Stepwise modifications of genetic parts reinforce the secretory production of nattokinase in Bacillus subtilis.
Microb Biotechnol
; 11(5): 930-942, 2018 09.
Article
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| MEDLINE
| ID: mdl-29984489
ABSTRACT
Nattokinase (NK) is an important serine-protease with direct fibrinolytic activity involving the prevention of cardiovascular disease as an antithrombotic agent. Dozens of studies have focused on the characterization of intrinsic novel promoters and signal peptides to the secretory production of recombinant proteins in Bacillus subtilis. However, intrinsic genetic elements have several drawbacks, which cannot mediate the production of NK to the desired level. In this study, the genetic elements, which were used to overproduce the recombinant secretory NK, were rationally modified in B. subtilis in a stepwise manner. The first step was to select a suitable signal peptide for the highly efficient secretion of NK. By comparison of the secretory levels mediated by two different signal peptides, which were encoded by the genes of a minor extracellular protease epr (SPepr ) and cell-wall associated protease wapA (SPwapA ), respectively, SPwapA was verified as the superior secretory element. Second, P04, which was a synthetic promoter screened from an array of mutants based on the promoter cloned from the operon of a quorum-sensing associated gene srfA (PsrfA ), was paired to SPwapA. The secretory level of NK was obviously augmented by the combination of these two genetic elements. Third, the cis-acting element CodY-binding sequence positioned at the 5'UTR was deleted (yielding P08), and thus the secretory level was significantly elevated. The activity of NK, which was defined as fibrinolytic units (FU), reached to a level of 270 FU ml-1 . Finally, the superior genetic element composed of P08 and SPwapA was utilized to overproduce NK in the host B. subtilis WB800, which was able to produce the secretory NK at 292 FU ml-1 . The strategy established in this study can not only be used to overproduce NK in B. subtilis but also might be a promising pipeline to modify the genetic element for the synthetic secretory system.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Bacillus subtilis
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Proteínas Recombinantes
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Subtilisinas
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Ingeniería Metabólica
Idioma:
En
Revista:
Microb Biotechnol
Año:
2018
Tipo del documento:
Article
País de afiliación:
China