Production of succinate from colon bacillus
A technology of Escherichia coli and succinic acid, which is applied in the field of succinic acid fermentation and production by Escherichia coli, can solve the problems of low succinic acid productivity and specific production rate, and achieve high specific production rate and high production rate
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Embodiment 1
[0029] The wild-type Escherichia coli TG1 is a strain publicly sold by Pharmacia, and it can be provided by strain collection institutions such as the China Microorganism Collection Center;
[0030] 1ml of Escherichia coli TG1 preserved in the frozen glycerol tube was inserted into a 250ml Erlenmeyer flask containing 30ml of LB (1% tryptone, 0.5% yeast extract, 0.5% sodium chloride) medium, at 37°C, and the rotating speed was 220rpm. Overnight aerobic culture activation. Take 2ml of the culture solution and insert it into a 500ml Erlenmeyer flask containing 100ml of culture medium. The culture medium is LB with 40mM malic acid (to adjust the pH to 7.0), 80mM sodium acetate, and 5g / L glucose (as a control), respectively. Cultivate in an aerobic atmosphere for 7 hours.
[0031] After aseptic centrifugation, the bacteria were collected and resuspended in M9 (Na 2 HP 4 12H 2 O15.12g / L, KH 2 PO 4 3.0g / L, NaCl 0.5g / L, NH 4 Cl 1.0g / L, MgSO 4 ·7H 2 O 0.5g / L, CaCl 2 0.011g / ...
Embodiment 2
[0034] Escherichia coli NZN111 lacks pyruvate formate lyase and lactate dehydrogenase (Microbiology 143:187-95, 1997). Under anaerobic conditions, NZN111 basically does not grow or consume glucose even if sodium acetate is added. Escherichia coli NZN111 can be provided by CGSC (The Coli Genetic Stock Center, MCDB Department, Yale University), CGSC number 7726.
[0035] Take 1ml of NZN111 preserved in glycerol tube and put it into a 250ml Erlenmeyer flask filled with 30ml LB medium, and activate it by aerobic culture at 37°C overnight at 220rpm. Take 2ml of the culture solution and insert it into a 500ml Erlenmeyer flask containing 100ml of culture medium. LB with sodium α-ketoglutarate as the sole carbon source, and LB without carbon source as a control, were incubated at 37°C for 7 hours under aerobic conditions. At this time, the dry weight of the cells cultured in LB without carbon source was only 1.60g / L, while the LB cultured with sodium malate, sodium fumarate, sodium α...
Embodiment 3
[0037] NZN111 was cultivated aerobically as in Example 2, and the medium used was 100 ml LB containing 40 mM malic acid. After the cultivation, the cells were collected by aseptic centrifugation, and resuspended in a solution containing 15g / L glucose and 20g / L MgCO 3 In 50ml of LB, at this time the cell concentration was 8.68g / L, and anaerobically fermented in a 100ml Schott bottle in a closed 100ml Schott bottle at 37°C in a water bath shaker at 150rpm. After 2.5 hours of anaerobic fermentation, glucose was completely consumed, and pyruvate was accumulated, and the concentration of succinic acid was 9.7g / L. At 5 hours, the concentration of succinic acid reached 11.5g / L, no accumulation of pyruvate, the yield of succinic acid was 1.15mol / mol, the average rate of glucose consumption was 3.04g / L·h, and the average production rate of succinic acid was 2.30 g / L·h, the average specific production rate reached 267mg / g(DCW)·h.
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