Next Article in Journal
Toxicological Assessment of a Lignin Core Nanoparticle Doped with Silver as an Alternative to Conventional Silver Core Nanoparticles
Next Article in Special Issue
Streptomyces Differentiation in Liquid Cultures as a Trigger of Secondary Metabolism
Previous Article in Journal
Antibiotic Prescribing for Oro-Facial Infections in the Paediatric Outpatient: A Review
Previous Article in Special Issue
Specificity of Induction of Glycopeptide Antibiotic Resistance in the Producing Actinomycetes
Article Menu

Export Article

Antibiotics 2018, 7(2), 39; doi:10.3390/antibiotics7020039

Unraveling Nutritional Regulation of Tacrolimus Biosynthesis in Streptomyces tsukubaensis through omic Approaches
Área de Microbiología, Departamento de Biología Molecular, Universidad de León, León 24071, Spain
Instituto de Biotecnología de León, INBIOTEC, Avda. Real no. 1, León 24006, Spain
Departamento de Biología Funcional, Universidad de Oviedo, Oviedo 33006, Spain
Author to whom correspondence should be addressed.
Received: 27 February 2018 / Accepted: 26 April 2018 / Published: 1 May 2018


Streptomyces tsukubaensis stands out among actinomycetes by its ability to produce the immunosuppressant tacrolimus. Discovered about 30 years ago, this macrolide is widely used as immunosuppressant in current clinics. Other potential applications for the treatment of cancer and as neuroprotective agent have been proposed in the last years. In this review we introduce the discovery of S. tsukubaensis and tacrolimus, its biosynthetic pathway and gene cluster (fkb) regulation. We have focused this work on the omic studies performed in this species in order to understand tacrolimus production. Transcriptomics, proteomics and metabolomics have improved our knowledge about the fkb transcriptional regulation and have given important clues about nutritional regulation of tacrolimus production that can be applied to improve production yields. Finally, we address some points of S. tsukubaensis biology that deserve more attention.
Streptomyces tsukubaensis; tacrolimus; FK506; omics

1. Discovery of S. tsukubaensis and Tacrolimus Use in Current Clinics

Streptomyces tsukubaensis and its secondary metabolite tacrolimus were discovered in 1984, during a screening performed by the Fujisawa Pharmaceutical Co. (since 2005 merged to Yamanouchi Pharmaceutical Co. to form Astellas Pharma). S. tsukubaensis was isolated from a soil sample in the Tsukuba region (Japan) and tacrolimus was identified in its culture broths, becoming the first immunosuppressant discovered with macrolide structure [1,2]. The strain, patented as S. tsukubaensis No. 9993, is currently known as S. tsukubaensis NRRL 18488 and is the parental strain of most of the strains used for the industrial production of tacrolimus.
Macrolides such as erythromycin are composed of 14–16 C-membered macrolactone rings to which one or more deoxysugars are attached. Tacrolimus, a 23-carbon macrolide (822 Da), was initially named as compound FR900506 but, later on, it received other names such as FK506 or fujimycin. The name of tacrolimus was established as an acronym of “Tsukuba Macrolide Immunosuppressant” [3]. The first reference to tacrolimus was made at the 11th International Congress of the Transplantation Society, held in Helsinki in 1986, one year before the first publications by Kino and coworkers. The first clinical assays, focused on hepatic transplantation, were developed at the University of Pittsburgh in 1989. Two years later the first international congress on tacrolimus was celebrated in that city [3]. Tacrolimus acts as a calcineurin inhibitor, showing a mechanism of action very similar to that of cyclosporine (Figure 1) [4]. When tacrolimus interacts with its cytosolic receptors, mainly FKBP12- [5], the calmodulin-dependent serine/threonine phosphatase activity of calcineurin is inhibited, resulting in the arrest of T cell proliferation [6]. The mechanism of action is conserved in human T cells and yeast and thus, tacrolimus also has antifungal activity [7,8]. This activity is useful for the qualitative detection of tacrolimus by bioassay against susceptible strains such as Saccharomyces cerevisiae TB23 [9].
Since its approval by the FDA for the treatment of hepatic transplantation in 1994, tacrolimus has been also applied to medulla, kidney and heart transplantation [10,11,12]. This macrolide is also used for the treatment of other diseases such as atopic dermatitis [13,14] and is applied to the stents implanted in coronary arteries [15]. Several works have been published about its use in immune diseases such as rheumatoid arthritis and intestinal inflammatory diseases [16,17]. Tacrolimus has shown antiviral activity against orthopoxvirus, HIV and feline immunodeficiency virus (FIV) [18,19,20,21] and has properties such as a hair growth stimulator [22]. Neuroprotective and neuroregenerative activities have been also reported [23,24,25] as well as its potential application in the treatment of cancer [26]. More recently, the efficacy of tacrolimus ointment in the treatment of allergic ocular diseases has been reported [27].
The efficacy of tacrolimus in the treatment of organ transplantation is the basis of its industrial importance. Tacrolimus is between 10 and 100 times more potent than cyclosporine and has been shown to be more effective in several clinical trials [28,29]. Tacrolimus generates important benefits for the pharmaceutical market; for example, the sales of tacrolimus under the commercial names ‘Prograf’ and “Protopic” yielded a total of $1727 million to Astellas Pharma in 2016 (data from

2. Biosynthetic Pathway and Gene Cluster

The first studies on the tacrolimus biosynthetic pathway were performed by researchers from the pharmaceutical company Merck (USA) during the 90’s [30,31,32,33]. Tacrolimus is a polyketide synthesized by a hybrid polyketide I synthase-non-ribosomal peptide synthase (PKSI-NRPS) system encoded by the fkb cluster, which encompasses a minimum of 19 genes (Figure 2). Until now, more than 15 tacrolimus-producing species have been reported [34], the last being S. tsukubaensis F601 [35]. There are two types of fkb clusters in the tacrolimus producing strains [36]: (i) A short version comprising the genes fkbQ, fkbN, fkbM, fkbD, fkbA, fkbP, fkbO, fkbB, fkbC, fkbL, fkbK, fkbJ, fkbI, fkbH, fkbG, allD, allR, allK and allA (found in Streptomyces tacrolimicus and Streptomyces kanamyceticus KCTC 9225) and; (ii) An extended version found in S. tsukubaensis NRRL 18488, S. tsukubaensis L19 and Streptomyces sp. KCTC 11604BP that includes 5 additional genes in the 5’ region of the fkbG gene (allMNPOS/tcs12345) and one or two extra genes (depending on the species) in the 3’ region (tcs6-fkbR/tcs67). Deletion of allMNPOS genes in Streptomyces sp. KCTC 11604BP does not significantly affect tacrolimus production; thus, it is dubious that they are involved in tacrolimus biosynthesis [37]. Actually, their transcription levels are low, which supports this assumption [38,39].
The first step in tacrolimus biosynthesis is the formation of (4R, 5R)-4,5-dihydroxycyclohex-1-enecarboxylic acid (abbreviated DHCHC) from chorismate through the so-called chorismatase activity of FkbO (Figure 3) [40]. DHCHC acts as starter unit for the subsequent formation of the carbon skeleton and corresponds to the cyclohexane ring in the final structure of tacrolimus. This ring is the most tolerant target for structural modifications that do not eliminate the immunosuppressant activity [41]. The polyketide synthases FkbA, FkbB and FkbC catalyze 10 elongation steps from DHCHC using as extender units malonyl-CoA (2 molecules), methylmalonyl-CoA (5 molecules), methoxymalonyl-ACP (2 molecules) and allylmalonyl-CoA (one molecule). The latter two extender units are unusual in the formation of polyketides and result in the methoxyl group of C13 and C15 and the allyl radical of C21, respectively [36,37,42,43]. The biosynthesis of methoxymalonyl-ACP from 1,3-biphosphoglycerate depends on the enzymes encoded by the fkbGHIJK subcluster [42,43,44,45]. The incorporation of allylmalonyl-CoA is the sole difference between tacrolimus and ascomycin (FK520), in which biosynthesis ethylmalonyl-CoA is used instead. The all subcluster is involved in the formation of allylmalonyl-CoA and encodes a polyketide synthase of unusual structure [46]. Nevertheless, ketoreductase and dehydratase activities encoded outside the fkb cluster might be involved in some steps of allylmalonyl-CoA formation and these activities could be shared with fatty acid synthases [37]. The tacrolimus cluster does not encode an ACP-CoA transacylase necessary for the final reaction leading to allylmalonyl-CoA [37], but the acyltransferase domain of the fourth module in FkbB (AT4FkbB) is able to transfer an allylmalonyl unit to the ACP domain [47].
For the cyclation of the macrolide, FkbL generates l-pipecolate from l-lysine [48], which is then incorporated into the carbon skeleton by NRPS FkbP [30,49,50]. Finally, two modification steps are necessary to achieve the final molecule with biological activity: a methylation of the hydroxyl group located at C31 and an oxidation at C9. Both groups are important for the binding of tacrolimus to FKBP12 [51,52]. The methylation is catalyzed by the S-adenosylmethione dependent O-methyltransferase FkbM and the oxidation by the cytochrome P450-oxidoreductase FkbD [31,53]. Both activities are encoded in the same operon and can occur in any order [31,45,54]. Interestingly, the reaction catalyzed by FkbD (a double step oxidation involving 4 electron transfers and the formation of the alcoholic intermediate 9-hydroxy-FK506) is known for terpenoid biosynthesis but was first described for polyketide biosynthesis [45].

3. Transcriptional Regulators and Recent Insights through Transcriptomic and RNAseq Studies

The first sequence analyses of the fkb cluster revealed three potential regulators: fkbN, fkbR and allN (belonging to the LAL, LysR and AsnC families, respectively). FkbN is a large regulatory protein of the LAL family (Large ATP binding regulators of the LuxR family). The LAL regulators are large proteins (872–1159 amino acids) that contain a LuxR-type HTH DNA binding region near the C-terminal end of the protein and an ATP binding motif in the N-terminal end [55,56]. Similar FkbN-like genes have been found in several other macrolide gene clusters including RapH of the rapamycin producer Streptomyces hygroscopicus [57], PikD of the pikromycin producer Streptomyces venezuelae [58], GdmR1 and GdmR2 of the geldanamycin producer Streptomyces hygroscopicus [59], FkbN of the ascomycin producer S. hygroscopicus var. ascomyceticus [44], FscRI in the candicidin producer Streptomyces griseus [60,61], PimM of the pimaricin producer Streptomyces natalensis [62,63], NysR from the nystatin producer Streptomyces noursei [64], AmphRIV in the amphotericin B producer Streptomyces nodosus [65] and PteF in the filipin producer Streptomyces avermitilis [66,67].
The second regulatory protein FkbR belongs to the family of the LysR-type transcriptional regulators, also named LTTR, which are very common autoregulatory genes in bacteria [68]. In fact, they are widely distributed in Streptomyces: genome sequencing revealed about 40 LTTRs in S. coelicolor [69]. FkbR, as occurs with other members of the LTTR family, is a relatively small protein of less than 325 amino acids that is characterized by an HTH DNA binding motif in the C-terminal and by a ligand (co-inducer) binding sequence in the N-terminal region [70,71]. Other LTTRs acting as pathway-specific regulators include SCLAV_p1262 of S. clavuligerus (77% identity), ThnI from Streptomyces cattleya (39% identity), AbaB from Streptomyces antibioticus or ClaR from S. avermitilis [72,73].
The third putative regulatory gene of the tacrolimus gene cluster is allN. This gene is located in the 5’ end of the extended version of the tacrolimus gene cluster and encodes a protein that has similarity with regulatory proteins involved in nitrogen metabolism, particularly with regulators of AsnC family [74]. This gene is included in a region that is involved in the formation of the precursor allylmalonyl-CoA (all genes) [37,46].
Functional analysis of the role of FkbN, FkbR and AllN in S. tsukubaensis was performed by gene disruption and complementation studies. Whilst the inactivation of fkbN resulted in the lack of tacrolimus production, disruption of fkbR reduced tacrolimus yields to 20% of that of the parental strain and the inactivation of allN did not affect tacrolimus production [36]. Thus, it was concluded that both fkbN and fkbR encode positive regulators whilst allN has no influence on tacrolimus production [36]. In addition, AllN (also named Tcs2) seems to be not involved in tacrolimus production in other strains such as S. tsukubaensis L19 [75]. Overexpression of fkbN or fkbR in the wild type strain using the ermE* promoter produced an increase of the final yield of tacrolimus of 55% and 30%, respectively, using a culture medium optimized for tacrolimus production. These results agree with the observations published by Mo and coworkers on the effect of FkbN in Streptomyces sp. KCTC 11604BP [76].
There are important differences between FkbN and FkbR that we summarize here as follows: (1) fkbN is present in both the extended and the short version of the fkb cluster but fkbR is only present in the extended cluster version [37]; (2) FkbN always shows a positive effect on tacrolimus production whilst FkbR can have positive or negative effects [36,76,77]; (3) A complete lack of tacrolimus production is only produced by inactivation of fkbN (but not with that of fkbR) [36,38]; (4) transcription of fkbR is constant and low throughout the culture whilst that of fkbN increases before the onset of tacrolimus production and is maintained during the production phase (Figure 4) [36,38,75].

3.1. Characterization of fkb Cluster Transcriptional Subunits

Early studies using the rppA chalcone synthase reporter systems and qRT-PCR showed that the inactivation of fkbR or fkbN prevents transcription of certain genes in the S. tsukubaensis fkb cluster such as fkbG or fkbB, implying that some fkb genes are regulated by FkbN while others are not [36]. However, more recent transcriptomic studies with the same fkbN inactivated mutant have confirmed that FkbN controls the expression of most of the genes of the fkb cluster [38]. Two types of gene expression were observed in response to fkbN inactivation: (a) Genes clearly induced by FkbN coinciding with the onset of tacrolimus biosynthesis (in the so called “induction phase”) and whose expression is significantly reduced in the fkbN mutant (i.e., fkbABC, fkbGHIJK, fkbL, allAKRD, fkbO, fkbP, fkbD and fkbM) and (b) Genes poorly expressed through the culture time and not affected by fkbN inactivation (i.e., allMNPOS and fkbR) (Figure 2). Thus, the complete transcriptional dependency of the fkb genes on FkbN, with the exception of allMNPOS and fkbR (only present in the extended versions of the fkb cluster), which are FkbN-independent, was demonstrated.
The use of tiling probes covering the fkb cluster allowed the identification of 6 transcriptional units: fkbR, tcs6-fkbQ-fkbN, fkbOPADM, fkbBCLKJIH, fkbG and allAKRD. It was concluded that fkbR is transcribed as a leaderless mRNA and that fkbN forms an operon along with tcs6 and fkbQ whose transcription depends on two different promoters, one FkbN-dependent and the other FkbN-independent [38]. These results are supported by the EMSAs performed with the FkbN-DNA binding domain in S. tsukubaensis L19 by Zhang and coworkers [75], who reported FkbN binding to the promoter regions of the same six transcriptional units and identified two new ones corresponding to allNPOS and allM. More recently, differential RNA-seq (dRNA-seq) transcriptional profiling has been performed in S. tsukubaensis by Bauer and coworkers [39], who identified 9 transcriptional units that are in good agreement with previous studies (Figure 2). The main finding is that allOS and allNP are transcribed as independent mRNAs [39].
fkbR seems to be transcribed as a leaderless mRNA and is not directly regulated by FkbN [38]. In fact, it is likely that FkbR regulates its own expression, although detailed information is not available. Recently, the binding of FkbR to the promoter regions of tcs6-fkbQ-fkbN and fkbR in S. tsukubaensis L19 has been reported [75].

3.2. Genes Located Outside of the Tacrolimus Gene Cluster Regulated by FkbN

It has been reported that cluster-situated regulators (CSR) can regulate genes located outside their own cluster [78,79] and, therefore, the utilization of transcriptomic studies is a good tool to identify them. The transcriptomic analysis performed with the fkbN mutant by Ordóñez-Robles and coworkers [38] revealed potential genes located outside the fkb cluster that might be targets of FkbN such as ppt1, encoding a 4′-phosphopantetheinyl transferase that is known to be involved in CDA formation in S. coelicolor [80]. This gene showed an FkbN-dependent profile and a putative FkbN binding sequence [38]. In agreement with these results it was reported that the orthologue of ppt1 is involved in tacrolimus production in S. tsukubaensis L19 [81] and later, it was observed that ppt1 and fkbN share a common transcriptional response to glucose, glycerol and N-acetylglucosamine additions (see below). The study identified acyl-CoA dehydrogenase and methoxymalonate biosynthesis coding genes that were negatively affected by the fkbN inactivation and thus, might be involved in tacrolimus biosynthesis. On the contrary, some PKS coding genes located in a chromosomal region that has been predicted to encode a cluster for the production of a bafilomycin-like compound [82] were upregulated after fkbN inactivation, which might reflect competition for precursors between these two clusters for the biosynthesis of secondary metabolites.
Using the information-theory of Schneider [83], a putative FkbN binding sequence would be composed by two 7 nt inverted repeats [38]. This sequence would be similar to that identified for binding of PimM in the genome of S. natalensis [63].
In-depth knowledge of the fkb cluster regulation is necessary to achieve higher tacrolimus production yields. In this sense, the identification of transcriptional start sites (TSS) is useful for the introduction of artificial promoters without affecting the structure of mRNAs. Bauer and coworkers [39] reported that 22% of the transcripts identified by dRNAseq are predicted to present long leader mRNAs (greater than 150 nt), which points out the importance of post-transcriptional regulation of the fkb cluster through the formation of RNA secondary structures [84]. In fact, the allAKRD operon was reported to be transcribed with a rather long untranslated 5’ region (5’-UTR; 247 bp) that is predicted to form a secondary structure.

4. Classical Strategies to Increase Tacrolimus Production

Despite the efficacy of tacrolimus in the treatment of organ transplantation, its use in clinical therapy is expensive. This is mainly due to the low production yields of the producer strains used but also to the formation of byproducts such as ascomycin (FK520) or FK525, which are structurally similar to tacrolimus but differ in the nature of some radical groups [85]. The presence of byproducts in the culture broths hampers extraction and purification of tacrolimus; thus, different approaches involving the use of organic solvents and/or chromatography have been developed to increase tacrolimus purity [86]. As an example, ascomycin production can represent 20% of tacrolimus production in S. tsukubaensis NRRL 18488 and 8% in Streptomyces clavuligerus KCTC 10561BP [86,87]. The chemical synthesis of tacrolimus was described in the 90’s but it is not applied in practice due to its low efficacy and high costs [88,89].
In the last decades, the research on tacrolimus production enhancement has been mainly focused on culture media optimization and genetic engineering of the strains. For a recent review on the improvement of tacrolimus biosynthesis through synthetic biology approaches see [90,91]. The optimization of culture media encompasses formulation of defined compositions, precursor supply and the addition of stressing agents. Defined media are highly necessary to perform nutritional studies in which the stimulating or inhibitory effect of a particular nutrient on growth and antibiotic production is tested. The first defined media for the growth of Streptomyces sp. MA6858 (ATCC 55098) was formulated by Yoon and Choi [92]; later, Martínez-Castro and coworkers [93] developed two additional media, MGm-2.5 and ISPz. MGm-2.5, which contain starch as the main carbon source and glutamate as carbon and nitrogen sources whilst ISPz, an optimization of ISP4 medium, contains glucose and corn dextrin as the main carbon source. MGm-2.5 has been further used to perform transcriptomic analyses on the carbon and phosphate control of S. tsukubaensis [94,95]. This medium supports dispersed growth and high tacrolimus production yields. Moreover, this medium permits an estimate of the onset of tacrolimus production since this process has been shown to take place when phosphate is depleted from this medium [93].
Considering that the availability of precursors is a limiting factor in the biosynthesis of secondary metabolites, precursor supply is a straightforward strategy to increase antibiotic yields [96]. A summary of the compounds that have been applied to increase tacrolimus production is shown in Table 1. At this point of the review and as a conclusion of all the mentioned work, it is interesting to note that (1) The effect of a precursor depends on its concentration; (2) The combination of positive additions does not always have an additive positive effect and (3) The positive effect can be exerted through growth promotion, production stimulation or both.
Nevertheless, the addition of precursors in industrial fermentations can be a non-efficient strategy from an economical point of view (i.e., shikimate, chorismate and pipecolate are expensive; [107]); thus, an alternative strategy is to increase the copy number of tacrolimus biosynthetic genes by genetic engineering. In this manner, the overexpression of genes coding for the synthesis of methylmalonyl-CoA, methoxymalonyl-ACP and allylmalonyl-CoA has been shown to have a positive impact on the tacrolimus production yields [104,108].
Finally, the addition of stressing agents, such as dimethylsulfoxide (DMSO) or sodium thiosulfate, has been shown to stimulate polyketide production in different bacteria [109,110] as well as tacrolimus production in S. tsukubaensis NRRL 18488 [90].

5. Omic Approaches in S. Tsukubaensis and Their Application in Tacrolimus Production

5.1. Metabolomic and Proteomic Studies

The inactivation or overexpression of a particular gene involved in a certain biosynthetic pathway can affect other metabolic pathways and also the growth of the microorganism. For this reason, global studies covering the whole transcriptome, proteome or metabolome are usually preferred. In S. tsukubaensis, several metabolomic studies have been performed in the last decade. Huang and coworkers [100,111] developed a genome-scale metabolic model (GSMM) for S. tsukubaensis D852 including 865 chemical reactions and 621 metabolites to predict targets for genetic manipulation. These models reconstruct the organism metabolism from the genome annotation, taking into account genes encoding enzymes and transporters. By this means it was predicted that some of those modifications in the primary metabolism pathways leading to the accumulation of erythrose-4-phosphate, α-ketoglutarate, fumarate, succinate, pyruvate, phosphoenolpyruvate, NADPH, chorismate and malonyl-CoA have a positive effect on tacrolimus production. This implies that both the pentose phosphate pathway and the TCA cycle are positively correlated with tacrolimus production. Regarding the biosynthetic cluster, the overexpression of genes involved in the formation of the starter unit DHCHC, pipecolate and in different modification reactions (fkbO, fkbL, fkbP, fkbM and fkbD; see Table 2) also has a positive effect. Interestingly, as mentioned before, the combination of positive mutations does not always have an additive effect, i.e., the combined overexpression of fkbL and fkbP reduced biomass formation due to the use of lysine for tacrolimus production. More recently, a metabolomic approach has been reported in which lysine, shikimate, malonate, and citrate (the last three ones in the form of sodium salts) were supplied to the culture media of S. tsukubaensis D852 [102]. In this study, the addition of compounds targeting different precursor pathways facilitates the comprehension of the metabolic switches that are positive for tacrolimus production, and the application of weighted correlation network analysis (WGCNA; [112]) allowed the identification of hub modules and key metabolites depending on the culture stage. For example, 48 h after the feeding, pyruvate, phosphoenolpyruvate and methylmalonate show a high degree of connectivity whilst 72 h after the feeding, shikimate and aspartate control tacrolimus production. Supporting previous results, it was reported that the pentose phosphate, shikimate and aspartate pathways are crucial for the biosynthesis of the immunosuppressant. Overexpression of aroC and dapA (involved in shikimate pathway and lysine biosynthesis, respectively) increased production of the macrolide by 40% and 23%, respectively. See a summary of the distinct gene modifications that produce a positive impact on tacrolimus production in Table 2.
The GSMM developed by Huang and coworkers [111] is a pseudo-steady metabolic model, that is to say, it assumes that there is no depletion or accumulation of intracellular metabolites. Dynamic flux balance analysis (DFBA) takes into consideration the fluctuations in metabolite concentrations and thus allows the study of the interaction between metabolism and environmental changes [114]. Wang C. and coworkers [113] developed a genome-scale DFBA (GS-DFBA) model for S. tsukubaensis NRRL 18488 which uncovered new targets for genetic manipulation (see Table 2) that resulted in increased tacrolimus production; i.e., inactivation of gcdh (glutaryl-CoA dehydrogenase) and overexpression of tktB (transketolase), msdh (methylmalonate semialdehyde dehydrogenase) and ask (aspartate kinase).
The approached used by Xia and coworkers [99] consisted of the growth of S. tsukubaensis TJ-04 in two media of similar composition but resulting in different tacrolimus productivity. They analyzed the concentration of a wide range of metabolites and compared them between the two media to identify key metabolites that correlate positively with tacrolimus production. In good agreement with the results of Huang and coworkers [100,111], intermediates of the TCA cycle such as oxaloacetate, citrate, α-ketoglutarate and, especially, succinyl-CoA and acetyl-CoA, showed a positive correlation with tacrolimus production. In addition, the intracellular levels of pentose phosphate pathway intermediates were lower in the high production media, supporting the assumption that this pathway is positively correlated with tacrolimus production. Regarding metabolites from the tacrolimus biosynthetic pathway, methylmalonyl-CoA showed the best correlation.
More recently, Wang and coworkers [103] performed a comparative proteomic and metabolomic approach in S. tsukubaensis NRRL 18488 grown under soybean oil feeding. The positive effect of this carbon source on growth and on tacrolimus production has been already reported in other producing strains [97,98,99,100,101] and, as expected, increased tacrolimus production by 89%. This work has unraveled the effect of soybean oil on tacrolimus production, which mainly affects primary metabolism proteins (42%), redox proteins (12.5%), transcriptional regulators, signal transduction components and translation proteins (11%). The key metabolites associated with tacrolimus production correlate well with those identified previously by Xia and coworkers [99] and include malic acid, gluconic acid, citric acid, α-ketoglutarate, hexadecanoic acid, threonine, fumaric acid, succinic acid, proline, valine, oleic acid, trehalose, pyruvate, ornithine, 10-undecenoic acid, shikimic acid, mannose, and lactate. Several enzymes involved in the lower glycolytic pathway and the TCA cycle (i.e., triosephosphate isomerase, phosphoglycerate mutase, pyruvate kinase or citrate synthase) were overproduced under the soybean oil condition, and the rate-limiting enzyme of the pentose phosphate pathway glucose-6-phosphate dehydrogenase showed higher amounts in the fed condition, which supports the above-mentioned positive correlation of the pentose phosphate and TCA cycle pathways with the tacrolimus production process. Finally, enzymes related to fatty acid, shikimic acid, valine and isoleucine metabolisms (which can be transformed in the extender units methylmalonyl-CoA and propionyl-CoA) were also upregulated (valine and isoleucine can be transformed in the extender units methylmalonyl-CoA and propionyl-CoA). Interestingly, higher amounts of the transcriptional regulators Crp and AfsQ1 were detected under the soybean oil feeding condition, pointing to their possible involvement in tacrolimus production regulation.

5.2. Transcriptomic Studies on Phosphate Regulation of the fkb Cluster

Understanding how a biosynthetic cluster is regulated is important to develop strategies to improve secondary metabolite production. Our group has studied the phosphate regulation of antibiotic production in different Streptomyces species in the last two decades, including S. tsukubaensis [94,115,116]. It is well known that high phosphate concentrations in the culture media downregulate antibiotic production [117]. This regulatory phenomenon is exerted, at least in part, through the two-component system PhoR-PhoP, which is formed by a sensor kinase and a response regulator, respectively [115,118]. When phosphate is depleted from the culture media, PhoR phosphorylates PhoP. The binding of phosphorylated PhoP (PhoP-P) to its target sequences (known as PHO boxes) can have a positive or negative transcriptional effect depending on the location of the PhoP-P binding site [118,119,120]. In S. tsukubaensis, the negative regulation of tacrolimus biosynthesis by phosphate was reported in 2013 [93] and later the PhoR-PhoP system was studied in detail [94]. In the work, transcriptomics were applied to identify genes that are transcriptionally activated after phosphate depletion. The study allowed the identification of not only common Pho members but also of potential new species-specific members, like, for example, three overlapping genes encoding a two component system and a small hydrophilic protein. In addition, a bioinformatic search for PHO boxes was developed [121]. Putative PHO boxes were identified in most of the genes responding to phosphate starvation, supporting the transcriptional results. A putative PHO box was identified in the promoter region of fkbN and also in primary metabolism genes that might be involved in tacrolimus precursor supply such as STSU_30046, encoding an acetoacetate-CoA ligase [94].

5.2.1. Transcriptomics of Carbon Catabolite Regulation of Tacrolimus Biosynthesis

A second regulatory mechanism governing secondary metabolite production is carbon repression. Similar to phosphate, the presence of ready-to-use carbon sources in the media reduces or blocks antibiotic production and this can happen at the transcriptional or at the posttranslational level [122,123]. The mechanisms involved in this nutritional regulation are not completely understood in streptomycetes and, as it can be deduced, its unveiling is very interesting in order to use easily assimilated carbon sources that allow faster growth in the culture broths without hampering tacrolimus biosynthesis. Regarding this subject, our group observed that glucose and glycerol, when added as carbon sources at a concentration of 0.22 M at the first growth phase (and before phosphate depletion), arrest tacrolimus production in S. tsukubaensis; the glucose effect being stronger than that of glycerol [95]. Both glucose and glycerol additions resulted in a lack of transcriptional activation of the fkb cluster; thus, it was concluded that transcriptional repression plays a role in this regulatory mechanism. In addition, the effect of these carbon sources can be exerted at the intermediary metabolism level: glucose addition increased transcription of genes involved in glycolysis, pyruvate and oxaloacetate formation but downregulated genes involved in the TCA cycle. These results are coherent with the previous assumption that the TCA cycle is positively correlated with tacrolimus production whilst glycolytic metabolites show a negative correlation [99].
In the MGm-2.5 medium used in the work, transcription of fkbN increases in a two-step fashion before tacrolimus is detected in the broths [38]: a slight increase in mRNA levels occurs between 80 h and 89 h and then it is followed by a higher increase from 92 h to 100 h (Figure 4). The first step coincides with phosphate depletion, supporting the proposal that fkbN is under phosphate control [95] (Figure 4). Taking into account that fkbN transcription is not strongly self-regulated [38], it seems that a key transcriptional regulator, co-activator molecule or sigma factor might be absent in the presence of glucose or glycerol. Therefore, the identification of this additional factor would be useful to trigger tacrolimus production under carbon repressing conditions. Actually, key sigma factors (i.e., hrdA or bldN) and transcriptional regulators (i.e., eshA, atrA, afsR) were downregulated under glucose or glycerol addition conditions [95]. HrdA might control secondary metabolism genes [124], and EshA and AtrA are both involved in antibiotic production in S. coelicolor and S. griseus [125,126,127,128]; thus, it seems interesting to analyze the effect of their inactivation and overexpression on tacrolimus production. Finally, AfsR is a very interesting candidate for these studies since it is overexpressed in an S. tsukubaensis strain that overproduces tacrolimus [101].

5.2.2. Transcriptomics of N-acetylglucosamine Addition in Tacrolimus Biosynthesis

A third example of the nutritional regulation of secondary metabolite production is that exerted by N-acetylglucosamine, the monomer of chitin. This compound shows a dual regulatory role, accelerating differentiation and antibiotic production under poor nutritional conditions and arresting them under rich nutritional conditions, which have been traditionally named as “famine” and “feast” conditions, respectively [129,130]. We observed a negative effect of N-acetylglucosamine addition on tacrolimus production when S. tsukubaensis was grown in MGm-2.5 medium, which might be due, at least in part, to the transcriptional repression of fkbN, since we observed a significant decrease in its transcription soon after N-acetylglucosamine addition (Ordóñez-Robles et al., unpublished data). The transcriptional response to N-acetylglucosamine addition is very similar to that exerted by glucose, which is not surprising since both carbon sources share a common catabolic pathway from fructose-6-phosphate.
Overall, the application of transcriptomics to nutritional studies in S. tsukubaensis unveils potential candidates for the rational engineering of industrial strains. It has also improved our knowledge about other aspects of its physiology such as the possible members of the PHO regulon in this species or the mechanisms operating in the presence of repressing carbon sources. These findings are worthy to detect potential targets for the bypass of nutritional repression of secondary metabolism in Streptomyces.

6. Conclusions and Future Prospective

It has been more than 30 years since S. tsukubaensis and its secondary metabolite tacrolimus were discovered. Despite the importance of this immunosuppressant macrolide in current clinics, there are still many aspects to be elucidated about the transcriptional and nutritional regulation of tacrolimus biosynthesis, and further studies are necessary to improve the yield and reduce the costs of its industrial production. In this sense, the omic approaches constitute an important basis to understand the producer microorganism physiology from a genome- [131], proteome- and metabolome-wide point of view. Initial omic studies performed in S. tsukubaensis have given important clues such as the positive correlation of the pentose phosphate pathway and TCA cycle with tacrolimus production or the identification of targets for genetic manipulation. These types of studies can be applied not only to the overproduction of tacrolimus but also to the awakening of cryptic clusters [132]. In fact, similar to most streptomycetes, S. tsukubaensis’ genome contains several clusters for the production of secondary metabolites which might encode useful compounds. One of the potential products encoded is predicted to be similar to bafilomycin [133] and two other clusters show homology to those for biosynthesis of nigericin and enduracidin [134,135]. Nevertheless, we must keep in mind the interpretation of the omic results in the framework of the strain and culture media used since there are important physiological differences depending on the strain and the culture conditions. Therefore, the comparison of different models can broaden our perspective of tacrolimus production and S. tsukubaensis’ physiology.
There are still some interesting points to address in the study of the fkb cluster such as the role of the allMNPOS subcluster in the strains that contain it. Although not strictly required for tacrolimus production, the all subcluster might be involved in the generation of macrolide variants with useful properties. Thus, the overexpression of these genes under promoters regulated by FkbN seems an interesting study. In addition, the ppt1 and scoT genes, which are affected by the inactivation of fkbN, might be potential targets for tacrolimus biosynthesis improvement. Considering the transcriptional regulation of eshA and atrA under tacrolimus producing and repressing conditions, both genes seem good candidates for genetic engineering of the strains.
The transcriptional regulation of fkbN is also interesting given that it is the main transcriptional activator of the fkb cluster. The identification of transcriptional regulators that bind to its promoter region is a good approach to identify new targets for genetic engineering of the strains that overexpress fkbN and therefore, to increase tacrolimus production. Finally, the post-transcriptional regulation of the fkb cluster deserves further attention. As reported by Bauer and coworkers [39], a high percentage of genes are transcribed with long leader sequences in S. tsukubaensis (i.e., allAKRD). Long 5’-UTRs might be involved in the formation of secondary structures that regulate transcription of the cistrons and might be potential targets for manipulation.

Author Contributions

Juan F. Martín wrote the sections on biosynthesis of tacrolimus and regulatory genes, corrected the text and supervised the final version. María Ordóñez-Robles wrote the other sections and Fernando Santos-Beneit corrected and improved the text.


We acknowledge Paloma Liras for helpful scientific discussion.

Conflicts of interest

The authors declare no conflict of interest.


  1. Kino, T.; Hatanaka, H.; Hashimoto, M.; Nishiyama, M.; Goto, T.; Okuhara, M.; Kohsaka, M.; Aoki, H.; Imanaka, H. FK-506, a novel immunosuppressant isolated from a Streptomyces. I. Fermentation, isolation, and physico-chemical and biological characteristics. J. Antibiot. 1987, 40, 1249–1255. [Google Scholar] [CrossRef] [PubMed]
  2. Kino, T.; Hatanaka, H.; Miyata, S.; Inamura, N.; Nishiyama, M.; Yajima, T.; Goto, T.; Okuhara, M.; Kohsaka, M.; Aoki, H. FK-506, a novel immunosuppressant isolated from a Streptomyces. II. Immunosuppressive effect of FK-506 in vitro. J. Antibiot. 1987, 40, 1256–1265. [Google Scholar] [CrossRef] [PubMed]
  3. Wallemacq, P.E.; Reding, R. FK506 (tacrolimus), a novel immunosuppressant in organ transplantation: Clinical, biomedical, and analytical aspects. Clin. Chem. 1993, 39, 2219–2228. [Google Scholar] [PubMed]
  4. Liu, J.; Farmer, J., Jr.; Lane, W.S.; Friedman, J.; Weissman, I.; Schreiber, S.L. Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell 1991, 66, 807–815. [Google Scholar] [CrossRef]
  5. Harding, M.W.; Galat, A.; Uehling, D.E.; Schreiber, S.L. A receptor for the immunosuppressant FK506 is a cis-trans peptidyl-prolyl isomerase. Nature 1989, 341, 758–760. [Google Scholar] [CrossRef] [PubMed]
  6. Tocci, M.J.; Matkovich, D.A.; Collier, K.A.; Kwok, P.; Dumont, F.; Lin, S.; Degudicibus, S.; Siekierka, J.J.; Chin, J.; Hutchinson, N.I. The immunosuppressant FK506 selectively inhibits expression of early T cell activation genes. J. Immunol. 1989, 143, 718–726. [Google Scholar] [PubMed]
  7. Foor, F.; Parent, S.A.; Morin, N.; Dahl, A.M.; Ramadan, N.; Chrebet, G.; Bostian, K.A.; Nielsen, J.B. Calcineurin mediates inhibition by FK506 and cyclosporin of recovery from alpha-factor arrest in yeast. Nature 1992, 360, 682–684. [Google Scholar] [PubMed]
  8. Kunz, J.; Hall, M.N.; Cyclosporin, A. FK506 and rapamycin: More than just immunosuppression. Trends Biochem. Sci. 1993, 18, 334–338. [Google Scholar] [CrossRef]
  9. Breuder, T.; Hemenway, C.S.; Movva, N.R.; Cardenas, M.E.; Heitman, J. Calcineurin is essential in cyclosporin A- and FK506-sensitive yeast strains. Proc. Natl. Acad. Sci. USA 1994, 91, 5372–5376. [Google Scholar] [CrossRef] [PubMed]
  10. Trede, N.S.; Warwick, A.B.; Rosoff, P.M.; Rohrer, R.; Bierer, B.E.; Guinan, E. Tacrolimus (FK506) in allogeneic bone marrow transplantation for severe aplastic anemia following orthotopic liver transplantation. Bone Marrow Transplant. 1997, 20, 257–260. [Google Scholar] [CrossRef] [PubMed]
  11. Meier-Kriesche, H.-U.; Li, S.; Gruessner, R.W.G.; Fung, J.J.; Bustami, R.T.; Barr, M.L.; Leichtman, A.B. Immunosuppression: Evolution in practice and trends, 1994–2004. Am. J. Transplant. 2006, 6, 1111–1131. [Google Scholar] [CrossRef] [PubMed]
  12. McCormack, P.L.; Keating, G.M. Tacrolimus: In heart transplant recipients. Drugs 2006, 66, 2269–2279. [Google Scholar] [CrossRef] [PubMed]
  13. Ingram, J.R.; Martin, J.A.; Finlay, A.Y. Impact of topical calcineurin inhibitors on quality of life in patients with atopic dermatitis. Am. J. Clin. Dermatol. 2009, 10, 229–237. [Google Scholar] [CrossRef] [PubMed]
  14. Remitz, A.; Reitamo, S. Long-term safety of tacrolimus ointment in atopic dermatitis. Expert Opin. Drug Saf. 2009, 8, 501–506. [Google Scholar] [CrossRef] [PubMed]
  15. Romano, A.; Jensen, M.R.; McAlpine, J. Toward the optimization of stent-based treatment for coronary artery disease. Curr. Opin. Drug Discov. Devel. 2010, 13, 157–158. [Google Scholar] [PubMed]
  16. Akimoto, K.; Kusunoki, Y.; Nishio, S.; Takagi, K.; Kawai, S. Safety profile of tacrolimus in patients with rheumatoid arthritis. Clin. Rheumatol. 2008, 27, 1393–1397. [Google Scholar] [CrossRef] [PubMed]
  17. Benson, A.; Barrett, T.; Sparberg, M.; Buchman, A.L. Efficacy and safety of tacrolimus in refractory ulcerative colitis and Crohn’s disease: A single-center experience. Inflamm. Bowel Dis. 2008, 14, 7–12. [Google Scholar] [CrossRef] [PubMed]
  18. Reis, S.A.; Moussatché, N.; Damaso, C.R.A. FK506, a secondary metabolite produced by Streptomyces, presents a novel antiviral activity against Orthopoxvirus infection in cell culture. J. Appl. Microbiol. 2006, 100, 1373–1380. [Google Scholar] [CrossRef] [PubMed]
  19. Karpas, A.; Lowdell, M.; Jacobson, S.K.; Hill, F. Inhibition of human immunodeficiency virus and growth of infected T cells by the immunosuppressive drugs cyclosporin A and FK 506. Proc. Natl. Acad. Sci. USA 1992, 89, 8351–8355. [Google Scholar] [CrossRef] [PubMed]
  20. Briggs, C.J.; Ott, D.E.; Coren, L.V.; Oroszlan, S.; Tözsér, J. Comparison of the effect of FK506 and cyclosporin A on virus production in H9 cells chronically and newly infected by HIV-1. Arch. Virol. 1999, 144, 2151–2160. [Google Scholar] [CrossRef] [PubMed]
  21. Mortola, E.; Endo, Y.; Ohno, K.; Watari, T.; Tsujimoto, H.; Hasegawa, A. The use of two immunosuppressive drugs, cyclosporin A and tacrolimus, to inhibit virus replication and apoptosis in cells infected with feline immunodeficiency virus. Vet. Res. Commun. 1998, 22, 553–563. [Google Scholar] [CrossRef] [PubMed]
  22. Yamamoto, S.; Jiang, H.; Kato, R. Stimulation of hair growth by topical application of FK506, a potent immunosuppressive agent. J. Investig. Dermatol. 1994, 102, 160–164. [Google Scholar] [CrossRef] [PubMed]
  23. Klettner, A.; Herdegen, T. FK506 and its analogs—Therapeutic potential for neurological disorders. Curr. Drug Targets CNS Neurol. Disord. 2003, 2, 153–162. [Google Scholar] [CrossRef] [PubMed]
  24. Sierra-Paredes, G.; Sierra-Marcuño, G. Ascomycin and FK506: Pharmacology and therapeutic potential as anticonvulsants and neuroprotectants. CNS Neurosci. Ther. 2008, 14, 36–46. [Google Scholar] [CrossRef] [PubMed]
  25. Konofaos, P.; Terzis, J.K. FK506 and nerve regeneration: Past, present, and future. J. Reconstr. Microsurg. 2013, 29, 141–148. [Google Scholar] [CrossRef] [PubMed]
  26. Periyasamy, S.; Warrier, M.; Tillekeratne, M.P.M.; Shou, W.; Sanchez, E.R. The immunophilin ligands cyclosporin A and FK506 suppress prostate cancer cell growth by androgen receptor-dependent and -independent mechanisms. Endocrinology 2007, 148, 4716–4726. [Google Scholar] [CrossRef] [PubMed]
  27. Barot, R.K.; Shitole, S.C.; Bhagat, N.; Patil, D.; Sawant, P.; Patil, K. Therapeutic effect of 0.1% Tacrolimus Eye Ointment in Allergic Ocular Diseases. JCDR 2016, 10, NC05-9. [Google Scholar] [CrossRef] [PubMed]
  28. Pirsch, J.D.; Miller, J.; Deierhoi, M.H.; Vincenti, F.; Filo, R.S. A comparison of tacrolimus (FK506) and cyclosporine for immunosuppression after cadaveric renal transplantation. FK506 Kidney Transplant Study Group. Transplantation 1997, 63, 977–983. [Google Scholar] [CrossRef] [PubMed]
  29. Jiang, H.; Kobayashi, M. Differences between cyclosporin A and tacrolimus in organ transplantation. Transplant. Proc. 1999, 31, 1978–1980. [Google Scholar] [CrossRef]
  30. Motamedi, H.; Shafiee, A. The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506. Eur. J. Biochem. 1998, 256, 528–534. [Google Scholar] [CrossRef] [PubMed]
  31. Motamedi, H.; Shafiee, A.; Cai, S.J.; Streicher, S.L.; Arison, B.H.; Miller, R.R. Characterization of methyltransferase and hydroxylase genes involved in the biosynthesis of the immunosuppressants FK506 and FK520. J. Bacteriol. 1996, 178, 5243–5248. [Google Scholar] [CrossRef] [PubMed]
  32. Motamedi, H.; Cai, S.J.; Shafiee, A.; Elliston, K.O. Structural organization of a multifunctional polyketide synthase involved in the biosynthesis of the macrolide immunosuppressant FK506. Eur. J. Biochem. 1997, 244, 74–80. [Google Scholar] [CrossRef] [PubMed]
  33. Shafiee, A.; Motamedi, H.; Chen, T. Enzymology of FK-506 biosynthesis. Purification and characterization of 31-O-desmethylFK-506 O:methyltransferase from Streptomyces sp. MA6858. Eur. J. Biochem. 1994, 225, 755–764. [Google Scholar] [CrossRef] [PubMed]
  34. Barreiro, C.; Martínez-Castro, M. Trends in the biosynthesis and production of the immunosuppressant tacrolimus (FK506). Appl. Microbiol. Biotechnol. 2014, 98, 497–507. [Google Scholar] [CrossRef] [PubMed]
  35. Zong, G.; Zhong, C.; Fu, J.; Qin, R.; Cao, G. Draft genome sequence of the tacrolimus-producing bacterium Streptomyces tsukubaensis F601. Genome Announc. 2017, 5, e00385-17. [Google Scholar] [CrossRef] [PubMed]
  36. Goranovič, D.A.; Blažič, I.M.; Magdevska, V.; Horvat, J.; Kuščer, E.; Polak, T.; Santos-Aberturas, J.; Martínez-Castro, M.; Barreiro, C.; Mrak, P.; et al. FK506 biosynthesis is regulated by two positive regulatory elements in Streptomyces tsukubaensis. BMC Microbiol. 2012, 12, 238. [Google Scholar] [CrossRef] [PubMed]
  37. Mo, S.; Kim, D.H.; Lee, J.H.; Park, J.W.; Basnet, D.B.; Ban, Y.H.; Yoo, Y.J.; Chen, S.-W.; Park, S.R.; Choi, E.A.; et al. Biosynthesis of the allylmalonyl-CoA extender unit for the FK506 polyketide synthase proceeds through a dedicated polyketide synthase and facilitates the mutasynthesis of analogues. J. Am. Chem. Soc. 2011, 133, 976–985. [Google Scholar] [CrossRef] [PubMed]
  38. Ordóñez-Robles, M.; Rodríguez-García, A.; Martín, J.F. Target genes of the Streptomyces tsukubaensis FkbN regulator include most of the tacrolimus biosynthesis genes, a phosphopantetheinyl transferase and other PKS genes. Appl. Microbiol. Biotechnol. 2016, 100, 8091–8103. [Google Scholar] [CrossRef] [PubMed]
  39. Bauer, J.S.; Fillinger, S.; Förstner, K.; Herbig, A.; Jones, A.C.; Flinspach, K.; Sharma, C.; Gross, H.; Nieselt, K.; Apel, A.K. dRNA-seq transcriptional profiling of the FK506 biosynthetic gene cluster in Streptomyces tsukubaensis NRRL18488 and general analysis of the transcriptome. RNA Biol. 2017, 14, 1617–1626. [Google Scholar] [CrossRef] [PubMed]
  40. Andexer, J.N.; Kendrew, S.G.; Nur-e-Alam, M.; Lazos, O.; Foster, T.A.; Zimmermann, A.-S.; Warneck, T.D.; Suthar, D.; Coates, N.J.; Koehn, F.E.; et al. Biosynthesis of the immunosuppressants FK506, FK520, and rapamycin involves a previously undescribed family of enzymes acting on chorismate. Proc. Natl. Acad. Sci. USA 2011, 108, 4776–4781. [Google Scholar] [CrossRef] [PubMed]
  41. Goulet, M.T.; Rupprecht, K.M.; Sinclair, P.J.; Wyvratt, M.J.; Parsons, W.H. The medicinal chemistry of FK-506. Perspect. Drug Discov. Des. 1994, 2, 145–162. [Google Scholar] [CrossRef]
  42. Carroll, B.J.; Moss, S.J.; Bai, L.; Kato, Y.; Toelzer, S.; Yu, T.-W.; Floss, H.G. Identification of a set of genes involved in the formation of the substrate for the incorporation of the unusual “glycolate” chain extension unit in ansamitocin biosynthesis. J. Am. Chem. Soc. 2002, 124, 4176–4177. [Google Scholar] [CrossRef] [PubMed]
  43. Kato, Y.; Bai, L.; Xue, Q.; Revill, W.P.; Yu, T.-W.; Floss, H.G. Functional expression of genes involved in the biosynthesis of the novel polyketide chain extension unit, methoxymalonyl-acyl carrier protein, and engineered biosynthesis of 2-desmethyl-2-methoxy-6-deoxyerythronolide B. J. Am. Chem. Soc. 2002, 124, 5268–5269. [Google Scholar] [CrossRef] [PubMed]
  44. Wu, K.; Chung, L.; Revill, W.P.; Katz, L.; Reeves, C.D. The FK520 gene cluster of Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) contains genes for biosynthesis of unusual polyketide extender units. Gene 2000, 251, 81–90. [Google Scholar] [PubMed]
  45. Chen, D.; Zhang, L.; Pang, B.; Chen, J.; Xu, Z.; Abe, I.; Liu, W. FK506 maturation involves a cytochrome p450 protein-catalyzed four-electron C-9 oxidation in parallel with a C-31 o-methylation. J. Bacteriol. 2013, 195, 1931–1939. [Google Scholar] [CrossRef] [PubMed]
  46. Goranovič, D.; Kosec, G.; Mrak, P.; Fujs, S.; Horvat, J.; Kuščer, E.; Kopitar, G.; Petković, H. Origin of the allyl group in FK506 biosynthesis. J. Biol. Chem. 2010, 285, 14292–14300. [Google Scholar] [CrossRef] [PubMed]
  47. Jiang, H.; Wang, Y.Y.; Guo, Y.Y.; Shen, J.J.; Zhang, X.S.; Luo, H.D.; Ren, N.N.; Jiang, X.H.; Li, Y.Q. An acyltransferase domain of FK506 polyketide synthase recognizing both an acyl carrier protein and coenzyme A as acyl donors to transfer allylmalonyl and ethylmalonyl units. FEBS J. 2015, 282, 2527–2539. [Google Scholar] [CrossRef] [PubMed]
  48. Byrne, K.; Shafiee, A.; Nielsen, J.; Arison, B.; Monaghan, R.; Kaplan, L. The biosynthesis and enzymology of an immunosuppressant, immunomycin, produced by Streptomyces hygroscopicus var. ascomyceticus. Dev. Ind. Microbiol. 1993, 32, 29–45. [Google Scholar]
  49. Gatto, G.J., Jr.; McLoughlin, S.M.; Kelleher, N.L.; Walsh, C.T. Elucidating the substrate specificity and condensation domain activity of FkbP, the FK520 pipecolate-incorporating enzyme. Biochemistry 2005, 44, 5993–6002. [Google Scholar] [CrossRef] [PubMed]
  50. Gatto, G.J., Jr.; Boyne, M.T., 2nd; Kelleher, N.L.; Walsh, C.T. Biosynthesis of pipecolic acid by RapL, a lysine cyclodeaminase encoded in the rapamycin gene cluster. J. Am. Chem. Soc. 2006, 128, 3838–3847. [Google Scholar] [CrossRef] [PubMed]
  51. Van Duyne, G.D.; Standaert, R.F.; Karplus, P.A.; Schreiber, S.L.; Clardy, J. Atomic structure of FKBP-FK506, an immunophilin-immunosuppressant complex. Science 1991, 252, 839–842. [Google Scholar] [CrossRef] [PubMed]
  52. Becker, J.W.; Rotonda, J.; McKeever, B.M.; Chan, H.K.; Marcy, A.I.; Wiederrecht, G.; Hermes, J.D.; Springer, J.P. FK-506-binding protein: Three-dimensional structure of the complex with the antagonist L-685,818. J. Biol. Chem. 1993, 268, 11335–11339. [Google Scholar] [PubMed]
  53. Shafiee, A.; Motamedi, H.; Dumont, F.J.; Arison, B.H.; Miller, R.R. Chemical and biological characterization of two FK506 analogs produced by targeted gene disruption in Streptomyces sp. MA6548. J. Antibiot. 1997, 50, 418–423. [Google Scholar] [CrossRef] [PubMed]
  54. Ban, Y.H.; Shinde, P.B.; Hwang, J.-Y.; Song, M.-C.; Kim, D.H.; Lim, S.-K.; Sohng, J.K.; Yoon, Y.J. Characterization of FK506 biosynthetic intermediates involved in post-PKS elaboration. J. Nat. Prod. 2013, 76, 1091–1098. [Google Scholar] [CrossRef] [PubMed]
  55. Schrijver, A.D.; Mot, R.D. A subfamily of MalT-related ATP-dependent regulators in the LuxR family. Microbiology 1999, 145, 1287–1288. [Google Scholar] [CrossRef] [PubMed]
  56. Bibb, M.J. Regulation of secondary metabolism in Streptomycetes. Curr. Opin. Microbiol. 2005, 8, 208–215. [Google Scholar] [CrossRef] [PubMed]
  57. Molnár, I.; Aparicio, J.F.; Haydock, S.F.; Khaw, L.E.; Schwecke, T.; König, A.; Staunton, J.; Leadlay, P.F. Organisation of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: Analysis of genes flanking the polyketide synthase. Gene 1996, 169, 1–7. [Google Scholar] [CrossRef]
  58. Wilson, D.J.; Xue, Y.; Reynolds, K.A.; Sherman, D.H. Characterization and analysis of the PikD regulatory factor in the pikromycin biosynthetic pathway of Streptomyces venezuelae. J. Bacteriol. 2001, 183, 3468–3475. [Google Scholar] [CrossRef] [PubMed]
  59. He, W.; Lei, J.; Liu, Y.; Wang, Y. The LuxR family members GdmRI and GdmRII are positive regulators of geldanamycin biosynthesis in Streptomyces hygroscopicus 17997. Arch. Microbiol. 2008, 189, 501–510. [Google Scholar] [CrossRef] [PubMed]
  60. Chen, S.; Huang, X.; Zhou, X.; Bai, L.; He, J.; Jeong, K.J.; Lee, S.Y.; Deng, Z. Organizational and Mutational Analysis of a Complete FR-008/Candicidin Gene Cluster Encoding a Structurally Related Polyene Complex. Chem. Biol. 2003, 10, 1065–1076. [Google Scholar] [CrossRef] [PubMed]
  61. Martín, J.F.; Aparicio, J.F. Biosynthesis Enzymology of the Polyenes Pimaricin and Candicidin. Methods Enzymol. 2009, 459, 215–242. [Google Scholar] [PubMed]
  62. Santos-Aberturas, J.; Payero, T.D.; Vicente, C.M.; Guerra, S.M.; Cañibano, C.; Martín, J.F.; Aparicio, J.F. Functional conservation of PAS-LuxR transcriptional regulators in polyene macrolide biosynthesis. Metab. Eng. 2011, 13, 756–757. [Google Scholar] [CrossRef] [PubMed]
  63. Santos-Aberturas, J.; Vicente, C.M.; Guerra, S.M.; Payero, T.D.; Martín, J.F.; Aparicio, J.F. Molecular control of polyene macrolide biosynthesis: Direct binding of the regulator PimM to eight promoters of pimaricin genes and identification of binding boxes. J. Biol. Chem. 2011, 286, 9150–9161. [Google Scholar] [CrossRef] [PubMed]
  64. Brautaset, T.; Sekurova, O.N.; Sletta, H.; Ellingsen, T.E.; Strøm, A.R.; Valla, S.; Zotchev, S.B. Biosynthesis of the polyene antifungal antibiotic nystatin in Streptomyces noursei ATCC 11455: Analysis of the gene cluster and deduction of the biosynthetic pathway. Chem. Biol. 2000, 7, 395–403. [Google Scholar] [CrossRef]
  65. Carmody, M.; Byrne, B.; Murphy, B.; Breen, C.; Lynch, S.; Flood, E.; Finnan, S.; Caffrey, P. Analysis andmanipulation of amphotericin biosynthetic genes by means of modified phage KC515 transduction techniques. Gene 2004, 343, 107–115. [Google Scholar] [CrossRef] [PubMed]
  66. Omura, S.; Ikeda, H.; Ishikawa, J.; Hanamoto, A.; Takahashi, C.; Shinose, M.; Hattori, M. Genome Sequence of an Industrial Microorganism Streptomyces avermitilis: Deducing the Ability of Producing Secondary Metabolites. Proc. Natl. Acad. Sci. USA 2001, 98, 12215–12220. [Google Scholar] [CrossRef] [PubMed]
  67. Ikeda, H.; Ishikawa, J.; Hanamoto, A.; Shinose, M.; Kikuchi, H.; Shiba, T.; Sakaki, Y.; Hattori, M.; Omura, S. Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis. Nat. Biotechnol. 2003, 21, 526–531. [Google Scholar] [CrossRef] [PubMed]
  68. Song, K.; Wei, L.; Liu, J.; Wang, J.; Qi, H.; Wen, J. Engineering of the LysR family transcriptional regulator FkbR1 and its target gene to improve ascomycin production. Appl. Microbiol. Biotechnol. 2017, 101, 4581–4592. [Google Scholar] [CrossRef] [PubMed]
  69. Bentley, S.D.; Chater, K.F.; Cerdeño-Tárraga, A.-M.; Challis, G.L.; Thomson, N.R.; James, K.D.; Harris, D.E.; Quail, M.A.; Kieser, H.; Harper, D.; et al. Complete genome sequence of the model actinomycete Streptomyces coelicolor A3. Nature 2002, 417, 141–147. [Google Scholar] [CrossRef] [PubMed]
  70. Schell, M.A. Molecular biology of the LysR family of transcriptional regulators. Annu. Rev. Microbiol. 1993, 47, 597–626. [Google Scholar] [CrossRef] [PubMed]
  71. Maddocks, S.E.; Oyston, P.C. Structure and function of the LysR-type transcriptional regulator (LTTR) family proteins. Microbiology 2008, 154 (Pt 12), 3609–3623. [Google Scholar] [CrossRef] [PubMed]
  72. Rodríguez, M.; Nuñez, L.E.; Braña, A.F.; Mendez, C.; Salas, J.A.; Blanco, G. Identification of transcriptional activators for thienamycin and cephamycin C biosynthetic genes within the thienamycin gene cluster from Streptomyces cattleya. Mol. Microbiol. 2008, 69, 633–645. [Google Scholar] [CrossRef] [PubMed]
  73. Pérez-Redondo, R.; Rodríguez-García, A.; Martín, J.F.; Liras, P. The claR gene of Streptomyces clavuligerus, encoding a LysR-type regulatory protein controlling clavulanic acid biosynthesis, is linked to the clavulanate-9-aldehyde reductase (car) gene. Gene 1998, 211, 311–321. [Google Scholar] [CrossRef]
  74. Kölling, R.; Lother, H. AsnC: An autogenlously regulated activator of asparragine syntetase A transcription in Eschericha coli. J. Bacteriol. 1985, 164, 310–315. [Google Scholar] [PubMed]
  75. Zhang, X.S.; Luo, H.D.; Tao, Y.; Wang, Y.Y.; Jiang, X.H.; Jiang, H.; Li, Y.Q. FkbN and Tcs7 are pathway-specific regulators of the FK506 biosynthetic gene cluster in Streptomyces tsukubaensis L19’. J. Ind. Microbiol. Biotechnol. 2016, 43, 1693–1703. [Google Scholar] [CrossRef] [PubMed]
  76. Mo, S.; Yoo, Y.J.; Ban, Y.H.; Lee, S.-K.; Kim, E.; Suh, J.-W.; Yoon, Y.J. Roles of fkbN in positive regulation and tcs7 in negative regulation of FK506 biosynthesis in Streptomyces sp. strain KCTC 11604BP. Appl. Environ. Microbiol. 2012, 78, 2249–2255. [Google Scholar] [CrossRef] [PubMed]
  77. Jones, A.C.; Gust, B.; Kulik, A.; Heide, L.; Buttner, M.J.; Bibb, M.J. Phage p1-derived artificial chromosomes facilitate heterologous expression of the FK506 gene cluster. PLoS ONE 2013, 8, e69319. [Google Scholar] [CrossRef] [PubMed]
  78. Liu, G.; Chater, K.F.; Chandra, G.; Niu, G.; Tan, H. Molecular regulation of antibiotic biosynthesis in Streptomyces. Microbiol. Mol. Biol. Rev. 2013, 77, 112–143. [Google Scholar] [CrossRef] [PubMed]
  79. Martínez-Burgo, Y.; Álvarez-Álvarez, R.; Rodríguez-García, A.; Liras, P. The Pathway-Specific Regulator ClaR of Streptomyces clavuligerus has a Global Effect on the Expression of Genes for Secondary Metabolism and Differentiation. Appl. Environ. Microbiol. 2015, 81, 6637–6648. [Google Scholar] [CrossRef] [PubMed]
  80. Lu, Y.W.; San Roman, A.K.; Gehring, A.M. Role of Phosphopantetheinyl Transferase Genes in Antibiotic Production by Streptomyces coelicolor. J. Bacteriol. 2008, 190, 6903–6908. [Google Scholar] [CrossRef] [PubMed]
  81. Wang, Y.Y.; Zhang, X.S.; Luo, H.D.; Ren, N.N.; Jiang, X.H.; Jiang, H.; Li, Y.Q. Characterization of discrete phosphopantetheinyl transferases in Streptomyces tsukubaensis L19 unveils a complicate phosphopantetheinylation network. Sci. Rep. 2016, 6, 24255. [Google Scholar] [CrossRef] [PubMed]
  82. Blazic, M.; Starcevic, A.; Lisfi, M.; Baranasic, D.; Goranovic, D.; Fujs, S.; Kuščer, E.; Kosec, G.; Petkovic, H.; Cullum, J.; et al. Annotation of the modular polyketide synthase and nonribosomal peptide synthetase gene clusters in the genome of Streptomyces tsukubaensis NRRL18488. Appl. Environ. Microbiol. 2012, 78, 8183–8190. [Google Scholar] [CrossRef] [PubMed]
  83. Schneider, T.D. Information content of individual genetic sequences. J. Theor. Biol. 1997, 189, 427–441. [Google Scholar] [CrossRef] [PubMed]
  84. Vockenhuber, M.P.; Sharma, C.M.; Statt, M.G.; Schmidt, D.; Xu, Z.; Dietrich, S.; Liesegang, H.; Mathews, D.H.; Suess, B. Deep sequencing based identification of small non-coding RNAs in Streptomyces coelicolor. RNA Biol. 2011, 8, 468–477. [Google Scholar] [CrossRef] [PubMed]
  85. Hatanaka, H.; Kino, T.; Asano, M.; Goto, T.; Tanaka, H.; Okuhara, M. FK-506 related compounds produced by Streptomyces tsukubaensis No. 9993. J. Antibiot. 1989, 42, 620–622. [Google Scholar] [CrossRef] [PubMed]
  86. Kosec, G.; Goranovič, D.; Mrak, P.; Fujs, S.; Kuščer, E.; Horvat, J.; Kopitar, G.; Petković, H. Novel chemobiosynthetic approach for exclusive production of FK506. Metab. Eng. 2012, 14, 39–46. [Google Scholar] [CrossRef] [PubMed]
  87. Park, J.W.; Mo, S.-J.; Park, S.R.; Ban, Y.-H.; Yoo, Y.J.; Yoon, Y.J. Liquid chromatography-mass spectrometry characterization of FK506 biosynthetic intermediates in Streptomyces clavuligerus KCTC 10561BP. Anal. Biochem. 2009, 393, 1–7. [Google Scholar] [CrossRef] [PubMed]
  88. Nakatsuka, M.; Ragan, J.A.; Sammakia, T.; Smith, D.B.; Uehling, D.E.; Schreiber, S.L. Total synthesis of FK506 and an FKBP probe reagent, [C,C-13C2]-FK506. J. Am. Chem. Soc. 1990, 112, 5583–5601. [Google Scholar] [CrossRef]
  89. Ireland, R.E.; Gleason, J.L.; Gegnas, L.D.; Highsmith, T.K. A Total Synthesis of FK-506. J. Org. Chem. 1996, 61, 6856–6872. [Google Scholar] [CrossRef] [PubMed]
  90. Ban, Y.H.; Park, S.R.; Yoon, Y.J. The biosynthetic pathway of FK506 and its engineering: From past achievements to future prospects. J. Ind. Microbiol. Biotechnol. 2016, 43, 389–400. [Google Scholar] [CrossRef] [PubMed]
  91. Fu, L.F.; Tao, Y.; Jin, M.Y.; Jiang, H. Improvement of FK506 production by synthetic biology approaches. Biotechnol. Lett. 2016, 38, 2015–2021. [Google Scholar] [CrossRef] [PubMed]
  92. Yoon, Y.J.; Choi, C.Y. Nutrient Effects on FK-506, a New Immunosuppressant, Production by Streptomyces sp. in a Defined Medium. J. Ferment. Bioeng. 1997, 83, 599–603. [Google Scholar] [CrossRef]
  93. Martínez-Castro, M.; Salehi-Najafabadi, Z.; Romero, F.; Pérez-Sanchiz, R.; Fernández-Chimeno, R.I.; Martín, J.F.; Barreiro, C. Taxonomy and chemically semi-defined media for the analysis of the tacrolimus producer ‘Streptomyces tsukubaensis’. Appl. Microbiol. Biotechnol. 2013, 97, 2139–2152. [Google Scholar] [CrossRef] [PubMed]
  94. Ordóñez-Robles, M.; Santos-Beneit, F.; Rodríguez-García, A.; Martín, J.F. Analysis of the PHO regulon in Streptomyces tsukubaensis. Microbiol. Res. 2017, 205, 80–87. [Google Scholar] [CrossRef] [PubMed]
  95. Ordóñez-Robles, M.; Santos-Beneit, F.; Albillos, S.M.; Liras, P.; Martín, J.F.; Rodríguez-García, A. Streptomyces tsukubaensis as a new model for carbon repression: Transcriptomic response to tacrolimus repressing carbon sources. Appl. Microbiol. Biotechnol. 2017, 101, 8181–8195. [Google Scholar] [CrossRef] [PubMed]
  96. Reeves, A.R.; Cernota, W.H.; Brikun, I.A.; Wesley, R.K.; Weber, J.M. Engineering precursor flow for increased erythromycin production in Aeromicrobium erythreum. Metab. Eng. 2004, 6, 300–312. [Google Scholar] [CrossRef] [PubMed]
  97. Singh, B.P.; Behera, B.K. Regulation of tacrolimus production by altering primary source of carbons and amino acids. Lett. Appl. Microbiol. 2009, 49, 254–259. [Google Scholar] [CrossRef] [PubMed]
  98. Mishra, A.; Verma, S. Optimization of process parameters for tacrolimus (FK 506) production by new isolate of Streptomyces sp. using response surface methodology. J. Biochem. Technol. 2012, 3, 419–425. [Google Scholar]
  99. Xia, M.; Huang, D.; Li, S.; Wen, J.; Jia, X.; Chen, Y. Enhanced FK506 production in Streptomyces tsukubaensis by rational feeding strategies based on comparative metabolic profiling analysis. Biotechnol. Bioeng. 2013, 110, 2717–2730. [Google Scholar] [CrossRef] [PubMed]
  100. Huang, D.; Xia, M.; Li, S.; Wen, J.; Jia, X. Enhancement of FK506 production by engineering secondary pathways of Streptomyces tsukubaensis and exogenous feeding strategies. J. Ind. Microbiol. Biotechnol. 2013, 40, 1023–1037. [Google Scholar] [CrossRef] [PubMed]
  101. Du, W.; Huang, D.; Xia, M.; Wen, J.; Huang, M. Improved FK506 production by the precursors and product-tolerant mutant of Streptomyces tsukubaensis based on genome shuffling and dynamic fed-batch strategies. J. Ind. Microbiol. Biotechnol. 2014, 41, 1131–1143. [Google Scholar] [CrossRef] [PubMed]
  102. Wang, C.; Liu, J.; Liu, H.; Liang, S.; Wen, J. Combining metabolomics and network analysis to improve tacrolimus productionin Streptomyces tsukubaensis using different exogenous feedings. J. Ind. Microbiol. Biotechnol. 2017, 44, 1527–1540. [Google Scholar] [CrossRef] [PubMed]
  103. Wang, J.; Liu, H.; Huang, D.; Jin, L.; Wang, C.; Wen, J. Comparative proteomic and metabolomic analysis of Streptomyces tsukubaensis reveals the metabolic mechanism of FK506 overproduction by feeding soybean oil. Appl. Microbiol. Biotechnol. 2017, 101, 2447–2465. [Google Scholar] [CrossRef] [PubMed]
  104. Mo, S.; Ban, Y.-H.; Park, J.W.; Yoo, Y.J.; Yoon, Y.J. Enhanced FK506 production in Streptomyces clavuligerus CKD1119 by engineering the supply of methylmalonyl-CoA precursor. J. Ind. Microbiol. Biotechnol. 2009, 36, 1473–1482. [Google Scholar] [CrossRef] [PubMed]
  105. Turlo, J.; Gajzlerska, W.; Klimaszewska, M.; Król, M.; Dawidowski, M.; Gutkowska, B. Enhancement of tacrolimus productivity in Streptomyces tsukubaensis by the use of novel precursors for biosynthesis. Enzyme Microb. Technol. 2012, 51, 388–395. [Google Scholar] [CrossRef] [PubMed]
  106. Gajzlerska, W.; Kurkowiak, J.; Turlo, J. Use of three-carbon chain compounds as biosynthesis precursors to enhance tacrolimus production in Streptomyces tsukubaensis. New Biotechnol. 2014, 32, 32–39. [Google Scholar] [CrossRef] [PubMed]
  107. Zhu, X.; Zhang, W.; Chen, X.; Wu, H.; Duan, Y.; Xu, Z. Generation of high rapamycin producing strain via rational metabolic pathway-based mutagenesis and further titer improvement with fed-batch bioprocess optimization. Biotechnol. Bioeng. 2010, 107, 506–515. [Google Scholar] [CrossRef] [PubMed]
  108. Chen, D.; Zhang, Q.; Zhang, Q.; Cen, P.; Xu, Z.; Liu, W. Improvement of FK506 production in Streptomyces tsukubaensis by genetic enhancement of the supply of unusual polyketide extender units via utilization of two distinct site-specific recombination systems. Appl. Environ. Microbiol. 2012, 78, 5093–5103. [Google Scholar] [CrossRef] [PubMed]
  109. Chen, G.; Wang, G.Y.; Li, X.; Waters, B.; Davies, J. Enhanced production of microbial metabolites in the presence of dimethyl sulfoxide. J. Antibiot. 2000, 53, 1145–1153. [Google Scholar] [CrossRef] [PubMed]
  110. Butler, A.R.; Cundliffe, E. Influence of dimethylsulfoxide on tylosin production in Streptomyces fradiae. J. Ind. Microbiol. Biotechnol. 2001, 27, 46–51. [Google Scholar] [CrossRef] [PubMed]
  111. Huang, D.; Li, S.; Xia, M.; Wen, J.; Jia, X. Genome-scale metabolic network guided engineering of Streptomyces tsukubaensis for FK506 production improvement. Microb. Cell. Fact. 2013, 12, 52. [Google Scholar] [CrossRef] [PubMed]
  112. Pei, G.; Chen, L.; Zhang, W. Chapter nine-WGCNA application to proteomic and metabolomic data analysis. Methods Enzymol. 2017, 585, 135–158. [Google Scholar] [PubMed]
  113. Wang, C.; Liu, J.; Liu, H.; Wang, J.; Wen, J. A genome-scale dynamic flux balance analysis model of Streptomyces tsukubaensis NRRL18488 to predict the targets for increasing FK506 production. Biochem. Eng. J. 2017, 123, 45–56. [Google Scholar] [CrossRef]
  114. Höffner, K.; Harwood, S.; Barton, P. A reliable simulator for dynamic flux balance analysis. Biotechnol. Bioeng 2013, 110, 792–802. [Google Scholar] [CrossRef] [PubMed]
  115. Sola-Landa, A.; Moura, R.S.; Martín, J.F. The two-component PhoR-PhoP system controls both primary metabolism and secondary metabolite biosynthesis in Streptomyces lividans. Proc. Natl. Acad. Sci. USA 2003, 100, 6133–6138. [Google Scholar] [CrossRef] [PubMed]
  116. Sola-Landa, A.; Rodríguez-García, A.; Franco-Domínguez, E.; Martín, J.F. Binding of PhoP to promoters of phosphate-regulated genes in Streptomyces coelicolor: Identification of PHO boxes. Mol. Microbiol. 2005, 56, 1373–1385. [Google Scholar] [CrossRef] [PubMed]
  117. Martín, J.F. Phosphate control of the biosynthesis of antibiotics and other secondary metabolites is mediated by the PhoR-PhoP system: An unfinished story. J. Bacteriol. 2004, 186, 5197–5201. [Google Scholar] [CrossRef] [PubMed]
  118. Martín, J.F.; Santos-Beneit, F.; Rodríguez-García, A.; Sola-Landa, A.; Smith, M.C.M.; Ellingsen, T.E.; Nieselt, K.; Burroughs, N.J.; Wellington, E.M.H. Transcriptomic studies of phosphate control of primary and secondary metabolism in Streptomyces coelicolor. Appl. Microbiol. Biotechnol. 2012, 95, 61–75. [Google Scholar] [CrossRef] [PubMed]
  119. Santos-Beneit, F. The Pho regulon: A huge regulatory network in bacteria. Front. Microbiol. 2015, 6, 402. [Google Scholar] [CrossRef] [PubMed]
  120. Martín, J.F.; Rodríguez-García, A.; Liras, P. The master regulator PhoP coordinates phosphate and nitrogen metabolism, respiration, cell differentiation and antibiotic biosynthesis: Comparison in Streptomyces coelicolor and Streptomyces avermitilis. J. Antibiot. 2017, 70, 534–541. [Google Scholar] [CrossRef] [PubMed]
  121. Makino, K.; Shinagawa, H.; Amemura, M.; Kimura, S.; Nakata, A.; Ishihama, A. Regulation of the phosphate regulon of Escherichia coli Activation of pstS transcription by PhoB protein in vitro. J. Mol. Biol. 1988, 203, 85–95. [Google Scholar] [CrossRef]
  122. Ruiz, B.; Chávez, A.; Forero, A.; García-Huante, Y.; Romero, A.; Sánchez, M.; Rocha, D.; Sánchez, B.; Rodríguez-Sanoja, R.; Sánchez, S.; et al. Production of microbial secondary metabolites: Regulation by the carbon source. Crit. Rev. Microbiol. 2010, 36, 146–167. [Google Scholar] [CrossRef] [PubMed]
  123. Sánchez, S.; Chávez, A.; Forero, A.; García-Huante, Y.; Romero, A.; Sánchez, M.; Rocha, D.; Sánchez, B.; Avalos, M.; Guzmán-Trampe, S.; et al. Carbon source regulation of antibiotic production. J. Antibiot. 2010, 63, 442–459. [Google Scholar] [CrossRef] [PubMed]
  124. Strakova, E.; Zikova, A.; Vohradsky, J. Inference of sigma factor controlled networks by using numerical modeling applied to microarray time series data of the germinating prokaryote. Nucl. Acids Res. 2014, 42, 748–763. [Google Scholar] [CrossRef] [PubMed]
  125. Kawamoto, S.; Watanabe, M.; Saito, N.; Hesketh, A.; Vachalova, K.; Matsubara, K.; Ochi, K. Molecular and functional analyses of the gene (eshA) encoding the 52-kilodalton protein of Streptomyces coelicolor A3 required for antibiotic production. J. Bacteriol. 2001, 183, 6009–6016. [Google Scholar] [CrossRef] [PubMed]
  126. Saito, N.; Xu, J.; Hosaka, T.; Okamoto, S.; Aoki, H.; Bibb, M.J.; Ochi, K. EshA accentuates ppGpp accumulation and is conditionally required for antibiotic production in Streptomyces coelicolor A3. J. Bacteriol. 2006, 188, 4952–4961. [Google Scholar] [CrossRef] [PubMed]
  127. Uguru, G.C.; Stephens, K.E.; Stead, J.A.; Towle, J.E.; Baumberg, S.; McDowall, K.J. Transcriptional activation of the pathway-specific regulator of the actinorhodin biosynthetic genes in Streptomyces coelicolor. Mol. Microbiol. 2005, 58, 131–150. [Google Scholar] [CrossRef] [PubMed]
  128. Vujaklija, D.; Horinouchi, S.; Beppu, T. Detection of an A-factor-responsive protein that binds to the upstream activation sequence of strR, a regulatory gene for streptomycin biosynthesis in Streptomyces griseus. J. Bacteriol. 1993, 175, 2652–2661. [Google Scholar] [CrossRef] [PubMed]
  129. Rigali, S.; Nothaft, H.; Noens, E.E.E.; Schlicht, M.; Colson, S.; Müller, M.; Joris, B.; Koerten, H.K.; Hopwood, D.A.; Titgemeyer, F.; et al. The sugar phosphotransferase system of Streptomyces coelicolor is regulated by the GntR-family regulator DasR and links N-acetylglucosamine metabolism to the control of development. Mol. Microbiol. 2006, 61, 1237–1251. [Google Scholar] [CrossRef] [PubMed]
  130. Rigali, S.; Titgemeyer, F.; Barends, S.; Mulder, S.; Thomae, A.W.; Hopwood, D.A.; van Wezel, G.P. Feast or famine: The global regulator DasR links nutrient stress to antibiotic production by Streptomyces. EMBO Rep. 2008, 9, 670–675. [Google Scholar] [CrossRef] [PubMed]
  131. Barreiro, C.; Prieto, C.; Sola-Landa, A.; Solera, E.; Martínez-Castro, M.; Pérez-Redondo, R.; García-Estrada, C.; Aparicio, J.F.; Fernández-Martínez, L.T.; Santos-Aberturas, J.; et al. Draft genome of Streptomyces tsukubaensis NRRL 18488, the producer of the clinically important immunosuppressant tacrolimus (FK506). J. Bacteriol. 2012, 194, 3756–3757. [Google Scholar] [CrossRef] [PubMed]
  132. Martín, J.F.; Liras, P. Novel Antimicrobial and other Bioactive Metabolites obtained from Silent Gene Clusters. In Antibiotics: Current Innovations and Future Trends; Demain, A.L., Sánchez, S., Eds.; Horizon Scientific Press and Caister Academic Press: Norkfolk, UK, 2015; pp. 275–292. ISBN 978-1-908230-54-6. [Google Scholar]
  133. Werner, G.; Hagenmaier, H.; Drautz, H.; Baumgartner, A.; Zähner, H. Metabolic products of microorganisms. 224. Bafilomycins, a new group of macrolide antibiotics. Production, isolation, chemical structure and biological activity. J. Antibiot. 1984, 37, 110–117. [Google Scholar] [CrossRef] [PubMed]
  134. Harvey, B.M.; Mironenko, T.; Sun, Y.; Hong, H.; Deng, Z.; Leadlay, P.F.; Weissman, K.J.; Haydock, S.F. Insights into polyether biosynthesis from analysis of the nigericin biosynthetic gene cluster in Streptomyces sp. DSM4137. Chem. Biol. 2007, 14, 703–714. [Google Scholar] [CrossRef] [PubMed]
  135. Yin, X.; Zabriskie, T.M. The enduracidin biosynthetic gene cluster from Streptomyces fungicidicus. Microbiology 2006, 152 Pt 10, 2969–2983. [Google Scholar] [CrossRef] [PubMed]
Figure 1. Mechanism of action of tacrolimus (FK506). Tacrolimus interacts with cytosolic receptors such as FKBP12. The complex FKBP12-FK506 inhibits the calmodulin-dependent serine/threonine phosphatase activity of calcineurin. In this situation, calcineurin can no longer dephosphorylate transcriptional factors (e.g., NFAT). The dephosphorylated TFs are required for governing T cell proliferation. L: ligand; R: receptor; CM: calmodulin; CN: calcineurin; TF: transcription factor; P: phosphate group; FKBP-12: FK506 binding protein 12.
Figure 1. Mechanism of action of tacrolimus (FK506). Tacrolimus interacts with cytosolic receptors such as FKBP12. The complex FKBP12-FK506 inhibits the calmodulin-dependent serine/threonine phosphatase activity of calcineurin. In this situation, calcineurin can no longer dephosphorylate transcriptional factors (e.g., NFAT). The dephosphorylated TFs are required for governing T cell proliferation. L: ligand; R: receptor; CM: calmodulin; CN: calcineurin; TF: transcription factor; P: phosphate group; FKBP-12: FK506 binding protein 12.
Antibiotics 07 00039 g001
Figure 2. Tacrolimus biosynthesis cluster (fkb). Genes present in both the short and extended version of the fkb cluster are depicted in black. Genes present only in the extended version are depicted in red. These groups also correspond to their FkbN transcriptional dependence (Black) or independence (Red). The transcriptional units identified to date are indicated by boxes.
Figure 2. Tacrolimus biosynthesis cluster (fkb). Genes present in both the short and extended version of the fkb cluster are depicted in black. Genes present only in the extended version are depicted in red. These groups also correspond to their FkbN transcriptional dependence (Black) or independence (Red). The transcriptional units identified to date are indicated by boxes.
Antibiotics 07 00039 g002
Figure 3. Scheme representing the assembly of the tacrolimus polyketide and the early and late biosynthetic steps. In the upper part the arrows represent the three PKS genes (fkbA, fkbB, fkbC) of the cluster. Note that the fkbA gene is physically separated from fkbB and fkbC genes in the fkb cluster (see Figure 2). The modules of the PKSs are boxed and indicated as M1 to M10. Domains in the modules are indicated by circles: ACP, acyl carrier protein; AT, acyltransferase; ER, enoyl reductase; CAS, CoA synthetase; KR, 3-oxoacyl (ACP) reductase; DH, 3-oxoacyl thioester dehydratase; KS, 3-oxoacyl (ACP) synthase. DHCHC: (4R, 5R)-4,5-dihydroxycyclohex-1-enecarboxylic acid. Biosynthetic and late modification steps, and the encoding genes for the starter (fkbO), elongation units (fkbL, fkbP) and late modification reactions (fkbM, fkbD). Based on data from Motamedi and Shafiee [30].
Figure 3. Scheme representing the assembly of the tacrolimus polyketide and the early and late biosynthetic steps. In the upper part the arrows represent the three PKS genes (fkbA, fkbB, fkbC) of the cluster. Note that the fkbA gene is physically separated from fkbB and fkbC genes in the fkb cluster (see Figure 2). The modules of the PKSs are boxed and indicated as M1 to M10. Domains in the modules are indicated by circles: ACP, acyl carrier protein; AT, acyltransferase; ER, enoyl reductase; CAS, CoA synthetase; KR, 3-oxoacyl (ACP) reductase; DH, 3-oxoacyl thioester dehydratase; KS, 3-oxoacyl (ACP) synthase. DHCHC: (4R, 5R)-4,5-dihydroxycyclohex-1-enecarboxylic acid. Biosynthetic and late modification steps, and the encoding genes for the starter (fkbO), elongation units (fkbL, fkbP) and late modification reactions (fkbM, fkbD). Based on data from Motamedi and Shafiee [30].
Antibiotics 07 00039 g003
Figure 4. Transcriptional profiles of genes encoding transcriptional regulators of the fkb cluster. Transcription of fkbN, fkbR and allN in S. tsukubaensis NRRL 18488 grown in MGm-2.5 production media. As indicated in the graph, phosphate depletion occurs between 80 h and 89 h and tacrolimus is detected from 89 h. The cultures were performed in duplicated flasks. Error bars have been omitted to facilitate the visualization of the results.
Figure 4. Transcriptional profiles of genes encoding transcriptional regulators of the fkb cluster. Transcription of fkbN, fkbR and allN in S. tsukubaensis NRRL 18488 grown in MGm-2.5 production media. As indicated in the graph, phosphate depletion occurs between 80 h and 89 h and tacrolimus is detected from 89 h. The cultures were performed in duplicated flasks. Error bars have been omitted to facilitate the visualization of the results.
Antibiotics 07 00039 g004
Table 1. Common precursors used for tacrolimus production enhancement in different S. tsukubaensis strains. The precursor, S. tsukubaensis strain used and bibliographic reference are indicated.
Table 1. Common precursors used for tacrolimus production enhancement in different S. tsukubaensis strains. The precursor, S. tsukubaensis strain used and bibliographic reference are indicated.
Soybean oilStreptomyces sp. MA6858 B3178[97,98,99,100,101]
S. tsukubaensis TJ-04
S. tsukubaensis D852
l-lysineStreptomyces sp. MA6858 B3178[93,97,98,100,101,102,103]
S. tsukubaensis D852
S. tsukubaensis NRRL18488
Methyl-oleateS. clavuligerus CKD1119[98,104]
Pipecolic acidS. tsukubaensis NRRL18488[100,101,105]
S. tsukubaensis D852
Picolinic acidS. tsukubaensis NRRL18488[105]
NicotinamideS. tsukubaensis NRRL18488[105]
Nicotinic acidS. tsukubaensis NRRL18488[105]
ChorismateS. tsukubaensis D852[100,101]
ShikimateS. tsukubaensis D852[99,100,101,102,103]
S. tsukubaensis TJ-04
S. tsukubaensis NRRL18488
LactateS. tsukubaensis D852[92,100,101,102]
Streptomyces sp. MA6858
SuccinateS. tsukubaensis D852[99,100,101,103]
S. tsukubaensis TJ-04
S. tsukubaensis NRRL18488
IsoleucineS. tsukubaensis D852[100,101]
ValineS. tsukubaensis D852[99,100,101]
S. tsukubaensis TJ-04
ProlineS. tsukubaensis TJ-04[99]
LeucineS. tsukubaensis TJ-04[99]
ThreonineS. tsukubaensis TJ-04[99]
PropilenglycolS. tsukubaensis FERM BP-927[106]
PropanolS. tsukubaensis FERM BP-927[106]
Propionic acidS. tsukubaensis FERM BP-927[106]
MalonateS. tsukubaensis D852[102,103]
S. tsukubaensis NRRL18488
CitrateS. tsukubaensis D852[102,103]
S. tsukubaensis NRRL18488
Table 2. Genetic modifications predicted through metabolic modelling in S. tsukubaensis to improve tacrolimus production. The target gene, type of modification, strain and bibliographic reference are indicated.
Table 2. Genetic modifications predicted through metabolic modelling in S. tsukubaensis to improve tacrolimus production. The target gene, type of modification, strain and bibliographic reference are indicated.
fkbO/overexpressionS. tsukubaensis D852[100]
fkbL/overexpressionS. tsukubaensis D852[100]
fkbM/overexpressionS. tsukubaensis D852[100]
fkbP/overexpressionS. tsukubaensis D852[100]
fkbD/overexpressionS. tsukubaensis D852[100]
gdhA/inactivationS. tsukubaensis D852[111]
ppc/inactivationS. tsukubaensis D852[111]
dahp/overexpressionS. tsukubaensis D852[111]
pntAB/overexpressionS. tsukubaensis D852[111]
accA2/overexpressionS. tsukubaensis D852[111]
zwf2/overexpressionS. tsukubaensis D852[111]
fkbD/overexpressionS. tsukubaensis D852[111]
aroC/overexpressionS. tsukubaensis D852[102]
dapA/overexpressionS. tsukubaensis D852[102]
gcdh/inactivationS. tsukubaensis NRRL 18488[113]
tktB/overexpressionS. tsukubaensis NRRL 18488[113]
msdh/overexpressionS. tsukubaensis NRRL 18488[113]
ask/overexpressionS. tsukubaensis NRRL 18488[113]

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (
Antibiotics EISSN 2079-6382 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
Back to Top