Ketoolivosyl-tetracenomycin C: a new ketosugar bearing tetracenomycin reveals new insight into the substrate flexibility of glycosyltransferase ElmGT

Abstract

A new tetracenomycin analogue, 8-demethyl-8-(4′-keto)-α-l-olivosyl-tetracenomycin C, was generated through combinatorial biosynthesis. Streptomyces lividans TK 24 (cos16F4) was used as a host for expression of a “sugar plasmid” (pKOL) directing the biosynthesis of NDP-4-keto-l-olivose. This strain harbors all of the genes necessary for production of 8-demethylt-etracenomycin C and the sugar flexible glycosyltransferase ElmGT. To the best of our knowledge, this report represents the first characterization of a tetracenomycin derivative decorated with a ketosugar moiety. Also, as far as we know, 4-keto-l-olivose has only been described as an intermediate of oleandomycin biosynthesis, but has not been described before as an appendage for a polyketide compound. Furthermore, this report gives further insight into the substrate flexibility of ElmGT to include an NDP-ketosugar, which is unusual and is rarely observed among glycosyltransferases from antibiotic biosynthetic pathways.

Streptomycetes produce a variety of glycosylated polyketide compounds with antibiotic and antitumor properties. The appended sugar moiety/moieties contribute tremendously to the biological activity of parent compounds. The alteration of these appended sugar(s) can improve or diversify the biological activities of these compounds. As such, combinatorial biosynthesis is one promising strategy for altering deoxysugar moieties. This can be achieved through either gene deletion or heterologous expression of deoxysugar gene clusters from other pathways. Provided that the endogenous glycosyltransferase is flexible enough to accommodate the altered NDP-deoxysugar donor substrates, novel derivatives can be generated.¹

Elloramycin (1) and the structurally-related tetracenomycins are anthracycline-like polyketide compounds with mild antitumor activity.^2,3 Elloramycin is produced by Streptomyces olivaceus Tü2353, and it features 12a-O-methyl moiety and 2′, 3′, 4′-tri-O-methyl-α-l-rhamnose O-glycosidically linked at 8-position (Figure 1). In previous studies, the entire gene cluster responsible for production of 8-demethyl-tetracenomycin C (2) (8-DMTC) was cloned into cosmid 16F4, and by expressing the cosmid in various Streptomyces sp., tetracenomycins with novel deoxysugar moieties were produced.^4–6 It was then discovered that the elloramycin glycosyltransferase, ElmGT, was responsible for appending these foreign NDP-deoxysugars to 8-DMTC. Recent studies by Salas and co-workers have involved cloning several “deoxysugar plasmids,” which combine deoxysugar genes from different pathways into a single operon for expression in Streptomyces species. By expression of these sugar plasmids in a Streptomyces host harboring cos16F4, it was discovered that ElmGT demonstrated remarkable flexibility towards accepting L- and D- neutral and branched sugar substrates in generating a library of tetracenomycin derivatives.^7–12 Recently, mutation of active site residues of ElmGT has been shown to modulate transfer of specific deoxysugars.¹³ Here we report the isolation and characterization of a new tetracenomycin decorated with a 4′-keto-l-olivose moiety.

Figure 1.

Elloramycin and tetracenomycin analogues discussed in this communication. Elloramycin (1); 8-demethyl-tetracenomycin C (2); 8-demethyl-8-(4'-keto)-α-l-olivosyl-tetracenomycin C (3); 8-demethyl-8-α--olivosyl-tetracenomycin C (4).

In order to generate a plasmid for expression of a ketosugar, the 4-ketoreductase gene (oleU) was removed from plasmid pLN2 (Rodriguez, et al., 2002). NheI and SpeI were used to digest pLN2, thereby releasing the oleU fragment. DNA gel electrophoresis was carried out to remove the oleU gene and the resulting fragment (~14kb) was rescued and re-ligated to generate pKOL. In this construct, all of the sugar genes are under strong ermE* promotion. The plasmid map for this construct is depicted in Figure S1 (see Supplementary Information). This plasmid was introduced into the S. lividans (cos16F4) strain through protoplast transformation as described previously.^14–20

The activities of OleS (NDP-glucose-synthase), OleE (NDP-glucose-4, 6-dehydratase), OleV (2, 3-dehydratase), OleL (3, 5-epimerase), and OleW (3-ketoreductase) catalyze the conversion of NDP-d-glucose to NDP-4-keto-2, 6-dideoxy-d-glucose during the biosynthesis of NDP-l-olivose (Figure 2). The 4-ketoreduction step catalyzed by OleU represents the last step of the NDP-l-olivose biosynthetic pathway. Thus, the deletion of oleU from pLN2 in pKOL would lead to the accumulation of NDP-4-keto-l-olivose, which could be utilized by ElmGT as an alternative donor substrate when the natural substrate (TDP-l-rhamnose) is not available. To test this hypothesis, the pKOL. plasmid was expressed in the S. lividans (cos16F4) strain.

Figure 2.

Hypothesized deoxysugar biosynthetic pathway of pKOL.. OleU, which was encoded in the parent plasmid pLN2 but not encoded in this construct, is indicated in brackets.

Interestingly, expression of pKOL in S. lividans (cos16F4) resulted in the accumulation of two major peaks with R_t (10.76 and 11.26 min., respectively), in addition to 8-DMTC, when ethyl acetate extracts were analyzed by HPLC/MS (Figure 3). Both peaks showed UV absorption typical for a tetracenomycin-type compound. Low resolution ESI/MS revealed a peak of 585 amu (−ve mode) pertaining to the molecular ion of the compound and another pseudomolecular ion of a hydrated species at 603 amu (−ve mode) indicating the addition of water to the molecule. Under aqueous conditions, ketosugars can easily interconvert from the keto form to a hydrate form. The second peak possessed an m/z value of 587 in (−ve) ESI mode. This mass data suggested the presence of a glycosylated tetracenomycin in which the sugar was fully reduced. Both peaks possessed a fragmentation ion corresponding to the 8-DMTC aglycone (m/z 457, M-H⁻).

Figure 3.

HPLC analyses of the metabolites: trace A, 8-demethyl-tetracenomycin C (2) (Rt. 12.98 min.) isolated from S. lividans (cos16F4); trace B, metabolites isolated from the S. lividans (cos16F4)/pKOL mutant, 8-demethyl-(4’-keto)-α-l-olivosyl-tetracenomycin C (3 (Rt. 10.76 min.) and (Rt. 12.26 min.) 3* denote hydrated and keto forms, respectively) and 8-demethyl-α-l-olivosyltetracenomycin C (4) (Rt. 11.26 min.)

This strain was fermented in a large scale fermentation (7.2 L) for isolation and spectroscopic characterization of the metabolites. The structure of 8-demethyl-8-(4′-keto)-α-l-olivosyl-tetracenomycin C (3) was solved through NMR and mass spectral analyses. The (−) HR-ESI MS of 3 showed two peaks at 585.1267 amu and 603.1363 amu, which corresponded to the molecular formula of its keto form (C₂₈H₂₆O₁₄, calcd. molecular weight 585.1317 [M-H⁻]) and its hydrate form (C₂₈H₂₈O₁₅, calcd. molecular weight 603.1423 amu [M-H⁻]), respectively. ¹H NMR data of 3 revealed 2 singlets for two aromatic protons (δ 7.94 and δ 7.66) and two methoxy signals corresponding to the 3-OCH₃ (δ 3.81) and 9-OCH₃ (δ 3.97) of 8-DMTC, respectively (Table 1). The anomeric proton of the sugar appeared as a broad singlet (δ 6.16), which suggested its α configuration. A pair of protons at δ 2.13 and δ 2.24 corresponded to the C-2′ methylene protons. A 4′-H signal was not observed, indicating the presence of keto or a hydrated-keto group. The splitting of the 3′-H (dd, J= 12.0, 6.5 Hz) at δ 4.68 indicated a large diaxial coupling with 2′-H_a and an axial-equatorial coupling with 2′-H_e. The 5′-H appeared as a quartet (J= 6.5 Hz) at δ 4.39, which indicated coupling with 6′-CH₃. The ¹H, ¹H-COSY exhibited two spin systems for the sugar moiety, one stretching from 1′-H to 3′-H, and the other stretching from 5′-H to 6′-CH₃, which strongly indicates the presence of a carbonyl at C-4′ (Figure 4). The ¹³C showed a carbonyl signal at δ 207.4, which indicates that 3 is present predominantly in the ketosugar form when measured in Methanol-d₄. The 2D-HMBC demonstrated a correlation between the 6’-CH₃ protons (δ 1.12) and the 4’-C carbonyl moiety (δ 207.4), which unambiguously assigned it to this position (Figure 4). These data suggested the structure of 3 as 8-demethyl-8-(4′-keto)-α-l-olivosyl-tetracenomycin C. Compound 4 was eluted at the identical retention time when co-injected with standard 8-demethyl-8-α-l-olivosyl-tetracenomycin. The identity of 4 as 8-demethyl-8-α-l-olivosyl-tetracenomycin was further confirmed through the comparison of ¹H NMR data with published ¹H NMR data.⁹

Table 1.

¹H and ¹³C NMR of 8-Demethyl-8-(4’-keto)-α-L-olivosyl-tetracenomycin C (3) in comparison with the reported data of 8-Demethyl-8-α-l-olivosyl-tetracenomycin C (4),9 δ in ppm relative to TMS, (multiplicity, J/Hz).

Position	8-Demethyl-8-(4'-keto)-α-l-olivosyl- tetracenomycin C (3)a		8-Demethyl-8-α-l-olivosyl-tetracenomycin C (4)b
	δC (125 MHz)	δH (500 MHz)	δC (100 MHz)	δH (400 MHz)
1	193.0	-	190.8	-
2	100.8	5.62 (s)	100.2	5.66 (s)
3	176.1	-	175.6	-
3-OCH3	57.8	3.81 (s)	57.5*	3.87 (s)
4	70.7	4.88 (d, 1.5)	70.6	5.11 (d, 7.0)
4-OH	-	-	-	4.96 (d, 7.0)
4a	85.9	-	85.2	-
4a-OH	-	-	-	5.18 (s)
5	194.8	-	194.0	-
5a	141.5	-	141.2	-
6	122.1	7.91 (s)	121.6	8.10 (s)
6a	129.6	-	131.0**	-
7	112.4	7.55 (s)	112.0	7.80 (s)
8	155.4	-	155.6	-
9	130.9	-	129.3	-
9-OCH3	53.4	3.97 (s)	53.0*	4.00 (s)
9-CO	169.5	-	168.0	-
10	139.5	-	138.6	-
10-CH3	21.3	2.79 (s)	21.3	2.87 (s)
10a	122.6	-	122.0**	-
11	167.8	-	167.8	-
11-OH	-	-	-	14.02 (s)
11a	110.6	-	110.2	-
12	198.2	-	198.0	-
12a	84.6	-	83.8	-
12a-OH	-	-	-	5.81 (s)
1′	96.4	6.16 (s)	97.0	6.07 (d, 3.0)
2′	37.3	2.24 (ddd, 15.0, 12.0, 3.0, He),	38.2	2.32 (ddd, 16.0, 6.0, 2.0, He),
		2.13 (dd, 15.0, 6.5, Ha)		1.88 (ddd, 14.0, 12.0, 3.0, Ha)
3′	70.6	4.68 (dd, 12.0, 6.5)	69.0	3.95 (dddd, 11.0, 6.0, 6.0, 6.0)
3′-OH	-	-	-	4.25 (s)
4′	207.4	-	70.6	3.14 (dd, 9.0, 9.0)
4′-OH	-	-	-	4.38 (s)
5′	72.5	4.39 (q, 6.5)	78.4	3.67 (dq, 10.0, 6.0)
6′	14.3	1.12 (keto, d, 6.5)	18.3	1.22 (d, 6.0)
		1.16 (hydrate, d, 6.5)

^a)Methanol-d₄,

^c)Acetone-d₆,

* and ** signals were mutually wrongly assigned in cited publication, however, assignments were corrected here for better comparison

Figure 4.

¹H-¹H-COSY (), and selected HMBC (→) correlations of 8-demethyl-8-(4'-keto)-α-l-olivosyl-tetracenomycin C (3).

To evaluate the microbiological activity of 3, 1 mg mL⁻¹ methanolic solutions of 1 and 3 were prepared for disc diffusion assays against E. coli XL1-blue, Streptomyces prasinus, and Mucor meihei strains. Streptomyces prasinus NRRL B-2712 was chosen because it was previously shown to be the most susceptible gram positive organism to 1.² 3 demonstrated no detectable activity against E. coli or Mucor meihei, but demonstrated a diameter halo against Streptomyces prasinus of 12±2 mm as compared to the 25±2 mm of 1. This finding indicates that the 4’-ketosugar modification of 3 diminishes some of the antimicrobial effect observed with 1. This is consistent with similar modifications of the sugar moiety of 8-demethyl tetracenomycin C, as many derivatives have resulted in poorer antimicrobial activity.¹⁰ Very possibly, the O-methyl groups of the L-rhamnose moiety of 1 are essential for its cytotoxicity. The possible anticancer activity of 3 is currently being evaluated against several cancer cell lines in our lab for a future report.

ElmGT represents one of the most flexible glycosyltransferases with respect to its ability to accommodate a large number of sugar donor substrates as compared to glycosyltransferases in other secondary metabolite biosynthetic pathways. ElmGT reportedly utilizes various NDP-d-sugars: d-olivose, d-mycarose, d-diolivose, d-amicetose, d-boivinose, d-digitoxose, and d-glucose. ElmGT also accepts a number of NDP-l-sugars: l-rhamnose, l-rhodinose, l-digitoxose, l-olivose, l-amicetose, l-mycarose and 4-deacetyl-l-chromose B. However, ElmGT has not been previously shown to accommodate NDP-ketosugars. In this context, production of 3 through the expression of pKOL in S. lividans (cos16F4) was interesting, especially, as earlier efforts to glycosylate 8-DMTC with NDP-4-keto-l-rhamnose or NDP-4-keto-l-mycarose were unsuccessful.^9,10

The observed production of 4 along with 3 by S. lividans (cos16F4)/pKOL was unanticipated. However, we assume that a pathway-independent ketoreductase of S. lividans TK 24 might be responsible for the partial conversion of NDP-4-keto-l-olivose to NDP-l-olivose, and the latter is utilized by ElmGT as an alternate substrate to yield 4. Pathway independent reductions have been reported previously in the literature. Earlier in the pikromycin biosynthetic pathway, in which a d-quinovosyl macrolide was accumulated instead of the anticipated 4-keto-6-deoxy-d-glucosyl analogue when desI was inactivated in Streptomyces venezuelae.²¹ To search for possible sugar ketoreductases in Streptomyces lividans TK 24 that could be responsible for reduction of NDP-4-keto-l-olivose, thus possibly explaining the presence of 4, the amino acid sequence for OleU was compared to encoded proteins in the Streptomyces lividans genome using the protein BLAST public database. One such candidate was indicated in the search, SSPG_00655 (30% sequence identity/42% sequence similarity). This candidate has a domain that shows similarity to RfbD, which is a 4-ketohexulose reductase responsible for equatorial 4-ketoreduction of NDP-4’-keto-l-rhamnose in the NDP-l-rhamnose pathway (Figure S2, Supporting Information). This enzyme may be responsible for the 4-ketoreduction witnessed in 4. Despite the ability of SSPG_00655 or some other promiscuous reductase to reduce NDP-4-keto-l-olivose to NDP-l-olivose in S. lividans (Figure 2), enough of the ketosugar substrate was available to be accepted by ElmGT to flux towards 3. It is equally interesting that none of the elm pathway sugar O-methyltransferases (ElmMI, ElmMII, and ElmMIII) recognized the 4-keto-l-sugar moiety, even though these enzymes acted on an appended 8-O-l-olivose previously.

Despite the many glycosyltransferases discovered and explored, those which can attach ketosugars to their acceptor co-substrates are relatively scarce. MtmGIV (and possibly MtmGIII) from the mithramycin pathway has/have been shown to handle NDP-4-keto-d-olivose and NDP-4-keto-d-mycarose to generate novel premithramycin and mithramycin analogues in Streptomyces argillaceus strains in which mtmC (3-C-methyltransferase) and mtmTIII (4-ketoreductase) genes were disrupted.²² Like 3, the ketomithramycin derivative demonstrated weaker biological activity as compared to the parent mithramycin compound, possibly due to the ketosugar substitution.²² EryBV from the erythromycin pathway has been shown to accommodate NDP-4-keto-l-mycarose.²³ Similarly, BgtfA from the balhimycin pathway is capable of utilizing NDP-4-keto-l-vancosamine as a natural sugar donor substrate.²⁴ The unprecedented flexibility of ElmGT towards NDP-4′-keto-l-olivose reported in this communication is particularly encouraging for further studies towards understanding the structural role of ElmGT in binding deoxysugar substrates.