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合成气/富CO废气发酵制乙醇是一项新的乙醇生产技术。该技术能以生物质、有机废物气化产生的合成气(主要成分CO,CO2和H2)或炼钢厂的废气为原料,利用特殊的微生物将其发酵为乙醇,因而在生物质、富CO废气及一些有毒或难降解的有机废物利用上将发挥重要作用。Clostridium ljungdahlii是最早发现也是研究得最多的合成气乙醇发酵菌株,其全基因组序列已报道。乙醛铁氧还蛋白氧化还原酶是Clostridiumljungdahlii乙醇合成过程中催化乙酸/乙醛转化的一个重要的酶。本研究根据Clostridium ljungdahlii乙醛铁氧还蛋白氧化还原酶基因序列设计了Ⅱ型内含子插入识别位点及相关引物,其最适插入位点为基因的有义链第360与361位核苷酸之间,之后采用重叠延伸PCR法扩增了内含子的IBS,EBS1d和EBS2序列,在质粒pMTL007的基础上构建了用于Clostridium ljungdahlii乙醛铁氧还蛋白氧化还原酶基因插入失活的质粒pMTL007-Clj-aor-360s,经酶切和DNA测序验证,再转化含质粒pMCljS的大肠杆菌E.coli XL1-blue,利用氯霉素和奇霉素双抗性筛选获得了经甲基化修饰的目的质粒,可用于下一步电转化Clostridium ljungdahlii以构建Clostridiumljungdahlii乙醛铁氧还蛋白氧化还原酶基因失活的基因工程菌。
Syngas / CO-rich waste gas to ethanol production is a new ethanol production technology. The technology can use biomass, organic waste gasification syngas (main components CO, CO2 and H2) or steel mill waste gas as raw material, the use of special microorganisms to ferment it into ethanol, which in the biomass, CO Exhaust gas and some toxic or non-degradable organic waste will play an important role. Clostridium ljungdahlii is the earliest discovered and most studied synthetic ethanol fermentation strains, the whole genome sequence has been reported. Acetaldehyde ferredoxin oxidoreductase is an important enzyme for the conversion of acetic acid / acetaldehyde during the ethanol synthesis of Clostridium ljungdahlii. In this study, based on the Clostridium ljungdahlii acetaldehyde ferredoxin oxidoreductase gene sequence designed type II intronic insertion recognition sites and related primers, the optimal insertion site for the gene’s sense strand 360 and 361 nucleosides Acid, the IBS, EBS1d and EBS2 sequences of intron were then amplified by overlap extension PCR. Based on the plasmid pMTL007, a gene for insertion and inactivation of the acetaldehyde ferredoxin oxidoreductase gene of Clostridium ljungdahlii The plasmid pMTL007-Clj-aor-360s was verified by restriction enzyme digestion and DNA sequencing. The plasmid pMCljS was transformed into E.coli XL1-blue. The methylated The modified plasmid can be used for the next electrotransformation of Clostridium ljungdahlii to construct the genetically engineered bacteria inactivated the Clostridium ljungdahlii acetaldehyde ferredoxin oxidoreductase gene.