Genetic Engineering & Fermentation Technology, Instituto de Biología Celular y Molecular de Rosario-CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, Rosario, 2000, República Argentina.
Microb Cell Fact. 2012 Nov 7;11:147. doi: 10.1186/1475-2859-11-147.
Synthetic biology approaches can make a significant contribution to the advance of metabolic engineering by reducing the development time of recombinant organisms. However, most of synthetic biology tools have been developed for Escherichia coli. Here we provide a platform for rapid engineering of C. glutamicum, a microorganism of great industrial interest. This bacteria, used for decades for the fermentative production of amino acids, has recently been developed as a host for the production of several economically important compounds including metabolites and recombinant proteins because of its higher capacity of secretion compared to traditional bacterial hosts like E. coli. Thus, the development of modern molecular platforms may significantly contribute to establish C. glutamicum as a robust and versatile microbial factory.
A plasmid based platform named pTGR was created where all the genetic components are flanked by unique restriction sites to both facilitate the evaluation of regulatory sequences and the assembly of constructs for the expression of multiple genes. The approach was validated by using reporter genes to test promoters, ribosome binding sites, and for the assembly of dual gene operons and gene clusters containing two transcriptional units. Combinatorial assembly of promoter (tac, cspB and sod) and RBS (lacZ, cspB and sod) elements with different strengths conferred clear differential gene expression of two reporter genes, eGFP and mCherry, thus allowing transcriptional "fine-tuning"of multiple genes. In addition, the platform allowed the rapid assembly of operons and genes clusters for co-expression of heterologous genes, a feature that may assist metabolic pathway engineering.
We anticipate that the pTGR platform will contribute to explore the potential of novel parts to regulate gene expression, and to facilitate the assembly of genetic circuits for metabolic engineering of C. glutamicum. The standardization provided by this approach may provide a means to improve the productivity of biosynthetic pathways in microbial factories for the production of novel compounds.
合成生物学方法可以通过减少重组生物的开发时间，为代谢工程的发展做出重大贡献。然而，大多数合成生物学工具都是为大肠杆菌开发的。在这里，我们提供了一个快速工程化谷氨酸棒杆菌的平台，谷氨酸棒杆菌是一种具有重要工业价值的微生物。几十年来，该细菌一直被用于发酵生产氨基酸，最近由于其比传统细菌宿主如大肠杆菌具有更高的分泌能力，已被开发为生产几种具有经济重要性的化合物的宿主，包括代谢物和重组蛋白。因此，现代分子平台的发展可能会极大地促进谷氨酸棒杆菌作为一个强大而通用的微生物工厂的建立。
创建了一个基于质粒的平台，命名为 pTGR，其中所有的遗传元件都被独特的限制酶切位点包围，以方便评估调控序列和构建多个基因表达的构建体。该方法通过使用报告基因来测试启动子、核糖体结合位点，以及组装双基因操纵子和包含两个转录单元的基因簇来验证。不同强度的启动子（ tac 、 cspB 和 sod ）和 RBS （ lacZ 、 cspB 和 sod ）元件的组合组装赋予了两个报告基因，即 eGFP 和 mCherry 的明确的差异基因表达，从而实现了多个基因的转录“微调”。此外，该平台允许快速组装操纵子和基因簇，以实现异源基因的共表达，这一特性可能有助于代谢途径工程。
我们预计 pTGR 平台将有助于探索新型调控基因表达的元件的潜力，并促进谷氨酸棒杆菌代谢工程中基因电路的组装。该方法提供的标准化可能为提高微生物工厂中生物合成途径的生产力提供一种手段，以生产新型化合物。
