Expression of a Bacterial 3-dehydroshikimate dehydratase (QsuB) Reduces Lignin Content and Improves Biomass Saccharification Efficiency

Outcomes• Arabidopsis plants expressing QsuB show drastic lignin reductions (~50%), enrichment of H-units, and reduced lignin DP. • The biomass from the engineered Arabidopsis lines display a two-fold increase of saccharification efficiency.

Eudes, A., Noppadon, S., Baidoo, E., George, A., Liang, Y., Yang, F., Singh, S., Keasling, J., Simmons, B., Loque, D. (2014). Expression of a bacterial 3‐dehydroshikimate dehydratase reduces lignin content and improves biomass saccharificationefficiency. Plant Biotechnology Journal.

Background• Lignin is a polymer that

confers recalcitrance to plant biomass.

• Novel approaches to reduce lignin in bioenergy crops without affecting biomass yield are desired for economical production of second-generation biofuels

Approach• Identify enzymes whose

expression in Arabidopsis leads to lignin reduction. Such enzymes will be expressed in a tissue-specific manner in bioenergy crops to avoid the adverse effects of lignin reduction.

Significance• Expression of 3-dehydroshikimate dehydratase is a novel strategy to reduce lignin.• Crops expressing QsuB under the control of tissue-specific promoters are being developed.

3-dehydroshikimate

shikimate

phenylalanine

p-coumaroyl-CoA

p-coumaroyl-shikimate

feruloyl-CoA

p-coumaryl alcohol

coniferyl alcohol

sinapyl alcohol

H-unit

G-unit

HCT shikimate

PCA QsuB

phenylalanine

coniferaldehyde

sinapaldehydeS-unit

phosphoenol pyruvate+

p-coumaraldehyde

erythrose-4-phosphate

1) The lignin pathway and expression of  QsuBPCA, protocatechuate

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WT

qsuB

‐1qsuB

‐3qsuB

‐6qsuB

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3) Lignin content, composition (2D‐NMR), and structure (SEC) in qsuB transgenic lines

qsuB-1 WT qsuB-3 qsuB-6 qsuB-7

2) QsuB plants do not show drastic biomass reduction

Lignin (%

 Cell w

all)

H: 3.8% S: 20.0%G: 76.2%

H: 27.2% S: 30.2%G: 42.6%

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450 WT qsuB‐1qsuB‐3 qsuB‐6qsuB‐7

4) Biomass saccharification

Sugars  (µg

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ass)

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5 10 15 20 25Elution time (min)

Normalized

 intensity

m > 22 kDa 22 kDa > m > 0.74 kDa

WT

qsuB-1

m < 0.74 kDa

OsSERK1 Regulates Rice Development but not Immunity to Rice Bacterial Blight or Fungal Blast

Outcomes• OsSERK1 silencing results in a reduced angle of the lamina

joint, but does not affect immunity.• OsSERK1 overexpression complements brassinosteroid (BR)

signaling defects of OsSERK2-silencing plants.

Zuo, et al.,OsSERK1 regulates rice development but not immunity to Xanthomonas oryzae pv. oryzae or Magnaporthe oryzae. JIPB 56:1179-1192 (2014). DOI: 10.1111/jipb.12290

Background• Somatic embryogenesis receptor

kinase (SERK) proteins play pivotal roles in regulation of plant development and immunity.

• The rice genome contains two SERKgenes, OsSERK1 and OsSERK2. We previously demonstrated that OsSERK2 is required for rice immunity and for normal development. Here we report the function of OsSERK1.

Approach• We use a transgenic approach to

silence or over-express OsSERK1in rice and investigate its influence on plant immunity and development.

Significance• Unlike OsSERK2, OsSERK1 contributes to plant development, but not immunity.

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• Transgenic rice over-expressing OsSERK1 are semi-dwarf, but complement OsSERK2-silencing effects on BR-signaling.

• OsSERK1-silencing results in reduced lamina joint angles.

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• OsSERK1 silencing does not affect rice immunity to Xanthomonas oryza pv oryzae.

Background• Root hairs are model single cells that develop by tip growth, a

process shared with pollen tubes, axons, and fungal hyphae. • Prolyl-4-Hydroxylases (P4H) are enzymes that modify

proteins prior to glycosylation. Many plant proteins have glycans attached to HyP and P4Hs are important for root hair growth.

Approach• To understand how different P4H proteins work together in

cell wall protein hydroxylation and root hair growth, we analyzed P4H localization, protein-protein interactions, and mutant phenotypes.

Outcomes• Among of Prolyl-4-Hydroxylases, P4H5 was pivotal for root

hair tip growth. • P4H5 had in vitro preferred specificity for hydroxylation of

extensin, a cell wall protein essential for root hair expansion.• P4H2 and P4H13 are present in root hair cells and have

interchangeable functions but cannot replace P4H5. • The P4Hs are shown to be targeted to the secretory pathway,

where P4H5 forms dimers with P4H2 and P4H13.

Complex Regulation of Prolyl-4-Hydroxylases Impacts Root Hair Expansion

Velasquez, Silvia M., Ricardi, Martiniano M., Poulsen, Christian P., Oikawa, A., Dilokpimol, A., Halim, A., Mangano, S., DenitaJuarez, Silvina P., Marzol, E., Salgado Salter, Juan D., Dorosz, Javier G., Borassi, C., Möller, Svenning R., Buono, R., Ohsawa, Y., Matsuoka, K., Otegui, Marisa S., Scheller, Henrik V., Geshi, N., Petersen, Bent L., Iusem, Norberto D., & Estevez, José M. (2014). "Complex Regulation of Prolyl-4-Hydroxylases Impacts Root Hair Expansion". Molecular Plant(0). doi, 10.1016/j.molp.2014.11.017

Significance• The effect of protein-protein interactions on localization of

proteins in Golgi is an important phenomenon that must to be understood for engineering of biomass traits in crops for biofuel production

Deficient extensin prolinehydroxylation by P4H5 impacts on root hair cell wall architecture.

P4H2 localization can shift by associating with P4H5.

P4H-P4H protein interaction assessed by Bimolecular Fluorescence Complementation

Background• To optimize greenhouse and field breeding trials, we used a variety

of compositional assays to identify biomass compositional trends between rice varieties and tissue types that translate successfully from greenhouse to field grown plants.

Approach• Researchers from GLBRC and JBEI participated in a study that

examined 16 compositional traits of stem and leaves from 20 diverse rice varieties

• An array of compositional assays were used to identify compositional trends between varieties and tissue types across growth conditions. Compositional assays include analysis of cell wall polysaccharides (cellulose and hemicellulose), lignin, ash, enzyme saccharification, hydroxyproline, and bulk density.

Outcomes/Impacts• Rice variety had the least effect on traits measured in this study,

while growth environment (greenhouse versus field) had the greatest effect.

• Yield of greenhouse-grown biomass was significantly higher than field grown biomass, which indicates that studies utilizing greenhouse grown biomass overestimate yield.

• The authors conclude that glucose yield of greenhouse-grown plants is a good predictor of glucose yield in field grown plants and could be used to optimize greenhouse and field breeding trials.

Cell Wall Composition and Bioenergy Potential of Rice Straw Tissues Are Influenced by Environment, Tissue Type, and Genotype

Tanger, P., Vega-Sánchez, M. E., Fleming, M., Tran, K., Singh, S., Abrahamson, J. B., Jahn, C. E., Santoro, N., Naredo, E. B., Baraoidan, M., Danku, J. M. C., Salt, D. E., McNally, K. L., Simmons, B. A., Ronald, P. C., Leung, H., Bush, D. R., McKay, J. K., & Leach, J. E. (2015). "Cell Wall Composition and Bioenergy Potential of Rice Straw Tissues Are Influenced by Environment, Tissue Type, and Genotype". BioEnergy Research, 1-18. doi, 10.1007/s12155-014-9573-y

Background• Reduced cell wall recalcitrance and increased C6

monosaccharide content are desirable traits for future biofuel crops

• New approaches are needed to achieve this goal with minimal impact to phenotype

Approach• Mixed-linkage glucans (MLGs), are comprised of glucose

monomers linked by both β-1,3 and β-1,4 bonds• Previous studies that expressed in planta have shown

decreased recalcitrance, but can negatively impact plant growth

• Use of senescence-associated promoters may yield higher MLG content while maintaining desirable plant growth phenotype

Outcomes• 4x more glucose in the matrix cell wall fraction• 42% increase in saccharification yields

Engineering Temporal Accumulation of a Low Recalcitrance Polysaccharide leads to Increased C6 Sugar Content in Plant Cell Walls

Vega-Sanchez, M. E., Loque, D., Lao, J., Catena, M., Verhertbruggen, Y., Herter, T., Yang, F., Harholt, J., Ebert, B., Baidoo, E. E., Keasling, J. D., Scheller, H. V., Heazlewood, J. L., & Ronald, P. C. (2015). "Engineering temporal accumulation of a low recalcitrance polysaccharide leads to increased C6 sugar content in plant cell walls". Plant Biotechnol J. doi, 10.1111/pbi.12326

Significance• Induction of cell wall polysaccharide biosynthesis in

senescing tissues offers a novel engineering alternative to enhance cell wall properties of lignocellulosic biofuel crops

Accumulation of MLG in Arabidopsis lines expressing OsCslF6 via the senescence‐associated promoter SAG12 without causing growth defects. (a) Morphology of mature rosettes (top) and whole plants (bottom) of Col‐0 and three independent pSAG12::CslF6 lines. Scale bars: 20 mm (rosettes), 15 mm (whole plants). (b) Cell wall immunolabelling of senescing leaf cross sections using an anti‐MLG monoclonal antibody; scale bar: 10 lm; m: mesophyll; sm: spongy mesophyll; vb: vascular bundle. (c) MLG content of senesced rosettes

High-throughput Prediction of Acacia and Eucalypt Lignin Syringyl/Guaiacyl Content using FT-Raman Spectroscopy and Partial Least Squares Modeling

Summary/Impacts • This study examined the usefulness of Raman model

to gauge the lignin S/G ratio in a large unknown data set. Specifically, we demonstrated the application of a partial least squares model comprised of Raman spectral data and lignin S/G ratios measured using pyrolysis/molecular beam mass spectrometry (pyMBMS) for the prediction of S/G ratios in an unknown data set.

• Pairwise comparisons within each data set were employed to assess statistical differences between each biomass species.

• Predicted S/G ratios of Acacias and eucalypts were statistically similar with those measured using pyMBMS.

• This study shows the power of using Raman spectroscopy to replace tedious, destructive methods for the evaluation of the lignin S/G ratio of diverse plants.

Background• HTP method to evaluate

phenotypic traits that correlate to plant cell wall structure and recalcitrance are needed.

• The ratio of lignin syringyl (S)-to-guaiacyl (G) moieties has been shown as an indirect means to evaluate biomass recalcitrance.

• Mid-IR and Raman spectroscopy models are shown to be capable of predicting lignin S/G ratios. However, more robust models to accurately predict S/G are needed.

Comparison of the first derivative + EMSC Raman spectra of low S/G Acacia microbotrya (black spectrum, S/G=1.2) and higher S/G Eucalyptus globulus subspecies maidenii (red spectrum, S/G=2.8), as measured by pyrolysis/molecular beam mass spectrometry (A), Scores plot showing classification by plant genus (B). The x and y-axes represent the second and third factors respectively. The blue squares represent the genus Acacia, the red circles depict the genus Corymbia, and the green triangles show the genus Eucalyptus

Lupoi, J. S., Healey, A., Singh, S., Sykes, R., Davis, M., Lee, D. J., Shepherd, M., Simmons, B. A., & Henry, R. J. (2015). "High‐Throughput Prediction of Acacia and Eucalypt Lignin Syringyl/Guaiacyl Content Using FT‐Raman Spectroscopy and Partial Least Squares Modeling". BioEnergy Research. doi, 10.1007/s12155‐015‐9578‐1 

Reference versus predicted lignin S/G ratios using FT-Raman spectra and pyMBMS reference data

A B

Background• Isoprenoids have been identified and used as

natural pharmaceuticals, fragrances, solvents, and, more recently, advanced biofuels.

• Although isoprenoids are most commonly found in plants, researchers have successfully engineered both the eukaryotic and prokaryotic isoprenoid biosynthetic pathways to produce them in microorganisms at high yields.

Approach• In this review, we highlighted the metabolic

engineering strategies that were used to successfully produce valuable isoprenoid compounds including antimalarial drug artemisinin and various biofuel compounds in microbial hosts.

• In addition, we presented a current outlook on microbial isoprenoid production, with an eye towards the many challenges that must be addressed to achieve higher yields and industrial-scale production.

Significance• This review provides a good summary of the

synthetic biology efforts for microbial isoprenoids production for drugs, biofuels, and chemicals

Isoprenoid Drugs, Biofuels, and Chemicals-Artemisinin, Farnesene, and Beyond

George, K. W., Alonso-Gutierrez, J., Keasling, J. D., & Lee, T. S. (2015). Isoprenoid Drugs, Biofuels, and Chemicals-Artemisinin, Farnesene, and Beyond Adv Biochem Eng Biotechnol (2015 Jan 11 ed.): Springer Berlin Heidelberg