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首页> 外文期刊>Biotechnology Progress >Biosynthetic burden and plasmid burden limit expression of chromosomallyintegrated heterologous genes (pdc, adhB) in Escherichia coli
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Biosynthetic burden and plasmid burden limit expression of chromosomallyintegrated heterologous genes (pdc, adhB) in Escherichia coli

机译：生物合成负担和质粒负担限制了大肠杆菌中染色体整合的异源基因（pdc，adhB）的表达

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摘要

Previous studies have shown an unexpectedly high nutrient requirement for efficient ethanol production by ethanologenic recombinants of Escherichia coli B such as LY01 which contain chromosomally integrated Zymomonas mobilis genes (pdc,adhB) encoding the ethanol pathway. The basis for this requirement has been identified as a media-dependent effect on the expression of the Z. mobilis genes rather than a nutritional limitation. Ethanol production was substantially increased without additional nutrients simply by increasing the level of pyruvate decarboxylase activity. This was accomplished by adding a multicopy plasmid containing pdc alone (but not adhB alone) to strain LY01, and by adding multicopy plasmids which express pde and adhB from strong promoters. New strong promoters were isolated from random fragments of Z. mobilis DNA and characterized but were not used to construct integrated biocatalysts. These promoters contained regions resembling recognition sites for 3 different E. coli sigma factors: sigma(70), sigma(38), and sigma(28). The most effective plasmid-based promoters for fermentation were recognized by multiple sigma factors, expressed both pde and adhB at high levels, and produced ethanol efficiently while allowing up to 80% reduction in complex nutrients as compared to LY01. The ability to utilize multiple sigma factors may be advantageous to maintain the high levels of PDC and ADH needed for efficient ethanol production throughout batch fermentation. From this work, we propose that the activation of biosynthetic genes in nutrient-poor media creates a biosynthetic burden that reduces the expression of chromosomal pdc and adhB by competing for transcriptional and translational machinery. This reduced expression can be viewed as analogous to the effect of plasmids (plasmid burden) on the expression of native chromosomal genes.
机译：先前的研究表明，通过大肠杆菌B的产醇重组子（例如LY01）产生高效乙醇生产​​所需要的出乎意料的高营养需求，所述重组子包含编码乙醇途径的染色体整合的运动发酵单胞菌基因（pdc，adhB）。已经确定了该要求的基础是对运动发酵单胞菌基因表达的介质依赖性作用，而不是营养限制。仅通过增加丙酮酸脱羧酶活性水平，就可以在不增加营养素的情况下大幅提高乙醇产量。这是通过向菌株LY01中添加仅包含pdc的多拷贝质粒（但不单独包含adhB），以及添加从强启动子表达pde和adhB的多拷贝质粒来实现的。从运动发酵单胞菌DNA的随机片段中分离出新的强启动子，并对其进行了表征，但并未用于构建整合的生物催化剂。这些启动子包含类似于3个不同大肠杆菌sigma因子识别位点的区域：sigma（70），sigma（38）和sigma（28）。多个sigma因子可识别最有效的基于质粒的发酵启动子，可高水平表达pde和adhB，可高效生产乙醇，同时与LY01相比，复合营养素最多可减少80％。利用多个sigma因子的能力可能有利于在批量发酵过程中维持高效乙醇生产​​所需的高水平PDC和ADH。通过这项工作，我们建议在营养贫乏的培养基中激活生物合成基因会产生生物合成负担，从而通过竞争转录和翻译机制来降低染色体pdc和adhB的表达。可以将这种减少的表达视为类似于质粒对天然染色体基因表达的影响（质粒负担）。

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