Hespell R B, Wyckoff H, Dien B S, Bothast R J
Fermentation Biochemistry Research Unit, U.S. Department of Agriculture, Peoria, Illinois 61604, USA.
Appl Environ Microbiol. 1996 Dec;62(12):4594-7. doi: 10.1128/aem.62.12.4594-4597.1996.
In the last decade, a major goal of research in biofuels has been to metabolically engineer microorganisms to ferment multiple sugars from biomass or agricultural wastes to fuel ethanol. Escherichia coli strains genetically engineered to contain the pet operon (Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase B genes) produce high levels of ethanol. Strains carrying the pet operon in plasmid (e.g., E. coli B/pLOI297) or in chromosomal (e.g., E. coli KO11) sites require antibiotics in the media to maintain genetic stability and high ethanol productivity. To overcome this requirement, we used the conditionally lethal E. coli strain FMJ39, which carries mutations for lactate dehydrogenase and pyruvate formate lyase and grows aerobically but is incapable of anaerobic growth unless these mutations are complemented. E. coli FBR1 and FBR2 were created by transforming E. coli FMJ39 with the pet operon plasmids pLOI295 and pLOI297, respectively. Both strains were capable of anaerobic growth and displayed no apparent pet plasmid losses after 60 generations in serially transferred (nine times) anaerobic batch cultures. In contrast, similar aerobic cultures rapidly lost plasmids. In high-cell-density batch fermentations, 3.8% (wt/vol) ethanol (strain FBR1) and 4.4% (wt/vol) ethanol (strain FBR2) were made from 10% glucose. Anaerobic, glucose-limited continuous cultures of strain FBR2 grown for 20 days (51 generations; 23 with tetracycline and then 28 after tetracycline removal) showed no loss of antibiotic resistance. Anaerobic, serially transferred batch cultures and high-density fermentations were inoculated with cells taken at 57 generations from the previous continuous culture. Both cultures continued to produce high levels of ethanol in the absence of tetracycline. The genetic stability conferred by selective pressure for pet-containing cells without requirement for antibiotics suggests potential commercial suitability for E. coli FBR1 and FBR2.
在过去十年中，生物燃料研究的一个主要目标是对微生物进行代谢工程改造，使其能够将生物质或农业废弃物中的多种糖类发酵为燃料乙醇。经过基因工程改造含有pet操纵子（运动发酵单胞菌丙酮酸脱羧酶和乙醇脱氢酶B基因）的大肠杆菌菌株能产生高水平的乙醇。携带pet操纵子的质粒（如大肠杆菌B/pLOI297）或染色体（如大肠杆菌KO11）位点的菌株需要在培养基中添加抗生素以维持遗传稳定性和高乙醇生产率所需。为克服这一需求，我们使用了条件致死性大肠杆菌菌株FMJ39，该菌株携带乳酸脱氢酶和丙酮酸甲酸裂解酶的突变体且在需氧条件下生长，但除非这些突变得到互补，否则无法进行厌氧生长。大肠杆菌FBR1和FBR2分别通过用pet操纵子质粒pLOI295和pLOI297转化大肠杆菌FMJ39而构建成功。在连续转移（九次）的厌氧分批培养60代后，这两种菌株都能够进行厌氧生长且未出现明显的pet质粒丢失。相比之下，类似的需氧培养物会迅速丢失质粒。在高细胞密度分批发酵中，由10%葡萄糖制得3.8%（重量/体积）乙醇（菌株FBR1）和4.4%（重量/体积）乙醇（菌株FBR2）。对菌株FBR2进行厌氧、葡萄糖限制的连续培养20天（51代；23代添加四环素，之后去除四环素培养28代），结果显示未出现抗生素抗性丢失。厌氧、连续转移的分批培养和高密度发酵均接种取自前一次连续培养57代的细胞。在不添加四环素的情况下，两种培养物均继续产生高水平的乙醇。对含pet细胞无需抗生素的选择性压力所赋予的遗传稳定性表明大肠杆菌FBR1和FBR2具有潜在的商业适用性。
