Production of the Bacillus licheniformis SubC protease using Lactococcus lactis NICE expression system

Area of Science:

• * Microbial biotechnology
• * Protein engineering
• * Enzyme production

Background:

• * Bacillus licheniformis subtilisin (subC gene) is a valuable protease.
• * Extracellular secretion is crucial for enzyme applications like rennet production.
• * Lactococcus lactis is a common host for food-grade protein expression.

Purpose of the Study:

• * To clone and express the subC gene in Lactococcus lactis using the nisin-controlled expression (NICE) system.
• * To evaluate the efficiency of different signal peptides (SP Usp45 and SubC signal) for extracellular secretion.
• * To optimize protease production and activity for potential industrial applications.

Main Methods:

• * Cloning of the subC gene into NICE vectors (pNZ8048, pNZ8148).
• * Expression in Lactococcus lactis NZ9000 using nisin induction.
• * Testing extracellular protease activity with purified nisin and L. lactis NZ970 supernatant.
• * Optimization of induction time, nutrient supplementation, and calcium stabilization.
• * Verification in fed-batch bioreactor cultures.

Main Results:

• * SP Usp45 effectively directed extracellular secretion of active and stable subtilisin.
• * The native SubC signal peptide was recognized by L. lactis Sec pathway but with lower efficiency (3-fold lower activity).
• * Protease production and activity were significantly improved by optimizing induction, nutrients, and calcium ions.
• * Similar expression levels were achieved using purified nisin or L. lactis NZ970 supernatant.

Conclusions:

• * Lactococcus lactis is a suitable host for extracellular production of Bacillus licheniformis subtilisin.
• * The SP Usp45 signal peptide is highly effective for Sec-mediated extracellular secretion in L. lactis.
• * Optimized expression conditions and calcium stabilization enhance protease yield and stability.
• * The system shows potential for scalable rennet production.