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Synthetic Inducible Regulatory Systems Optimized for the Modulation of Secondary Metabolite Production in Streptomyces Chang-Hun Ji, Hiyoung Kim, and Hahk-Soo Kang ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.9b00001 • Publication Date (Web): 26 Feb 2019 Downloaded from http://pubs.acs.org on February 27, 2019

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ACS Synthetic Biology

Synthetic Inducible Regulatory Systems Optimized for the Modulation of Secondary Metabolite Production in Streptomyces Chang-Hun Ji, Hiyoung Kim, Hahk-Soo Kang* Department of Biomedical Science and Engineering, Konkuk University, Seoul 05029, Republic of Korea *Corresponding author : Prof. Hahk-Soo Kang, Department of Biomedical Science and Engineering, Konkuk university, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Republic of Korea; Telephone: +82-2450-4136; Email [email protected]

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Abstract Biosynthesis of secondary metabolites is a highly complex process that often requires tight control of their production, as over-production of metabolites could be toxic and also may cause metabolic burden to their hosts. Tight control of metabolite production could be achieved by expressing key biosynthetic genes under control of an inducible regulatory system. In this study, we employed the modular design approach to build a high performance synthetic inducible regulatory systems that displays a large dynamic range, and thus is well-suited for the modulation of secondary metabolite production in Streptomyces. To this end, an inducible regulatory system was divided into three separate functional modules: 1) the induction module, 2) the target expression module, and 3) the repressor expression module. Then, these three separate modules were individually optimized in a step-wise manner and assembled to a new system. First, the cumate (CMT) induction module was chosen as the best performing induction module based on the large dynamic range and moderate inducer sensitivity. Then, the CMT induction module maintained its performance when combined with diverse constitutive target expression modules, in which overall dynamic ranges varied depending on maximum promoter strengths. Lastly, the repressor expression module was optimized to achieve complete elimination of leaky expression, further increasing the dynamic range of the system. We also demonstrate that any strong constitutive regulatory system could be converted into an inducible regulatory system by simple CRISPR/Cas9-aided marker-less insertion of an operator sequence whenever tight control of gene expression is required. We believe that the synthetic inducible regulatory system we report here would become an useful tool in modulating secondary metabolite production in Streptomyces. Keyword: secondary metabolites, inducible regulatory system, Streptomyces, production control

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Streptomyces produce a structurally diverse array of secondary metabolites, many of which possess promising biologically activities.1-3 However, utilization of these active metabolites as new drug leads has been hampered by the difficulty of acquiring sufficient quantity for in vivo efficacy studies due to their low titers in normal laboratory culture conditions.4,

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In addition, recent genome sequencing has revealed the

presence of a large number of cryptic secondary metabolite biosynthetic gene clusters in Streptomyces genomes.6-8 However, characterization of their metabolites has been challenging, as their production level in normal culture conditions is significantly lower than the detection limits of most analytical methods.9 Low titers of secondary metabolites in normal culture conditions are mainly due to tight control of their biosynthesis mostly in a transcription level because their biosynthesis requires the consumption of a significant level of cellular resources and energy.10 The promoter regions of biosynthetic operons are an integral part of transcriptional control of secondary metabolite biosynthetic genes,11 and most of their regulation mechanisms remain un-characterized hampering their optimization. Therefore, the replacement of tightly regulated native promoters with well-characterized synthetic promoters could provide a potential solution to this problem.12, 13 Promoter engineering of secondary metabolite biosynthetic gene clusters has so far been performed using strong constitutive promoters. However, the insertion of strong constitutive promoters upstream of biosynthetic genes can result in significantly impaired cell growth due to metabolic burden caused by overexpression of biosynthetic enzymes.14 Strong expression of biosynthetic enzymes can also led to the large accumulation of toxic metabolites inside cells that negatively affect cellular physiologies of production hosts.15 This indicates that tight control of gene expression is essential to maximize secondary metabolite production without causing the inhibition of cell growth. Optimized expression of biosynthetic genes by promoter engineering could be achieved by repeated cycles of trial-and-error using the library of promoters with a diverse range of transcriptional strengths. Since each cycle involves the characterization of both metabolite production and growth rate, this trial-and-error process is highly time-consuming, making it difficult to do in a systematic way. The use of an inducible regulatory system would therefore provide an attractive means to expedite the optimization process,16 since the level of metabolite production could be modulated simply by adjusting inducer concentration. In this case, tight control of metabolite production is premised on the predictable and reproducible relationship between inducer concentration and gene expression level.17 Although several inducible regulatory systems have been previously described in Streptomyces, none of them are suitable for the modulation of gene expression primary due to their low dynamic ranges, and thus they are rather used as an on-off switch.18-20 Here, we report the development of a high-performance synthetic inducible regulatory system with a large 3



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dynamic range that enables the predictable modulation of metabolite production in Streptomyces. To this end, we employed the modular design approach (Figure 1), which has been the key design concept in synthetic biology.21 An inducible regulatory system was divided into three separate functional genetic modules: 1) the induction module, 2) the target expression module, and 3) the repressor expression module. These three separate modules were optimized in a step-wise manner using secondary metabolite biosynthetic genes, including the indigoidine non-ribosomal peptide synthetase (NRPS) and the actinorhodin (ACT) type II polyketide synthase (PKS) genes. Using this approach, we created the synthetic cumatebased inducible system that possesses a large dynamic range in which the level of metabolite production varies in response to cumate concentrations. We also demonstrate that strong constitutive RSs (Regulatory Sequences) could be converted into inducible RSs by simple insertion of an operator sequence via CRISPR/Cas9-aided marker-less homologous recombination in yeast.

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Results and Discussion Design principle of synthetic inducible regulatory system: modular design approach To build a high performance inducible regulatory system that is well-suited for the modulation of secondary metabolite production in Streptomyces, we employed the modular design approach in which a biological system is divided into separate functional genetic modules that are re-designed and assembled into a new system with an improved and predictable function. Applying this concept, we divided an inducible regulatory system into three separate functional modules: 1) the induction module (part composition: repressor and operator), 2) the target expression module (part composition: promoter and RBS); and 3) the repressor expression module (part composition: promoter and RBS). These three functional modules are likely to independently affect the overall performance of an inducible regulatory system. The induction module would determine the sensitivity of an inducible system to varying inducer concentrations, whereas an overall dynamic range would be affected by both target and repressor expression modules, which determine the achievable maximum expression and repression, respectively. There are two different types of inducible regulatory systems, repressor- and activator-based systems.22, 23 Of these, repressor-based systems were chosen for the induction module because repressor/operator pairs could be combined with constitutive RSs for which transcription is initiated by the housekeeping sigma factor HrdB that is prevalent in most Streptomyces species.24,

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Well-characterized repressor-based induction

modules in Streptomyces include anhydrotetracycline (TET: TetR/tetO), oxytetracycline (OTR: OtrR/otrO), cumate (CMT: CymR/cmtO) and resorcinol (ROL: RolR/rolO) induction modules.26-28 These induction modules were chosen because their operator sequences were relatively short and unambiguously characterized. To compare their performances, we combined these four induction modules with the same target and repressor expression modules (kasO*RS and SF14RS, respectively) to create the four IRCs (Inducible Regulatory Cassettes). First, the repressor genes, which were all codon-optimized to allow for the optimal expression in Streptomyces, were positioned immediate downstream of the SF14RS repressor expression module. Next, the operator sequences were inserted between the promoter and RBS sequences in the kasO*RS target expression module. Lastly, we inserted the 25 bp random spacer sequences between the oppositely placed repressor and target expression modules to eliminate any possible transcriptional collision between two elongating RNA polymerases during transcription (Figure S1). Dynamic ranges of the induction modules: characterization of leakiness and inducibility 5



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The designed four IRCs were synthesized as double stranded DNA fragments (Figure 2A) and cloned into the promoter assay vector pIJPT1 immediate upstream of the indC (indigoidine synthetase) gene using homologous recombination in yeast. These four pIJPT1/IRC constructs were shuttled into S. albus J1074, and integrated into the genome for heterologous expression. Indigoidine production was induced by adding appropriate inducers into the cultures to final concentrations known to drive full induction. The indC gene with no promoter was used as a negative control, and the indC gene transcribed by the constitutive kasO*RS was used as a positive control. Performance of each IRC could be characterized using two measurable factors, a dynamic range and inducer sensitivity. A dynamic range reflects the difference in expression levels between the fully repressed and fully induced states (Figure 2B), whereas the range of inducer concentrations in which the level of gene expression could be modulated indicates an inducer sensitivity. First, a dynamic range of each IRC was characterized by the fold-increase (induced state/repressed state), which is governed by the two measurable factors: 1) leakiness (repressed state/induced state) and 2) inducibility (induced state/positive control). Of the four IRCs tested, the CMT induction module demonstrated the largest dynamic range (10.7 fold-increase) due to the lowest leakiness (9.3%) and the highest inducibility (107%). In contrast, the dynamic ranges were noticeably low for the OTR (2.2 fold-increase) and ROL (1.2 fold-increase) induction modules. Since we used the same repressor expression (SF14RS) and target expression (kasO*RS) modules for all four IRCs, the degree of repression and inducibility would only rely on the binding affinity of repressor proteins to their operator sequences. The ROL induction module showed the highest leakiness (84.9%), which indicates that the binding affinity of the RolR repressor was not strong enough to drive full repression by SF14RS. Upon full induction, significantly low indigoidine production (52.9% inducibility) was observed for the OTR induction module. One possibility for this could be due to the strong binding affinity of the OtrR repressor, causing a significant portion of the repressors remaining bound to the operator sites. However, this is not likely to be the case as the OTR induction module showed the high leakiness (44.7%). Another possibility could be due to the growth inhibition effect of the OTR inducer. Since oxytetracycline has been known to be a strong antibiotic,29 its addition to the culture at high concentration could negatively affect the cell growth, eventually lowering the dynamic range of the OTR induction module. To test this possibility, we monitored both indigoidine production and cell growth for 5 days (Figure S2) upon addition of the OTR inducer, and compared the results with those of the other inducers. The results indicated that TET, CMT and ROL had no effect on the cell growth; however significant growth inhibition was observed for the OTR inducer. This explains the low inducibility of the OTR module (52.9%) compared to those of the TET (90.1%), CMT (107%) and ROL (97.8%) modules. Our result suggests that although the OTR concentration (5 µM) used for full induction was lower than the reported minimum inhibitory concentration (MIC, 10 µM ~ 30 µM), it still could inhibit cell growth, limiting the use of 6


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the OTR induction module as an inducible system for production control. Unlike the OTR inducer, the TET inducer showed no toxicity,30 and thus it is likely that S. albus is self-resistant to TET at the concentration tested.31 Inducer sensitivities of the induction modules: characterization of inducer concentration-dependent indigoidine production The ultimate goal of promoter engineering is to achieve high-titer production of metabolites without affecting the growth of a production host. In this sense, the ability to precisely modulate gene expression would be the most crucial factor when developing a synthetic inducible regulatory system for use in the modulation of metabolite production.32 To identify the induction module best suited for this purpose, we next characterized the relationship between the level of metabolite production and inducer concentration for each induction module. The ROL induction module was excluded from the test due to its small dynamic range. Six different concentrations were prepared for each inducer by serial 4-fold dilution starting from the concentration used for full induction (Figure 3A). Of the three induction modules tested, the OTR and CMT modules displayed the change in indigoidine production in response to inducer concentration. When the logarithmic scale was used for x-axis, both OTR and CMT modules showed the linear relationships within the dynamic ranges that determine the highest and lowest levels of indigoidine production (Figure 3B). The range of inducer concentration, in which the linearity was observed, was almost identical between the OTR and CMT modules (75 ~ 6,250 nM), indicating that both inducers have similar binding sensitivities to their repressors. In contrast, the TET induction module showed nearly identical indigoidine production in all the concentrations tested, and this suggest that the TET module has relatively high inducer sensitivity compared to those of the OTR and CMT modules. Thus, we further diluted the TET inducer solution by serial 2-fold dilutions, making another six different concentrations ranging between 1 - 40 nM. In this concentration range, indigoidine production varied in response to the TET concentration. Although the linearity was also observed for the TET induction module, the narrow concentration range indicates the high inducer sensitivity, and thus raises the possibility of low reproducibility and high error occurrence, limiting its application as a tool for the modulation of metabolite production. The overall results suggest that the CMT module is best suited for use in the modulation of metabolite production in Streptomyces among the four induction modules tested due to its largest dynamic range and moderate inducer sensitivity. Module compatibility: the CMT induction module combined with diverse constitutive RSs 7



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The induction module that is compatible with a diverse range of constitutive RSs (promoter/RBS) would offer a special advantage when applied as a tool for promoter engineering.33 The diverse libraries of constitutive promoters has been previously developed for use in Streptomyces.34, 35 Secondary metabolite biosynthetic gene clusters are commonly comprised of multiple operons, and therefore their promoter engineering requires the replacement of multiple RSs to achieve complete refactoring of their transcriptional regulatory networks. However, strong expression of multiple biosynthetic genes often causes the formation of a biosynthetic bottleneck due to the presence of a rate limiting step, resulting in the accumulation of shunt products.36 In this case, it would be desirable to tune expression of a biosynthetic gene that participate in a rate-limiting step to reduce the formation of toxic and difficult to separate shunt products, and this could be achieved simply by converting a constitutive RS to an inducible RS by inserting an operator sequence between the promoter and RBS sequences. To make this possible, an operator sequence should exhibit a similar and predictable performance when combined with diverse constitutive RSs. To test if the CMT induction module is compatible with diverse constitutive RSs, we designed five additional constructs in which the CMT module was combined with five different constitutive RSs, including two native (ermE* and otrB) and three synthetic (A12, A26 and A35) RSs (Figure 4A). These constructs were synthesized as DNA fragments and cloned into pIJPT1, and then their dynamic ranges were compared with that of the CMT-inducible kasO* RS. Overall, the CMT induction module generally performed well when combined with constitutive RSs and showed dynamic ranges ranging from 5X to as high as 14.8X (Figure 4B). The difference in dynamic ranges stem from the difference in transcriptional strengths of combined RSs as the level of indigoidine production upon full induction was similar to those of the positive controls (inducibility near 100%). Only exception found was the construct in which the CMT module was combined with otrBRS. This construct displayed unexpectedly weak inducibility (30.8%) even upon full induction. The careful examination of the DNA sequence of the construct found the presence of a short palindromic (red highlighted in the Figure S3) sequence between the otrBp and cmtO sequences, suggesting that the formation of a secondary structure probably interfered with binding of a sigma factor to the promoter site. Refactoring of the ACT cluster: CRISPR/Cas9-aided insertion of the operator sequence Next, we applied the CMT-based inducible regulatory system to the more complex, multi-operoncontaining ACT biosynthetic gene cluster. ACT is an aromatic polyketide synthesized by the type II PKS biosynthetic gene cluster, and its biosynthesis is initiated by the minPKS enzyme complex that generates a polyketide chain with a pre-defined length.37 We have previously engineered the ACT cluster by replacing the native P1 promoter, which drives the transcription of the minPKS genes, with the strong RS cassette.34 8


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This engineered construct produced both ACT and shunt products. To enable the modulation of ACT production using the inducible regulatory system (Figure 5A), we converted the strong constitutive RS into the CMT-inducible RS by inserting the cmtO operator sequence using the CRISPR/Cas9-aided marker-less homologous recombination in yeast. First, the S. albus strain constitutively expressing the CymR repressor was created: the pIJPT1 assay vector that contains the CMT IRC was modified to remove the indC reporter gene from the vector, transformed to S. albus and integrated into the genome using the ΦBT1 integrase. Then, the strong constitutive RS that drives transcription of the minPKS genes in the ACT-P1(S1) construct was converted into the inducible RS by inserting the cmtO operator sequence between the promoter and RBS sequences using the CRISPR/Cas9-aided marker-less homologous recombination in yeast.38 To this end, the two CRISPR target sequences were selected from the S1 RS region in the ACT cluster (Figure S4), and cloned into the yeast CRISPR vector pCRCT creating the plasmids that express the iCas9 protein as well as the guide RNA. The pCRCT/S1 plasmid was co-transformed with the ACT-P1(S1) construct and the bridge DNAs into yeast. The bridge DNA contained the cmtO operator sequence flanked by the two 40 bp homologous sequences designed to place cmtO into the immediate downstream (+1 position) of the transcription start site. Then, the engineered ACT-P1(S1/cmtO) construct was isolated from yeast, transferred to the CymR-expressing S. albus strain via intergenic conjugation and integrated into the genome using the ΦC31 integrase (Figure 5B). Optimization of the repressor expression: further improvement of the dynamic range S. albus bearing the ACT-P1(S1/cmtO) construct was cultured with or without the CMT inducer, and then the level of ACT production was measured by recording OD640 of the culture supernatant. Upon full induction, the ACT-P1(S1/cmtO) construct showed ACT production comparable to that of the ACT-P1(S1) construct, indicating that transcriptional and translational strength of the S1 RS remain the same after the insertion of the cmtO operator sequence. However, leaky ACT production (28.4% leakiness) was observed for the culture without the CMT inducer, lowering its dynamic range to the 3.52X fold-increase. Higher leakiness observed is probably due to the higher detection sensitivity of ACT compared to that of indigoidine. This suggest that the SF14 RS, used as the repressor expression module, might not be strong enough to drive full repression. In an attempt to achieve complete repression,39 we replaced the SF14 RS with the stronger RSs present in our synthetic constitutive RS library,34 including A13RS, A51RS or A26RS (Figure 6A). The three S. albus strains that strongly express the CymR repressors showed nearly complete repression with the 2.26% (A13RS), 1.51% (A51RS) and 1.76% (A26RS) leakiness in the absence of the CMT 9



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inducer (Figure 6B). For these strains, ACT production was also fully induced upon addition of the CMT inducer at the maximum concentration, thus all displaying 10 times higher dynamic ranges (>40X foldincrease) than the one observed with the SF14 RS (3.5X fold-increase). Unexpectedly, the S. albus strain expressing CymR repressor gene under the A51 RS showed maximum ACT production increased by approximately 60% compared to that of the positive control. Although the exact reason for this is unknown, the increased level of maximum production (162% inducibility) further extended the dynamic range of the CMT-inducible RS to the 66.4X fold-increase, making it the largest dynamic range observed in the study. To test the utility of the CMT-inducible RS for the modulation of ACT production, we next measured the change in the level of ACT production in response to varying CMT concentrations (Figure 6C). The four S. albus strains that vary only in the repressor expression module all produced ACT in the CMT concentration dependent manner with the concentration range of 0.1–100 μM, which is consistent with the result observed with indigoidine as a reporter. This results indicate that the predictable modulation of metabolite production could be achievable using the synthetic CMT-inducible RS, and the range of modulation would be determined by the dynamic range of a system which is governed by the target expression (maximum production) and repressor expression (maximum repression) modules.

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Conclusions Production of secondary metabolites is tightly controlled in native Streptomyces hosts as their biosynthesis is a highly energy-demanding process that negatively affects the cell growth. The advance in genomics technology has provided us with tools that enable rapid cloning of complex, large secondary metabolite biosynthetic gene clusters. As a result, heterologous expression is being increasingly used as a platform not only to optimize production of existing high-value secondary metabolites, but also to characterize metabolites encoded by yet-unidentified cryptic biosynthetic gene clusters. Transcriptional control of most of biosynthetic gene clusters are complex and difficult to generalize, and therefore promoter engineering provides an opportunity to bypass their native regulatory systems and put their transcription under our control. Therefore, a well-developed inducible regulatory system could be an ideal tool for this purpose, in which the expression of biosynthetic genes is tightly controlled using inducer concentration. In this study, we employed the modular design approach to construct the high-performing synthetic inducible regulatory system characterized by the large dynamic range and moderate inducer sensitivity, thus allowing for the predictable modulation of gene expression. An inducible regulatory system was divided into three separate functional modules, the induction module, the target expression module, and the repressor expression module. These three separate modules were individually optimized stepwise. First, we fixed the target and repressor expression modules the same, and tested the four different Streptomyces induction modules, of which the CMT induction module demonstrated the highest performance. Next, we proved that the CMT module shows the highly reproducible and uniform performance when combined with diverse constitutive RSs as the target expression modules. Lastly, the repressor expression module was further optimized to achieve complete elimination of the leaky expression. Through this process, we were able to build the cumate-based synthetic inducible regulatory system with the large dynamic range (66.4X fold increase), which could be used as a metabolic modulator that allows for optimized production of bioactive secondary metabolites displaying toxicity to heterologous hosts. We also demonstrated that the operator sequence could be combined with any constitutive RS by CRISPR/Cas9aided homologous recombination in yeast. This allows for the simple conversion of a constitutive RS to an inducible RS whenever the tight control of gene expression is required. We believe that the inducible regulatory system and the modular design approach we report here would become an useful tool for promoter engineering of secondary metabolite biosynthetic gene clusters that will facilitate genomics-based natural product research in the post-genomic era.

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Materials and methods Strains and culture conditions. Bacterial and yeast strains used in this study were listed in Table S1. The strain Saccharomyces cerevisiae BY4727 was used for all DNA engineering experiments.40 Yeast was maintained on YPD agar plates and grown overnight at 30 °C with shaking (200 rpm) prior to transformation. The LiAc/ss carrier DNA/PEG yeast transformation protocol was used to introduce DNA into yeast,41 and selection was made on the appropriate amino acid dropout SC (Synthetic Complete) media (Sigma). Recombinant DNAs were maintained in E. coli with appropriate antibiotic selection, and transferred to Streptomyces via intergenic conjugation using the helper strain E. coli S17. Streptomyces albus J1074 was used as a heterologous host for metabolite production.42 S. albus J1074 was maintained on ISP4 agar plates and cultured in R5A liquid media at 30 °C with shaking (200 rpm) for metabolite production. All plasmids used in this study were listed in Table S2. DNA synthesis and molecular cloning All primers and double stranded DNA fragments used in this study were synthesized by IDT (Integrated DNA Technology) and listed in Table S3 - S11. The designed IRCs (Inducible Regulatory Cassettes), synthesized as gBlock DNA fragments (Table S4 - S7), were cloned upstream of the indigoidine synthetase gene in the pIJPT1 promoter assay vector

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using homologous recombination in yeast.43 Briefly,

approximately 100 ng of each IRC was transformed into yeast together with the equimolar amount of the HpaI-digested pIJPT1 plasmid, and the transformants were selected on the URA dropout SC agar plates. The recombinant DNAs were isolated from yeast using a Zymolyase lysis protocol, and the correct cloning was confirmed by Sanger sequencing using the primers that were designed to bind the flanking sequences on both sides (pIJPT_screen_FW and pIJPT_screen_RV, Table S3). To replace the target expression module in the pIJPT1/IRC construct, the DNAs were digested with AflII and HindIII and then transformed into yeast together with a newly synthesized module (gBlock DNA fragment, Table S8). The correct replacement was confirmed by Sanger sequencing using the same protocol as described above. Measurement of indigoidine production

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For heterologous expression, the pIJPT1/IRC constructs were transformed into E. coli S17, shuttled into S. albus via intergenic conjugation, and integrated into its genome using the ΦBT1 integrase. The resulting S. albus pIJPT1/IRCs were maintained and sporulated on the ISP4 agar plates containing hygromycin B (100 µg/ml) for selection. All spore stocks were prepared using the standard method (OD600 0.3 ~ 0.5 in 25% aqueous glycerol). For indigoidine production, 10 µL of each spore stock was inoculated into 20 mL of R5A liquid media, and cultured in a shaking incubator (30 °C, 200 rpm) with or without a chemical inducer: anhydrotetracycline (5 μM), oxytetracycline (5 μM), cumate (100 μM) or resorcinol (50 μM). Indigoidine production was measured after 5 days of the culture. A 1 mL aliquot of each culture was centrifuged at 15,000 g for 10 min, and 200 μL of the supernatant was diluted with 900 μL of DMSO (dimethyl sulfoxide) and used for measuring OD600. To monitor cell density during the growth, cell pellets were collected from 20 ml cultures by centrifugation at 15,000 g, washed with dH2O twice, and re-suspended in 1 ml of dH2O for the measurement of OD600. All experiments were performed in triplicate. Creation of CymR-expressing Streptomyces The pIJPT1 plasmid containing the cumate IRC was digested with both AflII and AvrII to delete the indigoidine synthetase gene and the upstream inducible regulatory sequence region. The resulting DNA breaks were repaired by homologous recombination using the stitching DNAs that contain various RSs for the repressor expression and 40 bp homology sequences to the plasmid (gBlock from IDT, Table S9). Errorfree modification of the plasmid was confirmed by Sanger sequencing using the same protocol as described above. The resulting plasmid was transformed into E. coli S17, transferred to S. albus J1074 via intergenic conjugation and integrated into its genome using the ΦBT1 integrase, creating the S. albus strain constitutively expressing the CymR repressor. Refactoring of the ACT cluster The ACT cluster with the P1 site replaced with the S1 constitutive RS cassette that contains the HIS3 auxotrophic marker flanked by the two strong RSs (A26 and kasO*) was described previously.34 To convert the constitutive RS to the inducible RS in the ACT cluster, the CRISPR-aided homologous recombination was used. The two gRNA sequences containing the 20 bp target sequences to the S1 region was synthesized as the gBlock DNA fragments (Table S10) and cloned into the pCRCT vector using homologous recombination in yeast.44 The pCRCT/S1 plasmid was co-transformed with the ACT-P1(S1) and the double stranded gBlock DNA fragment (Table S11) containing the cmtO operator sequence flanked 13



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by the 40 bp homology sequences to the S1 region, and the transformants were selected on the TRP, URA and HIS dropout SC agar plates. The correct insertion of the cmtO operator sequence was confirmed by Sanger sequencing using the same protocol as described above. Measurement of ACT production For heterologous expression, the ACT-P1(S1/cmtO) construct in pTARa was transformed into the E. coli S17 helper strain, transferred to S. albus :: CymR via intergenic conjugation and integrated into its genome using the ΦC31 integrase. The resulting S. albus :: CymR / ACT-P1(S1/cmtO) were maintained and sporulated on the ISP4 agar plates containing hygromycin B (100 µg/ml) and Apramycin (50 µg/ml) for selection. For ACT production, 10 µl of each spore stock was inoculated into 20 ml of R5A liquid media and cultured in a shaking incubator (30 °C, 200 rpm) with or without the cumate inducer at various concentrations. After 5 days, a 500 µl aliquot of each culture was mixed with an equal volume of the 2N KOH solution. The resulting mixture was incubated at room temperature for 10 min to allow for cell lysis. The mycelium was removed by centrifugation (4,000 g, 10 min), and the level of ACT production was determined by measuring OD640 of the supernatant using the UV/visible spectrophotometer (Thermo scientific, USA).45

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Supporting information The Supporting information is available free of charge on the ACS Publications website at DOI: Additional figures and graphs; the list of strains; the list of plasmids; the list of primers; the sequences of synthetic regulatory cassettes Author Information Corresponding Author: *Prof. Hahk-Soo Kang, Department of Biomedical Science and Engineering, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea; Telephone: +82-2-450-4136; E-mail: [email protected] Author contributions: C.-H.J., H.K. and H.-S.K. designed experiments and analyzed data. C.-H.J. conducted all experiments. C.-H.J. and H.-S.K. wrote the manuscript. Note: The authors declare no competing financial interest. Acknowledgments This work was supported by Next-Generation BioGreen21 Program (SSAC grant: PJ01318901) from Rural Development Administration, Young Researcher Program (NRF-2018R1C1B3001028) from National Research Foundation, and the KU Scholarships for Outstanding Research Papers funded by Konkuk University Graduate School.

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Figure captions Figure 1. Modular design approach for the construction of synthetic inducible regulatory systems. An inducible regulatory system is composed of three separate modules: a. induction module (part composition: repressor + operator), b. target expression module (part composition: promoter + RBS), and c. repressor expression module (part composition: promoter + RBS). These three separate modules indipendently affect the overall performance of an inducible regulatory system. The induction module would determine the sensitivity of a system to varying inducer concentrations. Target expression and repressor expression modules are likely to affect the level of maximum production and the degree of full repression, respectively. Figure 2. Characterization of induction modules. (A) The cassete design. Four different IRCs were designed by combining the SF14RS repressor expression and kasO*RS target expression modules with the TET, OTR, CMT or ROL induction module. The four designed IRCs were synthesized and cloned into pIJPT1 to characterize their performances. (B) Indigoidine production in the un-induced and full-induced states. Leakiness, inducibility and dynamic ranges were used to characterize the performance of each IRC. Percent leakiness represents relative indigoidine production in the un-induced state (white bar) to indigoidine production in the full-induced state (colored bar). Inducer concentrations used for full induction were TET (5µM), OTR (5µM), CMT (100µM) and ROL (50µM). Indigoidine production in the full-induced state (colored bar) relative to that of the positive control (black bar) was designated percent inducibility. Dynamic ranges were calculated as a fold-increase by dividing OD600 in the full-induced state with OD600 in the un-induced state. Figure 3. Indigodidine production as a function of inducer concentration. (A) Heat map showing the inducer sensitivity of the TET, OTR and CMT induction modules. The level of indigoidine production was measured at six different inducer concentrations that were made by serial 4-fold dilutions starting from the concentrations used for full induction. (B) Relationships between indigoidine production and inducer concentrations for the TET (red), OTR (yellow) and CMT (green) induction modules. Concentration ranges, in which indigoidine production linearly increases in reponse to increasing inducer concentrations, represent the inducer sensitvity of each induction module. Inducer concentrations (x-axis) were ploted on a log4 scale in the graph.

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Figure 4. Compatibility test of the CMT induction module. (A) The cassette design. Five IRCs were designed to test compatibility of the CMT induction module, in which the SF14RS repressor expression module was fixed the same, and the CMT induction module was combined with five different constitutive RSs that include two native (ermE*RS and otrBRS) and three synthetic (A12RS, A26RS and A35RS) RSs. (B) Indigoidine production in the un-induced and full-induced states. Percent leakiness, percent inducibility and fold-increase were compared between the IRCs. The black bars in each graph represent negative control (no RS) and positive control (no operator). Leakiness, inducibility and fold-increase were calculated in the same way as described above. Figure 5. CMT-inducible regulatory system as a modulator of ACT production. (A) Strategy for the modulation of ACT production. ACT biosynthesis is initiated by a minPKS complex, and thus we assumed that it would be possible to modulate ACT production by expressing minPKS genes (KSα, KSβ and ACP) under the control of an inducible RS. (B) CRISPR/Cas9-aided insertion of an operator sequence. Using the ACT cluster, we show that any constitutive RS could be converted into an inducibe RS by inserting an operator sequence between the promoter and RBS sequences using CRISPR/Cas9-aided markerless homologous recombination in yeast. The promoter engineered ACT cluster was integrated into the genome of S. albus using the ΦC31 integrase, and the ΦBT1 integrase was used to integrate the CymR-expressing cassette. Figure 6. Optimization of the repressor expression module for the CMT-inducible RS and modulation of ACT production. (A) The cassette design. To further extend the dynamic range of the CMT-inducible RS, the SF14 RS, used to express the CymR repressor, was replaced with three strong synthetic constitutive RSs including A13RS, A51RS and A26RS. The designed CymR gene-expressing cassettes were integrated into the genome of S. albus using the ΦBT1 integrase, creating the three different CymR gene-expressing S. albus strains. (B) ACT production in the un-induced and full-induced states. The promoter-engineered ACT cluster, in which the minPKS genes are expressed under the control of the CMT-inducible RS, was transformed into the three different CymR gene-expressing S. albus strains. Then, percent leakiness, percent inducibility and fold-increase were compared in the same way as described for indogoidine production. (C) ACT production as a function of inducer concentration. ACT production was measured at six different CMT concentrations that were made by serial 4-fold dilution from the concentration used for full induction. CMT concentrations (x-axis) were ploted on a log4 scale in the graph.

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Synthetic Inducible Regulatory Systems Optimized ... - ACS Publications

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