Cloning and Overexpression of the als, pflA, and adhB Genes in Streptococcusthermophilus and Their Effects on Metabolite Formation

• Published in: Molecular biotechnology Vol. 57; no. 10; pp. 923 - 930
• Main Authors: Akyol, Ismail, Ozcelik, Fatma Gul, Karakas-Sen, Asuman, Ozkose, Emin, Gezginc, Yekta, Ekinci, M. Sait
• Format: Journal Article
• Language: English
• Published: New York Springer US 18-08-2015
• Subjects: Biochemistry; Biological Techniques; Biotechnology; Cell Biology; Chemistry; Chemistry and Materials Science; Human Genetics; Protein Science; Gene overexpression; Metabolic pathways
• ISSN: 1073-6085; 1559-0305
• DOI: 10.1007/s12033-015-9882-1

Streptococcus
thermophilus
is a lactic acid bacterium and used as starter culture in the dairy industry, mainly in the manufacture of yoghurt, with
Lactobacillus delbrueckii
subsp
. bulgaricus
. It produces lactic acid as a major fermentation end product and some carbonyl compounds through sugar metabolism. The level of metabolites could be improved using molecular biotechnology. The genes of
als
, encoding α-acetolactate synthase (Als), the
pflA
, encoding pyruvate-formate lyase activating enzyme (PflA), and the
adhB
which encodes alcohol dehydrogenase (AdhB) of
S. thermophilus
NCFB2393 strain were amplified by polymerase chain reaction and separately cloned into the overexpression vector pNZ276 under the control of the
lacA
promoter. The strains were transformed individually with the constructed plasmids. Their abilities to generate important metabolites such as pyruvate, lactate, formate, acetaldehyde, acetoin, ethanol, and 2,3-butanediol in LM17 medium were analyzed using high-performance liquid chromatography. High level of 2,3-butanediol was obtained by overexpressing the
als
gene. The level of formate increased slightly by overexpressing the
pfl
A gene. The overexpression of the
adhB
gene, on the other hand, resulted in a significant increase in the ethanol level.