State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.
Biotechnol Prog. 2013 Jul-Aug;29(4):862-9. doi: 10.1002/btpr.1759. Epub 2013 Jun 27.
The production of complex compounds from technically convenient microorganisms is an emerging route to the chemical diversity found in the surrounding environment. In this study, the antibiotic compound erythromycin A is produced from Escherichia coli as an alternative to native production through the soil bacterium Saccharopolyspora erythraea. By doing so, there is an opportunity to apply and refine engineering strategies for the manipulation of the erythromycin biosynthetic pathway and for the overproduction of this and other complex natural compounds. Previously, E. coli-derived production was enabled by the introduction of the entire erythromycin pathway (20 genes total) using separately selectable expression plasmids which demonstrated negative effects on final biosynthesis through metabolic burden and plasmid instability. In this study, improvements to final production were made by altering the design of the expression plasmids needed for biosynthetic pathway introduction. Specifically, the total number of genes and plasmids was pruned to reduce both metabolic burden and plasmid instability. Further, a comparison was conducted between species-specific (E. coli vs. S. coelicolor) protein chaperonins. Results indicate improvements in growth and plasmid retention metrics. The newly designed expression platform also increased erythromycin A production levels 5-fold. In conclusion, the steps outlined in this report were designed to upgrade the E. coli erythromycin A production system, led to improved final compound titers, and suggest additional forms of pathway engineering to further improve results from heterologous production attempts.
从技术上方便的微生物中生产复杂化合物是一种新兴途径，可以获得周围环境中存在的化学多样性。在这项研究中，通过大肠杆菌生产红霉素 A 等抗生素化合物，替代了土壤细菌红球菌中的天然生产。这样，就有机会应用和完善工程策略，用于操纵红霉素生物合成途径，并过量生产这种和其他复杂天然化合物。以前，通过使用分别可选择的表达质粒引入整个红霉素途径（总共 20 个基因），使大肠杆菌能够进行衍生生产，但这会通过代谢负担和质粒不稳定性对最终生物合成产生负面影响。在这项研究中，通过改变引入生物合成途径所需的表达质粒的设计，对最终生产进行了改进。具体来说，减少了基因和质粒的总数，以降低代谢负担和质粒不稳定性。此外，还比较了种特异性（大肠杆菌与变铅青链霉菌）蛋白伴侣素。结果表明，在生长和质粒保留指标方面有所改善。新设计的表达平台还将红霉素 A 的产量提高了 5 倍。总之，本报告中概述的步骤旨在升级大肠杆菌红霉素 A 生产系统，提高最终化合物的产量，并提出了进一步改进异源生产尝试结果的其他途径工程形式。
