Research Team 4 - Results & Status

Judson Sheridan

Results and Status as of October 2010

Sub-team A

The joint activities focus on CBP21, a protein produced by Serratia marcescens that acts synergistically with chitinases in the degradation of crystalline chitin (see picture and Vaaje-Kolstad et al., 2005). CBP21 is a member of the so-called CBM33 family of proteins. Using directed evolution (at UMN) we want to find out (1) how this protein works and (2) if we can engineer variants of this protein that work on cellulose (i.e. act synergistically with cellulases). Since the start of the project, it has become clear that there exist another family of proteins, called GH61, that act synergistically with cellulases and that are structurally similar to CBM33 proteins. There are also indications that there exist CBM33 variants that work on cellulose. All in all, this shows that our goals are realistic.

In the initial phase of the project, much time has been spent (at UMN) on developing screening methods for the directed evolution experiments. We screen both for binding (to chitin, cellulose) and for synergistic activity (i.e. enhancing the activity of e.g chitinases). Both types of assays have now been developed. Mutant libraries of CBP21 have been made (mainly by saturation mutagenesis) and several mutants have been picked-up (by a binding screen). These mutants are currently being characterized. Several interesting mutants have been identified.

Both UMB and UMN are now carrying out structural work on CBP21 using NMR techniques. The main goal here is to get insight into the role of dynamics in CBP21 function.

After initial observations concerning the role of metal ions in CBP21 functionality in summer 2009, the UMB group has recently found that CBP21, and, by analogy, GH61 in fact are enzymes that introduce chain breaks on the crystalline surfaces of their substrates (Vaaje-Kolstad et al., 2010). In this way, the crystalline surface is disrupted and further degradation by normal hydrolytic enzymes (chitinases) is facilitated. It was also shown that activity of CBP21 can be increased to previously unseen very high levels by adapting the reaction conditions. These novel findings are of great importance for the joint UMB-UMN project and will boost interest in CBP21-like and GH61 proteins. Clearly the ongoing joint project is very “hot”.

Zarah Forsberg has started her Ph.D. project by cloning, expressing and characterizing several naturally occurring CBM33 proteins (i.e natural variants of CBP21). We are particularly interested in finding CBM33 proteins that work on cellulose. Later on, she will carry out directed evolution experiments.

Image of CBP21

Sub-team B

In regard to the ligninolytic enzymes in white-rot fungal secretomes, the mechanistic basis of lignin cleavage during lignocellulose biodegradation has been provisionally identified. The activity of an enzyme exhibiting such behavior is being studied in regard to its effect on native lignin preparations with molecular weights in the hundreds of thousands. Under favorable conditions near neutrality, the radius of gyration of the native lignin substrate can be reduced by 30% within 12 hours.

To investigate the genomic basis of the ligninolytic enzymes, we are mapping the Trametes cingulata RNAs to the Trametes genome using next-generation sequencing data which tests our gene prediction that were made strictly using computational methods. We are also defining the transcriptomes of Trametes and Herterobasidion parviporum by de novo transcriptome assembly. The transcriptome results can be used to identify genes and in Trametes to define their boundaries. We are also growing these two white-rot fungi on a variety of carbon sources, such as glucose, cellulose and wood. The RNAs from these cultures will be extracted and analyzed by massive next-generation sequencing to determine the gene expression patterns. Those genes that are highly expressed as the fungus grows on wood as compared to their expression as the fungi grow on glucose or cellulose will be identified. These genes and one of more of their products may be intimately involved in the ligninolytic activity.

Updated: 29.10.10
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Cooperation UMN-UMB

Cooperation between University of Minnesota (UMN) and the Norwegian University of Life Sciences (UMB). Contact persons:

Claudia Schmidt-Dannert

Professor, Biochemistry, Molecular Biology and Biophysics

College of Biological Sciences, St. Paul, UMN

Office Phone: 612/625-5782

Ragnhild Solheim

Research Director, UMB

Office Phone: +47 6496 6191