A new tool for engineering Phaeodactylum tricornutum: the METE promoter drives both high expression and B12-tuneable regulation of transgenes.

For advanced metabolic engineering strategies, it is crucial to be able to regulate transgene expression, to prevent potential deleterious effects in the host organism during growth and allow optimisation of production levels. Here, we identified vitamin B₁₂ (cobalamin)-responsive promoters in the diatom Phaeodactylum tricornutum, a promising biotechnological chassis that readily absorbs this metabolite with minimal physiological impact. Using promoter-reporter constructs, the promoters of the cobalamin acquisition protein 1 (CBA1) and the B₁₂-independent form of methionine synthase (METE) were shown to regulate transgene expression in a B₁₂-dependent manner. Further characterisation of the METE promoter (P_METE) demonstrated that it exhibited significantly higher expression levels than several previously characterised promoters, but could be repressed by nanomolar amounts of B₁₂, with a dynamic range >100-fold. Tight regulation was demonstrated by the suppression of the lethal ribonuclease, barnase at 1 μg L^-1 B₁₂. Reporter expression was doubled when P_METE was paired with its cognate terminator, compared with the widely used FCPA terminator. Promoter truncations resulted in decreased expression, but no loss of B₁₂ regulation. A 14 nucleotide motif, present in four copies in P_METE, was found to be necessary for expression, and when fused to the constitutive FCPA promoter, enhanced expression levels. Transgenic lines expressing the heterologous diterpenoid enzyme, casbene synthase, produced casbene titres of approximately 2 mg L^-1 and this was tuneable by B₁₂. This demonstrates the utility of P_METE in efforts to establish P. tricornutum as an industrial biotechnology production platform.