Optimization of fusion proinsulin production by high cell-density fermentation of recombinantE. coli

The optimum conditions for mass production of fusion proinsulin were studied in recombinantEscherichia coli strain BL21 (DE3) [pT7-PI] using fed-batch culture employing pH-stat method. Yeast extract was found to enhance both the growth rate of recombinantE. coli strain BL21 (DE3) [pT7-PI] and its cell mass yield. When the glucose concentration was 10 g/L in the initial medium, 10 g/L concentration of yeast extract was found to be optimal to control the acetate production and to augment both the cell mass yield and the growth rate. Optimum ratio of glucose to yeast extract to minimize the cost of the feeding medium in the fed-batch culture was calculated to be 1.225 and verified by the subsequent experiments. The appropriate inducer concentration and induction time were examined with isopropyl-β-D-thiogalactopyranoside (IPTG). Irrespective of the induction time, IPTG induction resulted in the reduction of growth rate, but the expression level of the fusion protein was maintained at the level of about 20% of the total proteins. Since the volumetric productivity was well maintained in the range between 0.15 and 0.18 g/L.hr at the inducer concentration of above 0.025 mM, the appropriate inducer concentration, in relation to the inducer cost, is considered to be about 0.025 mM.     

Combined use of growth rate correlated and growth rate independent promoters for recombinant glucoamylase production in Fusarium venenatum

Fusarium venenatum JeRS 325, a strain which produces recombinant glucoamylase under control of a growth rate independent promoter was transformed with a plasmid carrying the Aspergillus niger glucoamylase gene under control of its own growth rate correlated promoter. Some disruption of the original recombinant genes occurred and at pH 5.8 the double transformant did not produce as much glucoamylase as JeRS 325 in batch culture. However, the double transformant still produced as much glucoamylase as JeRS 325 in fed-batch cultures, illustrating the potential for the combined use of growth rate independent and dependent promoters to improve production of recombinant proteins in fed-batch culture systems.     

Production of antithrombotic hirudin in <Emphasis Type="Italic">GAL1</Emphasis>-disrupted <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A gratuitous strain was developed by disrupting the GAL1 gene (galactokinase) of recombinant Saccharomyces cerevisiae harboring the antithrombotic hirudin gene in the chromosome under the control of the GAL10 promoter. A series of glucose-limited fed-batch cultures were carried out to examine the effects of glucose supply on hirudin expression in the gratuitous strain. Controlled feeding of glucose successfully supported both cell growth and hirudin expression in the gratuitous strain. The optimum fed-batch culture done by feeding glucose at a rate of 0.3 g h^–1 produced a maximum hirudin concentration of 62.1 mg l^–1, which corresponded to a 4.5-fold increase when compared with a simple batch culture done with the same strain.     

Fuzzy neural network for the control of high cell density cultivation of recombinant Escherichia coli

A five-layer fuzzy neural network (FNN) was developed for the control of fed-batch cultivation of recombinant Escherichia coli JM103 harboring plasmid pUR 2921. The FNN was believed to represent the membership functions of the fuzzy subsets and to implement fuzzy inference using previous experimental data. This FNN was then used for compensating the exponential feeding rate determined by the feedforward control element. The control system is therefore a feedforward-feedback type. The change in pH of the culture broth and the specific growth rate were used as the inputs to FNN to calculate the glucose feeding rate. A cell density of 84 g DWC/l in the fed-batch cultivation of the recombinant E. coli was obtained with this control strategy. Two different FNNs were then employed before and after induction to enhance plasmid-encoded β-galactosidase production. Before induction the specific growth rate was set as 0.31 h⁻¹, while it was changed to 0.1 h⁻¹ after induction. Compared to when only one FNN was used, the residual glucose concentration could be tightly controlled at an appropriate level by employing two FNNs, resulting in an increase in relative activity of β-galactosidase which was about four times greater. The present investigation demonstrates that a feedforward-feedback control strategy with FNN is a promising control strategy for the control of high cell density cultivation and high expression of a target gene in fed-batch cultivation of a recombinant strain.     

Disruption of hexokinase II (HXK2) partly relieves glucose repression to enhance production of human kringle fragment in gratuitous recombinant Saccharomyces cerevisiae

The GAL1 gene encoding galactokinase was disrupted in a recombinant Saccharomyces cerevisiae strain in which production of LK8 protein, a kringle fragment of human apolipoprotein, is under the control of GAL1 promoter. Null mutation of the HXK2 gene was introduced further in the gal1Delta strain to relieve glucose repression. A pattern for LK8 expression was compared for the two recombinant S. cerevisiae systems in continuous and fed-batch cultivations. A critical dilution rate in continuous cultivation that repressed LK8 expression was significantly higher for the gal1Deltahxk2Delta strain than that for the gal1Delta strain to sustain the LK8 production even at high glucose consumption rate. Expressed LK8 for the gal1Delta strain was not detectable when the dilution rate exceeded 0.05 h(-1). Maximum LK8 concentration of 57 mgl(-1) was obtained in glucose-limited fed-batch cultivation of the gal1Deltahxk2Delta strain, corresponding to a 13.8-fold enhancement compared with the gal1Delta strain grown under the same conditions.     

Physiological and genetic strategies for enhanced subtilisin production by Bacillus subtilis.

Defined minimal media conditions were used to assess and subsequently enhance the production of subtilisin by genetically characterized Bacillus subtilis strains. Subtilisin production was initiated by the exhaustion or limitation of ammonium in batch and fed-batch cultures. Expression of the subtilisin gene (aprE) was monitored with a chromosomal aprE::lacZ gene fusion. The beta-galactosidase production driven by this fusion reflected subtilisin accumulation in the culture medium. Subtilisin gene expression was temporally extended in sporulation-deficient strains (spoIIG), relative to co-genic sporogenous strains, resulting in enhanced subtilisin production. Ammonium exhaustion not only triggered subtilisin production in asporogenous spoIIG mutants but also shifted carbon metabolism from acetate production to acetate uptake and resulted in the formation of multiple septa in a significant fraction of the cell population. Fed-batch culture techniques, employing the spoIIG strain, were investigated as a means to further extend subtilisin production. The constant provision of ammonium resulted in linear growth, with doubling times of 11 and 36 h in each of two independent experiments. At the lower growth rate, the responses elicited (subtilisin production, glucose metabolism, and morphological changes) during the feeding regime closely approximated the ammonium starvation response, while at the higher growth rate a partial starvation response was observed.     

Development of high cell density cultures of engineeredSaccharomyces cerevisiae cells able to grow on lactose

Saccharomyces cerevisiae cells transformed with a multicopy expression vector bearing an in frameSTA2-LacZ gene fusion under the control of the galactose inducibleUAS _GAL/CYC1 promoter, secrete -galactosidase in the periplasmic space. Fermentation studies showed that with this transformed strain it is possible to reach on lactose high cell densities with both high productivity and high growth yield by means of fed-batch fermentations.     

Novel approach of high cell density recombinant bioprocess development: Optimisation and scale-up from microlitre to pilot scales while maintaining the fed-batch cultivation mode of E. coli cultures

Background

Bioprocess development of recombinant proteins is time consuming and laborious as many factors influence the accumulation of the product in the soluble and active form. Currently, in most cases the developmental line is characterised by a screening stage which is performed under batch conditions followed by the development of the fed-batch process. Performing the screening already under fed-batch conditions would limit the amount of work and guarantee that the selected favoured conditions also work in the production scale.

Results

Here, for the first time, high throughput multifactorial screening of a cloning library is combined with the fed-batch technique in 96-well plates, and a strategy is directly derived for scaling to bioreactor scale. At the example of a difficult to express protein, an RNase inhibitor, it is demonstrated that screening of various vector constructs and growth conditions can be performed in a coherent line by (i) applying a vector library with promoters and ribosome binding sites of different strength and various fusion partners together with (ii) an early stage use of the fed-batch technology. It is shown that the EnBase^® technology provides an easy solution for controlled cultivation conditions in the microwell scale. Additionally the high cell densities obtained provide material for various analyses from the small culture volumes. Crucial factors for a high yield of the target protein in the actual case were (i) the fusion partner, (ii) the use of of a mineral salt medium together with the fed-batch technique, and (iii) the preinduction growth rate. Finally, it is shown that the favorable conditions selected in the microwell plate and shake flask scales also work in the bioreactor.

Conclusions

Cultivation media and culture conditions have a major impact on the success of a screening procedure. Therefore the application of controlled cultivation conditions is pivotal. The consequent use of fed-batch conditons from the first screening phase not only shortens the developmental line by guarantying that the selected conditions are relevant for the scale up, but in our case also standard batch cultures failed to select the right clone or conditions at all.     

Fermentation strategies for 1,3-propanediol production from glycerol using a genetically engineered Klebsiella pneumoniae strain to eliminate by-product formation

We generated a genetically engineered Klebsiella pneumoniae strain (AK-VOT) to eliminate by-product formation during production of 1,3-propanediol (1,3-PD) from glycerol. In the present study, the glycerol-metabolizing properties of the recombinant strain were examined during fermentation in a 5 L bioreactor. As expected, by-product formation was completely absent (except for acetate) when the AK-VOT strain fermented glycerol. However, 1,3-PD productivity was severely reduced owing to a delay in cell growth attributable to a low rate of glycerol consumption. This problem was solved by establishing a two-stage process separating cell growth from 1,3-PD production. In addition, nutrient co-supplementation, especially with starch, significantly increased 1,3-PD production from glycerol during fed-batch fermentation by AK-VOT in the absence of by-product formation.     

Variability of the response of Saccharomyces cerevisiae strains to lignocellulose hydrolysate

The development of tolerant microorganisms is needed for the efficient fermentation of inhibitory lignocellulose hydrolysates. In the current work, the fermentation performance of six selected strains of Saccharomyces cerevisiae in dilute-acid spruce hydrolysate was compared using two different modes of fermentation; either single pulse addition of hydrolysate to exponentially growing cells or continuous feeding of the same amount of hydrolysate in a controlled fed-batch fermentation was made. All strains performed better in fed-batch mode than when all hydrolysate was added at once. However, the difference between strain performances varied significantly in the two fermentation modes. Large differences were observed between strains during the fed-batch experiments in the in vitro ability to reduce the furan compounds furfural and 5-hydroxymethyl furfural (HMF). A common feature among the strains was the induction of NADPH-coupled reduction of furfural and HMF, with the exception of strain CBS 8066. This strain also performed relatively poorly in both batch and fed-batch fermentations. Strain TMB3000--previously isolated from spent sulphite liquor fermentation--was by far the most efficient strain with respect to specific fermentation rate in both pulse addition and fed-batch mode. This strain was the only strain showing a significant constitutive NADH-coupled in vitro reduction of HMF. The ability to induce NADPH-coupled reduction together with the level of the apparently constitutive NADH-coupled reduction appeared to be key factors for selecting a suitable strain for fed-batch conversion of lignocellulose hydrolysate.     

Mixed feeds of glycerol and methanol can improve the performance of Pichia pastoris cultures: A quantitative study based on concentration gradients in transient continuous cultures

Transient continuous cultures constitute a means to speed up strain characterization, by avoiding the need for many time-consuming steady-state experiments. In this study, mixed substrate growth on glycerol and methanol of a Pichia pastoris strain expressing and secreting recombinant avidin was characterized quantitatively by performing a nutrient gradient with linear increase of the methanol fraction in the feed medium from 0.5 to 0.93 C-mol C-mol(-1) at a dilution rate of 0.06 h(-1). The influence of the methanol fraction in the feed medium on recombinant avidin productivity and on specific alcohol oxidase activity were also examined. Results showed that, compared with cultures on methanol as sole carbon source, the specific recombinant avidin production rate was the same provided the methanol fraction in the feed medium was higher than 0.6 C-mol C-mol(-1). The volumetric avidin production rate was even 1.1-fold higher with a methanol fraction in the feed medium of 0.62 C-mol C-mol(-1) as a result of the higher biomass yield on mixed substrate growth compared with methanol alone. Moreover, since heat production and oxygen uptake rates are lower during mixed substrate growth on glycerol and methanol, mixed substrate cultures present technical advantages for the performance of high cell density P. pastoris cultures. Results obtained in a high cell density fed-batch culture with a mixed feed of 0.65 C-mol C-mol(-1) methanol and 0.35 C-mol C-mol(-1) glycerol were in agreement with results obtained during the transient nutrient gradient.     

Enhancement of Candida rugosa lipase production by using different control fed-batch operational strategies

Simulation studies have predicted that maximum lipase activity is reached with fed-batch operation strategies. In this work, two different fed-batch operational strategies have been studied: constant substrate feeding rate and specific growth rate control. A constant substrate feeding rate strategy showed that maximum aqueous lipolytic activity (55 U/mL) was reached at low substrate feeding rates, whereas lipase tends to accumulate inside the cell at higher rates of substrate addition. In the second fed-batch strategy studied, a feedback control strategy has been developed based on the estimation of state variables (X and mu) from the measurement of indirect variables such as CER by means of mass spectrometry techniques. An on-off controller was then used to maintain the specific growth rate at the desired value by adjusting the substrate feeding rate. A constant specific growth rate strategy gave higher final levels of aqueous lipolytic activity (117 U/mL) at low specific growth rates. At higher specific growth rates the enzyme remained accumulated inside the cell, as was observed with a constant feeding fed-batch strategy. With a constant specific growth rate strategy, lipase production by Candida rugosa was enhanced 10-fold compared to a batch operation. Purification studies have demonstrated that lipolytic and esterasic specific activity ratios of Candida rugosa isoenzymes can be modified by using different operational conditions. These studies have also showed that the isoenzymes obtained in a controlled growth rate strategy are around three- to four-fold more active than those obtained in a constant feeding rate strategy.     

Performance of the auxotrophic Saccharomyces cerevisiae BY4741 as host for the production of IL-1β in aerated fed-batch reactor: role of ACA supplementation, strain viability, and maintenance energy

Background  

Saccharomyces cerevisiae BY4741 is an auxotrophic commonly used strain. In this work it has been used as host for the expression and secretion of human interleukin-1β (IL1β), using the cell wall protein Pir4 as fusion partner. To achieve high cell density and, consequently, high product yield, BY4741 [PIR4-IL1β] was cultured in an aerated fed-batch reactor, using a defined mineral medium supplemented with casamino acids as ACA (auxotrophy-complementing amino acid) source. Also the S. cerevisiae mutant BY4741 Δyca1 [PIR4-IL1β], carrying the deletion of the YCA1 gene coding for a caspase-like protein involved in the apoptotic response, was cultured in aerated fed-batch reactor and compared to the parental strain, to test the effect of this mutation on strain robustness. Viability of the producer strains was examined during the runs and a mathematical model, which took into consideration the viable biomass present in the reactor and the glucose consumption for both growth and maintenance, was developed to describe and explain the time-course evolution of the process for both, the BY4741 parental and the BY4741 Δyca1 mutant strain.     

Fed-batch xylitol production with two recombinant Saccharomyces cerevisiae strains expressing XYL1 at different levels, using glucose as a cosubstrate: a comparison of production parameters and strain stability

Xylitol production with two recombinant Sacharomyces cerevisiae strains expressing the XYL1 gene, coding for xylose reductase (XR), at different levels, the 'low XR strain' at 0.51 U/mg and the 'high XR strain' at 10.8 U/mg, was compared in batch and fed-batch culture. Xylose was not consumed in the presence of high glucose concentrations, because both sugars are transported by the glucose transport system, which has a higher affinity for glucose than for xylose. When glucose was fed gradually to the culture, high concentrations were avoided, and xylose was converted to xylitol with a specific productivity of 0.10 g g(-1) h(-1) attained with the low XR strain and 0.19 g g(-1) h(-1) with the high XR strain, indicating that factors other than the XR-activity control the rate of xylose conversion.The overproduction of XR put a substantial protein burden on the high XR strain, contributing to a 50% decrease in specific growth rate and reduced biomass yield compared with the low XR strain. Despite the use of selective medium, the stability of the high XR strain was poor in long fed-batch and chemostat cultures, whereas the low XR strain was stable. The high XR strain lost its XR activity almost completely in some fed-batch cultures and in chemostat culture. In chemostat cultivation, part of the population lost the plasmid harboring the XR gene. This was due to the fact that leucine was released into the broth from plasmid containing cells, which enabled some cells to grow without the plasmid containing the LEU2 auxotrophic complementation selection marker. Furthermore, isolation and analysis of plasmids from a population that had lost its XR activity, showed that in addition to the original plasmid, a rearranged form of the plasmid, retaining the selection marker but not the expression of active XR, was present. However, these observations could only partly explain the decrease in XR activity. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 391-399, 1997.     

Production of toluene cis-glycol using recombinant Escherichia coli strains in glucose-limited fed batch culture

Production of toluene cis-glycol (TCG) was investigated using recombinant Escherichia coli strains that express toluene dioxygenase under the tac promoter. E. coli TG2 was selected as the host for the recombinant plasmid, pTAC365, because the TCG yield was 64% higher than with strain JM105 as the host. By using fed-batch culture, TCG production could be improved by 4.4-fold compared with batch cultures, with a toluene vapor feed. A further improvement of 1.2-fold was obtained by using a two-liquid phase culture system, but the improvement was 1.6-fold when the feed rate of toluene vapor was reduced by 75%. When the period of growth in fed-batch culture was extended and the reduced vapor feed was used, the maximum TCG concentration increased to 4 g/l, an overall improvement of 10-fold compared with the batch culture system used initially. However, product consumption was observed during the late stages of fed-batch growth and in stationary phase, resulting in the formation of o-cresol, 3-methylcatechol and benzyl alcohol. We conclude that the key to optimizing TCG production by recombinants is to prolong growth of the cells to ensure sustained growth-linked product formation, and to optimize the supply of the toxic substrate, toluene, but further work is needed to eliminate by-product formation.     

过量表达自身hemA基因的重组大肠杆菌发酵生产5-氨基乙酰丙酸的研究

研究了优化重组大肠杆菌产5-氨基乙酰丙酸（ALA）的条件,提高大肠杆菌发酵生产AL气的产量。在测定重组大肠杆菌GT48的生长曲线的基础上,确定诱导时间,优化摇瓶发酵条件。然后,进一步在5L发酵罐上进行间歇和流加发酵研究。摇瓶实验表明,细胞培养最佳初始pH为6．5,最佳诱导时间为稳定期前期,最佳接种量为2％,过高的葡萄糖浓度对细胞生长和产物合成均有一定的抑制作用。在5L发酵罐间歇发酵中,重组菌产ALA能力达到47．8mg／L。采用流加发酵可以进一步将产物产量提高到63．8mg／L。构建的过量表达自身的hemA基因的大肠杆菌具有较高的产ALA能力,通过发酵条件优化和采用流加发酵可以提高AL气产量。