Programming Cells by Multicopy Chromosomal Integration Using CRISPR-Associated Transposases

Abstract

Directed evolution and targeted genome editing have been deployed to create genetic variants with usefully altered phenotypes. However, these methods are limited to high-throughput screening methods or serial manipulation of single genes. In this study, we implemented multicopy chromosomal integration using CRISPRassociated transposases (MUCICAT) to simultaneously target up to 11 sites on the Escherichia coli chromosome for multiplex gene interruption and/or insertion, generating combinatorial genomic diversity. The MUCICAT system was improved by replacing the isopropyl-beta-D-thiogalactoside (IPTG)-dependent promoter to decouple gene editing and product synthesis and truncating the right end to reduce the leakage expression of cargo. We applied MUCICAT to engineer and optimize the N-acetylglucosamine (GlcNAc) biosynthesis pathway in E. coli to overproduce the industrially important GlcNAc in only 8 days. Two rounds of transformation, the first round for disruption of two degradation pathways related gene clusters and the second round for multiplex integration of the GlcNAc gene cassette, would generate a library with 1-11 copies of the GlcNAc cassette. We isolated a best variant with five copies of GlcNAc cassettes, producing 11.59 g/L GlcNAc, which was more than sixfold than that of the strain containing the pET-GNAc plasmid. Our multiplex approach MUCICAT has potential to become a powerful tool of cell programing and can be widely applied in many fields such as synthetic biology.

Transposon mutagenesis libraries reveal novel molecular requirements during CRISPR RNA-guided DNA integration

“…We also further investigated the importance of a palindromic sequence found 97–107 bp from the transposon right end boundary. Previous work suggested that this sequence might affect integration orientation, possibly by promoting transcription of the tnsABC operon, which would be consistent with empirical expression data and the AT-richness of the transposon end (48). To test this possibility, we mutated the palindromic sequence and found that variants with this sequence shifted the orientation preference towards T-LR, with just one arm of the palindrome (P B ) being sufficient to shift the orientation bias (Figure S4D-E).…”

Whole‐Cell P450 Biocatalysis Using Engineered Escherichia coli with Fine‐Tuned Heme Biosynthesis

“…To enhance plasmid‐free heme synthesis, the fragment of pT7‐ hemH ‐pT7‐ ADB1‐hemB‐ADB3‐hemD‐ADB2‐hemC were multicopy chromosomal integrated using the CRISPR‐associated transposases (MUCICAT). [ 63,64 ] At first, plasmids pRE57I‐ADB‐hemBDC‐hemH, pTnsABC, and pQCascade were co‐transformed into E. coli C41(DE3) by electro‐transformation, and the transformants were obtained by incubation on triple antibiotic LB‐agar plate (50 µg mL −1 kanamycin, 100 µg mL −1 ampicillin, and 100 µg mL −1 streptomycin) for 16 h at 37 °C. In the following, the transformants were induced by incubation on the triple antibiotic LB‐agar plate containing different concentrations of anhydrotetracycline (100 ng mL −1 and 1000 ng mL −1 ) for 16 h at 37 °C, [ 64 ] and the copy numbers of pT7‐ hemH ‐pT7‐ ADB1‐hemB‐ADB3‐hemD‐ADB2‐hemC in obtained colonies were verified by colony PCR.…”

“…To enhance plasmid-free heme synthesis, the fragment of pT7-hemH-pT7-ADB1-hemB-ADB3-hemD-ADB2-hemC were multicopy chromosomal integrated using the CRISPR-associated transposases (MUCICAT). [63,64] At first, plasmids pRE57I-ADB-hemBDC-hemH, pTnsABC, and pQCascade were co-transformed into E. coli C41(DE3) by electro-transformation, and the transformants were obtained by incubation on triple antibiotic LB-agar plate (50 μg mL −1 kanamycin, 100 μg mL −1 ampicillin, and 100 μg mL −1 streptomycin) for 16 h at 37 °C. In the following, the transformants were induced by incubation on the triple antibiotic LB-agar plate containing different concentrations of anhydrotetracycline (100 ng mL −1 and 1000 ng mL −1 ) for 16 h at 37 °C, [64] and the copy numbers of pT7-hemH-pT7-ADB1-hemB-ADB3-hemD-ADB2-hemC in obtained colonies were verified by colony PCR.…”

Whole-cell P450 biocatalysts using engineered Escherichia coli with fine-tuned heme supply

“…The hemA gene was deleted using the one-step gene inactivation method 53 . The CRISPR-Cas9 system 54 and the CRISPR-associated transposases (MUCICAT) 55 were used to overexpress endogenous heme biosynthetic genes. The plasmids pTetQCas-8 + IS186 (MC_0101250), pRE57I-GNAc (MC_0101251), pTnsABC (MC_0101189), and pCutamp (MC_0101104) were obtained from Molecular Cloud.…”