Wang Miaomiao, Yu Huimin, He Xin, Li Yanmei, Yang Huaiyu
Key Laboratory of Industrial Biocatalysis Ministry of Education, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China.
Daqing Refining & Petrochemical Company, Daqing 163000, Heilongjiang, China.
Sheng Wu Gong Cheng Xue Bao. 2020 Nov 25;36(11):2377-2386. doi: 10.13345/j.cjb.200156.
Surfactin has great potential applications in enhancing oil recovery, agriculture, pharmaceuticals, foods and beverages, and cosmetics due to its extraordinary surface activity, biodegradability, anti-bacterial activity and biocompatibility. Enhancing surfactin production by engineering surfactin-producer and optimizing culture conditions is the key of its industrial production and subsequent applications. In this study, the effect of fatty acid synthesis pathway on surfactin synthesis was investigated, and Bacillus subtilis THBS-2 and THBS-8 with high surfactin titer were constructed by overexpressing key genes involved in the fatty acid synthesis pathway. To optimize culture condition, the amount and adding time of isopropyl-beta-D-thiogalactopyranoside (IPTG) and amino acids were studied, and a two-stage culture method was obtained: IPTG (final concentration: 1.25 mmol/L) and leucine (final concentration: 5 g/L) were added at 3 h, leucine (final concentration 5 g/L) and condensed culture medium (5 mL) were added at 24 h. Applying this strategy, the surfactin titer of B. subtilis THBS-2 reached to 24 g/L in shake flask at 48 h and up to 34 g/L after 68 h fermentation in a 30-L fermentor. The results provide basis for large-scale production and broad application of surfactin.
由于表面活性素具有非凡的表面活性、生物降解性、抗菌活性和生物相容性，它在提高石油采收率、农业、制药、食品饮料及化妆品等领域具有巨大的潜在应用价值。通过对表面活性素产生菌进行工程改造以及优化培养条件来提高表面活性素的产量，是其工业化生产及后续应用的关键。本研究考察了脂肪酸合成途径对表面活性素合成的影响，并通过过表达脂肪酸合成途径中的关键基因构建了表面活性素产量较高的枯草芽孢杆菌THBS-2和THBS-8。为优化培养条件，研究了异丙基-β-D-硫代半乳糖苷（IPTG）和氨基酸的用量及添加时间，得到了一种两阶段培养方法：在3 h时添加IPTG（终浓度：1.25 mmol/L）和亮氨酸（终浓度：5 g/L），在24 h时添加亮氨酸（终浓度5 g/L）和浓缩培养基（5 mL）。应用该策略，枯草芽孢杆菌THBS-2在摇瓶中48 h时表面活性素产量达到24 g/L，在30-L发酵罐中发酵68 h后产量高达34 g/L。这些结果为表面活性素的大规模生产和广泛应用提供了依据。
