Optimum conditions for production and purification of human papillomavirus type 16 L1 protein from Saccharomyces cerevisiae

Several prophylactic human papillomavirus (HPV) vaccines have been developed based on virus-like particles (VLPs) made from viral L1 proteins. A substantial number of VLPs is necessary for biochemical characterization and diagnostic test development. To establish the optimum conditions for production and purification of HPV L1 in the yeast expression system we varied the amount and nature of the carbon source and evaluated HPV 16 L1 recovery by three purification methods. Maximally threefold more HPV 16 L1 was produced with a 4% carbon source than with a 2% carbon source. In addition, the productivity of HPV 16 L1 varied by 25% depending on the combination of glucose and galactose in the 4% carbon source. We introduced an ammonium sulfate precipitation step in place of the ultracentrifugation using a sucrose cushion routinely used for HPV L1 purification, and optimized the purification by cation-exchange chromatography. Overall L1 protein recovery using the ammonium sulfate precipitation method was 30%, the highest recovery achieved so far. The purified HPV 16 L1 protein successfully self-assembled into VLPs. Purification by ammonium sulfate precipitation was maximally 15 times greater than ultracentrifugation on a sucrose cushion. We anticipate that our procedures for production and purification will reduce the cost, time and labor involved in obtaining sufficient yields of VLPs.     

Reduced Lysis upon Growth of Lactococcus lactis on Galactose Is a Consequence of Decreased Binding of the Autolysin AcmA

When Lactococcus lactis subsp. lactis IL1403 or L. lactis subsp. cremoris MG1363 is grown in a medium with galactose as the carbon source, the culture lyses to a lesser extent in stationary phase than when the bacteria are grown in a medium containing glucose. Expression of AcmA, the major autolysin of L. lactis, is not influenced by the carbon source. Binding studies with a fusion protein consisting of the MSA2 protein of Plasmodium falciparum and the C-terminal peptidoglycan-binding domain of AcmA revealed that cell walls of cells from both subspecies grown on galactose bind less AcmA than cell walls of cells grown on glucose. Cells grown on glucose or galactose and treated with trichloroacetic acid prior to AcmA binding bind similar amounts of AcmA. Analysis of the composition of the lipoteichoic acids (LTAs) of L. lactis IL1403 cells grown on glucose or galactose showed that the LTA composition is influenced by the carbon source: cells grown on galactose contain LTA with less galactose than cells grown on glucose. In conclusion, growth of L. lactis on galactose changes the LTA composition in the cell wall in such a way that less AcmA is able to bind to the peptidoglycan, resulting in a decrease in autolysis.     

益生菌体外增殖因子的初步研究

目的研究单糖、pH、温度及时间对青春双歧杆菌、长双歧杆菌和类干酪乳杆菌体外增殖的影响。方法用甘露糖、半乳糖、山梨醇及果糖代替MRS中的葡萄糖,筛选出每种细菌的最适碳源。以此为基础,选择其最佳初始pH、培养温度、碳源添加量及培养时间。结果青春双歧杆菌、长双歧杆菌和类干酪乳杆菌的最适碳源分别为葡萄糖、甘露糖和半乳糖;最佳初始pH为6.0、7.0和6.0;培养温度为42、30和30℃;碳源添加量为20、15和25 g/L;培养时间都为28-48 h。结论益生菌具有不同的最适增殖条件,本文研究结果为优化益生菌的生长条件提供了基础数据。     

Expression, purification, and antibody binding activity of human papillomavirus 16 L1 protein fused to maltose binding protein

Genetic human papillomavirus type 16 L1 (HPV16 L1) has been widely studied for cervical cancer vaccine development. For the enzyme-linked immunosorbent assay (ELISA) screening of these vaccines, HPV16 L1 protein, which is required as a coating protein, has previously been expressed from costly and laborious recombinant baculovirus-infected insect cells. For a novel HPV16 L1 expression system characterized by a high yield of soluble form with simple purification steps, we have cloned and expressed two different types of HPV16 L1, both fused to maltose binding protein (MBP) or glutathione-S-transferase (GST) in Escherichia coli. The yield of soluble HPV16 L1 was influenced by the cultivation temperature. The yield of soluble form in the total MBP-fused HPV16 L1 protein (MBP-HPV16 L1) was 35% at 37 degrees C, but increased to 85% at 22 degrees C. Among the fusion partners, MBP provided higher yields of total and soluble HPV16 L1 than did GST. MBP-HPV16 L1 showed a 4.9-fold higher yield of the soluble form over insoluble inclusion bodies under optimized culture conditions. The soluble form of MBP-HPV16 L1 was purified via MBP affinity chromatography in a recovery yield of 9.7%. After fusion with MBP, HPV16 L1 showed binding activity to HPV16 L1-specific monoclonal antibody comparable to HPV16 L1 from the insect cells in ELISA tests. These results demonstrate that the use of MBP as a fusion partner may generate a high yield of soluble HPV16 L1 under optimized temperature conditions, and that MBP-fused HPV16 L1 might be applied further in evaluations of the immune responses of HPV16 L1-based cervical cancer vaccines.     

Exopolysaccharides, proteins and lipids in Pleurotus pulmonarius submerged culture using different carbon sources

For many years mushrooms have been consumed and appreciated by their nutritional value, and medicinal properties. The traditional mushroom cultivation takes too long and the macrofungi biotechnology has not been explored in its full potential yet. The goal of this work was to observe if different carbon sources could improve the yield and diversify fungi nutrient composition in submerged culture.Pleurotus pulmonarius mycelia and exopolysacharide productions were evaluated using glucose, galactose, xylose and arabinose. The mycelia yield varied depending on the culture medium, and galactose showed to be the best carbon source to produce EPS. Samples that showed the highest protein contents were grown with xylose (19.44%) and arabinose (26.05%). Furthermore, the biomass cultivated with these carbohydrates and with galactose showed five essential amino acids. All cultured biomass showed low lipid contents (∼1%), being composed mainly of unsaturated fatty acids. All EPS fractions showed as main structures glucans and mannogalactans.     

Application of a gratuitous induction system in Kluyveromyces lactis for the expression of intracellular and secreted proteins during fed-batch culture

A gratuitous induction system in the yeast Kluyveromyces lactis was evaluated for the expression of intracellular and extracellular products during fed-batch culture. The Escherichia coli lacZ gene (beta-galactosidase; intracellular) and MFalpha1 leader-BPTI cassette (bovine pancreatic trypsin inhibitor; extracellular) were placed under the control of the inducible K. lactis LAC4 promotor, inserted into partial-pKD1 plasmids, and transformed into a ga1-209 K. lactis strain. To obtain a high level of production, culture conditions for growth and expression were initially evaluated in tube cultures. A selective medium containing 5 g/L glucose (as carbon source) and 0.5 g/L galactose (as inducer) demonstrated the maximum activity of both beta-galactosidase and secreted BPTI. This level of expression had no significant effect on the growth of the recombinant cells; growth rate dropped by approximately 11%, whereas final biomass concentrations remained the same. In shake-flask culture, biomass concentration, beta-galactosidase activity, and BPTI secreted activity were 4 g/L, 7664 U/g dry cell, and 0.32 mg/L, respectively. Fed-batch culture (with a high glucose concentration and a low galactose [inducer] concentration feed) resulted in a 6.5-fold increase in biomass, a 23-fold increase in beta-galactosidase activity, and a 3-fold increase in BPTI secreted activity. The results demonstrate the success of gratuitous induction during high-cell-density fed-batch culture of K. lactis. A very low concentration of galactose feed was sufficient for a high production level.     

Modeling of Xanthophyllomyces dendrorhous growth on glucose and overflow metabolism in batch and fed-batch cultures for astaxanthin production

An astaxanthin-producing yeast Xanthophyllomyces dendrorhous ENM5 was cultivated in a liquid medium containing 50 g/L glucose as the major carbon source in stirred fermentors (1.5-L working volume) in fully aerobic conditions. Ethanol was produced during the exponential growth phase as a result of overflow metabolism or fermentative catabolism of glucose by yeast cells. After accumulating to a peak of 3.5 g/L, the ethanol was consumed by yeast cells as a carbon source when glucose in the culture was nearly exhausted. High initial glucose concentrations and ethanol accumulation in the culture had inhibitory effects on cell growth. Astaxanthin production was partially associated with cell growth. Based on these culture characteristics, we constructed a modified Monod kinetic model incorporating substrate (glucose) and product (ethanol) inhibition to describe the relationship of cell growth rate with glucose and ethanol concentrations. This kinetic model, coupled with the Luedeking-Piret equation for the astaxanthin production, gave satisfactory prediction of the biomass production, glucose consumption, ethanol formation and consumption, and astaxanthin production in batch cultures over 25-75 g/L glucose concentration ranges. The model was also applied to fed-batch cultures to predict the optimum feeding scheme (feeding glucose and corn steep liquor) for astaxanthin production, leading to a high volumetric yield (28.6 mg/L) and a high productivity (5.36 mg/L/day).     

The production and immunogenicity of human papillomavirus type 58 virus-like particles produced in Saccharomyces cerevisiae

Human papillomavirus (HPV) is the cause of most cases of cervical cancer. HPV type 58 (HPV58) is the second most frequent cause of cervical cancer and high-grade squamous intraepithelial lesions (HSIL) in Asia and South / Central America, respectively. However, there is no vaccine against HPV58, although there are commercially available vaccines against HPV16 and 18. In this study, we produced HPV58 L1 protein from Saccharomyces cerevisiae, and investigated its immunogenicity. We first determined the optimum period of culture for obtaining HPV58 L1. We found that a considerable portion of the HPV58 L1 resulting from 48 h culture cannot be recovered by purification, while the HPV58 L1 resulting from 144 h culture is recovered efficiently: the yield of HPV58 L1 finally recovered from 144 h culture was 2.3 times higher than that from 48 h culture, although the production level of L1 protein from 144 h culture was lower than that from 48 h culture. These results indicate that the proportion of functional L1 protein from 144 h-cultured cells is significantly higher than that of 48 h-cultured cells. The HPV58 L1 purified from the 144 h culture was correctly assembled into structures similar to naturally occurring HPV virions. Immunization with the HPV58 L1 efficiently elicited anti-HPV58 neutralizing antibodies and antigen-specific CD4+ and CD8+ T cell proliferations, without the need for adjuvant. Our findings provide a convenient method for obtaining substantial amounts of highly immunogenic HPV58 L1 from S. cerevisiae.     

Growth Profile of Crown Gall Cells of Tobacco in Suspension Culture

In order to obtain a basic information of plant cell suspension culture as a step toward the development of large scale culture, culture conditions of crown gall cells (auxin non-requiring cells) were investigated. Addition of yeast extract to culture medium was significantly effective for the growth and cell dispersion.

In experiments on the ability of the cultured cells to utilize sugars as the carbon source, it was observed that galactose, added to the culture medium, markedly inhibited the cell growth.

Pasteurization of the medium containing fructose as carbon source made it brownish by Maillard reaction and the medium apparently restrained the cell growth. However, the fructose medium sterilized by filtration was excellent for the cell growth as well as sucrose or glucose medium. In a jar fermentor, even the glucose medium became brownish by heat sterilization and the brown colored medium restrained the cell growth. Under optimum conditions, the doubling time was 1.1 day in exponential phase and 2.0 g of cell (dry weight) per 100 ml culture was obtained as the maximum yield.     

Glycan Residues Balance Analysis - GReBA: A novel model for the N-linked glycosylation of IgG produced by CHO cells

The structure of N-linked glycosylation is a very important quality attribute for therapeutic monoclonal antibodies. Different carbon sources in cell culture media, such as mannose and galactose, have been reported to have different influences on the glycosylation patterns. Accurate prediction and control of the glycosylation profile are important for the process development of mammalian cell cultures. In this study, a mathematical model, that we named Glycan Residues Balance Analysis (GReBA), was developed based on the concept of Elementary Flux Mode (EFM), and used to predict the glycosylation profile for steady state cell cultures. Experiments were carried out in pseudo-perfusion cultivation of antibody producing Chinese Hamster Ovary (CHO) cells with various concentrations and combinations of glucose, mannose and galactose. Cultivation of CHO cells with mannose or the combinations of mannose and galactose resulted in decreased lactate and ammonium production, and more matured glycosylation patterns compared to the cultures with glucose. Furthermore, the growth rate and IgG productivity were similar in all the conditions. When the cells were cultured with galactose alone, lactate was fed as well to be used as complementary carbon source, leading to cell growth rate and IgG productivity comparable to feeding the other sugars. The data of the glycoprofiles were used for training the model, and then to simulate the glycosylation changes with varying the concentrations of mannose and galactose. In this study we showed that the GReBA model had a good predictive capacity of the N-linked glycosylation. The GReBA can be used as a guidance for development of glycoprotein cultivation processes.     

Decoupling cell growth and product formation in Chinese hamster ovary cells through metabolic control.

The development of a strategy for the culture of Chinese hamster ovary (CHO) cells producing tissue plasminogen activator (t-PA) is investigated. This strategy is based on the replacement of the main carbon source, glucose, by another compound that is slowly metabolizable, particularly galactose. The introduction of this change allows for acute change in cell behavior at various levels. Cell growth is stopped after this nutrient shift, and the cells can be kept in long-duration culture at a low growth rate and high viability as compared with a culture strategy based solely on glucose utilization. Moreover, the capability of cells to produce recombinant proteins (t-PA in this work) can be maintained over the entire period of galactose feeding. From the metabolic point of view, use of a slowly metabolizable carbon source (galactose) introduces important changes in the production of lactate, ammonia, and some amino acids. The use of this metabolic shift enables the generation of biphasic processes, with a first phase with cell growth on glucose and a second stationary phase on galactose, which is particularly suited to perfusion systems.     

Development of a fed-batch cultivation for antibody-producing cells based on combined feeding strategy of glucose and galactose

In order to achieve enhanced cell mass and productivity with less lactate accumulation, a fed-batch culture based on a combined feeding strategy of glucose and galactose was developed. Cell performance was first examined with feeding of galactose alone. While cell growth was improved compared with glucose-feeding culture, cell maintenance was inefficient with rapid lactate depletion and considerable ammonium accumulation. Subsequently, to improve cell maintenance, a combined feeding strategy of glucose and galactose was proposed focusing on optimizing the ratio of glucose to galactose and feeding time. In addition, the compositions of amino acids and vitamins in feeding medium were refined for balanced supply of nutrients. With the combined feeding strategy, the metabolic shift of lactate from production to consumption occurred, but not accompanied by rapid lactate depletion and ammonium production. Furthermore, energy metabolism was more efficient and better utilization of carbon sources was achieved. Compared with the glucose-feeding culture in bioreactor, maximum lactate concentration was reduced by 55%; IVCC and the specific production rate of antibody were increased by 45% and 143%, respectively.     

The influence of conditions of growth on the endogenous metabolism of Saccharomyces cerevisiae: effect on protein,carbohydrate, sterol and fatty acid content and on viability

The nature of the endogenous reserves of Saccharomyces cerevisiae was examined with respect to conditions of growth, specifically extremes of oxygen tension and carbon source. Cells were grown in batch culture at 30 C under aerobic conditions on a galactose or glucose carbon source and under anaerobic conditions on glucose. The greatest effect of growth conditions on the chemical composition of the cells was on their fatty acid and sterol content.Cells grown under both aerobic and anaerobic conditions mobilised concurrently protein, glycogen, trehalose and fatty acids during a period of 72 hours' starvation under aerobic conditions. The viability of both types of the aerobically grown cells declined to 75% during this period and was not influenced by the initial fatty acid and sterol content of the cells. Cells grown anaerobically showed a more rapid decline in viability which was only 17% after 72 hours' starvation. This loss of viability was not due to a lack of available endogenous reserves but was probably due to an impaired membrane function caused by a deficiency of sterols and unsaturated fatty acids.     

Evaluation of Arthrospira platensis extracellular polymeric substances production in photoautotrophic, heterotrophic and mixotrophic conditions

The kinetic study of Arthrospira platensis extracellular polymeric substances (EPS) production under different trophic modes??photoautotrophy (100???mol photons m^?2?s^?1), heterotrophy (1.5?g/L glucose), and mixotrophy (100???mol photons m^?2?s^?1 and 1.5?g/L glucose)??was investigated. Under photoautotrophic and heterotrophic conditions, the maximum EPS production 219.61?±?4.73 and 30.30?±?1.97?mg/L, respectively, occurred during the stationary phase. Under a mixotrophic condition, the maximum EPS production (290.50?±?2.21?mg/L) was observed during the early stationary phase. The highest specific EPS productivity (433.62?mg/g per day) was obtained under a photoautotrophic culture. The lowest specific EPS productivity (38.33?mg/g per day) was observed for the heterotrophic culture. The effects of glucose concentration, light intensity, and their interaction in mixotrophic culture on A. platensis EPS production were evaluated by means of 32 factorial design and response surface methodology. This design was carried out with a glucose concentration of 0.5, 1.5, and 2.5?g/L and at light levels of 50, 100, and 150???mol photons m^?2?s^?1. Statistical analysis of the model demonstrated that EPS concentration and EPS yield were mainly influenced by glucose concentration and that conditions optimizing EPS concentration were dissimilar from those optimizing EPS yield. The highest maximum predicted EPS concentration (369.3?mg/L) was found at 150???mol photons m^?2?s^?1 light intensity and 2.4?g/L glucose concentration, while the highest maximum predicted EPS yield (364.3?mg/g) was recorded at 115???mol photons m^?2?s^?1 light intensity and 1.8?g/L glucose concentration.     

小球藻高密度培养及油脂提取条件的优化

【目的】高密度培养小球藻及优化油脂提取条件。【方法】通过进行单因素实验研究不同培养基组成及环境因子对其细胞生长影响,并采用超声波提取法进行正交实验对藻粉油脂提取条件进行研究。【结果】对椭圆小球藻Y4进行异养培养,最适培养条件为:葡萄糖50 g/L,硝酸钾2 g/L,适宜的培养温度、摇床转速和接种量分别为29°C、180 r/min和20%。在此基础上,进行了1 L发酵罐培养实验,获得了干重18.25 g/L的生物量。通过对油脂提取条件进行优化,Y4的油脂提取率由优化前的25.0%提高到60.2%,提高了35.2%。【结论】优化了小球藻的培养条件及油脂提取条件,促进了小球藻的开发和利用。     

Butanol production from corn fiber xylan using Clostridium acetobutylicum

Acetone, butanol, and ethanol (ABE) were produced from corn fiber arabinoxylan (CFAX) and CFAX sugars (glucose, xylose, galactose, and arabinose) using Clostridium acetobutylicum P260. In mixed sugar (glucose, xylose, galactose, and arabinose) fermentation, the culture preferred glucose and arabinose over galactose and xylose. Under the experimental conditions, CFAX (60 g/L) was not fermented until either 5 g/L xylose or glucose plus xylanase enzyme were added to support initial growth and fermentation. In this system, C. acetobutylicum produced 9.60 g/L ABE from CFAX and xylose. This experiment resulted in a yield and productivity of 0.41 and 0.20 g/L x h, respectively. In the integrated hydrolysis, fermentation, and recovery process, 60 g/L CFAX and 5 g/L xylose produced 24.67 g/L ABE and resulted in a higher yield (0.44) and a higher productivity (0.47 g/L x h). CFAX was hydrolyzed by xylan-hydrolyzing enzymes, and ABE were recovered by gas stripping. This investigation demonstrated that integration of hydrolysis of CFAX, fermentation to ABE, and recovery of ABE in a single system is an economically attractive process. It is suggested that the culture be further developed to hydrolyze CFAX and utilize all xylan sugars simultaneously. This would further increase productivity of the reactor.     

碳源对重组大肠杆菌发酵生产GLP-1融合蛋白的影响

以一株表达人胰高血糖素样肽-1融合蛋白的重组大肠杆菌为研究对象,首先通过摇瓶实验对碳源种类进行了初步选择,发现葡萄糖和甘油对菌体生长以及GLP-1融合蛋白表达较为适宜。进一步在5 L反应器上对初始葡萄糖及甘油浓度进行了考察,发现高浓度碳源有利于菌体生长却抑制GLP-1融合蛋白表达,但能提高GLP-1融合蛋白的体积得率。在0.25%初始葡萄糖或甘油存在的条件下,在培养过程中流加葡萄糖或甘油维持其在发酵液中的浓度,比较了两者对菌体生长以及GLP-1融合蛋白表达的影响,结果发现,以甘油为碳源时,菌体生长以及GLP-1融合蛋白的表达量均高于以葡萄糖为碳源的结果,最终发酵液的菌浓(OD_(600))可达到25.4,较葡萄糖为碳源时19.1提高了33.0%,GLP-1融合蛋白表达水平和体积得率分别可达到22.4%和1.051 g/L,较葡萄糖为碳源的15.8%和0.504 g/L分别提高41.8%和108.5%。该结果对GLP-1融合蛋白表达菌株发酵条件的进一步优化提供了依据。     

Comparison of productivity and quality of bacterial nanocellulose synthesized using culture media based on seven sugars from biomass

Komagataeibacter xylinus ATCC 23770 was statically cultivated in eight culture media based on different carbon sources, viz. seven biomass-derived sugars and one sugar mixture. The productivity and quality of the bacterial nanocellulose (BNC) produced in the different media were compared. Highest volumetric productivity, yield on consumed sugar, viscometric degree of polymerization (DP_v, 4350–4400) and thermal stability were achieved using media based on glucose or maltose. Growth in media based on xylose, mannose or galactose resulted in lower volumetric productivity and DP_v, but in larger fibril diameter and higher crystallinity (76–78%). Growth in medium based on a synthetic sugar mixture resembling the composition of a lignocellulosic hydrolysate promoted BNC productivity and yield, but decreased fibril diameter, DP_v, crystallinity and thermal stability. This work shows that volumetric productivity, yield and properties of BNC are highly affected by the carbon source, and indicates how industrially relevant sugar mixtures would affect these characteristics.     

碳源和氮源对5-酮基-葡萄糖酸生成的影响

氧化葡萄糖杆菌Gluconobacter oxydans可以将葡萄糖氧化成葡萄糖酸,并进一步氧化成2-酮基-葡萄糖酸(2KGA)和5-酮基-葡萄糖酸(5KGA),其中5KGA在催化剂的作用下能够转化为L(+)-酒石酸。为了提高5-酮基-葡萄糖酸产量,以仅生成5KGA的氧化葡萄糖杆菌Gluconobacter oxydans HGI-1为出发菌株,研究不同碳源(蔗糖、乳糖、麦芽糖、淀粉、葡萄糖)和有机氮源(酵母浸粉、鱼粉、玉米浆、黄豆饼粉、棉籽饼粉)对5KGA产量的影响。500 mL摇瓶试验结果表明,当葡萄糖浓度为100 g/L时,5KGA产量最高为98.20 g/L;当有机氮源为酵母浸粉、鱼粉和玉米浆,其添加量的蛋白含量为1.60%时,5KGA产量分别为100.20 g/L、109.10 g/L和99.83 g/L,其中,使用鱼粉的5KGA产量最高,使用玉米浆的5KGA产量比酵母浸粉略低。出于经济考虑,文中选择玉米浆作有机氮源,并在5 L发酵罐中进行分批发酵放大试验,5KGA的产量为93.80 g/L,最大生成速率为3.48 g/(L·h),平均生成速率为1.56 g/(L·h)。结果表明,葡萄糖和玉米浆分别为Gluconobacter oxydans HGI-1规模化生产5KGA的最适碳源和氮源,可利用葡萄糖几乎全部(85.93%)转化为5KGA。     

High-yield production of lutein by the green microalga Chlorella protothecoides in heterotrophic fed-batch culture

The green microalga Chlorella protothecoides was grown heterotrophically in batch mode in a 3.7-L fermenter containing 40 g/L glucose and 3.6 g/L urea. In the late exponential phase, concentrated nutrients containing glucose and urea were fed into the culture, in which the nitrogen source was sufficient compared to carbon source. As a result, a maximum cell dry weight concentration of 48 g/L was achieved. This cell dry weight concentration was 28.4 g/L higher than that obtained in batch culture under the same growth conditions. In another cultivation run, the culture was provided with the same initial concentrations of glucose (40 g/L) and urea (3.6 g/L) as in the batch mode, followed by a relatively reduced supply of nitrogen source in the fed-batch mode to establish a nitrogen-limited culture. Such a modification resulted in an enhanced lutein production without significantly lowering biomass production. The cellular lutein content was 0.27 mg/g higher than that obtained in the N-sufficient culture. The improvements were also reflected by higher maximum lutein yield, lutein productivity, and lutein yield coefficient on glucose. This N-limited fed-batch culture was successfully scaled up from 3.7 L to 30 L, and a three-step cultivation process was developed for the high-yield production of lutein. The maximum cell dry weight concentration (45.8 g/L) achieved in the large fermenter (30 L) was comparable to that in the small one (3.7 L). The maintenance of the culture at a higher temperature (i.e., 32 degrees C) for 84 h resulted in a 19.9% increase in lutein content but a 13.6% decrease in cell dry weight concentration as compared to the fed-batch culture (30 L) without such a treatment. The enhancement of lutein production resulted from the combination of nitrogen limitation and high-temperature stress.