Construction of Co-Expressed N-Acetyltransferase and Phospholipase Gene in Recombinant Strain and Optimization of Fermentation Conditions

Abstract:

To increase the production of phospholipase，a strain of P. pastoris KM71 containing the gene co-expressing the N-cetyltransferase（Mpr1） and phospholipase was constructed. Mpr1 is an intracellular enzyme that catalyzes the transfer of acetyl groups between acetyl CoA and amines. Mpr1 has significant physiological functions in ROS oxidative stress resistance ability. Phospholipase，which come from Fusarium oxysporum is a group of hydrolases that hydrolyze phospholipids，releasing a variety of products，such as lysophospholipids，free fatty acids. Constructing the recombinant P. pastoris KM71/pPIC9K-phospholipase/pPICZA-Mpr1，the impact of Mpr1 on yeast cell growth and production of phospholipase from Fusarium oxysporum was investigated. In order to obtain the optimal condition of phospholipase fermentation，we observed the influence of temperature，initial induction cell density and methanol concentration on the high density fermentation process in 3.6 L fermentor of P. pastoris KM71/pPIC9K-phospholipase/ pPICZA-MPR1. By using glycerol feeding strategy with DO control in high-density fermentation and maintained the residual methanol concentration constantly with a methanol sensor. The result showed that the optimum condition were listed as follows：the temperature was 28 ℃，the initial induction cell density was 40 g/L，the methanol concentration was 1.0%. Under the condition，the phospho- lipase activity reached 8 100 U/mL and the protein concentration（8.3 mg/mL） at cultivation of 120 h，which was 1.33 times than before.