Fig 1.
Biosynthesis and metabolism of triterpenes in Botryococcus braunii, race B.
Squalene synthase (SS) converts two molecules of farnesyl pyrophosphate (FPP) into one molecule of squalene via presqualene pyrophosphate (PSPP). Squalene synthase-like protein (SSL) -1 (SSL-1) catalyzes formation of PSPP from two FPPs, SSL-2 converts PSPP into squalene, and SSL-3 synthesizes C30 botryococcene from PSPP. Squalene and C30 botryococcene are methylated and excreted outside cells as free hydrocarbons that can be used as biofuels. Squalene is epoxidized into squalene 2,3-epoxide that is the precursor of membrane sterols or into squalene 10,11-epoxide that is further converted into hydrophobic secondary metabolites including biopolymers.
Fig 2.
Amino acid sequences of BbSQEs-I and -II aligned with those from the other organisms.
A. The amino acid sequences of land plant SQEs that complemented Saccharomyces cerevisiae erg1 and BbSQEs were aligned using CLUSTAL W (ver.1.83) multiple sequence alignment tool (www.ebi.ac.uk/Tools/msa/clustalw2/help/faq.html, accessed Nov. 14, 2014) and adjusted manually. Amino acid residues that are 100% identical in the alignment are highlighted in black and those which are more than 50% identical are highlighted in grey. Boxes show conserved domains, hyphens denote the gaps in aligned sequences and asterisks indicate amino acid residues whose point mutations could result in the loss of complementation of erg1 [26]. B. Sequences of motifs I and II in BbSQEs-I and -II aligned with plant consensus sequence. The top sequence shows the consensus among seven plant sequences from panel A. Numbers denote amino acid residue positions in AtSQE1. Hyphens indicate varieties in plant sequences. Asterisks are the same as in panel A. BbSQE sequences that are identical to the plant consensus are shown with dots.
Fig 3.
Complementation of KLN1 by BbSQEs-I and -II.
The BbSQE cDNA clones were cloned into expression vector pWV3, and introduced into Saccharomyces cerevisae erg1 mutant, KLN1. Under anaerobic conditions, transformants were selected on leucine-deficent synthetic minimum medium plates which were supplemented with ergosterol and re-streaked twice more on fresh plates. Subsequently, three lines of SQE transformants were streaked on solidified YPD medium and incubated for four days under aerobic conditions, along with an empty vector control.
Fig 4.
Southern blot analyses of BbSQEs-I and -II using genomic DNA.
The cDNA fragments of 325-bp BbSQE-I, or 1477-bp BbSQE-II were labeled with digoxigenin, and used as probes for detecting corresponding genomic fragments. Upper panels show the Southern blots. After stripping probe of BbSQE-I, the same membrane was re-hybridized with that of BbSQE-II. The representative drawings at the bottom show positions of probe regions (arrows), as well as PstI and EcoRI sites (vertical bars with a letter) in the ORF (rectangles). Scale bar for cDNA length is indicated at the bottom.
Table 1.
RNA-seq analysis of BbSQEs-I and -II at days 0 and 17 after inoculation into fresh culture medium.
Fig 5.
Expression of BbSQEs-I and -II during a culture peroid.
Botryococcus culture was inoculated into fresh liquid medium and cultured for 42 days. Aliquots were harvested every 6 days and total RNA was extracted from each sample. Using qRT-PCR, relative amounts of BbSQE-I and-II transcripts were determined using that of GAPDH as a reference gene. The relative amounts at day 0 are shown as 1.0. Values are the mean of three technical replicates ± S.D. in samples collected every 6 days of a representative algal culture.