Tacrolimus Biosynthesis Research Articles

Streptomyces tsukubensis VKM Aс-2618D-an Effective Producer of Tacrolimus.

The Streptomyces sp. VKM Ac-2618D strain has been identified, and its morphological and physiological features have been studied in relation to the production of the immunosuppressant tacrolimus. The phenotypic variability of the strain was analyzed, and a dissociant with a high level of tacrolimus production was selected. Based on a comprehensive study of morphological, physiological, and chemotaxonomic properties and on phylogenetic analysis, the strain was named Streptomyces tsukubensis VKM Ac-2618D. The strain genome contains the full version of the tacrolimus biosynthetic gene cluster. The advantages of fed-batch cultivation mode for tacrolimus biosynthesis are shown. The results broaden the understanding of the characteristics of polyketide biosynthesis and can be used in the development of technology for tacrolimus production.

THE SPORADIC DYSRHYTHMIA OF TACROLIMUS IN AN ORTHOTOPIC HEART

TOPIC: Cardiovascular Disease TYPE: Medical Student/Resident Case Reports INTRODUCTION: Immunosuppressive therapy is essential for organ transplanted patients. One such broadly used agent is the commonly used macrolide antibiotic tacrolimus. Tacrolimus has several well-known adverse effects including nephrotoxicity, neurotoxicity, infection, diarrhea. Reports of supraventricular tachycardia as a side effect of tacrolimus are rare. CASE PRESENTATION: We present a 48-year-old male with a history of non-ischemic cardiomyopathy status post orthotopic heart transplant who presented to the hospital due to sudden onset palpitations with mild diaphoresis which lasted 30 minutes in duration. Patient reported some mild diaphoresis but no shortness of breath, chest pain, lightheadedness, or dizziness. The patient stated that he had been experiencing episodes of tachycardia for the last few months but none that had persisted to this extent. He reported medication compliance. His vitals were Temp 98.4, BP 140/88, HR 260, RR 20. After review of his EKG, he was diagnosed with supraventricular tachycardia (SVT) (referenced EKG is after adenosine administration). The patient was hospitalized a few months prior to admission for COVID 19 infection and acute pulmonary embolism. His transplant antirejection regimen was changed from sirolimus to tacrolimus during this prior hospitalization due to the increased risk of thrombosis associated with sirolimus in view of the recognized prothrombic effects of COVID-19. Incidentally, the patient started experiencing sporadic episodes of tachycardia since this change in his medication regimen. In the emergency department, diagnostic studies such as troponins, CBC, BMP, magnesium, hepatic panel, TSH, CMV DNA were within normal parameters for the patient. Tacrolimus blood concentration level was found to be 17 with a goal normal level between 4 to 6. The patient had a right/left heart catheterization with endomyocardial biopsy which showed no evidence of coronary artery vasculopathy or acute cellular rejection, thereby ruling out other potential causes of his SVT. His tacrolimus dose was subsequently decreased with significant improvement in the frequency of his symptoms and no further reported episodes of SVT (with further tacrolimus levels not exceeding 7). DISCUSSION: The mechanism by which tacrolimus leads to SVT is not well understood. One purported mechanism is through the inhibition of the calcineurin-calcium-calmodulin complex. Calcium calmodulin is coincidentally also used by smooth muscle cells to contract. It is thought that by inhibiting this system, there is a subsequent increase in vasodilation which leads to the increased sympathetic and adrenergic response, thereby increasing the risk of arrhythmias such as SVT. CONCLUSIONS: Incessant dysrhythmias caused by tacrolimus can be life-threatening, whereby we seek to raise awareness of the importance of carefully monitoring patients on this drug. REFERENCE #1: Kim BR, Shin HS, Jung YS, Rim H. A case of tacrolimus-induced supraventricular arrhythmia after kidney transplantation. Sao Paulo Med J. 2013;131(3):205-207. doi:10.1590/1516-3180.2013.1313472 REFERENCE #2: Pires SDS, Oliveira R, Moradas-Ferreira P, Mendes MV. The Onset of Tacrolimus Biosynthesis in Streptomyces tsukubaensis Is Dependent on the Intracellular Redox Status. Antibiotics (Basel). 2020;9(10):703. Published 2020 Oct 15. doi:10.3390/antibiotics9100703 REFERENCE #3: Wojciechowski D, Wiseman A. Long-Term Immunosuppression Management: Opportunities and Uncertainties [published online ahead of print, 2021 Apr 14]. Clin J Am Soc Nephrol. 2021;CJN.15040920. doi:10.2215/CJN.15040920 DISCLOSURES: No relevant relationships by Jamarcus Brider, source=Web Response No relevant relationships by Ahamed Khalyfa, source=Web Response No relevant relationships by Navkiran Randhawa, source=Web Response

Streptomyces tsukubensis ВКМ Ac-2618Д - эффективный продуцент такролимуса

The identification of the Streptomyces sp. VKM Ac-2618D strain had been carried out and its morphological and physiological features have been studied in relation to the immunosuppressant tacrolimus production. The phenotypic variability of the strain was analyzed and the dissociant with a high level of tacrolimus production was identified. Based on a comprehensive study of morphological, physiological and chemotaxonomic properties, as well as phylogenetic analysis, the strain was named Streptomyces tsukubensis VKM Ac-2618D. The strain genome contains the full version of the tacrolimus biosynthetic gene cluster. The advantages of fed-batch cultivation mode for tacrolimus biosynthesis were shown. The results obtained expand the understanding of the peculiarities of polyketide biosynthesis and can be used in the development of a tacrolimus production technology. Key words: Streptomyces tsukubensis, tacrolimus, FK-506, dissociant, genome, biosynthesis The research was carried out within the framework of the state assignment АААА-А16-116062110077-6.

Study of the Effect of Lower Eukaryotes on Tacrolimus (FK-506) Biosynthesis by the Streptomyces tsukubensis Strain VKM Ac-2618D

The effect of inactivated yeast cells, individual components of the yeast cells, and the inactivated biomass of filamentous fungi (micromycetes) of various taxonomic positions on the biosynthesis of immunosuppressant tacrolimus (FK-506) by Streptomyces tsukubensis VKM Ac-2618D cells has been studied. The fractions of soluble and insoluble components of yeast cells positively influenced tacrolimus biosynthesis; the FK-506 concentrations reached 447.1± 22.4 mg/L and 514.3 ± 25.7 mg/L, respectively. The insoluble cell components, including the cell walls and cytoplasmic membranes, had a greater effect than other fractions. The presence of whole baker’s yeast cells and the filamentous fungus Aspergillus ochraceus biomass in the complex nutrient medium stimulated tacrolimus production by S. tsukubensis VKM Ac-2618D (667.5 ± 33.4 mg/L and 633.5 ± 31.7 mg/L, respectively). The biomass of deuteromycete Curvularia lunata had no effect on FK‑506 biosynthesis (288.4 ± 14.4 mg/L). Conversely, the biomass of the Gongronella butleri fungus fully inhibited growth and tacrolimus production in S. tsukubensis. The increase in FK-506 biosynthesis in the presence of the cells of the tested micromycetes is more likely due to the polysaccharide composition of their cell walls than to the ergosterol content. The results expand the knowledge of S. tsukubensis physiology and may be useful for the development of the biotechnological process of tacrolimus production.

Biosynthesis of Tacrolimus by the Streptomyces tsukubensis VKM Ac-2618D Strain in the Presence of Polymeric Sorbents and Development of a Method for Its Isolation and Purification

Tacrolimus is known as the most effective immunosuppressive drug which is synthesized by actinobacteria of the genus Streptomyces. Its biosynthesis is accompanied by the formation of the structural analogs. A new method for the production of tacrolimus by Streptomyces tsukubensis BKM Ac-2618D with the use of brominated styrene-divinylbenzene sorbent SP-207 was developed. This approach enabled to increase the yield, prevent undesirable destruction, and simplify the tacrolimus isolation procedure from the cultivation media. The preliminary separation step was optimized by means of the usage of regenerable sorbents such as methylmethacrylate (HP2MG) and chemically modified silica gel (Diasorb-100-Diol). By implementation of the silica gel-based cation exchange resins pretreated with silver ions for the final purification stage, it was possible to increase its productivity. The overall yield of tacrolimus by this method was found to around 60–70%. The results could be implemented for the full-cycle production development of the pharmaceutical grade tacrolimus.

The Onset of Tacrolimus Biosynthesis in Streptomyces tsukubaensis Is Dependent on the Intracellular Redox Status.

The oxidative stress response is a key mechanism that microorganisms have to adapt to changeling environmental conditions. Adaptation is achieved by a fine-tuned molecular response that extends its influence to primary and secondary metabolism. In the past, the role of the intracellular redox status in the biosynthesis of tacrolimus in Streptomyces tsukubaensis has been briefly acknowledged. Here, we investigate the impact of the oxidative stress response on tacrolimus biosynthesis in S. tsukubaensis. Physiological characterization of S. tsukubaensis showed that the onset of tacrolimus biosynthesis coincided with the induction of catalase activity. In addition, tacrolimus displays antioxidant properties and thus a controlled redox environment would be beneficial for its biosynthesis. In addition, S. tsukubaensis ∆ahpC strain, a strain defective in the H2O2-scavenging enzyme AhpC, showed increased production of tacrolimus. Proteomic and transcriptomic studies revealed that the tacrolimus over-production phenotype was correlated with a metabolic rewiring leading to increased availability of tacrolimus biosynthetic precursors. Altogether, our results suggest that the carbon source, mainly used for cell growth, can trigger the production of tacrolimus by modulating the oxidative metabolism to favour a low oxidizing intracellular environment and redirecting the metabolic flux towards the increase availability of biosynthetic precursors.

Evaluation of carbon:nitrogen ratio in semi-defined culture medium to tacrolimus biosynthesis by Streptomyces tsukubaensis and the effect on bacterial growth

Tacrolimus is an immunosuppressant produced by several Streptomyces species, widely used in transplantolgy and dermatology. In this report, attempts have been made to enhance tacrolimus productivity by altering the primary carbon source on culture media. In order to better understand the effects of glucose and maltose on tacrolimus biosynthesis, different carbon contents of these sugars were evaluated in the culture media, thus altering the carbon:nitrogen ratio. The highest specific production achieved on this report was 10.46 mg/g at a carbon:nitrogen ratio of 8.7:1.0 and maltose as carbon source. Using glucose as primary carbon source, the highest specific production achieved was 9.06 mg/g at a carbon:nitrogen ratio of 8.5:1.0. The range of carbon:nitrogen ratios evaluated for each sugar presented distinct behaviors. Glucose containing-media exhibited an initial linear downward trend, by the other hand, maltose containing-media exhibited an increasing tendency. Both glucose and maltose presented the highest specific production at 3 % of carbon source on media. Media containing more than 3 % of carbon content exerted carbon catabolite repression.The range of carbon:nitogen ratios evaluated presented an increasing tendency on tacrolimus biosynthesis up to 3 % of glucose or maltose in the media, both carbon sources exerted the phenomenon of carbon catabolite repression with more than 3 % of carbon source on the media.

Effect of Starch Composition on the Biosynthesis of Immunosuppressant Tacrolimus (FK-506) by Streptomyces tsukubaensis VKM Ac-2618D Strain

The dependence of biosynthesis of macrolide immunosuppressive agent tacrolimus (FK-506) in starch-containing media by the Streptomyces tsukubaensis VKM Ac-2618D strain on the molecular weight and composition of potato starch polysaccharides was studied. It was shown that the use of high-molecular branched starches as a carbon source provided a higher tacrolimus yield as compared to linear low-molecular polysaccharides at the similar culture growth indicators. The approaches providing the highest rate of tacrolimus biosynthesis by using high- and low-molecular starches and dextrins have been proposed. The biosynthesis process carrying out under the periodic batch cultivation with fractional starch feeding increased the tacrolimus yield significantly (up to 2 times), maintained biomass at a high level and reduced destructive processes rates. The maximum tacrolimus titer was observed when high-molecular potato starches were added to the culture in the retardation growth phase, while the best positive effect for low-molecular starches and dextrins additives was noted at stationary growth phase. The results could be useful in creating biotechnology for the production of pharmaceutical tacrolimus.

Enhancing the production of tacrolimus by engineering target genes identified in important primary and secondary metabolic pathways and feeding exogenous precursors.

Tacrolimus has been widely used as a powerful novel immunosuppressant. The objective of this study was to improve the production of tacrolimus by engineering the target genes of important primary and secondary metabolic pathways and feeding exogenous precursors. Based on the metabonomics analysis, the shikimic acid pathway is an important primary metabolic pathway for the producing tacrolimus. Combined overexpression of shikimate kinase and dehydroquinic acid synthetase genes led to a 33.1% enhancement of tacrolimus production compared to parent strain. To predict the most efficient targets in secondary metabolic pathways for improving the production of tacrolimus, a genome-scale dynamic metabolic network model was used. A knockout of the D-lactate dehydrogenase gene, combined with the overexpression of tryptophane synthase and aspartate 1-decarboxylase genes, led to a 29.8% enhancement of tacrolimus production compared to the parent strain. Finally, we investigated the impact of the genetic manipulations on transcription levels, cell growth, cell morphology and production of tacrolimus by qRT-PCR and scanning electron microscopy to reveal the relationship between the growth of strains, the effects of engineering and fermentation. As the efficient synthesis of tacrolimus requires a rich supply of external substrates, the efficiency of the metabolic pathways that convert these substances is extremely important. The combined addition of three external substrates such as shikimic acid, alanine and the n-dodecane increased tacrolimus production by 49.5%. The insights obtained in this study will help further elucidate the mechanisms by which the identified target genes promote the activity of important primary and secondary metabolic pathways for tacrolimus biosynthesis and provide a new feeding strategy to improve tacrolimus production.

Влияние низших грибов на биосинтез такролимуса (FK-506) штаммом Streptomyces tsukubensis BKM Ас-2618Д

The effect of inactivated yeast cells, cell components of yeast cells, and inactivated biomass of the lower fungi of various taxonomic positions on the immunosuppressant tacrolimus (FK-506) biosynthesis by Streptomyces tsukubensis VKM Ac-2618D has been studied. The fractions of soluble and insoluble components of yeast cells positively influenced the tacrolimus biosynthesis with its concentrations reaching 447.1±22.4 mg/L and 514.3±25.7 mg/L, respectively. Wherein, the insoluble cell components including cell walls and cytoplasmic membranes had a greater effect. The presence of the baker's yeast whole cells and filamentous fungus Aspergillus ochraceus biomass in the complex nutrient medium stimulated the tacrolimus production by S. tsukubensis VKM Ac-2618D (667.5±33.4 mg/L and 633.5±31.7 mg/L, respectively). The biomass of deuteromycete Curvularia lunata had no effect on the FK-506 biosynthesis (288.4±14.4 mg/L). Conversely, the biomass of fungus Gongronella butleri fully inhibited the growth and tacrolimus production by S. tsukubensis. The increase in the FK-506 production in media containing cells of the lower fungi is mainly related to the polysaccharide composition of their cell walls rather than the ergosterol content. The results extend the knowledge on the S. tsukubensis physiology and could be useful for the development of the tacrolimus biotechnological production. tacrolimus, biosynthesis, Streptomyces tsukubensis, yeasts, fungi, ergosterol The study was supported by the FASIE program UMNIK 16-12 (Project no. 12081GU/2017).

Genome-wide transcriptome response of Streptomyces tsukubaensis to N-acetylglucosamine: effect on tacrolimus biosynthesis

Chitin is the second most abundant carbohydrate biopolymer present in soils and is utilized by antibiotic–producing Streptomyces species. Its monomer, N-acetylglucosamine (GlcNAc), regulates the developmental program of the model organism Streptomyces coelicolor. GlcNAc blocks differentiation when growing on rich medium whilst it promotes development on poor culture media. However, it is unclear if the same GlcNAc regulatory profile observed in S. coelicolor applies also to other industrially important Streptomyces species. We report here the negative effect of GlcNAc on differentiation and tacrolimus (FK506) production by Streptomyces tsukubaensis NRRL 18488. Using microarrays technology, we found that GlcNAc represses the transcription of fkbN, encoding the main transcriptional activator of the tacrolimus biosynthetic cluster, and of ppt1, encoding a phosphopantheteinyltransferase involved in tacrolimus biosynthesis. On the contrary, GlcNAc stimulated transcription of genes related to amino acid and nucleotide biosynthesis, DNA replication, RNA translation, glycolysis and pyruvate metabolism. The results obtained support those previously reported for S. coelicolor, but some important differences were observed; for example genes involved in GlcNAc transport and metabolism and genes encoding transcriptional regulators such as crr, ptsI, nagE1, nagE2, nagB, chiA, chiJ, ngcE, dasR or atrA are not significantly induced in S. tsukubaensis by GlcNAc addition. Differences in the GlcNAc transport systems, in the physiology of S. tsukubaensis and S. coelicolor and/or the different composition of the culture media used are likely to be responsible for the discrepancies observed between these species.

Unraveling Nutritional Regulation of Tacrolimus Biosynthesis in Streptomyces tsukubaensis through omic Approaches.

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.

Manipulating the expression of SARP family regulator BulZ and its target gene product to increase tacrolimus production.

Tacrolimus (FK506), an effective immunosuppressant, is widely used in the treatment of autoimmune diseases. In this study, we identified that BulZ, a Streptomyces antibiotic regulatory protein (SARP) family regulator, acted as a positive regulator for spore differentiation and tacrolimus production. A knockout of bulZ resulted in a 47.5% decrease of tacrolimus production and a delay of spore differentiation. Using quantitative real-time PCR (qRT-PCR) analysis and electrophoretic mobility shift assays (EMSAs), it was found that BulZ directly activated the transcriptions of bulZ and bulS2, a putative γ-butyrolactone (GBL) synthetase, and bulS2 was shown to play a positive role in tacrolimus biosynthesis. Meanwhile, BulZ was able to indirectly regulate the transcriptions of the cluster-linked activator genes tcs7 and fkbN, as well as the GBL receptor gene bulR1. STSU_RS22595, which encoded a WhiB family transcriptional regulator, was found to be a previously unknown potential target gene of BulZ based on a whole-genome search of the conserved sequence (5'-TSVAVVVNVNBTSRAGNN-3') of the SARP-binding motifs. Although overexpression of STSU_RS22595 did not result in an obvious enhancement of tacrolimus yield, STSU_RS22595 might have important effects on the spore differentiation process. Finally, co-overexpression of bulZ and its target gene bulS2 improved tacrolimus production by 36% compared to the control strain, reaching 324mg/L. The insights obtained in this study will help further elucidate the regulatory mechanism of tacrolimus biosynthesis and provide new avenues for further improvement of tacrolimus production.

Combining metabolomics and network analysis to improve tacrolimus production in Streptomyces tsukubaensis using different exogenous feedings.

Tacrolimus is widely used as an immunosuppressant in the treatment of various autoimmune diseases. However, the low fermentation yield of tacrolimus has thus far restricted its industrial applications. To solve this problem, the time-series response mechanisms of the intracellular metabolism that were highly correlated with tacrolimus biosynthesis were investigated using different exogenous feeding strategies in S. tsukubaensis. The metabolomic datasets, which contained 93 metabolites, were subjected to weighted correlation network analysis (WGCNA), and eight distinct metabolic modules and seven hub metabolites were identified to be specifically associated with tacrolimus biosynthesis. The analysis of metabolites within each metabolic module suggested that the pentose phosphate pathway (PPP), shikimate and aspartate pathway might be the main limiting factors in the rapid synthesis phase of tacrolimus accumulation. Subsequently, all possible key-limiting steps in the above metabolic pathways were further screened using a genome-scale metabolic network model (GSMM) of S. tsukubaensis. Based on the prediction results, two newly identified targets (aroC and dapA) were overexpressed experimentally, and both of the engineered strains showed higher tacrolimus production. Moreover, the best strain, HT-aroC/dapA, that was engineered to simultaneously enhanced chorismate and lysine biosynthesis was able to produce 128.19mg/L tacrolimus, 1.64-fold higher than control (78.26mg/L). These findings represent a valuable addition to our understanding of tacrolimus accumulation in S. tsukubaensis, and pave the way to further production improvements.

Draft Genome Sequence of the Tacrolimus-Producing Bacterium Streptomyces tsukubaensis F601.

ABSTRACTStreptomyces tsukubaensis strain F601 was found to be a producer of the immunosuppressive drug tacrolimus. The draft genome sequence of this strain was approximately 8.52 Mbp. Genes involved in the biosynthesis of tacrolimus were identified in the genome. This draft genome sequence will provide insights into the genetic basis of tacrolimus biosynthesis and regulation.

Target genes of the Streptomyces tsukubaensis FkbN regulator include most of the tacrolimus biosynthesis genes, a phosphopantetheinyl transferase and other PKS genes.

Tacrolimus (FK506) is a 23-membered macrolide immunosuppressant used in current clinics. Understanding how the tacrolimus biosynthetic gene cluster is regulated is important to increase its industrial production. Here, we analysed the effect of the disruption of fkbN (encoding a LAL-type positive transcriptional regulator) on the whole transcriptome of the tacrolimus producer Streptomyces tsukubaensis using microarray technology. Transcription of fkbN in the wild type strain increases from 70h of cultivation reaching a maximum at 89h, prior to the onset of tacrolimus biosynthesis. Disruption of fkbN in S. tsukubaensis does not affect growth but prevents tacrolimus biosynthesis. Inactivation of fkbN reduces the transcription of most of the fkb cluster genes, including some all (for allylmalonyl-CoA biosynthesis) genes but does not affect expression of allMNPOS or fkbR (encoding a LysR-type regulator). Disruption of fkbN does not suppress transcription of the cistron tcs6-fkbQ-fkbN; thus, FkbN self-regulates only weakly its own expression. Interestingly, inactivation of FkbN downregulates the transcription of a 4'-phosphopantetheinyl transferase coding gene, which product is involved in tacrolimus biosynthesis, and upregulates the transcription of a gene cluster containing a cpkA orthologous gene, which encodes a PKS involved in coelimycin P1 biosynthesis in Streptomyces coelicolor. We propose an information theory-based model for FkbN binding sequences. The consensus FkbN binding sequence consists of 14 nucleotides with dyad symmetry containing two conserved inverted repeats of 7nt each. This FkbN target sequence is present in the promoters of FkbN-regulated genes.

Use of three-carbon chain compounds as biosynthesis precursors to enhance tacrolimus production in Streptomyces tsukubaensis

Tacrolimus, a 23-membered polyketide macrolide, is isolated as a metabolite from the whole fermentation broth of Streptomyces species. This potent immunosuppressive calcineurin inhibitor has been widely used in various fields of medicine, including transplantology, dermatology and pharmacotherapy of autoimmune diseases. The current study was focused on optimisation of tacrolimus biosynthesis by Streptomyces tsukubaensis through the use of culture media supplements. Enrichment of the fermentation medium with propionic acid, propylene glycol or propanol resulted in a 5.5-, 3.5- and 1.8-fold, respectively, increase in tacrolimus production. The optimal concentration of the precursors was 0.25% for both propanol and propionic acid and 0.75% for propylene glycol. The mode of action of each media supplement tested was unique. For instance, propionic acid acted as a tacrolimus biosynthesis precursor while propylene glycol induced mycelial growth of S. tsukubaensis. Results from the current study clearly demonstrate that a set of novel culture medium supplements considerably increased tacrolimus production. Application of such promoters of tacrolimus biosynthesis may lead to a substantial improvement in the production of tacrolimus by S. tsukubaensis in industrial fermentation processes.

Trends in the biosynthesis and production of the immunosuppressant tacrolimus (FK506)

The current off-patent state of tacrolimus (FK506) has opened the hunting season for new generic pharmaceutical formulations of this immunosuppressant. This fact has boosted the scientific and industrial research on tacrolimus for the last 5 years in order to improve its production. The fast discovery of tacrolimus producer strains has generated a huge number of producers, which presents the biosynthetic cluster of FK506 as a high promiscuous genetic region. For the first time, the current state-of-the-art on the tacrolimus biosynthesis, production improvements and drug purification is reviewed. On one hand, all the genes involved in the tacrolimus biosynthesis, in addition to the traditional PKS/NRPS, as well as their regulation are analysed. On the other hand, tacrolimus direct and indirect precursors are reviewed as a straight manner to improve the final yield, which is a current trend in the field. Twenty years of industrial and scientific improvements on tacrolimus production are summarised, whereas future trends are also drafted.

Taxonomy and chemically semi-defined media for the analysis of the tacrolimus producer ‘Streptomyces tsukubaensis’

'Streptomyces tsukubaensis' was the first tacrolimus producer strain identified. Although it has been included in the Streptomyces genus, its taxonomic position has not been rigorously determined. By using a polyphasic approach, we have established that the tacrolimus producer strain 'S. tsukubaensis' NRRL 18488 represents a unique species in the Streptomyces genus, which is phylogenetically distant from other subsequently described producers. This fact means a horizontal transference of the tacrolimus-producing gene cluster. Physiology, nutrient requirement, and molecular genetics analyses of tacrolimus biosynthesis in 'S. tsukubaensis' necessitate chemically defined or semi-defined media, which work as a jigsaw puzzle and allow for pieces (nutrients) exchange. To date, studies related to 'S. tsukubaensis' have been mainly focused in the improvement of tacrolimus production using complex industrial fermentation media, which difficulty allows testing of tacrolimus overproduction enhancers or inhibitors because of the presence of non-defined substances. In the present work, two semi-defined media were developed in order to study the main factors involved in tacrolimus production in 'S. tsukubaensis'.

Enhancement of tacrolimus productivity in Streptomyces tsukubaensis by the use of novel precursors for biosynthesis

In this report the optimization of biosynthesis of tacrolimus, the immunosupressant widely used in transplantology and dermatology was described. The enhancement of the productivity of Streptomyces tsukubaensis strain was achieved by development of new precursors of tacrolimus biosynthesis, which should allow to reduce the costs of the process.The enrichment of the fermentation medium in pyridine-2-carboxylic acid (picolinic acid), piperidine-2-carboxylic acid (pipecolic acid), pyridine-3-carboxylic acid (nicotinic acid) or pyridine-3-carboxylic acid amide (nicotinamide) caused significant growth of the productivity of tacrolimus: 7-fold, 6-fold, 3-fold and 5-fold, respectively. The optimum concentration of the precursors in medium was 0.0025–0.005%. The investigation of the kinetics of tacrolimus biosynthesis together with the analysis of the impact of tested compounds on the culture growth and NAD (nicotinamide adenine dinucleotide) concentration in S. tsukubaensis cells enables to put forward a hypothesis concerning the mechanism of action of tested culture medium additives. The compounds active as tacrolimus precursors (pipecolic and picolinic acids) are more effective than these active mainly as the growth promoters (nicotinamide and nicotinic acid). Nicotinamide and nicotinic acid – vitamin B3 components – promote S. tsukubaensis growth most probably due to the stimulation of NAD/NADP biosynthesis.