Polymerase chain reaction based cloning of acetate kinase in propionibacterium acidipropionici
Year: 2004
Type: article
Abstract: Currently, the U.S. market for propionate is about 400 million pounds per year, and the demand is increasing at 3 ~ 4 % annually. Fermentation process can use byproducts from the food industry, such as corn steep liquor and cheese whey, to produce propionic acid. It is thus desirable to convert these low-grade byproducts to a marketable product, such as propionic acid by fermentation. However, propionic acid fermentation with Propionibacterium acidipropionici is limited by the low product yield due to its heterofermetation nature. Metabolic engineering to modify the dicarboxylic acid pathway can be an effective method to alter carbon flow to improve propionate yield by knocking-out the acetate formation. Acetate kinase (ACK) is a key enzyme involving acetate formation in the pathway, and was the knockout target in this research. Integrational mutagenesis by homologous recombination can be used to disrupt ack gene by first constructing a non-replicative plasmid with partial ack and then transforming it into cells. Integration of the non-replicative plasmid can result in duplicate repeats of the homologous regions flanking DNA sequences, and thus cause mutagenesis. In this work, partial ack, encoding ACK, was first cloned and sequenced by PCR based cloning. A 477 bp of partial ack sequence was obtained. However, low yield of genomic DNA preparation from P. acidipropionici might have attributed to failure to clone the ack gene from the genomic DNA using the degenerate
Author: Yuhua Liao
Institution: The Ohio State University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2244290654