Promoter and N-Terminus Engineering of the Chlamydomonas Chloroplast Protein for Taxadiene Production

Taxadiene, a diterpene hydrocarbon, is the first committed intermediate in the biosynthesis of paclitaxel, a crucial anticancer drug. Its efficient production remains a key bottleneck for heterologous paclitaxel production. Although chloroplasts provide abundant terpenoid precursors, heterologous enzymes expressed in this compartment are rapidly degraded. Here we show that promoter engineering combined with N-terminal modification enables biosynthesis of taxadiene in the chloroplast of Chlamydomonas reinhardtii and accumulation in the dodecane overlay of a biphasic culture. Expressing TS under the psaA promoter rather than atpA increased taxadiene production 5.6-fold (1.9 to 10.7 μg/L). Adding methionine to the N-terminus of taxadiene synthase (TS) raised the titer to 26.7 ± 1.3 μg/L, a 14-fold increase over the atpA strain; the modified strain retained about 30% of TS at day 6, whereas the unmodified strains retained less than 10%, indicating that the modification slowed protein turnover. Optimizing light, acetate, temperature, inoculation density and methyl jasmonate supplementation increased the titer a further 6.9-fold to 185.5 ± 4.4 μg/L, about 98-fold above the initial atpA strain. N-terminal methionine engineering therefore provides a simple strategy to stabilize chloroplast-expressed enzymes.