• Journal of Microbiology and Biotechnology
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• v.30 no.5
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• pp.762-769
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• 2020

Vitamin K2 (menaquinone) is an essential vitamin existing in the daily diet, and menaquinone-7 (MK-7) is an important form of it. In a recent work, we engineered the synthesis modules of MK-7 in Bacillus subtilis, and the strain BS20 could produce 360 mg/l MK-7 in shake flasks, while the methylerythritol phosphate (MEP) pathway, which provides the precursor isopentenyl diphosphate for MK-7 synthesis, was not engineered. In this study, we overexpressed five genes of the MEP pathway in BS20 and finally obtained a strain (BS20DFHG) with MK-7 titer of 415 mg/l in shake flasks. Next, we optimized the fermentation process parameters (initial pH, temperature and aeration) in an 8-unit parallel bioreactor system consisting of 300-ml glass vessels. Based on this, we scaled up the MK-7 production by the strain BS20DFHG in a 50-l bioreactor, and the highest MK-7 titer reached 242 mg/l. Here, we show that the engineered strain BS20DFHG may be used for the industrial production of MK-7 in the future.

• Journal of Applied Biological Chemistry
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• v.58 no.3
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• pp.273-279
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• 2015

Phaeodactylum tricornutum is a model diatom that its genomic information and biological tools are well established. In this study, a gene encoding (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (PtHDR), a terminal enzyme of the methylerythritol phosphate pathway regulating chlorophyll and carotenoid biosynthesis, was isolated from P. tricornutum. The isolated gene was cloned into pPha-T1 vector containing fcpA promoter to prepare pPha-T1-HDR plasmid. As a positive control, pPha-T1-eGFP plasmid was constructed with egfp gene. Stable nuclear transformation was carried out with these plasmids by particle bombardment method and zeocin resistant colonies of P. tricornutum were selected on f/2 agar plate. In result, transformation efficiency was evaluated according to the amount of plasmid DNA coated with gold particles. Integration of introduced plasmids was confirmed with genomic DNA of each transformant by polymerase chain reaction. The eGFP fluorescence was visible in the cytoplasm, indicating that eGFP was successively expressed in P. tricornutum system. The transcript level of exogenous Pthdr gene was evaluated with the obtained transformants. The results presented here demonstrated that introduction of Pthdr gene into P. tricornutum chromosome succeeded and expression of PtHDR was enhanced under the fcpA promoter.

• BMB Reports
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• v.40 no.6
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• pp.911-920
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• 2007

Tuberculosis, caused by Mycobacterium tuberculosis, continues to be one of the leading infectious diseases to humans. It is urgent to discover novel drug targets for the development of antitubercular agents. The 2-C-methyl-Derythritol-4-phosphate (MEP) pathway for isoprenoid biosynthesis has been considered as an attractive target for the discovery of novel antibiotics for its essentiality in bacteria and absence in mammals. MEP cytidyltransferase (IspD), the third-step enzyme of the pathway, catalyzes MEP and CTP to form 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) and PPi. In the work, ispD gene from M. tuberculosis H37Rv (MtIspD) was cloned and expressed. With N-terminal fusion of a histidine-tagged sequence, MtIspD could be purified to homogeneity by one-step nickel affinity chromatography. MtIspD exists as a homodimer with an apparent molecular mass of 52 kDa. Enzyme property analysis revealed that MtIspD has high specificity for pyrimidine bases and narrow divalent cation requirements, with maximal activity found in the presence of CTP and $Mg^{2+}$. The turnover number of MtIspD is $3.4 s^{-1}$. The Km for MEP and CTP are 43 and $92{\mu}M$, respectively. Furthermore, MtIspD shows thermal instable above $50^{\circ}C$. Circular dichroism spectra revealed that the alteration of tertiary conformation is closely related with sharp loss of enzyme activity at higher temperature. This study is expected to help better understand the features of IspD and provide useful information for the development of novel antibiotics to treat M. tuberculosis.

• BMB Reports
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• v.39 no.5
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• pp.502-510
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• 2006

2C-methyl-D-erythritol 2, 4-cyclodiphosphate synthase (MECPS, EC: 4.6.1.12) is the fifth enzyme of the non-mevalonate terpenoid pathway for isopentenyl diphosphate biosynthesis and is involved in the methylerythritol phosphate (MEP) pathway for ginkgolide biosynthesis. The full-length mecps cDNA sequence (designated as Gbmecps) was cloned and characterized for the first time from gymnosperm plant species, Ginkgo biloba, using RACE (rapid amplification of cDNA ends) technique. The full-length cDNA of Gbmecps was 874 bp containing a 720 bp open reading frame (ORF) encoding a peptide of 239 amino acids with a calculated molecular mass of 26.03 kDa and an isoelectric point of 8.83. Comparative and bioinformatic analyses revealed that GbMECPS showed extensive homology with MECPSs from other species and contained conserved residues owned by the MECPS protein family. Phylogenetic analysis indicated that GbMECPS was more ancient than other plant MECPSs. Tissue expression pattern analysis indicated that GbMECPS expressed the highest in roots, followed by in leaves, and the lowest in seeds. The color complementation assay indicated that GbMECPS could accelerate the accumulation of $\beta$-carotene. The cloning, characterization and functional analysis of GbMECPS will be helpful to understand more about the role of MECPS involved in the ginkgolides biosynthesis at the molecular level.