Cloning Strategies

The development of synthetic biology tools have enhanced cloning of intact BGCs in heterologous hosts (Table 1). Some of these methods are based on exonucleases to “chew back” one of the strands of double-stranded DNAs, thereby exposing complementary single-stranded DNA sequences that can anneal to each other in vitro (Figures 1A,B), such as Gibson isothermal assembly (Jiang et al., 2015; Greunke et al., 2018), sequence- and ligation-independent cloning (SLIC) (D’Agostino et al., 2018). Blunt-end ligation have also been emplyed to ligate the CRISPR/Cas9 digested product into a universal vector for λ packaging into phage and transfecting into E. coli (Tao et al., 2019) (Figure 1A).

This “chew-back and repair” mechanism has also been applied to clone intact BGCs leveraging on in vivo homologous recombination (Figure 1C and Table 1). Several hosts have been widely used for cloning purpose, such as the TAR method in Saccharomyces cerevisiae (Kouprina and Larionov, 2019), linear-linear homologous recombination (LLHR) or linear plus circular homologous recombination (LCHR) in E. coli (Fu et al., 2012), and exonuclease combined with RecET recombination (ExoCET) (Wang et al., 2018). In these methods, partially digested or randomly sheared DNA was co-transformed into the recombinant host with linearized and pathway-specific vectors containing homology arms that flank the upstream and downstream of the target BGCs. Also, ExoCET can be used to promote homologous recombinations between a linear DNA molecule and a circular plasmid (Fu et al., 2012).

Another type of approach employs site-direct recombination to clone intact BGCs by first integrating specific-vector with the integrase recognition sites in the native host, then the targeted BGCs together with the integrated vector are captured and circularized for heterologous expression (Figures 1D,E and Table 1). This approach requires the native host to have high efficiency of homologous recombination. For example, Dai et al. (2015) intergrated plasmid pEry-up and pEry-down with the BT1 integrase recognition sites BattP and BattB via single- or double- crossover at both ends of erythromycin BGC, after which genome DNA was carefully isolated and treated with the BT1 integrase to circularize at att recombination sequences as a plasmid via in vitro site-specific recombination. Similarily, iCatch intergrates homing endonucleases I-SceI and PI-PspI recognition sites flanking the region of interest, after which the genome is isolated and digested with I-SceI or PI-PspI and then self-ligated to clone the target BGC in vitro (Wang et al., 2019). Moreover, several groups have developed methods that express recombinases to extract DNA fragments between two integrase recognition sites and circularize the plasmid in vivo (Figure 1D), such as phage ϕBT1 integrase-mediated site-specific recombination (Du et al., 2015), Cre/loxP plus BAC (Hu et al., 2016). These plasmids can then be isolated from the native host for heterologous expression.

As shown in Table 1, compared with in vivo methods (e.g., TAR, LLHR), in vitro cloning methods (e.g., DiPaC, CATCH, iCatch) require carefully preparation of DNA via pre-treatment and purification. Moreover, site-directed recombination methods are suitable for the host with high efficient homologous recombination system. Direct cloning are clearly valuable methods that are well-suited for mining the vast amount of genome for applications in natural product discovery.