The Research Of Genetic Engineering Strain Of Streptococcus Zooepidemicus Construction And Its Fermentation For High Efficiently Producing Low Molecular Hyaluronic Acid

Hyaluronic acid（HA）is a uniformly repetitive,linear glycosaminoglycan composedof glucuronic acid and N-acetylglucosamine joined alternately byβ-1-3 andβ-1-4 glycosidic bonds.HA has strong ability to bind with water,which weight is about 500 times than the weight of HA.Therefore,HA has a strong water retention property and is commonly used in cosmetics as a moisturizer,Especially,HA with low molecular weight and oligosaccharides HA can be more widely used in the medicinetherapy.However,at present,the efficient and inexpensive preparation of such HA is still urgent to be explored.In this study,we used different methods such as genetic modification,NTG mutatgenesis and rifampicin selecetion,optimum the fermentation conditions and add the hyaluronidase into the cultures during the fermentation to get a better strain and fermentation method for the production of low molecular weight of HA:1.Constitutive expressionhaemoglobin of Vitreoscilla（Vhb）can enhance the oxygen usage efficiency,which can stimulate the NADH oxidase（NOX）activity to get more energy and inhibit the production of lactic acid during the fermentation.2.Analysethe fermentation conditions,the fed-batch of sucrose can enhance the production of low molecular of HA in this strain.3.Using the NTG mutagenesis and rifampicinselection method,we have found a hyaluronidasemutant strain（SZ042）,which has strong ability to synthesis the low molecular of HA.Compared to the key factors for the HA production,the enzyme activity of NOX and lactate dehydrogenase（LDH）in mutant strain is higher,conversely the wild type strainis lower.Analyse the optimum fermentation condition for this strain,the best carbon source for the production of HA is sucrosetomaltose 3:1 during the fed-batch.During the fermentarion the optimum of the concentration of carbon source is only the half time than the wild type strain.4.Using the Zn²⁺induction promoter to co-expression of HasA,HasD and HasC can improve the efficiency of low molecular of HA production in the SZ042.During the induction,increased ratio of HasA to HasB can promote the low molecular of HA production.Additionally,the high level of HasD and HasC can decrease the energy expenditure,which can promote the low molecular of HA production.5.Adding the hyaluronidase into the cultures during the fermentation can induce the high production of the low molecular of HA.Using the K.lactis protein expression system,we successfullyget the the hyaluronidase expression from Leech.In the supernatant of recombinant K.lactis fermentation,we detected very high hyaluronidase（6×10⁴U/ml）in it.Adding 100U/L of the recombinant hyaluronidase from Leech can promote the low molecular weight of HA production greatly.The maximum production of HA is 29.5g/L,which is about 15 times than the wild type strain.In conclusion,we use new genetic modification and optimum the fermentation conditions to get a proper strain and fermentation methods for the production of low molecular weight of HA by NTG mutagenesis and rifampicinselection.The new strain can reduce the usage of carbon source,energy consumption during the fermentation process and increase the yield of HA during the fermentation.Adding the hyaluronidase into the cultures during the fermentation can be used to obtain low molecular weight HA or HAoligomer,and it can greatly increase the yield of hyaluronic acid.Therefore,these improved technologies will make great contribute to extensive application of HA.