Random Chromosomal Integration and Screening Yields E. coli K-12 Derivatives Capable of Efficient Sucrose Utilization,ACS Synthetic Biology

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Random Chromosomal Integration and Screening Yields E. coli K-12 Derivatives Capable of Efficient Sucrose Utilization
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2020-11-25 , DOI: 10.1021/acssynbio.0c00392
David N. Carruthers ₁ , Tatyana E. Saleski ₁ , Scott A. Scholz ₂ , Xiaoxia Nina Lin ₁

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Chromosomal expression of heterologous genes offers stability and maintenance advantages over episomal expression, yet remains difficult to optimize through site-specific integration. The challenge has in large part been due to the variability of chromosomal gene expression, which has only recently been shown to be affected by multiple factors, including the local genomic context. In this work we utilize Tn5 transposase to randomly integrate a three-gene csc operon encoding nonphosphotransferase sucrose catabolism into the E. coli K-12 chromosome. Isolates from the transposon library yielded a range of growth rates on sucrose as the sole carbon source, including some that were comparable to that of E. coli K-12 on glucose (μ_max = 0.70 ± 0.03 h^–1). Narrowness of the growth rate distributions and faster growth compared to plasmids indicate that efficient csc expression is attainable. Furthermore, enhanced growth rate upon transduction into strains that underwent adaptive laboratory evolution indicate that sucrose catabolism is not limiting to cellular growth. We also show that transduction of a csc fast-growth locus into an isobutanol production strain yields high titer (7.56 ± 0.25 g/L) on sucrose as the sole carbon source. Our results demonstrate that random integration is an effective strategy for optimizing heterologous expression within the context of cellular metabolism for both fast growth and biochemical production phenotypes.

中文翻译：

随机染色体整合和筛选产量能够有效利用蔗糖的大肠杆菌K-12衍生物

异源基因的染色体表达相对于游离表达具有稳定性和维持优势，但仍难以通过位点特异性整合来优化。挑战在很大程度上是由于染色体基因表达的可变性，直到最近才证明它受多种因素的影响，包括局部基因组环境。在这项工作中，我们利用Tn5转座酶将编码非磷酸转移酶蔗糖分解代谢的三基因csc操纵子随机整合到大肠杆菌K-12染色体中。转座子文库中的分离物以蔗糖为唯一碳源产生了一系列的生长速率，其中包括一些与大肠杆菌K-12的葡萄糖相当的生长速率（_最大= 0.70±0.03 h ^–1）。与质粒相比，生长速率分布较窄且生长较快，表明可获得有效的csc表达。此外，转导到适应性实验室进化的菌株中后，生长速率的提高表明蔗糖分解代谢不仅限于细胞生长。我们还表明，将csc快速生长基因座转导到异丁醇生产菌株中，在蔗糖作为唯一碳源的情况下可产生高滴度（7.56±0.25 g / L）。我们的结果表明，随机整合是在细胞代谢范围内优化异源表达的有效策略，可用于快速生长和生化产生表型。

更新日期：2020-12-18

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