基于NADPH和ATP再生提高枯草芽孢杆菌Surfactin合成研究

Surfactant is a new type of green biosurfactant. Because of its unique cyclic lipopeptide structure, it has strong surface activity, antibacterial activity, strong emulsification and biodegradability, and has a broad application prospect in the fields of oil exploration, medicine, food, and daily chemicals. Based on the biosynthesis pathway of Surfactin, the synthesis process of Surfactin needs to consume a lot of NADPH and ATP. Therefore, this study is the first to study the effect of NADPH and ATP regeneration on the synthesis of Surfactan from the level of intracellular energy and cofactor regeneration. The results showed that enhancing the key enzymes zwf and gndA of the intracellular NADPH regeneration pathway, or heterologous expression of Escherichia coli transhydrogenase sthA, had a certain effect on cell growth and Surfactin synthesis, while heterologous expression of Escherichia coli transhydrogenase pntAB had no significant effect on growth and yield. Among them, the overexpression of zwf increased the most significantly, the yield of the original strain increased from 0.91 g/L to 1.19 g/L, increased by 30.77%, the cell growth slightly increased, and the OD600 increased from 3.60 to 4.16, which had no significant effect on the maximum specific growth rate; Secondly, by enhancing the ATP level of the key enzymes pgk and pyk in the phosphorylation pathway of the intracellular substrate, the maximum specific growth rate was increased from 0.41 /h to 0.48 /h and 0.47 /h respectively, and the OD600 was increased from 3.60 to 4.97 and 5.10, but the yield of Surfactan was only slightly increased; In addition, the co-expression of NADPH regeneration and ATP regeneration enzyme genes can improve cell growth and Surfactin production. Among them, the co-expression of zwf and pyk cells has the most significant increase in growth and Surfactin synthesis. Compared with the initial bacteria, the maximum specific growth rate has increased from 0.41 /h to 0.56 /h, and the yield of Surfactin has increased from 0.91 g/L to 1.90 g/L, 2.08 times of the initial bacteria. 7.5 L fermentation tank ferments in batches, OD600 reaches 8.05, and the yield of Surfactan is 3.00 g/L.