Natural products (NPs) and their derivatives lead a huge pharmaceutical market share comprising 61% of anticancer drugs and 49% of anti-infection medicine in the past 30 years (Newman and Cragg, 2012). Especially, actinomycetes NPs are a major resource for drug discovery and development, mainly due to their structural novelty, diversity, and complexity (Donadio et al., 2007). Isolation and characterization of NP biosynthetic gene clusters (BGCs) have further accelerated our understanding of their molecular biosynthetic mechanisms, leading to the rational redesign of novel NPs through BGC manipulation (Fischer et al., 2003; Castro et al., 2015).

Some of these potentially valuable BGCs are, however, derived from non-culturable meta-genomes or genetically recalcitrant microorganisms. Moreover, many of these BGCs are expressed poorly or not at all under laboratory culture conditions, which makes it challenging to characterize the target NPs (Galm and Shen, 2006). Since efficient expression of actinomycetes NP BGCs present a major bottleneck for novel NP discovery, various cryptic BGC awakening strategies such as regulatory genes control, ribosome engineering, co-culture fermentation, and heterologous expression have been pursued for NP development (Tang et al., 2000; Flinspach et al., 2014; Martinez-Burgo et al., 2014; Miyamoto et al., 2014).

A traditional method for BGC cloning involves cosmid library construction by partial digestion or random shearing of chromosomal DNA. A typical size of NP BGC is usually larger than 20 kb (sometimes over 100 kb), and a cosmid vector system can only accept a relatively small BGC (up to 40 kb) or only a part of a large BGC. Therefore, cloning and efficient overexpression of an entire BGC still remains challenging due to the ineffectiveness of current host cells including the genetic and metabolic characteristics in manipulating large BGCs for heterologous expression. This mini review summarizes the list of the actinomycetes NP BGCs that have been successfully cloned and expressed in Streptomyces heterologous hosts (Table 1). In addition, three cloning and heterologous expression systems, which are quite suitable for large NP BGCs, such as transformation-associated recombination (TAR) system, integrase-mediated recombination (IR) system, and plasmid Streptomyces bacterial artificial chromosome (pSBAC) system are introduced (Figure 1).

We summarized about 90 actinomycetes NP BGCs that have been successfully expressed in Streptomyces heterologous hosts from the last several decades (Table 1). Relatively small BGCs encoding Type II polyketide were first to be isolated at the beginning of heterologous expression research. Many of the listed BGCs (about 83%) were isolated by cosmid/fosmid library construction and some of these BGCs were cloned into replicative or integrative vector by linear-plus-linear (recombination between two linear DNAs) or linear-plus-circular (recombination between linear and replicating circular DNA) homologous recombination. Approximately 60% of BGCs were integrated into the heterologous host chromosome and only 37% of BGCs existed in the heterologous host via replicative plasmid. Cosmid vectors such as pOJ446 and SuperCos1 were used to be replicative or integrative in the heterologous host, so the production level of the heterologously expressed NP BGC varied significantly. Some BGCs were isolated with two different vector systems, followed by heterologous expression via both integrative and replicative systems. For example, the epothilone BGC was expressed by both pSET152-based integration vector and SCP2^*-based replication vectors, so that its expression level was increased from 0.1 mg/L in the original Sorangium cellulosum system to 20 mg/L in the epothilone BGC-expressing Streptomyces host (Tang et al., 2000). S. coelicolor and S. lividans were two major strains for heterologous expression, thanks to their well-characterized genetic and biochemical properties. About 12% BGCs were expressed in another popular heterologous host, S. albus, which has fast growth and an efficient genetic system (Zaburannyi et al., 2014). Comparing with the original NP producing strains, approximately 14% of NPs had a higher expression level and 12% lower when they were expressed in the heterologous hosts. When bernimamycin BGC was heterologously expressed both in S. lividans and S. venezuelae, its production yield was increased 2.4-fold in S. lividans with no production in S. venezuelae (Malcolmson et al., 2013).

The Streptomyces genus is suitable for heterologous expression of large NP BGCs due to its intrinsic ability to produce various valuable secondary metabolites. Well-studied Streptomyces strains such as S. coelicolor, S. lividans, and S. albus have been mainly used as heterologous expression surrogate hosts (Table 1). The regulatory networks of secondary metabolite production have been well characterized in these strains, and thus several NP high-level producing strains have been constructed (Baltz, 2010; Gomez-Escribano and Bibb, 2011). In addition, some of these Streptomyces host genomes have been further engineered to eliminate precursor-competing biosynthetic BGCs, so that the extra precursors such as malonyl-CoA and acetyl-CoA could be funneled into the target polyketide NP biosynthesis (Gomez-Escribano and Bibb, 2011).

As shown in Table 1, most of the heterologously expressed NPs were detected as a final product, but some were detected as an intermediate due to their partial BGC expression. The NP production yield was similar to or slightly lower than that in WT. To increase the production level in heterologous hosts, it was devised to substitute with strong promoters or to increase the copy number of BGCs (Montiel et al., 2015; Nah et al., 2015). In case of pSBAC system, the tautomycetin production yield in the heterologous hosts was similar to that in the original producing strain. The selection marker on the tautomycetin BGC was changed and re-introduced into the heterologous host by tandem repeat, resulting in further yield increase from 3.05 to 13.31 mg/L in comparison with the heterologous host harboring only single copy of tautomycetin BGC. The heterologous host harboring tandem copies of tautomycetin BGC was proved to stably maintain two BGCs in the presence of appropriate antibiotic selection (Nah et al., 2015).

Meanwhile, the TAR system used yeast homologous recombination-based promoter engineering for the activation of silent natural product BGCs (Montiel et al., 2015). Bi-directional promoter cassettes were generated by PCR amplification of varied yeast selectable markers, which contains promoter-insulator-RBS combinations, and they were co-transformed with the cosmid or BAC clone harboring the target BGC into yeast. The rebeccamycin BGC was used as a model BGC. The promoter-replaced rebeccamycin BGC was transferred into S. albus by conjugation, and the production of rebeccamycin was examined in the heterologous host (Montiel et al., 2015). Using the TAR-based promoter engineering strategy, multiple promoter cassettes could be inserted simultaneously into the target BGC, thereby expediting the re-engineering process. The TAR-based promoter engineering strategy was also used to capture the silent tetarimycin BGC and the silent, cryptic pseudogene-containing, environmental DNA-derived lazarimide BGC (Montiel et al., 2015).

In conclusion, Streptomyces heterologous expression systems have been proved to be a very attractive strategy to awaken cryptic NP BGCs, and could also be applied to overexpression of a variety of large NP BGCs in actinomycetes.

HN, SK, SC, and EK planned, outlined, and revised the manuscript. HN, HP, and EK wrote and revised the manuscript.