For the generation of therapeutic proteins in cell culture, high producing clones are used. These clones have a high demand in amino acids to support cell growth and productivity. l-cysteine (Cys) is critical in highly concentrated feeds due to low stability of Cys and low solubility of the oxidation product cystine at neutral pH. S-sulfocysteine (SSC) was developed to substitute the Cys source and fed-batch experiments using SSC showed good cellular performance regarding viable cell density and titer, indicating uptake and metabolization of SSC by Chinese hamster ovary cells. However, the responsible transporter allowing cellular uptake remains unclear and was studied in this work. Due to the structure similarity of SSC with cystine and glutamate, it was proposed that the cystine/glutamate antiporter (xc-) allows cellular uptake of SSC. The uptake was assessed via transporter inhibition using sulfasalazine and transporter overexpression using either sulforaphane or sulforaphane-N-acetylcysteine during fed-batch experiments. Following daily addition of 50 μM and 100 μM sulfasalazine, the extracellular SSC concentration was increased by 65 % and 177 % respectively, suggesting a reduced uptake due to xc- inhibition. In contrast, enhanced transporter activity through 15 μM sulforaphane and sulforaphane-N-acetylcysteine treatment, induced a 60 % and 52 % reduced extracellular SSC concentration, respectively. These inverse uptake results strongly suggest that xc- is facilitating the transport of SSC.

Softwood is of interest as a renewable carbon source for production of lactic acid. Softwood hydrolysate contains a high content of mannose. Lactic acid production from mannose by two modified strains, L-lactic acid producing Pediococcus acidilactici TY112 and D-lactic acid producing ZP26, was investigated in the current work. The two strains efficiently converted mannose to L- and D-lactate isomers with an optical purity exceeding 99 %, although the mannose utilization rates were lower than the glucose utilization rates. The mannose conversion to L- and D-lactic acids by P. acidilactici was also confirmed in dilute spruce hemicellulose hydrolysate. The present study provides important knowledge on utilization of the spectrum of fermentable sugars in softwood for future production of chiral lactic acid from lignocellulose feedstocks.

Haloalkane dehalogenase DhaA can catalyze the hydrolytic cleavage of carbonhalogen bonds, along with production of the corresponding alcohol, a proton and a halide. However, DhaA suffers from poor environmental tolerance, such as sensitivity to high temperature, low pH and hypersaline. Arabinogalactan (AG) is a hydrophilic polysaccharide with highly branched long chains. DhaA was conjugated with AG to improve the environmental stability of DhaA in the present study. Each DhaA was averagely conjugated with 4∼5 AG molecules. Conjugation of AG essentially maintained the enzymatic activity of DhaA (91.4 %) without apparent structural alteration. The hydration layer formed by AG could reduce the solvent accessible area of DhaA and slow the protonation process, thereby improving the pH and high salt stability of DhaA. In particular, the remaining activities of the conjugate (AG-DhaA) were 35.3 % after treatment at pH4.0 for 1 h, and 80.8 % in 1 M NaCl after treatment for 16 h. As compared with DhaA, AG-DhaA showed slightly different kinetic parameters (K M of 1.90 μmol/L and k cat of 2.60 s -1).

The green alga Chlamydomonas reinhardtii serves as a model organism for plant and photosynthesis research due to many commonalities in metabolism and to the fast growth rate of C. reinhardtii which accelerates experimental turnaround time. In addition, C. reinhardtii is a focus of research efforts in metabolic engineering and synthetic biology for the potential production of biofuels and value-added chemicals. Here, we report that the C. reinhardtii cia5 mutant, which lacks a functional carbon-concentrating mechanism (CCM), can produce substantial amounts of glycolate, a high-value cosmetic ingredient, when the mutant is cultured under ambient air conditions. In order to reveal the metabolic basis of glycolate accumulation by the cia5 mutant, we investigated the metabolomes of the cia5 mutant and a wild type strain CC-125 (WT) through the global metabolic profiling of intracellular and extracellular fractions using gas chromatography and mass spectrometry. We observed the intracellular and extracellular metabolic profiles of the WT and the cia5 mutant were similar during the mixotrophic phase at 30 h. However, when the cells entered the photoautotrophic phase (i.e., 96 h and 120 h), both the intracellular and extracellular metabolic profiles of cia5 mutant differed significantly when compared to WT. In the cia5 mutant strain, a group of photorespiration pathway intermediates including glycolate, glyoxylate, glycine, and serine accumulated to significantly higher levels compared to WT. In the photorespiration pathway, glycolate is metabolized to glyoxylate and glycine leading to NH3 and CO2 generation during the mitochondrial conversion of glycine to serine. This result provides further evidence that the CIA5 mutation increased the photorespiration rate. Because the cia5 mutant lacks a CCM, and C. reinhardtii might harbor an inefficient or incomplete photorespiration pathway, glycolate may accumulate when the CCM is not functional. We envision that investigating photorespiration controls in C. reinhardtii provides tools for producers to use the cia5 mutant to produce glycolate as well as platform to engineer alternative pathways for glycolate metabolism.

Varicella-zoster virus (VZV), the causative agent of varicella and herpes zoster, is highly cell-associated and spreads via cell-to-cell contact in tissue culture. The lack of cell-free VZV hampers studies on VZV biology as well as antiviral and vaccine development. In the present study, a poly(methylmethacrylate) microfluidic device integrated with arrays of microelectrode was fabricated to continuously electrolyse VZV-infected cells to produce cell-free viruses. By designing multiple constrictions and microelectrode arrays, a high electric field is focused on the constricted region of the microchannel to disrupt large numbers of virus-infected cells with high-throughput on a microfluidic platform. Plaque assay and scanning electron microscopy were conducted to quantify and characterize cell-free VZV produced using the microfluidic continuous-flow electrical cell lysis device. The process of microfluidic electrical cell lysis followed by subsequent filtration and virus concentration process yielded a 1.4-2.1 × 104 plaque-forming units (PFUs) per mL of cell-free VZV from 7.0 × 106 VZV-infected human foreskin fibroblasts (HFF) cells. The high electric field formed inside a microfluidic channel combined with the continuous-flow of virus-infected cells within the microchannel enabled the rapid and efficient production of high-titer cell-free virus in large quantities with relatively low input of the voltage.

Biocleaning of cultural heritage items is mainly performed using living microorganisms. Approaches utilizing the enzymes of isolated microorganisms have not been frequently investigated. To find an enzymatic alternative for the removal of an oil-based overpainting, we focused on the characterization and use of a yeast Extracellular Enzymatic Mixture (EEM). A historical silk yeast was selected for its lipolytic properties and its EEM was extracted after cultivation on a medium supplemented with linseed oil. The EEM protein content was visualized by SDS-PAGE, its concentration assessed by fluorimeter and the enzymatic activity evaluated by p-NPP spectrophotometric lipase assay. The yeast growth was suppressed by adding diverse metal ions (Cd, Zn, Cr and Cu) in Reasoner's 2A (R2A) broth, while the quantity and activity of EEM were affected by adding Fe and Pb. Various delivery systems (agar-agar, tylose and klucel G) alone or in a combination with EEM were assayed on the historical painting surface. The colorimetric measurements and the ATR-FTIR analysis indicated that the combinations tylose-EEM and klucel G-EEM can be easily and effectively applied as biocleaning procedures to remove oil-based overpainting from fragile and valuable historical painting surfaces.

Metabolism is the sum of all chemical reactions that sustain life. There is an ongoing effort to control metabolic rate, which correlates with the maximum lifespan potential and constitutes one of the oldest scientific questions. Herein, we report on the complete reversible arrest of cellular metabolism and cell growth in a series of organisms, from microalgae to yeast upon exposure to a 100 % hydrogen atmosphere. We also report a tolerance of the microalgae under these conditions against extreme stress conditions, like high salt concentrations. The addition of oxygen or air almost completely restores the metabolic rate and cell growth. Molecular dynamics simulations are employed to decipher this phenomenon at atomic scale. Various proteins, including photosynthetic and respiratory complexes (LHCII, cytochrome c5) are probed in the interaction with hydrogen. Exposure to hydrogen, as opposed to oxygen, decreases the fluctuations of protein residues indicating thermostability. According to the above mechanism, an absolute hydrogen atmosphere can preserve biological products (e.g. fruits) for a long time without consuming any energy. By combining biological, chemical and computational methods, in this research we provide the basis for future innovative studies and advances in the field of biotechnology.

Current CHO cell production processes require an optimized space-time-yield. Process intensification can support achieving this by enhancing the productivity and improving facility utilization. The use of perfusion at the last stage of the seed train (N-1) for high cell density inoculation of the fed-batch N-stage production culture is a relatively new approach with few industry applicable examples. Within this work, the impact of the cell-specific perfusion rate (CSPR) of the N-1 perfusion and the relevance of its control for the quality of generated inoculation cells was evaluated using an automated perfusion rate (PR) control based on online biomass measurements. Precise correlations (R² = 0.99) between permittivity and viable cell counts were found up to the high densities of 100⋅106 c·mL-1. Cells from N-1 perfusion were cultivated at a high and low CSPR with 50 and 20 pL·(c·d)-1, respectively. Lowered cell growth and an increased apoptotic reaction was found as a consequence of the latter due to nutrient limitations and reduced uptake rates. Subsequently, batch cultivations (N-stage) from the different N-1 sources were inoculated to evaluate the physiological state of the inoculum. Successive responses resulting from the respective N-1 condition were uncovered. While cell growth and productivity of approaches inoculated from high CSPR and a conventional seed were comparable, low CSPR inoculation suffered significantly in terms of reduced initial cell growth and impaired viability. This study underlines the importance to determine the CSPR for the design and implementation of an N-1 perfusion process in order to achieve the desired performance at the crucial production stage.