Performance of recombinant fermentation and evaluation of gene expression efficiency for gene product in two-stage continuous culture system.
In order to develop a general methodology for evaluation of the gene expression efficiency for gene product, theoretical and experimental studies were undertaken using a recombinant Escherichia coli K12DeltaH1Deltatrp/ pPLc23trpA1 as a "gene-host cell" model system in a two-stage continuous-culture system. For this, a genetically structured kinetic model proposed earlier for biosynthesis of gene product in batch cultivation was extended to the two-stage continuous-culture system. A partial list of key parameters of the model includes the rate of plasmid segregation, specific growth rate of recombinant cell, plasmid content, rates of transcription and translation, and other parameters related to product biosynthesis. The dynamics of heterogeneous cell population containing plasmid-harboring and plasmid-free cells were also studied. Theoretical analysis of cell population dynamics shows that the recombinant cells could be maintained stably for a prolonged time in a two-stage continuous-culture system. Fermentation performance of the recombinant E. Coli cells in a two-stage continuous bioreactor system was examined experimentally, and the gene expression efficiency of a cloned gene product was determined based on the genetically structured kinetic model proposed. Based on our experimental results, the gene expression efficiency of the model gene-host cell system was found to be about twofold more efficient (i. e., 41.8 mg TrpA protein/mg plasmid DNA) as compared to the average rate of protein biosynthesis by E. coli cells. The performance of two-stage recombinant fermentation was also simulated using a mathematical model developed. General trends obtained from the model simulation agree reasonably well with the currently available experimental data, although further refinements need to be made. The methodology illustrated in this article could be used for evaluation of the gene expression efficiency of other genetically engineered recombinants once such recombinants with certain gene-host cell systems are constructed.
Lee
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SB
Korea Advanced Insititute of Science and Technology, P.O. Box 131, Seoul, Korea.
Ryu
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DD
Seigel
R
R
Park
S H
SH