The complete nucleotide sequence (155 844 bp) of tobacco (Nicotiana tabacum var. Bright Yellow 4) chloroplast DNA has been determined. It contains two copies of an identical 25 339 bp inverted repeat, which are separated by a 86 684 bp and a 18 482 bp single‐copy region. The genes for 4 different rRNAs, 30 different tRNAs, 39 different proteins and 11 other predicted protein coding genes have been located. Among them, 15 genes contain introns. Blot hybridization revealed that all rRNA and tRNA genes and 27 protein genes so far analysed are transcribed in the chloroplast and that primary transcripts of the split genes hitherto examined are spliced. Five sequences coding for proteins homologous to components of the respiratory‐chain NADH dehydrogenase from human mitochondria have been found. The 30 tRNAs predicted from their genes are sufficient to read all codons if the ‘two out of three’ and ‘U:N wobble’ mechanisms operate in the chloroplast. Two sequences which autonomously replicate in yeast have also been mapped. The sequence and expression analyses indicate both prokaryotic and eukaryotic features of the chloroplast genes.
A cDNA corresponding to a putative phosphatidylinositol-specific phospholipase C (PI-PLC) in the higher plant Arabidopsis thaliana was cloned by use of the polymerase chain reaction. The cDNA, designated cAtPLCl, encodes a putative polypeptide of 561 aa with a calculated molecular mass of 64 kDa. The putative product includes so-called X and Y domains found in all PI-PLCs identified to date. In mammalian cells, there are three types of PI-PLC,
Isoprenoids, which include over 23,000 known metabolites, are the most chemically diverse family of naturally occurring compounds. The essential and major biosynthetic step in all isoprenoid metabolism is the elongation of isoprene units by prenyltransferases ( Fig. 1) (1). These enzymes, which consecutively mediate alkylation of isopentenyl diphosphate (IPP, 1 by allylic diphosphates, are classified according to the chain length of the final product and the stereochemistry of double bond formed by the condensations. So far, a number of prenyltransferases have been determined from various organisms.For example, farnesyl diphosphate (FPP) synthase (EC 2.5.1.1) catalyzes the sequential condensations of two molecules of IPP (C-5) with dimethylallyl diphosphate (DMAPP, C-5) to give a C-15 compound with E-stereochemistry. The product, FPP, occupies a central point leading to several branches of the pathway for the synthesis of important classes of compounds, including sterols, farnesylated proteins, hemes, respiratory quinones, sesquiterpenes, and dolichols. On the other hand, geranylgeranyl diphosphate (GGPP, C-20) synthase (EC 2.5.1.29) catalyzes the condensation of IPP to give (all-E)-GGPP, which plays as a precursor for carotenoids, chlorophylls, geranylgeranylated proteins, and archaebacterial membrane lipids.These prenyltransferases catalyze the same sort of condensation and have a similarity in amino acid sequences (2, 3). However, every enzyme does not catalyze a further condensation of IPP than the general ultimate product. Until now it has been left in question how the consecutive condensations precisely stop at a destined step.Recently, our group succeeded in converting FPP synthase from Bacillus stearothermophilus to GGPP synthase using chemical random mutagenesis followed by an in vivo color selection (4). From the analysis of the mutations in the FPP synthases whose product specificities had become the same as GGPP synthase, we defined three amino acids that could determine the final chain length; leucine at position 34, tyrosine at position 81, and valine at position 157. In particular, the mutated enzyme that has a substitution of histidine for tyrosine at position 81, which is situated at the fifth amino acid before the first aspartate rich consensus motif, the most effectively produces GGPP. Moreover, our group also showed that, in the case of Sulfolobus acidocaldarius GGPP synthase, the amino acid at the same position also determines the chain length of the product, GGPP (5). Thus, in this paper, we precisely analyze the role of the amino acid at position 81 of B. stearothermophilus FPP synthase on chain length determination.
(E, E, E)-Geranylgeraniol (GGOH) is a valuable starting material for perfumes and pharmaceutical products. In the yeast Saccharomyces cerevisiae, GGOH is synthesized from the end products of the mevalonate pathway through the sequential reactions of farnesyl diphosphate synthetase (encoded by the ERG20 gene), geranylgeranyl diphosphate synthase (the BTS1 gene), and some endogenous phosphatases. We demonstrated that overexpression of the diacylglycerol diphosphate phosphatase (DPP1) gene could promote GGOH production. We also found that overexpression of a BTS1-DPP1 fusion gene was more efficient for producing GGOH than coexpression of these genes separately. Overexpression of the hydroxymethylglutaryl-coenzyme A reductase (HMG1) gene, which encodes the major rate-limiting enzyme of the mevalonate pathway, resulted in overproduction of squalene (191.9 mg liter ؊1 ) rather than GGOH (0.2 mg liter ؊1 ) in test tube cultures. Coexpression of the BTS1-DPP1 fusion gene along with the HMG1 gene partially redirected the metabolic flux from squalene to GGOH. Additional expression of a BTS1-ERG20 fusion gene resulted in an almost complete shift of the flux to GGOH production (228.8 mg liter ؊1 GGOH and 6.5 mg liter ؊1 squalene). Finally, we constructed a diploid prototrophic strain coexpressing the HMG1, BTS1-DPP1, and BTS1-ERG20 genes from multicopy integration vectors. This strain attained 3.31 g liter ؊1 GGOH production in a 10-liter jar fermentor with gradual feeding of a mixed glucose and ethanol solution. The use of bifunctional fusion genes such as the BTS1-DPP1 and ERG20-BTS1 genes that code sequential enzymes in the metabolic pathway was an effective method for metabolic engineering.(E,E,E)-Geranylgeraniol (GGOH) can be used as an important ingredient for perfumes and as a desirable raw material for synthesizing vitamins A and E (4, 13). It is also known to induce apoptosis in various cancer and tumor cell lines (24,36). GGOH is the dephosphorylated derivative of (E,E,E)-geranylgeranyl diphosphate (GGPP) (Fig. 1). GGPP is a significant intermediate of ubiquinone and carotenoid biosyntheses, especially in carotenoid-producing microorganisms and plant cells. It is also utilized as the lipid anchor of geranylgeranylated proteins. In the yeast Saccharomyces cerevisiae, GGPP is synthesized by GGPP synthase (GGPS), encoded by the BTS1 gene, which catalyzes the condensation of farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP) rather than the successive addition of IPP molecules to dimethylallyl diphosphate, geranyl diphosphate, and FPP that is detected in mammalian tissues (14). Biologically synthesized GGOH comprises only (E,E,E)-geometric isomers, and only the (E,E,E)-isomers have significant biological activities (23). The chemically synthesized form is usually obtained as mixtures of (E)-and (Z)-isomers and thus has lower potency. Therefore, there is a greater possibility of attaining efficient production of (E,E,E)-GGOH through fermentative production.Some yeast strains accumulate ergosterol up to 4.6% d...
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