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首页> 外文期刊>Applied Microbiology and Biotechnology >Constitutive high-level expression of a codon-optimized β-fructosidase gene from the hyperthermophile Thermotoga maritima in Pichia pastoris
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Constitutive high-level expression of a codon-optimized β-fructosidase gene from the hyperthermophile Thermotoga maritima in Pichia pastoris

机译：巴斯德毕赤酵母中嗜热嗜热菌的密码子优化β-果糖苷酶基因的组成型高水平表达。

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摘要

Enzymes for use in the sugar industry are preferred to be thermotolerant. In this study, a synthetic codon-optimized gene encoding a highly thermostable β-fructosidase (BfrA, EC 3.2.1.26) from the bacterium Thermotoga maritima was expressed in the yeast Pichia pastoris. The gradual increase of the transgene dosage from one to four copies under the control of the constitutive glyceraldehyde 3-phosphate dehydrogenase promoter had an additive effect on BfrA yield without causing cell toxicity. Maximal values of cell biomass (115 g/l, dry weight) and overall invertase activity (241 U/ml) were reached at 72 h in fed-batch fermentations using cane sugar as the main carbon source for growth. Secretion driven by the Saccharomyces cerevisiae α-factor signal peptide resulted in periplasmic retention (44 %) and extracellular release (56 %) of BfrA. The presence of N-linked oligosaccharides did not influence the optimal activity, thermal stability, kinetic properties, substrate specificity, and exo-type action mode of the yeast-secreted BfrA in comparison to the native unglycosylated enzyme. Complete inversion of cane sugar at initial concentration of 60 % (w/v) was achieved by periplasmic BfrA in undisrupted cells reacting at pH 5.5 and 70 C, with average productivity of 4.4 g of substrate hydrolyzed per grams of biomass (wet weight) per hour. The high yield of fully active glycosylated BfrA here attained by recombinant P. pastoris in a low-cost fermentation process appears to be attractive for the large-scale production of this thermostable enzyme useful for the manufacture of inverted sugar syrup.
机译：制糖工业中使用的酶优选是耐热的。在这项研究中，在酵母毕赤酵母中表达了一种合成的密码子优化基因，该基因编码了一种来自海洋嗜热菌的高度稳定的β-果糖苷酶（BfrA，EC 3.2.1.26）。在组成型3-磷酸甘油醛脱氢酶启动子的控制下，转基因剂量逐渐增加至一到四个拷贝，对BfrA产量产生累加效应，而不会引起细胞毒性。使用蔗糖作为主要生长碳源，在分批补料发酵中72小时达到了细胞生物量（115 g / l，干重）和总转化酶活性（241 U / ml）的最大值。由酿酒酵母α-因子信号肽驱动的分泌导致BfrA的周质保留（44％）和细胞外释放（56％）。与天然非糖基化酶相比，N-连接寡糖的存在不影响酵母分泌的BfrA的最佳活性，热稳定性，动力学特性，底物特异性和外型作用模式。通过周质BfrA在pH 5.5和70 C下反应的未破裂细胞中，蔗糖在初始浓度为60％（w / v）时完全转化，每克生物质（湿重）每克生物质能水解4.4克底物。小时。重组巴斯德毕赤酵母在低成本的发酵过程中获得的高活性的全活性糖基化BfrA，似乎对大规模生产这种热稳定酶具有吸引力，这种酶可用于生产倒糖浆。

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