Elucidation of genes enhancing natural product biosynthesis through co-evolution analysis.

Streptomyces has the largest repertoire of natural product biosynthetic gene clusters (BGCs), yet developing a universal engineering strategy for each Streptomyces species is challenging. Given that some Streptomyces species have larger BGC repertoires than others, we proposed that a set of genes co-evolved with BGCs to support biosynthetic proficiency must exist in those strains, and that their identification may provide universal strategies to improve the productivity of other strains. We show here that genes co-evolved with natural product BGCs in Streptomyces can be identified by phylogenomics analysis. Among the 597 genes that co-evolved with polyketide BGCs, 11 genes in the 'coenzyme' category have been examined, including a gene cluster encoding for the cofactor pyrroloquinoline quinone. When the pqq gene cluster was engineered into 11 Streptomyces strains, it enhanced production of 16,385 metabolites, including 36 known natural products with up to 40-fold improvement and several activated silent gene clusters. This study provides an innovative engineering strategy for improving polyketide production and finding previously unidentified BGCs.

Biosynthesis of ansamitocin P-3 incurs stress on the producing strain Actinosynnema pretiosum at multiple targets.

Microbial bioactive natural products mediate ecologically beneficial functions to the producing strains, and have been widely used in clinic and agriculture with clearly defined targets and underlying mechanisms. However, the physiological effects of their biosynthesis on the producing strains remain largely unknown. The antitumor ansamitocin P-3 (AP-3), produced by Actinosynnema pretiosum ATCC 31280, was found to repress the growth of the producing strain at high concentration and target the FtsZ protein involved in cell division. Previous work suggested the presence of additional cryptic targets of AP-3 in ATCC 31280. Herein we use chemoproteomic approach with an AP-3-derived photoaffinity probe to profile the proteome-wide interactions of AP-3. AP-3 exhibits specific bindings to the seemingly unrelated deoxythymidine diphosphate glucose-4,6-dehydratase, aldehyde dehydrogenase, and flavin-dependent thymidylate synthase, which are involved in cell wall assembly, central carbon metabolism and nucleotide biosynthesis, respectively. AP-3 functions as a non-competitive inhibitor of all three above target proteins, generating physiological stress on the producing strain through interfering diverse metabolic pathways. Overexpression of these target proteins increases strain biomass and markedly boosts AP-3 titers. This finding demonstrates that identification and engineering of cryptic targets of bioactive natural products can lead to in-depth understanding of microbial physiology and improved product titers.