Escherichia coliBinary Culture Engineered for Direct Fermentation of Hemicellulose to a Biofuel

Abstract

Metabolic engineering has created several Escherichia coli biocatalysts for production of biofuels and other useful molecules. However, the inability of these biocatalysts to directly use polymeric substrates necessitates costly pretreatment and enzymatic hydrolysis prior to fermentation. Consolidated bioprocessing has the potential to simplify the process by combining enzyme production, hydrolysis, and fermentation into a single step but requires a fermenting organism to multitask by producing both necessary enzymes and target molecules. We demonstrate here a binary strategy for consolidated bioprocessing of xylan, a complex substrate requiring six hemicellulases for complete hydrolysis. An integrated modular approach was used to design the two strains to function cooperatively in the process of transforming xylan into ethanol. The first strain was engineered to coexpress two hemicellulases. Recombinant enzymes were secreted to the growth medium by a method of lpp deletion with over 90% efficiency. Secreted enzymes hydrolyzed xylan into xylooligosaccharides, which were taken in by the second strain, designed to use the xylooligosaccharides for ethanol production. Cocultivation of the two strains converted xylan hemicellulose to ethanol with a yield about 55% of the theoretical value. Inclusion of other three hemicellulases improved the ethanol yield to 70%. Analysis of the culture broth showed that xylooligosaccharides with four or more xylose units were not utilized, suggesting that improving the use of higher xyloogligomers should be the focus in future efforts. This is the first demonstration of an engineered binary culture for consolidated bioprocessing of xylan. The modular design should allow the strategy to be adopted for a broad range of biofuel and biorefinery products.

Functional Characterization of Corynebacterium alkanolyticum β-Xylosidase and Xyloside ABC Transporter in Corynebacterium glutamicum

“…For genetic manipulations, Escherichia coli strains were grown at 33°C in Luria-Bertani medium (16). For aerobic growth conditions, C. alkanolyticum and C. glutamicum R (JCM18229) (17) and recombinant strains were precultured at 33°C overnight in nutrient-rich medium (A medium) containing (per liter) 2 g of urea, 2 g of yeast extract, 7 g of Casamino Acids, 7 g of (NH 4 (10). Where appropriate, the medium was supplemented with antibiotics.…”

Homologous overexpression of xylanase in Fusarium oxysporum increases ethanol productivity during consolidated bioprocessing (CBP) of lignocellulosics

“…As a result, the corresponding yields and productivities are also higher. Of particular importance, is the very recent study of Shin et al (2010) where a co-culture of two E. coli strains, engineered to express several xylanolytic enzymes was able to transform birchwood xylan into ethanol at a yield equal to 54% of the theoretical in only 24 h, in a partially optimized process. Of course the system has to be evaluated with real life complex substrates where a complete enzyme system for lignocellulose hydrolysis is required.…”

Developing Organisms for Consolidated Bioprocessing of Biomass to Ethanol

“…This enabled E. coli to secrete more than 50% of the recombinant Cel5Z it produced. Recently, Shin et al (2010) demonstrated a binary strategy for CBP of xylan. Two E. coli strains were designed to function cooperatively in the process of transforming xylan into ethanol.…”