One signal is enough: Stepwise transport of two distinct passenger proteins by the Tat pathway across the thylakoid membrane

The twin-arginine translocation (Tat) pathway, one of four protein transport pathways operating at the thylakoid membrane of chloroplasts, shows remarkable substrate flexibility. Here, we have analyzed the thylakoid transport of chimeric tandem substrates that are composed of two different passenger proteins fused to a single Tat transport signal. The chimera 23/23-EGFP in which the reporter protein EGFP is connected to the C-terminus of the OEC23 precursor shows that a single Tat transport signal is sufficient to mediate transport of two distinct passenger proteins in a row. Replacing the transit peptide of OEC23 in 23/23-EGFP by its homolog from OEC16 yields the chimera 16/23-EGFP, which can likewise be fully translocated by the Tat pathway across the thylakoid membrane. However, transport of 16/23-EGFP is retarded at specific steps in the transport process leading to the temporary and consecutive accumulation of three translocation intermediates with distinct membrane topology. They are associated with two oligomeric membrane complexes presumably representing TatBC-receptor complexes. The composition of the translocation intermediates as determined by immunoprecipitation experiments suggests that the two passenger proteins are translocated in a stepwise manner across the membrane.     

Identified biosynthetic pathway of aspergiolide A and a novel strategy to increase its production in a marine-derived fungus Aspergillus glaucus by feeding of biosynthetic precursors and inhibitors simultaneously

Aspergiolide A is a novel anti-tumor anthraquinone derivant produced by marine-derived fungus Aspergillus glaucus. To identify its biosynthetic pathway and further improve the production, the effects of biosynthetic pathway specific inhibitors and precursors were investigated. Cerulenin and iodoacetamide, the specific inhibitors of polyketide pathway, could completely inhibit the aspergiolide A accumulation. Putative precursors of polyketide pathway could increase aspergiolide A production greatly, such as 6 mM acetate increased production by 135%. Simvastatin and citrate, the inhibitors of mevalonate pathway, stimulated the production by 63% and 179%, respectively. Considering that acetyl-CoA is the common starter unit in both polyketide and mevalonate pathway, a novel strategy was designed to stimulate the aspergiolide A accumulation. Combinations of 12 mM acetate with 0.3 mM simvastatin could increase the production by 151%, while the supplementation with 12 mM acetate and 12 mM citrate brought a 262% increase of aspergiolide A production. The strategy might be very useful to enhance the production of other secondary metabolites derived from polyketide pathway.