INVESTIGATOR(S): Liu, C.-J.
INSTITUTION: Brookhaven National Laboratory
NON-TECHNICAL SUMMARY: The goal of GTL is to use newly acquired genomic data to better understand fundamental biological processes and enhance the translation of that scientific knowledge into new technologies for energy and environmental applications. Our project is going to characterize the O-acylation reactions participated in lignocellulosic biosyntheses using poplar genomics resources. O-acylation is involved in the cell-wall component polysaccharide and lignin biosyntheses. It affects the cell-wall sugar's solubility and the lignocellulosic bio-digestibility. O-acylation is also responsible for the structural modification and sequestration of a variety of polyphenolic metabolites required for wood formation. Understanding the mechanism of O-acylation at molecular level implicates biotechnological applications in genetic modification of lignocellulosic structures to facilitate biomass to bioethanol conversion, and in improvement of feedstock biomass production.
OBJECTIVES: 1) genome-wide identify acyl-CoA dependent acyltransferase genes from poplar genomics database; 2) systemically explore the tissue specific and stress-responsible expression patterns of O-acyltransferase genes to identify the enzymes specifically involved in lignocellulosic biosynthesis; 3)systemically characterize the biochemical functions of acyltransferases responsible for polysaccharide acetylation, lignol biosynthesis and phenolic compound modification.
APPROACH: Obj. 1. tblastn algorithm will be applied to search poplar genomics resources (P. trichocarpa V1.0, http://genome.jgi-psf.org) by using the highly conserved sequence motifs (HXXXD and DFGWG) of acy-CoA dependent acyltransferases. In order to distinguish the potential soluble and membrane bound proteins, the encoded polypeptides of the identified gene candidates (at least ~50 gene models) will be subjected to the computational topology and post-translational modification analyses by using PSORT and SignalP webservices to predict the protein sorting signal, subcellular localization site and the location of signal peptide cleavage sites in amino acid sequences. Obj. 2. The transcriptional profiling of putative acyltransferases will be analyzed both by "in silico" northern, based upon the high resolution poplar EST/microarray databases, and by QRT-PCR against mRNAs from different types of tissues (leaf, shoot, root, stem etc.) and the tissue sections representing different stages of developing wood, including the early expansion, late expansion, secondary cell wall formation, and programmed cell death (sapwood/heartwood) from poplars under normal growing conditions, the drought, salt stresses, and insect damage or physical wounding. Obj. 3. The recombinant proteins of the gene candidates that are highly expressed in wood-forming tissues will be produced using either E. coli, yeast, or Drosophila Gateway expression systems. Subsequently the combinatorial in vitro assays will be conducted by using different acyl-CoA donors and potential substrates including sugars, oligosaccharides, hydrolyzed and pre-deacylated pectin and xyloglucan, lignols, and other phenolics. Products detection and identification will be performed by LC-UV-ESI-MSn, HPAEC-MS and MALDI-TOF-MS analyses.
Name: Liu, C.-J.