产丁二酸运动发酵单胞菌的构建和发酵性能

Construction and preliminary fermentation of succinate-producing recombinant ethanologenic Zymomonas mobilis

丁二酸是一种重要的有机化工原料及中间体,微生物发酵法产丁二酸具有十分显著的经济和环境优势,但目前研究中的产丁二酸野生菌或工程菌发酵副产物较多,且大多数发酵过程中需严格厌氧,限制了在丁二酸工业化生产中的应用.本研究以运动发酵单胞菌(Zymomonas mobilis ZM4)为出发菌株,利用同源重组原理分别敲除其ED代谢途径2个关键酶基因——丙酮酸脱羧酶基因(pdc,ZMO1360)和乙醇脱氢酶基因(adh B,ZMO1596),以期获得能产丁二酸的重组工程菌株.通过同源重组成功获得敲除丙酮酸脱羧酶基因及乙醇脱氢酶基因的2株突变菌株,分别命名为ZM1360和ZM1596.发酵试验结果显示:在23 g/L葡萄糖的培养基发酵30 h,ZM1360和ZM1596丁二酸产量分别达2.8 g/L和0.67 g/L,乙醇产量分别下降16.7%和2.33%;而出发菌株Z.mobilis ZM4发酵液中则未能检测到丁二酸.与出发菌株Z.mobilis ZM4相比,ZM1360和ZM1596中丙酮酸脱羧酶(PDC)和乙醇脱氢酶(ADH)酶活也分别下降35.85%和69.51%;相应地,pdc基因表达下降了82%,基本无adh B基因表达.本研究表明,通过阻断Z.mobilis乙醇合成代谢途径,有利于丁二酸的积累,为构建新型高效的产丁二酸基因工程菌株奠定了一定基础.

Succinic acid is an important organic chemical raw material and intermediate, and its production based on microbial fermentation has economic and environmental advantages. Currently, different natural succinate-producing or genetically modified strains have been used for bio-based succinic acid production. Although these strains showed some advantages for succinic acid production, the process of fermentation is anaerobic with many byproducts, severely limiting industrialized production of succinic acid. In order to construct high-yield succinate producing engineering strains of Zymomonas mobilis, two key genes pdc and adhB of Z. mobilis in ED metabolic pathway were deleted using homologous recombination, with the recombinant strains named ZM1360 and ZM1596 found to produce 2.8 g/L and 0.67 g/L succinate from 23 g/L glucose in 30 h fermentation. Their ethanol production declined by 16.7% and 2.33%, respectively. Interestingly, there was no succinate production in the fermentationbroth of Z. mobilis ZM4. Meanwhile, PDC and ADH activity of Z. mobilis strains ZM1360, ZM1596 were 35.85% and 69.51%, respectively, lower than the parent strain. The gene expression level of pdc in ZM1360 decreased by 82%, and no expression was detected for adhB in ZM1596. Thisstudysuggests that disturbing synthesis pathways of ethanol in Z. mobilis is conducive to the accumulation of succinic acid, and laysafoundationfor theconstructionof gene engineering succinate-producing strains.