A novel feeding strategy for enhanced plasmid stability and protein production in recombinant yeast fedbatch fermentation

A novel feeding strategy in fedbatch recombinant yeast fermentation was developed to achieve high plasmid stability and protein productivity for fermentation using low-cost rich (non-selective) media. In batch fermentations with a recombinant yeast, Saccharomyces cerevisiae, which carried the plasmid pSXR125 for the production of beta-galactosidase, it was found that the fraction of plasmid-carrying cells decreased during the exponential growth phase but increased during the stationary phase. This fraction increase in the stationary phase was attributed to the death rate difference between the plasmid-free and plasmid-carrying cells caused by glucose starvation in the stationary phase. Plasmid-free cells grew faster than plasmid-carrying cells when there were plenty of growth substrate, but they also lysed or died faster upon the depletion of the growth substrate. Thus, pulse additions of the growth substrate (glucose) at appropriate time intervals allowing for significant starvation period between two consecutive feedings during fedbatch fermentation should have positive effects on stabilizing plasmid and enhancing protein production. A selective medium was used to grow cells in the initial batch fermentation, which was then followed with pulse feeding of concentrated non-selective media in fedbatch fermentation. Both experimental data and model simulation show that the periodic glucose starvation feeding strategy can maintain a stable plasmid-carrying cell fraction and a stable specific productivity of the recombinant protein, even with a non-selective medium feed for a long operation period. On the contrary, without glucose starvation, the fraction of plasmid-carrying cells and the specific productivity continue to drop during the fedbatch fermentation, which would greatly reduce the product yield and limit the duration that the fermentation can be effectively operated. The new feeding strategy would allow the economic use of a rich, non-selective medium in high cell density recombinant fedbatch fermentation. This new feeding strategy can be easily implemented with a simple IBM-PC based control system, which monitors either glucose or cell concentration in the fermentation broth.     

Simultaneous fructose and ethanol production from sucrose,usingZymomonas mobilis 2864 co-immobilised with invertase

Summary Fructokinase negativeZymomonas mobilis UQM 2864, was co-immobilised with invertase in alginate and incubated on sucrose-based media in batch and fedbatch culture. The highest fructose concentration achieved was 138 g/l using fed-batch cultivation with sugar-cane syrup-simultaneously producing 79.9 g/l or 10.1% (v/v) ethanol in less than 24 hours. The ethanol and fructose yields were 95 and 84% respectively. Co-immobilisation resulted in faster fermentation times, particularly for the batch fermentations, and complete utilisation of substrate.     

Acetate production from lactate and glucose fermentation by Gluconobacter oxydans

Summary The production of acetate from the fermentation of lactate by Gluconobacter oxydans was studied. Batch experiments showed that glucose was the preferred substrate compared to lactate. A fed-batch culture was fed with a mixture of glucose and lactate followed by periodic addition of lactate. The maximum productivity of acetate was 0.16 g/l h but this value decreased during the fedbatch culture due to growth inhibition by acetate.     

On-line estimation of viable biomass of a microaerobic culture using exit gas analysis

Summary A method is proposed to estimate the concentration of metabolically active cells of a microaerobic culture on-line from the measurement of oxygen uptake rate (OUR) and respiratory quotient (RQ). With the cultivation ofEnterobacter aerogenes in a fedbatch mode the estimated active cell concentration agrees well with the viable cell concentration determined by microscopic count and agar plate incubation.     

Production of poly-β-hydroxybutyrate by fed-batch culture of recombinantEscherichia coli

Summary A recombinantEscherichia coli strain harboring the PHB biosynthesis genes fromAlcaligenes eutrophus was used to produce poly--hydroxybutyrate (PHB) by pH-stat fedbatch culture. Initial glucose concentration for optimal growth was found to be 20g/L from a series of flask cultures. A final PHB concentration of 88.8 g/L could be obtained after 42 hrs of cultivation.     

Best practices in heterotrophic high-cell-density microalgal processes: achievements,potential and possible limitations

Microalgae of numerous heterotrophic genera (obligate or facultative) exhibit considerable metabolic versatility and flexibility but are currently underexploited in the biotechnological manufacturing of known plant-derived compounds, novel high-value biomolecules or enriched biomass. Highly efficient production of microalgal biomass without the need for light is now feasible in inexpensive, well-defined mineral medium, typically supplemented with glucose. Cell densities of more than 100 g l⁻¹ cell dry weight have been achieved with Chlorella, Crypthecodinium and Galdieria species while controlling the addition of organic sources of carbon and energy in fedbatch mode. The ability of microalgae to adapt their metabolism to varying culture conditions provides opportunities to modify, control and thereby maximise the formation of targeted compounds with non-recombinant microalgae. This review outlines the critical aspects of cultivation technology and current best practices in the heterotrophic high-cell-density cultivation of microalgae. The primary topics include (1) the characteristics of microalgae that make them suitable for heterotrophic cultivation, (2) the appropriate chemical composition of mineral growth media, (3) the different strategies for fedbatch cultivations and (4) the principles behind the customisation of biomass composition. The review confirms that, although fundamental knowledge is now available, the development of efficient, economically feasible large-scale bioprocesses remains an obstacle to the commercialisation of this promising technology.     

Characterisation of the <Emphasis Type="Italic">Escherichia coli</Emphasis> membrane structure and function during fedbatch cultivation

BACKGROUND: Important parameters during recombinant protein production in Escherichia coli, such as productivity and protein activity, are affected by the growth rate. This includes the translocation of protein over the membrane to gain better folding capacity or reduced proteolysis. To vary the growth rate two techniques are available: fedbatch and continuous cultivation, both controlled by the ingoing feed rate. RESULTS: During fedbatch cultivation, E. coli contains phosphatidylethanolamine, phosphatidylglycerol, cardiolipin and saturated fatty acids in amounts which are stable with growth rate. However, the levels of cardiolipin are very high compared to continuous cultivation. The reason for fedbatch triggering of this metabolism is not known but hypothesised to result from an additional need for carbon and energy. The reason could be the dynamic and sometimes rapid changes in growth rate to which the fedbatch cell has at all times to adjust. The membrane flexibility, essential for translocation of various components, is however to some degree sustained by production of increased amounts of unsaturated fatty acids in phosphatidylglycerol. The result is a functionally stiff membrane which generally promotes low cell lysis and is constant with respect to protein leakage to the medium. At comparatively high growth rates, when the further stabilising effect of cyclic fatty acids is gone, the high level of unsaturated fatty acids results in a pronounced effect upon sonication. This is very much in contrast to the membrane function in continuous cultivation which shows very specific characteristics as a function of growth rate. CONCLUSIONS: The stiff and unchanging fedbatch membrane should promote a stable behaviour during downstream processing and is less dependent on the time of harvest. However, optimisation of protein leakage can only be achieved in the continuously cultivated cell where leakage is twice as high compared to the constant leakage level in fedbatch. If leakage is undesired, continuous cultivation is also preferred since it can be designed to lead to the lowest values detected. Induction at low growth rate (<0.2 h-1) should be avoided with respect to productivity, in any system, since the specific and total protein production shows their lowest values at this point.     

Effect of substrate feed rate on recombinant protein secretion, degradation and inclusion body formation in Escherichia coli

The effect of changes in substrate feed rate during fedbatch cultivation was investigated with respect to soluble protein formation and transport of product to the periplasm in Escherichia coli. Production was transcribed from the P_malK promoter; and the cytoplasmic part of the production was compared with production from the P_lacUV5 promoter. The fusion protein product, Zb-MalE, was at all times accumulated in the soluble protein fraction except during high-feed-rate production in the cytoplasm. This was due to a substantial degree of proteolysis in all production systems, as shown by the degradation pattern of the product. The product was also further subjected to inclusion body formation. Production in the periplasm resulted in accumulation of the full-length protein; and this production system led to a cellular physiology where the stringent response could be avoided. Furthermore, the secretion could be used to abort the diauxic growth phase resulting from use of the P_malK promoter. At high feed rate, the accumulation of acetic acid, due to overflow metabolism, could furthermore be completely avoided.     

Improvement of hEGF production with enhanced cell division ability using dissolved oxygen responses to pulse addition of tryptone

Tryptone has multiple and complex effects on cell physiology and process performance in pulse fed-batch cultivation of recombinant Escherichia coli. By applying feedback control of dissolved oxygen signal responding to pulse in the feed rate, the production of acetate was avoided and the optimization of production of recombinant human epidermal growth factor (hEGF) was successfully achieved. With the addition of an optimum amount of tryptone along with glucose in the pulse fedbatch cultivation of E. coli, the ability of the cell to divide and the stability of the plasmid within the bacteria were improved. Consequently, segregation of the cells into a viable but non-culturable physiological state was alleviated. Addition of tryptone also enhanced cell respiration before and after hEGF expression and thus further benefited the production of recombinant hEGF. Excessive addition of tryptone resulted in low sensitivity of the oscillation of dissolved oxygen signal and poor operability of pulse fed-batch cultivation as this led to an accumulation of acetate, which weakened the dissolved oxygen signal responses. Consequently, the production of recombinant protein was considerably reduced. By combining the process performance and the positive effect of complex media pulse addition on bacterial metabolism, the optimal production conditions of hEGF were successfully determined. A high cell density of 91 g/L dry cell weight was obtained under these optimal production conditions. Furthermore, a high level of 0.24 g/L hEGF was attained leading to a 32.6% increase in product yield as compared to the controls.     

Model-based design of different fedbatch strategies for phenol degradation in acclimatized activated sludge cultures

Microbial degradation of phenol was studied using batch and fedbatch cultures of acclimatized activated sludge under a wide range of phenol (0-793 mg l⁻¹) and biomass (0.74-6.7 g l⁻¹) initial concentrations. As cell growth continued after total phenol removal, the production and later consumption of a main metabolic intermediate was considered the step governing the biodegradation kinetics. A model that takes explicitly into account the kinetics of the intermediate was developed by introducing a specific growth rate model associated with its consumption and the incorporation of a dual-substrate inhibitory effect on phenol degradation. Biomass growth and phenol removal were adequately predicted in all the cultures. Moreover, the model-based design of the fedbatch feeding strategies allowed driving separately the phenol degradation under substrate-limitation and substrate-inhibition modes. A sensitivity analysis was also performed in order to establish the importance of the parameters in the accuracy of model predictions.     

Flux response of glycolysis and storage metabolism during rapid feast/famine conditions in Penicillium chrysogenum using dynamic 13C labeling

The scale‐up of fermentation processes frequently leads to a reduced productivity compared to small‐scale screening experiments. Large‐scale mixing limitations that lead to gradients in substrate and oxygen availability could influence the microorganism performance. Here, the impact of substrate gradients on a penicillin G producing Penicillium chrysogenum cultivation was analyzed using an intermittent glucose feeding regime. The intermittent feeding led to fluctuations in the extracellular glucose concentration between 400 μM down to 6.5 μM at the end of the cycle. The intracellular metabolite concentrations responded strongly and showed up to 100‐fold changes. The intracellular flux changes were estimated on the basis of dynamic ¹³C mass isotopomer measurements during three cycles of feast and famine using a novel hybrid modeling approach. The flux estimations indicated a high turnover of internal and external storage metabolites in P. chrysogenum under feast/famine conditions. The synthesis and degradation of storage requires cellular energy (ATP and UTP) in competition with other cellular functions including product formation. Especially, 38% of the incoming glucose was recycled once in storage metabolism. This result indicated that storage turnover is increased under dynamic cultivation conditions and contributes to the observed decrease in productivity compared to reference steady‐state conditions.     

Bioreactor operating strategy inThalictrum rugosum plant cell culture for the production of berberine

Optimal substrate feeding strategy in bioreactor operation was investigated to increase the production of secondary metabolite in a high density culture of plant cell. It was accomplished by the previously proposed structured kinetic model that describes the cell growth and synthesis of the secondary metabolite, berberine, in a batch suspension culture ofThalictrum rugosum. Four types of operation strategies for sugar feeding intoT. rugosum culture were proposed based on the model, which were the periodic fedbatch operations to maintain the cell activity, the cell viability, and the specific production rate, and the perfusion operation to maintain the specific production rate. From the simulation results of these strategies, it could be found that the periodic fed-batch operation and the perfusion operation could achieve the higher volumetric production of berberine (mg berberine/L) and specific production yield (mg berberine/g dry cell weight) than those of batch cultures. Although the highest productivity (mg berberine/day) of berberine could be achieved by the periodic fed-batch operation to maintain the cell activity compared with the other strategies in the periodic fed-batch operations, the specific production yield was low due to the higher maximum dry cell weight than other cases. The periodic fed-batch operation to maintain cell viability resulted in the highest volumetric production of berberine and specific production yield compared with the other strategies. In the cases of maintaining the specific production rate, the per-formance of the periodic fed-batch operation was better than that of the perfusion operation in the respect of the volumetric production and productivity of berberine. In order to increase the volumetric production of berberine and to get the highest specific production yield, the periodic fed-batch operation to maintain cell viability could be chosen as the optimal operating strategy in high density, culture ofT. rugosum plant cell.     

Variable production windows for porcine trypsinogen employing synthetic inducible promoter variants in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Natural tools for recombinant protein production show technological limitations. Available natural promoters for gene expression in Pichia pastoris are either constitutive, weak or require the use of undesirable substances or procedures for induction. Here we show the application of deletion variants based on the well known methanol inducible AOX1 promoter and small synthetic promoters, where cis-acting elements were fused to core promoter fragments. They enable differently regulated target protein expression and at the same time to replace methanol induction by a glucose or glycerol feeding strategy. Trypsinogen, the precursor of the serine protease trypsin, was expressed using these different promoters. Depending on the applied promoter the production window (i.e. the time of increasing product concentration) changed significantly. In fedbatch processes trypsinogen yields before induction with methanol were up to 10 times higher if variants of the AOX1 promoter were applied. In addition, the starting point of autoproteolytic product degradation can be predetermined by the promoter choice.     

<Emphasis Type="Italic">Agaricus blazei</Emphasis> production on non-composted substrates based on sunflower seed hulls and spent oyster mushroom substrate

Agaricus blazei Murrill is usually cultivated using the same biphasic composting method employed for A. bisporus. Because cultivation of A. blazei on traditional A. bisporus composts poses some disadvantages, non-composted substrates were studied for A. blazei cultivation. Mycelial growth rate and productive performance of A. blazei were evaluated on substrates containing sunflower seed hulls, Pleurotus spp. spent mushroom substrate, or their combination, in the absence or in the presence of different supplements (vermicompost, peat or brewery residues). Substrates were prepared by initially soaking them and then they were sterilized (1 atm for 120 min). In addition, each substrate’s degradation was measured after cultivation by obtaining the lignin, cellulose, hemicellulose, organic matter, total fiber, ash, carbon and nitrogen contents before spawn-run and at the end of two flushes of A. blazei. The cultivation of A. blazei on non-composted substrates is possible and with a low rate of contamination when using the spent mushroom substrate as the main component or combined 50:50 with sunflower seed hulls. In addition, the best yields were obtained on those substrates containing spent Pleurotus mushroom substrate with supplements and those mixtures with sunflower seed hulls and vermicompost. These yields were similar to those reported on composted substrates. Substrate changes in composition measured at the end of two flushes indicate that the lignin-hemicellulose fraction was preferentially used and that the substrates exhibiting the best yield showed greater biodegradation of lignin-hemicellulose fraction than the others did.     

Comparative study of L-phenylalanine production in the growing and stationary phases during high cell density cultivation of an auxotrophic <Emphasis Type="Italic">Escherichia coli</Emphasis>

High cell density cultivation was investigated for L-phenylalanine (L-Phe) production by an L-tyrosine (L-Tyr) auxotrophic Escherichia coli WSH-BR165 (pAPB03). Dual exponential feeding of L-Tyr and glucose was adopted to achieve high cell density cultivation. The control was completed without dual feeding. The processes where dual feeding and induction were commenced together and those where induction began after dual feeding were studied and compared. The results indicated that the former dual feeding mode was most favorable for enhanced L-Phe production. With an optimal specific growth rate of 0.09/h during the dual exponential feeding period, the maximum dry cell weight reached 43.16 g/L (3.04 times that of the control) with a final L-Phe titer of 44.53 g/L (1.06 times that of the control) and an L-Phe productivity of 1.484 g/L/h (1.69 times that of the control). High cell density cultivation via the feeding of L-Tyr and glucose exponentially after the induction point proved to be an efficient approach to enhance L-Phe production.     

Scale-down of oxygen and glucose fluctuations in a tubular photobioreactor operated under oxygen-balanced mixotrophy

Oxygen-balanced mixotrophy (OBM) is a novel type of microalgal cultivation that improves autotrophic productivity while reducing aeration costs and achieving high biomass yields on substrate. The scale-up of this process is not straightforward, as nonideal mixing in large photobioreactors might have unwanted effects in cell physiology. We simulated at lab scale dissolved oxygen and glucose fluctuations in a tubular photobioreactor operated under OBM where glucose is injected at the beginning of the tubular section. We ran repeated batch experiments with the strain Galdieria sulphuraria ACUF 064 under glucose pulse feeding of different lengths, representing different retention times: 112, 71, and 21 min. During the long and medium tube retention time simulations, dissolved oxygen was depleted 15–25 min after every glucose pulse. These periods of oxygen limitation resulted in the accumulation of coproporphyrin III in the supernatant, an indication of disruption in the chlorophyll synthesis pathway. Accordingly, the absorption cross-section of the cultures decreased steeply, going from values of 150–180 m² kg⁻¹ at the end of the first batch down to 50–70 m² kg⁻¹ in the last batches of both conditions. In the short tube retention time simulation, dissolved oxygen always stayed above 10% air saturation and no pigment reduction nor coproporphyrin III accumulation were observed. Concerning glucose utilization efficiency, glucose pulse feeding caused a reduction of biomass yield on substrate in the range of 4%–22% compared to the maximum levels previously obtained with continuous glucose feeding (0.9 C-g C-g⁻¹). The missing carbon was excreted to the supernatant as extracellular polymeric substances constituted by carbohydrates and proteins. Overall, the results point out the importance of studying large-scale conditions in a controlled environment and the need for a highly controlled glucose feeding strategy in the scale-up of mixotrophic cultivation.     

Regulation of Amylase Synthesis in Bacillus circulans F-2

In the usual batch cultivation, Bacillus circulans F-2 produced amylase only when granular carbon sources such as raw starch or crosslinked starches (CLP) were added. In the dialysis cultivation, where CLP and partially purified amylase were incubated inside the dialysis tubing, the bacterium inoculated outside of the tubing grew and produced the amylase. Amylase production of this bacterium was further investigated in feeding cultivation, in which maltose was fed to the cultivation medium at various rates. The bacterial growth increased with the increase of the feeding rate of maltose, but maximum amylase production was observed at a feeding rate of 45 mg/hr/1. No amylase was produced on the media containing monosaccharides, sucrose, lactose, or isomaltose in the feeding cultivation although bacterial growth was observed. The amylase of this bacterium was found to be inducible. Replacement of 20% of the maltose with glucose resulted in a great decrease (70%) in the amylase production. This shows that the amylase synthesis of B. circulans F-2 is severely repressed by glucose.     

High cell density fed-batch cultivation of<Emphasis Type="Italic"> Escherichia coli</Emphasis> using exponential feeding combined with pH-stat

A new feeding strategy in fed-batch culture, exponential feeding combined with pH-stat is suggested to avoid the accumulation of substrate in culture broth. Exponential feeding was stopped whenever a predetermined amount of limiting substrate was supplied and then pH change was observed. When pH rose above an upper limit due to the depletion of substrate, feeding was restarted. With this feeding strategy, recombinant Escherichia coli could be grown to 101 g/l by controlling the specific growth rate at 0.1 h^–1.An erratum to this article can be found at     

Growth rate control in fed-batch cultures of recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg)

Summary A recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg) exhibited growth-assciated product formation. By controlling the medium feed rate, based on the calculated amount of medium required for 1 h, a constant specific growth rate was obtained in the range of 1.12-0.18 h^–1. In order to prolong the exponential growth phase, the medium feed rate was increased exponentially. A fedbatch cultivation method based on the production kinetics of batch culture enhanced HBsAg production ten times more than in batch culture. The reason for the increase can be explained by the fact that the production of HBsAg is expressed as an exponential function of time when the specific growth rate is controlled to a constant value in growth-associated product fromation kinetics. In the scale-up of this culture to 91, the specific growth rate could also be maintained constant and the HBsAg production trend was similar to that in a 1-l culture. However, ethanol accumulation occurred at a late stage in fed-bach culture. Ethanol produced was not reutilized and inhibited further cell growth. Offprint requests to: M. B. Gu