N-terminal sequences affect expression of triterpene biosynthesis enzymes in Chlamydomonas chloroplasts

Metabolic engineering is an emerging technology to modify the biochemical properties of living cells. In microalgae, metabolic engineering has often been directed towards optimizing the production of desirable lipids or related bioproducts. Here we describe efforts to engineer the green alga Chlamydomonas reinhardtii for the production of botryococcene, a drop-in biofuel precursor. Genes encoding farnesyl diphosphate synthase (FPS) and squalene synthase-like (SSL)-1 and -3, were introduced into the chloroplast genome using biolistic transformation. Through a series of construct modifications, we identified intergenic sequences that promote expression of stable, discrete transcripts. We also found amino acids that dramatically increased the accumulation of SSL-3 when they were inserted at the N-terminal penultimate position, and similar manipulation of the N-terminal sequence of FPS appeared to improve its protein level as well. However, SSL-1 only accumulated to detectable levels when expressed as a chimera with SSL-3. In vitroassays showed that chloroplast-expressed SSL-3 was enzymatically active, but not SSL-1, although the SSL-1-SSL-3 chimeras were active when expressed in yeast. Taken together, our results suggest that the N-terminal sequence and other cellular factors are important when heterologous proteins are expressed in this model algal species.