„Whole Genome Sequencing of Lactobacillus delbrueckii ssp. bulgaricus
2038: Structural and Functional Analysis”, Hua-Jun ZHENG, Bo-Fei
WANG, Shuang-Xi REN, Sheng-Yue WANG, Hui HAN, Munehiro ODA,
Kazuo SHIN, Gang FU, Zai Si JI, Guo-Ping ZHAO
The following genetic systems for yogurt starter bacteria have been developed in
our laboratory.
1) We succeeded for the first time in transformation of Lactobacillus bulgaricus
using pBUL1 plasmid from L. bulgaricus M-878. Efficient transformation was
achieved using a restriction and modification (R/M) deficient mutant of M-878,
which had been obtained by a spontaneous deletion of the R/M genes. Several
vectors including pX3 and pSYE2 were developed and foreign gene expression
was achieved.
2) Two methods were developed to integrate gene into the chromosome of L.
bulgaricus: The first method uses pAM
as the integration vector, and the
second uses a temperature-sensitive replication vector, pSG+E2. The lactate
dehydrogenase gene (ldh) was replaced with that from Streptococcus
thermophilus, resulting in a gene-convertant of L. bulgaricus that produces
solely L-lactate. Some other genes have been successfully integrated, knockedout or replaced. We analyzed the R/M genes in M-878, and an R/M-deficient
knock-out deletant was constructed.
3) We also developed food-grade vectors using DNA exclusively from industrial
strains of LAB with either a -galactosidase gene or a thymidilate synthase
gene as the selection marker. Gene conversion of ldh of L. bulgaricus and
-amylase gene secretion by S. thermophilus were successful using
these food-grade vectors.