In B. Neritina, bryostatin biosynthesis is carried out through a type I polyketide synthase cluster, bry. BryR is the secondary metabolism homolog of HMG-CoA synthase, which is the PKS in bacterial primary metabolism. In the bryostatin pathway, the BryR module catalyzes β-Branching between a local acetoacetyl acceptor acyl carrier protein (ACP-a) and an appropriate donor BryU acetyl-ACP (ACP-d).
The first step involves the loading of a malonyl unit onto a discrete BryU ACP-d within an initial BryA module. The extended BryU product in BryA is then loaded onto a cysteine sidechain of BryR for interaction with ACP-a. Upon interaction, BryR then catalyzes β-Branching, facilitating an aldol reaction between the alpha-carbon of the BryU unit and the β-ketone of ACP-a, yielding a product similar to HMGS products in primary metabolism. After β-Branching, subsequent dehydration by a BryT enoyl-CoA hydratase homolog (ECH), as well as BryA O-methylation and BryB double bond isomerization of the generated HMGS product, are carried out in specific domains of the bry cluster. These post-β-Branching steps generate the vinyl methylester moieties which are found in all natural product bryostatins. Finally, BryC and BryD are responsible for further extension, pyran ring closure, and cyclization of the HMGS product to produce the novel bryostatin product.
In the presence of BryR, ACP-d conversion to holo-ACP-d was observed prior to β-Branching. BryR was shown to have high specificity for ACP-d only after this conversion. Specificity for these protein-bound groups is a feature that differentiates the HMGS homologs found in primary metabolism, where HMGS typically acts on substrates linked to Coenzyme A, from those found in non-ribosomal peptide synthase (NRPS) or PKS pathways such as the bryostatin pathway.