Recombinant protein production by sucrose‐utilizing Escherichia coli W: untreated beet molasses‐based feeding strategy development

BACKGROUND Untreated beet molasses feeding strategy was developed for recombinant thermostable glucose isomerase ( GI ) production by sucrose‐utilizing Escherichia coli W. RESULTS pRSETA :: xyl A plasmid carrying GI encoding gene of Thermus thermophilus ( xyl A) was transferred into E. coli W, and the effects of carbon sources, i.e. glucose, sucrose, and beet molasses containing 50% sucrose, on GI production were investigated. 32 g L −1 beet molasses‐based medium resulted in the highest recombinant GI production ( A = 4364.1 U L −1 ) and cell concentration ( C X = 4.2 g L −1 ) in laboratory‐scale shake‐bioreactors. Using beet molasses as the carbon source, pulse and exponential feeding strategies were designed. The highest recombinant GI production was achieved as 35 265 U L −1 at t = 16 h in V = 3.0 L controlled bioreactors, where the cell concentration was C X = 17 g L −1 , by the application of a three‐stage strategy: Stage I (0< t <5 h): batch operation with the beet molasses and (NH 4 ) 2 HPO 4 initial concentrations of C M0 = 32 g L −1 and C N0 ,( NH4 ) 2HPO4 = 5 g L −1 , respectively; Stage II (5≤ t <11 h): semi‐batch operation by consecutive pulse feeding of (NH 4 ) 2 HPO 4 containing molasses at t = 5 h and t = 8 h, to increase C M to 32 g L −1 and C (NH4)2HPO4 to 5 g L −1 ; and Stage III ( t ≥11 h): semi‐batch operation by continuous feeding of molasses with the pre‐determined specific growth rate of μ 0 = 0.05 h −1 , starting at t = 11 h. CONCLUSIONS This work demonstrates the significance of the use of beet molasses containing high concentrations of sucrose by designing semi‐batch feeding strategies for the over‐production of recombinant proteins by sucrose‐utilizing E. coli W. © 2014 Society of Chemical Industry