Screening of Selenium/Glutathione-Enriched Candida utilis and Its Anti-inflammatory and Antioxidant Activities in Mice.

This study aimed to screen a mutant of Candida utilis SE-172 with high selenite tolerance and glutathione (GSH) biosynthesis capability via 60Co γ-radiation mutagenesis to prepare selenium (Se)-enriched yeast. The maximal intracellular contents of GSH and organic Se of 22.94 mg/g and 1308.1 µg/g were obtained, respectively, under a batch culture of SE-172. The physiological mechanism underlying increased GSH and organic Se contents in Se/GSH-enriched C. utilis SE-172 was revealed based on assaying activities of γ-glutamylcysteine synthase (γ-GCS) involved in GSH biosynthesis and selenophosphate synthase (SPS) related to organic Se bioconversion, and by determining intracellular ATP and NADH contents and ATP/ADP and NADH/NAD+ ratios associated with energy supply and regeneration. Moreover, the effect of this selenized yeast on anti-inflammatory and antioxidant activities in mice with colitis was investigated. The supplementation of Se/GSH-enriched yeast decreased the dextran sodium sulfate-induced damage to colon tissues, reduced the expression of pro-inflammatory factors [interleukin (IL)-6, IL-1ß, and tumor necrosis factor-α (TNF-α)] in serum, increased the antioxidant-related enzyme [superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px)] activities, and decreased the malondialdehyde content in colon. The Se/GSH-enriched C. utilis SE-172 showed potent anti-inflammatory and antioxidant activities in mice with colitis.

Metabolic Engineering of Escherichia coli for Production of α-Santalene, a Precursor of Sandalwood Oil.

α-Santalene belongs to a class of natural compounds with many physiological functions and medical applications. Advances in metabolic engineering enable non-native hosts (e.g., Escherichia coli) to produce α-santalene, the precursor of sandalwood oil. However, imbalances in enzymatic activity often result in a metabolic burden on hosts and repress the synthetic capacity of the desired product. In this work, we manipulated ribosome binding sites (RBSs) to optimize an α-santalene synthetic operon in E. coli, and the best engineered E. coli NA-IS3D strain could produce α-santalene at a titer of 412 mg·L-1. Concerning the observation of the inverse correlation between indole synthesis and α-santalene production, this study speculated that indole-associated amino acid metabolism would be competitive to the synthesis of α-santalene rather than indole toxicity itself. The deletion of tnaA could lead to a 1.5-fold increase in α-santalene production to a titer of 599 mg·L-1 in E. coli tnaA- NA-IS3D. Our results suggested that the optimization of RBS sets of the synthetic module and attenuation of the competitive pathway are promising approaches for improving the production of terpenoids including α-santalene.

The mechanism of improved intracellular organic selenium and glutathione contents in selenium-enriched Candida utilis by acid stress.

Batch culture of Candida utilis CCTCC M 209298 for the preparation of selenium (Se)-enriched yeast was carried out under different pH conditions, and maximal intracellular organic Se and glutathione (GSH) contents were obtained in a moderate acid stress environment (pH 3.5). In order to elucidate the physiological mechanism of improved performance of Se-enriched yeast by acid stress, assays of the key enzymes involved in GSH biosynthesis and determinations of energy supply and regeneration were performed. The results indicated that moderate acid stress increased the activity of γ-glutamylcysteine synthetase and the ratios of NADH/NAD+ and ATP/ADP, although no significant changes in intracellular pH were observed. In addition, the molecular mechanism of moderate acid stress favoring the improvement of Se-yeast performance was revealed by comparing whole transcriptomes of yeast cells cultured at pH 3.5 and 5.5. Comparative analysis of RNA-Seq data indicated that 882 genes were significantly up-regulated by moderate acid stress. Functional annotation of the up-regulated genes based on gene ontology and the Kyoto Encyclopedia of Genes and Genome (KEGG) pathway showed that these genes are involved in ATP synthesis and sulfur metabolism, including the biosynthesis of methionine, cysteine, and GSH in yeast cells. Increased intracellular ATP supply and more amounts of sulfur-containing substances in turn contributed to Na2SeO3 assimilation and biotransformation, which ultimately improved the performance of the Se-enriched C. utilis.

The mechanism of improved pullulan production by nitrogen limitation in batch culture of Aureobasidium pullulans.

Batch culture of Aureobasidium pullulans CCTCC M 2012259 for pullulan production at different concentrations of ammonium sulfate and yeast extract was investigated. Increased pullulan production was obtained under nitrogen-limiting conditions, as compared to that without nitrogen limitation. The mechanism of nitrogen limitation favoring to pullulan overproduction was revealed by determining the activity as well as gene expression of key enzymes, and energy supply for pullulan biosynthesis. Results indicated that nitrogen limitation increased the activities of α-phosphoglucose mutase and glucosyltransferase, up-regulated the transcriptional levels of pgm1 and fks genes, and supplied more ATP intracellularly, which were propitious to further pullulan biosynthesis. The economic analysis of batch pullulan production indicated that nitrogen limitation could reduce more than one third of the cost of raw materials when glucose was supplemented to a total concentration of 70 g/L. This study also helps to understand the mechanism of other polysaccharide overproduction by nitrogen limitation.

Selenium-enriched Candida utilis: Efficient preparation with l-methionine and antioxidant capacity in rats.

Selenium-enriched Candida utilis has attracted much attention due to its expanding application in food and feed additives. The objective of this study was to efficiently prepare selenium-enriched C. utilis and to investigate the effects of the prepared yeast on antioxidant capacity in rats. A batch culture of selenium-enriched C. utilis was first carried out, and the addition of sodium selenite (Na(2)SeO(3)) after all glucose had been consumed was found to favor higher intracellular glutathione and organic selenium content. Moreover, l-methionine boosted yeast cell growth and glutathione biosynthesis, and prevented glutathione from leaking to the extracellular space that can be caused by Na(2)SeO(3). We therefore developed a two-stage culture strategy involving supplementation with l-methionine and Na(2)SeO(3) at separate culture phases to improve the performance of selenized C. utilis. Using this two-stage culture strategy, intracellular glutathione content reached 18.6 mg/g and 15.5mg/g, respectively, in batch and fed-batch systems, and organic selenium content reached 905.2 µg/g and 984.7 µg/g, respectively. The effects of selenium-enriched C. utilis on the activities of antioxidant related enzymes in rats were investigated, and the prepared selenium-enriched C. utilis was shown to be an optimal dietary supplement for enhancing antioxidant capacity in rats.

Efficient preparation of selenium/glutathione-enriched Candida utilis and its biological effects on rats.

The main purpose of this study was to prepare selenium/glutathione-enriched Candida utilis and investigate its effect on growth performance, antioxidant capacity, and immune response in rats. The preparation of the selenium/glutathione-enriched yeast was conducted using fed-batch culture for high cell density. The optimal culture conditions for increased intracellular organic selenium and glutathione contents were as follows: the concentrated medium was fed beginning at 12 h using a polynomial feeding strategy until a total glucose concentration of 150 g/l was reached, and sodium selenite was continuously added together with glucose to a total concentration of 60 mg/l. As a result, 81 % of sodium selenite was assimilated and transformed into organic selenium by C. utilis under optimal conditions, which in turn resulted in greater glutathione accumulation and lower malondialdehyde cellular content in the yeast. To investigate and compare the effects of the prepared selenized C. utilis and other dietary supplements, 40 female rats were divided into five groups of eight rats each, following a randomized block design. Experimental feeding was conducted for a period of 6 weeks. Selenium supplementation with inorganic selenium (sodium selenite) and organic selenium (selenized C. utilis) showed better results than the control and other groups supplemented with yeast with or without glutathione. The body mass of rats, selenium deposition, and oxidative enzymes activities in both serum and liver samples, and immunity responses were all significantly improved by selenium supplementation, and between the two sources, organic selenium was more effective than inorganic selenium.