(341e) Production of Biofuels Using Genetically Engineered Escherichia Coli

A whole-cell biocatalyst system was developed in our research for the production of ethanol from cellulosic substrate in a single step. The whole cell biocatalyst was constructed with Escherichia coli LY01, which is the most ethanologenic strain. The cellulase genes, cel5A, cel9E and beta-glucosidase, were cloned from the mesophilic strain Clostridium cellulolyticum, and displayed on the surface of LY01 using the anchor protein PgsA. The pure cellulosic substrate phosphoric acid swollen cellulose (PASC) and the industrial substrate, dilute acid pretreated corn stover (PCS) were fermented. The yield of produced ethanol from PASC was 3.20 ± 0.15 g/L from 10 g/L PASC. 0.30 ± 0.02 g/L ethanol was produced from PCS equivalent of 1 g/L glucose. During the fermentation of the whole-cell biocatalyst, it was observed that the saccharification product glucose was uptaken immediately by the host cell and metabolized into ethanol. Currently, butanol has been considered as a potential liquid biofuel, and the butanol production has long been studied using clostridia strains. One of the difficulties of using clostridia strains is the formation of mixed-fermentation products, such as butyrate, acetate, acetone and ethanol. In this study, we engineered E. coli by introducing recombinant butanol pathway from multiple clostridia strains. The key enzyme coding genes: thiolase (thl), 3-hydroxybutyryl-CoA dehydrogenase (hbd), crotonase (crt), butyryl-CoA dehydrogenase (bcd), and the bifunctional butyraldehyde/butanol dehydrogenase (adhE), which are involved in the butanol production pathway in C. acetobutylicum were expressed in E. coli BL21(DE3). In the same E. coli, some of the carbon competitive pathway genes, e.g. phosphotransacetylase (pta), lactate dehydrogenase A (ldhA), bifunctional acetaldehyde/ethanol dehydrogenase (adhE), fumarate reductase (frdBC), and pyruvate oxidase B (poxB) were disrupted to maximize the carbon flux to butanol pathway with the expectation of increasing specific butanol productivity. Additionally, the transcriptional regulator FNR protein gene was also deleted. The engineered E. coli produced 830.1 mg/L butanol from 20 g/L glucose in 48 h fermentation.