Production of pyruvate and lactate by metabolically engineering Escherichia coli
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Pyruvic acid and lactic acid have a wide range of applications in the food, chemical, and pharmaceutical industries. The primary goal of this research is to improve pyruvate and lactate production through bacterial strain engineering and fermentation process development. Lactate production was studied using E. coli strain YYC202 (aceEF pfl poxB pps), whichgenerated 90 g/L lactate in a two-phase fermentation (aerobic growth phase followed by anaerobic phase for lactate production). This process also generated over 7 g/L succinate. Ca(OH)2 was found to be superior to NaOH for pH control. Strain ALS961 (YYC202 ppc) prevented succinate accumulation but growth was very poor. Strain ALS974 (YYC202 13frdABCD) reduced succinate formation by 70% to less than 3 g/L.C-NMR using uniformly labeled acetate demonstrated that succinate formation by ALS974 was biochemically derived from acetate in the medium. The two-phase process with ALS974 achieved 138 g/L lactate (1.55 M, 97% of carbon products) with a yield of 0.99 g/g and productivity of 6.3 g/L·h during the anaerobic phase. By using the dissolved oxygen (DO) during the initial cell-growth phase to monitor residual acetate, and timing the switch to the anaerobic production phase with acetate depletion, succinate production was reduced to less than 1 g/L, and ethanol production was eliminated. Furthermore, by using a cell-recycle fermentation with ultrafiltration, the anaerobic lactate production phase was prolonged from 22 h to 34 h, and an overall lactate productivity of 4.2 g/L·h was achieved. The productivity of this process was nearly 20% greater than the productivity of the fed-batch process.Pyruvate was studied using E. coli strain ALS929 (pflB aceEF poxB pps ldhA). First, a series of steady-state (chemostat) experiments were conducted to evaluate pyruvate formationunder four different nutrient-limited conditions: glucose, acetate, nitrogen (ammonia), and phosphorus (phosphate). The greatest specific glucose consumption rate of 1.60 g/g·h and specific pyruvate formation rate of 1.10 g/g·h were found under conditions of acetate-limited growth. The specific glucose consumption rate and therefore pyruvate productivity were further increased to 2.67 g/g·h and 2.01 g/g·h respectively by introducing ATP synthase knockout (atpFH), an arcA knockout and introducing heterologous NADH oxidase. A fed-batch process at -1a constant specific growth rate of 0.15 h using ALS1059 (pflB aceEF poxB pps ldhA atpFH arcA) resulted in the highest pyruvate yield and productivity. In a defined medium with 5mM betaine, a final pyruvate concentration of 89.7 g/L, a yield of 0.68 g/g and a productivity of 2.04 g/L·h were achieved.