Rau Martin Holm, Calero Patricia, Lennen Rebecca M, Long Katherine S, Nielsen Alex T
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
Microb Cell Fact. 2016 Oct 13;15(1):176. doi: 10.1186/s12934-016-0577-5.
Economically viable biobased production of bulk chemicals and biofuels typically requires high product titers. During microbial bioconversion this often leads to product toxicity, and tolerance is therefore a critical element in the engineering of production strains.
Here, a systems biology approach was employed to understand the chemical stress response of Escherichia coli, including a genome-wide screen for mutants with increased fitness during chemical stress. Twelve chemicals with significant production potential were selected, consisting of organic solvent-like chemicals (butanol, hydroxy-γ-butyrolactone, 1,4-butanediol, furfural), organic acids (acetate, itaconic acid, levulinic acid, succinic acid), amino acids (serine, threonine) and membrane-intercalating chemicals (decanoic acid, geraniol). The transcriptional response towards these chemicals revealed large overlaps of transcription changes within and between chemical groups, with functions such as energy metabolism, stress response, membrane modification, transporters and iron metabolism being affected. Regulon enrichment analysis identified key regulators likely mediating the transcriptional response, including CRP, RpoS, OmpR, ArcA, Fur and GadX. These regulators, the genes within their regulons and the above mentioned cellular functions therefore constitute potential targets for increasing E. coli chemical tolerance. Fitness determination of genome-wide transposon mutants (Tn-seq) subjected to the same chemical stress identified 294 enriched and 336 depleted mutants and experimental validation revealed up to 60 % increase in mutant growth rates. Mutants enriched in several conditions contained, among others, insertions in genes of the Mar-Sox-Rob regulon as well as transcription and translation related gene functions.
The combination of the transcriptional response and mutant screening provides general targets that can increase tolerance towards not only single, but multiple chemicals.
从经济可行性角度来看，基于生物基生产大宗化学品和生物燃料通常需要高产物滴度。在微生物生物转化过程中，这往往会导致产物毒性，因此耐受性是生产菌株工程改造中的关键因素。
在此，采用系统生物学方法来了解大肠杆菌的化学应激反应，包括对化学应激期间适应性增强的突变体进行全基因组筛选。选择了12种具有显著生产潜力的化学品，包括类似有机溶剂的化学品（丁醇、羟基-γ-丁内酯、1,4-丁二醇、糠醛）、有机酸（乙酸、衣康酸、乙酰丙酸、琥珀酸）、氨基酸（丝氨酸、苏氨酸）以及膜插入化学品（癸酸、香叶醇）。对这些化学品的转录反应揭示了化学组内和化学组间转录变化的大量重叠，能量代谢、应激反应、膜修饰、转运蛋白和铁代谢等功能受到影响。调控子富集分析确定了可能介导转录反应的关键调控因子，包括CRP、RpoS、OmpR、ArcA、Fur和GadX。因此，这些调控因子、其调控子内的基因以及上述细胞功能构成了提高大肠杆菌化学耐受性的潜在靶点。对遭受相同化学应激的全基因组转座子突变体（Tn-seq）进行适应性测定，鉴定出294个富集突变体和336个缺失突变体，实验验证表明突变体生长速率提高了60%。在几种条件下富集的突变体中，除其他外，还包括Mar-Sox-Rob调控子基因以及转录和翻译相关基因功能的插入。
转录反应和突变体筛选的结合提供了可提高对单一化学品以及多种化学品耐受性的通用靶点。
