Effect Of Dissolved Oxygen On Curdlan Production And Physiological Metabolism In Agrobacterium Sp. ATCC 31749

Curdlan is produced by Agrobacterium sp. ATCC 31749 under the nitrogen limiting condition. The biosynthesis of crudlan is a typical aerobic bioprocess, and the production of curdlan would be severely limited under micro-aerobic and anoxic conditions. In order to elucidate the influence of the dissolved oxygen on the metabolic pathway and regulatory mechanism of the biosynthesis of curdlan, textural analyzer was used to investigate the gel properties of curdlan under various dissolved oxygen. Subsequently, proteomic analysis of Agrobacterium sp. was conducted to investigate the effect of the dissolved oxygen on the expression profiles of the crucial regulatory proteins and enzymes involved in curdlan biosynthesis. In addition, transcriptomics was conducted to clarify the metabolic regulation and control mechanism, attempting to elucidate the preliminary mechanism of crudlan biosynthesis affected by dissolved oxygen, and then providing a theoretical basis for further improving of the extracellular polysaccharide synthetic rate in curdlan fermentation process.Curdlan fermentation process was characterized at 5%, 25%, 50%, 75% of dissolved oxygen levels. The results suggested that the production of curdlan were promoted with DO increased from 5% to 50%, but declined at 75% of oxygen level. Textural analysis illustrated that the gel characteristics of strength, springiness, hardness, and frangibility of curdlan were influenced by various dissolved oxygen levels.Proteomic analysis of Agrobacterium sp. was conducted to investigate the influence of the dissolved oxygen on the crucial enzymes involved in curdlan biosynthesis. Twodimensional gel electrophoresis was performed to separate and visualize the differential expression of the intracellular proteins extracted from Agrobacterium sp. ATCC 31749 cultured under various dissolved oxygen levels(5%, 25%, 50%, 75%). In addition, a comparative proteomic analysis of the intracellular proteins expression level under various dissolved oxygen levels was carried out. The significant differently expressed proteins were selected and identified by MALDI-TOF/TOF. Finally, forty-eight differently expressed proteins were identified which were involved in polysaccharide synthesis, fatty acid synthesis, amino acid synthesis pathway. Among these proteins, phosphoglucomutase and orotidine 5-phosphate decarboxylase were the key metabolic enzymes directing curdlan biosynthesis. It was indicated that oxygen could affect the expression of the proteins taking charge of curdlan synthesis significantly. High dissolved oxygen level could enhance the metabolic flux of the key precursor biosynthesis processes, such as UDP-glucose accumulation, resulting the increase of curdlan producing. Furthermore, Agrobacterium sp. enhanced curdlan biosynthesis through promoting fatty acid synthesis and energy accumulation under the higher dissolved oxygen level.Real-time reverse transcription PCR was used to analysis transcriptional levels of the key genes involved in curdlan synthesis, energy metabolism, central metabolic pathway, oxidation stress, transcriptional regulation factors. The results indicated that the dissolved oxygen could significantly affect the transcriptional levels of the gene related to curdlan synthesis and transport proteins to regulate curdlan production. Under 50% of DO, the transcriptional levels of the genes about the central metabolic pathway and energy metabolism were markedly increased, leading to a large amounts of energy to promote the synthesis of curdlan. Furthermore, appropriate dissolved oxygen level would improve the transcriptional level of sigma factor to enhance curdlan accumulation.