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.
Food Functionality Research Institute, Meiji Dairies Corporation, Odawara, Kanagawa 250-0862, Japan. YASUKO_SASAKI@MEIJI-MILK.COM
Yasuko
Y
Sasaki
Yoshiyuki
Y
Ito
Takashi
T
Sasaki
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