Parallel Integration and Chromosomal Expansion of Metabolic Pathways,ACS Synthetic Biology

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Parallel Integration and Chromosomal Expansion of Metabolic Pathways
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-10-23 00:00:00 , DOI: 10.1021/acssynbio.8b00243
Garima Goyal _{1,

2} , Zak Costello _{1,

2} , Jorge Alonso-Gutierrez ₂ , Aram Kang ₂ , Taek Soon Lee ₂ , Hector Garcia Martin _{1,

2} , Nathan J. Hillson _{1,

2}

Affiliation

Robust fermentation of biomass-derived sugars into bioproducts demands the reliable microbial expression of metabolic pathways. Plasmid-based expression systems may suffer from instability and result in highly variable titers, rates, and yields. An established mitigation approach, chemical induced chromosomal expansion (CIChE), expands a singly integrated pathway to plasmid-like copy numbers while maintaining stability in the absence of antibiotic selection pressure. Here, we report parallel integration and chromosomal expansion (PIACE), extensions to CIChE that enable independent expansions of pathway components across multiple loci, use suicide vectors to achieve high-efficiency site-specific integration of sequence-validated multigene components, and introduce a heat-curable plasmid to obviate recA deletion post pathway expansion. We applied PIACE to stabilize an isopentenol pathway across three loci in E. coli DH1 and then generate libraries of pathway component copy number variants to screen for improved titers. Polynomial regressor statistical modeling of the production screening data suggests that increasing copy numbers of all isopentenol pathway components would further improve titers.

中文翻译：

代谢途径的平行整合和染色体扩展

从生物质衍生的糖到生物产品的强劲发酵需要代谢途径的可靠微生物表达。基于质粒的表达系统可能会不稳定，并导致滴度，速率和产量高度可变。一种成熟的缓解方法是化学诱导的染色体扩增（CIChE），可将单整合途径扩展至质粒样拷贝数，同时在没有抗生素选择压力的情况下保持稳定性。在这里，我们报告了平行整合和染色体扩展（PIACE），CIChE的扩展，使跨多个基因座的途径组分能够独立扩展，使用自杀载体实现了序列验证的多基因组分的高效位点特异性整合，并引入了热量可固化质粒消除recA途径扩展后缺失。我们应用PIACE稳定了大肠杆菌DH1中三个位点的异戊烯醇途径，然后生成途径组分拷贝数变异体的文库，以筛选提高的滴度。生产筛选数据的多项式回归统计模型表明，增加所有异戊烯醇途径组分的拷贝数将进一步提高效价。

更新日期：2018-10-23

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