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

Abstract

BACKGROUND: Untreated beet molasses feeding strategy was developed for recombinant thermostable glucose isomerase (GI) production by sucrose-utilizing Escherichia coli W. RESULTS: pRSETA::xylA plasmid carrying GI encoding gene of Thermus thermophilus (xylA) 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 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.

Improvement and characterization of a hyperthermophilic glucose isomerase from Thermoanaerobacter ethanolicus and its application in production of high fructose corn syrup

“…The lifetime productivity of TNGI at 90 °C is 1169 kg fructose/kg enzyme, which is 5.6 times higher than that of SMGI [5]. In recent years, over-production of recombinant hyperthermophilic GIs has been achieved in Escherichia coli strains [1,2,4]. Akdağ and Çalık reported the highest production of GI reached 4364.1 U/L using the exponential feeding strategy [1].…”

“…In recent years, over-production of recombinant hyperthermophilic GIs has been achieved in Escherichia coli strains [1,2,4]. Akdağ and Çalık reported the highest production of GI reached 4364.1 U/L using the exponential feeding strategy [1]. To meet the requirement of the industry, it is imminent to exploit novel GIs of class II with better activities and stabilities at higher temperature to produce 55 % of HFCS by one-step conversion [18].…”

A new approach for separation and recovery of betaine from beet molasses based on cloud point extraction technique

“…Molasses is usually produced at about 4 to 5% on beet (OB). It contains about 50% sugars and 80% dry substance (Asadi 2007) and is a highly attractive because of its low price and its high annual production (Akdag and Calık 2015).…”

Beet molasses–based feeding strategy enhances recombinant thermostable glucose isomerase production by Escherichia coli BL21 (DE3)

“…Several studies have been performed to produce GI using various strains of E. coli , namely E. coli BL21 (DE3) pLysS carrying pRSETA:: xyl A , E. coli JC1553 carrying pRAC:: xyl A , E. coli W595 carrying pCG138 , E. coli BL21(DE3) carrying pRAC:: xylA , and E. coli W (NRRL B‐766) carrying pRSETA:: xyl A .…”