Oxygen requirements for growth and citric acid production of Yarrowia lipolytica

During continuous cultivation of Yarrowia lipolytica N 1, oxygen requirements for growth and citric acid synthesis were found to depend on the iron concentration in the medium. A coupled effect of oxygen and iron concentrations on the functioning of the mitochondrial electron transport chain in Y. lipolytica N 1 was established. Based on the results obtained in continuous culture, conditions for citric acid production in a batch culture of Y. lipolytica N 1 were proposed. At relatively low pO(2) value and a high iron concentration, citric acid accumulation was as high as 120 g l(-1); the specific rate of citric acid synthesis reached 120 mg citric acid (g cells h)(-1). The mass yield coefficient was 0.87 and the energy yield coefficient was 0.31.     

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).     

Development of bacterial cytochrome P-450(cam) (cytochrome m) production

Cytochrome P-450(cam) monooxygenase is an important bacterial redox enzyme system with potential commercial value for detoxifying trace hydrocarbon contaminants, catalyzing regiospecific hydroxylations, and amperometric biosensing. The present study was undertaken to increase productivity of this enzyme, which is induced in its host, pseudomonas putida PpG 786, by D(+)-camphor. Culture processes were studied in batch, fed-batch, and continuous modes to evaluate metabolic behavior and develop constitutive equations for specific rate of growth (mu), camphor utilization (q(p)). Fed-batch culture was characterized by an extended linear growth phase which is often encountered in hydrocarbon fermentations. Inhibition by the camphor solvent, dimethylformamide, was assessed. Production of the terminal protein of the p-450(cam) enzyme system, cytochrome m, was shown to depend on growth medium iron content in fed-batch culture and was increased by 130% over previously protocols by eliminating iron deficiency. A continuous process that enables greater production rates was developed by using oxygen enrichment while simultaneously reducing gas throughput. Camphor and oxygen requirements were determined for fedbatch and continuous growth. (c) 1993 John Wiley & Sons, Inc.     

Production of tissue plasminogen activator (t-PA) in Aspergillus niger

A protease-deficient strain of Aspergillus niger has been used as a host for the production of human tissue plasminogen activator (t-PA). In defined medium, up to 0.07 mg t-PA (g biomass)(-1) was produced in batch and fed-batch cultures and production was increased two- to threefold in two-phase batch cultures in which additional glucose was provided as a single pulse at the end of the first batch growth phase. Production was increased [up to 1.9 mg t-PA (g biomass)(-1)] by the addition of soy peptone to the defined medium. The rate of t-PA production in batch cultures supplemented with soy peptone (0.2 to 0.6 mg t-PA L(-1) h(-1)) was comparable to rates observed previously in high-producing mammalian or insect cell cultures. In glucose-limited chemostat culture supplemented with soy peptone, t-PA was produced at a rate of 0.7 mg t-PA L(-1) h(-1). Expression of t-PA in A. niger resulted in increased expression of genes (bipA, pdiA, and cypB) involved in the unfolded protein response (UPR). However, when cypB was overexpressed in a t-PA-producing strain, t-PA production was not increased. The t-PA produced in A. niger was cleaved into two chains of similar molecular weight to two-chain human melanoma t-PA. The two chains appeared to be stable for at least 16 h in culture supernatant of the host strain. However, in general, <1% of the t-PA produced in A. niger was active, and active t-PA disappeared from the culture supernatant during the stationary phase of batch cultures, suggesting that the two-chain t-PA may have been incorrectly processed or that initial proteolytic cleavage occurred within the proteolytic domain of the protein. Total t-PA (detected by enzyme-linked immunoassay) also eventually disappeared from culture supernatants, confirming significant extracellular proteolytic activity, even though the host strain was protease-deficient.     

Regulation of Cell Component Production by Growth Rate in the Group B Streptococcus

Group B Streptococcus (GBS) is the leading cause of bacterial sepsis and meningitis among neonates. While the capsular polysaccharide (CPS) is an important virulence factor of GBS, other cell surface components, such as C proteins, may also play a role in GBS disease. CPS production by GBS type III strain M781 was greater when cells were held at a fast (1.4-h mass-doubling time [td]) than at a slow (11-h td) rate of growth. To further investigate growth rate regulation of CPS production and to investigate production of other cell components, different serotypes and strains of GBS were grown in continuous culture in a semidefined and a complex medium. Samples were obtained after at least five generations at the selected growth rate. Cells and cell-free supernatants were processed immediately, and results from all assays were normalized for cell dry weight. All serotypes (Ia, Ib, and III) and strains (one or two strains per serotype) tested produced at least 3.6-fold more CPS at a td of 1. 4 h than at a td of 11 h. Production of beta C protein by GBS type Ia strain A909 and type Ib strain H36B was also shown to increase at least 5.5-fold with increased growth rate (production at a td of 1. 4 h versus 11 h). The production of alpha C protein by the same strains did not significantly change with increased growth rate. The effect of growth rate on other cell components was also investigated. Production of group B antigen did not change with growth rate, while alkaline phosphatase decreased with increased growth rate. Both CAMP factor and beta-hemolysin production increased fourfold with increased growth rate. Growth rate regulation is specific for select cell components in GBS, including beta C protein, alkaline phosphatase, beta-hemolysin, and CPS production.     

High cell density culture of Yarrowia lipolytica using a one-step feeding process

Yarrowia lipolytica is a potentially useful host for heterologous protein production. To develop an efficient culture method for high cell density cultivation and heterologous gene expression of Y. lipolytica, the effects of medium components and their concentrations on the growth of Y. lipolytica have been investigated. Addition of yeast extract to the culture media was found to significantly reduce the long lag phase encountered when Y. lipolytica was cultivated in synthetic culture media containing high concentrations of glycerol. Therefore, by enriching with 0.3% yeast extract the synthetic culture medium containing 15% glycerol, we could cultivate Y. lipolytica up to 83 g/L dry cell weight in a batch culture. Furthermore, over 100 g/L and 88 units/mL of rice alpha-amylase activity were obtained in less than 50 h with a one-step feeding process in which a recombinant Y. lipolytica expressing rice alpha-amylase was cultivated in the 10% glycerol medium enriched with 0.3% yeast extract and fed only once with the concentrated feeding medium (60% glycerol). The easy cultivation of recombinant Y. lipolytica to a high cell density may strengthen its position as a host for heterologous protein production.     

Is the host or the parasitoid in control?: effects of host age and temperature on pseudoparasitization by Microplitis rufiventris in Spodoptera littoralis

We compared the production of pseudoparasitization by Microplitis rufiventris females in most (third) and less (fourth) preferred instars of Spodoptera littoralis larvae at 20+/-1 and 27+/-1 degrees C. The parasitized hosts were classified into hosts producing parasitoids (type A hosts) and hosts producing no parasitoids, i.e., pseudoparasitized hosts (type B hosts). The latter were further classified into: (a) pseudoparasitized hosts with "well" arrested development (type B1 hosts); (b) pseudoparasitized hosts with partially arrested development (type B2 hosts); and (c) pseudoparasitized hosts that successfully pupated to apparently normal host pupae (type B3 hosts). The present series of experiments showed that parasitization by M. rufiventris was clearly affected by host instar, age within an instar and rearing temperature. Production of type B hosts was less when third instar S. littoralis larvae were exposed to the wasp females than when the host larvae were in fourth instar. The production of type A hosts was much greater when early or mid ages of an instar was stung by the wasp females comparing with stung late age of the same instar. Production of type B hosts may be due to one or overall of the following: (a) dosage dilution of M. rufiventris female's factors in the different age classes of the instar; (b) endocrine system (physiological state) at parasitization time, i.e., early vs late age of the instar; (c) growth rate of host larvae. The lowest production of type B hosts was at highest growth rate; and (d) temperature, larger proportions of type B hosts were produced at 27+/-1 than at 20+/-1degrees C. The three types host development (B1, B2 and B3) are possibly representing three levels of host resistance (host control) resulting in partial or complete failure of parasitoid control. Type A hosts represent complete success of parasitoid control. The results suggest that the impact of parasitoid factor(s) on developmental arrest is affected by host age at the time of parasitism and/or by temperature.     

Adaptation of Escherichia coli growth rates to the presence of pBR322

Changes in the growth rate of Escherichia coli K12 J62-1 in response to the presence of plasmid pBR322 have been investigated. Plasmid-free and plasmid-containing strains were grown in batch culture and their maximum specific growth rate (μ_max) determined. The acquisition of pBR322 by the host resulted in a decreased μ_max. Following repeated subculturing of the plasmid-containing strain on selective medium, restoration in μ_max was observed. The copy number and structure of the plasmid were not significantly altered during the experiment Growth rate measurements for a series of strains constructed using a combination of host cells and plas-mids with and without culture histories, indicated that the site of the adaptive mutation was located on the host chromosome rather than on the plasmid.     

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.     

Effect of pH on high-temperature production of bacterial penicillin acylase in Escherichia coli

High-temperature-oriented production of bacterial penicillin acylase (PAC), which is usually expressed at low temperatures (less than 30 degrees C), was demonstrated in this study via heterologous expression of the Providencia rettgeri (P. rettgeri) pac gene in Escherichia coli (E. coli). While it is possible to produce PAC at a temperature as high as 37 degrees C, the environmental condition (specifically, culture pH) critically affected culture performance. Production of PAC at 37 degrees C was feasible only when culture pH was close to neutral (i.e., 6.5-7.5). Outside this pH range, cell physiology for the host/vector system was seriously affected, resulting in poor culture performance. In acidic culture environments, temperature significantly affected the pac expression level and specific PAC activity decreased with an increase in culture temperature. In basic culture environments, cell growth was seriously inhibited though the pac expression level was minimally affected by temperature. Such unusual types of pH and temperature effects on pac expression were never reported for bacterial PACs. The results suggest that culture pH should be precisely controlled for the current host/vector systems being applied on the overproduction of P. rettgeri PAC in E. coli at high temperatures.     

The use of the tetrazolium salt XTT for the estimation of biological activity of activated sludge cultivated under steady-state and transient regimes

The tetrazolium salt 3′-{1-[(phenylamino)-carbonyl]-3,4-tetrazolium}-bis (4-methoxy-6-nitro) benzenesulfonic acid hydrate (XTT) was used as a tool for estimating the activity of the electron transport system (ETS) in activated sludge cultivated under steady-state and transient regimes in chemostat culture. Production of formazan by reduction of XTT depended on the initial concentration of the XTT following a saturation law and was proportional to live cell biomass. Addition of cyanide (KCN) to activated sludge gave an initial 1.5-fold increase in XTT reduction, while addition of 3,5-dichlorophenol (3,5-DCP) reduced this value drastically. At steady-state and transient regimes of an activated sludge chemostat, oxygen uptake rate (OUR) and XTT reduction rate were highly correlated and indicated significant variations depending on the growth conditions.     

少量制备大肠杆菌感受态细胞条件探索

目的:为了获得重复性好、转化率高的少量制备感受态细胞的方法,利用不同生长时期的大肠杆菌感受态细胞,进行转化比较。方法:根据普通实验室的实验条件,常规方法提取质粒,氯化钙法转化不同生长时期的大肠杆菌感受态细胞,比较转化率。结果:大肠杆菌感受态细胞的转化率与OD值显著相关,在OD600nm为0.39和0.55时转化率最高,在OD600nm为0.28~0.55之间均可得到理想的转化效果。结论:少量制备感受态细胞方法操作中无需添加任何保护试剂和细胞复苏培养,操作简便、重复性好,实验成本低廉。     

Transformation of the Pseudonocardiaceae Amycolatopsis sp. strain HR167 is highly dependent on the physiological state of the cells

The Pseudonocardiaceae Amycolatopsis sp. strain HR167 is used in a biotransformation process to produce vanillin from ferulic acid. To make this strain accessible for genetic engineering, a direct mycelium transformation system developed for Amycolatopsis mediterranei [Madon and Hotter (1991) J Bacteriol 173: 6325-6331] was applied and optimized for Amycolatopsis sp. strain HR167. The physiological state of the cells had a major influence on the transformation rate. The highest transformation rate of about 7x10(5) transformants per microgram of DNA was obtained with mycelium harvested 6.5-7.5 h after the culture has reached the stationary growth phase. When cells were harvested outside of this time slot, the transformation rate drastically decreased. The density of the mycelium suspensions used in the transformation mixture and the methylation state of the plasmid DNA used for the transformation were also crucial parameters. With plasmid DNA isolated from Escherichia coli ET12567, transformation rates were 3,500-fold higher than those obtained with DNA isolated from E. coli XL1-Blue.     

The kinetics of Lagenidium giganteum growth in liquid and solid cultures

AIMS: Production of the mosquito biolarvacide Lagenidium giganteum in solid culture has been proposed as an economic alternative to production in liquid culture because of observations of improved shelf life and efficacy upon storage. Understanding the differences between these production systems and estimating growth rate in solid culture are important for commercialization. In order to address these needs a logistic model was developed to describe the growth kinetics of L. giganteum produced in solid and liquid cultures. METHODS AND RESULTS: Kinetic parameters in the logistic model were estimated by nonlinear regression of CO2 evolution rate (CER) and biomass data from solid and liquid cultivation experiments. Lagenidium giganteum biomass was measured using DNA extracted directly from samples. The logistic model was fit to experimental biomass and CER data with low standard errors for parameter estimates. The model was validated in two independent experiments by examining prediction of biomass using on-line CER measurements. CONCLUSIONS: There were significant differences between maximum biomass density, maintenance coefficients, and specific growth rates for liquid and solid cultures. The maximum biomass density (mg dw ml-1) was 11 times greater for solid cultivation compared with liquid cultivation of L. giganteum; however, the maintenance coefficient (mg CO2 h-1 (mg dw)-1) was six times greater for liquid cultivation than in solid cultivation. The specific growth rate at 30 degrees C was approximately 30% greater in liquid cultivation compared with solid cultivation. Slower depletion of substrate and lower endogenous metabolism may explain the longer shelf life of L. giganteum produced in solid culture. SIGNIFICANCE AND IMPACT OF THE STUDY: A simple logistic model was developed which allows real-time estimation of L. giganteum biomass from on-line CER measurements. Parameter estimates for liquid and solid cultivation models also elucidated observations of longer shelf life for production in solid culture.     

Physiological regulation of competence induction for natural transformation in Acinetobacter calcoaceticus

Acinetobacter calcoaceticus induced competence for natural transformation maximally after dilution of a stationary culture into fresh medium. Competence was gradually lost during prolonged exponential growth and after entrance into the stationary state. Growth cessation and nutrient upshift were involved in the induction of competence. The level of competence of a chemostat culture of A. calcoaceticus was dependent on the nature of the growth limitation. Under potassium limitation a transformation frequency of ±1x10^-4 was obtained. This frequency was independent of the specific growth rate. In phosphate-, nitrogen-, and carbon-limited chemostat cultures, in contrast, the transformation frequency depended on the specific growth rate; the transformation frequency equalled±10^-4 at dilution rates close to µ_max of 0.6h^-1 and decreased to ±10^-7 at a dilution rate of 0.1 h^-1. We conclude that (1) DNA uptake for natural transformation in A. calcoaceticus does not serve a nutrient function and (2) competence induction is regulated via a promoter(s) that resembles the fis promoter from Escherichia coli.     

Vancomycin production in batch and continuous culture

Production of the glycopeptide antibiotic vancomycin by two Amycolatopsis orientalis strains was examined in batch shake flask culture in a semidefined medium with peptone as the nitrogen source. Different growth and production profiles were observed with the two strains; specific production (Y(p/x)) was threefold higher with strain ATCC 19795 than with strain NCIMB 12945. A defined medium with amino acids as the nitrogen source was developed by use of the Plackett-Burman statistical screening method. This technique identified certain amino acids (glycine, phenylalanine, tyrosine, and arginine) that gave significant increased specific production, whereas phosphate was identified as inhibitory for high specific vancomycin production. Experiments made with the improved medium and strain ATCC 19795 showed that vancomycin production kinetics were either growth dissociated or growth associated, depending on the amino acid concentration. In chemostat culture at a constant dilution rate (0.087 h(-1)), specific vancomycin production rate (q(vancomycin)) decreased linearly as the medium phosphate concentration was increased from 2 to 8 mM. In both phosphate and glucose limited chemostats, q(vancomycin) was a function of specific growth rate; the maximum value was observed at D = 0.087 h(-1) (52% of the maximum specific growth rate). Under phosphate limited growth conditions, q(vancomycin) was threefold higher (0.37 mg/g dry weight/h) than under glucose limitation (0.12 mg/g dry weight/h). (c) 1996 John Wiley & Sons, Inc.     

Lipase location in Yarrowia lipolytica cells

Lipase production by Yarrowia lipolytica was growth associated and, at the beginning of cultivation, it was mainly cell-bound. Lipase release into the culture medium started when about 50% of the carbon source (olive oil or glucose) was consumed reaching its maximum concentration in the late stationary phase.     

Plasmid stability and alpha-amylase production in batch and continuous cultures of Bacillus subtilis TN106[pAT5

Cell growth and enzyme (alpha-amylase) production characteristics of Bacillus subtilis TN106 containing the recombinant plasmid pAT5 are investigated in batch and continuous cultures using a defined medium with glucose as the limiting nutrient. The batch culture studies demonstrate that the recombinant plasmid, reported earlier(1) to be stably maintained in the host, suffers from segregational and structural instabilities. The structural instability of this strain occurred during culture storage and can be eliminated in bioreactor experiments by using a modified inoculum preparation procedure. Such elimination allows an unbiased investigation of segregational instability via continuous culture studies. Such studies conducted with this fast growing microorganism, in the absence of antibiotic selection pressure, indicate a very efficient glucose utilization (very low residual glucose concentrations) over a wide range of dilution rates (0.16 h(-1) - 0.94 h(-1)). The nearly time-invariant and low residual glucose concentrations at each such dilution rate enable convenient estimation of growth parameters of the host and recombinant cells and frequency of segregational instability from transients in the resulting mixed cultures. The specific alpha-amylase activity exhibits an inverse relationship to the specific growth rate of recombinant cells. The growth of recombinant cells is not affected by the presence of antibiotic (kanamycin). The growth advantage of host cells over recombinant cells diminishes with increasing dilution rate.