• The Korean Journal of Food And Nutrition
• /
• v.23 no.4
• /
• pp.526-530
• /
• 2010

Isopentenyl diphosphate(IPP) isomerization to dimethylallyl diphosphate(DMAPP) is an important step for the efficient production of isoprenoids such as lycopene, ${\beta}$-carotene, astaxanthin, etc. The type II isopentenyl diphosphate isomerase gene from Synechocystis sp. PCC6803(sll1556, Syidi2) was cloned and expressed in Escherichia coli $DH5{\alpha}$. When E. coli $DH5{\alpha}$ harboring lycopene synthesis genes, crtE, crtB, and crtI and mevalonate pathway genes, MvK1, MvK2, and Mvd, was cultured on LB medium containing mevalonate, the strain grew very slowly be due to the toxicity of isopentenyl diphosphate derived from mevalonate. When Syidi2 was introduced to E. coli $DH5{\alpha}$ harboring the lycopene synthesis genes and mevalonate pathway genes, growth on mevalonate medium was fully restored and the colony showed red color indicating lycopene formation. The growth rate of the mutant strain, E. coli $DH5{\alpha}$(idi::${\Delta}km$), was very slow because of IPP accumulation and DMAPP deprivation. Ultimately the idi mutant was complemented by introducing the Syidi2 gene.

• BMB Reports
• /
• v.40 no.6
• /
• pp.911-920
• /
• 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
• /
• v.40 no.6
• /
• pp.861-869
• /
• 2007

The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR; EC1.1.1.34) catalyzes the first committed step of isoprenoids biosynthesis in MVA pathway. Here we report for the first time the cloning and characterization of a full-length cDNA encoding HMGR (designated as CgHMGR, GenBank accession number EF206343) from hazel (Corylus avellana L. Gasaway), a taxol-producing plant species. The full-length cDNA of CgHMGR was 2064 bp containing a 1704-bp ORF encoding 567 amino acids. Bioinformatic analyses revealed that the deduced CgHMGR had extensive homology with other plant HMGRs and contained two transmembrane domains and a catalytic domain. The predicted 3-D model of CgHMGR had a typical spatial structure of HMGRs. Southern blot analysis indicated that CgHMGR belonged to a small gene family. Expression analysis revealed that CgHMGR expressed high in roots, and low in leaves and stems, and the expression of CgHMGR could be up-regulated by methyl jasmonate (MeJA). The functional color assay in Escherichia coli showed that CgHMGR could accelerate the biosynthesis of $\beta$-carotene, indicating that CgHMGR encoded a functional protein. The cloning, characterization and functional analysis of CgHMGR gene will enable us to further understand the role of CgHMGR involved in taxol biosynthetic pathway in C. avellana at molecular level.

• Journal of Life Science
• /
• v.27 no.6
• /
• pp.726-737
• /
• 2017

Carotenoids are isoprenoids with a long polyene chain containing 3 to 15 conjugated double bonds, which determines their absorption spectrum. They typically consist of a $C_{40}$ hydrocarbon backbone often modified by different oxygen-containing functional groups, to yield cyclic or acyclic xanthophylls. Much work has also been focused on the identification, production, and utilization of natural sources of carotenoid (plants, microorganisms and crustacean by-products) as an alternative to the synthetic pigment which currently covers most of the world markets. Nevertheless, only a few carotenoids (${\beta}-carotene$, lycopene, astaxanthin, canthaxanthin, and lutein) can be produced commercially by fermentation or isolation from the small number of abundant natural sources. The market and demand for carotenoids is anticipated to increase dramatically with the discovery that carotenoids exhibit significant anti-carcinogenic activities and play an important role in the prevention of chronic diseases. The increasing importance of carotenoids in the feed, nutraceutical food and pharmaceutical markets has renewed by efforts to find ways of producing additional carotenoid structures in useful quantities. Because microorganisms and plants synthesize hundreds of different complex chemical carotenoid structures and a number of carotenoid biosynthetic pathways have been elucidated on a molecular level, metabolic and genetic engineering of microorganisms can provide a means towards economic production of carotenoid structures that are otherwise inaccessible. The aim of this article is to review our current understanding of carotenoid formation, to explain the perceived benefits of carotenoid in the diet and review the efforts that have been made to increase carotenoid in certain microorganisms.