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1.
Journal of Southern Medical University ; (12): 287-293, 2023.
Article in Chinese | WPRIM | ID: wpr-971527

ABSTRACT

OBJECTIVE@#To explore the molecular mechanisms of Porphyromonas gingivalis infection-induced umbilical vein endothelial barrier dysfunction in vitro.@*METHODS@#Human umbilical vein endothelial cells (HUVECs) were cultured in vitro, and after the formation of the endothelial barrier, the cells were infected with P. gingivals at a multiplicity of infection (MOI). The transepithelial electrical resistance (TEER) of the cell barrier was measured, and FITC-dextran trans-endothelial permeability assay and bacterial translocation assay were performed to assess the endothelial barrier function. The expression levels of cell junction proteins including ZO-1, occludin and VE-cadherin in the cells were examined by qRT-PCR and Western blotting.@*RESULTS@#In freshly seeded HUVECs, TEER increased until reaching the maximum on Day 5 (94 Ωcm2), suggesting the formation of the endothelial barrier. P. gingivals infection caused an increase of the permeability of the endothelial barrier as early as 0.5 h after bacterial inoculation, and the barrier function further exacerbated with time, as shown by significantly lowered TEER, increased permeability of FITC-dextran (40 000/70 000), and increased translocation of SYTO9-E. coli cross the barrier. MTT assay suggested that P. gingivals infection did not significantly affect the proliferation of HUVECs (P>0.05), but in P. gingivalsinfected cells, the expressions of ZO-1, occludin and VE-cadherin increased significantly at 24 and 48 h after bacterial inoculation (P < 0.05).@*CONCLUSION@#P. gingivals may disrupt the endothelial barrier function by down-regulating the expressions of the cell junction proteins (ZO-1, occludin, VE-cadherin) and increasing the permeability of the endothelial barrier.


Subject(s)
Humans , Cadherins/metabolism , Escherichia coli/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Occludin , Porphyromonas gingivalis/metabolism , Umbilical Veins/metabolism
2.
Chinese Journal of Biotechnology ; (12): 1142-1162, 2023.
Article in Chinese | WPRIM | ID: wpr-970429

ABSTRACT

Lysis is a common functional module in synthetic biology and is widely used in genetic circuit design. Lysis could be achieved by inducing expression of lysis cassettes originated from phages. However, detailed characterization of lysis cassettes hasn't been reported yet. Here, we first adopted arabinose- and rhamnose-inducible systems to develop inducible expression of five lysis cassettes (S105, A52G, C51S S76C, LKD, LUZ) in Escherichia coli Top10. By measuring OD600, we characterized the lysis behavior of strains harboring different lysis cassettes. These strains were harvested at different growth stages, induced with different concentrations of chemical inducers, or contained plasmids with different copy numbers. We found that although all five lysis cassettes could induce bacterial lysis in Top10, lysis behaviors differed a lot at various conditions. We further found that due to the difference in background expression levels between strain Top10 and Pseudomonas aeruginosa PAO1, it was hard to construct inducible lysis systems in strain PAO1. The lysis cassette controlled by rhamnose-inducible system was finally inserted into the chromosome of strain PAO1 to construct lysis strains after careful screen. The results indicated that LUZ and LKD were more effective in strain PAO1 than S105, A52G and C51S S76C. At last, we constructed an engineered bacteria Q16 using an optogenetic module BphS and the lysis cassette LUZ. The engineered strain was capable of adhering to target surface and achieving light-induced lysis by tuning the strength of ribosome binding sites (RBSs), showing great potential in surface modification.


Subject(s)
Rhamnose/pharmacology , Plasmids/genetics , Pseudomonas aeruginosa , Escherichia coli/metabolism
3.
Chinese Journal of Biotechnology ; (12): 2730-2742, 2023.
Article in Chinese | WPRIM | ID: wpr-981229

ABSTRACT

c-Myc protein encoded by c-Myc (cellular-myelocytomatosis viral oncogene) gene regulates the related gene expression through the Wnt/β-catenin signaling pathway, and has received extensive attention in recent years. The purpose of this study was to express Helicoverpa armigera c-Myc gene (Ha-c-Myc) by using prokaryotic expression system, prepare the polyclonal antibody, examine the spatio-temporal expression profile of Ha-c-Myc, and investigate the possible function of Ha-c-Myc in regulating H. armigera sterol carrier protein-2 (SCP-2) gene expression. The Ha-c-Myc gene was amplified by PCR and cloned into a prokaryotic expression plasmid pET-32a(+). The recombinant plasmid pET-32a-Ha-c-Myc was transformed into Escherichia coli BL21. IPTG was used to induce the expression of the recombinant protein. Protein was purified by Ni2+-NTA column and used to immunize New Zealand rabbits for preparing the polyclonal antibody. The Ha-c-Myc expression levels in different developmental stages (egg, larva, prepupa, pupa, and adult) of H. armigera and different tissues (midgut, fat body, head, and epidermis) of the prepupa were determined by real-time quantitative reverse transcription PCR (qRT-PCR). Ha-c-Myc siRNA was synthesized and transfected into H. armigera Ha cells. The relative mRNA levels of Ha-c-Myc and HaSCP-2 in Ha cells were detected by qRT-PCR. Results showed that the pET-32a-Ha-c-Myc recombinant plasmid was constructed. The soluble Ha-c-Myc protein of about 65 kDa was expressed in E. coli. The polyclonal antibody was prepared. Western blotting analysis suggested that the antibody had high specificity. Enzyme linked immunosorbent assay (ELISA) showed that the titer of the antibody was high. Ha-c-Myc gene expressed at all developmental stages, with high levels in the early and late instars of larva, and the prepupal stage. Tissue expression profiles revealed that Ha-c-Myc expressed in various tissues of prepupa, with high expression level in the midgut, but low levels in the epidermis and fat body. RNAi results showed that the knockdown of Ha-c-Myc expression significantly affected transcription of HaSCP-2, leading to a 50% reduction in HaSCP-2 mRNA expression level. In conclusion, the Ha-c-Myc was expressed through a prokaryotic expression system, and the polyclonal anti-Ha-c-Myc antibody was obtained. Ha-c-Myc may promote the expression of HaSCP-2 and play an important role in the lipid metabolism of H. armigera. These results may facilitate further study on the potential role and function mechanism of Ha-c-Myc in H. armigera and provide experimental data for exploring new targets of green pesticides.


Subject(s)
Animals , Rabbits , Escherichia coli/metabolism , Enzyme-Linked Immunosorbent Assay , Moths/genetics , Blotting, Western , Larva/genetics , Isoantibodies/metabolism , Antibody Specificity
4.
Chinese Journal of Biotechnology ; (12): 2375-2389, 2023.
Article in Chinese | WPRIM | ID: wpr-981207

ABSTRACT

Adipic acid is a high-value-added dicarboxylic acid which is primarily used in the production of nylon-66 for manufacturing polyurethane foam and polyester resins. At present, the biosynthesis of adipic acid is hampered by its low production efficiency. By introducing the key enzymes of adipic acid reverse degradation pathway into a succinic acid overproducing strain Escherichia coli FMME N-2, an engineered E. coli JL00 capable of producing 0.34 g/L adipic acid was constructed. Subsequently, the expression level of the rate-limiting enzyme was optimized and the adipic acid titer in shake-flask fermentation increased to 0.87 g/L. Moreover, the supply of precursors was balanced by a combinatorial strategy consisting of deletion of sucD, over-expression of acs, and mutation of lpd, and the adipic acid titer of the resulting E. coli JL12 increased to 1.51 g/L. Finally, the fermentation process was optimized in a 5 L fermenter. After 72 h fed-batch fermentation, adipic acid titer reached 22.3 g/L with a yield of 0.25 g/g and a productivity of 0.31 g/(L·h). This work may serve as a technical reference for the biosynthesis of various dicarboxylic acids.


Subject(s)
Escherichia coli/metabolism , Metabolic Engineering , Bioreactors , Fermentation , Adipates/metabolism
5.
Chinese Journal of Biotechnology ; (12): 2359-2374, 2023.
Article in Chinese | WPRIM | ID: wpr-981206

ABSTRACT

As an essential amino acid, l-tryptophan is widely used in food, feed and medicine sectors. Nowadays, microbial l-tryptophan production suffers from low productivity and yield. Here we construct a chassis E. coli TRP3 producing 11.80 g/L l-tryptophan, which was generated by knocking out the l-tryptophan operon repressor protein (trpR) and the l-tryptophan attenuator (trpL), and introducing the feedback-resistant mutant aroGfbr. On this basis, the l-tryptophan biosynthesis pathway was divided into three modules, including the central metabolic pathway module, the shikimic acid pathway to chorismate module and the chorismate to tryptophan module. Then we used promoter engineering approach to balance the three modules and obtained an engineered E. coli TRP9. After fed-batch cultures in a 5 L fermentor, tryptophan titer reached to 36.08 g/L, with a yield of 18.55%, which reached 81.7% of the maximum theoretical yield. The tryptophan producing strain with high yield laid a good foundation for large-scale production of tryptophan.


Subject(s)
Escherichia coli/metabolism , Tryptophan , Metabolic Engineering , Bioreactors , Metabolic Networks and Pathways
6.
Chinese Journal of Biotechnology ; (12): 2070-2080, 2023.
Article in Chinese | WPRIM | ID: wpr-981190

ABSTRACT

5-aminovalanoic acid (5AVA) can be used as the precursor of new plastics nylon 5 and nylon 56, and is a promising platform compound for the synthesis of polyimides. At present, the biosynthesis of 5-aminovalanoic acid generally is of low yield, complex synthesis process and high cost, which hampers large-scale industrial production. In order to achieve efficient biosynthesis of 5AVA, we developed a new pathway mediated by 2-keto-6-aminohexanoate. By combinatory expression of L-lysine α-oxidase from Scomber japonicus, α-ketoacid decarcarboxylase from Lactococcus lactis and aldehyde dehydrogenase from Escherichia coli, the synthesis of 5AVA from L-lysine in Escherichia coli was achieved. Under the initial conditions of glucose concentration of 55 g/L and lysine hydrochloride of 40 g/L, the final consumption of 158 g/L glucose and 144 g/L lysine hydrochloride, feeding batch fermentation to produce 57.52 g/L of 5AVA, and the molar yield is 0.62 mol/mol. The new 5AVA biosynthetic pathway does not require ethanol and H2O2, and achieved a higher production efficiency as compared to the previously reported Bio-Chem hybrid pathway mediated by 2-keto-6-aminohexanoate.


Subject(s)
Nylons , Lysine/metabolism , Hydrogen Peroxide/metabolism , Metabolic Engineering , Plastics/metabolism , Fermentation , Escherichia coli/metabolism , Aminocaproates/metabolism
7.
Chinese Journal of Biotechnology ; (12): 1621-1632, 2023.
Article in Chinese | WPRIM | ID: wpr-981158

ABSTRACT

The widespread of tigecycline resistance gene tet(X4) has a serious impact on the clinical efficacy of tigecycline. The development of effective antibiotic adjuvants to combat the looming tigecycline resistance is needed. The synergistic activity between the natural compound β-thujaplicin and tigecycline in vitro was determined by the checkerboard broth microdilution assay and time-dependent killing curve. The mechanism underlining the synergistic effect between β-thujaplicin and tigecycline against tet(X4)-positive Escherichia coli was investigated by determining cell membrane permeability, bacterial intracellular reactive oxygen species (ROS) content, iron content, and tigecycline content. β-thujaplicin exhibited potentiation effect on tigecycline against tet(X4)-positive E. coli in vitro, and presented no significant hemolysis and cytotoxicity within the range of antibacterial concentrations. Mechanistic studies demonstrated that β-thujaplicin significantly increased the permeability of bacterial cell membranes, chelated bacterial intracellular iron, disrupted the iron homeostasis and significantly increased intracellular ROS level. The synergistic effect of β-thujaplicin and tigecycline was identified to be related to interfere with bacterial iron metabolism and facilitate bacterial cell membrane permeability. Our studies provided theoretical and practical data for the application of combined β-thujaplicin with tigecycline in the treatment of tet(X4)-positive E. coli infection.


Subject(s)
Humans , Tigecycline/pharmacology , Escherichia coli/metabolism , Reactive Oxygen Species/therapeutic use , Plasmids , Anti-Bacterial Agents/metabolism , Escherichia coli Infections/microbiology , Bacteria/genetics , Microbial Sensitivity Tests
8.
Chinese Journal of Biotechnology ; (12): 1596-1608, 2023.
Article in Chinese | WPRIM | ID: wpr-981156

ABSTRACT

Fusobacterium nucleatum is an opportunistic pathogenic bacterium that can be enriched in colorectal cancer tissues, affecting multiple stages of colorectal cancer development. The two-component system plays an important role in the regulation and expression of genes related to pathogenic resistance and pathogenicity. In this paper, we focused on the CarRS two-component system of F. nucleatum, and the histidine kinase protein CarS was recombinantly expressed and characterized. Several online software such as SMART, CCTOP and AlphaFold2 were used to predict the secondary and tertiary structure of the CarS protein. The results showed that CarS is a membrane protein with two transmembrane helices and contains 9 α-helices and 12 β-folds. CarS protein is composed of two domains, one is the N-terminal transmembrane domain (amino acids 1-170), the other is the C-terminal intracellular domain. The latter is composed of a signal receiving domain (histidine kinases, adenylyl cyclases, methyl-accepting proteins, prokaryotic signaling proteins, HAMP), a phosphate receptor domain (histidine kinase domain, HisKA), and a histidine kinase catalytic domain (histidine kinase-like ATPase catalytic domain, HATPase_c). Since the full-length CarS protein could not be expressed in host cells, a fusion expression vector pET-28a(+)-MBP-TEV-CarScyto was constructed based on the characteristics of secondary and tertiary structures, and overexpressed in Escherichia coli BL21-Codonplus(DE3)RIL. CarScyto-MBP protein was purified by affinity chromatography, ion-exchange chromatography, and gel filtration chromatography with a final concentration of 20 mg/ml. CarScyto-MBP protein showed both protein kinase and phosphotransferase activities, and the MBP tag had no effect on the function of CarScyto protein. The above results provide a basis for in-depth analysis of the biological function of the CarRS two-component system in F. nucleatum.


Subject(s)
Humans , Histidine Kinase/metabolism , Fusobacterium nucleatum/metabolism , Automobiles , Protein Kinases/genetics , Escherichia coli/metabolism , Colorectal Neoplasms
9.
Journal of Southern Medical University ; (12): 1013-1018, 2022.
Article in Chinese | WPRIM | ID: wpr-941034

ABSTRACT

OBJECTIVE@#To construct an adenovirus vector expressing artificial splicing factor capable of regulating alternative splicing of Yap1 in cardiomyocytes.@*METHODS@#The splicing factors with different sequences were constructed against Exon6 of YAP1 based on the sequence specificity of Pumilio1. The PCR fragment of the artificially synthesized PUF-SR or wild-type PUFSR was cloned into pAd-Track plasmid, and the recombinant plasmids were transformed into E. coli DH5α for plasmid amplification. The amplified plasmids were digested with Pac I and transfected into 293A cells for packaging to obtain the adenovirus vectors. Cultured neonatal rat cardiomyocytes were transfected with the adenoviral vectors, and alternative splicing of YAP1 was detected using quantitative and semi-quantitative PCR; Western blotting was performed to detect the signal of the fusion protein Flag.@*RESULTS@#The transfection efficiency of the adenovirus vectors was close to 100% in rat cardiomyocytes, and no fluorescent protein was detected in the cells with plasmid transfection. The results of Western blotting showed that both the negative control and Flag-SR-NLS-PUF targeting the YAPExon6XULIE sequence were capable of detecting the expression of the protein fused to Flag. The results of reverse transcription-PCR and PCR demonstrated that the artificial splicing factor constructed based on the 4th target sequence of YAP1 effectively regulated the splicing of YAP1 Exon6 in the cardiomyocytes (P < 0.05).@*CONCLUSION@#We successfully constructed adenovirus vectors capable of regulating YAP1 alternative splicing rat cardiomyocytes.


Subject(s)
Animals , Rats , Adenoviridae/metabolism , Alternative Splicing , Animals, Newborn , Escherichia coli/metabolism , Genetic Vectors , Myocytes, Cardiac/metabolism , Plasmids , RNA Splicing Factors/metabolism , Transfection
10.
Chinese Journal of Biotechnology ; (12): 4719-4730, 2022.
Article in Chinese | WPRIM | ID: wpr-970343

ABSTRACT

Vibrio parahaemolyticus, the main pathogen causing seafood related food poisoning worldwide, has strong biofilm formation ability. ToxR is a membrane binding regulatory protein, which has regulatory effect on biofilm formation of V. parahaemolyticus, but the specific mechanism has not been reported. c-di-GMP is an important second messenger in bacteria and is involved in regulating a variety of bacterial behaviors including biofilm formation. In this study, we investigated the regulation of ToxR on c-di-GMP metabolism in V. parahaemolyticus. Intracellular c-di-GMP in the wild type (WT) and toxR mutant (ΔtoxR) strains were extracted by ultrasonication, and the concentrations of c-di-GMP were then determined by enzyme linked immunosorbent assay (ELISA). Three c-di-GMP metabolism-related genes scrA, scrG and vpa0198 were selected as the target genes. Quantitative real-time PCR (q-PCR) was employed to calculate the transcriptional variation of each target gene between WT and ΔtoxR strains. The regulatory DNA region of each target gene was cloned into the pHR309 plasmid harboring a promoterless lacZ gene. The recombinant plasmid was subsequently transferred into WT and ΔtoxR strains to detect the β-galactosidase activity in the cellular extracts. The recombinant lacZ plasmid containing each of the target gene was also transferred into E. coli 100λpir strain harboring the pBAD33 plasmid or the recombinant pBAD33-toxR to test whether ToxR could regulate the expression of the target gene in a heterologous host. The regulatory DNA region of each target gene was amplified by PCR, and the over-expressed His-ToxR was purified. The electrophoretic mobility shift assay (EMSA) was applied to verify whether His-ToxR directly bound to the target promoter region. ELISA results showed that the intracellular c-di-GMP level significantly enhanced in ΔtoxR strain relative to that in WT strain, suggesting that ToxR inhibited the production of c-di-GMP in V. parahaemolyticus. qPCR results showed that the mRNA levels of scrA, scrG and vpa0198 significantly increased in ΔtoxR strain relative to those in WT strain, suggesting that ToxR repressed the transcription of scrA, scrG and vpa0198. lacZ fusion assay showed that ToxR was able to repress the promoter activities of scrA, scrG and vpa0198 in both V. parahaemolyticus and E. coli 100λpir. EMSA results showed that His-ToxR was able to bind to the regulatory DNA regions of scrA and scrG, but not to the regulatory DNA region of vpa0198. In conclusion, ToxR inhibited the production of c-di-GMP in V. parahaemolyticus via directly regulating the transcription of enzyme genes associated with c-di-GMP metabolism, which would be beneficial for V. parahaemolyticus to precisely control bacterial behaviors including biofilm formation.


Subject(s)
Vibrio parahaemolyticus/metabolism , Escherichia coli/metabolism , Bacterial Proteins/metabolism , Transcription Factors/genetics , Gene Expression Regulation, Bacterial
11.
Chinese Journal of Biotechnology ; (12): 4681-4691, 2022.
Article in Chinese | WPRIM | ID: wpr-970340

ABSTRACT

Semiconductor nanoparticles generate photoelectrons and photo-induced holes under light excitation, and thus may influence the growth of microbial cells. The highly oxidative holes may severely damage the cells, while the photoelectrons may promote microbial metabolism. In this study, we evaluated the effect of exogenous cadmium sulfide (CdS) nanoparticles on bacterial growth using OD600 and colony forming unit (CFU) as indicators. The oxidase activities, the concentration of pyruvate and malondialdehyde, and the expression of relevant genes assessed by real-time fluorescent quantitative PCR were analyzed to investigate the effect of excited CdS on cellular metabolism. The results showed that the OD600 and pyruvate accumulation of E. coli increased by 32.4% and 34.6%, respectively, under light conditions. Moreover, the relative expression level of the division protein gene ftsZ was increased more than 50%, and the tricarboxylic acid cycle pathway gene icdA and gltA increased by 86% and 103%, respectively. The results indicated that photoelectrons could be used by microorganisms, resulting in promoted growth and metabolism. This study gives a deep insight into the interaction between nanoparticles and bacteria.


Subject(s)
Escherichia coli/metabolism , Nanoparticles , Cadmium Compounds/metabolism , Quantum Dots
12.
Chinese Journal of Biotechnology ; (12): 4658-4668, 2022.
Article in Chinese | WPRIM | ID: wpr-970338

ABSTRACT

Trehalase is widely used in industrial fermentation, food, medicine and other fields. There is a lack of industrial varieties of trehalase with excellent performance in China. Moreover, the applied research on trehalase was not well conducted. In this study, a strain of Pectobacterium cypripedii was screened from nature, and the gene PCTre encoding an acidic trehalase was cloned and expressed in E. coli BL21(DE3). The highest enzyme activity reached 4130 U/mL after fermenting in a 5 L fermenter for 28 h. The enzymatic properties study showed that PCTre hydrolyzed trehalose specifically. The optimum pH and temperature were 5.5 and 35 ℃, respectively. 80% of the enzyme activity was retained after being treated at pH 4.0, 4.5, and 5.0 for 8 h, showing good acid tolerance. Moreover, it has good tolerance to organic solvents, 60% enzyme activity was retained after being treated with 20% (V/V) ethanol solution for 24 h. Furthermore, trehalose could be completely hydrolyzed within 16 h in a simulated fermentation system containing 20% (V/V) ethanol and 7.5% trehalose, with 500 U/L PCTre added. This indicated a good application potential for industrial ethanol fermentation.


Subject(s)
Trehalase/metabolism , Trehalose/metabolism , Escherichia coli/metabolism , Ethanol/metabolism , Cloning, Molecular
13.
Chinese Journal of Biotechnology ; (12): 4644-4657, 2022.
Article in Chinese | WPRIM | ID: wpr-970337

ABSTRACT

β-glucosidase has important applications in food, medicine, biomass conversion and other fields. Therefore, exploring β-glucosidase with strong stability and excellent properties is a research hotspot. In this study, a GH3 family β-glucosidase gene named Iubgl3 was successfully cloned from Infirmifilum uzonense. Sequence analysis showed that the full length of Iubgl3 was 2 106 bp, encoding 702 amino acids, with a theoretical molecular weight of 77.0 kDa. The gene was cloned and expressed in E. coli and the enzymatic properties of purified IuBgl3 were studied. The results showed that the optimal pH and temperature for pNPG hydrolysis were 5.0 and 85 ℃, respectively. The enzyme has good thermal stability, and more than 85% of enzyme activity can be retained after being treated at 80 ℃ for2 h. This enzyme has good pH stability and more than 85% of its activity can be retained after being treated at pH 4.0-11.0 for 1 h. It was found that the enzyme had high hydrolysis ability to p-nitrophenyl β-d-glucoside (pNPG) and p-nitrophenyl β-d-xylopyranoside (pNPX). When pNPG was used as the substrate, the kinetic parameters Km and Vmax were 0.38 mmol and 248.55 μmol/(mg·min), respectively, and the catalytic efficiency kcat/Km was 6 149.20 s-1mmol-1. Most metal ions had no significant effect on the enzyme activity of IuBgl3. SDS completely inactivated the enzyme, while EDTA increased the enzyme activity by 30%. This study expanded the β-glucosidase gene diversity of the thermophilic archaea GH3 family and obtained a thermostable acid bifunctional enzyme with good industrial application potential.


Subject(s)
beta-Glucosidase/chemistry , Archaea/metabolism , Escherichia coli/metabolism , Hydrogen-Ion Concentration , Temperature , Glucosides , Enzyme Stability , Substrate Specificity , Kinetics
14.
Chinese Journal of Biotechnology ; (12): 4587-4600, 2022.
Article in Chinese | WPRIM | ID: wpr-970333

ABSTRACT

Ultrashort peptides have higher stability, tissue penetrability, biocompatibility, and less immunogenicity, and are widely applied in biology and medicine. GHK (glycyl-l-histidyl-l-lysine) and GQPR (glycyl-l-glutamyl-l-prolyl-l-arginine) can stimulate collagen renewal and inhibit collagen degradation. GHK and GQPR have been used in cosmetic anti-wrinkle skincare and make-up products. The most common approach for ultrashort peptide production is the solid-phase synthesis, which is eco-unfriendly due to heavy usage of organic chemical reagents during the manufacturing process. Here we report a new approach to the production of ultrashort peptides. Recombinant expression of ultrashort peptides is usually unfeasible because of the short amino acid sequences. A vector pET28a-Trxm harboring the thioredoxin gene was first constructed for subsequent fusion expression. The tandem repeats of GHK and GQPR genes were used as the templates for rolling circle amplification (RCA). The RCA reaction was tuned to incorporate noncanonical nucleotides 5-methylcytosine to obtain long DNA fragments. Gene sequences with various lengths were generated through double digestion of Acc65 Ⅰ and Apa Ⅰ. The resulting digestion products were gel recovered by size (from 500 bp to 1 500 bp) and cloned into pET28a-Trxm to obtain the recombinant vector pET28a-Trxm-(TRSP)n. The pET28a-Trxm-(TRSP)n was introduced into E. coli BL21(DE3) to generate a library of Trxm-(TRSP)n sequences with a controlled distribution of lengths. Through double digestion and sequencing, positive clones with tandem repeats n=1, 2, 3, 4, 6, 7, 8, 9 were obtained. Protein expression results showed protein bands with corresponding molecular weight, and the protein expression level decreased as the tandem repeats increased. The expression level of Trxm-(TRSP)1 achieved 50% of the total protein, while the expression level of Trxm-(TRSP)2 was 30% of the total protein. The crude extracts from cell pellets were further treated with enterokinase cleavage, and the supernatants containing (TRSP)1 were collected after ultrafiltration and then subjected to trypsin cleavage. HPLC analysis indicated that the ultrashort peptides GHK and GQPR were successfully obtained through two-step cleavage. This study may facilitate the commercial production of ultrashort peptides.


Subject(s)
Escherichia coli/metabolism , Peptides/chemistry , Amino Acid Sequence , Gene Library , Tandem Repeat Sequences
15.
Chinese Journal of Biotechnology ; (12): 4567-4586, 2022.
Article in Chinese | WPRIM | ID: wpr-970332

ABSTRACT

l-cysteine is an important sulfur-containing α-amino acid. It exhibits multiple physiological functions with diverse applications in pharmaceutical cosmetics and food industry. Here, a strategy of coordinated gene expression between carbon and sulfur modules in Escherichia coli was proposed and conducted for the production of l-cysteine. Initially, the titer of l-cysteine was improved to (0.38±0.02) g/L from zero by enhancing the biosynthesis of l-serine module (serAf, serB and serCCg) and overexpression of CysB. Then, promotion of l-cysteine transporter, increased assimilation of sulfur, reduction or deletion of l-cysteine and l-serine degradation pathway and enhanced expression of cysEf (encoding serine acetyltransferase) and cysBSt (encoding transcriptional dual regulator CysB) were achieved, resulting in an improved l-cysteine titer (3.82±0.01) g/L. Subsequently, expressions of cysM, nrdH, cysK and cysIJ genes that were involved in sulfur module were regulated synergistically with carbon module combined with utilization of sulfate and thiosulfate, resulting in a strain producing (4.17±0.07) g/L l-cysteine in flask shake and (11.94±0.1) g/L l-cysteine in 2 L bioreactor. Our results indicated that efficient biosynthesis of l-cysteine could be achieved by a proportional supply of sulfur and carbon in vivo. This study would facilitate the commercial bioproduction of l-cysteine.


Subject(s)
Escherichia coli/metabolism , Cysteine/metabolism , Bioreactors , Sulfur/metabolism , Serine/metabolism
16.
Chinese Journal of Biotechnology ; (12): 4403-4419, 2022.
Article in Chinese | WPRIM | ID: wpr-970323

ABSTRACT

1, 5-diaminopentane, also known as cadaverine, is an important raw material for the production of biopolyamide. It can be polymerized with dicarboxylic acid to produce biopolyamide PA5X whose performances are comparable to that of the petroleum-based polyamide materials. Notably, biopolyamide uses renewable resources such as starch, cellulose and vegetable oil as substrate. The production process does not cause pollution to the environment, which is in line with the green and sustainable development strategy. The biosynthesis of 1, 5-diaminopentane mainly includes two methods: the de novo microbial synthesis and the whole cell catalysis. Lysine decarboxylase as the key enzyme for 1, 5-diaminopentane production, mainly includes an inducible lysine decarboxylase CadA and a constituent lysine decarboxylase LdcC. Lysine decarboxylase is a folded type Ⅰ pyridoxal-5' phosphate (PLP) dependent enzyme, which displays low activity and unstable structure, and is susceptible to deactivation by environmental factors in practical applications. Therefore, improving the catalytic activity and stability of lysine decarboxylase has become a research focus in this field, and molecular engineering and immobilization are the mainly approaches. Here, the mechanism, molecular engineering and immobilization strategies of lysine decarboxylase were reviewed, and the further strategies for improving its activity and stability were also prospected, with the aim to achieve efficient production of 1, 5-diaminopentane.


Subject(s)
Escherichia coli/metabolism , Carboxy-Lyases/metabolism , Catalysis , Cadaverine/metabolism
17.
Chinese Journal of Biotechnology ; (12): 4385-4402, 2022.
Article in Chinese | WPRIM | ID: wpr-970322

ABSTRACT

L-homoserine and its derivatives (O-succinyl-L-homoserine and O-acetyl-L-homoserine) are precursors for the biosynthesis of L-methionine, and various C4 compounds (isobutanol, γ-butyrolactone, 1, 4-butanediol, 2, 4-dihydroxybutyric acid) and L-phosphinothricin. Therefore, the fermentative production of L-homoserine and its derivatives became the research hotspot in recent years. However, the low fermentation yield and conversion rate, and the unclear regulation mechanism for the biosynthesis of L-homoserine and its derivatives, hamper the development of an efficient production process for L-homoserine and its derivatives. This review summarized the advances in the biosynthesis of L-homoserine and its derivatives by metabolic engineering of Escherichia coli from the aspects of substrate uptake, redirection of carbon flow at the key nodes, recycle of NADPH and export of target products. This review may facilitate subsequent metabolic engineering and biotechnological production of L-homoserine and its derivatives.


Subject(s)
Escherichia coli/metabolism , Metabolic Engineering , Homoserine/metabolism , Escherichia coli Proteins/metabolism , Fermentation
18.
Chinese Journal of Natural Medicines (English Ed.) ; (6): 527-536, 2022.
Article in English | WPRIM | ID: wpr-939917

ABSTRACT

Phenylalanine ammonia-lyase (PAL), which catalyzes the conversion from L-phenylalanine to trans-cinnamic acid, is a well-known key enzyme and a connecting step between primary and secondary metabolisms in the phenylpropanoid biosynthetic pathway of plants and microbes. Schisandra chinensis, a woody vine plant belonging to the family of Magnoliaceae, is a rich source of dibenzocyclooctadiene lignans exhibiting potent activity. However, the functional role of PAL in the biosynthesis of lignan is relatively limited, compared with those in lignin and flavonoids biosynthesis. Therefore, it is essential to clone and characterize the PAL genes from this valuable medicinal plant. In this study, molecular cloning and characterization of three PAL genes (ScPAL1-3) from S. chinensis was carried out. ScPALs were cloned using RACE PCR. The sequence analysis of the three ScPALs was carried out to give basic characteristics followed by docking analysis. In order to determine their catalytic activity, recombinant protein was obtained by heterologous expression in pCold-TF vector in Escherichia coli (BL21-DE3), followed by Ni-affinity purification. The catalytic product of the purified recombinant proteins was verified using RP-HPLC through comparing with standard compounds. The optimal temperature, pH value and effects of different metal ions were determined. Vmax, Kcat and Km values were determined under the optimal conditions. The expression of three ScPALs in different tissues was also determined. Our work provided essential information for the function of ScPALs.


Subject(s)
Cloning, Molecular , Escherichia coli/metabolism , Phenylalanine/metabolism , Phenylalanine Ammonia-Lyase/chemistry , Recombinant Proteins , Schisandra/genetics
19.
China Journal of Chinese Materia Medica ; (24): 906-912, 2022.
Article in Chinese | WPRIM | ID: wpr-928008

ABSTRACT

As an important active ingredient in the rare Chinese herb Gastrodiae Rhizoma and also the main precursor for gastrodin biosynthesis, 4-hydroxybenzyl alcohol has multiple pharmacological activities such as anti-inflammation, anti-tumor, and anti-cerebral ischemia. The pharmaceutical products with 4-hydroxybenzyl alcohol as the main component have been increasingly favored. At present, 4-hydroxybenzyl alcohol is mainly obtained by natural extraction and chemical synthesis, both of which, however, exhibit some shortcomings that limit the long-term application of 4-hydroxybenzyl alcohol. The wild and cultivated Gastrodia elata resources are limited. The chemical synthesis requires many steps, long time, and harsh reaction conditions. Besides, the resulting by-products are massive and three reaction wastes are difficult to treat. Therefore, how to artificially prepare 4-hydroxybenzyl alcohol with high yield and purity has become an urgent problem facing the medical researchers. Guided by the theory of microbial metabolic engineering, this study employed the genetic engineering technologies to introduce three genes ThiH, pchF and pchC into Escherichia coli for synthesizing 4-hydroxybenzyl alcohol with L-tyrosine. And the fermentation conditions of engineering strain for producing 4-hydroxybenzyl alcohol in shake flask were also discussed. The experimental results showed that under the conditions of 0.5 mmol·L~(-1) IPTG, 15 ℃ induction temperature, and 40 ℃ transformation temperature, M9 Y medium containing 200 mg·L~(-1) L-tyrosine could be transformed into(69±5)mg·L~(-1) 4-hydroxybenzyl alcohol, which has laid a foundation for producing 4-hydroxybenzyl alcohol economically and efficiently by further expanding the fermentation scale in the future.


Subject(s)
Benzyl Alcohols , Escherichia coli/metabolism , Gastrodia/chemistry , Metabolic Engineering , Tyrosine/metabolism
20.
Chinese Journal of Biotechnology ; (12): 1267-1294, 2022.
Article in Chinese | WPRIM | ID: wpr-927780

ABSTRACT

This article summarized the reviews and research articles published in Chinese Journal of Biotechnology in the field of biomanufacturing in 2021. The article covered major chassis cells such as Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, filamentous fungi, non-model bacteria and non-conventional yeasts. Moreover, this article summarized the advances in the production of amino acids, organic acids, vitamins, higher alcohols, natural compounds (terpenoids, flavonoids, alkaloids), antibiotics, enzymes and enzyme-catalyzed products, biopolymers, as well as the utilization of biomass and one-carbon materials. The key technologies used in the construction of cell factories, such as regulation, evolution, and high-throughput screening, were also included. This article may help the readers better understand the R & D trend in biomanufacturing driven by engineered microbes.


Subject(s)
Biomass , Biotechnology , Corynebacterium glutamicum/metabolism , Escherichia coli/metabolism , Metabolic Engineering , Saccharomyces cerevisiae/genetics
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