Challenges and Advances in Genome Editing Technologies in Streptomyces.

Guoquan Li,Yunliang Chen,Yinhua Lu,Yawei Zhao

doi:10.3390/biom10050734

Abstract

The genome of Streptomyces encodes a high number of natural product (NP) biosynthetic gene clusters (BGCs). Most of these BGCs are not expressed or are poorly expressed (commonly called silent BGCs) under traditional laboratory experimental conditions. These NP BGCs represent an unexplored rich reservoir of natural compounds, which can be used to discover novel chemical compounds. To activate silent BGCs for NP discovery, two main strategies, including the induction of BGCs expression in native hosts and heterologous expression of BGCs in surrogate Streptomyces hosts, have been adopted, which normally requires genetic manipulation. So far, various genome editing technologies have been developed, which has markedly facilitated the activation of BGCs and NP overproduction in their native hosts, as well as in heterologous Streptomyces hosts. In this review, we summarize the challenges and recent advances in genome editing tools for Streptomyces genetic manipulation with a focus on editing tools based on clustered regularly interspaced short palindrome repeat (CRISPR)/CRISPR-associated protein (Cas) systems. Additionally, we discuss the future research focus, especially the development of endogenous CRISPR/Cas-based genome editing technologies in Streptomyces.

Highlights

Streptomyces are Gram-positive bacteria that can produce high amounts of secondary metabolites, such as antibiotics, immunosuppressants, insecticides and anti-tumor drugs, which are widely used in agriculture and veterinary/human medicine
Various strategies have been developed to activate these silent biosynthetic gene clusters (BGCs) and trigger natural product (NP) overproduction, which has contributed to scientific research on the identification of potential chemical compound resources in streptomycetes [12]
Several clustered regularly interspaced short palindrome repeat (CRISPR)/CRISPR-associated protein (Cas)-derived technologies, such as the CRISPR interference (CRISPRi)-mediated gene repression tool based on dCas9 [40] or ddCpf1 [37] and the base editors (BEs) for targeted base mutagenesis based on dCas9 or Cas9 nickase (Cas9n) have recently been developed [41,42]

Summary

Introduction

Streptomyces are Gram-positive bacteria that can produce high amounts of secondary metabolites, such as antibiotics (e.g., pristinamycin [1] and daptomycin [2]), immunosuppressants (e.g., rapamycin [3] and FK506 [4]), insecticides (e.g., avermectin [5] and milbemycin [6]) and anti-tumor drugs (e.g., daunorubicin [7] and bleomycin [8]), which are widely used in agriculture and veterinary/human medicine. Various strategies have been developed to activate these silent BGCs and trigger NP overproduction, which has contributed to scientific research on the identification of potential chemical compound resources in streptomycetes [12]. These strategies can be broaBdiolmyoclelcauslessi2fi0e20d, 9i,nxto the following two main categories: (i) the induction of BGCs expressio2nofin15native. The PCR-targeting system has the following three limitations, which have limited its wide application: (i) a scar (FRT or loxP site) is left on the genome of the mutant strain; (ii) a cosmid library of Streptomyces must be constructed in advance; (iii) the procedure involves four steps, which are cumbersome and time-consuming

Cre-loxP Recombination System

I-SceI Meganuclease-Promoted Recombination System

Cas9-Based Genome Editing

Cpf1-Assisted Genome Editing

Findings

Conclusions and Perspectives