Genetic algorithm-based medium optimization for a toxic dinoflagellate microalga

A genetic algorithm stochastic search strategy was used to optimize the culture medium for producing the toxic marine dinoflagellate microalga Protoceratium reticulatum. The optimized medium contained 26 different components (macronutrients, trace elements, vitamins). The use of this medium allowed a 60% enhancement in the final cell concentration relative to the control culture based on L1 medium. The final titer of yessotoxins was improved by 40% relative to the control medium.     

Nutritional Requirement for the Expression of Nitrogenase Activity by Rhizobium sp. in Agar Culture

The effect of various carbon sources, nitrogen sources, vitamins and trace elements on the ability of three strains (32H1, CB627, CB744) of a slow-growing Rhizobium sp. to develop nitrogenase activity in agar culture was studied. Strains 32H1 and CB627 developed nitrogenase but showed differences with respect to the nature and concentrations of carbon sources required for optimum activity. Strain 32H1 had less specific requirements than CB627 in this respect and could sustain high nitrogenase activity over a wider range of phosphate concentration (5 to 60 mmol/1) in the medium than CB627. There were only minor differences between these two strains with respect to the nitrogen source [glutamine, asparagine, histidine or (NH₄SO₄] required in the medium for nitrogenase induction, and nitrogenase activity in both strains was unaffected by changes in the concentration of vitamins or trace elements supplied. Strain CB744 did not develop nitrogenase activity under any of the conditions tested. Adenosine 3', 5'-cyclic monophosphate (1 mmol/1) was found to accelerate derepression of nitrogenase synthesis in agar cultures of strains 32H1 and CB627.     

Application of medium optimization tools for improving recombinant human interferon gamma production from Kluyveromyces lactis

The present study is focused upon improving biomass of Kluyveromyces lactis cells expressing recombinant human interferon gamma (hIFN-γ), with the aim of augmenting hIFN-γ concentration using statistical and artificial intelligence approach. Optimization of medium components viz., lactose, yeast extract, and trace elements were performed with Box–Behnken design (BBD) and artificial neural network linked genetic algorithm (ANN-GA) for maximizing biomass of recombinant K. lactis (objective function). The studies resulted over 1.5-fold improvement in the biomass concentration in a medium composed of 80?g/L lactose, 10.353?g/L yeast extract, and 15?mL/L trace elements as compared with initial biomass value. In the same study hIFN-γ concentration reached 881?µg/L which was 2.28-fold higher as compared with initial hIFN-γ concentration obtained in unoptimized medium. Further the batch fermentation study displayed mixed growth associated kinetics with the maximum hIFN-γ production rate of 1.1?mg/L. BBD and ANN-GA, both optimization techniques predicted a higher lactose concentration was clearly beneficial for augmenting K. lactis biomass which in turn increased hIFN-γ concentration.     

Optimization of culture medium for growth of Haematococcus pluvialis

A central composite rotatable design was used to examine the effects of five components of the medium on the growth of Haematococcus pluvialis in batch culture. The medium components considered were: sodium acetate,potassium nitrate, major elements, trace elements and vitamins. Within the range of the concentrations tested, a moderate concentration of the major elements significantly enhanced algal growth, both in terms of specific growth rate and cell dry weight, whereas the vitamins had no significant effect. Based on the response surface contour plots and the results of numerical analyses, the optimal nutrient concentrations for growth in terms of specific growth rate were 0.51 g L^-1 sodium acetate, 0.25 g L^-1 potassium nitrate, 0.63 mL L^-1 of the major element stock solution and 0.2 mL L^-1 of the trace element stock solution. The optimal nutrient concentrations for biomass production were 1.64 g L^-1 sodium acetate, 0.37 g L^-1potassium nitrate, 2.52 mL L^-1 of the major element stock solution and 0.03 mL L^-1 of the trace element stock solution. This revised version was published online in August 2006 with corrections to the Cover Date.     

Production of HBsAg by growth rate control with recombinantSaccharomyces cerevisiae in fed-batch

Summary To maintain a constant specific growth rate for a recombinantS.cerevisiae in fed-batch, the medium feeding rate has been controlled with respect to the hourly calculated glucose uptake rate. The recombinant yeast producing HBsAg showed the exponential production trend in proportion to the exponential cell growth. Total cell yield in fed-batch was about 0.402 g cells/g glucose, and HBsAg was produced about ten times more than in batch. Decrease of growth rate by HBsAg produced was not shown.     

Optimization of expression of hepatitis B surface antigen gene in yeasts

A series of recombinant plasmids has been constructed for expression of the hepatitis B viral surface antigen gene (HBsAg) under the control of the regulatory elements of the yeast acidic phosphatase gene (PHO5). The obtained plasmids possess the high mitotic and structural stability in the transformant yeast cells. The effect of different structural modifications of the vector on the level of HBsAg synthesis in yeasts has been studied. Optimal construction devoid of the bacterial DNA sequences and pre-S region has been selected.     

Transient expression of hepatitis B virus surface antigen (HBsAg) gene in monkey cells by a SV40-based virus vector

Summary We have constructed a recombinant SV40-based vector carrying the S gene coding for the hepatitis B virus surface antigen (HBsAg). This vector replicates as an episome in monkey COS7 cells, producing high levels of hepatitis B virus surface antigen (HBsAg), which is liberated in the cell medium, probably as a membrane vesicule. The vector also carries the SV40-late genes and produces recombinant viruses. These viruses were used to infect fresh cell culture, with detection of HBsAg in the medium. Thus, this virus vector can efficiently transduce the gene for HBsAg.     

Growth rate control in fed-batch cultures of recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg).

A recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg) exhibited growth-associated product formation. By controlling the medium feed rate, based on the calculated amount of medium required for 1 h, a constant specific growth rate was obtained in the range of 0.12-0.18 h-1. In order to prolong the exponential growth phase, the medium feed rate was increased exponentially. A fed-batch cultivation method based on the production kinetics of batch culture enhanced HBsAg production ten times more than in batch culture. The reason for the increase can be explained by the fact that the production of HBsAg is expressed as an exponential function of time when the specific growth rate is controlled to a constant value in growth-associated product formation kinetics. In the scale-up of this culture to 91, the specific growth rate could also be maintained constant and the HBsAg production trend was similar to that in a 1-1 culture. However, ethanol accumulation occurred at a late stage in fed-bach culture. Ethanol produced was not reutilized and inhibited further cell growth.     

Optimization of chemically defined medium for recombinant Pichia pastoris for biomass production

A chemically defined medium was optimized for the maximum biomass production of recombinant Pichia pastoris in the fermentor cultures using glycerol as the sole carbon source. Optimization was done using the statistical methods for getting the optimal level of salts, trace metals and vitamins for the growth of recombinant P. pastoris. The response surface methodology was effective in optimizing nutritional requirements using the limited number of experiments. The optimum medium composition was found to be 20 g/L glycerol, 7.5 g/L (NH4)2SO4, 1 g/L MgSO4.7H2O, 8.5 g/L KH2PO4, 1.5 mL/L vitamin solution and 20 mL/L trace metal solution. Using the optimized medium 11.25 g DCW/L biomass was produced giving a yield coefficient of 0.55 g biomass/g of glycerol in a batch culture. Chemostat cultivation of recombinant P. pastoris was done in the optimized medium at different dilution rates to determine the kinetic parameters for growth on glycerol. Maximum specific growth rate of 0.23 h(-1) and Monod saturation constant of 0.178 g/L were determined by applying Monod model on the steady state data. Products of fermentation pathway, ethanol and acetate, were not detected by HPLC even at higher dilution rates. This supports the notion that P. pastoris cells grow on glycerol by a respiratory route and are therefore an efficient biomass and protein producers.     

Expression of HBV surface antigen or HIV envelope protein using recombinant adenovirus vectors

Recombinant adenoviruses were constructed that contained either the HBsAg coding sequence or the HIV envelope protein coding sequence. The recombinant adenoviruses can replicate normally in cultured human cells. Cells infected with the adenovirus-HBV recombinant secreted HBsAg into the tissue culture medium. This HBsAg had immunological and physical properties similar to those of the 22-nm particles found in human serum. Expression of HIV envelope protein in cells infected with the adenovirus-HIV recombinant was demonstrated using cytoimmunofluorescence and immunoprecipitation. A hamster model was developed to evaluate the immunogenic properties of adenovirus-HBV recombinants. Hamsters inoculated intranasally with live adenovirus-HBV recombinant produced antibody against both adenovirus and hepatitis B virus surface antigen.     

Influence of medium components on growth kinetics of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

Starting with the standard complex medium HL-5C, the influence of different medium components on the growth behavior of Dictyostelium discoideum was investigated. For this purpose, each component was individually deleted from the complex medium HL-5C, and the overall concentration of all components as well as the kinds and concentrations of carbon source were varied. The effects of the supplementation with major components of synthetic medium like vitamins, trace metals, inorganic salts were also investigated. When glucose being the carbon source was deleted from the standard HL-5C medium, the maximal cell density then had a drastic drop. Deletion of yeast extract also caused lower maximal cell density. Of all the carbohydrates investigated only maltose and glucose led to higher final cell concentration. Adding inorganic salts such as CaCl₂ and MgCl₂ to HL-5C medium significantly improved the cell growth, whereas addition of vitamins or trace metals had little effect on cell growth. A semi-empirical model is employed to simulate the cell growth.     

Glycosylation of prourokinase produced by Pichia pastoris impairs enzymatic activity but not secretion

Both glycosylated and nonglycosylated forms of recombinant human prourokinase were produced to the level of 20 mg/L by yeast Pichia pastoris in BMMY medium after 2 days of culture. The expressed pro-UK was 98% secreted into the culture medium and easily purified by carboxymethyl cellulose chromatography. More than 99% of pro-UK in the culture medium was found in single-chain form. This was contradictory to a previous finding which found that glycosylation of pro-UK by yeast inhibited its secretion. The absence of glycosylation at Asn302 of pro-UK has no measurable effect on its secretion from the yeast cells. However, the nonglycosylated pro-UK was much less stable in the culture medium, probably due to proteolysis. Nonglycosylated pro-UK from yeast had a clot lysing activity comparable to that of Escherichia coli-derived or mammalian cell-derived recombinant pro-UK. By contrast, the glycosylated yeast pro-UK was less activatable by plasmin and had a lower enzymatic activity against plasminogen and a lower clot lysing activity than nonglycosylated pro-UK from yeast, while their amidolytic activity against S2444 was equivalent. It was concluded that glycosylation of pro-UK by yeast P. pastoris interferes with the catalytic site but not secretion of this protein.     

Isolation and culture of the terminally differentiated adult mammalian ventricular cardiac muscle cell

Summary We have carried out systematic studies to optimize and standardize methodology to isolate and culture the adult rat ventricular cardiac muscle cell. Four hearts were perfused simultaneously with a calcium-free medium containing collagenase. The ventricular tissue was then minced and further digested to liberate individual cells. Approximately 16 million rod-shaped muscle cells were obtained. The plating efficiency has been greatly improved by culturing the cells in a conditioned medium prepared from a rabbit corneal cell line. This medium also contained added fetal bovine serum, essential and nonessential amino acids, vitamins, insulin, transferrin, and 25 trace minerals. The culture flasks were precoated with rat-tail collagen. Fibroblast contamination was virtually eliminated by including cytosine arabinoside in the medium during the first 7 d of culture. After this time the cells could be cultured in the absence of serum in a chemically defined medium composed of MEM, vitamins, nonessential amino acids, and trace minerals. They continued to contract spontaneously and do well in this medium for at least 3 d thereafter. This improved methodology resulted in a reproducible culture system with improved plating efficiency. It provided a new and unique system to study the structure and function of the adult mammalian ventricular cardiac muscle cell. This investigation was supported by Grant HL 25873 from the National Institutes of Health, Bethesda, MD.     

Preparation of selenium yeasts I. Preparation of selenium-enriched Saccharomyces cerevisiae

Selenium (Se) is an essential micronutrient for human and animal organisms. Organic selenium complexes and selenium-containing amino acids are considered the most bioavailable.Under appropriate conditions yeasts are capable of accumulating large amounts of trace elements, such as selenium, and incorporating them into organic compounds. It has been found that introduction of water-soluble selenium salt as a component of the culture medium for yeasts produced by conventional batch processing results in a substantial amount of selenium being absorbed by the yeast.Using a culture medium supplemented with 30 μg/mL sodium-selenite added during the exponential growth phase results in selenium-accumulation in the range of 1200–1400 μg/g dried baker's yeast (Saccharomyces cerevisiae) measured by ICP-AES method. In our previous studies it was shown that higher amounts of sodium-selenite in the culture medium have a strong inhibitory effect on the growth of this yeast. As a consequence of variations in cultivation conditions we obtained selenium yeast with different inorganic selenium content. The most important parameters influencing incorporated forms of selenium are pH value and dissolved oxygen level in the culture medium, and depending on these the selenium consumption rate of the yeast. A 0.40–0.50 mg/g h-1 specific selenium consumption rate was found to be appropriate to obtain selenium-enriched bakers' yeast of a high quality. Under suitable conditions the undesirable inorganic selenium content of the yeast could be suppressed to as low as 5–6% at the expense, however, of approximately a 20% decrease in the final biomass.     

Preparation of selenium yeasts I. Preparation of selenium-enriched Saccharomyces cerevisiae.

Selenium (Se) is an essential micronutrient for human and animal organisms. Organic selenium complexes and selenium-containing amino acids are considered the most bioavailable. Under appropriate conditions yeasts are capable of accumulating large amounts of trace elements, such as selenium, and incorporating them into organic compounds. It has been found that introduction of water-soluble selenium salt as a component of the culture medium for yeasts produced by conventional batch processing results in a substantial amount of selenium being absorbed by the yeast. Using a culture medium supplemented with 30 microg/mL sodium-selenite added during the exponential growth phase results in selenium-accumulation in the range of 1200-1400 microg/g dried baker's yeast (Saccharomyces cerevisiae) measured by ICP-AES method. In our previous studies it was shown that higher amounts of sodium-selenite in the culture medium have a strong inhibitory effect on the growth of this yeast. As a consequence of variations in cultivation conditions we obtained selenium yeast with different inorganic selenium content. The most important parameters influencing incorporated forms of selenium are pH value and dissolved oxygen level in the culture medium, and depending on these the selenium consumption rate of the yeast. A 0.40-0.50 mg/g h-1 specific selenium consumption rate was found to be appropriate to obtain selenium-enriched bakers' yeast of a high quality. Under suitable conditions the undesirable inorganic selenium content of the yeast could be suppressed to as low as 5-6% at the expense, however, of approximately a 20% decrease in the final biomass.     

Growth rate control in fed-batch cultures of recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg)

Summary A recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg) exhibited growth-assciated product formation. By controlling the medium feed rate, based on the calculated amount of medium required for 1 h, a constant specific growth rate was obtained in the range of 1.12-0.18 h^–1. In order to prolong the exponential growth phase, the medium feed rate was increased exponentially. A fedbatch cultivation method based on the production kinetics of batch culture enhanced HBsAg production ten times more than in batch culture. The reason for the increase can be explained by the fact that the production of HBsAg is expressed as an exponential function of time when the specific growth rate is controlled to a constant value in growth-associated product fromation kinetics. In the scale-up of this culture to 91, the specific growth rate could also be maintained constant and the HBsAg production trend was similar to that in a 1-l culture. However, ethanol accumulation occurred at a late stage in fed-bach culture. Ethanol produced was not reutilized and inhibited further cell growth. Offprint requests to: M. B. Gu     

特异性表达siRNA的新型乙肝核酸药物的研究

旨在研究靶向于HBsAg的肝脏特异性siRNA,实现核酸药物高靶向性、高特异性基因治疗乙肝。通过重组PCR的方法构建靶向于HBsAg的肝脏特异性表达的siRNA的核酸药物。将目标载体转染HepG2 2.2.15细胞,用ELISA的方法测定转染细胞培养液内HBsAg的含量。PCR鉴定和测序确定重组PAAV-MCS载体PApoE1-ApoE2-hAAT-siHBsAg-U1 3’Box构建成功。重组质粒对HepG2 2.2.15细胞分泌的HBsAg所产生的抑制作用自转染后5 d达到高峰,抑制率达到(30±0.28)%(P<0.01),siRNA特异性表达单元能显著降低HepG2 2.2.15细胞HBV DNA的拷贝数。成功构建的肝脏特异性、siRNA高表达的核酸药物可以有效地抑制HBV基因的表达和复制,为乙肝的进一步治疗研究做了关键性工作。     

海带水提液和酵母提取物促大肠杆菌生长研究*

通过细菌培养液DD值测量和平板计数技术观察不同物质对大肠杆菌生长的影响;对微量元素、稀土元素、动、植物激素、细菌提取物、真菌提取物、藻类提取物、植物提取物和动物提取物等13大类120余种物质在一定浓度范围的促大肠杆菌生长作用进行了观察。海带水提液和酵母提取物对大肠杆菌生长具有明显的促进作用;两的最佳作用浓度分别为20g/L和2％,DD值最大可分别达到对照组的2．93倍和5．06倍;平板计数可达3．94倍和5．39倍。     

人源性抗HBsAg抗体Fab段在酵母中的表达

通过分步整合的方式,将人源性抗乙肝表面抗原（HBsAg）抗体Fab的轻、重链基因分步整合到巴斯德毕赤（Pichia pastoris）酵母GS115菌株的染色体上,经甲醇诱导,成功地分泌表达出抗HBsAg抗体的Fab片段,表达量达50～80mg/L。ELISA结果显示重组酵母分泌表达出的Fab具有较强的结合HBsAg的能力。通过抗Fab的抗体柱亲和层析,纯化出了纯度较高的Fab产品。     

EFFECTS OF CERTAIN LIMITING CONDITIONS ON THE SYNTHESIS OF B VITAMINS BY YEAST

In yeast crops which were grown in the presence of various inhibitors, there was considerable variation in content of the various B vitamins. A higher degree of parallelism in variation in content was found to exist between thiamine and niacin than between any other pair of vitamins; this has been interpreted as indicating that the predominant functions of these two vitamins are their established rôles in fermentation. Values for inositol indicate that it may be involved in fermentation processes, but this is not the case for other members of the B complex. Biotin appears to be unique since in no case did the biotin content of yeast grown in the presence of an inhibitor fall below that of the control yeast. There was some evidence of synthesis of biotin, or a material with biotin activity, in the presence of certain inhibitors, the most striking instance being with sulfaguanidine. An exogenous supply of biotin was essential for extensive proliferation of F. B. yeast, and yeast grown in a medium to which biotin was the only added vitamin contained the B vitamins in amounts very similar to those found in the control yeast, the most marked differences being in increased vitamin B₆ and p-aminobenzoic acid contents. In the absence of biotin, significant amounts of all of the B vitamins except biotin were synthesized, both in the presence and absence of certain other members of the B complex. The addition of thiamine, pyridoxine, inositol, and β-alanine to the culture medium caused a reduction in the amounts of vitamin B₆ and p-aminobenzoic acid synthesized. F. B. yeast was able to grow in a xylose medium only when certain of the B vitamins were present, and even then growth was limited. Evidence was obtained for some synthesis of all of the vitamins investigated except biotin and vitamin B₆. The most significant differences in vitamin content between galac yeast and the parent F. B. strain were in folic acid and vitamin B₆, the former being considerably reduced in amount, the latter being increased.