Fed-batch Phase Research Articles

Glucose limited feed strategy leads to increased production of fusicocca-2,10(14)-diene by Saccharomyces cerevisiae

Fusicocca-2,10(14)-diene (FCdiene) is a diterpene which is interesting as a precursor of the anti-cancer drug fusicoccin A and therefore for pharmaceutical applications. Production of FCdiene using a genetically modified Saccharomyces cerevisiae has been previously demonstrated with batch cultivations with a product concentration up to 10 mg/L. However, it is widely known that fed-batch processes can significantly improve product titer in yeast fermentations. This study focuses on the establishment of fed-batch fermentation for FCdiene production because fed-batch cultivations using FeedBeads® indicated that limiting glucose supply could increase yields of biomass (1.07 gCDW/gGlucose instead of 0.20 gCDW/gGlucose) and FCdiene (21.54 mgFCdiene/gGlucose instead of 9.74 mgFCdiene/gGlucose) in shake flask scale and may have implications for larger scale processes. We implemented a new exponential glucose feed profile in a 1.8 L stirred tank reactor. This reduced overfeeding and the consequent, ethanol production. As a result improvements in cell concentrations up to 246% could be achieved and FCdiene yield increased up to 2.8X in the first 28 h. FCdiene concentration reached 161 mg/L and 320 mg/L at 44 h. Fed-batch and batch mode were combined to examine dynamics of bi-modal cultivation where a fed-batch phase was used for biomass production and a batch phase used for FCdiene production potentially supported by ethanol consumption as reported on production of betulinic acid. The present study highlights the potential of process development improvements which increase high-value heterologous diterpene yields from S. cerevisiae.

Fate of the UPR marker protein Kar2/Bip and autophagic processes in fed-batch cultures of secretory insulin precursor producing Pichia pastoris

BackgroundSecretory recombinant protein production with Pichia (syn. Komagataella) pastoris is commonly associated with the induction of an unfolded protein response (UPR) usually apparent through increased intracellular levels of endoplasmic reticulum (ER) resident chaperones such as Kar2/Bip. During methanol-induced secretory production of an insulin precursor (IP) under industrially relevant fed-batch conditions the initially high level of intracellular Kar2/Bip after batch growth on glycerol unexpectedly declined in the following methanol fed-batch phase misleadingly suggesting that IP production had a low impact on UPR activation.ResultsAnalysis of the protein production independent level of Kar2/Bip revealed that high Kar2/Bip levels were reached in the exponential growth phase of glycerol batch cultures followed by a strong decline of Kar2/Bip during entry into stationary phase. Ultra-structural cell morphology studies revealed autophagic processes (e.g. ER phagy) at the end of the glycerol batch phase most likely responsible for the degradation of ER resident chaperones such as Kar2/Bip. The pre-induction level of Kar2/Bip did not affect the IP secretion efficiency in the subsequent methanol-induced IP production phase. During growth on methanol intracellular Kar2/Bip levels declined in IP producing and non-producing host cells. However, extracellular accumulation of Kar2/Bip was observed in IP-producing cultures but not in non-producing controls. Most importantly, the majority of the extracellular Kar2/Bip accumulated in the culture supernatant of IP producing cells as truncated protein (approx. 35 kDa).ConclusionsRapid growth leads to higher basal levels of the major UPR marker protein Kar2/Bip independent of recombinant protein production. Entry into stationary phase or slower growth on poorer substrate, e.g. methanol, leads to a lower basal Kar2/Bip level. Methanol-induced secretory IP production elicits a strong UPR activation which counteracts the reduced UPR during slow growth on methanol. The major ER chaperone Kar2/Bip is found together with recombinant IP in the culture medium where full-length Kar2/Bip accumulates in addition to large amounts of truncated Kar2/Bip. Thus, for judging UPR activating properties of the produced protein it is important to additionally analyze the medium not only for intact Kar2/Bip but also for truncated versions of this UPR reporter protein.

Improvement of a fermentation process for the production of two PfAMA1-DiCo-based malaria vaccine candidates in Pichia pastoris

Pichia pastoris is a simple and powerful expression platform that has the ability to produce a wide variety of recombinant proteins, ranging from simple peptides to complex membrane proteins. A well-established fermentation strategy is available comprising three main phases: a batch phase, followed by a glycerol fed-batch phase that increases cell density, and finally an induction phase for product expression using methanol as the inducer. We previously used this three-phase strategy at the 15-L scale to express three different AMA1-DiCo-based malaria vaccine candidates to develop a vaccine cocktail. For two candidates, we switched to a two-phase strategy lacking the intermediate glycerol fed-batch phase. The new strategy not only provided a more convenient process flow but also achieved 1.5-fold and 2.5-fold higher space-time yields for the two candidates, respectively, and simultaneously reduced the final cell mass by a factor of 1.3, thus simplifying solid–liquid separation. This strategy also reduced the quantity of host cell proteins that remained to be separated from the two vaccine candidates (by 34% and 13%, respectively), thus reducing the effort required in the subsequent purification steps. Taken together, our new fermentation strategy increased the overall fermentation performance for the production of two different AMA1-DiCo-based vaccine candidates.

Systematic evaluation of characteristics of the membrane-based fed-batch shake flask

BackgroundThe initial part of process development involves extensive screening programs to identify optimal biological systems and cultivation conditions. For a successful scale-up, the operation mode on screening and production scale must be as close as possible. To enable screening under fed-batch conditions, the membrane-based fed-batch shake flask was developed. It is a shake flask mounted with a central feed reservoir with an integrated rotating membrane tip for a controlled substrate release. Building on the previously provided proof of principle for this tool, this work extends its application by constructive modifications and improved methodology to ensure reproducible performance.ResultsThe previously limited operation window was expanded by a systematic analysis of reservoir set-up variations for cultivations with the fast-growing organism Escherichia coli. Modifying the initial glucose concentration in the reservoir as well as interchanging the built-in membrane, resulted in glucose release rates and oxygen transfer rate levels during the fed-batch phase varying up to a factor of five. The range of utilizable membranes was extended from dialysis membranes to porous microfiltration membranes with the design of an appropriate membrane tip. The alteration of the membrane area, molecular weight cut-off and liquid volume in the reservoir offered additional parameters to fine-tune the duration of the initial batch phase, the oxygen transfer rate level of the fed-batch phase and the duration of feeding. It was shown that a homogeneous composition of the reservoir without a concentration gradient is ensured up to an initial glucose concentration of 750 g/L. Finally, the experimental validity of fed-batch shake flask cultivations was verified with comparable results obtained in a parallel fed-batch cultivation in a laboratory-scale stirred tank reactor.ConclusionsThe membrane-based fed-batch shake flask is a reliable tool for small-scale screening under fed-batch conditions filling the gap between microtiter plates and scaled-down stirred tank reactors. The implemented reservoir system offers various set-up possibilities, which provide a wide range of process settings for diverse biological systems. As a screening tool, it accurately reflects the cultivation conditions in a fed-batch stirred tank reactor and enables a more efficient bioprocess development.

High Expression of Human Cathepsin S by Recombinant Pichia pastoris with Cod Skin as an Organic Co-Nitrogen Source

Human cathepsin S production by recombinant Pichia pastoris using cod skin as the co-nitrogen source was investigated in this study. The addition of carbon sources of glycerol in the fed-batch phase and of methanol in the induction stage was also investigated. A new approach to the highly expression of human cathepsin S was developed using 90 g/L of cod skin (wet weight). After 24 h of the initial fermentation, 4% glycerol (v/v, glycerol/culture) was added once to enhance the cell density (OD₆₀₀) in the cultivation. Then, adding and maintaining methanol at 0.5% (v/v, methanol/cultivation) after about 48 h of fermentation achieved a high expression of human cathepsin S in a 5-L bioreactor. The results demonstrate that the maximum activity of human cathepsin S in the fermentation supernatant reached 7,152 U/L after 96 h of methanol induction. The methylotrophic yeast P. pastoris grown in the medium containing cod skin (90 g/L) as the co-nitrogen source provided a 21% higher cell density (OD₆₀₀) and 18.3% higher human cathepsin S yield than P. pastoris grown in BMGY medium. For the first time, human cathepsin S was successfully expressed by P. pastoris with cod skin as the co-nitrogen source. The glycerol fed-batch controlling strategy and method of maintaining methanol at a constant concentration of 0.5% (v/v, methanol/cultivation) in the induction stage was efficient for P. pastoris growth and the expression of human cathepsin S.

A Novel Application for Low Frequency Electrochemical Impedance Spectroscopy as an Online Process Monitoring Tool for Viable Cell Concentrations.

New approaches in process monitoring during industrial fermentations are not only limited to classical pH, dO2 and offgas analysis, but use different in situ and online sensors based on different physical principles to determine biomass, product quality, lysis and far more. One of the very important approaches is the in situ accessibility of viable cell concentration (VCC). This knowledge provides increased efficiency in monitoring and controlling strategies during cultivations. Electrochemical impedance spectroscopy—EIS—is used to monitor biomass in a fermentation of E. coli BL21(DE3), producing a recombinant protein using a fed batch-based approach. Increases in the double layer capacitance (Cdl), determined at frequencies below 1 kHz, are proportional to the increase of biomass in the batch and fed batch phase, monitored in offline and online modes for different cultivations. A good correlation of Cdl with cell density is found and in order to get an appropriate verification of this method, different state-of-the-art biomass measurements are performed and compared. Since measurements in this frequency range are largely determined by the double layer region between the electrode and media, rather minor interferences with process parameters (aeration, stirring) are to be expected. It is shown that impedance spectroscopy at low frequencies is a powerful tool for cultivation monitoring.

Oxygen transfer as a tool for fine-tuning recombinant protein production by Pichia pastoris under glyceraldehyde-3-phosphate dehydrogenase promoter.

Effects of oxygen transfer on recombinant protein production by Pichia pastoris under glyceraldehyde-3-phosphate dehydrogenase promoter were investigated. Recombinant glucose isomerase was chosen as the model protein. Two groups of oxygen transfer strategies were applied, one of which was based on constant oxygen transfer rate where aeration rate was Q O/V=3 and 10 vvm, and agitation rate was N=900min(-1); while the other one was based on constant dissolved oxygen concentrations, C DO=5, 10, 15, 20 and 40% in the fermentation broth, by using predetermined exponential glucose feeding with μ o=0.15h(-1). The highest cell concentration was obtained as 44gL(-1) at t=9h of the glucose fed-batch phase at C DO=20% operation while the highest volumetric and specific enzyme activities were obtained as 4440UL(-1) and 126Ug(-1) cell, respectively at C DO=15% operation. Investigation of specific enzyme activities revealed that keeping C DO at 15% was more advantageous with an expense of relatively higher by-product formation and lower specific cell growth rate. For this strategy, the highest oxygen transfer coefficient and oxygen uptake rate were K L a=0.045s(-1) and OUR=8.91mmolm(-3)s(-1), respectively.

On-line specific growth rate control for improving reduced glutathione production in Saccharomyces cerevisiae

Reduced glutathione (GSH), the abundant bioactive tripeptide in most living cells, is widely used in pharmaceutical, food, and cosmetic industries. Specific growth rate (μ) is a key physiological parameter for GSH high-cell-density cultivation using microbial cell factories. Here, based on a biomass probe, an on-line μ feedback control was developed to regulate glucose feeding rate during the fed-batch phase for overproducing GSH in Saccharomyces cerevisiae. Compared with real-time μ controlled at 0.15/h, μ controlled at 0.2/h achieved yeast dry weight (120 g/L), GSH concentration (1.5 g/L), and intracellular GSH content (1.25%), which improved by 9, 150, and 129.1%, respectively. To our knowledge, this is the first report about on-line μ feedback control for GSH production. On-line μ control led to 59.38 mg/L/h of GSH productivity and 3.52 mg/g of GSH yield on glucose, which improved by 107.6 and 7.2%, respectively, in comparison with those of traditional ethanol feedback control (maintaining ethanol concentration at 1%). Taken together, the on-line μ feedback control is a promising method as an efficient alternative to conventional feed control techniques presently practiced in the GSH industry, and has the potential for the production of other valuable chemicals.

Rapid start-up of mesophilic dry anaerobic digestion of kitchen waste in a continuous stirred tank reactor (CSTR)

Under mesophilic conditions (37 °C), a bench-scale experiment of dry fermentation start-up using kitchen waste was conducted in a fed-batch single phase reactor over a period of 40 days. The results showed that (1) comparing biogas production, efficiencies, and methane concentrations, three apparent different stages emerged: adaption, growth, and stability; (2) in adaption and growth stages, daily biogas production was 0–0.2 L/day and 0.2–0.4 L/day, respectively, and the concentration of methane in the biogas was 22.91% and 48.79%, respectively, with a biodegradation rate of approximately 34.72% in the adaption phase and 27.60% during the growth phase; (3) in the stability stage, the biogas production efficiency attained the maximal level and stabilized at approximately 0.6–0.4 L/day and the concentration of methane was 54.32%. Furthermore, the pH fell to 4.15, and the concentration of chemical oxygen demand increased from 51.139 to 119.295 mg/ml.

Real-time monitoring of glycerol and methanol to enhance antibody production in industrial Pichia pastoris bioprocesses

Monoclonal antibody production in glycoengineered Pichia pastoris was optimized for high cell density fed-batch fermentations. Results show that antibody productivity was 1.72-fold higher when maintaining oxygen-limited conditions compared to methanol-limited conditions during the protein production phase of the bioprocess. Under oxygen-limited conditions with a 30mmolL−1h−1 of oxygen uptake rate, an optimum methanol dosage level of 10gL−1 was established by comparing antibody titers under different methanol dosages during protein production. Real-time on-line NIR monitoring of glycerol concentration, methanol concentration, and relative cell density provided valuable data for process characterization and optimization. Real-time profiles of glycerol concentration and relative cell density documented run-to-run consistency during the batch and fed-batch phases by analyses of the glycerol consumption and cell growth kinetics. The volumetric methanol consumption rate was characterized for the induction process following the end of the glycerol fed-batch phase. Under oxygen-limited conditions, the volumetric methanol consumption rate increased over a 60-h period and this increase was independent of the methanol concentration over a range of 2.5–30gL−1 when the oxygen utilization rate was maintained at 30mmolL−1h−1. Utility of the on-line NIR monitor is demonstrated over the one-year period of this investigation by accurately tracking biomass, glycerol concentration, and methanol concentration without user required recalibration as well as by documenting abnormal events during high cell density bioprocesses.

Efficient and cost-reduced glucoamylase fed-batch production with alternative carbon sources.

Glucoamylase is an important industrial enzyme. Glucoamylase production by industrial Aspergillus niger strain featured with two major problems: (i) empirical substrate feeding methods deteriorating the fermentation performance; and (ii) the high raw materials cost limiting the economics of the glucoamylase product with delegated specification. In this study, we first proposed a novel three-stage varied-rate substrate feeding strategy for efficient glucoamylase production in a 5 L bioreactor using the standard feeding medium, by comparing the changing patterns of the important physiological parameters such as DO, OUR, RQ, etc., when using different substrate feeding strategies. With this strategy, the glucoamylase activity and productivity reached higher levels of 11,000 U/ml and 84.6 U/ml/h, respectively. The performance enhancement in this case was beneficial from the following results: DO and OUR could be controlled at the higher levels (30%, 43.83 mmol/l/h), while RQ was maintained at a stable/lower level of 0.60 simultaneously throughout the fed-batch phase. Based on this three-stage varied-rate substrate feeding strategy, we further evaluated the economics of using alternative carbon sources, attempting to reduce the raw materials cost. The results revealed that cornstarch hydrolysate could be considered as the best carbon source to replace the standard and expensive feeding medium. In this case, the production cost of the glucoamylase with delegated specification (5,000 U/ml) could be saved by more than 61% while the product quality be ensured simultaneously. The proposed strategy showed application potential in improving the economics of industrial glucoamylase production.

A Soft Sensor for Bioprocess Control Based on Sequential Filtering of Metabolic Heat Signals

Soft sensors are the combination of robust on-line sensor signals with mathematical models for deriving additional process information. Here, we apply this principle to a microbial recombinant protein production process in a bioreactor by exploiting bio-calorimetric methodology. Temperature sensor signals from the cooling system of the bioreactor were used for estimating the metabolic heat of the microbial culture and from that the specific growth rate and active biomass concentration were derived. By applying sequential digital signal filtering, the soft sensor was made more robust for industrial practice with cultures generating low metabolic heat in environments with high noise level. The estimated specific growth rate signal obtained from the three stage sequential filter allowed controlled feeding of substrate during the fed-batch phase of the production process. The biomass and growth rate estimates from the soft sensor were also compared with an alternative sensor probe and a capacitance on-line sensor, for the same variables. The comparison showed similar or better sensitivity and lower variability for the metabolic heat soft sensor suggesting that using permanent temperature sensors of a bioreactor is a realistic and inexpensive alternative for monitoring and control. However, both alternatives are easy to implement in a soft sensor, alone or in parallel.

Impact of glycerol and nitrogen concentration on Enterobacter A47 growth and exopolysaccharide production

Enterobacter A47 produces a fucose-containing exopolysaccharide (EPS) by cultivation in mineral medium supplemented with glycerol. EPS synthesis by Enterobacter A47 was shown to be influenced by both the initial glycerol and nitrogen concentrations and by the nutrients’ feeding rate during the fed-batch phase. Initial nitrogen concentrations above 1.05g/L were detrimental for EPS synthesis: the productivity was reduced to 0.35–0.62g/Ld (compared to 1.89–2.04g/Ld under lower nitrogen concentrations) and the polymer had lower fucose content (14–17%mol, compared to 36–38%mol under lower nitrogen concentrations). On the other hand, EPS productivity was improved to 5.66g/Ld by increasing the glycerol and nitrogen feeding rates during the fed-batch phase. However, the EPS thus obtained had lower fucose (26%mol) and higher galactose (34%mol) contents, as well as lower average molecular weight (7.2×105). The ability of Enterobacter A47 to synthesize EPS with different physico-chemical characteristics may be useful for the generation of biopolymers with distinct functional properties suitable for different applications.

Decrease of UPR- and ERAD-related proteins in Pichia pastoris during methanol-induced secretory insulin precursor production in controlled fed-batch cultures

BackgroundPichia pastoris is a popular yeast preferably employed for secretory protein production. Secretion is not always efficient and endoplasmic retention of proteins with aberrant folding properties, or when produced at exaggerated rates, can occur. In these cases production usually leads to an unfolded protein response (UPR) and the induction of the endoplasmic reticulum associated degradation (ERAD). P. pastoris is nowadays also an established host for secretory insulin precursor (IP) production, though little is known about the impact of IP production on the host cell physiology, in particular under industrially relevant production conditions. Here, we evaluate the cellular response to aox1 promoter-controlled, secretory IP production in controlled fed-batch processes using a proteome profiling approach.ResultsCells were first grown in a batch procedure using a defined medium with a high glycerol concentration. After glycerol depletion IP production was initiated by methanol addition which was kept constant through continuous methanol feeding. The most prominent changes of the intracellular proteome after the onset of methanol feeding were related to the enzymes of central carbon metabolism. In particular, the enzymes of the methanol dissimilatory pathway - virtually absent in the glycerol batch phase - dominated the proteome during the methanol fed-batch phase. Unexpectedly, a strong decrease of UPR and ERAD related proteins was also observed during methanol-induced IP production. Compared to non-producing control strains grown under identical conditions the UPR down-regulation was less pronounced indicating that IP production elicits a detectable but non prominent UPR response which is repressed by the general culture condition-dependent UPR down-regulation after the shift from glycerol to methanol.ConclusionsThe passage of IP through the secretory pathway using an optimized IP vector and growing the strain at fed-batch conditions with a high initial glycerol concentration does not impose a significant burden on the secretory machinery even under conditions leading to an extracellular accumulation of ~ 3 g L-1 IP. The glycerol batch pre-induction culture conditions are associated with a high constitutive - recombinant protein production independent - induction of the UPR and ERAD pathways probably preconditioning the cells for effective IP secretion in the methanol fed-batch phase.

Enhanced Spore Production of <i>Bacillus subtilis</i> Grown in a Chemically Defined Medium

Spores of Bacillus subtilis are being used as probiotics and competitive exclusion agents for animal consumption. Commercial production media often include relatively expensive components of animal origin that are a potential source for the presence of adventious agents, therefore undesirable for use in production scale. In this study a new animal-free component, chemically defined medium, was tested for B. subtilis spore production. Medium composition was optimized with respect to vitamin composition, carbon, nitrogen and calcium concentrations. A fed-batch bioprocess was developed, being the effect on sporulation of the carbon to nitrogen ratio at the end of the exponential growth phase studied. The developed strategy consisted of an initial and a final batch phase and an intermediate fed-batch phase with the addition of a feeding solution containing glucose and calcium and the addition of a feeding solution of ammonium sulphate, using an exponential and a constant feeding profile, respectively. Using the fed-batch strategy, it was possible to achieve a maximum spore production of 3.6 × 1010 spores/mL, corresponding to a 5 folds increase when compared to the preliminary batch experiments.

Substrate oscillations boost recombinant protein release from Escherichia coli

Intracellular production of recombinant proteins in prokaryotes necessitates subsequent disruption of cells for protein recovery. Since the cell disruption and subsequent purification steps largely contribute to the total production cost, scalable tools for protein release into the extracellular space is of utmost importance. Although there are several ways for enhancing protein release, changing culture conditions is rather a simple and scalable approach compared to, for example, molecular cell design. This contribution aimed at quantitatively studying process technological means to boost protein release of a periplasmatic recombinant protein (alkaline phosphatase) from E. coli. Quantitative analysis of protein in independent bioreactor runs could demonstrate that a defined oscillatory feeding profile was found to improve protein release, about 60%, compared to the conventional constant feeding rate. The process technology included an oscillatory post-induction feed profile with the frequency of 4min. The feed rate was oscillated triangularly between a maximum (1.3-fold of the maximum feed rate achieved at the end of the fed-batch phase) and a minimum (45% of the maximum). The significant improvement indicates the potential to maximize the production rate, while this oscillatory feed profile can be easily scaled to industrial processes. Moreover, quantitative analysis of the primary metabolism revealed that the carbon dioxide yield can be used to identify the preferred feeding profile. This approach is therefore in line with the initiative of process analytical technology for science-based process understanding in process development and process control strategies.

Fed-batch operational strategies for recombinant Fab production with Pichia pastoris using the constitutive GAP promoter

Carbon source and growth rate are two major parameters affecting recombinant protein production in Pichia pastoris. The effect of the most commonly used carbon sources (glycerol or glucose) and the specific growth rate (μ) has been studied on the production of a human antigen-binding fragment (Fab) in this cell factory under the constitutive GAP promoter in fed-batch cultures.Glycerol for batch phase and glucose for fed-batch phase was the most successful carbon source combination. During batch phase, by-products were detected when glucose was used, despite maintaining DO at values higher than 35%. Also, the presence of cell aggregates was detected affecting the reproducibility and operability of the bioprocess.Conversely, glucose was the best substrate for fed-batch phase. When working at C-limiting conditions, neither by-products nor aggregates were detected and Fab production levels were comparable to those obtained with glycerol. In addition, the lower heat yield (YQ/X) and oxygen to biomass yield (YO2/X) for glucose-supported cultures made this substrate the best alternative from an industrial operational point of view.In addition, the effect of specific growth rate on fed-batch Fab production was studied. Medium and high μ (0.10 and 0.15h−1) set-points showed similar Fab production yield. However, in terms of total and volumetric productivity, higher μ was the best process condition.

A high-throughput media design approach for high performance mammalian fed-batch cultures

An innovative high-throughput medium development method based on media blending was successfully used to improve the performance of a Chinese hamster ovary fed-batch medium in shaking 96-deepwell plates. Starting from a proprietary chemically-defined medium, 16 formulations testing 43 of 47 components at 3 different levels were designed. Media blending was performed following a custom-made mixture design of experiments considering binary blends, resulting in 376 different blends that were tested during both cell expansion and fed-batch production phases in one single experiment. Three approaches were chosen to provide the best output of the large amount of data obtained. A simple ranking of conditions was first used as a quick approach to select new formulations with promising features. Then, prediction of the best mixes was done to maximize both growth and titer using the Design Expert software. Finally, a multivariate analysis enabled identification of individual potential critical components for further optimization. Applying this high-throughput method on a fed-batch, rather than on a simple batch, process opens new perspectives for medium and feed development that enables identification of an optimized process in a short time frame.

Combined Strategies for Improving the Production of Recombinant Rhizopus oryzae Lipase in Pichia pastoris

We have developed a yeast Pichia pastoris system for the high-level expression of recombinant Rhizopus oryzae lipase (ROL), which is a potentially effective catalyst in the solvent-free production of biodiesel fuel. In the glycerol fed-batch phase, the combination of the dissolved-oxygen-stat and gradient-control glycerol feeding strategies resulted in a higher cell biomass in the P. pastoris culture, with shorter feed times. In the methanol fed-batch phase, a constant methanol concentration of 0.3-0.5% (v/v) was found to be optimal for high ROL activity, maximum protein concentration, and a maximum biomass. Cells were grown at 30 °C and induced by methanol at 22 °C and a pH of 6.0 with the addition of 0.5% (w/v) casein, which reduced the proteolytic degradation of ROL. Using a combined glycerol step-increasing, on-line methanol feeding strategy and these reducing proteolytic degradation methods, a maximum biomass of 96 g DCW/l, a maximum concentration of 2.0 g/L, and an activity of 1302.2 U/mL ROL were attained. The simple culture process is the highest level of ROL expression reported in P. pastoris. Our results strongly demonstrate that the efficiency of the recombinant ROL expression is directly dependent on feed strategies, lower induction temperature, maintaining the proper pH, and the addition of casein.

Optimization of the pilot-scale production of an ice-binding protein by fed-batch culture of Pichia pastoris

Ice-binding proteins (IBPs) can bind to the ice crystal and inhibit its growth. Because this property of IBPs can increase the freeze-thaw survival of cells, IBPs have attracted the attention from industries for their potential use in biotechnological applications. However, their use was largely hampered by the lack of the large-scale recombinant production system. In this study, the codon-optimized IBP from Leucosporidium sp. (LeIBP) was constructed and subjected to high-level expression in methylotrophic Pichia pastoris system. In a laboratory-scale fermentation (7 L), the optimal induction temperature and pH were determined to be 25 °C and 6.0, respectively. Further, employing glycerol fed-batch phase prior to methanol induction phase enhanced the production of recombinant LelBP (rLeIBP) by ∼100 mg/l. The total amount of secreted proteins at these conditions (25 °C, pH 6.0, and glycerol fed-batch phase) was ∼443 mg/l, 60 % of which was rLeIBP, yielding ∼272 mg/l. In the pilot-scale fermentation (700 L) under the same conditions, the yield of rLeIBP was 300 mg/l. To our best knowledge, this result reports the highest production yield of the recombinant IBP. More importantly, the rLeIBP secreted into culture media was stable and active for 6 days of fermentation. The thermal hysteresis (TH) activity of rLeIBP was about 0.42 °C, which is almost the same to those reported previously. The availability of large quantities of rLeIBP may accelerate further application studies.