Effect of Genomic Integration Location on Heterologous Protein. Expression and Metabolic Engineering in E. coli. Supporting Information

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1 Effect of Genomic Integration Location on Heterologous Protein Expression and Metabolic Engineering in E. coli. Supporting Information

2 Supporting Figures Table S1. DNA primers used in this study Number Primer ID Primer Sequence (5 -->3 ) 1 mcherrypktipfor cgcgcgggcccatgcgtccggcgtagccta 2 mcherryptkiprev cgccaatccggatatagtcgac 3 ptkscs-laczfor tctcgttgctgcataaaccgactacacaaatcagcgattttacggccccaag GTCCAAAC 4 ptkscs-laczrev gtgccttcttccgcgtgcagcagatggcgatggctggtttttggcttcaggg ATGAGGCG 5 ptkscs-atpgidfor CAAAAAGCGGTCAAATTATACGGTGCGCCCCCGTGATTTCAAA CAATAAGTACGGCCCCAAGGTCCAAACGGTGA 6 ptkscs-atpgidrev ATAACGTGGCTTTTTTTGGTAAGCAGAAAATAAGTCATTAGTG AAAATATTTGGCTTCAGGGATGAGGCGCCATC 7 ptkscs-recafor aaaatcttcgttagtttctgctacgccttcgctatcatcttacggccccaagg TCCAAAC 8 ptkscs-recarev atggctatcgacgaaaacaaacagaaagcgttggcggcagttggcttcagg GATGAGGCG 9 ptkscs-ybbdfor ACTGAGAAAAGACATGTCGGCTATTGTGTAAAGCCATATAGC TCAGACGATACGGCCCCAAGGTCCAAACGGTGA 10 ptkscs-ybbdrev TTCTATGTAAACTCTCTGACTGTTCATTTTATTTGTTGTTTCAGG GTCGGTTGGCTTCAGGGATGAGGCGCCATC 11 laczchkfor CTGGCTGGCATAAATATCTC 12 laczchkrev GAAACCGCCAAGACTGTTAC 13 atpichkfor CAGTAACTGAACGAGCAGAAG 14 atpichkrev CTTCGTCAGGTGCAACATGAGC 15 recachkfor GCCGTAGCATCAATCTCTTC 16 recachkrev ATGCATTGCAGACCTTGTGG 17 ybbdchkfor ATTGGAGCTGGATTGCCTGATGCTTG 18 ybbdchkrev CTCACATTAAACACGTAACATTTTAATTAATG 19 tetachkfor CAGGTTATCTTTGCTCCTTG 20 tetachkrev CAAGGAGCAAAGATAACCTG 21 neochkfor CTGAATGAACTGCAGGACGA 22 neochkrev TGACAACGTCGAGCACAGCT 23 mcherrychkfor GGCGAAGAAGACAACATGGC 24 mcherrychkrev CGGATGCTTAACGTACGCTTTCG

3 25 vioafor GCATTCAGTTTCAATTGTCGGAG 26 vioarev CACAGTTTCACGATGTGTAGGC 27 viobfor CTCGGGTTAATATACCAACCGC 28 viobrev GGTCAGCCAGTTGTTGTTG 29 vioefor CGTAAAGTAGATAGAGTTCCGCC 30 vioerev GGAAAGTCTCTTACAGAGGCG 31 viocfor CAGCCTGACGGCAATCTATC 32 viocrev CATCCCGAAGCTCGCTATAC 33 viodfor CTGCCGGATTAATGTTTGCT 34 viodrev GCGACTTGTTTGTGCATGTTG 35 TALchkFOR GTTGAGCCTGTTGAGCCAATC 36 TALchkREV GATTGGCTCAACAGGCTCAAC 37 viobsalfor TAATTCGGGCATTAAAAGACTCTGTgGACCTAGAGTTGTCGAT TATGCTGC 38 viobsalrev GCAGCATAATCGACAACTCTAGGTCcACAGAGTCTTTTAATGC CCGAATTA 39 viocsalfor ACTATCACTATCTACAACGCTGTGTgGACGTTGATATTGAAAG AGCGTCAG 40 viocsalrev CTGACGCTCTTTCAATATCAACGTCcACACAGCGTTGTAGATA GTGATAGT 41 viobmidfor1 GCAAGGATTTCTTATTGAATCAATTGGGCTTGC 42 viobmidfor2 CCAAACCTAATCGAGTTGCAAAAAAGCAAGC 43 viobmidfor3 GCATTAAAAGACTCTGTCGACCTAGAGTTGTCG

4 Figure S1. Metabolic pathways for the conversion of (A) L-tryptophan to violacein and its byproducts, and (B) the conversion of L-phenylalanine to trans-cinnamic acid. Figure S2. Impact of genomic integration of mcherry into four genomic loci on cellular growth. Optical density was measured at 650 nm with (+) and without (-) induction of mcherry expression by

5 addition of 1 mm IPTG. Error bars represent standard deviation of triplicate measuremrents. Top shows all plots combined, bottom four show individual strains. Figure S3. Colony PCR to verify the integration of the violacein pathway into four genomic loci. Colony PCR was performed on 8 colonies containing vioabecd integrated into the lacz (a), atpi-gidb (b), reca (c), and ybbd-ylbg (d) loci. PCR was performed with a forward primer upstream of the integration event, and a reverse primer inside vioa. Figure S4. PCR analysis to examine recombination between genes of the integrated violacein pathway. PCR was performed to amplify the DNA sequence between vioa and viob (a), viob and vioe (b), vioe and vioc (c), and vioc and viod (d) in 8 colonies of MG1655(DE3) ΔlacZ::vioABECD.

6 Figure S5. Verification of integration of TAL into four genomic loci. Colony PCR to amplify the integration junction of TAL integrated into lacz (a), atpi-gidb (b), reca (c), and ybbd-ylbg (d). Figure S6. HPLC chromatogram of products of strains harboring vioabecd integrated into the genomic reca locus. Violacein standard was produced from E. coli containing petm6-vioabecd. vioabe standard produced in strain containing petm6-vioabe. Peaks in the standard are labeled based on their retention time.

7 trans-cinnamic Acid Titer (mg/l) Hr 3 Hr 4 Hr 5 Hr atpi::lp lacz::lp reca::lp ybbd::lp petm6-tal Figure S7. Impact of landing-pad integration into genomic loci on production of trans-cinnamic acid. trans-cinnamic acid production was measured using HPLC. Strains were induced by addition of IPTG at designated time. Positive control is E. coli MG1655(DE3) harboring petm6-tal syn. Landing-pad (LP) strains are the same, except they have the indicated genomic locus replaced with the tetracyclineresistant landing-pad trans-cinnamic Acid Titer (mg/l) MG1655(DE3) petm6 mg1655(de3) petm6-tal ybbd1 ybbd3 ybbd5 ybbd7 Figure S8. Production of trans-cinnamic acid in four colonies with the gene encoding TAL integrated into the ybbd-ylbg genomic locus. The ability to produce trans-cinnamic acid was determined for four individual colonies. Negative control was wild-type E. coli MG1655(DE3) containing empty petm6 vector. Positive control was the same strain containing PETM6-TAL syn. Error bars represent standard deviation of biological duplicate.

8 Cinnamic Acid (mg/l) LacZ-1 LacZ-2 LacZ-3 LacZ-5 LacZ-6 LacZ-7 LacZ-8 Figure S9. trans-cinnamic acid production when the gene encoding TAL is integrated into the lacz locus on the genome of E. coli MG1655(DE3). Seven individual colonies were screen for production of trans-cinnamic acid. Error bars represent standard deviation of biological duplicates % Yield mM 2mM 1 hr 2 hr 3 hr 4 hr 5 hr Induction Time IPTG Concentration Figure S10. 2-Dimensional analysis of the effect of varying inducer concentration and time of induction on trans-cinnamic acid production in cells harboring the TAL gene integrated into the atpigidb genomic locus. Percent yield based on feed concentration of phenylalanine.