Production of Bioethanol and Biobutanol Using Genetically Engineered
In this dissertation, the different Escherichia coli strains were engineered for producing bioethanol from the lignocellulosic biomass, and biobutanol from glucose.
The recombinant whole-cell biocatalyst was developed by expressing three cellulases from Clostridium cellulolyticum: endoglucanase (Cel5A), exoglucanase (Cel9E), and β-glucosidase (BGL), onto the Escherichia coli LY01 cell surface (LY01/pRE1H-AEB). The above-mentioned cellulases were expressed by fusing with the anchor protein PgsA. The developed whole cell biocatalyst was used for single-step ethanol fermentation using the phosphoric acid swollen cellulose (PASC), and the dilute acid pretreated corn stover. The ethanol production was 3.59 ± 0.15 g/L using 10 g/L of phosphoric acid swollen cellulose (PASC) which corresponds to 95.4 ± 0.15% of the theoretical yield. The ethanol production was 0.30 ± 0.02 g/L when the cellulosic fraction of the dilute sulfuric acid pretreated corn stover (PCS) equivalent of 1 g/L glucose, was fermented for 84 h. Total 0.71 ± 0.12 g/L ethanol was obtained in 48 h when the fermentation of PCS were performed in the simultaneous saccharification and co-fermentation (SSCF) process using the hemicellulosic (1 g/L total soluble sugar) as well as cellulosic (1 g/L glucose equivalent) parts of PCS. In a control experiment, 0.48 g/L ethanol was obtained from 1 g/L of hemicellulosic PCS. It was concluded that the whole cell biocatalyst could convert both cellulosic and hemicellulosic substrate into ethanol in a single reactor.
A new recombinant E. coli (KO11) was constructed by expressing the extracellularly secreted as well as the cell surface displayed endoglucanase. The expression vector pHR5A was constructed with the (EAAAK)5 peptide linker between the pgsA and the endoglucanase cel5A genes. Due to the auto-cleavage characteristic of the (EAAAK)5 peptide linker, Cel5A was partially detached from the PgsA anchor protein during the fermentation. The enzymatic hydrolysis efficiency of the recombinant KO11/pHR5A was investigated with various pure insoluble cellulosic substrates such as PASC, Solka-Floc 200NF, and Avicel.. Compared to the control strain, KO11/pH5A, with the recombinant strain, KO11/pHR5A, the saccharification efficiency was increased by 37%, 29%, and 17%, with substrates PACS, Solka-Floc 200NF, and Avicel, respectively. Ethanol production, supplemented by Novozyme 188 (commercial β-glucosidase), was found to be greater with the KO11/pHR5A than with the KO11/pH5A over 48 h fermentation. It was concluded that the extracellularly secreted single enzyme component enhanced the saccharification efficiency. For the butanol production, Escherichia coli BL21 was engineered. The mutant E. coli BM322 was constructed by disrupting phosphotransacetylase (pta), lactate dehydrogenase A (ldhA), bifunctional acetaldehyde/ethanol dehydrogenase (adhEB), fumarate reductase (frdBC), and pyruvate oxidase B (poxB) genes. Additionally, the transcriptional regulator FNR protein gene was also deleted. The recombinant plasmid pBUOH was constructed by cloning the key enzyme coding genes that are involved in the butanol production pathway in Clostridium acetobutylicum. These genes include: thiolase (thl), 3-hydroxybutyryl-CoA dehydrogenase (hbd), crotonase (crt), butyryl-CoA dehydrogenase (bcd), electron transfer flavoprotein (etfAB), and the bifunctional butyraldehyde/butanol dehydrogenase (adhEC). The recombinant E. coli BM322/pBUOH produced 1.2 g/L butanol with 10.2 g/L lactate as a by-product for 60 h. The obtained butanol yield and productivity was 0.07 g butanol/g glucose and 0.04 g/L•h, respectively.