Engineering of Streptoalloteichus tenebrarius 2444 for Sustainable Production of Tobramycin.

Tobramycin is a broad-spectrum aminoglycoside antibiotic agent. The compound is obtained from the base-catalyzed hydrolysis of carbamoyltobramycin (CTB), which is naturally produced by the actinomycete Streptoalloteichus tenebrarius. However, the strain uses the same precursors to synthesize several structurally related aminoglycosides. Consequently, the production yields of tobramycin are low, and the compound's purification is very challenging, costly, and time-consuming. In this study, the production of the main undesired product, apramycin, in the industrial isolate Streptoalloteichus tenebrarius 2444 was decreased by applying the fermentation media M10 and M11, which contained high concentrations of starch and dextrin. Furthermore, the strain was genetically engineered by the inactivation of the aprK gene (∆aprK), resulting in the abolishment of apramycin biosynthesis. In the next step of strain development, an additional copy of the tobramycin biosynthetic gene cluster (BGC) was introduced into the ∆aprK mutant. Fermentation by the engineered strain (∆aprK_1-17L) in M11 medium resulted in a 3- to 4-fold higher production than fermentation by the precursor strain (∆aprK). The phenotypic stability of the mutant without selection pressure was validated. The use of the engineered S. tenebrarius 2444 facilitates a step-saving, efficient, and, thus, more sustainable production of the valuable compound tobramycin on an industrial scale.

Concentrated biosynthesis of tobramycin by genetically engineered Streptomyces tenebrarius.

Tobramycin is an important broad spectrum aminoglycoside antibiotic widely used against severe Gram-negative bacterial infections. It is produced by base-catalyzed hydrolysis of carbamoyltobramycin (CTB) generated by S. tenebrarius. We herein report the construction of a genetically engineered S. tenebrarius for direct fermentative production of tobramycin by disruption of aprK and tobZ. A unique putative NDP-octodiose synthase gene aprK was disrupted to optimize the production of CTB, resulting in the blocking of apramycin biosynthesis and the obvious increase in CTB production of aprK disruption mutant S. tenebrarius ST316. Additional mutation on the carbamoyltransferase gene tobZ in S. tenebrarius ST316 generated a strain ST318 that produces tobramycin as a single metabolite. ST318 could be used for industrial fermentative production of tobramycin.

Engineering Streptomyces tenebrarius to synthesize single component of carbamoyl tobramycin.

AIMS: To engineer Streptomyces tenebrarius for producing carbamoyl tobramycin as a main component. METHODS AND RESULTS: The aprH-M gene fragment (apramycin biosynthetic gene from GenBank) in S. tenebrarius Tt49 was knocked out by genetic engineering to form S. tenebrarius T106 (ΔaprH-M). Compared to the wild-type strain, mutant strain T106 (ΔaprH-M) no longer produced apramycin, while mainly synthesize carbamoyl tobramycin. TLC and HPLC-MS analyses indicated that the mutant strain significantly increased the production of carbamoyl tobramycin. CONCLUSIONS: The metabolic flow for the apramycin and its analogues biosynthesis was blocked by disrupting the aprH-M gene clusters. The aprH-M gene clusters might be essential for the biosynthesis of apramycin. The mutant strain T106 mainly synthesized carbamoyl tobramycin. SIGNIFICANCE AND IMPACT OF STUDY: The mutant T106 mainly produces carbamoyl tobramycin without synthesizing apramycin, which will reduce cost of postextraction from fermentation products. Therefore, it has good prospects for industrial application.

Identification and functional analysis of dTDP-glucose-4,6-dehydratase gene and its linked gene cluster in an aminoglycoside antibiotics producer of Streptomyces tenebrarius H6.

Streptomyces tenebrarius H6 produces a variety of aminoglycoside antibiotics, such as apramycin, tobramycin, and kanamycin B. Primers were designed according to the highly conserved sequences of the dTDP-glucose-4,6-dehydratase genes, and a 0.6-kb PCR product was obtained from S. tenebrarius H6 genomic DNA. With the 0.6-kb PCR product as a probe, a BamHI 7.0-kb fragment was isolated. DNA sequence analysis of the 7.0-kb fragment revealed four ORFs and an incomplete ORF. In search of databases, the deduced product of one ORF (orfE) showed 62% identity to the dTDP-glucose-4,6-dehydratase, StrE of S. griseus. Three other ORFs (orfG1, orfG2, and orfGM) showed 55%, 62%, and 42% similarities, respectively, to glycosyltransferase from Clostridium acetobutylicum and mannosyltransferase from Xanthomonas axonopodis pv. citri str. 306 and glycosyltransferase from Pseudomonas putida KT2440. Upstream of the orfE was an incomplete ORF, and the deduced product showed 56% similarity to dTDP-4-dehydrorhamnose, StrL from S. griseus. The function of the orfE gene was studied by targeted gene disruption. The resulting mutant failed to produce tobramycin and kanamycin B, but still produced apramycin, suggesting that the orfE gene and linked gene cluster are essential for the biosynthesis of tobramycin and kanamycin B in S. tenebrarius H6.

Isolation and characterization of the tobramycin biosynthetic gene cluster from Streptomyces tenebrarius.

The biosynthetic gene cluster for tobramycin, a 2-deoxystreptamine-containing aminoglycoside antibiotic, was isolated from Streptomyces tenebrarius ATCC 17920. A genomic library of S. tenebrarius was constructed, and a cosmid, pST51, was isolated by the probes based on the core regions of 2-deoxy-scyllo-inosose (DOI) synthase, and L-glutamine:DOI aminotransferase and L-glutamine:scyllo-inosose aminotransferase. Sequencing of 33.9 kb revealed 24 open reading frames (ORFs) including putative tobramycin biosynthetic genes. We demonstrated that one of these ORFs, tbmA, encodes DOI synthase by in vitro enzyme assay of the purified protein. The catalytic residues of TbmA and dehydroquinate synthase were studied by homology modeling. The gene cluster found is likely to be involved in the biosynthesis of tobramycin.

[Fermentative hydrolysate of mycelial waste of aminoglycoside antibiotics production as component of culture media used in biosynthesis of tobramycin]. / Fermentativnyi gidrolizat mitselial'nykh otkhodov proizvodstva aminoglikozidnykh antibiotikov kak komponent pitatel'nykh sred, ispol'zuemykh pri biosinteze tobramitsina.

New nutrient media for cultivation of the tobramycin-producing organism were developed. As an additional source of nitrogen the media contain fermentative hydrolysate of the mycelial waste of manufacture of aminoglycoside antibiotics (tobramycin and apramycin). The use of the media provided a 20 to 50% decrease of consumption of soybean meal, an essential food raw material, and design of a low-waste technology for biosynthesis of tobramycin.

Physiology and genetics of antibiotic production and resistance.

Actinomycetes have the genetic capability to synthesize many different biologically active secondary metabolites and of these compounds, antibiotics predominate in therapeutic and commercial importance. Intensive research often centres on the use of molecular techniques to investigate the physiology and genetics of antibiotic biosynthesis with a view to improving production. The isolation of clones of Streptomyces hygroscopicus, the producer of geldanamycin, which synthesizes geldanamycin in S. lividans, is reported. Molecular approaches using genes for elongation factors (tuf) were used in attempts to increase the fermentation yield of kirromycin, whilst probes for aphD and sph, genes for streptomycin phosphotransferases, were used to gather information on streptomycin genes in soil. Actinomycete populations in soil and earthworms may help in developing a strategy for discovering additional antimicrobials in soil. The relationship of proline metabolism to the secondary metabolite undecylprodigiosin and the carbon regulation of spiramycin biosynthesis in S. ambofaciens is also reported.

[Identification of idiotrophs for 3'-deoxykanamycin B synthesis in Streptomyces tenebrarius (Higgens a. Kastner)]. / Identifikatsiia idiotrofov po sintezu 3'-dezoksikanamitsina B u Streptomyces tenebrarius (Higgens a. Kastner).

Mutants of Streptomyces tenebrarius with the blocked synthesis of 3'-deoxykanamycin B were obtained by treating the producer with NTG and chloramphenicol, or after gamma-irradiation. These mutants (idiotrophs) were distributed into three groups by means of the cosynthesis experiments on agar plates: convertors, secretors and "neutral" strains. Five idiotrophs represented five complementation groups for biosynthesis of the antibiotic. Three of these were defective in 2-deoxystreptamine synthesis, the fourth was defined as neamine-negative, and the fifth was probably blocked in regulation of enzymes responsible for conversion of neamine or paromamine into kanamycins. Localization of mutations has been shown on the scheme of kanamycins' biosynthesis.

[Stability of the antibiotic formation trait of the tobramycin producer Streptomyces cremeus subsp. tobramycini]. / Izuchenie stabil'nosti produtsenta tobramitsina Streptomyces cremeus subsp. tobramycini po priznaku antibiotikoobrazovaniia.

The stability of the tobramycin-producing organism was studied by the property of the antibiotic production. The organism was stored under conditions of different exposures to light and subculture on slants. The culture was also subjected to long-term storage with various methods. The organism was stable in preserving its antibiotic activity for 1.5 months when stored on the Gauze organic agar No. 2. Subcultures of the organism on this medium provided preservation of the antibiotic activity throughout 4 passages. The culture storage on millet provided preservation of the antibiotic activity for 6 months.

[Effect of culture parameters on tobramycin biosynthesis]. / Vliianie nekotorykh parametrov kul'tivirovaniia na biosintez tobramitsina.

The effect of temperature, aeration and active acidity of the medium on biosynthesis of tobramycin was studied. The optimal temperature conditions (37 degrees C) were developed. It was shown that biosynthesis of tobramycin depended on the aeration conditions, especially at the beginning of the fermentation process. The initial pH 4.9-6.35 and 6.35-7.1 were found to be optimal for the growth of the tobramycin-producing organism and biosynthesis of the antibiotic, respectively.

[Effect of several components of the nutrient medium on the biosynthesis of tobramycin]. / Vliianie nekotorykh komponentov pitatel'noi sredy na biosintez tobramitsina.

The optimal concentrations of mineral phosphorus for the growth of the tobramycin-producing organism and for the production of the antibiotic by it in the synthetic medium were determined. Introduction of an additional source of mineral phosphorus into the rich soybean medium resulted in decreased levels of antibiotic production. The stimulating effect of manganese sulfate on the biosynthesis of tobramycin in the rich medium was shown. The stimulating effect of soybean, linseed and palm oils on the production of tobramycin was evident when the fermentation period was longer.

[Optimization of the nutrient medium composition for the biosynthesis of the antibiotic tobramycin using a mathematical experimental design method]. / Optimizatsiia sostava pitatel'noi sredy dlia biosinteza antibiotika tobramitsina s pomoshch'iu matematicheskogo metoda planirovaniia éksperimenta.

The mathematical design of the experiment was used for optimization of the nutrient medium for biosynthesis of tobramycin by Streptomyces cremeus var. tobramycini. The use of the orthogonal Latin rectangles provided development of the fermentation medium composition and definition of the optimal concentration of its ingredients. The level of tobramycin production on this medium was 2 times higher than that on the initial one.

[Effect of the inoculum on tobramycin biosynthesis]. / Vliianie posevnogo materiala na biosintez tobramitsina.

The effect of the quality and quantity of the inoculum on the biosynthesis of tobramycin was studied. The medium with corn steep liquor and glycerol was found to be the optimal seed medium for the tobramycin-producing organism. It is required that the fermentation medium be supplemented with 2 per cent of the vegetative inoculum at the stationary growth phase or with an increased amount of the seed material at the exponential growth phase or at the early stage of the culture dying. Subculturing of the inoculum decreases the level of tobramycin production at the fermentation stage: when the culture of the 5th--6th generation is used, the activity of the fermentation broth decreases by 30--50 per cent.

[Obtaining mutants that yield tobramycin in a culture of the producer of a complex of aminoglycoside antibiotics, Streptomyces cremeus var. tobramycini var. nov. 2242]. / Poluchenie mutantov, obrazuiushchikh tobramitsin, v kul'ture produtsenta kompleksa aminoglikozidnykh antibiotikov Streptomyces cremeus var. tobramycini var. nov. 2242.

The organism producing an aminoglycoside antibiotic complex was subjected to selection with a purpose of obtaining a strain producing tobramycin. Spontaneous and gamma-radiation-induced variation of the strains obtained as a result of a step-wise selection of the tobramycin-producing organism was studied. Correlation between the antibiotic activity levels and morphological features of these strains was shown. 8 physiological groups of mutants were differentiated and characterized as dependent on the ratio of the components in the aminoglycoside antibiotic complex produced by them. A highly productive strain 3406 was selected. The level of tobramycin synthesis by this strain is 10 times higher than that of the initial culture.

[Synthetic medium for the biosynthesis of tobramycin]. / Sinteticheskaia sreda dlia biosinteza tobramitsina.

The effect of the sources of carbon and inorganic nitrogen on the biosynthesis of tobrmycin complex was studied. Maltose and ammonium chloride were shown tobe optimal. A synthetic medium for Streptomyces cremeus var. nov. 2242, to tobramyc in producing organism, was elaborated and its optimization was performed using the experiment mathematical modelling, i. e. the method of latin squares. Tobramycin production on the new medium was 6 to 7 times higher than that on the initial medium.