Ethanol Using Yeast Based Simultaneous Saccharification Biology Essay

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These studies accentuate the utilization of genetic engineering and demonstrate that the down-regulation or silencing of the lignin pathway genes could reduce the lignin content and alter its composition. Such genetic modification of genes and transcription factors controlling the developmental processes may lead to an increase in biomass production. The feasibility of these plants also need to be further assessed by utilizing them for ethanol production using various pretreatments and fermentation procedures used commercially. Research is also warranted on whether the increase in saccharification efficiency is due to the ease in cellulose availability or a reduction in enzyme inhibition in these modified plants.

There might be many more genes and transcription factors that may lead to reduction in lignin content and composition. The identification of these may further aid lignocellulose digestibility. There is also a need to focus research attention on other aspects such as increasing the enzymatic hydrolysis rate, ready availability of cellulose, reduction in crystallinity of cellulose, removal of hemicelluloses as well as the improvement of enzyme efficacies of cellulases and hemicellulases. Research on the expression of cellulases, in planta, under extreme conditions and its thermal stability also needs to be carried out. The cost of lignocellulosic ethanol production may also be reduced by genetically modifying switchgrass to produce the enzymes that are either required or will be useful during fermentation. Devising strategies for recycling these enzymes will also lead to reduction in cost.

Altering switchgrass development Improvement in the rate of saccharification efficiency, which is inhibited by the complex structure of the plant cell wall, is an important objective in developing a competent and lucrative biofuel industry [79,80]. The potential of enhancing biomass yield by manipulation of gene regulatory factors such as microRNAs (miRNAs), that regulate transcription factors controlling growth and development in plants, has been tested [68,80,81,82]. Less lignifications and variations in biomass accumulation have been observed in juvenile plants and these properties may enhance the production of biofuels by decreasing the recalcitrance of biomass [80,83]. Belonging to the miR156 class, the maize Corngrass1 (Cg1) gene is targets the SQUAMOSA PROMOTER BINDING LIKE (SPL) family and reduces lignification while promoting juvenile characteristics in plants [84,85]. To study how juvenile characters improve the biofuel potential of switchgrass, the Cg1 gene was constitutively expressed in 'Alamo' by Agrobacterium-mediated transformation [80]. In another study, to generate over-expressed mature PvmiR156 in switchgrass, the OsmiR156b precursor fragment was introduced using Agrobacterium-mediated transformation containing the pANIC6APre-OsmiR156b vector [81]. In both these studies, the transgenic plants were differentiated based on the variations in their morphology, biomass accumulation, cell-wall carbohydrate accessibility and forage digestibility. Based on the level of expression of the introduced gene and the morphology of the transgenic plants, they were divided into three categories. Transgenic plants expressing low levels of the introduced gene were similar to wild type with no effect on flowering time but had juvenile cell identity. Moderate levels affected the plant height and flowering but improved the tiller number and biomass accumulation with plants having bigger leaves and thicker stems. High levels induced severe dwarfism and reduced biomass accumulation [80,81]. Both the studies also reported an increase in the amount of fermentable sugar, improved forage digestibility and biomass yield. These studies highlighted that the genetic manipulation of miR156 controlled apical dominance, biomass composition, digestibility, and flowering in switchgrass and also presented a proficient approach for transgene containment. Biofuel production may be further augmented by transgene pyramiding by crossing the miR156 over-expression lines with other transgenic lines with lower recalcitrance transgenic events [81]. These studies highlight the potential utility of this approach for the domestication of new switchgrass cultivars and the lack or delay in flowering will have important implications for the limitation or prevention of transgene flow into native/wild switchgrass plants. Recently, it was demonstrated that the expression levels of miR156 and miR162 could be changed under drought condition in switchgrass [126]. Introduction of such genes into switchgrass will lead to its further improvement. The specificity of the target silencing and the stable inheritance of miRNA-mediated transgenes in the progenies make this technology highly promising and applicable for switchgrass improvement. Genetic engineering can also be used to increase the biomass by modifying the plant growth regulators such as increasing the biosynthesis of gibberellins [86] to improve the growth and increase the biomass in switchgrass. Though all these studies are and will be significant in their own way, it is very important to observe the effect of these modifications on the biomass production potential of switchgrass. Not only the reduction in recalcitrance but the biomass yield is also crucial for making switchgrass a better biofuel feedstock. Hence, those genes which reduce the lignin content and composition without compromising the biomass yield and can enhance the conversion properties for better utilization in biorefineries will be more preferable. Transgene pyramiding by breeding various transgenic lines showing higher saccharaification efficiency may provide another effective strategy for improving the production of lignocellulosic biofuels.

Genetic and genomics resources

Molecular markers A number of DNA marker systems such as restriction fragment length polymorphism, chloroplast DNA, randomly amplified polymorphic DNA, amplified frag­ment length polymorphism and simple sequence repeats (SSRs) have been developed for the genetic diversity assessment and phylogenetic studies in switchgrass [20,87,88,89,90,91,92,93]. These studies were able to reveal the two prevalent ecotypes of switchgrass and will be useful in developing germplasm conservation and breeding programs [94]. Genetic linkage maps have also been constructed using single dose restriction fragments, SSRs, sequence-tagged sites (STS) markers, expressed sequence tags (EST) derived SSRs, gene derived STS, and diversity array technology markers [95,96,97,98,99]. These linkage maps will aid in the identification of quantitative trait loci linked with biomass yield, plant composition and other important agronomic traits. They will also help in understanding the genome structure and may provide genetic framework to facilitate marker-assisted breeding and genomics research in switchgrass.

Over the last few years, even though various technologies have become proficient for whole genome sequencing, it is still technically difficult and expensive to completely sequence complex polyploid species such as switchgrass [100,101]. ESTs are randomly arrayed cDNA clones that give information on the sequence and are valuable in discovering gene transcripts, and sequence determination [102,103]. Having high functional information, ESTs offer a practical analytical option for gene annotation and gene discovery in complex non-model systems with big genomes wherein whole genome sequencing is daunting [101,104,105]. ESTs have been successfully used for identification of molecular markers, analysis of tissue-specific patterns of expression or for comparative genomics [103,106]. cDNA libraries derived from leaf, stem, crown, and callus of 'Kanlow' were utilized for generating 11,990 individual sequences of which 7,810 were unique gene clusters [103]. The blast similarity and functional classification of these unique gene clusters was also performed. EST sequence information can also be mined for DNA sequence polymorphisms such as single nucleotide polymorphisms (SNPs) and SSRs that can be used for genome characterization and genetic diversity assessment [92]. For developing SSR markers, Tobias et al. assessed the unique gene clusters and reported the occurrence of short tandem repeats in 3.8% of the ESTs tested [103]. ESTs were also produced by end-sequencing of callus, crown, and seedling tissue derived cDNA libraries of 'Kanlow' and the assembled consensus sequences was aligned with sorghum genome [106]. They observed that 3.3% of the sequences were similar to potential cell-wall related genes. Millions of ESTs from tissue or xylem cell-specific EST libraries of 'Alamo' are also now available (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=switchgrass) [55].

SSRs and EST-SSRs are significant resources for developing dense linkage maps and identifying economically important traits for utilization in molecular breeding programs intended to develop superior switchgrass cultivars [92]. EST-SSR markers were identified and assessed for the production of fragment length polymorphisms in the two individual parents of a mapping population [106]. To identify SSR sequences longer than 20 bp, available sequence data from switchgrass were assessed using the program SSRIT and approximately 32 genic di-, tri- and tetranucleotide repeat SSRs were characterized [107,108]. When used to differentiate 'Alamo' and 'Kanlow' individuals, these SSRs exhibited a high degree of polymorphism consistent with their tetraploid, allogamous behavior [108]. Using genomic DNA of 'SL93 7 x 15', Wang et al. reported the construction of five genomic SSR-enriched libraries and identified 1,300 unique SSR-containing clones [92]. These switchgrass SSRs can be extensively used for identification and diversity analysis of germplasm, development of dense linkage maps, identification and development of quantitative trait loci for marker-assisted selection, and gene cloning for introduction of specific traits via genetic engineering. Thus, the expanding capabilities of genomics and bioinformatics have the potential to revolutionize the entire field of switchgrass biology and genetics, and they offer promise of greatly improving the cultivars that are grown using precise and targeted manipulations of the genome. Development of these markers will facilitate the on-going mapping efforts and population diversity analysis in switchgrass.

BAC libraries and physical mapping Efforts to map important traits for enhancing the breeding programs, and utilizing map-based cloning for the isolation of target genes are dependent on the availability of an extensive physical and genetic maps. This necessitates the development of physical map of switchgrass and will also provide the required framework for precise assembly of the genome [109]. Also, before the whole genome sequencing and assembly can be accomplished, it is imperative to approximate the structure and composition of switchgrass genome by producing and sequencing bacterial artificial chromosome (BAC) libraries [94,110,111]. 'Alamo' has been extensively used in switchgrass breeding programs and is the parent of several mapping populations, so therefore it follows that the current whole-genome sequencing effort is focused on an 'Alamo' clone: AP13, which is a heterozygous tetraploid with two subgenomes [94]. Saski et al. assembled the first BAC library, by incomplete digestion of nuclear DNA of the 'Alamo'-derived genotype, SL93 2001-1 with EcoRI, which had approximately ten-fold coverage of the total nuclear content and five-fold of each of the two genomes based on a genome size of 3.2 gigabases (~1.6 Gb per genome) [109]. They also successfully carried out comparative sequence analysis of the rice OsBRI1 gene, which is correlated with biomass. Since the study was restricted to a single locus and restriction enzyme, it warranted the need of additional libraries to attain fair and near-complete depiction for genome-wide studies. Recently, two BAC libraries were constructed from the AP13 clone of 'Alamo' and characterized using HindIII and BstYI [111]. The BAC-end sequences (BES) produced led to the detection of SSRs, known and novel repeat elements and will be highly significant for the inundation of linkage, physical and genetic maps of switchgrass [111]. Comparative analysis with other grass genomes such as foxtail millet, sorghum, rice, maize, and Brachypodium revealed high levels of homology with switchgrass exhibiting high microcolinearity with foxtail millet as compared to sorghum [112]. In addition, HudsonAlpha/Joint Genome Institute (JGI) has generated BES from randomly selected BACs (http://genomicscience.energy.gov/). These studies provided a precise BAC-based physical platform which offers a definitive approach for sequencing and assembly of switchgrass genome. They will also be able to give a precise estimate of the GC content, distribution of known, novel and repeat elements, and, thus, of the genome structure and composition of switchgrass.

Sub-organelle genome sequencing Chloroplasts are invaluable for genetic and phylogenetic studies. They are highly conserved, have small size, are maternally inherited and exhibit high potential in transgene expression studies [113,114]. To differentiate genetic diversity in whole chloroplast genomes and a large number of nuclear loci in switchgrass, an unique strategy utilizing high-throughput sequencing of multiplexed restriction-digested reduced-representation libraries was used for the identification of SNPs [115]. The SNPs identified were able to characterize eight haplogroups. Switchgrass chloroplast genomes were also sequenced from individuals of the upland ('Summer Lin2') and lowland ('Kanlow Lin1') ecotypes giving an insight regarding the amount of variation within the two ecotypes, and facilitated comparisons within the ecotypes as well as among other sequenced plastid genomes [116]. These studies emphasize the use of chloroplast genome for comparing genetic variation between the upland and lowland ecotypes, are highly desirable for robust phylogenetic studies and can be used in differentiating mixed population into up- or lowland ecotypes. The complete chloroplast genome will facilitate the generation of species specific transformation vectors [117] and will create an opportunity for the utilization of plastid genetic engineering in switchgrass.

Whole genome sequencing Basic characterization of the switchgrass genome indicates that the tetraploid lowland cultivars have a nuclear DNA content of 3.07 ± 0.06 pg per nucleus on an average [118]. This makes the effective genome size to be ~1600 Mb for 'Alamo' derived genotypes, which is approximately twice that of sorghum and about three and a half times that of rice [109,119]. Even with the availability of new and modern technologies, whole genome sequencing (WGS) of switchgrass would be difficult to achieve due to its large genome size and polyploidy. A practical solution to Sanger sequencing may be provided by pyrosequencing or other such next generation sequencing (NGS) technologies that offer quick and inexpensive technologies for transcriptomics by avoiding extensive and comparatively low throughput steps [120,121,122]. For de novo sequencing and transcriptomics of complex genomes, 454 pyrosequencing is the most extensively exploited NGS technology. GS FLX Titanium, the latest 454-sequencing platform, can produce a typical read length of approximately 330-700 bases [123,124].

'Alamo' AP13 has been chosen for genome sequencing by Joint Genome Institute (http://genome.jgi.doe.gov/genome-projects/). Sequencing of cDNA libraries produced from various tissues of switchgrass utilizing GS-FLX Titanium technology produced large number of reads for de novo assembly, and EST and SSR identification [101]. The accessibility to the foxtail millet draft genome also enhanced the switchgrass EST assembly and has almost doubled the EST information in the public domain. US DOE-JGI has been using a combination of Roche 454-based and Illumina-based sequencing to produce the switchgrass genomic sequence [94]. Initial investigations on assembly of switchgrass genome onto the foxtail millet framework led to the identification of paralogous assemblies from homoeologous ones [112]. However, autonomous assembly of both the subgenomes to achieve chromosome-scale contiguity for the reference will be daunting [94]. Though dihaploid lines may simplify sequence assembly in switchgrass, they are not preferred for whole genome sequencing due to their elevated infertility and instability [124,125]. The genome of switchgrass will help the biologists to determine the function and biotechnological potential of all the genes especially those responsible for increasing the biofuel potential such as biomass yield, decreasing the lignin content and improving saccharification efficiency. Furthermore, comparative analysis of switchgrass with other sequenced grass genomes such as foxtail millet and sorghum will enable a more detailed annotation, and will play an important role in understanding how gene networks evolved and function (National Plant Genome Initiative:2009-2013; http://www.nsf.gov/bio/pubs/reports/). This will also lead to marker-selection and genomic selection studies in switchgrass.

Gene expression studies Joint BioEnergy Institute scientists enriched the switchgrass Affymetrix chip by the addition of cell-wall and stress-related genes from full-length sequences of BACs. Assembly of unigenes from transcript and cDNA sequences was achieved by the utilization of Newbler and PAVE while RNASeq analysis will further improve the switchgrass Gene Expression Atlas (http://genomicscience.energy.gov/). Information on the fundamental biology and the regulatory mechanisms of gene expression in switchgrass under abiotic stress conditions are required for determining the consequences of genetic improvements and for detection and manipulation of stress tolerance related gene candidates [126]. Mature miRNAs inhibit gene expression at the post-transcriptional levels by either targeting mRNAs for degradation or inhibiting protein translation [82,127]. Utilizing comparative analysis, switchgrass miRNAs that potentially control regulatory genes including those engaged in cellulose biosynthesis and regulation, sucrose and fat metabolism, signal transduction, and plant development have been identified [128]. Gene annotation also detected miRNAs with possible role in biofuel-related metabolic pathways [128]. Investigations on the effect of salt and drought stress on the expression of miRNAs revealed an altered expression pattern of miRNAs in a dose-dependent manner [126]. Transgenic plants expressing the miR156 gene were produced using Agrobacterium-mediated transformation and the transgenic line, T-44, exhibiting severe morphological alterations, was used to investigate the effects of miR156 over-expression on its downstream genes using Affymetrix microarray analysis [81]. The study discovered that transcript abundance reduced in eight SPL gene probe sets, leading to the expression analysis of the corresponding cDNA sequences which showed that the transgenic lines with the maximum miR156 level had the most reduction in PvSPLs transcript abundance [81]. An atlas of gene expression has been developed for switchgrass [Zhang et al. 2013] and P. hallii [Meyer et al. 2012]. Such gene expression analyses will further augment the characterization and expression of genes controlling the biofuel traits, enhance the functional genomics studies and molecular breeding, and may further help in the assembly of the switchgrass genome. To take advantage of the new DNA sequence information and to investigate the functions of specific genes, targeting induced local lesions in genomes (TILLING) was developed. TILLING is a non-transgenic technology which utilizes a reverse genetics approach for the production and detection of mutation [129]. EcoTILLING is a variation of TILLING that investigates the natural variation among cultivar/ inbred line/accession when aligned with a sequenced reference genome for the identification of SNPs [130,131]. In switchgrass, TILLING, EcoTILLING, or a permutation of both are being utilized [131]. This will lead to the identification of multiple SNPs within a target region of switchgrass accessions and when compared to a reference genome will be able to define the relatedness and differences among the target region. Traits such as biomass yield, saccharification efficiency, and flowering time may be potentially identified in switchgrass using these techniques. The limitations being that the mutations may be introduced randomly throughout the genome, and a large number of individuals need to be screened to identify the mutants having the trait of interest. It will also be difficult to identify recessive mutants due to the polyploidy nature of switchgrass.

Switchgrass has been the topic of important discoveries and relevance in genomics and biotechnology in the last decade [29,46,47,62]. Significant trait improvement via biotechnology [e.g. 68,72,73] with increased transformation efficiency [50] has been demonstrated in switchgrass. This suggests that genetic improvements of biofuel properties of switchgrass through expression and down-regulation of transgenes is a practical way to rapidly establish it as a viable bioenergy crop on a commercial level and will be achieved with growing reliability in the coming years [59,80,81]. Though transgenic approaches are considered imperative for the development of switchgrass and other biofuel crops, their cost-effectiveness will be dependent on their domestication, productivity and biofuel properties [44]. However, a regulatory necessity will likely be bioconfinement [10,132,133].

Transgene escape has been considered as a major environmental, ecological and regulatory concern. Hence, for commercialization of transgenic switchgrass, efficient and reliable transgene bioconfinement strategies would be enabling, especially in US and where there are wild stands of switchgrass. While transgenes can be vectored in pollen or seed and less commonly asexually, the prospective for long-distance pollination has made pollen-dispersed transgenes a major concern [134]. One strategy to control geneflow in switchgrass would be to introduce male sterility using transgene-encoded ribonucleases that inhibit pollen formation [135,136]. With switchgrass being wind-pollinated, the excision of transgenes from the pollen genomes using site-specific recombination systems will also be desirable [137,138]. Another strategy would be to use plastid (chloroplasts or mitochondria) transformation for the introduction of cytoplasmic male sterility into switchgrass, and thus developing plastid transformation for switchgrass would be helpful. Since the pollen of most plant species contain no chloroplasts, pollen spread will not introduce the foreign genes into wild or non-transgenic switchgrass populations [139,140]. Thus, strategies for transgene bioconfinement and alleviation of gene flow and research that facilitates the utilization of information and proper regulatory guidelines for transgenic feedstocks are essential in developing the biofuel industry's infrastructure [10], including that for switchgrass [133]. The challenge is to generate efficient methods and procedures to accomplish elevated levels of agricultural productivity while conserving the environment and natural resources [7].

Recent advances in switchgrass genomics will further facilitate biotechnological interventions as well as its germplasm improvements via conventional and molecular breeding. The close collinearity of the switchgrass genome with other grasses will aid in the elucidation of gene function, regulation, and expression by leveraging off other resources. The application of new knowledge and tools developed from genomic resources such as identification of genes like those involved in the lignin pathway, saccharification efficiency, biomass yield, nutritional quality, and pest resistance will help geneticists and plant genetic improvement managers to overcome the limitations associated with conventional breeding, make sexual hybridization more efficient and manipulate various traits effectively. It is important to keep in mind, however, that the utility of new genetic combinations must be demonstrated ultimately by field trials and the value to consumers. Although plant biotechnology will play an important role to the successful generation of energy crops, it should be followed up with breeding programs aimed at sustaining or improving the significant agronomic attributes which made these plants imperative for biofuel generation to start with, namely resistance to abiotic and biotic factors, and low fertilization requirements [141]. The critical issue to be dealt with is how to improve the conversion efficiency from the solar energy to biofuel energy such that biofuels can meet anthropogenic energy consumption demand

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