Isolation and characterization of 4-hydroxy-3-methylbut-2-enyl diphosphate reductase gene from Botryococcus braunii, race B.

The B race of a green microalga Botryococcus braunii Kützing produces triterpene hydrocarbons that is a promising source for biofuel. In this algal race, precursors of triterpene hydrocarbons are provided from the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. The terminal enzyme of this pathway, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is regarded as one of the key enzymes that affect yields of products in terpene biosynthesis. In order to better understand the MEP pathway of the alga, cDNA and genomic clones of HDR were obtained from B. braunii Showa strain. B. braunii HDR (BbHDR) is encoded on a single copy gene including a 1509-bp open reading frame that was intervened by 6 introns. The exon-intron structure of BbHDR genes did not show clear relation to phylogeny, while its amino acid sequence reflected phyla and classes well. BbHDR sequence was distinctive from that of the HDR protein from Escherichia coli in the residues involved in hydrogen-bond network that surrounds substrate. Introduction of BbHDR cDNA into an E. coli HDR deficient mutant resulted in recovery of its auxotrophy. BbHDR expression level was upregulated from the onset of liquid culture to the 24th day after inoculation with a 2.5-fold increase and retained its level in the subsequent period.

Isolation and characterization of two squalene epoxidase genes from Botryococcus braunii, race B.

The B race of the green microalga Botryococcus braunii produces triterpene hydrocarbons, botryococcenes and methylsqualenes that can be processed into jet fuels with high heating values. In this alga, squalene is also converted into membrane sterols after 2,3-epoxidation. In the present study, cDNA clones of two distinct squalene epoxidases (BbSQE-I and -II) were isolated. Predicted amino acid sequences encoded on these genes are 45% identical with each other. Introduction of BbSQE-I or -II into Saccharomyces cerevisie erg1 mutants resulted in the complementation of ergosterol auxotrophy. The relative expression level of SQE-II increased 3.5-fold from the early stage to the middle phase of a culture period of 42 days, while that of SQE-I was almost constant throughout the culture period. Southern blot analyses suggested that these genes are single-copied genes. This is the first report on the isolation of functional SQEs that are encoded in duplicated loci in the algal genome.

The single cellular green microalga Botryococcus braunii, race B possesses three distinct 1-deoxy-D-xylulose 5-phosphate synthases.

Green algae exclusively use the methylerythritol 4-phosphate (MEP) pathway for the biosynthesis of isoprenoids. The first enzyme of this pathway is 1-deoxy-D-xylulose 5-phosphate synthase (DXS, EC 2.2.1.7). Green algae have been thought to possess only a single DXS, in contrast to land plants, which have at least two isoforms that serve different roles in metabolism. The green microalga Botryococcus braunii has an extraordinary isoprenoid metabolism, as it produces large amounts of triterpene hydrocarbons. Here, we did cDNA cloning of DXSs from B. braunii and examined enzyme activities of the heterologously expressed proteins. Three distinct DXS isoforms were identified, all of which were functional and had similar kinetic properties, whereas the temperature dependence of enzyme activity showed considerable differences. Transcription of the genes was examined by real time quantitative RT-PCR. The three DXS genes were simultaneously expressed, and the expression levels were highest on day six after subculturing. B. braunii is the first green microalga demonstrated to have multiple DXS isoforms like land plants. This difference to other microalgae seems to mirror its special needs for extensive triterpene production by increasing the metabolic flow through the MEP pathway.