One-Pot Combinatorial Biosynthesis of Glycosylated Anthracyclines by

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Letter

One-pot combinatorial biosynthesis of glycosylated anthracyclines by co-cultivation of Streptomyces strains producing aglycones and nucleotide deoxysugars Eunji Kim, Myoung Chong Song, Myoun Su Kim, Ji Yoon Beom, Jin A Jung, Hang Soo Cho, and Yeo Joon Yoon ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.6b00194 • Publication Date (Web): 13 Feb 2017 Downloaded from http://pubs.acs.org on February 14, 2017

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ACS Combinatorial Science

One-pot combinatorial biosynthesis of glycosylated anthracyclines by co-cultivation of Streptomyces strains producing aglycones and nucleotide deoxysugars

Eunji Kim,§ Myoung Chong Song,§ Myoun Su Kim, Ji Yoon Beom, Jin A Jung, Hang Soo Cho, and Yeo Joon Yoon* Department of Chemistry and Nanoscience, Ewha Womans University, Seoul, Republic of Korea

Abstract Anthracyclines such as doxorubicin are effective anticancer drugs composed of a tetracyclic polyketide aglycone and one or more deoxysugar moieties, which play a critical role in their biological activity. A facile one-pot combinatorial biosynthetic system was developed for the generation of a range of glycosylated derivatives of anthracyclines. Co-cultivation of Streptomyces venezuelae mutants producing two anthracycline aglycones with eight different nucleotide deoxysugar-producing S. venezuelae mutants that co-express a substrate-flexible glycosyltransferase led to the generation of sixteen aklavinone or εrhodomycinone glycosides containing diverse deoxysugar moieties, seven of which are new. This demonstrates the potential of the one-pot combinatorial biosynthetic system based on co-cultivation as a facile biological tool capable of combining diverse aglycones and deoxysugars to generate structurally diverse polyketides carrying engineered sugars for drug discovery and development. 1

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Keywords Co-cultivation, Streptomyces, Glycosylation, Anthracycline, Deoxysugar

Polyketides are a structurally diverse class of natural products and include many clinically important compounds with antibacterial, anticancer, antiparasitic and immunosuppressive activities.1 Many of them possess diverse deoxysugar moieties attached to the polyketide aglycone.2,3 These sugar moieties influence the biological activities or pharmacological properties of their respective polyketides and are essential for binding their molecular targets.4,5 For example, the effective anthracycline anticancer drugs, such as doxorubicin (also known as adriamycin) produced by Streptomyces peucetius ATCC 27952 and aclacinomycin A (aclarubicin) produced by Streptomyces galilaeus ATCC 31615 (Figure 1), have a tetracyclic polyketide ring aglycone glycosylated with one or more deoxysugar residues, and the important relationship between the sugar moiety and biological activity or toxicity is well-exemplified by the reduced cardiotoxicity of the semisynthetic analogue of doxorubicin, epirubicin (4′-epi-doxorubicin; Figure 1). This therapeutic improvement can be attributed solely to the appended sugar as the only difference between doxorubicin and epirubicin is the opposite configuration of the C-4 hydroxy group on the deoxysugar daunosamine.6 Anthracyclines have long been the targets for combinatorial biosynthesis and one of the classical examples is the direct production of epirubicin from the S. peucetius mutant where the dnmV gene involved in the determination of the stereochemistry of the C-4′ hydroxy group was replaced by Streptomyces avermitilis avrE or Saccharopolyspora erythraea eryBIV.7 Therefore, by altering the sugar portion of these polyketides, next generation antibacterial or anticancer agents with improved activities and reduced toxicities can be developed. However, the successful production of these novel polyketide glycosides requires significant chemical effort, and these synthetic difficulties have triggered the use of 2


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combinatorial biosynthesis to generate novel polyketide glycosides through the modification of their glycosylation patterns.4,5,8-10 The common in vivo combinatorial biosynthesis strategies involve inactivating specific sugar biosynthesis gene(s) and expressing heterologous sugar biosynthesis genes or using the substrate-flexible glycosyltransferase (GT) gene to facilitate the glycosylation of an endogenously produced aglycone with an engineered nucleotide-activated sugar.5,8,11,12 Alternatively, by deleting the genes involved in the biosynthesis of the native aglycone and sugar and expressing the recombinant sugar biosynthesis pathway and a GT, a nonproducer strain can be constructed and used as a bioconversion host to promote the glycosylation of exogenously fed aglycones.5,8,13,14 In the former method, the endogenouslyproduced aglycone is fixed and different sugars can be combined specifically with a desired aglycone, thus limiting the generation of more diverse structures. In the latter method, the aglycone to be supplemented should be available. An alternative strategy to facilitate this latter method involves co-expressing the large gene cluster encoding the biosynthesis of the polyketide aglycone in the surrogate bioconversion host along with the genes for the biosynthesis and transfer of the desired nucleotide sugar. However, the assembly of large gene clusters such as polyketide synthase (PKS) genes and the co-expression with several sugar biosynthetic genes or the reconstitution of the whole polyketide biosynthetic pathway in the heterologous host are challenging tasks.9 The fast growth and relative ease of genetic manipulation make the pikromycinproducing Streptomyces venezuelae ATCC 15439 a favorable host for combinatorial biosynthesis.15 In our previous study, an S. venezuelae-based combinatorial biosynthetic system, S. venezuelae YJ183, was constructed to convert the exogenously fed doxorubicin aglycone ε-rhodomycinone (ε-RHO) into glycosylated doxorubicin analogues. To engineer this strain, the entire pikromycin biosynthetic gene cluster was deleted, doxorubicin 3



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resistance genes (drrA, drrB, and drrC) were introduced into the chromosome, and different combinations of genes for the biosynthesis of various nucleotide deoxysugars were expressed together with the flexible GT AknS and its auxiliary helper protein AknT from the aclacinomycin A gene cluster.13 AknS can recognize aklavinone (AKV) and ε-RHO as sugar acceptors and a variety of sugar donors, and AknT are required for efficient glycosylation.13,16,17 In this study, we employed S. venezuelae YJ183 to construct two aglycone-producing strains, AKV and ε-RHO. The conditions for co-cultivation were optimized using these two aglycone-donor strains and a daunosamine-attaching strain followed by co-cultivation with additional seven S. venezuelae mutants producing different sugars. In this manner, sixteen anthracyclines containing non-natural sugar moieties (seven of these are new) were produced in good to moderate conversion yields. This one-pot combinatorial biosynthesis based co-cultivation system provides a facile tool for generating diverse combinations of aglycones and deoxysugars without the need to supplement a purified aglycone separately to each biotransformation host and avoiding the expression of the large gene clusters required for both aglycone and sugar biosynthesis. AKV is a biosynthetic intermediate of ε-RHO (Figure 2a) and is the aglycone of another clinically approved anthracycline aclacinomycin A. The biosynthesis of ε-RHO has been reviewed in detail.18,19 The carbon skeleton of ε-RHO is biosynthesized by a type II PKS encoded by the dpsABCDGEFY genes and further modified by the products of the dnrGCDEF genes (Figure 2a). Thus, the expression plasmid pRHO was constructed to contain all thirteen genes required for ε-RHO biosynthesis, whereas pAKV was constructed without the DnrF needed to convert AKV to ε-RHO using a replicative Escherichia coliStreptomyces shuttle vector pSE3420 containing the strong constitutive ermE* promoter.21 Although the heterologous ermE* promoter was used to express these genes efficiently in the heterologous host, the native intergenic regions, which may contain native promoter and 4


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the ribosome-binding sites of each gene, were maintained in these constructs. Therefore, the gene encoding the pathway-specific transcription regulator DnrI which controls the transcription of most of the biosynthetic and resistance genes for doxorubicin in S. peucetius22-24 was also included in the final construct to possibly increase the transcription of the cloned genes (Figure S1a, b) as overexpression of dnrI led to a significant increase of εRHO production in S. peucetius.23 These engineered plasmids were separately introduced into S. venezuelae YJ183,13 to provide YJ183/pAKV and YJ183/pRHO, respectively. High-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) analysis of the organic extract of YJ183/pRHO grown on R2YE25 medium at 30 °C for 5 days revealed a peak that was consistent with authentic ε-RHO selected at a mass transition from m/z 428 to 393 in multiple-reaction-monitoring (MRM) mode with a retention time (Rt) of 49.0 min (Figure 3a). Similarly, a peak corresponding to AKV (m/z 412 > 377 in MRM) was detected at an Rt of 44.3 min from the extract of the culture of YJ183/pAKV (Figure 3b). The titers of ε-RHO and AKV from YJ183/pRHO and YJ183/pAKV were approximately 0.2 mg/L and 0.5 mg/L, respectively, based on the calibration curve generated using a ε-RHO standard. To the best of our knowledge, this is the first report of the heterologous production of AKV and ε-RHO, although auramycinone, the aglycone of auramycin which is structurally similar to aclacinomycin, was produced in Streptomyces lividans by expression of nine genes from nogalamycin-, daunomycin-, and aclacinomycin-producing Streptomyces species.26 AKV and ε-RHO produced by aglycone-producing strains must be secreted efficiently for glycosylation by the sugar-producing and attaching strain. HPLC-ESI-MS/MS analysis revealed that the majority of AKV and ε-RHO is secreted to the culture medium (data not shown) probably by the ATP-dependent efflux pump formed by DrrA and DrrB, which together form an ATP-dependent efflux pump.27 Taken together, these results show 5



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that both the YJ183/pAKV and YJ183/pRHO strains can be used as aglycone-donors in a co-cultivation system. In order to develop the co-cultivation system for high levels of anthracycline glycoside production, three major variables in the co-culture process were optimized: the pre-cultivation period for each of the aglycone- and sugar-producing strains to determine the optimal time to combine the cultures, the mixing ratio required for combining the aglyconeand sugar-producing strains, and the ultimate co-cultivation period of the two strains. To optimize these, a model system for each of the two aglycone-producers was set up using the previously constructed sugar-producing YJ183 strain harboring pDNS1 (YJ183/pDNS1)13 to express the TDP-L-daunosamine biosynthetic genes (Figure 2b and Table S1) along with the GT genes aknS/aknT. The relative amount of each compound produced was compared using the peak intensity obtained from the MRM mode via ESI-MS/MS by selecting the two mass ions set to detect a transition of the parent ion to the product ion. First, to determine the pre-cultivation period of YJ183/pAKV and YJ183/pRHO required for maximum aglycone production, the production of AKV and ε-RHO were monitored throughout a 7 day incubation period (Figure 4a). The highest production of AKV for the YJ183/pAKV strain was approximately 3.78 mg/L at day 2, whereas the production of ε-RHO by YJ183/pRHO increased until day 3 reaching a maximum of approximately 0.68 mg/L and decreasing thereafter. Thus, the optimal pre-cultivation periods for YJ183/pAKV and YJ183/pRHO achieving the highest productions were determined to be 2 and 3 days, respectively. Additionally, to determine the optimal pre-cultivation period for the sugarproducing strains, a bioconversion experiment was performed where exogenously supplemented AKV was utilized to produce L-daunosaminyl-AKV (compound 1, Figure 2c). Seven cultures of YJ183/pDNS1 that had been allowed to grow for different periods of time ranging between 1 to 7 days were supplemented with 2.4 µM (1 mg/L) of purified AKV and 6


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incubated for an additional 3 days to determine the relative conversion yield (Figure 4b). The HPLC-ESI-MS/MS analysis showed a peak corresponding to compound 1 selected at the mass transition from m/z 542 to 130 (the oxonium ion fragment derived from the daunosamine moiety) with an Rt of 37.0 min (Figure 5a and see Figure S2a for its MS/MS fragmentation pattern). The conversion yield was highest when AKV was supplemented at day 5 (Figure 4b) establishing a 5 day optimal pre-cultivation period for the sugar-producing strains. Second, to select the optimal mixing ratio for the aglycone- and sugar-producing cultures, the culture broths of 2 day-old aglycone-producer YJ183/pAKV and 5 day-old sugar-producer YJ183/pDNS1 were mixed at varying ratios of 1:1, 2:1, and 1:2 by combining the culture broth of each strain directly to make total volume of 50 mL. After 3 days additional incubation, HPLC-ESI-MS/MS analysis showed the peak corresponding to compound 1. Similarly, 3 days co-cultivation of 3 day-old YJ183/pRHO and 5 day-old YJ183/pDNS1 produced L-daunosaminyl-ε-RHO (rhodomycin D) (compound 2, Figure 2c), which was detected at an Rt of 39.3 min selected at a mass transition from m/z 558 to 130 (Figure 5b and see Figure S2b for its MS/MS fragmentation pattern). The co-cultivations where equal amounts of the aglycone- and sugar-producing culture broths were mixed gave the highest conversions of compounds 1 and 2 (Figure 4c). Lastly, 25 mL of the 2 day-old YJ183/pAKV culture broth or the 3 day-old YJ183/pRHO culture broth was mixed with 25 mL of the 5 day-old YJ183/pDNS1 culture broth, co-incubated for an additional 1 to 7 days, and analyzed to determine the optimal cocultivation period for the highest production of glycosylated anthracyclines. The highest production of compounds 1 and 2 were observed with the 2 days co-cultivation of YJ183/AKV-DNS1 and YJ183/RHO-DNS1 (Figure 4d). The conversion yields of AKV to glycoside 1 and ε-RHO to glycoside 2 were approximately 56% and 63%, respectively, under 7



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these optimized conditions (Table 1). To generate diverse anthracycline glycosides using this co-cultivation system, both the AKV-producing strain YJ183/pAKV and the ε-RHO-producing strain YJ183/pRHO were separately cultured with each one of additional seven sugar-producing strains. The previously constructed plasmids expressed in these sugar-producing strains include pMDNS1, pRDS, pEDNS1, pRST1, and pEVCS expressing diverse TDP-deoxysugar biosynthetic genes with the GT genes aknS/aknT13 (Table S1). Two new plasmids pDDSS2 and pNDDA, which respectively direct the biosynthesis of TDP-D-desosamine and TDP-4-Ndemethyl-D-amosamine and their transfer, were constructed (Figure S1c, d) and separately introduced into S. venezuelae YJ183 (Table S1). Each combination of the aglycone- and sugar-producing strains was cultured under the optimized culture conditions and the culture extract was analyzed for the presence of glycosylated anthracyclines by HPLC-ESI-MS/MS in MRM mode by selecting the two mass ions set to detect a transition of the parent glycoside ion to the product ion of the sugar moiety (Figure 5). The anthracycline glycosides produced were characterized by the fragmentation patterns corresponding to the loss of the sugar moiety from the glycosides, dehydration of the aglycones, and the oxonium ion fragment derived from the sugar moieties (Figure S2). The calibration curves using standard ε-RHO and doxorubicin were used to determine the amount and conversion yield of each compound. The glycosylated anthracyclines produced and the conversion yield of each cocultivation are summarized in Table 1. The sugar-producing strain YJ183/pMDNS1 was designed to direct the biosynthesis of TDP-L-rhodosamine by expressing the designed 3-N-methyl-L-daunosamine biosynthetic genes plus the N-methyltransferase AknX2 from the aclacinomycin A-producing S. galilaeus ATCC 3161513 (Figure 2b and Table S1). The co-cultivation of YJ183/pAKV with YJ183/pMDNS1 (YJ183/AKV-MDNS1) mainly produced 3′-N-methyl-L-daunosaminyl-AKV (3 8


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in Figure 2c) which was produced by the transfer of TDP-3-N-methyl-L-daunosamine, the biosynthetic intermediate of TDP-L-rhodosamine, at a conversion yield of approximately 84.3%. Similarly, the co-cultivation of YJ183/RHO-MDNS1 produced 3′-N-methyl-Ldaunosaminyl-ε-RHO (3′-N-methyl-rhodomycin D) (4 in Figure 2c) as a major product at a conversion yield of approximately 78.3% (Figure 5c, d and see Figure S2c, d for their MS/MS fragmentation patterns). Low levels (~3.4% conversion) of L-daunosaminyl-AKV (1) were also produced by the transfer of TDP-L-daunosamine, and trace amounts of Lrhodosaminyl-AKV (5 in Figure 2c) were detected probably by the weak dimethylation activity of 3-N-methyltransferase AknX213 (Figure 2b) from the co-cultivation of YJ183/AKVMDNS1. Analogously, L-daunosaminyl-ε-RHO (2) and L-rhodosaminyl-ε-RHO (6 in Figure 2c) were produced at low conversion yields (2.4% and 0.3%, respectively) from YJ183/RHOMDNS1 (Figure 5c, d and see Figure S2a, b, e, f for their MS/MS fragmentation patterns). The sugar-synthesis plasmid pRDS was constructed to express an additional Nmethyltransferase gene aclP from another aclacinomycin A-producing S. galilaeus ATCC 31133 with the same gene sets cloned in pMDNS113 (Figure 2b and Table S1). The HPLCESI-MS/MS analysis of the co-cultured YJ183/AKV-RDS extract showed the peaks corresponding to glycosides 1, 3, and 5 in a conversion yield of approximately 2.4%, 23.4%, and 34.3%, respectively (Figure 5e). The co-culture of YJ183/RHO-RDS also showed three peaks arising from RHO glycosides corresponding to glycosides 2, 4, and 6 in conversion yields of 2.7%, 27.6%, and 34.7%, respectively (Figure 5f). Utilization of the sugar-producing strain YJ183/pRDS expressing the N-methyltransferase gene aclP in addition to aknX2 produced increased amounts of L-rhodosamine-attached aglycones (compounds 5 and 6) when compared to YJ183/pMDNS1 which contains only the aknX2 gene. This is consistent with the previous observation that both of these N-methyltransferases are required to efficiently produce TDP-L-rhodosamine in an S. venezuelae heterologous host.13 The 9



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increased production of 5 and 6 could simply due to the increased copy number of the genes whose products catalyze the same N-methyltransfer reaction. However, it is at present unclear how the additional expression of aclP enhanced the dimethylation activity because its function was not verified in vitro yet. Plasmid pEDNS1 was designed to direct the biosynthesis of 4-epi-L-danunosamine by replacing the TDP-4-ketohexose reductase dnmV gene in the daunosamine biosynthetic gene cluster of S. peucetius with the avrE from the L-oleandrose biosynthetic pathway of Streptomyces avermitilis13 (Figure 2b and Table S1). The HPLC-ESI-MS/MS analysis showed that 4′-epi-L-daunosaminyl-AKV (7 in Figure 2c) was produced with a conversion yield of ~43% from the co-cultivation of YJ183/AKV-EDNS1. Similarly, 4′-epi-Ldaunosaminyl-ε-RHO (4′-epi-rhodomycin D) (8 in Figure 2c) was detected from the cocultivation of YJ183/RHO-EDNS1 in a conversion yield of 50% (Figure 5g, h and see Figure S2g, h for their MS/MS fragmentation patterns). In pRST1, avrE was replaced by jadV, another TDP-4-ketohexose reductase gene from the jadomycin B cluster in S. venezuelae ISP5230 for the biosynthesis of TDP-Lristosamine13 (Figure 2b and Table S1). Co-cultivation of YJ183/AKV-RST1 and YJ183/RHORST1 respectively produced L-ristosaminyl-AKV (9 in Figure 2c) and L-ristosaminyl-ε-RHO (10 in Figure 2c) as main products with a conversion yield of approximately 55%. Trace amounts of glycoside 7 and glycoside 8 were produced from YJ183/AKV-RST1 and YJ183/RHO-RST1, respectively (Figure 5i, j and see Figure S2g − j for their MS/MS fragmentation patterns), which is consistent with the previous study in which JadV was also able to produce small amounts of TDP-4-epi-L-daunosamine13 (Figure 2b). The plasmid pEVCS contains combinations of the genes for the biosynthesis of TDP-4-epi-L-vancosamine13 (Figure 2b and Table S1). As expected, 4′-epi-L-vancosaminylAKV (11 in Figure 2c) was detected primarily from the organic extract of the YJ183/AKV10


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EVCS co-cultivation in a conversion yield of approximately 45% with small amounts of glycoside 7 (Figure 5k and see Figure S2g, k for their MS/MS fragmentation patterns). In the YJ183/RHO-EVCS co-cultivation, 4′-epi-L-vancosaminyl-ε-RHO (12 in Figure 2c) was produced as a major product with a similar conversion yield, along with small amounts of glycoside 8 (Figure 5l and see Figure S2h, l for their MS/MS fragmentation patterns). Glycosides 7 and 8 were probably produced due to the skipping of the C-3 methylation step catalyzed by EvaC as has been previously observed13 (Figure 2b). The newly constructed sugar-producing strains YJ183/pDDSS2 and YJ183/pNDDA were designed to direct the biosynthesis of TDP-D-desosamine and its intermediate TDP-4N-demethyl-D-amosamine, respectively (Figure 2b and Table S1). The HPLC-ESI-MS/MS analyses of YJ183/AKV-DDSS2 and YJ183/RHO-DDSS2 showed the peaks corresponding to D-desosaminyl-AKV (13 in Figure 2c) in a conversion yield of ~25% and D-desosaminyl-εRHO (14 in Figure 2c) in a conversion yield of ~30%, respectively (Figure 5m, n and see Figure S2m, n for their MS/MS fragmentation patterns). In addition, the co-cultures of YJ183/AKV-NDDA and YJ183/RHO-NDDA produced 4′-N-demethyl-D-amosaminyl-AKV (15 in Figure 2c) and 4′-N-demethyl-D-amosaminyl-ε-RHO (16 in Figure 2c), respectively, at a conversion yield of approximately >45% (Figure 5o, p and see Figure S2o, p for their MS/MS fragmentation patterns). Through the production of glycosylated derivatives of AKV and ε-RHO achieved in this co-cultivation system, the glycosylation efficiencies of the two structurally similar polyketide aglycones with each sugar donor could be seen. The efficiencies of the two different aglycones with the same sugars were usually similar, but the glycosylation efficiency varied when different sugars were used depending on the structure of the sugar (approximately 25% to 84% for the major products, Table 1). This suggests that the glycosylation efficiency catalyzed by AknS/AknT in the sugar-producing strain varies with the 11



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structure of the sugar for a given aglycone. Unfortunately, the sugar donor’s intracellular concentration cannot be measured, and thus, the exact glycosylation efficiency regarding each sugar donor and acceptor cannot be determined for this in vivo system. However, because each sugar gene cassette was expressed by the same plasmid and cloning strategy, the expression level for each gene cassette should be similar. This has been confirmed by semi-quantitative reverse transcriptase PCR (RT-PCR) analysis of the expression levels of the genes in three sugar-producing strains showing different conversion yields: YJ183/pMDNS1 (~85% conversion), YJ183/pDNS1 (~60% conversion), and YJ183/pDDSS2 (~30% conversion) (Figure S3). Therefore, any difference in the anthracycline glycoside production rate is most likely due to the varying catalytic efficiency of the GT with respect to each substrate rather than a difference in sugar gene cassette expression levels. The one-pot combinatorial biosynthesis system developed here through the cocultivation of S. venezuelae mutants producing aglycones and nucleotide-activated deoxysugars has facilitated the generation of a range of glycosylated derivatives of AKV and ε-RHO by promoting the facile combination of aglycones with various sugars. A total of sixteen glycosylated anthracyclines were produced, among which seven compounds (7, 9, 11, 13, 14, 15, and 16) are new with their structures characterized by HPLC-ESI-MS/MS. Because the focus of this study was developing a combinatorial biosynthesis system, we did not fully determined the structures of these newly obtained compounds by NMR. However, it is likely that the designed sugars were biosynthesized and introduced to the oxygen at C-7 of AKV or ε-RHO based on the previously established functions of AknS and the sugar biosynthetic enzymes as well as the previously confirmed structures of compounds 2, 4, 6, 8, 10, and 12, which were obtained by feeding ε-RHO to the S. venezuelae YJ183 expressing AknS and the same deoxysugar biosynthetic gene sets.13 Although only the basic conditions 12


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for co-cultivation were optimized in this study, the current conversion yields and the titer of some of the engineered glycosides show a potential for this system to produce moderate amounts of engineered compounds for further development by the additional systematic optimization of each aglycone- and sugar-producing host and its expression system. For example, the highest conversion yield observed in YJ183/AKV-MDNS1 and YJ183/RHOMDNS1 was approximately 80%, which corresponds to the production of ~3 mg/L of 3′-Nmethyl-L-daunosaminyl-AKV (3) and ~0.5 mg/L of 3′-N-methyl-L-daunosaminyl-ε-RHO (4) although these compounds were not purified. The lowest conversion yields observed in YJ183/AKV-DDSS2 and YJ183/RHO-DDSS2 were ~25% − 30% producing ~0.9 mg/L of Ddesosaminyl-AKV (13) and ~0.2 mg/L of D-desosaminyl-ε-RHO (14). Taken together, this cocultivation system provides a potentially easy-to-use tool for the creation of novel glycosylated polyketides or other natural products by combining various aglycones and deoxysugars.

Experimental Procedures Bacterial strains, culture conditions, and genetic manipulation. E. coli DH5α and plasmid Litmus28 (New England Biolabs) were used for routine subcloning. S. venezuelae YJ183 was used as a heterologous host,13 and S. venezuelae mutant strains were propagated on SPA medium.25 Protoplast preparation and transformation procedures of S. venezuelae were performed following standard protocols and transformants of S. venezuelae were selected on R2YE agar plates supplemented with thiostrepton.25 The genomic DNAs were prepared from S. peucetius ATCC 29050 and S. venezuelae ATCC 15439 to construct expression plasmids carrying the biosynthetic genes of AKV, ε-RHO, TDP-D-desosamine, TDP-4-N-demethyl-D-amosamine.

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Construction of expression plasmids and S. venezuelae mutant strains. Construction of pDNS1, pMDNS1, pRDS, pEDNS1, pRST1, and pEVCS was previously described13 (Table S1). The genes involved in the biosynthesis of AKV (pAKV), ε-RHO (pRHO), and TDP-Ddesosamine and TDP-4-N-demethyl-D-amosamine and their transfer (pDDSS2 and pNDDA, respectively) were cloned into the replicative E. coli-Streptomyces shuttle vector pSE3420 containing the ermE* promoter21 (Table S1 and Figure S1). Details regarding the construction of these plasmids are described in the Supporting Information. Production and analysis of aglycones and anthracycline glycosides. The production levels and conversion yields of each compound are the averages of two series of triplicate separate cultivations and extractions. Details regarding the production and analysis of intracellular and extracellular aglycones and glycosides are provided in the Supporting Information.

Associated Content Supporting Information. Experimental details; tables for bacterial strains, plasmids, and primers used in this study; ESI-MS/MS fragmentation pattern of 1 – 16.

Author Information Corresponding Author * Tel: +82-2-3277-4082. Fax: +82-2-3277-3419. E-mail: [email protected]. Notes §

Kim, E. and Song, M. C. contributed equally to this work.

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Funding Sources This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (MISP) (2016R1A2A1A05005078), Ministry of Education (2016R1A6A3A11930649), the Intelligent Synthetic Biology Center of the Global Frontier Project funded by MISP (20110031961), and High Value-added Food Technology Development Program, Ministry of Agriculture, Food and Rural Affairs (114019052SB010), Republic of Korea.

Acknowledgments We thank Dr Ah Reum Han for initiating this work and Dr Kris Rathwell for critically reading this manuscript.

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Table 1. Glycosylated anthracyclines produced and their conversion yields from cocultivation systems.

Co-cultivation system

Conversion yield (%)a

YJ183/AKV-DNS1

L-daunosaminyl-AKV

YJ183/RHO-DNS1

L-daunosaminyl-ε-RHO

YJ183/AKV-MDNS1

L-daunosaminyl-AKV

(1) 3′-N-methyl-L-daunosaminyl-AKV (3) L-rhodosaminyl-AKV (5)

3.4 ± 0.19 84.3 ± 5.82 0.3 ± 0.02

YJ183/RHO-MDNS1

L-daunosaminyl-ε-RHO

(2, rhodomycin D) 3′-N-methyl-L-daunosaminyl-ε-RHO (4, 3′-N-methyl-rhodomycin D) L-rhodosaminyl-ε-RHO (6)

2.4 ± 0.10 78.3 ± 2.69 0.3 ± 0.01

YJ183/AKV-RDS

L-daunosaminyl-AKV

(1) 3′-N-methyl-L-daunosaminyl-AKV (3) L-rhodosaminyl-AKV (5)

2.4 ± 0.21 23.4 ± 1.86 34.3 ± 2.74

YJ183/RHO-RDS

L-daunosaminyl-ε-RHO (2, rhodomycin D) 3′-N-methyl-L-daunosaminyl-ε-RHO (4, 3′-N-methyl-rhodomycin D) L-rhodosaminyl-ε-RHO (6)

2.7 ± 0.11 27.6 ± 1.13 34.7 ± 1.38

YJ183/AKV-EDNS1

4′-epi-L-daunosaminyl-AKV (7)

42.8 ± 6.41

YJ183/RHO-EDNS1

4′-epi-L-daunosaminyl-ε-RHO (8, 4′-epi-rhodomycin D)

50.2 ± 6.52

YJ183/AKV-RST1

4′-epi-L-daunosaminyl-AKV (7) L-ristosaminyl-AKV (9)

4.2 ± 0.39 55.8 ± 5.01

L-ristosaminyl-ε-RHO

4′-epi-L-daunosaminyl-ε-RHO (8, 4′-epi-rhodomycin D) (10)

4.6 ± 0.52 61.4 ± 6.08

YJ183/AKV-EVCS

4′-epi-L-daunosaminyl-AKV (7) 4′-epi-L-vancosaminyl-AKV (11)

0.4 ± 0.02 45.1 ± 2.35

YJ183/RHO-EVCS

4′-epi-L-daunosaminyl-ε-RHO (8, 4′-epi-rhodomycin D) 4′-epi-L-vancosaminyl-ε-RHO (12)

0.6 ± 0.04 46.4 ± 2.91

YJ183/AKV-DDSS2

D-desosaminyl-AKV

25.3 ± 2.48

YJ183/RHO-DDSS2

D-desosaminyl-ε-RHO

YJ183/AKV-NDDA

4′-N-demethyl-D-amosaminyl-AKV (15)

48.7 ± 2.42

YJ183/RHO-NDDA

4′-N-demethyl-D-amosaminyl-ε-RHO (16)

45.2 ± 2.71

YJ183/RHO-RST1

a

Products (1) (2, rhodomycin D)

(13) (14)

The conversion yield is expressed as the mean (%) ± the standard errors of the mean.

19


56.2 ± 2.81 63.3 ± 1.32

30.5 ± 2.14


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Figure 1. The structures of doxorubicin, epirubicin, and aclacinomycin A.

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O

a 1x propionyl-CoA dpsABCDG 9x malonyl-CoA (minimal PKS)

O

OH O

dpsE

dpsFY

(9-KR)

(cyclase)

O

dnrG

OH O

O

O

O

O

O

(O-MT) OH O

OH O

O

dnrD

OH OH O

OH O OH O aklanonic acid methyl ester

O

dnrE

OH O

OH O

OH OH

OH O

aklavinone (AKV)

evaD O

O

OTDP

OTDP

(4-KR) O NH2 TDP-4-epi-L-vancosamine NH2

O

(EP) NH2 OTDP

OH

OH desIII HO HO (NT) HO OPO 23 glucose-1-phosphate O

dnmT O O evaA O desIV HO (2,3-DH) (4,6-DH) HO O HO OTDP OTDP TDP-4-keto-6-deoxy-D-glucose

O

O

dnmJ evaB

O

(AT)

H2N

(4-KR) jadV

(EP) OTDP

O HO

desV

desII

O

(AT)

O

OTDP

HO

(DA) OTDP

O

H2N HO

7 or 8

NH2

OTDP

N

OTDP TDP-D-desosamine

HO TDP-L-rhodosamine

13 or 14

aknX 2 aclP

c

O

R1

O

OTDP

NH2

aknX2 aclP

O OTDP (N-MT) NH (N-MT) HO NH2 HO TDP-L-daunosamine TDP-3-N-methyl-Ldaunosamine O

5 or 6 O

OTDP avrE (4-KR) O

dnmU evaD

(4-KR) dnmV

O

15 or 16

HO

OTDP

O

TDP-4-epi-L-daunosamine

O N

O

HO

HO

OTDP TDP-4-N-demethylD-amosamine

(N-MT) desVI

NH2 O

(EP)

(AT) desI

H2N

OTDP

TDP-L-ristosamine

dnmU evaD

O

OTDP

NH2 O

HO

(MT) evaC

HO HO

OH OH

-rhodomycinone ( -RHO)

9 or 10

evaE

O

HO

O OH

11 or 12

b

O OH

O dnrF OH (C11-hydroxylase)

(reductase)

aklaviketone

OH O

aklanonic acid O

O

(cyclase)

dnrC

(C12-oxygenase) 12-deoxyaklanonic acid

O

OH

O

OTDP

3 or 4

1 or 2

O OH

OH O

1 R1 = H, R2 = 2 R1 = OH, R2 =

3 R1 = H, R2 = 4 R1 = OH, R2 =

O HO

NH2

9 R1 = H, R2 = 10 R1 = OH, R2 = HO

OH OR2

NH2 O

5 R1 = H, R2 = 6 R1 = OH, R2 =

O HO

NH

11 R1 = H, R2 = 12 R1 = OH, R2 = HO

O NH2

13 R1 = H, R2 = 14 R1 = OH, R2 =

7 R1 = H, R2 = 8 R1 = OH, R2 = HO

O HO

N

O N HO

15 R1 = H, R2 = H2N 16 R1 = OH, R2 = HO

Figure 2. (a) Biosynthetic pathway of ε-RHO, (b) different deoxysugars directed by the 21


O NH2

O HO


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plasmids described in this study, and (c) the proposed structures of glycosylated AKV and εRHO. The functions of the proteins carrying out each step are shown in brackets: aminotransferase (AT), deaminase (DA), 2,3-dehydratase (2,3-DH), 4,6-dehydratase (4,6DH), epimerase (EP), 4-ketoreductase (4-KR), 9-ketoreductase (9-KR), methyltransferase (MT), N-methyltransferase (N-MT), O-methyltransferase (O-MT), nucleotidyl transferase (NT).

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Figure 3. HPLC-ESI-MS/MS chromatograms of cultures of aglycone-producing S. venezuelae strains. (a) ε-RHO (m/z 428 > 393) detected from culture of YJ183/pRHO. (b) AKV (m/z 412 > 377) detected from culture of YJ183/pAKV.

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Figure 4. Optimization of co-cultivation system. (a) Time course of AKV and ε-RHO titers in YJ183/pAKV and YJ183/pRHO strains, respectively. (b) Relative glycosylation yields at different pre-cultivation periods of the sugar-producing strain, (c) at different mixing ratios of aglycone- and sugar-producing strains, and (d) at different co-cultivation periods for the combined aglycone- and sugar-producing strains. All data are expressed as the mean ± the standard errors of the productivity (mg/L) or peak intensity (%).

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Figure 5. HPLC-ESI-MS/MS chromatograms of co-cultures of aglycone- and sugarproducing S. venezuelae strains. (a) AKV (Rt, 44.3 min; m/z 412 > 377) and L-daunosaminylAKV (1; Rt, 37.0 min; m/z 542 > 130) detected from the co-culture of YJ183/AKV-DNS1. (b) ε-RHO (Rt, 49.0 min; m/z 428 > 393) and L-daunosaminyl-ε-RHO (2; Rt, 39.3 min; m/z 558 > 25



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130) detected from the co-culture of YJ183/RHO-DNS1. (c) AKV, 1, 3′-N-methyl-Ldaunosaminyl-AKV (3; Rt, 38.3 min; m/z 556 > 144), and L-rhodosaminyl-AKV (5; Rt, 40.6 min; m/z 570 > 158) detected from the co-culture of YJ183/AKV-MDNS1. (d) ε-RHO, 2, 3′-Nmethyl-L-daunosaminyl-ε-RHO (4; Rt, 41.5 min; m/z 572 > 144), and L-rhodosaminyl-ε-RHO (6; Rt, 43.5 min; m/z 586 > 130) detected from the co-culture of YJ183/RHO-MDNS1. (e) AKV, 1, 3, and 5 detected from the co-culture of YJ183/AKV-RDS. (f) ε-RHO, 2, 4, and 6 detected from the co-culture of YJ183/RHO-RDS. (g) AKV and 4′-epi-L-daunosaminyl-AKV (7; Rt, 38.5 min; m/z 542 > 130) detected from the co-culture of YJ183/AKV-ENDS1. (h) εRHO and 4′-epi-L-daunosaminyl-ε-RHO (8; Rt, 41.3 min; m/z 558 > 130) detected from the co-culture of YJ183/RHO-ENDS1. (i) AKV, 7, and L-ristosaminyl-AKV (9; Rt, 45.3 min; m/z 542 > 130) detected from the co-culture of YJ183/AKV-RST1. (j) ε-RHO, 8, and Lristosaminyl-ε-RHO (10; Rt, 49.8 min; m/z 558 > 130) detected from the co-culture of YJ183/RHO-RST1. (k) AKV, 7, and 4′-epi-L-vancosaminyl-AKV (11; Rt, 39.3 min; m/z 556 > 144) detected from the co-culture of YJ183/AKV-EVCS. (l) ε-RHO, 8, and 4′-epi-Lvancosaminyl-ε-RHO (12; Rt, 42.8; m/z 572 > 144) detected from the co-culture of YJ183/RHO-EVCS. (m) AKV and D-desosaminyl-AKV (13; Rt, 46.4 min; m/z 570 > 158) detected from the co-culture of YJ183/AKV-DDSS2. (n) ε-RHO and D-desosaminyl-ε-RHO (14; Rt, 50.6 min; m/z 586 > 158) detected from the co-culture of YJ183/RHO-DDSS2. (o) AKV and 4′-N-demethyl-D-amosaminyl-AKV (15; Rt, 34.0 min; m/z 558 > 146) detected from the co-culture of YJ183/AKV-NDDA. (p) ε-RHO and 4′-N-demethyl-D-amosaminyl-ε-RHO (16; Rt, 35.2 min; m/z 574 > 146) detected from the co-culture of YJ183/RHO-NDDA. Magnification factors of peaks’ intensities are displayed at the tops of the chromatograms.

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Table of Contents (TOC)/Abstract graphic

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One-Pot Combinatorial Biosynthesis of Glycosylated Anthracyclines by

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