BGC, BMMB, PLBIO
The Tien Lab has two main research areas. Initial impression is that they are not related; however, they share the common theme of increasing the efficiency of biofuel production - one through understanding the synthesis of biomass and the other in understanding how it is degraded.
The Tien Lab also does research into dissimilatory iron reduction, with applications in alternative energy and bioremediation.
The study of cellulose synthesis
Cellulose is the most abundant biopolymer on earth. Every day, a vast quantity of solar energy is stored in the form of chemical bonds through the process of photosynthesis. One major sink for this carbon biopolymer is the cell wall, which is comprised of 20 to 90% cellulose, making cellulose an abundant, renewable energy source just waiting to be utilized. However, the natural crystallinity of cellulose, along with other cell wall components like lignin, make plant biomass recalcitrant to degradation, and the amount of energy input required to bread down cellulose significantly reduces the yield. But what if one could modify the synthesis of cellulose to better suit our energy, and other needs?
Our research attempts to uncover the fundamental nature of cellulose synthesis and its obligate proteins. We are trying to better understand how these proteins function so that informed decisions can be made on how to modify cellulose synthesis. Cellulose synthesis research is currently the main focus of Tien Lab.
Our lab studies cellulose synthesis in three main model organisms: the cellulose synthesizing bacteria Gluconacetobacter hansenii, the moss Physcomitrella patens, and the model plant, Arabidopsis thaliana. In Gluconacetobacter, the cellulose synthase operon encodes 3 proteins - AcsAB, AcsC, and AcsD, which work in conjunction to synthesize and extrude cellulose outside of the cell. In higher plants, including Arabidopsis, there are multiple cellulose synthase proteins (CesAs) that assemble at the plasma membrane to form a cellulose generating mega complex (theorized to be up to 4 Megadaltons in size). Evidence suggests that different isoforms of the CesA proteins are required in a specific arrangement and stoichiometry in order to successfully synthesize cellulose. To approach our research we use many classical biochemical techniques such as western blotting, Blue Native PAGE, formaldehyde crosslinking, immunoprecipatation, in vitro cellulose synthesis assays, column purification of cellulose synthase proteins, heterologous expression, X-ray crystallography, isothermal titration calorimetry, plant transgenics, analytical ultracentrifugation, 2-D PAGE, proteomics, and other various ways to assess, purify, and characterize proteins biochemically.
Fungal lignin biodegradation
The degradation of lignin plays a key role in carbon recycling on earth. Lignin, an aromatic polymer, is second only to cellulose in abundances as a renewable carbon source and accounts for approximately 20% of all the carbons fixed by photosynthesis. Lignin is nature’s plastic imparting rigidity to woody biomass and conferring resistance to wood from most forms of microbial attack. The degradation of lignin is brought about predominantly by filamentous fungi. Due to the heterogeneity of woody biomass, an ensemble of enzymes is required to degrade this substrate to carbon dioxide. Both hydrolytic and oxidative enzymes are involved. The hydrolytic enzymes are used for depolymerization of cellulose whereas the oxidative enzymes are used for depolymerization of lignin. Our past research efforts have focused on the enzymology and regulation of the oxidative enzymes. These oxidative enzymes generate free radicals in lignin resulting in its depolymerization.
Despite years of research by many labs, the identity of the enzymes involved in wood degradation is still not known. Our past efforts have focused on mechanistic studies of peroxidases, Mn peroxidase and lignin peroxidase. However, to degrade wood, an ensemble of enzymes is required. The recent completion of the sequencing of a fungal genome, Phanerochaete chrysosporium and advances in methodology/instrumentation now allows us to identify all of the proteins produced to degrade wood. We are using a proteomics approach toward identifying all of the extracellular proteins produced by P. chrysosporium when grown on wood. Protein spots are excised from 2-dimensional gels, digested with trypsin and the peptides are sequenced by LC/MS/MS. Using this method, we are in the process of identifying the greater than 40 proteins produced when P. chrysosporium degrades oak. Ongoing research involves determining the role of the wood substrate on enzyme production and the succession of enzymes involved in degradation of wood.
Dissimilatory Iron reduction
Micro-organisms are known to use over 20 elemental systems other than O2 to accept electrons during respiration. Of these, only 6 are known to be respired in solid form: S, As, Se, U, Fe, Mn. Of these six, the ability to reduce iron is the most common among deeply branching members of Archaea and Bacteria. Microbial Fe respiration is important because it dominates reduction of iron in a large number of natural systems today. Interest in this process, largely focused on Shewanella and Geobacter, has intensified recently because bioreduction of iron oxides releases co-precipitated or sorbed contaminants in the subsurface. Using the Shewanella oneidensis, we aim to elucidate the biochemical mechanism of iron reduction. Collaborating with Dr. Susan Brantley at Penn State’s Department of Geoscience, we have utilized a protemics approach toward understanding the enzymology of this process. Using 2-D gel electrophoresis, we have identified a number of proteins that are uniquely expressed under iron-reducing growth conditions. Because S. oneidensis has been recently sequenced, we have used trypsin fingerprinting using MALDI/TOF mass spectroscopy. In addition, we are the first team to develop an in vitro (cell-free) model system derived from Shewanella wherein membrane fractions directly reduce solid-phase Fe minerals. Ongoing research aims to purify the enzymes involved in this process.