补加前体L-蛋氨酸对高密度发酵生产S-腺苷-L-蛋氨酸的影响

将高密度发酵技术成功应用于S-腺苷-L-蛋氨酸的生产。考察了补加前体L-蛋氨酸的量以及补加策略对酿酒酵母G14发酵生产S-腺苷-L-蛋氨酸的影响。实验发现补加前体L-蛋氨酸能明显促进S-腺苷-L-蛋氨酸的积累。同时还发现不同的补加策略对菌体浓度以及S-腺苷-L-蛋氨酸的产量和浓度有不同的影响。确定了补加L-蛋氨酸不应低于0.7g/10g菌体干重。比较了五种不同的补加前体L-蛋氨酸的方式。结果表明在菌体干重达到高密度的情况下(120g/L)补加前体L-蛋氨酸进行转化生产S-腺苷-L-蛋氨酸能达到比较好的效果一次性补加9g L-蛋氨酸,SAM的积累量在补加后的18h达到最高,为4.31g/L;采取流加方式补加L-蛋氨酸,流加速率为2g/h,共流加5h,流加结束28h后SAM达到最高积累量后者达到4.98g/L。两者最终的生物量均可达到130g/L以上。     

Batch and fed-batch growth of Pichia pastoris under increased air pressure

Pichia pastoris CBS 2612 behavior under air pressures of 1, 3 and 5 bar in culture media of glycerol (pure and crude) and methanol was studied. Generally, the increase in oxygen transfer rate due to the increase of total pressure improved cellular growth for all carbon sources and for batch and fed-batch processes with different feeding rate strategies. In batch cultures, 1.4-, 1.2-, and 1.5-fold improvement in biomass production was obtained with the increase of air pressure up to 5 bar, using methanol, pure glycerol, and crude glycerol, respectively. The increase of air pressure to 5 bar using exponential feeding rate led to 1.4-fold improvement in biomass yield per glycerol mass consumed, for crude and pure glycerol. The current low cost of crude glycerol from the biodiesel production together with the present results shows the possibility of improving cell mass production of P. pastoris using increased air pressure.     

Isolation of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> mutants for heterologous protein production from a double proteinase gene disruptant

The recombinant Pichia pastoris harboring an improved methionine adenosyltransferase (MAT) shuffled gene was employed to biosynthesize S-adenosyl-l-methionine (SAM). Two l-methionine (l-Met) addition strategies were used to supply the precursor: the batch addition strategy (l-Met was added separately at three time points) and the continuous feeding strategies (l-Met was fed continuously at the rate of 0.1, 0.2, and 0.5 g l⁻¹ h⁻¹, respectively). SAM accumulation, l-Met conversion rate, and SAM productivity with the continuous feeding strategies were all improved over the batch addition strategy, which reached 8.46 ± 0.31 g l⁻¹, 41.7 ± 1.4%, and 0.18 ± 0.01 g l⁻¹ h⁻¹ with the best continuous feeding strategy (0.2 g l⁻¹ h⁻¹), respectively. The bottleneck for SAM production with the low l-Met feeding rate (0.1 g L⁻¹ h⁻¹) was the insufficient l-Met supply. The analysis of the key enzyme activities indicated that the tricarboxylic acid cycle and glycolytic pathway were reduced with the increasing l-Met feeding rate, which decreased the adenosine triphosphate (ATP) synthesis. The MAT activity also decreased as the l-Met feeding rate rose. The reduced ATP synthesis and MAT activity were probably the reason for the low SAM accumulation when the l-Met feeding rate reached 0.5 g l⁻¹ h⁻¹.     

Effect of methanol feeding strategies on production and yield of recombinant mouse endostatin from Pichia pastoris

Pichia pastoris, a methylotrophic yeast, is an efficient producer of recombinant proteins in which the heterologous gene is under the control of the methanol-induced AOX1 promoter. Hence, the accepted production procedure has two phases: In the first phase, the yeast utilizes glycerol and biomass is accumulated; in the second phase, the yeast utilizes methanol which is used both as an inducer for the expression of the recombinant protein and as a carbon source. Since the yeast is sensitive to methanol concentration, the methanol is supplied gradually to the growing culture. Three methanol addition strategies were evaluated for the purpose of optimizing recombinant endostatin production. Two strategies were based on the yeast metabolism; one responding to the methanol consumption using a methanol sensor, and the other responding to the oxygen consumption. In these two strategies, the methanol supply is unlimited. The third strategy was based on a predetermined exponential feeding rate, controling the growth rate at 0.02 h(-1), in this strategy the methanol supply is limited. Throughout the induction phase glycerol, in addition to methanol, was continuously added at a rate of 1 g L h(-1). Total endostatin production was similar in all three strategies, (400 mg was obtained from 3 L initial volume), but the amount of methanol added and the biomass produced were lower in the predetermined rate method. This caused the specific production of endostatin per biomass and per methanol to be 2 times higher in the predetermined rate than in the other two methods, making the growth control strategy not only more efficient but also more convenient for downstream processing.     

毕赤酵母发酵产S-腺苷蛋氨酸的甘油-甲醇混合流加策略

在毕赤酵母发酵生产S-腺苷蛋氨酸(SAM)的诱导阶段,以不同甘油-甲醇比例的甘油-甲醇混合培养基进行诱导培养,结果表明以10%(w/v)甘油含量的甘油-甲醇混合培养基进行诱导培养时最有利于SAM的表达,SAM产量达6.09 g/L,比0%甘油含量条件下的SAM产量提高了20.4%。对诱导方式进行优化,先以100%甲醇诱导24 h,然后再连续流加10%(w/v)甘油含量的甘油-甲醇混合培养基,SAM产量可达7.94 g/L,在此基础上,进一步改进诱导方式,SAM产量得到进一步的提高,达到9.80 g/L。     

Depletion of S-adenosyl-l-methionine with cycloleucine potentiates cytochrome P450 2E1 toxicity in primary rat hepatocytes

S-Adenosyl-l-methionine (SAM) is the principal biological methyl donor. Methionine adenosyltransferase (MAT) catalyzes the only reaction that generates SAM. Hepatocytes were treated with cycloleucine, an inhibitor of MAT, to evaluate whether hepatocytes enriched in cytochrome P450 2E1 (CYP2E1) were more sensitive to a decline in SAM. Cycloleucine decreased SAM and glutathione (GSH) levels and induced cytotoxicity in hepatocytes from pyrazole-treated rats (with an increased content of CYP2E1) to a greater extent as compared to hepatocytes from saline-treated rats. Apoptosis caused by cycloleucine in pyrazole hepatocytes appeared earlier and was more pronounced than control hepatocytes and could be prevented by incubation with SAM, glutathione reduced ethyl ester and antioxidants. The cytotoxicity was prevented by treating rats with chlormethiazole, a specific inhibitor of CYP2E1. Cycloleucine induced greater production of reactive oxygen species (ROS) in pyrazole hepatocytes than in control hepatocytes, and treatment with SAM, Trolox, and chlormethiazole lowered ROS formation. In conclusion, lowering of hepatic SAM levels produced greater toxicity and apoptosis in hepatocytes enriched in CYP2E1. This is due to elevated ROS production by CYP2E1 coupled to lower levels of hepatoprotective SAM and GSH. We speculate that such interactions e.g. induction of CYP2E1, decline in SAM and GSH may contribute to alcohol liver toxicity.     

The reduction of xylose to xylitol by Candida guilliermondii and Candida parapsilosis: Incidence of oxygen and pH

Summary The ability of C. guilliermondii and C. parapsilosis to ferment xylose to xylitol was evaluated under different oxygen transfer rates in order to enhance the xylitol yield. In C. guilliermondii, a maximal xylitol yield of 0.66 g/g was obtained when oxygen transfer rate was 2.2 mmol/l.h. Optimal conditions to produce xylitol by C. parapsilosis (0.75 g/g) arose from cultures at pH 4.75 with 0.4 mmoles of oxygen/l.h. The response of the yeasts to anaerobic conditions has shown that oxygen was required for xylose metabolism.Nomenclature max maximum specific growth rate (per hour) - qSmax maximum specific rate of xylose consumption (g xylose per g dry biomass per hour) - qpmax maximum specific productivity of xylitol (g xylitol per g dry biomass per hour) - Qp average volumetric productivity of xylitol (g xylitol per liter per hour) - Y_P/S xylitol yield (g xylitol per g substrate utilized) - Y_P'/S glycerol yield (g glycerol per g substrate utilized) - Y_X/S biomass yield (g dry biomass per g substrate utilized)     

A rational approach to improving productivity in recombinant Pichia pastoris fermentation

A Mut(S) Pichia pastoris strain that had been genetically modified to produce and secrete sea raven antifreeze protein was used as a model system to demonstrate the implementation of a rational, model-based approach to improve process productivity. A set of glycerol/methanol mixed-feed continuous stirred-tank reactor (CSTR) experiments was performed at the 5-L scale to characterize the relationship between the specific growth rate and the cell yield on methanol, the specific methanol consumption rate, the specific recombinant protein formation rate, and the productivity based on secreted protein levels. The range of dilution rates studied was 0. 01 to 0.10 h(-1), and the residual methanol concentration was kept constant at approximately 2 g/L (below the inhibitory level). With the assumption that the cell yield on glycerol was constant, the cell yield on methanol increased from approximately 0.5 to 1.5 over the range studied. A maximum specific methanol consumption rate of 20 mg/g. h was achieved at a dilution rate of 0.06 h(-1). The specific product formation rate and the volumetric productivity based on product continued to increase over the range of dilution rates studied, and the maximum values were 0.06 mg/g. h and 1.7 mg/L. h, respectively. Therefore, no evidence of repression by glycerol was observed over this range, and operating at the highest dilution rate studied maximized productivity. Fed-batch mass balance equations, based on Monod-type kinetics and parameters derived from data collected during the CSTR work, were then used to predict cell growth and recombinant protein production and to develop an exponential feeding strategy using two carbon sources. Two exponential fed-batch fermentations were conducted according to the predicted feeding strategy at specific growth rates of 0.03 h(-1) and 0.07 h(-1) to verify the accuracy of the model. Cell growth was accurately predicted in both fed-batch runs; however, the model underestimated recombinant product concentration. The overall volumetric productivity of both runs was approximately 2.2 mg/L. h, representing a tenfold increase in the productivity compared with a heuristic feeding strategy.     

Overproduction of alkaline polygalacturonate lyase in recombinant Escherichia coli by a two-stage glycerol feeding approach

This work aims to achieve the overproduction of alkaline polygalacturonate lyase (PGL) with recombinant Escherichia coli by a two-stage glycerol feeding approach. First, the PGL coding gene from Bacillus subtilis WSHB04-02 was expressed in E. coli BL21 (DE3) under the strong inducible T7 promoter of the pET20b (+) vector. And then the influence of media composition, induction temperature, and inducer isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration on cell growth and PGL production was investigated. Finally, a two-stage glycerol feeding strategy was proposed and applied in a 3-L fermenter, where cultivation was conducted at a controlled specific growth rate (μset=0.2) during pre-induction phase, followed by a constant glycerol feeding rate of 12 ml h(-1) at post-induction phase. The total PGL yield reached 371.86 U mL(-1), which is the highest PGL production by recombinant E. coli expression system.     

Effect of oxygen transfer on glycerol biosynthesis by an osmophilic yeast Candida magnoliae I(2)B

The influence of oxygen on glycerol production by an osmophilic yeast, Candida magnoliae I(2)B, was studied in a bioreactor. Oxygen transfer rates (OTRs) and volumetric oxygen transfer coefficients (k(L)a) were determined at different aeration and agitation rates. Cell growth as well as glycerol production was strongly affected by oxygen supply. Improvement in OTRs resulted in increased cell growth and glycerol yield. However, at high OTRs, there was a reduction in glucose uptake rate, indicating Pasteur Effect, and glycerol accumulation was also reduced at k(L)a of 253 h(-1). The availability of oxygen per unit of cell mass was found to be the most important factor that controlled cell growth, glucose uptake, and glycerol yield. The overall productivity and yield of glycerol could be related with k(L)a. The biosynthesis of glycerol was found to both growth- and non-growth-associated, although glycerol was mainly produced in post-exponential phase.     

Enhanced truncated-t-PA (CT-b) expression in high-cell-density fed-batch cultures of <Emphasis Type="Italic">Pichia pastoris</Emphasis> through optimization of a mixed feeding strategy by response surface methodology

Recently, Pichia pastoris has been the focal point of interest as an expression system for production of many recombinant proteins. The study and optimization of feeding strategy are of major importance to achieve maximum volumetric productivity in fed-batch cultivations. Among different feeding strategies used in P. pastoris fed-batch cultures, those trying to maintain a constant specific growth rate have usually resulted in superior productivities. The objective of the present study was to investigate and optimize the co-feeding of glycerol and methanol to attain maximum expression of t-PA in P. pastoris fed-batch cultures with constant specific growth rate. The experiments were designed by response surface methodology, considering the specific feeding rates of methanol and glycerol as independent variables. In each experiment, glycerol and methanol were fed according to a predetermined equation to maintain a constant specific growth rate. It was found that with glycerol feeding for higher specific growth rates, the inhibitory properties of glycerol are more pronounced, while the best expression level was achieved when the ratio of µ _set glycerol to that of methanol was around 1.67. In all specific growth rates tested, almost a similar ratio of the specific glycerol feeding rate to that of methanol led to the maximum protein production and activity. The statistical model predicted the optimal operating conditions for µ _set glycerol and that of methanol to be 0.05 and 0.03 h^?1, respectively. Applying the optimum strategy, maximum of 52 g/L biomass, 300 mg/L t-PA and 340,000 IU/mL enzyme activity were obtained.     

Bioprocess strategies for mass multiplication of and metabolite synthesis by plant growth promoting pseudomonads for agronomical applications

The exponential substrate feeding (open-loop) and automated feedback substrate feeding (closed loop) strategies were developed to obtain high cell densities of fluorescent pseudomonad strains R62 and R81 and enhanced production of antifungal compound 2,4-diacetylphloroglucinol (DAPG) from glycerol as a sole carbon source. The exponential feeding strategy resulted in increased glycerol accumulation during the fed-batch cultivation when the predetermined specific growth rate (μ) was set at 0.10 or 0.20 h^?1 (<μ_m = 0.29 h^?1). Automated feeding strategies using dissolved oxygen (DO) or pH as feedback signals resulted in minimal to zero accumulation of glycerol for both the strains. In case of DO-based feeding strategy, biomass productivity of 0.24 g/(L h) and 0.29 g/(L h) was obtained for R62 and R81, respectively. Using pH-based feeding strategy, biomass productivity could be increased to a maximum of 0.51 and 0.54 g/(L h), for the strains R62 and R81, respectively, whereas the DAPG concentration was enhanced to 298 mg/L for R62 and 342 mg/L for R81 strains. These yields of DAPG are thus far the highest reported from GRAS organisms.     

Effect of agitation and aeration on the citric acid production by Yarrowia lipolytica grown on glycerol

The effects of agitation rates from 400 to 900 rpm and aeration rates ranging from 0.18 to 0.6 vvm on biomass and citric acid production on glycerol media by acetate-negative mutants of Yarrowia lipolytica, Wratislavia 1.31 and Wratislavia AWG7, in batch culture were studied. The agitation rates of 800 and 900 rpm (at a constant aeration rate of 0.36 vvm) and aeration rates within the range of 0.24-0.48 vvm (at a constant agitation rate of 800 rpm), which generated dissolved oxygen concentration (DO) higher than 40%, were found the best for citric acid biosynthesis from glycerol. An increase in agitation rate (higher than 800 rpm) and aeration rate (higher than 0.36 vvm) had no impact on DO and citric acid production. The highest citric acid concentration (92.8 g/L) and yield (0.63 g/g) were obtained with Wratislavia 1.31 strain at 0.24 vvm. The highest volumetric citric acid production rate (1.15 g/Lh) and specific citric acid production rate (0.071 g/gh) were reached at 0.48 vvm.     

Optimization of the high-level production of Rhizopus oryzae lipase in Pichia pastoris

The lipases of the Rhizopus species family are important and versatile enzymes that are mainly used in fat and oil modification due to their strong 1,3-regiospecificity. Inexpensive synthetic medium was used for the production of Rhizopus oryzae lipase in the methylotrophic yeast Pichia pastoris. Methanol accumulation inside the bioreactor has previously been shown to negatively influence the production level. Three different methanol fed-batch strategies for maintaining the methanol concentration within optimal limits have been assayed in high-density cultures. One methanol feeding strategy, which is based on the monitoring of the methanol concentration by gas chromatography, resulted in a 2.5-fold higher productivity compared to an initial cultivation, where the feeding rate was adjusted according to the dissolved oxygen concentration (DO) in the supernatant. Finally, productivity could be further increased by introducing a transition phase that involved the simultaneous feeding of glycerol and methanol followed by a single methanol feed. This optimized strategy resulted in the highest productivity (12888 U l(-1) h(-1)), which is 13.6-fold higher than the DO-based strategy.     

Fed-batch cultivation of Wautersia eutropha

Batch kinetics of polyhydroxybutyrate (PHB) synthesis in a bioreactor under controlled conditions of pH and dissolved oxygen gave a biomass of 14 g l(-1) with a PHB concentration of 6.1 g l(-1) in 60 h. The data of the batch kinetics was used to develop a mathematical model, which was then extrapolated to fed-batch by incorporating the dilution due to substrate feeding. Offline computer simulation of the fed-batch model was done to develop the nutrient feeding strategies in the fed-batch cultivation. Fed-batch strategies with constant feeding of only nitrogen and constant feeding of both nitrogen and fructose were tried. Constant feeding strategy for nitrogen and fructose gave a better PHB production rate of 0.56 g h(-1) over the value obtained in batch cultivation (PHB production rate - 0.4 g h(-1)).     

Effect of dilution rate and methanol‐glycerol mixed feeding on heterologous Rhizopus oryzae lipase production with Pichia pastoris Mut+ phenotype in continuous culture

The induction using substrate mixtures is an operational strategy for improving the productivity of heterologous protein production with Pichia pastoris. Glycerol as a cosubstrate allows for growth at a higher specific growth rate, but also has been reported to be repressor of the expression from the AOX1 promoter. Thus, further insights about the effects of glycerol are required for designing the induction stage with mixed substrates. The production of Rhizopus oryzae lipase (ROL) was used as a model system to investigate the application of methanol‐glycerol feeding mixtures in fast metabolizing methanol phenotype. Cultures were performed in a simple chemostat system and the response surface methodology was used for the evaluation of both dilution rate and methanol‐glycerol feeding composition as experimental factors. Our results indicate that productivity and yield of ROL are strongly affected by dilution rate, with no interaction effect between the involved factors. Productivity showed the highest value around 0.04–0.06 h^?1, while ROL yield decreased along the whole dilution rate range evaluated (0.03–0.1 h^?1). Compared to production level achieved with methanol‐only feeding, the highest specific productivity was similar in mixed feeding (0.9 UA g‐biomass^?1 h^?1), but volumetric productivity was 70% higher. Kinetic analysis showed that these results are explained by the effects of dilution rate on specific methanol uptake rate, instead of a repressor effect caused by glycerol feeding. It is concluded that despite the effect of dilution rate on ROL yield, mixed feeding strategy is a proper process option to be applied to P. pastoris Mut⁺ phenotype for heterologous protein production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:707–714, 2015     

High-cell-density fermentation of recombinant Saccharomyces cerevisiae using glycerol

To obtain a high cell density of recombinant Saccharomyces cerevisiae (INVSc 1 strain bearing a 2 microm plasmid, pYES2 containing a GAL1 promoter for expression of the beta-galactosidase gene), the yeast was grown with glycerol as the substrate by fed-batch fermentation. The feeding strategy was based on an on-line response of the medium pH to the consumption of glycerol. The approach was to feed excess carbon into the medium to create a benign environment for rapid biomass buildup. During cell growth in the presence of glycerol, the release of protons in the medium caused a decrease in pH and the consumption rate of ammonium phosphate served as an on-line indicator for the metabolic rate of the organism. The extent of glycerol feeding in a fed-batch mode with pH control at 5.0 +/- 0.1 was ascertained from the automatic addition of ammonium phosphate to the medium. The glycerol feeding to ammonium phosphate addition ratio was found to be 2.5-3.0. On the basis of the experiments, a maximum dry cell biomass of 140 g per liter and a productivity of 5.5 g DCW/L/h were achieved. The high cell density of S. cerevisiae obtained with good plasmid stability suggested a simple and efficient fermentation protocol for recombinant protein production.     

High-level production of 3-hydroxypropionic acid from glycerol as a sole carbon source using metabolically engineered Escherichia coli

As climate change is an important environmental issue, the conventional petrochemical-based processes to produce valuable chemicals are being shifted toward eco-friendly biological-based processes. In this study, 3-hydroxypropionic acid (3-HP), an industrially important three carbon (C3) chemical, was overproduced by metabolically engineered Escherichia coli using glycerol as a sole carbon source. As the first step to construct a glycerol-dependent 3-HP biosynthetic pathway, the dhaB1234 and gdrAB genes from Klebsiella pneumoniae encoding glycerol dehydratase and glycerol reactivase, respectively, were introduced into E. coli to convert glycerol into 3-hydroxypropionaldehyde (3-HPA). In addition, the ydcW gene from K. pneumoniae encoding γ-aminobutyraldehyde dehydrogenase, among five aldehyde dehydrogenases examined, was selected to further convert 3-HPA to 3-HP. Increasing the expression level of the ydcW gene enhanced 3-HP production titer and reduced 1,3-propanediol production. To enhance 3-HP production, fed-batch fermentation conditions were optimized by controlling dissolved oxygen (DO) level and employing different feeding strategies including intermittent feeding, pH-stat feeding, and continuous feeding strategies. Fed-batch culture of the final engineered E. coli strain with DO control and continuous feeding strategy produced 76.2 g/L of 3-HP with the yield and productivity of 0.457 g/g glycerol and 1.89 g·L⁻¹·h⁻¹, respectively. To the best of our knowledge, this is the highest 3-HP productivity achieved by any microorganism reported to date.     

Linear correlation between online capacitance and offline biomass measurement up to high cell densities in Escherichia coli fermentations in a pilot-scale pressurized bioreactor

To yield high concentrations of protein expressed by genetically modified Escherichia coli, it is important that the bacterial strains are cultivated to high cell density in industrial bioprocesses. Since the expressed target protein is mostly accumulated inside the E. coli cells, the cellular product formation can be directly correlated to the bacterial biomass concentration. The typical way to determine this concentration is to sample offline. Such manual sampling, however, wastes time and is not efficient for acquiring direct feedback to control a fedbatch fermentation. An E. coli K12-derived strain was cultivated to high cell density in a pressurized stirred bioreactor on a pilot scale, by detecting biomass concentration online using a capacitance probe. This E. coli strain was grown in pure minimal medium using two carbon sources (glucose and glycerol). By applying exponential feeding profiles corresponding to a constant specific growth rate, the E. coli culture grew under carbon-limited conditions to minimize overflow metabolites. A high linearity was found between capacitance and biomass concentration, whereby up to 85 g/L dry cell weight was measured. To validate the viability of the culture, the oxygen transfer rate (OTR) was determined online, yielding maximum values of 0.69 mol/l/h and 0.98 mol/l/h by using glucose and glycerol as carbon sources, respectively. Consequently, online monitoring of biomass using a capacitance probe provides direct and fast information about the viable E. coli biomass generated under aerobic fermentation conditions at elevated headspace pressures.     

Methanol induction optimization for scFv antibody fragment production in Pichia pastoris

Fibronectin splice variant ED B (extracellular domain B) is a promising marker for angiogenesis in growing solid tumors. Currently, recombinant antibodies against ED B are being investigated concerning their potential use, for either therapeutic or diagnostic purposes. Single-chain antibody fragments directed against the ED B can be efficiently expressed in Pichia pastoris; thus, a recombinant strain of the methylotropic yeast P. pastoris was used for this work. Three different forms of scFv antibody fragment are found in the supernatant from this fermentation: covalent homodimer, associative homodimer, and monomer. Both homodimeric forms can be converted to the monomeric form (under reducing conditions) and be efficiently radiolabeled, whereas the monomeric form of scFv already present in the supernatant cannot. It was also found that the fraction of protein in the monomeric form is highly dependent on the mode of induction rather than scFv concentration. This suggests that the monomeric form of the scFv present in the supernatant might be a result of events occurring at the expression, secretion, or folding level. A high cell density fermentation protocol was developed by optimizing methanol induction, yielding the highest scFv antibody fragment production rate and product quality; cell concentration at the induction point and specific methanol uptake rate were found to be the most important control variables. A decrease in specific methanol uptake rate led to a higher specific production rate for the scFv antibody fragment (5.4 microg g(cell) h(-1)). Product quality, i.e., percentage of product in a homodimeric form, also increased with the decrease in methanol uptake rate. Furthermore, the volumetric productivity depended on cell concentration at the induction point, increasing with the increase of cell concentration up to 320 g L(-1) wet cell weight (WCW). The reduction of the methanol feeding rate for induction, and consequently of the oxygen uptake rate, have important consequences for optimizing product titers and quality and thus on the scale-up of this production process; hence one of the major limitations upon high cell density cultivation in bioreactors is keeping the high oxygen transfer rate required. From the results obtained, a scale-up strategy was developed based on the available oxygen transfer rates at larger scales, allowing the definition of the optimum biomass concentration for induction and methanol feeding strategy for maximization of product titer and quality.