Heterologous Expression and CRISPR/Cas9-Assisted Manipulation of the Hybrid Gene Cluster Specifying the Biosynthesis of Meroterpenoids and Phenazines

A hybrid gene cluster, mfq, predicted to govern the biosynthesis of both meroterpenoids and phenaziterpenes, was cloned from the genome of Streptomyces sp. S4.7 and introduced into the heterologous host Streptomyces coelicolor M1154. The biosynthesis of the meroterpenoids marfuraquinocins C and D, previously isolated from Streptomyces niveus SCSIO 3406, as well as a new congener, marfuraquinocin E, which exhibited antibacterial activity, was activated upon overexpression of the regulatory protein MfqF. However, production of neither phenaziterpenes nor phenazines was detected. The structure of marfuraquinocin E was elucidated, revealing the attachment of a terpene moiety at C-2, in contrast to C-6 as seen in the known congeners A–D. Using the CRISPR/Cas9 system, several genes in the mfq cluster were inactivated, confirming the role of MfqW as a prenyltransferase specific to the meroterpenoid pathway. Both gene overexpression and further knockouts provided the first insights into the complex regulation of this hybrid gene cluster. To restore the presumably deficient phenazine biosynthetic pathway, a gene encoding a PhzF homologue from another gene cluster in S4.7 was heterologously expressed alongside the mfq cluster, leading to the production of 1,6-phenazine dicarboxylic acid upon MfqF overexpression. This work lays the foundation for elucidating the complete biosynthetic pathway of marfuraquinocins and its potential coregulation with that of phenazines.