An autoregulated fine-tuning strategy for titer improvement of secondary
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1 Supporting Information An autoregulated fine-tuning strategy for titer improvement of secondary metabolites using native promoters in Streptomyces Shanshan Li #,,, Junyang Wang #,,, Wensheng Xiang, Keqian Yang, Zilong Li, * and Weishan Wang, * State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No.1 West Beichen Road, Chaoyang District, Beijing , China State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuan West Road, Haidian District, Beijing , China University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing , China # S. L. and J. W. contributed equally to this work. * Correspondence: Weishan Wang, wangws@im.ac.cn; Zilong Li, lizl@im.ac.cn. 1
2 Content Supplementary figures... 4 Figure S1 Map of plasmids designed for fine-tuning the expression of actinorhodin (Act) and oxytetracycline (OTC) biosynthetic gene clusters (BGCs)... 4 Figure S2 Optimization of the expression of Act and jadomycin B (JdB) BGCs using constitutive promoters with different strengths... 5 Figure S3 Determination of appropriate range of induction time and dosage by single factor test in M145-OA... 6 Figure S4 Sampling timepoints for transcriptome experiments... 7 Figure S5-1 Comparison of transcription profile of sfgfp controlled by native temporal promoter and the inducible promoter under optimal induction condition... 8 Figure S5-2 Comparison of transcription profile of sfgfp controlled by native temporal promoter and the inducible promoter under optimal induction condition... 9 Supplementary tables Table S1 Results of the central composite design of M145-OA Table S2 Description of the quadratic response surface model obtained from central composite design Table S3 Information of the selected native temporal promoters Table S4 Performance of native temporal promoters on Act production in S. coelicolor Table S5 Investigation of the relationships between behaviors of native temporal promoter and the cumate-inducible promoter by correlation analysis Table S6 Performance of native temporal promoters on OTC production in S. coelicolor Table S7 Strains and plasmids Table S8 Primers and oligonucleotides used in this work
3 Reference
4 Supplementary figures Figure S1 Map of plasmids designed for fine-tuning the expression of actinorhodin (Act) and oxytetracycline (OTC) biosynthetic gene clusters (BGCs). (a) Map of plasmid constructed for the fine-tuning of expression of Act BGC. (b) Map of plasmid constructed for the fine-tuning of expression of OTC BGC. 4
5 Figure S2 Optimization of the expression of Act and jadomycin B (JdB) BGCs using constitutive promoters with different strengths. (a) Relationship between promoter strength and Act production in S. coelicolor M145. (b) Relationship between promoter strength and JdB production in S. venezuelea ATCC Control was the strain integrated with a promoter-less plasmid pactii-sfgfp. The relative strength was determined by Bai et al. 1 Results shown are averages of three independent experimental replicates and s.d. 5
6 Figure S3 Determination of appropriate range of induction time and dosage by single factor test in M145-OA. (a) Relationship between the induction timepoint and Act titer. The concentration of cumate was 3 μm. (b) Relationship between the dosage of inducer cumate and Act titer. Cumate inducer was added at 20 h. Results shown are averages of three independent experimental replicates and s.d. 6
7 Figure S4 Sampling timepoints for transcriptome experiments. Cell growth was determined by diphenylamine colorimetric method. 2 Results shown are averages of three independent experimental replicates and s.d. 7
8 Figure S5-1 Comparison of transcription profile of sfgfp controlled by native temporal promoter and the inducible promoter under optimal induction condition. Results shown are averages of three independent experimental replicates and s.d. 8
9 Figure S5-2 Comparison of transcription profile of sfgfp controlled by native temporal promoter and the inducible promoter under optimal induction condition. Results shown are averages of three independent experimental replicates and s.d. 9
10 Supplementary tables Table S1 Results of the central composite design of M145-OA Test No a Induction timepoint (h) Induction dosage (cumate/μm) Titer of Act (mg/l) b Level of design experiment data Level of design experiment data ± ± ± ± ± ± ± ± Control / / / / 61 ± 6 a Control was cultivated in the condition without cumate inducer. b Test 3, 4, 5, 7, and 13 were carried out under the same induction condition. Results of the rest tests are averages of three independent experimental replicates and s.d. 10
11 Table S2 Description of the quadratic response surface model obtained from central composite design Term a Estimate b Std Error t Ratio Prob> t Significance c Intercept * xd * xt *** xd*xd ** xt*xt *** xd*xt a xd and xt were the two parameters of the quadratic response surface model, indicating induction dosage and induction timepoint, respectively. b The quadratic response surface model was as follows: f(x d,x t ) = x d x t x d x t x d x t c Asterisks indicate the statistically significant differences (*, p < 0.05; **, p < 0.01; ***, p < 0.001). 11
12 Table S3 Information of the selected native temporal promoters Native temporal promoters Strand Location of the selected promoter region in the genome of M145 Start Stop Psco Psco Psco Psco Psco Psco Psco Psco Psco Psco Psco Psco Psco Psco Psco Psco Psco Psco Psco Psco Psco Psco Psco Psco
13 Table S4 Performance of native temporal promoters on Act production in S. coelicolor Candidate promoters Act production comparison a vs. Control vs. SP26 vs. Pcumate Psco0477 *** * ns Psco0475 *** *** * Psco0476 *** *** ns Psco1200 *** ns *** Psco1803 *** *** ns Psco2230 *** ns *** Psco2505 *** ** ns Psco2817 *** ns *** Psco2937 *** *** ns Psco3132 *** *** ns Psco3424 *** *** ns Psco3428 *** *** ns Psco3429 *** ns *** Psco3800 *** ns *** Psco4280 *** ** ns Psco4297 ns *** *** Psco4789 *** *** ns Psco7676 *** ns *** Psco7677 *** * * Psco7681 *** *** ** Psco7682 *** *** ns Psco7692 *** *** ns Psco7693 *** *** ns Psco7729 *** ns *** Number of promoters with comparable effect on Act production a Red and blue asterisk indicates the significant improved and reduced production, respectively, while ns means no significance. Significance analysis was conducted by one-way analysis of variance (ANOVA), ***, p < 0.001; **, p < 0.01; *, p <
14 Table S5 Investigation of the relationships between behaviors of native temporal promoter and the cumate-inducible promoter by correlation analysis Promoter region Correlation analysis of transcript level of sfgfp a R 2 Slope Psco ± 0.03 Psco ± 0.05 Psco ± 0.10 Psco ± 0.06 Psco ± 0.05 Psco ± 0.08 Psco ± 0.11 Psco ± 0.03 Psco ± 0.03 Psco ± 0.05 Psco ± 0.12 Psco ± 0.11 Psco ± 0.11 Psco ± 0.19 Psco ± 0.09 Psco ± 0.29 Psco ± 0.28 Psco ± 0.05 Psco ± 0.04 Psco ± 0.06 Psco ± 0.34 Psco ± 0.34 Psco ± 0.14 Psco ± 0.22 a R 2 and the slope of the fitted line were calculated based on the transcript level of sfgfp obtained from three independent experiments. Data specified in blue indicates the behaviors of corresponding native temporal promoters are not well consistent with that of the cumate-inducible promoter. 14
15 Table S6 Performance of native temporal promoters on OTC production in S. coelicolor Candidate promoters Comparison of OTC titer (vs. Pcumate) Psco6718 *** Psco3538 ns Psco6206 *** Psco3366 ** Psco5758 *** Psco4544 ns Psco2790 ** Psco2217 ns Psco6655 ns Psco2643 ns Psco2330 *** Psco4748 *** Psco7628 *** Psco6654 ns Psco1802 ** a Blue asterisk indicates the significant reduced production, while ns means no significance. Significance analysis was conducted by one-way ANOVA, ***, p < 0.001; **, p < 0.01; *, p <
16 Table S7 Strains and plasmids Name Description Source Strain Escherichia coli E. coli JM109 General cloning host for plasmid manipulation Novagen ET12567 (puz8002) Donor strain for conjugation between E. coli and Streptomycetes Kieser et al. 3 Streptomyces coelicolor M145 M1146 SCP1- SCP2- derivative from S. coelicolor A3(2) Derivative of M145, completely lacks the biosynthetic clusters of Act, Red, CDA and ycpk Gomez-Escribano and Bibb 4 M145C Integrated with plasmid pactii-sfgfp M145-Pc M145-OA M145-Pn Integrated a copy of actii-orf4 driven by different constitutive promoters in the genome of M145, c indicates erme*, SP8, SP18, SP24, SP26, SP31, kaso*, SP42, and SP44, respectively Integrated a copy of actii-orf4 driven by the cumate-inducible promoter in the genome of M145 Integrated a copy of actii-orf4 driven by the selected native temporal promoter regions in the genome of M145, n indicates sco0477, sco0475, sco0476, sco1200, sco1803, sco2230, sco2505, sco2817, sco2973, sco3132, sco3424, sco3428, sco3429, sco3800, sco4280, sco4297, sco4789, sco7676, sco7677, sco7681, sco7682, sco7692, sco7693 and sco7729, respectively M1146-OTC Integrated the OTC biosynthetic gene cluster in M
17 M1146-OTCK Controlled the expression of otcr by constitutive promoter kaso* in M1146 M1146-OTCC Controlled the expression of otcr by the cumate-inducible promoter in M1146 M1146-Pn Streptomyces venezuelae Controlled the expression of otcr by selected native temporal promoter regions in M1146, n indicates sco1802, sco2217, sco2330, sco2643, sco2790, sco3366, sco3538, sco4544, sco4748, sco5758, sco6206, sco6654, sco6655, sco6718, sco7628, respectively ATCC10712 Wild-type strain for jadomycin B production ATCC Sv-Pc Sv-Potr Plasmids pgcymrp21 Controlled the expression of jadomycin biosynthetic gene cluster by the constitutive promoter, c indicates erme*, SP8, SP18, SP24, SP26, SP31, kaso*, SP42, and SP44 Controlled the expression of jadomycin biosynthetic gene cluster by the OTCinducible promoter Potr* Amp r, the cumate inducible expression system containing p21 promoter and CymR operator Horbal et al. 5 pij8660::bsai-sfgfp Am r, derived from pset152, containing the sfgfp reporter gene Bai et al. 1 pactii-sfgfp Am r, derived from pij8660, the promoter-less plasmid with the co-transcribed actiiorf4 and sfgfp Pc-actII-sfgfp Am r, derived from pactii-sfgfp, controlling the expression of both actii-orf4 and sfgfp by the constitutive promoter erme*, SP8, SP18, SP24, SP26, SP31, kaso*, SP42, and SP44, respectively piw01-jad Am r, controlling the expression of jadj by the OTC-inducible promoter Potr* 17
18 ppc-jad pcumate-actii-sfgfp potcr-sfgfp pcumate-otcr-sfgfp ppn-actii-sfgfp ppn-otcr-sfgfp Am r, derived from piw01-jad, controlling the expression of jadj by the constitutive promoter erme*, SP8, SP18, SP24, SP26, SP31, kaso*, SP42, and SP44, respectively Am r, derived from pactii-sfgfp, controlling the expression of both actii-orf4 and sfgfp by the cumate inducible promoter Am r, derived from pij8660, the promoter-less plasmid with the co-transcribed otcr and sfgfp Am r, derived from potcr-sfgfp, controlling the expression of both otcr and sfgfp by the cumate inducible promoter Am r, a series of plasmids controlling the expression of actii-orf4 and sfgfp by promoter region of gene sco0477, sco0475, sco0476, sco1200, sco1803, sco2230, sco2505, sco2817, sco2973, sco3132, sco3424, sco3428, sco3429, sco3800, sco4280, sco4297, sco4789, sco7676, sco7677, sco7681, sco7682, sco7692, sco7693, sco7729, respectively Am r, a series of plasmids controlling the expression of otcr and sfgfp by promoter region of gene sco1802, sco2217, sco2330, sco2643, sco2790, sco3366, sco3538, sco4544, sco4748, sco5758, sco6206, sco6654, sco6655, sco6718, sco7628, respectively 18
19 Table S8 Primers and oligonucleotides used in this work Name P1-F P1-R actii4-f actii4-r otcr-f otcr-r cumate-f cumate-r IW01-F IW01-R ermep* kasop* SP8 SP18 SP24 SP26 SP31 SP42 Sequence (5'-3') TGACAAGAGACAGGATGAGGATCGTTTCGC ATGCGTAAAGGCGAAGA GGTCTCCGATATCAGATCTCGA GAGATCTGATATCGGAGACCCGAGCAACGGAGGTACGGA CATGAGATTCAACTTATTGGGACGTG CTTCGCCTTTACGCATCTACACGAGCACCTTCTCACCG GAGATCTGATATCGGAGACCCGAGCAACGGAGGTACGG ACATGGACTTCAAGGCACTCGGC CCTCATCCTGTCTCTTGTCATCAAGACGCCGACCTCAACA CTAGAGATCTTTATCACCGCTTGAACTTG TACAGATATCATAATACAAACAGACCAGATT GAAGCGCCGGCCAGGAGAGTGAG TATCAGATCTCGAGGATCTAAAGTTTTGTCGTC CTAGAGATCTGCGGTCGATCTTGACGGCTGGCGAGAGGT GCGGGGAGGATCTGACCGACGCGGTCCACACGTGGCAC CGCCATGCTGTTGTGGGCACAATCGTGCCGGTTGGTAGA TATCTGTA CTAGAGATCTTGTTCACATTCGAACCGTCTCTGCTTTGAC AACATGCTGTGCGGTGTTGTAAAGTCGTGGCCAGATATC TGTA CTAGAGATCTTGTTCACATTCGAACCGTCTCTGCTTTGAC ACCCGCGGATTACCTCCTGTAAAGTCGTGGCCAGATATC TGTA CTAGAGATCTTGTTCACATTCGAACCGTCTCTGCTTTGAC ACCCGCGGATTACCTCCTGTAAAGTCGTGGCCAGATATC TGTA CTAGAGATCTTGTTCACATTCGAACCGTCTCTGCTTTGAC AGGCTCTGCTGTGAACGTGTAAAGTCGTGGCCAGATATC TGTA CTAGAGATCTTGTTCACATTCGAACCGTCTCTGCTTTGAC AACCAAGGCACATCTAATGTAAAGTCGTGGCCAGATATC TGTA CTAGAGATCTTGTTCACATTCGAACCGTCTCTGCTTTGAC AGCTCACTGGGCATGGGTGTAAAGTCGTGGCCAGATATC TGTA CTAGAGATCTTGTTCACATTCGAACCGTCTCTGCTTTGAC AACATGCTGTGCGGTGTTGTAAAGTCAGTAACAGATATC TGTA 19
20 20 SP44 CTAGAGATCTTGTTCACATTCGAACCGTCTCTGCTTTGAC AACATGCTGTGCGGTGTTGTAAAGTCGGGTGAAGATATC TGTA Psco0477-F CTAGAGATCTGGCTGATCGTGGACTCG Psco0475-F CTAGAGATCTTGACGCGGCGCGAGGT Psco0476-F CTAGAGATCTCCGAGCACGGAGACGA Psco1200-F CTAGAGATCTCTCGTCCCTGAGACCGGTGG Psco1803-F CTAGAGATCTGGTGGCCTCGTGGAGCATC Psco2230-F CTAGAGATCTGCGGCCACAACCTCTC Psco2505-F CTAGGGATCCGCCCCTTCTTCTCCTCGTACGC Psco2817-F CTAGAGATCTCAGGCCCTCGGAGTGGTG Psco2937-F CTAGAGATCTGAGGGCGATGGCGGAGACT Psco3132-F CTAGAGATCTCGCCGGTCGCTGGGGT Psco3424-F CTAGAGATCTTCCCAACCCGCTGGACCG Psco3428-F CTAGAGATCTTTACGCGTGGGGCTCGCG Psco3429-F CTAGAGATCTGCTCATGCCCCGTCCTGC Psco3800-F CTAGGGATCCCGATCACCTCCGCCCCG Psco4280-F CTAGAGATCTGACCAGGTTGACGCACGC Psco4297-F CTAGAGATCTCGGCGTCGTCGTGGAGAAG Psco4789-F CTAGAGATCTGGTGATCCTCTCCGTGGT Psco7676-F CTAGAGATCTGTATCTGCACGCACACCTCG Psco7677-F CTAGAGATCTCCGGAACCCTCCCGAAGC Psco7681-F CTAGAGATCTTCGTGGCCAGCGGGAAG Psco7682-F CTAGAGATCTTCTCCAGGCGCCGGTGA Psco7692-F CTAGAGATCTGGACCCTGGGACGCCTT Psco7693-F CTAGAGATCTATGGCGTCCGTGACCAGTTC Psco7729-F CTAGAGATCTGCTCCTGCCGCGTGGTTAAG Psco0477-R TACAGATATCTCTTCAGGAAGCCCAGGGTCT Psco0475-R TACAGATATCGGCCAGGGCGCAGATG Psco0476-R TACAGATATCCGCACAGCTCGGTCAG Psco1200-R TACAGATATCTCCCGCTCCTTGCGCACCT Psco1803-R TACAGATATCGTCCAGCACCAGGTCCCA Psco2230-R TACAGATATCAGTGCTTCTGGTAGCCGTCTTTC Psco2505-R TACAGATATCGCTGGAGCAGGCCGAGAGG Psco2817-R TACAGATATCCGGCACGAGGAGCCAGGC Psco2937-R TACAGATATCGCGGCTCAGCACGGTCTC Psco3132-R TACAGATATCGGCGAGGAGGTCGGTGAA Psco3424-R TACAGATATCCGAGTTGCTGCGCCGAC Psco3428-R TACAGATATCGTCGTTGCGGCGGTTCTT
21 21 Psco3429-R TACAGATATCGGATGTTGGGGTCGAAGCG Psco3800-R TACAGATATCCTTGTCGCGGCCGAGCAC Psco4280-R TACAGATATCGCGACGGCAACGGGGA Psco4297-R TACAGATATCCGATGCGGTTGGGCACGGT Psco4789-R TACAGATATCGAGGAAGATCCGCAGGGG Psco7676-R TACAGATATCCGGCGAGGACGCACTG Psco7677-R TACAGATATCAGCACGCCGCCAGCAGG Psco7681-R TACAGATATCACCCCCTGCCAGTGCCCG Psco7682-R TACAGATATCCGTCGTCCAGCTCCTCCGG Psco7692-R TACAGATATCGCACCGCGGCCCGTC Psco7693-R TACAGATATCTCACGATCGCGCGGACACC Psco7729-R TACAGATATCCGACCCGGGGGGAACC Psco6718-F CTAGAGATCTGACGTCGGCCTCACCTACC Psco1802-F CTAGAGATCTCGTTGCTGACCAGCAGATC Psco2217-F CTAGAGATCTAAGGACCGGGTGTTCGACGC Psco2330-F CTAGAGATCTGACGCAGACGGACGTGA Psco2643-F CTAGAGATCTAGGGCGGGCGCGAAGT Psco2790-F CTAGGGATCCCAGCAGCAGACGGCACGC Psco3366-F CTAGAGATCTGGCCTGGTTCTCCTGCAT Psco3538-F CTAGAGATCTCAGGTCGCTGGGGTAC Psco4544-F CTAGAGATCTGGCAGAATTCCTCGGAAC Psco4748-F CTAGAGATCTCGTGACCTCCCCGACCTTC Psco5758-F CTAGAGATCTACACCACCGCCGTCCA Psco6206-F CTAGAGATCTGTCCGGCGGCAGCATCT Psco6654-F CTAGAGATCTGAGGTCGTCGAGGATTC Psco6655-F CTAGAGATCTACGCGCCGTCTCCCAGT Psco7628-F CTAGAGATCTGTTCTCCTCGACCCTCC Psco6718-R TACAGATATCCAGGCCGTGATCGACCTTCTC Psco1802-R TACAGATATCCTGCTCATGGCCAGCAGC Psco2217-R TACAGATATCCCGCCCCGGCTACGATCTT Psco2330-R TACAGATATCTGTAGGTGTTCTGGTAGTCGACG Psco2643-R TACAGATATCCGCCGGTGAGGTCGAGATC Psco2790-R TACAGATATCACGACTGGCGTGGCTCTT Psco3366-R TACAGATATCGCATCATCGCGATCATGAG Psco3538-R TACAGATATCCTTCTTGCGTCCCCGTCT Psco4544-R TACAGATATCAAGAGTGCGGCGGCAGA Psco4748-R TACAGATATCCGCAGGAGGCGAGCAG Psco5758-R TACAGATATCACAGGTCGTCCCGCACG Psco6206-R TACAGATATCTCCAGGAGCGGGAGTTCC
22 Psco6654-R Psco6655-R Psco7628-R sfgfp-f sfgfp-r sco3183-f sco3183-r TACAGATATCCGGAAGTCCCGAGCCC TACAGATATCGGAATTTCCCGTACACGGT TACAGATATCATGTGCGGGACTCGTCG TCGGTGATGGTCCTGTTCT ATGATCGCGTTTCTCGTTC GGGCACCCTCGCGCTCC TACTCGCCCCAGTCCAGGTCG 22
23 Reference 1. Bai, C., Zhang, Y., Zhao, X., Hu, Y., Xiang, S., Miao, J., Lou, C., and Zhang, L. (2015) Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in Streptomyces, Proc. Natl. Acad. Sci. USA 112, Zhao, Y., Xiang, S., Dai, X., and Yang, K. (2013) A simplified diphenylamine colorimetric method for growth quantification, Appl. Microbiol. Biotechnol. 97, Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F., and Hopwood, D. A. (2000) Practical Streptomyces Genetics, The John Innes Foundation, Norwich, UK. 4. Gomez-Escribano, J. P., and Bibb, M. J. (2011) Engineering Streptomyces coelicolor for heterologous expression of secondary metabolite gene clusters, Microb. Biotechnol. 4, Horbal, L., Fedorenko, V., and Luzhetskyy, A. (2014) Novel and tightly regulated resorcinol and cumate-inducible expression systems for Streptomyces and other actinobacteria, Appl. Microbiol. Biotechnol. 98,
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