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  • Molecular Systems Biology: 7 (1)

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Article

Proteome‐wide systems analysis of a cellulosic biofuel‐producing microbe

Andrew C Tolonen, Wilhelm Haas, Amanda C Chilaka, John Aach, Steven P Gygi, George M Church
DOI 10.1038/msb.2010.116 | Published online 18.01.2011
Molecular Systems Biology (2011) 7, 461
Andrew C Tolonen
Department of Genetics, Harvard Medical School, Boston, MA, USA
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Wilhelm Haas
Department of Cell Biology, Harvard Medical School, Boston, MA, USA
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Amanda C Chilaka
Department of Biology, Northeastern University, Boston, MA, USA
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John Aach
Department of Genetics, Harvard Medical School, Boston, MA, USA
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Steven P Gygi
Department of Cell Biology, Harvard Medical School, Boston, MA, USA
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George M Church
Department of Genetics, Harvard Medical School, Boston, MA, USA
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Author Affiliations

  1. Andrew C Tolonen*,1,
  2. Wilhelm Haas*,2,
  3. Amanda C Chilaka3,
  4. John Aach1,
  5. Steven P Gygi2 and
  6. George M Church1
  1. 1 Department of Genetics, Harvard Medical School, Boston, MA, USA
  2. 2 Department of Cell Biology, Harvard Medical School, Boston, MA, USA
  3. 3 Department of Biology, Northeastern University, Boston, MA, USA
  1. ↵*Corresponding authors. Department of Genetics, Harvard Medical School, NRB 238, 77 Avenue Louis Pasteur, Boston, MA 02115, USA. Tel.: +1 617 432 6510; Fax: +1 617 432 6510; E-mail: tolonen{at}alum.mit.eduDepartment of Cell Biology, Harvard Medical School, Boston, MA 02115, USA. Tel.: +1 617 432 3155; Fax: +1 617 432 1144; E-mail: wilhelm_haas{at}hms.harvard.edu
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  • Figure 1.
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    Figure 1.

    Integrated systems biology strategy to study cellulosic bioconversion. Cultures metabolizing different biomass substrates were examined for (A) growth and biomass consumption rates (Figure 2A–C), (B) fermentation production rates and yields (Figure 2D–F), and (C) ability of the microbe to adhere to cellulosic substrates (Figure 2G–I). (D) Supernatant and cellular protein samples were taken for reductive dimethylation (ReDi) proteomics and analyzed for enzyme secretion (Figure 4), abundances of cellulolytic enzymes (Figure 5), and proteome‐wide changes (Figure 6). (E) These data were integrated to identify key enzymes for each step in biomass deconstruction and fermentation (Table I, Figure 7).

  • Figure 2.
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    Figure 2.

    Growth (A–C), fermentation (D–F), and cell morphology (G–I) of C. phytofermentans on different carbon sources. Data points are means of triplicate cultures; error bars show one s.d. and are smaller than the symbols where not apparent. Gray bars show when samples were taken for mass spectrometry. Growth on glucose (A) and hemicellulose (B) was quantified as OD600. Growth on cellulose (C) was measured as dry mass of cellulose in culture. Production of ethanol and acetate, the two most abundant fermentation products, and glucose consumption in the glucose treatment was measured by HPLC. Dotted lines show maximum theoretical yield of ethanol. Scanning electron microscopy shows cells growing on glucose (G), hemicellulose (H), and cellulose (I). White scale bar is 1 μm.

  • Figure 3.
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    Figure 3.

    Protein identification (A–C) and quantification (D–F) by mass spectrometry. (A) Venn diagram of proteins identified in each treatment. Protein subsets in the hemicellulose and cellulose culture supernatants are shown with dashed ellipses. In total, 2567 of 3926 (65%) putative proteins were detected. (B) The 65% overall protein identification rate is conserved across Clusters of Orthologous Genes (COG) functional categories. (C) The percent of the proteome shown as summed Absolute Protein EXpression (APEX) values in each COG category for cells growing on glucose and cellulose. (D) Relative protein expression in different cultures quantified by ReDi labeling. The fraction of proteins expressed within twofold levels for the glucose treatment compared with a duplicate glucose culture (94%), hemicellulose (80%), and cellulose (49%) cultures. (E) Fold change in protein expression (MS1 peak area ratio, MPA ratio) for cellulose versus glucose duplicate cultures is highly correlated (r2=0.82). (F) Scatter plot of mRNA versus protein expression of 40 carbohydrate‐active enzymes on cellulose (orange circles, r2=0.77) and hemicellulose (turquoise triangles, r2=0.71) versus glucose. The mRNA fold change was measured by qRT–PCR (−ΔΔCt).

  • Figure 4.
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    Figure 4.

    The C. phytofermentans secretome. (A) Proteins with high‐scoring N‐terminal signal peptides have a greater probability of being in culture supernatants. Fraction of proteins in proteome at each SignalP‐NN D value observed in the supernatants of hemicellulose or cellulose cultures. Data were fit to a piecewise linear regression with the leftmost regression to a horizontal line. Consensus sequences of (B) type I and (C) type II lipoprotein N‐terminal signal peptides for proteins found in the supernatant of cellulose cultures. (D) Functional categories of proteins in culture supernatants: rust, flagellum; turquoise, cell wall/surface; gray, proteases; orange, transport; green, CAZy; purple, other; gold, unknown. (E) Transmission electron micrograph of a C. phytofermentans cell cross section showing the cell membrane, cell wall, and surface layer.

  • Figure 5.
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    Figure 5.

    Carbohydrate‐active enzyme (CAZy) expression and activities in glucose, hemicellulose, and cellulose cultures. (A) Secreted and cellular cellulolytic enzyme activities. Protein lysates from cultures grown on glucose (purple), hemicellulose (turquoise), or cellulose (orange) prepared from the cellular fraction (C), supernatant (S), or whole‐culture lystates (L). Proteins were incubated with hemicellulose (hemicellulase assay) or carboxymethylcellulose substrate (cellulase assay), reducing sugars were assayed using dinitrosalicyclic acid, and were normalized to protein concentration. (B) CAZy expression changes (MS1 peak area ratio, MPA ratio) on hemicellulose and cellulose versus glucose showing differentially expressed proteins (P<0.01) on hemicellulose (turquoise), cellulose (orange), or both (green). Symbols show cellular proteins (circles) and supernatant proteins (triangles). (C–E) Shifts in the relative abundances of CAZy proteins in glucose (C) hemicellulose (D), and cellulose (E) treatments by Absolute Protein EXpression (APEX) show acclimation to different carbon sources. Fraction of proteome comprised of CAZy proteins in each treatment is shown.

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    Figure 6.

    Proteome‐wide expression changes on cellulose versus glucose visualized as a Cytoscape interaction network (Shannon et al, 2003). Nodes are proteins (circles) or KEGG/carbohydrate‐active enzyme (CAZy) categories (yellow diamonds); edges are protein interactions defined by KEGG or CAZy databases. Protein node sizes show expression on cellulose as log2 (Absolute Protein EXpression, APEX). Node colors are expression changes as cellulose/glucose log2 protein ratios (MS1 peak area ratio, MPA ratio). Proteins less than twofold changed are white, higher on cellulose are graded orange, and higher on glucose are graded purple (see legend).

  • Figure 7.
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    Figure 7.

    Model of the key secreted and intracellular proteins for the degradation and fermentation of plant biomass. Protein ID numbers are colored by highest Absolute Protein EXpression (APEX) expression on glucose (purple), hemicellulose (turquoise), cellulose (orange). Number in parentheses show the number of proteins of related function.

Tables

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  • Table 1. Highly expressed enzymes for each step in the degradation of hemicellulose and cellulose
    ProteinFunctionCAZyHemicelluloseCellulosePsortSignalp‐NN
    Cphy2108Cell surface 1,4‐β‐d‐xylanaseGH10, CBM225737.9(S)563.8(S)Cell wall0.9
    Cphy21051,4‐β‐d‐XylanaseGH113120.7(S)6007.9Extracellular0.97
    Cphy15101,4‐β‐d‐XylanaseGH102074.5(S)29559.8(S)Extracellular0.9
    Cphy06241,4‐β‐d‐XylanaseGH10, CBM221055.6(S)4767.9(S)Extracellular0.65
    Cphy30101,4‐β‐d‐XylanaseGH10933.10.0Extracellular—
    Xylosaccharides to xylose
    Cphy3009Non‐reducing end 1,4‐β‐xylosidaseGH35982.0542.6Cytoplasmic—
    Cphy3207Reducing end 1,4‐β‐xylosidaseGH82473.20.0Unknown—
    Remove hemicellulose side groups
    Cphy3158α‐1,2‐GlucuronosidaseGH673508.982.4Cytoplasmic—
    Cphy3160β‐Glucuronidase, galactosidaseGH2813.31701.1Cytoplasmic—
    Cphy2632ArabinaseGH43703.40.0Cytoplasmic—
    Cphy2848α‐Glucuronidase, galactosidaseGH4701.1199.9(S)Cytoplasmic—
    Cphy3862Xylanase, carboxylesteraseGH10, CE15466.8(S)1511.7(S)Extracellular0.9
    Cphy2730Acetyl xylan esteraseCE4155.4225.6Cytoplasmic—
    Cellulose
    Hydrolysis of cellulose to cellodextrins
    Cphy3368ExocellulaseGH48, CBM31911.7(S)9730.3(S)Extracellular0.65
    Cphy3367Bifunctional endo, exocellulaseGH9, CBM32181.4(S)9277.7(S)Extracellular0.87
    Cphy3202CellulaseGH5, CBM2, CBM461550.3(S)3485.3(S)Unknown0.81
    Cphy2058CellulaseGH5424.5(S)1199.0(S)Unknown0.62
    Cphy1163CellulaseGH5136.9(S)525.1Unknown0.82
    Hydrolysis of cellodextrins
    Cphy0220Non‐reducing end β‐glucosidaseGH3688.93469.6Cytoplasmic—
    Cphy1169Endo‐1,4‐β‐d‐glucanaseGH51112.0615.3Cytoplasmic—
    Phosphorolytic cleavage of cellodextrins to glucose‐1‐phosphate
    Cphy3854Cellodextrin, cellobiose phosphorylaseGH94136.47602.4Membrane—
    Cphy0430Cellodextrin, cellobiose phosphorylaseGH94166.24421.4Membrane—
    Cphy1929Cellodextrin, cellobiose phosphorylaseGH94133.62743.0Membrane—
    Other highly expressed CAZy
    Cphy1799ChitinaseGH18, CBM12175.3(S)43042.0Unknown0.77
    Cphy1800ChitinaseGH18, CBM12101.9(S)31498.1(S)Extracellular0.8
    Cphy0218α‐GlucosidaseGH31184.53366.0Cytoplasmic—
    Cphy1687Polysaccharide deacetylaseCE4, CBM360.02551.3(S)Extracellular0.858
    Cphy1888Pectin lyasePL90.02334.6Extracellular0.72
    Cphy1071β‐MannanaseGH26, CBM3, CBM35764.5(S)1664.3(S)Extracellular0.68
    Cphy1652ChitinaseGH18, CBM500.01329.0(S)Cytoplasmic—
    Cphy2128β‐MannanaseGH26, CBM3, CBM35153.7(S)956.0(S)Extracellular0.78
    Cphy1943ChitinaseGH19625.6(S)875.4Unknown0.77
    • Protein ID, putative function, CAZy category, APEX expression, localization (PsortB v2.0), and significant signal peptide predictions (SignalP3.0‐NN D‐value>0.45) are shown. APEX values followed by (S) are supernatant proteins.

    • Hemicellulose

    • Xylan backbone to xylosaccharides

Supplementary Materials

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  • Supplementary Information

    Supplementary information, Supplementary figures S1–15, Supplementary tables SI–VIII [msb2010116-sup-0001.zip]

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Volume 7, Issue 1
01 January 2011
Molecular Systems Biology: 7 (1)
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