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中国农学通报

中国农学通报 ›› 2018, Vol. 34 ›› Issue (27): 89-96.doi: 10.11924/j.issn.1000-6850.casb17070007

所属专题： 生物技术

• 生物技术科学 • 上一篇    下一篇

2,3-丁二醇脱氢酶基因(BudC)过表达菌株的构建和初步鉴定

王长丽,孙 雯,宋 刚,葛菁萍   

  1. 黑龙江大学 生命科学学院,黑龙江大学 生命科学学院,黑龙江大学 生命科学学院,黑龙江大学 生命科学学院
  • 收稿日期:2017-07-03 修回日期:2017-08-12 接受日期:2017-08-24 出版日期:2018-09-19 发布日期:2018-09-19
  • 通讯作者: 葛菁萍
  • 基金资助:
    国家自然科学基金“从2,3-丁二醇代谢角度构建工程微生物群体及其生态学机制研究”(31570492)，“菌群演替与温水沤麻系统关键酶产生菌代谢组学特征的耦合机制”(31270534)，“副干酪乳杆菌与枯草芽孢杆菌等菌群种间关系及协同合作策略的研究”(31470537)

2,3-Butanediol Dehydrogenase Gene (BudC) Overexpress K. oxytoca HD79 Strain: Construction and Identification

  • Received:2017-07-03 Revised:2017-08-12 Accepted:2017-08-24 Online:2018-09-19 Published:2018-09-19

摘要： 旨在过表达BudC进而上调2,3-丁二醇（2,3-Butanediol, 2,3-BD）的合成，通过基因工程手段构建整合载体pR1SW-BudC，经PCR和酶切双重验证后将其电转化至产酸克雷伯氏菌(Klebsiella oxytoca) HD79。结果经卡那霉素筛选，获得一株菌株K. oxytoca HD79-01。以原始菌株为对照，摇瓶发酵检测2,3-BD含量和相关酶活，q RT-PCR检测BudC表达差异，最终BudC在HD79-01中转录水平的表达量为HD79的45.25倍（p<0.01）。摇瓶发酵显示2,3-BD的最高产量、转化率和生产强度分别提高了24.54%、10.97%、45.08%（分别为30.818 ± 0.003 g/L、0.253 ± 0.013 g/g、0.43 ± 0.01 g/(L·h)），酶活提高了3.61倍(0.563 ± 0.003 U/mg)。因此，2,3-丁二醇脱氢酶基因(BudC)过表达菌株构建不仅成功的提高了2,3-BD的产量也为实现2,3-BD的工业化生产奠定基础。

关键词: 沼气, 沼气, 产业发展, 可再生能源, 政策法律框架

Abstract: This study aims at overexpressingBudC and then upregulating the synthesis of 2,3-butanediol (2,3- BD). We constructed a recombinant overexpression vector named pR1SW-BudC by genetic engineering technology, after the PCR and enzyme digestion identification, pR1SW-BudC was transformed intoKlebsiella oxytoca HD79 by electric conversion, K. oxytoca HD79- 01 was obtained by kanamycin screening. Using original strain as the control, 2,3-BD content and the relative enzyme activity was detected with shaking flask fermentation. Furthermore, expression difference of BudC was detected with qRT- PCR. Finally, BudC expression in HD79-01 was 45.25 times as much as that of HD79 (P <0.01). The shaking flask fermentation showed that the maximum of yield, conversion rate and production strength increased by 24.54%, 10.97% and 45.08%, respectively (30.818 ± 0.003 g/L, 0.253 ± 0.013 g/g, 0.43 ± 0.01 g/(L·h)). The enzyme activity increased by 3.61 times (0.563 ± 0.003 U/mg). Thus, the construction ofBudC overexpressK. oxytoca HD79 strain not only increased 2,3-BD yield, but also laid the foundation for the industrial production of 2,3-BD.

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