1

Supporting Information for

TREX – A Universal Tool for the Transfer and Expression of Biosynthetic Pathways in Bacteria

Anita Loeschcke1,3, Annette Markert1,3, Susanne Wilhelm1, Astrid Wirtz1, Frank Rosenau2, Karl-Erich

Jaeger1*

, Thomas Drepper1*

1Institute of Molecular Enzyme Technology, Heinrich-Heine-University Düsseldorf, Research Center

Jülich, Jülich, Germany.

2Institute of Pharmaceutical Biotechnology, Ulm University, Ulm, Germany.

3These authors contributed equally to this work.

*e-mail: k.-e.jaeger@fz-juelich.de or t.drepper@fz-juelich.de

Supporting information contains:

- Supplementary Figures S1 – S5 pp. 2-7

- Supplementary Tables S1, S2 pp. 8-11

- Supplementary Methods pp. 12 - 15

- References p. 16

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SUPPLEMENTARY FIGURES

Supplementary Figure S1. Characterization of neurosporene producing R. capsulatus mutant strain ∆crtC. (A)

Pigmentation of R. capsulatus wild type B10S and mutant strain Rc∆crtC after 3 days of growth. Droplets (20 µl) of

cell suspension adjusted to a cell density of OD660 = 0.8 are shown. (B) Absorption spectra of extracts from R.

capsulatus B10S wild type and Rc∆crtC. Pigments of cell material corresponding to OD660 = 0.2 were extracted with

1 ml EtOH. λ (max) spheroidene: 416, 440, 469 nm; λ (max) neurosporene: 429, 454, 487 nm. λ (max)

bacteriochlorophyll a: 360, 600, 775 nm. (C) HPLC chromatogram of Rc∆crtC showing neurosporene accumulation.

Pigments were extracted and analyzed as described in the methods section.

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Supplementary Figure S2. Verification of β-carotene accumulation in PpTREXcrt∆Z. Total carotenoid

accumulation in P. putida strain PpTREXcrt∆Z was assayed photometrically (A) and β-carotene was detected using

HPLC (B). Values represent means from three independent measurements. Error bars indicate the respective standard

deviations.

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Supplementary Figure S3. Verification of prodigiosin accumulation in PpTREXpig. (A) Total pigment content in

P. putida strain PpTREXpig was assayed photometrically as described in the methods section. In order to verify

prodigiosin accumulation in P. putida strain PpTREXpig under T7RP expression conditions, UV/Vis spectra of cell

extracts were recorded from 450 to 600 nm. (B) The mass of the extracted pigment was determined by ESI-MS using

mass spectrometer LTQ FS Ultra (Thermo Fisher). Mass spectrometry analysis revealed a molecular ion peak at

324.2 Da, which exactly corresponds to theoretical mass of prodigiosin (C20H25N3O).

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Supplementary Figure S4

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Supplementary Figure S4. Construction of the TREX cassettes. (A) Construction of the L-TREX cassette.

Conventional restriction and ligation based cloning was used to combine a tetracycline resistance gene (TcR) with an

origin of transfer (oriT) as well as a aphII gene and a T7 promoter (PT7) together with one outside end of transposon

Tn5 (OE-L). (B) Construction of the R-TREX cassette. A yfp gene and T7 promoter as well as a gentamicin

resistance gene (GmR) together with a transposase gene (tnp) and one corresponding outside end of transposon Tn5

(OE-R) have been combined to construct the R-TREX cassette. (C) Plasmid map of pIC20H-RL encompassing the

divergently orientated L-TREX (orange) and R-TREX (green) cassettes.

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Supplementary Figure S5. Plasmids pUC18crt∆X and pUC18crt∆Z. Plasmids pUC18crt∆X (A) and pUC18crt∆Z

(B) encompass two variants of P. ananatis crt cluster. Variant crt∆X carries a truncated crtX gene resulting in 5’-

fragment crtX’ and 3’-fragment crtX’’ (indicated with grey color) whereas in variant crt∆Z gene crtZ is fully deleted.

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SUPPLEMENTARY TABLES

Supplementary Table S1. Vectors and relevant recombinant plasmids

Name Relevant genotypic information Reference

Construction of TREX cassettes

pBBR1MCS-3 TcR, lacZα 58

pBSL15 ApR, aphII 59

pUCPSK ApR, lacZα 60

pUC18 ApR, lacZα 61

pL-TREX pUC18, TcR, oriT, OE-L, aphII, PT7 this study

pFF19-EYFP aphII, yfp 62

pWKR202 CmR, aphII, Gm

R 63

pSVB10 pUC18 derived 64

pSUP2021 CmR, Tc

R, Tn5 incl. aphII 51

pUC19 ApR, lacZα 61

pR-TREX pUC19, lacO, PT7, yfp, GmR, tnp, OE-R this study

pIC20H pUC18 derived, ApR 65

pIC20H-RL pIC20H, L- and R-TREX cassette this study

Construction of TREX-crt vectors

pUC18crt∆Z crtEXYIB this study

pUC18crt∆X crtE(∆X)YIBZ this study

pTREX-crt∆X pUC18crt∆X, L- and R-TREX cassette this study

pKD46 ori101, repA101ts, ApR, exo, bet, gam 66

pUC18ts pUC18 MCS & ApR, pKD46 ori101 & repA101ts this study

ptsTREX-crt∆Z pUC18ts, crtEXYIB this study

Construction of TREX-pig vector

pPIG pUC19, pig cluster this study

pTREX-pig pPIG, L- and R-TREX cassette this study

Construction of mutant strain R. capsulatus ∆crtC

pRc∆crtC pET22b, crtC, Ω-SpcR, oriT-Tc

R This study

TREX expression

pML5 RSF1010, TcR, lacZYA 42

pML5-T7 pML5, TcR, Plac>T7, lacIq 11

pML5-PfruT7 pML5, TcR, Pfru>T7, lacIq 11

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SupplementaryTable S2. Oligonucleotides used for cloning and expression analysis.

Name Sequence (5’ → 3’) Application

Construction of L-TREX

5’ oriT BclI TGATCAGGGCCGAGCGAAGCGAGCCAG PCR TcR + oriT

3’ TcR ScaI BclI TGATCAAGTACTGTTAGCGAGGTGCCGCCGGCT PCR TcR + oriT

5’ aphII PT7 ScaI AGTACTCCTATAGTGAGTCGTATTAATTGATCAAGA

GACAGGATGAGGA PCR aphII + PT7

3’ aphII BglII AGATCTGTCATTTCGAACCCCAGAGTC PCR aphII + PT7

OEL BglII GATCTCTGACTCTTATACACAAGTTAACTATAACGG

TCCTAAGGTAGCGAG Oligo OE-L

OEL BamHI GATCCTCGCTACCTTAGGACCGTTATAGTTAACTTG

TGTATAAGAGTCAGA Oligo OE-L

Construction of R-TREX

3’ yfp BglII ATATAGATCTATTACCCTGTTATCCCTA

TTACTTGTACAG PCR PT7 + yfp

5’ yfp PT7 SmaI BglII

ATATAGATCTCCCGGGGAATTGTTATCCGCTCA

CAATTCCCCTATAGTGAGTCGTATTAATTTAAG

GAGAATGGTGAGCAAGGGCGA

PCR PT7 + yfp

5’ GmR BamHI GGATCCGACGCACACCGTGGAAACGGATG PCR GmR

3’ GmR BclI TGATCAATTACCCTGTTATCCCTAGGAAGCCGA

TCTCGGCTTGAACG PCR GmR

3’ tnp TAACTATAACGGTCCTAAGGTAGCGAGCCG

GCCCCTGTCTCTTGATCAG PCR tnp + OE-R

5’ tnp -OER

½ SmaI GGGCTGACTCTTATACACAAGTAGCG PCR tnp + OE-R

Construction of TREX-crt vectors

Crt1 XbaI-for ATATTCTAGAGGTACCGCACGGTCTGCCAATC PCR crtEXYI’

Crt1(BamHI)-rev ATATGGATCCCCGCAGCTTGTAGACGAATTG PCR crtEXYI’

Crt2 (BamHI)-for ATATGGATCCCCGTCTTACTGCTTGAAC PCR crt’’IBZ

Crt2 EcoRI-rev ATATGAATTCAAGCTTTAAAAAGCCTGGCG PCR crt’’IBZ

Crt2∆Z EcoRI-rev ATATGAATTCCTAGAGCGGGCGCTGCCAG PCR crt’’IB

pUC18 MCS NcoI ATATCCATGGGATAACAATTTCACAC PCR MCS + ApR

pUC18 ApR BstBI TATATTCGAAATATGAGTAAACTTGG PCR MCS + ApR

pKD46 ori101 BstBI ATATTTCGAAACCCCGTTGATGATACC PCR ori101 + repA

pKD46 rep NcoI TATACCATGGAACCTCAGATCCTTCCG PCR ori101 + repA

Construction of mutant strain R. capsulatus ∆crtC

crtC1 AACGCTTTGAAATCATCATGAAC PCR crtC

crtC2 CTGACTCTCTTTGCCGTCTATTTC PCR crtC

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Construction of TREX-pig vector

pig-1.I XbaI ATATTCTAGAATCGGCACCCGCGCCACG PCR cueR - pigABC

pig -1.II SpeI HindIII ATATAAGCTT ACTAGTCTAGCCATCGGCACGTTCTCCGC PCR cueR - pigABC

pig -2.I XbaI ATATTCTAGATCAGCACCACATCCGGCGAAGGTAAA PCR pigDEF

pig -2.II SpeI HindIII ATATAAGCTT ACTAGTTTATTTTTCGCCGACGATCAGGGTG PCR pigDEF

pig -3.I XbaI ATATTCTAGACCAGTTAGCAACGGGAGCTCGC PCR pigGHIJKLMN

pig -3.II HindIII ATATAAGCTT

ACTAGTTTACAGCACGAAAGGAATGAAACAC PCR pigGHIJKLMN

crt cluster: Reverse transcription & qPCR Size [bp] / Tm [°C]

crtE-for GCGGTTTCTGAACTGTCAAA 20 / 59

crtE-rev TCCCCTTCAGACAGATCCTT 20 / 59

crtX-for CGTTACCGGCTTGTTTTCAT 20 / 60.00

crtX-rev AACGGGATGAAGACGTTGAC 20 / 60

crtY-for CCTACCACCGGCTATTCACT 20 / 59

crtY-rev GAGGCCGACGTAAAGACATC 20 / 60

crtI-for GATCCCCGTCTTACTGCTTG 20 / 60

crtI-rev ACCCCTGATCCTCGTAGACA 20 / 60

crtB-for ACCAGGCGTAGAGCATCAGT 20 / 60

crtB-rev CGAAAAGTTTTGCGACAGC 19 / 60

crtZ-for ATCATGAACCGCGTAAAGGT 20 / 60

crtZ-rev CAGGATCGATAATGCAGCAA 20 / 60

Ec rpoD-for ATATCAACCGTCGTATGTCCATC 23 / 60

Ec rpoD-rev TAAGTTCGCTTCAACCATCTCTT 23 / 60

Rc rpoD-for GCTACATCACCATCGACCAG 20 / 59

Rc rpoD-rev ACATCACATCCTCGATCTGC 20 / 59

Pp rpoD-for TCGCCAAGAAGTACACCAAC 20 / 59

Pp rpoD-rev TTTCATCAGACCGATGTTGC 20 / 60

pig cluster: Reverse transcription & analytic PCR

cueR-for AGTTCGCTGATGTGCCTTTC 20 / 60

cueR-rev AGCTGGTGGCGTTATTCAAC 20 / 60

pigA-for GCCTGTCGGCTCTGTTTATC 20 / 62

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pigA-rev CTGTTCCAGACGCAGTTTCA 20 / 60

pigB-for CGGGACAACTATTCCTGCAT 20 / 60

pigB-rev GGTATCCAGGAAGGCGTACA 20 / 62

pigC-for GTTGAACTTTCCGGCGATAA 20 / 58

pigC-rev ACGGCGAATTCGATAAACTG 20 / 58

pigD-for CAGGAACGCATTCTGTTCAA 20 / 58

pigD-rev ATGCGTAAGCGTTTTGCTCT 20 / 58

pigE-for ATTCGGATTCATCGCTCATC 20 / 58

pigE-rev AGGTTTTGTCGTTCCCACAG 20 / 60

pigF-for CATGGGGTATCTCGACCAAC 20 / 62

pigF-rev GCGGATACAGGAAGGTATCG 20 / 62

pigG-for GCATATCGCCACCCAGTATC 20 / 62

pigG-rev CATGAGACTCCTGACGCAGA 20 / 62

pigH-for CGGTTTTGACCTTGAGCATT 20 / 58

pigH-rev ACCACGGTCTGGCAGAATAG 20 / 62

pigI-for CGACATGGTGAAAATCAACG 20 / 58

pigI-rev GATCACGGCGATCAGTTTTT 20 / 58

pigJ-for TCAGGTTTTCTGCCATCTCC 20 / 60

pigJ-rev GAAGCCGTCGGTTTTAATGA 20 / 58

pigK-for GTCATCGAAGTCAGCCCTTC 20 / 62

pigK-rev TTCCGCCAACTGATTAAAGC 20 / 58

pigL-for ACGTTTCCCTCAGCCATTC 20 / 58

pigL-rev ATGTGTCCCAATCGCTGTTC 20 / 60

pigM-for AGCATTCAAGCGCTTCTTTC 20 / 58

pigM-rev TTTCGTCGTTCAGACACAGC 20 / 60

pigN-for GCCGTTGTTTTGTTGATCCT 20 / 58

pigN-rev CCTCGCCGAAATAGCAATAG 20 / 60

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SUPPLEMENTARY METHODS

Construction of the TREX cassettes

For the construction of the L-TREX cassette (Figure S4A), a DNA fragment encompassing the

tetracycline resistance gene (TcR) and origin of transfer (oriT) was PCR-amplified using vector

pBBR1MCS-3 as a template. To insert a Bcll site at both ends of the resulting PCR fragment as well as a

ScaI site at the 3’-end of TcR, corresponding recognition sites were added to the 5’-ends of the used

primers. In addition, aphII gene was amplified from vector pBSL15. To insert a BglII site as well as the

PT7 promoter into the resulting PCR fragment, corresponding sequences were added to the primers. Both

PCR products were cloned into the SmaI sites of the vectors pUCPSK and pUC18, respectively, resulting

in plasmids pUCPSKTcR.oriT and pUC18aphII.PT7. A synthetic DNA comprising outside end OE-L from

transposon Tn5 as well as a BglII and BamHI restriction site was cloned as a BglII-BamHI fragment into

the BglII site of pUC18aphII.PT7. The resulting plasmid was designated as pUC18 aphII.PT7.OE-L.

Subsequently, the TcR.oriT fragment was isolated from the pUCPSK derivative using BclI and inserted

into the BglII site of vector pUC18 aphII.PT7.OE-L. The final vector carrying the complete L-TREX

cassette was designated as pL-TREX.

For the construction of the R-TREX cassette (Figure S4B), the yfp gene was PCR-amplified using the

recombinant plasmid pFF19-EYFP as a template. In order to add the lac operator the PT7 promoter, and a

SmaI site to one end as well as BglII sites to both ends of the PCR product, the corresponding sequences

were included in the primers. The PT7.yfp fragment was cloned into the SmaI site from pSVB10 resulting

in plasmid pSVB10PT7.yfp. In parallel, the GmR gene was amplified from pWKR202 using primers

harboring a BamHI and BclI restriction site, respectively, and subsequently cloned into the SmaI site of

pSVB10. In addition, the transposase gene tnp was PCR amplified together with its OE-R using vector

pSUP2021 as a template. The used primers encompassed additional sequences for inserting a BamHI and a

SmaI site into the product, which was cloned into the SmaI site of pUC19 resulting in vector pUC19 OE-

R.tnp. The subcloned GmR fragment was isolated from pSVB10-GmR using BamHI and BclI and was

further inserted into the BamHI site of recombinant vector pUC19 OE-R.tnp. Afterwards, the second

subcloned fragment comprising lacO, PT7 and yfp was excised from vector pSVB10-derivative using BglII

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and ligated into the BamHI site of pUC19 derivative containing GmR, tnp and OE-R. The final vector

carrying the complete R-TREX cassette was designated as pR-TREX.

In summary, the TREX cassettes contain selection markers (TcR, GmR) for gene cluster labeling, an origin

of transfer (oriT) for conjugational gene delivery, transposon elements (OE’s and tnp) for chromosomal

integration and T7 promoters (PT7) for bidirectional expression. In addition, we included yfp and aphII as

promoter-less reporter genes, which may be used to verify full length gene cluster expression, if needed.

In order to further simplify the use of the TREX cassettes, <L-TREX-R> module was constructed.

Therefore, both cassettes were isolated from vectors pL-TREX and pR-TREX using SmaI and ScaI,

respectively. The R-TREX cassette was inserted into the EcoRV site of pIC20H followed by the L-TREX

cassette that was integrated into the SmaI site of the recombinant vector which resulted in vector pIC20H-

RL carrying both TREX cassettes in an inside-out assembly (Figure S4C). In this vector, the <L-TREX-

R> module is flanked by two XbaI recognition sites which allow one-step isolation of all TREX elements.

Detailed sequence information on pIC20H-RL is deposited in Genbank (Accession number: JX668229).

Construction of vectors pTREX-crt∆X, pTREX-crt∆Z and ptsTREX-crt∆Z. The crt gene cluster was

amplified from P. ananatis genomic DNA by PCR. Therefore, genes crtEXYI’ were amplified by PCR.

For the following cloning step, a XbaI site was introduced at the 5’ end of crtE, whereas a naturally

occurring BamHI site inside the crtI gene was used at the 3’ end. For crtX deletion, the resulting PCR

product crtEXYI’ was subcloned via XbaI and BamHI into pASK-IBA3 and subsequently deletion was

achieved by removing an internal part of crtX using EcoRV and PvuII. A second PCR fragment covering

genes crt’’IBZ was amplified thereby adding an EcoRI site to the 3’ end of crtZ. Both parts of the crt

cluster, the XbaI-crtE∆XYI’-BamHI and the BamHI-crt’’IBZ-EcoRI fragment, were subsequently brought

together on pUC18, resulting in pUC18crt∆X (Figure S5A). To obtain variant crt∆Z, genes crt’’IB were

amplified thereby adding an EcoRI site to the 3’ end of crtB. According to the strategy used for

pUC18crt∆X, both fragments (XbaI-crtE∆XYI’-BamHI and BamHI-crt’’IB-EcoRI) were subsequently

cloned into pUC18 (Figure S5B).

To obtain pTREX-crt∆X and pTREX-crt∆Z, respectively, the <L-TREX-R> module was isolated from

plasmid pIC20H-RL using XbaI and cloned into the respective site of the two recombinant pUC18

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derivatives. For further experiments we used plasmid pTREX-crt∆X and pTREX-crt∆Z, where PT7 of L-

TREX is adjacent to the crtE gene.

To generate a temperature sensitive pTREX-crt∆Z derivative for E. coli, we basically combined vectors

pUC18 and pKD46. Two DNA fragments encompassing (i) the multiple cloning site and ApR from pUC18

as well as (ii) the temperature sensitive origin of replication ori101 and rep gene from pKD46 were

amplified by PCR. During both PCRs, NcoI and BstBI sites were inserted into the resulting fragments.

Thus, the obtained fragments could be ligated to one vector called pUC18ts. Afterwards, the truncated crt

gene cluster was isolated from pUC18crt∆Z and introduced into pUC18ts using XbaI and EcoRI. The <L-

TREX-R> cassette was added via its XbaI restriction site thereby creating ptsTREX-crt∆Z.

Construction of vector pTREX-pig. As described for the crt gene cluster, the prodigiosin gene cluster

(including all pig genes and the regulatory gene cueR) was stitched together by PCR amplification and

ligation. Therefore, three fragments with appropriate restriction sites were created and merged on one

vector. The first fragment comprises genes cueR and pigA-C where XbaI was added to the cueR gene and

SpeI and HindIII to pigC. The second fragment contains pigD-F with sites for XbaI at pigD and for SpeI

and HindIII at pigF. The third fragment encompasses pigG-N with sites for XbaI at pigG and for HindIII

at pigN. All three fragments were separately cloned into pUC19 using XbaI and HindIII. Subsequently, the

second pig fragment was isolated from pUC19 using XbaI and HindIII and cloned into the pUC19

derivative carrying the first pig fragment that was hydrolyzed with SpeI and HindIII. In the last step, the

third pig fragment was isolated from pUC19 with XbaI and HindIII and was cloned into the sites of SpeI

and XbaI of the pUC19 derivative carrying the first and second pig fragment. The resulting plasmid

containing the complete pig gene cluster was called pPIG. The <L-TREX-R> module was isolated from

the plasmid pIC20H-RL using XbaI and cloned into the respective site of the recombinant vector pPIG

thereby creating plasmid pTREX-pig (Figure 5B), where the T7 promoter from L-TREX reads into the pig

cluster.

Construction of deletion plasmid pRc∆crtC. Neurosporene hydroxylase encoding crtC gene was

deleted via homologous recombination. Therefore, wild type crtC gene was initially PCR amplified

together with up- and down-stream regions from genomic DNA of R. capsulatus B10S. The PCR product

was digested with NcoI isolating gene crtC. The fragment was cloned into NcoI site of pET22b. The gene

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sequence was disrupted by insertion of an Ω-SpcR-cassette using a SmaI site occurring within crtC. The

cassette was obtained from vector pHP45Ω using SmaI. An oriT-TcR cassette was isolated from

pWKR459 using EcoRI and inserted into the respective site of the recombinant construct pET22b-crtC-Ω-

SpcR. The final deletion construct was designated as pRc∆crtC.

Generation of mutant strain R. capsulatus ∆crtC. The deletion construct pRc∆crtC was transferred to R.

capsulatus wild type strain B10S by conjugation. As the vector cannot replicate in R. capsulatus, use of

spectinomycin enabled selection for recombination mutants. Single-cross-over mutants were excluded by

replicaplating on tetracycline. The strain Rc∆crtC was successfully obtained by double-cross-over

resulting in exchange of the intact genomic version of crtC with the disrupted version on the vector.

Characterization of R. capsulatus mutant strain ∆crtC. The crtC deletion strain showed a different

phenotype compared to the wild type (Figure S3A). After phototrophic growth the wild type is typically

dark red while the mutant showed green color. After aerobic growth the wild type shows a pink

pigmentation while the mutant is light yellow. Under aerobic conditions bacteriochlorophyll does not

accumulate thus the yellow color corresponded to neurosporene. Neurosporene accumulation in Rc∆crtC

can be verified spectrophotometrically as wild type and mutant pigments show specific absorption spectra

(Figure S3B). HPLC analysis confirmed neurosporene accumulation in Rc∆crtC. By comparison of

retention times and spectra with the authentic standard, neurosporene can be identified as the main

pigment in Rc∆crtC (Figure S3C).

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