RT Dissertation/Thesis
T1 Food-grade Lactobacilli expression systems for recombinant enzymes
A1 Böhmer,Nico
WP 2014/02/12
AB Lactobacilli are Gram-positive bacteria used throughout the food industry as traditional starters for various fermented foods. Lactobacilli would be superior for recombinant enzyme production regarding the food safety demands since most of them are Generally Recognised As Safe (GRAS) organisms. The major advantages of Lactobacilli as food-associated microorganisms used for recombinant enzyme production are their safe and sustainable use as overall safety food-grade expression systems. In the work presented, Lactobacilli were studied in detail as food-grade expression systems for recombinant enzyme production. In a first analysis, the two pSIP expression systems, pSIP403 and pSIP409, were investigated to produce a hyper-thermophilic Beta-glycosidase (CelB) from Pyrococcus furiosus in Lactobacillus plantarum NC8 and Lactobacillus casei as hosts, respectively. Both Lactobacilli harbouring the pSIP409-celB vector produced active CelB in batch bioreactor cultivations, while the specific CelB activity of the cell-free extract was about 44% higher with Lb. plantarum (1,590 ± 90 nkatpNPGal/mgprotein) than with Lb. casei (1,070 ± 66 nkatpNPGal/mgprotein). A fed-batch bioreactor cultivation of Lb. plantarum NC8 pSIP409-celB resulted in a specific CelB activity of 2,500 ± 120 nkatpNPGal/mgprotein. A basal whey medium with supplements was developed as an alternative to the cost intensive MRS medium used. About 556 ± 29 nkat pNPGal/mgprotein of CelB activity was achieved in bioreactor cultivations using this medium. It was shown that both Lactobacilli were potential expression hosts for recombinant enzyme production. 
An additional approach was performed to produce a metagenome-beta-galactosidase using Lb. plantarum NC8 with the pSIP expression system. Using this system, a quite low maximal galactosidase activity of only 0.18 nkatoNPGal/mgprotein was detected. A 13 times higher activity of 2.42 nkatoNPGal/mgprotein was produced after the knock out of the interfering native Kluyveromyces lactis Beta-galactosidase in the well-known food-grade K. lactis pKLAC2 expression system. Nevertheless, the best performing expression system for the recombinant production of the metagenome-derived enzyme was the Escherichia coli BL21 strain with a pET vector, resulting in the highest Beta-galactosidase of 82.01 nkatoNPGal/mgprotein.
Beside the use of the pSIP expression system, a novel expression system for Lb. plantarum was developed. This system is based on the manganese starvation-inducible promoter from the specific manganese transporter of Lb. plantarum NC8 which was cloned for the first time. The expression of CelB was achieved by cultivating Lb. plantarum NC8 at low manganese concentrations with MRS medium and the pmntH2-celB expression vector. A CelB activity of 8.52 µkatoNPGal/L was produced in a bioreactor. The advantages of the novel expression system are that no addition of an external inducing agent was required, and additionally, no further introduction of regulatory genes was necessary. The new promoter meets the general demands of food-grade expression systems. 
The glutamic acid racemase of Lb. plantarum NC8 was cloned and characterized in this work for the first time as a possible target for a food-grade selection system for this species. Glutamic acid racemases (MurI, E.C. 5.1.1.3) catalyse the racemisation of L- and D-glutamic acid. MurIs are essential enzymes for bacterial cell wall synthesis, which requires D-glutamic acid as an indispensable building block. Therefore, these enzymes are suitable targets for antimicrobial drugs as well as for the potential design of auxotrophic selection markers. A high expression system in E. coli BL21 was constructed to produce and characterize the biochemical properties of the MurI from Lb. plantarum NC8. The recombinant, tag-free Murl was purified by an innovative affinity chromatography method using L-glutamic acid as the relevant docking group, followed by an anion exchange chromatography step (purification factor 9.2, yield 11%). This two-step purification strategy resulted in a Murl sample with a specific activity of 34.06 µkatD-Glu/mgprotein, comprising a single protein band in SDS-PAGE. The purified Murl was used for biochemical characterization to gain in-depth knowledge about this enzyme. Only D- and L-glutamic acid were recognised as substrates for the Murl with similar kcat/Km ratios of 3.6 sec-1/mM for each enantiomer. 
The findings in this study may contribute to further development and implementation of food-grade Lactobacilli expression systems for recombinant enzyme production. Furthermore, the results obtained may help to optimise and select hosts and expression systems for industrial enzyme production for the needs of the food industry. 


K1  
K1 Lactobacillus
K1 rekombinant
K1 Enzyme
K1 Produktion
PP Hohenheim
PB Kommunikations-, Informations- und Medienzentrum der Universität Hohenheim
UL http://opus.uni-hohenheim.de/volltexte/2014/925