Additional file 11 of Farnesyl pyrophosphate compartmentalization in the green microalga Chlamydomonas reinhardtii during heterologous (E)-α-bisabolene production
Creators: name: Wichmann, Julian
name: Eggert, Annibel
name: Elbourne, Liam D. H.
name: Paulsen, Ian T.
name: Lauersen, Kyle J.
name: Kruse, Olaf
Publisher: name: figshare
Year: 2022
Subjects: subject: Plant Biology
subject: FOS: Biological sciences

description: Additional file 11: Figure S1. Attempts to increase flux towards cytosolic FPP by CrFPPS accumulation. Different orientations with the mCerulean3 reporter and co-expression of AgBS and corresponding bisabolene yields (fg cell−1) are depicted (constructs indicated on the left). Error bars represent 95% confidence intervals. Figure S2. Detection of full-length protein products of 3 independent cell lines transformed with various constructs. Each cell line is denoted by individual numbers, constructs are specified at the top, and corresponding bisabolene yields are depicted in Fig. 2. Total cellular proteins were run on a 10% SDS PAGE gel and subjected to Western blotting with α-GFP antibody. M marker (PageRuler prestained protein ladder, ThermoFisher Scientific). WT parental strain UVM4. P positive control expressing AgBS-mVenus, AgBS-mCerulean3, and AgBS-mRuby2 to the cytosol. Positive control and ERG20 protein products show previously observed pattern of protein breakage in the reporter [7]. Predicted protein sizes are: ~ 124.0 kDa for AgBS-mVenus, ~ 70.2 kDa for ERG20-mCerulean3, and ~ 67.7 kDa for mCerulean3-ERG20 which lacks a StrepII tag on its C terminus. Figure S3. Detection of full-length protein products of 3 independent cell lines transformed with various constructs. Each cell line is denoted by individual numbers, constructs are specified at the top, and corresponding bisabolene yields are depicted in Fig. 3. Total cellular proteins were run on a 10% SDS PAGE gel and subjected to Western blotting with α-GFP antibody. M marker (PageRuler prestained protein plus ladder, ThermoFisher Scientific). WT parental strain UVM4. Y mVenus expressed to the cytosol. Predicted protein sizes are: ~ 124.0 kDa for AgBS-mVenus, ~ 161.9 kDa for AgBS-mVenus-ERG20. Ponceau S stain is shown as loading control on the nitrocellulose membrane. Signal for construct vi, strain #3 was below detection limit, but bisabolene could be detected in GC–MS measurements, indicating full-length expression. Figure S4. Detection of full-length protein products of 3 independent cell lines transformed with various constructs. Each cell line is denoted by individual numbers, constructs are specified at the top, and corresponding bisabolene yields are depicted in Fig. 4. Total cellular proteins were run on a 10% SDS PAGE gel and subjected to Western blotting with α-GFP and α-StrepII antibodies. M marker (PageRuler prestained protein ladder, ThermoFisher Scientific). WT parental strain UVM4. P positive control expressing AgBS-mVenus, AgBS-mCerulean3, and AgBS-mRuby2 to the cytosol. Positive control and ERG20 protein products show previously observed pattern of protein breakage in the reporter [7]. Predicted protein sizes are: ~ 124.0 kDa for AgBS-mVenus, ~ 123.1 kDa for AgBS-mCerulean3, ~ 120.9 kDa for AgBS-mRuby2, ~ 123.7 kDa for AgBS-aadA, ~ 67.7 kDa for mCerulean3-ERG20 which lacks a StrepII tag on its C terminus, ~ 161.9 kDa for AgBS-mVenus-ERG20, and ~ 162.2 kDa for AgBS-mCerulean3-ERG20. Figure S5. Detection of full-length protein products of 3 independent cell lines transformed with various constructs. Each cell line is denoted by individual numbers, constructs are specified at the top, and corresponding bisabolene yields are depicted in Fig. 3 and Additional file 11 Fig. S1. Total cellular proteins were run on a 10% SDS PAGE gel and subjected to Western blotting with α-GFP antibodies. M marker (PageRuler prestained protein ladder, ThermoFisher Scientific). WT parental strain UVM4. CrFPPS protein products show previously observed pattern of protein breakage in the reporter [7]. Predicted protein sizes are: ~ 160.9 kDa for AgBS-mVenus-CrFPPS, ~ 124.0 kDa for AgBS-mVenus, ~ 68.5 kDa for CrFPPS-mCerulean3, and ~ 67.0 kDa for mCerulean3-CrFPPS which lacks a StrepII tag on its C terminus. Figure S6. Calibration line and correspondent equation for bisabolene quantification. GC/MS peak areas were normalized to the internal standard peak area, and plotted against the bisabolene concentration in dodecane.