Addition of Genes for Cellobiase and Pectinolytic Activity in Escherichia coli for Fuel Ethanol Production from Pectin-Rich Lignocellulosic Biomass

Abstract

Ethanologenic Escherichia coli strain KO11 was sequentially engineered to contain the Klebsiella oxytoca cellobiose phosphotransferase genes (casAB) as well as a pectate lyase (pelE) from Erwinia chrysanthemi, yielding strains LY40A (casAB) and JP07 (casAB pelE), respectively. To obtain an effective secretion of PelE, the Sec-dependent pathway out genes from E. chrysanthemi were provided on a cosmid to strain JP07 to construct strain JP07C. Finally, oligogalacturonide lyase (ogl) from E. chrysanthemi was added to produce strain JP08C. E. coli strains LY40A, JP07, JP07C, and JP08C possessed significant cellobiase activity in cell lysates, while only strains JP07C and JP08C demonstrated extracellular pectate lyase activity. Fermentations conducted by using a mixture of pure sugars representative of the composition of sugar beet pulp (SBP) showed that strains LY40A, JP07, JP07C, and JP08C were able to ferment cellobiose, resulting in increased ethanol production from 15 to 45% in comparison to that of KO11. Fermentations with SBP at very low fungal enzyme loads during saccharification revealed significantly higher levels of ethanol production for LY40A, JP07C, and JP08C than for KO11. JP07C ethanol yields were not considerably higher than those of LY40A; however, oligogalacturonide polymerization studies showed an increased breakdown of biomass to small-chain (degree of polymerization, <6) oligogalacturonides. JP08C achieved a further breakdown of polygalacturonate to monomeric sugars, resulting in a 164% increase in ethanol yields compared to those of KO11. The addition of commercial pectin methylesterase (PME) further increased JP08C ethanol production compared to that of LY40A by demethylating the pectin for enzymatic attack by pectin-degrading enzymes.

Molecular Cloning and Expression of Cellulase and Polygalacturonase Genes in E. coli as a Promising Application for Biofuel Production

“…Based on the available conserved domain sequences in the NCBI website, the deduced amino acid sequences of the 3 cloned ORFs were found to belong to 8,28 for the cloned celB, celC and peh, respectively [45,46]. The enzymes belonging to GH-28 family are classified into several categories in accordance with their catalytic hydrolysis mechanism and are given specific E.C.…”

“…The production of several types of fuel through direct lignocellulosic biomass conversions has been demonstrated by various studies [6,7]. A genetically engineered E. coli capable of degrading pectin-rich lignocellulosic biomass by cellulolytic and pectinolytic activities has been developed [8]. E. coli has been considered a convenient biocatalyst in biofuel production for its fermentation of glucose into a wide range of short-chain alcohols [9,10], and production of highly deoxygenated hydrocarbon through fatty acid metabolism [11,12].…”

Examining the feasibility of bulk commodity production in Escherichia coli

“…Engineering was subsequently performed to increase tolerance to furfural, a fermentation inhibitor found in feedstocks derived from lignocellulose (Wang et al 2011c). In an attempt to develop a strain that can both break down lignocellulosic carbon sources and produce ethanol, cellobiase and pectinolytic activities have been introduced into KO11 (Edwards et al 2011). These strains were tested using synthetic sugar beet pulp medium; the best strain exhibited and increase in ethanol yield of 164% compared to KO11 on the same medium.…”

Improved ethanol production from biomass by a rumen metagenomic DNA fragment expressed in Escherichia coli MS04 during fermentation

“…Escherichia coli offers a good platform for the CBP because there are many genetic tools available to facilitate its genetic modification, can grow in mineral salt media without complex supplements, and can ferment a wide variety of fermentable sugars, such as hexoses, pentoses, and uronic acids (Muñoz-Gutiérrez and Martínez 2013;Orencio-Trejo et al 2010). Several strategies involving expression of heterologous cellulases in fermentative ethanologenic strains are currently being pursued (Kojima et al 2012;Linger et al 2010;Muñoz-Gutiérrez et al 2014;Zheng et al 2012) including the development of ethanologenic E. coli strains engineered to heterologously express saccharolytic enzymes to produce ethanol directly from lignocellulosic biomass (Edwards et al 2011;Ryu and Karim 2011).…”