[Expression in Lactococcus lactis of catalytically active phenylalanine ammonia-lyase from parsley].
The phenylalanine ammonia-lyase (PAL) cDNA of parsley (Petroselinum crispum) was subcloned into constitutive expression vector pMG36e downstream of the P32 promoter. The resulting plasmid pMG36ePAL was introduced into Lactococcus lactis subsp. lactis MG1363 by electroporation. The recombinant strain showed its PAL activity conversing the L-phenylalanine in the culture medium into trans-cinnamic acid. A new secretory vector pXHS was constructed by recombination of pMG36e with a Lactococcal usp45 secretion leader coding sequence and a translational coupling sequence. Then the pXHSPAL was constructed and used for expression of PAL in L. lactis, the PAL activity was also detectable. The L. lactis dnaJ promoter sequence was cloned and used to construct a heat inducible vector pXHJ. PAL cDNA was cloned into pXHJ and the L. lactis IL1403 was transformed with the recombinant plasmid pXHJPAL. After a heat shock from 30 degrees C to 37 degrees C, the PAL activity of the pXHJPAL strain could increase approximately onefold. The prospect of using these engineering L. lactis strains for PKU therapy was also discussed.
Xiang
H
H
Beijing Red Cross Chaoyang Hospital, Beijing 100020.
Liu
J
J
Hu
W
W
Zhu
J
J
Zhu
Z
Z
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D013294:Lactococcus lactis / Q000235:genetics*; D008969:Molecular Sequence Data; D028528:Petroselinum / Q000201:enzymology* / Q000235:genetics; D010650:Phenylalanine Ammonia-Lyase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D010661:Phenylketonurias / Q000628:therapy; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D010650:Phenylalanine Ammonia-Lyase