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

## TL;DR

Scientists cloned a gene cluster from a soil bacterium and used CRISPR to study how it makes antibiotic compounds, revealing new insights into their biosynthesis.

## Contribution

The study demonstrates CRISPR/Cas9-assisted manipulation of a hybrid gene cluster to activate and dissect meroterpenoid biosynthesis and partial phenazine production.

## Key findings

- Overexpression of MfqF activated biosynthesis of marfuraquinocins C, D, and a new antibacterial compound, marfuraquinocin E.
- CRISPR/Cas9 confirmed MfqW's role as a prenyltransferase in the meroterpenoid pathway.
- Heterologous expression of a PhzF homologue restored partial phenazine biosynthesis.

## Abstract

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.