Production of the Bacillus licheniformis SubC protease using Lactococcus lactis NICE expression system.pdf
RESEARCH Open Access Production of the Bacillus licheniformis SubC protease using Lactococcus lactis NICE expression system Aleksandra M Mirończuk1,2, Anna Krasowska2, Anna Murzyn2, Małgorzata Płachetka2 and Marcin Łukaszewicz1,2,3* 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. Keywords: Lactic acid bacteria, Lactococcus lactis, Nisin-controlled expression system, NICE, Bacillus licheniformis, SubC protease Introduction Lactococcus lactis is a Gram-positive, lactic acid bacte- rium that is commonly used in traditional food indus- tries such as in cheese and butter production. In addition, it is increasingly used in modern biotechno- logical applications. Many recent studies have investi- gated the physiology and genetic of this bacterium, therefore a wide variety of genetic tools have been deve- loped. 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 exten- sive study on heterologous protein expression in L. lactis. Since L. lactis is generally recognized as safe (GRAS) it could be used for large-scale production of heterologous proteins (Mierau et al. 2005a; Morello et al. 2008). Most of the laboratory scale examples consist of intracellular ex- pression (Blatny et al. 2003; Kunji et al. 2003) or cell wall bound enzymes (Cibik et al. 2001; Miyoshi et al. 2002; Nouaille et al. 2003). Much less in known about extracel- lular production of the proteins in L. lactis, moreover the protein yield might differ significantly and are strongly case-dependent (Mierau & Kleerebezem 2005). In 1995 Kuipers et al. (1995) published a study on the autoinduction of the expression of nisin in lactococci. This study allowed for the construction of a food grade expression system based on the regulation mechanism of the nisinA operon of L. lactis (Platteeuw et al. 1996), named NICE (nisin-controled gene expression). In this operon the gene product, a small 34 amino acid bac- teriocin, induces its own transcription at very low con- centrations (0.5 – 5 ng/mL) (Kuipers et al. 1998). In this system, nisin induces the regulatory cascade starting with binding to the membrane-bound receptor NisK. Next, the phosphate group from the activated NisK is * Correspondence: marcin.lukaszewicz@uni.wroc.pl 1Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, Chełmońskiego 37/41, Wrocław 51-630, Poland 2Department of Biotransformation, Faculty of Biotechnology, University of Wroclaw, Przybyszewskiego 63-77, Wroclaw 51-148, Poland Full list of author information is available at the end of the article a SpringerOpen Journal © 2012 Mironczuk et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mirończuk et al. SpringerPlus 2012, 1:54 http://www.springerplus.com/content/1/1/54 transferred to the intracellular response regulator NisR, activating this regulator. Subsequently, NisR, induces the nisin operon at the promoter nisA (Kleerebezem & Quadri 2001). The NisA promoter controls the expres- sion of the genes involved in the nisin biosynthesis (or the gene of interest). Genes of this regulatory system have been inserted in a suitable host strain L. lactis NZ900. The nisin-producing strain L. lactis NZ9700 secretes the nisin into the medium (Kuipers et al. 1995). It was shown that the NICE system can be developed to “food-grade” production of heterologous protein, by replacing the antibiotic resistant gene by another select- able marker (Olempska-Beer et al. 2006), for example lacF, which has been deleted from the host strain, and is essential for growth on lactose (Mierau et al. 2005b). Although for large-scale production nisin usage remains costly, a good alternative is the addition of NZ9700 supernatant. The NICE system is often used in the laboratories for research, while the data on large-scale application of the NICE system for secreted proteins is still very limited. Up to now, only a few reports present usage of this system in industry (Mierau et al. 2005a; Mierau et al. 2005b; Berlec & Strukelj 2009), moreover, efficient systems for the industrial scale production of secrete heterologous proteins have never been described. In this study, we describe the production of secrete heterologous protein SubC in L. lactis using the NICE expression system. SubC is the industrially important Carlsberg-type subtilisin (Jacobs 1995) produced by Bacillus licheniformis. Bacterial subtilisins are multipur- pose alkaline proteases that are frequently used in indus- try (Gupta et al. 2002): common variants include subtilisin from B. amyloliquefaciens, highly alkalophilic B. lentus or from B. licheniformis (Rao et al. 1998; von der Osten et al. 1993). Furthermore, Flavobacterium also produces subtilisin (Morita et al. 1998). Interestingly, al- most two-third of commercial proteases produced in the world originate from microorganisms (Kumar & Takagi 1999; Tremacoldi et al. 2004). Microbial proteases are classified into different groups, according to their activ- ity in acid, neutral or alkaline conditions, and on the characteristics of the active site group of the enzyme (Saeki et al. 2007). Subtilisin-like serine proteases are usually secreted extracellulary for searching nutrients (Aehle et al. 2009). One of the features of this class of proteases (subtilases) is an aromatic or hydrophobic residue, such as leucine, tyrosine or phenylalanine. The highest proteolytic activity is around pH 10, with a molecular weight range of 15–40 kDa and an isoelectric point around pI 9. Moreover, the native form of SubC remains fully stable up to 60°C (Hirata et al. 2003) and it possesses two calcium binding site(s), therefore bound Ca2+ contributes to enzyme stability (Briedigkeit & Frömmel 1989). Our main objective in this study was an improvement of the expression condition for secreted heterologous protein production and the comparison of the gene expression efficiencies using two different NICE expres- sion plasmids. We compared two different NICE vectors and the ability of L. lactis for secretion of heterologous protein. The activity of protease production was tested on milk plates. Subsequently the proteolytic activity assays were performed to investigate the functionality of the secreted protease in the medium. In addition, we present modification of the NICE system, by changing the growth conditions, the induction point, and by extending the logarithmic phase growth of bacteria by supplying further nutrients. Methods Strains and growth conditions The strains and plasmids used in this study are listed in Table 1. Lactococcus lactis strains were grown in M17 broth (Terzaghi & Sandine 1975) supplemented with 0.5% glucose (GM17). Additionally, for strains carrying plas- mid, medium was supplemented with chloramphenicol (5 μg mL-1). Cultures were incubated at 30°C. If required, medium was supplemented with 10% milk or different concentrations of CaCl2, MnCl2, MgCl2 and MgSO4. Additionally, samples were supplemented with 5 μg mL-1 chloramphenicol, if required. Bacterial growth was determined by measuring the optical density (OD) at 600 nm. The cultures were inoculated at optical dens- ity 0.1 or at a different point if mentioned. At the begin- ning of the incubation or when the bacterial growth reached the required cell density, the cultures were stimulated to produce protease by the addition of nisin (Sigma) or by the dilutions of the culture supernatant of the nisin producing strain L. lactis NZ9700 (the range of concentrations or dilutions indicated in Results). The Table 1 Strains used in this study Strain/plasmid Relevant characteristics Reference ATCC 10716 B. licheniformis Laboratory stock NZ9000 L. lactis MG1363 pepN::nisRK (Kuipers et al. 1998) NZ9700 L. lactis Nisin producer (Kuipers et al. 1995) pNZ8048 CmR, inducible expression vector containing the nisA promoter (Kuipers et al. 1998) pNZ8148 PnisA, CmR; replicon of rolling circle plasmid pSH71, basic NICE vector, derivative of pNZ8048 NIZO pNZ45 pNZ8048 carrying SP Usp45 under nisA promoter This study pNZ45subC pNZ45 carrying subC gene This study pNZ48subC pNZ8148 carrying subC gene This study Mirończuk et al. SpringerPlus 2012, 1:54 Page 2 of 10 http://www.springerplus.com/content/1/1/54 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. Moreover, for L. lactis the genome sequence has been published (Wegmann et al. 2007) and many genetic tools have been developed for LAB, which simplifies the usage of these microorganisms as a cell factory. Add- itionally they do not require aeration and only very limited mixing that significantly reduces production and reactor costs. One of the most popular expression systems in L. lactis is the NICE system (Mierau & Kleerebezem 2005; Kuipers et al. 1995). In this study, we present the modification of the NICE system for heterologous secretion protease production in L. lactis, with possible usage at industrial scale. We used two different NICE vectors; we compared different ranges of nisinA concentration for induction and nu- trient requirements. For that purpose, the subC gene encoding B. licheniformis extracellular alkaline protease was cloned downstream of the strong inducible pro- moter nisA and ranges of diluted supernatant of NZ9700 L. lactis (nisin producer) were used for the induction of secreted enzyme production. In this study, we optimized the growth and NICE- related parameters. Strikingly, laboratory strains of L. lactis possess only 24h 0 50 100 150 200 250 300 350 400 450 proteolytic activity % 2h 3h                    4h  Figure 5 The proteolytic activity of L. lactis carrying pNZ45subC. The cultures were grown in GM17 medium supplemented with 0.5 mM CaCl2 and 5 μg mL-1 of chloramphenicol. The cultures were induced at OD600 0.7. The samples were taken as indicated on the picture: 2, 3, 4 and 24 hours after induction with 10 000 NZ9700 supernatant. Pale gray bars- strain without induction (values below 1). Black bars- control strain without additional nutrients. Dark gray bars- the strain supplemented with an additional nutrients in two hours after induction. All data are mean values of three independent experiments; error bars indicate standard deviation. 0 20 40 60 80 100 120 140 160 0.0E+00 1.0E+09 2.0E+09 3.0E+09 4.0E+09 5.0E+09 6.0E+09 7.0E+09 8.0E+09 0h 2h 4h 6h 24h 48h Activity U/l Cell number Cell number Activity Figure 6 The proteolytic activity of L. lactis carrying pNZ45subC in 5 L bioreactor (working volume 1.5 L). The culture was grown in GM17 medium supplemented with 10% milk and 5 μg mL-1 of chloramphenicol. The culture was induced at OD600 0.2. The samples were taken as indicated on the picture. Mirończuk et al. SpringerPlus 2012, 1:54 Page 8 of 10 http://www.springerplus.com/content/1/1/54 one exported housekeeping protease, HtrA involved in protein quality control at the cell surface. Moreover, HtrA is responsible for clearing anomalous proteins from the surface, and is induced under several stress conditions (Morello et al. 2008). The expression of heterologous protease might be lethal for L. lactis, however it was recently shown that the use of SP Usp45 also allows the secretion of bacte- riocins which are toxic for cells (Borrero et al. 2009). Therefore to avoid toxicity we added signal peptide of Usp45 to secrete SubC via Sec transporters. Indeed higher protease expression and accumulation within the cell resulted in growth inhibition (data not shown). Here we observed that the addition of lactococcal SP usp45 to the native signal peptide of SubC leads to better secretion, and more importantly, secreted protein remains stable, since both signal peptides are properly cleaved during protein translocation to the medium. Interestingly, a con- struct with two signal peptides results in higher accumu- lation of the protease in extracellular medium. Subsequently, we investigated the media preferences for optimal protein production. The previously described medium (Mierau et al. 2005a) did not increase the pro- tein level. In contrast, medium supplementation with calcium chloride resulted in superior activity of protease in the extracellular environment. Interestingly, we have also noticed that supplementing GM17 medium with other two positive ions, results in elevated proteolytic activity of SubC producer, nonetheless the highest acti- vity was observed in samples supplemented with 0.5 mM CaCl2. After establishing the medium preferences for SubC production, we tested various induction time points. Hitherto, the strains carrying overexpression constructs were induced at the midlog growth phase or at high OD (Mierau et al. 2005a; Maischberger et al. 2010) although we have not confirmed the reports show- ing the results of induction at start point. To this end, the overnight cultures of L. lactis carrying pNZ45subC or pNZ48subC were inoculated into fresh medium, sup- plemented as aforementioned, and induced at OD600 0.1 and 0.6 and with various ranges of NZ9700 super- natant. The samples were tested for proteolytic activity 24 hours after induction. We did not observe significant differences in the proteolytic activity between all sam- ples, which might suggest that the level of functional SubC protease was the same in all cultures. In contrast to other reports (Berlec et al. 2008), we did not observe a correlation between increasing OD and protein pro- duction. One of the most interesting parts of this phenomenon is the problem of the stability/activity of SubC in the extracellular environment. Strikingly, the SDS-PAGE data showed that the level of protein pro- duction in various cultures was unchanged; however the proteolytic activity was different. This could result from various proportions of mature versus immature protease as seen from SubC amino acid sequence determination. Noticeably, the overproduction of protein might also cause stress responses and consequently degradation of the target protein in the cells (Thumm & Gotz 1997). We compared different nisinA concentrations for L. lactis induction. The studied nisin concentration range was from 0.1 to 10.0 ng mL-1 and the highest pro- teolytic activity was obtained when 1 ng mL-1 of nisinA or 10 000 – 20 000 diluted supernatant of L. lactis NZ9700 was used. In comparison, nisin concentration in published data varies from 0.5 to 40 ng mL-1 (Mierau et al. 2005b). Thus induction during inoculation of the bioreactor enables reduction of the inducer amount and simplifies the production process. One of the most inter- esting issues of this study was an extension of the loga- rithmic phase growth of L. lactis, since during this phase the highest activity was observed. Initial experiments showed that L. lactis utilizes most of the 0.5% glucose present in GM17 medium. Conse- quently, the next step was to supply more nitrogen and carbon sources. The cultures were induced at OD600 0.6, the additional nutrients were added in two hours after induction. Interestingly, we observed increased proteo- lytic activity already 2 hours after supplementing with nutrients. The differences between the target strain and the control were between 30-40%, the highest differences were observed in 6 hours after induction, and remained stable up to 24th hour after inductions. In summary, we optimized the NICE expression sys- tem for heterologous secretion protease production. The use of a GRAS expression host for secreted enzyme pro- duction will make it possible to use these proteins much more easily and economically, than in the case of intra- cellular production. The described expression system might be used for industrial production of rennet or direct application of such strain in dairy. Competing interests The authors declare that they have no competing interests. Authors’ contributions AMM carried out the molecular genetic studies, performed the experiments, analyzed the data, wrote the manuscript. AK analyzed the data, drafted the manuscript. AM performed the experiments, analyzed the data, drafted the manuscript. MP performed the experiments, analyzed the data, participated in the sequence alignment, drafted the manuscript. MŁ Contributed reagents/materials/analysis tools, analyzed the data, wrote the manuscript. All authors read and approved the final manuscript. Acknowledgements We thank Oscar P. Kuipers for the gift of L. lactis vectors pNZ8048 and L. lactis NZ9700, and Katarzyna Bednarz for technical support. This work was supported by grant KB/48/13639/IT1-B/U/08 from the Polish National Centre for Research and Development and EU POIG.01.01.02-00- 016/2008. Mirończuk et al. SpringerPlus 2012, 1:54 Page 9 of 10 http://www.springerplus.com/content/1/1/54 Author details 1Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, Chełmońskiego 37/41, Wrocław 51-630, Poland. 2Department of Biotransformation, Faculty of Biotechnology, University of Wroclaw, Przybyszewskiego 63-77, Wroclaw 51-148, Poland. 3Faculty of Chemistry, Wrocław University of Technology, Gdańska 7/9, Wrocław 50-344, Poland. Received: 4 September 2012 Accepted: 8 November 2012 Published: 29 November 2012 References Aehle W, Bott R, Graycar T, Flickinger MC (2009) Proteolytic Cleavage, Reaction Mechanisms. 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Title: Production of the Bacillus licheniformis SubC protease using Lactococcus lactis NICE expression system ​
Authors: Aleksandra M. Mirończuk, Anna Krasowska, Anna Murzyn, Małgorzata Płachetka, Marcin Łukaszewicz ​
Publisher: SpringerPlus
Date: November 29, 2012 ​
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 were 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 were 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 a fed-batch bioreactor for further scale-up of the expression system. ​
Keywords: Lactic acid bacteria, Lactococcus lactis, Nisin-controlled expression system, NICE, Bacillus licheniformis, SubC protease ​