Identification and metabolomic analysis of chemical elicitors for tacrolimus accumulation in Streptomyces tsukubaensis

Biotech & Bioengineering

Yuan

et al. 2019

Self Cite

Ascomycin (FK520) is a macrocyclic antibiotic that also exhibits antifungal and immunosuppressive activity. However, its relatively low titer and yield have hampered commercial application. Here, we have successfully constructed an efficient ascomycinproducing strain of Streptomyces hygroscopicus with high titer and yield, using a novel combinatorial engineering approach based on the identification of targets involved in both metabolic and transcriptional regulation. First, we investigated the effects of different chemicals on ascomycin accumulation and found that dimethyl sulfoxide best stimulated ascomycin overproduction. We next compared intracellular metabolic and transcriptional profiles after dimethyl sulfoxide and control treatments and identified potential target genes (zwf and aroA, involved in metabolic precursor pathways; and luxR, iclR, fadR, and fkbN, involved in transcriptional regulation). These candidate genes were then engineered to produce strains with individual and combinatorial overexpression. Combined overexpression of aroA, fkbN, and luxR resulted in the highest yield of ascomycin (1258.30 ± 33.49 mg/L), 4.12-fold higher than the control yield (305.60 ± 16.90 mg/L). This integrative multilevel approach identified novel determinants involved in both metabolic and transcriptional regulation, resulting in the diversion of carbon flux towards ascomycin accumulation. This approach could be applied to boost the production of a variety of useful bacterial metabolites.

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Generation of Streptomyces hygroscopicus cell factories with enhanced ascomycin production by combined elicitation and pathway‐engineering strategies

Biotech & Bioengineering

Yuan

et al. 2019

Self Cite

“…For the biosynthesis of the secondary metabolites, the supply of precursors from the primary metabolism pathways is very important [44]. FK520 is a secondary metabolite produced by Streptomyces hygroscopicus var.…”

Section: Discussionmentioning

confidence: 99%

“…To exclude DNA contamination, the RNA sample which treated by gDNA Eraser but not reverse transcription was used as a template for negative control. The 16S rRNA was used as the internal reference gene, and the change folds of the selected genes were quanti ed relatively using comparative C T method [44].…”

Section: Quantitative Real-time Pcr (Qrt-pcr) Analysismentioning

confidence: 99%

Increasing Ascomycin Yield in Streptomyces Hygroscopicus var. Ascomyceticus by Using Polyhydroxybutyrate as an Intracellular Carbon Supply Station

Yin

et al. 2020

Preprint

Background: Ascomycin is a multifunctional antibiotic produced by Streptomyces hygroscopicus var. ascomyceticus. As a secondary metabolite, the production of ascomycin is often limited by the shortage of precursors during the late fermentation phase. Polyhydroxybutyrate is an intracellular polymer accumulated by various microorganisms. The development of polyhydroxybutyrate as a useful carbon reservoir for precursor synthesis is of great significance for improving the yield of ascomycin.Results: The fermentation characteristics of the parent strain S. hygroscopicus var. ascomyceticus FS35 showed that the accumulation and decomposition of polyhydroxybutyrate were respectively correlated with the growth of strain and the production of ascomycin. The co-overexpression of exogenous polyhydroxybutyrate synthesis gene phaC and native polyhydroxybutyrate decomposition gene fkbU increased both strain biomass and ascomycin yield. Comparative transcription analysis showed that the storage of polyhydroxybutyrate during the exponential phase accelerated biosynthesis processes by stimulating the utilization of carbon sources, and the decomposition of polyhydroxybutyrate during the stationary phase increased the biosynthesis of ascomycin precursors by enhancing metabolic flux of primary pathways. The comparative analysis of cofactor concentration reflected that the biosynthesis of polyhydroxybutyrate depended on the supply of NADH pool. Under the condition of low sugar content in the later exponential phase, the optimization of carbon source addition further strengthened the polyhydroxybutyrate metabolism by increasing total concentration of cofactors. Finally, in the fermentation medium with 22 g/L starch and 52 g/L dextrin, the ascomycin yield of co-overexpression strain was increased to 626.30 mg/L, 2.11-fold higher than that of parent strain in the initial medium (296.29 mg/L). Conclusions: This paper reported for the first time that the polyhydroxybutyrate was beneficial to the growth of strain and the production of ascomycin by working as an intracellular carbon reservoir, storing as polymers when carbon sources are abundant and depolymerizing to monomers for the biosynthesis of precursors when carbon sources are insufficient. The successful application of polyhydroxybutyrate in increasing the output of ascomycin provided a new strategy for the high yield of other secondary metabolites.

“…11,13,15 The biomass production reached its highest values from 120 to 168 hours, which probably corresponds to the death phase, characterized by the unavailability of nutrients resulting in cellular death. 26,27 From 48 to 96 hours, the biomass production almost remained constant because biomass formation competes with secondary metabolites biosynthesis. 28 The amount of proteins increased during the tacrolimus biosynthesis.…”

Section: Discussionmentioning

confidence: 99%

Streptomyces tsukubaensis Fermentation Using Brazil Nut Oil to Enhance Tacrolimus Production

Silva

Ferrari

Moreira

et al. 2019

J Apple Biotechnol Rep

Introduction Tacrolimus, also known as FK-506, is a drug first isolated in 1984, that has been widely used to prevent the rejection of transplanted organs, and to also treat dermatoses and eye diseases. 1-3 It is a 23 membered polyketide macrolide produced during fermentation through different Streptomyces species. The most commonly used is the Streptomyces tsukubaensis. 4 This medication is interesting to dermatology because it is rapidly absorbed by the epidermis and has less side effects than commonly used drugs. 5 Besides, as an immunosuppressant agent it is 10-100 times more potent than cyclosporine. 6 Nevertheless, the treatment cost using tacrolimus is high because of its low productivity. 7 Up to now, different strategies have been used to solve this problem, including the use of mutant bacteria, 8-11 and exogenous feeding of precursors in the biosynthesis. 12-14 Literature reports that S. tsukubaensis is capable of metabolizing different carbon sources, such as glucose, maltose, dextrin, soybean oil, cottonseed oil and groundnut oil. 14-16 Exogenous feeding of oil has played an important role in this bioprocess, since it is a complex source of nutrients which has increased the tacrolimus production. 12,14,17 The Brazil nut oil is rich in fatty acids and are broken in order to produce coenzymes (acetyl or propionyl) which are known as precursors for the polyketide production. 17 This study has investigated the influence of the Brazil nut (Bertholletia excelsa) oil on the tacrolimus production. The fermentative process was carried for 168 h using S. tsukubaensis. Materials and Methods Organism The Strain of S. tsukubaensis was acquired from the Leibniz Institute DSMZ. Culture Conditions Streptomyces tsukubaensis culture was maintained on petri dishes containing glucose 4 g.L-1 , yeast extract 1 g.L-1 , malt extract 10 g.L-1 , CaCO 3 2 g.L-1 and agar 12 g.L-1 , pH 7.2, and incubated at 28 o C. The seed culture medium and the production medium were based on Turlo et al 14 which are presented in Table 1 and Table 2, respectively. The seed culture medium was prepared, sterilized at 120 o C