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Supplementary material for

Johan O. Westman, Jonas Nyman, Richard M.A. Manara,Valeria Mapelli and Carl Johan Franzén:

A novel chimaeric flocculation protein enhances flocculation in Saccharomyces cerevisiae

Metabolic Engineering Communications, https://doi.org/10.1016/j.meteno.2018.04.001

https://doi.org/10.1016/j.meteno.2018.04.001

Westman et al: A novel chimaeric flocculation protein enhances flocculation in Saccharomyces cerevisiae. Metabolic Engineering Communications, https://doi.org/10.1016/j.meteno.2018.04.001. Supplementary material.

Supplementary Figure S1.

A) Agarose gel with the PCR products of PCR reactions with genomic DNA of CCUG 53310 and S288c and primers specific for FLO1 in S288c. The strongest band in the lane with product using CCUG 53310 as template was the FLONF gene. The two weak bands of larger size are likely allelic variants of FLO1. B) Ploidy determination by flow cytometry. The three reference strains used were: haploid, S. cerevisiae BY4741; diploid, S. cerevisiae CEN.PK 122 MDS; and tetraploid, S. cerevisiae G26. The results indicated that S. cerevisiae CCUG 53310 is tetraploid.

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Supplementary Table 1.

Identity between sequences flanking FLONF and known flocculation genes. The reverse (RV) and forward (FW) primers give the sequence upstream and downstream of the gene, respectively.

Restriction enzyme and primer

Length

(bp)

FLO1

(%)

FLO5

(%)

FLO9

(%)

FLO10

(%)

FLO11

(%)

HindIII + IPCR-RV1 428 99.5 48.0 40.1 45.3 42.7

BstXI + IPCR-RV2 729 99.5 45.3 43.3 49.7 41.5

BstXI + IPCR-RV1 40 100 46.0 35.7 51.1 29.8

BstXI + IPCR-FW 725 95.4 99.6 91.4 43.4 42.4

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Supplementary Figure S2. The effect of Ca2+ on the flocculation behaviour of the recombinant strain carrying the FLONF gene. Average values of duplicate experiments with duplicate technical replicates, shown with ± one standard deviation, n = 2.

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Supplementary Figure S3. Alignment of Flo-family proteins. The alignment of the N-terminals of FloNFp, the major flocculation proteins and Lg-Flo1p was made with Muscle (Edgar, 2004). Initial sequence comparisons showed that the gene was considerably less similar to FLO10 and FLO11 than to the chosen genes. Therefore these two genes were not included in the analysis. The important carbohydrate binding loops (CBL) as well as the Flo5 subdomain and the outer loops, L1, L2 and L3 are highlighted. The figure was prepared using Jalview (Waterhouse et al., 2009).

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ST1 Alignment and phylogenetic analysis

Alignments and phylogenetic analysis of FLONF and related genes/proteins retrieved from the Saccharomyces Genome Database (Cherry et al., 2012) were made using MEGA6 (Tamura et al., 2013).

The alignment revealed differences between FloNFp and Flo1p, e.g. the replacement of a serine residue with an alanine residue in carbohydrate binding loop 2 of FloNFp as well as numerous changes in the L3 outer loop (Supplementary Fig. S3).

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Supplemental Figure S4. A) Homology modelled structure of the N-terminal part of FloNFp. The Flo5 subdomain, the outer loops flanking the carbohydrate binding site, L1 and L3, as well as CBL1 and CBL2 are highlighted. Parts of the protein are named as defined in (Sim et al., 2013; Veelders et al., 2010). B) Ramachandran plot of the predicted protein structure. The backbone torsion angle distribution suggests that the predicted structure for FloNFp is close to the native structure. C) RMSD analysis of molecular dynamics trajectory. The root mean square deviation of the backbone carbon atoms’ positions is plotted for each pair of frames in the molecular dynamics simulation. The predicted protein structure showed minimal motion throughout the simulation, demonstrating the stability of the predicted structure.

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ST2 Homology modelling

FLONF encodes for a protein homologous to Flo5p. The N-terminal amino acid-sequence, from residue 23 to 271, closely matches the sequence of the Flo5p 2XJP X-ray crystallography structure available in the Protein Data Bank (Veelders et al., 2010). Using the on-line service SWISS-MODEL (http://swissmodel.expasy.org), an automated homology modelling was performed to predict the structure of the N-terminal domain, including the sugar binding site, of FloNFp using the 2XJP structure as template (Arnold et al., 2006). The predicted structure of the protein (Supplementary Fig. S4A) was evaluated by the QMEAN Z and QMEANscore4 to estimate its quality (Benkert et al., 2011). The QMEAN4 score was 0.794 and the QMEAN Z-score was 0.093, indicating that the predicted structure is likely to be close to the native configuration (Benkert et al., 2011). Only residues Gln197 – Asp204 reached slightly elevated local QMEAN scores. The Ramachandran plot, obtained via MolProbity, also indicated that the homology modelling resulted in a plausible native structure (Supplementary Fig. S4B) (Chen et al., 2010). During molecular dynamics-simulation, the protein changed configuration to a minor extent (Cα root-mean-square deviation (RMSD) ≈ 0.2 nm) after 40 ns. By plotting the RMSD of the protein’s Cα positions vs. the original structure, the time-evolution of the configuration can be visualized (Supplementary Fig. S4C). The small RMSD is evidence supporting that the predicted structure is of good quality and that the 2XJP crystal structure is representative of the in vivo structure.

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ST3 Molecular dynamics and normal modes

To confirm that the predicted structure is stable under physiological conditions, i.e. in the native state, it was subjected to a long isothermal-isobaric (NPT)–ensemble molecular dynamics simulation. Using GROMACS (Hess et al., 2008) the protein was solvated in 1.0 M NaCl and TIP3P water in a periodic cubic box at 300 K. After equilibration, the simulation was run for 100 ns using the AMBER99 force field. Temperature and pressure were held constant by the Nosé-Hoover thermostat and the Parrinello-Rahman barostat. The Leap-frog algorithm was used for integrating the equations of motion with a time step of 2 fs. Electrostatic interactions were calculated using particle mesh Ewald (PME) with the short range cut off for van der Waals and electrostatics at a distance of 1.0 nm. The flexibility of the protein was studied in two ways; first by analysing the simulation trajectory utilizing standard tools in the GROMACS software package (Hess et al., 2008). Secondly, elastic network model normal mode analysis of the AMBER99 energy-minimized structure was performed (with and without the Ca2+ ion), through the ElNémo online service (http://www.igs.cnrs-mrs.fr/elnemo) (Suhre and Sanejouand, 2004). ElNémo uses a coarse-graining normal mode analysis to efficiently model larger proteins. The atom masses are normalized to 1 and a Hookean potential is introduced between atoms closer than 0.8 nm. The large scale motion of the system can be considered linear combinations of the vibrational modes. The lowest-frequency modes correspond to collective motions of large groups of atoms, usually whole structural domains (Krebs et al., 2002; Tama and Sanejouand, 2001).

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Supplementary Figure S5. Detail of G198E (corresponding to position 200 in FloNFp) mutated (A) and wild type (B) Flo5p structure 2XJP, showing the possible hydrogen bond between the glutamate in L3 and the hexose ligand C6-hydroxy group in FloNFp.

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Supplementary Figure S6. The sequence of the chimaeric flocculation gene with colour coded restriction sites that were used in the determination of the sequence. The underlined sequence originates from S. cerevisiae CCUG 53310, while the two other parts originate from the strongly flocculating FLO1 variant, created from the S. cerevisiae S288c FLO1 sequence (Westman et al., 2014). The first repeat of the gene is marked in bold.

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Supplementary Figure S7. A) The repeats identified by Pfam (numbered from the first repeat N-terminally) for FloNF, Flo1 strongly flocculating variant (SFV) and Flow were aligned by ClustalW and a rooted phylogenetic tree was built. The repeats were grouped based on their sequence similarity, as shown by the colored frames. B) Domain analysis by Pfam. The carbohydrate binding PA14 domains are shown in dark grey close to the N-terminus of the proteins. The flocculin repeats are color coded based on their origin, they are all classified as flocculin repeats by Pfam. Repeats from FloNF are dark grey squares, whereas those from Flo1 are white squares. Furthermore, the frame of the squares are colored as in the phylogenetic tree above. Close to the C-terminus there are three more repeats in all proteins, shown in light grey. These are classified as Flocculin type 3 repeats by Pfam. A block representing the C-terminal part of the proteins has been added (in black), this part is similar in all proteins. Flo1 (S288c) is shown as reference.

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References for supplemental texts

Arnold, K., Bordoli, L., Kopp, J., Schwede, T., 2006. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics. 22, 195-201.

Benkert, P., Biasini, M., Schwede, T., 2011. Toward the estimation of the absolute quality of individual protein structure models. Bioinformatics. 27, 343-350.

Cherry, J. M., Hong, E. L., Amundsen, C., Balakrishnan, R., Binkley, G., Chan, E. T., Christie, K. R., Costanzo, M. C., Dwight, S. S., Engel, S. R., Fisk, D. G., Hirschman, J. E., Hitz, B. C., Karra, K., Krieger, C. J., Miyasato, S. R., Nash, R. S., Park, J., Skrzypek, M. S., Simison, M., Weng, S., Wong, E. D., 2012. Saccharomyces Genome Database: the genomics resource of budding yeast. Nucleic Acids Res. 40, D700-5.

Edgar, R. C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792-1797.

Hess, B., Kutzner, C., van der Spoel, D., Lindahl, E., 2008. GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation. J. Chem. Theory. Comput. 4, 435-447.

Krebs, W. G., Alexandrov, V., Wilson, C. A., Echols, N., Yu, H., Gerstein, M., 2002. Normal mode analysis of macromolecular motions in a database framework: developing mode concentration as a useful classifying statistic. Proteins. 48, 682-95.

Sim, L., Groes, M., Olesen, K., Henriksen, A., 2013. Structural and biochemical characterization of the N-terminal domain of flocculin Lg-Flo1p from Saccharomyces pastorianus reveals a unique specificity for phosphorylated mannose. FEBS Journal. 280, 1073-1083.

Suhre, K., Sanejouand, Y. H., 2004. ElNemo: a normal mode web server for protein movement analysis and the generation of templates for molecular replacement. Nucleic Acids Res. 32, W610-4.

Tama, F., Sanejouand, Y.-H., 2001. Conformational change of proteins arising from normal mode calculations. Protein Eng. 14, 1-6.

Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 30.

Waterhouse, A. M., Procter, J. B., Martin, D. M. A., Clamp, M. l., Barton, G. J., 2009. Jalview Version 2-a multiple sequence alignment editor and analysis workbench. Bioinformatics. 25, 1189-1191.

Veelders, M., Brückner, S., Ott, D., Unverzagt, C., Mösch, H.-U., Essen, L.-O., 2010. Structural basis of flocculin-mediated social behavior in yeast. Proc. Natl. Acad. Sci. U. S. A. 107, 22511-22516.

Westman, J. O., Mapelli, V., Taherzadeh, M. J., Franzén, C. J., 2014. Flocculation causes inhibitor tolerance in Saccharomyces cerevisiae for second-generation bioethanol production. Appl. Environ. Microbiol. 80, 6908-6918.

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