2,3-丁二醇脱氢酶基因(BudC)过表达菌株的构建和初步鉴定

旨在过表达Bud C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p R1SW-Bud C,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Bud C表达差异,最终Bud C在HD79-01中转录水平的表达量为HD79的45.25倍(P0.01)。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(Bud C)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。

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

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.