基于表达元件和宿主优化促地衣芽胞杆菌高效表达l-天冬酰胺酶

Efficient production of l-asparaginase in Bacillus licheniformis by optimizing expression elements and host

摘要:

l-天冬酰胺酶(l-asparaginase, l-ASN)广泛用于恶性肿瘤治疗及低丙烯酰胺食品生产,然而其较低的表达水平限制了应用推广。异源蛋白表达是提高目标酶表达水平的有效策略,芽胞杆菌广泛用于酶蛋白的高效生产,本研究拟通过表达元件及宿主优化提高芽胞杆菌(Bacillus)中l-天冬酰胺酶产量。首先,筛选了5种信号肽(SP_SacC、SP_AmyL、SP_AprE、SP_YwbN、SP_WapA)用于l-天冬酰胺酶的分泌表达,其中SP_SacC介导下l-天冬酰胺酶分泌效果最好,酶活达到157.61 U/mL。随后,选取了4种芽胞杆菌强启动子(P43、P_ykzA-P43、P_Ubay、P_bacA),其中串联启动子P_ykzA-P43介导的l-天冬酰胺酶表达量最高,较对照菌株提高了52.94%。最后,筛选了3种芽胞杆菌表达宿主:地衣芽胞杆菌(Bacillus licheniformis) Δ0F3和BL10、枯草芽胞杆菌(B. subtilis) WB800,其中,地衣芽胞杆菌BL10作为宿主时,l-天冬酰胺酶酶活最高,达到了438.3 U/mL,较对照菌株提高了81.83%,为目前报道的l-天冬酰胺酶摇瓶酶活最高水平。综上所述,本研究成功构建了一株高产l-天冬酰胺酶的地衣芽胞杆菌工程菌株BL10/P_ykzA-P43-SP_sacC-ansZ,为l-天冬酰胺酶工业化生产奠定了基础。

Abstract:

l-asparaginase (l-ASN) is widely applied in the treatment of malignant tumor and low-acrylamide food production, however, the low expression level hampers its application. Heterologous expression is an effective strategy to increase the expression level of target enzymes, and Bacillus is generally used as the host for efficient production of enzymes. In this study, the expression level of l-asparaginase in Bacillus was enhanced through optimization of expression element and host. Firstly, five signal peptides (SP_SacC, SP_AmyL, SP_AprE, SP_YwbN and SP_WapA) were screened, among which SP_SacC showed the best performance, reaching an activity of 157.61 U/mL. Subsequently, four strong promoters (P43, P_ykzA-P43, P_Ubay and P_bacA) from Bacillus were screened, and tandem promoter P_ykzA-P43 showed the highest yield of l-asparaginase, which was 52.94% higher than that of control strain. Finally, three Bacillus expression hosts (B. licheniformis Δ0F3 and BL10, B. subtilis WB800) were investigated, and the maximum l-asparaginase activity, 438.3 U/mL, was reached by B. licheniformis BL10, which was an 81.83% increase compared with that of the control. This is also the highest level of l-asparaginase in shake flask reported to date. Taken together, this study constructed a B. licheniformis strain BL10/P_ykzA-P43-SP_SacC-ansZ capable of efficiently producing l-asparaginase, which laid the foundation for industrial production of L-asparaginase.