P128: A pyrrole and pyrazine synthetase discovered from genome mining of Aspergillus terreus

Although filamentous fungi have a strong track record in producing blockbuster drugs such as penicillin and lovastatin, these microorganisms are widely considered to be underachievers in natural product biosynthesis. The vast biosynthetic potential of species in the Aspergillus, Penicillium, Gibberella, etc genera is far from being realized. Mining these uncharacterized pathways is therefore an important objective towards discovery of new bioactive molecules and novel enzymatic machineries. A cryptic, nonribosomal peptide synthetase-like enzyme (NRPS325) mined from Aspergillus terreus was reconstituted in vitro from Escherichia coli and was shown to synthesize pyrroles and pyrazines via unprecedented mechanisms. In the presence of a β-ketoacyl precursor such as acetoacetyl-S-N-acetylcysteamine and L-Leucine, NRPS325 synthesized 5-isobutyl-2-methyl-1H-pyrrolyl-3-carboxyl-S-NAC. On the other hand, in the presence of L-Leucine and a free thiol such as SNAC, NRPS325 can synthesize a trisubstituted pyrazine in which the SNAC is attached to the C-2 of the pyrazine via an aryl sulfide linkage. The remarkable substrate promiscuity of NRPS325 towards different amino acids, free thiols and β-ketoacyl substrates were explored to produce hundreds of new compounds. Our work here demonstrated the power of heterologous expression as a method to awake and harvest the biocatalytic power of silent biosynthetic enzymes.