Biological Science Research, Kao Corporation, 1334 Minato, Wakayama, Wakayama, 640-8580, Japan.
Biological Science Research, Kao Corporation, Haga , Tochigi, 2606 Akabane, Ichikai321-3497, Japan.
Microb Cell Fact. 2021 Dec 28;20(1):231. doi: 10.1186/s12934-021-01724-x.
Most of the proteases classified into the M23 family in the MEROPS database exhibit staphylolytic activity and have potential as antibacterial agents. The M23 family is further classified into two subfamilies, M23A and M23B. Proteases of the M23A subfamily are thought to lack the capacity for self-maturation by auto-processing of a propeptide, which has been a challenge in heterologous production and application research. In this study, we investigated the heterologous expression, in Bacillus subtilis, of the Lysobacter enzymogenes beta-lytic protease (BLP), a member of the M23A subfamily.
We found that B. subtilis can produce BLP in its active form. Two points were shown to be important for the production of BLP in B. subtilis. The first was that the extracellular proteases produced by the B. subtilis host are essential for BLP maturation. When the host strain was deficient in nine extracellular proteases, pro-BLP accumulated in the supernatant. This observation suggested that BLP lacks the capacity for self-maturation and that some protease from B. subtilis contributes to the cleavage of the propeptide of BLP. The second point was that the thiol-disulfide oxidoreductases BdbDC of the B. subtilis host are required for efficient secretory production of BLP. We infer that intramolecular disulfide bonds play an important role in the formation of the correct BLP conformation during secretion. We also achieved efficient protein engineering of BLP by utilizing the secretory expression system in B. subtilis. Saturation mutagenesis of Gln116 resulted in a Q116H mutant with enhanced staphylolytic activity. The minimum bactericidal concentration (MBC) of the wild-type BLP and the Q116H mutant against Staphylococcus aureus NCTC8325 was 0.75 μg/mL and 0.375 μg/mL, respectively, and the MBC against Staphylococcus aureus ATCC43300 was 6 μg/mL and 3 μg/mL, respectively.
In this study, we succeeded in the secretory production of BLP in B. subtilis. To our knowledge, this work is the first report of the successful heterologous production of BLP in its active form, which opens up the possibility of industrial use of BLP. In addition, this study proposes a new strategy of using the extracellular proteases of B. subtilis for the maturation of heterologous proteins.
MEROPS 数据库中大多数分类为 M23 家族的蛋白酶具有溶葡萄球菌活性，具有作为抗菌剂的潜力。M23 家族进一步分为 M23A 和 M23B 两个亚家族。据认为，M23A 亚家族的蛋白酶缺乏通过原肽的自动加工进行自我成熟的能力，这在异源生产和应用研究中是一个挑战。在这项研究中，我们研究了枯草芽孢杆菌中 Lysobacter enzymogenes β-溶菌酶（BLP）的异源表达，BLP 是 M23A 亚家族的成员。
我们发现枯草芽孢杆菌可以以其活性形式产生 BLP。有两个方面被证明对枯草芽孢杆菌中 BLP 的生产很重要。首先，枯草芽孢杆菌宿主产生的细胞外蛋白酶对于 BLP 的成熟是必不可少的。当宿主菌株缺乏九种细胞外蛋白酶时，原 BLP 会在上清液中积累。这一观察结果表明，BLP 缺乏自我成熟的能力，枯草芽孢杆菌中的某些蛋白酶有助于 BLP 原肽的切割。其次，枯草芽孢杆菌宿主的硫醇-二硫键氧化还原酶 BdbDC 是高效分泌生产 BLP 的必需条件。我们推断，在分泌过程中，分子内二硫键在形成正确的 BLP 构象中起着重要作用。我们还通过利用枯草芽孢杆菌的分泌表达系统实现了 BLP 的高效蛋白质工程。对 Gln116 的饱和诱变导致 Q116H 突变体的溶葡萄球菌活性增强。野生型 BLP 和 Q116H 突变体对金黄色葡萄球菌 NCTC8325 的最小杀菌浓度（MBC）分别为 0.75μg/mL 和 0.375μg/mL，对金黄色葡萄球菌 ATCC43300 的 MBC 分别为 6μg/mL 和 3μg/mL。
在这项研究中，我们成功地在枯草芽孢杆菌中实现了 BLP 的分泌生产。据我们所知，这是首次成功以其活性形式异源生产 BLP 的报告，这为 BLP 的工业用途开辟了可能性。此外，本研究提出了一种利用枯草芽孢杆菌细胞外蛋白酶促进异源蛋白成熟的新策略。
