Fermentation kinetics of recombinant yeast in batch and fed-batch cultures

Fed-batch cultures of recombinant microorganisms have attracted attention as they can separate cell growth stage from cloned-gene expression phase during fermentations. In this work, the effect of different glucose feeding strategies on cell growth and cloned gene expression was studied during aerobic fed-batch fermentations of recombinant yeast, containing the plasmid pRB58. The plasmid contains the yeast SUC2 gene, which codes for the enzyme invertase. Some feeding policies resulted in a constant glucose concentration inside the fermentor, while others deliberately introduced a cyclic variation. The cell mass yield was found to be higher at low glucose concentrations, thus indicating a shift to the more energy-efficient respiratory pathway. The SUC2 gene expression was derepressed at glucose levels below 2 g/L. The response of specific invertase activity to changes in the medium glucose concentration was found to be almost immediate.     

Optimization of fed-batch production of the model recombinant protein GFP in Lactococcus lactis

Optimization of recombinant protein production using lactic acid bacteria (LAB) remains an important obstacle on the road to realizing LAB as oral vaccine delivery vehicles. Despite this, there have been few published investigations to explore the higher limits of LAB recombinant protein expression in fed-batch fermentations. In this study, results from response surface experiments suggested an optimal set of conditions for expression of green fluorescent protein (GFP), a model recombinant protein, in bench-scale, fed-batch Lactococcus lactis IL1403 fermentations. The 48 4-L fed-batch fermentations in this set of experiments, along with preliminary studies, investigated the effects of pH, temperature, hemin concentration, concentration of the nisin inducer per cell, and time of induction. Cell densities in this data set ranged from 2.9 to 7.4 g/L and maximum GFP expression per cell ranged from 0.1 to 4.4 relative fluorescence units (RFU)/g. The optimal 4-L, fed-batch fermentation process found here yields growth and protein expression values that dramatically improve upon results from traditional test tube and flask processes. Relative to the traditional process, the experimental optimum conditions yield 4.9 times the cell density, 1.6 times the protein per cell mass, and 8 times the total protein concentration. Unexpectedly, experiments also revealed that the compound hemin, known previously to improve growth and survival of Lactococcus lactis (L. lactis), negatively impacted recombinant protein production when added in concentrations from 5 to 20 microg/mL with this strain. The improvement in protein expression over traditional processes demonstrated here is an important step toward commercial development of LAB for oral delivery of recombinant vaccines and therapeutic proteins.     

Modelling the effects of glucose feeding on a recombinant E. coli fermentation

A simple unstructured model is described and compared with experimental data for fed-batch fermentations. The process studied is typical of the most common industrial recombinant fermentation in which temperature induced E. coli produces high yields of a heterologous protein (4?g met-asp-Bovine Somatotropin/l) using a complex peptone feed and a glucose feed. The model accurately predicts the effect of glucose feeding and shows that there is an optimal glucose feedrate to maximize productivity. At higher glucose feedrates, acetate levels become inhibitory and at lower levels glucose starvation occurs. The model is novel since it combines terms for acetate production and inhibition, effect of peptone feeding and temperature induction into a single relatively simple unstructured model. However the present model is unable to predict the effect of induction at different cell densities and reasons for this are suggested.     

A simple feedback control of Escherichia coli growth for recombinant aldolase production in fed-batch mode

Fed-batch production of recombinant fuculose-1-phosphate aldolase (FucA) by Escherichia coli XL1 Blue MRF′ (pTrcfuc) has been automated by using a simple feedback specific growth rate control strategy. Non-induced continuous cultures were conducted in order to characterize substrate consumption and carbon dioxide production yields and rates. In fed-batch cultures, substrate feeding rate was adjusted using on-line biomass estimation based on exhaust gas analysis and macroscopic mass balances. Overexpression of recombinant protein induced by isopropyl-β-d-thiogalactopyranoside (IPTG) under trc promoter did not affect significantly the control of specific growth rate during 7 h after induction. Growth and protein production curves were parallel until high level of protein expression started to inhibit cell growth. The proposed specific growth rate control strategy has been successfully applied to both non-induced and induced fed-batch cultures that do not exhibit severe growth rate depression.     

Process development for an inducible rituximab-expressing Chinese hamster ovary cell line

Inducible mammalian expression systems are becoming increasingly available and are not only useful for the production of cytotoxic/cytostatic products, but also confer the unique ability to uncouple the growth and production phases. In this work, we have specifically investigated how the cell culture state at the time of induction influences the cumate-inducible expression of recombinant rituximab by a GS-CHO cell line. To this end, cells grown in batch and fed-batch cultures were induced at increasing cell densities (1 to 10 × 10 ⁶ cells/mL). In batch, the cell specific productivity and the product yield were found to reduce with increasing cell density at induction. A dynamic feeding strategy using a concentrated nutrient solution applied prior and postinduction allowed to significantly increase the integral of viable cells and led to a 3-fold increase in the volumetric productivity (1.2 g/L). The highest product yields were achieved for intermediate cell densities at induction, as cultures induced during the late exponential phase (10 × 10 ⁶ cells/mL) were associated with a shortened production phase. The final glycosylation patterns remained however similar, irrespective of the cell density at induction. The kinetics of growth and production in a 2 L bioreactor were largely comparable to shake flasks for a similar cell density at induction. The degree of galactosylation was found to decrease over time, but the final glycan distribution at harvest was consistent to that of the shake flasks cultures. Taken together, our results provide useful insights for the rational development of fed-batch cell culture processes involving inducible CHO cells. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2742, 2019     

Effects of temperature shift strategies on human preproinsulin production in the fed-batch fermentation of recombinant<Emphasis Type="Italic">Escherichia coli</Emphasis>

Preproinsulin is a well-known precursor of human insulin for the regulation of blood glucose levels. In this study, fed-batch fermentations of recombinantEscherichia coli JM109/pPT-MRpi were carried out for the overexpression of human preproinsulin. The expression of human preproinsulin was controlled by the temperature inducibleP2 promoter. The time-course profiles of fed-batch fermentation and SDS-PAGE analysis showed that human insulin expression was triggered by a culture temperature change from 30 to 37°C. Fermentation shift strategies, including the multi-step increase of temperature and the modulation of initiation time, were optimized to obtain high titers of cell mass and preproinsulin. The optimized fed-batch fermentation, consisting of a three-step shift of culture temperature from 30 to 37°C for 2 h, gave the best results of 43.1 g/L of dry cell weight and 33.3% preproinsulin content, which corresponded to 2.0- and 1.2-fold increases, respectively, as compared to those of fed-batch culture at a constant temperature of 37°C.     

Induction of T4 DNA ligase in a recombinant strain of Escherichia coli

The kinetics of cell growth and foreign protein production, as well as factors affecting protein stability, were studied and optimized in batch and fed-batch fermentations of a recombinant strain of Escherichia coli. The pL promoter from bacteriophage lambda under the control of a temperature-sensitive cl represser, with the entire construct integrated into the E. coli chromosome through the use of a defective bacteriophage lambda lysogen, was used to direct the synthesis of T4 DNA ligase. The biphasic fermentations consisted of a primary growth phase at 30 degrees C followed by an induction phase which was initiated by shifting the temperature to 42 degrees C. In the fed-batch fermentations, additional nutrients were added at the time of initiating induction. Maintenance of sufficiently high concentrations of the organic substrates (glucose and casamino acids) during the induction phase was required for continued cell growth at 42 degrees C. Such growth was essential for T4 DNA ligase formation and in vivo stability. Hence, fed-batch fermentations produced the highest yield of the foreign protein Commensurate with providing lower total amounts of substrates. In such cases, high cell densities (6 g dry wt/L) with substantial intracellular levels of T4 DNA ligase (4.6% total cellular protein, or 2.7% of the dry biomass) were achieved.     

Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 in fed-batch fermentation: Effects of sucrose concentration in a complex medium and process modeling

Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 was investigated in semi-aerobic, pH-controlled, batch and fed-batch fermentations using a complex medium containing sucrose as the main source of carbon. The effects of sucrose concentration were studied in fed-batch fermentations in which a sucrose solution was added at stable feeding rates (5, 7, 9 and 10 g/l/h). The results showed that pediocin is produced as a product of the primary metabolism and its titer could be greatly improved by adjusting the sucrose feeding rate in fed-batch fermentation. The maximum titer of pediocin of 145 AU/ml was obtained in the fed-batch culture with 7 g/l/h feeding rate and that was 119% higher compared to the titer obtained in batch culture. Higher feeding rates (9 and 10 g/l/h) resulted in decreased pediocin yields while biomass levels appeared to be rather unaffected. The specific rate of pediocin formation was also sensitive to sucrose concentration levels. A mathematical model developed on the basis of well-known rate equations for batch and fed-batch cultures and growth associated production, described successfully cell growth, sucrose assimilation, lactate production and pediocin production in fed-batch culture.     

Kinetics of inclusion body production in batch and high cell density fed-batch culture of Escherichia coli expressing ovine growth hormone.

A process for maximizing the volumetric productivity of recombinant ovine growth hormone (r-oGH) expressed in Escherichia coli during high cell density fermentation process has been devised. Kinetics of r-oGH expression as inclusion bodies and its effect on specific growth rates of E. coli cells were monitored during batch fermentation process. It was observed that during r-oGH expression in E. coli, the specific growth rate of the culture became an intrinsic property of the cells which reduced in a programmed manner upon induction. Nutrient feeding during protein expression phase of the fed-batch process was designed according to the reduction in specific growth rate of the culture. By feeding yeast extract along with glucose during fed-batch operation, high cell growth with very little accumulation of acetic acid was observed. Use of yeast extract helped in maintaining high specific cellular protein yield which resulted in high volumetric productivity of r-oGH. In 16 h of fed-batch fermentation, 3.2 g l-1 of r-oGH were produced at a cell OD of 124. This is the highest concentration of r-oGH reported to date using E. coli expression system. The volumetric productivity of r-oGH was 0.2 g l-1 h-1, which is also the highest value reported for any therapeutic protein using IPTG inducible expression system in a single stage fed-batch process.     

毕赤酵母高密度发酵工艺的研究

高密度发酵是毕赤酵母提高蛋白表达量的一种重要策略,发酵工艺是高密度发酵的一个重要因素。采用下列措施均可以有效地提高表达水平：调节基础培养基,采用变pH和变温发酵,提高DO,选择最适的诱导前菌体密度和比生长速率并降低甘油初始浓度和采用分段式指数流加进行调控。选择合适的甲醇补料策略：甲醇限制补料（MLFB）、氧气限制补料（OLFB）、甲醇不限制补料（MNLFB）和温度限制补料（TLFB）。采用两种方式调控补料：诱导阶段菌体生长时,甲醇比消耗速率(qMeOH)为0.02-0.03gg-1h-1,而菌体不生长时,qMeOH采用较高值。     

Plasmid DNA fermentation strategies: influence on plasmid stability and cell physiology

In order to provide sufficient pharmaceutical-grade plasmid DNA material, it is essential to gain a comprehensive knowledge of the bioprocesses involved; so, the development of protocols and techniques that allow a fast monitoring of process performance is a valuable tool for bioprocess design. Regarding plasmid DNA production, the metabolic stress of the host strain as well as plasmid stability have been identified as two of the key parameters that greatly influence plasmid DNA yields. The present work describes the impact of batch and fed-batch fermentations using different C/N ratios and different feeding profiles on cell physiology and plasmid stability, investigating the potential of these two monitoring techniques as valuable tools for bioprocess development and design. The results obtained in batch fermentations showed that plasmid copy number values suffered a pronounced increase at the end of almost all fermentation conditions tested. Regarding fed-batch fermentations, the strategies with exponential feeding profiles, in contrast with those with constant feeding, showed higher biomass and plasmid yields, the maximum values obtained for these two parameters being 95.64 OD₆₀₀ and 344.3 mg plasmid DNA (pDNA)/L, respectively, when using an exponential feed rate of 0.2 h⁻¹. Despite the results obtained, cell physiology and plasmid stability monitoring revealed that, although higher pDNA overall yields were obtained, this fermentation exhibited lower plasmid stability and percentage of viable cells. In conclusion, this study allowed clarifying the bioprocess performance based on cell physiology and plasmid stability assessment, allowing improvement of the overall process and not only plasmid DNA yield and cell growth.     

Phytase production by high cell density culture of recombinant Bacillus subtilis

Bacillus subtilis BD170, harboring a plasmid pGT44[phyC] carrying the phytase gene (phyC) and a phosphate-depletion inducible pst-promoter, was grown in a 2 l bioreactor. Using a controlled feeding of glucose, high cell densities of 32 and 56 g dry cell weight l^–1 were achieved with peptone and yeast extract, respectively, as the complex nitrogen sources in a semi-defined growth medium. The fed-batch protocol was applied to production of recombinant phytase and a high extracellular phytase activity (48 U ml^–1) was reached with peptone. Although the yeast extract feeding resulted in a higher cell density, it was unsuitable as a medium component for phytase expression due to its relatively high phosphate content.     

Impeller types and feeding modes influence the morphology and protein expression in the submerged culture of<Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

The influences of impeller types on morphology and protein expression were investigated in a submerged culture ofAspergillus oryzae. The impeller types strongly affected mycelial morphology and protein production in batch and fed-batch fermentations. Cells that were cultured by propeller agitation grew in the form of a pellet, whereas cells that were cultured by turbine agitation grew in a freely dispersed-hyphal manner and in a clumped form. Pellet-grown cells showed high levels of protein production for both the intracellular heterologous protein (β-glucuronidase) and the extracellularly homologous protein (α-amylase). The feeding mode of the carbon source also influenced the morphological distribution and protein expression in fed-batch fermentation ofA. oryzae. Pulsed-feeding mainly showed high protein expression and homogeneous distribution of pellet whereas continuous feeding resulted in less protein expression and heterogeneous distribution with pellet and dispersed-hyphae. The pellet growth with propeller agitation paralleling with the pulsed-feeding of carbon source showed a high level of protein production in the submerged fed-batch fermentation of recombinantA. oryzae.     

Fed-batch high-cell-density fermentation strategies for Pichia pastoris growth and production

Pichia pastoris is extensively used to produce various heterologous proteins. Amounts of biopharmaceutical drugs and industrial enzymes have been successfully produced by fed-batch high-cell-density fermentation (HCDF) of this cell factory. High levels of cell mass in defined media can be easily achieved and therefore large quantities of recombinant proteins with enhanced activities and lower costs can be obtained through HCDF technology. A robust HCDF process makes a successful transition to commercial production. Recently, efforts have been made to increase the heterologous protein production and activity by the HCDF of P. pastoris. However, challenges around selecting a suitable HCDF strategy exist. The high-level expression of a specific protein in P. pastoris is still, at least in part, limited by optimizing the methanol feeding strategy. Here, we review the progress in developments and applications of P. pastoris HCDF strategies for enhanced expression of recombinant proteins. We focus on the methanol induction strategies for efficient fed-batch HCDF in bioreactors, mainly focusing on various stat-induction strategies, co-feeding, and the limited induction strategy. These processes control strategies have opened the door for expressing foreign proteins in P. pastoris and are expected to enhance the production of recombinant proteins.     

Using a kinetic model that considers cell segregation to optimize hEGF expression in fed-batch cultures of recombinant Escherichia coli

Growth inhibition of recombinant Escherichia coli during the expression of human epidermal growth factor was observed. The recombinant cells could be segregated into three populations based on their cell division and plasmid maintenance abilities: dividing and plasmid-bearing cells, dividing and plasmid-free cells, and viable-but-non-culturable (VBNC) cells. Fed-batch fermentations were performed to investigate the effect of cell segregation on the kinetics of growth and foreign protein production. The results showed that a low concentration of inducer caused weak induction, whereas high levels cause strong induction, resulting in cells segregating into VBNC bacteria and producing a low foreign protein yield. A kinetic model for cell segregation was proposed and its predictions correlated well with experimental data for cell growth and protein expression. The optimal induction strategy could then be predicted by the model, and this prediction was then verified by experimentally deriving the conditions necessary for maximum expression of recombinant protein.     

Growth-associated synthesis of recombinant human glucagon and human growth hormone in high-cell-density cultures of Escherichia coli

Synthesis of two recombinant proteins (human glucagon and human growth hormone) was investigated in fed-batch cultures at high cell concentrations of recombinant Escherichia coli. The glucose-limited growth was achieved without accumulation of metabolic by-products and hence the cellular environment is presumed invariable during growth and recombinant protein synthesis. Via exponential feeding in the two-phase fed-batch operation, the specific cell growth rate was successfully controlled at the desired rates and the fed-batch mode employed is considered appropriate for examining the correlation between the specific growth rate and the efficiency of recombinant product formation in the recombinant E. coli strains. The two recombinant proteins were expressed as fusion proteins and the concentration in the culture broth was increased to 15 g fusion growth hormone l⁻¹ and 7 g fusion glucagon l⁻¹. The fusion growth hormone was initially expressed as soluble protein but seemed to be gradually aggregated into inclusion bodies as the expression level increased, whereas the synthesized fusion glucagon existed as a cytoplasmic soluble protein during the whole induction period. The stressful conditions of cultivation employed (i.e. high-cell-density cultivation at low growth rate) may induce the increased production of various host-derived chaperones and thereby enhance the folding efficiency of synthesized heterologous proteins. The synthesis of the recombinant fusion proteins was strongly growth-dependent and more efficient at a higher specific growth rate. The mechanism linking specific growth rate with recombinant protein productivity is likely to be related to the change in cellular ribosomal content. Received: 27 May 1997 / Received last revision: 31 October 1997 / Accepted: 21 November 1997     

Influences of yeast extract on specific cellular yield of Ovine growth hormone during fed-batch fermentation of E. coli

In most cases of E. coli high cell density fermentation process, maximizing cell concentration helps in increasing the volumetric productivity of recombinant proteins usually at the cost of lower specific cellular protein yield. In this report, we describe a process for maintaining the specific cellular yield of Ovine growth hormone (oGH) from E. coli by optimal feeding of yeast extract during high cell density fermentation process. Recombinant oGH was produced as inclusion bodies in Escherichia coli. Specific cellular yield of recombinant oGH was maintained by feeding yeast extract along with glucose during fed-batch fermentation. Glucose to yeast extract ratio of 0.75 was found to be optimum for maintaining the specific cellular oGH yield of 66 mg/g of E. coli cells. Continuous feeding of yeast extract along with glucose helped in reducing acetic acid secretion and promoted higher cell growth during fed-batch fermentation. High cell growth of E. coli and high specific yield of recombinant oGH thus helped in achieving high volumetric productivity of the expressed protein. A maximum of 2 g/l of ovine growth hormone was expressed as inclusion bodies in 12 h of fed-batch fermentation.     

High cell density cultivation of <Emphasis Type="Italic">Escherichia coli</Emphasis> with surface anchored transglucosidase for use as whole-cell biocatalyst for α-arbutin synthesis

A fed-batch culture strategy for the production of recombinant Escherichia coli cells anchoring surface-displayed transglucosidase for use as a whole-cell biocatalyst for α-arbutin synthesis was developed. Lactose was used as an inducer of the recombinant protein. In fed-batch cultures, dissolved oxygen was used as the feed indicator for glucose, thus accumulation of glucose and acetate that affected the cell growth and recombinant protein production was avoided. Fed-batch fermentation with lactose induction yielded a biomass of 18 g/L, and the cells possessed very high transglucosylation activity. In the synthesis of α-arbutin by hydroquinone glucosylation, the whole-cell biocatalysts showed a specific activity of 501 nkat/g cell and produced 21 g/L of arbutin, which corresponded to 76% molar conversion. A sixfold increased productivity of whole cell biocatalysts was obtained in the fed-batch culture with lactose induction, as compared to batch culture induced by IPTG.     

Two-stage glycerol feeding for enhancement of recombinant hG-CSF production in a fed-batch culture of Pichia pastoris

Recombinant hG-CSF was expressed in Pichia pastoris under the control of the AOX1 promoter. In this study, the glycerol feeding rate was adjusted to achieve the maximum attainable specific growth rate before induction. Using a two-stage glycerol feeding method, the specific growth rate was changed from a maximum value of 0.21 h⁻¹ (at the beginning of feeding) to 0.15 h⁻¹ prior to induction. With this approach, the final dry cell wt and rhG-CSF yield achieved was close to 120 g l⁻¹ and 320 mg l⁻¹, respectively. Our study found that the two-stage feeding method allowed the overall productivity of rhG-CSF to increase 2.9 times that of the conventional fed-batch method.