Cloning and Overexpression of the als, pflA, and adhB Genes in Streptococcus thermophilus and Their Effects on Metabolite Formation.
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 pflA gene. The overexpression of the adhB gene, on the other hand, resulted in a significant increase in the ethanol level.
Biotechnology and Gene Engineering Laboratory, Agricultural Biotechnology Department, Agriculture Faculty, Kahramanmaras Sutcu Imam University, Avsar, 46060, Kahramanmaras, Turkey, ismailakyol@ksu.edu.tr.
Ismail
I
Akyol
Fatma Gul
FG
Ozcelik
Asuman
A
Karakas-Sen
Emin
E
Ozkose
Yekta
Y
Gezginc
M Sait
MS
Ekinci
D000095:Acetolactate Synthase / Q000235:genetics* / Q000378:metabolism; D000123:Acetyltransferases; D000426:Alcohol Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002072:Butylene Glycols / Q000032:analysis; D003001:Cloning, Molecular / Q000379:methods*; D004798:Enzymes / Q000235:genetics* / Q000378:metabolism; D000431:Ethanol / Q000032:analysis; D005561:Formates / Q000032:analysis; D015964:Gene Expression Regulation, Bacterial; D060847:Metabolic Engineering; D010957:Plasmids / Q000235:genetics; D048249:Streptococcus thermophilus / Q000201:enzymology* / Q000235:genetics; D015854:Up-Regulation
D001426:Bacterial Proteins; D002072:Butylene Glycols; D004798:Enzymes; D005561:Formates; C030544:formic acid; D000431:Ethanol; C026978:2,3-butylene glycol; D000426:Alcohol Dehydrogenase; D000095:Acetolactate Synthase; D000123:Acetyltransferases; C040285:pyruvate formate-lyase activating enzyme