发酵透明质酸寡糖兽疫链球菌工程菌株的构建
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摘要
透明质酸(HA)广泛应用于医学、化妆品、食品等领域。HA的生物活性取决于其分子量(M_w)。透明质酸寡糖由于具有重要的生理活性与特殊生理功能,在医药领域具有重要的应用前景。兽疫链球菌因其发酵周期短、生产强度较强的特点,在商业生产HA上具有广泛的应用。为了高效发酵合成透明质酸寡糖和解决发酵过程的溶氧问题,文中通过在兽疫链球菌WSH-24中过表达透明质酸合酶HasA以及优化表达水蛭来源的透明质酸酶LHAase。重组菌株摇瓶发酵24h,透明质酸寡糖积累至0.97g/L,比野生菌提高了182.0%。在3L发酵罐中发酵24 h,透明质酸寡糖生产强度为294.2 mg/(L·h),HA积累至7.06 g/L,比野生菌的罐上水平提高了112.4%。文中所构建的发酵合成透明质酸寡糖的兽疫链球菌重组菌株具有重要的应用前景。
Hyaluronic acid(HA) is widely used in many fields, such as medicine, cosmetics and food. The bioactivity of HA depends on its molecular weight(M_w). Owing to the important physiological activities and special physiological functions, HA oligosaccharides have important application prospects in medicine fields. Streptococcus zooepidemicus has wide applications in commercial production of HA, due to its short fermentation cycle and strong production intensity. In order to efficiently synthesize HA oligosaccharides and solve the dissolved oxygen in the fermentation process, in this study, we overexpressed HA synthase(HasA) and introduced and optimized the leech hyaluronidase LHAase in Streptococcus zooepidemicus WSH-24. As a result, HA oligosaccharides were efficiently produced with improved dissolved oxygen. After 24 h, HA oligosaccharides production intensity reached to 294.2 mg/(L·h), and the concentration accumulated to 0.97 g/L in flask cultures, which was 1.82 times of the wild strain. Impressively, HA oligosaccharides were increased to 7.06 g/L in 3 L fermentor. The constructed Streptococcus zooepidemicus strain for producing HA oligosaccharides would have broad application prospects.
Hyaluronic acid(HA) is widely used in many fields, such as medicine, cosmetics and food. The bioactivity of HA depends on its molecular weight(M_w). Owing to the important physiological activities and special physiological functions, HA oligosaccharides have important application prospects in medicine fields. Streptococcus zooepidemicus has wide applications in commercial production of HA, due to its short fermentation cycle and strong production intensity. In order to efficiently synthesize HA oligosaccharides and solve the dissolved oxygen in the fermentation process, in this study, we overexpressed HA synthase(HasA) and introduced and optimized the leech hyaluronidase LHAase in Streptococcus zooepidemicus WSH-24. As a result, HA oligosaccharides were efficiently produced with improved dissolved oxygen. After 24 h, HA oligosaccharides production intensity reached to 294.2 mg/(L·h), and the concentration accumulated to 0.97 g/L in flask cultures, which was 1.82 times of the wild strain. Impressively, HA oligosaccharides were increased to 7.06 g/L in 3 L fermentor. The constructed Streptococcus zooepidemicus strain for producing HA oligosaccharides would have broad application prospects.
引文
[1]Meyer K,Palmer JW.The polysaccharide of the vitreous humor.J Biol Chem,1934,107(3):629-634.
[2]Weissmann B,Meyer K.The structure of hyalobiuronic acid and of hyaluronic acid from umbilical cord.J Am Chem Soc,1954,76(7):1753-1757.
[3]Kang Z,Zhou ZX,Wang Y,et al.Bio-based strategies for producing glycosaminoglycans and their oligosaccharides.Trends Biotechnol,2018,36(8):806-818.
[4]Cheng FY,Gong QY,Yu HM,et al.High-titer biosynthesis of hyaluronic acid by recombinant Corynebacterium glutamicum.Biotechnol J,2016,11(4):574-584.
[5]Knudson CB,Knudson W.Hyaluronan-binding proteins in development,tissue homeostasis,and disease.FASEBJ,1993,7(13):1233-1241.
[6]Toole BP.Proteoglycans and hyaluronan in morphogenesis and differentiation//Hay ED,Ed.Cell Biology of Extracellular Matrix.2nd ed.Boston,MA:Springer,1991:305-341.
[7]Zakeri A,Rasaee MJ,Pourzardosht N.Enhanced hyluronic acid production in Streptococcus Zooepidemicus by over expressing HasA and molecular weight control with Niscin and glucose.Biotechnol Rep,2017,16:65-70.
[8]De Oliveira JD,Carvalho LS,Gomes AMV,et al.Genetic basis for hyper production of hyaluronic acid in natural and engineered microorganisms.Microb Cell Fact,2016,15(1):119.
[9]Yamada T,Kawasaki T.Microbial synthesis of hyaluronan and chitin:new approaches.J Biosci Bioeng,2005,99(6):521-528.
[10]Liu L,Liu YF,Li JH,et al.Microbial production of hyaluronic acid:current state,challenges,and perspectives.Microb Cell Fact,2011,10(1):99.
[11]Cimini D,Iacono ID,Carlino E,et al.Engineering S.equi subsp.zooepidemicus towards concurrent production of hyaluronic acid and chondroitin biopolymers of biomedical interest.AMB Express,2017,7(1):61.
[12]Stern R,Asari AA,Sugahara KN.Hyaluronan fragments:an information-rich system.Eur J Cell Biol,2006,85(8):699-715.
[13]Johnson ME,Murphy PJ,Boulton M.Effectiveness of sodium hyaluronate eyedrops in the treatment of dry eye.Graef’s Arch Clin Exp Ophthalmol,2006,244(1):109-112.
[14]Sand BB,Marner K,Norn MS.Sodium hyaluronate in the treatment of keratoconjunctivitis sicca.A double masked clinical trial.Acta Ophthalmol,1989,67(2):181-183.
[15]Patel,Sanjay R,Malhotra,et al.The mechanism of action for hyaluronic acid treatment in the osteoarthritic knee:a systematic review.Bmc Musculoskel Dis,2015,16(1):321.
[16]Chen LH,Xue JF,Zheng ZY,et al.Hyaluronic acid,an efficient biomacromolecule for treatment of inflammatory skin and joint diseases:a review of recent developments and critical appraisal of preclinical and clinical investigations.Int J Biol Macromo,2018:572-584.
[17]West DC,Kumar S.The effect of hyaluronate and its oligosaccharides on endothelial cell proliferation and monolayer integrity.Exp Cell Res,1989,183(1):179-196.
[18]Trabucchi E,Pallotta S,Morini M,et al.Low molecular weight hyaluronic acid prevents oxygen free radical damage to granulation tissue during wound healing.Int JTissue React,2002,24(2):65-71.
[19]Termeer C,Sleeman JP,Simon JC.Hyaluronan-magic glue for the regulation of the immune response?Trends Immunol,2003,24(3):112-114.
[20]Benitez A,Yates TJ,Lopez LE,et al.Targeting hyaluronidase for cancer therapy:antitumor activity of sulfated hyaluronic acid in prostate cancer cells.Cancer Res,2011,71(12):4085-4095.
[21]Karbownik MS,Nowak JZ.Hyaluronan:towards novel anti-cancer therapeutics.Pharmacol Rep,2013,65(5):1056-1074.
[22]Miyazaki T,Yomota C,Okada S.Ultrasonic depolymerization of hyaluronic acid.Polym Degrad Stab,2001,74(1):77-85.
[23]RehákováM,Bako?D,Soldán M,et al.Depolymerization reactions of hyaluronic acid in solution.Int J Biol Macromol,1994,16(3):121-124.
[24]Tokita Y,Okamoto A.Hydrolytic degradation of hyaluronic acid.Polym Degrad Stab,1995,48(2):269-273.
[25]Hawkins CL,Davies MJ.Degradation of hyaluronic acid,poly-and mono-saccharides,and model compounds by hypochlorite:evidence for radical intermediates and fragmentation.Free Radic Biol Med,1998,24(9):1396-1410.
[26]Tawada A,Masa T,Oonuki Y,et al.Large-scale preparation,purification,and characterization of hyaluronan oligosaccharides from 4-mers to 52-mers.Glycobiology,2002,12(7):421-426.
[27]Armstrong DC,Johns MR.Culture conditions affect the molecular weight properties of hyaluronic acid produced by Streptococcus zooepidemicus.Appl Environ Microbiol,1997,63(7):2759-2764.
[28]Pummill PE,DeAngelis PL.Alteration of polysaccharide size distribution of a vertebrate hyaluronan synthase by mutation.J Biol Chem,2003,278(22):19808-19814.
[29]Keasling JD.Manufacturing molecules through metabolic engineering.Science,2010,330(6009):1355-1358.
[30]Jin P,Kang Z,Yuan PH,et al.Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168.Metab Eng,2016,35:21-30.
[31]Zhang YL,Luo KL,Zhao QS,et al.Genetic and biochemical characterization of genes involved in hyaluronic acid synthesis in Streptococcus zooepidemicus.Appl Microbiol Biotechnol,2016,100(8):3611-3620.
[32]TlustáM,Krahulec J,Pepeliaev S,et al.Production of hyaluronic acid by mutant strains of group CStreptococcus.Mol Biotechnol,2013,54(3):747-755.
[33]Jin P,Kang Z,Zhang N,et al.High-yield novel leech hyaluronidase to expedite the preparation of specific hyaluronan oligomers.Sci Rep,2014,4:4471.
[34]Chen WY,Marcellin E,Hung J,et al.Hyaluronan molecular weight is controlled by UDP-N-acetylglucosamine concentration in Streptococcus zooepidemicus.J Biol Chem,2009,284(27):18007-18014.
[2]Weissmann B,Meyer K.The structure of hyalobiuronic acid and of hyaluronic acid from umbilical cord.J Am Chem Soc,1954,76(7):1753-1757.
[3]Kang Z,Zhou ZX,Wang Y,et al.Bio-based strategies for producing glycosaminoglycans and their oligosaccharides.Trends Biotechnol,2018,36(8):806-818.
[4]Cheng FY,Gong QY,Yu HM,et al.High-titer biosynthesis of hyaluronic acid by recombinant Corynebacterium glutamicum.Biotechnol J,2016,11(4):574-584.
[5]Knudson CB,Knudson W.Hyaluronan-binding proteins in development,tissue homeostasis,and disease.FASEBJ,1993,7(13):1233-1241.
[6]Toole BP.Proteoglycans and hyaluronan in morphogenesis and differentiation//Hay ED,Ed.Cell Biology of Extracellular Matrix.2nd ed.Boston,MA:Springer,1991:305-341.
[7]Zakeri A,Rasaee MJ,Pourzardosht N.Enhanced hyluronic acid production in Streptococcus Zooepidemicus by over expressing HasA and molecular weight control with Niscin and glucose.Biotechnol Rep,2017,16:65-70.
[8]De Oliveira JD,Carvalho LS,Gomes AMV,et al.Genetic basis for hyper production of hyaluronic acid in natural and engineered microorganisms.Microb Cell Fact,2016,15(1):119.
[9]Yamada T,Kawasaki T.Microbial synthesis of hyaluronan and chitin:new approaches.J Biosci Bioeng,2005,99(6):521-528.
[10]Liu L,Liu YF,Li JH,et al.Microbial production of hyaluronic acid:current state,challenges,and perspectives.Microb Cell Fact,2011,10(1):99.
[11]Cimini D,Iacono ID,Carlino E,et al.Engineering S.equi subsp.zooepidemicus towards concurrent production of hyaluronic acid and chondroitin biopolymers of biomedical interest.AMB Express,2017,7(1):61.
[12]Stern R,Asari AA,Sugahara KN.Hyaluronan fragments:an information-rich system.Eur J Cell Biol,2006,85(8):699-715.
[13]Johnson ME,Murphy PJ,Boulton M.Effectiveness of sodium hyaluronate eyedrops in the treatment of dry eye.Graef’s Arch Clin Exp Ophthalmol,2006,244(1):109-112.
[14]Sand BB,Marner K,Norn MS.Sodium hyaluronate in the treatment of keratoconjunctivitis sicca.A double masked clinical trial.Acta Ophthalmol,1989,67(2):181-183.
[15]Patel,Sanjay R,Malhotra,et al.The mechanism of action for hyaluronic acid treatment in the osteoarthritic knee:a systematic review.Bmc Musculoskel Dis,2015,16(1):321.
[16]Chen LH,Xue JF,Zheng ZY,et al.Hyaluronic acid,an efficient biomacromolecule for treatment of inflammatory skin and joint diseases:a review of recent developments and critical appraisal of preclinical and clinical investigations.Int J Biol Macromo,2018:572-584.
[17]West DC,Kumar S.The effect of hyaluronate and its oligosaccharides on endothelial cell proliferation and monolayer integrity.Exp Cell Res,1989,183(1):179-196.
[18]Trabucchi E,Pallotta S,Morini M,et al.Low molecular weight hyaluronic acid prevents oxygen free radical damage to granulation tissue during wound healing.Int JTissue React,2002,24(2):65-71.
[19]Termeer C,Sleeman JP,Simon JC.Hyaluronan-magic glue for the regulation of the immune response?Trends Immunol,2003,24(3):112-114.
[20]Benitez A,Yates TJ,Lopez LE,et al.Targeting hyaluronidase for cancer therapy:antitumor activity of sulfated hyaluronic acid in prostate cancer cells.Cancer Res,2011,71(12):4085-4095.
[21]Karbownik MS,Nowak JZ.Hyaluronan:towards novel anti-cancer therapeutics.Pharmacol Rep,2013,65(5):1056-1074.
[22]Miyazaki T,Yomota C,Okada S.Ultrasonic depolymerization of hyaluronic acid.Polym Degrad Stab,2001,74(1):77-85.
[23]RehákováM,Bako?D,Soldán M,et al.Depolymerization reactions of hyaluronic acid in solution.Int J Biol Macromol,1994,16(3):121-124.
[24]Tokita Y,Okamoto A.Hydrolytic degradation of hyaluronic acid.Polym Degrad Stab,1995,48(2):269-273.
[25]Hawkins CL,Davies MJ.Degradation of hyaluronic acid,poly-and mono-saccharides,and model compounds by hypochlorite:evidence for radical intermediates and fragmentation.Free Radic Biol Med,1998,24(9):1396-1410.
[26]Tawada A,Masa T,Oonuki Y,et al.Large-scale preparation,purification,and characterization of hyaluronan oligosaccharides from 4-mers to 52-mers.Glycobiology,2002,12(7):421-426.
[27]Armstrong DC,Johns MR.Culture conditions affect the molecular weight properties of hyaluronic acid produced by Streptococcus zooepidemicus.Appl Environ Microbiol,1997,63(7):2759-2764.
[28]Pummill PE,DeAngelis PL.Alteration of polysaccharide size distribution of a vertebrate hyaluronan synthase by mutation.J Biol Chem,2003,278(22):19808-19814.
[29]Keasling JD.Manufacturing molecules through metabolic engineering.Science,2010,330(6009):1355-1358.
[30]Jin P,Kang Z,Yuan PH,et al.Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168.Metab Eng,2016,35:21-30.
[31]Zhang YL,Luo KL,Zhao QS,et al.Genetic and biochemical characterization of genes involved in hyaluronic acid synthesis in Streptococcus zooepidemicus.Appl Microbiol Biotechnol,2016,100(8):3611-3620.
[32]TlustáM,Krahulec J,Pepeliaev S,et al.Production of hyaluronic acid by mutant strains of group CStreptococcus.Mol Biotechnol,2013,54(3):747-755.
[33]Jin P,Kang Z,Zhang N,et al.High-yield novel leech hyaluronidase to expedite the preparation of specific hyaluronan oligomers.Sci Rep,2014,4:4471.
[34]Chen WY,Marcellin E,Hung J,et al.Hyaluronan molecular weight is controlled by UDP-N-acetylglucosamine concentration in Streptococcus zooepidemicus.J Biol Chem,2009,284(27):18007-18014.
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