Additional file 1 of Biosensor-driven, model-based optimization of the orthogonally expressed naringenin biosynthesis pathway
Creators: name: Van Brempt, Maarten
name: Peeters, Andries Ivo
name: Duchi, Dries
name: De Wannemaeker, Lien
name: Maertens, Jo
name: De Paepe, Brecht
name: De Mey, Marjan
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Genetics
subject: Molecular Biology
subject: Immunology
subject: FOS: Clinical medicine
subject: 69999 Biological Sciences not elsewhere classified
subject: 80699 Information Systems not elsewhere classified
subject: FOS: Computer and information sciences
subject: Cancer
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: Plant Biology
subject: Computational Biology

description: Additional file 1: Table S1. Pathway architecture of the strains selected through the biosensor-driven combinatorial engineering process. The naringenin titer determined by UPLC analysis and the biosensor output is also given for each strain (n = 1). (TAL: Tyrosine ammonia-lyase; 4CL: 4-coumaroyl-CoA ligase; CHS: Chalcone synthase; CHI: Chalcone isomerase; Nar.: naringenin; Fluo: fluorescence; a.u.: arbitrary units CDS: coding DNA sequence; NA: not applicable, failed sequencing). Table S2. NUCLEOTIDE SEQUENCE OF THE CODON OPTIMIZED FJTAL, NEWLY SYNTHESIZED FOR THIS STUDY. Table S3. output of the artificial neural network (ANN) ensemble in the format described by Zhou et al. [30]. Pathway configurations are included in the Top x list if, after 1000 ANN train and predict iterations, the frequency of their occurrence in the predicted top x strains is higher than half the frequency of the most occurring strain in that top list ( f(top x) &gt; 0.5*fmax(top x) ). Identical colors are used to indicate identical strains. (P_X: promoter variant of enzymatic step X; CDS_X: enzyme variant; freq: frequency (f)). Figure. S1 Characteristics of the naringenin-responsive biosensor (pSynSens1.100 [36]) in the conditions used in this study. (A) The responsive curve and fitted Hill function for a supplied naringenin concentration range of 0-100 mg/L and the corresponding Hill parameters. Also the operational range and Noise parameter are given, as determined with the method described by De Paepe et al. (2018) [36] and depicted in (B). (a: the basal normalized fluorescent signal (a.u., arbitrary units); M: the maximum normalized fluorescent signal (a.u.); n: Hill coefficient (cooperativity); K: Hill constant (transcription factor – ligand affinity, mg/L); error bars: standard errors for 5 biological replicates, n = 5). Figure. S2 Linlog transformation of the sigma B promoter library promoters as input for the created models. (A) Original data, displayed as sfGFP corrected mKate values [8]. (B) Linlog transformed promoters. (C) Used linlog transformation and properties of the transformed data. (TIF: transcription initiation frequency; P = untransformed promoter TIF, X = linlog transformed promoter TIF). Figure. S3 Correlation (Pearson, ρ) between the continuous variables (promoter transcription initiation frequency and titer) in the dataset, shown as the generated output of the corr.test() function of the R psych package [71]. The top matrix shows the correlation between the variables with -1 and 1 indicating a perfect (inverse) correlation and 0, no correlation. The bottom matrix shows the corresponding probability values (Null hypothesis = ‘H0: 2 variables are not correlated (ρ = 0)’). The Holm-Bonferroni method is used to adjust for multiple testing. (Rg: Rhodotorula glutinis; Fj: Flavobacterium johnsoniae; Pc: Petroselinum crispum; At: Arabidopsis thaliana; Ph: Petunia hybrida; Gh: Gerbera hybrida; Ms: Medicago sativa; TAL: Tyrosine ammonia-lyase; 4CL: 4-coumaroyl-CoA ligase; CHS: Chalcone synthase; CHI: Chalcone isomerase). Figure. S4 Ordinary least squares regression output from the lm() function in R. The input formula is obtained by a limitted sequential removal of terms holding the highest p-value, starting from the full quadratic regression model. (Q: quadrant; P_X: promoter transcription initiation frequency for expression of enzyme X; CDS_X: coding sequence variant of enzyme X; P_X:P_X: interaction term; I(P_X^2): quadratic term; Rg: Rhodotorula glutinis; Fj: Flavobacterium johnsoniae; Pc: Petroselinum crispum; At: Arabidopsis thaliana; Ph: Petunia hybrida; Gh: Gerbera hybrida; Ms: Medicago sativa; TAL: Tyrosine ammonia-lyase; 4CL: 4-coumaroyl-CoA ligase; CHS: Chalcone synthase; CHI: Chalcone isomerase). Figure. S5 Cross-validated (CV) root mean squared error of prediction (RMSEP) curve. A model only including the first two components (i.e. latent variables, LV) shows the lowest prediction error. A model with two LV predicts 78.92% of the product titer by using 38.82% of the predictors’ variance. (adjCV: adjusted CV). Figure. S6 Biplot of the first two components of the partial least squares (PLS) regression model. (P_X: promoter transcription initiation frequency (TIF) of enzymatic step X; CDS_X: enzyme variant; I(P_X^2): quadratic term of promoter transcription initiation frequency; ‘P_X:CDS_X’ and ‘P_X: P_Y’: promoter TIF interaction terms with enzyme variants or between the promoter TIFs of two different enzymatic pathway reaction steps; TAL: Tyrosine ammonia-lyase; 4CL: 4-coumaroyl-CoA ligase; CHS: Chalcone synthase; CHI: Chalcone isomerase). Figure. S7 The estimated partial least squares (PLS) regression coefficients of all pathway features, including quadratic and interaction terms. (P_X: promoter transcription initiation frequency (TIF) of enzymatic step X; CDS_X: enzyme variant; I(P_X^2): quadratic term of promoter TIF; ‘P_X:CDS_X’ and ‘P_X: P_Y’: promoter TIF interaction terms with enzyme variants or between the promoter TIFs of two different enzymatic pathway reaction steps; TAL: Tyrosine ammonia-lyase; 4CL: 4-coumaroyl-CoA ligase; CHS: Chalcone synthase; CHI: Chalcone isomerase). Figure. S8: Cross sections of the multidimensional production landscape, predicted by the partial least squares (PLS) model. For each cross section, two pathway features (promoter transcription initiation frequencies, TIF) are varied while the remaining part of the pathway configuration is fixed. The fixed input values, other than the two variables depicted on the x- and y-axes, are set according to the predicted optimal producer (see legend, P_X: promoter TIF of enzymatic step X; CDS_X: enzyme variant; TAL: Tyrosine ammonia-lyase; 4CL: 4-coumaroyl-CoA ligase; CHS: Chalcone synthase; CHI: Chalcone isomerase). Figure. S9 The UPLC-UV chromatogram of one of the ethyl acetate-extracted biological replications of NarRef, Top10.4, Top10.5 and Top10.4 grown on the glycerol supplied medium. The Top10.5 + glycerol profile is similar to the Top10.4 + glycerol profile and the depicted profiles are also representative for the other biological replications, but are left out for visual clarity. As a reference, also the chromatographic profile of the strain bearing only heterologous sigma factor (σ) B in the genome but no plasmid is included. Figure. S10 Batch fermentation with production strain Top10.4. In the upper part the production profiles of cell dry weight (CDW), naringenin and p-coumaric acid, and the substrate usage of glycerol are given. In the lower part, the process parameter profiles for base and acid addition and dissolved oxygen (PO2) are given, together with events of process parameter(-change) indications for airflow, stirrer speed, temperature and antifoam addition. Stirrer speed spikes were used to break accumulated foam. Figure. S11 (A) Plating of the first sample taken of the batch fermentation with production strain Top10.4, compared to an empty LB-agar plate. The yellow coloration is found to indicate product formation, most likely coming from intermediate metabolite naringenin chalcone. (B) Gram-stained sample of the performed batch fermentation with strain Top10.4. Filamentation of the production organism is an indication for stress [52]. Figure. S12 Annotated genbank file of the optimized naringenin biosynthesis pathway (pTop10.4, Figure 6). The expression vector originates from Coussement et al. (2017) [34] (Table 1). The promoters driving the pathway are created in Bervoets et al. (2018) [8]. More information about the enzymes and source of CDSs can be found in Table S2. The used transcription terminators are from the BIOFAB collection [72].