Additional file 1 of Synthesis of (−)−deoxypodophyllotoxin and (−)−epipodophyllotoxin via a multi-enzyme cascade in E. coli
Creators: name: Decembrino, Davide
name: Raffaele, Alessandra
name: Knöfel, Ronja
name: Girhard, Marco
name: Urlacher, Vlada B.
Publisher: name: figshare
Year: 2021
Subjects: subject: Biophysics
subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: Genetics
subject: Pharmacology
subject: Biotechnology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: Ecology
subject: Developmental Biology
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: 60506 Virology

description: Additional file 1: Supplementary materials include DNA-sequences of the genes used. Supplementary methods include detailed information about individual genes expression and reaction analytics. Supplementary results support the conclusions drawn in the main manuscript. Table S1. Summary of generated cyp71cu1 and cyp82d61 gene variants. Table S2. Summary of genes used in establishing of multi-enzyme cascade. Table S3. Summary of plasmids used within this study. Table S4. Oligonucleotides used for generation of cyp71cu1 and cyp82d61 gene variants. Table S5. LC/MS methods used within this study. Table S6. Qualitative metabolite analysis.Table S7. Quantitative metabolite analysis. Table S8. Expression levels of CYP71CU1 variants in E. coli C41(DE3) at 25°C after 48 h. Table S9. Expression levels of N-terminally truncated CYP71CU1 variants in E. coli C41(DE3) at various incubation temperatures after 48 h. Table S10. Expression levels of codon optimized WT and N-terminally truncated CYP82D61 variants in E. coli C41(DE3) at various incubation temperatures after 48 h. Figure S1. 12.5% SDS-gel of OMT3 (41 kDa; marked by red arrow) expression in various E. coli strains. Figure S2. 12.5% SDS-gel of OMT1 (38 kDa; marked by red arrow) expression in various E. coli strains. Figure S3. 12.5% SDS-gel of 2-ODD (35 kDa; marked by red arrow) expression in various E. coli strains. Figure S4. 12.5% SDS-gel of CYP71CU1 (56 kDa; marked by red arrow) expression in various E. coli strains. Figure S5. CO-difference spectra of soluble CYP71CU1OC_WT in E. coli BL21 (DE3) (orange line), C41 (DE3) (blue line), and C43 (DE3) (green line) after 24 h expression. Figure S6. CO-difference spectra of soluble CYP71CU1nat_WT (blue line) and CYP71CU1OC_WT (orange line) after 48 h expression. Figure S7. Expression analysis of CYP71CU1 variants incubated at different temperatures. Figure S8. LC/MS analysis (method 4) of one-cell biotransformations of (−)−matairesinol 1 to (−)−deoxypodophyllotoxin 6. Figure S9. LC/MS analysis (method 1) of OMT3 activity in the reaction cascade with CYP719A23. Figure S10. (A) CO-difference spectra of single expressed CYP71CU1∆20nat (orange line) and CYP71CU1∆20nat co-expressed with OMT3 (blue line). (B) 12.5% SDS-gel of CYP71CU1∆20nat (54 kDa) co-expression with OMT3 (41 kDa), and OMT1 (38 kDa) with 2-ODD (35 kDa). Figure S11. Schematic overview on the plasmid-based modular strategy developed to synthesize (−)−deoxypodophyllotoxin 6 from (−)−matairesinol 1 in E. coli via a one-cell 5-steps 6-enzyme reaction cascade. Figure S12. Hypothesized side reaction of 2-ODD occurring in the two-cell approach. Figure S13. Schematic overview on the plasmid-based modular strategy developed to synthesize (−)−epipodophyllotoxin 7 from (−)−matairesinol 1 in E. coli via a two-cell 6-steps 7-enzyme reaction cascade.