Improvement of heterologous protein productivity using recombinant Yarrowia lipolytica and cyclic fed-batch process strategy

A cyclic fed-batch bioprocess is designed and a significant improvement of rice alpha-amylase productivity of recombinant Yarrowia lipolytica is illustrated. A bioprocess control strategy developed and reported here entails use of a genetically stable recombinant cloned for heterologous protein, use of optimized media for cell growth and enzyme production phases, and process control strategy enabling high cell-density culture and high alpha-amylase productivity. This process control can be achieved through maintaining a constant optimal specific cell growth rate at a predetermined value (i.e., 0.1 h-1), controlling medium feed rate commensurate with the cell growth rate, and maintaining a high cell-density culture (i.e., 60-70 g/L) for high productivity of cloned heterologous protein. The volumetric enzyme productivity (1, 960 units/L. h) achieved from the cyclic fed-batch process was about 3-fold higher than that of the fed-batch culture process (630 units/L. h).     

Upstream process optimization of polyhydroxybutyrate (PHB) by Alcaligenes latus using two-stage batch and fed-batch fermentation strategies

This research focused on optimizing the upstream process time for production of polyhydroxybutyrate (PHB) from sucrose by two-stage batch and fed-batch fermentation with Alcaligenes latus ATCC 29714. The study included selection of strain, two-stage batch fermentations with different time points for switching to nitrogen limited media (14, 16 or 18?h) and fed-batch fermentations with varied time points (similar to two stage) for introducing nitrogen limited media. The optimal strain to produce PHB using sucrose as carbon source was A. latus ATCC 29714 with maximum-specific growth rate of 0.38?±?0.01?h^?1 and doubling time of 1.80?±?0.05?h. Inducing nitrogen limitation at 16?h and ending second stage at 26?h gave optimal performance for PHB production, resulting in a PHB content of 46.7?±?12.2?% (g PHB per g dry cell weight) at the end of fermentation. This was significantly higher (P?≤?0.05) (approximately 7?%) than the corresponding fed batch run in which nitrogen limitation was initiated at 16?h.     

Production of flavonoids and polysaccharide by adding elicitor in different cellular cultivation processes of Glycyrrhiza uralensis Fisch

In this study, callus and cell suspension were induced from seedlings of licorice (G. uralensis). In addition, it was revealed that the appropriate concentration of sucrose could promote the callus growth and increase the content of polysaccharide. The methyl jasmonate (MJ) and phenylalanine (PHE) could enhance the callus growth and content of flavonoids for G. uralensis. For producing more flavonoids and polysaccharide, two-stage cultivation was performed. In the first step, 30 g L^?1 sucrose was fed into a 5-L balloon-type bubble bioreactor on 8th day of culture to enhance cell production and metabolite production. In a two-stage cultivation process, PHE (2 mM) and MJ (5 mg L^?1) were added into a 5-L balloon-type bubble bioreactor after 10 days of culture. Using a fed-batch cultivation strategy (30 g L^?1 sucrose was fed into a 5-L balloon-type bubble bioreactor on 8th day), polysaccharide production was enhanced to 1.19 g L^?1, which was 2.12-fold greater than that in batch cultivation. The flavonoids yield (55.42 mg L^?1) which was about 22 % higher than that in batch cultivation was obtained on 21st day. In a two-stage cultivation process, the polysaccharide content was increased by 1.14- and 2.12-fold compared with fed-batch cultivation and batch cultivation on 15th day. Meanwhile, total flavonoids yield (132.36 mg L^?1) on 15th day, was increased by 2.26- and 2.67-fold compared with fed-batch cultivation and batch cultivation. In conclusion, two-stage cultivation process combined with the sucrose and elicitor treatment could promote both the callus growth and the secondary metabolites accumulation.     

Hybrid On-Line Optimal Control Strategy for Producing α-Amylase by Bacillus subtilis

The combined effect of macronutrients in the extraction medium on α-amylase produced by Bacillus subtilis were studied by using response surface methodology in shaken flask cultures. The production of amylase was significantly affected by the interaction between wheat bran and the cotton seed extract in the extraction medium and by the interaction between the cotton seed extract and starch. The optimal combination in the extraction medium for maximum α-amylase production was determined as 10.80 g·L^?1 of wheat bran, 9.90 g·L^?1 of cotton seed extract, 0.5 g·L^?1 of starch, 2.0 g·L^?1 of yeast extract, 5.00 g·L^?1 of NaCl and 2.00 g·L^?1 of CaCl₂. A 12.55-fold increase of enzyme activity was recorded in the optimized medium compared to the result acquired in a minimum essential medium. The optimized medium was used to compare different cultivation strategies in fermenters. The pH-stat strategy for reducing cellular stress response and the substrate concentration-stat strategy for reducing substrate inhibition were independently investigated. The temperature-limited strategy has been proposed to solve the proteolytic digestion problem, although the high-pressure strategy resulted in high productivity. A hybrid strategy simultaneously controlling pH, temperature, substrate concentration and pO₂ was finally investigated to enhance the efficiency of the process. This hybrid strategy resulted in high activity of α-amylase, increasing the productivity almost three-fold as compared to an ordinary fed-batch culture.     

Development and optimization of two-stage cyclic fed-batch culture for hG-CSF production using L-arabinose promoter of Escherichia coli

The long-term process for producing human granulocyte-colony stimulating factor (hG-CSF) was developed using two-stage cyclic fed-batch culture, in which hG-CSF expressing-recombinant Escherichia coli was directed by an L-arabinose promoter system. For the optimization, the preinduction growth rate during the growth stage and the feeding strategy during the production stage were investigated. The maximum harvest volume during the production stage was predicted before long-term cyclic operation. Based on those optimized strategies, the two-stage cyclic fed-batch culture was performed for 12 cycles (86 h). The cell growths in both stages were maintained at 45-50 g/L and 71-77 g/L, respectively. hG-CSF was stably produced at a level of 8-9 g/L and the plasmid stability was maintained at more than 90%. Volumetric productivity by the two-stage cyclic fed-batch culture was 0.643 g/L/h, which was about 280% higher than that of conventional DO-stat fed-batch culture.     

The effect of degree and timing of nitrogen limitation on lipid productivity in Chlorella vulgaris

Improvements in lipid productivity would enhance the economic feasibility of microalgal biodiesel. In order to optimise lipid productivity, both the growth rate and lipid content of algal cells must be maximised. The lipid content of many microalgae can be enhanced through nitrogen limitation, but at the expense of biomass productivity. This suggests that a two-stage nitrogen supply strategy might improve lipid productivity. Two different nitrogen supply strategies were investigated for their effect on lipid productivity in Chlorella vulgaris. The first was an initial nitrogen-replete stage, designed to optimise biomass productivity, followed by nitrogen limitation to enhance lipid content (two-stage batch) and the second was an initial nitrogen-limited stage, designed to maximise lipid content, followed by addition of nitrogen to enhance biomass concentration (fed-batch). Volumetric lipid yield in nitrogen-limited two-stage batch and fed-batch was compared with that achieved in nitrogen-replete and nitrogen-limited batch culture. In a previous work, maximum lipid productivity in batch culture was found at an intermediate level of nitrogen limitation (starting nitrate concentration of 170 mg L^?1). Overall lipid productivity was not improved by using fed-batch or two-stage culture strategies, although these strategies showed higher volumetric lipid concentrations than nitrogen-replete batch culture. The dilution of cultures prior to nitrogen deprivation led to increased lipid accumulation, indicating that the availability of light influenced the rate of lipid accumulation. However, dilution did not lead to increased lipid productivity due to the resulting lower biomass concentration.     

Optimization of erythritol production by Candida magnoliae in fed-batch culture

A two-stage fed-batch process was designed to enhance erythritol productivity by the mutant strain of Candida magnoliae. The first stage (or growth stage) was performed in the fed-batch mode where the growth medium was fed when the pH of the culture broth dropped below 4.5. The second stage (or production stage) was started with addition of glucose powder into the culture broth when the cell mass reached about 75 g dry cell weight l⁻¹. When the initial glucose concentration was adjusted to 400 g l⁻¹ in the production stage, 2.8 g l⁻¹ h⁻¹ of overall erythritol productivity and 41% of erythritol conversion yield were achieved, which represented a fivefold increase in erythritol productivity compared with the simple batch fermentation process. A high glucose concentration in the production phase resulted in formation of organic acids including citrate and butyrate. An increase in dissolved oxygen level caused formation of gluconic acid instead of citric acid. Journal of Industrial Microbiology & Biotechnology (2000) 25, 100–103. Received 25 February 2000/ Accepted in revised form 08 June 2000     

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.     

Utilization of cane molasses towards cost-saving astaxanthin production by a Chlorella zofingiensis mutant

The aim of the present study was to survey the growth and astaxanthin production of E17, an astaxanthin-rich mutant of Chlorella zofingiensis, through feeding the low-cost carbon source cane molasses. In heterotrophic batch cultivation, E17 fed with pretreated molasses achieved biomass (1.79 g L^?1 day^?1) and astaxanthin (1.99 mg L^?1 day^?1) productivities comparable to those with glucose, which were about 2- and 2.8-fold of those fed with untreated molasses, respectively. Molasses-induced astaxanthin accumulation may be attributed to the elicited expression of carotenogenic genes, in particular the genes specifically responsible for the ketolation and hydroxylation of β-carotene to form astaxanthin. A two-stage fed-batch strategy was employed to grow E17 and induce astaxathin accumulation, resulting in 45.6 g L^?1 biomass and 56.1 mg L^?1 astaxanthin, the highest volumetric astaxanthin yield ever reported for this alga. In addition, the astaxanthin production by E17 was tested with a semi-continuous culture method, where the directly diluted raw molasses (giving 5 g L^?1 sugar) was used as the carbon source. Little growth inhibition of E17 was observed in the semi-continuous culture with a biomass productivity of 1.33 g L^?1 day^?1 and an astaxanthin productivity of 0.83 mg L^?1 day^?1. The mixotrophic semi-continuous cultures enhanced the biomass and astaxanthin productivities by 29.3 % and 42.2 %, respectively. This study highlights the potential of using the industrially cheap cane molasses towards large-scale cost-saving production of the high-value ketocarotenoid astaxanthin.     

Enhanced riboflavin production by recombinant Bacillus subtilis RF1 through the optimization of agitation speed

Dissolved oxygen is one of the most important bioprocess parameters that could affect cell growth and product formation, and it is easy to control by changing agitation speed. In this work, the effects of agitation speed on the performance of riboflavin production by recombinant Bacillus subtilis RF1 was investigated in fed-batch fermentation. The lower agitation speed (600 rpm) was beneficial for cell growth and riboflavin biosynthesis in the initial phase of fermentation process. While, during the later phase, higher agitation speed (900 rpm) was favor for cell growth and riboflavin biosynthesis. Thus, a two-stage agitation speed control strategy was proposed based on kinetic analysis, in which the agitation speed was controlled at 600 rpm in the first 26 h and then switched to 900 rpm to maintain high μ for cell growth and high q _p for riboflavin production during the entire fermentation process. However, it was observed that a sharp increase of agitation speed resulted in an adverse effect on cell growth and riboflavin synthesis within a short time. To avoid this phenomenon, a multi-stage agitation speed control strategy was set up based on the two-stage control strategy, the maximum concentration of riboflavin reached 9.4 g l^?1 in 48 h with the yield of 0.051 g g^?1 by applying this strategy, which were 20.5 and 21.4 % over the best results controlled by constant agitation speeds.     

Transient responses of <Emphasis Type="Italic">Wickerhamia</Emphasis> sp. yeast continuous cultures to qualitative changes in carbon source supply: induction and catabolite repression of α-amylase synthesis

The purpose of this study was to evaluate the inductive effect of starch and maltose, and the repressive/inhibitory effect of glucose, on amy-1 gene expression and α-amylase production by Wickerhamia sp., using continuous culture under transient-state conditions at a dilution rate (D) of 0.083 h^?1. Induction and repression kinetics of α-amylase were studied by changing the medium feed from glucose to maltose or starch in the induction experiments and vice versa in the repression experiments. Expression levels of amy-1 gene were measured by RT-qPCR. Results showed that starch was a more efficient inducer of α-amylase synthesis compared to maltose, with maximum accumulation rate constants of 0.424 and 0.191 h^?1, respectively. In contrast, α-amylase synthesis in starch and maltose cultures was partially repressed by glucose as indicated by a specific activity close to basal levels and a decay constant rate (??0.065 and ??0.069 h^?1, respectively) higher than ??D. A linear dependence of the specific rate of α-amylase production on mRNA relative abundance of amy-1 gene was observed. An inhibitory effect of glucose was not observed even at a concentration of 30 g L^?1. In conclusion, the transient continuous culture is a useful tool to determine the qualitative and quantitative effects of maltose and starch on α-amylase induction and of glucose on enzyme repression, as well as to obtain a detailed understanding of the dynamic behavior of the yeast culture. Furthermore, results showed that amylaceous substrates can be very effective carbon sources for the production of α-amylase without being inhibited by glucose.     

Batch, fed-batch and repeated fed-batch fermentation processes of the marine thraustochytrid Schizochytrium sp. for producing docosahexaenoic acid

Different fermentation processes, including batch, fed-batch and repeated fed-batch processes by Schizochytrium sp., were studied and compared for the effective DHA-rich microbial lipids production. The comparison between different fermentation processes showed that fed-batch process was a more efficient cultivation strategy than the batch process. Among the four different feeding strategies, the glucose concentration feed-back feeding strategy had achieved the highest fermentation results of final cell dry weight, total lipids content, DHA content and DHA productivity of 72.37, 48.86, 18.38 g l^?1 and 138.8 mg l^?1 h^?1, respectively. The repeated fed-batch process had the advantages of reducing the time and cost for seed culture and inoculation between each fermentation cycles. The results of fermentation characteristics and lipid characterization of the repeated fed-batch process indicated that this repeated fed-batch process had promising industrialization prospect for the production of DHA-rich microbial lipids.     

Poly(β-hydroxybutyric acid) thermoplastic production by Alcaligenes latus: Behavior of fed-batch cultures

Fed-batch culture of Alcaligenes latus, ATCC 29713, was investigated for producing the intracellular bioplastic poly(β–hydroxybutyric acid), PHB. Constant rate feeding, exponentially increasing feeding rate, and pH-stat fed batch methods were evaluated. pH-stat fed batch culture reduced or delayed accumulation of the substrate in the broth and led to significantly enhanced PHB productivity relative to the other modes of feeding. Presence of excessive substrate appeared to inhibit PHB synthesis, but not the production of cells. In fed-batch culture, the maximum specific growth rate (0.265?h^?1) greatly exceeded the value (0.075?h^?1) previously observed in batch culture of the same strain. Similarly, the maximum PHB production rate (up to 1.15?g?·?l^?1?·?h^?1) was nearly 8-fold greater than values observed in batch operations. Fed-batch operation was clearly superior to batch fermentation for producing PHB. A low growth rate was not a prerequisite for PHB accumulation, but a reduced or delayed accumulation of substrate appeared to enhance PHB accumulation. Under the best conditions, PHB constituted up to 63% of dry cell mass after 12?h of culture. The average biomass yield coefficient on sucrose was about 0.35, or a little less than in batch fermentations. The highest PHB concentrations attained were about 18?g?·?l^?1.     

Nutrient modulation based process engineering strategy for improved butanol production from Clostridium acetobutylicum

The present study demonstrates a process engineering strategy to achieve high butanol titer and productivity from wild type Clostridium acetobutylicum MTCC 11274. In the first step, two different media were optimized with the objectives of maximizing the biomass and butanol productivity, respectively. In the next step, attributes of these two media compositions were integrated to design a two-stage fed-batch process which resulted in maximal butanol productivity of 0.55 g L⁻¹ h⁻¹ with titer of 13.1 g L⁻¹. Further, two-stage fed-batch process along with combinatorial use of magnesium limitation and calcium supplementation resulted in the highest butanol titer and productivity of 16.5 g L⁻¹ and 0.59 g L⁻¹ h⁻¹, respectively. Finally, integration of the process with gas stripping and modulation of feeding duration resulted in a cumulative butanol titer of 54.3 g L⁻¹ and productivity of 0.58 g L⁻¹ h⁻¹. The strategy opens up possibility of developing a viable butanol bioprocess. © 2019 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2771, 2019.     

Cytokinin Reversal of Abscisic Acid Inhibition of Growth and α-Amylase Synthesis in Barley Seed

The effect of cytokinin, kinetin, on abscisic acid (dormin) inhibition of α-amylase synthesis and growth in intact barley seed was investigated. Abscisic acid at 5 × 10^?5M nearly completely inhibited growth response and α-amylase synthesis in barley seed. Kinetin reversed to a large extent abscisic acid inhibition of α-aniylase synthesis and coleoptile growth. The response curves of α-amylase synthesis and coleoptile growth in presence of a fixed amount of abscisic acid (6 × l0^?6M) and increasing concentrations of kinetin (from 5 × l0^?7M to 5 × 10^?5 M) showed remarkable similarity. Kinetin and abscisic acid caused synergistic inhibition of root growth. Gibberellic acid was far less effective than kinetin in reversing abscisic acid inhibition of α-amylase synthesis and coleoptile growth. A combination of kinetin and gibberellic acid caused nearly complete reversal of abscisic acid inhibition of α-amylase synthesis but not the abscisic acid inhibition of growth. The results suggest that factors controlling α-amylase synthesis may not have a dominant role in all growth responses of the seed. Kinetin possibly acts by removing the abscisic acid inhibition of enzyme specific sites thereby allowing gibberellic acid to function to produce α-amylase.     

Integrated production of lactic acid and biomass on distillery stillage

The possibilities of parallel lactic acid and biomass production in batch and fed-batch fermentation on distillery stillage from bioethanol production were studied. The highest lactic acid yield and productivity of 92.3 % and 1.49 g L^?1 h^?1 were achieved in batch fermentation with initial sugar concentration of 55 g L^?1. A significant improvement of the process was achieved in fed-batch fermentation where the concentration of lactic acid was increased to 47.6 % and volumetric productivity for 21 % over the batch process. A high number of Lactobacillus rhamnosus ATCC 7469 viable cells of 10⁹ CFU ml^?1 was attained at the end of fed-batch fermentation. The survival of 92.9 % of L. rhamnosus cells after 3 h of incubation at pH 2.5 validated that the fermentation media remained after lactic acid removal could be used as a biomass-enriched animal feed thus making an additional value to the process.     

Production of recombinant beta-amylase of Bacillus aryabhattai

In this study, the effects of carbon source, nitrogen source, and metal ions on cell growth and Bacillus aryabhattai β-amylase production in recombinant Brevibacillus choshinensis were investigated. The optimal medium for β-amylase production, containing glucose (7.5?g·L^?1), pig bone peptone (40.0?g·L^?1), Mg²⁺ (0.05?mol·L^?1), and trace metal elements, was determined through single-factor experiments in shake flasks. When cultured in the optimized medium, the β-amylase yield reached 925.4?U mL^?1, which was 7.2-fold higher than that obtained in the initial medium. Besides, a modified feeding strategy was proposed and applied in a 3-L fermentor fed with glucose, which achieved a dry cell weight of 15.4?g L^?1. Through this cultivation approached 30?°C with 0?g·L^?1 initial glucose concentration, the maximum β-amylase activity reached 5371.8?U mL^?1, which was 41.7-fold higher than that obtained with the initial medium in shake flask.     

Effect of adrenergic agents on α-amylase release and adenosine 3′,5′-monophosphate accumulation in rat parotid tissue slices

The role of cyclic AMP in stimulus-secretion coupling was investigated in rat parotid tissue slices in vitro. Isoproterenol and norepinephrine stimulated a rapid intracellular accumulation of cyclic AMP, which reached a maximum level of 20–30 times the control value by 5 to 10 min after addition of the drug. Isoproterenol was approximately ten times more potent in stimulating both α-amylase release and cyclic AMP accumulation than were norepinephrine and epinephrine, which had nearly equal effects on these two parameters. Salbutamol and phenylephrine were less effective. A parallel order of potency and sensitivity was observed for the stimulation of adenylate cyclase activity in a washed particulate fraction. The results suggest that these drugs are acting on the parotid acinar cell through a β₁-adrenergic mechanism.At the lowest concentrations tested, each of the adrenergic agonists stimulated significant α-amylase release with no detectable stimulation of cyclic AMP accumulation. Even in the presence of theophylline, phenylephrine at several concentrations increased α-amylase release without a detectable increase in cyclic AMP levels. However, phenylephrine did stimulate adenylate cyclase. These data suggest that, under certain conditions, large increases in the intracellular concentration of cyclic AMP may not be necessary for stimulation of α-amylase release by adrenergic agonists. Also consistent with this idea was the observation that stimulation of cyclic AMP accumulation by isoproterenol was much more sensitive to inhibition by propranolol than was the stimulation of α-amylase release by isoproterenol.Stimulation of α-amylase release by phenylephrine was only partially blocked by either α- or β-adrenerg blocking agents, whereas stimulation of adenylate cyclase by phenylephrine was blocked by propranolol and not by phentolamine. Phenoxybenzamine and phentolamine potentiated the effects of norepinephrine and isoproterenol on both cyclic AMP accumulation and α-amylase release. However, phenoxybenzamine also potentiated the stimulation of α-amylase release by N⁶,O^2′-dibutyryl adenosine 3′,5′-monophosphate. These observations may indicate a non-specific action of phenoxybenzamine, and demonstrate the need for caution in interpreting evidence obtained using α-adrenergic blocking agents as tools for investigation of α- and β-adrenergic antagonism.