Methods Used for Activation of Silent Biosynthetic Genes: Comparison
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Antibiotic resistance is becoming a burning issue due to the frequent use of antibiotics for curing common bacterial infections, indicating that we are running out of effective antibiotics. Enhancement of antimicrobial resistance (AMR) is strengthening the pathogenicity and virulence of infectious microbes. Endophytes have shown expression of various new many bioactive compounds with significant biological activities. Specifically, in endophytic fungi, bioactive metabolites with unique skeletons have been identified which could be helpful in the prevention of increasing antimicrobial resistance. The major classes of metabolites reported include anthraquinone, sesquiterpenoid, chromone, xanthone, phenols, quinones, quinolone, piperazine, coumarins and cyclic peptides. Various methods including epigenetic modifications, co-culture, and OSMAC to induce silent gene clusters for the production of noble bioactive compounds in endophytic fungi were discussed.

  • endophytic fungi
  • antibacterial compound
  • natural product
  • drug resistance

1. Introduction

Over the decades since the discovery of the first antibiotics, resistance to those has been a curse that is being dragged along with every discovery of new antibiotics. This has kept all scientists, professionals, and clinical specialists working on antibiotics on their toes. The quest for new antibiotics scaffolds and repurposing of existing molecules has been persistent for the past nine decades. Getting a new and right scaffold is a herculean task, especially with the least ability to induce mutations in the target bacteria. As examined in some of the earlier reviews [1,2][1][2] there are several ways of getting new scaffolds and classes of antimicrobial bioactive compounds. In the domain of natural products, one of the most demonstrated ways is studying less explored species and genera of microbes [3,4,5][3][4][5].
It has been reported that fungi have various unexpressed gene clusters related to bioactive secondary metabolites, which do not express in mass multiplications of the axenic form [213,214][6][7]. The expression of such gene clusters directly or indirectly depends on the surrounding environment of the microorganism. In axenic form, various induction or activation signals are or may be absent for some bioactive molecule production in the culture, which are usually present in natural habitats [215][8]. Such biosynthetic gene clusters (BGC) are part of the heterochromatin of fungal chromosomes, which do not express at laboratory conditions [216][9]. To induce such silent biosynthetic gene clusters two major approaches have been reported, including pleiotropic- and pathway-specific approaches, which include various techniques like knocking down, mutation induction [217][10], co-culture methods [218][11], heterologous expression [219[12][13],220], interspecies crosstalk [221][14], one strain many compounds (OSMAC) [222][15] and epigenetic manipulation [223][16]. Changes in media composition and physical factors like pH, temperature, light, salt concentration, metal and elicitor also support the induction of silent BGC and improve production of secondary metabolites in microbes. The generation of various types of stresses significantly affects the metabolic activities of growing culture and microbes to release compounds for their survival under stress conditions. Changes in physical conditions or stresses impacted gene regulation by upregulating or downregulating the gene expression [126,224][17][18]. Nowadays, high throughput elicitor screening technique (HiTES) is also employed to save time in exposing culture against various types of elicitors. In this technique selected culture is grown in 96 well plates with various elicitors in each well and after the incubation period metabolites are identified by mass spectrometry or assay system. The mutation is one of the other approaches to induce silent biosynthetic gene clusters (BGC). Mutation in RNA polymerase genes and ribosomal proteins changes the transcription and translational process and upregulates the expression of biosynthetic gene clusters. Some of the genes related to biosynthetic gene clusters are silent from decades and overexpression of adpA, a global regulatory gene, induced the expression of silent lucensomycin in Streptomyces cyanogenus S136 [225][19]. Cloning is another type of molecular technique used to express the silent BGC incompatible strains. In the cloning method, isolation of high-quality DNA, fragmentation, library construction and development of suitable expression vectors for large sequences of BGC is a challenging task and many groups are working on this aspect [226][20]. In addition to this, use of bioinformatics also helps in direct cloning of silent BGCs and their expression for secondary metabolites production. Development of various bioinformatics tools such as PRISM3, BiG-SCAPE and anti-SMASH etc facilitated the scientist to identify bioactive gene clusters in unknown strains without time consumption used in identification of active BGC sites [227][21]. The CRISPR-Cas system is also a excellent tool for cloning system or genome editing that provides better expression of silent BGC in comparison to conventional molecular techniques [228][22]. Similarly, promoter engineering, transcriptional regulation engineering and ribosome engineering also support the activation of silent BGC through molecular approaches [229][23]. Recent use of Cpf1 nuclease in genome editing was also found to be a suitable tool for induction of silent BGC [230][24].

2. Epigenetic Modification

On the other hand, epigenetic modification played a great role to induce the silent genes related to bioactive molecules, which are actively produced under symbiotic interactions. Epigenetics refers to the study of DNA sequences that do not changes in mutation but change in gene function [231][25]. The epigenetic regulations such as methylation, demethylation, acetylation, deacetylation and phosphorylation of histones also regulate the transcription of biosynthetic genes of fungi and are helpful in silencing or expression of such genes related to the production of secondary metabolites [232][26]. The importance of epigenetic regulation in secondary metabolite production by fungi has been shown in a few reports published [231,233,234,235,236][25][27][28][29][30]. Modification or alteration in DNA or chromatin changes the expression level of the selected genes, which directly impacted the biosynthesis of the metabolites in the strain.

3. The Co-Culture Strategy

The co-culture is another method to induce the silent biosynthetic gene clusters by interspecies cross-talking of microorganisms. In this method, various combinations of inducers with producer microbial strains are screened for the production of novel molecules. In co-culture technique real-time bioactivity screening can also be measured by the growth of pathogen as co-culture [218][11]. Recently, Kim et al. [237][31] reviewed the co-culture interactions of fungi with various actinomycetes for induction of silent biosynthetic gene clusters and reported upregulation and production of novel antibiotics and bioactive compounds. Co-culturing of microbes provides the habitat type environment to producers and helps to promote silent BGCs by producing signal molecules. Exchange of chemical signals of growing organisms is helpful in the induction of defense molecules and other silent BGC, and usually results in the production of new natural products or secondary metabolites in the culture [238][32]. Another concept has also been introduced to elicit the production of silent secondary metabolites by scaffold technique. In this technique, two types of scaffold named cotton and talc powder are introduced in the medium which physically interacts with the grown culture and elicit chemical signaling of the culture and activate the production of silent BGC. The addition of scaffold in the medium supports the grown culture in formation of biofilm and provides a mimic architecture of natural habitat [239,240][33][34]. The addition of scaffold in medium affects the morphology of growing culture and sporulation pattern like an agglomeration of spores, oxygen diffusion in comparison to non-scaffold containing medium and then facilitates more metabolites production [241][35].

4. OSMAC

In the OSMAC technique different cultivation approaches are applied to induce silent bioactive gene clusters to promote more production of secondary metabolites including media variations, variation in media composition, co-cultivation with other strains and variations in cultivations strategy [222,242][15][36]. Variation in growth conditions also supports the induction of silent biosynthetic gene clusters and the production of novel compounds. Scherlach and Hertweck [243][37] and Scherlach et al. [244][38] reported the production of novel aspoquinolone and aspernidine alkaloid compounds from Aspergillus nidulans by variation in growth conditions.

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