sanguineus encodes at least 19 biosynthetic gene clusters for secondary metabolites, including a terpene cluster for biosynthesis of the antitumor clavaric acid. Seven laccases were identified, while 22 genes were found to be involved in the kynurenine pathway in which the intermediate metabolite 3-hydroxyanthranilic acid were catalyzed by laccases into cinnabarinic acid. This study represented the third genome of the genus Pycnoporus, and wound facilitate the exploration of useful sources from Pycnoporus sanguineus for future industrial applications.


Assuntos
Proteínas Fúngicas/genética , Genoma Fúngico/genética , Microbiologia Industrial/métodos , Lacase/genética , Pycnoporus/genética , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Cinurenina/metabolismo , Lacase/metabolismo , Engenharia Metabólica , Oxazinas/metabolismo , Estabilidade Proteica , Pycnoporus/enzimologia , Metabolismo Secundário/genética
7.
Arch Microbiol ; 202(5): 1203-1209, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32077990

RESUMO

Selenium nanoparticles (Se NPs) were synthesized using Saccharomyces cerevisiae yeast. Influences of different amounts of sodium selenite (5.0, 10.0, 15.0, 20.0, and 25 µg) were evaluated on growth of yeast during incubation at 32 °C, during 4 days. UV-Vis spectroscopy results have shown that synthesized Se NPs had broad emission peak (λmax) in the wavelength around 350 nm which demonstrated that formation of Se NPs occurred in intracellular manner. Physico-chemical characteristics of the synthesized Se NPs using dynamic light scattering particle-size analyzer indicated that the fabricated Se NPs had particle size, polydispersity index, and zeta potential ranging from 75 to 709 nm, 0.189 to 0989, and -7.06 to -10.3 mV, respectively. Obtained results revealed that intracellular Se NPs with minimum particle size (75 nm), maximum zeta potential (-10.3 mV), and antioxidant activity (48.5%) were synthesized using minimum amount of selenium salt (5 µg). However, most uniform Se NPs were formed using maximum amount of selenium salt (25 µg). Results also indicated that by increasing amount of sodium selenite in the culture media, from 5.0 to 25 µg, antioxidant activity of the formed Se NPs decreased from 48.5 to 20.8, respectively.


Assuntos
Biotecnologia/métodos , Microbiologia Industrial/métodos , Nanopartículas/química , Saccharomyces cerevisiae/metabolismo , Selênio/química , Meios de Cultura , Tamanho da Partícula
8.
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
9.
Food Chem ; 313: 126138, 2020 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31931424

RESUMO

The fermentation products of edible fungi are rich in anthraquinones and have a variety of activities, including the antioxidant activity. Because of the large number of combinations, it is very difficult to obtain the optimal multi-strains co-fermentation to improve the yield of anthraquinone. In the present study, an intelligent model based on artificial neural networks (ANNs) using backpropagation (BP) and radial basis function (RBF) algorithms was developed and validated to predict the anthraquinone contents in 136 two fungi and 680 three fungi co-fermented products. After experimental validation of the anthraquinone contents, the mean absolute error and the mean bias error of the results from RBF ANN were lower than those from BP ANN. The results indicated that the anthraquinone contents in A. bisporus, C. comatus and H. erinaceus co-fermentation product was the highest (2.11%). Furthermore, this co-fermentation product showed strong antioxidant activity.


Assuntos
Antraquinonas/metabolismo , Antioxidantes/metabolismo , Fungos/metabolismo , Microbiologia Industrial/métodos , Redes Neurais de Computação , Algoritmos , Fermentação , Reprodutibilidade dos Testes
10.
Nat Chem Biol ; 16(2): 113-121, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31974527

RESUMO

Microbial chemical production is a rapidly growing industry, with much of the growth fueled by advances in synthetic biology. New approaches have enabled rapid strain engineering for the production of various compounds; however, translation to industry is often problematic because native phenotypes of model hosts prevent the design of new low-cost bioprocesses. Here, we argue for a new approach that leverages the native stress-tolerant phenotypes of non-conventional microbes that directly address design challenges from the outset. Growth at high temperature, high salt and solvent concentrations, and low pH can enable cost savings by reducing the energy required for product separation, bioreactor cooling, and maintaining sterile conditions. These phenotypes have the added benefit of allowing for the use of low-cost sugar and water resources. Non-conventional hosts are needed because these phenotypes are polygenic and thus far have proven difficult to recapitulate in the common hosts Escherichia coli and Saccharomyces cerevisiae.


Assuntos
Bactérias/metabolismo , Fungos/metabolismo , Microbiologia Industrial/métodos , Bactérias/genética , Fungos/genética , Engenharia Genética , Concentração de Íons de Hidrogênio , Microbiologia Industrial/economia , Engenharia Metabólica , Microrganismos Geneticamente Modificados/fisiologia , Pressão Osmótica , Fenótipo , Solventes , Estresse Fisiológico
11.
Proc Natl Acad Sci U S A ; 117(3): 1404-1413, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31915296

RESUMO

Bio-based production technologies may complement or replace petroleum-based production of chemicals, but they face a number of technical challenges, including product toxicity and/or water insolubility. Plants and microorganisms naturally biosynthesize chemicals that often are converted into derivatives with reduced toxicity or enhanced solubility. Inspired by this principle, we propose a bioderivatization strategy for biotechnological chemicals production, defined as purposeful biochemical derivatization of intended target molecules. As proof of principle, the effects of hydrophobic (e.g., esterification) and hydrophilic (e.g., glycosylation) bioderivatization strategies on the biosynthesis of a relatively toxic and poorly soluble chemical, 1-octanol, were evaluated in Escherichia coli and Synechocystis sp. PCC 6803. The 1-octanol pathway was first optimized to reach product titers at which the host displayed symptoms of toxicity. Solvent overlay used to capture volatile products partially masked product toxicity. Regardless of whether solvent overlay was used, most strains with bioderivatization had a higher molar product titer and product yield, as well as improved cellular growth and glucose consumption, compared with strains without bioderivatization. The positive effect on bioproduction was observed with both the hydrophobic and hydrophilic strategies. Interestingly, in several combinations of genotype/induction strength, bioderivatization had a positive effect on productivity without any apparent effect on growth. We attribute this to enhanced product solubility in the aqueous or solvent fraction of the bioreactor liquid phase (depending on the derivative and medium used), with consequent enhanced product removal. Overall, under most conditions, a benefit of bioproduction was observed, and the bioderivatization strategy could be considered for other similar chemicals as well.


Assuntos
1-Octanol/metabolismo , Microbiologia Industrial/métodos , Biodegradação Ambiental , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Synechocystis/crescimento & desenvolvimento , Synechocystis/metabolismo
12.
Prep Biochem Biotechnol ; 50(1): 74-81, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31517565

RESUMO

Ectoine has fostered the development of products for skin care and cosmetics. In this study, we employed the marine bacterial strain Marinococcus sp. MAR2 to increase ectoine production by optimizing medium constituents using Response Surface Methodology (RSM) and a fed-batch strategy. The results from the steepest ascent and central composite design indicated that 54 g/L of yeast extract, 14.0 g/L of ammonium acetate, 74.4 g/L of sodium glutamate, and 6.2 g/L of sodium citrate constituted the optimal medium with maximum ectoine production (3.5 g/L). In addition, we performed fed-batch culture in the bioreactor, combining pH and dissolved oxygen to produce ectoine by Marinococcus sp. MAR2. The ectoine production, content, and productivity of 5.6 g/L, 10%, and 3.9 g/L/day were further reached by a fed-batch culture. Thus, the ectoine production by Marinococcus sp. MAR2 using RSM and fed-batch strategy shows its potential for industrial production.


Assuntos
Diamino Aminoácidos/metabolismo , Bacillaceae/metabolismo , Técnicas de Cultura Celular por Lotes/métodos , Microbiologia Industrial/métodos , Acetatos/análise , Acetatos/metabolismo , Bacillaceae/crescimento & desenvolvimento , Técnicas de Cultura Celular por Lotes/instrumentação , Reatores Biológicos , Meios de Cultura/química , Meios de Cultura/metabolismo , Desenho de Equipamento , Fermentação , Microbiologia Industrial/instrumentação , Citrato de Sódio/análise , Citrato de Sódio/metabolismo , Glutamato de Sódio/análise , Glutamato de Sódio/metabolismo
13.
Prep Biochem Biotechnol ; 50(1): 91-97, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31517567

RESUMO

Xylanases have gained increasing importance due to their diverse applications in the food, paper, and pharmaceutical industries, however, the production of these enzymes currently uses expensive substrates. It has already been estimated that more than 30% of the enzyme production cost originates from the substrate. The present study aimed to optimize the production of extracellular xylanases by the Bacillus sp. TC-DT 13 using solid-state fermentation with agro-industrial residues, with a view at reducing the production cost of these enzymes. All the agro-industrial residues were tested in submerged fermentation to select the best inductor to produce xylanase. Among these residues, wheat bran was selected as the best inducer of xylanase production with 1500 U/mL. Regarding solid-state fermentation, the use of wheat bran as the only fermentation substrate was used and a ratio of 1:4 moisture over a time of 144 hours induced higher amount of xylanase reaching 2943 U/g. The use of carbon and nitrogen sources did not result in the increase in production of xylanolitic enzymes. The use of agro-industrial residues in the solid-state fermentation, besides increasing the production of xylanase, reduces the cost of production and is an environmentally friendly alternative.


Assuntos
Bacillus/enzimologia , Fibras na Dieta/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Bacillus/metabolismo , Carbono/metabolismo , Fermentação , Microbiologia Industrial/economia , Microbiologia Industrial/métodos , Nitrogênio/metabolismo , Temperatura
14.
Prep Biochem Biotechnol ; 50(1): 10-17, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31430215

RESUMO

Due to wide application of laccase, many researchers have shown great interest in over production of white-rot fungi laccase by co-culture. In this study, a white-rot fungus Trametes hirsuta SSM-3, and a yeast Sporidiobolus pararoseus SSM-8 were isolated and identified from Mulberry fruit. The capacity of S. pararoseus to enhance laccase production was remarkable in T. hirsuta, yielding 31777 ± 742 U/L, about 9.9 times higher than the result from the monoculture. The stimulatory factor in the S. pararoseus cells might be temperature-sensitive. The laccase production was enhanced by oil-extract of S. pararoseus and ß-carotene induction. The amylase activity was decreased rapidly when strain S. pararoseus SSM-8 was inoculated. The glucose deprivation was occurred both in the mono-culture and co-culture process, and S. pararoseus propagated slowly in co-culture all the time. Native-PAGE revealed an increase of laccase-1(lac-1) level and a laccase-3 (lac-3) in the co-culture. Therefore, it was concluded that competition for resources between the co-cultured microbes leaded to amylase decreasing and the enhanced production of laccase. This conclusion was helpful for the development of laccase fermentation industry because it provided an effective, simple and economic method to improve the yield of laccase.


Assuntos
Proteínas Fúngicas/metabolismo , Lacase/metabolismo , Trametes/metabolismo , Leveduras/metabolismo , Técnicas de Cocultura/métodos , Fermentação , Microbiologia Industrial/métodos , Morus/microbiologia , beta Caroteno/metabolismo
15.
Prep Biochem Biotechnol ; 50(1): 66-73, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31502910

RESUMO

Response surface methodology was applied to maximize the yield and production of carotenoids by Sporidiobolus pararoseus WZ012 using citrus juice. A high concentration of yeast extract and citrus juice favored carotenoid production and biomass concentration, respectively. Under optimal conditions, a more than 51 percent (from 860 to 1300 µg/g) and 62 percent (from 17.05 to 27.66 mg/L) respective enhancement in intracellular and total carotenoid production was achieved. Finally, this process was successfully upscaled in a 5-L fermentor. A comparison of the carotenoid distributions revealed that torulene (61.3%) was the dominant carotenoid when using the citrus based medium, while the main carotenoid was ß-carotene (62.5%) when using the glucose medium. The present work provides an alternative method to produce high-value products derived from waste and low-grade citrus.


Assuntos
Basidiomycota/metabolismo , Citrus/metabolismo , Microbiologia Industrial/métodos , Biomassa , Reatores Biológicos , Carotenoides/metabolismo , Fermentação , Glucose/metabolismo , Microbiologia Industrial/instrumentação
16.
Enzyme Microb Technol ; 133: 109463, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31874686

RESUMO

Penicillium subrubescens is an ascomycete fungus with an enriched content of specific carbohydrate-active enzyme families involved in plant biomass degradation, which makes this strain a promising industrial cell factory for enzyme production. The development of tools that allow genetic manipulation is crucial for further strain improvement and the functional characterization of its genes. In this context, the CRISPR/Cas9 system represents an excellent option for genome editing due to its high efficiency and versatility. To establish CRISPR/Cas9 genome editing in P. subrubescens, first a method for protoplast generation and transformation was developed, using hygromycin as selection marker. Then the CRISPR/Cas9 system was established in P. subrubescens by successfully deleting the ku70 gene, which is involved in the non-homologous end joining DNA repair mechanism. Phenotypic characterization of the mutants showed that ku70 mutation did not affect P. subrubescens growth at optimal temperature and Δku70 strains showed similar protein production pattern to the wild type.


Assuntos
Sistemas CRISPR-Cas , Enzimas/biossíntese , Edição de Genes , Penicillium/enzimologia , Penicillium/genética , Genoma Fúngico , Microbiologia Industrial/métodos , Fenótipo
17.
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
18.
World J Microbiol Biotechnol ; 36(1): 1, 2019 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-31811524

RESUMO

Metabolome defines a set of metabolites present in a biological sample, which provides an immediate and dynamic recording of microbes in response to genetic and/or environmental perturbations. In recent years, metabolomics in combination with other omics diagnostic tools such as genomics, transcriptomics and proteomics is focused on addressing open biological questions that accelerate our understanding of the system as a whole and boost the use of systems metabolic engineering tools in industrial settings. In this review article, we summarize the applications of metabolomics to industrial microbial fermentations with respect to the bulk production of organic acids, amino acids, enzymes, antibiotics and therapeutic proteins. In addition, future prospects regarding metabolomics-assisted research are provided.


Assuntos
Microbiologia Industrial/métodos , Engenharia Metabólica , Metabolômica/métodos , Aminoácidos/metabolismo , Antibacterianos/metabolismo , Biocombustíveis , Biologia Computacional , Bases de Dados Factuais , Enzimas/metabolismo , Fermentação , Genômica , Metaboloma , Proteômica , Biologia de Sistemas
19.
BMC Biotechnol ; 19(1): 98, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31842877

RESUMO

BACKGROUND: Rapeseed cake (RSC), as the intermediate by-product of oil extraction from the seeds of Brassica napus, can be converted into rapeseed meal (RSM) by solvent extraction to remove oil. However, compared with RSM, RSC has been rarely used as a raw material for microbial fermentation, although both RSC and RSM are mainly composed of proteins, carbohydrates and minerals. In this study, we investigated the feasibility of using untreated low-cost RSC as nitrogen source to produce the valuable cyclic lipopeptide antibiotic iturin A using Bacillus amyloliquefaciens CX-20 in submerged fermentation. Especially, the effect of oil in RSC on iturin A production and the possibility of using lipases to improve the iturin A production were analyzed in batch fermentation. RESULTS: The maximum production of iturin A was 0.82 g/L at the optimal initial RSC and glucose concentrations of 90 and 60 g/L, respectively. When RSC was substituted with RSM as nitrogen source based on equal protein content, the final concentration of iturin A was improved to 0.95 g/L. The production of iturin A was further increased by the addition of different lipase concentrations from 0.1 to 5 U/mL into the RSC medium for simultaneous hydrolysis and fermentation. At the optimal lipase concentration of 0.5 U/mL, the maximal production of iturin A reached 1.14 g/L, which was 38.15% higher than that without any lipase supplement. Although rapeseed oil and lipase were firstly shown to have negative effects on iturin A production, and the effect would be greater if the concentration of either was increased, their respective negative effects were reduced when used together. CONCLUSIONS: Appropriate relative concentrations of lipase and rapeseed oil were demonstrated to support optimal iturin A production. And simultaneous hydrolysis with lipase and fermentation was an effective way to produce iturin A from RSC using B. amyloliquefaciens CX-20.


Assuntos
Bacillus amyloliquefaciens/metabolismo , Brassica napus/microbiologia , Fungicidas Industriais/metabolismo , Microbiologia Industrial/métodos , Lipase/química , Peptídeos Cíclicos/biossíntese , Biocatálise , Meios de Cultura/metabolismo , Fermentação , Sementes/microbiologia , Resíduos/análise
20.
Int J Mol Sci ; 21(1)2019 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-31877855

RESUMO

Carbonic anhydrase (CA) is a diffusion-controlled enzyme that rapidly catalyzes carbon dioxide (CO2) hydration. CA has been considered as a powerful and green catalyst for bioinspired CO2 capture and utilization (CCU). For successful industrial applications, it is necessary to expand the pool of thermostable CAs to meet the stability requirement under various operational conditions. In addition, high-level expression of thermostable CA is desirable for the economical production of the enzyme. In this study, a thermostable CA (tdCA) of Thermosulfurimonas dismutans isolated from a deep-sea hydrothermal vent was expressed in Escherichia coli and characterized in terms of expression level, solubility, activity and stability. tdCA showed higher solubility, activity, and stability compared to those of CA from Thermovibrio ammonificans, one of the most thermostable CAs, under low-salt aqueous conditions. tdCA was engineered for high-level expression by the introduction of a point mutation and periplasmic expression via the Sec-dependent pathway. The combined strategy resulted in a variant showing at least an 8.3-fold higher expression level compared to that of wild-type tdCA. The E. coli cells with the periplasmic tdCA variant were also investigated as an ultra-efficient whole-cell biocatalyst. The engineered bacterium displayed an 11.9-fold higher activity compared to that of the recently reported system with a halophilic CA. Collectively these results demonstrate that the highly expressed periplasmic tdCA variant, either in an isolated form or within a whole-cell platform, is a promising biocatalyst with high activity and stability for CCU applications.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Dióxido de Carbono/metabolismo , Anidrases Carbônicas/metabolismo , Escherichia coli/metabolismo , Periplasma/enzimologia , Sequência de Aminoácidos , Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Anidrases Carbônicas/química , Anidrases Carbônicas/genética , Estabilidade Enzimática , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica , Fontes Hidrotermais , Microbiologia Industrial/métodos , Periplasma/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Solubilidade , Temperatura
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