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 lignocellulosisa. Secretion of cellulases and xylanases to process cellulose and xylansb. Fermentation of glucose and xylose for production of biofuels and chemicals

Objective: Engineer Bacillus subtilis for CBP of methylglucurono(arabino)xylans (MeG(A)Xn)c. Engineer B. subtilis to convert MeGAXn to lactate for production of bioplasticsd. Engineer B. subtilis to convert MeGXn 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

Cellulose fiber32-48%

MeGAXn 18-35% MeGXn

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, MeGAXn

Methylglucuronoxylan, MeGXn

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

Schematic for bioconversion of MeGXn to U-XOS by B. subtilis strains.

MeGX3, MeGX4 or MeGX4-18 accumulate in the cultures of B.s. 168,

MR42(ΔxynC) or MR44(ΔxynA), allowing production of XOS and/or U-XOS

MeGXn

(Sweetgum xylan)

MeGX4-12MeGX4

MeGX3

XOSX2, X3

XynC (GH30) XynA (GH11)

XynA (GH11)

MR44(168, ΔxynA )

MeGX3

XynC (GH30)

MR42(168, ΔxynC )

168

X2,X3XOS

Hemicellulose, xylan

Xylanase GH11Xylanase GH30

GH11 GH30

Acidic XOS

XOSPrebiotics

PharmaceuticalsNutraceuticals

Xylanase GH10Xylanase GH67

XOS

GH10 GH67

Fermentation

Biofuel and Biochemicals

Engineering B. subtilis as Biocatalyst for Production of Biofuel and Value-added Products

Bacillus subtilis

Bacillus subtilis

Monocot : MeGAXnDicot : MeGXn

A, XOS