| Summary: | 碩士 === 國立中正大學 === 化學工程研究所 === 104 === As the global population is rapidly rising, the demand for energy, food, daily necessities, plastics and chemicals is also increasing. The development of new energies/chemicals from non-petroleum sources becomes important. This study aims at the use of genetic engineering to improve the microbial strain for the production of biochemicals like lactic acid or succinic acid. Escherichia coli CCU-8 that can produce high level lactic acid and succinic acid was previously isolated from bovine rumen. In order to enhance the yield and optical purity of lactic acid, we used λ-Red homologous recombination system to knockout genes of phosphotransacetylase (pta) and methylglyoxal synthase (mgsA) for producing D-lactic acid. A strain with double knockout of D-lactate dehydrogenase (ldhA) and L-lactate dehydrogenase (lldD) genes were also established for producing L-lactic acid. In order to enhance the yield of succinic acid, we also tried to knockout both genes of D-lactate dehydrogenase (ldhA) and methylglyoxal synthase (mgsA) using the λ-Red homologous recombination system.
In the fermentation starting with 50 g/L glucose, E. coli CCU-8 (Δpta, ΔmgsA) could produce 44.9 g/L of lactic acid, 3.98 g/L of succinic acid, 1.31 g/L of acetic acid, and 0.86 g/L of ethanol. The lactic acid yield could achieve 1.15 g/g-glucose. This lactic acid product is made of 78% D-lactic acid and 22% L-lactic acid. Compared with the recombinant strain E. coli CCU-8 (Δpta), the fraction of D-lactic acid increased from 66% to 78% by the double knockout strain. However, the total yield of lactic acid did not decrease, indicating that the knockout of mgsA gene could reduce the formation of L-lactic acid. Another double knockout strain E. coli CCU-8 (ΔldhA, ΔlldD) could produce 5.61 g/L of lactic acid, 0.2 g/L of succinic acid, 5.13 g/L of acetic acid, and 6.75 g/L of ethanol, and the lactic acid was optically pure L-lactic acid. In addition, this strain could produce pyruvate, suspecting that lldD gene is not only in charge of reaction from L-lactic acid to pyruvate, but also that from pyruvate to L-lactic acid in the metabolism. Because the lldD gene was deleted, the strain could not metabolize pyruvate to L-lactic acid and thus accumulate pyruvate in the cell. Surprisingly, the double knockout strain E. coli CCU-8 (ΔldhA, ΔmgsA) could produce only 3.31 g/L of lactic acid, 0.136 g/L of succinic acid, 5.05 g/L of acetic acid, and 5.02 g/L of ethanol. We expected this strain can produce succinic acid, but the results shows that no succinic acid was produced. The consumption of glucose became slowly, and no other by-product was observed in HPLC analysis.
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