Identifizierung und Charakterisierung von Komponenten der Mannan Synthese zur Steigerung der Biokraftstoff Gewinnung aus Lignocellulose
Zusammenfassung der Projektergebnisse
Lignocellulosic feedstock derived from energy crops is of particular importance for the production of biofuels and bio-based products. To realize the plan to particularly liberate the economy and transport sector from the reliance on fossil fuels renewable and sustainable biofuels and bio-based products are expected to gain importance in the near future. To meet the highly increasing demand and to circumvent conflicts of food vs. energy crops growing on the limited area of arable land biofuel production from lignocellulosic feedstock needs to become highly effective. Currently, commercial production of second generation biofuels is in its infancy and just entering commercialization. There is tremendous room for optimization. Therefore, research on lignocellulosic material as a feedstock for biofuels is fundamental and should be highly prioritized. There are several points of optimization to create plants with increased lignocellulosic feedstocks characteristics. One promising approach is to increase non-crystalline C6 sugars in plant cell walls. As a DFG Fellow, I set up a co-expression screen with genes known to be involved in cell wall bio synthesis to generate candidates for increased amount of non-crystalline C6 sugars in plant cell walls. The co-expression approach yielded 13 candidates and after screening all genes I proceeded to single gene analysis with the two most promising candidates 6 and 13. Candidate 6 showed strong interaction with Csla14 that is involved in mannan synthesis (Probable mannan synthase 14) decreased mannan in stem and different mannan structure in candidate 6 mutant plants and Candidate 6 protein was successfully expressed heterologously in Echerichia coli. Ongoing experiments include stable overexpression in plants, identification of protein structure and function, and identification of additional interaction partners. Candidate 13 mutant plants showed WT like mannan content and structure but revealed an increase in overall fucose content. Fucose is present in three different structures in plants (i) xyloglucan fucosylation, (ii) arabinogalactan protein fucosylation, and (iii) rhamnogalactan (RG) II. I tested all three possibilities and only xyloglucan fucosylation seemd to be different in both mutant lines compared to WT. Matrix-assisted laser desorption/ionization (MALDI) coupled to time-of-flight mass spectrometer (TOF) was used to identify effected oligosaccharide and this method is currently under investigation. Candidate 13 protein was successfully heterologous expressed in E. coli and purified protein was submitted within JBEI for further characterization and to identify the effected polysaccharide in xyloglucan. Completing both single gene analyses is subject of ongoing projects and further promising candidates that resulted from the co-expression screen will be analyzed in the future.
