(63d) Refactored Biosynthetic Pathways: Delineating Pathway Expression From Sophisticated Regulation Cascades Via Synthetic Biology

Microorganisms have been evolved to produce a myriad array of complex molecules known as natural products or secondary metabolites of pharmaceutical and biological importance.  The production of natural product is subjected to sophisticated regulation cascades in which target gene and multiple regulators interact with each other, resulting in a complex regulatory network, and responding to a variety of physiological and environmental signals.  This highly regulated process complicates the discovery and characterization of new compounds with important biological activities and hampers sustainable production of the ones with high-value.  Here we developed a general synthetic biology-based strategy to resolve the complexity and delineate the pathway expression from sophisticated regulation cascades. 

Three important biosynthetic pathways from Streptomyces including the spectinabilin gene cluster, the fosfomycin gene cluster, and the FR900098 gene cluster were chosen for study.  Spectinablin, isolated from Streptomyces spectabilis and Streptomyces orinoci, is a nitro-phenyl substituted polyketide exhibiting antimalarial and antiviral activities.  Although both hosts were reported to produce spectinabilin, only the pathway from S. spectabilis can be functionally expressed in S. lividans, making the silent spectinabilin biosynthetic pathway a perfect model for studies of cryptic pathways.  These two clusters undergo different regulations as the cluster from S. spectabilis contains SpnD as an activator and the cluster from S. orinoci contains NorD as a repressor.  Real-time PCR was used to analyze the expression level of each nor gene in both the native host and the heterologous host.  It was shown that 7 out of the 10 enzymes involved in spectinabilin biosynthesis were expressed at an extremely low level in the heterologous host.  This may have resulted in the silence of the spectinabilin biosynthesis in S. lividans. 

To activate the spectinabilin pathway in S. lividans, we propose to construct a refactored pathway by inserting a constitutive promoter in front of each gene.  Approximately 40 constitutive promoter candidates in front of house-keeping genes were cloned from the genomes of Streptomyces and other related actinobacteria and fused with reporter genes.  Currently, they are being evaluated in different growth conditions.  The strong constitutive promoters will be inserted in front of the nor genes.  The success in isolating strong and constitutive promoters will be very useful in activating numerous uncharacterized biosynthetic pathways.

In addition, the proposed strategy for refactoring biosynthetic pathways can also be used to improve the production of valuable natural products in Streptomyces.  Previous effort mainly focused on testing various growth conditions to optimize pathway expression and precursor availability.  The overall process is tedious and labor-intensive as there is no universal solution and pathways have to be examined one by one in an array of media.  Here we aim to demonstrate that the optimization process can be greatly shortened if the sophisticated regulation is removed.  Two example pathways, the fosfomycin biosynthetic pathway and the FR900098 biosynthetic pathway, both having important biomedical applications are chosen and will be reconstructed with strong and constitutive promoters for higher production levels.