Senger Jana, Seitl Ines, Pross Eva, Fischer Lutz
Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany.
Microb Cell Fact. 2024 Jun 12;23(1):170. doi: 10.1186/s12934-024-02445-7.
The gram-positive bacterium Bacillus subtilis is widely used for industrial enzyme production. Its ability to secrete a wide range of enzymes into the extracellular medium especially facilitates downstream processing since cell disruption is avoided. Although various heterologous enzymes have been successfully secreted with B. subtilis, the secretion of cytoplasmic enzymes with high molecular weight is challenging. Only a few studies report on the secretion of cytoplasmic enzymes with a molecular weight > 100 kDa.
In this study, the cytoplasmic and 120 kDa β-galactosidase of Paenibacillus wynnii (β-gal-Pw) was expressed and secreted with B. subtilis SCK6. Different strategies were focused on to identify the best secretion conditions. Tailormade codon-optimization of the β-gal-Pw gene led to an increase in extracellular β-gal-Pw production. Consequently, the optimized gene was used to test four signal peptides and two promoters in different combinations. Differences in extracellular β-gal-Pw activity between the recombinant B. subtilis strains were observed with the successful secretion being highly dependent on the specific combination of promoter and signal peptide used. Interestingly, signal peptides of both the general secretory- and the twin-arginine translocation pathway mediated secretion. The highest extracellular activity of 55.2 ± 6 µkat/L was reached when secretion was mediated by the PhoD signal peptide and expression was controlled by the P promoter. Production of extracellular β-gal-Pw was further enhanced 1.4-fold in a bioreactor cultivation to 77.5 ± 10 µkat/L with secretion efficiencies of more than 80%.
For the first time, the β-gal-Pw was efficiently secreted with B. subtilis SCK6, demonstrating the potential of this strain for secretory production of cytoplasmic, high molecular weight enzymes.
革兰氏阳性细菌枯草芽孢杆菌被广泛用于工业酶生产。其将多种酶分泌到细胞外培养基中的能力尤其有利于下游加工，因为无需进行细胞破碎。尽管多种异源酶已成功通过枯草芽孢杆菌分泌，但分泌高分子量的胞质酶具有挑战性。只有少数研究报道了分子量大于100 kDa的胞质酶的分泌情况。
在本研究中，温氏类芽孢杆菌的胞质120 kDaβ-半乳糖苷酶（β-gal-Pw）在枯草芽孢杆菌SCK6中表达并分泌。我们着重采用不同策略来确定最佳分泌条件。对β-gal-Pw基因进行定制密码子优化导致细胞外β-gal-Pw产量增加。因此，使用优化后的基因测试了四种信号肽和两种启动子的不同组合。观察到重组枯草芽孢杆菌菌株之间细胞外β-gal-Pw活性存在差异，成功分泌高度依赖于所使用的启动子和信号肽的特定组合。有趣的是，一般分泌途径和双精氨酸转运途径的信号肽均介导了分泌。当由PhoD信号肽介导分泌且由P启动子控制表达时，细胞外活性最高可达55.2±6 μkat/L。在生物反应器培养中，细胞外β-gal-Pw的产量进一步提高了1.4倍，达到77.5±10 μkat/L，分泌效率超过80%。
首次成功利用枯草芽孢杆菌SCK6高效分泌β-gal-Pw，证明了该菌株在分泌生产胞质高分子量酶方面的潜力。
