Additional file 1 of Engineering cascade biocatalysis in whole cells for bottom-up synthesis of cello-oligosaccharides: flux control over three enzymatic steps enables soluble production
Creators: name: Schwaiger, Katharina N.
name: Voit, Alena
name: Wiltschi, Birgit
name: Nidetzky, Bernd
Publisher: name: figshare
Year: 2022
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Pharmacology
subject: Biotechnology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: Developmental Biology
subject: Inorganic Chemistry
subject: Computational Biology

description: Additional file 1: Table S1. Parts used for the construction of the co-expression plasmids. Table S2. Enzymatic synthesis activities corresponding to 2.6 gCDW/L reaction mixtures used in whole-cell COS productions with single-plasmid catalysts (Figure S4). Activities were measured in corresponding cell-free extracts (see the "Methods" section in "Expression analysis and enzyme activity assays" sub section). Table S3. COS synthesis with different pPOLY_2 whole-cell catalyst preparations. Table S4. Enzyme activities measured in the supernatant of the different pPOLY_2 catalyst preparations; cell suspensions had a concentration of 25 mgCDW/mL. A cell-free extract preparation was measured as the reference for 100% activity. Table S5. Primers used to construct the co-expression plasmids containing CcCdP. Table S6. Specific activities of purified CcCdP on cellobiose and pNP-G2 at two temperatures. Figure S1. SDS PAGE gel (top) shows soluble protein of co-expression catalysts. Two-plasmid approach (p15A + pBICI med ori) strongly expressed CcCdP but not CuCbP nor BaScP. After purification and restriction analysis of both plasmids, agarose gel electrophoresis (bottom) revealed a reduced size of pBICI med ori (~3000 bp instead of 7379 bp). Sample 1 is the overnight preculture (not induced), samples 2 and 3 are duplicates of induced expression cultures. Figure S2. SDS-PAGE analysis of expression optimization. (A) Tested parameters were expression durations (after 4 hours and 18 hours) and IPTG concentrations (0.1 mM and 1 mM) at 25 °C expression temperature. (B) pPOLY_2 expression was further analysed at two different temperatures (a, b) and four different IPTG concentrations (1-4). The cyan arrow marks 6xHis-CcCdP (112.8 kDa); dark green arrow, 6xHis-CuCbP (92.7 kDa); light green arrow, Strep II-BaScP (57.7 kDa). NI, non-induced sample. Figure S3. Plasmid maps of one-plasmid approaches (A-C) and two-plasmid approaches (D-F). The plasmid maps and full sequences are stored in the add gene database, accessible with the numbers 179272 to 179276 ( https://www.addgene.org ). Figure S4. Conversions of one-plasmid cell catalysts: (A) pDUBI, (B) pPOLY_1, (C) pPOLY_2. Figure S5. Temperature dependence of the COS composition. Comparison of COS species (DP 2-6) after 8 hours reaction at 30 °C (first bar; see also Figure 4, pPOLY_2) and after 1.5 h and 2 h reaction at 45 °C (second and third bar). Reactions were performed with freeze-thaw treated whole cell catalysts carrying the plasmid pPOLY_2. Figure S6. HPLC chromatograms of product solutions. Mono- and disaccharides were measured with an YMC-Pack Polyamine II/S-5 µm/12 nm column (A) and oligosaccharides (cellodextrins DP 3 to DP 6) were measured with a Luna 5 µm NH2 column (B).