Research ArticleBIOENGINEERING

Unique genetic cassettes in a Thermoanaerobacterium contribute to simultaneous conversion of cellulose and monosugars into butanol

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Science Advances  23 Mar 2018:
Vol. 4, no. 3, e1701475
DOI: 10.1126/sciadv.1701475
  • Fig. 1 Fermentation of sugars by T. thermosaccharolyticum TG57.

    (A) Cellulose, (B) cellobiose, (C) glucose, and (D) xylan as the sole carbon source. Error bars represent SD of biological triplicates.

  • Fig. 2 Strain TG57 exhibits resistance to product inhibition.

    (A) Cellulose conversion in the presence of a typical co-substrate glucose or cellobiose. Substrate concentration: control, cellulose (10 g/liter); co-substrates, cellulose (10 g/liter) + glucose (30 g/liter) or cellobiose (30 g/liter). (B) Transcriptional expression of genes responsible for the catabolite control protein and the cellulase secretory system of strain TG57. ccp encodes the catabolite control protein, sec encodes general secretion pathway protein A, t2ssE and t2ssF encode type II secretion system proteins E and F, and pilT encodes the Tfp pilus assembly protein.

  • Fig. 3 Co-fermentation and resistance to CCR.

    (A) Co-fermentation of a sugar mixture containing xylose (18.7 g/liter) and cellobiose (18.3 g/liter). (B) Co-fermentation of a sugar mixture containing glucose (19.2 g/liter), xylose (8.8 g/liter), and arabinose (9.3 g/liter). Acetate and butyrate data are shown in the Supplementary Materials (see fig. S4). (C) Transcriptional levels of the ccp gene (encoding the catabolite control protein) and the xylR gene (encoding the transcriptional repressor of the xylose operon). (D) Comparison of transcriptional levels of genes related to the catabolism of glucose and xylose in strain TG57 at the early exponential phase (12 hours of fermentation) and the late exponential phase (36 hours of fermentation). Functional genes gptA, gptB, gluT1, gkA, gkB, g6pd, gpi, xylT1, xylT2, xylm, xylk1, and xylk2 encode the following: glucose-specific phosphotransferase enzyme IIA components, PTS system glucose transporter subunit IIB, a sugar ABC transporter permease, the glucokinase ROK family, glucose-6P dehydrogenase, a glucose-6-phosphate isomerase, a xylose transporter, a xylose isomerase, and xylulokinase. Error bars indicate the SDs calculated from triplicates.

  • Fig. 4 Genomic and transcriptomic analysis of T. thermosaccharolyticum strain TG57.

    (A) Genome overview of strain TG57 with previously genome-sequenced isolates [C. acetobutylicum ATCC 824 (accession no. AE001437), C. beijerinckii NCIMB 8052 (accession no. CP000721), Clostridium sp. MF28 (accession no. CP014331), and T. thermosaccharolyticum DSM 571 (accession no. CP002171) ] deposited in the GenBank database. (B) Circos plot represents similarity between strain TG57 and C. acetobutylicum ATCC 824 based on gene sequence and biological annotation. Track (green) on the left, strain TG57; track (black) on the right, strain ATCC 824. Number represents CDS. The links are color-coded by similarity. Red, 100% similarity; blue, 0% similarity. (C) Transcriptomic analysis of strain TG57. Tracks from the inside to outside: tracks 1 and 2, the transcription levels of all genes in culture on xylan and cellulose; track 3, cellulosome genes (red, forward strand; blue, reverse strand); track 4, genes for carbohydrates in glycoside hydrolase family (green, forward strand; orange, reverse strand); track 5, CDS (red, forward strand; blue, reverse strand); track 6, genome sequence of T. thermosaccharolyticum TG57. (D) Butanol synthesis and NADH (reduced form of nicotinamide adenine dinucleotide)/NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) regenerations in T. thermosaccharolyticum TG57.

  • Fig. 5 Distinct central metabolic pathways and relative abundances of transcripts in cellulose and xylan cultures.

    Solid arrows indicate genes for the reaction. Gray dotted arrows and gray letters indicate genes for the inactive reaction. Red dotted arrows and red letters indicate the absence of genes and reactions in the metabolic pathway.

  • Fig. 6 The secretome and transportome features in T. thermosaccharolyticum strain TG57.

    (A) Relative abundances of transcripts of the secretory system, transport and efflux, exopolysaccharide, and carbohydrate-binding protein for biofilm formation. (B) Consensus sequences of signal peptide motifs for the secretory system. (C) Secreted exopolysaccharide, protein, and extracellular polymeric substance (EPS) contents in culture TG57. (D) qPCR analysis of carbohydrate-binding modules and exopolysaccharide biosynthesis proteins in culture TG57. (E) Morphology of a fluorescence micrograph showing the adhesion of cells to a crystalline cellulose particle (scale bar, 20 μm).

  • Table 1 Performance comparison of strain TG57 with previous microorganisms capable of converting lignocellulose to biofuels.

    Avicel, crystalline cellulose; PASC, phosphoric acid–swollen cellulose.

    OrganismGenotypeCarbon sourceProductConcentration
    (g/liter)
    Yield
    (g/g)
    Productivity
    [g/(liter·hour)]
    Reference
    Caldicellulosiruptor besciiRecombinantSwitchgrassEthanol0.590.02940.0049(15)
    C. thermocellumRecombinantCrystalline celluloseEthanol22.40.390.162(16)
    C. cellulolyticumRecombinantAvicelIsobutanol0.660.066(17)
    C. thermocellumRecombinantAvicelIsobutanol5.40.170.072(18)
    C. acetobutylicumRecombinantAvicelButanolNilNilNil(19)
    C. cellulovoransRecombinantCrystalline celluloseButanol1.420.20.0059(23)
    Ethanol1.600.190.0067
    C. phytofermentans ATCC 700394Wild typeBirchwood xylanEthanol0.460.1530.0192(27)
    CelluloseEthanol2.760.4380.0041
    Saccharomyces cerevisiaeRecombinantPASCEthanol2.10.5250.035(28)
    C. cellulolyticumRecombinantCrystalline celluloseEthanol2.70.270.008(29)
    C. thermocellumRecombinantAvicelEthanol5.460.14(30)
    Cellvibrio japonicusRecombinantAvicelEthanol2.60.260.052(31)
    S. cerevisiaeRecombinantPASCEthanol1.00.3720.0052(32)
    C. phytofermentans ATCC 700394Wild typeAFEX-pretreated corn stoverEthanol7.00.1910.0265(33)
    S. cerevisiaeRecombinantPASCEthanol2.70.27(34)
    C. cellulolyticumRecombinantAvicelEthanol0.80.025(35)
    Clostridium sp. strain MF28Wild typeBeechwood xylanButanol3.20.1770.0222(41)
    T. thermosaccharolyticum TG57Wild typeCrystalline celluloseButanol1.930.2010.0045This study
    Beechwood xylanButanol3.630.2290.0189

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/3/e1701475/DC1

    section S1. Taxonomic identification and phylogenetic analysis of strain TG57

    section S2. Confirmation of culture purity of T. thermosaccharolyticum strain TG57

    section S3. Carbohydratases

    fig. S1. Neighbor-joining tree of the housekeeping gene sequences after global alignment.

    fig. S2. Relative growth of culture TG57 when fed on cellulose at various temperatures and pHs.

    fig. S3. Transcription levels of genes for polysaccharide and other carbon utilization in T. thermosaccharolyticum TG57.

    fig. S4. Bioacid (acetic acid and butyric acid) production in the co-fermentation broth by T. thermosaccharolyticum TG57.

    fig. S5. Comparative genomic characterization of T. thermosaccharolyticum TG57 with previously sequenced isolates.

    table S1. Specific activities of functional enzymes involved in T. thermosaccharolyticum TG57.

    table S2. Carbon balance of fermenting different carbohydrates by T. thermosaccharolyticum TG57.

    table S3. Comparison of glycoside hydrolases found in T. thermosaccharolyticum TG57 with T. thermosaccharolyticum DSM 571.

    data set S1. Genes in strain TG57 were present in isoforms.

    data set S2. Gene-specific primers used for RT-qPCR.

  • Supplementary Materials

    This PDF file includes:

    • section S1. Taxonomic identification and phylogenetic analysis of strain TG57
    • section S2. Confirmation of culture purity of T. thermosaccharolyticum strain TG57
    • section S3. Carbohydratases
    • fig. S1. Neighbor-joining tree of the housekeeping gene sequences after global alignment.
    • fig. S2. Relative growth of culture TG57 when fed on cellulose at various temperatures and pHs.
    • fig. S3. Transcription levels of genes for polysaccharide and other carbon utilization in T. thermosaccharolyticum TG57.
    • fig. S4. Bioacid (acetic acid and butyric acid) production in the co-fermentation broth by T. thermosaccharolyticum TG57.
    • fig. S5. Comparative genomic characterization of T. thermosaccharolyticum TG57 with previously sequenced isolates.
    • table S1. Specific activities of functional enzymes involved in T. thermosaccharolyticum TG57.
    • table S2. Carbon balance of fermenting different carbohydrates by T. thermosaccharolyticum TG57.
    • table S3. Comparison of glycoside hydrolases found in T. thermosaccharolyticum TG57 with T. thermosaccharolyticum DSM 571.
    • Legends for data sets S1 and S2

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    Other Supplementary Material for this manuscript includes the following:

    • data set S1 (Microsoft Excel format). Genes in strain TG57 were present in isoforms.
    • data set S2 (Microsoft Excel format). Gene-specific primers used for RT-qPCR.

    Files in this Data Supplement:

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