质粒
生物
代谢途径
生物制品
计算生物学
遗传学
代谢工程
基因
生物技术
生物燃料
作者
Garima Goyal,Zak Costello,Jorge Alonso-Gutiérrez,Aram Kang,Taek Soon Lee,Héctor García Martín,Nathan J. Hillson
标识
DOI:10.1021/acssynbio.8b00243
摘要
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.
科研通智能强力驱动
Strongly Powered by AbleSci AI