Acid Adaptive Mechanism Of The Bacterial Cellulose Biosynthesis

Bacterial cellulose, a kind of exopolysaccharides secreted by Komagataeibacter hansenii, has peculiar properties such as exquisite net structure, high crystallinity, high intensity, excellent bio-affinity, biocompatibility, biocompatibility and biodegradability. As a new type of nano material, BC had already become into one of research hotspots, and wildly applied in many fields. However, during the BC biosynthesis process, the producer generated many by-products such as gluconic acid and acetic acid that made p H of the medium into acidic circumstance, and the acid stress effect the growth and survival of the producer, and then influence the BC biosynthesis. Hence, understand the survival of producer under acid stress and the adaptive mechanism has important meaning to both fundamental research and application. In this research, anti-oxidant of glutathione pathway, metabolism adaptive of cytomembrane fatty acid and compensatory pathway of glyoxylate cycle are mainly studied, and illustrated the acid adaptive mechanism through anti-oxidant system, resistance of cytomembrane structure and energy metabolism. At the same time, combine DES and 60Co-γ to compound mutation, and screen the strains with high yield and low acid with proton suicide method. To provide science basis for follow-up metabolism engineering of high yield synthesis of BC, improvement of BC yield also contribute to the rapid development and the survival of the industry. Main research contents are as follow:Acid stress caused the excessive accumulation of ROS, ATP over consumption, reduced the activity of H+-ATPase, aggravated the damage of H+ in intracellular, and then resulted in the decline of survival rate, total glutathione and GSH, increase the GSSG, and GSH/GSSG significant reduce. Enzymology analyses demonstrated that the activity of GSH still high-efficiency expression, however the activity of key enzyme that catalyze GSSG into GSH significant restrained. At the same time, the NADPH that necessary to the catalysis is also excessive consumption. All the results demonstrated that under acid stress, partial death of thallus maybe caused by the imbalance of redox, high-efficiency expression of NADP-GDH to catalyze the reaction of deamination of glutamic acid into α-oxoglutarate and ammoniac, and generate the NADPH and ATP demonstrated that thallus can resistant the oxidative stress through the energy and reduce power provided by catabolism of glutamic acid under severe acid stress circumstance.Compare the fluidity of cell membrane and change rule of fatty acid of Komagataeibacter hansenii under acid stress circumstance with the environment with p H value 6.5. The results demonstrated that decline of the fluidity, increase the unsaturated fatty acid, the degree of unsaturation and the chain length. At the same time, alone with the decline of the p H value, saturated fatty acid decrease, unsaturated fatty acid increase especially C18-1w9 c and C19-Cyc during the fermentation. RT-PCR demonstrated that gene des and cfa are activated under acid stress. The results of exogenous additive group demonstrated that fatty acid synthesis was participated in the regulation of formation of fatty acid structure of cell membrane, and the regulation has the feedback inhibition. The inhibition of the synthesis of fatty acid will enhance the sensitivity of Komagataeibacter hansenii to acid stress circumstance and result in the lower survival rate. All results demonstrated that the conversation of the fatty acid of cell membrane is a kind of survival mechanism of acid resistance, the process of synthesis of fatty acid was participated in the conversation and improvement the adaptation of acid resistance.Enzyme activity of G6 PDH and IDH are decreased resulted in metabolism inhibition of ppp pathway and TCA pathway, and decrease the synthesis of NADPH under acid stress. Key enzymes of ICL and MS are increased significantly which belongs to the glyoxylate cycle. With the corresponding, the products of glyoxylate cycle are significant increased such as malic acid, succinic acid, and α-oxoglytarate with anti-oxidant effect, however the accumulation of pyruvic acid and citric acid are decreased. Stress proteins such as aat A, grp E, dna K and dna J are activated under acid stress demonstrated that glyoxylate cycle used as compensation way exert important role in anaplerosis of gluconeogenesis and supply energy in TCA, at the same time, the generated α-oxoglytarate and stress proteins are participated in the oxidative stress against the acid stress to ensure the survival of thallus under acid stress.Combined DES and 60Co-γ to compound mutation, and obtained the strains Br-3 and Co-5 with high yield and low acid by proton suicide method, and strain Br-12 with low yield. Gene polymorphism analyzed of the mutation strains by AFLP, and sequenced 5 difference fragments found that 3 difference fragments of Br-12 encode dependency receptor protein Ton B, exopolysaccharides exportin(Pe Pr) and conserved hypothetical protein, respectively and have highly homologous gene sequence, however, one of the 2 difference gene fragments from Co-5 homology to acs D, and the other one homology to epimerase of UDPG. Metabolism analyze to mutations strains demonstrated that, gluconic acid had greater influence on the yield of BC, and present negative correlation, this also demonstrated that differences of genes influenced the metabolism and yield directly, and combine the metabolism and gene information could rapid screen and identification the corresponding mutation strains.