Carbon-free production of 2-deoxy-scyllo-inosose (DOI) in cyanobacterium Synechococcus elongatus PCC 7942.

Owing to their photosynthetic capabilities, there is increasing interest in utilizing cyanobacteria to convert solar energy into biomass. 2-Deoxy-scyllo-inosose (DOI) is a valuable starting material for the benzene-free synthesis of catechol and other benzenoids. DOI synthase (DOIS) is responsible for the formation of DOI from d-glucose-6-phosphate (G6P) in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics such as neomycin and butirosin. DOI fermentation using a recombinant Escherichia coli strain has been reported, although a carbon source is necessary for high-yield DOI production. We constructed DOI-producing cyanobacteria toward carbon-free and sustainable DOI production. A DOIS gene derived from the butirosin producer strain Bacillus circulans (btrC) was introduced and expressed in the cyanobacterium Synechococcus elongatus PCC 7942. We ultimately succeeded in producing 400 mg/L of DOI in S. elongatus without using a carbon source. DOI production by cyanobacteria represents a novel and efficient approach for producing benzenoids from G6P synthesized by photosynthesis.

X-ray analysis of butirosin biosynthetic enzyme BtrN redefines structural motifs for AdoMet radical chemistry.

The 2-deoxy-scyllo-inosamine (DOIA) dehydrogenases are key enzymes in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics. In contrast to most DOIA dehydrogenases, which are NAD-dependent, the DOIA dehydrogenase from Bacillus circulans (BtrN) is an S-adenosyl-l-methionine (AdoMet) radical enzyme. To examine how BtrN employs AdoMet radical chemistry, we have determined its structure with AdoMet and substrate to 1.56 Å resolution. We find a previously undescribed modification to the core AdoMet radical fold: instead of the canonical (ß/α)6 architecture, BtrN displays a (ß5/α4) motif. We further find that an auxiliary [4Fe-4S] cluster in BtrN, thought to bind substrate, is instead implicated in substrate-radical oxidation. High structural homology in the auxiliary cluster binding region between BtrN, fellow AdoMet radical dehydrogenase anSME, and molybdenum cofactor biosynthetic enzyme MoaA provides support for the establishment of an AdoMet radical structural motif that is likely common to ~6,400 uncharacterized AdoMet radical enzymes.

Epimerization at C-3'' in butirosin biosynthesis by an NAD(+) -dependent dehydrogenase BtrE and an NADPH-dependent reductase BtrF.

Butirosin is an aminoglycoside antibiotic consisting two epimers at C-3'' of ribostamycin/xylostasin with a unique 4-amino-2-hydroxybutyrate moiety at C-1 of the aminocyclitol 2-deoxystreptamine (2DOS). To date, most of the enzymes encoded in the biosynthetic gene cluster for butirosin, from the producing strain Bacillus circulans, have been characterized. A few unknown functional proteins, including nicotinamide adenine dinucleotide cofactor-dependent dehydrogenase/reductase (BtrE and BtrF), are supposed to be involved in the epimerization at C-3'' of butirosin B/ribostamycin but remain to be characterized. Herein, the conversion of ribostamycin to xylsostasin by BtrE and BtrF in the presence of NAD(+) and NADPH was demonstrated. BtrE oxidized the C-3'' of ribostamycin with NAD(+) to yield 3''-oxoribostamycin. BtrF then reduced the generated 3''-oxoribostamycin with NADPH to produce xylostasin. This reaction step was the last piece of butirosin biosynthesis to be described.

Biosynthesis of butirosin: transfer and deprotection of the unique amino acid side chain.

Butirosin, an aminoglycoside antibiotic produced by Bacillus circulans, bears the unique (S)-4-amino-2-hydroxybutyrate (AHBA) side chain, which protects the antibiotic from several common resistance mechanisms. The AHBA side chain is advantageously incorporated into clinically valuable antibiotics such as amikacin and arbekacin by synthetic methods. Therefore, it is of significant interest to explore the biosynthetic origins of this useful moiety. We report here that the AHBA side chain of butirosin is transferred from the acyl carrier protein (ACP) BtrI to the parent aminoglycoside ribostamycin as a gamma-glutamylated dipeptide by the ACP:aminoglycoside acyltransferase BtrH. The protective gamma-glutamyl group is then cleaved by BtrG via an uncommon gamma-glutamyl cyclotransferase mechanism. The application of this pathway to the in vitro enzymatic production of novel AHBA-bearing aminoglycosides is explored with encouraging implications for the preparation of unnatural antibiotics via directed biosynthesis.

Characterization and mechanistic study of a radical SAM dehydrogenase in the biosynthesis of butirosin.

BtrN encoded in the butirosin biosynthetic gene cluster possesses a CXXXCXXC motif conserved within the radical S-adenosyl methionine (SAM) superfamily. Its gene disruption in the butirosin producer Bacillus circulans caused the interruption of the biosynthetic pathway between 2-deoxy-scyllo-inosamine (DOIA) and 2-deoxystreptamine (DOS). Further, in vitro assay of the overexpressed enzyme revealed that BtrN catalyzed the oxidation of DOIA under the strictly anaerobic conditions along with consumption of an equimolar amount of SAM to produce 5'-deoxyadenosine, methionine, and 3-amino-2,3-dideoxy-scyllo-inosose (amino-DOI). Kinetic analysis showed substrate inhibition by DOIA but not by SAM, which suggests that the reaction is the Ordered Bi Ter mechanism and that SAM is the first substrate and DOIA is the second. The BtrN reaction with [3-2H]DOIA generated nonlabeled, monodeuterated and dideuterated 5'-deoxyadenosines, while no deuterium was incorporated by incubation of nonlabeled DOIA in the deuterium oxide buffer. These results indicated that the hydrogen atom at C-3 of DOIA was directly transferred to 5'-deoxyadenosine to give the radical intermediate of DOIA. Generation of nonlabeled and dideuterated 5'-deoxyadenosines proved the reversibility of the hydrogen abstraction step. The present study suggests that BtrN is an unusual radical SAM dehydrogenase catalyzing the oxidation of the hydroxyl group by a radical mechanism. This is the first report of the mechanistic study on the oxidation of a hydroxyl group by a radical SAM enzyme.

Biosynthesis of the unique amino acid side chain of butirosin: possible protective-group chemistry in an acyl carrier protein-mediated pathway.

Butirosins A and B are naturally occurring aminoglycoside antibiotics that have a (2S)-4-amino-2-hydroxybutyrate (AHBA) side chain. Semisynthetic addition of AHBA to clinically valuable aminoglycoside antibiotics has been shown both to improve their pharmacological properties and to prevent their deactivation by a number of aminoglycoside-modifying enzymes involved in bacterial resistance. We report here that the biosynthesis of AHBA from L-glutamate, encoded within a previously identified butirosin biosynthetic gene cluster, proceeds via intermediates tethered to a specific acyl carrier protein (ACP). Five components of the pathway have been purified and characterized, including the ACP (BtrI), an ATP-dependent ligase (BtrJ), a pyridoxal phosphate-dependent decarboxylase (BtrK), and a two-component flavin-dependent monooxygenase system (BtrO and the previously unreported BtrV). The proposed biosynthetic pathway includes a gamma-glutamylation of an ACP-derived gamma-aminobutyrate intermediate, possibly a rare example of protective group chemistry in biosynthesis.

The ribostamycin biosynthetic gene cluster in Streptomyces ribosidificus: comparison with butirosin biosynthesis.

A cluster of genes for ribostamycin (Rbm) biosynthesis was isolated from Streptomyces ribosidificus ATCC 21294. Sequencing of 31.892 kb of the genomic DNA of S. ribosidificus revealed 26 open reading frames (ORFs) encoding putative Rbm biosynthetic genes as well as resistance and other genes. One of ten putative Rbm biosynthetic genes, rbmA, was expressed in S. lividans TK24, and shown to encode 2-deoxy-scyllo-inosose (DOI) synthase. Acetylation of various aminoglycoside-aminocyclitol (AmAcs) by RbmI confirmed it to be an aminoglycoside 3-N-acetyltransferase. Comparison of the genetic control of ribostamycin and butirosin biosynthesis pointed to a common biosynthetic route for these compounds, despite the considerable differences between them in genetic organization.

Extended sequence and functional analysis of the butirosin biosynthetic gene cluster in Bacillus circulans SANK 72073.

Butirosin produced by Bacillus circulans is among the clinically important 2-deoxystreptamine containing aminoglycoside antibiotics and its unique structure is found in (S)-4-amino-2-hydroxyburyric acid substituted at C-1 of 2-deoxystreptamine. Recently, the key part of the butirosin biosynthetic gene cluster has been identified from Bacillus circulans SANK 72073, however the whole gene for the biosynthesis awaited for identification. In the present study, we undertook extended analysis of the butirosin biosynthetic gene cluster and found nine additional open reading flames (ORFs), btrQ, btrR1, btrR2, btrT, btrU, btrV, btrW, btrX and orf1 in the cluster. In addition, we constructed disruption mutants of btrR1 and btrP-V, and found that the btr genes (ca. 24Kb) between btrR1 and btrP-V are at least required for the butirosin biosynthesis.

Biosynthesis of butirosin in Bacillus circulans NRRL B3312: identification by sequence analysis and insertional mutagenesis of the butB gene involved in antibiotic production.

As an approach to an analysis of the biosynthesis of the aminoglycoside antibiotic butirosin (But), we investigated the chromosomal regions flanking the ButR gene (aphA4/butA) of Bacillus circulans NRRL-B3312, and have identified, by nucleotide sequence analysis, a large open reading frame (ORF; ButB) upstream from the ButR gene. Hybridization was detected between butB and chromosomal DNA from other Bacillaceae that produce But-like compounds (but not from non-producers). Interruption of this sequence by insertion of an erythromycin-resistance-encoding gene (erm) at either of two distinct sites eliminated the production (biosynthesis or export) of But, thus indicating a role for butB in antibiotic production. Gene butB is transcribed in the same direction as butA and encodes a protein of 1616 amino acid (aa) residues with a 30-aa N-terminal signal peptide. Comparison of the sequence for the translation product (ButB) with the aa compositions and sequences of known bacterial surface proteins, such as S-layer proteins, suggests that this protein is cell-wall associated. It is proposed that ButB plays a role in the export of But from the producing organism.

The sequence of an antibiotic resistance gene from an antibiotic-producing bacterium. Homologies with transposon genes.

The APH gene of a butirosin-producing Bacillus circulans has been cloned and sequenced; a comparison of the translated protein sequence with those from TN5 and TN903 indicates that they may have a common origin.

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.

Purification and characterization of 2-deoxy-scyllo-inosose synthase derived from Bacillus circulans. A crucial carbocyclization enzyme in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics.

The biosynthesis of 2-deoxystreptamine, the central aglycon of a major group of clinically important aminoglycoside antibiotics, commences with the initial carbocycle formation step from D-glucose-6-phosphate to 2-deoxy-scyllo-inosose. This crucial step is known to be catalyzed by 2-deoxy-scyllo-inosose synthase, which has not yet been characterized so far. Reported in this paper is the first purification of 2-deoxy-scyllo-inosose synthase from butirosin-producing Bacillus circulans SANK 72073 to electrophoretic homogeneity. The enzyme was isolated as a heterodimeric protein comprising from a 23 kDa- and a 42 kDa polypeptide chains. The Km of the enzyme for D-glucose-6-phosphate was estimated to be 9.0 x 10(-4) M and that for NAD+ 1.7 x 10(-4) M, kcat for D-glucose-6-phosphate being 7.3 x 10(-2) s(-1). The presence of Co2+ was essential for the enzyme activity, but Zn2+ was totally inhibitory. While the reaction mechanisms are quite similar, 2-deoxy-scyllo-inosose synthase appears to be distinct from dehydroquinate synthase in the shikimate pathway, with respect to the quaternary structure, metal ion requirement, and the kinetic parameters.

Aminoglycoside 3'-phosphotransferases I and II in Pseudomonas aeruginosa.

Aminoglycoside 3'-phosphotransferases I and II in three strains of Pseudomonas aeruginosa were studied in comparison with those in two strains of R factor-carrying Escherichia coli. The strain TI-13 of P. aeruginosa produced the former and strain H-9 the latter. Strain B-13 produced the both enzymes. The 3'-phosphotransferases of type I in P. aeruginosa TI-13, B-13 and E. coli K12 J5 R11-2 were different from each other in chromatographic behavior, molecular weight, pH optimum, and Ii. The 3'-phosphotransferase of type II in P. aeruginosa H-9 and E. coli JR66/W677 showed the same behavior.

Ribostamycin production by a mutant of butirosin producing bacteria.

By the use of our improved colony selection technique, xylostasin and ribostamycin producing mutants were isolated from nitrosoguanidine treated Bacillus circulans B15M, a producer of butirosins A and B. Among these structurally related aminoglycosides, ribostamycin is the well-known product of a Steptomyces and has not been isolated as a bacterial metabolite. A selected mutant of strain 306, which produces xylostasin and ribostamycin, was futher mutagenized in expectation of getting an improved strain having the ability to accumulate a large amount of ribostamycin in the culture broth. One mutant, strain 451, derived from strain 306, produced ribostamycin free of xylostasin.

A plasmid which does not encode the aminoglycoside phosphotransferase in the butirosin-producing strain of Bacillus circulans.

Bacillus circulans NRRL B-3312 produces the aminoglycoside antibiotic butirosin and encodes an aminoglycoside 3'-phosphotransferase. We detected a 48 kilobase plasmid, pIP850, in this strain; this was analyzed by agarose gel electrophoresis following digestion with EcoRI restriction endonuclease and by nucleic acid hybridization. The results obtained indicate that plasmid pIP850 does not carry the structural gene for the aminoglycoside modifying enzyme.

Butirosin-biosynthetic gene cluster from Bacillus circulans.

Butirosin is an interesting 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotic produced by non-actinomycete Bacilli. Recently we were successful in purification of 2-deoxy-scyllo-inosose synthase from butirosin-producer Bacillus circulans as the key enzyme for the biosynthesis of DOS, in cloning of the responsible gene (btrC), and in its overexpression in Escherichia coli. The present study involved gene-walking approach, which allowed us to find a gene cluster around btrC. The function of each gene was further investigated by gene disruption, and the disruptants of btrB, btrC, btrD and btrM showed no antibiotic producing activity. Therefore, the gene cluster found so far was determined to be a part of the butirosin biosynthetic gene cluster. Functions of some ORFs are also discussed in terms of butirosin biosynthesis on the basis of database search.

Mutational biosynthesis of butirosin analogs. I. Conversion of neamine analogs into butirosin analogs by mutants of Bacillus circulans.

By N-methyl-N'-nitro-N-nitrosoguanidine treatment, neamine-negative mutants which required neamine for biosynthesis of butirosins were obtained from a butirosin-producing organism Bacillus circulans. These mutants also produced butirosins from paromamine and could be divided into two types I and II. Mutants of type I could not produce butirosins from 2-deoxystreptamine, whereas those of type II could. Two typical mutants MCRL 5003 (type I) and MCRL 5004 (type II) could produce butirosin analogs, 3', 4'-dideoxybutirosins, 6'-N-methylbutirosins, 3', 4'-dideoxy-6'-N-methylbutirosins and 3', 4'-dideoxy-6'-C-methyl-butirosins from neamine analogs, gentamine Cla, 6'-N-methylneamine, 6'-N-methylgentamine Cla and gentamine C2, respectively.

Mutational biosynthesis of butirosin analogs. II. 3', 4'-Dideoxy-6'-N-methylbutirosins, new semisynthetic aminoglycosides.

A pair of new butirosin analogs was isolated from the fermentation broth obtained by cultivating a neamine-negative mutant of the butirosin-producing organism Bacillus circulans in the medium supplemented with 6'-N-methylgentamine C1a. These antibiotics were characterized and elucidated as 3', 4'-dideoxy-6'-N-methylbutirosins A and B (DMB-A & DMB-B), by chemical and spectroscopic studies. DMB-A and DMB-B exhibited broad-spectrum antibacterial activities with in vitro potency similar to or slightly less than that for the butirosin A, with the exception of strains of Pseudomonas aeruginosa and Serratia marcescens against which they exhibited activities equal to or slightly greater than that for butirosin A. As expected, they exhibited stronger activities against butirosin-resistant organisms which contain acetylating enzymes AAC(6')-I and AAC(6')-IV, and phosphorylating enzyme APH(3')-II. They were also active against some of the clinical isolates resistant to butirosins, dibekacin and/or gentamicin. The acute intravenous toxicity in mice of the DMB complex (B:70 APPROXIMATELY 80%) was somewhat less than that of the butirosin A.

Mutational biosynthesis of butirosin analogs. III. 6'-N-methylbutirosins and 3', 4'-dideoxy-6'-c-methylbutirosins, new semisynthetic aminoglycosides.

Two pairs of butirosin analogs were isolated from the fermentation broths obtained by cultivating a neamine-negative mutant of the butirosin-producing organism Bacillus circulans in the medium supplemented with 6'-N-methylneamine and gentamine C2, respectively. These amtibiotics were characterized as 6'-N-mentylbutirosins A and B (NMB-A & NMB-B), and 3', 4'-dideoxy-6'-C-methylbutirosins A and B (DCB-A & DCB-B), respectively, by chemical and spectroscopic studies. NMB-A and NMB-B exhibited broad-spectrum antibacterial activities with in vitro potency similar to or slightly less than that for butirosin A, but with greater activities against butirosin-resistant strain which contains acetylating enzyme AAC-(6')-I. The activities of NMB-A and NMB-B against Pseudomonas aeruginosa strains were relatively weak. DCB-B had broad-spectrum activity, and exhibited greatly improved activity against butirosin-resistant organisms which contain acetylating enzymes AAC(6')-I and AAC(6')-IV, and phosphorylating enzyme APH(3')-II. These new butirosin analogs were also active against some of the clinical isolates resistant to butirosins, dibekacin and/or gentamicin.

Identification of L-glutamine: 2-deoxy-scyllo-inosose aminotransferase required for the biosynthesis of butirosin in Bacillus circulans.

Using inverse PCR, two new genes (btrN and btrS) were identified upstream of the putative glycosyltransferase gene btrM in the butirosin-biosynthetic btr gene cluster of Bacillus circulans. The upstream gene btrS showed significant homology with stsC of Streptomyces griseus, which encodes L-glutamine:scyllo-inosose aminotransferase in the biosynthesis of streptomycin. The function of BtrS was further confirmed by heterologous expression in Escherichia coli and chemical identification of the conversion of 2-deoxy-scyllo-inosose into 2-deoxy-scyllo-inosamine. The identification of BtrS as L-glutamine:2-deoxy-scyllo-inosose aminotransferase is the first report of the aminotransferase gene responsible for 2-deoxystreptamine biosynthesis.