Fermentation of biomass-derived glucuronic acid by pet expressing recombinants of E. coli B.

Abstract

The economics of large-scale production of fuel ethanol from biomass and wastes requires the efficient utilization of all the sugars derived from the hydrolysis of the heteropolymeric hemicellulose component of lignocellulosic feedstocks. Glucuronic and 4-O-methyl-glucuronic acids are major side chains in xylans of the grasses and hardwoods that have been targeted as potential feedstocks for the production of cellulosic ethanol. The amount of these acids is similar to that of arabinose, which is now being viewed as another potential substrate in the production of biomass-derived ethanol. This study compared the end-product distribution associated with the fermentation of D-glucose (Glc) and D-glucuronic acid (GlcUA) (as sole carbon and energy sources) by Escherichia coli B (ATCC 11303) and two different ethanologenic recombinants--a strain in which pet expression was via a multicopy plasmid (pLOI297) and a chromosomally integrated construct, strain KO11. pH-stat batch fermentations were conducted using a modified LB medium with 2% (w/v) Glc or GlcUA with the set-point for pH control at either 6.3 or 7.0. The nontransformed host culture produced only lactic acid from glucose, but fermentation of GlcUA yielded a mixture of ethanol, acetic, and lactic acids, with acetic acid being the predominant end-product. The ethanol yield associated with GlcUA fermentation by both recombinants was similar, but acetic acid was a significant by-product. Increasing the pH from 6.3 to 7.0 increased the rate of glucuronate fermentation, but it also decreased the ethanol mass yield from 0.22 to 0.19 g/g primarily because of an increase in acetic acid production. In all fermentations there was good closure of the carbon mass balance, the exception being the recombinant bearing plasmid pLOI297 that produced an unidentified product from GlcUA. The metabolism of GlcUA by this metabolically engineered construct remains unresolved. The results offered insights into metabolic fluxes and the regulation of pyruvate catabolism in the wild-type and engineered strains. End-product distribution for metabolism of glucuronic acid by the nontransformed, wild-type E. coli B and recombinant strain KO11 suggests that the enzyme pyruvate-formate lyase is not solely responsible for the production of acetylCoA from pyruvate and that derepressed pyruvate dehydrogenase may play a significant role in the metabolism of GlcUA.