液化沙雷氏菌磷脂酶A1的克隆表达及乳糖自诱导发酵

为了利用大肠杆菌高效生产重组磷脂酶,克隆了液化沙雷氏菌磷脂酶A1的编码基因pla,分别使用pET-28a(+)和pET-20b(+)载体,实现了磷脂酶A1在大肠杆菌BL21(DE3)中的功能表达.重组菌利用载体pET-28a(+)在原始信号肽的介导下胞外PLA1酶活达40.8 U/mL,占总酶活的91％.重组菌转接至优化后的发酵诱导培养基:蛋白胨10 g/L,酵母粉5g/L,葡萄糖0.8 g/L,乳糖5 g/L,25 mmol/L Na2HPO4,25 mmol/L KH2PO4和1 mmol/L MgSO4;菌体生长6h后,添加7.5 g/L的甘氨酸,37℃恒温发酵24 h,重组菌胞外PLA1酶活达到128.7 U/mL.     

Advanced microscale bioreactor system: a representative scale-down model for bench-top bioreactors

In recent years, several automated scale-down bioreactor systems have been developed to increase efficiency in cell culture process development. ambr™ is an automated workstation that provides individual monitoring and control of culture dissolved oxygen and pH in single-use, stirred-tank bioreactors at a working volume of 10–15 mL. To evaluate the ambr™ system, we compared the performance of four recombinant Chinese hamster ovary cell lines in a fed-batch process in parallel ambr™, 2-L bench-top bioreactors, and shake flasks. Cultures in ambr™ matched 2-L bioreactors in controlling the environment (temperature, dissolved oxygen, and pH) and in culture performance (growth, viability, glucose, lactate, Na⁺, osmolality, titer, and product quality). However, cultures in shake flasks did not show comparable performance to the ambr™ and 2-L bioreactors.     

Improved production of human type II procollagen in the yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis> in shake flasks by a wireless-controlled fed-batch system

Background  

Here we describe a new technical solution for optimization of Pichia pastoris shake flask cultures with the example of production of stable human type II collagen. Production of recombinant proteins in P. pastoris is usually performed by controlling gene expression with the strong AOX1 promoter, which is induced by addition of methanol. Optimization of processes using the AOX1 promoter in P. pastoris is generally done in bioreactors by fed-batch fermentation with a controlled continuous addition of methanol for avoiding methanol toxification and carbon/energy starvation. The development of feeding protocols and the study of AOX1-controlled recombinant protein production have been largely made in shake flasks, although shake flasks have very limited possibilities for measurement and control.     

Studies on the production of human parathyroid hormone by recombinant Escherichia coli

Expression of human parathyroid hormone, hPTH(-1-84), by Escherichia coli N4830: pEX-PPTH was studied in controlled bioreactors. The hPTH is expressed as a fusion protein under control of the bacteriophage p_R promoter. In batch runs, low biomass concentrations but high specific hPTH productivities were obtained with complex TY (bactotryptone and yeast extract) medium whereas high biomass concentration and low specific productivities were found when fructose was used instead of bactotryptone (YF medium). The preinduction temperature was always 30°C; the temperature shift to induce production of fusion protein was varied from 36 to 42°C. Formation of hPTH passed a pronounced maximum as a function of induction temperature when using YF medium. However, the optimum temperature shift was 38°C for both media used. For this temperature increase both media yielded about the same volumetric hPTH productivity (approx. 30 mg hPTH/l per hour). By applying a fedbatch strategy for the YF medium, the productivity of the recombinant protein could be further increased more than fourfold. Compared to shake-flask experiments, the hPTH yield could be increased by a factor larger than 20.     

Process performance of parallel bioreactors for batch cultivation of Streptomyces tendae

Batch cultivations of the nikkomycin Z producer Streptomyces tendae were performed in three different parallel bioreactor systems (milliliter-scale stirred-tank reactors, shake flasks and shaken microtiter plate) in comparison to a standard liter-scale stirred-tank reactor as reference. Similar dry cell weight concentrations were measured as function of process time in stirred-tank reactors and shake flasks, whereas only poor growth was observed in the shaken microtiter plate. In contrast, the nikkomycin Z production differed significantly between the stirred and shaken bioreactors. The measured product concentrations and product formation kinetics were almost the same in the stirred-tank bioreactors of different scale. Much less nikkomycin Z was formed in the shake flasks and MTP cultivations, most probably due to oxygen limitations. To investigate the non-Newtonian shear-thinning behavior of the culture broth in small-scale bioreactors, a new and simple method was applied to estimate the rheological behavior. The apparent viscosities were found to be very similar in the stirred-tank bioreactors, whereas the apparent viscosity was up to two times increased in the shake flask cultivations due to a lower average shear rate of this reactor system. These data illustrate that different engineering characteristics of parallel bioreactors applied for process development can have major implications for scale-up of bioprocesses with non-Newtonian viscous culture broths.     

Effect of bioreactor configuration on the growth and maturation of Picea sitchensis somatic embryo cultures

Somatic embryo suspension cultures of Picea sitchensis (Sitka spruce) derived from two cell lines, SS03 and SS10, were grown in shake flasks, air-lift, bubble, stirred tank and hanging stirrer bar bioreactors. Cell line SS03 yielded freely suspended and individual stage 1 embryos, while the embryos of SS10 were present in large aggregates. Compared to shake flasks, proliferation in bioreactors resulted in increased biomass; however, cell line morphology influenced the effect of different bioreactor configurations on growth and maturation of embryo cultures. Somatic embryos grown in shake flasks and bioreactors were matured on gelled solid medium and in submerged culture where gelled solid medium was covered with a layer of liquid medium. The number of stage 3 (mature) embryos produced from SS03 in the bubble bioreactor was significantly higher than those from stirred tank and hanging stirrer bar bioreactors with both solid medium and submerged culture. Submerged culture was unsuitable for SS10 embryo maturation. This revised version was published online in June 2006 with corrections to the Cover Date.     

beta-Carotene production in sugarcane molasses by a Rhodotorula glutinis mutant

Several wild strains and mutants of Rhodotorula spp. were screened for growth, carotenoid production and the proportion of -carotene produced in sugarcane molasses. A better producer, Rhodotorula glutinis mutant 32, was optimized for carotenoid production with respect to total reducing sugar (TRS) concentration and pH. In shake flasks, when molasses was used as the sole nutrient medium with 40 g l⁻¹ TRS, at pH 6, the carotenoid yield was 14 mg l⁻¹ and -carotene accounted for 70% of the total carotenoids. In a 14-l stirred tank fermenter, a 20% increase in torulene content was observed in plain molasses medium. However, by addition of yeast extract, this effect was reversed and a 31% increase in -carotene content was observed. Dissolved oxygen (DO) stat fed-batch cultivation of mutant 32 in plain molasses medium yielded 71 and 185 mg l⁻¹ total carotenoids in double- and triple-strength medium, respectively. When supplemented with yeast extract, the yields were 97 and 183 mg l⁻¹ total carotenoid with a 30% increase in -carotene and a simultaneous 40% decrease in torulene proportion. Higher cell mass was also achieved by double- and triple-strength fed-batch fermentation. Journal of Industrial Microbiology & Biotechnology (2001) 26, 327–332. Received 18 September 2000/ Accepted in revised form 02 March 2001     

Medium optimization for glucoamylase production by a yeast,Pichia subpelliculosa ABWF-64, in submerged cultivation

An amylolytic yeast strain Pichia subpelliculosawas shown to produce glucoamylase in submerged cultivation. The yeast strain produced the enzyme optimally at 30 °C and pH 5.6 in shake flasks agitated at 200 rev min^–1 in the optimized glucoamylase production medium containing 1% starch, 0.2% yeast extract, 0.4% K₂HPO₄, 0.035% NaCl and 0.1% MgCl₂. Maximum enzyme production was attained during early growth of 11 h in shake flasks, and 6 h in a laboratory fermenter. By optimizing media components and cultivation parameters, a 15-fold increase in glucoamylase secretion was achieved.     

Secretion of the sweet-tasting protein thaumatin by recombinant strains of Aspergillus niger var. awamori

A recombinant form of the sweet-tasting protein thaumatin has been produced in the filamentous fungus Aspergillus niger var. awamori. Expression cassettes containing a synthetic gene encoding thaumatin II were prepared and used to transform Aspergillus niger var. awamori strain NRRL312. Several fungal strains capable of synthesizing and secreting thaumatin into the culture medium were generated, and their production capabilities were determined, first in shake flasks and later in a laboratory fermentor. We report the expression and secretion of thaumatin in concentrations of 5–7 mg/l. This recombinant thaumatin is sweet. Received: 7 October 1997 / Received revision: 21 November 1997 / Accepted: 21 November 1997     

Oxygen limitation is a pitfall during screening for industrial strains

Oxygen supply is a key parameter in aerobic fermentation processes like the industrial production of amino acids. Although the oxygen transfer rate (OTR; or the volumetric oxygen transfer coefficient k_La) is routinely analyzed by engineers during stirred tank fermentations, it is often not taken into account by biologists conducting screening experiments in shake flasks. To show the importance of knowing how to avoid oxygen transfer limitations during primary screenings, Corynebacterium glutamicum ATCC 13032 (wild-type strain) and DSM 12866 (lysine-producing strain) were cultivated in shake flasks with different culture liquid volumes and under different shaking conditions. With the Respiration Activity Monitoring System, the OTR was determined quasi-continuously. Optical density as well as concentrations of lysine and byproducts (lactate, acetate, succinate) were determined off-line and correlated with the OTR signal. From the results, design criteria for improved screening in shaken bioreactors that help to avoid selection of suboptimal strains during early process development steps can be derived. Finally, the suitability of DSM 12866 as a strain for industrial processes with a high space–time yield is discussed.     

Selection of a high-biomass, chromium-rich yeast strain and optimization of cultivation conditions

Saccharomyces cerevisiae LZ-53 was selected from 240 primary yeast strains from different genera and species, whose chromium (Cr) resistance and biomass were higher than others were. The highest biomass and Cr content of the strain was obtained in 30 h at 28°C and 200 rpm, when 20 ml of the culture in 250-ml shake flasks was grown in wort containing 1200 μg/ml Cr. The initial pH was adjusted to 6.0. The optimal inoculum volume was 10% (v/v). The Cr content of the cells was determined by neutron activation analysis. Under the optimized cultivation conditions, the Cr content reached 3248 μg/g. Journal of Industrial Microbiology & Biotechnology (2001) 27, 195–198. Received 28 January 2001/ Accepted in revised form 11 June 2001     

Sophorose lipids produced from sucrose

Summary Torulopsis bombicola produced sophorose lipids when growing on sucrose in similar yields as if growing on glucose. Besides yield the composition of sophorose lipids characterized by TLC/FID was greatly influenced by yeast extract concentration in the shake flasks in contrary to highly aerated fermenter.     

Stimulation of indirubin production by KNO3 depletion in indole-supplemented suspension culture of Polygonum tinctorium

Summary Indole when added at 20 mM to shake flask cultures of Polygonum tinctorium cells on 16th day increased indirubin production from 41 mg/l to 126 mg/l. Use of KNO₃ depleted media stimulated indirubin formation up to 88% and 26% in shake flasks and air-lift bioreactors, respectively.     

The influence of cultivation methods on Shewanella oneidensis physiology and proteome expression

High-throughput analyses that are central to microbial systems biology and ecophysiology research benefit from highly homogeneous and physiologically well-defined cell cultures. While attention has focused on the technical variation associated with high-throughput technologies, biological variation introduced as a function of cell cultivation methods has been largely overlooked. This study evaluated the impact of cultivation methods, controlled batch or continuous culture in bioreactors versus shake flasks, on the reproducibility of global proteome measurements in Shewanella oneidensis MR-1. Variability in dissolved oxygen concentration and consumption rate, metabolite profiles, and proteome was greater in shake flask than controlled batch or chemostat cultures. Proteins indicative of suboxic and anaerobic growth (e.g., fumarate reductase and decaheme c-type cytochromes) were more abundant in cells from shake flasks compared to bioreactor cultures, a finding consistent with data demonstrating that “aerobic” flask cultures were O₂ deficient due to poor mass transfer kinetics. The work described herein establishes the necessity of controlled cultivation for ensuring highly reproducible and homogenous microbial cultures. By decreasing cell to cell variability, higher quality samples will allow for the interpretive accuracy necessary for drawing conclusions relevant to microbial systems biology research.     

Optimizing conditions for poly(β-hydroxybutyrate) production by <Emphasis Type="Italic">Halomonas boliviensis</Emphasis> LC1 in batch culture with sucrose as carbon source

Halomonas boliviensis LC1 is able to accumulate poly(β-hydroxybutyrate) (PHB) under conditions of excess carbon source and depletion of essential nutrients. This study was aimed at an efficient production of PHB by growing H. boliviensis to high cell concentrations in batch cultures. The effect of ammonium, phosphate, and yeast extract concentrations on cell concentration [cell dry weight (CDW)] and PHB content of H. boliviensis cultured in shake flasks was assayed using a factorial design. High concentrations of these nutrients led to increments in cell growth but reduced the PHB content to some extent. Cultivations of H. boliviensis under controlled conditions in a fermentor using 1.5% (w/v) yeast extract as N source, and intermittent addition of sucrose to provide excess C source, resulted in a polymer accumulation of 44 wt.% and 12 g l⁻¹ CDW after 24 h of cultivation. Batch cultures in a fermentor with initial concentrations of 2.5% (w/v) sucrose and 1.5% (w/v) yeast extract, and with induced oxygen limitation, resulted in an optimum PHB accumulation, PHB concentration and CDW of 54 wt.%, 7.7 g l⁻¹ and 14 g l⁻¹, respectively, after 19 h of cultivation. The addition of casaminoacids in the medium increased the CDW to 14.4 g l⁻¹ in 17 h but reduced the PHB content in the cells to 52 wt.%.     

Optimization of lactic acid production by immobilized <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IO-1

Production of lactic acid from glucose by immobilized cells of Lactococcus lactis IO-1 was investigated using cells that had been immobilized by either entrapment in beads of alginate or encapsulation in microcapsules of alginate membrane. The fermentation process was optimized in shake flasks using the Taguchi method and then further assessed in a production bioreactor. The bioreactor consisted of a packed bed of immobilized cells and its operation involved recycling of the broth through the bed. Both batch and continuous modes of operation of the reactor were investigated. Microencapsulation proved to be the better method of immobilization. For microencapsulated cells at immobilized cell concentration of 5.3 g l⁻¹, the optimal production medium had the following initial concentrations of nutrients (g l⁻¹): glucose 45, yeast extract 10, beef extract 10, peptone 7.5 and calcium chloride 10 at an initial pH of 6.85. Under these conditions, at 37 °C, the volumetric productivity of lactic acid in shake flasks was 1.8 g l⁻¹ h⁻¹. Use of a packed bed of encapsulated cells with recycle of the broth through the bed, increased the volumetric productivity to 4.5 g l⁻¹ h⁻¹. The packed bed could be used in repeated batch runs to produce lactic acid.     

Development of a shaking bioreactor system for animal cell cultures

The feasibility of using shake flasks to culture animal cells was evaluated using various sizes of cylindrical shaped vessels as bioreactors. It was found that conditions can be optimized so that hybridoma, Chinese Hamster Ovary cells, and insect cells can be efficiently cultured in the shaking reactors to cell densities comparable to that obtained with stirred-jar bioreactors, and the system is scalable to larger volumes for the production of recombinant proteins or cell mass production in the laboratory.     

Efficient production of intact human parathyroid hormone in a Saccharomyces cerevisiae mutant deficient in yeast aspartic protease 3 (YAP3)

When human parathyroid hormone (hPTH) is expressed as a secretory product in yeast, the main problem is the aberrant proteolytic cleavage that reduces the yield of intact protein. To overcome this problem, we developed an hPTH expression system using a host strain in which the YAP3 gene encoding yeast aspartic protease 3 (YAP3) was disrupted. After 48 h of culture, most of the hPTH secreted by the yap3 disruptant remained intact, whereas more than 90% of the hPTH secreted by the wild-type strain was cleaved. When the authentic hPTH was incubated in each of the culture supernatants of untransformed yap3 disruptant and wild-type strain, the proteolysis proceeded much more slowly in the culture supernatant of yap3 disruptant than in that of the wild type. The extent of hPTH proteolysis was also significantly reduced by the addition of pepstatin A, a specific aspartic protease inhibitor. The results suggest that YAP3 is involved in the internal cleavage of hPTH expressed in yeast. The correct processing of the intact hPTH secreted in the yap3 disruptant demonstrates that the yeast mutant lacking the YAP3 activity is a suitable host for the high-level expression of intact hPTH. Received: 8 December 1997 / Received last revision: 3 March 1998 / Accepted: 19 April 1998