过表达核苷二磷酸激酶对透明质酸的产量和相对分子质量的影响
Effect of homologous overexpression of nucleoside diphosphate kinase on the yield and molecular weight of hyaluronic acid
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摘要: 为了考察核苷二磷酸激酶（NDK）对透明质酸合成过程的影响，将核苷二磷酸激酶基因（ndk）和透明质酸合成酶基因在重组枯草芽孢杆菌细胞内共同进行过表达，成功构建了两株工程菌株Hp8tg及Pn8tg，均获得了均一相对分子质量透明质酸（HA）。通过响应面分析试验对诱导条件进行了优化，通过单糖组成分析、傅里叶红外光谱分析及核磁共振波谱分析对HA进行了鉴定。ndk过表达使HA产量提高了1.3倍，相对分子质量提高了1.1倍。研究结果表明，核苷二磷酸激酶的过表达可消除尿苷二磷酸（UDP）蓄积对Ⅱ型透明质酸合成酶的抑制作用，从而提高了HA的产量及相对分子质量。此策略可以有效提高HA的产量和相对分子质量，也为其他多糖的制备提供了新的思路。Abstract: To investigate the effect of nucleoside diphosphate kinase (NDK) on the synthesis of hyaluronic acid, nucleoside diphosphate kinase gene (ndk) was overexpressed along with the hyaluronic acid-producing genes in recombinant B.subtilis. Two engineered strains named Hp8tg and Pn8tg were constructed.Uniform hyaluronic acid (HA) could be obtained from both engineered strains.HA produced by both recombinant strains was confirmed by monosaccharide composition analysis, Fourier transform infrared spectometry and nuclear magnetic resonance spectroscopy.Inducing conditions of HA fermentation were optimized by response surface methodology.Overexpression of ndk could increase the production and molecular weight of HA by 1.3-fold and 1.1-fold, respectively. This study revealed for the first time that overexpression of ndk could relieve the inhibition effect of uridine diphosphate (UDP) on Class II HA synthase and increase the production and molecular weight of HA, which proves to be an efficient strategy for the production of HA, and the preparation of other polysaccharides.
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[1] . Biotechnol Lett，2007，29（1）：17-25. [2] Price RD，Berry MG，Navsaria HA. Hyaluronic acid：the scientific and clinical evidence［J］. J Plast Reconstr Aesthet Surg，2007，60（10）：1110-1119. [3] Li WH，Zhou CC，Fu Y，et al. Targeted delivery of hyaluronic acid nanomicelles to hepatic stellate cells in hepatic fibrosis rats［J］. Acta Pharm Sin B，2020，10（4）：693-710. [4] Galassi G，Brun P，Radice M，et al. In vitro reconstructed dermis implanted in human wounds：degradation studies of the HA-based supporting scaffold［J］. Biomaterials，2000，21（21）：2183-2191. [5] Zeng C，Toole BP，Kinney SD，et al. Inhibition of tumor growth in vivo by hyaluronan oligomers［J］. Int J Cancer，1998，77（3）：396-401. [6] Meyer K. The biological significance of hyaluronic acid and hyaluronidase［J］. Physiol Rev，1947，27（3）：335-359. [7] Jing W，DeAngelis PL. Synchronized chemoenzymatic synthesis of monodisperse hyaluronan polymers［J］. J Biol Chem，2004，279（40）：42345-42349. [8] Yu HM，Stephanopoulos G. Metabolic engineering of Escherichia coli for biosynthesis of hyaluronic acid［J］. Metab Eng，2008，10（1）：24-32. [9] Chien LJ，Lee CK. Enhanced hyaluronic acid production in Bacillus subtilis by coexpressing bacterial hemoglobin［J］. Biotechnol Prog，2007，23（5）：1017-1022. [10] Jeong E，Shim WY，Kim JH. Metabolic engineering of Pichia pastoris for production of hyaluronic acid with high molecular weight［J］. J Biotechnol，2014，185：28-36. [11] Hmar RV，Prasad SB，Jayaraman G，et al. Chromosomal integration of hyaluronic acid synthesis （has） genes enhances the molecular weight of hyaluronan produced in Lactococcus lactis［J］. Biotechnol J，2014，9（12）：1554-1564. [12] Jia YN，Zhu J，Chen XF，et al. Metabolic engineering of Bacillus subtilis for the efficient biosynthesis of uniform hyaluronic acid with controlled molecular weights［J］. Bioresour Technol，2013，132：427-431. [13] O''Regan M，Martini I，Crescenzi F，et al. Molecular mechanisms and genetics of hyaluronan biosynthesis［J］. Int J Biol Macromol，1994，16（6）：283-286. [14] Pummill PE，DeAngelis PL. Evaluation of critical structural elements of UDP-sugar substrates and certain cysteine residues of a vertebrate hyaluronan synthase［J］. J Biol Chem，2002，277（24）：21610-21616. [15] Baggenstoss BA，Weigel PH. Size exclusion chromatography-multiangle laser light scattering analysis of hyaluronan size distributions made by membrane-bound hyaluronan synthase［J］. Anal Biochem，2006，352（2）：243-251. [16] Sikarwar J，Kaushik S，Sinha M，et al. Cloning，expression，and purification of nucleoside diphosphate kinase from Acinetobacter baumannii［J］. Enzyme Res，2013，2013：597028. [17] DeAngelis PL. Enzymological characterization of the Pasteurella multocida hyaluronic acid synthase［J］. Biochemistry，1996，35（30）：9768-9771. [18] Wang ZY，Ly M，Zhang FM，et al. E. coli K5 fermentation and the preparation of heparosan，a bioengineered heparin precursor［J］. Biotechnol Bioeng，2010，107（6）：964-973. -
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