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.

Two Cryptic Self-Resistance Mechanisms in Streptomyces tenebrarius Reveal Insights into the Biosynthesis of Apramycin.

Apramycin is a clinically promising aminoglycoside antibiotic (AGA). To date, mechanisms underlying the biosynthesis and self-resistance of apramycin remain largely unknown. Here we report that apramycin biosynthesis proceeds through unexpected phosphorylation, deacetylation, and dephosphorylation steps, in which a novel aminoglycoside phosphotransferase (AprU), a putative creatinine amidohydrolase (AprP), and an alkaline phosphatase (AprZ) are involved. Biochemical characterization revealed that AprU specifically phosphorylates 5-OH of a pseudotrisaccharide intermediate, whose N-7' acetyl group is subsequently hydrolyzed by AprP. AprZ is located extracellularly where it removes the phosphate group from a pseudotetrasaccharide intermediate, leading to the maturation of apramycin. Intriguingly, 7'-N-acetylated and 5-O-phosphorylated apramycin that were accumulated in ΔaprU and ΔaprZ respectively exhibited significantly reduced antibacterial activities, implying Streptomyces tenebrarius employs C-5 phosphorylation and N-7' acetylation as two strategies to avoid auto-toxicity. Significantly, this study provides insight into the design of new generation AGAs to circumvent the emergence of drug-resistant pathogens.

Characterization of a C3 Deoxygenation Pathway Reveals a Key Branch Point in Aminoglycoside Biosynthesis.

Apramycin is a clinically interesting aminoglycoside antibiotic (AGA) containing a highly unique bicyclic octose moiety, and this octose is deoxygenated at the C3 position. Although the biosynthetic pathways for most 2-deoxystreptamine-containing AGAs have been well characterized, the pathway for apramycin biosynthesis, including the C3 deoxygenation process, has long remained unknown. Here we report detailed investigation of apramycin biosynthesis by a series of genetic, biochemical and bioinformatical studies. We show that AprD4 is a novel radical S-adenosyl-l-methionine (SAM) enzyme, which uses a noncanonical CX3CX3C motif for binding of a [4Fe-4S] cluster and catalyzes the dehydration of paromamine, a pseudodisaccharide intermediate in apramycin biosynthesis. We also show that AprD3 is an NADPH-dependent reductase that catalyzes the reduction of the dehydrated product from AprD4-catalyzed reaction to generate lividamine, a C3' deoxygenated product of paromamine. AprD4 and AprD3 do not form a tight catalytic complex, as shown by protein complex immunoprecipitation and other assays. The AprD4/AprD3 enzyme system acts on different pseudodisaccharide substrates but does not catalyze the deoxygenation of oxyapramycin, an apramycin analogue containing a C3 hydroxyl group on the octose moiety, suggesting that oxyapramycin and apramycin are partitioned into two parallel pathways at an early biosynthetic stage. Functional dissection of the C6 dehydrogenase AprQ shows the crosstalk between different AGA biosynthetic gene clusters from the apramycin producer Streptomyces tenebrarius, and reveals the remarkable catalytic versatility of AprQ. Our study highlights the intriguing chemistry in apramycin biosynthesis and nature's ingenuity in combinatorial biosynthesis of natural products.

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.

Glycine origin of the methyl substituent on C7'-N of octodiose for the biosynthesis of apramycin.

Apramycin is unique in the aminoglycoside family due to its octodiose moiety. However, either the biosynthesis process or the precursors involved are largely unknown. Addition of glycine, as well as serine or threonine, to the Streptomyces tenebrabrius UD2 fermentation medium substantially increases the production of apramycin with little effect on the growth of mycelia, indicating that glycine and/or serine might be involved in the biosynthesis of apramycin. The 13C-NMR analysis of [2-13C] glycine-fed (25% enrichment) apramycin showed that glycine specifically and efficiently incorporated into the only N-CH3 substituent of apramycin on the C7' of the octodiose moiety. We noticed that the in vivo concentration of S-adenosyl methionine increased in parallel with the addition of glycine, while the addition of methione in the fermentation medium significantly decreased the productivity of apramycin. Therefore, the methyl donor function of glycine is proposed to be involved in the methionine cycle but methionine itself was proposed to inhibit the methylation and methyl transfer processes a previously reported for the case of rapamycin. The 15N NMR spectra of [2-13C,15N]serine labeled apramycin indicated that serine may also act as a limiting precursor contributing to the -NH2 substituents of apramycin.

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.

[Development of new technology for isolation of nebramycin complex antibiotics from the cultural fluid]. / Razrabotka novoi tekhnologii vydeleniia antibiotikov nebramitsinovogo kompleksa iz kurtural'noi zhidkosti.

A new technology for nebramycin complex antibiotics isolation from non-filtered culture fluid was developed. The industrial use of the technology permitted to exclude the stage of the culture fluid filtration, to increase the yield by more than 33% and to reduce the time of the process by 1.7 times.

[Cloning of the sugar related biosynthesis gene cluster from Streptomyces tenebrarius H6].

Streptomyces tenebrarius H6 produces a complex of aminoglycoside antibiotics, such as apramycin, tobramycin and kanamycin B etc. To study the apramycin biosynthetic genes the genomic library from the Streptomyces tenebrarius H6 was established using E. coli/Streptomyces shuttle vector pKC505 by in vitro packing. The probability of finding a specific gene from the library composed of 3,000 colonies was over 99.9%. According to the highly conserved sequence of the genes involved in 6-deoxyhexose biosynthesis, primers were designed and 0.6 kb fragment homologous to strE gene was obtained by PCR. 30 positive clones were found from the genomic library of S. tenebrarius H6 with the 0.6 kb fragment as a probe. Overlapped regions were localized by Southern hybridization and putative sugar related biosynthetic gene cluster was mapped by restriction enzyme digestions. An ORF of dTDP-glucose-4,6-dehydratase gene consisted of 1,132 bp, designated as aprE, was obtained and submitted to GenBank under the accession number of AF306787. A DNA sequence highly homologous to strL coding dTDP-4-dehydrorhamnose reductase was found linked with aprE gene.

[Effect of protoplast formation and regeneration on the production of nebramycin complex in Streptomyces cremeus subsp. tobramycini]. / Vliianie obrazovaniia i regeneratsii protoplastov na produktsiiu nebramitsinovogo kompleksa u Streptomyces cremeus subsp. tobramycini.

Production of the nebramycin complex in Streptomyces cremeus subsp. tobramycini before and after the protoplast formation and regeneration was comparatively studied. The antibiotic production was estimated by the total activity and component composition of the nebramycin complex. It was found that formation and regeneration of the protoplast led to lowering of the activity and changing of the complex component composition. Strains mainly synthesizing each one of the three basic components of the nebramycin complex were isolated. The strains proved to be unstable by the antibiotic production property and after three subcultures lost the differences in the complex component composition.

Regulation of nebramycin biosynthesis by inorganic phosphate.

The effect of inorganic phosphate on the biosynthesis of nebramycin factors 2, 4 and 5' was studied in Streptomyces tenebrarius strain A (forming 2, 4 and 5' in natural ratios) and its mutants B (forming predominantly 2), C (forming 2 as the only major product) and D (forming predominantly 5'). In phosphate-supplemented complex media, the production of 2 in A, B and C was reduced by 20-70%, while the yields of 5' remained unchanged in A and decreased by 30-60% in B. The production of 4 increased by 50-90% in A and was fully suppressed in B. In D the biosynthesis of the three factors was inhibited completely.

[Effect of antibiotic resistance on the appearance of mutants in a culture of Streptomyces cremeus subsp. tobramycini producing an aminoglycoside complex]. / Vliianie ustoichivosti k antibiotikam na vozniknovenie mutantov v kul'ture Streptomyces cremeus subsp. tobramycini, obrazuiushchei kompleks aminoglikozidov.

Possible formation of auxotrophs and changing of the antibiotic production property connected with resistance to antibiotics of different modes of action were studied in Streptomyces cremeus subsp. tobramycini producing the nebramycin complex of 2-desoxystreptamine derivatives. Four hundred and five spontaneous and 1800 gamma-radiation induced antibiotic resistant mutants of the culture were studied. The frequency of the auxotrophs was shown to be increasing. Correlation between formation of strains producing monocomponent aminoglycosides and antibiotic resistance was observed. The frequency of mutants with preferable synthesis of the tobramycin component among strr-, rifr- and rubr-mutants was 3--10 times higher than among the sensitive portion of the population when total selection was used. Therefore, the spontaneous mutation of antibiotic resistance is selective with respect to both isolation of auxotrophs and strains producing separate aminoglycoside antibiotics.

Mechanism of resistance to aminoglycoside antibiotics in nebramycin-producing Streptomyces tenebrarius.

Streptomyces tenebrarius ISP 5477, which produces nebramycins, was highly resistant to the following aminoglycoside antibiotics: neamine, ribostamycin, butirosin A, neomycin B, paromomycin, kanamycin A, dibekacin, gentamicin C complex, lividomycin A, istamycin B and streptomycin. Polyphenylalanine synthesis on the ribosomes of this strain was highly resistant to neamine, ribostamycin, butirosin A, kanamycins A, B and C, dibekacin, gentamicin C complex and istamycin B, moderately resistant to lividomycin A and streptomycin, but sensitive to neomycin B and paromomycin. Moreover, cell free extract of the strain contained phosphotransferase and N-acetyltransferase. The former enzyme was confirmed to be an aminoglycoside 6-phosphotransferase which inactivated streptomycin; the latter inactivated kanamycins B and C, dibekacin, neamine, neomycin B, paromomycin, lividomycin A, butirosin A and ribostamycin, but did not inactivate kanamycin A, gentamicin C complex and sagamicin, suggesting an aminoglycoside 2'-acetyltransferase. These results indicated that the high resistance of S. tenebrarius ISP 5477 to a wide range of aminoglycoside antibiotics is due to ribosomal resistance and to the inactivating enzymes, aminoglycoside N-acetyltransferase(s) and aminoglycoside 6-phosphotransferase.

[Antibiotic formation from a pyrimidine base group and an aminoglycoside group by a Streptomyces coeruleoaurantiacus sp. nov. culture]. / Obrazovanie antibiotikov iz gruppy pirimidinovykh osnovanii i gruppy aminoglikozidov kul'turoi Streptomyces coeruleoaurantiacus sp. nov.

An actinomycete strain 4009 was isolated from a soil sample collected in Volgograd. The strain showed a broad antibacterial spectrum. It produced antibiotics belonging to 2 different groups of chemical compounds, i.e. amycetin from the group of pyrimidine bases and nebramycin from the group of aminoglycosides. The antibiotic-producing organism is described as a type strain of Streptomyces coeruleoaurantiacus sp. nov. The new species is characterized by spiral spore chains, wart-like spore surface, blue aerial mycelium and dark yellow, orange-yellow or brown-orange-yellow substrate mycelium, the absence of soluble pigments on synthetic media and secretion of melanin pigments on the Tresner medium. Various conditions for submerged production of the antibiotic were studied.