Production of extracellular heterologous proteins in Streptomyces rimosus, producer of the antibiotic oxytetracycline

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1 Applied Microbiology and Biotechnology SUPLEMENTARY INFORMATION Production of extracellular heterologous proteins in Streptomyces rimosus, producer of the antibiotic oxytetracycline Andrés Felipe Carrillo Rincón 3,4, Vasilka Magdevska 1,2, Luka Kranjc 1, Štefan Fujs 1,2, Rolf Müller 4 and Hrvoje Petković 1,2,3 * 1 Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia 2 Acies Bio, d.o.o. Tehnološki park 21, SI-1000 Ljubljana, Slovenia 3 Universidad de Cantabria, Instituto de Biomedicina y Biotecnología de Cantabria, Parque Científico y Tecnológico de Cantabria (PCTCAN), C/Albert Einstein, Santander, Spain 4 Department of Microbial Natural Products, Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Centre for Infection Research (HZI) and Pharmaceutical Biotechnology, Saarland University, Campus C2 3, Saarbrücken, Germany * Corresponding author: Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, Ljubljana, Slovenia, hrvoje.petkovic@bf.uni-lj.si ; Tel: ; Orcid ID:

2 Table S1. Plasmids used in this study Name Promoter ORF Origin of Integrase Selective Source replication marker ppz pij101 NA Thiostrepton (Chambers and Hunter 1984) puc pmb1 NA Invitrogen pvf - - pij101 NA Thiostrepton pmb1 pts repsa ΦC31attP Thiostrepton (Smokvina et al. 1990) pbluescript-perm*- P erme* xyle ColE1 NA xyle pvmx3 P erme* xyle repsa ΦC31attP Thiostrepton Apramycin pex-p nita /NitR PnitA/NitR - pmb1 NA pex-tcp830 tcp830 - pmb1 NA pex-pnita-1 PnitA/NitR xyle pmb1 NA pex-tcp830-1 tcp830 xyle pmb1 NA pex-tcp830-1/tetris tcp830/sf14 xyle/ tetris pmb1 NA pex-pnita-2 PnitA/NitR srt/appa pmb1 NA pex-tcp830-2 tcp830 srt/appa pmb1 NA 2

3 pex-sf14/tetris SF14 tetris pmb1 NA pblue-xyle - xyle ColE1 NA pblue- P erme* - P erme* amy/xyle ColE1 NA amy/xyle pblue- P erme* - P erme* srpb/xyle ColE1 NA srpb/xyle pblue- P erme* - P erme* aml/xyle ColE1 NA aml/xyle pblue- P erme* - P erme* xysa/xyle ColE1 NA xysa/xyle pblue- P erme* - P erme* aml*/xyle ColE1 NA amy*/xyle pblue- P erme* -lip/xyle P erme* lip/xyle ColE1 NA pblue- P erme* - P erme* srt/xyle ColE1 NA srt/xyle pvm5 P erme* vsi/xyle pmb1 psam2 Thiostrepton attp pvm6 P erme* amy/xyle pmb1 psam2 attp Thiostrepton 3

4 pvm7 P erme* srpb/xyle pmb1 psam2 Thiostrepton attp pvm8 P erme* aml/xyle pmb1 psam2 Thiostrepton attp pvm11 P erme* xysa/xyle pmb1 psam2 Thiostrepton attp pvm12 P erme* aml*/xyle pmb1 psam2 Thiostrepton attp pvm13 P erme* srt/xyle pmb1 psam2 Thiostrepton attp pvm14 P erme* lip/xyle pmb1 psam2 Thiostrepton attp pblue-appasyn - appa ColE1 NA pblue-appasyn-aml P erme* aml/appa ColE1 NA pblue-appasyn-lip P erme* lip/appa ColE1 NA pblue-appasyn-srt P erme* srt/appa ColE1 NA pvm4 P erme* aml/appa repsa ΦC31attP Thiostrepton 4

5 pvm15 P erme* srt/appa repsa ΦC31attP Thiostrepton pvm16 P erme* lip/appa repsa ΦC31attP Thiostrepton pab pmb1 ΦC31attP Thiostrepton Apramycin pab04-1 P erme* xyle pmb1 ΦC31attP Thiostrepton Apramycin pab04-2 PnitA/NitR xyle pmb1 ΦC31attP Thiostrepton Apramycin pab04-3 tcp830/sf14 xyle/ tetris pmb1 ΦC31attP Thiostrepton Apramycin pab04-4 P erme* srt/appa pmb1 ΦC31attP Thiostrepton Apramycin pvf-1 P erme* xyle pij101 NA Thiostrepton pmb1 pvf-2 PnitA/NitR xyle pij101 NA Thiostrepton pmb1 pvf-3 tcp830/sf14 xyle/ tetris pij101 NA Thiostrepton pmb1 5

6 pvf-4 PnitA/NitR srt/appa pij101 NA Thiostrepton pmb1 pvf-5 tcp830 srt/appa pij101 NA Thiostrepton pmb1 6

7 Table S2. Oligonucleotides used in this study a) Oligonucleotides used to sequence pvf vector Primer name Sequence (5 to 3 ) pvf_fw_fw_15 CACCGAGTCCCACGCCA pvf_fw_fw_13 GACGCCTTCCGCGAT pvf_puc4 GTAACGCCAGGGTTTTCCCAGT pvf_puc2 GCTTATCAGTGAGGCACCT pvf_puc1 CGACACGGAAATGTTGAATACTCATAC pvf_puc2 GTATATATGAGTAAACTTGGTCTGACAG pvf10 ACCCCCGACAGCGGATC pvf_puc3 CTGTAGGTATCTCAGTTCGGTGTA pvf_14 CTAAATACATTCAAATATGTATCCGCTCAT pvf_15 GCAGTGCTGCCATAACCATGAGT pvf_fw_fw_19 GCCGCGGGAGTAATCCT pvf13 GCCCGGCTCGCAAGT pvf_16 ACTCAAGACGATAGTTACCGGATAAG pvf11 GATCGTCGCCACCTTCGA pvf_fw_fw_17 ACCGCAATTGCCCACACAC pvf12 TACTCGTGCCAGCGCGAG b) Oligonucleotides used to amplify selected genes and signal sequences Primer name Sequence (5 To 3 ) XylE_NdeI AAAACATATGAACAAAGGTGTAATGCGACCG XylE_XbaI AAAATCTAGACATCAGGTCAGCACGGTC XylE_PstI AAAACTGCAGCCAACAAAGGTGTAATGCGACCG Fw-srT-appA- AAAAACATATGTTGCAGAGCTACCTGAAGCACCT NdeI Rv-appA-XbaI AAAAATCTAGAGTCAGTGGTGGTGGTGGTGGTGGAGGGA GCACGCCGGGATGC aml*_ndei AAAACATATGCGCAGAAAGACCGTGGCAGCTGCACTCG aml*_psti AAAGGCTGCAGCCGGGACGGCCTGCGCC srt_ndei AAAACATATGTTGCAGAGCTACCTGAAGCACCTGCGCAGA 7

srt_psti lip_ndei lip_psti xysa_ndei xysa_psti GTC AAAACTGCAGTGGGGGCGGCGGCGGTG AAAACATATGAGACTGTCCCGACGCGCGGCCACG AAAACTGCAGCCTGGATTCGCGGCGCGGACAC AAAACATATGGCTCAGAATCCCCCGGTCGGC

8 srt_psti lip_ndei lip_psti xysa_ndei xysa_psti GTC AAAACTGCAGTGGGGGCGGCGGCGGTG AAAACATATGAGACTGTCCCGACGCGCGGCCACG AAAACTGCAGCCTGGATTCGCGGCGCGGACAC AAAACATATGGCTCAGAATCCCCCGGTCGGC AAAACTGCAGCGCCGGCGGCTTGGGCGGT Table S3. Proportion of the transformants with AppA activity when pts55-based vectors were used in S. rimosus M4018 Plasmid Streptomyces rimosus M4018 Phytase producing colonies Total number of colonies evaluated Positive colonies for AppA activity (%) pvm pvm pvm Figure S1. A. Map of the E. coli Streptomyces shuttle replicative vector pvf. B. Segregation instability of pvf plasmid in S. rimosus cultivated in TSB medium. DNA sequencing of the pvf plasmid revealed a size of 8787 bp with mean G+C content of 64.5%. By applying the frame plot software (Ishikawa and Hotta 1999), we identified six putative open reading frames (ORFs), two from the puc19 vector (amp r and rep), and four corresponding to the ppz12 vector. As expected, one ORF that belonged to the ppfz12 8

backbone encodes the thiostrepton resistance gene, while the remaining putative ORFs (rep, korb, orf56) have homologues in the pij101 plasmid (Kieser et al. 1982).

9 backbone encodes the thiostrepton resistance gene, while the remaining putative ORFs (rep, korb, orf56) have homologues in the pij101 plasmid (Kieser et al. 1982). The segregation instability of the pvf vector, which was typical for pij101 type of Streptomyces vectors, was evaluated to establish the rate at which pvf-containing transformants lose the plasmid in the absence of antibiotic, and thus become thiostrepton sensitive, as described in Figure S1. The frequency was calculated by subculturing S. rimosus that contained the pvf transformant every 24 h in TSB liquid medium without the selection marker (thiostrepton). Each day, serial dilutions of 24-h-old cultures were plated on the TSB agar medium that contained the antibiotic thiostrepton and TSB plates without antibiotic. CFU were counted after 48 hours incubation. Based on the collected data, unless thiostrepton was added to the TSB liquid medium, around 30% of the cells lost the pvf plasmid at every step of sub-cultivation (Figure S1B). Figure S2. Schematic presentation of the signal-sequences testing cassette that includes the promoter, signal sequence and (reporter) gene. Unique restriction sites were introduced to facilitate the construction of different components of the transcriptional unit. 9

10 Detailed description of the enzymatic assays for measurement of XylE and AppA activity: Additional information on the measurement of XylE activity: The catechol 2,3-dioxygenase XylE activity was assayed as described previously (Ingram et al. 1989). Here, 1 ml sample was taken after the biosynthetic process was complete and the cells were harvested at 4 C, g and 10 min. The pellet and supernatant were frozen at -20 C for further enzymatic analysis. Intracellular activity was evaluated using the pellet while extracellular activity was evaluated directly from the supernatant. Briefly, the cell pellet was washed with 1 ml 2 mm potassium phosphate buffer (ph 7.5), and centrifuged for 10 min at g, and the cells were resuspended in lysis buffer (0.5 ml 100 mm potassium phosphate buffer ph 7.5, 20 mm EDTA, 10% acetone) and sonicated on ice 4 15 s, with 30 s intervals at 100% amplitude with Ultrasonic homogenisator (LABSONIC M, Sartorius). After sonication, 5 µl 10% Triton X-100 was added, and the extracts were incubated on ice for 15 min. Cell debris was removed by centrifugation for 5 min at g at 4 C, 10 µl of cell extract or supernatant was mixed with 190 µl assay buffer (100 mm potassium phosphate buffer (ph 7.2), 0.2 mm catechol) and incubated for 10 min at 37 C. Catechol 2,3-dioxygenase activity was evaluated spectrophotometrically at 375 nm. One mu of XylE activity was calculated as the rate of change in absorbance at 375 nm per min and the specific activity (mu/mg total protein) was calculated as the rate of catechol 2,3-dioxygenase activity per milligram of total protein (Ingram et al. 1989). Protein concentration was determined using by Bradford method (Bradford 1976) using Bovine serum albumin as a standard. Additional information on the measurement of AppA activity: Phytase activity was determined using the ammonium molybdate method as described previously (Lee et al. 2005) with minor modifications. Briefly, 1 ml cell culture was centrifuged for 5 minutes at g and the cell pellet was sonicated on ice for 4 15 s, with 30 s intervals at 100% amplitude with Ultrasonic homogenisator (LABSONIC M, Sartorius) using 50 mm Tris, ph 7 buffer to measure the intracellular AppA activity. Extracellular AppA activity was deduced directly from the supernatant. 30 µl of cell extracts or supernatant (dilutions were made, when necessary) were mixed with 120 µl sodium phytate reagent (0.1 M acetic acid buffer, ph 5, 2 mm phytic acid sodium salt) and incubated for 15 min at 37 C. The reaction was stopped by adding 150 µl 15% trichloroacetic acid and the samples were mixed by vortexing. Blanks were made by adding trichloroacetic acid to the samples and incubating for 15 min at 37 C before adding the sodium phytate reagent. Afterwards, 300 µl of colour reagent solution (3:1:1 mixture of 1 M sulfuric acid, 2.5% 10

11 ammonium molybdate [w/v], and 10% ascorbic acid [w/v] respectively) were added, followed by incubation for 15 min at 50 C. The phosphorous released from phytic acid by AppA was measured spectrophotometrically at 820 nm. One phytase unit (FTU) was defined as the amount of enzyme required to release 1 µmol of inorganic phosphorus per min from sodium phytate at 37 C and specific activity (mu/mg of total proteins) was calculated as the rate of phytase activity per milligram of total proteins. Protein concentration was determined using by Bradford method (Bradford 1976) using Bovine serum albumin as a standard. Sequence data: Intensity coverage: 27.3% Sequence coverage MS: 44.5% pi (Isoelectric point):

12 Figure S3. LC-MS/MS analysis to confirm phytase AppA production after His-tag purification. 12

FAQSEPELKLESVVIVSRHGVRAPTKATQLMQDVTPDAWPTWPVKLGWLTPRGGELI AYLGHYQRQRLVADGLLAKKGCPQSGQVAIIADVDERTRKTGEAFAAGLAPDCAIT VHTQADTSSPDPLFNPLKTGVCQLDNANVTDAILSRAGGSIADFTGHRQTAFRELERV

13 FAQSEPELKLESVVIVSRHGVRAPTKATQLMQDVTPDAWPTWPVKLGWLTPRGGELI AYLGHYQRQRLVADGLLAKKGCPQSGQVAIIADVDERTRKTGEAFAAGLAPDCAIT VHTQADTSSPDPLFNPLKTGVCQLDNANVTDAILSRAGGSIADFTGHRQTAFRELERV LNFPQSNLCLKREKQDESCSLTQALPSELKVSADNVSLTGAVSLASMLTEIFLLQQAQ GMPEPGWGRITDSHQWNTLLSLHNAQFYLLQRTPEVARSRATPLLDLIKTALTPHPP QKQAYGVTLPTSVLFIAGHDTNLANLGGALELNWTLPGQPDNTPPGGELVFERWRR LSDNSQWIQVSLVFQTLQQMRDKTPLSLNTPPGEVKLTLAGCEERNAQGMCSLAGFT QIVNEARIPACSLHHHHHH Figure S4. Phytase amino-acid sequence (MW: ). Fig. S5. A. Recombinant AppA purified with a His-tag affinity column from the supernatant of S. rimosus pvf-tcp830/srt/appa cultures. The AppA band was digested in-gel and analysed by LC-MS/MS, thus confirming the identity of the phytase AppA (Fig. S3). B. Enzymatic assay to confirm AppA activity using: 1. Purified phytase AppaSyn-his and the following controls; 2. Purified phytase treated with TCA; 3. Elution buffer; and 4. H 2 O. 13

14 References Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: Chambers A, Hunter I (1984) Construction and use of a bifunctional streptomycete cosmid. Biochem SocTrans 12: doi: /bst Ingram C, Brawner M, Youngman P, Westpheling J (1989) xyle functions as an efficient reporter gene in Streptomyces spp.: use for the study of galp1, a catabolite-controlled promoter. J Bacteriol 171: Ishikawa J, Hotta K (1999) FramePlot: a new implementation of the frame analysis for predicting protein-coding regions in bacterial DNA with a high G + C content. FEMS Microbiol Lett 174: Kieser T, Hopwood DA, Wright HM, Thompson CJ (1982) pij101, a multi-copy broad hostrange Streptomyces plasmid: functional analysis and development of DNA cloning vectors. Mol Gen Genet 185: Lee S, Kim T, Stahl CH, Lei XG (2005) Expression of Escherichia coli AppA2 phytase in four yeast systems. Biotechnol Lett 27: doi: /s Smokvina T, Mazodier P, Boccard F, Thompson CJ, Guerineau M (1990) Construction of a series of psam2-based integrative vectors for use in actinomycetes. Gene 94:

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