High-level expression of Lactobacillus beta-galactosidases in Lactococcus lactis using the food-grade, nisin-controlled expression system NICE.
In this work the overlapping genes (lacL and lacM) encoding heterodimeric beta-galactosidases from Lactobacillus reuteri , Lb. acidophilus , Lb. sakei , and Lb. plantarum were cloned into two different nisin-controlled expression (NICE) vectors and expressed using Lactococcus lactis NZ9000 and NZ3900 as hosts. The lacL gene, encoding the large subunit of the beta-galactosidases, was fused translationally downstream of the nisin-inducible promoter nisA. Chloramphenicol was employed as selection marker for the standard system using L. lactis NZ9000, whereas lactose utilization based on the complementation of the lacF gene was used as a dominant selection marker for the food-grade system employing L. lactis NZ3900. Comparison of the standard and the food-grade expression system, differing only in their selection markers, gave considerable differences in volumetric beta-galactosidase activity, ranging from 1.17 to 14 kU/L of fermentation broth, depending on both the origin of the lacLM genes and the selection marker used. The occurrence of codons less frequently used by L. lactis especially at the beginning of the lacL gene could be an explanation for the significant differences between the expression levels of lacLM from different origins, while plasmid stability might cause the difference obtained when employing the different selection markers.
Maischberger
Thomas
T
Research Centre Applied Biocatalysis, Petersgasse 14, A-8010 Graz, Austria.
Mierau
Igor
I
Peterbauer
Clemens K
CK
Hugenholtz
Jeroen
J
Haltrich
Dietmar
D
D001234:Aspergillus niger / Q000201:enzymology / Q000235:genetics; D001236:Aspergillus oryzae / Q000201:enzymology / Q000235:genetics; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D017931:DNA Primers; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005816:Genetic Complementation Test; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics; D009561:Nisin / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D009561:Nisin; D001616:beta-Galactosidase