Synthetic biology era: Improving antibiotic’s world

Biochemical Pharmacology 134 (2017) 99–113

Contents lists available at ScienceDirect

Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm

Review

Synthetic biology era: Improving antibiotic’s world Silvia Guzmán-Trampe a,⇑, Corina D. Ceapa b, Monserrat Manzo-Ruiz a, Sergio Sánchez a a b

Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico University of Chicago, Laboratory of Microbiology, 5801 South Ellis Avenue, Chicago, IL 60637, USA

a r t i c l e

i n f o

Article history: Received 26 August 2016 Accepted 26 January 2017 Available online 31 January 2017 Keywords: Synthetic biology Synthetic biological systems Antibiotics Artemisinin Protein engineering Drug delivering

a b s t r a c t The emergence of antibiotic-resistant pathogen microorganisms is problematic in the context of the current spectrum of available medication. The poor specificity and the high toxicity of some available molecules have made imperative the search for new strategies to improve the specificity and to pursue the discovery of novel compounds with increased bioactivity. Using living cells as platforms, synthetic biology has counteracted this problem by offering novel pathways to create synthetic systems with improved and desired functions. Among many other biotechnological approaches, the advances in synthetic biology have made it possible to design and construct novel biological systems in order to look for new drugs with increased bioactivity. Advancements have also been made in the redesigning of RNA and DNA molecules in order to engineer antibiotic clusters for antibiotic overexpression. As for the production of these antibacterial compounds, yeasts and filamentous fungi as well as gene therapy are utilized to enhance protein solubility. Specific delivery is achieved by creating chimeras using plant genes into bacterial hosts. Some of these synthetic systems are currently in clinical trials, proving the proficiency of synthetic biology in terms of both pharmacological activities as well as an increase in the biosafety of treatments. It is possible that we may just be seeing the tip of the iceberg, and synthetic biology applications will overpass expectations beyond our present knowledge. Ó 2017 Published by Elsevier Inc.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Introduction to the concept of synthetic biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Synthetic biology for drug research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Synthetic gene circuits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Synthetic gene circuits for drug discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Synthetic RNA molecules (therapeutic RNA devices) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. Phage devices and therapeutic applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Targeted engineering for awakening or overexpressing known secondary metabolite biosynthetic cluster. . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Activation of cryptic or silent gene clusters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Overexpressing pathway-specific regulatory factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3. Changing the cultivation conditions (OSMAC approach) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.4. Microbes co-cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.5. Engineering microorganisms with plant metabolic pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Protein engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1. Directed evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2. Modifications of substrate specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Mutasynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bacterial drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biomedical applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Convergence with synthetic genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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⇑ Corresponding author at: Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México D.F. 04510, Mexico E-mail addresses: [email protected] (S. Guzmán-Trampe), [email protected] (C.D. Ceapa), [email protected] (M. ManzoRuiz), [email protected] (S. Sánchez). http://dx.doi.org/10.1016/j.bcp.2017.01.015 0006-2952/Ó 2017 Published by Elsevier Inc.

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Examples of pharmaceutically active compounds Main concerns – biosafety . . . . . . . . . . . . . . . . . . . Future outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

discovered by ............ ............ ............ ............

synthetic biology ............... ............... ............... ...............

1. Introduction Large scale antibiotic use is widespread not only in human therapy, but also for farm animals and for aquaculture. As result of their wide applications, antibiotic related ecological pressure led to the arise of multi-resistant pathogenic bacteria. Therefore, new and more effective antibiotics are continuously required to fight antibiotic-resistant bacteria and pathogenic yeast. Antibiotic resistance progressively limits the efficiency of the current antimicrobial drugs. The incidence of resistant bacteria is highly increased in hospitals [1] and the infections caused by them kill many people around the world. In addition to antibiotic resistance, the appearance of an increasing number of multidrug-resistant pathogens makes the panorama more difficult to resolve [2]. Among them, Staphylococcus aureus causes half of the hospitalacquired infections and causes deaths of many people around the world [1]. In addition to the antibiotic resistance, new antibiotics are required to face new diseases caused by evolving pathogens. From 1980 to 1995 at least 30 new diseases were detected which are growing in prevalence. The picture is worst considering reemerging diseases such as the novel varieties of influenza and hepatitis B. The costs of combating such diseases are more than $120 billion per year. Many pharmaceutical companies moved away from natural product research programs [3], especially from antibiotics. This was reflected in the number of natural drug approvals by FDA, which dropped from 36 in 2004 to 7 between 2003 and 2012. Nevertheless, the antimicrobial pharmaceuticals still amount for a significant percent of the drug market and the search for new active molecules is continuing, in both academia and industry. According to Baltz [4], we can no longer depend on the pharmaceutical companies alone to isolate and produce new antibiotics. The effort will need to come from medical research by the academia in collaboration with the biotechnology and pharmaceutical companies. Finding new leads is clearly a priority. Traditionally, the new drugs have been obtained from natural microbial products, but with the increase in the knowledge from microbial physiology and with the technological developments, new avenues are opening. For example, new screening approaches, including the search for novel targets [5] and the exploration of non-conventional places as sources of the producer microorganisms are being implemented. In this regard, plant endophytes [6], springs/geysers [7] and caves [8], have been successfully explored. Most of the clinically used antibiotics have been derived from the bacterial small molecules produced by dedicated biosynthetic gene clusters, 90% of which remain unexplored. Therefore, the modern metagenomic and genome-mining that have recently been introduced show a strong potential for the discovery of new antibiotics [9,10]. As a discipline meant to design and construct organisms with desired properties, synthetic biology has generated rapid progresses in the last decade. This review will cover the strategies for synthetic biology applications, and some examples of pharmaceutical active compounds discovered by this modern discipline. 1.1. Introduction to the concept of synthetic biology Finding an accurate definition of synthetic biology has been challenging. However, one plausible option is to understand

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synthetic biology as an engineering approach to improve or completely create systems and organisms with specific or desirable functions. This field of science incorporates different knowledge areas such as biology, chemistry, biotechnology, engineering, genetics and informatics to better comprehended living cells as working factories capable to evolve into anything imaginable [11–13]. According to the Presidential Commission of the USA [14], there are two main types of synthetic biology research, bottom-up and top-down. The first one creates new systems from nothing but complex organic chemicals. The second one uses living organisms as models, but rearranges their enzymes, genes and chemical molecules in a new puzzle. Despite its inherently challenging and complex nature, synthetic biology has allowed scientists to develop new strategies to exert control over the cellular behavior through this large library of building blocks. Origins of synthetic biology have been differently reported. Some reports refer to the discovery and the study of the lac operon as the breakthrough in molecular biology, which allowed scientists to learn how regulatory circuits are controlled by specific conditions. This milestone discovery leads to the understanding of regulation and of gene expression of the cellular components [15,16]. Some of the most impressive highlights achieved thanks to synthetic biology could be consulted in other reviews [7,17–19]. One of the finest examples is the engineering of an Escherichia coli strain able to respond as a biological film, projecting different patterns of light to create a chemical image [20]. This goal was achieved by creating a chimera in which a synthetic sensor kinase from a cyanobacterial photoreceptor was fused to an E. coli intracellular histidine kinase domain. This experiment required the creation of a genetic circuit never reported before, using iGEM building blocks [21], thus attesting for the possibilities of synthetic biology. Another outstanding example involves the creation of the first self-replicating synthetic genome in bacteria by scientists at the J. Craig Venter Institute [22]. Synthetic biology has accelerated research in many scientific areas. Metabolic and microbial engineering has been one of the most benefited fields, since every metabolic capability (including cellular metabolism and gene regulatory and signaling networks) may be increased, for example by directing the metabolic fluxes to produce novel compounds with a specific biological activity [23]. In this review we will focus on the advances of the antibiotic drug discovery generated by synthetic biology. These advances have led to the discovery of novel and safer medicines. Among others, some examples include the development of genetic circuits, the enhancement of metabolite production, the awakening of silent clusters and the use of bacteria as drug delivery agents. Synthetic biology brings many benefits meant to improve the clean energy, agriculture, food and medicine industries. Therefore, the applications are beyond our present knowledge, and they will most probably change the biological sciences as we know them. 2. Synthetic biology for drug research In the following pages, we will review examples of how synthetic biology has improved the development of drugs, especially antibiotics. The aforementioned new emerging pathogens and the prevalence of antibiotic-resistant strains have become an unresolved health problem of global dimensions. Despite many efforts, the

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trend of compound rediscovery decreased the investments into new drug research by pharmaceutical industries, by focusing some resources on designing synthetic derivatives and by reducing prices and toxicity of known compounds. Recently, access to large datasets of complete sequenced bacterial genomes revealed a new lane for research, allowing for a better understanding of the production and the regulation of secondary metabolites’ pathways. This new period known as the ‘‘omics era” has revealed how promising microorganisms are for new compound discovery [24,25]. Technological progress in bioinformatics, DNA synthesis, pathway regulation, Gibson assembly, microbial engineering and systems biology has enhanced productivity and progress in antibiotic research. Synthetic biology however has begun to make discoveries that soon changed the course of the antibiotics research. Modularity of most of the secondary metabolite pathways enabled combinatorial biosynthesis to translate antibiotics into puzzles whose pieces can be interchanged to obtain novel molecules with desirable bioactivities. Later in this review we will discuss engineering of antibiotic clusters [26]. A major challenge for production of secondary metabolites continues to be the expression of gene clusters, which sometimes may result difficult. Current strategies to express such clusters include enhancing production through strong promoters, cultivation in different media, metabolomics and proteomics. Synthetic biology combined with RNA technology and mammalian systems for drug testing has allowed the introduction of synthetic gene circuits to simulate biological pathways for a new model of antibiotics discovery. This led to the design of new biological systems which are tightly controlled and regulated with respect to an input controlled signal [27]. Synthetic genetic circuits built in this manner enable the simulation of biological pathways inside biological factories to bring theory into action [18]. Thanks to synthetic biology efforts, the bio production of artemisinin [28], and different kind of terpenelike compounds [29] in microorganisms such as E. coli and Saccharomyces cerevisiae has been possible [12]. 2.1. Synthetic gene circuits Genetic elements of cells may be organized as single entities or as operons. A wide variety of basic genetic elements such as promoters, regulators, genes and more have been synthesized as

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Fig. 2. Chemical structure of artemisinin.

Fig. 3. Chemical structure of taxol (paclitaxel).

single units, and included as a module registered by BioBricksÒ, thus facilitating direct synthetic biology design of new entities [30]. To better understand a synthetic circuit, we should think about synthetic cells as electrical circuits composed of standard sections: an inducer, a ligand or receptor and an output signal (meaning generally an enzymatic reaction like the one emitted by GFP or the

Fig. 1. 2-Phenylethyl-butyrate as an antituberculosis compound revealed by gene circuits. Bacterial cells transfected with EthR-based synthetic gene circuit: A) Gene circuit turns on SEAP expression when ethR binds to its operator. EthR causes ethionamide resistance. B) When 2-phenylethyl-butyrate is administrated, ethR dissociates from its operator and the expression of SEAP decreases to a basal level. Under this effect, ethA is derepressed and the cytotoxicity of ethionamide increases. C) When tested in a in vivo system, mice treated with 2-phenylethyl-butyrate conserve the capability for regulate EthR, reducing levels of SEAP to a basal level.

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Lux- operon). Another important aspect involves multicellularity and the complex biochemical environment that allows one cell to converge and communicate with other cells. Even when the scope of synthetic circuits focusses on single cells, the final task is to adjust the complete system in order to obtain the final result [31,32]. 2.1.1. Synthetic gene circuits for drug discovery As biological networks may be seen as electrical circuits, a tight control over the expression of a desired molecule can be achieved. When a certain biological function is required, it can be de novo engineered through synthetic pathways [25]. This model involves prediction of the most suitable pathway after pooling of a large set of computational databases for pathway prediction, design and construction. If the best prediction model for a desired synthetic circuit has been obtained, genetic elements that are intrinsic to this blueprint must be isolated from nature. Final proof involves the use of molecular biology tools for cloning in a host organism, followed by verification in a heterologous or homologous host [32,33]. Gene circuit systems applied to the discovery of novel antibiotics has been broadly developed by Fussenegger laboratory [34–36]. They introduced the human-compatible gene regulation technology, centered in the construction of different antibioticrepressible systems which fusses different biosensors such as PIP (pristinamycin-induced), TetR and ScbR with VP16 transactivation domain or the PSV40 viral promoter. This investigation led to the design of the MAST system (mammalian antibiotic sensor technology), which is able to evaluate the presence of a specific class of antibiotic structure through a chemiluminescence response. Both assays are based on determining the production of the SEAP reporter protein, which is turned on in presence of the required antibiotic. One of the most recent examples of synthetic circuits involves the use of genetic circuits to potentiate the sensitivity of Mycobacterium tuberculosis to the antibiotic ethionamide. This goal was achieved by fusing the repressor EthR with a viral transactivation domain. The model allowed for the screening of a library of hydrophilic ester-like compounds capable of trigger the release of EthRVP16 from its operator OethR, leading to repression of SEAP expression (Fig. 1). When testing this model in mice, 2-phenylethylbutyrate increased the sensibility of M. tuberculosis to ethionamide, indicating that lower antibiotic doses may be required and reducing the neurotoxic and hepatotoxic effects of this treatment [36]. However, this approach is limited by the typically large size of the encoding sequences. 2.1.2. Synthetic RNA molecules (therapeutic RNA devices) Synthetic biology seeks to exert control over synthetic biological systems and this goal could be achieved by using DNA or RNA synthetic devices. As cells behave just like electrical networks, in which tight control may be achieved by regulating gene expression, one of the most popular antibiotic targets are ribosomal RNA and riboswitches [37]. Riboswitches are RNA sequences found in 50 (UTR) region of mRNA of some bacteria that selectively bind to small molecules. Each riboswitch class usually consists of two regions, an aptamer domain and an expression platform. The aptamer is a conserved receptor that particularly recognizes the ligand. The expression platform region assumes different conformations and its final folded structure establishes whether the associated genes are expressed or not. They can adopt different structures with various predicted stability, and can be engineered to perform functions such as the regulation of gene expression and biomolecules detection [38], or have evolved to specifically respond to certain types of metabolites [39], offering new RNA-based targets for antibacterial compound development.

Most of the known riboswitches are engaged in feedback control mechanisms, wherein the accumulation of an essential metabolite triggers the down-regulation of genes whose protein products make or import more of the metabolite. In these regard, it is predicted that analogues of the natural ligand could bind to a riboswitch and exert repression of genes that are necessary for maintaining an adequate concentration of the metabolite. Therefore, riboswitch-mediated repression of genes necessary to synthesize or import essential metabolites may cause inhibition of the bacterial growth or even cause bacterial cell death. One experiment proving the RNA control over expression of endogenous genes involved the design of three different RNA aptamers (bl-RSETA, bl-kan1 and bl-tob1) that bound aminoglycoside antibiotics. This RNA aptamers regulate expression when their specific ligand was available, both in vitro as well as in mammalian cells [40]. In a different case, the effect of fluoroquinolone antibiotics was tested when the toxic bacterial protein CcdB expression was post-transcriptionally regulated by using engineered riboregulators, in which the 50 -UTR region of their mRNA sequence form a hairpin structure that prevents ribosome to bind its target site (gyrase), thus letting the protein be translated [41]. This experiment allowed for identification of the sequence of events conducting to CcdB-induced cell death [26,32,38,40]. Kim and colleagues described 16 different riboswitches that selectively regulate gene expression throughout guanine recognition [42]. Guanine riboswitches are found in Gram-positive bacteria and they regulate the expression of purine transport and metabolism genes and the complete deletion of all these genes has been associated with growth inhibition. When analyzing the aptamer sequences, the group found the cytosine residue (C74) as essential, since its elimination led to an inactive riboswitch. When different growth media was used, the activity of guanine riboswitch binding compounds inhibited B. subtilis differently, proving that media conditions may affect antibiotic effectiveness. Finally, they found a specific purine analogue (6-N-hydroxylaminopurine, or G7) that binds to the xpt aptamer, inhibiting B. subtilis by binding to guanine riboswitches and repressing purine biosynthesis and/or transport genes [42]. Although the action of G7 suggests that guanine riboswitches can function as targets for antibacterial agents, new guanine analogues must be examined to create compounds that exhibit more drug-like properties. 2.1.3. Phage devices and therapeutic applications In addition to synthetic genes and regulators, bacteriophages, which are bacterial viruses, represent a feasible antimicrobial therapy since they hold a wide range of mechanisms of action and host specificity and present no harm to mammalian cells. Recent studies revealed that a combination of phage therapy with antibiotic administration notably improves cell death of pathogenic bacteria [43,44]. When administered linezolid simultaneously with the phage MR-10, Kaur and colleagues found a maximum reduction in bacterial growth as well as a diminishing skin ulceration when compared to controls in diabetic mice infected by methicillinresistant S. aureus (MRSA) [45] a multidrug resistant bacteria responsible for the largest number of hospital acquired infections. When Hochberg et al. [46] tracked the bacterial population density of an opportunistic Pseudomonas aeruginosa PAO1 over 70 h, they separately exposed bacterial cells with phage LUZ7, with streptomycin and with phage plus streptomycin. They established that the synergistic activity of the third treatment strongly reduces the bacterial density independently of streptomycin concentration [46]. In parallel, they showed how the addition of the streptomycin at 12 h after phage introduction reduces the streptomycinresistant phenotype of tested bacteria. In a different type of experiment, the in vivo model of larvae of Galleria mellonella infected

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with strains of Burkholderia cepacia displayed increased survival when treated with phage KS12 and minocycline or meropenem than when treated with the antibiotics alone [47]. The findings listed above represent examples of the scope of using phages to increase the potency of antibiotics against a defined pathogen. There are some limitations associated with phage therapy, for example bacterial resistance against phages, over-induction of antibodies or immune response and biosafety concerns. 2.2. Targeted engineering for awakening or overexpressing known secondary metabolite biosynthetic cluster With the introduction of genomics and a decrease in the cost of DNA sequencing, the genome analysis of microorganisms involved in the production of commercially important bioactive compounds became possible. One of the first findings derived from the genome mining of actinomycetes was that this group of microorganisms, responsible for over 50% of all the antibiotics production contained more biosynthetic gene clusters for secondary metabolites than those previously detected or reported [48]. Therefore, the biosynthetic potential of the actinomycetes for the production of new and useful bioactive compounds increased dramatically. This finding was also observed in fungi, especially for the genus Aspergillus [49]. 2.2.1. Activation of cryptic or silent gene clusters In recent years, there has been a notable increase in the sequenced genomes of actinomycetes, fungi and other microorganisms. Their study has revealed that natural-bioactive producers contain unknown clusters to produce more compound classes than were previously reported. Detailed analysis of the new biosynthetic secondary metabolism clusters has demonstrated inactivity or silencing of the great majority of them under the standard laboratory growth conditions [50–52]. Considering that bioactive natural products have been a major source of therapeutic molecules, and because the gene clusters coding for them constitute an almost inexhaustible natural resource of secondary metabolites [51,53– 55], reasonable methods directed to activate these cryptic (encoding unknown products) clusters are having a strong impact on drug discovery. The recent advances in genome synthesis [56–58] are bringing such strategies to reality. In addition, the modular nature of the biosynthetic clusters responsible for the synthesis of secondary metabolites makes them an attractive target for the synthetic biology strategies. In order to awake the silent gene clusters involved in the production of natural bioactive compounds, several strategies have been developed [49,59–61]. In one of these strategies, transcription from gene clusters is directed to bypass the original regulatory mechanisms by deleting or adding genetic control elements of the silent cluster [62]. The genetic control elements amenable for modification can be either cluster of specific genes regulating the expression of genes coding for one or several catalytic steps in a specific pathway, or transcriptional regulators that can activate or repress the expression of clusters responsible for secondary metabolites’ formation. These modifications can be done on the original producer microorganisms or in the entire cluster previously cloned. The production of a non-ribosomal peptide can be cited as an example of this strategy. Computational analysis of the draft genome of Saccharomonospora sp. CNQ49 evidenced this nonribosomal peptide as putatively silent. When deleting the negative repressor Tar20 (LuxR-type regulator) of this cluster, the resulting mutant produced the novel lipopeptide taromycin A (structurally similar to the antibiotic daptomycin), and several analogues, which differed in the nature of their lipid side chains [63,64]. In addition to this cluster, 18 more putative silent clusters were detected in this microorganism.

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The replacement of promoters in cluster-specific biosynthetic silent genes or replacement of their transcriptional factors promoters by an inducible one can be an alternative strategy to awake silent gene clusters. As expected, this strategy made the silent genes expression controllable. The same strategies have been reported for the production of the cryptic polyketides in Aspergillus nidulans. For this purpose, Ahuja et al. [65] used an arabinose-inducible promoter to replace the native PKS promoter in this fungus. This approach turned on the PKS genes and allowed the production of new polyketides. Similar examples include the activation of a novel macrolactam production in Streptomyces griseus [66]. Examples of compounds in which promoters of transcription factors have been replaced by inducible promoters include the production of new polyketides in A. nidulans. For this purpose, the fungal transcriptional activator CtnR was replaced with the inducible alcohol dehydrogenase alkA promoter, which enables the production of the new polyketide asperfuranone [67]. Exchange of the native promoters of silent gene clusters from Photorhabdus luminescens and Xenorhabdus budapestensis with the strong arabinose-inducible PBAD promoter allowed the production of the novel mevlanapeptide xenothabdin-2, a compound similar to holomycin from Streptomyces clavuligerus [68]. This strategy cannot be utilized for clusters, which lack transcriptional regulators [67]. This type of examples demonstrates the utility of the awakening of silent or cryptic clusters toward expressing known or discovering novel therapeutic molecules. However, more progress has to be made in increasing and refining the general procedures for silent gene activation as well as for the discovery of cryptic gene products. 2.2.2. Overexpressing pathway-specific regulatory factors Some bacteria and fungi can undertake a complex morphological program, which goes from vegetative mycelium to spore formation and production of antibiotics and other secondary metabolites. These processes are precisely regulated by proteins capable of binding DNA known as transcriptional factors (TFs) in order to repress or activate the transcription of certain genes [69,70]. TFs activities are dependent on the threshold concentration of extracellular signaling molecules and other environmental stimuli. Many gene clusters encoding antibiotic biosynthetic pathways contain pathway-specific regulatory genes, which in turn can be regulated by TFs. Modification of these physiological signals and regulatory mechanisms may be of practical importance for activation and overexpression of the silent secondary metabolic gene cluster pathways, which are continuously revealed by an increasing number of sequenced microorganisms. Several examples can be cited as representatives of this strategy. For instance, the glycopeptide antibiotic teicoplanin is a last resort drug used for the treatment of pathogenic antibiotic resistant bacteria. It is produced by Actinoplanes teichomyceticus and is encoded in a cluster of 89 kb which includes 49 ORFs including 5 putative regulatory genes, predicted to participate in its biosynthesis and regulation. Antibiotic production is switched on by the pathway specific regulatory genes tei15 and tei16. It has been reported that tei15, a Str-type regulator positively regulates the transcription of at least 17 genes in the cluster, whereas the targets of Tei16⁄ are not known. Overexpression of these genes was obtained under the control of the apramycin resistance gene promoter aac(3)IVp expression. In regard to the wild type strain, recombinant strains produced increments in teicoplanin production from 100 mg L1 to 1 g L1 [71]. Another example is the antiinfective streptomycin, produced by S. griseus. Streptomycin production is induced by A-factor (2-isoca pryloyl-3R-hydroxymethyl-gamma-butyrolactone) [72]. This factor

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allows expression of the pathway-specific transcriptional activator strR, in the A-factor regulatory cascade of S. griseus. StrR activates nine transcriptional units within the streptomycin biosynthesis gene cluster. Overexpression of strR under the control of a constitutive promoter resulted in a high antibiotic production [72]. In the same line of research, Streptomyces lividans 1326 is known not to produce the antibiotic actinorhodin, even though it contains the entire actinorhodin gene cluster. However, this bacterium can be forced to produce this antibiotic in sufficient concentrations by introducing act II-ORF4, the actinorhodin pathwayspecific activator gene from Streptomyces coelicolor, on a multicopy plasmid [73]. A similar effect was observed with another Streptomyces avermitilis putative regulatory gene of 8 kb, whose expression stimulates both actinorhodin and undecylprodigiosin formation in S. lividans [74]. Avermectin production by S. avermitilis represents another interesting example of increased production by manipulation of the copy number of the regulator gene. This compound is also used in the treatment of parasitic worms. Four proteins are encoded by the biosynthetic cluster (AveT, PepD2, AveM, and Sav_7491) of the parasiticide biosynthesis. Its expression is regulated by the specific AveT regulatory protein. Overexpression of aveT in strains of S. avermitilis led to increases in avermectin production [75]. The AveT targets are aveT, pepD2, aveM, and sav_7491. Another example is related to the production of the macrolide antibiotic tacrolimus. This compound is produced by Streptomyces tsukubaensis and also has a profound immunosuppressive effect, particularly affecting T cells and the cellular immune response. Its production is regulated by two pathway-specific regulatory proteins encoded by bulZ and bulY. These regulators are involved in the gamma-butyrolactone biosynthetic gene cluster in S. tsukubaensis. Overexpression of both proteins exerted a 1.6-fold increase on the tacrolimus production [76]. An additional regulatory protein is FkbN, which is encoded by fkbN. This gene, located inside the tacrolimus gene cluster, also plays a positive role in tacrolimus production [77]. In S. coelicolor M1146, overexpression of FkbN resulted in a 5-fold increase in the production titer of this pharmaceutical compound, as compared to the heterologous strain without fkbN overexpression [78]. One interesting situation occurs in Streptomyces ambofaciens, producer of two antibiotics, the pyrrole-amide congocidine and the macrolide spiramycin. Genome sequence of this microbe has revealed the presence of a type II polyketide synthase. The genes of this cluster are responsible for the biosynthesis of a new compound with antibacterial activity designated as alpomycin (alp cluster) and an orange pigment. In this microorganism, alpV is the essential regulator gene required for activation of the biosynthetic alpomycin genes. When alpV is introduced into an S. coelicolor mutant with deletions in actinorhodin and undecylprodigiosin regulatory proteins actII-ORF4 and red, alpV was able to restore only the actinorhodin production [79]. This is a very promising methodology for pre-existing or silent secondary metabolites’ operons. 2.2.3. Changing the cultivation conditions (OSMAC approach) In addition to genome mining, many secondary metabolites have been discovered by modifications in the growth conditions [80]. It is well known that media composition and nutrient concentrations can impact the growth rate and also determine a series of changes in the global gene regulation. Small changes in the growth nutrients of Actinobacteria can have an effect on the production of secondary metabolites, even to the extent of facilitating the discovery of novel secondary metabolites. During the last decades, reports about the wide range of natural conditions determining the production of different antimicrobials have dramatically increased in numbers [81]. In agreement with this, it is often

observed that limitations in some of the nutrients of the culture media trigger secondary metabolites production [82]. The OSMAC approach (one strain/many compounds) is a powerful way to search for new microbial secondary metabolites. This technique requires changes in the microbial growth conditions like media composition and concentration, aeration rate, illumination, temperature, [83] and addition of enzyme inhibitors [84]. Although addition of chemical elicitors is not traditionally considered to be part of the OSMAC approach, it is known that the presence of certain compounds in the growth media wake silent or cryptic gene clusters for the synthesis of alternative secondary metabolites [85]. Concerning this, Rateb et al. [86] found a strong dependence between the used culture media and the metabolic profile of a Streptomyces sp. strain C34, isolated from soil of a Chilean hyperarid Atacama Desert. By testing eight different culture media in strain C34, the same group discovered three new chaxalactins, a rare class of 22-membered macrolactone polyketides. These compounds showed strong activity against the Gram-positive S. aureus ATCC 25923, Listeria monocytogenes ATCC 19115 and Bacillus subtilis NCTC 2116 [87]. Furthermore, when glucose was substituted by glycerol in the growth media of the same microorganism, four new ansamycin-type polyketides (chaxamycins A-D), were discovered [87]. At 100 lM concentration, one of these new chaxamycins (D) exhibited a selective and high antibacterial activity against S. aureus ATCC 25923 and methicillin-resistant S. aureus isolates with MIC values lower than 1 lg mL1. Three of the new chaxamycins (A-C) showed activity against the intrinsic ATPase activity of the human chaperone Hsp90, a well-established mechanism of the antitumor effects of ansamycin-type compounds. By using the chemical screening of microbial fermentation broths, three new armeniaspirol compounds (A-C) were discovered in Streptomyces armeniacus grown in a medium containing maltyeast extract [88]. These compounds exhibit a chlorinated spiro (4,4)non-8-ene scaffold. From these compounds, armeniaspirol B exhibited the best in vivo antibiotic activity against MRSA in a MRSA sepsis model. Besides, no development of resistance in serial passaging experiments was observed. On the contrary, S. armeniacus cultures grown in the absence of malt-yeast extract produced only the antibiotic streptopyrrole with an unusual benzopyranopyrrole scaffold [88]. Addition of limiting concentrations of rare earth elements have been also reported to induce activation of cryptic or silent biosynthetic gene clusters in bacteria and to stimulate antibiotics’ production [89]. For instance, a new NRPS tetrapeptide coelichelin was discovered by growing S. coelicolor M145 in an iron deficient medium [90]. This compound, previously predicted by genome mining, is a tris-hydroxamate tetrapeptide with iron-chelating function. In a recent paper, the role of sub-inhibitory concentrations of secondary metabolites as inducers of silent gene clusters was reported. By applying a high-throughput screen with genetic reporter fusions, natural elicitors of silent gene clusters for antibiotic production were disclosed [91]. Besides discovery of new metabolites, the OSMAC approach allowed enhancement in the production of known secondary metabolites. For example, addition of the rare earth scandium to the fermentation medium stimulated actinorhodin, actinomycin and streptomycin production in S. coelicolor, Streptomyces antibioticus and S. griseus, respectively [92]. Actually, for S. coelicolor, addition of low scandium or lanthanum concentrations activated the expression of nine genes belonging to nine secondary metabolite biosynthetic gene clusters [93]. Furthermore, a combination of fermentation conditions with genomic techniques allowed uncovering of novel compounds, as reported for Streptomyces flaveolus through screening in six different media in four-day fermentations [94].

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Despite these advances, there are limitations to the use of this approach, stressed by the strain-specific variation observed in the quantity of metabolite production as well as the variable behavior of fungi to alter metabolite profiles when re-cultured [95] 2.2.4. Microbes co-cultivation Extending functional capabilities by co-culture techniques, from single-cell behaviors to multicellular microbial consortia, represents a new frontier of synthetic biology [96] for studying natural or synthetic interactions between cell populations [97]. Such interactions in the microbial consortia can be influenced by the extracellular environment present in the experimental conditions. This environment contains specific signals or nutritional components, likely required for the activation of silent gene clusters. By these means, cell-consortia may induce the expression of silent gene clusters through stimulation of the natural competition between the microbial species present in the system [85,98]. In this line, co-cultivation of Streptomyces endus with Tsukamurella pulmonis (a mycolic acid-containing bacteria), resulted in production and identification of the new antibiotic alchivemycin A [99]. In addition, Streptomyces rapamycinicus can specifically induce expression of silent biosynthetic gene clusters in A. nidulans, by triggering fungal histone acetylation modifications [100]. Furthermore, the production of lobocompactol, a diterpene with antifouling activity, was induced in Streptomyces cinnabarinus when grown with Alteromonas sp. [101]. All these are classical examples of a cross talk between two different genera Streptomyces and other species, whose understanding may provide more strategies for the activation of cryptic clusters of antimicrobials. In 2012, Watrous et al. [102] described an interesting approach to detect the metabolic profile of microbial colonies seeded together in a Petri dish. They used nanoscale mass spectroscopy (MS) combined with alignment of MS data and molecular networking to visualize small molecular changes within bacterial interactions. By using this strategy, they were able to demonstrate that Pseudomonas sp. SH-C52 protects the sugar beet plants from the soil-borne fungi infection by producing thanamycin. This is a monochlorinated lipopeptide belonging to the syringomycin family of antifungal agents. One limitation of this technique is that it is only suitable for cultivable microorganisms. However, the cocultivation approach represents a solid and promising strategy for the discovery of new bioactive metabolites. The underlying factors responsible for the activation of silent biosynthetic gene clusters during co-cultivation are likely species and combination specific. 2.2.5. Engineering microorganisms with plant metabolic pathways Plants represent a rich source of secondary metabolites with human therapeutic activity. They have represented the only alternative for the treatment of many diseases for thousands of years. They are a rich source of secondary metabolites with human therapeutic activity. The secondary metabolites of higher plants include diverse chemicals, such as isoprenoids, alkaloids, and phenolic compounds. However, their isolation from plants is often limited by low abundance and the influence of environmental, seasonal, and regional variations [103]. In addition, the total chemical synthesis of these compounds, which are frequently complex structures, is usually commercially infeasible. A recent wave in the genomics of ‘unknown’ therapeutic natural bioactive products’ aims, has been the sequencing of genomes and transcriptomes of plants used in traditional medicine. With this information, several research groups have designed synthetic biology strategies to incorporate genomic sequences coding for plant secondary metabolites pathways into microorganisms or algae. Therefore, microbial cell cultures can act as workhorse biofactories, offering their metabolic machinery for the expression and further optimization of the production conditions of a selective

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compound. Microbial production of plant secondary metabolites offers several advantages since they are relatively easy to manipulate, their fermentation can be better controlled, they have rapid growth rates and generally use inexpensive substrates [104]. Plant synthetic biology requires strong and effective methods for assembling multigene constructs. For this purpose, several genetic tools like Golden Gate cloning [105] have been designed. As with other secondary metabolites, achieving expression and good yields of plant secondary metabolites in microbes is a challenging task [106]. Among the factors impairing productivity, the most noticeable include the functionality of the biosynthetic pathway and the availability of precursors. Additional factors are the concentration of its constituent enzymes, which is also influenced by translation-related mechanisms, protein degradation rate [107] and enzyme efficiency (kcat/KM). Codon usage is known to affect heterologous gene expression [108] and should be part of every metabolic engineering approach. Enzyme stability in the heterologous host, cofactor requirements, post-translational modification and regulation, subcellular compartmentation and feedback inhibition are other factors to consider. Eukaryotic expression systems offer the possibility of posttranslational modifications and are often used as heterologous hosts for plant secondary metabolites production. The most commonly used eukaryotic expression systems are yeast and insects. 2.2.5.1. The artemisinin case. An exceptional example of engineering secondary metabolite biosynthesis is the overproduction of artemisinin Fig. 2. Artemisinin is a sesquiterpene lactone isolated from the plant Artemisia annua, very effective against malaria disease, caused by Plasmodium falciparum infections. In addition to its antiparasitic effect, artemisinin shows anticancer and antiviral properties. However, because of its plant origin, problems with its production and reduced drug levels have been observed. This has been reflected in a high fluctuation cost (350–1200 US Dlls per kilogram of the active ingredient). Microbial artemisinin production has been reported in S. ambofaciens. The compound is encoded by a cryptic type I polyketide synthase (PKS) gene cluster. The production of semi-synthetic artemisinin has been successful by combining metabolic engineering and synthetic biology. Genes encoding for the enzymes in the artemisinin biosynthetic pathways have been recruited from S. cerevisiae, A. annua, S. aureus and E. coli. They were assembled into two operons and transformed into an E. coli host strain. After optimization, good production levels (25 g per liter) of artemisinic acid (an artemisinin precursor), were obtained [28,109,110]. Artemisinic acid can precipitate out of the growth medium and the precipitate is solubilized with isopropyl myristate. Finally, artemisinic acid can be chemically converted to artemisinin. Semi-synthetic artemisinin has been produced at an industrial scale and the final product is functionally equivalent to the plant-derived drug. Artemisinin has been approved by the WHO for the preparation of derivatives (such as artesunate) for incorporation into artemisinin-based combination therapies (ACTs). 2.2.5.2. The taxol production case. An additional example of multivariate modular synthetic biology it is related to the synthesis of taxadiene, precursor of taxol (paclitaxel) Fig. 3, a terpenoid drug used clinically for ovarian, breast, lung, pancreatic and other cancers’ treatment. In this example, a native upstream methylerythritol-phosphate (MEP) pathway forming isopentenyl pyrophosphate (key intermediate in terpenoid biosynthesis) as the first module and a heterologous downstream terpenoid-forming pathway was engineered in E. coli to produce high IPP. To convert IPP to taxadiene in the second module, two plant genes from Taxus species, GGP synthase (geranylgeranyl diphosphate synthase) and taxadiene synthase,

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were added (modified to function in E. coli) to perform the reactions. The number of copies of the genes to find the most efficient combination was also varied [29]. Both pathways were placed under the control of inducible promoters in order to control their relative gene expression. Maximum taxadiene production (up to 1 g per liter) with a minimal accumulation of indol, an inhibitory compound was achieved by balancing both modules. Finally, the conversion of taxadiene to taxadien-5a-ol, was engineered to further increase its production by 2400-fold over the level reported for yeast [29]. The microbial production of taxadien-5a-ol represents a good success. However, there are still several additional steps to go before achieving synthesis of the intermediate baccatin III, from which Taxol can be chemically synthesized. 2.2.5.3. Additional examples. Other examples of this synthetic biology strategy include production of plant-derived functional terpenoids in E. coli at high yield, using native plant P450 s [111]. A similar modular approach has been used for the production of the nutraceutical resveratrol in E. coli [112]. Furthermore, the plant alkaloid (S)-reticuline has been produced by engineered cell cultures of E. coli [113] and yeast. This compound is the key intermediate in benzylisoquinoline alkaloid biosynthesis. Furthermore, by using different combination cultures of transgenic E. coli and S. cerevisiae cells, this research group reported the synthesis of an aporphine alkaloid, magnoflorine, or a protoberberine alkaloid, scoulerine, from dopamine via reticuline. 2.3. Protein engineering Full chemical synthesis of molecules is an exhausting and complicated task since it requires many steps. The site-specificity, stereospecificity, and reactivity of functional groups are difficult to manage, thus resulting in a different compound than that expected and/or in poor yields. If the goal is to obtain molecules as complex as many antibiotics, the technological hurdles could become unsurmountable. On the other hand, enzymes are capable to perform a wide variety of chemical reactions useful for natural products production, making it easier to obtain a compound with fewer steps, in a short time-lapse, and sometimes in high yields, giving them advantage over synthetic chemistry in compounds production [106,114]. Due to all the advantages mentioned above, many natural products, included antibiotics, are produced industrially by enzymes. However, since novel antibiotics face the increasing development of antibiotic resistance, and enzymes’ features are limited in terms of region-, stereo- and substrate specificity, there are some limitations for natural products diversification [115]. In order to elude such enzyme limitations and extend natural product enrichment, random and rational design approaches have been developed for engineering biosynthetic and tailoring enzymes. 2.3.1. Directed evolution One random design approach for protein refinement is directed evolution, where a library of mutants is created and then screened to identify for improved versions of the protein [116,117]. In this approach, many methods for gene diversification may be applied, which include but are not limited to random mutagenesis by error-prone PCR or DNA damaging agents and recombination of related sequences [116]. There are also several methods for library screening, from colorimetric methods to the use of biosensors and reporters [117]. This strategy has been used for engineering polyketide synthase (PKS) and non-ribosomal peptide synthase (NRPS) enzymes [117,118]; both are multi-modular enzymes responsible for production of a wide variety of compounds with diverse biological activities, such as antibacterial, antiparasitic and antitumoral

[119]. As an example, the improvement of the production of the antibiotic andrimid [120] and the generation of three andrimid derivatives in Pantoea agglomerans [121] have been achieved by evolving the adenylation domain of Admk, a NRPS protein. Despite the utility of directed evolution, in some cases various rounds of evolution and screening are needed, consuming effort and time. Therefore, rational design approaches, where protein structure and dynamics are analyzed to perform site-directed mutagenesis are preferred over random approaches [122]. 2.3.2. Modifications of substrate specificity The biosynthesis of many important therapeutic polyketides is performed by type I PKSs [123], making this sort of enzymes attractive objects of study for chemical derivatization. This enzyme is constituted by three catalytic domains responsible for the polyketide backbone elongation. The first module to participate in polyketides biosynthesis is the acyltransferase (AT) domain, recognizing the extender units to be incorporated in the polyketide chain. This module has a certain level of substrate promiscuity and determines the type of molecule to be produced. While the erythromycin loading AT domain accepts propionyl-, acetyl- and butyryl-CoA, the loading AT domain of lipomycin PKS admits propionyl-, isobutyryl, 2-methylbutyryl- and isovaleryl-CoA. Despite this promiscuity, the kcat/KM for similar substrates are up to forty-fold lower than the preferential substrate [117]. Approaches such as domain swaps, performance of hybrid AT domains and site-directed mutagenesis (Fig. 4A) are used to modify AT substrate specificity and derivatize polyketides. However, poor yields of production of the novel derivatives are obtained [119,125,126]. Despite these results, engineering of the AT domain is still a promising approach. Another module that has been engineered is the ketoreductase (KR) domain, which is in charge of catalysing the reduction of the b-ketone produced [124,127]. Variants of the KR domain from the lipomycin synthase recombinant were obtained as a result of module exchange of the KR domains using as donors the KRs from amphotericin, concanamycin, spinosyn, borrelidin, avermectin and pikromycin pathways. In this work, products with either syn or anti stereochemistries were obtained in a substrate dependent manner, some of them in similar yields compared to the original product [127]. This result highlights the importance of substrate promiscuity for chemical derivatization of natural products. 2.4. Mutasynthesis Mutasynthesis has been developed as a tool for the derivatization of natural products, which takes advantage of natural or induced enzymatic promiscuity. In this approach, a mutant or promiscuous protein is built with synthetic precursors recognized by biosynthetic enzymes to produce analogues (Fig. 4B) and avoid chemical derivatization [128]. Sansanmycin, another type of antibiotic analogues, have been produced by semi-synthesis, but such analogues showed less antibiotic activity in contrast to parent antibiotics. Sansanmycin, an uridyl peptide antibiotic (UPA) produced by a NRPS, is used for P. aeruginosa and M. tuberculosis infection treatments. Shi and co-workers [129] obtained sansanmycin derivatives by deleting the ssaX gene from Streptomyces sp. SS, responsible for adding mTyr to the N-terminal of the tetrapeptide chain of this antibiotic, and then feeding this mutant with non-proteinogenic amino acids analogues. In the wild type strain, they increased the production of derivatives that bear Phe and Tyr at N-terminal by feeding it with those respective amino acids. All these analogues showed different grades of stability and antibiotic activity [129]. Furthermore, by taking advantage of substrate promiscuity of the sansanmycin

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Fig. 4. Representation of some strategies in protein engineering and mutasynthesis for derivatization of antibiotics. A) Structural modification of a biosynthetic protein that changes substrate specificity to achieve antibiotics derivatization. B) Steps followed in mutasynthesis starting in either protein mutagenesis or deletion of an important protein in biosynthetic pathway (e.g., a transcriptional regulator) for antibiotic analogues production.

biosynthesis pathway, more derivatives were obtained from Streptomyces sp. SS by adding phenylalanine derivatives [130]. Another example of mutasynthesis is the generation of cahuitamycin analogues from Streptomyces gandocaensis [131]. In this Streptomyces species, Park and co-workers [131] performed a streptomycin-induced point mutation of the rpsL gene to restore active molecules production and increase their yields. The ribosome engineering approach is applied in bacteria isolated from non-conventional environments either to improve yields of metabolites production or to ‘‘wake up” the production of a lost metabolite. From this strain, a DcahI mutant obtained by mutasynthesis with substituted benzoic acid substrates produced two new analogues with improved activity against biofilm [131]. These compounds could be implemented in the future to prevent biofilm formation in surgical devices. Bioinformatics tools are very useful for mutasynthesis. For instance, molecular dynamics simulations are used to predict locations of amino acids related to substrate binding specificity and to predict suitable non-native substrates. This strategy has been used successfully for obtaining premonensin derivatives in Streptomyces cinnamonensis A495 [132]. Similar predictions could be used to perform site-directed mutagenesis in various types of bacteria, leading to the production of a new generation of antimicrobials, much needed in medical applications nowadays.

3. Bacterial drug delivery New therapies and ways to deliver them are needed to gain control over the spread of microbiological agents and indeed, progress in drug delivery technologies over the past three decades has led to the development of increasingly refined systems. For instance, targeted release of drugs rather than systemic was shown to increase the bacterial sensitivity to the same medication [133].

Recent reports indicate that bacteria could constitute suitable drug delivery systems, for one, because they are capable of delivery solely at a target location, thus enhancing the therapeutic index of a drug while minimizing its adverse effects [133–135]. Narrowspectrum molecules such as fidaxomicin for Clostridium difficile [135–137] or amoxicillin for pneumococcal infections [138] are suitable for killing a specific pathogen with minimal disruption of the resident microbiota. Secondly, drug administration can be less invasive. For example, gastrointestinal tract infections could be treated by oral administration of bacteria that can cross the epithelium to the desired location and start delivering medication [139]. While the idea is not new – Rubinstein reviewed 40 years of use of drug pre-incubated bacteria to treat digestive illnesses in 1990 [140] – modern development of molecular biology tools and safety features enhanced bacteria as attractive vectors for drug delivery [141,142]. Antibody or peptide therapy that would not otherwise survive crossing the acidic stomach environment would be best suitable for such delivery. Third, a combination therapy can easily be achieved, if more agents are needed, as it is the case for P. aeruginosa [143], coagulase-negative staphylococci [144] or pneumococci [145]. Finally, costs of production would decrease as the biotechnological processes for bacterial production of active molecules are being optimized by the probiotic/fermentation and drug industry [21–23,146–148]. Bacterial drug delivery ranges its activity from detection to prevention and finally to treatment. In terms of treatment, heterologous expression of the active molecules, alone or in congruence with detection, is one method of choice. For instance, live Lactococcus lactis were used for both recognition and inhibition of the pathogen Enterococcus faecalis [149]. Enterococci are a major cause of hospital-acquired infections. The system uses a pheromonemediated intercellular detection system and the production of bacteriocins enterocin A, hiracin JM79, and enterocin P, specific for E. faecalis, for inhibition of the pathogen’s growth [149]. While not

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yet tested in vivo, this system has shown much promise due to the specificity that both the detection system as well as the antibacterial agents have for the intended target. Other detection systems can be triggered under various conditions such as oxidative damage [150], temperature [151,152] or pH shift [153,154], or the presence of specific sugars or receptors [141,155,156]. Intracellular pathogens present a challenging target for an antimicrobial treatment due to the additional barrier to drug access provided by the mammalian cell membrane. Existing efforts toward developing intracellular bacterial delivery agents have focused on using virulence-attenuated pathogenic bacteria [157–159] as well as modification of nonpathogenic strains with invasion genes [160,161]. In another approach, cargo can be loaded onto nanoparticles, which are carried on the bacteria surface [142,162] or inside an emptied envelope [163]. Demir Akin of Stanford University and his colleagues used antibodies and nanoparticles to attach molecules of DNA to weakened Listeria monocytogenes, which is a bacterium responsible for many cases of food poisoning. When incubated with cells, the cargo-carrying bacteria (termed ’microbots’) are internalized by the cells, and the genes released from the nanoparticles were expressed in the cells [162]. Mice injected with microbots also successfully expressed the genes, as seen by the luminescence in different organs. This new approach may be used to deliver different types of cargo into live animals and a variety of cells in culture without the need for genetic manipulations. A key feature of live bacteria is their capacity to stimulate the mucosal as well as the humoral and/or cellular systemic immunity [164]. This facilitates the use of vaccinations to prevent pathogen colonization of the mucosal tissues, the first barrier for many infectious agents. Furthermore, DNA vaccines and immune system stimulatory molecules, such as cytokines, can be delivered using bacteria, whose adjuvant properties and, sometimes, invasive capacities enhance the immune response [165,166]. A gradual change toward the use of commensal and probiotic bacteria that can naturally colonize the target niches is also expected [167]. Cellular targeting of antimicrobials using bacteria is an advancing field of research that provides novel strategies in the treatment of pathogenic infections in general and intracellular pathogens in particular, but future mechanistic and epidemiological studies are required to assess the effectiveness of these novel methodologies in disease control in vivo.

4. Biomedical applications While proof of principle studies involving bacteria as drug carriers have reached both success and sophistication, only a limited number were confirmed in clinical studies. The Belgian enterprise ActoGeniX applied the non-pathogenic food bacterium L. lactis as a delivery vehicle for DNA-based polypeptide - antigens, allergens, cytokines and antibodies [168] to clinical and preclinical development, with potential applications in gastrointestinal, immunological and metabolic diseases. Their first tested product, Ag013, showed a 35% reduction in oral mucositis in subjects with locally advanced head and neck cancer receiving induction chemotherapy [169]. In another Phase I clinical study, a small number of Crohn’s disease patients observed a decrease in disease activity when treated with genetically modified L. lactis in which the thymidylate synthase gene was replaced with a synthetic sequence encoding human interleukin-10. The research also proved that the use of genetically modified bacteria for mucosal delivery of proteins in humans is feasible and safe [170]. An additional aspect of drug delivery using bacteria that has reached the clinical phase relates to cancer therapy [135,171]. Bacteria anticancer therapy was documented around hundred years

ago, when german scientists W. Busch and F. Fehleisen separately observed that some cancer types regressed following accidental Streptococcus pyogenes infections. Since then, many bacteria such as Salmonella, Listeria, Escherichia and Clostridium have proved to have tumor targeting and in some cases even tumordestroying phenotypes [172–176]. Bacillus Calmette-Guerin (BCG), attenuated bovine tuberculosis bacteria (Mycobacterium bovis), appears to be one of the most successful cancer immunotherapeutics. BCG, in the form of repeated intravesical instillations, has been in use for over 30 years as a standard method to prevent cancer recurrence after endoscopic surgery of intermediate and high-risk non-muscle invasive bladder cancer [176]. It is also protective against inoperable bladder carcinoma in situ resulting in a 70–75% complete response rate [177]. Clinical studies using live attenuated L. monocytogenes immunotherapy bioengineered to secrete a human papilloma virus E7 fusion protein targeting HPV-E7 transformed cells established safety [178] as well as efficacy of the therapy in women with cervical cancer [179]. To sum up, bacterial therapeutics became novel delivery agents and in particular cancer targeting remedies, merging tumorrelated molecular targeting, natural bacterial invasion features and genetic engineering. Bacteria meet all the requirements for ideal disease-targeting agents and complement the existing tools in the anticancer toolbox.

5. Convergence with synthetic genomics Synthetic biology has emerged as a powerful tool to analyze the structure and relationships between fundamental units of highly complex biological networks. We are reaching an unprecedented understanding of cellular systems at the levels of RNA, proteins and metabolites. By using an engineer’s view in biology, synthetic biology aims to create plug-and-play concepts that make innovation much simpler than even few years ago [27]. The rapid progress of genome-sequencing ventures has revealed thousands of yet unexplored secondary metabolite biosynthetic pathways [24], many of which are expected to produce new biologically active compounds such as anti-tumor drugs, cholesterol-lowering agents and antibiotics. Mimicking the combinatorial chemistry approach, chemical diversity can be explored by genetic shuffling or individual modification of corresponding biosynthetic modules [24]. Many public resources are available, if not dedicated, to this purpose. Among them, the Registry of Standard Biological Parts supporting the iGEM competition (iGEM Registry), the American Type Culture Collection (ATCC), Addgene, the Coli Genetic Stock Center (CGSC) and the Synthetic Biology Engineering Resource Center (SynBERC) Registry can be mentioned. Besides, the Joint BioEnergy Institute Public Registry (JBEI-ICE Public), the European Saccharomyces cerevisiae Archive for Functional Analysis (EUROSCARF), the Agricultural Research Service NRRL collection (ARS/ NRRL) and the BIOFAB: International Open Facility Advancing Biotechnology (BIOFAB) are also included. Finally the DanaFarber/Harvard Cancer Center (DF/HCC) PlasmID Repository, the DNASU Plasmid Repository (DNASU), the Belgian Coordinated Collections of Micro-organisms (BCCM), and the Leibniz-Institut DSMZ – German Collection of Microorganisms and Cell Cultures (DSMZ) [180] should also be mentioned. As an epitome for synthetic biology, researchers were able to create a synthetic organism for the first time in 2010, by following a path laid down by recent successes in reducing the size of existing genomes [181–183]. This breakthrough was undertaken by Synthetic Genomics Inc., which synthesized a 600 kbp genome resembling that of Mycoplasma genitalium [22] and afterwards, a living synthetic Mycoplasma mycoides [184]. The first synthetic

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designer eukaryotic chromosome is currently being built [185]. Synthetic organisms may soon enable scientists to build biological production systems dedicated for medical applications – real biological factories. Using genome-minimized production hosts seems to be an efficient approach, given that they waste only a minimal amount of cellular resources on reactions other than their designed purpose [182,186]. In addition, the successful incorporation of a third base pair is a significant breakthrough toward the goal of greatly expanding the number of amino acids which can be encoded by DNA, from the existing 20 amino acids to a theoretically possible 172, thereby expanding the potential for living organisms to produce novel proteins [187,188]. The artificial DNA does not yet deliver information or produce proteins, but scientists speculate they could be designed to manufacture new proteins which could have industrial or pharmaceutical uses, while insuring safety as the new bioalphabet could not be used by natural systems. The basis for this hypothesis is only theoretical as of yet, and there are likely unforeseen limitations in the implementation of such artificial proteins. Unlike the natural aminoacyl-tRNA synthetases and ligases that were highly selected through evolution to activate a set of only 20 amino acids, the newly designed ones could have properties that make them unsuitable as building blocks or that prevent their recognition by ribosomes, such that an artificial cellular protein production machinery could be necessary for this strategy to be feasible. Synthetic biology sets the grounds for engineering biotechnologies for wide-spectrum applications in treatment and prevention of disease, and for designing novel materials and devices for environmental improvement, all in a setting of increased biosafety.

6. Examples of pharmaceutically active compounds discovered by synthetic biology Interestingly, genes encoding for secondary metabolite biosynthetic pathways are typically closely grouped in gene clusters that often also contain the specific regulators and transport systems. This allows for their easy detection in silico. The difficulty stands in expressing the drug of interest in a heterologous host, where not all precursors or signaling events are present, as well as in obtaining sufficient material for biochemical characterization and for therapeutic doses. Synthetic biology approaches envisage solving all these difficulties by creating suitably pre-engineered microbial hosts with an optimized machinery for overproduction of drugs from specific pathway classes [24]. As a step in this direction, sequentially acting iterative polyketide synthases subunits were relocated into in a yeast heterologous host to create a diverse library of benzendiol lactone polyketides derivatives [189]. One of these remarkable new compounds was a polyketide presenting a unique skeleton and heat shock response-inducing activity. Most pharmaceutically active compounds used in human clinical practice today are extracted from plants [190]. Synthetic biology aims to express plants secondary metabolites in microorganisms or algae. One of the best-known applications linked to synthetic biology is the production of the anti-malaria compound artemisinin through assembled complex metabolic pathways [191]. The new metabolic pathway was assembled partially from host enzymes from yeast and E. coli, and a heterologous downstream part composed of plant enzymes. The production was increased by upregulation of several rate-limiting pathway components [110]. In another approach, CRISPR/Cas9 technology was used to create antimicrobials with a designed spectrum of activity. RNAguided nucleases were delivered to microbial populations through transmissible plasmids carried by a bacteriophage. These RNA-

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guided nucleases target DNA genes that induce antibiotic resistance or virulence determinants in carbapenem-resistant Enterobacteriaceae (CRE) and enterohemorrhagic E. coli. Such an approach induces selective pressure at the DNA level and can be used as a tool to modulate the composition of complex microbial communities [192]. Lastly, screening for drugs against specific disease targets can be greatly improved using synthetic biology approaches. For instance, Klein et al. [193] reported the discovery of 74 novel compounds belonging to various classes, including type III polyketide and flavonoids, by using of the concept of coevolution with the target protein [193].

7. Main concerns – biosafety Despite the many advantages, bacterial therapy raises issues surrounding safety, containment, and the public opinion on using genetically modified organisms. Especially in the case of pathogenic bacteria, inherent risk of reversal to the native pathogenic form [194] and of horizontal gene transfer to increase resistance or virulence of related, commensal microorganisms [195,196] exists. Thus, implementation of practices and policies to ensure that these technologies remain safe is paramount. While in the examples of clinical trials using transgenic bacteria presented above, a record of safety toward patients was achieved, the use of pathogens or synthetic organisms in therapy still presents itself as a biosafety concern, as long term effects have not been evaluated. To prevent transgenic organisms from persisting in the human body, non-colonizing commensal organisms could be used. On the downside, therapeutic microorganisms will need to be administered regularly for the desired effect. One alternative is to include a repression module into the therapeutic cells to ‘‘switch off” or eradicate the microorganism either when the strain is no longer necessary after the treatment is completed or when it leaves its target location. An example of a repression mechanism has been designed for L. lactis by deleting the thymidylate synthase gene from its genome. When exogenous thymine or thymidine is no longer added, the auxotrophic strain dies [197]. In another study, a protein-based piston capable of puncturing membranes in a pH-dependent manner was used to puncture E. coli membranes to release the trapped content [198]. Limitations of these techniques are based on reversal, as bacteria could reacquire the necessary pathway by horizontal transfer or evolution. While the bacteria would retain the ability to cure, our control over production of the active molecule or location would be lost. Alternatively, the use of inactivated or dead bacterial cells was attempted. For instance, the inner and outer membranes of Gramnegative bacteria were fused to create an empty bacterial shell named bacterial ‘‘ghosts” (BG) [163], used to transport vaccines [199,200], DNA [201,202] and drugs [203,204] in mouse experiments. In another line of study, mouse neutrophils loaded exvivo with dead M. luteus containing chlorhexidine were used as vehicles for targeted therapy of liver abscesses in mice caused by Fusobacterium necrophorum. Neutrophils, the targeting component of this drug delivery system, were recruited via chemotaxis into infected tissue. The additional stability provided by the bacterial cell wall was crucial to the successful transport of the antibacterial drug, which otherwise would have a cytotoxic effect on the neutrophils and other tissues [134]. Only a limited number of bacteria were shown to be suitable for this approach. To decrease the danger that bacteria pose, other microorganisms can be used for drug delivery. In one case, the unicellular, biflagellated algae Chlamydomonas reinhardtii served as transporter for polystyrene beads which could be controlled to move by phototaxis and to release their load by photochemistry [205]. Control

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over a drug’s distribution and release in the body could significantly diminish side effects and increase efficiency of treatment. While the examples presented above have been tested solely in vitro or in animal experiments, superior proof of containment control over bacterial therapeutics in human patients need to be completed in order to convince government agencies and the public opinion to allow for their use in medicine. 8. Future outlook Undoubtedly, synthetic biology has a bright and promising future for the production of new antibiotics and other medicines on cellular, molecular and genetic level models. In this way, health problems derived from the antibiotic resistance, antibiotic toxicity and appearance of evolving pathogens will be able to be addressed and reduced. The design of microbes that will seek and destroy specific pathogens in the body before self-destructing or without affecting the mammalian cells is another feasible endeavor to be explored. Even personalized genome-specific medications for the treatment of particular infections, in cases of personal susceptibility to certain family of drugs is now feasible. Additional challenges for the future include the advancement in the number of genome sequences of bioactive producing biological collections and the development of high-throughput screening technologies to facilitate their use. One key area of synthetic biology research for the future concerns redesigning of living cells into ‘synthetic cells’, for the production of bioactive products, including antibiotics. For unknown natural products, research efforts in synthetic biology will likely create improved computational methods to gain a better characterization of antibiotic biosynthetic clusters from genomic and metagenomic DNA sequences. Due to its large scale applications and potential of new gene editing techniques, synthetic biology has gained industrial interest in the last few years. Pharmaceutical and healthcare companies have started investing in synthetic biology and in the near future we can expect traditional medicines to be replaced by genetically engineered products. The high potential of these new technologies will need to be complemented by advanced government biosafety and biosecurity regulations and policies, as well as their implementation by regulatory bodies and research institutions. Synthetic biology is a relatively young field, however it has already shown a great influence in the design and production of natural products like antibiotics from microorganisms and plants. The application of synthetic biology approaches such as those discussed in this review are at the beginning of what might be a new era in antibiotic drug discovery. However, despite the explosion in genome sequencing and the strong developments in computational predictive programs, there still remains an amount of genetic information that has yet to be visualized and, likely, new paradigms for sequence analysis are waiting to be discovered [206]. Acknowledgements This work was supported by the grant IN202216 from PAPIIT, DGAPA, UNAM, México. SGT is supported by a doctoral scholarship from Consejo Nacional de Ciencia y Tecnología CONACYT, Mexico. We thank Betsabe Linares-Ferrer for chemical structure drawings. We also thank Beatriz Ruiz-Villafán and Marco A. Ortíz-Jiménez for critical reading and manuscript preparation. References [1] N. Balaban, G.A. Dell’, Barriers on the road to new antibiotics, Scientist 19 (2005) 42–43.

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