Shikimic acid (SA) is the key synthetic material for the chemical synthesis of Oseltamivir, which is prescribed as the front-line treatment for serious cases of influenza. Multi-gene expression vector can be used for expressing the plurality of the genes in one plasmid, so it is widely applied to increase the yield of metabolites. In the present study, on the basis of a shikimate kinase genetic defect strain Escherichia coli BL21 (ΔaroL/aroK, DE3), the key enzyme genes aroG, aroB, tktA and aroE of SA pathway were co-expressed and compared systematically by constructing a series of multi-gene expression vectors. The results showed that different gene co-expression combinations (two, three or four genes) or gene orders had different effects on the production of SA. SA production of the recombinant BL21-GBAE reached to 886.38 mg·L(-1), which was 17-fold (P < 0.05) of the parent strain BL21 (ΔaroL/aroK, DE3).
Escherichia coli ; enzymology ; genetics ; metabolism ; Escherichia coli Proteins ; genetics ; metabolism ; Plasmids ; genetics ; metabolism ; Shikimic Acid ; metabolism

Shikimic acid is an intermediate metabolite in the synthesis of aromatic amino acids in Escherichia coli and a synthetic precursor of Tamiflu. The biosynthesis of shikimic acid requires blocking the downstream shikimic acid consuming pathway that leads to inefficient production and cell growth inhibition. In this study, a dynamic molecular switch was constructed by using growth phase-dependent promoters and degrons. This dynamic molecular switch was used to uncouple cell growth from shikimic acid synthesis, resulting in the production of 14.33 g/L shikimic acid after 72 h fermentation. These results show that the dynamic molecular switch could redirect the carbon flux by regulating the abundance of target enzymes, for better production.
Escherichia coli/genetics* ; Escherichia coli Proteins/genetics* ; Industrial Microbiology/methods* ; Metabolic Engineering ; Shikimic Acid/metabolism*

Shikimic acid (SA), as a hydroaromatic intermediate in the common pathway of aromatic amino acid biosynthesis, is the starting material for the synthesis of neuraminidase inhibitors and other useful compounds. The fermentative production of SA by metabolically engineered microorganisms is an excellent alternative to the extraction from fruits of the Illicium plant. In this study, Escherichia coli was metabolically engineered by rational design and genetic manipulation for fermentative production of SA. Firstly, blocking the aromatic amino acid pathway after the production of SA was carried out by deletion of aroL and aroK genes encoding SA kinase. Secondly, the ptsG gene encoding protein EIICBglc were removed in the aroL/aroK mutant strain to make the phosphotransferase system (PTS) system default. In the resulting strain, the phosphoenolpyruvate-dependent PTS pathway, a main pathway for glucose transport, were replaced by ATP-dependent GalP (galactose permease). Thus, more PEP flux was used to produce SA as a critical precursor of SA. Furthermore, ydiB gene (encoding quinic acid/SA dehydrogenase) was deleted to prevent SA precursors of 3-dehyroquinic acid into the byproduct of quinic acid. Thus, the engineered strain with four genes deletion was constructed and 576 mg/L SA was produced in the shake flask fermentation. Results show that SA produciton was increased 90 times compared to the parent strain E. coli CICIM B0013.
Escherichia coli ; enzymology ; genetics ; metabolism ; Gene Knockout Techniques ; Metabolic Engineering ; methods ; Recombinant Proteins ; genetics ; metabolism ; Shikimic Acid ; metabolism

OBJECTIVETo study the plasma protein binding rate of isopropylidene-shikimic acid.

METHODThe ultrafiltration was employed to determine the plasma protein binding rate of isopropylidene-shikimic acid. The plasma concentrations of isopropylidene-shikimic acid were measured by HPLC.

RESULTThe plasma protein binding rate of isopropylidene-shikimic acid with dog plasma at the concentration of 0.3, 0.15 g x L(-1) and 0.5 mg x L(-1) were (4.36 +/- 0.02)%, (4.12 +/- 0.19)% and (2.23 +/- 0.59)%, respectively. While the plasma protein binding rate of isopropylidene-shikimic acid with normal human plasma at the above concentrations were (11.23 +/- 0.01)%, (10.06 +/- 0.69)% and (9.72 +/- 0.59)%, respectively.

CONCLUSIONThe binding rate of isopropylidene-shikimic acid with plasma protein is low.

Alkenes ; chemistry ; Animals ; Blood Proteins ; metabolism ; Chromatography, High Pressure Liquid ; Dogs ; Humans ; Protein Binding ; Shikimic Acid ; chemistry ; metabolism ; Species Specificity

In the aromatic amino acid biosynthetic pathway 3-dehydroshikimate (DHS) is a key intermediate. As a potent antioxidant and important feedstock for producing a variety of important industrial chemicals, such as adipate and vanillin, DHS is of great commercial value. Here, in this study, we investigated the effect of the co-expression of aroFFBR (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase mutant with tyrosine feedback-inhibition resistance) and tktA (Transketolase A) at different copy number on the production of DHS. The increased copy number of aroFFBR and tktA would enhance the production of DHS by the fold of 2.93. In order to further improve the production of DHS, we disrupted the key genes in by-product pathways of the parent strain Escherichia coli AB2834. The triple knockout strain of ldhA, ackA-pta and adhE would further increase the production of DHS. The titer of DHS in shake flask reached 1.83 g/L, 5.7-fold higher than that of the parent strain E. coli AB2834. In 5-L fed-batch fermentation, the metabolically engineered strain produced 25.48 g/L DHS after 62 h. Metabolically engineered E. coli has the potential to further improve the production of DHS.
3-Deoxy-7-Phosphoheptulonate Synthase ; genetics ; Amino Acids, Aromatic ; biosynthesis ; Biosynthetic Pathways ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Metabolic Engineering ; Shikimic Acid ; analogs & derivatives ; metabolism ; Transketolase ; genetics

Based on the transcriptome data, we cloned the open reading frame of IiHCT gene from Isatis indigotica, and then performed bioinformatic analysis of the sequence. Further, we detected expression pattern in specific organs and hairy roots treated methyl jasmonate( MeJA) by RT-PCR. The IiHCT gene contains a 1 290 bp open reading frame( ORF) encoding a polypeptide of 430 amino acids. The predicted isoelectric point( pI) was 5.7, a calculated molecular weight was about 47.68 kDa. IiHCT was mainly expressed in stem and undetectable in young root, leaf and flower bud. After the treatment of MeJA, the relative expression level of IiHCT increased rapidly. The expression level of IiHCT was the highest at 4 h and maintained two fold to control during 24 h. In this study, cloning of IiHCT laid the foundation for illustrating the biosynthesis mechanism of phenylpropanoids in I. indigotica.
Acyltransferases ; chemistry ; genetics ; metabolism ; Amino Acid Sequence ; Cloning, Molecular ; Gene Expression Regulation, Plant ; Isatis ; chemistry ; classification ; enzymology ; genetics ; Models, Molecular ; Molecular Sequence Data ; Open Reading Frames ; Phylogeny ; Plant Proteins ; chemistry ; genetics ; metabolism ; Quinic Acid ; metabolism ; Sequence Alignment ; Shikimic Acid ; metabolism

AIMTo study the effect of 3,4-oxo-isopropylidene-shikimic acid (ISA) on H2O2 (200 mol.L-1, 4 h) injured human umbilical vein endothelial cells (HUVEC).

METHODSMorphological change was observed under microscop. Cell viability was assessed by MTT assay. The release of intracellular lactate dehydrogenase (LDH) and NO was assessed by colorimetry. Radioimmunoassay was used to assess 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha).

RESULTSPretreatment with ISA for 6 h alleviated the morphological damage of H2O2 induced HUVECs. At the concentration of 1-100 mumol.L-1, ISA prevented the inhibitory effect on cell viability induced by H2O2 in dose-dependent manner, but increased the ratio of cell viability from 60.4% to 84.3%. ISA reduced LDH release and increased the level of NO and 6-keto-PGF1 alpha in H2O2 induced HUVECs.

CONCLUSIONISA exerted protective effect on H2O2 injured HUVEC.

6-Ketoprostaglandin F1 alpha ; metabolism ; Cell Survival ; drug effects ; Cells, Cultured ; Endothelial Cells ; metabolism ; pathology ; Humans ; Hydrogen Peroxide ; antagonists & inhibitors ; Infant, Newborn ; L-Lactate Dehydrogenase ; metabolism ; Nitric Oxide ; metabolism ; Protective Agents ; pharmacology ; Shikimic Acid ; analogs & derivatives ; pharmacology ; Umbilical Veins ; cytology

The key and crucial step of metabolic engineering during quinic acid biosynthesize using shikimic acid pathway is high expression of quinate 5-dehydrogenase. The gene qa-3 which code quinate 5-dehydrogenase from Neurospora crassa doesn't express in Escherichia coli. By contrast with codon usage in Escherichia coli, there are 27 rare codons in qa-3, including eight AGG/AGA (Arg) and nine GGG (Gly). Two AGG are joined together (called box R) and some GGG codons are relative concentrate (called box G). Along with the secondary structure of mRNA analysed in computer, the free energy of mRNA changes a lot from -374.3 kJ/mol to least -80.5 kJ/mol when some bases in the end of qa-3 were transformed, and moreover, the change of free energy is quite small when only some bases in the box G and box R transformed. After the change of rare codon and optimization of some bases in the end, qa-3 was expression in E. coli and also the enzyme activity of quinate 5-dehydrogenase can be surveyed accurately. All the work above benefit the further research on producing quinic acid engineering bacterium.
Alcohol Oxidoreductases ; biosynthesis ; genetics ; Base Sequence ; Codon ; chemistry ; genetics ; Escherichia coli ; genetics ; metabolism ; Hydro-Lyases ; genetics ; Molecular Sequence Data ; Neurospora crassa ; enzymology ; genetics ; RNA, Messenger ; chemistry ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Shikimic Acid ; metabolism ; Transformation, Bacterial

The obstacle to successful remyelination in demyelinating diseases, such as multiple sclerosis, mainly lies in the inability of oligodendrocyte precursor cells (OPCs) to differentiate, since OPCs and oligodendrocyte-lineage cells that are unable to fully differentiate are found in the areas of demyelination. Thus, promoting the differentiation of OPCs is vital for the treatment of demyelinating diseases. Shikimic acid (SA) is mainly derived from star anise, and is reported to have anti-influenza, anti-oxidation, and anti-tumor effects. In the present study, we found that SA significantly promoted the differentiation of cultured rat OPCs without affecting their proliferation and apoptosis. In mice, SA exerted therapeutic effects on experimental autoimmune encephalomyelitis (EAE), such as alleviating clinical EAE scores, inhibiting inflammation, and reducing demyelination in the CNS. SA also promoted the differentiation of OPCs as well as their remyelination after lysolecithin-induced demyelination. Furthermore, we showed that the promotion effect of SA on OPC differentiation was associated with the up-regulation of phosphorylated mTOR. Taken together, our results demonstrated that SA could act as a potential drug candidate for the treatment of demyelinating diseases.
Animals ; Apoptosis ; drug effects ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Demyelinating Diseases ; prevention & control ; Encephalitis ; prevention & control ; Encephalomyelitis, Autoimmune, Experimental ; prevention & control ; Female ; Mice, Inbred C57BL ; Myelin Basic Protein ; metabolism ; Neuroprotective Agents ; administration & dosage ; Oligodendrocyte Precursor Cells ; drug effects ; metabolism ; Rats ; Remyelination ; drug effects ; Shikimic Acid ; administration & dosage ; TOR Serine-Threonine Kinases ; metabolism