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A BioBricks Metabolic Engineering Platform for the Biosynthesis of Anthracyclinones in Streptomyces coelicolor

Actinomycetes produce a variety of clinically indispensable molecules, such as antineoplastic anthracyclines. However, the actinomycetes are hindered in their further development as genetically engineered hosts for the synthesis of new anthracycline analogues due to their slow growth kinetics associated with their mycelial life cycle and the lack of a comprehensive genetic toolbox for combinatorial biosynthesis. In this report, we tackled both issues via the development of the BIOPOLYMER (BIOBricks POLYketide Metabolic EngineeRing) toolbox: a comprehensive synthetic biology toolbox consisting of engineered strains, promoters, vectors, and biosynthetic genes for the synthesis of anthracyclinones. An improved derivative of the production host Streptomyces coelicolor M1152 was created by deleting the matAB gene cluster that specifies extracellular poly-β-1,6-N-acetylglucosamine (PNAG). This resulted in a loss of mycelial aggregation, with improved biomass accumulation and anthracyclinone production. We then leveraged BIOPOLYMER to engineer four distinct anthracyclinone pathways, identifying optimal combinations of promoters, genes, and vectors to produce aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone at titers between 15–20 mg/L. Optimization of nogalamycinone production strains resulted in titers of 103 mg/L. We structurally characterized six anthracyclinone products from fermentations, including new compounds 9,10-seco-7-deoxy-nogalamycinone and 4-O-β-d-glucosyl-nogalamycinone. Lastly, we tested the antiproliferative activity of the anthracyclinones in a mammalian cancer cell viability assay, in which nogalamycinone, auramycinone, and aklavinone exhibited moderate cytotoxicity against several cancer cell lines. We envision that BIOPOLYMER will serve as a foundational platform technology for the synthesis of designer anthracycline analogues.

Synthetic biology is a discipline that has been defined as the "engineering-driven building of increasingly complex biological entities for novel applications". 16 Indeed, the field of synthetic biology has resulted in new genetic systems and organisms useful for filling in critical gaps in the pharmaceutical, biofuels, and cosmetics industries. 17 Synthetic biology boasts the development of a variety of genetic tools, including chassis hosts, promoters, terminators, genes, and vectors useful for reprogramming model organisms. Synthetic biology is also a promising discipline for increasing access to indispensable natural products, such as polyketides. 18 Synthetic biology has also contributed new tools for the refinement of idiosyncratic model organisms, such as Streptomyces spp., that are difficult to transform or feature morphological limitations, such as the formation of mycelial clumps during submerged liquid fermentation. 19,20 Streptomyces produce valuable bioactive molecules, such as anthracyclines and tetracyclines. Streptomyces spp. exhibit a highly complex life cycle, including sporulation, branching, fragmentation, and adhesion that is regulated and correlated with specialized metabolism, which limits their use as industrial hosts. Recently, the matAB gene cluster was discovered to encode novel glycosyltransferases responsible for the synthesis of extracellular poly-β-1,6-N-acetylglucosamine (PNAG) that leads to cellular clumping. 21 Van Dissel et al. inactivated the matAB gene cluster in Streptomyces coelicolor M145 and characterized strains that lost the mycelial aggregation phenotype and exhibited improved biomass accumulation. Here, we developed the derivative strain Streptomyces coelicolor M1152ΔmatAB as an improved cell In this report, we developed a BioBricks toolkit of promoters, expression vectors, and engineered Streptomyces hosts for the metabolic engineering of anthracyclinones from type II PKS pathways. All genes were assembled according to the BioBricks [RFC10] standard. We designed minimal PKS (minPKS) cassettes based on the snoa123 (C-20 nogalamycin), aknBC-DE2F (C-21 aclacinomycin), dpsABGCD (C-21 doxorubicin), and oxyABCD (C-19N oxytetracycline) biosynthetic pathways. First, we compared the production titer resulting from expressing minPKS gene cassettes in different Streptomyces hosts: Streptomyces lividans K4−114, Streptomyces coelicolor M1146, M1152, and M1154, and Streptomyces coelicolor M1152ΔmatAB. These experiments resulted in the production of the expected aromatic minimal polyketides. Second, we performed combinatorial biosynthesis experiments by coexpressing the minPKS gene cassettes with different combinations of KR/ARO/CYC/OXY genes to optimize metabolic flux toward tricyclic anthracyclinones. Third, we coexpressed different orthologs of O-methyltransferases, fourth ring cyclases, and 7-ketoreductases to generate the expected anthracyclinones. Finally, the optimum gene combinations were cloned onto one plasmid to determine the production yields in S. coelicolor M1152ΔmatAB. This work resulted in the production of eight anthracyclinone analogues, including the new compounds 9,10seco-7-deoxy-nogalamycinone and 4-β-D-glucosyl-nogalamycinone and the unexpected 7-deoxy analogues ( Figure 2). Finally, the anticancer activity of the different anthracyclinone derivatives was assessed in a mammalian cell viability assay, which revealed that nogalamycinone, auramycinone, and aklavinone had moderate antiproliferative activity (<30 μM IC 50 ) against several human cancer cell lines.

■ RESULTS AND DISCUSSION
Engineering of the snoa123 MinPKS into Different Streptomyces spp. Hosts. We first synthesized a codonoptimized version of the Streptomyces nogalater minimal polyketide synthase (snoa123) responsible for the synthesis of the nogalamycin C-20 poly-β-ketothioester ( Figure 1). The codon optimization was based on the native codon preference for S. coelicolor (Genscript, OptimumGene). Previously, the involvement of snoa123 in the synthesis of nogalamycin has been confirmed via heterologous expression studies and genetic complementation studies in Streptomyces spp. strains. 6,22 The snoa123 operon was designed based on principles from the BioBricks [RFC-10] standard: (1) the genes were decoupled from their native translational coupling and synthesized as individual ORFs; (2) the strong BBa_B0034 ribosome binding site was incorporated into the 5′-untranslated region (5′-AAAGAGGAGAAA-3′) to control the rate of translation initiation for each gene in the operon; 23,24 (3) the genes were cloned to lack internal EcoRI, PstI, SpeI, and XbaI restriction sites, and if necessary, silent mutations were engineered in these restriction sites to make the genes compatible with the BioBricks [RFC-10] standard; (4) the genes were given BioBricks prefix (5′-GAATTCGCGGCCGCTTCTAGAG-3′) and suffix (5′-TACTAGTAGCGGCCGCTGCAG-3′) sequences to enable isocaudomer cloning 25 (Table 1). To facilitate gene expression, we generated a BioBricks compatible version of the ϕC31-based integrating expression vector pSET152 26 (e.g., pSET152BB) to allow for cloning into EcoRI/PstI sites and transformation into S. coelicolor via intergeneric conjugation (Table 1). 27 The genes were expressed from the strong constitutive promoter kasOp*. 28,29 The resulting pSET152BB derivative featured the snoa123 fused to the kasOp* promoter and was transformed into three heterologous hosts: (1) S. lividans K4−114 (lacking the actinorhodin type II polyketide synthase gene cluster); 30 (2) S. coelicolor M1146 (lacking the prodiginine, actinorhodin, coelimycin, and cryptic polyketide gene clusters); 31 and (3) S. coelicolor M1152 (an RNA polymerase B up-regulated mutant of S. coelicolor M1146) ( Table 2).
The heterologous expression of the codon-optimized snoa123 operon resulted in significant production of yellow-orange pigments on SFM agar plates. Each of the recombinant strains was plated in triplicate on R5 agar plates for 5 days and then extracted to analyze the production of polyketides ( Figure 3). Each strain produced copious amounts of known polyketides SEK15 (e.g., C 20 Figures S11 and S12). S. lividans K4−114 also produced large quantities of undesired prodiginines, based on HRESI-MS total ion counts, and therefore this strain was excluded from further experimentation. The production titer of SEK15 was straindependent and media-dependent. On R5 agar plates, the strains transformed with empty pSET152BB vector produced no detectable SEK15; however, S. lividans K4−114 expressing the construct produced 39.6 mg/L SEK15, S. coelicolor M1146 produced 39.7 mg/L SEK15, and S. coelicolor M1152 produced 34.8 mg/L SEK15 ( Figure 3). In SG liquid media, S. coelicolor M1146 and M1152 strains transformed with empty pSET152BB produced no SEK15, whereas S. coelicolor M1152 expressing the snoa123 operon produced 55 mg/L SEK15 and S. coelicolor M1146 produced the highest SEK15 titer at 79 mg/L ( Figure 3).
Engineering of aknBCDE2F, dpsABCDG, and oxyABCD MinPKS Operons into S. coelicolor. To compare the biosynthetic capacity of S. coelicolor M1152 and the S. coelicolor M1152ΔmatAB mutant, heterologous expression experiments were conducted to produce minimally cyclized aromatic polyketides using propionyl-CoA, acetyl-CoA, or malonamyl-CoA starter units. We anticipated that the strains resulting from these experiments would serve as a proof-of-concept for the use of S. coelicolor M1152ΔmatAB as a production host for pathway engineering of anthracyclines. The original aknBCDE2F gene annotations from Streptomyces galilaeus ATCC 31615 (Accession No. AF257324.2) were revised using next-generation sequencing (Supporting Information, Table S1). Next-generation sequencing of pSnogaori, which encodes the majority of the nogalamycin biosynthetic pathway, identified several mutations in the original annotations of snoaD, snoaE, snoaM, and snoaB genes. The revised sequence was deposited in the NCBI database (Accession No. OM832358). Despite several attempts to express codon-optimized versions of the aknBC-DE2F, dpsABCDG, and oxyABCD minPKS operons, attempts to produce the expected minimal aromatic polyketides were unsuccessful. These attempts also included the preservation of the native translational coupling and ribosome binding site of the KSα-KSβ subunits, which Liu et al. previously demonstrated to be indispensable for the expression of the alpABC and whiE− III−IV−V type II PKS subunits in E. coli. 35 As a result, we focused further construct development on the expression of wild-type versions of the snoa123, aknBCDE2F, dpsABCDG, and oxyABCD minPKS operons. 36 The expression of aknBCDE2F and dpsABCDG operons was expected to produce the C-21 polyketide UWM7 and the oxyABCD operon was expected to produce the amidated polyketide WJ85. 37,38 We cloned the aknBCDE2F, snoa123, and oxyABCD genes under the control of the intermediate strength ermE*p promoter or the strong kasOp* promoter and spliced these cassettes into pSET152BB. 28,39 The resulting plasmids were transformed into S. coelicolor M1152 and S. coelicolor M1152ΔmatAB via intergeneric conjugation. S. coelicolor M1152/pSET152BB and S. coelicolor M1152ΔmatAB/pSET152BB were included in the analysis as negative controls. Expression of the wild-type snoa123 minPKS in S. coelicolor M1152 resulted in the production of 9.2 mg/L SEK15, S. coelicolor M1152ΔmatAB harboring the same construct produced 3-fold more SEK15 at 41.8 mg/L (p < 0.0001) ( Figure  3). The codon-optimized version of snoa123 was more productive than the wild-type version of snoa123 (e.g., 79 mg/ L), which indicates that codon-optimization likely enhances translation of the Snoa123 minPKS complex and results in greater metabolic flux toward SEK15 via mass action. We previously observed a similar result when comparing wild-type and codon-optimized versions of the valerena-1,10-diene synthase, VoTPS1, in E. coli. 40 Expression of the codonoptimized version resulted in a 3-fold greater production titer of valerena-1,10-diene than the wild-type version. Additionally, this result demonstrates that the dispersed growth phenotype of S. coelicolor M1152ΔmatAB greatly enhanced polyketide production.
We are only beginning to unravel the mechanisms that control morphogenesis of streptomycetes in submerged cultures. Productivity of streptomycetes in industrial fermentation depends on the mycelial morphology in a product-dependent manner; in other words, less favorable growth conditions may have to be accepted to obtain good productivity. 41−44 Considering that it is hard to predict how production responds to changes in growth and morphology, we need to have more tools at our disposal to change the growth characteristics and thus optimize the chance of success. Overexpression of the cell division activator SsgA increases fragmentation of streptomycetes, which leads to faster growth. A strain of S. coelicolor overexpressing SsgA produced large amounts of prodigionines, but hardly any actinorhodin. 45 However, SsgA affects the intracellular architecture, making the hyphae less robust. Deletion of matAB prevents the production of poly-Nacetylglucosamine (PNAG), an EPS that "glues" the hyphae together, promoting pellet formation. 19,21 Deletion of matAB in S. coelicolor M1152 significantly resulted in a dispersed growth phenotype (data not shown) and led to better productivity. The more PNAG is produced the larger the pellets, and thus the technology may be widely applicable.  Figure S13). 38 In each case, the corresponding S. coelicolor M1152ΔmatAB resulted in a statistically significant increase in UWM7 production titers as compared to S. coelicolor  M1152 (p ≤ 0.01), and the aknBCDE2F operon was twice as productive as the dpsABCDG operon. The best construct featured the kasOp* promoter fused to the aknBCDE2F operon (7.5 mg/L UWM7), which resulted in 33% greater UWM7 as compared to the ermE*p promoter fused to the aknBCDE2F operon (p ≤ 0.05) (Figure 4).
Engineering of oxyABCD into the host strains resulted in the production of WJ85 as determined via HPLC-MS analysis (Supporting Information, Figure S14). WJ85 exhibited a retention time of 9.54 min with a UV maximum at 290 nm, and it exhibited a parental mass of [M + H] + = 368 m/z, as previously described. 37 The ermE*p-oxyABCD construct resulted in the production of 2.3 mg/L WJ85 in S. coelicolor M1152ΔmatAB, as compared to the kasOp*-oxyABCD construct, which resulted in the production of 2.0 mg/L WJ85, although the difference between these two promoters was not statistically significant in this case ( Figure 4). Due to the deletion of the ΔmatAB operon, the S. coelicolor M1152ΔmatAB strain accumulated 1 mg/L SEK15, which was not detected in S. coelicolor M1152. This result was interpreted to mean that S. coelicolor M1152ΔmatAB strain exhibited improved production characteristics for minimal aromatic polyketides. The differences in production titer of WJ85 and SEK15 between M1152 and M1152ΔmatAB were determined to be statistically significant based on ANOVA statistical analysis ( Figure 4). Due to the low production yields of the oxyABCD operon, we did not pursue its use any further. In summation, we chose S. coelicolor M1152ΔmatAB for further experiments to build out anthracyclinone pathways.
Engineering of Ketoreductases, Aromatases, and Cyclases. We next engineered reducing ketoreductase (KR), aromatase (ARO), and cyclase (CYC) gene cassettes into S. coelicolor M1152ΔmatAB to generate tricyclic anthracyclinones ( Figure 5). Starting from the enzyme-tethered poly-βketothioester synthesized by Snoa123, the 3-oxoacyl-[acyl carrier protein] ketoreductase (SnoaD/AknA) reduces the poly-β-ketothioester at 9-position, followed by C7−C12 first ring cyclization and aromatization (SnoaE/AknE1), C5−C14 s ring cyclization and C3−C16 third ring cyclization by the second-third ring cyclase (SnoaM/AknW), and C-12 oxidation by the anthrone oxygenase (SnoaB/AknX) ( Figure 1). Genetic cassettes for aknAE1W, aknAE1WX, snoaDEM, and snoaDEMB were codon-optimized and cloned as described above under the control of the strong p15 promoter from Streptomyces albus and cloned into a separate integrating vector pENSV1 for two plasmid coexpression or spliced into the pSET152BB-kasOp*-snoa123 construct for expression of the entire cassette on a single plasmid. 24,44 Heterologous expression of snoa123+snoaDEM, snoa123+snoaDEMB, snoa123+aknAE1W, and snoa123+a-knAE1WX on two plasmids resulted in the production of SEK15, and C7−C12 cyclized metabolites SEK43 and SEK43b, C7−C12 and C9−C14 cyclized metabolite S2502, and tricyclic anthracyclinones nogalonic acid and its rearrangement product decarboxy-nogalonic acid, as expected ( Figure 5, Supporting Information, Figure S10). These metabolites were confirmed based on a comparison to authenticated biosynthetic standards from S. lividans TK24/pSY21c. 11 Percent conversion was calculated based on the integration of the peak areas at λ = 290 nm since the polyketides exhibit similar molar absorptivity coefficients at this wavelength ( Figure 5). 11 This finding supports the role of AknA as a ketoreductase, AknE1 as an ARO/CYC, AknW as a second and third-ring cyclase, and AknX as an anthrone oxygenase. Previously, Chung et al. demonstrated that the AknX anthrone oxygenase enhances the oxidation of emodin anthrone to emodin anthraquinone, although C-12 oxidation can also occur spontaneously. 10 Expression of the C-12 anthraquinol oxidase increased the formation of correctly cyclized tricyclic anthracyclinone intermediate, which confirms its essential role in the oxidation of nogalonic acid. Coexpression of the snoa123 minPKS genes with the KR/ARO/CYC cassettes on one plasmid resulted in higher metabolic flux toward nogalonic acid ( Figure 5). One possible explanation for this was offered by Yang et al., who observed that expression of type II PKS genes on the same plasmid in E. coli resulted in higher production of carminic acid. 45 Yang et al. postulated that colocalization of the minPKS genes and cyclase genes enhances the formation of the transient polyketide synthase complex, enhancing the production titer. Interestingly, the coexpression of snoa123+aknAE1WX on the same plasmid construct resulted in approximately 15% conversion to nogalonic acid. Expression of the snoa123+snoa-DEMB construct resulted in a 50% conversion to nogalonic acid ( Figure 5). These results demonstrate that the systematic testing of different orthologous gene combinations can provide additional insight into the compatibility of heterologous polyketide synthase components from related pathways. In addition, this approach can be used to provide biochemical evidence for gene products from uncharacterized pathways or for which only bioinformatic description is provided (e.g., AknA, AknE1, AknW).
We next decided to test the potential of BIOPOLYMER for combinatorial biosynthesis by carrying out a full factorial experiment for the biosynthesis of aklanonic acid. We designed this experiment by cloning combinations of the aknBCDE2F and dpsABCDG minPKS operons with the aknAE1WX, snoaDEMB, and dpsEFY+dnrG KR/ARO/CYC/OXY operons. The KR/ ARO/CYC/OXY operons were all cloned under the control of the strong kasOp* promoter since the strong transcriptional regulation of tailoring genes correlates with improved cyclic product formation and diminished shunt product accumulation in the actinorhodin pathway. 46 Expression of aknBCDE2F and dpsABCDG with KR/ARO/CYC/OXY genes resulted in the accumulation of three aklanonic acid-derived shunt products (AA-1, AA-2, and AA3), as previously described ( Figure 6). 4 Mass spectroscopy confirmed the production of the three degradation products:  Figure S85). All combinations assessed resulted in the production of aklanonic acid, which indicates that there is a significant biosynthetic collaboration between minPKS enzymes and ketoreductase/cyclase enzymes between the nogalamycin, daunorubicin, and aclacinomycin biosynthetic pathways ( Figure  6). The production titers of aklanonic acid varied between 20 mg/L to 35 mg/L with the best combinations consisting of recombinant PKS systems dpsABCDG+snoaDEMB and dpsABCDG+aknAE1WX and the native aknBCDE2F +aknAE1WX pathway ( Figure 6). We performed an analysis of variance (ANOVA) of all 12 strains to determine if the differences in production titer between the different strains were statistically significant ( Figure 6). The ANOVA indicated that the observed results between many of the comparisons were statistically significant, which provides good evidence that the combinatorial biosynthesis of the minPKS and cyclase gene cassettes was responsible for the observed differences in production titer.

Engineering of Nogalamycinone and Structural
Characterization of Anthracyclinones. We next decided to complete the aglycone engineering pathway by introducing the nogalonic acid methyltransferase, fourth-ring cyclase, and 7ketoreductase from the nogalamycin biosynthetic pathway (Figure 1). The resulting construct, pSET152BB-ermE*p-snoa123+kasOp*-aknAE1WX+sp44-snoaLCF was transformed into S. coelicolor M1152ΔmatAB and assessed for production of nogalamycinone. The order of genes in the snoaLCF operon was determined to be important for the complete conversion of nogalonic acid to nogalamycinone. Initially, cloning of the snoaC before snoaL and subsequent expression of the resulting plasmid yielded incomplete conversion from nogalonic acid to nogalamycinone. The resulting strains produced six compounds that could be detected via HPLC-MS, including nogalamycinone (1) which could be confirmed via comparison to an authentic HPLC standard ( Figure S18). Interestingly, a new early eluting product was also detected that corresponded to a glucosylated derivative of nogalamycinone, as determined by mass spectrometric analysis in ESI− mode ([M − H] − = 559 m/ z). The resulting strain was scaled up in a 5 L SG shake flask fermentation, extracted with 3 × 5 L of ethyl acetate, and fractionated via SiO 2 column chromatography in chloroform:methanol systems on a Teledyne Combiflash 100 auto purification system. A second 2 L fermentation of the strain was carried out to isolate the unknown glucosylated nogalamycinone derivative. Compounds 1−6 were purified from additional prep-HPLC experiments.
It is generally accepted that the codon-optimization of genes is known to improve translational efficiency by substituting rare codons in an mRNA sequence for preferred codons with abundant tRNAs, which leads to an increase in the concentration of the protein. 48 What is less understood is whether codon-optimization disrupts information that is present in wild-type mRNAs (e.g., rare codons that slow down the translation or that influence translation via binding to rRNA), which could impact protein conformation and function. 49 Hu et al. verified 342 synonymous codon variants of the scFv antibody and observed widely varying production titers, solubility, and binding affinity (e.g., ranging from no binding affinity to 10 −8 M), while all proteins encoded the same original amino acid sequence. 50 Sander et al. demonstrated that synonymous codon changes in a fluorescent protein impacted the fluorescence and can alter the folded structure due to differences in the rate of translation and folding of the N-and C-termini. 51 It could be hypothesized that the production of 3 and 4 by codon optimized SnoaL could be due to protein conformational differences from ACS Synthetic Biology

pubs.acs.org/synthbio
Research Article the wild-type SnoaL which impact the binding of NAME in the active site, but this must be examined further. The formation of 3, 4, and 5 requires the elimination of the 7hydroxyl group, possibly due to the presence of a promiscuous CytA-like enzyme that is present in S. coelicolor. Gui et al. have characterized CytA as a promiscuous anthracycline-inactivating enzyme that reduces the C-7 position for a broad variety of anthracyclines in the cytorhodin pathway according to an NADH-dependent mechanism. 52 Compound 5 was obtained as a yellow solid and displayed UV−vis characteristics like metabolites 1−4. The molecular formula of 5 was established as C 21 H 20 O 7 based on (+)-and (−)-HRESIMS indicative of two additional protons as compared to compounds 3 and 4 (Supporting Information, Figure S64 and S65). Comparison of the 1 H and 13 C NMR of 5 and 3/4 (Supporting Information, Tables S3 and S4) revealed 5 to lack the C-9/C-10 bond connection and the methine proton signal at C-10 position (CH-10) in compounds 3−4 was replaced by CH 2 at δ H 3.83 (CH 2 -10). In addition, an additional oxygenated methine signal was observed at δ H 3.80 (m, CH-9) in compound 5. Furthermore, the singlet methyl signals in compounds 3/4 were replaced by a doublet methyl signal in 5 at δ H 1.22 (d, 6.2). This was consistent with the presence of 1 H− 1 H COSY correlations CH 3 -13/H-9, H-9/CH 2 -8, CH 2 -8/ CH 2 -7 for 5 (Supporting Information, Figure S16). All the remaining 2D-NMR ( 1 H, 1 H-COSY, HMBC, and NOESY) correlations are in full agreement with structure 5 (Supporting Information, Figures S63−S72). Based on the cumulative spectroscopic data, 5 differed solely from those of compounds 3-4 via their C-9/C-10 bond connection. Consistent with the cumulative 1D and 2D NMR data analysis, structure 5 was established as depicted in Figure 1 and named 9,10-seco-7deoxy-nogalamycinone (5).  Figures S74 and S75). Based on the 1 H NMR, 13 C NMR, NOESY, and 1 H, 1 H-COSY data, the carbohydrate was unambiguously assigned as D-glucose (Supporting Information, Table S5 and Figure S17). Compared to 1, the 13 C/ 1 H/HSQC NMR of 6 (Supporting Information, Table S1−S3) highlighted the presence of additional signals for the O-glycoside moiety in compound 6. Based on the 1 H NMR, 13 C NMR, NOESY, and 1 H, 1 H-COSY data, the carbohydrate was unambiguously assigned as D-glucose (Supporting Information, Table S5, Figure S17 and Figures S76−S82). The connection of the sugar moiety at the 4-position was established based on the observed critical HMBC correlation from H-1′ (δ H 5.12, d, J = 7.7 Hz) to C-4 (δ C 160.2). All the remaining 2D-NMR ( 1 H, 1 H-COSY, HMBC, and NOESY) correlations are in full agreement with structure 6 (Supporting Information, Figure  S17 and Figures S76−S82). As a new natural product and closely related to 1, compound 6 was designated as 4-β-D-glucosylnogalamycinone (6).
Full Pathway Engineering of Anthracyclinones. We decided to complete the pathway engineering of other anthracyclinones, including auramycinone (2), aklavinone (7), and 9-epi-aklavinone (8). Fujiwara et al. isolated auramycinone as a C-20 anthracyclinone from S. galilaeus OBB-111, and aklavinone is a C-21 anthracyclinone that serves as the backbone for doxorubicin and aclacinomycin A. 53,54 9-epi-Aklavinone was reported as a hybrid anthracyclinone resulting from the heterologous expression of snoaL from the nogalamycin biosynthetic pathway in a mutant strain of S. peucetius M18. 5 Using the strains S. coelicolor M1152ΔmatAB::pS2S5 (nogalonic acid producer) and S. coelicolor M1152ΔmatAB::pA2A5 (aklanonic acid producer) as hosts, we cloned different combinations of O-methyltransferase genes (aknG, dnrC, snoaC), fourth-ring cyclases (dnrD, kyc34, aknH), and 7ketoreductases (snoaF, dnrE, aknU) under the control of the strong sp44 promoter and cloned them into the TG1actinophage integrating vector pENTG1. 24 In brief, pENTG1 is a vector that encodes bla and vph for ampicillin and viomycin selection, respectively, and a codon-optimized version of the TG1 integrase and corresponding attP site for integration into the S. coelicolor TG1 attB chromosomal locus at a single copy. 55,56 The resulting vectors were expected to result in production of 9(S)-configured anthracyclinones (e.g., pENTG1-sp44-snoaC+kyc34+snoaF, pENTG1-sp44-snoCLF) or 9(R)-configured anthracyclinones (e.g., pENTG1-sp44-dnrCDE and pENTG1-sp44-aknGHU). Coexpression of these vectors in either of the two strains resulted in the production of the expected anthracyclinones (Figure 7). To produce nogalamycinone, snoaLCF and snoaC+kyc34+snoaF both resulted in the complete conversion of nogalonic acid. This result also provides experimental proof that kyc34 from the keyicin biosynthetic pathway encodes a NAME cyclase, like snoaL. 57 Similarly, coexpression of aknGHU in S. coelicolor M1152ΔmatAB::pS2S5 resulted in the complete conversion of nogalonic acid to 2. For the production of 7, when S. coelicolor M1152ΔmatAB::pA2A5 was complemented with aknGHU, complete conversion of aklanonic acid was observed. Lastly, the complementation of the aklanonic acid producer with either snoaLCF or snoaC+kyc34+snoaF resulted in approximately 80% conversion to 9-epi-aklavinone. This experiment demonstrates that the BIOPOLYMER system can be used to produce all the naturally occurring reduced anthracyclinone analogues.
The dose−response relationships of 1, 2, 6, 7, and 9−13 were established for A549 and PC3 cells and Merkel cells MKL1 and MCC26 (Supporting Information, Figure S83 and S84). The half-maximal inhibitory concentration values (IC 50 ) were determined for the entire cancer cell panel (Table 3). Previously reported compound 9 was the most cytotoxic among the set tested, with IC 50 values ranging from 21 to 95 nM. In contrast, compound 7 was the most active among the analogues derived from the current study, with IC 50 values ranging from 8 to 26 μM. This is consistent with previous studies highlighting the contribution of anthracycline glycosylation to potency, selectivity, and ADMET. 67

■ CONCLUSIONS
The "mixing and matching" of PKS components for the reconstitution of non-native aromatic polyketide synthases has been the subject of numerous in vivo studies, which provided the foundational basis for the biosynthetic logic of these enzymes. 68−72 However, these experiments often utilized PKS enzymes from disparate pathways, which resulted in recombinant PKS systems that produced low yields of the expected novel  polyketides, or that failed. 73 This resulted in the observation that the failure of polyketide combinatorial biosynthesis derives from the inflexibility of downstream enzymes to recognize novel substrates or lack of enzyme solubility. 74 In contrast, focusing on enzymes from evolutionarily related pathways might provide more fertile ground for combinatorial biosynthesis efforts and improved substrate turnover to produce novel polyketides. More recently, the use of cell-free biosynthesis (also known as "combinatorial biosynthetic enzymology") has demonstrated the success of this "mix-and-match" approach via the use of PKS enzymes from closely related pathways for the synthesis of defucogilvocarcin M and steffimycinone in vitro. 75,76 A "Design-Build-Test-Learn" (DBTL) approach has been suggested to uncover the logic of recombinant PKS systems. 77 This is the approach that we have currently undertaken with the development of the BIOPOLYMER toolbox, via the identification of PKS gene orthologs that interact positively and result in the production of expected polyketides in S. coelicolor M1152ΔmatAB. In this work, the "design phase" resulted in the selection of different S. coelicolor hosts, strong promoters, vector combinations, and gene orthologs. The "build phase" was facilitated by the use of the BioBricks-[RFC 10] synthetic biology standard and straightforward transformation of Streptomyces spp. using intergeneric conjugation. The "test phase" carried out several different comparisons of anthracyclinone genes to identify those with the best conversion percentage and production yield of the expected polyketide. Lastly, the "learn phase" resulted in the identification of an optimal production host, S. coelicolor M1152ΔmatAB, promoter and gene combinations, and the structure elucidation of several anthracyclinones (1−4) and new compounds 9,10-seco-7deoxy-nogalamycinone (5) and 4-O-β-D-glucosyl-nogalamycinone (6). Furthermore, the production platform was used to generate newly engineered metabolites for testing in mammalian cancer cell viability assays. These assays confirmed that the 7-Oglycoside is important for the anticancer activity of anthracyclines, including nogalamycin A (9), 3′,4′-demethoxy-nogalose-1-hydroxy-nogalamycinone (12), and aclacinomycin T (13). Nevertheless, the observation that nogalamycinone, auramycinone, and aklavinone exhibited moderate cytotoxicity is encouraging for further efforts to generate diverse anthracycline analogues incorporating these pharmacophores.
The pathway engineering studies described here resulted in the unexpected generation of 7-deoxygenated metabolites, 3 and 4, and two new anthracycline metabolites, 9,10-seco-7-deoxynogalamycinone (5) and 4-O-β-D-glucosyl-nogalamycinone (6). Despite being a genome minimalized "superhost", the genome S. coelicolor M1152ΔmatAB still encodes hypothetical proteins that can interact with heterologous pathways in an unanticipated fashion. Compounds 3, 4, and 5 are thought to derive from the dehydration of the 7-hydroxyl group via an ancillary CytA-like enzyme. 52 CytA has been shown to catalyze the 7-reduction of a wide variety of anthracycline saccharide chains within the cytorhodin pathway. CytA could be part of a larger family of promiscuous enzymes encoded within actinomycete genomes, perhaps as an evolutionary self-defense strategy against xenobiotics. Studies are ongoing to identify this reductase within S. coelicolor M1152.
Compound 6 is thought to derive from a hypothetical glucosyltransferase that catalyzes the transfer of NDP-D-glucose to the 4-position of nogalamycinone. Studies are ongoing to identify the glucosyltransferase responsible for the transfer of NDP-D-glucose to nogalamycinone. The macrolide glucosyl-transferase OleD has been mutagenized via directed evolution to afford one of the most substrate-promiscuous glycosyltransferase catalysts for the glycorandomization of a variety of diverse natural products. 78−81 Notably, 6 has a similar 4-O-glycosylation pattern to the previously discovered mutactimycin PR, andicoquinones A−D, komodoquinone A, and histomodulin. 82 In the cancer cell line cytotoxicity assays, the 4-β-D-glucose substitution was deleterious to the topoisomerase II inhibition of 6 since this compound was only slightly cytotoxic ( Figure 9). Instead, the closely related 4-O-β-D-glucopyranuronosyl-εrhodomycinone (histomodulin) has been shown to exhibit upregulation of major histocompatibility class-I molecules on the surface of T-cells. 83 This class of 4-O-glucosides could be further investigated for unique immunomodulatory activities as potential new anticancer or anti-infective agents.
In summation, BIOPOLYMER is a flexible synthetic biology platform for the rational programming of S. coelicolor to produce aromatic polyketide natural products. We anticipate that BIOPOLYMER will be useful for providing access to known and new anthracycline natural products for antiproliferative activity studies. We also expect that BIOPOLYMER could be useful for studying cryptic type II PKS pathways and for providing a robust toolset for unraveling their biosynthetic logic.
S. lividans and S. coelicolor derivative strains were routinely maintained on Soya-Mannitol Flour (SFM) agar supplemented with 10 mM MgCl 2 and International Streptomyces Project medium #4 (ISP4) (BD Difco) at 30°C as described previously. 85 S. lividans K4−114 was a gift from Prof. Dr. Lou Charkoudian (Haverford College, PA). S. coelicolor M1146 and S. coelicolor M1152 were gifts from Prof. Dr. Mervyn Bibb's laboratory (John Innes Centre, Norwich, UK). S. coelicolor M1152ΔmatAB was generated via replacement of the sco2963-sco2962 matAB locus via PCR-ReDirect mutagenesis as previously described (Supporting Information, Method 1). 19 For liquid culturing, S. coelicolor derivative strains were grown in TSB media (3 mL) to ferment the seed culture and then grown in a modified 50 mL SG-TES liquid medium (soytone 10 g, glucose 20 g, yeast extract 5 g, TES free acid 5.73 g, CoCl 2 1 mg, per liter) or 50 mL E1 medium for production for 7 days. 86 All media and reagents were purchased from Thermo-Fisher Scientific.
General Manipulations. Routine genetic cloning and plasmid manipulation were carried out in E. coli JM109 (New England Biolabs). E. coli ET12567/pUZ8002 was used as the host for intergeneric conjugation with S. coelicolor as previously described. 85   Statistical Analyses. The statistical significance of the impact of genetic manipulations and combinatorially assessed variables on production was assessed via post hoc analysis. Oneway ANOVA, two-way ANOVA, and Student's t test analyses were performed using GraphPad Prism version 9.4.1 for Mac OS X, GraphPad Software, San Diego, CA, USA, www.graphpad. com.
Cancer Cell Line Viability Assay. A549 and PC3 cells were obtained from ATCC (Manassas, VA). Merkel cells MKL1 and MCC26 were a gift from Dr. Isaac Brownell's laboratory (NIH/ NIAMS, Bethesda, MD). All other reagents used were reagent grade and purchased from Sigma-Aldrich (Saint Louis, MO). Human cell line cytotoxicity [A549 (non-small-cell lung) and PC3 (prostate) human cancer cell lines, Merkel cells MKL1 and MCC26] assays were accomplished in triplicate following our previously reported protocols. 87−90 Vehicle (DMSO) was used as the negative control, and actinomycin D and H 2 O 2 (A549 and PC3; MKL1 and MCC26) were used as positive control at 20 μM and 1 mM concentration, respectively.
Isolation of Compounds 1−6. S. coelicolor M1152Δma-tAB::pRW10000 was grown on a soya mannitol flour (SFM) agar plate until well-sporulated for 4−5 days. Spores from the SFM agar plate were used to inoculate a seed culture of 50 mL tryptic soy broth in a 250 mL Erlenmeyer shake flask and were grown for 48 h in an orbital shaker at 30°C at 220 rpm. One milliliter of seed culture was used to inoculate each of the 40 × 100 mL SG liquid media shake flasks (1% v/v inoculum) which were fermented for 5 days. The 4 L culture was extracted 4 × 4 L of ethyl acetate +0.1% formic acid and was dried in vacuo on a rotary evaporator. The resulting 2.3 g crude extract was dissolved in 9:1 chloroform−methanol and was loaded onto a 25 g SiO 2 solid load cartridge and chromatographed on a 24 g RediSep Gold Normal Phase Silica cartridge (Teledyne-ISCO). The extract was fractionated using the gradient setting and chloroform−methanol systems [Method E: chloroform; solvent B: methanol; flow rate: 40.0 mL min −1 ; 0−15 min, 0−10% B.] Compounds 1−5 eluted in combined fraction at a retention time of 3.0 min. The compounds were individually resolved and purified via preparatory HPLC.
The same strain was separately grown in 2 L of E1 media to produce substantive amounts of compound 6. The 2 L fermentation was centrifuged to separate the cell pellet from the fermentation broth. The fermentation broth was extracted with 40 g Amberlite XAD-7 resin for 16 h, washed with 2 L of water, eluted with 1 L of methanol, and dried in vacuo on a rotary evaporator. Compound 6 was isolated via preparatory HPLC.
Experimental procedure for generation of S. coelicolor M1152ΔmatAB strain, plasmid maps for vectors, sequences of BioBricks parts and coding sequences, high-resolution mass spectrometry data, NMR tables, NMR spectra, HPLC-UV−vis chromatograms, and dose−response data for human cancer cell lines (PDF)