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Modification of plant cell wall properties in an effort to improve biofuel crops
Kenneth Keegstra

DOE Plant Research Laboratory, DOE Great Lakes Bioenergy Research Center, Michigan State University, East Lansing, Michigan, USA.
This lecture will briefly review the efforts underway in the DOE Great Lakes Bioenergy Research Center aimed at improving biofuel crops by modifying the properties of plant cell walls. These efforts are being pursued via two major strategies; each will be explained briefly and recent progress summarized. The first is to modify the composition of lignin in such a way that lignin can be degraded by mild pretreatment conditions thereby allowing easier access to the polysaccharides present in plant cell walls. The second is to modify the polysaccharide content of biomass such that it will have increase the levels of hexose-containing hemicelluloses. The latter efforts build on research from our lab to understand and manipulate glucomannan biosynthesis. Our current understanding of glucomannan biosynthesis will be reviewed and the important unsolved questions will be described.
* E-mail:keegstra@msu.edu



Beta-glucan Biosynthesis in Grasses
A. Bacic1, R.A. Burton2, M.S. Doblin1, F.A. Pettolino1, S. Wilson1, H. Collins2, G.B. Fincher2
1. Australian Centre for Plant Functional Genomics and Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Victoria, 3010, Australia;
2. Australian Centre for Plant Functional Genomics , University of Adelaide, SA, Australia.
Grass cell walls are distinguished by the presence of the non-cellulosic polysaccharide (1,3;1,4)-b-D-glucan. These polysaccharides act as both carbon storage reserves and structural elements during plant growth and development. They are also of immense commercial interest as they constitute an important component of the soluble fibre component of the grain. Soluble fibre is known to be beneficial in the human diet reducing the occurrence and risk of coronary heart disease, colorectal cancer, obesity and type ii diabetes.
We have recently shown that the proteins encoded by the cellulose synthase-like gene families, CslF (Burton et al, 2006) and CslH (Doblin et al, 2009) gene families, are able to catalyse the assembly of these b-glucans. Manipulation of the expression levels of these genes has provided insights into both their molecular mechanisms of assembly and the capacity to redirect the flux of carbon between different non-cellulosic polysaccharides in the wall. The results of these studies will be presented.
The work was funded in part by a CSIRO Food Futures Flagship grant to the High Fibre Grains Cluster.
[1] Burton RA, Wilson SM, Hrmova M, Harvey AJ, Shirley NJ, Medhurst A, Stone BA, Newbigin EJ, Bacic A, Fincher GB (2006) Cellulose synthase-like CslF genes mediate the synthesis of cell wall (1,3;1,4)-b-D-glucans. Science 311: 1940-1942.
[2] Doblin M, Pettolino F, Wilson S, Campbell R, Burton R, Fincher GBF, Newbigin E, Bacic A (2009) A barley cellulose synthase-like CSLH gene mediates (1,3;1,4)-β-D-glucan synthesis in transgenic Arabidopsis. Proc Natl Acad Sci USA 106: 5996-6001.
* E-mail:



Genetic engineering of Hemicelluloses, a major component of lignocellulosic biomass
Florian Kraemer1, Sarah Hake2, Jacob Jensen3, Sascha Gille1, Markus Pauly1
1 Energy Biosciences Institute, University of California, Berkeley, USA;
2 Plant Genome Expression Center, USDA, Albany, USA;
3 DOE Plant Research Lab, Michigan State University, East Lansing, USA..
Among plant cell wall polymers cross-linking glycans (commonly referred to as hemicelluloses) are a major component of lignocellulosic biomass where they tightly associate with cellulose microfibrils. Unlike cellulose, crosslinking glycans are substituted with sidechains of various composition and length making them water soluble and thus enzyme accessible. How these sidechains are spaced along the backbone and what the precise role of their patterning remains to be discovered. Hence, as the most complex polysaccharides in lignocellulosic biomass in terms of composition and interaction with other polymers they are an important factor when considering the use of plant biomass for the biorefinery.
The structure of a crosslinking glycan found in the wall is biochemically defined by two processes, synthesis of the polymer in the cells Golgi-apparatus, and after binding to cellulose-microfibrils upon secretion into the apoplast further enzymatic modification by apoplastic hydrolases and transglycosylases. Here, we present various approaches including forward and reverse genetics as well as deep sequencing approaches to identify key cellular components that are involved in shaping the structure of various hemicelluloses. Genetic engineering utilizing these key genes can lead to altered hemicelluloses structure in the wall leading to a variety of effects on biomass processing.
* E-mail:



Study on rice brittleness mutants, a way to open the ‘black box’ in monocot cell wall biosynthesis
Baocai Zhang, Guangyan Xiong, Rui Li, Lifeng Liu, Yihua Zhou*
State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
Rice is a model organism for studying the mechanism of cell wall biosynthesis and remolding in Gramineae. Mechanical strength is an important agronomy trait of rice plants (Oryza sativa L.) that affects crop lodging and gain yield. As a prominent physical property of cell walls, mechanical strength reflects upon the structure of different wall polymers and how they interact. Studies on the mechanisms that regulate the mechanical strength therefore depend on uncovering the functions of corresponding genes in cell wall biosynthesis and remodeling. Our group focuses on the studies of systematical isolation of brittle culm (bc) mutants and functional characterization of their corresponding genes. Till now, we have reported five bc mutants. The identified genes cover several pathways of cell wall biosynthesis, including cellulose biosynthesis and deposition, membrane trafficking, and matrix polysaccharides formation. All of those have revealed many secrets in monocot cell wall biosynthesis and remodeling, which are helpful for harnessing the waste rice straws for biofuel production.
Key words: cell wall biosynthesis, brittleness, mechanical strength, rice



Cellulose biosynthesis in higher plants
Vincent Bulone
Royal Institute of Technology (KTH), School of Biotechnology, AlbaNova University Centre, SE-106 91 Stockholm, Sweden .
Cell wall polysaccharide biosynthesis is a complex process involving numerous glycosyltransferases. This large family of enzymes is poorly characterized despite the identification of a high proportion of the corresponding putative genes in plants for which the full genome has been sequenced. A main objective of our group is to uncover the biochemical aspects related to cell wall formation, with particular emphasis on cellulose biosynthesis. This presentation will summarise our latest progress in the area of cell wall biosynthesis (cellulose biosynthesis in particular) based on the use of a combination of biochemical and biophysical approaches, expression of a recombinant catalytic subunit of cellulose synthase and proteomics on subcellular membrane compartments.
* E-mail:



A Barley Brittle Stem Mutant has a Lesion in
a Cellulose Synthase Gene
Geoffrey B. Fincher
Australian Centre for Plant Functional Genomics, School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Glen Osmond, SA 5064, Australia
Two barley brittle stem mutants have reduced levels of crystalline cellulose compared with their parental lines. A custom-designed microarray revealed that transcript levels for the HvCesA4 cellulose synthase gene decreased in both mutants in the lower regions of stem internodes. Altered abundance of several hundred transcripts in the upper, maturation zones of stem internodes reflected pleiotropic responses to a weakened cell wall that resulted from the primary genetic lesion. Sequencing the HvCesA4 genes reveals the presence of a retroelement in an intron of the HvCesA4 gene; the retroelement interferes with transcription of the gene or with processing of the mRNA. These mutants offer potential as a biomass source in bioethanol production.
* E-mail:

Metabolisms Mediated by 4-Coumarate:Coenzyme A Ligases in Monocot Rice
Jinshan Gui, Junhui Shen, Laigeng Li*
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Rd, Shanghai 200032
4-coumarate:CoA ligase (4CL, EC is a key enzyme involved in phenylpropanoid metabolisms, such as monolignol and flavonoid biosynthesis. 4CL has been muchstudied in dicotyledons, but its function is not completely understood in monocotyledons which display different monolignol composition and phenylpropanoid profiles. In present study, the 4CL gene family of 5 members in rice genome were identified and characterized. Biochemical analysis revealed that the five rice 4CLs displayed different catalytic properties with diverse substrate specificity. Expression of the five 4CLs was distinct in the tissue specificity and transcript abundance. Suppression of the 4CL expression led to significant lignin reduction and shorted plant phenotype. Interestingly, wall-bound and free phenolic compounds were found being changed in the 4CL suppression plants, which could contribute to the decrease of panicles fertility. Together, these results support a model in which present study demonstrated that the monocot rice 4CLs in vitro exhibited catalytic properties different from those in dicots and in vivo also regulated other phenolic compound metabolism which played a critical role in regulating plant growth in addition to controlling lignin biosynthesis.
* Tel 021-54924151 ; Fax 021-54924015 ; Email:



Identifying novel transcripts involved in secondary cell wall from vascular tissues of switchgrass ( Panicum virgatum L. cv Alamo ) using genomics tools
Avinash C Srivastava1,2, Ji-Yi Zhang1,2, Yanbin Yin2,3 , Junying Ma1,2, Christa Pennacchio4, Erika Lindquist4, Ying Xu2,3, Elison B. Blancaflor1,2, Michael Udvardi 1,2 Yuhong Tang 1, 2
1 Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, OK 73401, USA;
2 BESC - The BioEnergy Science Center of U.S. Department of Energy;
3 Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602;
4 DOE Joint Genome Institute, Walnut Creek, CA 95598.
Lignin is an integral component of secondary plant cell walls that strongly interferes in the hydrolytic process during conversion of cellulose to fermentable sugars. Thus, various efforts are in progress worldwide to reduce lignin in plants for efficient production of cellulosic ethanol. To understand the complex genetic network that governs secondary cell wall formation, and to uncover novel genes involved in lignification, we used a targeted approach to isolate transcripts of genes from vascular tissues using laser-capture microdissection (LCM). This enabled us to identify vascular tissue (VT) specific candidate genes by comparing transcription profiles between the VT with that of the whole stem (WS). By using PAVE software and combined assembly, we determined that 846 consensus sequences were putatively VT-specific with at least 5 EST reads from the VT and none from the WS. Tissue-specific expression of these genes is being confirmed using real time qRT-PCR and in situ hybridization. Genes that show preferential expression in lignified versus non-lignified tissues are being considered as targets to reduce recalcitrance. In the mean time, more than 10 million ESTs were generated from Alamo using 454 and Sanger sequencing technology. The assembly efforts and application of these sequence data for swtichgrass genomics research will be presented.
* E-mail:




Comprehensive Analysis of NAC Domain Transcription Factor
Gene Family in Populus trichocarpa
Ruibo Hu, Guang Qi, Yingzhen Kong, Gongke Zhou*
Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences
NAC (NAM, ATAF1/2 and CUC2) domain proteins are plant-specific transcriptional factors known to play diverse roles in various plant developmental processes. NAC transcription factors comprise of a large gene family represented by more than 100 members in Arabidopsis, rice and soybean etc. Recently, a preliminary phylogenetic analysis was reported for NAC gene family from 11 plant species. However, no comprehensive study incorporating phylogeny, chromosomal location, gene structure, conserved motifs, and expression profiling analysis has been presented thus far for the model tree species Populus. In the present study, a comprehensive analysis of NAC gene family in Populus was performed. A total of 163 full-length NAC genes were identified in Populus, and they were phylogeneticly clustered into 18 distinct subfamilies. The gene structure and motif compositions were considerably conserved among the subfamilies. The distributions of 120 Populus NAC genes were non-random across the 19 linkage groups (LGs), and 87 genes (73%) were preferentially retained duplicates that located in both duplicated regions. The majority of NACs showed specific temporal and spatial expression patterns based on EST frequency and microarray data analyses. However, the expression patterns of a majority of duplicate genes were partially redundant, suggesting the occurrence of subfunctionalization during subsequent evolutionary process. Furthermore, quantitative real-time RT-PCR (RT-qPCR) was performed to confirm the tissue-specific expression patterns of 25 NAC genes. Based on the genomic organizations, we can conclude that segmental duplications contribute significantly to the expansion of Populus NAC gene family. The comprehensive expression profiles analysis provides first insights into the functional divergence among members in NAC gene family. In addition, the high divergence rate of expression patterns after segmental duplications indicates that NAC genes in Populus are likewise to have been retained by substantial subfunctionalization. Taken together, our results presented here would be helpful in laying the foundation for functional characterization of NAC gene family and further gaining an understanding of the structure-function relationship between these family members.
Acknowledgement: The work is supported by grants from the National High-Tech Research and Development Program of China (to G.Z., 2009AA10Z101), the Program of 100 Distinguished Young Scientists of the Chinese Academy of Sciences (to Gongke Zhou), and National Natural Science Foundation of China (No.30901157 and No. 31000311 ).
* E-mail:




Giant King Grass: A New Dedicated Energy Crop
Carl Kukkonen
Viaspace Inc, USA
Giant King Grass is a high yield perennial crop that is being considered for bioenergy applications. Independent analyses by potential partners have shown the suitability of Giant King Grass for direct combustion in power plants, energy pellets to be co-fired in existing coal power plants, and bio methane production through anaerobic digestion. Tests as a feedstock for cellulosic ethanol and other liquid biofuels are in process.110 ha of Giant King Grass are being grown in Guangdong province China.
* E-mail:




Genome-based genetic improvement of sweet sorghum
(Sorghum bicolor)as a dedicated biofuel crop

Haichun Jing
Institute of Botany, Chinese Academy of Sciences, 20 Nanxincun, Xiangshan, Beijing, 100093.
Developing and searching for alternate energy sources is an imperative challenge for human beings. Chinese government is committed to tackle the energy crisis by strengthening research on energy crops. This is well reflected in the recently released ‘China White Book of Energy Status and Strategies’ and ‘the National Energy Laws’. In the mid- and long-term energy roadmap, renewable energy will increase to occupy 15% of the total national energy supply, equivalent to 10 million ton crude oil by 2020.
Sweet sorghum (Sorghum bicolor) is a C4 annual crop with a high photosynthetic rate, high biomass, high tolerance to adverse conditions such as drought, salinity, poor nutrient supplies. Across the whole country in China, sweet sorghum has a long history of cultivation and has been as an important source of folder, feed, fibre and food. Recently, sweet sorghum is amongst the priority list of biofuel crops and considered the crop of choice for the first and second generation of biofuel. This is due to its distinctive bioenergy related traits: high directly fermentable stalk sugars, high combustible fibres and high stress tolerance.
However, many bottlenecks still need to be overcome before sweet sorghum is transformed into a dedicated biofuel crop. For instance, how to balance the partitioning of energy for biomass production and stress tolerance, how to generate more genetic variation in maturation and hence harvesting time and how to improve post-harvest storage time are some of the key traits for further genetic improvement. We take a genome-based breeding approach and aim to design molecularly sweet sorghum into a dedicated biofuel crop. By using the next-generation high-throughput sequencing technology, three sorghum genomes with different origins were analysed, aligned and compared with that of the published grain sorghum BTx623 genome. Large amounts of SNPs, 1-6bp InDels and genome structure variation including insertion, deletion and duplication were observed. For example, over three million SNPs and Indels were detected for the three genomes. An asymmetric distribution of these SNPs and InDels were found amongst chromosomes and in different regions within a chromosome. These results allow the exploitation of the synteny and divergence between these sweet sorghums and the grain sorghum as well as the candidate genome regions for further characterization.
* E-mail:




Research Progress of Jatropha curcas L. in CAS
Guojiang Wu
South China Botanical Garden, CAS, China
Jatropha curcas L. grows as a large shrub or small tree. Recently, the high yield of seed from the tree and the high oil content of its seeds attracted global attention for the development of jatropha as a source for bio-fuel. It is also in China. We established of energy plant research platform: Energy-plant garden; Jatropha L. resources garden and Comprehensive experimental station for energy plants. In order to determine the mechanisms involved in the biosynthesis of fatty acids in J. curcas seeds, we isolated several key enzymes involved in fatty acid desaturation, chain elongation and termination, and detected the expression patterns of these genes at various tissues and different seed development stages.
We also introduce the mutant collection, Proteomics researches and so on.
* E-mail:




Large-scale identification of rice cell wall mutants for
energy crop breeding

Guosheng Xie1, 2, ,Fengcheng Li1, 2, Kai Guo1, 2, Mingliang Zhang1, 2, Lingqiang Wang1, 2, Weihua Zou1, 2, Zhengdan Xu1, 2, Yu Li1, 2, Shuangfeng Ren1, 2, Bo Yang1, 2, Rui Zhang1, 3, Yuanyuan Tu1, 2, Yanting Wang1, 2, Liangcai Peng1, 2,3
1 National Key Laboratory of Crop Genetic Improvement, Biomass and Bioenergy Research Centre, Huazhong Agricultural University, Wuhan 430070, China;
2 College of Plant Sciences and Technology, Huazhong Agricultural University, Wuhan 430070, China;
3 College of Life Sciences and Technology, Huazhong Agricultural University, Wuhan 430070, China.
Rice is the major food crop that can provide large lignocelluloses in China. As the second generation biofuel, lignocellulosic ethanol from rice residues is increasingly considered to use in the near future. Because of its recalcitrance, however, lignocellulosic ethanol production is under developing. Discovery of energy crops is a bottle-neck-breaking solution, and the ideal energy crops are defined with a high yield of grain and an easy-to-destruction of straw. To meet the need, we defined the strategy that includes three major steps: mutagenesis of the high-yield-grain crops, selection of the cell-wall-altered mutants with good agronomic traits and high biomass yield, and test of the mutants with efficient biomass degradation. Here we screened out large mutagenesis pools of rice T-DNA mutants, and further generated the chemical (EMS-induced) and physical (Cobalt60 irradiation) mutagenesis pools for any typical rice mutant discovery. With different pretreatments, most selected mutants showed an increased rate in the biomass degradation by 2-3 folds compared with wild type. Several candidate mutants could be directly used for the bioethanol production, and the related gene identification is underway in our lab.
*Tel: +86 27 8728 1765; Fax: +86 27 8728 0016; E-mail:



Genetic engineering of cassava and sweetpotato for
industrial applications

Jun Yang, Shanshan Zhao, Dong An, Huiping Bi, Jia Xu, Qiuxiang Ma, Min Zhang, Jia Liu, Peng Zhang*
Institute of Plant Physiology & Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China
Cassava (Manihot esculenta Crantz) and sweetpotato (Ipomoea batatas) accumulate a lot of starch in their storage roots which can be processed to food, feed and bio-ethanol. The high yield potential and robustness against unfavorable environmental conditions make them the suitable energy crops for margin land cultivation, which will not highly compete with other food crops on arable lands. There are several key biological constraints in cassava and sweetpotato for biofuel development. For example, the rapid post-harvest physiological deterioration of cassava storage roots after harnest is the biggest disadvantage during starch and bio-ethanol process world-wide. The stem nematode disease causes great losses on sweetpotato yield. Different types of starches from cassava and sweetpotato are also demanding by starch companies. Conventional breeding efforts have attempted to address the constraints to cassava and sweetpotato production, but with limited success due to their nature of heterozygousity and inbreeding depression. The new tools of biotechnology can change this situation by offering new approaches to the challenges of cassava and sweetpotato. These new technologies have the potential to make them much more productive, a better source of bio-industrial products, and profitable to grow. Here we present our recently research progresses on genetic transformation, prolongation of leaf life, modification of starch biosynthesis, and enhancement of low-temperature/salinity tolerance. Our ultimate objective is to promote cassava and sweetpotato as the major starch biomass for bio-industrial applications through the generation of novel germplasms by regulation of gene expression.
Keywords: Cassava, sweetpotato, genetic improvement, transgenesis, industrialization
Acknowledgement: This work was supported by grants from the Chinese Academy of Sciences (No. KSCX2-YW-G-035), the National Natural Science Foundation of China (No. 30771366), the National Basic Research Program (2010CB126605) and National High Technology Research and Development Program (2009AA10Z102) of China and the Earmarked Fund for Modern Agro-industry Technology Research System (nycytx-17).
* E-mail:




Chloroplast genetic engineering for enhanced biomass production and hydrolysis
Henry Daniell
Department of Molecular Biology & Microbiology, College of Medicine, University of Central Florida, Orlando FL 32816-2364, USA.
Biofuel production from lignocellulosic materials is limited by the lack of technology to efficiently and economically release fermentable sugars from the complex multi-polymeric raw materials. Therefore, mixtures of enzymes containing endoglucanases, exoglucanase, pectate lyases, cutinase, swollenin, xylanase, acetyl xylan esterase, beta glucosidase and lipase genes from bacteria or fungi have been expressed in tobacco chloroplasts. Homoplasmic transplastomic lines showed normal phenotype and were fertile. Chloroplast-derived crude-extract enzyme cocktails yielded more (up to 3,625%) glucose from pine wood or citrus peel than commercial cocktails produced via fermentation and 1000-10,000 fold less expensive than recombinant commercial enzymes. Limitations of higher cost and lower production capacity of fermentation systems are addressed by chloroplast-derived enzyme cocktails.
Hormones play an important regulatory role in plant growth and development. Most of the steps in hormone biosynthesis and metabolism are irreversible except for the formation of inactive conjugates which can release active hormones by enzymatic hydrolysis. Transplastomic tobacco lines expressing β-glucosidase (Bgl-1) flower one month earlier with an increase in biomass (190%), height (150%), and leaf area (160%) than untransformed plants. Trichome density on the upper and lower leaf surface of Bgl-1 plants increase by 1033% and 740%, respectively, harboring 427-510% more glandular trichomes as determined by rhodamine B staining, suggesting that the Bgl-1 lines produce more sugar esters than control plants. Gibberellin levels were measured because it is a known regulator of flowering time, plant size, and trichome development. Both GA1 and GA4 levels were twice as high in Bgl-1 leaves than untransformed plants but did not change in other organs. In addition, elevated levels of other plant hormones, including zeatin and IAA, were observed in Bgl-1 lines. Protoplasts from Bgl-1 lines divide and form calli without exogenous hormones. Cell division in protoplasts is enhanced 670% in the presence of exogenously applied zeatin-O-glucoside conjugate, all indicating the release of free hormones from their conjugates stored within chloroplasts. The whitefly and aphid population in control plants was 18- and 15-times higher than in transplastomic lines, respectively. These data confirm that increase in sugar ester levels in BGl-1 lines is likely to function as an effective biopesticide. This study provides a novel strategy for designing plants with enhanced biomass production and insect control by releasing plant hormones from their conjugates stored within their chloroplasts.Recent advancements in this field will be presented.
* E-mail:




Enhanced biomass through improved agronomic traits engineered
via the chloroplast genome

Jihong Liu Clarke1 and Henry Daniell2
1 Department of Genetics and Biotechnology, Plant Health and Protection Division,Bioforsk- Norwegian Institute for Agricultural & Environmental Research, Hoegskoleveien 7, N-1432 Aas, Norway;
2 Department of Molecular Biology & Microbiology, University of Central Florida,College of Medicine, 336 Biomolecular Science Building, Orlando, FL 32816-2364, USA..
The world population is expected to reach an estimated 9.2 billion by year 2050. Therefore, food production globally should increase by 70 percent in order to feed the world, while total arable land, which has reached its maximal utilization, may even decrease. Climate change adds yet another challenge to the food security problem. In order to feed the world in 2050, biotechnological advances in modern agriculture are essential. After the first green revolution, plant genetic engineering has offered a new tool to incease global crop production and this will continue to play an important role in modern agriculture to meet global challenges. To enhance crop biomass through improved agronomic traits and increase photosynthetic capacity via the chloroplast genome will be of importance in the years ahead.
Chloroplast genome engineering, with several unique advantages, especially transgene containment, has made significant progress in the last two decades in various biotechnology applications including development of crops with high levels of resistance to insects, bacterial, fungal and viral diseases, different types of herbicides, drought, salt and cold tolerance, cytoplasmic male sterility, metabolic engineering, phytoremediation of toxic metals and production of many vaccine antigens, biopharmaceuticals and biofuels. Chloroplast genomes of several major crops have been transformed. These advances should promote crop productivity under a changing climate. This talk will give a brief insight into the current state of the art of plastid engineering in relation to agricultural production, especially for engineering of agronomic traits and improvement of crop biomass. The future direction of this technology and challenges for improvement of cereal crops to enhance their biomass for food security in a changing climate with special emphasis on elevated CO2 will be presented.
* E-mail:
* E-mail: daniell@mail.ucf.edu




Increased efficiency in hydrolysis of lignocellulose of poplar wood
by down-regulation of lignin synthesis genes

Mengzhu Lu, Shaozong Yang, Shutang Zhao
Lab of biotechnolgy, Rerch Institute of Forestry, Chinese Academy of Forestry, Beijing 100091
Forest trees are one of the major sources for renewable bioenergy, and China has the world's largest poplar plantation. The high productivity of poplar plantation has provided an attractive source for biofuel production.However, efficient hydrolysis of lignocellulose is the biggest technical challenge on converting lignocellulose to ethanol in poplar due to its high lignin content.
We are attempting to genetically modify poplar to decrease lignin content in order to enhance fermentable sugar s which can be converted to ethanol. To do this, we targeted at coumaroyl shikimate 3-hydroxylase(C3H), cinnamate 4-hydroxylase(C4H)and hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase(HCT)involved in lignin biosynthesis. Four full gene-length cDNAs of C3H1, C4H1, HCT1 and HCT5 in Populus tomentosa Carr.were cloned and RNA interfering vectors were constructed using their individule or combinational sequences. Poplar was transformed via the Agrobactria-mediated leaf-disc method. Transgenic poplar lines harboring the C3H1 and HCT5 RNAi constructs were obtained and vegetatively propagated by cutting for each lines in the greenhouse.
The transcription level of C3H1 and HCT5 in both non- and transgenic lines were analyzed by real-time PCR, showing 89.04%, 82.22% and 68.38% reduction in C3H1 RNAi inhibition lines 323, 325, 322 and 67.64%, 56.35%, 49.88%, 45.05% reduction in HCT5 RNAi inhibition lines 312, 308, 502, 307 respectively in comparison with the non-transgenic controls. Stem cross-section observations showed that cell layers of the secondary xylem in transgenic plants were increased, but the cells became smaller with cell wall collapsed and irregular thickened. This indicates that the xylem development and lignin deposition pattern in transgenic plants are changed. Lignin and cellulose content in transgenic lines showed that transgenic plants with reduced lignin content generally in accordance with the transcript level of the target gene, and lignin reduction in transgenic plants also led to a higher cellulose content.
To determine relationships between lignin content and the efficiency of chemical/enzymatic saccharification, stem materials with modified lignin content were analyzed. Plants with the least lignin had the highest total carbohydrate levels in untreated biomass, reflecting compensation for the reduction in lignin level on a mass balance basis. After 72 h incubation, saccharification efficiency was higher in C3H and HCT reduced lines compared with controls. More than 90% of the released sugar from most lines was glucose, indicating enzymatic hydrolysis of cellulose. In addition, enzymatic hydrolysis released more xylose from transgenic lines than that from control lines, suggesting that lignin modification increases the accessibility of residual hemicellulose by enzymes.
This study tested the effectiveness of reduction of lignin content by RNAi strategy using the key genes involed in lignin biosynthesis. The results further indicate that lignin is probably the major factor in recalcitrance of cell walls to saccharification. Moreover, it demonstrates that genetic reduction of lignin content effectively overcame cell wall recalcitrance to bioconversion. This approach could obviate the need for acid pretreatment, as indicated by that the saccharification efficiency of untreated biomass of the 312 and 323 lines were even greater than that of control plants with pretreatment. This approach thus would be applicable to other bioenergy trees for improving saccharification.
Keywords: Poplar ,RNAi ,Lignin, Saccharification Efficiency
* E-mail:





Systems Biology-based Exploration of Natural Biomass Utilization Systems for Reverse Design of Biorefinery
Weibing Shi1,2, Ugur Uzunner1,2, Ryan Syrenne1,2, Peng Gao1,2, Yixiang Zhang1,2, Nikos C. Kyrpides3, Eun-gyu No2, Sanmin Liu4, 5, Jianzhong Sun6, Lantao Liu1, 5, Susie Y. Dai4,7, Joshua S. Yuan1,2,8*
1 Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX, 77843;
2 Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station, TX, 77843;
3 Genome Biology Group, Joint Genome Institute, Walnut Greek, CA, 94598;
4 Department of Veterinary Pathobiology, Texas A&M University, TX, 77843;
5 Department of Computer Sciences, Texas A&M University, TX 77843;
6 Center for Bioenergy Research, Jiangsu University, Zhenjiang, Jiangsu, China;
7 Office of Texas State Chemist, Texas A&M University, TX 77843;
8 Advanced Research Institute for Sustainable Energy (ARISE), Texas A&M University, College Station, TX 77845.

One of the key issues for lignocellulosic ethanol to become a viable option for transportation fuel is the biomass conversion efficiency in biorefinery [1]. Many natural biomass utilization systems have evolved to convert lignocellulosic biomass with high efficiency and in a simultaneous pretreatment, saccharification and fermentation pattern. We therefore integrated the latest systems biology approaches with tranditional biochemical assay to explore various natural biomass utilization systems for the biorefinery applications [2]. Both insect guts and cattle rumen have been explored for biocatalyst activity, microbiota composition, metabolic capacity, microbial strain isolation, coordinative expression of key enzymes, enzyme discovery and characterization. The gut contents from four insect species including grasshopper, silkworm, termite and cutworm were explored as model biomass utilization systems. We carried out a series of work to analyze the lignocellulosic degrading enzyme activities, to study the microbial diversity with DGGE, to select the proper species for comprehensive metagenome analysis with Illumina Genome Analyzer, to perform the metaproteomics profiling, to isolate biomass degrading microbial strains, to clone and characterize key biocatalysts based on the systems biology analysis, and to express these enzymes in bacteria, plants and yeast for various applications. The enzyme assay and microbial diversity analysis both indicated that grasshopper is a powerful source to discover novel cellulytic enzymes [3, 4]. Metagenome sequencing has been carried out to study the gut symbionts from grasshopper and cutworm representing insects feeding on two types of the food. The phylogenetic analysis revealed diverse group bacterial species in the insect gut and the existence of many microbe species relevant to biomass degradation. The functional annotation revealed that gut symbionts from both species contain many glycosyl hydrolases from different families and enzymes related to lignin degradation. Strong microbiota composition-function relevance has been identified by phylogenetic analysis and metabolic pathway reconstruction [5]. More importantly, we have cloned and characterized multiple cellulase and xylanase genes from the gut of grasshopper and cutworm. Latest HDX mass spectrometry platform has been employed to study the structure dynamics and determinants for the new enzymes [6]. High throughput in planta expression activity screening and compatibility with yeast expression system were explored to investigate the potential for these biocatalysts to be used in CBP and other applications. Besides the metagenome analysis, we also carried out metaproteome profiling for different parts of termite gut. The results highlighted the potential different function for mid-gut and hindgut, indicating that midgut is the main component for lignin degradation. Overall, our study indicated insect guts are superior natural biomass utilization systems for biocatalyst discovery and microbial strain isolation to develop the next generation biorefinery.
In parallel to the insect gut analysis, we carried out the similar comprehensive analysis for cattle rumen integrating enzyme assay, metagenome sequencing, and metaproteomics analysis to study the rumen material from cattle fed with lignocellulosics with different lignin and carbonhydrate content and composition [7]. The enzyme assays revealed that cattle rumen symbiotic microbiotaresponds to the different type of biomass at the biochemical level. More cellulytic enzyme activities
have been found in the rumen fed with lignocellulosic material with higher lignin and cellulose composition. The metaproteomics analysis revealed substantial differences in enzyme profile in response to the lignocellulosic content and composition. Moreover, we analyzed the total protein from both the fiber and the supernatant part, which shows that both enzymes in the biofilm and the free enzymes in the solution are important for the complete degradation of lignocellulosic biomass. Overall, the microbial diversity, enzyme profile, and cellulytic activity coordinately respond to the different content and composition of lignocellulosic biomass. We are exploiting the dynamics of the natural biomass utilization system to design the enzyme mixtures for different types of biomass material. Our research for both insect guts and cattle rumen highlighted the potential of natural biomass utilization systems for the reverse design of next generation biorefiner
[1] Joshua S. Yuan, Kelly H. Tiller, Hani Al-Ahmad, Nathan R. Stewart, and C. Neal Stewart Jr., Plants to Power: Bioenergy to Fuel the Future, 2008, Trends in Plant Sciences, 13:421-429.
[2] Weibing Shi, Jianzhong Sun, Ryan Syrenne, and Joshua S. Yuan, Molecular Approaches to Study the Insect Gut Symbiotic Microbiota at the ‘Omics’ Age, Insect Science, 17: 199-219.
[3] Weibing Shi, Shiyou Ding, and Joshua S. Yuan, Comparison of Insect Gut Cellulase and Xylanase Activity across Different Insect Species with Distinct Food Sources, BioEnergy Research, In Press, DOI: 10.1007/s12155-010-9096-0.
[4] Weibing Shi, Ugur Uzuner, Palmy R. Jesudhasan, Suresh D. Pillai, and Joshua S. Yuan, Comparative Analysis of Insect Gut Symbiotic Diversity as the Adaptation to Different Food Types, Submitted.
[5] Weibing Shi, Xin Zhou, Lantao Liu, Peng Gao, Xueyan Chen, Nikos Kyprides, En-Gyu No, Susie Y. Dai, and Joshua S. Yuan, Comparative Metagenomic Analysis of Herbivore Insect Symbionts Revealed Composition-Function Relationship Important for Eco-environmental Adaptations and Biotechnology Applications, Manuscript Drafted.
[6] Ugur Uzunner, Weibing Shi, Lantao Liu, Sanmin Liu, Susie Y. Dai, and Joshua S. Yuan, Enzyme Structure Dynamics of Xylanase I from Trichoderma longibrachiatum, BMC Bioinformatics, S6, S12
[7] Weibing Shi, Peng Gao, Yixiang Zhang, Sanmin Liu, Susie Y. Dai, and Joshua S. Yuan, The Dynamic and Coordinative Function of Biocatalysts in Cattle Rumen as Revealed by Metaproteomics, In Preparation.
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Recent Advances in Biorefining and Pretreatment Chemistry
Arthur J. Ragauskas
BioEnergy Science Center, Institute of Paper Science and Technology, School of Chemistry and Biochemistry, Georgia Institute of Technology, 500 10th St., Atlanta, GA, 30332, USA
Over the past several years we have examined the chemistry of acid pretreatment focusing on the ultra-structure of cellulose, lignin functionality and hemicellulose in the bulk and surface of poplar and switchgrass.  From these studies a re-occurring theme is that acid pretreatments typically increases overall crystallinity and changes the relevant amounts of amorphous, paracrystalline cellulose along with Iα and Iβ while decreasing the DP of cellulose.  The polysaccharide component of biomass has also been recently shown to generate pseudo-lignin which increases the residual Klason content after pretreatment which may impact enzymatic deconstruction. Accompanying these changes in carbohydrate chemistry, lignin is changed during pretreatment leading to increases in polydispersity, increases in condensed lignin and decreases in β–O-aryl ether content.  Surface studies by ToF-SIMS have shown that surface pretreatment chemistry is uniquely different from bulk chemistry especially in residual hemicelluloses. This presentation will examine these chemical reactions and identifying what established reactions suggest about acid pretreatment chemistry and what select reaction chemistries need further investigation to refine the efficiency pretreatment and facilitate the next generation of low-recalcitrance engineered crops.
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Progress on lignocellulose refining process engineering

Xiaowei Peng, and Hongzhang Chen*
State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China
The characteristics of lignocellulose, such as complex composition, heterogeneity structure and dispersive resources determine it difficult to be used. Any of the traditional disciplines can not solve the problems for its industrial utilization. It need reset many disciplines and form a new disciplinary system for lignocellulose biomass conversion. Moreover, to overcome the economic bottleneck of lignocellulose biomass industry we must give full consideration to the diversities of raw materials, conversion methods and products then forming a system engineering including the whole process from raw materials to products.
Based on analyzing the above problems and fully absorbing the concept of process engineering, we have proposed the research ideas of integration of raw materials, process and products to break through the traditional lignocellulose use patterns of using single technology to produce single product from single component of the lignocellulose and established a series of lignocellulose conversion research platforms and industrial demonstrations. Firstly, a comprehensive complex lignocellulose component separation technology platform based on the core technology of the steam explosion has been created. The steam-explosion system and the correlative technology and the industrial equipments from 5 to 50 m3 have also been set up. These solved the problems of lignocellulose raw material pretreatment for conversion such as production of bio-fuel, organic acids and biomaterials from straw. Secondly, the modern large-scale energy and water saving solid-state fermentation technology platform and relative processes coupling with technology platforms have been created which overcome the essential problem of pure-blood cultivation for solid state fermentation, And the largest international 100 m3 of pure-blood solid state fermentation industrial equipment have been built and had been used for the production of enzymes, bio-pesticides and other products. Thirdly, a new technology platform for fermentation coupling with product extraction has been built for the production of ethanol, butanol, succinic acid, bio-gas and lactic acid from lignocellulose. After the above key technologies being broken through, some demonstration projects had been built up, such as "Industrial demonstrations of cellulase and new bio-pesticide production by solid-state fermentation", " Demonstration of 3,000 tons of fuel ethanol production per year from straw by enzymatic hydrolysis and fermentation", " Demonstration of 600 tons of butanol production per year from straw hemicellulose integrating lignin and cellulose utilization”. Moreover, some eco-industrial models had been set up, such as “The eco-industrial models of straw refining by ethanol, butanol, cellulase, paper and eco-board respectively as the leading products” and “Co-production of ethanol and feed produced from sweet sorghum eco-industrial model”.
This August, a product line for straw refining of 300 thousand tons per year was set up based on the demonstration of 600 tons of butanol production per year from straw hemicellulose integrating lignin and cellulose utilization. The product line has the capability of producing 50 thousand tons of butanol and acetone, 30 thousand tons of high purity lignin and 120 thousand tons of cellulose. Furthermore, another product line for production of 50 thousand tons of polyols and 20 thousand tons of phenolic aldehyde glue using the high purity lignin and cellulose from straw refining was also set up. This project will attain 1.2 billion RMB sales income and 0.1 billion profit per year.
*Tel / Fax: 86-10-82627071; E-mail:
hzchen@home.ipe.ac.cn (HZ. Chen)

Ethanol production from soft biomass: test on lab and pilot scale
Yueqin Tang1, Zeshen Liu1, Guoli Lai1, Kenji Kida2, Xiaolei Wu1