thyA as a Selection Marker in Construction of Food-Grade Host-Vector and Integration Systems for Streptococcus thermophilus

Abstract

ABSTRACT 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.