Molecular characterization of squalene synthase from the green microalga Botryococcus braunii, race B

The green microalga Botryococcus braunii produces large amounts of liquid hydrocarbons and is classified into three races, depending on the type of the hydrocarbon produced. The B race produces two types of triterpenoid hydrocarbons, squalene and botryococcene, both of which are putative condensation products of farnesyl diphosphate. In an attempt to better understand the regulation involved in the production of squalene and botryococcene, we have isolated and characterized a squalene synthase (SS) gene from the B race of B. braunii, A 366-bp cDNA fragment was initially obtained from the B race utilizing a reverse transcription/polymerase chain reaction and degenerate primers based on conserved amino acid sequences found in all SS enzymes. Using this putative SS fragment as a probe, a 2632-bp cDNA clone was isolated from a cDNA library. This cDNA contained an open reading frame coding for a protein with 461 amino acids and a predicted molecular mass of 52.5 kDa, Comparison of the Botryococcus SS (BSS) with SS from different organisms showed 52% identity with Nicotiana tabacum, 51% with Arabidopsis thaliana, 48% with Zea mays, 40% with rat, 39% with yeast, and 26% with Zymomonas mobilis, Expression of full-length and carboxy-terminus truncated BSS cDNA in Escherichia coli resulted in significant levels of bacterial SS enzyme activity but no botryococcene synthase activity. RNA blot hybridization analysis of algal cultures during a culture cycle indicated that BSS gene expression is preferential during rapid growth. Given that the DNA blot analysis indicated only a single copy of the SS gene in the algal genome, these results suggest either that there exists coordinate expression of separate synthase genes for squalene and botryococcene biosynthesis or that there might be unique physiological conditions controlling the SS vs botryococcene synthase activity of a single peptide species. (C) 2000 Academic Press.