Metabolic engineering of bacteria for ethanol production

Abstract

Technologies are available which will allow the conversion of lignocellulose into fuel ethanol using genetically engineered bacteria. Assembling these into a cost‐effective process remains a challenge. Our work has focused primarily on the genetic engineering of enteric bacteria using a portable ethanol production pathway. Genes encoding Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase have been integrated into the chromosome of Escherichia coli B to produce strain KO11 for the fermentation of hemicellulose‐derived syrups. This organism can efficiently ferment all hexose and pentose sugars present in the polymers of hemicellulose. Klebsiella oxytoca M5A1 has been genetically engineered in a similar manner to produce strain P2 for ethanol production from cellulose. This organism has the native ability to ferment cellobiose and cellotriose, eliminating the need for one class of cellulase enzymes. The optimal pH for cellulose fermentation with this organism (pH 5.0–5.5) is near that of fungal cellulases. The general approach for the genetic engineering of new biocatalysts has been most successful with enteric bacteria thus far. However, this approach may also prove useful with Gram‐positive bacteria which have other important traits for lignocellulose conversion. Many opportunities remain for further improvements in the biomass to ethanol processes. These include the development of enzyme‐based systems which eliminate the need for dilute acid hydrolysis or other pretreatments, improvements in existing pretreatments for enzymatic hydrolysis, process improvements to increase the effective use of cellulase and hemicellulase enzymes, improvements in rates of ethanol production, decreased nutrient costs, increases in ethanol concentrations achieved in biomass beers, increased resistance of the biocatalysts to lignocellulosic‐derived toxins, etc. To be useful, each of these improvements must result in a decrease in the cost for ethanol production. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:204–214, 1998.

Microbial Cellulose Utilization: Fundamentals and Biotechnology

“…Utilization of hemicellulose-derived monomeric sugars, and xylose in particular, has received intensive investigation for over a decade (137,237,272,299,301,310,433,775). With the exception of dilute-acid pretreatment, a substantial fraction of dissolved hemicellulose is present as oligomers for most processes in which associated hydrolysis products have been characterized, including steam explosion (248,546), hydrothermal pretreatment (372), AFEX (B. Dale, personal communication) and countercurrent very-dilute-acid pretreatment (R. W. Torget, personal communication).…”

Isolation and Characterization of Shigella flexneri G3, Capable of Effective Cellulosic Saccharification under Mesophilic Conditions

“…Various types of cellulase enzymes could be inhibited by soluble products (glucose, cellobiose, cellotriose, etc.) via feedback mechanisms (28). HPLC analysis showed that oligosaccharides were mainly composed of glucose (26%), cellobiose (70%), and trace amounts of cellotriose and cellotetraose (less than 3%) ( Fig.…”

Ethanol fermentation in an immobilized cell reactor using Saccharomyces cerevisiae

“…The phosphorylation of carbohydrates is carried out through the metabolic pathway; the end products are two moles of ethanol and carbon dioxide (Ingram et al, 1998).…”