Biomass Depolymerization programs
Biocatalysis and Conversion of Plant Cell Wall Polysaccharides to Biofuels
A major limitation of microorganisms used in fermentation of carbohydrates to ethanol is their lack of enzymes required for the efficient conversion of cellulose to sugars. Nature, however, has evolved such enzymes that degrade cell walls in places like the stomach of cows. This work is identifying the genes that encode for such enzymes, with a view to development of more highly active enzyme systems for use in the bioenergy-crops-to-fuels conversion.
We have expressed and demonstrated plant cell wall degrading activities in several enzymes derived from mesophilic and thermophilic bacteria. These include enzymes from the cow rumen and very high-temperature hot springs. However, these enzymes are produced at the milligram-per-liter culture level, and plant cell wall degradation for industrial production of value-added products, such as biofuels, will require large amounts of enzymes at very high costs. In order to produce enzymes at large quantities and at cheaper costs, we aim at producing these hydrolytic enzymes in filamentous fungi. It has been demonstrated that filamentous fungi, such as Trichoderma reesei, secrete enzymes or proteins at gram-per-liter levels. However, it is also known that heterologous expression of genes in filamentous fungi often leads to very low protein production. We are therefore investigating the molecular determinants of high gene expression in 2 filamentous fungi, Neurospora crassa and Aspergillus nidulans. We have cloned and expressed several glycoside hydrolase genes of the same family in the 2 fungi and we are currently using molecular methods to trace the path of the highly expressed and the lowly expressed genes from DNA transformation to recombinant protein secretion. It is anticipated that by understanding the molecular bases for the high expression of some proteins, we can manipulate the cells to increase the levels of production of the lowly expressed genes, especially the heterologous ones derived from bacteria. We are also using a plant and the mega-bacterium Bacillus megaterium as expression systems in an attempt to express enzymes that have been recalcitrant to expression in the common E. coli systems. These new enzymes should help augment our enzyme library aimed at creating designer enzyme cocktails for depolymerization of bioenergy feedstock of interest to the EBI.
We have assembled enzyme cocktails from mesophilic to hyperthermophilic bacteria for deconstruction of plant cell wall polysaccharides to simple sugars. We aim at expressing these enzymes in large quantities. To achieve this goal, we are using Neurospora crassa and Aspergillus nidulans as models to gain insight into the molecular basis for high expression of genes in filamentous fungi. We have cloned several glycoside hydrolase family 10 (GH10) genes in the two model fungi and followed the steps from transformation to protein secretion. Two of the genes, Ncu05924 (N. crassa gene) and Ani01818 (A. nidulans gene), expressed homologously and heterologously, have been classified as highly expressed genes based on our molecular analyses. In contrast, by using the same strategy, Ncu07130 and Ani02356 were classified as poorly expressed genes. Glycoside hydrolase genes encoding gene products of larger molecular masses than the GH10 proteins are being analyzed for highly and poorly expressed homologs. Our next step is to carry out RNAseq and proteomic analyses on the two groups of recombinant filamentous fungi (highly expressing and poorly expressing). It is anticipated that these analyses will identify molecular determinants that are unique to high recombinant protein production in filamentous fungi. Ultimately, the acquired knowledge should aid us in expressing our enzymes at the level required for industrial application.
We tested expression of glycoside hydrolase genes in Neurospora crassa and Aspergillus nidulans. The initial success in production of recombinant glycoside hydrolases in the two filamentous fungi was achieved through expression of Ncu05924, a protein that originates from N. crassa. The gene for Ncu05924 was successfully expressed in the two fungi, although the expression levels were quite low. By purifying the recombinant proteins, we determined that active Ncu05924 could be produced at approximately 1 mg/L. Further gene expression experiments demonstrated that a higher yield (about 3-fold) of recombinant proteins can be achieved if the gene was expressed in the presence of its introns. Consistent with these results, an A. nidulans glycoside hydrolase gene (Ani01818) that was neither expressible in N. crassa nor A. nidulans, in the absence of its introns, was successfully expressed in A. nidulans in the presence of its introns. Therefore, it appears that for successful expression of recombinant genes in the two filamentous fungi, the presence of introns in the target gene is important.
Gene expression based on the Gram-positive bacteria Bacillus subtilis and Bacillus megaterium was also successfully introduced to the lab. We demonstrated that a large endoglucanase, containing a GH48 catalytic module, could be readily expressed in B. megaterium. The gene celA is highly secreted in the cellulolytic and thermophilic bacterium C. bescii. In contrast, several attempts to express this gene in E. coli and B. subtilis failed. These gene expression systems expand the capacity of EBI scientists to express recombinant proteins. The gene expression systems, based on the two strains of Bacillus, are currently in use by other members of the EBI. Our group also used biochemical and structural analyses to characterize a large xylan degradation gene cluster from C. polysaccharolyticus. Furthermore, we used biochemical approaches to characterize several multi-domain cellulose-targeting enzymes from C. bescii.
Isaac Cann’s group in Illinois isolated lignocellulose-digesting enzymes from microbes found in hot springs. High-temperature bioconversion minimizes microbial contamination, increases enzyme activity and reduces cooling costs. They isolated genes from Caldicelluosiruptor bescii and engineered a hemicellulase “cocktail” that is stable at 70 degrees Centigrade and retains the majority of its activity after 24 hours. They also isolated cellulose-degrading enzymes from thermophiles that produce cellobiose that can be fed directly into fermentation systems developed by other EBI scientists.
The Cann group used transcriptomics analysis (microarray and RNAseq) to determine, for the first time, the strategy used by a hemicellulose degrading bacterium to deconstruct xylan. They also characterized a xylanolytic gene cluster containing all enzymes in the pentose phosphate pathway from a thermophilic bacterium. The DNA (18.4 kb) fragment containing the gene cluster can be used to transform relevant microbes into xylan utilizers. They assembled a hemicellulase mix that functions efficiently at 65 – 80 °C.
The genomes of two thermophilic bacteria were sequenced using 454 sequencing technology. Analysis of the genetic blueprints of the two organisms shows that T. bryantii is mostly equipped to break down hemicellulose, whereas C. polysaccharolyticus is well equipped with the enzymes required to break down both cellulose and hemicellulose. Based on the architecture of the two plant cell wall polysaccharides, the group used computer analysis to extract from the genomes the genes that target their hydrolysis. Through gene expression and biochemical analysis, they successfully assembled a thermostable hemicellulase mix composed of five synergistically acting enzymes (endoxylanase, beta-xylosidase, arabinofuranosidase, alpha-glucuronidase, and acetyl xylan esterase). This enzyme mix represents a minimal hemicellulase, which is able to release mainly xylose and arabinose from wheat arabinoxylan, a hemicellulosic model substrate. Transcriptomic analyses (microarrays and RNA sequence) were also used to identify enzymes in Prevotella bryantii that have the potential to enhance the efficacy of low-temperature (37 C)-acting hemicellulase.
Published in 2014
Xylan Utilization in Human Gut Commensal Bacteria is Orchestrated by Unique Modular Organization of Polysaccharide-Degrading Enzymes, Meiling Zhang, Jonathan R. Chekan, Dylan Dodd, Pei-Ying Hong, Lauren Radlinski, Vanessa Revindran, Satish K. Nair, Roderick I. Mackie, and Isaac Cann, Proceedings of the National Academy of Sciences, V. 111 (35), PMID: 25136124, doi: 10.1073/pnas.1406156111, April 2014.
Molecular Dynamics Study of Enhanced Man5B Enzymatic Activity, R. C. Bernardi, I. Cann, K. Schulten, Biotechnology for Biofuels, V. 7, pp. 83, June 5, 2014.
Published in 2013
Insights into Lignin Degradation and its Potential Industrial Applications, A. M. Abdel-Hamid, J. O. Solbiati, I. K. Cann, Advances in Applied Microbiology, 82: pp. 1-28, doi: 10.1016/B978-0-12-407679-2.00001-6, 2013.
Reconstitution of a Thermostable Hemicellulase Mixture from the Bacterium Caldicellulosiruptor bescii. Xiaoyun Su, Yejun Han, Dylan Dodd, Young-Hwan Moon, Shosuke Yoshida, Roderick I., and Isaac K.O. Cann, Applied and Environmental Microbiology, 79: pp. 1481-1490.
Mutational and Structural Analyses of Caldanaerobius polysaccharolyticus Man5B Reveal Novel Active Site Residues for Family 5 Glycoside Hydrolases. Takuji Oyama, E. Schmitz, Dylan Dodd, Yejun Han, Alanna Burnett, Naoko Nagasawa, Roderick I. Mackie, Haruki Nakamura, Kosuke Morikawa, and Isaac Cann, PLoS One 8:11, e80448.
Molecular and Biochemical Analyses of Cbcel9a/Cel48a, a Highly Secreted Multi-Modular Cellulase by Caldicellulosiruptor Bescii During Growth on Crystalline Cellulose, Zhoulin Yi, Xiaoyun Su, Vanessa Revindran, Roderick I. Mackie, Isaac Cann, PLoS One, 8:12, E84172, doi: 10.1371/journal.pone.0084172, December 2013
Published in 2012
Heterologous Gene Expression in Filamentous Fungi, Xiaoyun Su, George E. Schmitz, Meiling Zhang, Roderick I. Mackie, and Isaac K. O. Cann, Advances in Applied Microbiology, http://dx.doi.org/10.1016/B978-0-12-394382-8.00001-0, Sept. 11, 2012.
Supplementing with Non-Glycoside Hydrolase Proteins Enhances Enzymatic Deconstruction of Plant Biomass, Xiaoyun Su, Jing Zhang, Roderick I. Mackie, and Isaac K. O. Cann, PLos One, http://dx.doi.org/10.1371/journal.pone.0043828, Sept. 7, 2012.
Biochemical and Structural Insights into Xylan Utilization by the Thermophilic Bacterium Caldanaerobius Polysaccharolyticus, Yejun Han, Vinayak Agarwal, Dylan Dodd, Jason Kim, Brian Bae, Roderick I. Mackie, Satish K. Nair, and Isaac K.O. Cann, Journal of Biological Chemistry, http://dx.doi.org/10.1074/jbc.M112.391532, August 25, 2012.
Molecular and biochemical analyses of the GH44 module from CbMan5B/Cel44A, a 2 bifunctional enzyme from the hyperthermophilic bacterium Caldicellulosiruptor bescii, LiBin Ye, Xiaoyun Su, George E. Schitz, Yang Hwan Moon, Jing Zhang, Roderick I. Mackie, and Isaac K.O. Cann, Journal of Environmental Microbiology, http://doi.org/10.1128/AEM.02009-12, July 27, 2012.
Biochemical and Mutational Analyses of a Multi-Domain Cellulase/Mannanase from Caldicellulosiruptor bescii, Xiaoyun Su, Roderick Mackie, Isaac Cann, Applied and Environmental Microbiology, doi: 10.1128/AEM.06814-11, January 2012.
Published in 2011
Functional Analyses of Multiple Lichenin Degrading Enzymes from the Rumen Bacterium Ruminococcus, Michael Iakiviak, Roderick Mackie, Isaac Cann, Applied and Environmental Microbiology, doi:10.1128/AEM.06088-11, September 2, 2011.
Biochemical Analysis of Multiple Endoxylanases from the Rumen Bacterium Ruminococcus albus and their Synergistic Activities with Accessory Hemicellulose Degrading Enzymes, Young Hwan Moon, Michael Iakiviak, Stefan Bauer, Roderick Mackie, Isaac Cann, Applied and Environmental Microbiology, doi: 10.1128/AEM.00353-11, June 10, 2011.
Structural and Functional Analyses of a Glycoside Hydrolase Family 5 Enzyme with an Unexpected Beta-Fucosidase Activity, Shosuke Yoshida, David Park, Brian Bae, Roderick Mackie, Isaac Cann, Satish Nair, Biochemistry 50(16), pp. 3369-3375, doi: 10.1021/bi200222u, March 16, 2011.
Xylan Degradation, a Metabolic Property Shared By Rumen and Human Colonic Bacteroidetes, Dylan Dodd, Robert Mackie, Isaac Cann, Molecular Microbiology, 79(2), pp. 1365-2958, doi:10.1111/j.1365-2958.2010.07473.x, January 2011.
Published in 2010
Mutational Insights into the Roles of Amino Acid Residues in Ligand Binding for Two Closely Related Family 16 Carbohydrate Binding Modules, Xiaoyun Su, Vinayak Agarwal, Dylan Dodd, Brian Bae, Roderick Mackie, Satish Nair, Isaac Cann, Journal of Biological Chemistry, DOI: 10.1074/jbc.M110.168302, August 24, 2010.
Domain Analysis of a Modular (alpha)-L-arabinofuranosidase with a unique carobhydrate Binding Strategy from the Fiber Degrading Bacterium Fibrobacter succinogenes S85, Shosuke Yoshida, Charles W. Hespen, Robert Beverly, Roderick Mackie, and Isaac Cann, Journal of Bacteriology, DOI: 10.1128/JB.00503-10, August 13, 2010.
Transcriptomic Analyses of Xylan Degradation by Prevotella bryantii and Insights into Energy Acquisition by Xylanolytic Bacteroidetes, Dylan Dodd, Young-Hwan Moon, Kankishita Swaminathan, Roderick Mackie, Isaac Cann, Journal of Biological Chemistry, DOI: 10.1074/jbcM110.14178, July 9, 2010.
Thermostable Enzymes as Biocatalysts in the Biofuel Industry, Carl J. Yeoman, Yejun Han, Dylan Dodd, Charles M. Schroeder, Roderick Mackie, Isaac Cann, Advances in Applied Microbiology, 70: pp. 1-55, March 6, 2010.
Biochemical and Domain Analyses of FSUAxe6B, a Modular Acetyl Xylan Esterase, Identify a Unique Carbohydrate Binding Molecule in Fibrobacter succinogenes S85, Shosuke Yoshida, Roderick Mackie, Isaac Cann, Journal of Bacteriology, 192(2): pp. 483-493, January 9-13, 2010.
Comparative Analyses of Two Thermophilic Enzymes Exhibiting both beta-1,4 Mannosidic and beta-1,4 Glucosidic Cleavage Activities from Caldanaerobius polysaccharolytus, Yujan Han, Dylan Dodd, Charles Hesen, Samuel Ohene-Adjei, Charles Schroeder, Roderick Mackie, Isaac Cann, Journal of Bacteriology, 192(16): pp. 4111-4121, August 2010.
Published in 2009
Enzymatic Deconstruction of Xylan for Biofuel Production, Dylan Dodd and Isaac Cann, Global Change Biology - Bioenergy, 1: pp. 2-17, February 18, 2009