Cloning and
stable expression of the alpha-acetolactate decarboxylase gene from Bacillus
Licheniformis
in yeast.
Xiaoming Bao (1),
Yujing Qin (2), Huajun Zheng (2), Aihua Hou (2), Guoliang Yang (2), Dong Gao
(2)
(1) State Key Laboratory of Microbial Technology, College of Life Sciences,
Shandong University, Jinan City, 250100, P.R. China (bxm@sdu.edu.cn); (2)
Shandong University, State Key Lab. of Microb Techn, Shandananlu 27#, Jinan,
250100, P. R. China
The gene
encoding alpha-acetolactate decarboxylase (alpha-ALDC) of Bacillus
licheniformis
was isolated from the genomic library by detecting enzyme activity on the agar
plate. After sequencing of a 1.6-kb DNA fragment, an open reading frame
(GenBank accession number AF428095) of the alpha-ALDC gene was got. The
alpha-ALDC from native srtain was purified and characters of the enzyme were
studied, the results showed that the enzyme optimal activity was at pH 6 and
40° and its pI was 4.5. The ORF of the alpha-ALDC was placed under the control
of the PGK promoter of the Saccharomyces cerevisiae. Then the expression
cassette of alpha-ALDC gene was ligated to the multiple integration plasmid
pMIRY2 which has the rDNA region as the integrating site. The new recombinant
plasmid pMIRY2-ALDC, which was linearized at the SmaI site in the rDNA region,
was co-transformed into the beer yeast industrial strain together which a
plasmid containing G418 gene. The multiple integration of the plasmid in the
transformants was proved by Southern blot. Yeast cells containing the
recombinant plasmids showed alpha-ALDC activity. The result of lab-scale
fermentation reveals that the diacetyl concentration in wort was significantly
lower than in wort that fermented by the host strain. This indicates in
lab-level that it was feasible to degrade the diacetyl production in industrial
scale by constructing an engineering strain having alpha-ALDC activity.