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1.
Chinese Journal of Biotechnology ; (12): 4266-4276, 2021.
Article in Chinese | WPRIM | ID: wpr-921504

ABSTRACT

Dopamine is the precursor of a variety of natural antioxidant compounds. In the body, dopamine acts as a neurotransmitter that regulates a variety of physiological functions of the central nervous system. Thus, dopamine is used for the clinical treatment of various types of shock. Dopamine could be produced by engineered microbes, but with low efficiency. In this study, DOPA decarboxylase gene from Sus scrofa (Ssddc) was cloned into plasmids with different copy numbers, and transformed into a previously developed L-DOPA producing strain Escherichia coli T004. The resulted strain was capable of producing dopamine from glucose directly. To further improve the production of dopamine, a sequence-based homology alignment mining (SHAM) strategy was applied to screen more efficient DOPA decarboxylases, and five DOPA decarboxylase genes were selected from 100 candidates. In shake-flask fermentation, the DOPA decarboxylase gene from Homo sapiens (Hsddc) showed the highest dopamine production (3.33 g/L), while the DOPA decarboxylase gene from Drosophila Melanogaster (Dmddc) showed the least residual L-DOPA concentration (0.02 g/L). In 5 L fed-batch fermentations, production of dopamine by the two engineered strains reached 13.3 g/L and 16.2 g/L, respectively. The residual concentrations of L-DOPA were 0.45 g/L and 0.23 g/L, respectively. Finally, the Ssddc and Dmddc genes were integrated into the genome of E. coli T004 to obtain genetically stable dopamine-producing strains. In 5 L fed-batch fermentation, 17.7 g/L of dopamine was produced, which records the highest titer reported to date.


Subject(s)
Animals , Dopa Decarboxylase/genetics , Dopamine/biosynthesis , Drosophila melanogaster/genetics , Escherichia coli/metabolism , Humans , Metabolic Engineering
2.
Chinese Journal of Biotechnology ; (12): 1998-2009, 2021.
Article in Chinese | WPRIM | ID: wpr-887777

ABSTRACT

Aromatic compounds make up a large part of fragrances and are traditionally produced by chemical synthesis and direct extraction from plants. Chemical synthesis depends on petroleum resources and has disadvantages such as causing environment pollutions and harsh reaction conditions. Due to the low content of aromatic compounds in plants and the low yield of direct extraction, plant extractions require large amounts of plant resources that occupy arable land. In recent years, with the development of metabolic engineering and synthetic biology, microbial synthesis of aromatic compounds from renewable resources has become a promising alternative approach to traditional methods. This review describes the research progress on the synthesis of aromatic fragrances by model microorganisms such as Escherichia coli or yeast, including the synthesis of vanillin through shikimic acid pathway and the synthesis of raspberry ketone through polyketide pathway. Moreover, this review highlights the elucidation of native biosynthesis pathways, the construction of synthetic pathways and metabolic regulation for the production of aromatic fragrances by microbial fermentation.


Subject(s)
Biosynthetic Pathways , Metabolic Engineering , Odorants , Shikimic Acid , Synthetic Biology
3.
Chinese Journal of Biotechnology ; (12): 1771-1793, 2021.
Article in Chinese | WPRIM | ID: wpr-878666

ABSTRACT

Metabolic engineering has been developed for nearly 30 years since the early 1990s, and it has given a great impetus to microbial strain breeding and improvement. Aromatic chemicals are a variety of important chemicals that can be produced by microbial fermentation and are widely used in the pharmaceutical, food, feed, and material industry. Microbial cells can be engineered to accumulate a variety of useful aromatic chemicals in a targeted manner through rational engineering of the biosynthetic pathways of shikimate and the derived aromatic amino acids. This review summarizes the metabolic engineering strategies and biosynthetic pathways for the production of aromatic chemicals developed in the past 30 years, with the aim to provide a valuable reference and promote the research in this field.


Subject(s)
Biosynthetic Pathways , Fermentation , Metabolic Engineering , Shikimic Acid
4.
Chinese Journal of Biotechnology ; (12): 2387-2397, 2020.
Article in Chinese | WPRIM | ID: wpr-878495

ABSTRACT

Recently, fast-growing Vibrio natriegens, as the great potential chassis, has shown a wide application in synthetic biology. Genome editing is an indispensable tool for genetic modification in synthetic biology. However, genome editing tools with high efficiency and fidelity are still to be developed for V. natriegens synthetic biology. To deal with this problem, the physiological characteristics of 6 V. natriegens strains were evaluated, and CICC 10908 strain with fast and stable growth was selected as the host strain for genome editing study. Then, the natural transformation system of V. natriegens was established and optimized. The efficiencies of optimized natural transformation that integrates antibiotic resistance marker cat-sacB or Kan(R) onto the chromosome of V. natriegens could reach 4×10⁻⁵ and 4×10⁻⁴, respectively. Based on the optimized natural transformation, a double-selection cassette was used to achieve seamless genome editing with high efficiency and fidelity. The positive rates of four different types of genetic manipulation, including gene deletion, complementation, insertion and substitution, were 93.8%, 100%, 95.7% and 100%, respectively. Finally, transformation and elimination of the recombinant plasmid could be easily achieved in V. natriegens. This work provides a seamless genome editing system with high efficiency and fidelity for V. natriegens synthetic biology.


Subject(s)
Gene Editing , Plasmids/genetics , Synthetic Biology , Vibrio/genetics
5.
Chinese Journal of Biotechnology ; (12): 2367-2376, 2020.
Article in Chinese | WPRIM | ID: wpr-878493

ABSTRACT

p-coumaric acid is an important natural phenolic compound with a variety of pharmacological activities, and also a precursor for the biosynthesis of many natural compounds. It is widely used in foods, cosmetics and medicines. Compared with the chemical synthesis and plant extraction, microbial production of p-coumaric acid has many advantages, such as energy saving and emission reduction. However, the yield of p-coumaric acid by microbial synthesis is too low to meet the requirements of large-scale industrial production. Here, to further improve p-coumaric acid production, the directed evolution of tyrosine ammonia lyase (TAL) encoded by Rhodotorula glutinis tal gene was conducted, and a high-throughput screening method was established to screen the mutant library for improve the property of TAL. A mutant with a doubled TAL catalytic activity was screened from about 10,000 colonies of the mutant library. There were three mutational amino acid sites in this TAL, namely S9Y, A11N, and E518A. It was further verified by a single point saturation mutation. When S9 was mutated to Y, I or N, or A11 was mutated to N, T or Y, the catalytic activity of TAL increased by more than 1-fold. Through combinatorial mutation of three types of mutations at the S9 and A11, the TAL catalytic activity of S9Y/A11N or S9N/A11Y mutants were significantly higher than that of other mutants. Then, the plasmid containing S9N/A11Y mutant was transformed into CP032, a tyrosine-producing E. coli strain. The engineered strain produced 394.2 mg/L p-coumaric acid, which is 2.2-fold higher than that of the control strain, via shake flask fermentation at 48 h. This work provides a new insight for the biosynthesis study of p-coumaric acid.


Subject(s)
Ammonia-Lyases/genetics , Escherichia coli/genetics , Propionates , Rhodotorula , Tyrosine/genetics
6.
Chinese Journal of Geriatrics ; (12): 311-314, 2018.
Article in Chinese | WPRIM | ID: wpr-709246

ABSTRACT

Objective To evaluate the value of hysteroscopy in elder women with abnormal uterine bleeding (AUB) and asymptomatic postmenopausal women with a thickened endometrium.Methods Fifty-three cases in the AUB group and seventy-eight cases in the endometrial hyperplasia group underwent hysteroscopy examination and hysteroscopy-guided biopsy,then the hysteroscopic and histopathological results were compared between the two groups.Results Of the 131 cases,the normal endometrium accounted for 29.8% (n=39),endometrial polyp for 49.6% (n=65),submucous myomas for 4.6% (n=6),hyperplasia endometrii for 6.1%(n=8) and endometrial carcinoma for 9.9% (n=13).Both the AUB group and theendometrial hyperplasia group had 8 cases of endometrial carcinoma (15.1%,6.4%,respectively).For the diagnosis of normal endometrium with hysteroscopy,the sensitivity,specificity,positive predictive value (PPV) and negative predictive value (NPV) were 88%,97%,94% and 95%,respectively,in the AUB group,versus 82%,95%,86% and 93%,respectively,in the endometrial hyperplasia group.For the endometrial polyps,hysteroscopy showed a sensitivity,specificity,PPV and NPV of 100%,79%,74%,100%,respectively,in the AUB group and 98%,88%,92%,97%,respectively,in the endometrial hyperplasia group.For the endometrial cancer,hysteroscopy had a sensitivity,specificity,PPV and NPV of 75%,100%,100% and 96%,respectively,in the AUB group;while in the endometrial hyperplasia group,the sensitivity was 80%,the specificity and PPV were 100%,and the NPV was 99%.Conclusions In elder females,hysteroscopy allows for an accurate diagnosis in endometrial disease,and hysteroscopically directed sampling is mandatory,even if the uterine cavity appears normal at hysteroscopy,to rule out endometrial neoplasms.

7.
Chinese Journal of Geriatrics ; (12): 192-196, 2018.
Article in Chinese | WPRIM | ID: wpr-709218

ABSTRACT

Objective To investigate the expression of different inflammatory variables,such as procalcitonin(PCT),C-reactive protein (CRP),D-dimer (DD),fibrinogen (FIB),white blood cell (WBC),neutrophils and platelet(PLT)in septic elderly female patients with bacterial bloodstream infection,in order to assess the early diagnostic value of these variables.Methods A total of 308 elderly female patients with systemic inflammatory response syndrome(SIRS)were enrolled for this prospective study in Beijing Hospital between January 2014 and December 2015.Patients were divided into the sepsis group(n=210)and non-sepsis group(n=98)based on the diagnostic criteria of sepsis.The early inflammatory variables in blood,including PCT,CRP,DD,FIB,WBC,neutrophils and PLT,were detected within 6 hours of bloodstream infection,and their correlations were analyzed.The receiver operating characteristic(ROC)curve of inflammatory variables for the diagnosis of bloodstream infection was plotted,and the area under ROC curve (AUC)was calculated and used to evaluate diagnostic value for bloodstream infection.The best diagnostic cut-off points were identified based on the best(largest)AUC and the best sensitivity and specificity of inflammatory variables for bloodstream infection.Results The levels of all the inflammatory variables were significantly higher in the sepsis group than in non-sepsis group(all P<0.05).Additionally,PCT and CRP were independent factors for diagnosis of blood stream infection.AUC of the combination of two biomarkers of PCT and CRP was 0.694 for diagnosis of sepsis,which was higher than the either biomarkers alone with AUC of 0.628 for PCT and 0.627 for CRP.The combination group of PCT and CRP showed better values of sensitivity,specificity,positive predictive,and negative predictive (86.2 %,59.1%,65.1 %,81.3 %),as compared with those used individually(63.4%,58.2%,60.3%,61.4% for PCT;and 62.4%,58.2%,59.9%,60.7% for CRP,respectively).Conclusions The combination assay of PCT and CRP enhances the diagnostic ability for bacterial bloodstream infection.

8.
Article in Chinese | WPRIM | ID: wpr-454361

ABSTRACT

Objective To investigate the diagnostic value of serum tumor markers carcinoembryonic antigen (CEA) ,neuron spe-cific enolase(NSE) ,cytokeratin-19 fragments(CYFRA21-1) ,squamous cell carcinoma antigen(SCCA)in the lung cancer .Methods By using electro chemiluminescence immunoassay ,the concentrations of CEA ,NSE ,CYFRA21-1 and SCCA were determined in 98 cases of lung cancer and 30 cases of healthy control group ,and the positive rate ,sensitivity ,specificity ,accuracy were calculated .Re-sults In different lung cancer pathological groups ,the concentrations of the 4 serum tumor markers were significantly higher than the control group(P<0 .05) .CEA had the highest diagnostic sensitivity for detecting adeno carcinoma ,NSE had the highest diag-nostic sensitivity for detecting small cell lung cancers ,CYFRA21-1 had the highest diagnostic sensitivity for detecting squamous carcinoma .The combined tests of four serum tumor markers had higher diagnostic sensitivity and accuracy than single test for lung cancers .Conclusion The four serum tumor markers are valuable in the pathological classification ,diagnosis and differential diagno-sis of lung cancer .The diagnostic sensitivity and the accuracy are obviously higher in combined tests .

9.
Chinese Journal of Geriatrics ; (12): 1334-1337, 2014.
Article in Chinese | WPRIM | ID: wpr-469795

ABSTRACT

Objective To investigate the changes and significances of vaginal microecology in elderly postmenopausal women.Methods 50 postmenopausal women and 68 reproductive age women with no subjective symptoms and normal vaginal microecological who had health examination in Beijing Hospital were selected.The contents and strains of vaginal lactobacilli in both groups were detected by polymerase chain reaction (PCR),and their differences between the two groups were analyzed.Results 92% (46 cases) of postmenopausal women had only one vaginal lactobacillus strain in vaginal,and 8%(4 cases) of them had two lactobacillus strains in vaginal.But 4%(3 cases) of reproductive-age women had only one vaginal lactobacillus strain in vaginal,and 96% (65 cases) of them had two and more lactobacillus strains in vaginal.There was a significant difference in the strains of vaginal lactobacilli between the two groups (x2=91.035,P=0.000).The strains of vaginal lacmbacilli ranking top 4 detection rate was Lactobaeillus jensenii (30.0%),Lactobacillus gasseri (22.6%),Lactobacillus crispatus (16.9%) and Lactobacillus inert (13.2%) in postmenopausal women,while Lactobacillus inert (80.8 %),Lactobacillus crispatus (70.5 %),Lactobacillus gasseri (67.6%) and Lactobacillus jensenii (39.7%) in reproductive-age women.The content of 16s rDNA from vaginal lactobacillus was more in reproductive age women than in postmenopausal women [(87.3 ±0.8) ng/g total DNA vs.(35.1±3.6) ng / g total DNA,t=6.514,P=0.048].Conclusions Lactobacillus strains colonizing in vaginal are different between postmenopausal women and reproductive age women,and the content of the vaginal lactobacillus is reduced significantly in postmenopausal women.

10.
Article in Chinese | WPRIM | ID: wpr-398000

ABSTRACT

Objective To determine the effects of sexual hormone therapy at varied doses on prevention of bone mineral loss in Chinese postmenopausal women. Methods From March 2002 to March 2003, 90 Chinese postmenopausal women were randomly divided into three groups, each given one of the following regiments for 12 months, estradiol valerate (EV) 1 mg plus medroxyprogesterone (MPA) 2 mg for group A (31 subjects), eonjngated estradiol (ethinylestradiol-3-cyclopentylether, CEE) 0.45 mg plus MPA 2 mg for group B (29 subjects) and livial 1.25 mg for group C (30 subjects), respectively. In addition, 400 mg of elemental calcium were given daily to all those women. Bone mineral density (BMD) of the 2nd to 4th lumbar vertebra (L2~4) and biochemical markers of bone turnover, urine N-telopeptide of type Ⅰ collagen/creatinine(NTX/Cr) and serum total alkaline phnsphatase (ALP), were measured before and after drug administration. Results After treatment for 12 months, BMD of the L2~4 increased significantly by 0.039 g/cm2(P<0.01) in group B, but not significantly in group A or group C (P<0.05). Increases in BMD of the L2~4 was more in group B than that in group A and group C, respectively (P < 0.05), but no significant difference in BMD increase between group A and group C was found (P >0.05). After treatment for 6 months, urine NTX/Cr reduced from the baseline for all the three groups (P < 0.05), but no significant difference among group A, group B and group C was found (P >0.05). After treatment for 12 months, serum ALP significantly reduced from the baseline for all the three groups (P <0.01), but no significant difference among group A, group B and group C wag found (P > 0.05). Conclusions Sexual hormone therapy at varied doses lower than regular one for 12 months was effective in preventing bone mineral less in postmenopausal women.

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