Amino acid metabolism, demand and supply in Chinese Hamster ovary cell culture - A comprehensive literature review.

Enhanced anthraquinone biosynthesis in Damnacanthus major cells via medium optimization and fed-batch bioreactor culture.

Expansins, whose cell wall-loosening function contributes to plant cell growth, biomass deconstruction, and substrate accessibility, have attracted increasing attention in agricultural biotechnology and bioprocessing. However, their predominant sourcing from plants, with limited low abundance and inconsistency, presents a challenge for industrial production. In this study, we developed a multi-level engineering strategy in Bacillus subtilis to enhance the extracellular production of two expansins: B. subtilis expansin-like group X1 (BsEXLX1) and Solanum lycopersicum α-expansin (LeEXP2). By systematically screening temporal promoters, optimizing ribosome-binding site (RBS) and signal peptides (SPs), and expressing secretion machinery components, the expression levels of BsEXLX1 and LeEXP2 reached 232.3mg/L and 15.6mg/L, respectively, in shake flasks. Further fed-batch culture increased the extracellular BsEXLX1 and LeEXP2 to 1.3g/L and 43.7mg/L in a 5-L bioreactor, representing the highest level reported in B. subtilis to date. Furthermore, both microbial-generated expansins exhibited a strong synergistic effect on cellulose degradation, enhancing total sugar release by 42.6% (BsEXLX1) and 5.7% (LeEXP2) when combined with commercial cellulase. Collectively, this study establishes a scalable B. subtilis-based secretion platform for the high-level production of functional expansins and provides a transferable framework for efficient extracellular protein production in B. subtilis.

Fed-batch processes using Chinese hamster ovary (CHO) cells are primarily employed to manufacture biologics. In these cultures, cells transition from exponential growth to the stationary phase, often resulting in a viability decline. This decline along, with similar reductions in cell growth and productivity is attributed to changes in cellular metabolism, the cell cycle, and apoptosis, potentially caused by nutrient imbalance and the accumulation of inhibitory byproducts. To enhance productivity in fed-batch processes, understanding the interactions among these factors is crucial for identifying intervention strategies to sustain cell culture performance. In this study, we evaluated the dynamics of the cell cycle and apoptosis in a mAb-producing cell line during a fed-batch process. Results showed that the cells transitioned to the G0/G1 phase during the stationary phase. Although viability remained above 80% throughout the culture, early signs of apoptosis were observed as early as Day 5, coinciding with product accumulation. Our investigation revealed that early apoptotic cells were not productive and that removing apoptotic stimuli or adding fresh nutrients did not rescue these cells. This underscores the necessity for early process interventions. Media exchange after Day 7, when cells are in stationary phase, resulted in higher viability, fewer early apoptotic cells, and approximately a 26% increase in productivity compared to conditions with no or earlier media exchange. This study highlights the significance of timely interventions to remove the inhibitory byproducts and replenish the culture with fresh media, based on detailed process characterization.

Haemophilus influenzae b (Hib) is a pathogenic bacterium that causes meningitis worldwide, mainly in children less than two years old. The capsular polysaccharide b (PRP) is an essential antigen for vaccine formulation. This study aimed to develop a high-yield, technically accessible production strategy for PRP production to facilitate vaccine manufacturing in non-profit laboratories. Various fed-batch cultivation strategies were evaluated to address metabolic limitations and identify a robust, simplified process suitable for seamless scale-up to pilot scale. Glucose limitation strategies did not reduce inhibitory acetic acid accumulation due to deficiencies in Hib's respiratory chain, whereas oxygen availability was identified as critical parameter. Increasing the specific air flow from 0.5 to 1.0 vvm in constant fed-batch (Cfb) resulted in a 33% yield increase, reaching 1706.40 mg PRP.L-1. However, the highest PRP concentration was achieved using exponential fed-batch with cell recycling (EfbCR), resulting in 1879.28 mg PRP.L-1. Although EfbCR offered high productivity, the Cfb strategy emerged to be the most technically feasible and robust solution and was successfully scaled up to an 80 L bioreactor, achieving 1885 mg PRP.L-1. These results advance understanding of PRP production by Hib and provides valuable insight into an efficient and simplified strategy for producing this key/vital vaccine antigen. The findings support the potential for cost-effective local production in public health initiatives.

Improved neutral lipid production from Tetradesmus obliquus through fed-batch mixotrophic cultivation at high pH using potato peel hydrolysate

The methanogenic archaeon Methanothermobacter marburgensis offers a promising alternative to traditional bacterial systems for the sustainable production of proteinogenic amino acids (AAs), eliminating the need for sugar-based feedstock. In this study, we quantitatively examined AA excretion and consumption in fed-batch cultivation mode in bioreactors under varying ammonium (NH4+) concentrations and gas compositions. M. marburgensis demonstrated excretion of a wide spectrum of AAs with distinct profiles shaped by nitrogen availability. While high NH4+ concentrations suppressed total AA excretion, NH4+ limited conditions triggered alanine accumulation followed by its re-assimilation, suggesting a regulatory mechanism linked to nitrogen stress. Moreover, carbon limitation and nitrogen excess resulted in the production of an AA pattern including asparagine. Despite lower overall productivity compared to engineered bacterial strains, M. marburgensis exhibited the unique ability to simultaneously excrete multiple AAs without requiring organic carbon input. These findings advance the feasibility of using methanogens for AA bioprocessing and the development of archaea as next-generation microbial cell factories.

Effect of culture media and fermentation process on the refolding and purification of rh-GCSF.

Buprenorphine can assist in the treatment of opiate addiction but the current methods of synthesis for this alkaloid are suboptimal. N-demethylation of the precursor oripavine by bioconversion could present an alternative route for the first steps of synthesis but requires an N-demethylase that can efficiently produce nororipavine at sufficient yields, together with a process that can be controlled and scaled within a bioreactor. An N-demethylase acting on oripavine was sourced from insect pest species living on the alkaloid-containing parts of poppy that naturally contain oripavine. When coupled with a cytochrome P450 oxidoreductase and purine permease for transport, the enzyme expressed in Saccharomyces cerevisiae could N-demethylate oripavine to nororipavine. Bioconversion examined in batch/fed-batch mode gave a greater understanding of the optimal concentration of oripavine substrate and kinetics of nororipavine production. The highest titre of 1.59g/L was achieved with fed-batch cell growth and batch bioconversion, due to greater cell biomass and improved process control, while continuous processing gave a productivity of 1.01g/L/day at 0.7 L scale. This study presents an alternative approach to achieving an environmentally friendly, industrially scalable whole-cell conversion of high-value benzylisoquinoline alkaloids using a novel N-demethylase. Yield and productivity are promising, with potential for further optimization of both enzyme and bioconversion process, including further exploration of a continuous process for process intensification. The N-demethylation step described here could prove useful in the production of buprenorphine or be coupled with other described whole cell pathways for more complex conversions of therapeutic alkaloids.

Using pure oxygen aeration to increase recombinant protein production by an Aspergillus oryzae hyphal dispersion strain.

Enhancing heterotrophic lutein production in Chlorella protothecoides through combined phytohormone and nitrogen strategies.

Selective vanillate production from sugarcane bagasse-derived aromatic compounds using an engineered Pseudomonas sp. NGC7-based strain.

Saccharomyces cerevisiae is an established production host for therapeutic proteins; many of those are small proteins such as insulin or glucagon-like peptide-1 (GLP-1) analogs. Contrastingly, proteins of higher molecular weight, foremost antibodies, did not reach the market due, among other factors, to limiting productivity. Here we addressed the loss of product to protein degradation through a combination of genetic engineering of the host and medium optimization. We screened target genes that either directly or indirectly can lead to proteolytic degradation. We identified four deletions that are beneficial for expression: PEP1 and VPS30, which both can channel proteins to the vacuole for degradation; MON2, which can lead to the re-uptake of secreted proteins; and ALG3, which can affect the permeability of the cell wall. In parallel, we developed a small-scale fed-batch cultivation system for 24-well deep well plate cultivations and using an amino acid-rich medium. To stabilize secreted proteins, we screened chemical chaperones and osmolytes. We fortified the medium with arginine, 4-phenylbutyrate (4-PBA), and Tween-20. Using the engineered yeast strain, which features VPS30, PEP1, and ALG3 deletions, and the small-scale fed-batch system, we obtained 2.5µg/mL of a secreted chimeric fusion of a nanobody to the crystallizable fragment (Fc) of a human immunoglobulin. Instrumental to the increase in the final titer were the reduced losses. This was achieved by a combination of complementary measures: improving diffusion through the cell wall, achieved through genetic engineering, and reducing losses to proteolytic degradation through medium optimization and genetic engineering. Moreover, we showed that the engineered strain and cultivation set-up are suitable for the production of different antibodies. KEY POINTS: • Chemical chaperones and amino acid-rich medium increased secreted protein titers. •Medium and host engineering are instrumental for improving productivity. •Small-scale cultivation system enables production levels suitable for characterization.

Euglena gracilis cells grown in the dark accumulate a β-1,3-glucan called paramylon, synthesized from organic carbon sources. Paramylon has potential applications as a raw material for bioplastics and nanofibers. Strain SM-ZK, a streptomycin-bleached mutant of E. gracilis, lacks chloroplasts permanently and accumulates more paramylon than the wild-type strain. However, data are limited on the fermentation characteristics of this mutant strain. This study compares the cultivation characteristics of E. gracilis strains Z (wild-type strain) and SM-ZK under fed-batch cultivation conditions. Strain SM-ZK showed significantly higher productivity in terms of both biomass yield and glucose consumption than strain Z. Moreover, strain SM-ZK cells appeared to be gradually enlarged during fed-batch cultivation compared with strain Z that grows in uniform sizes. These novel findings support the potential use of strain SM-ZK for industrial-scale production of paramylon.

Chitin valorization through microbial bioprocessing relies on efficient utilization of its monomeric units as fermentation substrates. In this study, the effects of salt concentration and the mixing ratio of N-acetylglucosamine (GlcNAc) to glucosamine hydrochloride (GlcN·HCl) on the specific growth rate of our previously isolated V. natriegens N5.3 was investigated in the shake-flask. Batch and fed-batch fermentations using chitin-derived amino sugars were further performed to assess high-cell-density cultivation potential.Although the maximum specific growth rate ( ) at 60 g/L NaCl was nearly two-fold lower than that at the optimal concentration of 15 g/L, strain N5.3 retained robust growth with values of 0.37 h-1 on GlcN·HCl and 0.66 h-1 on GlcNAc. Fed-batch cultivation yielded a maximum cell dry weight (CDW) of 42.3 g/L within 9 h on GlcNAc, with of 0.53 h-1, but with a low biomass yield ( = 0.16 g/g). In contrast, a substrate mixture containing 5% (w/w) GlcNAc and 95% (w/w) GlcN·HCl maintained a high (0.49 h-1) while substantially improving (0.29 g/g), resulting in a CDW of 35.5 g/L after 9 h. Due to low solubility of both amino sugars, exponential feeding with non-sterilized powders was successfully applied. The absence of contamination demonstrate the feasibility of this approach. These results demonstrate that the mixture of GlcNAc:GlcN·HCl (1:19 ratio) is effective substrate for cultivation of V. natriegens N5.3. This provides a promising foundation for the microbial conversion of chitin-derived feedstocks into high-value products.

Chinese Hamster Ovary (CHO) cells are widely used for the production of recombinant therapeutics due to their ability to carry out human-like post-translational modifications. Media adaptation represents a key step in large-scale production to ensure optimal safety and cost efficiency. As DNA methylation is a central epigenetic mechanism underlying adaptive modulation of gene expression, we report here, for the first time, the use of high-coverage whole-genome bisulfite sequencing to generate single-base-resolution maps of CHO cells at different phases of growth in a fed-batch culture and undergoing media adaptation across four different media. A CHO cell line was adapted to four commercially available media, and their growth rates and productivity were compared with those obtained using the control medium in a 7-day batch culture. This approach resulted in the generation of n = 57 high-quality whole-genome DNA methylation datasets, which were subjected to differential DNA methylation and gene association analyses. In addition, we developed a novel DNA methylation array comprising more than 63,000 CpG methylation sites across the CHO genome, enabling streamlined and efficient DNA methylation profiling of CHO cells. Analysis of n = 57 high-quality DNA methylation datasets revealed altered DNA methylation patterns across different phases of growth in a fed-batch culture and in response to distinct media adaptations. Specifically, adaptation to four different media resulted in highly specific methylation changes that were associated with distinct functional outcomes, including protein productivity. Finally, the customized DNA methylation microarray platform was used to validate all media adaptation-dependent epigenetic changes identified by whole-genome bisulfite sequencing (WGBS). These findings identify and characterize dynamic DNA methylation changes occurring during media adaptation and support their potential use as predictive indicators of CHO cell phenotypic changes in response to a dynamic culture environment. Furthermore, this work represents a valuable resource for the development of DNA methylation-based biomarkers for the optimization of CHO cell culture.

Engineering of endogenous plasmids in probiotic Escherichia coli Nissle 1917 for autonomous accumulation of 5‑aminolevulinic acid.

Beyond tyrosine feeding: A novel fed-batch cultivation strategy based on tyrosine metabolic engineering in recombinant CHO cells.

Systematic optimization of fed-batch fermentation for enhanced heparosan production in engineered Corynebacterium glutamicum.

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.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-28316-8.