Optimization of the extracellular production of a bacterial phytase with Escherichia coli by using different fed-batch fermentation strategies

Abstract

The extracellular production of Escherichia coli phytase was studied in fed-batch fermentations. Two different feeding strategies were compared: control by keeping the glucose concentration constant, and control by keeping a low constant oxygen level in the medium. For the feeding control based on glucose concentration, a recently developed rapid glucose controlling system was tested for the first time in bacterial cultivations and used to establish the fermentative production of extracellular phytase with E. coli. High activity levels (120 U ml(-1)) at short cultivation times (14 h) were obtained. Even higher activity levels - albeit at longer cultivation times - were reached by applying a feeding control, the main characteristic of which was a constant low oxygen concentration. The optimum oxygen level for the production of phytase was in the range of 5-10% saturation.

Production of recombinant proteins by high cell density culture of Escherichia coli

“…Fusion partners such as the PelB, protein A and OmpA leader peptides as well as dedicated translocators such as the hemolysin and pululanase systems have been used for the excretion of some recombinant proteins Li et al, 2002;Matsumoto et al, 2002). Co-expression of the kil gene, tolAIII gene, or the bacteriocin release protein gene also allowed certain degree of protein excretion (Fu et al, 2003;Kleist et al, 2003;Wan and Baneyx, 1998). In another study, fusing the protein (peptide) of interest to the OmpF allowed excretion of the fusion protein into the culture medium when E. coli BL21 host was used (Jeong and Lee, 2002).…”

“…The use of L-form E. coli strains, which lack in cell walls, is an alternative way of achieving excretory protein production (Kujau et al, 1998). The HCDC of E. coli for the excretory production of recombinant proteins has been reported for limited cases Jeong and Lee, 2002;Kleist et al, 2003). Even though it is possible to use E. coli as a host strain for excretory protein production, researchers seem to prefer other hosts for this purpose; especially, those microorganisms that lack outer membranes (such as gram-positive bacteria) or those equipped with good excretory mechanisms (such as yeasts) are often employed.…”

A thermostable phytase from Bacillus sp. MD2: cloning, expression and high-level production in Escherichia coli

“…The stirrer speed started to increase at about 7 h of cultivation to compensate for the decrease in dissolved oxygen and reached a maximum of 1,000 rpm after 14 h cultivation time. Glucose concentration was reduced to <3 g/l after 8.5 h, and thereafter a continuous feed of glucose was applied to the reactor according to a programmed feeding proWle to maintain the glucose concentration at 2-3 g/l [1,18,31]. This resulted in an extension of the exponential phase of the cultivation, max of 0.47 h ¡1 between 4-16 h of the cultivation, and increase in cell density to an OD 620 of 117.6 (cell dry weight of 33.7 g/l) at 16 h (Fig.…”

“…Most of them are related to controlling the substrate concentration at certain levels in order to avoid overXow metabolism and the resultant acetate accumulation under glucose excess conditions [1,10,18,31]. Åkesson and coworkers [1] have previously proposed a feeding strategy controlled automatically by the dissolved oxygen level such that aerobic conditions are maintained in spite of the limitations in oxygen transfer, i.e., the feeding rate is decreased when maximum oxygen transfer capacity is reached in the bioreactor.…”

Enhanced secretion of adhesive recognition sequence containing hirudin III mutein in E. coli

“…To improve expression level, appropriate signal peptide, promotor, and host strains usually need to be selected and optimized [4,8,10]. This work has been done in our previous report [21], and E. coli L-ASP signal peptide has been selected because of its typical characteristics of signal peptide, which is composed of 22 residues [5] and contains less hydrophobic residues at C-terminal, as well as easily forms so-called Ala-X-Ala box which can be recognized and efficiently cleaved by signal peptidase I [15].…”