重组枯草芽孢杆菌全细胞高效催化合成D-甘露糖

Efficient whole-cell biosynthesis of D-mannose by recombinant Bacillus subtilis

摘要:

D-甘露糖是一种在食品、医疗、化妆品等领域具有很大经济价值和应用价值的天然己糖。但是目前大部分生物合成法都是以大肠杆菌作为宿主，生产过程中存在安全性问题，对后续应用产生了诸多限制。本研究通过比较多个来源的甘露糖异构酶的酶学性质，筛选出最优来源甘露糖异构酶。以食品安全级菌株枯草芽孢杆菌(Bacillus subtilis) 168为底盘细胞，将最优来源的甘露糖异构酶进行异源表达，获得了重组菌株B. subtilis 168/pMA5-EcMIaseA，用于以D-果糖为底物全细胞催化高效合成D-甘露糖。通过优化转化温度、pH、底物浓度等条件，提高其转化合成D-甘露糖的效率。结果表明，重组枯草芽孢杆菌B. subtilis 168/pMA5-EcMIaseA在最适转化条件下进行5 L发酵罐全细胞转化，分别以500 g/L果糖和600 g/L果糖为底物，转化6 h后D-甘露糖的产量分别为138.74 g/L和163.30 g/L，转化率分别达到27.75%和27.22%，这是目前报道的以食品级菌株进行D-甘露糖生产的最高产量。本研究为D-甘露糖的工业化安全生产及应用奠定了重要基础。

Abstract:

D-mannose is a natural hexose with great economic and application values in the food, medicine, and cosmetic fields. However, most biosynthesis methods of D-mannose rely on Escherichia coli as the host, which poses safety issues during the production process and imposes limitations on subsequent applications. This study compared the enzyme properties of mannose isomerases from multiple sources to select the most suitable source. B. subtilis 168/pMA5-EcMIaseA was constructed with “generally recognized as safe” (GRAS) Bacillus subtilis as the host and used as a whole-cell catalyst to synthesize D-mannose from D-fructose. Optimizing the conversion conditions such as culture temperature, pH, and substrate concentration increased the yield of D-mannose. The results showed that the conversion rates reached 27.75% and 27.22% and the yields of D-mannose were 138.74 g/L and 163.30 g/L after 6 h whole-cell transformation with D-fructose at the concentrations of 500 g/L and 600 g/L, respectively, in a 5 L fermentor. This study achieves the highest yield of D-mannose produced under the catalysis by recombinant B. subtilis that has ever been reported and provides a basis for the industrial production and application of D-mannose.