Explore the vast range of titles available.

Nearly 80% of the approved human therapeutic antibodies are produced by Chinese Hamster Ovary (CHO) cells. To achieve better cell growth and high-yield recombinant protein, fed-batch culture is typically used for recombinant protein production in CHO cells. According to the demand of nutrients consumption, feed medium containing multiple components in cell culture can affect the characteristics of cell growth and improve the yield and quality of recombinant protein. Fed-batch optimization should have a connection with comprehensive factors such as culture environmental parameters, feed composition, and feeding strategy. At present, process intensification (PI) is explored to maintain production flexible and meet forthcoming demands of biotherapeutics process. Here, CHO cell culture, feed composition in fed-batch culture, fed-batch culture environmental parameters, feeding strategies, metabolic byproducts in fed-batch culture, chemostat cultivation, and the intensified fed-batch are reviewed.Key points• Fed-batch culture in CHO cells is reviewed.• Fed-batch has become a common technology for recombinant protein production.• Fed batch culture promotes recombinant protein production in CHO cells.

There is currently much interest in fucoxanthin due to its broad beneficial health effects. The major commercial source of fucoxanthin is marine seaweed, which has many shortcomings, and has thus restricted its large-scale production and more diversified applications. In this study, growth characteristics and fucoxanthin accumulation were evaluated to explore the potential of the marine diatom Nitzschia laevis in fucoxanthin production. The results suggested that heterotrophic culture was more effective for cell growth, while the mixotrophic culture was favorable for fucoxanthin accumulation. A two-stage culture strategy was consequently established. A model of exponential fed-batch culture led to a biomass concentration of 17.25 g/L. A mix of white and blue light significantly increased fucoxanthin content. These outcomes were translated into a superior fucoxanthin productivity of 16.5 mg/(L·d), which was more than 2-fold of the best value reported thus far. The culture method established herein therefore represents a promising strategy to boost fucoxanthin production in N. laevis, which might prove to be a valuable natural source of commercial fucoxanthin.

There is increasing industrial demand for five-carbon platform chemicals, particularly glutaric acid, a widely used building block chemical for the synthesis of polyesters and polyamides. Here we report the development of an efficient glutaric acid microbial producer by systems metabolic engineering of an L-lysine–overproducing Corynebacterium glutamicum BE strain. Based on our previous study, an optimal synthetic metabolic pathway comprising Pseudomonas putida L-lysine monooxygenase (davB) and 5-aminovaleramide amidohydrolase (davA) genes and C. glutamicum 4-aminobutyrate aminotransferase (gabT) and succinate-semialdehyde dehydrogenase (gabD) genes, was introduced into the C. glutamicum BE strain. Through system-wide analyses including genome-scale metabolic simulation, comparative transcriptome analysis, and flux response analysis, 11 target genes to be manipulated were identified and expressed at desired levels to increase the supply of direct precursor L-lysine and reduce precursor loss. A glutaric acid exporter encoded by ynfM was discovered and overexpressed to further enhance glutaric acid production. Fermentation conditions, including oxygen transfer rate, batch-phase glucose level, and nutrient feeding strategy, were optimized for the efficient production of glutaric acid. Fed-batch culture of the final engineered strain produced 105.3 g/L of glutaric acid in 69 h without any byproduct. The strategies of metabolic engineering and fermentation optimization described here will be useful for developing engineered microorganisms for the high-level bio-based production of other chemicals of interest to industry.

Background Pectinase enzymes present a high priced category of microbial enzymes with many potential applications in various food and oil industries and an estimated market share of $ 41.4 billion by 2020. Results The production medium was first optimized using a statistical optimization approach to increase pectinase production. A maximal enzyme concentration of 76.35 U/mL (a 2.8-fold increase compared with the initial medium) was produced in a medium composed of (g/L): pectin, 32.22; (NH 4 ) 2 SO 4 , 4.33; K 2 HPO 4 , 1.36; MgSO 4 .5H 2 O, 0.05; KCl, 0.05; and FeSO 4 .5H 2 O, 0.10. The cultivations were then carried out in a 16-L stirred tank bioreactor in both batch and fed-batch modes to improve enzyme production, which is an important step for bioprocess industrialization. Controlling the pH at 5.5 during cultivation yielded a pectinase production of 109.63 U/mL, which was about 10% higher than the uncontrolled pH culture. Furthermore, fed-batch cultivation using sucrose as a feeding substrate with a rate of 2 g/L/h increased the enzyme production up to 450 U/mL after 126 h. Conclusions Statistical medium optimization improved volumetric pectinase productivity by about 2.8 folds. Scaling-up the production process in 16-L semi-industrial stirred tank bioreactor under controlled pH further enhanced pectinase production by about 4-folds. Finally, bioreactor fed-batch cultivation using constant carbon source feeding increased maximal volumetric enzyme production by about 16.5-folds from the initial starting conditions.

Advances in the ethanol fermentation process are essential to improving the performance of bioethanol production. Fed-batch fermentation is a promising approach to increase the final ethanol titer, which benefits the recovery in the bioethanol industry’s downstream process. However, the development of feeding strategies, a crucial control variable in the fed-batch approach, is limited. Thus, in the present work, different modes of substrate delivery—fixed feeding, adapted feeding—were investigated in fed-batch cultures of Saccharomyces cerevisiae in a 5-L bioreactor. Evolved gas production, which was positively correlated with glucose consumption, was used to adjust the sugar feed rate in fed-batch fermentations under an adapted feeding strategy. The adapted feeding strategy enhanced ethanol productivity by 21% compared to the fixed feeding strategy, in which the sugar feed rate was stable, and the ethanol titer reached 91 g/L (~ 11.5%, v/v) at the end of fermentation. Moreover, cell biomass accumulation and cell growth rate were significantly improved when using the adapted feeding strategy. The effect of nitrogen availability on the performance of the adapted feeding strategy was further explored using a low-nitrogen content medium. The results showed that, even under low nitrogen feeding conditions (N/C = 0.046:10), the adapted feeding strategy maintained the same ethanol productivity as nitrogen-rich medium feeding. Overall, these results suggest that sugar delivery with low nitrogen content using the adapted feeding strategy could help reduce medium costs and improve the productivity of current facilities in the ethanol industry.Future work will integrate adapted feeding strategies with other fermentation approaches to improve ethanol production. Graphical abstract Key points • Novel continuous sugar delivery was developed for fed-batch ethanol fermentation. • The adapted feeding strategy improved ethanol productivity by 21%. • The final ethanol concentration reached 91 g/L (11.5%, v/v) with no residual sugar.

Ergothioneine (ERG), a unique thiol compound, is suggested to function as an antioxidant and cytoprotectant. Despite several recent attempts to produce ERG using various organisms, its yield was still very low and the costs remained high. Since the level of ERG produced depends strictly on the availability of three distinct precursor amino acids ( l -cysteine (Cys), l -histidine, and l -methionine (Met)), metabolic engineering for enhancement of the flux toward ERG biosynthesis is required. Herein, we took advantage of a high-Cys production system using Escherichia coli cells, in which Cys biosynthesis and excretion were activated, and applied it to the fermentative production of ERG from glucose. The Cys overproduction in E . coli cells carrying the egtBCDE genes from Mycobacterium smegmatis was effective for ERG production. Furthermore, coexpression of the egtA gene, which encodes γ-glutamylcysteine synthetase that synthesizes the γ-glutamylcysteine used as a sulfur source of ERG biosynthesis, enhanced ERG production even though E . coli intrinsically has γ-glutamylcysteine synthetase. Additionally, disruption of the metJ gene that encodes the transcriptional repressor involved in Met metabolism was effective in further increasing the production of ERG. Finally, we succeeded in the high-level production of 1.31 g/L ERG in a fed-batch culture process using a jar fermenter.

Zeaxanthin is a xanthophyll pigment that is regarded as one of the best carotenoids for the prevention and treatment of degenerative diseases. In the worldwide natural products market, consumers prefer pigments that have been produced from biological sources. In this study, a Dunaliella tertiolecta strain that has 10–15% higher cellular zeaxanthin content than the parent strain (zea1), was obtained by random mutagenesis using ethyl methanesulfonate (EMS) as a mutagen. This mutant, mp3, was grown under various salinities and light intensities to optimize culture conditions for zeaxanthin production. The highest cellular zeaxanthin content was observed at 1.5 M NaCl and 65–85 μmol photons·m−2·s−1, and the highest daily zeaxanthin productivity was observed at 0.6 M NaCl and 140–160 μmol photons·m−2·s−1. The maximal yield of zeaxanthin from mp3 in fed-batch culture was 8 mg·L−1, which was obtained at 0.6 M NaCl and 140–160 μmol photons·m−2·s−1. These results suggest that random mutagenesis with EMS is useful for generating D. tertiolecta strains with increased zeaxanthin content, and also suggest optimal culture conditions for the enhancement of biomass and zeaxanthin production by the zeaxanthin accumulating mutant strains.

The general approach to industrial production of monoclonal antibodies is fed-batch culture using Chinese Hamster Ovary (CHO) cells. Perfusion culture is also attracting attention as a next-generation culture method. In these culture methods, optimization of amino acid and glucose concentration in the culture medium is essential, and influences cell proliferation, viability, productivity, and monoclonal antibody quality. Further, the maintenance of optimal nutrient levels – by avoiding both depletion and accumulation – is crucial. This study aimed to develop a dynamic feeding strategy based on specific indicators to maintain optimal amino acid and glucose concentrations. Multivariate correlation analysis confirmed a strong relationship between nutrient consumption and viable cell density (VCD). Regression analysis was used to establish a regression model to estimate amino acid and glucose consumption based on VCD. Using this model, the nutrient composition of feed media for both fed-batch and perfusion cultures was adjusted, and a dynamic feeding strategy guided by VCD was evaluated. The observed nutrient concentration trends closely matched the model’s predictions, confirming that VCD is a reliable indicator for implementing dynamic feeding. In both fed-batch and perfusion cultures, the VCD-guided dynamic feeding strategy enables the maintenance of multiple amino acids and glucose at target concentrations.

Chinese hamster ovary (CHO) cells are the most common cell line used for the production of therapeutic proteins including monoclonal antibodies (mAbs). Host cell proteins (HCPs), secreted and released from lysed cells, accumulate extracellularly during the cultures of recombinant CHO (rCHO) cells, potentially impairing product quality. In an effort to maintain good mAb quality during the cultures, HCPs accumulated extracellularly in batch and fed-batch cultures of a mAb-producing rCHO cell line were identified and quantified by nanoflow liquid chromatography-tandem mass spectrometry, followed by their gene ontology and functional analysis. Due to higher cell concentration and longer culture duration, more HCPs were identified and quantitated in fed-batch culture (2145 proteins identified and 1673 proteins quantified) than in batch culture (1934 proteins identified and 1486 proteins quantified). Clustering analysis of HCPs showed that the concentration profiles of HCPs affecting mAb quality (Lgmn, Ctsd, Gbl1, and B4galt1) correlated with changes in mAb quality attributes such as aggregation, charge variants, and N -glycosylation during the cultures. Taken together, the dataset of HCPs obtained in this study provides insights into determining the appropriate target proteins to be removed during both the cultures and purification steps for ensuring good mAb quality.

Background Methanol, synthesized from CO 2 , is a potentially sustainable one-carbon (C1) resource for biomanufacturing. The use of methanol as a feedstock to produce single cell protein (SCP) has been investigated for decades as an alternative to alleviate the high global demand for animal-derived proteins. The methylotrophic yeast Pichia pastoris is an ideal host for methanol-based SCP synthesis due to its natural methanol assimilation ability. However, improving methanol utilization, tolerance to higher temperature, and the protein content of P. pastoris are also current challenges, which are of great significance to the economical industrial application using methanol as a feedstock for SCP production. Results In the present work, adaptive laboratory evolution (ALE) has been employed to overcome the low methanol utilization efficiency and intolerance to a higher temperature of 33 °C in P. pastoris , associated with reduced carbon loss due to the lessened detoxification of intracellular formaldehyde through the dissimilation pathway and cell wall rearrangement to temperature stress resistance following long-term evolution as revealed by transcriptomic and phenotypic analysis. By strengthening nitrogen metabolism and impairing cell wall synthesis, metabolic engineering further increased protein content. Finally, the engineered strain via multi-strategy produced high levels of SCP from methanol in a pilot-scale fed-batch culture at 33 °C with a biomass of 63.37 g DCW/L, methanol conversion rate of 0.43 g DCW/g, and protein content of 0.506 g/g DCW. SCP obtained from P. pastoris contains a higher percentage of protein compared to conventional foods like soy, fish, meat, whole milk, and is a source of essential amino acids, including methionine, lysine, and branched-chain amino acids (BCAAs: valine, isoleucine, leucine). Conclusions This study clarified the unique mechanism of P. pastoris for efficient methanol utilization, higher temperature resistance, and high protein synthesis, providing a P. pastoris cell factory for SCP production with environmental, economic, and nutritional benefits.