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1.
Chinese Journal of Biotechnology ; (12): 1565-1575, 2022.
Article in Chinese | WPRIM | ID: wpr-927801

ABSTRACT

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.


Subject(s)
Dimethylallyltranstransferase/metabolism , Flavonoids/metabolism , Molecular Docking Simulation , Prenylation , Saccharomyces cerevisiae/metabolism , Sophora/metabolism
2.
Chinese Journal of Biotechnology ; (12): 719-736, 2022.
Article in Chinese | WPRIM | ID: wpr-927739

ABSTRACT

Gluconobacter oxydans are widely used in industrial due to its ability of oxidizing carbohydrate rapidly. However, the limited gene manipulation methods and less of efficient gene editing tools impose restrictions on its application in industrial production. In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR) system has been widely used in genome editing and transcriptional regulation which improves the efficiency of genome editing greatly. Here we constructed a CRISPR/dCpf1-mediated gene transcriptional repression system, the expression of a nuclease inactivation Cpf1 protein (dCpf1) in Gluconobacter oxydans together with a 19 nt direct repeats showed effective repression in gene transcription. This system in single gene repression had strong effect and the relative repression level had been increased to 97.9%. While it could be applied in multiplex gene repression which showed strong repression ability at the same time. Furthermore, this system was used in the metabolic pathway of L-sorbose and the regulatory of respiratory chain. The development of CRISPR transcriptional repression system effectively covered the shortage of current gene regulation methods in G. oxydans and provided an efficient gene manipulation tool for metabolic engineering modification in G. oxydans.


Subject(s)
CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Gene Editing , Gene Expression , Gluconobacter oxydans/genetics , Metabolic Engineering
3.
Chinese Journal of Biotechnology ; (12): 691-704, 2022.
Article in Chinese | WPRIM | ID: wpr-927737

ABSTRACT

Flavonoids have a variety of biological activities and have important applications in food, medicine, cosmetics, and many other fields. Naringenin is a platform chemical for the biosynthesis of many important flavonoids. Ubiquitination plays a pivotal role in the post-translational modification of proteins and participates in the regulation of cellular activities. Ubiquitinated proteins can be degraded by the ubiquitin-protease system, which is important for maintaining the physiological activities of cells, and may also exert a significant impact on the expression of exogenous proteins. In this study, a real-time in-situ detection system for ubiquitination modification has been established in Saccharomyces cerevisiae by using a fluorescence bimolecular complementation approach. The ubiquitination level of protein was characterized by fluorescence intensity. By using the approach, the potential ubiquitination sites of proteins involved in the naringenin biosynthesis pathway have been obtained. The lysine residues of the relevant ubiquitination sites were mutated to arginine to reduce the ubiquitination level. The mutants of tyrosine ammonia-lyase (FjTAL) and chalcone synthase (SjCHS, SmCHS) showed decreased fluorescence, suggested that a decreased ubiquitination level. After fermentation verification, the S. cerevisiae expressing tyrosine ammonia-lyase FjTAL mutant FjTAL-K487R accumulated 74.2 mg/L p-coumaric acid at 72 h, which was 32.3% higher than that of the original FjTAL. The strains expressing chalcone synthase mutants showed no significant change in the titer of naringenin. The results showed that mutation of the potential ubiquitination sites of proteins involved in the naringenin biosynthesis pathway could increase the titer of p-coumaric acid and have positive effect on naringenin biosynthesis.


Subject(s)
Biosynthetic Pathways , Flavanones/metabolism , Saccharomyces cerevisiae/metabolism , Ubiquitination
4.
Chinese Journal of Internal Medicine ; (12): 630-636, 2021.
Article in Chinese | WPRIM | ID: wpr-911425

ABSTRACT

Objective:To evaluate the efficacy and safety of anti-programmed cell death 1 (PD-1) receptor monoclonal antibody (MoAb) in patients with advanced hepatocellular carcinoma (HCC) after treatment of transcatheter arterial chemoembolization (TACE) combined with tyrosine kinase inhibitor (TKI).Methods:From February 2019 to February 2020, 56 HCC patients who relapsed after TACE-TKI treatment in Department of Interventional Radiology, The Second Affiliated Hospital of Guangzhou Medical University were enrolled. All patients received anti-PD-1 MoAb (sintilimab injection) and followed up every 6 weeks. According to mRECIST, the curative effect was evaluated as complete response (CR), partial response (PR), stable disease (SD) or progressive disease (PD). Objective response rate (ORR) and disease control rate (DCR), progression-free survival (PFS) and treatment-related adverse events (TRAEs) were recorded. Univariate analysis by Chi-square test and binary logistic regression model was used to determine the influencing factors of DCR. The Kaplan-Meier method and Cox proportional hazard regression model were used to analyze the survival data.Results:A total of 48 patients were enrolled in this study including 42 males and 6 females, with a median age of 55 years (29-71 years). ECOG scores comprised of 0 in 24 cases, 1-2 in 24 cases. Thirty-six patients were in Child-Pugh grade A of liver function and 12 cases were grade B. The median follow-up time was 4.5 months. There were 2 patients achieved CR, 12 patients with PR and 16 with SD. ORR was 29.2%, DCR was 62.5%. The independent influencing factors of DCR was ECOG score and AFP level ( P=0.031, P=0.012). Median PFS was 4.1 months (95% CI 2.7-5.4 months), and ECOG score was the independent influencing factor of PFS ( P=0.042). Treatment-related adverse events were reported in 70.8% (34/48) patients. Incidence of grade Ⅲ-Ⅳ TRAEs was 22.9% (11/48). Conclusion:In patients with HCC who relapse from TACE and TKI treatment, anti-PD-1 monoclonal antibody is efficacious safe especially in those with ECOG 0 score.

5.
Chinese Journal of Hepatobiliary Surgery ; (12): 570-574, 2021.
Article in Chinese | WPRIM | ID: wpr-910596

ABSTRACT

Objective:To study the use of radioactive I-125 seed implantation in the treatment of transarterial chemoembolization (TACE)-refractory hepatocellular carcinoma (HCC).Methods:A retrospective study was conducted on 70 patients with HCC who were initially treated with TACE between July 1, 2016 and August 31, 2019 at the Second Affiliated Hospital of Guangzhou Medical University. After these patients were found to be refractory to TACE, 29 patients were converted to radioactive I-125 seed implantation (the 125I seed group), and 41 patients were continued with TACE (the TACE group). The objective response rate, progression-free survival (PFS), overall survival (OS), total overall survival (TOS) of the two groups were compared. Results:There were 59 males and 11 females, aged (60.5±11.9 ) years in this study. At 1, 3, 6 months after treatment, the objective response rates of the 125I seed group were 20.7%, 40.7%, 34.6%, respectively, which were significantly higher than that of the TACE group of 2.6%, 3.3%, 5.0%, respectively. The PFS, OS, TOS in the 125I seed group were 7.6, 21.1, 32.1 months, respectively, which were significantly better when compared with the TACE group (3.5, 8.5, 14.8 months, respectively, all P<0.05). There was no significant difference in the embolization syndrome between the two groups [93.1%(27/29) vs 100.0%(41/41), P>0.05]. Child-Pugh B grading ( HR=0.311, 95% CI: 0.160-0.603, P=0.005) and TACE ( HR=0.308, 95% CI: 0.159-0.597, P=0.002) were independent risk prognostic factors for survival. Conclusion:This study showed better treatment efficacy and safety using radioactive I-125 seed implantation in TACE-refractory HCC and this treatment significantly improved survival of patients when compared with TACE alone.

6.
Chinese Journal of Biotechnology ; (12): 1827-1844, 2021.
Article in Chinese | WPRIM | ID: wpr-887766

ABSTRACT

Vitamin C is an essential vitamin for human beings. It has a huge market in the fields of food and pharmaceuticals. 2-keto-L-gulonic acid is an important precursor to produce vitamin C by microbial fermentation in industrial. In microbial fermentations, the L-sorbose pathway and the D-gluconate pathway have been the focus of research because of high yield. This article aims at stating recent research progress in dehydrogenases related to biosynthesis of vitamin C in the L-sorbose pathway and the D-gluconate pathway. The properties of dehydrogenase in terms of localization, substrate specificity, cofactors, and electron transport carrier are elaborated. And then, the main problems and strategies are reviewed in the L-sorbose pathway and in the D-gluconate pathway. Finally, future research on the dehydrogenases in the biosynthesis of vitamin C through L-sorbose pathway and D-gluconate pathway is discussed.


Subject(s)
Ascorbic Acid/biosynthesis , Fermentation , Gluconates , Oxidoreductases/metabolism , Sorbose
7.
Chinese Journal of Biotechnology ; (12): 950-965, 2021.
Article in Chinese | WPRIM | ID: wpr-878606

ABSTRACT

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.


Subject(s)
CRISPR-Cas Systems/genetics , Endonucleases/genetics , Gene Editing , Saccharomyces cerevisiae/genetics , Technology
8.
Chinese Journal of Biotechnology ; (12): 689-695, 2021.
Article in Chinese | WPRIM | ID: wpr-878594

ABSTRACT

Fermentation engineering is an industrial process that uses the transformation of microorganisms or other cells to produce a specific product in a specific bioreactor. Fermentation engineering has developed from an ancient food fermentation relying solely on experience accumulation to an important production mode of food, agriculture, medicine, chemical industry and other means of production and life. It has become a key technology to support the sustainable development of human beings, and is inseparable from the continuous progress of interdisciplinary technology. The interdisciplinary integration and the continuous upward movement of China's global industrial chain will inevitably put forward higher requirements for the cultivation of fermentation engineering composite talents in the new situation. In order to constantly improve the interdisciplinary fermentation engineering compound talent training system, in recent years, the research lab has been refining and improving the concept of talent training, and actively deepening the reform of talent training system. Systematic research and practice have been carried out around the aspects of training program, enrollment system, teacher background, subject setting, scientific research practice, evaluation system, etc., which has promoted the technological progress of fermentation engineering and related supporting industries, and contributed an important force to the transformation of China from a big fermentation country to a powerful fermentation country.


Subject(s)
Humans , Agriculture , China , Fermentation , Industry
9.
Chinese Journal of Biotechnology ; (12): 378-383, 2021.
Article in Chinese | WPRIM | ID: wpr-878568

ABSTRACT

In recent years, to solve the increasingly prominent problem of the contradiction between human social development and environmental resources, artificial meat has appeared in public view more and more. Generally speaking, the artificial meat can be divided into vegetable protein meat and cell cultured meat. Among them, vegetable protein meat has gradually begun to be commercialized, and cell cultured meat is cultured with animal cells, which is more similar to the real meat. Based on the analysis of the essence of cell cultured meat, we explore the positive significance of cell cultured meat technology for the meat production industry, consumer groups, and the sustainable development of mankind in the future. From the perspective of bioethics, the research, development and production of cell cultured meat can help ensure the sustainable development of human society, improve animal welfare, reduce resource demand, improve the nutritional function of meat products, and provide new growth points for the development of other industries. In addition, the ethical risks of food safety, technology abuse and technical supervision involved in cell cultured meat production are put forward for deep consideration, hoping to provide reference for the sustainable development of artificial meat industry from the perspective of bioethics.


Subject(s)
Animals , Humans , Animal Welfare , Food Safety , Meat , Meat Products
10.
Chinese Journal of Biotechnology ; (12): 207-217, 2021.
Article in Chinese | WPRIM | ID: wpr-878555

ABSTRACT

Scleroglucan is a high-molecular water-soluble microbial exopolysaccharide and mainly applied in the fields of petroleum, food, medicine and cosmetics. The high molecular weight of scleroglucan produced by microbial fermentation leads to low solubility, high viscosity and poor dispersibility, thus bringing a series of difficulties to extraction, preservation and application. It is important to explore suitable degradation method to adjust the molecular weight of scleroglucan for expanding its industrial application. Taking Sclerotium rolfsii WSH-G01 as a model strain, in which functional annotations of the glucanase genes were conducted by whole genome sequencing. Based on design of culture system for culture system for differential expression of β-glucanase, endogenous β-glucanase genes in S. rolfsii WSH-G01 were excavated by transcriptomics analysis. Functions of these potential hydrolases were further verified. Finally, 14 potential endogenous hydrolase genes were obtained from S. rolfsii. After heterologous overexpression in Pichia pastoris, 10 soluble enzymes were obtained and 5 of them had the activity of laminarin hydrolysis by SDS-PAGE and enzyme activity analysis. Further investigation of the 5 endogenous hydrolases on scleroglucan degradation showed that enzyme GME9860 has positive hydrolysis effect. The obtained results provide references not only for obtaining low and medium molecular weight of scleroglucan with enzymatic hydrolysis, but also for producing different molecular weight of scleroglucan during S. rolfsii fermentation process with metabolic engineering.


Subject(s)
Basidiomycota/genetics , Glucans , Hydrolysis , Saccharomycetales
11.
Chinese Journal of Nephrology ; (12): 618-624, 2020.
Article in Chinese | WPRIM | ID: wpr-870995

ABSTRACT

Objective:To investigate the effect of postoperative hypoalbuminemia on acute kidney injury (AKI) after cardiac surgery under cardiopulmonary bypass (CPB).Methods:The clinical data of adult patients undergoing cardiac surgery under CPB were retrospectively analyzed. The difference between preoperative and postoperative serum albumin level was compared. The patients were divided into hypoalbuminemia group (≤35 g/L) and non-hypoalbuminemia group (>35 g/L) according to the lowest serum albumin concentration within 48 hours after surgery. The incidence and severity of postoperative AKI were compared between the two groups. Univariate analysis and binary logistic regression analysis were used to evaluate the effect of postoperative hypoalbuminemia on the incidence of postoperative AKI.Results:Among the 749 patients, the serum albumin level after cardiac surgery was significantly lower than that before surgery ( Z=-15.739, P<0.001), and the proportion of patients with hypoalbuminemia increased from 9.6% to 27.6% ( χ2=83.516, P<0.001). Postoperative AKI occurred in 273 patients, including 109 cases (52.7%) in hypoalbuminemia group and 164 cases (30.3%) in non-hypoalbuminemia group. The incidence of AKI in hypoalbuminemia group was significantly higher than that in non-hypoalbuminemia group ( χ2=32.443, P<0.001), and the severity of AKI in hypoalbuminemia group increased than that in non-hypoalbuminemia group ( Z=-2.098, P=0.036), and the time of hospital stay extended ( Z=-2.442, P=0.015). After adjusted by gender, age, preoperative hypoalbuminemia, comorbidities (hypertension, hyperuricemia, diabetes mellitus, cerebrovascular disease), renal insufficiency, preoperative heart function, coronary angiography, CPB time, aorta blocking time, type of heart surgery and postoperative hypotension, binary logistic regression analysis revealed that postoperative hypoalbuminemia was an independent risk factor for CPB-associated AKI ( OR=2.319, 95% CI 1.586-3.392, P<0.001). Conclusions:AKI is a common complication following cardiac surgery under CPB. Serum albumin after CBP is significantly lower than that before CBP, and postoperative hypoalbuminemia within 48 hours after surgery is an independent risk factor for AKI.

12.
Chinese Journal of Biotechnology ; (12): 2838-2849, 2020.
Article in Chinese | WPRIM | ID: wpr-878533

ABSTRACT

(2S)-taxifolin is an important flavonoid that has anti-inflammatory and anti-oxidation effects. It is widely used in pharmaceutical and nutraceutical industries. Flavone 3-hydroxylase (F3H) can catalyze the synthesis of (2S)-taxifolin and other 3-hydroxylated flavonoids from (2S)-eriodictyol. Due to the low catalytic efficiency of F3H, the titer of many 3-hydroxyflavones, such as taxifolin, synthesized by microbial method is relatively low. In this study, a SmF3H was identified from the transcriptome of Silybum marianum (L.) Gaertn. The results of fermentation showed that SmF3H can catalyze the flavone 3-hydroxylation reaction, and its catalytic efficiency was significantly higher than that of commonly used SlF3H from Solanum lycopersicum. Six promoters with different transcription strength were selected to optimize the synthesis pathway from the flavonoid precursor (2S)-naringenin to (2S)-taxifolin. The results showed that the highest titer of (2S)-taxifolin (695.90 mg/L in shake flask) could be obtained when the P(GAL7) promoter was used to control the expression of SmF3H. The titer of (2S)-taxifolin was further improved to 3.54 g/L in a 5-L fermenter, which is the highest titer according to current available literatures.


Subject(s)
Antioxidants , Flavonoids , Silybum marianum , Quercetin/analogs & derivatives
13.
Chinese Journal of Emergency Medicine ; (12): 1178-1183, 2020.
Article in Chinese | WPRIM | ID: wpr-863848

ABSTRACT

Objective:To investigate the effect of hyperuricemia on acute kidney injury in sepsis patients.Methods:It is a retrospective cohort study of 459 adult sepsis patients who were admitted to the Department of Intensive Care Unit (ICU) of the First Affiliated Hospital of Guangxi Medical University from March 2014 to June 2019, and the Second Affiliated Hospital of Guangxi Medical University from January 2017 to June 2019. The patients were divided into the hyperuricemia group and the non-hyperuricemia group according to the first serum uric acid level within 24 h after ICU admission, and the incidence of AKI within 7 days after ICU admission was compared between the two groups. The effect of hyperuricemia on sepsis-associated AKI was analyzed by univariate analysis and binary logistic regression analysis.Results:Among the 459 sepsis patients, 81 patients (17.6%) had hyperuricemia, and 127 patients (27.7%) had AKI. The incidence of AKI in the hyperuricemia group and the non-hyperuricemia group were 60.5% (49/81) and 20.6% (78/378), respectively, which showed significantly statistical difference ( χ2=52.954, P<0.01). After adjusting for gender, associated diseases (diabetes, coronary heart disease), sequential organ failure score (SOFA) on the day of ICU admission, the use of diuretics within one week before and after ICU admission, invasive mechanical ventilation, basal renal function, lactic acid, and procalcitonin, binary logistic regression analysis showed that hyperuricemia was an independent risk factor for AKI in sepsis patients ( OR=5.091, 95% CI: 2.768-9.362, P<0.01); For every 1 mg/dL increase in serum uric acid in sepsis patients, the risk of developing AKI increased by 28.4% ( OR=1.284, 95% CI: 1.165-1.414, P<0.01). Conclusions:AKI is a common complication in sepsis patients admitted to ICU, and hyperuricemia is an independent risk factor for AKI in sepsis patients.

14.
Chinese Journal of Biotechnology ; (12): 1247-1255, 2019.
Article in Chinese | WPRIM | ID: wpr-771804

ABSTRACT

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.


Subject(s)
Escherichia coli , Escherichia coli Proteins , Gene Knockout Techniques , Metabolic Engineering , Tyrosine
15.
Chinese Journal of Biotechnology ; (12): 1256-1265, 2019.
Article in Chinese | WPRIM | ID: wpr-771803

ABSTRACT

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.


Subject(s)
Flavanones , Metabolism , Metabolic Engineering , Saccharomyces cerevisiae
16.
Chinese Journal of Biotechnology ; (12): 1374-1381, 2019.
Article in Chinese | WPRIM | ID: wpr-771792

ABSTRACT

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.


Subject(s)
Animals , Bioreactors , Cell Culture Techniques , Meat , Tissue Engineering , United States
17.
Chinese Journal of Biotechnology ; (12): 794-802, 2018.
Article in Chinese | WPRIM | ID: wpr-687736

ABSTRACT

Pyrroloquinoline quinone (PQQ) is a bacterial dehydrogenase coenzyme. PQQ can promote body growth and regulate the function of free radical level of the body. It could be applied in food, medicine and other fields. Due to the extremely high cost of chemical synthesis, the production of PQQ by microbial fermentation attracted more and more attention. At present, the production titer of PQQ by fermentation method is too low to achieve industrial application. Due to the lack of a thorough understanding of the PQQ biosynthesis and its regulation mechanisms, and the lack of necessary genetic engineering modification methods for wild type strains, metabolic engineering of microorganisms to enhance PQQ production still lacks essential requirements. In this study, a PQQ-producing bacterium, Methylobacterium extorquens I-F2, was employed as a model strain. By integration of Atmospheric and room temperature plasma (ARTP) mutagenesis, flow cytometry sorting and high-throughput screening strategies, optimization of sample preparation and flow sorting process, a high-titer PQQ mutant strain was obtained. The titer of PQQ was increased by 98.02% compared with that of M. extorqunens I-F2. The process described here showed that the combination of the flow cytometry with high-throughput screening method can be used to obtain high-titer mutants more simply and rapidly, compared with genetic engineering and traditional screening methods.

18.
Chinese Journal of Epidemiology ; (12): 374-377, 2017.
Article in Chinese | WPRIM | ID: wpr-737649

ABSTRACT

Objective To estimate the influence of the ambient PM10 and PM2.5 pollution on the hospital outpatient department visit due to respiratory diseases in local residents in Jinan quantitatively.Methods Time serial analysis using generalized addictive model (GAM) was conducted.After controlling the confotmding factors,such as long term trend,weekly pattern and meteorological factors,considering lag effect and the influence of other air pollutants,the excess relative risks of daily hospital visits associated with increased ambient PM10 and PM2.5 levels were estimated by fitting a Poisson regression model.Results A 10 μtg/m3 increase of PM10 and PM2.5 levels was associated with an increase of 0.36% (95% CI:0.30%-0.43%) and 0.50% (95% CI:0.30%-0.70%) respectively for hospital visits due to respiratory diseases.Lag effect of 6 days was strongest,the excess relative risks were 0.65% (95% CI:0.58%-0.71%) and 0.54% (95% CI:0.42%-0.67%) respectively.When NO2 concentration was introduced,the daily hospital visits due to respiratory disease increased by 0.83% as a 10 μg/m3 increase of PM10 concentration (95%CI:0.76%-0.91%).Conclusion The ambient PM10 and PM2.5 pollution was positively associated with daily hospital visits due to respiratory disease in Jinan,and ambient NO2 concentration would have the synergistic effect.

19.
Chinese Journal of Epidemiology ; (12): 374-377, 2017.
Article in Chinese | WPRIM | ID: wpr-736181

ABSTRACT

Objective To estimate the influence of the ambient PM10 and PM2.5 pollution on the hospital outpatient department visit due to respiratory diseases in local residents in Jinan quantitatively.Methods Time serial analysis using generalized addictive model (GAM) was conducted.After controlling the confotmding factors,such as long term trend,weekly pattern and meteorological factors,considering lag effect and the influence of other air pollutants,the excess relative risks of daily hospital visits associated with increased ambient PM10 and PM2.5 levels were estimated by fitting a Poisson regression model.Results A 10 μtg/m3 increase of PM10 and PM2.5 levels was associated with an increase of 0.36% (95% CI:0.30%-0.43%) and 0.50% (95% CI:0.30%-0.70%) respectively for hospital visits due to respiratory diseases.Lag effect of 6 days was strongest,the excess relative risks were 0.65% (95% CI:0.58%-0.71%) and 0.54% (95% CI:0.42%-0.67%) respectively.When NO2 concentration was introduced,the daily hospital visits due to respiratory disease increased by 0.83% as a 10 μg/m3 increase of PM10 concentration (95%CI:0.76%-0.91%).Conclusion The ambient PM10 and PM2.5 pollution was positively associated with daily hospital visits due to respiratory disease in Jinan,and ambient NO2 concentration would have the synergistic effect.

20.
Chinese Journal of Biotechnology ; (12): 1145-1149, 2016.
Article in Chinese | WPRIM | ID: wpr-242266

ABSTRACT

As a novel cofactor of oxidoreductase, pyrroloquinoline quinone (PQQ) has a great potential of application in medicine, food industries. In order to improve the efficiency of the PQQ production by Methylobacterium extorquens AM1, the strain was treated by atmospheric and room temperature plasma (ARTP). Positive mutants with changes in PQQ yield were obtained based on a high-throughput screening approach. After ARTP treatment, analysis data show that the positive mutation rate was 31.6%. Furthermore, we obtained an excellent positive mutant M. extorquens AM1 (E-F3) with the yield of 54.0 mg/L PQQ, which was approximately 3 times as much compared with that of the wild-type strain. The robust high-throughput screening method for mutagenesis by ARTP improves PQQ production. In addition, this method also provides a new strategy for further strain improvement.


Subject(s)
Bacterial Proteins , High-Throughput Screening Assays , Methylobacterium extorquens , Genetics , Mutagenesis , PQQ Cofactor , Plasma Gases , Temperature
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