Construction Of Engineered Strain Producing Pullulan With High Quality And High Yield,optimization Of Production Process And Its Application In Pharmaceutical Preparations

Pullulan is a non-toxic,edible and low calorific natural water-soluble polysaccharide produced by Aureobasidium pullulans.It is a regularly linear repeating glucan and viewed as a succession of α-1,6 linked maltotriose,which is formed by three glucoses linked by α-1,4 glycosidic bonds.Pullulan has unique properties,such as plasticity,stability,film forming ability,oxygen resistance and safety,which bring it a wide range of applications in medicine,food,cosmetics,environmental protection,and other fields.Pullulan was approved as a new food additive by the National Health Ministry of the People’s Republic of China in 2006.In contrast,pullulan was approved for use in the medical field relatively late,mainly in capsules.Now,vacant pullulan capsule has been recorded in the 2020 edition of the Chinese Pharmacopoeia as a new pharmaceutical adjuvant.Currently,the production of pullulan in China is mainly food grade.The high content of impurities,such as melanin,and the low molecular weight and viscosity of pullulan in food grade cannot meet the quality requirements of pullulan in pharmaceutical grade.With the in-depth progress of consistency evaluation of generic drugs and associated evaluation between drug and pharmaceutical adjuvant in China,the quality of pharmaceutical adjuvant needs to be improved.And it is urgent to further improve the quality of pullulan.In addition,due to the price of pullulan is much higher than that of gelatin,it is necessary to optimize the production process,increase the fermentation yield and purification recovery,and reduce the production cost.To date,there are still many problems in the production and application of pharmaceutical grade pullulan.Such as a large amount of melanin,a kind of by-product produced during the fermentation of A.pullulans brings great difficulties in the purification process.The yield of pullulan was low and the purification cost was high.The lack of research on the application of pullulan in the medical field leads to the lack of research data support in the market promotion.To solve problems mentioned above,we constructed engineered strain producing high yield pullulan with no melanin,optimized the fermentation and purification process of pullulan for industrial production,and carried out research on application of pullulan in the field of pharmaceutical preparations(probiotics,capsule).The main contents are as follows:(1)Construction of engineered strain producing high yield of pullulan with no melaninThe wild type A.pullulans CGMCC As3.3984 for pullulan production synthesized melanin during fermentation.It is proposed to achieve engineered strain without melanin synthesis by knocking out of melanin biosynthetic gene.In the process of the homologous recombination gene knocking out,the conditions of electrical transformation were improved and optimized.After comparison of several transformation methods,the attenuated cell wall pulsed electrical transformation method was chosen for further research.The discharge conditions including voltage,duty ratio,frequency and pulse number were optimized together with the culturing time of cells,lyzing enzyme dosage and plasmid dosage.Finally,an electrical transformation process with high efficiency for transformation of A.pullulans was established.According to the literature reports and the result of melanin inhibited by tricyclazole,DHN melanin was inferred as the main melanin produced in A.pullulans CGMCC As3.3984.The sequence of alb1,which had been reported to be important in the biosynthesis of DHN melanin in Sordaria macrospora,was blasted with the genome sequence of A.pullulans.A probable alb]gene was found out and cloned from CGMCC As3.3984,then expressed in pichia pastoris,and studied the fuctionThe key gene alb1 for the synthesis of DHN-melanin was knocked out from A pullulans CGMCC As3.3984 by homologous recombination.The alb1 deletion mutant As-△alb1 was a melanin-free strain with the yield of pullulan decreased by 41.01%than that in wild type strain CGMCC As3.3984.To further investigate the relationship between the synthesis of pullulan and melanin,a pullulan deletion mutant As-△pul was constructed by knocking out of pul from A.pullulans CGMCC As3.3984 by homologous recombination.As-△pul synthesized neither pullulan nor melanin.The supplementation of melanin in the culture of As-Δalb1 increased the production of pullulan.Meantime,Δs-Δpul recovered melanin synthesis by adding pullulan to the medium.The results suggested that a synergistic correlation existed between the synthesis of pullulan and melanin.The relative transcription levels of the genes relevant to the biosynthesis of pullulan were examined in both CGMCC As3.3984 and As-Δalb1.The transcription level of pul was significant reduced in As-△alb1 by knocking out of alb1To obtain melanin deletion A.pullulans with high yield of pullulan,an engineered strain As-△alb1-pul(AP-7)was constructed by overexpression of pul in As-Δalb1.The production of pullulan produceded by As-△alb1-pul(AP-7)was 29.33 g/L,which was 85.87%higher than that of As-△alb1 and 9.64%higher than that of wild type strain.Passage experiments showed that As-△alb1-pul(AP-7)had stable heredity and could be used in industral production of pullulan(2)Establishment of optimized preparation technology for industrial production of pullulan by engineered strain As-Aalb1-pul(AP-7)In order to rapidly and accurately detect the content of pullulan in large number of samples during the optimization of fermentation conditions,a method for rapid determination of pullulan content by step acidolysis was established.The method was designed according to the difference in hydrolysis degree between different types of glycosidic bonds in acid solution.Using the method mentioned above,the content of pullulan in a large number of samples could be accurately determined in about 40 minutes.The precision and accuracy of the method were high when the content of pullulan was below 100 g/L.Results indicated that the step acidolysis method was suitable for rapid determination of pullulan content in optimization of medium and fermentation conditions.Response surface method was used to optimize the fermentation medium.The optimal composition of fermentation medium was 95.89 g/L of sucrose,1.92 g/L of yeast powder,1.00 g/L of NaCl,0.20 g/L of MgSO4.7H2O,6.00 g/L of K2HPO4,and 0.60 g/L of(NH4)2SO4,pH 6.48.Under those conditions,the average yield of pullulan was 32.08 g/L,which was consistent with the predicted value(32.27g/L)Fermentation parameters,including optimum inoculation amount,dissolved oxygen,surfactant,feeding mode,and pH control,were investigated in 1 L-bioreactor.The optimized fermentation conditions in 1 L-bioreactor were as follows:inoculation amount of 5%,ventilation rate of 1 vvm,stirring at 1200 r/min,initial pH5.70,and fermentation time of 72 h.The yield of pullulan with Mw of 404.30 kDa reached 68.43 g/L.Then,100 L-fermentation experiments were carried out.The fermentation parameters needed to be adjusted with the increase of the size of the bioreactor.The optimized fermentation conditions were as follows:inoculation amount of 5%,ventilation rate of 1 vvm,stirring at 250 r/min,initial pH5.92,and fermentation time of 72 h.Under these conditions,the highest yield of pullulan(Mw:331.31 kDa)was 59.92 g/L.Finally,10 t-fermentation experiments were carried out.The maximum yield of pullulan(Mw:535,62 kDa～560.77 kDa)was 65.24 g/L.The downstream purification process was improved and optimized for production of pullulan by engineered strain As-Δalb1-pul(AP-7).The ultrafiltration desalinating and spray drying were used to replace the traditional ethanol precipitation Finally,an improved purification process of pullulan was established,which could be briefly descriped as "pretreatment of fermentation broth-decolorization by activated carbon-clarification by filtration-desalination by ultrafiltration-spray drying".Under the improved purification process,the recovery of pullulan reached 90.41%,and the purification cost was reduced by 79.30%when compared with the original process.(3)Application of pharmaceutical grade pullulan in drug preparationIn the preparation of probiotics,the effect of pullulan as freeze-drying protectant on the survival rate for lactic acid bacteria(Lactobacillus bulgaricus)was studied.The results showed that when pullulan was used as a single freeze-drying protectant,the survival rate of Lactobacillus bulgaricus was 57.3%.In contrast,the survival rate of Lactobacillus bulgaricus was 86.96%when the strain was frozen with compound freeze-drying protectant(3.0%glycerol,5.0%sucrose,5.5%trehalose and 2.0%pullulan).The pharmaceutical stability was investigated by application of pullulan capsule in vitamin C,mussel polysaccharide,and ibuprofen.Compared with gelatin capsules,pullulan capsule had better antioxidant effect and stability for oxidizable or hygroscopic drugs under high temperature and high humidity conditions.The bioequivalence of the pullulan and gelatin capsules containing model drugs,ibuprofen and oseltamivir,were found in beagle dogs,respectively.Results indicated that the replacement of gelatin capsules by pullulan capsules did not affect the bioavailability of ibuprofen and oseltamivir.In conclusion,we constructed an engineered strain producing high yield of pullulan with no melanin,established a preparation process suitable for industrial production through fermentation and purification process optimization,and carried out the application research of pullulan in probiotics and other pharmaceutical preparations.This project helps enterprises solve the technical problems of producing melanin in the fermentation process of pullulan,improves the fermentation yield and product quality of pullulan,reduces the production cost,and has positive significance for the promotion and application of pullulan in the pharmaceutical field.