Efficient co‐production of S‐adenosylmethionine and glutathione by Candida utilis: effect of dissolved oxygen on enzyme activity and energy supply

Abstract

BACKGROUND As both S‐adenosylmethionine (SAM) and glutathione (GSH) are biosynthesized with ATP consumption in yeast cells, their efficient co‐production not only depends on high activities of key enzymes, but also on sufficient energy supply. In this study, dissolved oxygen (DO) was optimized to improve enzyme activity and energy supply for high co‐production of SAM and GSH. RESULTS Batch culture of Candida utilis CCTCC M 209298 under different DO control modes was carried out in a 5 L stirred fermentor. Based on the analyses of kinetic parameters and metabolic fluxes of key nodes involved in the metabolism of SAM and GSH, a two‐stage DO control strategy with an initial agitation rate of 350 rpm followed by a constant DO level of 30% was designed for the overproduction of SAM and GSH. A maximum co‐production of SAM and GSH of 593.9 mg L−1 was achieved using the two‐stage DO control strategy, which was much higher than that obtained from other DO control modes. CONCLUSION Physiological mechanism analysis revealed that the improved co‐production of SAM and GSH was closely related to the increase in key enzyme activities and sufficient energy supply under the two‐stage DO control strategy. © 2017 Society of Chemical Industry

Improved S‐adenosylmethionine and glutathione biosynthesis by heterologous expression of an ATP6 gene in Candida utilis

“…However, overproduction of SAM and GSH is usually limited because of insufficient supply of intracellular ATP. Improved co-production of SAM and GSH has been achieved either by adding citrate as an auxiliary energy substrate [8] or by optimizing dissolved oxygen to increase energy supply [9]. In the present study, we tried to increase F 0 F 1 -ATPase activity to generate sufficient ATP needed for improved SAM and GSH biosynthesis.…”

“…Batch fermentation for SAM and GSH biosynthesis was performed at 30 °C, 350 rpm, and an aeration rate of 1.0 vvm. The pH was automatically controlled at 5.0 ± 0.02 by adding 3 mol L −1 of H 2 SO 4 or 3 mol L −1 of NaOH .…”

“…The activity of methionine adenosyltransferase (MAT, EC2.5.1.6) was measured using the method described previously . The activities of γ‐glutamylcysteine synthetase ( γ ‐GCS, EC 6.3.2.2), hexose kinase (HK, EC 2.7.1.1), NAD + ‐dependent isocitrate dehydrogenase (IDH, EC1.1.1.42), and F 0 F 1 ‐ATPase (EC 3.6.1.3) were assayed using commercial kits (Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer's instructions.…”

“…In eukaryotic cells, SAM and GSH are biosynthesized with the substrate amino acids and ATP . Nevertheless, in the presence of sufficient amino acids supply, intracellular ATP level and ATP regeneration easily act as limiting factors for the overproduction of SAM and GSH in Candida utilis . To overcome this bottleneck, exogenous addition of ATP is usually performed ; however, it increases the cost of raw materials for efficient production of SAM and GSH.…”

Enhanced synthesis of S‐adenosyl‐L‐methionine through combinatorial metabolic engineering and Bayesian optimization in Saccharomyces cerevisiae

“…The biosynthesis of SAM requires the participation of ATP, of which the intracellular supply level is an important factor that determines whether SAM can be excessively synthesized. [ 9 ] Because ATP not only effects the cell growth, but also provides an adenosine for SAM synthesis. ATP supply in microbial cells can be improved by a variety of approaches, such as addition of energy substrates, metabolic engineering to modulate pH, ATP production or ATP consumption pathways, and control of respiratory chain reactions.…”