Coussement Pieter, Bauwens David, Maertens Jo, De Mey Marjan
Department of Biochemical and Microbial Technology, Ghent University , Coupure Links 653, 9000 Gent, Belgium.
ACS Synth Biol. 2017 Feb 17;6(2):224-232. doi: 10.1021/acssynbio.6b00122. Epub 2016 Oct 11.
Combinatorial engineering approaches are becoming increasingly popular, yet they are hindered by the lack of specialized techniques for both efficient introduction of sequence variability and assembly of numerous DNA parts, required for the construction of lengthy multigene pathways. In this contribution, we introduce a new combinatorial multigene pathway assembly scheme based on Single Strand Assembly (SSA) methods and Golden Gate Assembly, exploiting the strengths of both assembly techniques. With a minimum of intermediary steps and an accompanying set of well-characterized and ready-to-use genetic parts, the developed workflow allows effective introduction of various libraries and efficient assembly of multigene pathways. It was put to the test by optimizing the lycopene pathway as proof-of-principle. The here constructed libraries yield ample variation in lycopene production. In addition, good-performing transformants with a significantly higher lycopene production were obtained as compared to previously published reference strains. The best selected producer yielded 3-fold improvement in lycopene titers up to 448 mg lycopene/g CDW. The proposed workflow in combination with the accompanying sets of ready-to-use expression and carrier plasmids, will allow the combinatorial assembly of increasingly lengthy product pathways with minimal effort.
组合工程方法正变得越来越流行，但它们受到缺乏专门技术的阻碍，这些技术既需要高效引入序列变异性，又需要组装构建冗长多基因途径所需的众多DNA部件。在本论文中，我们基于单链组装（SSA）方法和金门组装，引入了一种新的组合多基因途径组装方案，利用了这两种组装技术的优势。通过最少的中间步骤以及一组经过充分表征且随时可用的遗传部件，所开发的工作流程能够有效引入各种文库并高效组装多基因途径。通过优化番茄红素途径作为原理验证对其进行了测试。在此构建的文库在番茄红素产量方面产生了丰富的变异。此外，与先前发表的参考菌株相比，获得了番茄红素产量显著更高的优良转化体。最佳选择的生产者使番茄红素滴度提高了3倍，达到448毫克番茄红素/克细胞干重。所提出的工作流程与随附的随时可用的表达和载体质粒组合，将以最小的努力实现越来越长的产物途径的组合组装。
