# Recent Advances in the Heterologous Biosynthesis of Natural Products from _Streptomyces_
## 5 Strategies for the Construction of a Biosynthetic Gene Cluster  
### Vectors and Cloning Methods  
BGCs contain many genes, which encode one or some secondary metabolites. Some cloning methods were used to clone large BGCs. Traditionally based on a library, the large genomic DNA has been digested and ligated into high-capacity vectors, such as cosmids, fosmids, or BACs. Cosmids can insert DNA fragments ranging in size between 32 kb and 45 kb based on the bacteriophage vector [138]. Fosmids, which are similar to cosmids but contain an F-factor to control the copy number of vectors, can insert DNA fragments up to 100 kb in size [139]. For example, the _mat_ gene cluster used a fosmid vector to produce deshydroxymatlystains via heterologous expression in _S. coelicolor_ and _S. albus_[53]. The BAC vector contains the F-factor, multiple cloning sites, and is capable of inserting DNA that is approximately 490 kb in size [140]. PAC vector could insert DNA that was between 100 and 300 kb in size [141]. BGCs can be cloned in a directed manner by employing in vivo cloning methods, such as TAR, LLHR, ExoCET, and PISR. TAR constructs target BGC from a digested genome to an expression vector using yeast. This technique successfully cloned a DNA fragment with a size up to 250 kb [142]. As the recombinant vector was constructed in yeast, maintained in _E. coli_, and expressed in a host, it has to carry elements from each of the yeast, _E. coli_, and the host. For example, the 67 kb BGC of taromycin A was successfully expressed from _Saccharomyces_ sp. CNQ-490 in _S. coelicolor_ using the TAR cloning system [143]. The LLHR used _E. coli_ to clone a large gene cluster up to 100 kb to an expression vector based on the activity of RecE/RecT. [PERSON] et al. successfully used this method to clone and express PKS-NRPS BGCs from _P. luminescens_ in _E. coli_[144]. Recently, ExoCET was used to directly clone large selected regions (up to 106 kb) into a BAC vector in a single step. ExoCET combines an in vitro exonuclease of T4 polymerase with a full-length RecE/RecT to clone homology regions in _E. coli_. The efficiency of vector construction depends on the position of the homologous sequence in the digested genome. For increasing the efficiency of this method, Cas9 was applied to digest the genome. For instance, the salinomycin gene cluster was cloned to a BAC vector by using the ExoCET method [145]. PISR was used to clone and delete large BGCs directly from the _Streptomyces_ genome based on the activity of the \(\phi\)BT1 integrase. [PERSON] et al. applied this technique to delete and clone actinorhodin, nikkomycin, and gougerotin from _S. coelicolor_, _Streptomyces anochromogene_, and _Streptomyces graminearus_, respectively [146]. Besides in vivo cloning, some BGCs were also successfully cloned using PCR gene and DNA assembly in different ways. In vitro cloning includes DNA assembly methods, such as Gibson assembly, DiPAC, PfAgo-based AREs, SSRTA, SIRA, and CATCH. Gibson assembly was applied to clone DNA fragments with a size up to 14 kb, such as bicyclomycin [147], kocurin [23], and lasso peptides [148]. Gibson assembly needs DNA polymerases, exonucleases, and DNA ligases to assemble BGCs with vectors. DiPAC was applied to clone DNA fragments amplified from PCR based on HiFi DNA assembly. DiPAC is suitable for cloning short BGCs, with a capacity of up to 23 kb, such as _apt_[98], sodorifen [149], and haplosion [150] BGCs. SIRA used \(\Phi\)31 integrase and a pair of different orthogonal _attP/attB_ recombination sites to form _attL/attR_ for a recombational assembly and cloning strategy [151]. For example, [PERSON] et al. successfully cloned and expressed erythromycinycin BGC using SIRA [152]. The SSRTA method is based on the operation of _Streptomyces_\(\phi\)BT1 integrase and _attB/attP_ recombination sites for the tandem assembly of multiple DNA fragments. Some BGCs were cloned using this method, such as epothilone [28] and lycopene BGCs [153].