تولید فاکتور رشد اپیدرمال انسانی در فرهنگ دسته ای فدرال، قابلیت تحمل استات با اشرشیاکلی

An acetate-tolerant mutant of Escherichia coli DH5α, DA19, was used for secretory production of human epidermal growth factor (hEGF) whose expression was under the control of phoA promoter. The recombinant cells were cultured in a chemically defined medium, and glucose was added at different specific provision rates during the growth and expression phases. It was found that pH had a significant effect on the extracellular hEGF production. The extracellular hEGF concentration was 75.5mg.L−1, 5.2-fold of the level reached at pH 7.0, even though more acetate was produced. Nitrogen source was limited in the later growth phase. Supplementation of ammonium promoted the consumption of phosphate and reduced the time to exhaust phosphate, but the extracellular hEGF production was similar to that without supplementation of ammonium.

Escherichia coli is a valuable organism for the high level production of recombinant protein in high cell density culture. The expressed heterologous proteins are usually accumulated inside the cells, which can be easily degraded by cellular protease[ 11. Moreover, foreign proteins can form inactive inclusion bodies [2] caused by misfolding, and the refolding is quite inefficient and expensive. Secretion of the heterologous proteins reduces not only the toxic effects on the host cells, but also the difficulties and cost in recovery and purification. Another disadvantage of E. coli is accumulation of acetate during cultivation, which inhibits cell growth and the foreign gene expression [3,4], and it is more deleterious to the recombinant cells[S]. Many strategies have been adopted to reduce the formation of acetate by process control[3,6] and metabolic engineering[ 7-9]. An acetate-tolerant mutant of E. coli DHSa, DA19, was isolated by Zhu et aZ.[10], which forms less acetate on glucose than DHSa and grows better in the presence of acetate. This feature is especially useful in large-scale fermentors, where uniform distribution of the added concentrated glucose feed is more difficult than in small ones. Human epidermal growth factor (hEGF) is a peptide composed of 53 amino acids. It can promote proliferation of epidermal cells and inhibit the secretion of gastric acid, and has wide cosmetic and medical applications. We have studied hEGF production in E. coli YKS37 using complex media, and the secreted hEGF concentration reached 686mg.L - [ 11,121. A preliminary study has been carried out with DHSa and DA19 in shake flask cultures, which indicates that DA19 produces 7.54mg-L-' of secreted hEG7 in a complex medium, compared with 3.98mg.L- producedby DHSa[13]. The presence of large amount of proteins in complex medium increases the difficulty in downstream separation and purification of target protein, but study on foreign protein production under control of the phoA promoter in defined media is rare. In this study, hEGF was produced by the acetate-tolerant strain DA19 in a chemically defined culture medium. It was found that pH had a significant effect on phoA promoter-controlled gene expression, and hEGF production was improved remarkably at an alkaline pH

Initially, the cultivation was carried out in a batch mode. When glucose initially added in the medium was depleted, feeding of glucose was started to allow the total cell mass in the reactor to increase exponentially. To maintain a constant specific growth rate of 0.2h-', the feeding rate of limiting substrate, glucose, is given by F = XO"0 e x p w yXISsF where F is the volumetric feeding rate (L.h-'), SF is the glucose concentration in the feed solution (g.L-'), p is the specific growth rate (h-') to be controlled, Y X / S is the cell yield coefficient based on consumed glucose at the p to be controlled, XO and VO are the biomass concentration (g.L-') and culture volume (L) at the start of feeding. When the specific growth ratelwas 0.2h-', a specific glucose supply rate of 0.19g.g- .hwas obtained according to the above equation. The biomass concentration in the culture was measured every hour, and the quantity of glucose to be added until next sampling was calculated according to the above formula but was added at a constant volumetric rate to deliver the same amount of glucose. The preliminary fed-batch culture of DA19 (pAET8) was carried out in the defined main culture medium with pH controlled at 7.0 during the whole fermentation process. The initial glucose of 10g.L-l was exhausted at 8.25h indicated by a sudden increase of DO, and glucose was added with an average specific supply rate of 0.19g.g-'K1. At 14.85h, the p,hosphate concentration decreased to 1.6mmol.L - , at which point the specific growth rate of 0.2h-I could not be maintained any more despite continued addition of the glucose feed. Meanwhile, acetate started to accumulate, which indicated that nutrients other than glucose were limiting. In the expression phase, glucose was added at the same specific supply rate, and the residual glucose was gradually increased up to 6.1g.L-', while the NH4' concentration was very low. The hEGF production level was 16.lmg.L-' (data not shown) . This preliminary experiment indicated that in the expression phase the glucose feeding rate was too high. Fig.1 shows the typical trends of biomass (DCM), glucose, phosphate, ammonium, and extracellular hEGF concentrations in fed-batch culture with reduced glucose feeding rate in the expression phase. The process could be divided into four phases. Phase I is the batch culture period that lasted until exhaustion of the initial glucose; Phase I1 is an exponential growth period, during which glucose was added according to the above-mentioned method until the phosphate concentration decreased to about 1.6mmol~L-'; in Phase I11 glucose was added at a constant rate of 6.6g-h-' until phosphate in the medium was nearly consumed; and Phase IV was the expression period in which glucose was added at a constant specific rate of 0.16g.g-'.h-'. Because of the reduced feeding rate of glucose, no residual glucose was detected during theexpression phase. However, the extracellular hEGF concentration remained a very low level of 14.6mg.L-'. 3.2 The effect of pH on hEGF production Because alkaline phosphatase functions better in an alkaline environment and expression of hEGF was controlled by the promoter of alkaline phosphatase, hEGF production at higher pH was examined. The fermentation operation was the same as the above experiment except for pH in the expression phase being controlled at 7.5, 7.8, and 8.0, respectively. The results are summarized in Table 1. It can be seen that with the increase in pH, the hEGF production was improved and the highest level of 75.5mg.L-' was reached at pH 7.8 (Fig.2), 5.2-fold of that achieved at pH 7.0. The specific hEGF production (YEGF/xa)v, erage specific hEGF production rate (QEGF)a, nd average volumetric hEGF production rate (TEGF) also reachedtheir maximal values at pH 7.8. However, further increase in the pH to 8.0 resulted in decreased hEGF, YEGFIX, QEGF, and TEGF. Figure 3 shows the profiles of extracellular hEGF and acetate during the expression phase controlled at different pH values. At pH 7.5, acetate accumulation and hEGF production were similar to those at pH 7.0; the higher acetate concentration at the end of expression phase was caused by the longer expression phase. At pH 7.8, the EGF concentration significantly increased even though acetate accumulation was enhanced at the same time. At llh post induction, acetate reached 4g.L-', but hEGF still showed an increasing trend. When pH was controlled at 8.0, acetate accumulation further increased, and the hEGF concentration decreased during the period between 4 and 8h post induction. During the period between 8 and 10h, the acetate concentration rose from 3.9g.L-' to 5.6g.L-' and hEGF stopped increasing, while the hEGF concentration continued to increase within a similar acetate level when pH was controlled at 7.8. Therefore, the higher acetate concentration at pH 8.0 was not responsible for the observed lower hEGF concentration.Table 1 shows that the ammonia feeding rate increased with an increase in pH due to more acetate production. Because the concentration of ammonium was low in the expression phase (Fig.l), adding more ammonia solution should be beneficial to the expression of hEGF in the acetate-tolerant strain. When thepH was controlled at 8.0, the hEGF concentration was decreased even though more ammonium was provided. These facts suggested that pH 7.8 was most suitable for the expression of hEGF. Studies on phosphatases in Neurospora crassa show that pH is an important factor for enzyme production and secretion [18]. Northern analysis and enzyme activity assay confirm that the enzyme synthesis in Metarhizium anisopliae is considerably improved at the pH they function effectively, irrespective of whether the medium contains an inductive substrate[ 191. pH regulation is found in bacteria including Salmonella typhimurium, Vibrio cholera, E. coZi[20], as well as in fungi and animal cells, indicating that pH regulation of gene expression is a general phenomenon[ 21]. The present study indicated that at pH 7.8 best expression of the gene controlled by the phoA promoter was obtained. The hEGF production level in the minimal medium was not high compared with those obtained in complex media[ 113. However, when a mixture of tryptone and yeast extract was supplied, the overall hEGF level reached 765mq-Lp' but the secreted hEGF was only 78.7mg.L- , suggesting limitation of secretion [22]. 80 70 60 50 2 40 rE; 30 20 10 c - 0 3.3 Tolerance of DA19 to acetate One of the major drawbacks of E. coZi is formation of acetate that seriously inhibits the expression of foreign genes. In this study, an acetate-tolerant strain, DA19, was used. In order to examine the effect of acetate accumulation on hEGF production, glucose was added at a higher specific rate of 0.247gSg-'.h-' in the expression phase to produce more acetate with the pH controlled at 7.8. 13.7g.L-' of acetic acid were formed, but the hEGF concentration profile was similar to that when glucose was added at a lower specific rate of 0.159g.g-'.h;', where the highest acetate production was 5.6g.L- (Fig.4 and Table 1). This strongly indicated the advantage of this acetate-tolerant strain in expression of a recombinant protein.3.4 Factor limiting cell growth in phase III In complex media containing yeast extract and tryptone, cells of DA19 (pAET8) could grow at a high specific rate until phosphate was almost depleted (datanot shown), but in the defined medium, the phosphate consumption rate decreased when the phosphate concentration reduced to 1.6mmol.L-'. Examination of the fermentations in the defined medium suggested that the reduced phosphate consumption could be attributed to limitation of ammonium that was below 5mmol.L-' (Fig.1). To understand the effect of nitrogen source, NH4+ was added to reach 40rnm0l.L-~' when the concentration had decreased to 1Ommol.L- . As shown in Fig.5, supplementation of ammonium facilitated phosphate uptake. Phosphate was depleted within two hours after addition of ammonium, compared with six hours without ammonium supplementation. However, the secreted hEGF was only slightly improved (78.7mg.L-').The pH in the expression phase remarkably affected hEGF production controlled by the alkaline phosphatase (phoA) promoter. At pH 7.8, the extracellular hEGF increased by 5.2-fold compared with that at pH 7.0, even though more acetate was formed. During the expression phase, when DA19 (pAET8) was fed with glucose at a higher specific rate to result in an acetate concentration of 13.7g.L-', the hEGF production was similar to that obtained at an acetate concentration of 5.6g.L-', suggesting the advantage to use the acetate-tolerant strain, DA19.