Heterologous expression of the Bacillus subtilis (natto) alanine dehydrogenase in Escherichia coli and Lactococcus lactis

The major objective of the present study is to change the alanine production of Lactic acid bacteria by expression of Bacillus sub tills (natto) alanine dehydrogenase (AlaDH), the gene that is not present in Lactic acid. B. subtilis AlaDH gene (ald) was cloned into a pGEX6p-1 and expressed in E. coli JM109. Its enzyme activity was 48.3 U/mg at 30 degrees C and 45.2 U/mg at 42 degrees C. This aid gene was then cloned into a vector pNZ8148 to generate a vector pNZ8148/ald. The same aid gene was placed downstream of the ldh promoter from Streptococcus thermophilus to generate pNZ273/ldhp/ald. The pNZ8148/ald and pNZ273/ldhp/ald were introduced separately in Lactococcus lactis NZ9000. As a result of over-expressed aid, the production of alanine detected by HPLC in L. lactis NZ9000 carrying pNZ273/ldhp/ald reached 52 mu g/ml, an approximately 26-fold increase compared to the parent strain L. lactis NZ9000, but not in L. lactis NZ9000 carrying pNZ8148/ald. This study would help strain improvement to be used in dairy fermentation for developing healthy yogurts with sweet taste or other fermented dairy foods. (c) 2009 Elsevier GmbH. All rights reserved.