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1.
Trends Biotechnol ; 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-32005372

RESUMO

Based on the development of automatic devices and rapid assay methods, various high-throughput screening (HTS) strategies have been established for improving the performance of industrial microorganisms. We discuss the most significant factors that can improve HTS efficiency, including the construction of screening libraries with high diversity and the use of new detection methods to expand the search range and highlight target compounds. We also summarize applications of HTS for enhancing the performance of industrial microorganisms. Current challenges and potential improvements to HTS in industrial biotechnology are discussed in the context of rapid developments in synthetic biology, nanotechnology, and artificial intelligence. Rational integration will be an important driving force for constructing more efficient industrial microorganisms with wider applications in biotechnology.

2.
J Biotechnol ; 310: 6-12, 2020 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-31926982

RESUMO

3,4-Dihydroxyphenyl-L-alanine (L-DOPA) is the most important antiparkinsonian drug, and tyrosine phenol-lyase (TPL)-based enzyme catalysis process is one of the most adopted methods on industrial scale production. TPL activity and stability represent the rate-limiting step in L-DOPA synthesis. Here, 25 TPL mutants were predicted, and two were confirmed as exhibiting the highest L-DOPA production and named E313W and E313M. The L-DOPA production from E313W and E313M was 47.5 g/L and 62.1 g/L, which was 110.2 % and 174.8 % higher, respectively, than that observed from wild-type (WT) TPL. The Km of E313W and E313M showed no apparent decrease, whereas the kcat of E313W and E313M improved by 45.5 % and 36.4 %, respectively, relative to WT TPL. Additionally, E313W and E313M displayed improved thermostability, a higher melting temperature, and enhanced affinity between for pyridoxal-5'-phosphate. Structural analysis of the mutants suggested increased stability of the N-terminal region via enhanced interactions between the mutated residues and H317. Application of these mutants in a substrate fed-batch strategy as whole-cell biocatalysts allows realization of a cost-efficient short fermentation period resulting in high L-DOPA yield.

3.
J Agric Food Chem ; 68(4): 1015-1021, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31690080

RESUMO

(2S)-Naringenin, a (2S)-flavanone, is widely used in the food, chemical, and pharmaceutical industries because of its diverse physiological activities. The production of (2S)-naringenin in microorganisms provides an ideal source that reduces the cost of the flavonoid. To achieve efficient production of (2S)-naringenin in Saccharomyces cerevisiae (S. cerevisiae), we constructed a biosynthetic pathway from p-coumaric acid, a cost-effective and more efficient precursor. The (2S)-naringenin synthesis pathway genes were integrated into the yeast genome to obtain a (2S)-naringenin production strain. After gene dosage experiments, the genes negatively regulating the shikimate pathway and inefficient chalcone synthase activity were verified as factors limiting (2S)-naringenin biosynthesis. With fed-batch process optimization of the engineered strain, the titer of (2S)-naringenin reached 648.63 mg/L from 2.5 g/L p-coumaric acid. Our results indicate that the constitutive production of (2S)-naringenin from p-coumaric acid in S. cerevisiae is highly promising.

4.
J Chromatogr Sci ; 57(8): 950-960, 2020 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-31602470

RESUMO

Ionic liquid-based hollow-fiber liquid-liquid-liquid microextraction (IL-HF-LLLME) coupled to capillary electrophoresis (CE) has been developed for the determination of six sulfonamides (SAs) in aquaculture waters. A series of extraction parameters was optimized to enhance the extraction efficiency, which included type and pore size of hollow fiber, type and composition of extraction solvent, pH value of donor phase, the concentration of acceptor phase and the mass ratio of donor phase to acceptor phase along with extraction temperature and time. Under optimal conditions, the IL-HF-LLLME-CE method provided a wide liner range for six SAs from 2 to 1,000 µg L-1 (r2 ≥ 0.9995), the limits of the detection from 0.25 to 0.48 and the enrichment factors from 122 to 230, respectively. Relative standard deviations for intra- and interday precision were 1.4-5.3% and 1.8-7.5% (n = 5), respectively. The proposed method was successfully applied for the determination of trace-level SAs in seven real-world aquaculture water samples with good recoveries (80.4-100.7%). Also, sulfamerazine and sulfamethoxazole were detected at the level of 0.52-1.60 µg L-1 in two water samples. Due to its good sensitivity, simple operation, short analysis time and eco-friendliness, the developed method has a great application potential in analysis of trace SA residues in aquaculture waters.

5.
Bioresour Technol ; 295: 122248, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31627065

RESUMO

Dissolved oxygen (DO) supply plays essential roles in microbial organic acid production. Candida glabrata, as a dominant strain for producing pyruvic acid, principally converts glucose to pyruvic acid through glycolysis. However, this process relies excessively on high extracellular DO content. In this study, in combination with specific motif analysis of gene promoters, hypoxia-inducible factor 1 (HIF1) was engineered to improve the transcription level of some enzymes related to pyruvic acid synthesis under low DO level and directly led to increased pyruvic acid production and glycolysis efficiency. Moreover, the intracellular stability of HIF1 was further optimized from different aspects to maximize pyruvic acid accumulation. Finally, the pyruvic acid titer in a 5-L batch bioreactor with 10% DO level reached 53.1 g/L. As pyruvic acid is involved in the biosynthesis of various products, these findings suggest that HIF1-enabled regulation method has significant potential for increasing the synthesis of other chemicals in microorganisms.


Assuntos
Candida glabrata , Ácido Pirúvico , Reatores Biológicos , Glucose , Fator 1 Induzível por Hipóxia
6.
Enzyme Microb Technol ; 131: 109430, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31615664

RESUMO

L-tyrosine is an amino acid that has been widely used in the food, agriculture and pharmaceutical industries. In order to screen a tyrosine phenol-lyase (TPL) with excellent catalytic performance for L-tyrosine production, TPL genes from Citrobacter freundii (CfTPL), Erwinia herbicola (EhTPL) and Rhodobacter capsulatus (TutA) were codon-optimized and overexpressed in Escherichia coli. The results showed that EhTPL had the highest whole cell catalysis activity and tyrosine yield (3-fold that of CfTPL). The results of RT-qPCR and a stability analysis also revealed that EhTPL had a higher transcriptional level in whole cell catalysis, while CfTPL possessed greater stability. Conditions for the production by whole cell transformation were optimized in terms of reaction conditions and fed-batch strategy. Finally, the maximum production was obtained with a titer of 48.5 g·L-1 by intermittent feeding with a conversion ratio of 75%.

7.
ACS Synth Biol ; 8(11): 2514-2523, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31622552

RESUMO

Plants possess myriads of secondary metabolites with a broad spectrum of health-promoting benefits. To date, plant extraction is still the primary route to produce high-value natural products which inherently suffers from economics and scalability issues. Heterologous expression of plant biosynthetic gene clusters in microbial host is considered as a feasible approach to overcoming these limitations. Oleaginous yeast produces a large amount of lipid bodies, the abundant membrane structure and the lipophilic environment provide the ideal environment for the regioselectivity and stereoselectivity of many plant-derived P450 enzymes. In this work, we used modular method to construct, characterize, and optimize the flavonoid pathways in Yarrowia lipolytica. We also evaluated various precursor biosynthetic routes and unleashed the metabolic potential of Y. lipolytica to produce flavonoids and hydroxylated flavonoids. Specifically, we have identified that chalcone synthase (CHS) and cytochrome P450 reductases (CPR) were the bottlenecks of hydroxylated flavonoid production. We determined the optimal gene copy number of CHS and CPR to be 5 and 2, respectively. We further removed precursor pathway limitations by expressing genes associated with chorismate and malonyl-CoA supply. With pH and carbon-nitrogen ratio (C/N) optimization, our engineered strain produced 252.4 mg/L naringenin, 134.2 mg/L eriodictyol, and 110.5 mg/L taxifolin from glucose in shake flasks. Flavonoid and its hydroxylated derivatives are most prominently known as antioxidant and antiaging agents. These findings demonstrate our ability to harness the oleaginous yeast as the microbial workhorse to expand nature's biosynthetic potential, enabling us to bridge the gap between drug discovery and natural product manufacturing.

8.
ACS Omega ; 4(12): 15074-15080, 2019 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-31552350

RESUMO

Gluconobacter oxydans plays an important role in the conversion of d-sorbitol to l-sorbose, which is an essential intermediate for the industrial-scale production of vitamin C. In the fermentation process, some d-sorbitol could be converted to d-fructose and other byproducts by uncertain dehydrogenases. Genome sequencing has revealed the presence of diverse genes encoding dehydrogenases in G. oxydans. However, the characteristics of most of these dehydrogenases remain unclear. Therefore, the analyses of these unknown dehydrogenases could be useful for identifying those related to the production of d-fructose and other byproducts. Accordingly, dehydrogenases in G. oxydans WSH-003, an industrial strain used for vitamin C production, were examined. A nicotinamide adenine dinucleotide (NAD)-dependent dehydrogenase, which was annotated as xylitol dehydrogenase 2, was identified, codon-optimized, and expressed in Escherichia coli BL21 (DE3) cells. The enzyme exhibited a high preference for NAD+ as the cofactor, while no activity with nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, or pyrroloquinoline quinone was noted. Although this enzyme presented high similarity with NAD-dependent xylitol dehydrogenase, it showed high activity to catalyze d-sorbitol to d-fructose. Unlike the optimum temperature and pH for most of the known NAD-dependent xylitol dehydrogenases (30-40 °C and about 6-8, respectively), those for the identified enzyme were 57 °C and 12, respectively. The values of K m and V max of the identified dehydrogenase toward l-sorbitol were 4.92 µM and 196.08 µM/min, respectively. Thus, xylitol dehydrogenase 2 can be useful for the cofactor-reduced nicotinamide adenine dinucleotide regeneration under alkaline conditions, or its knockout can improve the conversion ratio of d-sorbitol to l-sorbose.

9.
J Ind Microbiol Biotechnol ; 46(12): 1631-1641, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31535250

RESUMO

L-DOPA is a key pharmaceutical agent for treating Parkinson's, and market demand has exploded due to the aging population. There are several challenges associated with the chemical synthesis of L-DOPA, including complicated operation, harsh conditions, and serious pollution. A biocatalysis route for L-DOPA production is promising, especially via a route catalyzed by tyrosine phenol lyase (TPL). In this study, using TPL derived from Erwinia herbicola (Eh-TPL), a mutant Eh-TPL was obtained by integrating enzyme evolution and high-throughput screening methods. L-DOPA production using recombinant Escherichia coli BL21 (DE3) cells harbouring mutant Eh-TPL was enhanced by 36.5% in shake flasks, and the temperature range and alkali resistance of the Eh-TPL mutant were promoted. Sequence analysis revealed two mutated amino acids in the mutant (S20C and N161S), which reduced the length of a hydrogen bond and generated new hydrogen bonds. Using a fed-batch mode for whole-cell catalysis in a 5 L bioreactor, the titre of L-DOPA reached 69.1 g L-1 with high productivity of 11.52 g L-1 h-1, demonstrating the great potential of Eh-TPL variants for industrial production of L-DOPA.


Assuntos
Levodopa/biossíntese , Tirosina Fenol-Liase/metabolismo , Biocatálise , Reatores Biológicos , Escherichia coli/genética , Escherichia coli/metabolismo
10.
Bioresour Technol ; 293: 122098, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31514118

RESUMO

As a stable microbial polysaccharide, scleroglucan has extensive application in the food, medicine, and cosmetics industries. However, its large-scale industrial application is limited by its high production cost, low yield, long production time, etc. This study aims to enhance scleroglucan production by Sclerotium rolfsii WSH-G01. Based on the analysis of batch fermentation kinetics parameters, a pH-shift strategy was adopted. Through systematic kinetics analysis, a 32.4 g/L scleroglucan was accomplished. The kinetic model of the pH-shift batch fermentation process was established using a logistic equation, Luedeking-Piret equation, and a Luedeking-Piret-like equation. As decreased glucose concentration could cause decreased scleroglucan synthesis rates during the batch fermentation process, 30 g/L glucose was fed in the later phase of fermentation. As a result, scleroglucan production increased to 42 g/L, with a productivity of 0.5 g/L·h. Thus, the pH-shift strategy and feeding approach could be useful for industrial scleroglucan production.


Assuntos
Basidiomycota , Glucanos , Fermentação , Concentração de Íons de Hidrogênio , Cinética
11.
Synth Syst Biotechnol ; 4(3): 134-141, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31384676

RESUMO

2-keto-d-gluconic acid (2-KGA) is a key precursor for synthesising vitamin C and isovitamin C. However, phage contamination is as constant problem in industrial production of 2-KGA using Pseudomonas fluorescens. Gluconobacter holds promise for producing 2-KGA due to impressive resistance to hypertonicity and acids, and high utilisation of glucose. In this study, the 2-KGA synthesis pathway was regulated to enhance production of 2-KGA and reduce accumulation of the by-products 5-keto-d-gluconic acid (5-KGA) and d-gluconic acid (D-GA) in the 2-KGA producer Gluconobacter japonicus CGMCC 1.49. Knocking out the ga5dh-1 gene from a competitive pathway and overexpressing the ga2dh-A gene from the 2-KGA synthesis pathway via homologous recombination increased the titre of 2-KGA by 63.81% in shake flasks. Additionally, accumulation of 5-KGA was decreased by 63.52% with the resulting G. japonicas-Δga5dh-1-ga2dh-A strain. Using an intermittent fed-batch mode in a 3 L fermenter, 2-KGA reached 235.3 g L-1 with a 91.1% glucose conversion rate. Scaling up in a 15 L fermenter led to stable 2-KGA titre with productivity of 2.99 g L-1 h-1, 11.99% higher than in the 3 L fermenter, and D-GA and 5-KGA by-products were completely converted to 2-KGA.

12.
Adv Mater ; 31(39): e1903852, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31397028

RESUMO

The Li-CO2 battery is a promising energy storage device for wearable electronics due to its long discharge plateau, high energy density, and environmental friendliness. However, its utilization is largely hindered by poor cyclability and mechanical rigidity due to the lack of a flexible and durable catalyst electrode. Herein, flexible fiber-shaped Li-CO2 batteries with ultralong cycle-life, high rate capability, and large specific capacity are fabricated, employing bamboo-like N-doped carbon nanotube fiber (B-NCNT) as flexible, durable metal-free catalysts for both CO2 reduction and evolution reactions. Benefiting from high N-doping with abundant pyridinic groups, rich defects, and active sites of the periodic bamboo-like nodes, the fabricated Li-CO2 battery shows outstanding electrochemical performance with high full-discharge capacity of 23 328 mAh g-1 , high rate capability with a low potential gap up to 1.96 V at a current density of 1000 mA g-1 , stability over 360 cycles, and good flexibility. Meanwhile, the bifunctional B-NCNT is used as the counter electrode for a fiber-shaped dye-sensitized solar cell to fabricate a self-powered fiber-shaped Li-CO2 battery with overall photochemical-electric energy conversion efficiency of up to 4.6%. Along with a stable voltage output, this design demonstrates great adaptability and application potentiality in wearable electronics with a breath monitor as an example.

13.
Bioresour Technol ; 292: 121897, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31398548

RESUMO

Co-production of α-ketoglutaric acid (KGA) and pyruvic acid (PYR) by Yarrowia lipolytica WSH-Z06 could significantly increase the final titer and yield of keto acids. However, efficient separation of KGA and PYR in an economic manner is a big challenge owing to their similar properties. In the present study, a separation process was established to convert PYR in the fermentation broth to l-tyrosine (TYR). Owing to its low solubility, TYR was easily precipitated out and could be easily removed from the reaction system. The whole-cell catalysis reaction solution was subjected to acid treatment, centrifugation, cation exchange column separation, rotary evaporation, Buchner funnel filtration, and dry separation method to obtain KGA and TYR powders. The purity/recovery rates of KGA and TYR were 98.16%/78.68% and 98.19%/73.46%, respectively. The use of biological pathways to separate KGA from the culture broth could make the separation process easier and further decrease the operation cost.


Assuntos
Yarrowia , Cetoácidos , Ácidos Cetoglutáricos , Ácido Pirúvico , Tirosina
14.
Sheng Wu Gong Cheng Xue Bao ; 35(8): 1374-1381, 2019 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-31441608

RESUMO

As one of the top 10 breakthrough and emerging technologies in the world in 2018, cultured meat has attracted extensive attention due to its advantages of traceable origin, food safety and green sustainable development. Europe and the United States have invested a lot of resources to focus on research about cultured meat, which will affect our domestic meat and food market in the future. At present, the challenge of cultured meat production is how to efficiently simulate the growth environment of animal muscle tissue and realize large-scale production in bioreactor. Although cell tissue engineering has been deeply studied and achieved varying successful application, it is still difficult to obtain large-scale cultured meat production due to the high cost and technical requirements. Therefore, the development of efficient and safe cell culture technology is an urgent problem for large-scale cultured meat production, which can effectively reduce costs and achieve industrial application. In this review, we summarize the research progress of animal cell tissue culture technology used for cultured meat, and highlighted the current challenges and possible strategies in further applications.


Assuntos
Técnicas de Cultura de Células , Carne , Animais , Reatores Biológicos , Engenharia Tecidual , Estados Unidos
15.
Sheng Wu Gong Cheng Xue Bao ; 35(7): 1247-1255, 2019 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-31328481

RESUMO

L-tyrosine is one of three aromatic amino acids that are widely used in food, pharmaceutical and chemical industries. The transport system engineering provides an important research strategy for the metabolic engineering of Escherichia coli to breed L-tyrosine producing strain. The intracellular transport of L-tyrosine in E. coli is mainly regulated by two distinct permeases encoded by aroP and tyrP genes. The aroP and tyrP gene knockout mutants were constructed by CRISPR-Cas technique on the basis of L-tyrosine producing strain HGXP, and the effects of regulating transport system on L-tyrosine production were investigated by fermentation experiments. The fermentation results showed that the aroP and tyrP knockout mutants produced 3.74 and 3.45 g/L L-tyrosine, respectively, which were 19% and 10% higher than that of the original strain. The optimum induction temperature was determined to be 38 °C. Fed-batch fermentation was carried out on a 3-L fermentor. The L-tyrosine yields of aroP and tyrP knockout mutants were further increased to 44.5 and 35.1 g/L, respectively, which were 57% and 24% higher than that of the original strain. The research results are of great reference value for metabolic engineering of E. coli to produce L-tyrosine.


Assuntos
Escherichia coli , Proteínas de Escherichia coli , Técnicas de Inativação de Genes , Engenharia Metabólica , Tirosina
16.
Sheng Wu Gong Cheng Xue Bao ; 35(7): 1256-1265, 2019 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-31328482

RESUMO

Naringenin is a natural flavonoid compound with anti-inflammatory, anti-oxidation, anti-viral, anti-atherosclerosis and other pharmacological activities. It is also an important precursor of other flavonoid synthesis and with great value of application. At present, the production of flavonoids such as naringenin by microbial methods has a low yield due to imbalance of metabolic pathways, which greatly limits its industrial application. In this study, a naringenin-producing strain of Saccharomyces cerevisiae Y-01 was used in the research object. The expression levels of 4-coumaric acid: CoA ligase (4CL), chalcone synthase (CHS) and chalcone isomerase (CHI) were controlled by promoter and copy numbers to investigate the quantitative effect of key enzyme expression level on the accumulation level of target products. The results showed that the correlation between naringenin production and 4CL or CHI expression was not significant while there was a positive correlation with the expression level of CHS. Strain Y-04 with high yield of naringenin was obtained by regulating the expression level of chs gene, and the yield was increased by 4.1-folds compared with the original strain Y-01. This study indicated that CHS is a key regulatory target of naringenin synthesis. Rational regulation of CHS expression can significantly promote the accumulation of naringenin. The related results provide an important theoretical reference for the use of metabolic engineering to strengthen microbial synthesis of important flavonoids such as naringenin.


Assuntos
Flavanonas/metabolismo , Engenharia Metabólica , Saccharomyces cerevisiae
17.
Appl Microbiol Biotechnol ; 103(16): 6449-6462, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31256230

RESUMO

Yeast is widely used in the baking, biocontrol, brewing, and bio manufacturing industries. In the baking industry alone, around two million tons of yeast are consumed worldwide every year. While yeast brings delicious and healthy lives to humans, we find that stress resistance of yeast is essential for the development of bioindustry. Whether during baking, biocontrol, brewing, bio manufacturing, or in other industries, yeast faces a variety of environmental stresses that have a great impact on its activity, transformation ability, etc., which make the production process uncertain. Therefore, robust yeast strains that can resist various environmental and endogenous stresses are needed. In recent years, many studies have investigated the stress resistance of laboratory strains and specific methods to improve stress resistance; however, applying these findings to industrial yeast is difficult. In this paper, based on summarizing the work of predecessors, we put forward the main steps to improve the stress resistance of industrial yeast systematically, which may provide a reference for researchers.


Assuntos
Biotecnologia/métodos , Genética Microbiana/métodos , Microbiologia Industrial/métodos , Engenharia Metabólica/métodos , Estresse Fisiológico , Leveduras/genética , Leveduras/fisiologia
18.
Bioresour Technol ; 289: 121612, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31203178

RESUMO

Herba Epimedii is a traditional Chinese herbal medicine that contains a mixture of bioactive flavonoid glycosides. Among them, icariin has the most outstanding bioactive functions, while epimedin C exhibits substantial toxicity. A recombinant α-L-rhamnosidase (synAnRhaE) from Aspergillus nidulans was expressed in Escherichia coli to promote the efficient bioconversion of epimedin C to icariin. A hydrolase activity of 574.5 U L-1 was acquired via optimized fed-batch fermentation in a 5-L bioreactor. The enzyme proved to be stable in an acidulous pH range below 55 °C with an optimal pH of 4.5 and optimal temperature of 55 °C. Epimedin C (1 g L-1) was 100% converted to icariin within 90 min using recombinant cells. The resting cells proved to be selective for epimedin C and 2″-O-rhamnosylicariside II in crude extracts of the epimedium plant. This work provides an original and efficient biocatalyst system that can be applied in industrialized production of icariin.


Assuntos
Aspergillus nidulans , Medicamentos de Ervas Chinesas , Cromatografia Líquida de Alta Pressão , Flavonoides , Glicosídeo Hidrolases
19.
Ann Hematol ; 98(8): 1813-1826, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31098739

RESUMO

Pregnant patients with ß-thalassemia are more likely to have progressive anemia which expose them to risk of adverse pregnancy outcomes, blood transfusion, and iron overload. Results from our previous study indicated that Colla corii asini (CCA, E'jiao), a natural ingredient of traditional Chinese medicine, could significantly increase hemoglobin level of pregnant women with ß- thalassemia, but the underlying molecular mechanism was unclear. Thus, we applied high-throughput transcriptome sequencing to study the transcriptomic change before and after the CCA treatment. Twenty eligible pregnant women were recruited and randomized to either the CCA treatment group or the blank control group in a 3:1 ratio. Patients in the treatment group orally received daily 15 g CCA powder for 4 weeks. We analyzed the therapeutic effect indexes and the transcriptomic change in subjects' peripheral blood before and after treatment. We found that ß CD 41-42(-TTCT)/ßA was the main genotype of the subjects. The regulatory impact of CCA treatment became more evident among the subjects of genotype ß CD 41-42(-TTCT)/ßA. Gene ontogenesis analysis revealed that the top five molecular functions of differentially expressed genes were involved in membrane functionality and cellular structure. We further identified two consistent upregulated genes ZNF471 and THOC5 in the effective treatment group, which were engaged in Kruppel-associated box (KRAB) domain-containing zinc-finger protein pathway and THOC5 pathway, respectively. Based on our current findings, we hypothesize that the anti-anemia effect of CCA on pregnant women with ß-thalassemia might be related to translation regulation of spectrin synthesis, membrane stability, and eventually prolonged the life span of erythrocytes.


Assuntos
Gelatina/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Fármacos Hematológicos/uso terapêutico , Medicina Tradicional Chinesa/métodos , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Talassemia beta/tratamento farmacológico , Administração Oral , Adulto , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Eritrócitos/patologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteínas Nucleares/agonistas , Proteínas Nucleares/metabolismo , Gravidez , Proteômica/métodos , Proteínas Repressoras/agonistas , Proteínas Repressoras/metabolismo , Transdução de Sinais , Espectrina/genética , Espectrina/metabolismo , Transcriptoma/efeitos dos fármacos , Talassemia beta/genética , Talassemia beta/metabolismo , Talassemia beta/patologia
20.
Microb Cell Fact ; 18(1): 91, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31133014

RESUMO

BACKGROUND: Self-assembling amphipathic peptides (SAPs) may improve protein production or induce the formation of inclusion bodies by fusing them to the N-terminus of proteins. However, they do not function uniformly well with all target enzymes and systematic research on how the composition of SAPs influence the production of fusion protein is still limited. RESULTS: To improve the efficiency of SAPs, we studied factors that might be involved in SAP-mediated protein production using S1 (AEAEAKAK)2 as the original SAP and green fluorescent protein (GFP) as the reporter. The results indicate that hydrophobicity and net charges of SAPs play a key role in protein expression. As hydrophobicity regulation tend to cause the formation of insoluble inclusion bodies of protein, an expression tag library composed of SAPs, which varied in net charge (from + 1 to + 20), was constructed based on the random amplification of S1nv1 (ANANARAR)10. The efficiency of the library was validated by polygalacturonate lyase (PGL), lipoxygenase (LOX), L-asparaginase (ASN) and transglutaminase (MTG). To accelerate preliminary screening, each enzyme was fused at the C-terminus with GFP. Among the four enzyme fusions, the SAPs with + 2 - + 6 net charges were optimal for protein expression. Finally, application of the library improved the expression of PGL, LOX, ASN, and MTG by 8.3, 3.5, 2.64, and 3.68-fold relative to that of the corresponding wild-type enzyme, respectively. CONCLUSIONS: This is the first report to study key factors of SAPs as an expression tag to enhance recombinant enzyme production. The SAP library could be used as a novel plug-and-play protein-engineering method to screen for enzymes or proteins with enhanced production.


Assuntos
Escherichia coli/genética , Biblioteca Gênica , Peptídeos/genética , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas de Fluorescência Verde/química , Ensaios de Triagem em Larga Escala , Interações Hidrofóbicas e Hidrofílicas , Corpos de Inclusão/metabolismo
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