## 4 Application of Rational Chemical Elicitors  
The regulation of the NP production involves multiple regulatory cascades and internal networking, which has immense implications for the control of biosynthesis and the production of such NPs [50]. In secondary metabolism, there are two levels of regulations: (1) specific regulation for the pathway of biosynthesis, and (2) pleiotropic regulation for controlling the multiple pathways of biosynthesis. Pleiotropic regulators indirectly control the production of NPs, often located in clusters, or modulate the pathway-specific genes [51; 52]. Elicitors are small molecules, acting as signal molecules, which can respond to many of the pleiotropic regulators [53]. The elicitors are very diverse. They can be antibiotics (chemical synthetic or biosynthetic compounds), inhibitors or activators of enzymes, or any compounds which can affect the metabolism of _Streptomyces_[54; 55; 56; 52]. In 1967, the first elicitor, A factor, a \(\gamma\)-butyrolactone, was reported, which controls the biosynthesis pathway of streptomycin and spore formation [53; 57]. Not only that, \(\gamma\)-butyrolactone regulate the production of a large class of antibiotics in different _Streptomyces_, including virginiamycins (_Streptomyces virginiae_) and showdomycin (_S. lavendulae_) (Figure 1B) [54; 52]. PI factor, a 2,3-diamino-2,3-bis(hydroxymethyl)-1,4-butanediol, can activate the production of pimaricin (Figure 1B) in _Streptomyces natalensis_. Additionally, hydromethylfuran was shown to induce methylenomycin variants (Figure 1B) in _S. coelicolor_[55]. Thus, the application of new elicitation strategies can be an effective strategy for streamlining the activation of cryptic biosynthesis gene clusters. Several attempts of conventional screening methods using elicitor have been attempted to activate cryptic pathways. In 2012, a new approach of High-Throughput Elicitor Screens (HiTESs) of cryptic gene clusters was utilized to activate the silent biosynthesis pathways, whereas the _S. coelicolor_ based model system was used. The production of the blue color compound (actinorhodin) and the red-colored compound (undecylprodigiosin) (Figure 1B) were used as the indicator compounds as they were easy to detect visually. Further 30,569 small molecules were used for screening the elicitors for activating the production of actinorhodin in _S. coelicolor_. Among them, 112 compounds affected, and of these, 19 compounds enhanced the production of actinorhodin. One of these compounds, ARC2, displayed pleiotropic regulations by enhancing the production of actinorhodin by 2 to 5-fold, and enhanced the production of germicidins (Figure 1B) to 3-fold, while decreasing the production of daptomycin-like calcium-dependent antibiotic by approximately 2-fold [58; 59; 52]. The major drawback of this method is that it is difficult to activate the particular silent gene cluster of interest. To solve this problem, a new strategy was developed by combining HiTESs with the reporter-guided genes. To test this idea, the cryptic BGC of malleilactone was activated in _Burkholderia mallei_ by integrating the _lacZ_ reporter gene to _malL_, a gene essential of the BGC of malleilactone. Screening elicitors by HiTESs based on LacZ activity from a library of 800 compounds, they successfully awakened the cryptic gene cluster to get different malleilactone analogs (Figure 1B) [60]. HiTESs combined with reported-guided strategy were also used in _Streptomyces_, for the first time for activating a _sur_ cluster, the nonribosomal peptide synthetase (NRPS) gene cluster in _Streptomyces albus_ and isolating different surugamide derivatives (Figure 1B). They used two reporter systems, XylE and eGFP, to apply HiTESs. They integrated the reported gene in the downstream of _Sur_ promoter (the promoter driving the expression of the _sur_ gene cluster). HiTESs were performed using 502 compounds as the elicitors, whereas two excellent elicitors, etoposide and ivermectin, could activate the _sur_ cluster.