Abstract
In this work the subC gene from Bacillus licheniformis encoding subtilisin was cloned into the nisin-controlled expression (NICE) vectors (pNZ8048 and pNZ8148) with or without the signal peptide SP Usp45 directing extracellular secretion via Sec machinery. Extracellular protease production and activity was tested using Lactococcus lactis NZ9000 as host, which could be used for rennet production. The efficiency of protein production was tested using purified nisin and the supernatant of L. lactis NZ970 nisin producer. Similar results were obtained for 1 ng/ml nisin and 10 000 diluted supernatant. SP Usp45 signal peptide effectively directed extracellular localization of active and stable protease. SubC signal for extracellular localization in B. licheniformis, was also recognized by L. lactis Sec pathway, although with lower efficiency, as shown by a 3-fold lower protease activity in the medium. Protease production and activity was optimized using parameters such as induction time, nutrients (glucose, casitone) supplementation during growth or protease stabilization by calcium ions. The results were also verified in fed-batch bioreactor for further scale-up of the expression system.
Highlights
Lactococcus lactis is a Gram-positive, lactic acid bacterium that is commonly used in traditional food industries such as in cheese and butter production
We describe the production of secrete heterologous protein SubC in L. lactis using the nisin-controlled expression (NICE) expression system
Proteins production is lactic acid bacteria (LAB) such as Lactococcus lactis (Hugenholtz 2008). They are widely used in industrial fermentations, so much information is available about nutrient requirements, growth conditions etc
Summary
Lactococcus lactis is a Gram-positive, lactic acid bacterium that is commonly used in traditional food industries such as in cheese and butter production. It is increasingly used in modern biotechnological applications. Many recent studies have investigated the physiology and genetic of this bacterium, a wide variety of genetic tools have been developed. Nowadays several genomes of L. lactis strains are completely sequenced (Bolotin et al 2001; Siezen et al 2010; Wegmann et al 2007). Genetic accessibility and the ease of working with this organism have led to extensive study on heterologous protein expression in L. lactis. Since L. lactis is generally recognized as safe (GRAS) it
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