两种策略实现1,3-丙二醇关键酶基因的共表达

Two strategies for coexpression of the key enzymes involved in 1,3-propanediol production

甘油脱水酶(GDHt)和1,3-丙二醇氧化还原酶(PDOR)是甘油歧化为1,3-丙二醇(1,3-PD)的两个关键酶。采用多顺反子重组和质粒共存两种策略,对来自克雷伯肺炎杆菌(Klebsiella pneumoniae)的两个关键酶进行共表达。构建表达载体pET-28a-dhaB1 B2 B3-dhaT将两酶基因dhaB1 B2 B3和dhaT用SD序列相隔,在E.coliBL21(DE3)高水平共表达了GDHt 3个亚基和PDOR,表达蛋白分别约占菌体总蛋白的18%、9%、7%和9%。质粒pET-28a-dhaB1 B2 B3和pET-22b-dhaT共转化E.coliBL21(DE3)得到稳定的双质粒系统,48 h后84%的细胞能同时含有两种质粒,GDHt 3亚基和PDOR分别约占菌体总蛋白的16%、8%、6%和14%。两种酶在两种表达方法下均显示高于原始菌株的酶活力。

Glycerol dehydratase （GDHt） and 1,3-propanediol oxidoreductase （PDOR） are the two key enzymes that catalyse the conversion of glycerol to 1, 3-propanediol （1, 3-PD）. In this study, PDOR and GDHt from Klebsiella pneumoniae were coexpressed by polycistronic expression and coexistence of incompatible plasmids. The recombinant plasmid pET-28a-dhaBIB2B3-dhaT harboring the genes encoding GDHt and PDOR with a Shine-Dalgarno （SD） sequence was transformed into E. coli BL21（DE3）. After induction with isopropyl-beta- D-thiogaiactoside （IPTG）, the three subunits of GDHt as well as PDOR were highly coexpressed, accounting for 18 %, 9 %, 7 % and 9 % respectively of the total protein in the host. A stable two plasmid system was obtained via cotransformation of pET-28a-dhaBIB2B3 and pET-22b-dhaT into E. coli BL21 （DE3）. Approximately 84 % of the daughter cells maintained the two plasmids after 48 fermentation hours and the proportion of expressed enzymes was 16 %, 8 %, 6 % and 14 %, respectively. The activities of the two enzymes are also discussed in this paper.