Genetic dissection of the biosynthetic route to gentamicin A2 by heterologous expression of its minimal gene set Je Won Park†, Jay Sung Joong Hong‡, Niranjan Parajuli†, Won Seok Jung‡, Sung Ryeol Park†, Si-Kyu Lim§, Jae Kyung Sohng¶, and Yeo Joon Yoon†ʈ †Division of Nano Sciences, Ewha Womans University, 11-1 Daehyung-dong, Seodaemun-gu, Seoul 120-750, Republic of Korea; ‡Interdisciplinary Program of Biochemical Engineering and Biotechnology, Seoul National University, San 56-1, Shilim-dong, Gwanak-gu, Seoul 151-742, Republic of Korea; §Genotech Corporations, 59-5 Jang-dong, Usung-gu, Daejon 305-343, Republic of Korea; and ¶Department of Pharmaceutical Engineering, Institute of Biomolecule Reconstruction, Sun Moon University, 100 Kalsanri, Tangjeonmyun, Asansi, Chungnam 336-708, Republic of Korea Communicated by Arnold L. Demain, Drew University, Madison, NJ, April 10, 2008 (received for review November 1, 2007) Since the first use of streptomycin as an effective antibiotic drug in minor components of gentamicin are also produced by Mi- the treatment of tuberculosis, aminoglycoside antibiotics have cromonospora echinospora, including gentamicin A2 (4a), the been widely used against a variety of bacterial infections for over precursor from which other forms of gentamicin are derived (4). six decades. However, the pathways for aminoglycoside biosyn- The recent discovery of gene clusters for biosynthesis of thesis still remain unclear, mainly because of difficulty in genetic butirosin and neomycin in Bacillus circulans and Streptomyces manipulation of actinomycetes producing this class of antibiotics. fradiae, respectively, has facilitated genetic and biochemical Gentamicin belongs to the group of 4,6-disubstituted aminogly- studies on the biosynthesis of 4,5-disubstituted aminoglycosides cosides containing a characteristic core aminocyclitol moiety, 2- (5–9). Concurrently, the biosynthetic gene clusters relating to the deoxystreptamine (2-DOS), and the recent discovery of its biosyn- 4,6-disubstituted aminoglycosides kanamycin, tobramycin, and thetic gene cluster in Micromonospora echinospora has enabled us gentamicin have been isolated from Streptomyces kanamyceticus, to decipher its biosynthetic pathway. To determine the minimal set Streptomyces tenebrarius, and M. echinospora, respectively (10– of genes and their functions for the generation of gentamicin A2, 13). However, little progress has been made to evaluate the the first pseudotrisaccharide intermediate in the biosynthetic path- proposed biosynthetic routes to these antibiotics, mainly because way for the gentamicin complex, various sets of candidate genes of difficulty in genetic manipulation of the producing microor- from M. echinospora and other related aminoglycoside-producing ganisms. Gentamicin-producing M. echinospora has innate re- strains were introduced into a nonaminoglycoside producing strain sistance to most antibiotics precluding the use of standard of Streptomyces venezuelae. Heterologous expression of different Streptomyces cloning vectors displays low transformation effi- combinations of putative 2-DOS biosynthetic genes revealed that ciency (Ͻ10Ϫ7 transformants per microgram of DNA) (14). a subset, gtmB-gtmA-gacH, is responsible for the biosynthesis of Current knowledge regarding the biosynthesis of 2-DOS con- this core aminocyclitol moiety of gentamicin. Expression of gtmG taining aminoglycosides was recently reviewed, and most of cited .(together with gtmB-gtmA-gacH led to production of 2؅-N- works relied on in vitro studies of individual gene products (15 acetylparomamine, demonstrating that GtmG acts as a glycosyl- When the biosynthetic route to the gentamicin complex was first transferase that adds N-acetyl-D-glucosamine (GLcNA) to 2-DOS. predicted, it was based on isolation of metabolites from cell -Expression of gtmM ina2؅-N-acetylparomamine-producing recom- culture extracts and precursor feeding studies of blocked mu binant S. venezuelae strain generated paromamine. Expression of tants (16, 17). However, functional studies of gentamicin bio- gtmE in an engineered paromamine-producing strain of S. ven- synthetic genes have not been possible, so that assignment of the ezuelae successfully generated gentamicin A2, indicating that roles of gene products has hitherto been based purely on amino GtmE is another glycosyltransferase that attaches D-xylose to acid sequence comparisons pending verification by direct exper- paromamine. These results represent in vivo evidence elucidating imental evidence. the complete biosynthetic pathway of the pseudotrisaccharide We show here that heterologous expression of different aminoglycoside. combinations of genes from the gentamicin biosynthetic gene cluster (refs. 11 and 12: GenBank accession nos. AJ575934 and aminoglycoside biosynthesis ͉ Micromonospora echinospora ͉ AY524043) in a nonaminoglycoside producing strain of S. ven- Streptomyces venezuelae ͉ pathway engineering ezuelae caused production of intermediates from the gentamicin biosynthetic pathway including 2-DOS (1a), 2Ј-N-acetylparo- entamicin is an aminoglycoside antibiotic complex pro- mamine (2a), paromamine (3a), and the first pseudotrisaccha- Gduced by various Micromonospora species (1). Aminogly- ride, gentamicin A2 (4a). This provided experimental evidence cosides act by binding to the bacterial ribosome inhibiting for assigning the functions of individual gene products, allowing protein synthesis and generating errors in the translation of the us to recognize a complete biosynthetic pathway for generation genetic code (2). This class of antibiotics has been used clinically for the treatment of severe bacterial infections for Ͼ60 years, beginning with the use of streptomycin to treat tuberculosis (3). Author contributions: J.W.P. and J.S.J.H. contributed equally to this work; J.W.P., N.P., S.-K.L., J.K.S., and Y.J.Y. designed research; J.W.P., J.S.J.H., N.P., W.S.J., and S.R.P. performed MICROBIOLOGY However, the genetics underlying the biosynthesis of aminogly- research; J.W.P. and J.K.S. contributed new reagents/analytic tools; J.W.P., J.S.J.H., N.P., cosides, including gentamicin, still remains unclear. Structurally, S.-K.L., J.K.S., and Y.J.Y. analyzed data; and J.W.P., J.S.J.H., and Y.J.Y. wrote the paper. gentamicin is a 4,6-disubstituted aminocyclitol composed of the The authors declare no conflict of interest. core aminocyclitol moiety, 2-deoxystreptamine (2-DOS, 1a), Data deposition: The sequences reported in this paper have been deposited in the GenBank which is decorated by purpurosamine and garosamine amin- database (accession nos. AM946392). osugars at positions C-4 and C-6, respectively. In its therapeutic ʈTo whom correspondence should be addressed. E-mail: [email protected]. form, gentamicin comprises a complex of gentamicin C1,C1a, This article contains supporting information online at www.pnas.org/cgi/content/full/ and C2, which differ only in the degree of methylation of the C-6Ј 0803164105/DCSupplemental. position of the sugar attached at C-4 of 2-DOS (Fig. 1). Other © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0803164105 PNAS ͉ June 17, 2008 ͉ vol. 105 ͉ no. 24 ͉ 8399–8404 Downloaded by guest on September 26, 2021 2- OPO3 6 O NH2 NH2 HO O HO HO HO 1 HO HO HO HO GtmA OH GtmB OH GacH OH OH OH OH OH O GtmB Glc- P-6 2-DOI 2-DOIA keto-2-DOIA NH2 4 2 HO 3 '6 OH 1 OH 6' OH '4 '6 HO 6 NH2 4' '4 5' O 5 5' HO OH HO '5 O HO O 2' 2' 2' HO '1 1' HO '1 NH 1a HO NH '3 HN 4 2 2 GtmG NH2 '3 2 3 3' H2N 4 2 H2N 4 2 O O 3 O 3 '7 1 1 1 HO 6 NH2 HO GtmE HO NH GtmM 5 6 NH2 6 2 O OH 5 O 5 OH '8 O "1 2a OH 3a "5 "2 "4 "3 OH HOHO "4 '2 '4 "5 O '3 H3C "2 H2N 4a "1 5' 6' H3CHN '1 O "3 OH R OH 6 4 R 2 O 5 O 1 H N 3 NH 2 1 2 2 R R 1 R 2 Gentamicin C1: CH3 NHCH3 Gentamicin C1a:H H N 2 Gentamicin C2: CH3 NH2 Fig. 1. Proposed biosynthetic pathway of gentamicin A2 (4a) and its biosynthetic intermediates 2-deoxystreptamine (1a), 2Ј-N-acetylparomamine (2a), and paromamine (3a). of gentamicin A2. This work has established an efficient in 2-DOS biosynthesis, based on its sequence homology to the heterologous expression platform based on a genetically ame- 2-DOIA dehydrogenase Neo5 [CAH58688, otherwise known as nable streptomycete to dissect the biosynthetic pathways of NeoA (BAD95818)] involved in neomycin biosynthesis. When other aminoglycoside antibiotics produced by less tractable the resultant plasmid pYJ495 harboring gtmD-gtmB-gtmA-gacH actinomycetes. was expressed in the heterologous host, 2-DOS (1a) was indeed produced (Fig. 2A). The two transamination reactions required Results and Discussion for biosynthesis of 2-DOS during butirosin production by B. Putative gentamicin biosynthetic genes, suggested by previous circulans are catalyzed by a single enzyme, BtrS [BAE07061, studies to encode the 2-DOS biosynthetic enzymes, two glyco- otherwise known as BtrR (CAD41947)] (23). To ascertain syltransferases and a deacetylase, were amplified by PCR (8, 10, whether the biosynthesis of 2-DOS during gentamicin produc- 18) and cloned into a thiostrepton-resistance vector, pSE34, tion likewise involves dual activity of a single aminotransferase, under control of the ermE* promoter (19). Plasmids harboring two separate constructs pYJ496 and pYJ497 expressing gtmB- different sets of genes necessary for the biosynthesis of genta- gtmA-gacH and gtmD-gtmA-gacH, respectively, were tested. The micin and its intermediates were heterologously expressed in a expression of gtmB, gtmA, and gacH produced Ϸ120 ␮g per liter mutant strain of S. venezuelae YJ003 that lacks the ability to of 2-DOS (1a) (corrected for Ϸ40% mean recoveries of 1a), biosynthesize its native deoxysugar (TDP-D-desosamine) (20). whereas, gtmD, gtmA, and gacH did not do so (Fig. 2 B and C), The culture extracts from the resulting strains were analyzed by indicating that GtmB catalyzes both transamination steps in the using HPLC-ESI-MS/MS to detect gentamicin-related com- synthesis of 2-DOS, whereas GtmD may act in a later step during pounds, the structures of which were then further identified by gentamicin production.