Highly efficient strategy for enhancing taxoid production by repeated elicitation with a newly synthesized jasmonate in fed-batch cultivation of Taxus chinensis cells

A highly efficient bioprocessing strategy was developed for enhancing the production of plant secondary metabolites by repeatedly eliciting a fed-batch culture with a newly synthesized powerful jasmonate analog, 2,3-dihydroxypropyl jasmonate (DHPJA). In suspension cultures of a high taxuyunnanine C (Tc)-producing cell line of Taxus chinensis, 100 microM DHPJA was added on day 7 to fed-batch cultures with feeding of 20 g L(-1) sucrose on the same day. The synergistic effect of elicitation and substrate feeding on Tc biosynthesis was observed, which resulted in higher Tc accumulation than that by elicitation or sucrose feeding alone. More interestingly, both specific Tc yield (i.e., Tc content) and volumetric yield was further improved by a second addition of 100 microM DHPJA (on day 12) to the fed-batch cultures. In particular, with repeated elicitation and sucrose feeding the Tc volumetric yield was increased to 827 +/- 29 mg L(-1), which was 5.4-fold higher than that of the nonelicited batch culture. Furthermore, the above novel strategy was successfully applied from shake flask to a 1-L airlift bioreactor. A high Tc production and productivity of 738 +/- 41 mg L(-1) and 33.2 +/- 1.9 mg L(-1) d(-1), respectively, was achieved, which is higher than previous reports on Tc production in bioreactors. The results suggest that the aforementioned bioprocessing strategy may potentially be applied to other cell culture systems for efficient production of plant secondary metabolites.     

Online detection of feed demand in high cell density cultures of Escherichia coli by measurement of changes in dissolved oxygen transients in complex media

A starvation-based dissolved oxygen (DO) transient controller was developed to supply growth-limiting substrate to high cell density fed-batch cultures of recombinant Escherichia coli. The algorithm adjusted a preexisting feed rate in proportion to the culture's oxygen demand, which was estimated from transients in the DO concentration after short periods of feed interruption. In this manner, the addition of glucose feed was precisely controlled at a rate that did not exceed the acetate production threshold, thus preventing acetate accumulation. In comparison to exponential feed algorithms commonly used in industry, the implementation of the new feeding strategy increased the final cell density from 32 to 44 g (dry cell weight).L(-1), with less than 16 mM acetate accumulated, producing an ideal culture for subsequent induction. Despite a constant starvation level and relatively low levels of acetate, experimental cultivations still tended to produce acetate towards the end of the process. The use of a simple Monod model provided an explanation as to why this may occur in high cell density cultivations and suggests how it may be overcome.     

Change of extracellular cAMP concentration is a sensitive reporter for bacterial fitness in high-cell-density cultures of Escherichia coli

Guanosine-3',5'-tetraphosphate (ppGpp) and sigmaS, two regulators of the starvation response of Escherichia coli, have received increasing attention for monitoring cell physiological changes in production processes, although both are difficult to quantify. The kinetics of cAMP formation and degradation were not yet investigated in such processes, although the complex regulation of cAMP by synthesis, release, and degradation in connection with straightforward methods for analysis renders it a highly informative target. Therefore, we followed the cAMP concentration in various nonrecombinant and in four different recombinant glucose-limited fed-batch processes in different production scales. The intracellular cAMP concentration increases strongly at the end of the batch phase. Most cAMP is released to the cultivation medium. The rates of accumulation and degradation of extracellular cAMP are growth-rate-dependent and show a distinct maximum at a growth rate of about 0.35 h(-1). At very low growth rates, below 0.05 h(-1), extracellular cAMP is not produced but rather degraded, independent of whether this low growth rate is caused by glucose limitation or by the high metabolic load of recombinant protein production. In contrast to intracellular cAMP, which is highly unstable, analysis of extracellular cAMP is simpler and the kinetics of accumulation and degradation reflect well the physiological situation, including unlimited growth, limitation, and severe starvation of a production host.     

不同耕作方式对内蒙古旱作农田土壤水热状况的影响

为了研究免耕留茬覆盖和传统耕作土壤水热状况的差异,20052008年在内蒙古呼和浩特市清水河县进行定位试验,结果表明:(1)免耕留茬覆盖能有效的调节土壤温度,出现高温时抑制温度上升,低温时抑制温度降低的现象;土壤温度24h变化的振幅随着土壤深度的增加呈现逐渐减小的趋势,表层010cm的变化幅度最大,变化范围为17.634℃;不同耕作方式的土壤温度均从09:00开始升高,至15:00达到最高值,之后缓慢下降,到06:00达到最低值;土壤温度随季节动态变化的趋势,受制于气候。(2)测定期内免耕留高茬覆盖平均土壤含水量较传统耕作增加了7.37%,免耕留低茬覆盖较传统耕作增加了5.93%;燕麦苗期,不同耕作方式的土壤含水量随土层深度增加,呈增大的态势。分蘖期,不同耕作方土壤含水量在020cm呈下降趋势,之后逐渐升高。燕麦花期,不同耕作方土壤含水量在040cm呈下降趋势,之后逐渐上升,至80100cm土层深度达到最大值;土壤含水量主要受季节影响,随降雨量变化而变化。(3)免耕留茬覆盖耕作方式增加土壤有机质和土壤含水量,其热容量增大,因此能调节和保持温度变化平缓。     

基于遗传算法和支持向量机的乳杆菌发酵过程建模

由于生化反应过程的复杂性和高度非线性,多数简单的数学模型不能准确描述。该文基于Matlab软件,利用改进的支持向量机(υ-SVR)对植物乳酸杆菌发酵这一典型生化过程进行研究,应用遗传算法估计模型最优参数,建立植物乳杆菌的菌体密度预测模型。同时建立传统的logistic动力学模型以进行比较。结果表明,采用结合遗传算法的υ-SVR预测模型拟合误差小,皮尔森相关系数(R)更高,可以较好地预测乳酸杆菌的发酵过程,为其优化控制及放大提供依据。     

Plasmid‐free T7‐based Escherichia coli expression systems

In order to release host cells from plasmid‐mediated increases in metabolic load and high gene dosages, we developed a plasmid‐free, T7‐based E. coli expression system in which the target gene is site‐specifically integrated into the genome of the host. With this system, plasmid‐loss, a source of instability for conventional expression systems, was eliminated. At the same time, system leakiness, a challenging problem with recombinant systems, was minimized. The efficiency of the T7 RNA polymerase compensates for low gene dosage and provides high rates of recombinant gene expression without fatal consequences to host metabolism. Relative to conventional pET systems, this system permits improved process stability and increases the host cell's capacity for recombinant gene expression, resulting in higher product yields. The stability of the plasmid‐free system was proven in chemostat cultivation for 40 generations in a non‐induced and for 10 generations in a fully induced state. For this reason plasmid‐free systems benefit the development of continuous production processes with E. coli. However, time and effort of the more complex cloning procedure have to be considered in relation to the advantages of plasmid‐free systems in upstream‐processing. Biotechnol. Bioeng. 2010. 105: 786–794. © 2009 Wiley Periodicals, Inc.     

Profiling of N‐glycosylation gene expression in CHO cell fed‐batch cultures

One of the goals of recombinant glycoprotein production is to achieve consistent glycosylation. Although many studies have examined the changes in the glycosylation quality of recombinant protein with culture, very little has been done to examine the underlying changes in glycosylation gene expression as a culture progresses. In this study, the expression of 24 genes involved in N‐glycosylation were examined using quantitative RT PCR to gain a better understanding of recombinant glycoprotein glycosylation during production processes. Profiling of the N‐glycosylation genes as well as concurrent analysis of glycoprotein quality was performed across the exponential, stationary and death phases of a fed‐batch culture of a CHO cell line producing recombinant human interferon‐γ (IFN‐γ). Of the 24 N‐glycosylation genes examined, 21 showed significant up‐ or down‐regulation of gene expression as the fed‐batch culture progressed from exponential, stationary and death phase. As the fed‐batch culture progressed, there was also an increase in less sialylated IFN‐γ glycoforms, leading to a 30% decrease in the molar ratio of sialic acid to recombinant IFN‐γ. This correlated with decreased expression of genes involved with CMP sialic acid synthesis coupled with increased expression of sialidases. Compared to batch culture, a low glutamine fed‐batch strategy appears to need a 0.5 mM glutamine threshold to maintain similar N‐glycosylation genes expression levels and to achieve comparable glycoprotein quality. This study demonstrates the use of quantitative real time PCR method to identify possible “bottlenecks” or “compromised” pathways in N‐glycosylation and subsequently allow for the development of strategies to improve glycosylation quality. Biotechnol. Bioeng. 2010;107: 516–528. © 2010 Wiley Periodicals, Inc.     

Model‐based high cell density cultivation of Rhodospirillum rubrum under respiratory dark conditions

The potential of facultative photosynthetic bacteria as producers of photosynthetic pigments, vitamins, coenzymes and other valuable products has been recognized for decades. However, mass cultivation under photosynthetic conditions is generally inefficient due to the inevitable limitation of light supply when cell densities become very high. The previous development of a new cultivation process for maximal expression of photosynthetic genes under semi‐aerobic dark conditions in common bioreactors offers a new perspective for utilizing the facultative photosynthetic bacterium Rhodospirillum rubrum for large‐scale applications. Based on this cultivation system, the present study aimed in determining the maximal achievable cell density of R. rubrum in a bioreactor, thereby providing a major milestone on the way to industrial bioprocesses. As a starting point, we focus on aerobic growth due to higher growth rates and more facile process control under this condition, with the option to extend the process by an anaerobic production phase. Process design and optimization were supported by an unstructured computational process model, based on mixed‐substrate kinetics. Key parameters for growth and process control were determined in shake‐flask experiments or estimated by simulation studies. For fed‐batch cultivation, a computer‐controlled exponential feed algorithm in combination with a pH‐stat element was implemented. As a result, a maximal cell density of 59 g cell dry weight (CDW) L^?1 was obtained, representing so far not attainable cell densities for photosynthetic bacteria. The applied exponential fed‐batch methodology therefore enters a range which is commonly employed for industrial applications with microbial cells. The biochemical analysis of high cell density cultures revealed metabolic imbalances, such as the accumulation and excretion of tetrapyrrole intermediates of the bacteriochlorophyll biosynthetic pathway. Biotechnol. Bioeng. 2010. 105: 729–739. © 2009 Wiley Periodicals, Inc.     

Polymeric and Compositional Properties of Novel Extracellular Microbial Polyglucosamine Biopolymer from New Strain of Citrobacter sp. BL-4

A novel polyglucosamine polymer, PGB-2, was produced extracellularly from a new strain Citrobacter sp. BL-4 using pH-stat fed batch cultivation. It was composed of 97.3% glucosamine and 2.7% rhamnose; its average molecular weight, solubility in 2% acetic acid and viscosity were 20 kDa, 5 g l⁻¹ and 2.9 cps, respectively. FT-IR and ¹H NMR spectra of PGB-2 revealed a close identity with chitosan from crab shells. Received 20 September 2005; Revisions requested 6 October 2005; Revisions received 16 November 2005; Accepted 16 November 2005     

Comparison of cell lines for stable production of fucose-negative antibodies with enhanced ADCC

Several methods have been described to enhance antibody-dependent cellular cytotoxicity (ADCC) using different host cells that produce antibody with reduced levels of fucose on their carbohydrates. We compared the suitability of these methods for the serum-free fed-batch production of antibody for clinical trials and commercial uses. Recombinant anti-human CD20 chimeric IgG1-producing clones were established from host-cells that have been shown to produce more than 90% fucose-negative antibody. The cell lines were a FUT8 (alpha-1,6-fucosyltransferase) knockout Chinese hamster ovary (CHO) cell line, Ms704, and two Lens culinaris agglutinin (LCA)-resistant cell lines, one derived from a variant CHO line, Lec13 and the other from a rat hybridoma cell line, YB2/0. The amount of fucose-negative antibody produced by Lec13 and YB2/0 significantly decreased with the culture. The increase in fucosylation was due to remaining synthesis of GDP-fucose via de novo pathway for the CHO line and the elevation of FUT8 expression by the YB2/0 cells. In contrast, Ms704 cells stably produced fucose-negative antibody with a consistent carbohydrate structure until the end of the culture. The productivity of the Ms704 cells reached 1.76 g/L with a specific production rate (SPR) of 29 pg/cell/day for 17 days in serum-free fed-batch culture using a 1 L spinner bioreactor. Our results demonstrate that FUT8 knockout has the essential characteristics of host cells for robust manufacture of fucose-negative therapeutic antibodies with enhanced ADCC.