Departmental Affiliation and City, State, Zip for All Authors

1. Department of Biology, Northridge, California, 91330; 2. Center for Medical Genetics, Institute for Molecular Medicine, Fort Worth, Texas, 76107

Scientific Abstract

Phenylketonuria (PKU) is an autosomal recessive disease that prevents metabolic breakdown of L-phenylalanine into L-tyrosine. If left untreated, PKU can result in severe neurological defects. An effective treatment is a diet in which phenylalanine intake is restricted. However, compliance to the diet is difficult due to its unpleasant taste and cost of supplemental medical foods. A possible alternative therapy is the use of a genetically engineered probiotic that could degrade ingested phenylalanine in the gut prior to its absorption. Preliminary work using Lactobacillus reuteri 100-23C carrying a plasmid borne copy of the avPAL gene encoding a modified phenylalanine ammonia lyase (PAL) enzyme capable of metabolizing phenylalanine resulted in reduced blood phenylalanine levels in a mouse model of PKU when the bacterium and gene were delivered as a probiotic. Due to issues related to plasmid transfer/loss and antibiotic resistance, a chromosomally inserted copy of avPAL is desired in an effort to move forward to human therapeutic trials. To this end, L. reuteri 100-23C cells were grown in MRS broth for 48 hours and prepared for electroporation with a transposon encoded copy of avPAL. After electroporation, the L. reuteri 100-23C cells were allowed to recover and then transformants were selected by playing on media containing erythromycin. DNA was then extracted from the resultant colonies and quantified. Primers were designed to amplify the regions of the transposon containing ermB and avPAL genes, and DNA from clones of L. reuteri 100-23C were screened by PCR. Gel electrophoresis of PCR amplicons was used to detect successful insertion of ermB and avPAL genes in the DNA of isolated colonies. Three L. reuteri 100-23C clones were found to contain avPAL and ermB, indicating that erythromycin resistance was not spontaneous. Future efforts will determine functional expression of the avPAL enzyme and map the insertion sites.

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Insertion of avPAL/ermB Transposon in Lactobacillus reuteri 100-23C (pHENOMMenal)

Phenylketonuria (PKU) is an autosomal recessive disease that prevents metabolic breakdown of L-phenylalanine into L-tyrosine. If left untreated, PKU can result in severe neurological defects. An effective treatment is a diet in which phenylalanine intake is restricted. However, compliance to the diet is difficult due to its unpleasant taste and cost of supplemental medical foods. A possible alternative therapy is the use of a genetically engineered probiotic that could degrade ingested phenylalanine in the gut prior to its absorption. Preliminary work using Lactobacillus reuteri 100-23C carrying a plasmid borne copy of the avPAL gene encoding a modified phenylalanine ammonia lyase (PAL) enzyme capable of metabolizing phenylalanine resulted in reduced blood phenylalanine levels in a mouse model of PKU when the bacterium and gene were delivered as a probiotic. Due to issues related to plasmid transfer/loss and antibiotic resistance, a chromosomally inserted copy of avPAL is desired in an effort to move forward to human therapeutic trials. To this end, L. reuteri 100-23C cells were grown in MRS broth for 48 hours and prepared for electroporation with a transposon encoded copy of avPAL. After electroporation, the L. reuteri 100-23C cells were allowed to recover and then transformants were selected by playing on media containing erythromycin. DNA was then extracted from the resultant colonies and quantified. Primers were designed to amplify the regions of the transposon containing ermB and avPAL genes, and DNA from clones of L. reuteri 100-23C were screened by PCR. Gel electrophoresis of PCR amplicons was used to detect successful insertion of ermB and avPAL genes in the DNA of isolated colonies. Three L. reuteri 100-23C clones were found to contain avPAL and ermB, indicating that erythromycin resistance was not spontaneous. Future efforts will determine functional expression of the avPAL enzyme and map the insertion sites.

Manuscript Number

1019