Chemical Elicitors of Antibiotic Biosynthesis in Actinomycetes

microorganisms Review Chemical Elicitors of Antibiotic Biosynthesis in Actinomycetes Anton P. Tyurin 1, * ID , Vera A. Alferova 1 and Vladimir A. Korshun 1,2 ID 1 2 * Gause Institute of New Antibiotics, Bolshaya Pirogovskaya 11, 119021 Moscow, Russia; (V.A.A.); (V.A.K.) Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia Correspondence: ; Tel.: +7-499-246-6983 Received: 2 May 2018; Accepted: 6 June 2018; Published: 8 June 2018



 Abstract: Whole genome sequencing of actinomycetes has uncovered a new immense realm of microbial chemistry and biology. Most biosynthetic gene clusters present in genomes were found to remain “silent” under standard cultivation conditions. Some small molecules—chemical elicitors—can be used to induce the biosynthesis of antibiotics in actinobacteria and to expand the chemical diversity of secondary metabolites. Here, we outline a brief account of the basic principles of the search for regulators of this type and their application. Keywords: actinomycetes; antibiotic biosynthesis; silent biosynthetic pathways; γ-butyrolactones; HiTES; translation inhibitors 1. Introduction Since the discovery of streptomycin by Selman A. Waksman, actinomycetes have become one of the most fruitful sources of new antibiotics. Most antibiotic classes in current clinical use were discovered during the “golden era”, 1940–1960s, by phenotypic screening of soil microorganisms. Moreover, since the 1960s, a significant number of approved drugs has been designed using chemical modifications of natural scaffolds. Due to an “innovation gap” in this area, society is now facing an emerging threat of microbial drug resistance. An urgent need for new effective antimicrobials has become an important social and political issue [1,2]. On the other hand, achievements in genome sequencing of actinomycetes has revealed a difference between their potential and observed biosynthetic gene expression. Biosynthetic gene clusters (BGCs) are several generally contiguous genes encoding enzymes responsible for a stepwise assembly of complex bioactive molecules. According to data from the majority of published genomes, most BGCs remain “silent” under standard cultivation conditions. These silent, or cryptic, BGCs represent a potential source of new scaffolds for the discovery of novel antimicrobials [3–5]. Several techniques for activation of silent BGCs have been developed in recent decades, e.g., direct identification of BGCs and their expression in heterologous hosts [6–10], and systematic alteration of cultivation parameters (the “one strain—many compounds” (OSMAC) approach) [11–13]. These strategies are extremely powerful although still remaining laborious and resource-intensive, especially for large (>40 kb) BGCs. Further techniques comprise co-cultivation [14,15], ribosome engineering [16–18], and the use of chemical elicitors—compounds that induce the synthesis of antibiotics in actinomycetes [14,19–21]. The last approach is accounted and discussed succinctly in this review, covering literature up to December 2017. Here, we focus on small organic molecules capable in nanomolar to micromolar minimum effective concentrations to induce biosynthesis of secondary metabolite in actinomycetes. Microorganisms 2018, 6, 52; doi:10.3390/microorganisms6020052 www.mdpi.com/journal/microorganisms Microorganisms 2018, 6, 52 Microorganisms 2018, 6, x FOR PEER REVIEW 2 of 10 2 of 11 2. γ-Butyrolactones γ‐Butyrolactones (GBL) and Related (auto)Regulators Historically, Historically, A-factor A‐factor (1, Chart 1) (A for “autoregulation”) “autoregulation”) was the first first compound compound that that revolutionized our ourviews viewsofofthe the secondary metabolism and development cycle of actinomycetes. secondary metabolism and development cycle of actinomycetes. This This γ-butyrolactone (GBL) derivative discovered by Prof. Khokhlov co-workers in 1967 γ‐butyrolactone (GBL) derivative was was discovered by Prof. Khokhlov andand co‐workers in 1967 [22].[22]. 3R 3R OH O O 2R 6S O O O 2a-f 1 R VB-A iso-hexyl b anteiso-hexyl VB-B VB-C c n-pentyl VB-D d n-hexyl VB-E e iso-pentyl Factor I f iso-heptyl OH a R OH R 3R 4R R' O O R R' H OH OH H a b O Factor II Factor III OH a b 2S O 4a-c Rc OH R Gräfe's iso-hexyl factor 1 iso-heptyl factor 2 iso-octyl factor 3 3a-b 3R O 2R 6R O 5a-i R n-propyl b iso-heptyl c n-heptyl d anteiso-octyl e anteiso-hexyl f iso-hexyl n-hexyl g n-octyl h n-pentyl i OH a OH R IM-2 SCB1 SCB2 SCB3 SCB4 SCB5 SCB6 SCB7 SCB8 R iso-butyl n-propyl iso-pentyl n-butyl n-pentyl Oa HO b c d Re HO2C 6a-e R1 OH O O 4S O O 10R 1 O O R 6R OH 8a-b R2 R3 7b-e O 7a HO O MMF1 MMF2 MMF3 MMF4 MMF5 2 3 R R R b OH H H c H OH H d H O O e H H H R a H SRB1 b CH3 SRB2 Chart 1. 1. GBL GBL and and closely closely related related regulators: regulators: butenolides butenolides (avenolide, (avenolide, S. S. rochei rochei butenolides butenolides or or SRB) SRB) and and Chart furans (methylenomycin (methylenomycin furans furans or or MMF). MMF). furans A‐factor acts as a pleiotropic regulator: it binds to the A‐factor receptor protein (ArpA) and A-factor acts as a pleiotropic regulator: it binds to the A-factor receptor protein (ArpA) and causes causes dissociation of this suppressor from DNA. This triggers the transcription of the adpA gene dissociation of this suppressor from DNA. This triggers the transcription of the adpA gene encoding the encoding the transcription activator AdpA, which in turn induces morphological differentiation, transcription activator AdpA, which in turn induces morphological differentiation, spore formation, spore formation, and biosynthesis of secondary metabolites [23,24]. and biosynthesis of secondary metabolites [23,24]. In further decades, many other closely related autoregulators were discovered, e.g., Streptomyces In further decades, many other closely related autoregulators were discovered, e.g., Streptomyces virginiae butanolides (VBs A‐E, 2a–e) [25–27], Factor I (2f) [28], Factors II and III (3a,b) [29], Gräfe’s virginiae butanolides (VBs A-E, 2a–e) [25–27], Factor I (2f) [28], Factors II and III (3a,b) [29], Gräfe’s factors (4a–c) from S. bikiniensis and S. cyaneofuscatus [30], IM‐2 (5a) [31,32], SCB1 (5b) [33,34], SCB2,3 factors (4a–c) from S. bikiniensis and S. cyaneofuscatus [30], IM-2 (5a) [31,32], SCB1 (5b) [33,34], (5c,d) [35] and SCB4–8 (5e–i) [36], methylenomycin furans (MMFs, 6) [37], avenolide (7a) from S. SCB2,3 (5c,d) [35] and SCB4–8 (5e–i) [36], methylenomycin furans (MMFs, 6) [37], avenolide (7a) avermitilis [38], related compounds (7b–e) from S. albus [39], and two S. rochei butenolides (SRBs, 8a,b) from S. avermitilis [38], related compounds (7b–e) from S. albus [39], and two S. rochei butenolides [40]. The stereoconfiguration of avenolide analogues 7b–e reported very recently has not been (SRBs, 8a,b) [40]. The stereoco (...truncated)