Effect Of Fumarate Reductase Amplification On Succinic Acid Production In Escherichia Coli

In silico aided simulation results of genetic modification was analyzed, and a key enzyme affecting succinic acid production, FRD, fumarate reductase, was determined. By constructing a recombinant plasmid with frd, combination of frd amplification and adhE knockout, as well as addition of fumarate as substrate, the metabolic flux in the pathway where fumarate is converted to succinate was amplified, and succinate yield was hence increased. The main results presented in this work are as follows:The sdh gene was amplified from Bacillus subtilis 168 by PCR and ligated into the expression plasmid pTrc99A with the suitable enzyme excision to give plasmid pTSXT. Expression of sdh has moderate effect on succinate production.When sdh was expressed in adhE-knockout mutant TUQ5, succinate production was enhanced to a greater extent.The addition of fumarate to medium under anaerobic conditions resulted in partial conversion from fumarate to succinate.The cause of the difference between simulation and experiment was investigated: the difference probably resulted from the complicated assembly of FRD subunits and complexity of metabolic network of Escherichia coli.