Production of glucaric acid from hemicellulose substrate by rosettasome enzyme assemblies

Authors: Lee, Charles; Kibblewhite, Rena; PAAVOLA, CHAD - National Aeronautics And Space Administration (NASA); Orts, William; Wagschal, Kurt

Submitted to: Molecular Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/30/2016
Publication Date: 6/4/2016
Citation: Lee, C.C., Kibblewhite, R.E., Paavola, C., Orts, W.J., Wagschal, K.C. 2016. Production of glucaric acid from hemicellulose substrate by rosettasome enzyme assemblies. Molecular Biotechnology. 58:489.

Interpretive Summary: Hemicellulose substrates are a rich source of chemical feedstocks that should be utilized for the overall economic efficiency of a biorefinery. We demonstrated that enzymes presented as a complex on a genetically-engineered protein scaffold can significantly enhance the yield of glucaric acid, a top-12 DOE platform chemical. We were able to quickly test a wide range of enzyme ratios to determine the optimum blend for our substrate. In applying this technology to other targets, it is anticipated that the best enzyme ratios will vary when applied to complex substrates such as biomass.

Technical Abstract: Hemicellulose biomass is a complex polymer with many different chemical constituents that can be utilized as industrial feedstocks. These molecules can be released from the polymer and transformed into value-added chemicals through multistep enzymatic pathways. Some bacteria produce cellulosomes which are assemblies composed of lignocellulolytic enzymes tethered to a large protein scaffold. Rosettasomes are artificial engineered ring scaffolds designed to mimic the bacterial cellulosome. Both cellulosomes and rosettasomes have been shown to facilitate much higher rates of biomass hydrolysis compared to the same enzymes free in solution. We investigated whether tethering enzymes involved in both biomass hydrolysis and oxidative transformation to glucaric acid onto a rosettasome scaffold would result in an analogous production enhancement in a combined hydrolysis and bioconversion metabolic pathway. Three different enzymes were used to hydrolyze birchwood hemicellulose and convert the substituents to glucaric acid, a top-12 DOE value added chemical feedstock derived from biomass. It was demonstrated that colocalizing the three different enzymes to the synthetic scaffold resulted in significantly higher levels of product compared to uncomplexed enzymes.