The Bacterial Cellulose Biosynthesis Of Komagataeibacter Rhaeticus 315

Bacterial cellulose(BC)is an extracellular polysaccharide produced by microbial strains.It is widely used in food,medical,chemical and other fields because of excellent physical and chemical properties.However,the high production cost limited its wide application.Therefore,we should obtain high-yield and high-quality BC production strains and give full play to the synthesis ability of them.In this paper,a genome-wide analysis of a new BC-producing strain Komagataeibacter rhaeticus 315 from Kombucha was studied.On the basis of understanding its metabolic characteristics,we optimized the energy supply,weaken branch metabolic pathways and improved the conversion rate of glucose to reduce the manufacturing cost of BC.The specific results were as follows:(1)The whole genome data showed that the size of K.rhaeticus 315 was 3.70 Mbp,the average coverage rate was 470X,the GC content was 63.98%and the number of predicted coding DNA sequences(CDS)was 3314.There was no coding gene of phosphofructokinase in the genome sequence,which cannot metabolize glucose to generate energy through the EMP pathway.In addition,the strain had a number of genes encoding glucose dehydrogenase.The analysis of metabolites showed that a large amount of gluconic acid accumulated in the system when glucose was the sole carbon source(the concentration of gluconic acid was32.25 g/L after 20 days of fermentation).The strain obtained energy by oxidation of glucose to gluconic acid.There,we constructed recombinant strain of glucose dehydrogenase gene deletion(gcd~-).The fermentation results showed that the production of gluconic acid was reduced by 77%,and the sugar consumption was reduced by 42.1%.The knockout of the gene was helpful to reduce the pathway from glucose to gluconic acid and the consumption of glucose in BC synthesis.(2)The effect of different carbon sources on BC synthesis was investigated.The results showed that glucose was the most favorable for the synthesis of BC,which the yield was 1.51g/L.Ethanol could be greatly improved the synthesis of BC.When 2%ethanol was added,the BC yield was up to 6.60 g/L,increased by 4.37 times.In the study on the mechanism of ethanol promoting BC synthesis,it was found that when glucose was the sole carbon source,the bacteria converted glucose into gluconic acid for energy supply,thus the precursor substance of BC synthesis was consumed heavily which affected BC yield.When ethanol was added,the bacteria could provide energy through the oxidation of ethanol.What's more,transcriptome data indicated that ethanol also affected the gene expression of key enzymes in the metabolic pathway.The gene expression of glucose dehydrogenase and the key enzymes in HMP pathway were down-regulated,the gene expression of key enzymes in the BC synthesis pathway was up-regulated,which were consistent with the trend of metabolic flux.(3)BC is an interfacial synthesis and requires oxygen.The way of oxygen supply and interface area had a great influence on BC yield.The results showed that shake flask fermentation improved the dissolved oxygen but violent agitation was not conducive to BC synthesis.BC can be synthesized continuously at the gas-liquid interface,so we used static intermittent submerged membrane culture which BC yield was increased by 2.3 times.The effect of batch fed fermentation was investigated.The results showed that when the initial concentration of glucose was 20 g/L and the initial amount of ethanol was 2%,5 g/L glucose and 2%ethanol were added every 4 days,BC yield reached 11.17 g/L after 12 days fermentation,which was 1.49 times higher.(4)The basic characteristics of BC membrane were characterized.The results showed that BC has typical type of cellulose?structure,the crystallinity was 88.23%.And BC has good thermal stability,tensile property,water reactivity and water holding capacity.