The metabolic pathways of polyhydroxyalkanoates and exopolysaccharides synthesized by Haloferax mediterranei in response to elevated salinity.

How polymer synthesis is mobilized or activated as a biological response of Haloferax mediterranei against hypertonic conditions remains largely unexplored. This study investigated the protein expression of H. mediterranei in response to high salinity by using isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis. The microbes were harvested at end of fermentation at the NaCl salinity of 75 and 250 g L-1. Among the identified 2123 proteins, 170 proteins were differentially expressed. Gene ontology annotation revealed that the highest number of proteins was annotated in biological process category, which was responsible for metabolic process, cellular component and catalytic activity. Differentially expressed proteins were belonged to the class of response to stimulus as well as catalytic activity and binding. Under high salinity conditions, three pathways were established as key responses of PHA and EPS production to hypertonic pressure. Two overexpressed proteins, beta-ketoacyl-ACP reductase and 3-hydroxyacyl-CoA dehydrogenase, enhanced the synthesis of PHAs. The serine-pyruvate transaminase and serine-glyoxylate transaminase were upregulated, thereby increasing the conversion of glucose to PHA. Downregulated levels of sulfate-adenylyl transferase and adenylyl-sulfate kinase could cause diminished EPS synthesis. This study could contribute to better understanding of the proteomic mechanisms of the synthesized polymers in defending against salt stress. SIGNIFICANCE: Haloferax mediterranei, a family member of halophilic archaea, is well known for its fermentative production of poly-ß-hydroxyalkanoates (PHAs). PHAs are natural polymers that exhibit great potential in a wide range of applications such as a good alternative to petroleum-based plastics and the biocompatible material. For decades, the functional role of PHAs synthesized by H. mediterranei is deemed to be carbon and energy reservations. The finding proved that differential production of PHA and EPS in H. mediterranei exposed to elevated salinity was caused by differential protein expression. This is the first report on how PHA and EPS synthesized by H. mediterranei is mobilized as the response of increased salinity, contributing to the understanding of halophilic archaea's response to hypertonic stress and the precise control of fermentation production. Despite its advantages as a PHA cell factory, H. mediterranei synthesized EPS simultaneously, thereby lowering the maximum yield of PHA production. Overall, salinity can be used as a vital microbial fermentation parameter to obtain the highest harvest of PHA, as well as the lowest EPS synthesis in industrial fermentation.

Preparation, characterization and in vitro release study of a glutathione-dependent polymeric prodrug Cis-3-(9H-purin-6-ylthio)-acrylic acid-graft-carboxymethyl chitosan.

In this work, an amphiphilic polymeric prodrug Cis-3-(9H-purin-6-ylthio)-acrylic acid-graft-carboxymethyl chitosan (PTA-g-CMCS) was designed and synthesized. In aqueous solution, this grafted polymer can self-assemble into spherical micelles with a size ranging from 104 to 285 nm and zeta potential ranging from -12.3 to -20.1 mV. For the release study, less than 24% of 6-Mercaptopurine (6-MP) was released from PTA-g-CMCS1 in the media containing 2 and 100 µM glutathione (GSH), whereas 37%, 54% and 75% of 6-MP was released from the media with GSH of 1, 2 and 10mM, respectively. Besides, pH and drug content of the polymeric prodrug only presented slight influence on the 6-MP release. MTT assay demonstrated that this system had higher inhibition ratio on HL-60 cells (human promyelocytic leukemia cells) in the presence of GSH and lower cytotoxicity on mouse fibroblast cell line (L929). Therefore, this nano-sized system is glutathione-dependent, and it can be employed as a potential carrier for the controlled release of 6-MP.