Study On Pyruvic Acid Accumulation Of Escherichia Coli

Lipoamide dehydrogenase (LPD) coded by lpdA gene is an important composition of pyruvate dehydrogenase complex (PDHc). Under aerobic condition, PDHc of Escherichia coli is the key enzyme which connects EMP pathway with TCA cycle. Pyruvic acid converts into acetyl-CoA under the catalysis of PDHc whose disfigurement would lead to pyruvic acid accumulation. In this dissertation, lpdA gene knockout Escherichia coli (Escherichia coli lpdA~-) was studied for the purpose of investigating pyruvic acid accumulation. This fermentation aimed at enhancing pyruvic acid concentration, yield and prdouctivity. Farther, primary study on polygene knockout was carried out in order to construct a better genetic strain.Firstly, comparison between wild type Escherichia coli and lpdA gene knockout Escherichia coli was carried out and the find was that Escherichia coli lpdA~- could accumulate pyruvic acid but wild type Escherichia coli could not. Free cell culture with glucose as carbon source was carried out and investigations about factor such as pH and concentration of glucose were done. pH=7 is a most important factors for lpdA gene knockout Escherichia coli, because the accumulation of pyruvic acid would lead to low pH which inhibited growth and metabolism of Escherichia coli. By the study on Escherichia coli lpdA~-, a series of parameters was obtained: pH=7, under aerobic condition with adding 30g/L glucose, the glucose was consumed in 20 hour and the final pyruvic acid concentration was 4.0g/L following the biomass raised to 3.5g/L ultimately.Secondly, the fermentation with fructose, xylose and their mixture as carbons respectively was investigated. The results showed that the mixture of two kind of monosaccharide would raise the production of pyruvic acid. For instance, the maximum of pyruvic acid concentration with 20g/L glucose singlly is about 2g/L, but the maximum of pyruvic acid concentration with 10g/L gluocose and 10g/L fructose is 8g/L which is the quadruple of single glucose fermentation.Third, according to capability of Escherichia coli lpdA~- to accumulate pyruvic acid, polygene knockout was investigated to construct a better genetic strain. One-step inactivation of chromosomal ldhA genes in Escherichia coli using PCR products were carried out. Construct linear kanamycin-resistant gene with ldhA gene homologous fragment first, and then transfer this DNA and vector pkD46 into the cell. After Screening by antibiotics and PCR, a mutant of ldhA knockout was obtained finally.