重组大肠杆菌高密度发酵研究进展

重组大肠杆菌的高密度发酵是提高基因工程产品产量的一个非常有效的手段,是现代发酵工程研究的一个热点。本文就高密度发酵中影响重组大肠杆菌发酵产率的几个因素,包括宿主菌、培养基、培养条件、补料方法以及高密度发酵过程中存在的问题和对策加以讨论,着重探讨了高密度下大肠杆菌产生的有害代谢副产物———乙酸的产生机制、抑制作用机理,以及控制乙酸积累的技术方法 。     

High cell density fed-batch fermentations for lipase production: feeding strategies and oxygen transfer

This review is focused on the production of microbial lipases by high cell density fermentation. Lipases are among the most widely used of the enzyme catalysts. Although lipases are produced by animals and plants, industrial lipases are sourced almost exclusively from microorganisms. Many of the commercial lipases are produced using recombinant species. Microbial lipases are mostly produced by batch and fed-batch fermentation. Lipases are generally secreted by the cell into the extracellular environment. Thus, a crude preparation of lipases can be obtained by removing the microbial cells from the fermentation broth. This crude cell-free broth may be further concentrated and used as is, or lipases may be purified from it to various levels. For many large volume applications, lipases must be produced at extremely low cost. High cell density fermentation is a promising method for low-cost production: it allows a high concentration of the biomass and the enzyme to be attained rapidly and this eases the downstream recovery of the enzyme. High density fermentation enhances enzyme productivity compared with the traditional submerged culture batch fermentation. In production of enzymes, a high cell density is generally achieved through fed-batch operation, not through perfusion culture which is cumbersome. The feeding strategies used in fed-batch fermentations for producing lipases and the implications of these strategies are discussed. Most lipase-producing microbial fermentations require oxygen. Oxygen transfer in such fermentations is discussed.     

毕赤酵母高密度发酵工艺的研究

高密度发酵是毕赤酵母提高蛋白表达量的一种重要策略,发酵工艺是高密度发酵的一个重要因素。采用下列措施均可以有效地提高表达水平：调节基础培养基,采用变pH和变温发酵,提高DO,选择最适的诱导前菌体密度和比生长速率并降低甘油初始浓度和采用分段式指数流加进行调控。选择合适的甲醇补料策略：甲醇限制补料（MLFB）、氧气限制补料（OLFB）、甲醇不限制补料（MNLFB）和温度限制补料（TLFB）。采用两种方式调控补料：诱导阶段菌体生长时,甲醇比消耗速率(qMeOH)为0.02-0.03gg-1h-1,而菌体不生长时,qMeOH采用较高值。     

补加前体L-蛋氨酸对高密度发酵生产S-腺苷-L-蛋氨酸的影响

将高密度发酵技术成功应用于S-腺苷-L-蛋氨酸的生产。考察了补加前体L-蛋氨酸的量以及补加策略对酿酒酵母G14发酵生产S-腺苷-L-蛋氨酸的影响。实验发现补加前体L-蛋氨酸能明显促进S-腺苷-L-蛋氨酸的积累。同时还发现不同的补加策略对菌体浓度以及S-腺苷-L-蛋氨酸的产量和浓度有不同的影响。确定了补加L-蛋氨酸不应低于0.7g/10g菌体干重。比较了五种不同的补加前体L-蛋氨酸的方式。结果表明在菌体干重达到高密度的情况下(120g/L)补加前体L-蛋氨酸进行转化生产S-腺苷-L-蛋氨酸能达到比较好的效果一次性补加9g L-蛋氨酸,SAM的积累量在补加后的18h达到最高,为4.31g/L;采取流加方式补加L-蛋氨酸,流加速率为2g/h,共流加5h,流加结束28h后SAM达到最高积累量后者达到4.98g/L。两者最终的生物量均可达到130g/L以上。     

代谢工程与重组大肠杆菌的发酵

利用代谢工程可以在重组大肠杆菌的改良中减少代谢副产物乙酸的累积,优化代谢系统,利于重组蛋白质的高表达以及重组菌的高密度发酵。应用代谢工程改良重组大肠杆菌主要包括阻断乙酸产生的主要途径、限制糖酵解途径上的碳代谢流、将过量的丙酮酸转化为其它低毒的副产物以及对碳代谢流进行分流等几个方面的工作。     

透析培养

本讨论了有效利用透析技术从发酵液中及时转移低分子杂质混合物,从而获得高密度发酵细胞的方法,章从反应系统、工艺策略、膜相关性能、应用我、生产性放大等方面说明了利用透析技术以达到高浓度细胞发酵的有效性和可靠性。透析技术不仅克服了微孔过滤和超滤中存在的膜孔堵塞弊端,而且如果应用“营养分离”补策略,还可以防止营养物质的损失而使培养基被高效利用,在实验条件下,透析培养的潜力通过两种反应模型进行演示：内置     

基因工程菌高密度发酵工艺研究进展

阐述了基因工程菌高密度发酵工艺的几个主要影响因素,包括重组菌构建、培养条件、生长抑制因子以及它们的控制技术。通过高密度发酵可以提高细胞生长密度、目的蛋白的表达含量。在高密度发酵过程中,会产生一些有害抑制代谢副产物,但通过分批补料可以降低影响。     

Effects of fermentation feeding strategies prior to induction of expression of a recombinant malaria antigen inEscherichia coli

Summary A variety of feeding strategies have been described for attaining high cell densities in fed-batch fermentors. Although cell density is an important component in the produtivity of recombinant fermentations, it must be achievable with high product expression levels. Experiments were conducted to study the influence of fermentation feeding strategies on the production of a recombinant malaria antigen inEscherichia coli. C-source feeding profiles were calculated to maintain specific growth rates at 0.1, 0.2, 0.35, and 0.5 l/h prior to induction in defined and complex media using an exponential growth model. Fed-batch fermentations employing these feeding profiles effectively controlled the specific growth rates prior to induction. Antigen yields per dry cell weight did not vary with specific growth rate. Antigen yields from fed-batch fermentations achieving high cell densities were similar to batch fermentations achieving low cell densities. These results show that C-feeding policies can limit growth without reducing expression levels in some systems, and suggest applications in managing oxygen demand and catabolic by-product formation during process scale-up.     

Dialysis cultures

Dialysis techniques are discussed as a means for effective removal of low-molecular-mass components from fermentation broth to reach high cell density. Reactor systems and process strategies, the relevant properties of membranes and examples for high-density fermentation with dialysis, and problems related to scale-up are addressed. The dialysis technique has turned out to be very efficient and reliable for obtaining high cell densities. As in dialysis processes the membranes are not perfused, membrane clogging is not a problem as it is for micro- and ultrafiltration. By applying a “nutrient-split” feeding strategy, the loss of nutrients can be avoided and the medium is used very efficiently. The potential of dialysis cultures is demonstrated on the laboratory scale in a membrane dialysis reactor with an integrated membrane and in reactor systems with an external dialysis loop. In dialysis cultures with different microorganisms (Staphylococci, Escherichia coli, extremophilic microorganisms, Lactobacilli) the cell densities achieved were up to 30 times higher than those of other fermentation methods. The technique enables high cell densities to be attained without time-consuming medium optimization. For animal cell cultures the concept of a fixed bed coupled with dialysis proved to be very effective. Received: 24 March 1998 / Received revision: 18 June 1998 / Accepted: 19 June 1998     

Stability of high cell density brewery fermentations during serial repitching

The volumetric productivity of the beer fermentation process can be increased by using a higher pitching rate (i.e. higher inoculum size). However, the decreased yeast net growth observed in these high cell density brewery fermentations can adversely affect the physiological stability throughout subsequent yeast generations. Therefore, different O₂ conditions (wort aeration and yeast preoxygenation) were applied to high cell density fermentation and eight generations of fermentations were evaluated together with conventional fermentations. Freshly propagated high cell density populations adapted faster to the fermentative conditions than normal cell density populations. Preoxygenating the yeast was essential for the yeast physiological and beer flavor compound stability of high cell density fermentations during serial repitching. In contrast, the use of non-preoxygenated yeast resulted in inadequate growth which caused (1) insufficient yield of biomass to repitch all eight generations, (2) a 10% decrease in viability, (3) a moderate increase of yeast age, (4) and a dramatic increase of the unwanted flavor compounds acetaldehyde and total diacetyl during the sequence of fermentations. Therefore, to achieve sustainable high cell density fermentations throughout the economical valuable process of serial repitching, adequate yeast growth is essential.     

Influences of yeast extract on specific cellular yield of Ovine growth hormone during fed-batch fermentation of E. coli

In most cases of E. coli high cell density fermentation process, maximizing cell concentration helps in increasing the volumetric productivity of recombinant proteins usually at the cost of lower specific cellular protein yield. In this report, we describe a process for maintaining the specific cellular yield of Ovine growth hormone (oGH) from E. coli by optimal feeding of yeast extract during high cell density fermentation process. Recombinant oGH was produced as inclusion bodies in Escherichia coli. Specific cellular yield of recombinant oGH was maintained by feeding yeast extract along with glucose during fed-batch fermentation. Glucose to yeast extract ratio of 0.75 was found to be optimum for maintaining the specific cellular oGH yield of 66 mg/g of E. coli cells. Continuous feeding of yeast extract along with glucose helped in reducing acetic acid secretion and promoted higher cell growth during fed-batch fermentation. High cell growth of E. coli and high specific yield of recombinant oGH thus helped in achieving high volumetric productivity of the expressed protein. A maximum of 2 g/l of ovine growth hormone was expressed as inclusion bodies in 12 h of fed-batch fermentation.     

A reliable method for detecting the intracellular accumulation of fermentation products: Application to intracellular ethanol analysis

Summary Intracellular ethanol concentrations inSaccharomyces cerevisiae were measured using a high cell density fermentation technique which permits samples to be fixed for analysis in less than two seconds. No accumulation of intracellular ethanol was detected, regardless of the stage of fermentation. The technique eliminates artifacts associated with concentrating the cell sample and provides a reliable means for detecting whether other fermentation products accumulate intracellularly.     

Production of a platelet aggregation inhibitor, salmosin, by high cell density fermentation of recombinant Escherichia coli

Optimal conditions for a high cell density fermentation were investigated in a recombinant Escherichia coli producing salmosin, a platelet aggregation inhibitor. The optimized carbon and nitrogen sources were glycerol 10 g/l, yeast extract 30 g/l, and bacto-tryptone 10 g/l, yielding the dry cell weight (DCW) of 10.61 g/l in a 500 ml flask culture. The late-stage induction with 1% L-arabinose in a 5 l jar fermentor showed the highest DCW of 65.70 g/l after 27 h of the fed-batch fermentation. Around 2,200 mg/l of the protein was expressed as an inclusion body that was then refolded to obtain the active salmosin of 96 mg/l. We also confirmed the inhibitory activity against platelet aggregation of the active salmosin from the high cell density fermentation.     

Recent advances in polyhydroxyalkanoate production by bacterial fermentation: mini-review.

Poly(3-hydroxybutyrate) [P(3HB)] and other polyhydroxyalkanoates (PHAs) have been drawing much attention as biodegradable substitutes for conventional nondegradable plastics. For the economical production of P(3HB), various bacterial strains, either wild-type or recombinant, and new fermentation strategies were developed for the production of P(3HB) with high concentration and productivity. To reduce the cost of carbon substrate, several processes for P(3HB) production from cheap carbon sources were also developed. P(3HB) can now be produced to a content of 80% of cell dry weight with the productivity greater than 4 g/l per h. Fermentation strategy was also developed for the efficient production of medium chain length PHA by high cell density culture. With all these advances, P(3HB) and PHAs can be produced by bacterial fermentation at a cost (ca. $2/kg) similar to that of other biodegradable polymers under development.     

The role of oxygen in yeast metabolism during high cell density brewery fermentations

The volumetric productivity of the beer fermentation process can be increased by using a higher pitching rate (i.e., higher inoculum size). However, the decreased yeast net growth observed in these high cell density fermentations can have a negative impact on the physiological stability throughout subsequent yeast generations. The use of different oxygen conditions (wort aeration, wort oxygenation, yeast preoxygenation) was investigated to improve the growth yield during high cell density fermentations and yeast metabolic and physiological parameters were assessed systematically. Together with a higher extent of growth (dependent on the applied oxygen conditions), the fermentation power and the formation of unsaturated fatty acids were also affected. Wort oxygenation had a significant decreasing effect on the formation of esters, which was caused by a decreased expression of the alcohol acetyl transferase gene ATF1, compared with the other conditions. Lower glycogen and trehalose levels at the end of fermentation were observed in case of the high cell density fermentations with oxygenated wort and the reference fermentation. The expression levels of BAP2 (encoding the branched chain amino acid permease), ERG1 (encoding squalene epoxidase), and the stress responsive gene HSP12 were predominantly influenced by the high cell concentrations, while OLE1 (encoding the fatty acid desaturase) and the oxidative stress responsive genes SOD1 and CTT1 were mainly affected by the oxygen availability per cell. These results demonstrate that optimisation of high cell density fermentations could be achieved by improving the oxygen conditions, without drastically affecting the physiological condition of the yeast and beer quality.     

Continuous production of acetone and butanol by Clostridium acetobutylicum growing in turbidostat culture

Turbidostat cultures of Clostridium acetobutylicum were analysed with respect to their fermentation products after steady states were obtained at various cell densities. It was found that at low densities the fermentation of glucose was essentially acidogenic in nature, whereas acetone and butanol were the major end-products when the cultures were maintained at a high cell density.     

代谢副产物乙酸对L-色氨酸发酵的影响

分析了重组大肠杆菌(E.coli TRTH/pSV-709)发酵生产L-色氨酸的发酵过程,检测结果表明发酵液中有大量代谢副产物乙酸的积累。利用外源添加试验研究了乙酸对L-色氨酸发酵的影响,结果表明乙酸浓度高于2g/L时对L-色氨酸生产菌的生长和产酸均有抑制作用。分析了乙酸的产生机制,并采取了调节溶氧水平、确定合适初始葡萄糖浓度、限制葡萄糖流加及控制菌体比生长速率等措施来减少乙酸的生成。在优化条件下,乙酸含量与原工艺相比降低了51.35%,菌体生物量和L-色氨酸产量分别提高了51.07%和46.54%,实现了高密度发酵培养的目的。     

酿酒酵母表达系统

酿酒酵母是单细胞真核微生物,人们以酿酒酵母为宿主菌,用载体表达外源基因的过程中,积累了丰富的经验,掌握了酿酒酵母表达的许多优缺点,如它繁殖速度快,可以大规模发酵生产,但在发酵过程中会产生乙醇,而乙醇在培养基中积累会影响酵母的生长代谢和基因产物的表达,尤其是进行高密度发酵时该效应更明显;针对这些缺点,可以采取积极的应对措施.主要就酿酒酵母表达系统的组成、优缺点及高效表达的策略等作一综述.     

Comparison of fluidized bed and ultrasonic cell-retention systems for high cell density mammalian cell culture

Economically viable biopharmaceutical production is to a high degree dependent on high product yields and stable fermentation systems that are easy to handle. In the current study we have compared two different fermentation systems for the production of recombinant protein from CHO cells. Both systems are fully scaleable and can be used for industrial high cell density bioprocesses. As a model cell line we have used a recombinant CHO cell line producing the enzyme arylsulfatase B (ASB). CHO cells were cultivated as adherent cell culture attached on Cytoline macroporous microcarrier (Amersham Biosciences, Sweden) using a Cytopilot Mini fluidized bed bioreactor (FBR, Vogelbusch-Amersham Biosciences, Austria) and as suspension culture using a stirred tank bioreactor equipped with a BioSep ultrasonic resonator based cell separation device (Applikon, The Netherlands). Both systems are equally well-suited for stable, long-term high cell density perfusion cell culture and provide industrial scalability and high yields. For products such as the recombinant ASB, high perfusion rates and therefore short product bioreactor residence times may be of additional benefit.