Background• Protein kinases are involved in diverse cellular and biological

processes, including cell wall biosynthesis and modification.• A major obstacle that hinders progress towards kinase

characterization is functional redundancy.

Approach• The rice kinase database (RKD), which integrated

omics-scale data within a phylogenetics context, isused to predict the function of protein kinases.

• Transcriptomic data of kinase-encoding genes in diverse ricetissues and in response to biotic and abiotic stresses andhormone treatments were integrated into RKD2.0.

Outcomes• An improved rice kinase database that integrates the most

recent publicly available omics-scale datasets.• The identification of 130 kinases that are significantly up-

regulated in response to biotic stress, 296 kinases inresponse to abiotic stress, and 260 kinases in response tohormones.

Significance• RKD2.0 enables efficient prediction of kinases involved in

grass cell wall biosynthesis and modification.

Rice kinase database RKD2.0: an efficienttool to identify genes regulating grass cell wall biosynthesis

Chandran et al.(2016) “Updated Rice Kinase Database RKD 2.0: enabling transcriptome and functional analysis of rice kinase genes” Rice, 9(1), 40. doi:10.1186/s12284-016-0106-5

Heatmap analysis of kinase genes related to abiotic-stress responses

A new role for glycoside hydrolase Family 12 proteins discovered using microbial consortia

Outcomes• Comparative community proteomics (with EMSL) identified a set of cellulases from Thermobispora bispora that were

highly abundant in the most active consortium. Among these cellulases, the abundance of a GH12 protein correlated withchanges in crystalline cellulose hydrolysis activity.

• Heterologous expression and biochemical characterization of the suite of T. bispora hydrolytic cellulases confirmed that theGH12 protein possessed the highest activity on multiple crystalline cellulose substrates and demonstrated that ithydrolyzes cellulose chains by a predominantly random mechanism.

Significance• May lead to more efficient enzyme mixtures for saccharificaiton of pretreated substrates.

Hiras, J. et al. (2015). “Comparative Community Proteomics Demonstrates the Unexpected Importance of ActinobacterialGlycoside Hydrolase Family 12 Protein for Crystalline Cellulose Hydrolysis.” mBio doi: 10.1128/mBio.01106-16

Background• GH12 proteins are thought to primarily

act on soluble cellulosic andhemicellulosic substrates

• Comparative community proteomicsallows differences in enzymaticactivties between different mixedconsortial cultures to be linked toindividual proteins

Approach• We measured cellulase activties in

thermophilic bacterial consortia derivedfrom compost and correlateddifferences in enzymatic activties withchanges in protein levels as measuredby comparative proteomics

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The need for integrated approachesin metabolic engineering

Lechner et al. (2016) “The Need for Integrated Approaches in Metabolic Engineering” Cold Spring Harbor Perspectives in Biology doi:10.1101/cshperspect.a023903

Integrated approaches in metabolic engineering. To achieve high metabolite flux through a biosynthetic pathway, it is essential to consider bottlenecks on the transcriptome, translatome, protein/proteome, and reactome level. Integrated metabolic engineering approaches need to occur both on the molecular-scale and system-scale level.

Approach• By bridging multiple disciplines, including molecular biology,

biochemistry, biophysics, and computational sciences, we cancreate an integral framework for the discovery and implementationof novel biosynthetic production routes.

Background• Recent developments in the field of metabolic engineering bring

promise to the design of biosynthetic pathways, in which entiremetabolic pathways can be (re)designed and expressed in hostorganisms.

• We attempt to give the reader a comprehensive view on theaspects pertinent to successful pathway engineering. Additionally,we emphasize that there remains a gap between optimizationefforts at the molecular level and those at the systems level.

Significance• This review highlights state-of-the-art procedures for heterologous

small-molecule biosynthesis, the associated bottlenecks, and newstrategies that have the potential to accelerate futureaccomplishments in metabolic engineering.

• We emphasize that a combination of different approaches overmultiple time and size scales must be considered for successfulpathway engineering in a heterologous host.

Outcomes• We have classified these optimization procedures based on the

“system” that is being manipulated: transcriptome, translatome,proteome, or reactome.

Effect of light and nitrogen on thephotosynthetic efficiency of Miscanthus

Outcomes• Photosynthetic activities were greater in high light and

increased with leaf nitrogen, suggesting limited nitrogenavailability can diminish the potential biochemicalacclimation of C4 photosynthesis to high-light conditions

• The response of CO2 leakiness to decreasing lightintensities was relatively small

1) Leaf cross sections of Miscanthus grown at two differentirradiancesThe mesophyll surface area exposed to the intercellular airspace per unit leafarea was greater in high light compared to low light.a) High light. B) Low light. Scale: 50 µm

Ma et al. (2016) “Influence of light and nitrogen on the photosynthetic efficiency in the C4 plant Miscanthus×giganteus” Photosynth. Res. doi: 10.1007/s11120-016-0281-7

Background• The CO2 concentrating mechanism in C4 plants generally

allows for high rates of net CO2 assimilation and biomassproduction. While growth conditions influence theefficiency of C4 photosynthesis, it remains unclear howchanges in the biochemical capacity versus leaf anatomydrives this acclimation.

Significance• CO2 concentrating mechanisms in Miscanthus are robust,

and that observed changes in leaf anatomy andbiochemistry likely help to maintain this efficiency.

2) Net CO2 assimilation rate under nitrogen and lightIn all plants, the net rate of CO2 assimilation increased with light intensity. In thelow light grown plants, there was not a significant difference between nitrogentreatments across all the measurement light intensities.

Approach• To test how growth light intensity and nitrogen availability

affect leaf anatomy, biochemistry and the efficiency of theCO2 concentrating mechanism in Miscanthus ×giganteus.

Activation of lignocellulosic biomass forhigher sugar yields using aqueous ionicliquid at low severity process conditions

Outcomes• Pretreatment with [TBA][OH] generated high glucose

yields (~95 %) after pretreatment at very mild processingconditions (50 oC)

• Glycome profiling (BESC) experiments and computationalresults indicate that removal of the non-cellulosicpolysaccharides occurs due to the ionic mobility of[TBA][OH] and is the key factor in determiningpretreatment efficiency

Effect of pretreatment severity on glucose and xylose yields obtained from saccharification of pretreated switchgrass

Parthasarathi et al. (2016) “Activation of lignocellulosic biomass for higher sugar yields using aqueous ionic liquid at low severity process conditions” Biotechnol Biofuels, 9, 160. doi:10.1186/s13068-016-0561-7

Background• Conventional ILs are relatively expensive in terms of

purchase price• The most effective imidazolium-based ILs also require

energy intensive processing conditions (>140 °C, 3 h) torelease >90 % fermentable sugar yields aftersaccharification

Significance• This approach to biomass pretreatment at lower

temperatures could be transformative in the affordabilityand energy efficiency of lignocellulosic biorefineries

Impact of pretreatment temperature on biorefineryenergy requirements at industrial scale (to process 2000

MT/day dry biomass)

Approach• Explore inexpensive IL comprised tetrabutylammonium

[TBA](+) and hydroxide [OH](-) ions• Determine impact of temperature and process severity on

sugar yields obtained after pretreatment

Role of organic matter on soil heating, organic acid accumulation, and bacterial communities in solarized soil

Outcomes• Diverse microbial communities developed under the harsh conditions

of solarization.• At lower soil depths, lignocellulose deconstruction and metabolisms

led to acid fermentation. Acetate was the primary fermentationproduct.

1) Non-metric multidimensional scaling of community dissimilarityLignocellulose amendment, soil heating, and soil depth affected adaptation ofmicrobial communities in the soil. Differences in community structure by soil depthillustrate the interaction effect between temperature and oxygen gradients ondeconstructive communities.

Simmons et al. (2016) “The role of organic matter amendment level on soil heating, organic acid accumulation, and development of bacterial communities in solarized soil”, Appl Soil Ecol, doi, http://dx.doi.org/10.1016/j.apsoil.2016.04.018

Background• Solarized soils provide a multi-stress environment to

observe development of robust lignocellulose-degradingmicrobial communities.

• These communities can provide insight into facultativeanaerobic, thermophilic, acidophilic, and high-solidstolerant communities capable of lignocellulosedeconstruction.

Significance• The robust lignocellulolytic microbial communities characterized here

can inform design of deconstructive communities for industrialbioprocessing.

2) Volatile fatty acid (VFA) production in amended soilsAnaerobic activity manifested as VFA fermentation inresponse to greater lignocellulose amendment and soil depth.

Approach• Soils were amended with varying levels of lignocellulose

and then solarized by covering with clear plastic tarp for15 days.

• At the end of treatment, soil samples were subjected to16S rRNA gene sequencing, volatile fatty measurement,and residual organic matter quantification.

Enhanced fatty acid production in engineered chemolithoautotrophic bacteria using reduced sulfur compounds as energy sources

OutcomesEngineered Thiobacillus denitrificans produced up to 52-foldmore fatty acids than the wild-type strain. The relativestrength of the two native promoters as assessed by fattyacid production in engineered strains was very similar to thatassessed by expression of the cognate genes in the wild-typestrain.

Beller et al. (2016) “Enhanced fatty acid production in engineered chemolithoautotrophic bacteria using reduced sulfur compounds as energy sources” Metabolic Engineering Comm., doi: 10.1016/j.meteno.2016.07.001

BackgroundChemolithoautotrophic bacteria that oxidize reduced sulfurcompounds, such as hydrogen sulfide, while fixing CO2 arean untapped source of renewable fuels from sulfide-ladenwaste, such as municipal or petroleum refineryhydrodesulfurization wastewater.

SignificanceThis proof-of-principle study suggests that engineeringsulfide-oxidizing chemolithoautotrophic bacteria tooverproduce fatty acid-derived products meritsconsideration as a technology that could simultaneouslyproduce renewable fuels as well as cost-effectivelyremediate sulfide-contaminated wastewater.

ApproachA modified thioesterase gene from E. coli (‘tesA) wasintegrated into the Thiobacillus denitrificans chromosomeunder the control of Pkan or one of two native T. denitrificanspromoters. Fatty acid production was tested during anaerobicgrowth on thiosulfate, nitrate, and CO2.

Up to 52-fold improvement in fatty acid titer in T. denitrificans growing on thiosulfate, nitrate, and carbon dioxide and expressing E. coli ‘tesAunder the control of native promoters (P2545 or P2726) or Pkan.