Karim Ann A, Gestaut Daniel R, Fincker Maeva, Ruth John C, Holmes Eric C, Sheu Wayne, Spormann Alfred M
Department of Civil and Environmental Engineering , Stanford University , Stanford , California 94305 , United States.
Department of Biology , Stanford University , Stanford , California 94305 , United States.
ACS Synth Biol. 2018 Aug 17;7(8):1874-1885. doi: 10.1021/acssynbio.8b00062. Epub 2018 Jul 13.
Methanogenic archaea can be integrated into a sustainable, carbon-neutral cycle for producing organic chemicals from C compounds if the rate, yield, and titer of product synthesis can be improved using metabolic engineering. However, metabolic engineering techniques are limited in methanogens by insufficient methods for controlling cellular protein levels. We conducted a systematic approach to tune protein levels in Methanosarcina acetivorans C2A, a model methanogen, by regulating transcription and translation initiation. Rationally designed core promoter and ribosome binding site mutations in M. acetivorans C2A resulted in a predicable change in protein levels over a 60 fold range. The overall range of protein levels was increased an additional 3 fold by introducing the 5' untranslated region of the mcrB transcript. This work demonstrates a wide range of precisely controlled protein levels in M. acetivorans C2A, which will help facilitate systematic metabolic engineering efforts in methanogens.
如果能够通过代谢工程提高产物合成的速率、产量和滴度，产甲烷古菌就可以被整合到一个可持续的、碳中和的循环中，用于从碳化合物生产有机化学品。然而，由于控制细胞蛋白质水平的方法不足，代谢工程技术在产甲烷菌中受到限制。我们采用了一种系统的方法，通过调节转录和翻译起始来调节模式产甲烷菌嗜乙酸甲烷八叠球菌C2A中的蛋白质水平。在嗜乙酸甲烷八叠球菌C2A中合理设计的核心启动子和核糖体结合位点突变导致蛋白质水平在60倍的范围内发生可预测的变化。通过引入mcrB转录本的5'非翻译区，蛋白质水平的总体范围又增加了3倍。这项工作证明了嗜乙酸甲烷八叠球菌C2A中蛋白质水平的广泛精确控制，这将有助于推动产甲烷菌的系统代谢工程研究。
