Strategies for improving plasmid stability in genetically modified bacteria in bioreactors.

Exploitation of recombinant organisms for the large-scale, commercial production of foreign proteins is often hampered by the problem of plasmid instability. A wide range of strategies have been reported for improving the stability of recombinant organisms. A combination of manipulating both the genetic design of recombinants and the conditions of culturing the organisms may be used to achieve stable host-vector associations during culture of recombinant organisms in bioreactors.

Integrated processing of biotechnology products.

Integrated bioprocessing in which a potentially inhibitory product is continuously removed from the fermentation broth as it is produced, has important advantages in improving yield and conversion relative to conventional processes. This review discusses integrated processing for ethanol, butanol, organic acids, antibiotics, and other products. A variety of recovery operations can be used to isolate the product, as discussed. Use of some of the available options is compared.

Liquid surfactant membrane emulsions. A new technique for enzyme immobilization.

Liquid membrane reactors are well known for metal extraction. This technology may also be applied to the immobilization of enzymes in enzyme emulsions. The use of liquid membrane reactors for enzymatic bioconversions has several advantages in comparison to solid membrane reactors and conventional immobilization techniques: there is no membrane fouling, enzyme emulsions can be used in cell-free fermentation broths, in complex mixtures the membrane can preselect the desired substrate for enzymatic reaction, and substances that might decrease the enzyme activity can be excluded. The separation effect is not based on differences in molecular weight but on the chemical behavior of the substances to be separated. Thus, it is not necessary to use cofactors with increased molecular weight for enzymatic reactions, since the coenzyme cannot permeate the liquid membrane. The three systems presented here indicate that enzyme systems can be easily immobilized in liquid surfactant membrane emulsions and there is a broad field of application for enzyme emulsions.

Comparison of the performances of stirred tank and airlift tower loop reactors.

Following a consideration of the prerequisites for reactor comparison and the fundamental differences between stirred tank and airlift tower loop reactors, their performances are compared for the production of secondary metabolites: penicillin V by Penicillium chrysogenum, cephalosporin C by Cephalosporium acremonium, and tetracycline by Streptomyces aureofaciens. In stirred tank reactors, cell mass concentrations, volumetric productivities, and specific power inputs are higher than in airlift tower loop reactors. In the latter, efficiencies of oxygen transfer are higher, and specific productivities with regard to power input, substrate and oxygen consumptions, and yield coefficients of product formation with regard to substrate and oxygen consumptions are considerably higher than in stirred tank reactors. The prerequisites for improved performance are discussed.

Progress in monitoring, modeling and control of bioprocesses during the last 20 years.

The paper gives a review on the recent development of bioprocess engineering. It includes monitoring of product formation processes by flow injection analysis, various types of chromatographic and spectroscopic methods as well as by biosensors. The evaluation of mycelial morphology and physiology by digital image analysis is discussed also. It deals with advanced control of indirectly evaluated process variables by means of state estimation/observer, with the use of structured and hybrid models, expert systems and pattern recognition for process optimization and gives a short report on the state of the art of metabolic flux analysis and metabolic engineering.

Immobilization of genetically engineered cells: a new strategy for higher stability.

The r-DNA clones improve the bioprocess and provide better economics, if and when properly developed. In recent times, many approaches were made to improve the stability of recombinants in a reactor which includes both genetic and environmental methods, but many of them were proved to be unsuccessful in the scale-up process. The immobilization technique, exploited recently for the cultivation of recombinants, in many cases gave high cell concentrations, better expression of cloned gene products and also maintained plasmid stability for longer periods in a host under continuous operation in comparison to a free cell system. Many plasmids and hosts were tested for improved stabilities. So far, no explanation was provided for higher stability in the immobilized system. However, it was observed to reduce the competition between the plasmid harboring and plasmid free cells in a matrix. The stability of recombinant strains under immobilization technique is affected by various factors, and these are important parameters for the commercial process. Thus, the immobilization system is promising for the successful cultivation and scale-up of genetically engineered cells.

An integrated process for the production and biotransformation of penicillin.

The extraction of Penicillin G (PG) from the filtered cultivation medium of Penicillium chrysogenum and its conversion into 6-amino penicillanic acid (6-APA) and phenyl acetic acid (PhA) at pH 8 was performed in a 10 l kühni extractor during the production by means of penicillin-G-amidase immobilized in a liquid membrane carrier system (LM). 6-APA was enriched in LM, and the PhA returned to the cultivation medium. After electrocoalescence of LM, the 6-APA was converted into ampicillin with the same enzyme at pH 6, while the liquid membrane phase and enzyme were recycled and reused.

Transient behaviour of baker's yeast during enforced periodical variation of dissolved oxygen concentration.

The aim of the investigation was to find out the influence of the variation of the dissolved oxygen concentration in the microenvironment of yeast cells on their physiological behaviour in small laboratory reactors and estimate their behaviour in large industrial reactors. Since the morphology of the laboratory and industrial yeasts differed considerably, their transient behaviour was investigated and compared. For this purpose, the strain Saccharomyces cerevisiae H620 and an industrial strain were cultivated on synthetic as well as on complex medium in batch operation during periodical variation of the dissolved oxygen concentration, monitoring the most important key parameters. Also the yeast was cultivated in batch as well as in continuous operation, the cell-containing culture medium was recirculated through a nonaerated loop at different recirculation rates (residence times of the cells in the loop), and the key operation variables were monitored. It was found that the transient behaviour of laboratory and industrial yeasts differed slightly. Since cells growing in batch culture are more sensitive to dissolved oxygen concentration variation than cells growing in continuous culture, the transient behavior of cells cultivated in batch operation varied from those in continuous operation. If the anaerobic phase was longer than 1 min, ethanol was produced. However, it was consumed during the aerobiosis again, provided that phase was considerably longer than the anaerobic phase. This means that the yeast cultivation was not influenced by the periodic operation of the dissolved oxygen. Judging from the measurements in large stirred tank and airlift tower loop reactors, in general, the cells would spend more time in the aerobic than in the anaerobic flow region, and they would spend less than 1 min in the anaerobic flow region. Therefore, no considerable effect of the periodically varied dissolved oxygen concentration on the cell cultivation can be expected in large-scale reactors. In the stirred tank-loop-system at high pumping rates/high frequencies of periodically varied dissolved oxygen concentration, unexpectedly, the formation of ethanol was observed, which might be caused by stress imposed on the cells.

The inhibitory mechanisms of glucose and carbon dioxide on the biosyntheses of penicillins and cephalosporins.

The inhibitory mechanism of glucose and CO2 on the biosyntheses of penicillins and cephalosporins is discussed in the present paper. 6-aminopenicillanic acid (6-APA) is considered to be an intermediate product, and the reaction between 6-APA and glucose may play an important role in the yield and rate of biosyntheses of beta-lactam antibiotics. According to this hypothesis the experimental phenomena taking place in biosynthesis of penicillin and cephalosporin, such as the inhibition by glucose and carbon dioxide and the reduction of the yield, can be satisfactorily explained. The stability of 6-aminopenicillanic acid (6-APA) in bicarbonate solution, the reaction of 6-APA with sugars, the determination of the concentration of the 6-APA-sugar compound and the effect of these reactions on the biosynthesis of penicillin G are investigated to present evidences for this hypothesis.

Influence of medium composition on the cephalosporin C production with a highly productive strain Cephalosporium acremonium.

Cephalosporin production by a highly productive Cephalosporium acremonium strain was carried out and optimized by fed-batch operation in a 40 l stirred tank reactor using a complex medium containing 30-120 g l-1 peanut flour. The concentrations of cephalosporin C (CPC) and its precursors: penicillin N (PEN N), deacetoxy cephalosporin C (DAOC), and deacetyl cephalosporin C (DAC) were monitored with an on-line HPLC. The concentrations of amino acids valine (VAL), cysteine (CYS), alpha-amino adipic acid (alpha-AAA), the dipeptide alpha-amino-adipyl-cysteine (AC), and the tripeptide alpha-amino-adipyl-cysteinyl-valine (ACV), were determined off-line by HPLC. The RNA content and dry weight of the sediment as well as the oxygen transfer rate (OTR) and the CO2 production rate (CPR) were used to calculate the cell mass concentration (X). The influences of peanut flour (PF) and the on-line monitored and controlled medium components: glucose (GLU), phosphate, methionine (MET) as well as the dissolved oxygen (DOC) on the cell growth, the product formation, and the pathway of cephalosporin C biosynthesis were investigated and evaluated. When the glucose fed-batch cycle was optimized and oxygen transfer limitation was avoided (DOC greater than 20% of the saturation value), high process performance (103.5 g l-1 X, 11.84 g l-1 CPC, a maximum CPC productivity of 118 mg l-1 h-1, and the whole concentration of the beta-lactam antibiotics CPC, DAC, DAOC, PEN N 17.34 g l-1) was achieved by using 100 g l-1 PF in the medium with the optimum concentration of phosphate (260-270 mg l-1) and a low glucose concentration (less than 0.5 g l-1). The cultivations with different medium concentrations demonstrated that the product formation was directly proportional to the cell mass concentration. On the average, the cell mass-based yield coefficient of CPC: YCPC/X amounted to 0.115 g CPC per g cell mass.

Influence of medium composition on penicillin V production in a stirred tank reactor.

Penicillin production with a high-producing strain Penicillium chrysogenum was investigated under well-controlled conditions in a stirred tank reactor with complex media containing lard oil and lactose on the one hand, and lactose on the other hand. With lard oil, cell growth and product formation rates were higher, and the production time was shorter by 40 h than without lard oil. On account of the longer production time without lard oil, the amount of beta-lactam compounds was higher (29.93 g l-1), but the mole fraction of the decomposed products (penicilloic acid and penilloic acid) was larger (0.282) than the amount of penicillin V (23.25 g l-1) and the decomposed mole fraction (0.0747) with lard oil. The final product concentrations were about the same (20.86 g l-1 or 35,462 IU ml-1 with lard oil, and 20.43 g l-1 or 34510 IU ml-1 without lard oil). The mole fractions of the by-product (p-OH-penicillin V) were 0.0365 and 0.066. The substitution of lard oil with lactose is possible without a considerable reduction of process performance.

Plasmid instabilities of single and three-plasmid systems in Escherichia coli during continuous cultivation.

Plasmid instabilities in E. coli JM103 carrying three plasmids (pRK248cI, pMTC48, pEcoR4) and a single plasmid system (pTG206) for the production of fusion EcoRI (SPA::EcoRI) and catechol 2,3-dioxygenase, respectively, were investigated in continuous cultures under selective and non-selective conditions. In a three-plasmid system, pRK248cI was lost gradually together with pMTC48 from the host under non-selective conditions. The selective pressure against pRK248cI stabilized the pMTC48. This indicates that the loss of pMTC48 under non-selective conditions was caused by the loss of cI857 gene (coded by pRK248cI) which resulted in the overproduction of the toxic gene product (coded by pMTC48). In the case of single plasmid (pTG206) system, the plasmid lost from the host under non-selective conditions. This plasmid was stabilized in the host growing under selective conditions. During this period we obtained some ampicillin resistant colonies which gave low levels of enzyme activities compared to the normal plasmid bearing cells. Plasmid analysis from the above cells showed that the plasmid has undergone structural instability. Further, restriction analysis of this plasmid exhibited an additional PvuII site in a 0.9 kbp fragment that was integrated near the tet promoter which controls the expression of the xyl E gene, thereby resulting low levels of enzyme activities. Our results indicate that some of the IS elements which are present in the host chromosome were responsible for such instabilities to turn off the synthesis by inserting into the tet promoter region to lower the protein formation during the bioprocess.

Segregated mathematical model for the fed-batch cultivation of a high-producing strain of Penicillium chrysogenum.

A new segregated mathematical model for the penicillin fed-batch process is presented and applied to the growth of the pellet-forming, industrially used high-producing strain Penicillium chrysogenum S2. The model comprises two kinds of biomass (growing and producing, nongrowing and still producing), cell lysis, and complex medium as an important substrate for primary growth. In accordance with our experimental observation, product formation is not inhibited by glucose, but related to the growth rate. Maintenance metabolism is cell age-dependent. The model was verified with two sets of experimental data including exhaust gas measurements while keeping the estimated parameters almost constant. The presented model derived from that of Bajpai and Reuss (1980, 1981) does not describe our data properly. The particular influence of the pellet structure on the model is discussed. The necessity of new model assumptions (e.g., different kinds of biomass, non-constant maintenance coefficient) is explained by cell damage by lysis and shear forces, and the succeeding repair processes.

Cephalosporin C production by a highly productive Cephalosporium acremonium strain in an airlift tower loop reactor with static mixers.

The production of cephalosporin C (CPC) and its precursors penicillin N (PEN N), deacetoxycephalosporin C (DAOC) and deacetylcephalosporin C (DAC), with a highly productive strain of Cephalosporin acremonium, was investigated in an 80-1 airlift tower loop reactor with four static mixer modules (Type SMV, Sulzer) (ATLRM) on a complex medium containing 50 g l-1 peanut flour (PF). The most important key parameters such as glucose concentration and cell mass concentration were monitored during a fed-batch cultivation process. The concentrations of products CPC, PEN N, DAOC an DAC were determined on line by HPLC. The influences of four motionless mixers on the dissolved oxygen concentration (DOC), oxygen transfer rate, the cell growth and the CPC production, as well as the reactor performance, were evaluated. The results were compared with the performance of an airlift tower loop reactor (ATLR) without static mixers as well as with a stirred tank reactor (STR). A comparison of cultivations in the ATLRM and ATLR with 50 g l-1 PF indicates that the obtained maximal CPC concentration and the (CPC + DAC + DAOC) concentration were 7% and 22% higher in the ATLRM (4.96 and 7.46 g l-1) than in the ATLR (4.63 and 6.13 g l-1) respectively. The maximal CPC volumetric productivity in the ATLRM (55.1 mg l-1 h-1) was also considerably higher than that in the ATLR (48.5 mg l-1 h-1). The specific power input was reduced from 2.36 to 1.5 kW m-3, the specific productivity pertaining to the power input was improved from 1.96 to 3.31 g W-1. On the other hand, cultivation in the ATLRM had a lower maximum CPC concentration and volumetric productivity than those in STR (7.2 g l-1 and 71.2 mg l-1 h-1) with the same medium due to the lower shear stress levels and the lower specific power input (1.5 vs. 3.0 kW m-3); but the specific power imput-based yield coefficient was in the ATLRM (3.31 g W-1) higher than in the STR (2.40 g W-1). By increasing the amount of PF, it was possible to enhance the CPC concentration and volumetric productivity in the STR. However, the performance of the ATLRM was limited to using a medium containing maximal 50 g l-1 PF because of the high viscosity of the medium, the limited energy input and thus the limited oxygen supply.

On-line determination of intracellular beta-galactosidase activity in recombinant Escherichia coli using flow injection analysis (FIA).

A flow injection analysis (FIA) system was developed for the determination of cytoplasmic beta-galactosidase activity in recombinant Escherichia coli. The FIA system and its application for on-line monitoring of beta-galactosidase production during cultivation of recombinant E. coli in a 60-l airlift tower loop reactor is described. The results demonstrate that an FIA assay in conjunction with a cell disintegration step can be applied successfully for on-line monitoring of intracellular protein formation.

Production of fusion protein SpA::EcoRI in batch culture in a 60-L airlift tower loop reactor.

Escherichia coli JM103 carrying the expression plasmid pMTC48, repressor plasmid pRK248, and protection plasmid pEcoR4 was grown in a 60-L working volume airlift tower loop reactor on M9 minimal medium. Production of fusion protein SpA::EcoRI was induced by a temperature shift from 30 to 38 (optimum), 40, or 42 degrees C. The following parameters were monitored: cell mass concentration (X), total cell counts (TCC), number of colony-forming units (CFU), concentrations of glucose, acetate, ethanol, pyruvate, lactate, succinate, amino acids, and ammonia, and soluble and total protein content, as well as product concentration (enzyme activity of the fusion protein), dissolved oxygen concentration, oxygen utilization rate (OUR), CO2 production rate (CPR), respiration quotient (RQ), and volumetric mass-transfer coefficients (kLa). Product formation by temperature shift was only observed if LB concentrate was added to the culture at the same time the aeration rate was increased to avoid oxygen-transfer limitation. No product accumulation was observed with glucose and ammonia supplementation. During gene expression, X and TCC increased, CFU decreased, acetate increased, and the primary metabolite (ethanol, pyruvate, lactate) concentrations as well as OUR and CPR passed a maximum while RQ changed only slightly. These facts indicate that, during gene expression, the metabolic activity of the cell passes a maximum, and after that it decreases. With increasing aeration rate, the volumetric productivity increased, but the specific productivity with respect to the cell concentration decreased.

Influence of lactate, ammonia, and osmotic stress on adherent and suspension BHK cells.

Adherent and suspension Baby Hamster Kidney (BHK) 21c13 cells were cultivated in a 2.5-1 stirred-tank reactor with indirect aeration. Cell concentration and viability as well as glucose, lactate, ammonia, and protein concentrations in the medium and intracellular and extracellular activities of the intracellular enzymes were determined off-line. The concentrations of glucose, lactate, ammonia, and the activity of lactate dehydrogenase in the culture medium were monitored on-line. The cell/cell fragment size distribution was determined by laser flow cytometer off-line. In several runs, the size distributions were ascertained on-line by a laser flow cytometer. The influence of lactate, ammonia, and osmotic pressure on the viability and biological parameters of the suspension cells was evaluated. In Roux flasks, lactate and ammonia had considerable influence on the cell properties; in stirred tank reactors, these influences were negligible up to 9.5 g l-1 lactate and 150 mg l-1 NH+4 ion concentrations. The influence of high osmolarity on the biological parameters of the cells was much less in the stirred-tank than in the Roux flasks. The adhesion of adherent cells on a surface was impeded neither by the lactate (up to 6 g l-1) nor by the ammonia concentration (up to 150 mg l-1). However, with increasing osmolarity, the fraction of the cells adhered to a surface reduced to below 5% (at 680 mOsmol l-1).

Determination of a "critical shear stress level" applied to adherent mammalian cells.

An apparatus for the detailed investigation of the influence of shear stress on adherent BHK cells was developed. Shear forces between 0.0 and 2.5 N m-2 were studied. The influence on cell viability, cell morphology, cell lysis, and cell size was determined. Increasing shear forces as well as increasing exposure duration caused increasing changes in cell morphology and cell death. A "critical shear stress level" was determined.

Development of a turbidimetric immunoassay for on-line monitoring of proteins in cultivation processes.

An on-line assay for a thermostable pullulanase and antithrombin III (AT III) is described. The assay is based on the formation of aggregates between the protein to be measured and antibodies raised against this protein. Assay automation was achieved by utilizing the flow injection analysis (FIA) principles. The apparatus, a stopped-flow, merging-zone manifold, is described in detail. Since the reaction used in an FIA system does not have to reach equilibrium, it was possible to reduce the time for an assay cycle to 2.5 min. A method for simulating cultivation conditions was developed for assay optimization. Using this method, a detection limit of 1 mg l-1 together with a standard deviation of 1.5 was found. A sandwich ELISA was used as reference assay in the case of AT III and an enzymatic activity assay in the case of pullulanase. Correlation coefficients of 0.988 (AT III) and 0.976 (pullulanase) were determined. The turbidimetric assay was successfully used for pullulanase monitoring during a 240-h cultivation of Clostridium thermosulfurogenes.

Influence of dissolved oxygen concentration on the biosynthesis of cephalosporin C.

Cephalosporin C was produced by a highly productive strain of Cephalosporium acremonium under industrial production conditions by fed-batch cultivation in a 40-l stirred-tank reactor using a complex medium containing 50 g l-1 peanut flour. The influence of dissolved oxygen concentration (pO2, DOC), which was maintained at different constant levels between 5 and 40% of its saturation value, during the production phase by means of a parameter-adaptive pO2-controller, on the cephalosporin C biosynthesis, was investigated. The concentrations of cephalosporin C (CPC) and its precursors penicillin N (PEN N), deacetoxycephalosporin C (DAOC), and deacetylcephalosporin C (DAC) were monitored by on-line HPLC. The concentrations of amino acids, valine (VAL), cysteine (CYS), alpha-amino-adipic acid (alpha-AAA), the dipeptide alpha-amino-adipyl-cysteine (AC), and the tripeptide alpha-amino-adipyl-cysteinyl-valine (ACV) were determined by off-line HPLC. By reducing the pO2 in the production phase from 40 to 5% of its saturation value, the CPC concentration diminished from 7.2 to 1.1 g l-1 and the PEN N concentration increased from 2.57 to 7.65 g l-1. The DAC concentration also dropped from 3.13 to 0.42 g l-1; however, the DAOC concentration was less influenced. The concentrations of AC and ACV were also less affected. The small DOC did not lead to an accumulation of the intermediate AC and ACV during the production phase. With increasing DOC in the range of 5-20%, the maximal specific production rate, the cell mass concentration-based and the substrate-based yield coefficients for CPC increased almost linearly, and fell back for PEN N.(ABSTRACT TRUNCATED AT 250 WORDS)