Production of tacrolimus using coconut oil as an alternative to glucose

Tacrolimus is an immunosuppressant used in the treatment of people who have undergone organ and tissue transplants, as well as in the treatment of dermatoses and eye diseases. Strategies have been carried out to increase the productivity of tacrolimus, since many compounds have in their composition tacrolimus precursors, and bacteria Streptomyces depend on a carbon source for their growth. One strategy is to change the carbon source of the fermentation medium. The present study aims to evaluate the use of coconut oil for the production of tacrolimus via Streptomyces tsukubaensis, as an alternative in the face of glucose, a traditional source of carbon. The batch fermentation process was done in an orbital shaker at 28°C and 130 rpm. Quantification of tacrolimus was performed by High Performance Liquid Chromatography (HPLC). The sugars and proteins, present in the medium, were measured from Somogyi-Nelson and Bradford methods, respectively. According to the results, coconut oil achieved the production of tacrolimus higher than glucose. This is due to the presence of a portion of monounsaturated and polyunsaturated fatty acids that act as precursors to tacrolimus. On the other hand, glucose is a quick carbon source for the bacteria and helps in its growth, but in high amounts it inhibits the production of the drug.

Improvement of FK506 production via metabolic engineering-guided combinational strategies in Streptomyces tsukubaensis

Background FK506, a macrolide mainly with immunosuppressive activity, can be produced by various Streptomyces strains. However, one of the major challenges in the fermentation of FK506 is its insufficient production, resulting in high fermentation costs and environmental burdens. Herein, we tried to improve its production via metabolic engineering-guided combinational strategies in Streptomyces tsukubaensis. Results First, basing on the genome sequencing and analysis, putative competitive pathways were deleted. A better parental strain L19-2 with increased FK506 production from 140.3 to 170.3 mg/L and a cleaner metabolic background was constructed. Subsequently, the FK506 biosynthetic gene cluster was refactored by in-situ promoter-substitution strategy basing on the regulatory circuits. This strategy enhanced transcription levels of the entire FK506 biosynthetic gene cluster in a fine-tuning manner and dramatically increased the FK506 production to 410.3 mg/mL, 1.41-fold higher than the parental strain L19-2 (170.3 mg/L). Finally, the FK506 production was further increased from 410.3 to 603 mg/L in shake-flask culture by adding L-isoleucine at a final concentration of 6 g/L. Moreover, the potential of FK506 production capacity was also evaluated in a 15-L fermenter, resulting in the FK506 production of 830.3 mg/L. Conclusion From the aspects of competitive pathways, refactoring of the FK506 biosynthetic gene cluster and nutrients-addition, a strategy for hyper-production and potentially industrial application of FK506 was developed and a hyper-production strain L19-9 was constructed. The strategy presented here can be generally applicable to other Streptomyces for improvement of FK506 production and streamline hyper-production of other valuable secondary metabolites.

Combination of Atmospheric and Room Temperature Plasma (ARTP) mutagenesis, Genome shuffling and Dimethyl sulfoxide (DMSO) feeding to improve FK506 production in Streptomyces tsukubaensis

FK506 is a clinically important macrocyclic polyketide with immunosuppressive activity produced by Streptomyces tsukubaensis . However, the production capacity of the strain is very low. To improve production, atmospheric and room temperature plasma (ARTP)mutagenesis was adopted to get the initial strains used in genome shuffling (GS). After three rounds of GS, S. tsukubaensis R3-C4 was the most productive strain, resultingin a FK506 concentration of 335 μg/mL, 2.6 times than that of the originalwild-type strain. Moreover, exogenous DMSO 4 % (v/v) addition could induce efflux of FK506 and increased FK506 production by 27.9% to 429 μg/mL . Finally, analyses of the differences in morphology, fermentation characteristics and specific gene expression levels between S. tsukubaensis R3-C4 and the wild-type strain revealed that R3-C4 strain: has hampered spore differentiation , thicker mycelia and more red pigment, which are likely related to the downregulation of bldD and cdgB expression. In addition, the expression levels of fkbO , fkbP , dahp , pccB and prpE all showed up-regulation at diverse degrees compared to the wild-type S. tsukubaensis . Overall, these results show that a combined approach involving classical random mutation and exogenous feeding canbe applied to increase FK506 biosynthesis andmay be applied also tothe improvement of other important secondary metabolites.

The novel ECF56 SigG1-RsfG system modulates morphological differentiation and metal-ion homeostasis in Streptomyces tsukubaensis

Extracytoplasmic function (ECF) sigma factors are key transcriptional regulators that prokaryotes have evolved to respond to environmental challenges. Streptomyces tsukubaensis harbours 42 ECFs to reprogram stress-responsive gene expression. Among them, SigG1 features a minimal conserved ECF σ2–σ4 architecture and an additional C-terminal extension that encodes a SnoaL_2 domain, which is characteristic for ECF σ factors of group ECF56. Although proteins with such domain organisation are widely found among Actinobacteria, the functional role of ECFs with a fused SnoaL_2 domain remains unknown. Our results show that in addition to predicted self-regulatory intramolecular amino acid interactions between the SnoaL_2 domain and the ECF core, SigG1 activity is controlled by the cognate anti-sigma protein RsfG, encoded by a co-transcribed sigG1-neighbouring gene. Characterisation of ∆sigG1 and ∆rsfG strains combined with RNA-seq and ChIP-seq experiments, suggests the involvement of SigG1 in the morphological differentiation programme of S. tsukubaensis. SigG1 regulates the expression of alanine dehydrogenase, ald and the WhiB-like regulator, wblC required for differentiation, in addition to iron and copper trafficking systems. Overall, our work establishes a model in which the activity of a σ factor of group ECF56, regulates morphogenesis and metal-ions homeostasis during development to ensure the timely progression of multicellular differentiation.

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.