UCLA

Nature products from filamentous fungi are extremely important sources of bioactive and structurally diverse compounds for agricultural and pharmaceutical applications. Understanding the biosynthetic machinery of fungal natural products will not only reveal novel enzymes that catalyze complicated chemical reactions, but also enable us to use these enzymes as powerful tools to make fine chemicals and high value drugs. Here, we revealed the biosynthetic pathways of a variety of fungal peptidyl alkaloids, including the anthranilate-containing tryptoquialanines (tqa), fumiquinazolines (fqa), asperlicins and ardeemin. By systematically inactivating every biosynthetic gene in the gene clusters, followed by isolation and characterization of the intermediates, we were able to establish the biosynthetic sequence of each pathway. Notably, the tqa pathway has been confirmed to go through an intermediate common to the fqa pathway, fumiquinazoline F (FQF), which originates from a fungal trimodular nonribosomal peptide synthetase (NRPS). Furthermore, cyclization of linear peptidyl precursors produced by NRPSs is an important step in the biosynthesis of bioactive cyclic peptides. Whereas bacterial NRPSs use thioesterase domains to perform the cyclization, fungal NRPSs producing macrocyclic peptides terminate with a condensation-like (C_T) domain. To probe the role of such a C_T domain, we reconstituted the activities of the Penicillium aethiopicum trimodular NPRS TqaA in Saccharomyces cerevisiae and in vitro. Extensive biochemical and mutational studies confirmed the essential role of the C_T domain in catalyzing cyclization in a thiolation domain-dependent fashion. After the formation of FQF, TqaH in tqa pathway and Af12060 in fqa pathway were demonstrated to be identical and perform indole ring oxidation. Annulation of the indole side chain of FQF with different stereochemistry was controlled by the condenstation domain of monomodular NRPS enzymes (Af12050 and TqaB) to form fumiquinazoline A (FQA) and epi-FQA, respectively. The bimodular NRPS AspA in the asperlicin biosynthetic pathways is also characterized and synthesize multicyclic fungal alkaloids asperlicin C and D in vitro. The first module of AspA iteratively activates two molecules of anthranilate, while the C_T domain cyclizes the linear peptide into a macrolactam which can undergo further transannular cyclization. Finally, we employed directed evolution to engineer an enzyme, LovD which was identified from the lovastatin biosynthetic pathway and can be used in the synthesis of the cholesterol-lowing drug, Simvastatin (Zocor^�). The best LovD mutant displayed an 11-foldincrease in an Escherichia coli-based biocatalytic platform. Seven X-ray crystal structures were determined to understand the structural basis of LovD enzymology.