Applied Microbiology and Biotechnology. De-bugging and maximizing plant cytochrome P450 production in Escherichia coli with C-terminal GFP-fusions

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1 Applied Microbiology and Biotechnology De-bugging and maximizing plant cytochrome P450 production in Escherichia coli with C-terminal GFP-fusions Ulla Christensen 1, Dario V. Albacete 1, Karina M. Søgaard 1, Tonja Wolff 1, Morten T. Nielsen 1, Scott James Harrison 1, Anders Holmgaard Hansen 1, Birger Lindberg Møller 2,3, Susanna Seppälä 1,4 and Morten H. H. Nørholm 1,4 1 Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark; 2 Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, Copenhagen, Denmark; 3 Center for Synthetic Biology: biosynergy, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, Copenhagen, Denmark. 4 Address correspondence to MHHN, morno@biosustain.dtu.dk. Phone: Fax: or SS, susse@biosustain.dtu.dk. 1

2 Supplementary figures Fig. S1 Mining of a library of membrane encoding genes. (A) Production levels of 520 different membrane proteins fused to GFP, based on whole cell fluorescence data previously published (Daley et al. 2005). SohB and YafU, selected based on their high expression levels, small size and topology, are highlighted in red color. (B) Production levels of 183 different membrane proteins fused to alkaline phosphate (PhoA), based on enzyme activity data previously published (Daley et al. 2005). LepB, selected based on the high expression level, small size and topology, is highlighted in red color. 2

3 Fig. S2 Expression of differently truncated variants of CYP79A1-TEV-GFP-his8 with different N-terminal modifications. (A) 3 h and (B) 22 h induction of expression of native CYP79A1, CYP79A1Δ1-35, 28-tag and SohB fused to CYP79A1: CYP79A1Δ1-69, CYP79A1Δ1-59, CYP79A1Δ1-43, CYP79A1Δ1-35 or CYP79A1 with an artificially extended linker region CYP79A1Δ Constructs were expressed in KRX and cell lysates analyzed by in-gel fluorescence. Quantification of full-length CYP79A1-TEV-GFP-his8 vs free GFP was performed using the Image J software. 3

4 Fig S3 Functional assaying of the highest expressing CYP71E1 and CYP79B2 constructs. (A) Microsomes from SohB 1-37-CYP71E1 and SohB 1-35-CYP79A1 expressing cell were mixed with purified SbCPR2b, NADPH and 14 C-tyrosine and the products were extracted by ethyl acetate and separated by thin layer chromatography (TLC). (B) Similarly, microsomes from SohB 1-45-CYP79B2 expressing cells were mixed with purified SbCPR2b, NADPH and 14 C-tryptophane and the product extracted by ethyl acetate and separated by TLC. 4

5 Fig. S4 Correlation between enzyme activity and fluorescence under short term (3h) expression conditions. Oxime production levels and whole-cell fluorescence was plotted from the different full-length CYP79A1 constructs (red squares) and the shortest (D1-35) truncations (green triangles). 5

6 Fig. S5 Effect of GFP fusions on the activity of different CYP79A1 variants. (A) Differently engineered versions of CYP79A1 with (green bars) or without (grey bars) fusions to GFP were co-produced with the compatible reductase electron donor CPR2b in the KRX strain and conversion of L-Tyrosine to (E)-p-hydroxyphenylacetaldoxime assayed by (A) adding the substrate to whole cells and analyzing the methanol-extracted product by HPLC or (B) by extracting the product without any externally added substrate and analysis by LC-MS. Cells were induced for 3h in A and 22 h in B. Empty vector control and 28-tag:native CYP79A1 expressed without CPR were included as references. Error bars indicate standard error of the mean (n=3). 6

7 Fig. S6. Effect of lysozyme on the production of CYP enzymes. BL21 (DE3) was transformed with the six different 28-tag:CYP:tev-gfp-his8 expression constructs in combination with plyssrare2, plyss or the corresponding plasmid after having deleted the lyss gene. Cells were grown in TB media and induced for 3 h, as cell cultures reached the exponential growing phase. Overall expression was monitored by whole cell fluorescence detection. Error bars indicate standard error of the mean (n=3). 7

8 Supplementary tables All pet28a(+)-based plasmids in this work had an AsiSI site removed like this Constructs Fwd oligonucleotide (5 - pet28a(+)-28tag-asisicastev-gfp-his8 3 ) ATCGCGUATTTCGTCT CGCTC ATCGCAAGCUTGCGAT CGCTGCTGAGGGGGTT ATATCTCCTTCTTGGA TCCAG Rev oligonucleotide (5-3 ) ACGCGAUCACTGTTAA AAGGACAATTACAAA CAGGAATC AGCTTGCGAUCGCCAG CTGAGGGAGAAAACC TGTACTTCCAGGGTC Template DNA See below pet28a(+)-28tag tev-gfp-his8 (Nørholm et al., 2013) ATCGCAAGCUTGCGAT AGCTTGCGAUCGCCAG pet28a(+)-malllavf- pet28a(+)-28tag-asisicas-tev- AsiSIcas-tev-gfp-his8 CGCTGCTGAGGGAAA CTGAGGGAGAAAACC gfp-his8 AACTGCTAATAACAG TGTACTTCCAGGGTC (this work) AGCCATATCATCCTCG AGTCTCCTTCTTAAAG pet28a(+)-sohb-asisicas-tev- ATCGCAAGCUTGCGAT AGCTTGCGAUCGCCAG pet28a(+)-sohb- -tev-gfp-his8 gfp-his8 CGCTGCTGAGGGGCTG CTGAGGGAGAAAACC (Daley et al., 2005) AGATTGTTGACCCGTA TGTACTTCCAGGGTC AC pet28a(+)-yafu-asisicas-tev- ATCGCAAGCUTGCGAT AGCTTGCGAUCGCCAG pet28a(+)-yafu-tev-gfp-his8 gfp-his8 CGCTGCTGAGGGGCTG CTGAGGGAGAAAACC (Daley et al., 2005) AGATTGTTGACCCGTA TGTACTTCCAGGGTC AC pet28a(+)-tev-gfp-his8 ATCCTCGAGUCTCCTT ACCTGGAUCCGAAAA pet28a(+)-28tag-asisicas-tev- CTTAAAG CCTGTACTTC gfp-his8 (this work) ACCAGTGCUTCATTGA AGCACTGGUCTAGCAT pet28a(+)-strep-hrv3c- pet28a(+)-strep-hrv3c- CPR2b-tev CCCTGGAAGTACAGGT AACCCCTTGGGGCCTC CPR2b-tev-gfp-his8 Table S1 DNA constructs made by single fragment PCR and uracil excision 8

9 Oligonucleotide name and use For cloning into AsiSI cassette CYP720B4_fwd CYP79A1 _fwd CYP79B2 _fwd CYP51G1 _fwd CYP71E1_fwd CYP51H10 _fwd CYP720B4_rev CYP79A1 _ rev CYP79B2 _ rev CYP51G1 _ rev CYP71E1_ rev CYP51H10 _rev For cloning truncated CYPs into AsiSI cassette tcyp720b4_fwd t1cyp79a1 _fwd t2cyp79a1 _fwd t3cyp79a1 _fwd t4cyp79a1 _fwd tcyp79b2_fwd tcyp51g1 _fwd tcyp71e1_fwd tcyp51h10 _fwd For cloning into PCR amplified pet28-variants ncyp720b4_fwd ncyp79a1 _fwd ncyp79b2 _fwd ncyp51g1 _fwd ncyp71e1_fwd ncyp51h10 _fwd ncyp720b4_rev ncyp79a1 _rev ncyp79b2 _rev CYP51G1 _rev ncyp71e1_rev ncyp51h10 _rev strepcpr2b_fwd CPR2b_rev Oligonucleotide sequence (5-3 ) GCAGCGAUGGCGCCCATGGCAGACCAAATAT GCAGCGAUGGCGACAATGGAGGTAGAGGCCG GCAGCGAUGAACACTTTTACCTCAAACTCTT GCAGCGAUGGATCTCGCCGACATCCCGCAGC GCAGCGAUGGCCACCACCGCCACCCCGCAGC GCAGCGAUGATGGACATGACAATTTGCGTCG GCTGGCGAUCCTTCATTCTCTACTCTACCATGA GCTGGCGAUCCGATGGAGATGGACGGGTAGAGG GCTGGCGAUCCCTTCACCGTCGGGTAGAGATGC GCTGGCGAUCCGTTGTCCACAACAAGCTTCCGC GCTGGCGAUCCGGCGGCGCGGCGGTTCTTGTAT GCTGGCGAUCCGTTTGCAGGCATACGACATCTC GCAGCGAUGCCTGGGTCGACTGGATGGCCGC GCAGCGAUGCCACGCAAAAGCACCACCAAGT GCAGCGAUGCCGGCCGGCGTTGGCAACCCGC GCAGCGAUGTACCTGGCCCGAGCCCTGAGGC GCAGCGAUGTACCTGGCCCGAGCCCTGAGGCGGCCATACCTGGCCCGAGCCCTGAGG GCAGCGAUGCCGGGTCCCACAGGATGGCCGA GCAGCGAUGCCGACGATCCCGGGGGCGCCGG GCAGCGAUGCCGGGCCCTGCGCAGCTGCCGA GCAGCGAUGCCTCCACCGGTGGTGACAGGAA ACTCGAGGAUGGCGGCGCCCATGGCAGACCAAATAT ACTCGAGGAUGGCGGCGACAATGGAGGTAGAGGCCG ACTCGAGGAUGGCGAACACTTTTACCTCAAACTCTT ACTCGAGGAUGGCGGATCTCGCCGACATCCCGCAGC ACTCGAGGAUGGCGGCCACCACCGCCACCCCGCAGC ACTCGAGGAUGGCGATGGACATGACAATTTGCGTCG ATCCAGGUACTTCATTCTCTACTCTACCATGA ATCCAGGUACGATGGAGATGGACGGGTAGAGG ATCCAGGUACCTTCACCGTCGGGTAGAGATGC ATCCAGGUACGTTGTCCACAACAAGCTTCCGC ATCCAGGUACGGCGGCGCGGCGGTTCTTGTAT ATCCAGGUACGTTTGCAGGCATACGACATCTC ACTCGAGGAUGGCGGCAAGCTGGAGCCACCCGCAGT ATCCAGGUACCCAGACATCACGCAGATAGCGG Table S2 Gene specific oligonucleotides used in this work 9

10 N-terminal tag Oligonucleotide sequence (5-3 ) ATGGAATTATCACAAGTTTGTACAAAAAAGGAGGCTGGCGCCGGAACCAATTCAGTCGACTGGA 28-tag TCCAAGAAGGAGATATAACC ATGGAATTGTTGTCTGAATATGGTTTGTTTTTGGCGAAAATCGTTACCGTTGTGCTAGCGATTGC SohB GGCGATTGCCGCCATTATTGTCAATGTTGCTCAACGTAATAAACGCCAGCGTGGCGAGTTACGG GTCAACAATCTCAGC MALLAVF ATGGCTCTGTTATTAGCAGTTTTT ATGAGTTCAGAGCGAGATCTGGTTAATTTTCTTGGCGATTTTTCAATGGATGTGGCCAAAGCAGT TATAGCCGGTGGTGTTGCAACCGCTATTGGAAGTCTGGCTTCTTTTGCCTGTGTTAGCTTTGGCTT YafU TCCAGTAATTCTTGTCGGAGGAGCAATTTTACTGACAGGGATAGTGTGTACAGTTGTTTTAAATG AAATCGATGCTCAATGCCATTTATCAGAAAAATTAAAATATGCAATTAGAGATGGACTAAAACG GCAA ATGGCGAATATGTTTGCCCTGATTCTGGTGATTGCCACACTGGTGACGGGCATTTTATGGTGCGT GGATAAATTCTTTTTCGCACCTAAACGGCGGGAACGTCAGGCAGCGGCGCAGGCGGCTGCCGGG GACTCACTGGATAAAGCAACGTTGAAAAAGGTTGCGCCGAAGCCTGGCTGGCTGGAAACCGGT GCTTCTGTTTTTCCGGTACTGGCTATCGTATTGATTGTGCGTTCGTTTATTTATGAACCGTTCCAG ATCCCGTCAGGTTCGATGATGCCGACTCTGAACTCTACTGATTTTATTCTGGTAGAGAAGTTTGC Lep TTATGGCATTAAAGATCCTATCTACCAGAAAACGCTGATCGAAACCGGTCATCCGAAACGCGGC GATATCGTGGTCTTTAAATATCCGGAAGATCCAAAGCTTGATTACATCAAGCGCGCGGTGGGTT TACCGGGCGATAAAGTCACTTACGATCCGGTCTCAAAAGAGCTGACGATTCAACCGGGATGCAG TTCCGGCCAGGCGTGTGAAAACGCGCTGCCGGTCACCTACTCAAACGTGGAACCGAGCGATTTC GTTCAGACCTTCTCACGCCGTAATGGTGGGGAAGCGACCAGCGGATTCTTTGAAGTGCCGAAAC AGGAAACCAAAGAAAATGGAATTCGTCTTTCCGAGACTAGTGGTGGTCCTGGA Table S3 Nucleotide sequences of N-terminal modifications used in this work 10

Design. Construction. Characterization

Design. Construction. Characterization Design Construction Characterization DNA mrna (messenger) A C C transcription translation C A C protein His A T G C T A C G Plasmids replicon copy number incompatibility selection marker origin of replication