1. Engineering Bacillus subtilis biocatalysts for production of biofuels and chemical feedstocks and biochemicals for pharmaceutical and nutraceutical applications. Mun Su Rhee, Lusha Wei, James F. Preston Department of Microbiology and Cell Science, University of Florida Goal: Develop bacterial biocatalysts for consolidated bioprocessing (CBP) of lignocellulosis a.Secretion of cellulases and xylanases to process cellulose and xylans b.Fermentation of glucose and xylose for production of biofuels and chemicals Objective: Engineer Bacillus subtilis for CBP of methylglucurono(arabino)xylans (MeG(A)X n ) a.Engineer B. subtilis to convert MeGAX n to lactate for production of bioplastics b.Engineer B. subtilis to convert MeGX n to xylooligosaccharides : acidic (U-XOS) for production of anti-inflamatory agents and neutral (XOS) for production of prebiotics Collaborators: L. O. Ingram, K. T. Shanmugam, W. Vermerris, J. Erickson, Support: FESC, USDA Biomass Research & Development Initiative

2. Cellulose fiber 32-48% MeGAX n 18-35% MeGX n Interaction of Major Polymeric Sugars in Lignocellulosic Biomass  Cellulose fibers form through hydrogen bonding interactions between individual cellulose stands  The noncarbohydrate polymer lignin embeds the interacting cellulose and glucuronoxylan through ester linkages to glucuronoxylan Monocots: grasses, e.g. sorghum, sugarcane Dicots: hardwoods, e.g.eucalyptus, sweetgum Methylglucuronoarabinoxylan, MeGAX n Methylglucuronoxylan, MeGX n

3. Pretreatment of O -acetyl-glucuronoarabinoxylan and enzymatic depolymerization of 4- O -methylglucuronoxylan

4. Schematic for bioconversion of MeGX n to U-XOS by B. subtilis strains. MeGX 3, MeGX 4 or MeGX 4-18 accumulate in the cultures of B.s. 168, MR42(ΔxynC) or MR44(ΔxynA), allowing production of XOS and/or U-XOS MeGX n (Sweetgum xylan) MeGX 4-12 MeGX 4 MeGX 3 XOS X 2, X 3 XynC (GH30) XynA (GH11) MR44 (168, ΔxynA ) MeGX 3 XynC (GH30) MR42 (168, ΔxynC ) 168 X 2, X 3 XOS

5. Monocot : MeGAX n Dicot : MeGX n A, XOS