Cell Physiology And Process Optimization In High-Cell-Density Cultivation Of GSH High-Yield Strain

Reduced glutathione (y-glutamyl-L-cysteinylglycine, GSH), the most abundant non-protein thiol compound, is distributed widely in living cells. Although GSH is involved in many physiological processes and plays important roles, it serves three key functions: an antioxidant which plays key roles in maintaining the redox homeostasis of the cell, an enhancer of immunity, and a detoxifier in higher eukaryotic organisms. These characteristics make GSH important in the treatment of many diseases, such as liver cirrhosis, pulmonary diseases, gastrointestinal and pancreatic inflammations, diabetes, neurodegenerative diseases, and aging. Thus, GSH is considered to be one of the most important self-generated defense molecules. GSH is now widely used as a medicine. It also has great potential as food additives and in the cosmetic industries, only if the production cost can be further decreased. The commercial demand for GSH is expanding. However, there is no fermentative production of GSH on an industrial scale in China. Based on multi-scale analysis, cell physiology, and process optimization of a GSH high-yield strain were investigated in this work.1. Identification and stress resistance of GSH high-yield strainThe 26S rDNA fragment of GSH high-yield strain was amplified by PCR with the template of its genomic DNA and fungal 26S rDNA universal primers. After blast the sequence of this 26S rDNA fragment in GSH high-yield strain using GenBank database of NCBI, it was identified as Saccharomyces cerevisiae. Moreover, stress resistance of GSH high-yield strain and Saccharomyces cerevisiae W303A to ethanol, glucose, nutrient starvation, temperature, osmotic pressure, oxidative pressure, and G418 and Zecin toxicity were studied by cell growth assay. The results showed that GSH high-yield strain is more resistant to the tested stress conditions than Saccharomyces cerevisiae W303A. Furthermore, GSH high-yield strain can grow on yeast SD-URA medium, suggesting that it is not a uracil autotrophic mutant. These results serve as useful reference data for further studies of GSH high-cell-density cultivation.2. Specific growth rates control strategy based on on-line viable-cell mass monitoring for GSH high-cell-density cultivationAn on-line monitoring of viable-cell mass in high-cell-density fed-batch cultivations of GSH high-yield strain grown on an industrial complex medium was performed with an in situ capacitance probe fitted to a 50-1 fermentor. Conventional off-line biomass determinations of several parameters, including dry cell weight (DCW), optical density at 600 nm wavelength (OD600 nm), packed mycelial volume (PMV) and number of colony forming units (CFU), were performed throughout the bioprocess and then compared with on-line viable-cell concentrations measured using a capacitance probe. Capacitance versus viable biomass and all off-line biomass assay values were compared during GSH fermentation in industrial complex culture media. As a result, the relationship between the number of colony forming units and capacitance with a correlation coefficient (R) of 0.995 was achieved. Simultaneously, compared with those determined by at-line indirect estimation methods including oxygen uptake rate (OUR) and carbon dioxide evolution rate (CER), the specific growth rates (μ) estimated by on-line capacitance measurement could be more reliable during GSH fermentation. An on-lineμfeedback control strategy based on capacitance measurement was developed for GSH high-cell-density fermentation.μcontrolled at 0.2 h"1 achieved yeast dry weight (120 g/1) and GSH yield (1.8 g/1), which improved by 86.7% and 200%, respectively, compared withμcontrolled at 0.15 h-1. Therefore, it is concluded that a capacitance probe is a practical tool for real-time viable biomass monitoring in GSH high-cell-density fed-batch cultivation in a complex medium.3. RQ feedback control for simultaneous improvement of GSH yield and GSH contentGSH production by high-cell-density fed-batch cultivation of GSH high-yield strain, a respiratory quotient (RQ) feedback control strategy was applied to determine glucose feeding rate(F) based on on-line off-gas monitoring. Glucose feed was manipulated by a classical proportional-integral-derivative (PID) controller to control RQ at its set-point. RQ controlled at 0.65 resulted in the highest GSH productivity (46.9 mg/1/h) and cell productivity (3.5 g/1/h) as well as improved GSH yield and intracellular GSH content. RQ feedback control achieved yeast dry weight (126 g/1), GSH yield (2.1 g/1), GSH content (1.67%) and GSH productivity (55.3 mg/1/h), which improved by 11.5%,75%,57.5% and 82.5%, respectively, compared with conventional ethanol feedback control in the GSH industry. Moreover, RQ feedback control reduced ethanol (byproduct) level to below 0.3 g/1. Advantages of RQ feedback control over the conventional ethanol feedback control in the GSH industry include lower byproduct concentration, shorter cultivation period, higher GSH content, and higher GSH yield. Based on metabolic flux analysis (MFA), it indicated that the reason of GSH over-production by RQ control was due to increase of metabolic flux of GSH biosynthesis pathway. Furthermore, GSH yield achieved 2.4 g/1 when feeding medium was changed from glucose (600 g/1) to glucose (600 g/1) and yeast extract (15 g/1). Hence, feedback control based on RQ is a promising method that poses as a simple and efficient alternative to conventional feed control techniques presently practiced in the GSH industry.4. A possible molecular mechanism for GSH high-yield synthesisy-glutamylcysteine synthetase (GSH1) catalytic activity of GSH high-yield strain achieved 4.1 mmol Pi/mg protein/min, which was-4-fold compared to that of wild-type strain (control). It may indicate that GSH1 mutation of GSH high-yield strain resulted in a significantly increased activity. DNA sequencing of GSH1 in GSH high-yield strain revealed that three amino acids were changed among GSH1 conserved domain and its catalytic subunit. The 3D structure of GSH1 in GSH high-yield strain was generated by homology modeling using Swiss-Model based on the crystal structure of GSH1 of S. cerevisiae (PDB code:3ig5A). The result showed that the structure of GSH1 was changed compared with control. Moreover, the Potential energy of GSH1 was lower than that of control, indicating that GSH1 is more stable than control, and may increase reaction rate with substrate. Examination of this model suggested that the structure change may affect substrate binding with GSH1, and regulate the enzymatic activity.5. Efficient extraction of GSH by ethanolGSH from fermentation broth of GSH high-yield strain was extracted with ethanol without disruption of the cells. The effects of ethanol concentration, extraction temperature and extraction time were assessed by using 23 full factorial designs (FFD). Preliminary studies showed that ethanol concentration had the most influence on GSH yield by ethanol extraction, based on the first order regression coefficients derived using MINITAB software, and an optimal ethanol concentration (25%, v/v) was obtained. However, compared to the conventional extraction technique (hot water extraction), there was no significant advantage in yield of GSH from yeast cells using ethanol extraction under these optimized conditions. But ethanol extraction has several advantages, such as lower energy consumption and lower protein concentration of extraction broth, which may reduce the complexity and cost of the purification process. Hence, ethanol extraction which does not disrupt yeast cells could be an inexpensive, simple and efficient alternative to conventional extraction techniques in the GSH industry.