In this study, Saccharomyces cerevisiae R0 was used as the chassis cell to synthesize oleanolic acid from scratch through the heterologous expression of β-amyrin synthase(β-AS) from Glycyrrhiza uralensis, cytochrome P450 enzyme CYP716A154 from Catharanthus roseus, and cytochrome P450 reductase AtCPR from Arabidopsis thaliana. The engineered strain R1 achieved shake flask titres of 5.19 mg·L~(-1). By overexpressing enzymes in the pentose phosphate pathway(PPP)(ZWF1, GND1, TKL1, and TAL), the NADH kinase gene in the mitochondrial matrix(POS5), truncated 3-hydroxy-3-methylglutaryl-CoA reductase(tPgHMGR1) from Panax ginseng, and farnesyl diphosphate synthase gene(SmFPS) from Salvia miltiorrhiza, the precursor supply and intracellular reduced nicotinamide adenine dinucleotide phosphate(NADPH) supply were enhanced, resulting in an 11.4-fold increase in squalene yield and a 3.6-fold increase in oleanolic acid yield. Subsequently, increasing the copy number of the heterologous genes tPgHMGR1, β-AS, CYP716A154, and AtCPR promoted the metabolic flow towards the final product, oleanolic acid, and increased the yield by three times. Shake flask fermentation data showed that, by increasing the copy number, precursor supply, and intracellular NADPH supply, the final engineered strain R3 could achieve an oleanolic acid yield of 53.96 mg·L~(-1), which was 10 times higher than that of the control strain R1. This study not only laid the foundation for the green biosynthesis of oleanolic acid but also provided a reference for metabolic engineering research on other pentacyclic triterpenoids in S. cerevisiae.
Oleanolic Acid/biosynthesis* ; Saccharomyces cerevisiae/metabolism* ; Industrial Microbiology ; Microorganisms, Genetically-Modified/metabolism* ; Plants/enzymology* ; Fermentation ; Metabolic Engineering

Many triterpenoid compounds have been successfully heterologously synthesized in Saccharomyces cerevisiae. To increase the yield of triterpenoids, various metabolic engineering strategies have been developed. One commonly applied strategy is to enhance the supply of precursors, which has been widely used by researchers. Squalene, as a precursor to triterpenoid biosynthesis, plays a crucial role in the synthesis of these compounds. This study primarily investigates the effect of different squalene levels in chassis strains on the synthesis of triterpenoids(oleanolic acid and ursolic acid), and the underlying mechanisms are further explored using real-time quantitative PCR(qPCR) analysis. The results demonstrate that the chassis strain CB-9-5, which produces high levels of squalene, inhibits the synthesis of oleanolic acid and ursolic acid. In contrast, chassis strains with moderate to low squalene production, such as Y8-1 and CNPK, are more conducive to the synthesis of oleanolic acid and ursolic acid. The qPCR analysis reveals that the expression levels of ERG1, βAS, and CrCYP716A154 in the oleanolic acid-producing strain CB-OA are significantly lower than those in the control strains C-OA and Y-OA, suggesting that high squalene production in the chassis strains suppresses the transcription of certain genes, leading to a reduced yield of triterpenoids. Our findings indicate that when constructing S. cerevisiae strains for triterpenoid production, chassis strains with high squalene content may suppress the expression of certain genes, ultimately lowering their production, whereas chassis strains with moderate squalene levels are more favorable for triterpenoid biosynthesis.
Squalene/analysis* ; Saccharomyces cerevisiae/genetics* ; Triterpenes/metabolism* ; Metabolic Engineering ; Oleanolic Acid/biosynthesis* ; Ursolic Acid

In this study, the synthetic pathway of β-amyrin was constructed in the pre-constructed Saccharomyces cerevisiae chassis strain Y0 by introducing β-amyrin synthase from Glycyrrhiza uralensis, resulting strain Y1-C20-6, which successfully produced β-amyrin up to 5.97 mg·L~(-1). Then, the mevalonate pyrophosphate decarboxylase gene(ERG19), mevalonate kinase gene(ERG12), 3-hydroxy-3-methylglutaryl-CoA synthase gene(ERG13), phosphomevalonate kinase gene(ERG8) and IPP isomerase gene(IDI1)were overexpressed to promoted the metabolic fluxto the direction of β-amyrin synthesis for further improving β-amyrin production, resulting the strain Y2-C2-4 which produced β-amyrin of 10.3 mg·L~(-1)under the shake flask fermentation condition. This is 100% higher than that of strain Y1-C20-6, illustrating the positive effect of the metabolic engineering strategy applied in this study. The titer of β-amyrin was further improved up to 157.4 mg·L~(-1) in the fed-batch fermentation, which was almost 26 fold of that produced by strain Y1-C20-6. This study not only laid the foundation for the biosynthesis of β-amyrin but also provided a favorable chassis strain for elucidation of cytochrome oxidases and glycosyltransferases of β-amyrin-based triterpenoids.
Fermentation ; Glycyrrhiza uralensis ; enzymology ; genetics ; Industrial Microbiology ; Intramolecular Transferases ; genetics ; Metabolic Engineering ; Oleanolic Acid ; analogs & derivatives ; biosynthesis ; Saccharomyces cerevisiae ; metabolism

OBJECTIVETo optimize the synthetic pathway and fermentation process of yeast cell factories for production of oleanoic acid.

METHODUsing the DNA assembler method, one copy of Glycyrrhiza glabra beta-amyrin synthase (GgbAS), Medicago truncatula oleanolic acid synthase (MtOAS) and Arabidopsis thaliana cytochrome P450 reductase 1 (AtCPR1) genes were introduced into Saccharomyces cerevisiae strain BY-OA, resulting in strain BY-20A. YPD medium with different glucose concentration were then used to cultivate strain BY-2OA.

RESULTIncreasing gene copies of GgbAS, MtOAS and AtCPR1 resulted in increased beta-amyrin and oleanolic acid production. The strain BY-2OA produced 136.5 mg x L(-1) beta-amyrin and 92.5 mg x L(-1) oleanolic acid, which were 54% and 30% higher than the parent strain BY-OA. Finally, the titer of oleanolic acid increased to 165.7 mg x L(-1) when cultivated in YPD medium with 40 mg x L(-1) glucose.

CONCLUSIONProduction of oleanoic acid increased significantly in the yeast strain BY-2OA, which can provide the basis for creating an alternative way for production of oleanoic acid in place of extraction from plant sources.

Biomass ; Biotechnology ; methods ; Dose-Response Relationship, Drug ; Fermentation ; Glucose ; pharmacology ; Oleanolic Acid ; biosynthesis ; Saccharomyces cerevisiae ; cytology ; drug effects ; metabolism

The ORF sequence of glycosyltransferase gene BcUGT1 cloned from Bupleurum chinense DC. was analyzed and its three dimentional structure was predicted. Using qRT-PCR method, the expression characteristics of BcUGT1 after methyl jasmonate (MeJA) induction and in different plant tissues were investigated. The results showed that BcUGT1 may be involved in saikosaponin biosynthesis in B. chinense. Thereafter, the recombinant vectors of BcUGT1 were constructed for its expression in E. coli. The target protein was successfully expressed and purified. In the present study, three vectors, pRSET-A, pET-28a (+) and pET-30a (+), and three isolates of E. coli, BL21 (DE3) plysS, BL21A1 and BL21-CodonPlus (DE3)-RIPL were used under different induction conditions, such as different concentrations and during times of inducers (L-arabinose and IPTG) and different inducing temperatures. The results showed that in the condition of 0.5 or 1 mmol x L(-1) IPTG, 16 degrees C, 20 h, target protein expressed in BL21-CodonPlus (DE3)-RIPL with pET-28a (+) or pET-30a (+) as vector. Using PrepEase His-tagged protein purification kit, the target protein was purified. The present work will be helpful for follow-up bio-function analysis of BcUGT1.
Amino Acid Sequence ; Base Sequence ; Bupleurum ; chemistry ; Cloning, Molecular ; DNA, Complementary ; genetics ; DNA, Plant ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Glycosyltransferases ; genetics ; isolation & purification ; metabolism ; Oleanolic Acid ; analogs & derivatives ; biosynthesis ; Open Reading Frames ; genetics ; Phylogeny ; Plants, Medicinal ; chemistry ; Protein Structure, Secondary ; Recombinant Fusion Proteins ; genetics ; metabolism ; Saponins ; biosynthesis

OBJECTIVETo investigate the effects of saikosaponin a (SSa) on Glu-activated hippocampal astrocytes of rats.

METHODSNeonatal rat (1-3 days) hippocampal astrocytes were obtained and divided into control group, L-Glu activation group and SSa groups with SSa treatment at 5, 2.5, and 1.25 mg/L. The cell proliferation, cell cycle changes, and expression of glial fibrillary acidic protein (GFAP) after the treatments were assessed with MTT assay, flow cytometry and Western blotting, respectively.

RESULTSIn comparison with Glu-activation group, SSa treatment resulted in significant inhibition of the cell proliferation, cell division and GFAP expression in the Glu-activated astrocytes (P < 0.05). SSa at 2.5 mg/L showed the strongest inhibitory effects against astrocyte activation and maintained nearly normal level of astrocyte activation in comparison with the control group (P > 0.05).

CONCLUSIONSGlu-induced activation of rat hippocampal astrocytes can be inhibited by SSa, whose antiepileptic effects is probably mediated by inhibition of hippocampal astrocyte activation.

Animals ; Animals, Newborn ; Astrocytes ; cytology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Female ; Glial Fibrillary Acidic Protein ; biosynthesis ; Glutamic Acid ; pharmacology ; Hippocampus ; cytology ; Male ; Oleanolic Acid ; analogs & derivatives ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Saponins ; pharmacology

OBJECTIVETo study the effect of saikosaponins, the active ingredients of Bupleurum chinense DC. on glutamate and GABA expressions in the hippocampus of slow kindling rats induced by pentetrazole.

METHODSForty-eight healthy Sprague-Dawley rats were randomly divided into 6 equal groups, namely the blank control group (group A), normal saline (NS) group (group B), sodium valproate group (group C), and 3 saikosaponins groups of high, medium and small doses (groups D, E, and F, respectively). The rats in each group other than group A were given corresponding treatments after slow kindling by pentetrazole. After 4 weeks of treatment, the rats were sacrifices and the brain tissues were sampled, sliced and stained by immunohistochemically, and the results were analyzed according to the positive cell number and gray scale.

RESULTSIn CA1 region, the glutamate-positive cell number and gray scale of group B was significantly different from the other groups (P<0.05), but such difference was not observed in the CA2 and DG (P>0.05); In CA1, CA2 and DG of the hippocampus, the GABA-positive cell number of group B was significantly greater but the gray scale lower than those of the other groups (P<0.05). In CA1 and CA2 regions of the hippocampus, the glutamate- and GABA-positive cell ratio of group B was significantly lower than that of the other groups (P<0.05), but in CA1, CA2, and DG region of the hippocampus, the ratio of gray scale between glutamate- and GABA-positive cells was comparable between the groups (P>0.05).

CONCLUSIONThe expression of glutamate and GABA, especially the latter, increased in chronic kindling rat hippocampus. Saikosaponins intervene in such changes of glutamate and GABA to contain their expressions within normal range, which may be one of the mechanisms of saikosaponins to inhibit slow kindling induced by pentetrazole.

Animals ; Female ; Glutamic Acid ; biosynthesis ; Hippocampus ; drug effects ; metabolism ; Immunohistochemistry ; Kindling, Neurologic ; metabolism ; Male ; Oleanolic Acid ; analogs & derivatives ; pharmacology ; Pentylenetetrazole ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Saponins ; pharmacology ; gamma-Aminobutyric Acid ; biosynthesis

OBJECTIVETo study the effect of saikosaponins, the active ingredients of Bupleurum chinense DC, on glial fibrillary acidic protein (GFAP) expression in hippocampal astrocytes of chronic kindling rats induced by pentetrazole (PTZ).

METHODSForty-eight healthy Sprague-Dawley rats were randomized into 6 equal groups, namely the blank control group (Group A), normal saline group (Group B), sodium valproate group (Group C), and 3 saikosaponins groups of high, medium and small doses (Groups D, E, and F, respectively). The rats (except those in Group A) received intraperitoneal injection of PTZ to induce chronic kindling 1 h after the respective agents as indicated were administered intragastrically on a daily basis for 4 consecutive weeks. Upon completion of the treatment course, the rats were sacrificed and the brain tissues were sampled, sliced and stained for immunohistochemical examination. The results were analyzed to calculate the positive cell count, cross-sectional area of the cells and the gray scale.

RESULTSIn group B, the positive cell population and cross-sectional area of the positive cells were the greatest among the groups (P<0.01), but the positive cell gray scale of the CA1 and CA2 regions and the dentate gyrus (DG) of the hippocampus was the lowest. The CA1 region of Group B was significantly different from that of groups A, C and D (P<0.01), and the CA2 region different from groups A, C, D and E (P<0.05), while the DG different from group F (P<0.05) and groups A, C, D and E (P<0.01).

CONCLUSIONIn chronic kindling rats induced by PTZ, GFAP overexpression can be inhibited by saikosaponins, which suppress the abnormal activation of hippocampal astrocyte of the kindling rats.

Animals ; Astrocytes ; metabolism ; Bupleurum ; chemistry ; Depression, Chemical ; Female ; Glial Fibrillary Acidic Protein ; biosynthesis ; genetics ; Hippocampus ; metabolism ; Kindling, Neurologic ; metabolism ; Male ; Oleanolic Acid ; analogs & derivatives ; pharmacology ; Pentylenetetrazole ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Saponins ; pharmacology

AIMTo investigate the anti-inflammatory mechanism of esculentoside A (EsA) and to observe the effects of EsA on cellular adhesion between human umbilical vein endothelial cell (VEC304) and human neutrophil and to further observe the mRNA expression of intercellular adhesion molecule-1 (ICAM-1) and cluster of differentiation 18(CD18).

METHODSThe hemocyte counting method was used for assaying the adhesion rate between VEC304 and neutrophil. The RT-PCR method was used for measuring the mRNA expression of ICAM-1 and CD18.

RESULTSThe adhesion rate between VEC304 and neutrophil was increased with treatment of lipopolysaccharide(LPS). EsA (3 - 12 x 10(-6) mumol.L-1) was shown to inhibit the high cellular adhesion induced by LPS. A further investigation of adhesion molecules mRNA expression was undertaken using semi-quantitative reverse transcribed polymerase chain reaction (RT-PCR). The results of RT-PCR from VEC304 and human neutrophil treating with LPS showed that ICAM-1 and CD18 mRNA expressions were higher than those of normal cells, while this increased expression of ICAM-1 and CD18 mRNA was remarkably attenuated by the addition of EsA.

CONCLUSIONEsA was found to inhibit the increased adhesion rate induced by LPS. Moreover, LPS induced high expression of ICAM-1 and CD18 was inhibited with treatment of EsA. It might be involved in the mechanisms of anti-inflammation of EsA.

Adult ; CD18 Antigens ; biosynthesis ; genetics ; Cell Adhesion ; drug effects ; Cell Line ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Endothelial Cells ; metabolism ; physiology ; Humans ; Intercellular Adhesion Molecule-1 ; biosynthesis ; genetics ; Neutrophils ; metabolism ; physiology ; Oleanolic Acid ; analogs & derivatives ; isolation & purification ; pharmacology ; Phytolacca ; chemistry ; Plants, Medicinal ; chemistry ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Saponins ; isolation & purification ; pharmacology ; Umbilical Veins ; cytology