ThyA as a selection marker in construction of food-grade host-vector and integration systems for Streptococcus thermophilus.
We constructed food-grade host-vector and integration systems for Streptococcus thermophilus by using a thymidylate synthase gene (thyA) as the selection marker. Two thyA genes, thyA(St) and thyA(Lb), were cloned from S. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, respectively. Thymidine-requiring mutants of S. thermophilus were obtained after successive cultures in the presence of trimethoprim, and one of them, TM1-1, was used as the host. Food-grade vectors were constructed by using either thyA(St) or thyA(Lb) as the selection marker. Transformants of TM1-1 created by using these vectors were selected for thymidine autotrophy as efficiently as for erythromycin resistance. By using the host-vector system developed in this way, a foreign amylase gene (amyA) was expressed in TM1-1 and was also integrated into the chromosome by use of a temperature-sensitive integration vector constructed with thyA(Lb) as the selection marker via a double-crossover event. The results obtained show that thyA is an efficient and safe selection marker for S. thermophilus that is suitable for food applications.
Sasaki
Yasuko
Y
Food Functionality Research Institute, Meiji Dairies Corporation, Odawara, Kanagawa 250-0862, Japan. YASUKO_SASAKI@MEIJI-MILK.COM
Ito
Yoshiyuki
Y
Sasaki
Takashi
T
D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D005516:Food Microbiology; D005524:Food Technology; D005798:Genes, Bacterial; D005819:Genetic Markers; D005822:Genetic Vectors; D006801:Humans; D007778:Lactobacillus / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015183:Restriction Mapping; D013291:Streptococcus / Q000201:enzymology* / Q000235:genetics*; D013940:Thymidylate Synthase / Q000235:genetics*; D014170:Transformation, Genetic; D015014:Yogurt / Q000382:microbiology
D004269:DNA, Bacterial; D005819:Genetic Markers; D013940:Thymidylate Synthase