CRISPR EnAbled Trackable genome Engineering for isopropanol production in Escherichia coli

Abstract

Isopropanol is an important target molecule for sustainable production of fuels and chemicals. Increases in DNA synthesis and synthetic biology capabilities have resulted in the development of a range of new strategies for the more rapid design, construction, and testing of production strains. Here, we report on the use of such capabilities to construct and test 903 different variants of the isopropanol production pathway in Escherichia coli. We first constructed variants to explore the effect of codon optimization, copy number, and translation initiation rates on isopropanol production. The best strain (PA06) produced isopropanol at titers of 17.5g/L, with a yield of 0.62 (mol/mol), and maximum productivity of 0.40g/L/h. We next integrated the isopropanol synthetic pathway into the genome and then used the CRISPR EnAbled Trackable genome Engineering (CREATE) strategy to generate an additional 640 individual RBS library variants for further evaluation. After testing each of these variants, we constructed a combinatorial library containing 256 total variants from four different RBS levels for each gene. The best producing variant, PA14, produced isopropanol at titers of 7.1g/L at 24h, with a yield of 0.75 (mol/mol), and maximum productivity of 0.62g/L/h (which was 0.22g/L/h above the parent strain PA07). We demonstrate the ability to rapidly construct and test close to ~1000 designer strains and identify superior performers.

Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape

“…RBS libraries integration for 5 genes involved in isopropanol production [64] CRISPRi Escherichia coli…”

“…For example, Shi et al specifically designed gRNAs to target multiple delta sites in the yeast genome, ultimately achieving 18-copy genomic integrations of a 24 kb combined xylose utilization and (R,R)-2,3-butanediol (BDO) production pathway in a single step, in S. cerevisiae [88]. DNA libraries, such as error-prone PCRs derived or double-stranded fragments obtained from DNA synthesizing companies, can be genomically integrated to find variants of a studied enzyme with enhanced catalytic activities or optimal level of expression [64,83]. Genomically integrated DNA libraries offer several advantages compared to plasmid based strategies, especially in terms of expression stability [83].…”

“…Genomically integrated DNA libraries offer several advantages compared to plasmid based strategies, especially in terms of expression stability [83]. Liang et al used that strategy to integrate 640 ribosome binding sites (RBS) for five different enzymes involved in the production of isopropanol in E. coli [64] (Fig. 3a).…”

“…After multiple rounds of screening for strains carrying the best RBS variants, i.e. optimal expression of the gene leading to a higher isopropanol titer, a final strain producing 7.1 g L −1 of isopropanol was obtained [64].…”

CRISPR /Cas9 recombineering‐mediated deep mutational scanning of essential genes in Escherichia coli

“…The editing cassettes on the plasmid also serve as the barcode to track the mutations before and after selection to assign fitness scores to each mutation (Garst et al , ). The technology has been used for directed evolution of E. coli proteins, pathways, and strains (Shalem et al , ; Cobb et al , ; Cho et al , ; Liang et al , ; Liu et al , ; Lu et al , ; Wu et al , ,b; Zhu et al , ; Bassalo et al , ). However, applying CREATE for DMS proved to be challenging.…”