Yang Xinyuan, Rao Yi, Zhang Mengxi, Wang Jiaqi, Liu Wenyuan, Cai Dongbo, Chen Shouwen
State Key Laboratory of Biocatalysis and Enzyme Engineering, Environmental Microbial Technology Center of Hubei Province, College of Life Sciences, Hubei University, Wuhan 430062, Hubei, China.
Sheng Wu Gong Cheng Xue Bao. 2023 Mar 25;39(3):1096-1106. doi: 10.13345/j.cjb.220642.
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 is generally used as the host for efficient production of enzymes. In this study, the expression level of L-asparaginase in was enhanced through optimization of expression element and host. Firstly, five signal peptides (SP, SP, SP, SP and SP) were screened, among which SP showed the best performance, reaching an activity of 157.61 U/mL. Subsequently, four strong promoters (P43, P, P and P) from were screened, and tandem promoter P showed the highest yield of L-asparaginase, which was 52.94% higher than that of control strain. Finally, three expression hosts ( Δ0F3 and BL10, WB800) were investigated, and the maximum L-asparaginase activity, 438.3 U/mL, was reached by 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 strain BL10/P-SP-ansZ capable of efficiently producing L-asparaginase, which laid the foundation for industrial production of L-asparaginase.
L-天冬酰胺酶（L-ASN）广泛应用于恶性肿瘤治疗及低丙烯酰胺食品生产中，然而，其低表达水平阻碍了它的应用。异源表达是提高目标酶表达水平的有效策略，且通常被用作高效生产酶的宿主。在本研究中，通过优化表达元件和宿主来提高L-天冬酰胺酶在[具体宿主]中的表达水平。首先，筛选了5种信号肽（SP1、SP2、SP3、SP4和SP5），其中SP5表现最佳，活性达到157.61 U/mL。随后，从[具体来源]筛选了4种强启动子（P43、P2、P3和P4），串联启动子P2显示出L-天冬酰胺酶的最高产量，比对照菌株高52.94%。最后，研究了3种[具体宿主]表达宿主（Δ0F3和BL10、WB800），[具体宿主]BL10达到了最大L-天冬酰胺酶活性438.3 U/mL，与对照相比提高了81.83%。这也是迄今为止摇瓶中报道的L-天冬酰胺酶的最高水平。综上所述，本研究构建了一种能够高效生产L-天冬酰胺酶的[具体宿主]菌株BL10/P2-SP5-ansZ，为L-天冬酰胺酶的工业化生产奠定了基础。
