Recent Advances in Synthetic Biology Approaches to Optimize Production of Bioactive Natural Products in Actinobacteria

Abstract

Actinobacteria represent one of the most fertile sources for the discovery and development of natural products (NPs) with medicinal and industrial importance. However, production titers of actinobacterial NPs are usually low and require optimization for compound characterization and/or industrial production. In recent years, a wide variety of novel enabling technologies for engineering actinobacteria have been developed, which have greatly facilitated the optimization of NPs biosynthesis. In this review, we summarize the recent advances of synthetic biology approaches for overproducing desired drugs, as well as for the discovery of novel NPs in actinobacteria, including dynamic metabolic regulation based on metabolite-responsive promoters or biosensors, multi-copy chromosomal integration of target biosynthetic gene clusters (BGCs), promoter engineering-mediated rational BGC refactoring, and construction of genome-minimized Streptomyces hosts. Integrated with metabolic engineering strategies developed previously, these novel enabling technologies promise to facilitate industrial strain improvement process and genome mining studies for years to come.

Keywords: BGC amplification; actinobacteria; dynamic regulation; genome-minimized host; natural product; pathway refactoring; synthetic biology.

FIGURE 1

Dynamic pathway regulation balances flux between bacterial growth and antibiotic biosynthesis in actinobacteria. (A) Autoregulated fine-tuning of the expression of an antibiotic (e.g., oxytetracycline) BGC based on metabolite-responsive native promoters without specific transcription factors or additional inducers. BGC, biosynthetic gene cluster. (B) Antibiotic-responsive biosensors. This strategy is engineered from cluster-situated regulators (e.g., PamR2) that bind to their associated promoters upon interaction with the corresponding secondary metabolites (e.g., pamamycins). Placing antibiotic biosynthetic genes under such promoters enables antibiotic dependent gene expression.

FIGURE 2

Two emerging synthetic biology approaches for multi-copy integration of target genes or natural products BGCs in actinobacteria. (A) Multiplex site-specific genome engineering (MSGE) for discrete amplification of target genes or BGCs. This method is based on the “one integrase-multiple attB sites” concept. The blue and blank triangles represent the native and artificial ΦC31 attB sites, respectively. (B) Advanced multiplex site-specific genome engineering (aMSGE) for multi-locus chromosomal integration of target genes or BGCs. This method is based on the “multiple integrases-multiple attB sites” concept. Particularly, these discrete attB sites are naturally occurring in the actinomycetal genomes. BGC, biosynthetic gene cluster.

FIGURE 3

Improved production titers of novel or important natural products by BGC refactoring strategy. Native BGCs can be obtained by in vivo or in vitro BGC cloning/assembly strategies. Preassembled BGC can be obtained by PCR amplification or CRISPR-mediated in vitro digestion of native BGCs. Refactored BGCs can be obtained by partial or complete replacement of native promoters with artificial promoters based on homologous recombination (HR) in S. cerevisiae or exonucleases combined with RecET recombination (ExoCET) in E. coli. Finally, different refactored BGCs will be integrated into native or heterologous actinomycetal hosts for activating silent BGCs or enhancing production titers of clinically important drugs. BGC, biosynthetic gene cluster.