# Recent Advances in the Heterologous Biosynthesis of Natural Products from _Streptomyces_
## 6 Conclusions  
The development of genome sequencing and bioinformatics tools has greatly facilitated the discovery of BGCs. Advances in heterologous expression have resulted in the development of novel secondary metabolites. The heterologous expression of natural products depends on two crucial factors: host engineering and BGC construction. Host engineering involves choosing hosts, deleting unwanted BGCs, inducing transcriptional terminators, riboswitches, engineering tRNAs, and deleting or inserting regulators. In comparison, BGC construction involves vectors, cloning methods, promoters, and RBSs.  
Many hosts were used for the heterologous expression of natural products from _Streptomyces_, such as _Streptomyces_ spp., _S. cerevisiae_, _B. subtilis_, _E. coli_, _C. glutamicum_, _P. putida_, and _R. erythropolis_. For easily detecting and purifying heterologous products, unwanted secondary metabolite BGCs from host strains should be deleted. Many genetic systems were developed for genome editing, such as CRISPR/Cas, meanuclease I-Sciel, site-specific recombination using Cre-loxP, and PCR-targeted systems. The transcriptional regulator and terminator are two factors that affect the transcription level. Negative regulator genes of heterologous BGCs should be deleted. Moreover, positive regulator genes and transcriptional terminator genes should be introduced in hosts to improve the heterologous expression level of natural products. Riboswitches could regulate the heterologous expression of BGCs in both transcription and translation levels. They are also crucial factors that can be used to control the heterologous expression. Furthermore, some active tRNAs are precursors for the biosynthesis of secondary metabolites. Therefore, inducing specific tRNA genes into hosts could also enhance the production achieved from the heterologous expression of BGCs.  
Two cloning method classes have been applied to clone the target BGCs, namely, in vitro and in vivo cloning methods. The vectors and the cloning methods were chosen depending on the heterologous hosts and the size of the target BGCs. In vitro cloning methods are often used for cloning the small- or medium-sized attractive BGCs, while in vivo cloning methods are usually applied for cloning the large-sized target BGCs. For the activation or enhancement of the expression products, recombinant vectors are also needed for the reconstruction. Robust and suitable promoters and RBSs were used to replace the native promoters and RBSs.  
In conclusion, the advances of synthetic biology and powerful genome mining techniques led to rapidly discovering and enhancing the production of natural products. Besides OSMAC, the overexpression/deletion of regulatory genes, ribosome engineering, and promoter replacement strategies that are utilized in heterologous expression provide robust strategies.  
Conceptualization, V.T.T.P., C.T.N. and J.K.S.; writing-original draft preparation, V.T.T.P. and C.T.N.; writing-review and editing, V.T.T.P., C.T.N., D.D., H.T.N., and T.-S.K.; supervision, J.K.S.; funding acquisition, J.K.S. All authors have read and agreed to the published version of the manuscript.  
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (NRF-2017R1A2A2A05000939.  
Not applicable.  
The authors declare no conflict of interest.