Product Selectivity in Baeyer-Villiger Monooxygenase-Catalyzed Bacterial Alkaloid Core Structure Maturation.

Baeyer-Villiger monooxygenases (BVMOs) play crucial roles in the core-structure modification of natural products. They catalyze lactone formation by selective oxygen insertion into a carbon-carbon bond adjacent to a carbonyl group (Baeyer-Villiger oxidation, BVO). The homologous bacterial BVMOs, BraC and PxaB, thereby process bicyclic dihydroindolizinone substrates originating from a bimodular nonribosomal peptide synthetase (BraB or PxaA). While both enzymes initially catalyze the formation of oxazepine-dione intermediates following the identical mechanism, the final natural product spectrum diverges. For the pathway involving BraC, the exclusive formation of lipocyclocarbamates, the brabantamides, was reported. The pathway utilizing PxaB solely produces pyrrolizidine alkaloids, the pyrrolizixenamides. Surprisingly, replacing pxaB within the pyrrolizixenamide biosynthetic pathway by braC does not change the product spectrum to brabantamides. Factors controlling this product selectivity have remained elusive. In this study, we set out to solve this puzzle by combining the total synthesis of crucial pathway intermediates and anticipated products with in-depth functional in vitro studies on both recombinant BVMOs. This work shows that the joint oxazepine-dione intermediate initially formed by both BVMOs leads to pyrrolizixenamides upon nonenzymatic hydrolysis, decarboxylative ring contraction, and dehydration. Brabantamide biosynthesis is enzyme-controlled, with BraC efficiently transforming all the accepted substrates into its cognate final product scaffold. PxaB, in contrast, shows only considerable activity toward brabantamide formation for the substrate analog with a natural brabantamide-type side chain structure, revealing substrate-controlled product selectivity.

Bypassing Biocatalytic Substrate Limitations in Oxidative Dearomatization Reactions by Transient Substrate Mimicking.

Enzymatic oxidative dearomatization is an efficient way to generate chiral molecules from simple arenes. One example is the flavin-dependent monooxygenase SorbC involved in sorbicillinoid biosynthesis. However, SorbC requires a long-chain keto substituent at its phenolic substrate, thus preventing its application beyond the synthesis of natural sorbicillinoids or close structural analogues. This work describes an approach to broaden the accessible product spectrum of SorbC by employing an ester functionality mimicking the natural substrate structure during enzymatic oxidation.

Direct Pathway Cloning (DiPaC) to unlock natural product biosynthetic potential.

Specialized metabolites from bacteria are an important source of inspiration for drug development. The genes required for the biosynthesis of such metabolites in bacteria are usually organized in so-called biosynthetic gene clusters (BGCs). Using modern bioinformatic tools, the wealth of genomic data can be scanned for such BGCs and the expected products can often structurally be predicted in silico. This facilitates the directed discovery of putatively novel bacterial metabolites. However, the production of these molecules often requires genetic manipulation of the BGC for activation or the expression of the pathway in a heterologous host. The latter necessitates the transplantation of the BGC into a suitable expression system. To achieve this goal, powerful cloning strategies based on in vivo homologous recombination have recently been developed. This includes LCHR and LLHR in E. coli as well as TAR cloning in yeast. Here, we present Direct Pathway Cloning (DiPaC) as an efficient complementary BGC capturing strategy that relies on long-amplicon PCR and in vitro DNA assembly. This straightforward approach facilitates full pathway assembly, BGC refactoring and direct transfer into any vector backbone in vitro. The broad applicability and efficiency of DiPaC is demonstrated by the discovery of a new phenazine from Serratia fonticola, the first heterologous production of anabaenopeptins from Nostoc punctiforme and the transfer of the native erythromycin BGC from Saccharopolyspora erythraea into Streptomyces. Due to its simplicity, we envisage DiPaC to become an essential method for BGC cloning and metabolic pathways construction with significant applications in metabolic engineering, synthetic biology and biotechnology.