Silent Escape: Induction Crack Unlock Code
Vida Hubbert
The finely tuned regulation of secondary metabolism poses a huge challenge to natural product researchers to identify conditions under which biosynthetic genes are expressed. In many cases, biosynthesis is downregulated, and the encoded structures escape detection. Therefore, efforts are required to induce the expression of silent genes and to link chemical structures to orphan biosynthesis gene clusters (Fig. 2 and 3).
Strategies for the activation of silent biosynthetic gene clusters in heterologous hosts and examples of natural products discovered through these methods. (BGC biosynthetic gene cluster). The color code of the boxes around the compound names and icons indicates the strategy that was applied for the discovery of the respective compound: Activation of natural product biosynthesis through heterologous expression of biosynthetic genes that are downregulated in the native producer (light orange) or from metagenomic DNA (green).
The genome of actinomycetes and several other microorganisms are endowed with many cryptic gene clusters that can code for previously undetected, a plethora of complex secondary metabolites. Under standard laboratory controlled conditions, the genes regulating these biosynthetic clusters are expressed at very low levels or remain phenotypically cryptic (silent). Over the past several decades, multi-drug-resistant bacteria have been observed with increased frequency, posing a significant threat to human health worldwide. The present alarming situation urgently calls for concerted global efforts for the discovery of new antimicrobials. The present situation, if not controlled, will take us again to the pre-antibiotic era. Today, in the post-genomic era, various new strategies such as the activation of cryptic gene clusters in microorganisms rejuvenate a new conviction in the field of natural product research that may lead to the identification of yet-unidentified novel secondary metabolites of therapeutic and other use. Decryptification of this versatile endogenous genetic reservoir may provide in the near future the more concrete rationale for antibiotic discovery. The present review is an attempt to provide a comprehensive detail, outlining current strategies that have been shown successful to activate cryptic biosynthetic gene clusters in microorganisms.
The insertion of inducible strong promoters has been also reported to lead to the activation of the cryptic antibiotic gene clusters. The biosynthetic genes for secondary metabolites that are usually silent or are expressed minimally can be cloned using strong promoters into suitable plasmid vectors. The silent spectinabilin pathway of S. orinoci and taromycin A pathway of Saccharomonospora sp. have been identified using this system (Shao et al. 2013; Yamanaka et al. 2014). Similarly, a cryptic antibiotic gene cluster SGR810-815 in S. griseus has been also reported to induce three novel polycyclic tetramate macrolactams (Luo et al. 2013). In E. coli, alterochromide lipopeptides of Pseudoalteromonas piscicida have been shown to heterologously expressed using native, E. coli T7 promoter (Ross et al. 2015). The expression of a silent antibiotic gene cluster has also been achieved in Streptomyces using a strong promoter ermE (Baltz 2010). The bbr gene (transcriptional activator) of Amycolatopsis balhimycina also exhibited the induction of balhimycin biosynthesis in the heterologous host A. japonicum (Spohn et al. 2014).
The silent antibiotic gene clusters in microorganisms are considered to be a potential source of secondary metabolites, but the environmental clues to induce their expression remain unknown (Abrudan et al. 2015; Zhu et al. 2014). It is highly important to understand the biological role of cryptic antibiotic gene clusters in antibiotic-producing microbes in a given niche before making attempts to activate them. The members of actinomycetes group grow as a branched multicellular network of hyphae and are known to reproduce through spores that are formed by an aerial mycelium. The detailed description of the control of morphological differentiation in actinomycetes is reviewed elsewhere (Chater 2006; Flardh and Buttner 2009; Hopwood 2006). In natural systems, many antibiotics are produced after specific signals are receiving from the surrounding environment. Specialized techniques are required to decode these clues that can activate the production of secondary metabolites in microorganisms. The antibiotics have classically been considered as antimicrobial weapons (Raaijmakers and Mazzola 2012; Ratcliff and Denison 2011). The studies conducted by Abrudan et al. (Abrudan et al. 2015) and Westhoff et al. (Westhoff et al. 2017) in Streptomyces also supported the observation that antibiotics play their major role in environment as defense molecules. The generalization that antibiotics have an antagonistic role in nature similar to its clinical role is a big question (Linares et al. 2006; Ratcliff and Denison 2011; Romero et al. 2011). The reason for this assumption is the low concentrations of antibiotics present in the soil environment that may not exhibit inhibitory effects. Subsequently, sub-inhibitory concentrations of antibiotics can induce a pleiotropic response in microorganisms such as quorum sensing, biofilm formation, and coordinated expression of virulence genes (Hoffman et al. 2005; Stevens et al. 2007; Yim et al. 2006).
The majority of mutations have neither negative nor positive effects on the organism in which they occur. These mutations are called neutral mutations. Examples include silent point mutations, which are neutral because they do not change the amino acids in the proteins they encode.
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