Iterative genome editing of Escherichia coli for 3-hydroxypropionic acid production

作者：Liu Rongming; Liang Liya; Choudhury Alaksh; Bassalo Marcelo C; Garst Andrew D; Tarasava Katia; Gill Ryan T^*

来源：Metabolic Engineering, 2018, 47: 303-313.

DOI：10.1016/j.ymben.2018.04.007

• Genome engineering
• Combinatorial engineering
• 3-hydroxypropionic acid
• High-throughput

Synthetic biology requires strategies for the targeted, efficient, and combinatorial engineering of biological subsystems at the molecular level. Here, we report the use of the iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method for the rapid construction of combinatorially modified genomes. We coupled this genome engineering strategy with high-throughput phenotypic screening and selections to recursively engineer multiple traits in Escherichia coil for improved production of the platform chemical 3-hydroxypropionic acid (3HP). Specifically, we engineered i) central carbon metabolism, ii) 3HP synthesis, and (iii) 3HP tolerance through design, construction and testing of similar to 162,000 mutations across 115 genes spanning global regulators, transcription factors, and enzymes involved in 3HP synthesis and tolerance. The iCREATE process required similar to 1 month to perform 13 rounds of combinatorial genome modifications with targeted gene knockouts, expression modification by ribosomal binding site (RBS) engineering, and genome-level site-saturation mutagenesis. Specific mutants conferring increased 3HP titer, yield, and productivity were identified and then combined to produce 3HP at a yield and concentration similar to 60-fold higher than the wild-type strain.

• 出版日期2018-5