Transformation Of Bacillus Subtilis Metabolic Pathway To Produce Hyaluronic Acid

Hyaluronic acid?HA?as an important medical application materials and cosmetics to add raw materials,now has been listed as emerging food resources.Since the first extraction of HA from the bovine vitreous in 1953 to the HA from the animal tissue to extract the production of HA to meet the market demand,and then in recent years to gradually replace the microbial fermentation of animal tissue extraction HA production of the main means,HA production sources gradually Has changed.Streptococcus is the preferred microbial strain for the production of HA,because Streptococcus is a specific enzyme known as hyaluronan synthase?HAS?which is necessary for the production of HA.But the streptococcus has ?-hemolytic and other shortcomings,researchers often use physical or chemical mutagenesis and other methods to reduce the toxicity of streptococcus.At the same time,such mutagenesis methods are risk factors such as randomness and genetic instability,and thus become the main limiting factor for the production of HA as a fermentative strain.With the development of synthetic biology,making the use of genetic background clear,high biosafety chassis cells to produce HA as possible,Bacillus subtilis as with HAS contains all HA synthetic metabolic pathways and key enzymes and is widely used in industrial production Which was selected as the object of transformation by this study.By using the synthetic biology principle and molecular biology technology to construct a new metabolic pathway,Bacillus subtilis can produce HA by self-metabolism by cloning the HAS gene.In this paper,the properties of cetyltrimethylammonium bromide?CTAB?and HA reaction were observed,and the CTAB turbidity method was optimized to determine the detection conditions and detection range: 2.5 g/L CTAB-Na Cl solution;detection wavelength of 400 nm;at room temperature,the test sample and CTAB solution reaction 5 min;measurement time error control within 5 s,through the production of standard curve to calculate the sample HA content.The results showed that the impurity had little effect on the determination of CTAB turbidity method.And the method is safe and easy to operate,and it is suitable for detecting HA content in a large amount of fermentation broth.The results showed that the yield of HA was 1.2 g/L,which was higher than that of domestic non-Streptococcus production.The relative molecular mass of HA produced by fermentation of Bacillus subtilis was measured by Ubbelohde viscometer.The relative molecular mass was about 350,000 Da.And after several passages,HA production and HA molecular weight remained stable,in order to further study the fermentation of Bacillus subtilis production HA established the foundation.The results showed that the composition of the culture medium was as follows: Yeast powder 5 g/L;peptone 20 g/L?2 g/L?was used to determine the efficiency of the synthesis of Bacillus subtilis in the construction of new metabolic pathways,and the recombinant medium was optimized by single factor test and orthogonal design experiment;Glucose 20 g/L;MgSO₄·7H₂O 100 mol/L;the best culture conditions: p H 7.5;37 ?;200 rpm/min;The medium was applied to 5 L fermentor and the yield was 5 g/L at 36 h.The separation and purification of HA in fermentation broth were established by CTAB precipitation method.The optimized separation conditions were as follows: the concentration of HA was 0.5-1 g/L,and the CTAB solution with the mass fraction of 10% was added to reach 15% of the total volume of the mixture.The mixture was dissociated with 2 mol/L Na Cl solution for 2 h with stirring in a magnetic stirrer.Finally,anhydrous ethanol was added to 70% of the total volume of precipitated protein.The protein removal rate was 94%.Based on the synthetic biology theory,the HA metabolic pathway was established by Bacillus subtilis.At the laboratory level,the HA production was preliminarily determined,and the preparation conditions and detection methods were optimized.The yield was higher than that of non-streptococcus Level,providing a possibility for the subsequent production of high biosafety levels.