Sun Xi, Yang Jun, Fu Xiaoping, Zhao Xingya, Zhen Jie, Song Hui, Xu Jianyong, Zheng Hongchen, Bai Wenqin
College of Biological Engineering, Tianjin Agricultural University, Tianjin 300384, China.
National Center of Technology Innovation for Synthetic Biology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
Bioengineering (Basel). 2023 Aug 18;10(8):977. doi: 10.3390/bioengineering10080977.
At present, the double-enzyme catalyzed method using maltooligosyltrehalose synthase (MTSase) and maltooligosyltrehalose trehalohydrolase (MTHase) is the mainstream technology for industrial trehalose production. However, MTSase and MTHase are prepared mainly using the heterologous expression in the engineered strains so far. In this study, we first proved that the addition of 3 U/g neutral pullulanase PulA could enhance the trehalose conversion rate by 2.46 times in the double-enzyme catalyzed system. Then, a CBM68 domain was used to successfully assist the secretory expression of MTSase and MTHase from S34 in SCK6. At the basis, an engineered strain PSH02 (::/pHT43-C68-ARS/pMC68-ARH), which co-expressed MTSase, MTHase, and PulA, was constructed. After the 24 h fermentation of PSH02, the optimum ratio of the extracellular multi-enzymes was obtained to make the highest trehalose conversion rate of 80% from 100 g/L maltodextrin. The high passage stability and multi-enzyme preservation stability made PSH02 an excellent industrial production strain. Moreover, trehalose production using these extracellular enzymes produced via the one-step fermentation of PSH02 would greatly simplify the procedure for multi-enzyme preparation and be expected to reduce production costs.
目前，使用麦芽寡糖基海藻糖合酶（MTSase）和麦芽寡糖基海藻糖海藻糖水解酶（MTHase）的双酶催化法是工业生产海藻糖的主流技术。然而，到目前为止，MTSase和MTHase主要是通过在工程菌株中的异源表达来制备的。在本研究中，我们首先证明，在双酶催化体系中添加3 U/g中性普鲁兰酶PulA可使海藻糖转化率提高2.46倍。然后，利用一个CBM68结构域成功地协助了S34来源的MTSase和MTHase在SCK6中的分泌表达。在此基础上，构建了共表达MTSase、MTHase和PulA的工程菌株PSH02（::/pHT43-C68-ARS/pMC68-ARH）。PSH02发酵24 h后，获得了细胞外多酶的最佳比例，使得100 g/L麦芽糖糊精的海藻糖转化率最高达到80%。高传代稳定性和多酶保存稳定性使PSH02成为一株优良的工业生产菌株。此外，使用通过PSH02一步发酵产生的这些细胞外酶生产海藻糖将大大简化多酶制备过程，并有望降低生产成本。
