Wilfred Vermerris

Replication Concepts for Bioenergy Research Experiments

While there are some large and fundamental differences among disciplines related to the conversion of biomass to bioenergy, all scientific endeavors involve the use of biological feedstocks. As such, nearly every scientific experiment conducted in this area, regardless of the specific discipline, is subject to random variation, some of which is unpredictable and unidentifiable (i.e., pure random variation such as variation among plots in an experiment, individuals within a plot, or laboratory samples within an experimental unit) while some is predictable and identifiable (repeatable variation, such as spatial or temporal patterns within an experimental field, a glasshouse or growth chamber, or among laboratory containers). Identifying the scale and sources of this variation relative to the specific hypotheses of interest is a critical component of designing good experiments that generate meaningful and believable hypothesis tests and inference statements. Many bioenergy feedstock experiments are replicated at an incorrect scale, typically by sampling feedstocks to estimate laboratory error or by completely ignoring the errors associated with growing feedstocks in an agricultural area at a field or farmland (micro- or macro-region) scale. As such, actual random errors inherent in experimental materials are frequently underestimated, with unrealistically low standard errors of statistical parameters (e.g., means), leading to improper inferences. The examples and guidelines set forth in this paper and many of the references cited are intended to form the general policy and guidelines for replication of bioenergy feedstock experiments to be published in BioEnergy Research.

Enhancing cellulose utilization for fuels and chemicals by genetic modification of plant cell wall architecture.

Cellulose from plant biomass can serve as a sustainable feedstock for fuels, chemicals and polymers that are currently produced from petroleum. In order to enhance economic feasibility, the efficiency of cell wall deconstruction needs to be enhanced. With the use of genetic and biotechnological approaches cell wall composition can be modified in such a way that interactions between the major cell wall polymers—cellulose, hemicellulosic polysaccharides and lignin—are altered. Some of the resulting plants are compromised in their growth and development, but this may be caused in part by the plants overcompensation for metabolic perturbances. In other cases novel structures have been introduced in the cell wall without negative effects. The first field studies with engineered bioenergy crops look promising, while detailed structural analyses of cellulose synthase offer new opportunities to modify cellulose itself.

The Structure and Catalytic Mechanism of Sorghum bicolor Caffeoyl-CoA O -Methyltransferase

The catalytic mechanism and substrate specificity of caffeoyl-CoA O-methyltransferase from Sorghum bicolor deduced from crystal structures, site-directed mutagenesis, and kinetic and thermodynamic analyses. Caffeoyl-coenzyme A 3-O-methyltransferase (CCoAOMT) is an S-adenosyl methionine (SAM)-dependent O-methyltransferase responsible for methylation of the meta-hydroxyl group of caffeoyl-coenzyme A (CoA) on the pathway to monolignols, with their ring methoxylation status characteristic of guaiacyl or syringyl units in lignin. In order to better understand the unique class of type 2 O-methyltransferases from monocots, we have characterized CCoAOMT from sorghum (Sorghum bicolor; SbCCoAOMT), including the SAM binary complex crystal structure and steady-state enzyme kinetics. Key amino acid residues were validated with site-directed mutagenesis. Isothermal titration calorimetry data indicated a sequential binding mechanism for SbCCoAOMT, wherein SAM binds prior to caffeoyl-CoA, and the enzyme showed allosteric behavior with respect to it. 5-Hydroxyferuloyl-CoA was not a substrate for SbCCoAOMT. We propose a catalytic mechanism in which lysine-180 acts as a catalytic base and deprotonates the reactive hydroxyl group of caffeoyl-CoA. This deprotonation is facilitated by the coordination of the reactive hydroxyl group by Ca2+ in the active site, lowering the pKa of the 3′-OH group. Collectively, these data give a new perspective on the catalytic mechanism of CCoAOMTs and provide a basis for the functional diversity exhibited by type 2 plant OMTs that contain a unique insertion loop (residues 208–231) conferring affinity for phenylpropanoid-CoA thioesters. The structural model of SbCCoAOMT can serve as the basis for protein engineering approaches to enhance the nutritional, agronomic, and industrially relevant properties of sorghum.

Using Genotyping by Sequencing to Map Two Novel Anthracnose Resistance Loci in Sorghum bicolor

Colletotrichum sublineola is an aggressive fungal pathogen that causes anthracnose in sorghum [Sorghum bicolor (L.) Moench]. The obvious symptoms of anthracnose are leaf blight and stem rot. Sorghum, the fifth most widely grown cereal crop in the world, can be highly susceptible to the disease, most notably in hot and humid environments. In the southeastern United States the acreage of sorghum has been increasing steadily in recent years, spurred by growing interest in producing biofuels, bio-based products, and animal feed. Resistance to anthracnose is, therefore, of paramount importance for successful sorghum production in this region. To identify anthracnose resistance loci present in the highly resistant cultivar ‘Bk7’, a biparental mapping population of F3:4 and F4:5 sorghum lines was generated by crossing ‘Bk7’ with the susceptible inbred ‘Early Hegari-Sart’. Lines were phenotyped in three environments and in two different years following natural infection. The population was genotyped by sequencing. Following a stringent custom filtering protocol, totals of 5186 and 2759 informative SNP markers were identified in the two populations. Segregation data and association analysis identified resistance loci on chromosomes 7 and 9, with the resistance alleles derived from ‘Bk7’. Both loci contain multiple classes of defense-related genes based on sequence similarity and gene ontologies. Genetic analysis following an independent selection experiment of lines derived from a cross between ‘Bk7’ and sweet sorghum ‘Mer81-4’ narrowed the resistance locus on chromosome 9 substantially, validating this QTL. As observed in other species, sorghum appears to have regions of clustered resistance genes. Further characterization of these regions will facilitate the development of novel germplasm with resistance to anthracnose and other diseases.

Structural and Biochemical Characterization of Cinnamoyl-CoA Reductases

In-depth investigation for CCRs from Sorghum bicolor provides information about the critical residuesfor substrate preference among CCRs. Cinnamoyl-coenzyme A reductase (CCR) catalyzes the reduction of hydroxycinnamoyl-coenzyme A (CoA) esters using NADPH to produce hydroxycinnamyl aldehyde precursors in lignin synthesis. The catalytic mechanism and substrate specificity of cinnamoyl-CoA reductases from sorghum (Sorghum bicolor), a strategic plant for bioenergy production, were deduced from crystal structures, site-directed mutagenesis, and kinetic and thermodynamic analyses. Although SbCCR1 displayed higher affinity for caffeoyl-CoA or p-coumaroyl-CoA than for feruloyl-CoA, the enzyme showed significantly higher activity for the latter substrate. Through molecular docking and comparisons between the crystal structures of the Vitis vinifera dihydroflavonol reductase and SbCCR1, residues threonine-154 and tyrosine-310 were pinpointed as being involved in binding CoA-conjugated phenylpropanoids. Threonine-154 of SbCCR1 and other CCRs likely confers strong substrate specificity for feruloyl-CoA over other cinnamoyl-CoA thioesters, and the T154Y mutation in SbCCR1 led to broader substrate specificity and faster turnover. Through data mining using our structural and biochemical information, four additional putative CCR genes were discovered from sorghum genomic data. One of these, SbCCR2, displayed greater activity toward p-coumaroyl-CoA than did SbCCR1, which could imply a role in the synthesis of defense-related lignin. Taken together, these findings provide knowledge about critical residues and substrate preference among CCRs and provide, to our knowledge, the first three-dimensional structure information for a CCR from a monocot species.

Enzyme activity and substrate specificity of the major cinnamyl alcohol dehydrogenases in sorghum

Substrate catalysis by cinnamyl alcohol dehydrogenases from Sorghum bicolor deduced from crystal structures, site-directed mutagenesis, and kinetics. Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step in monolignol biosynthesis, reducing sinapaldehyde, coniferaldehyde, and p-coumaraldehyde to their corresponding alcohols in an NADPH-dependent manner. Because of its terminal location in monolignol biosynthesis, the variation in substrate specificity and activity of CAD can result in significant changes in overall composition and amount of lignin. Our in-depth characterization of two major CAD isoforms, SbCAD2 (Brown midrib 6 [bmr6]) and SbCAD4, in lignifying tissues of sorghum (Sorghum bicolor), a strategic plant for generating renewable chemicals and fuels, indicates their similarity in both structure and activity to Arabidopsis (Arabidopsis thaliana) CAD5 and Populus tremuloides sinapyl alcohol dehydrogenase, respectively. This first crystal structure of a monocot CAD combined with enzyme kinetic data and a catalytic model supported by site-directed mutagenesis allows full comparison with dicot CADs and elucidates the potential signature sequence for their substrate specificity and activity. The L119W/G301F-SbCAD4 double mutant displayed its substrate preference in the order coniferaldehyde > p-coumaraldehyde > sinapaldehyde, with higher catalytic efficiency than that of both wild-type SbCAD4 and SbCAD2. As SbCAD4 is the only major CAD isoform in bmr6 mutants, replacing SbCAD4 with L119W/G301F-SbCAD4 in bmr6 plants could produce a phenotype that is more amenable to biomass processing.

Biochemical and structural analysis of substrate specificity of a phenylalanine ammonia-lyase

The major phenylalanine ammonia-lyases from Sorghum bicolor were characterized through crystal structures, molecular docking, site-directed mutagenesis, and kinetic and thermodynamic analyses. Phenylalanine ammonia-lyase (PAL) is the first enzyme of the general phenylpropanoid pathway catalyzing the nonoxidative elimination of ammonia from l-phenylalanine to give trans-cinnamate. In monocots, PAL also displays tyrosine ammonia lyase (TAL) activity, leading to the formation of p-coumaric acid. The catalytic mechanism and substrate specificity of a major PAL from sorghum (Sorghum bicolor; SbPAL1), a strategic plant for bioenergy production, were deduced from crystal structures, molecular docking, site-directed mutagenesis, and kinetic and thermodynamic analyses. This first crystal structure of a monocotyledonous PAL displayed a unique conformation in its flexible inner loop of the 4-methylidene-imidazole-5-one (MIO) domain compared with that of dicotyledonous plants. The side chain of histidine-123 in the MIO domain dictated the distance between the catalytic MIO prosthetic group created from 189Ala-Ser-Gly191 residues and the bound l-phenylalanine and l-tyrosine, conferring the deamination reaction through either the Friedel-Crafts or E2 reaction mechanism. Several recombinant mutant SbPAL1 enzymes were generated via structure-guided mutagenesis, one of which, H123F-SbPAL1, has 6.2 times greater PAL activity without significant TAL activity. Additional PAL isozymes of sorghum were characterized and categorized into three groups. Taken together, this approach identified critical residues and explained substrate preferences among PAL isozymes in sorghum and other monocots, which can serve as the basis for the engineering of plants with enhanced biomass conversion properties, disease resistance, or nutritional quality.

A cost-benefit analysis to select the most effective method for positional cloning: genotyping by sequencing versus allele-specific PCR

Next-generation sequencing technologies have enabled the rapid generation of high-resolution genetic maps to enable the identification of genomic regions associated with traits of interest. In certain cases these regions of interest need to be narrowed down via fine mapping to increase resolution to the level of the individual gene. In those cases, there is a choice of genotyping options, whereby cost, time, and information content need to be considered. We developed detailed, customizable models to compare the cost of genotyping by sequencing and allele-specific PCR for fine-mapping genomic regions of interest. The models were validated experimentally with data from a fine-mapping experiment designed to identify candidate genes within a 3.2-Mb disease resistance locus in the cereal crop sorghum (Sorghum bicolor (L.). Moench), which has a sequenced genome. The size of the mapping population, genetic diversity of the parental lines, choice of reagents, and labor cost were shown to influence the overall cost of the two methods, and, consequently, which method would be most cost-effective for a given experiment. The model can be easily customized to reflect experiments with different species, genetic populations, experimental and personnel costs to determine the most cost-effective procedure for a new positional cloning project.